pax_global_header00006660000000000000000000000064137066376320014527gustar00rootroot0000000000000052 comment=4a411a92213a5dd0a56e5f528bc994a7d831c3a7 hisat2-2.2.1/000077500000000000000000000000001370663763200127235ustar00rootroot00000000000000hisat2-2.2.1/.gitattributes000066400000000000000000000000351370663763200156140ustar00rootroot00000000000000*.pbxproj binary merge=union hisat2-2.2.1/.gitignore000066400000000000000000000013111370663763200147070ustar00rootroot00000000000000*~ *.dSYM .DS_Store tags *-debug *-s *-l hisat2.xcodeproj/project.xcworkspace hisat2.xcodeproj/xcuserdata hisat2.xcodeproj/xcshareddata *.patch build_automaton build_index clean_alignment determinize gcsa_alignment gcsa_test hisat2-repeat hisat2_test/*.bt2 hisat2_test/*.ht2 hisat2_test/*.sam hisat2_test/paper_example.malignment.automaton hisat2_test/paper_example.malignment.backbone hisat2_test/paper_example.malignment.gcsa hisat2_test/kim_example*.malignment.automaton hisat2_test/kim_example*.malignment.backbone hisat2_test/kim_example*.malignment.gcsa hisat2_test/genome* hisat2_test/2* hisat2_test/snp142* hisat2_test/testset* .idea .vscode .ht2lib-obj* *.a *.so docs/_site docs/*.lock docs/.*-cache hisat2-2.2.1/AUTHORS000066400000000000000000000024251370663763200137760ustar00rootroot00000000000000Ben Langmead wrote Bowtie 2, which is based partially on Bowtie. Bowtie was written by Ben Langmead and Cole Trapnell. Bowtie & Bowtie 2: http://bowtie-bio.sf.net A DLL from the pthreads for Win32 library is distributed with the Win32 version of Bowtie 2. The pthreads for Win32 library and the GnuWin32 package have many contributors (see their respective web sites). pthreads for Win32: http://sourceware.org/pthreads-win32 GnuWin32: http://gnuwin32.sf.net The ForkManager.pm perl module is used in Bowtie 2's random testing framework, and is included as scripts/sim/contrib/ForkManager.pm. ForkManager.pm is written by dLux (Szabo, Balazs), with contributions by others. See the perldoc in ForkManager.pm for the complete list. The file ls.h includes an implementation of the Larsson-Sadakane suffix sorting algorithm. The implementation is by N. Jesper Larsson and was adapted somewhat for use in Bowtie 2. TinyThreads is a portable thread implementation with a fairly compatible subset of C++11 thread management classes written by Marcus Geelnard. For more info check http://tinythreadpp.bitsnbites.eu/ Various users have kindly supplied patches, bug reports and feature requests over the years. Many, many thanks go to them. September 2011 hisat2-2.2.1/CMakeLists.txt000066400000000000000000000134271370663763200154720ustar00rootroot00000000000000cmake_minimum_required(VERSION 3.10) project(hisat2) # escape preprocessor definition cmake_policy(SET CMP0005 NEW) if(NOT CMAKE_BUILD_TYPE) set(CMAKE_BUILD_TYPE Release) endif() option(BOWTIE_MM "enable memory mapping" ON) option(BOWTIE_SHARED_MEM "enable shared memory mapping" OFF) option(USE_SRA "enable NCBI SRA Toolkit" OFF) set(CMAKE_CXX_STANDARD 11) set(CMAKE_VERBOSE_MAKEFILE true) set(CMAKE_EXPORT_COMPILE_COMMANDS on) set(NCBI_NGS_DIR ${NCBI_NGS_DIR}) # Common Flag set(CMAKE_CXX_FLAGS "${CMAKE_CXX_FLAGS} -m64 -g3 -Wall -msse2") # Debug/Release Flag set(CMAKE_CXX_FLAGS_DEBUG "${CMAKE_CXX_FLAGS_DEBUG} -O0") set(CMAKE_CXX_FLAGS_RELEASE "${CMAKE_CXX_FLAGS_RELEASE} -O3 -funroll-loops") set(INSTALL_DIR "${CMAKE_INSTALL_PREFIX}/bin" CACHE PATH "Installation directory for executables") if(MINGW OR CYGWIN) option(BOWTIE_MM "Memory mapped files not supported on Windows" OFF) option(BOWTIE_SHARED_MEM "Shared memory not supported on Windows" OFF) endif() if (USE_SRA AND APPLE) set(CMAKE_CXX_FLAGS "${CMAKE_CXX_FLAGS} -mmacosx-version-min=10.6") endif() set(HISAT2_BIN_LIST hisat2-build-s hisat2-build-l hisat2-align-s hisat2-align-l hisat2-inspect-s hisat2-inspect-l hisat2-repeat ) set(HISAT2_LIB_LIST hisat2lib ) # Source Codes set(SHARED_CPPS alphabet.cpp ccnt_lut.cpp ds.cpp edit.cpp gfm.cpp limit.cpp multikey_qsort.cpp random_source.cpp reference.cpp ref_read.cpp shmem.cpp tinythread.cpp ) set(SEARCH_CPPS aligner_bt.cpp aligner_cache.cpp aligner_driver.cpp aligner_result.cpp aligner_seed.cpp aligner_seed2.cpp aligner_seed_policy.cpp aligner_sw.cpp aligner_sw_driver.cpp aligner_swsse.cpp aligner_swsse_ee_i16.cpp aligner_swsse_ee_u8.cpp aligner_swsse_loc_i16.cpp aligner_swsse_loc_u8.cpp aln_sink.cpp dp_framer.cpp outq.cpp pat.cpp pe.cpp presets.cpp qual.cpp random_util.cpp read_qseq.cpp ref_coord.cpp mask.cpp scoring.cpp simple_func.cpp splice_site.cpp sse_util.cpp unique.cpp hisat2_main.cpp hisat2.cpp ) set(BUILD_CPPS diff_sample.cpp hisat2_build_main.cpp hisat2_build.cpp ) set(REPEAT_CPPS aligner_bt.cpp aligner_result.cpp aligner_sw_driver.cpp aligner_sw.cpp aligner_swsse_ee_i16.cpp aligner_swsse_ee_u8.cpp aligner_swsse_loc_i16.cpp aligner_swsse_loc_u8.cpp aligner_swsse.cpp bit_packed_array.cpp bit_packed_array.h dp_framer.cpp mask.cpp qual.cpp repeat_builder.cpp scoring.cpp simple_func.cpp diff_sample.cpp hisat2_repeat_main.cpp hisat2_repeat.cpp ) set(INSPECT_CPPS hisat2_inspect.cpp ) set(HT2LIB_CPPS hisat2lib/ht2_init.cpp hisat2lib/ht2_repeat.cpp hisat2lib/ht2_index.cpp ) string(TIMESTAMP BUILD_DATE) file(STRINGS VERSION HISAT2_VERSION) execute_process(COMMAND ${CMAKE_CXX_COMPILER} -v OUTPUT_VARIABLE COMPILER_VERSION) add_definitions( -DBOWTIE2 -DBUILD_HOST="${CMAKE_HOST_SYSTEM}" -DBUILD_TIME="${BUILD_DATE}" -DCOMPILER_VERSION="${CMAKE_CXX_COMPILER} ${CMAKE_CXX_COMPILER_VERSION}" -DHISAT2_VERSION="${HISAT2_VERSION}" -D_LARGEFILE_SOURCE -D_FILE_OFFSET_BITS=64 -D_GNU_SOURCE -DPOPCNT_CAPABILITY ) include_directories(third_party) link_libraries( pthread ) if(BOWTIE_MM) add_definitions(-DBOWTIE_MM) endif() if(BOWTIE_SHARED_MEM) add_definitions(-DBOWTIE_SHARED_MEM) endif() include_directories(${PROJECT_SOURCE_DIR}) add_definitions(-DCOMPILER_OPTIONS="${CMAKE_CXX_FLAGS}") add_library(hisat2lib_static STATIC ${HT2LIB_CPPS} ${SHARED_CPPS}) add_library(hisat2lib_shared SHARED ${HT2LIB_CPPS} ${SHARED_CPPS}) add_custom_target(ht2lib) add_dependencies(ht2lib hisat2lib_static hisat2lib_shared) set_target_properties(hisat2lib_static PROPERTIES OUTPUT_NAME hisat2lib) set_target_properties(hisat2lib_shared PROPERTIES OUTPUT_NAME hisat2lib) add_executable(hisat2-align-s ${SEARCH_CPPS} ${SHARED_CPPS}) add_executable(hisat2-align-l ${SEARCH_CPPS} ${SHARED_CPPS}) add_executable(hisat2-build-s ${BUILD_CPPS} ${SHARED_CPPS}) add_executable(hisat2-build-l ${BUILD_CPPS} ${SHARED_CPPS}) add_executable(hisat2-inspect-s ${INSPECT_CPPS} ${SHARED_CPPS}) add_executable(hisat2-inspect-l ${INSPECT_CPPS} ${SHARED_CPPS}) add_executable(hisat2-repeat ${REPEAT_CPPS} ${SHARED_CPPS}) set_target_properties(${HISAT2_BIN_LIST} PROPERTIES DEBUG_POSTFIX "-debug") set_target_properties(hisat2-align-l hisat2-build-l hisat2-inspect-l hisat2-repeat PROPERTIES COMPILE_FLAGS "${COMPILE_FLAGS} -DBOWTIE_64BIT_INDEX") target_compile_options(hisat2-inspect-s PUBLIC "-DHISAT2_INSPECT_MAIN") target_compile_options(hisat2-inspect-l PUBLIC "-DHISAT2_INSPECT_MAIN") target_compile_options(hisat2-build-s PUBLIC "-DMASSIVE_DATA_RLCSA") target_compile_options(hisat2lib_static PUBLIC "-fPIC") target_include_directories(hisat2lib_static PUBLIC hisat2lib) target_include_directories(hisat2lib_shared PUBLIC hisat2lib) if(USE_SRA) if(NOT NCBI_NGS_DIR) message(FATAL_ERROR "You should set NCBI_NGS_DIR path. cmake3 .... -DNCBI_NGS_DIR=/path/to/ngs") endif() #set(NCBI_NGS_DIR /home/parkch/.local/ngs) target_compile_options(hisat2-align-s PUBLIC "-DUSE_SRA") target_compile_options(hisat2-align-l PUBLIC "-DUSE_SRA") target_include_directories(hisat2-align-s PUBLIC "${NCBI_NGS_DIR}/include") target_include_directories(hisat2-align-l PUBLIC "${NCBI_NGS_DIR}/include") set(NCBI_LIBS ${NCBI_NGS_DIR}/lib64/libncbi-ngs-c++-static.a ${NCBI_NGS_DIR}/lib64/libngs-c++-static.a ${NCBI_NGS_DIR}/lib64/libncbi-vdb-static.a ${CMAKE_DL_LIBS} ) target_link_libraries(hisat2-align-s ${NCBI_LIBS}) target_link_libraries(hisat2-align-l ${NCBI_LIBS}) endif() add_custom_target(hisat2-all) add_dependencies(hisat2-all hisat2-align-s hisat2-align-l hisat2-build-s hisat2-build-l hisat2-inspect-s hisat2-inspect-l hisat2-repeat) # 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But first, please read . hisat2-2.2.1/MANUAL000066400000000000000000001774311370663763200136400ustar00rootroot00000000000000 Introduction ============ What is HISAT2? ----------------- HISAT2 is a fast and sensitive alignment program for mapping next-generation sequencing reads (whole-genome, transcriptome, and exome sequencing data) against the general human population (as well as against a single reference genome). Based on [GCSA] (an extension of [BWT] for a graph), we designed and implemented a graph FM index (GFM), an original approach and its first implementation to the best of our knowledge. In addition to using one global GFM index that represents general population, HISAT2 uses a large set of small GFM indexes that collectively cover the whole genome (each index representing a genomic region of 56 Kbp, with 55,000 indexes needed to cover human population). These small indexes (called local indexes) combined with several alignment strategies enable effective alignment of sequencing reads. This new indexing scheme is called Hierarchical Graph FM index (HGFM). We have developed HISAT 2 based on the [HISAT] and [Bowtie2] implementations. HISAT2 outputs alignments in [SAM] format, enabling interoperation with a large number of other tools (e.g. [SAMtools], [GATK]) that use SAM. HISAT2 is distributed under the [GPLv3 license], and it runs on the command line under Linux, Mac OS X and Windows. [HISAT2]: http://ccb.jhu.edu/software/hisat2 [HISAT]: http://ccb.jhu.edu/software/hisat [Bowtie2]: http://bowtie-bio.sf.net/bowtie2 [Bowtie]: http://bowtie-bio.sf.net [Bowtie1]: http://bowtie-bio.sf.net [GCSA]: http://ieeexplore.ieee.org/xpls/abs_all.jsp?arnumber=6698337&tag=1 [Burrows-Wheeler Transform]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform [BWT]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform [FM Index]: http://en.wikipedia.org/wiki/FM-index [SAM]: http://samtools.sourceforge.net/SAM1.pdf [SAMtools]: http://samtools.sourceforge.net [GATK]: http://www.broadinstitute.org/gsa/wiki/index.php/The_Genome_Analysis_Toolkit [TopHat2]: http://ccb.jhu.edu/software/tophat [Cufflinks]: http://cufflinks.cbcb.umd.edu/ [Crossbow]: http://bowtie-bio.sf.net/crossbow [Myrna]: http://bowtie-bio.sf.net/myrna [Bowtie paper]: http://genomebiology.com/2009/10/3/R25 [GPLv3 license]: http://www.gnu.org/licenses/gpl-3.0.html Obtaining HISAT2 ================== Download HISAT2 sources and binaries from the Releases sections on the right side. Binaries are available for Intel architectures (`x86_64`) running Linux, and Mac OS X. Building from source -------------------- Building HISAT2 from source requires a GNU-like environment with GCC, GNU Make and other basics. It should be possible to build HISAT2 on most vanilla Linux installations or on a Mac installation with [Xcode] installed. HISAT2 can also be built on Windows using [Cygwin] or [MinGW] (MinGW recommended). For a MinGW build the choice of what compiler is to be used is important since this will determine if a 32 or 64 bit code can be successfully compiled using it. If there is a need to generate both 32 and 64 bit on the same machine then a multilib MinGW has to be properly installed. [MSYS], the [zlib] library, and depending on architecture [pthreads] library are also required. We are recommending a 64 bit build since it has some clear advantages in real life research problems. In order to simplify the MinGW setup it might be worth investigating popular MinGW personal builds since these are coming already prepared with most of the toolchains needed. First, download the [source package] from the Releases section on the right side. Unzip the file, change to the unzipped directory, and build the HISAT2 tools by running GNU `make` (usually with the command `make`, but sometimes with `gmake`) with no arguments. If building with MinGW, run `make` from the MSYS environment. HISAT2 is using the multithreading software model in order to speed up execution times on SMP architectures where this is possible. On POSIX platforms (like linux, Mac OS, etc) it needs the pthread library. Although it is possible to use pthread library on non-POSIX platform like Windows, due to performance reasons HISAT2 will try to use Windows native multithreading if possible. For the support of SRA data access in HISAT2, please download and install the [NCBI-NGS] toolkit. When running `make`, specify additional variables as follow. `make USE_SRA=1 NCBI_NGS_DIR=/path/to/NCBI-NGS-directory NCBI_VDB_DIR=/path/to/NCBI-NGS-directory`, where `NCBI_NGS_DIR` and `NCBI_VDB_DIR` will be used in Makefile for -I and -L compilation options. For example, $(NCBI_NGS_DIR)/include and $(NCBI_NGS_DIR)/lib64 will be used. [Cygwin]: http://www.cygwin.com/ [MinGW]: http://www.mingw.org/ [MSYS]: http://www.mingw.org/wiki/msys [zlib]: http://cygwin.com/packages/mingw-zlib/ [pthreads]: http://sourceware.org/pthreads-win32/ [GnuWin32]: http://gnuwin32.sf.net/packages/coreutils.htm [Download]: https://sourceforge.net/projects/bowtie-bio/files/bowtie2/ [sourceforge site]: https://sourceforge.net/projects/bowtie-bio/files/bowtie2/ [source package]: http://ccb.jhu.edu/software/hisat2/downloads/hisat2-2.0.0-beta-source.zip [Xcode]: http://developer.apple.com/xcode/ [NCBI-NGS]: https://github.com/ncbi/ngs/wiki/Downloads Running HISAT2 ============= Adding to PATH -------------- By adding your new HISAT2 directory to your [PATH environment variable], you ensure that whenever you run `hisat2`, `hisat2-build` or `hisat2-inspect` from the command line, you will get the version you just installed without having to specify the entire path. This is recommended for most users. To do this, follow your operating system's instructions for adding the directory to your [PATH]. If you would like to install HISAT2 by copying the HISAT2 executable files to an existing directory in your [PATH], make sure that you copy all the executables, including `hisat2`, `hisat2-align-s`, `hisat2-align-l`, `hisat2-build`, `hisat2-build-s`, `hisat2-build-l`, `hisat2-inspect`, `hisat2-inspect-s` and `hisat2-inspect-l`. [PATH environment variable]: http://en.wikipedia.org/wiki/PATH_(variable) [PATH]: http://en.wikipedia.org/wiki/PATH_(variable) Reporting --------- The reporting mode governs how many alignments HISAT2 looks for, and how to report them. In general, when we say that a read has an alignment, we mean that it has a [valid alignment]. When we say that a read has multiple alignments, we mean that it has multiple alignments that are valid and distinct from one another. By default, HISAT2 may soft-clip reads near their 5' and 3' ends. Users can control this behavior by setting different penalties for soft-clipping (`--sp`) or by disallowing soft-clipping (`--no-softclip`). ### Distinct alignments map a read to different places Two alignments for the same individual read are "distinct" if they map the same read to different places. Specifically, we say that two alignments are distinct if there are no alignment positions where a particular read offset is aligned opposite a particular reference offset in both alignments with the same orientation. E.g. if the first alignment is in the forward orientation and aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, and the second alignment is also in the forward orientation and also aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, they are not distinct alignments. Two alignments for the same pair are distinct if either the mate 1s in the two paired-end alignments are distinct or the mate 2s in the two alignments are distinct or both. ### Default mode: search for one or more alignments, report each HISAT2 searches for up to N distinct, primary alignments for each read, where N equals the integer specified with the `-k` parameter. Primary alignments mean alignments whose alignment score is equal or higher than any other alignments. It is possible that multiple distinct alignments have the same score. That is, if `-k 2` is specified, HISAT2 will search for at most 2 distinct alignments. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS field. See the [SAM specification] for details. HISAT2 does not "find" alignments in any specific order, so for reads that have more than N distinct, valid alignments, HISAT2 does not guarantee that the N alignments reported are the best possible in terms of alignment score. Still, this mode can be effective and fast in situations where the user cares more about whether a read aligns (or aligns a certain number of times) than where exactly it originated. [SAM specification]: http://samtools.sourceforge.net/SAM1.pdf Alignment summary ------------------ When HISAT2 finishes running, it prints messages summarizing what happened. These messages are printed to the "standard error" ("stderr") filehandle. For datasets consisting of unpaired reads, the summary might look like this: 20000 reads; of these: 20000 (100.00%) were unpaired; of these: 1247 (6.24%) aligned 0 times 18739 (93.69%) aligned exactly 1 time 14 (0.07%) aligned >1 times 93.77% overall alignment rate For datasets consisting of pairs, the summary might look like this: 10000 reads; of these: 10000 (100.00%) were paired; of these: 650 (6.50%) aligned concordantly 0 times 8823 (88.23%) aligned concordantly exactly 1 time 527 (5.27%) aligned concordantly >1 times ---- 650 pairs aligned concordantly 0 times; of these: 34 (5.23%) aligned discordantly 1 time ---- 616 pairs aligned 0 times concordantly or discordantly; of these: 1232 mates make up the pairs; of these: 660 (53.57%) aligned 0 times 571 (46.35%) aligned exactly 1 time 1 (0.08%) aligned >1 times 96.70% overall alignment rate The indentation indicates how subtotals relate to totals. Wrapper ------- The `hisat2`, `hisat2-build` and `hisat2-inspect` executables are actually wrapper scripts that call binary programs as appropriate. The wrappers shield users from having to distinguish between "small" and "large" index formats, discussed briefly in the following section. Also, the `hisat2` wrapper provides some key functionality, like the ability to handle compressed inputs, and the functionality for `--un`, `--al` and related options. It is recommended that you always run the hisat2 wrappers and not run the binaries directly. Small and large indexes ----------------------- `hisat2-build` can index reference genomes of any size. For genomes less than about 4 billion nucleotides in length, `hisat2-build` builds a "small" index using 32-bit numbers in various parts of the index. When the genome is longer, `hisat2-build` builds a "large" index using 64-bit numbers. Small indexes are stored in files with the `.ht2` extension, and large indexes are stored in files with the `.ht2l` extension. The user need not worry about whether a particular index is small or large; the wrapper scripts will automatically build and use the appropriate index. Performance tuning ------------------ 1. If your computer has multiple processors/cores, use `-p` The `-p` option causes HISAT2 to launch a specified number of parallel search threads. Each thread runs on a different processor/core and all threads find alignments in parallel, increasing alignment throughput by approximately a multiple of the number of threads (though in practice, speedup is somewhat worse than linear). Command Line ------------ ### Setting function options Some HISAT2 options specify a function rather than an individual number or setting. In these cases the user specifies three parameters: (a) a function type `F`, (b) a constant term `B`, and (c) a coefficient `A`. The available function types are constant (`C`), linear (`L`), square-root (`S`), and natural log (`G`). The parameters are specified as `F,B,A` - that is, the function type, the constant term, and the coefficient are separated by commas with no whitespace. The constant term and coefficient may be negative and/or floating-point numbers. For example, if the function specification is `L,-0.4,-0.6`, then the function defined is: f(x) = -0.4 + -0.6 * x If the function specification is `G,1,5.4`, then the function defined is: f(x) = 1.0 + 5.4 * ln(x) See the documentation for the option in question to learn what the parameter `x` is for. For example, in the case if the `--score-min` option, the function `f(x)` sets the minimum alignment score necessary for an alignment to be considered valid, and `x` is the read length. ### Usage hisat2 [options]* -x {-1 -2 | -U | --sra-acc } [-S ] ### Main arguments -x The basename of the index for the reference genome. The basename is the name of any of the index files up to but not including the final `.1.ht2` / etc. `hisat2` looks for the specified index first in the current directory, then in the directory specified in the `HISAT2_INDEXES` environment variable. -1 Comma-separated list of files containing mate 1s (filename usually includes `_1`), e.g. `-1 flyA_1.fq,flyB_1.fq`. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in ``. Reads may be a mix of different lengths. If `-` is specified, `hisat2` will read the mate 1s from the "standard in" or "stdin" filehandle. -2 Comma-separated list of files containing mate 2s (filename usually includes `_2`), e.g. `-2 flyA_2.fq,flyB_2.fq`. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in ``. Reads may be a mix of different lengths. If `-` is specified, `hisat2` will read the mate 2s from the "standard in" or "stdin" filehandle. -U Comma-separated list of files containing unpaired reads to be aligned, e.g. `lane1.fq,lane2.fq,lane3.fq,lane4.fq`. Reads may be a mix of different lengths. If `-` is specified, `hisat2` gets the reads from the "standard in" or "stdin" filehandle. --sra-acc Comma-separated list of SRA accession numbers, e.g. `--sra-acc SRR353653,SRR353654`. Information about read types is available at http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?sp=runinfo&acc=sra-acc&retmode=xml, where sra-acc is SRA accession number. If users run HISAT2 on a computer cluster, it is recommended to disable SRA-related caching (see the instruction at [SRA-MANUAL]). [SRA-MANUAL]: https://github.com/ncbi/sra-tools/wiki/Toolkit-Configuration -S File to write SAM alignments to. By default, alignments are written to the "standard out" or "stdout" filehandle (i.e. the console). ### Options #### Input options -q Reads (specified with ``, ``, ``) are FASTQ files. FASTQ files usually have extension `.fq` or `.fastq`. FASTQ is the default format. See also: `--solexa-quals` and `--int-quals`. --qseq Reads (specified with ``, ``, ``) are QSEQ files. QSEQ files usually end in `_qseq.txt`. See also: `--solexa-quals` and `--int-quals`. -f Reads (specified with ``, ``, ``) are FASTA files. FASTA files usually have extension `.fa`, `.fasta`, `.mfa`, `.fna` or similar. FASTA files do not have a way of specifying quality values, so when `-f` is set, the result is as if `--ignore-quals` is also set. -r Reads (specified with ``, ``, ``) are files with one input sequence per line, without any other information (no read names, no qualities). When `-r` is set, the result is as if `--ignore-quals` is also set. -c The read sequences are given on command line. I.e. ``, `` and `` are comma-separated lists of reads rather than lists of read files. There is no way to specify read names or qualities, so `-c` also implies `--ignore-quals`. -s/--skip Skip (i.e. do not align) the first `` reads or pairs in the input. -u/--qupto Align the first `` reads or read pairs from the input (after the `-s`/`--skip` reads or pairs have been skipped), then stop. Default: no limit. -5/--trim5 Trim `` bases from 5' (left) end of each read before alignment (default: 0). -3/--trim3 Trim `` bases from 3' (right) end of each read before alignment (default: 0). --phred33 Input qualities are ASCII chars equal to the [Phred quality] plus 33. This is also called the "Phred+33" encoding, which is used by the very latest Illumina pipelines. [Phred quality]: http://en.wikipedia.org/wiki/Phred_quality_score --phred64 Input qualities are ASCII chars equal to the [Phred quality] plus 64. This is also called the "Phred+64" encoding. --solexa-quals Convert input qualities from [Solexa][Phred quality] (which can be negative) to [Phred][Phred quality] (which can't). This scheme was used in older Illumina GA Pipeline versions (prior to 1.3). Default: off. --int-quals Quality values are represented in the read input file as space-separated ASCII integers, e.g., `40 40 30 40`..., rather than ASCII characters, e.g., `II?I`.... Integers are treated as being on the [Phred quality] scale unless `--solexa-quals` is also specified. Default: off. #### Alignment options --n-ceil Sets a function governing the maximum number of ambiguous characters (usually `N`s and/or `.`s) allowed in a read as a function of read length. For instance, specifying `-L,0,0.15` sets the N-ceiling function `f` to `f(x) = 0 + 0.15 * x`, where x is the read length. See also: [setting function options]. Reads exceeding this ceiling are [filtered out]. Default: `L,0,0.15`. --ignore-quals When calculating a mismatch penalty, always consider the quality value at the mismatched position to be the highest possible, regardless of the actual value. I.e. input is treated as though all quality values are high. This is also the default behavior when the input doesn't specify quality values (e.g. in `-f`, `-r`, or `-c` modes). --nofw/--norc If `--nofw` is specified, `hisat2` will not attempt to align unpaired reads to the forward (Watson) reference strand. If `--norc` is specified, `hisat2` will not attempt to align unpaired reads against the reverse-complement (Crick) reference strand. In paired-end mode, `--nofw` and `--norc` pertain to the fragments; i.e. specifying `--nofw` causes `hisat2` to explore only those paired-end configurations corresponding to fragments from the reverse-complement (Crick) strand. Default: both strands enabled. #### Scoring options --mp MX,MN Sets the maximum (`MX`) and minimum (`MN`) mismatch penalties, both integers. A number less than or equal to `MX` and greater than or equal to `MN` is subtracted from the alignment score for each position where a read character aligns to a reference character, the characters do not match, and neither is an `N`. If `--ignore-quals` is specified, the number subtracted quals `MX`. Otherwise, the number subtracted is `MN + floor( (MX-MN)(MIN(Q, 40.0)/40.0) )` where Q is the Phred quality value. Default: `MX` = 6, `MN` = 2. --sp MX,MN Sets the maximum (`MX`) and minimum (`MN`) penalties for soft-clipping per base, both integers. A number less than or equal to `MX` and greater than or equal to `MN` is subtracted from the alignment score for each position. The number subtracted is `MN + floor( (MX-MN)(MIN(Q, 40.0)/40.0) )` where Q is the Phred quality value. Default: `MX` = 2, `MN` = 1. --no-softclip Disallow soft-clipping. --np Sets penalty for positions where the read, reference, or both, contain an ambiguous character such as `N`. Default: 1. --rdg , Sets the read gap open (``) and extend (``) penalties. A read gap of length N gets a penalty of `` + N * ``. Default: 5, 3. --rfg , Sets the reference gap open (``) and extend (``) penalties. A reference gap of length N gets a penalty of `` + N * ``. Default: 5, 3. --score-min Sets a function governing the minimum alignment score needed for an alignment to be considered "valid" (i.e. good enough to report). This is a function of read length. For instance, specifying `L,0,-0.6` sets the minimum-score function `f` to `f(x) = 0 + -0.6 * x`, where `x` is the read length. See also: [setting function options]. The default is `L,0,-0.2`. #### Spliced alignment options --pen-cansplice Sets the penalty for each pair of canonical splice sites (e.g. GT/AG). Default: 0. --pen-noncansplice Sets the penalty for each pair of non-canonical splice sites (e.g. non-GT/AG). Default: 12. --pen-canintronlen Sets the penalty for long introns with canonical splice sites so that alignments with shorter introns are preferred to those with longer ones. Default: G,-8,1 --pen-noncanintronlen Sets the penalty for long introns with noncanonical splice sites so that alignments with shorter introns are preferred to those with longer ones. Default: G,-8,1 --min-intronlen Sets minimum intron length. Default: 20 --max-intronlen Sets maximum intron length. Default: 500000 --known-splicesite-infile With this mode, you can provide a list of known splice sites, which HISAT2 makes use of to align reads with small anchors. You can create such a list using `python hisat2_extract_splice_sites.py genes.gtf > splicesites.txt`, where `hisat2_extract_splice_sites.py` is included in the HISAT2 package, `genes.gtf` is a gene annotation file, and `splicesites.txt` is a list of splice sites with which you provide HISAT2 in this mode. Note that it is better to use indexes built using annotated transcripts (such as genome_tran or genome_snp_tran), which works better than using this option. It has no effect to provide splice sites that are already included in the indexes. --novel-splicesite-outfile In this mode, HISAT2 reports a list of splice sites in the file : chromosome name `` genomic position of the flanking base on the left side of an intron `` genomic position of the flanking base on the right `` strand (+, -, and .) '.' indicates an unknown strand for non-canonical splice sites. --novel-splicesite-infile With this mode, you can provide a list of novel splice sites that were generated from the above option "--novel-splicesite-outfile". --no-temp-splicesite HISAT2, by default, makes use of splice sites found by earlier reads to align later reads in the same run, in particular, reads with small anchors (<= 15 bp). The option disables this default alignment strategy. --no-spliced-alignment Disable spliced alignment. --rna-strandness Specify strand-specific information: the default is unstranded. For single-end reads, use F or R. 'F' means a read corresponds to a transcript. 'R' means a read corresponds to the reverse complemented counterpart of a transcript. For paired-end reads, use either FR or RF. With this option being used, every read alignment will have an XS attribute tag: '+' means a read belongs to a transcript on '+' strand of genome. '-' means a read belongs to a transcript on '-' strand of genome. (TopHat has a similar option, --library-type option, where fr-firststrand corresponds to R and RF; fr-secondstrand corresponds to F and FR.) --tmo/--transcriptome-mapping-only Report only those alignments within known transcripts. --dta/--downstream-transcriptome-assembly Report alignments tailored for transcript assemblers including StringTie. With this option, HISAT2 requires longer anchor lengths for de novo discovery of splice sites. This leads to fewer alignments with short-anchors, which helps transcript assemblers improve significantly in computation and memory usage. --dta-cufflinks Report alignments tailored specifically for Cufflinks. In addition to what HISAT2 does with the above option (--dta), With this option, HISAT2 looks for novel splice sites with three signals (GT/AG, GC/AG, AT/AC), but all user-provided splice sites are used irrespective of their signals. HISAT2 produces an optional field, XS:A:[+-], for every spliced alignment. --no-templatelen-adjustment Disables template length adjustment for RNA-seq reads. #### Reporting options -k It searches for at most `` distinct, primary alignments for each read. Primary alignments mean alignments whose alignment score is equal or higher than any other alignments. The search terminates when it can't find more distinct valid alignments, or when it finds ``, whichever happens first. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS field. For reads that have more than `` distinct, valid alignments, `hisat2` does not guarantee that the `` alignments reported are the best possible in terms of alignment score. Default: 5 (HFM) or 10 (HGFM) Note: HISAT2 is not designed with large values for `-k` in mind, and when aligning reads to long, repetitive genomes large `-k` can be very, very slow. --max-seeds HISAT2, like other aligners, uses seed-and-extend approaches. HISAT2 tries to extend seeds to full-length alignments. In HISAT2, --max-seeds is used to control the maximum number of seeds that will be extended. HISAT2 extends up to these many seeds and skips the rest of the seeds. Large values for `--max-seeds` may improve alignment sensitivity, but HISAT2 is not designed with large values for `--max-seeds` in mind, and when aligning reads to long, repetitive genomes large `--max-seeds` can be very, very slow. The default value is the maximum of 5 and the value that comes with`-k`. --secondary Report secondary alignments. #### Paired-end options -I/--minins The minimum fragment length for valid paired-end alignments.This option is valid only with --no-spliced-alignment. E.g. if `-I 60` is specified and a paired-end alignment consists of two 20-bp alignments in the appropriate orientation with a 20-bp gap between them, that alignment is considered valid (as long as `-X` is also satisfied). A 19-bp gap would not be valid in that case. If trimming options `-3` or `-5` are also used, the `-I` constraint is applied with respect to the untrimmed mates. The larger the difference between `-I` and `-X`, the slower HISAT2 will run. This is because larger differences between `-I` and `-X` require that HISAT2 scan a larger window to determine if a concordant alignment exists. For typical fragment length ranges (200 to 400 nucleotides), HISAT2 is very efficient. Default: 0 (essentially imposing no minimum) -X/--maxins The maximum fragment length for valid paired-end alignments. This option is valid only with --no-spliced-alignment. E.g. if `-X 100` is specified and a paired-end alignment consists of two 20-bp alignments in the proper orientation with a 60-bp gap between them, that alignment is considered valid (as long as `-I` is also satisfied). A 61-bp gap would not be valid in that case. If trimming options `-3` or `-5` are also used, the `-X` constraint is applied with respect to the untrimmed mates, not the trimmed mates. The larger the difference between `-I` and `-X`, the slower HISAT2 will run. This is because larger differences between `-I` and `-X` require that HISAT2 scan a larger window to determine if a concordant alignment exists. For typical fragment length ranges (200 to 400 nucleotides), HISAT2 is very efficient. Default: 500. --fr/--rf/--ff The upstream/downstream mate orientations for a valid paired-end alignment against the forward reference strand. E.g., if `--fr` is specified and there is a candidate paired-end alignment where mate 1 appears upstream of the reverse complement of mate 2 and the fragment length constraints (`-I` and `-X`) are met, that alignment is valid. Also, if mate 2 appears upstream of the reverse complement of mate 1 and all other constraints are met, that too is valid. `--rf` likewise requires that an upstream mate1 be reverse-complemented and a downstream mate2 be forward-oriented. ` --ff` requires both an upstream mate 1 and a downstream mate 2 to be forward-oriented. Default: `--fr` (appropriate for Illumina's Paired-end Sequencing Assay). --no-mixed By default, when `hisat2` cannot find a concordant or discordant alignment for a pair, it then tries to find alignments for the individual mates. This option disables that behavior. --no-discordant By default, `hisat2` looks for discordant alignments if it cannot find any concordant alignments. A discordant alignment is an alignment where both mates align uniquely, but that does not satisfy the paired-end constraints (`--fr`/`--rf`/`--ff`, `-I`, `-X`). This option disables that behavior. #### Output options -t/--time Print the wall-clock time required to load the index files and align the reads. This is printed to the "standard error" ("stderr") filehandle. Default: off. --un --un-gz --un-bz2 Write unpaired reads that fail to align to file at ``. These reads correspond to the SAM records with the FLAGS `0x4` bit set and neither the `0x40` nor `0x80` bits set. If `--un-gz` is specified, output will be gzip compressed. If `--un-bz2` is specified, output will be bzip2 compressed. Reads written in this way will appear exactly as they did in the input file, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the input. --al --al-gz --al-bz2 Write unpaired reads that align at least once to file at ``. These reads correspond to the SAM records with the FLAGS `0x4`, `0x40`, and `0x80` bits unset. If `--al-gz` is specified, output will be gzip compressed. If `--al-bz2` is specified, output will be bzip2 compressed. Reads written in this way will appear exactly as they did in the input file, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the input. --un-conc --un-conc-gz --un-conc-bz2 Write paired-end reads that fail to align concordantly to file(s) at ``. These reads correspond to the SAM records with the FLAGS `0x4` bit set and either the `0x40` or `0x80` bit set (depending on whether it's mate #1 or #2). `.1` and `.2` strings are added to the filename to distinguish which file contains mate #1 and mate #2. If a percent symbol, `%`, is used in ``, the percent symbol is replaced with `1` or `2` to make the per-mate filenames. Otherwise, `.1` or `.2` are added before the final dot in `` to make the per-mate filenames. Reads written in this way will appear exactly as they did in the input files, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the inputs. --al-conc --al-conc-gz --al-conc-bz2 Write paired-end reads that align concordantly at least once to file(s) at ``. These reads correspond to the SAM records with the FLAGS `0x4` bit unset and either the `0x40` or `0x80` bit set (depending on whether it's mate #1 or #2). `.1` and `.2` strings are added to the filename to distinguish which file contains mate #1 and mate #2. If a percent symbol, `%`, is used in ``, the percent symbol is replaced with `1` or `2` to make the per-mate filenames. Otherwise, `.1` or `.2` are added before the final dot in `` to make the per-mate filenames. Reads written in this way will appear exactly as they did in the input files, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the inputs. --quiet Print nothing besides alignments and serious errors. --summary-file Print alignment summary to this file. --new-summary Print alignment summary in a new style, which is more machine-friendly. --met-file Write `hisat2` metrics to file ``. Having alignment metric can be useful for debugging certain problems, especially performance issues. See also: `--met`. Default: metrics disabled. --met-stderr Write `hisat2` metrics to the "standard error" ("stderr") filehandle. This is not mutually exclusive with `--met-file`. Having alignment metric can be useful for debugging certain problems, especially performance issues. See also: `--met`. Default: metrics disabled. --met Write a new `hisat2` metrics record every `` seconds. Only matters if either `--met-stderr` or `--met-file` are specified. Default: 1. #### SAM options --no-unal Suppress SAM records for reads that failed to align. --no-hd Suppress SAM header lines (starting with `@`). --no-sq Suppress `@SQ` SAM header lines. --rg-id Set the read group ID to ``. This causes the SAM `@RG` header line to be printed, with `` as the value associated with the `ID:` tag. It also causes the `RG:Z:` extra field to be attached to each SAM output record, with value set to ``. --rg Add `` (usually of the form `TAG:VAL`, e.g. `SM:Pool1`) as a field on the `@RG` header line. Note: in order for the `@RG` line to appear, `--rg-id` must also be specified. This is because the `ID` tag is required by the [SAM Spec][SAM]. Specify `--rg` multiple times to set multiple fields. See the [SAM Spec][SAM] for details about what fields are legal. --remove-chrname Remove 'chr' from reference names in alignment (e.g., chr18 to 18) --add-chrname Add 'chr' to reference names in alignment (e.g., 18 to chr18) --omit-sec-seq When printing secondary alignments, HISAT2 by default will write out the `SEQ` and `QUAL` strings. Specifying this option causes HISAT2 to print an asterisk in those fields instead. #### Performance options -o/--offrate Override the offrate of the index with ``. If `` is greater than the offrate used to build the index, then some row markings are discarded when the index is read into memory. This reduces the memory footprint of the aligner but requires more time to calculate text offsets. `` must be greater than the value used to build the index. -p/--threads NTHREADS Launch `NTHREADS` parallel search threads (default: 1). Threads will run on separate processors/cores and synchronize when parsing reads and outputting alignments. Searching for alignments is highly parallel, and speedup is close to linear. Increasing `-p` increases HISAT2's memory footprint. E.g. when aligning to a human genome index, increasing `-p` from 1 to 8 increases the memory footprint by a few hundred megabytes. This option is only available if `hisat2` is linked with the `pthreads` library (i.e. if `HISAT2_PTHREADS=0` is not specified at build time). --reorder Guarantees that output SAM records are printed in an order corresponding to the order of the reads in the original input file, even when `-p` is set greater than 1. Specifying `--reorder` and setting `-p` greater than 1 causes HISAT2 to run somewhat slower and use somewhat more memory then if `--reorder` were not specified. Has no effect if `-p` is set to 1, since output order will naturally correspond to input order in that case. --mm Use memory-mapped I/O to load the index, rather than typical file I/O. Memory-mapping allows many concurrent `hisat2` processes on the same computer to share the same memory image of the index (i.e. you pay the memory overhead just once). This facilitates memory-efficient parallelization of `hisat2` in situations where using `-p` is not possible or not preferable. #### Other options --qc-filter Filter out reads for which the QSEQ filter field is non-zero. Only has an effect when read format is `--qseq`. Default: off. --seed Use `` as the seed for pseudo-random number generator. Default: 0. --non-deterministic Normally, HISAT2 re-initializes its pseudo-random generator for each read. It seeds the generator with a number derived from (a) the read name, (b) the nucleotide sequence, (c) the quality sequence, (d) the value of the `--seed` option. This means that if two reads are identical (same name, same nucleotides, same qualities) HISAT2 will find and report the same alignment(s) for both, even if there was ambiguity. When `--non-deterministic` is specified, HISAT2 re-initializes its pseudo-random generator for each read using the current time. This means that HISAT2 will not necessarily report the same alignment for two identical reads. This is counter-intuitive for some users, but might be more appropriate in situations where the input consists of many identical reads. --version Print version information and quit. -h/--help Print usage information and quit. SAM output ---------- Following is a brief description of the [SAM] format as output by `hisat2`. For more details, see the [SAM format specification][SAM]. By default, `hisat2` prints a SAM header with `@HD`, `@SQ` and `@PG` lines. When one or more `--rg` arguments are specified, `hisat2` will also print an `@RG` line that includes all user-specified `--rg` tokens separated by tabs. Each subsequent line describes an alignment or, if the read failed to align, a read. Each line is a collection of at least 12 fields separated by tabs; from left to right, the fields are: 1. Name of read that aligned. Note that the [SAM specification] disallows whitespace in the read name. If the read name contains any whitespace characters, HISAT2 will truncate the name at the first whitespace character. This is similar to the behavior of other tools. 2. Sum of all applicable flags. Flags relevant to HISAT2 are: 1 The read is one of a pair 2 The alignment is one end of a proper paired-end alignment 4 The read has no reported alignments 8 The read is one of a pair and has no reported alignments 16 The alignment is to the reverse reference strand 32 The other mate in the paired-end alignment is aligned to the reverse reference strand 64 The read is mate 1 in a pair 128 The read is mate 2 in a pair Thus, an unpaired read that aligns to the reverse reference strand will have flag 16. A paired-end read that aligns and is the first mate in the pair will have flag 83 (= 64 + 16 + 2 + 1). 3. Name of reference sequence where alignment occurs 4. 1-based offset into the forward reference strand where leftmost character of the alignment occurs 5. Mapping quality 6. CIGAR string representation of alignment 7. Name of reference sequence where mate's alignment occurs. Set to `=` if the mate's reference sequence is the same as this alignment's, or `*` if there is no mate. 8. 1-based offset into the forward reference strand where leftmost character of the mate's alignment occurs. Offset is 0 if there is no mate. 9. Inferred fragment length. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if the mates did not align concordantly. However, size is non-0 if the mates aligned discordantly to the same chromosome. 10. Read sequence (reverse-complemented if aligned to the reverse strand) 11. ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the [Phred quality] scale and the encoding is ASCII-offset by 33 (ASCII char `!`), similarly to a [FASTQ] file. 12. Optional fields. Fields are tab-separated. `hisat2` outputs zero or more of these optional fields for each alignment, depending on the type of the alignment: AS:i: Alignment score. Can be negative. Only present if SAM record is for an aligned read. ZS:i: Alignment score for the best-scoring alignment found other than the alignment reported. Can be negative. Only present if the SAM record is for an aligned read and more than one alignment was found for the read. Note that, when the read is part of a concordantly-aligned pair, this score could be greater than `AS:i`. YS:i: Alignment score for opposite mate in the paired-end alignment. Only present if the SAM record is for a read that aligned as part of a paired-end alignment. XN:i: The number of ambiguous bases in the reference covering this alignment. Only present if SAM record is for an aligned read. XM:i: The number of mismatches in the alignment. Only present if SAM record is for an aligned read. XO:i: The number of gap opens, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read. XG:i: The number of gap extensions, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read. NM:i: The edit distance; that is, the minimal number of one-nucleotide edits (substitutions, insertions and deletions) needed to transform the read string into the reference string. Only present if SAM record is for an aligned read. YF:Z: String indicating reason why the read was filtered out. See also: [Filtering]. Only appears for reads that were filtered out. YT:Z: Value of `UU` indicates the read was not part of a pair. Value of `CP` indicates the read was part of a pair and the pair aligned concordantly. Value of `DP` indicates the read was part of a pair and the pair aligned discordantly. Value of `UP` indicates the read was part of a pair but the pair failed to aligned either concordantly or discordantly. MD:Z: A string representation of the mismatched reference bases in the alignment. See [SAM] format specification for details. Only present if SAM record is for an aligned read. XS:A: Values of `+` and `-` indicate the read is mapped to transcripts on sense and anti-sense strands, respectively. Spliced alignments need to have this field, which is required in Cufflinks and StringTie. We can report this field for the canonical-splice site (GT/AG), but not for non-canonical splice sites. You can direct HISAT2 not to output such alignments (involving non-canonical splice sites) using "--pen-noncansplice 1000000". NH:i: The number of mapped locations for the read or the pair. Zs:Z: When the alignment of a read involves SNPs that are in the index, this option is used to indicate where exactly the read involves the SNPs. This optional field is similar to the above MD:Z field. For example, `Zs:Z:1|S|rs3747203,97|S|rs16990981` indicates the second base of the read corresponds to a known SNP (ID: rs3747203). 97 bases after the third base (the base after the second one), the read at 100th base involves another known SNP (ID: rs16990981). 'S' indicates a single nucleotide polymorphism. 'D' and 'I' indicate a deletion and an insertion, respectively. [SAM format specification]: http://samtools.sf.net/SAM1.pdf [FASTQ]: http://en.wikipedia.org/wiki/FASTQ_format The `hisat2-build` indexer =========================== `hisat2-build` builds a HISAT2 index from a set of DNA sequences. `hisat2-build` outputs a set of 6 files with suffixes `.1.ht2`, `.2.ht2`, `.3.ht2`, `.4.ht2`, `.5.ht2`, `.6.ht2`, `.7.ht2`, and `.8.ht2`. In the case of a large index these suffixes will have a `ht2l` termination. These files together constitute the index: they are all that is needed to align reads to that reference. The original sequence FASTA files are no longer used by HISAT2 once the index is built. Use of Karkkainen's [blockwise algorithm] allows `hisat2-build` to trade off between running time and memory usage. `hisat2-build` has three options governing how it makes this trade: `-p`/`--packed`, `--bmax`/`--bmaxdivn`, and `--dcv`. By default, `hisat2-build` will automatically search for the settings that yield the best running time without exhausting memory. This behavior can be disabled using the `-a`/`--noauto` option. The indexer provides options pertaining to the "shape" of the index, e.g. `--offrate` governs the fraction of [Burrows-Wheeler] rows that are "marked" (i.e., the density of the suffix-array sample; see the original [FM Index] paper for details). All of these options are potentially profitable trade-offs depending on the application. They have been set to defaults that are reasonable for most cases according to our experiments. See [Performance tuning] for details. `hisat2-build` can generate either [small or large indexes]. The wrapper will decide which based on the length of the input genome. If the reference does not exceed 4 billion characters but a large index is preferred, the user can specify `--large-index` to force `hisat2-build` to build a large index instead. The HISAT2 index is based on the [FM Index] of Ferragina and Manzini, which in turn is based on the [Burrows-Wheeler] transform. The algorithm used to build the index is based on the [blockwise algorithm] of Karkkainen. [Blockwise algorithm]: http://portal.acm.org/citation.cfm?id=1314852 [Burrows-Wheeler]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform Command Line ------------ Usage: hisat2-build [options]* ### Notes If you use --snp, --ss, and/or --exon, hisat2-build will need about 200GB RAM for the human genome size as index building involves a graph construction. Otherwise, you will be able to build an index on your desktop with 8GB RAM. ### Main arguments A comma-separated list of FASTA files containing the reference sequences to be aligned to, or, if `-c` is specified, the sequences themselves. E.g., `` might be `chr1.fa,chr2.fa,chrX.fa,chrY.fa`, or, if `-c` is specified, this might be `GGTCATCCT,ACGGGTCGT,CCGTTCTATGCGGCTTA`. The basename of the index files to write. By default, `hisat2-build` writes files named `NAME.1.ht2`, `NAME.2.ht2`, `NAME.3.ht2`, `NAME.4.ht2`, `NAME.5.ht2`, `NAME.6.ht2`, `NAME.7.ht2`, and `NAME.8.ht2` where `NAME` is ``. ### Options -f The reference input files (specified as ``) are FASTA files (usually having extension `.fa`, `.mfa`, `.fna` or similar). -c The reference sequences are given on the command line. I.e. `` is a comma-separated list of sequences rather than a list of FASTA files. --large-index Force `hisat2-build` to build a [large index], even if the reference is less than ~ 4 billion nucleotides long. -a/--noauto Disable the default behavior whereby `hisat2-build` automatically selects values for the `--bmax`, `--dcv` and `--packed` parameters according to available memory. Instead, user may specify values for those parameters. If memory is exhausted during indexing, an error message will be printed; it is up to the user to try new parameters. --bmax The maximum number of suffixes allowed in a block. Allowing more suffixes per block makes indexing faster, but increases peak memory usage. Setting this option overrides any previous setting for `--bmax`, or `--bmaxdivn`. Default (in terms of the `--bmaxdivn` parameter) is `--bmaxdivn` 4. This is configured automatically by default; use `-a`/`--noauto` to configure manually. --bmaxdivn The maximum number of suffixes allowed in a block, expressed as a fraction of the length of the reference. Setting this option overrides any previous setting for `--bmax`, or `--bmaxdivn`. Default: `--bmaxdivn` 4. This is configured automatically by default; use `-a`/`--noauto` to configure manually. --dcv Use `` as the period for the difference-cover sample. A larger period yields less memory overhead, but may make suffix sorting slower, especially if repeats are present. Must be a power of 2 no greater than 4096. Default: 1024. This is configured automatically by default; use `-a`/`--noauto` to configure manually. --nodc Disable use of the difference-cover sample. Suffix sorting becomes quadratic-time in the worst case (where the worst case is an extremely repetitive reference). Default: off. -r/--noref Do not build the `NAME.3.ht2` and `NAME.4.ht2` portions of the index, which contain a bitpacked version of the reference sequences and are used for paired-end alignment. -3/--justref Build only the `NAME.3.ht2` and `NAME.4.ht2` portions of the index, which contain a bitpacked version of the reference sequences and are used for paired-end alignment. -o/--offrate To map alignments back to positions on the reference sequences, it's necessary to annotate ("mark") some or all of the [Burrows-Wheeler] rows with their corresponding location on the genome. `-o`/`--offrate` governs how many rows get marked: the indexer will mark every 2^`` rows. Marking more rows makes reference-position lookups faster, but requires more memory to hold the annotations at runtime. The default is 4 (every 16th row is marked; for human genome, annotations occupy about 680 megabytes). -t/--ftabchars The ftab is the lookup table used to calculate an initial [Burrows-Wheeler] range with respect to the first `` characters of the query. A larger `` yields a larger lookup table but faster query times. The ftab has size 4^(``+1) bytes. The default setting is 10 (ftab is 4MB). --localoffrate This option governs how many rows get marked in a local index: the indexer will mark every 2^`` rows. Marking more rows makes reference-position lookups faster, but requires more memory to hold the annotations at runtime. The default is 3 (every 8th row is marked, this occupies about 16KB per local index). --localftabchars The local ftab is the lookup table in a local index. The default setting is 6 (ftab is 8KB per local index). -p Launch `NTHREADS` parallel build threads (default: 1). --snp Provide a list of SNPs (in the HISAT2's own format) as follows (five columns). SNP ID `` snp type (single, deletion, or insertion) `` chromosome name `` zero-offset based genomic position of a SNP `` alternative base (single), the length of SNP (deletion), or insertion sequence (insertion) For example, rs58784443 single 13 18447947 T Use `hisat2_extract_snps_haplotypes_UCSC.py` (in the HISAT2 package) to extract SNPs and haplotypes from a dbSNP file (e.g. http://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/snp144Common.txt.gz). or `hisat2_extract_snps_haplotypes_VCF.py` to extract SNPs and haplotypes from a VCF file (e.g. ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/supporting/GRCh38_positions/ALL.chr22.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP_no_SVs.vcf.gz). --haplotype Provide a list of haplotypes (in the HISAT2's own format) as follows (five columns). Haplotype ID `` chromosome name `` zero-offset based left coordinate of haplotype `` zero-offset based right coordinate of haplotype `` a comma separated list of SNP ids in the haplotype For example, ht35 13 18446877 18446945 rs12381094,rs12381056,rs192016659,rs538569910 See the above option, --snp, about how to extract haplotypes. This option is not required, but haplotype information can keep the index construction from exploding and reduce the index size substantially. --ss Note this option should be used with the following --exon option. Provide a list of splice sites (in the HISAT2's own format) as follows (four columns). chromosome name `` zero-offset based genomic position of the flanking base on the left side of an intron `` zero-offset based genomic position of the flanking base on the right `` strand Use `hisat2_extract_splice_sites.py` (in the HISAT2 package) to extract splice sites from a GTF file. --exon Note this option should be used with the above --ss option. Provide a list of exons (in the HISAT2's own format) as follows (three columns). chromosome name `` zero-offset based left genomic position of an exon `` zero-offset based right genomic position of an exon Use `hisat2_extract_exons.py` (in the HISAT2 package) to extract exons from a GTF file. --seed Use `` as the seed for pseudo-random number generator. --cutoff Index only the first `` bases of the reference sequences (cumulative across sequences) and ignore the rest. -q/--quiet `hisat2-build` is verbose by default. With this option `hisat2-build` will print only error messages. -h/--help Print usage information and quit. --version Print version information and quit. The `hisat2-inspect` index inspector ===================================== `hisat2-inspect` extracts information from a HISAT2 index about what kind of index it is and what reference sequences were used to build it. When run without any options, the tool will output a FASTA file containing the sequences of the original references (with all non-`A`/`C`/`G`/`T` characters converted to `N`s). It can also be used to extract just the reference sequence names using the `-n`/`--names` option or a more verbose summary using the `-s`/`--summary` option. Command Line ------------ Usage: hisat2-inspect [options]* ### Main arguments The basename of the index to be inspected. The basename is name of any of the index files but with the `.X.ht2` suffix omitted. `hisat2-inspect` first looks in the current directory for the index files, then in the directory specified in the `HISAT2_INDEXES` environment variable. ### Options -a/--across When printing FASTA output, output a newline character every `` bases (default: 60). -n/--names Print reference sequence names, one per line, and quit. -s/--summary Print a summary that includes information about index settings, as well as the names and lengths of the input sequences. The summary has this format: Colorspace <0 or 1> SA-Sample 1 in FTab-Chars Sequence-1 Sequence-2 ... Sequence-N Fields are separated by tabs. Colorspace is always set to 0 for HISAT2. --snp Print SNPs, and quit. --ss Print splice sites, and quit. --ss-all Print splice sites including those not in the global index, and quit. --exon Print exons, and quit. -v/--verbose Print verbose output (for debugging). --version Print version information and quit. -h/--help Print usage information and quit. Getting started with HISAT2 =================================================== HISAT2 comes with some example files to get you started. The example files are not scientifically significant; these files will simply let you start running HISAT2 and downstream tools right away. First follow the manual instructions to [obtain HISAT2]. Set the `HISAT2_HOME` environment variable to point to the new HISAT2 directory containing the `hisat2`, `hisat2-build` and `hisat2-inspect` binaries. This is important, as the `HISAT2_HOME` variable is used in the commands below to refer to that directory. Indexing a reference genome --------------------------- To create an index for the genomic region (1 million bps from the human chromosome 22 between 20,000,000 and 20,999,999) included with HISAT2, create a new temporary directory (it doesn't matter where), change into that directory, and run: $HISAT2_HOME/hisat2-build $HISAT2_HOME/example/reference/22_20-21M.fa --snp $HISAT2_HOME/example/reference/22_20-21M.snp 22_20-21M_snp The command should print many lines of output then quit. When the command completes, the current directory will contain ten new files that all start with `22_20-21M_snp` and end with `.1.ht2`, `.2.ht2`, `.3.ht2`, `.4.ht2`, `.5.ht2`, `.6.ht2`, `.7.ht2`, and `.8.ht2`. These files constitute the index - you're done! You can use `hisat2-build` to create an index for a set of FASTA files obtained from any source, including sites such as [UCSC], [NCBI], and [Ensembl]. When indexing multiple FASTA files, specify all the files using commas to separate file names. For more details on how to create an index with `hisat2-build`, see the [manual section on index building]. You may also want to bypass this process by obtaining a pre-built index. [UCSC]: http://genome.ucsc.edu/cgi-bin/hgGateway [NCBI]: http://www.ncbi.nlm.nih.gov/sites/genome [Ensembl]: http://www.ensembl.org/ Aligning example reads ---------------------- Stay in the directory created in the previous step, which now contains the `22_20-21M` index files. Next, run: $HISAT2_HOME/hisat2 -f -x $HISAT2_HOME/example/index/22_20-21M_snp -U $HISAT2_HOME/example/reads/reads_1.fa -S eg1.sam This runs the HISAT2 aligner, which aligns a set of unpaired reads to the genome region using the index generated in the previous step. The alignment results in SAM format are written to the file `eg1.sam`, and a short alignment summary is written to the console. (Actually, the summary is written to the "standard error" or "stderr" filehandle, which is typically printed to the console.) To see the first few lines of the SAM output, run: head eg1.sam You will see something like this: @HD VN:1.0 SO:unsorted @SQ SN:22:20000001-21000000 LN:1000000 @PG ID:hisat2 PN:hisat2 VN:2.0.0-beta 1 0 22:20000001-21000000 397984 255 100M * 0 0 GCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU NH:i:1 2 16 22:20000001-21000000 398131 255 100M * 0 0 ATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:80A19 YT:Z:UU NH:i:1 Zs:Z:80|S|rs576159895 3 16 22:20000001-21000000 398222 255 100M * 0 0 TGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:16A83 YT:Z:UU NH:i:1 Zs:Z:16|S|rs2629364 4 16 22:20000001-21000000 398247 255 90M200N10M * 0 0 CAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAACCGCTGCACCAGGAAGTCGATCCAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU XS:A:- NH:i:1 5 16 22:20000001-21000000 398194 255 100M * 0 0 GGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:17A26A55 YT:Z:UU NH:i:1 Zs:Z:17|S|rs576159895,26|S|rs2629364 6 0 22:20000001-21000000 398069 255 100M * 0 0 CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU NH:i:1 7 0 22:20000001-21000000 397896 255 100M * 0 0 GTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:31G68 YT:Z:UU NH:i:1 Zs:Z:31|S|rs562662261 8 0 22:20000001-21000000 398150 255 100M * 0 0 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:61A26A11 YT:Z:UU NH:i:1 Zs:Z:61|S|rs576159895,26|S|rs2629364 9 16 22:20000001-21000000 398329 255 8M200N92M * 0 0 ACCAGGAAGTCGATCCAGATGTAGTGGGGGGTCACTTCGGGGGGACAGGGTTTGGGTTGACTTGCTTCCGAGGCAGCCAGGGGGTCTGCTTCCTTTATCT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU XS:A:- NH:i:1 10 16 22:20000001-21000000 398184 255 100M * 0 0 CTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:27A26A45 YT:Z:UU NH:i:1 Zs:Z:27|S|rs576159895,26|S|rs2629364 The first few lines (beginning with `@`) are SAM header lines, and the rest of the lines are SAM alignments, one line per read or mate. See the [HISAT2 manual section on SAM output] and the [SAM specification] for details about how to interpret the SAM file format. Paired-end example ------------------ To align paired-end reads included with HISAT2, stay in the same directory and run: $HISAT2_HOME/hisat2 -f -x $HISAT2_HOME/example/index/22_20-21M_snp -1 $HISAT2_HOME/example/reads/reads_1.fa -2 $HISAT2_HOME/example/reads/reads_2.fa -S eg2.sam This aligns a set of paired-end reads to the reference genome, with results written to the file `eg2.sam`. Using SAMtools/BCFtools downstream ---------------------------------- [SAMtools] is a collection of tools for manipulating and analyzing SAM and BAM alignment files. [BCFtools] is a collection of tools for calling variants and manipulating VCF and BCF files, and it is typically distributed with [SAMtools]. Using these tools together allows you to get from alignments in SAM format to variant calls in VCF format. This example assumes that `samtools` and `bcftools` are installed and that the directories containing these binaries are in your [PATH environment variable]. Run the paired-end example: $HISAT2_HOME/hisat -f -x $HISAT2_HOME/example/index/22_20-21M_snp -1 $HISAT2_HOME/example/reads/reads_1.fa -2 $HISAT2_HOME/example/reads/reads_2.fa -S eg2.sam Use `samtools view` to convert the SAM file into a BAM file. BAM is a the binary format corresponding to the SAM text format. Run: samtools view -bS eg2.sam > eg2.bam Use `samtools sort` to convert the BAM file to a sorted BAM file. The following command requires samtools version 1.2 or higher. samtools sort eg2.bam -o eg2.sorted.bam We now have a sorted BAM file called `eg2.sorted.bam`. Sorted BAM is a useful format because the alignments are (a) compressed, which is convenient for long-term storage, and (b) sorted, which is convenient for variant discovery. To generate variant calls in VCF format, run: samtools mpileup -uf $HISAT2_HOME/example/reference/22_20-21M.fa eg2.sorted.bam | bcftools view -bvcg - > eg2.raw.bcf Then to view the variants, run: bcftools view eg2.raw.bcf See the official SAMtools guide to [Calling SNPs/INDELs with SAMtools/BCFtools] for more details and variations on this process. [BCFtools]: http://samtools.sourceforge.net/mpileup.shtml [Calling SNPs/INDELs with SAMtools/BCFtools]: http://samtools.sourceforge.net/mpileup.shtml hisat2-2.2.1/MANUAL.markdown000066400000000000000000002326411370663763200154540ustar00rootroot00000000000000 Introduction ============ What is HISAT2? ----------------- HISAT2 is a fast and sensitive alignment program for mapping next-generation sequencing reads (whole-genome, transcriptome, and exome sequencing data) against the general human population (as well as against a single reference genome). Based on [GCSA] (an extension of [BWT] for a graph), we designed and implemented a graph FM index (GFM), an original approach and its first implementation to the best of our knowledge. In addition to using one global GFM index that represents general population, HISAT2 uses a large set of small GFM indexes that collectively cover the whole genome (each index representing a genomic region of 56 Kbp, with 55,000 indexes needed to cover human population). These small indexes (called local indexes) combined with several alignment strategies enable effective alignment of sequencing reads. This new indexing scheme is called Hierarchical Graph FM index (HGFM). We have developed HISAT 2 based on the [HISAT] and [Bowtie2] implementations. HISAT2 outputs alignments in [SAM] format, enabling interoperation with a large number of other tools (e.g. [SAMtools], [GATK]) that use SAM. HISAT2 is distributed under the [GPLv3 license], and it runs on the command line under Linux, Mac OS X and Windows. [HISAT2]: http://ccb.jhu.edu/software/hisat2 [HISAT]: http://ccb.jhu.edu/software/hisat [Bowtie2]: http://bowtie-bio.sf.net/bowtie2 [Bowtie]: http://bowtie-bio.sf.net [Bowtie1]: http://bowtie-bio.sf.net [GCSA]: http://ieeexplore.ieee.org/xpls/abs_all.jsp?arnumber=6698337&tag=1 [Burrows-Wheeler Transform]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform [BWT]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform [FM Index]: http://en.wikipedia.org/wiki/FM-index [SAM]: http://samtools.sourceforge.net/SAM1.pdf [SAMtools]: http://samtools.sourceforge.net [GATK]: http://www.broadinstitute.org/gsa/wiki/index.php/The_Genome_Analysis_Toolkit [TopHat2]: http://ccb.jhu.edu/software/tophat [Cufflinks]: http://cufflinks.cbcb.umd.edu/ [Crossbow]: http://bowtie-bio.sf.net/crossbow [Myrna]: http://bowtie-bio.sf.net/myrna [Bowtie paper]: http://genomebiology.com/2009/10/3/R25 [GPLv3 license]: http://www.gnu.org/licenses/gpl-3.0.html Obtaining HISAT2 ================== Download HISAT2 sources and binaries from the Releases sections on the right side. Binaries are available for Intel architectures (`x86_64`) running Linux, and Mac OS X. Building from source -------------------- Building HISAT2 from source requires a GNU-like environment with GCC, GNU Make and other basics. It should be possible to build HISAT2 on most vanilla Linux installations or on a Mac installation with [Xcode] installed. HISAT2 can also be built on Windows using [Cygwin] or [MinGW] (MinGW recommended). For a MinGW build the choice of what compiler is to be used is important since this will determine if a 32 or 64 bit code can be successfully compiled using it. If there is a need to generate both 32 and 64 bit on the same machine then a multilib MinGW has to be properly installed. [MSYS], the [zlib] library, and depending on architecture [pthreads] library are also required. We are recommending a 64 bit build since it has some clear advantages in real life research problems. In order to simplify the MinGW setup it might be worth investigating popular MinGW personal builds since these are coming already prepared with most of the toolchains needed. First, download the [source package] from the Releases section on the right side. Unzip the file, change to the unzipped directory, and build the HISAT2 tools by running GNU `make` (usually with the command `make`, but sometimes with `gmake`) with no arguments. If building with MinGW, run `make` from the MSYS environment. HISAT2 is using the multithreading software model in order to speed up execution times on SMP architectures where this is possible. On POSIX platforms (like linux, Mac OS, etc) it needs the pthread library. Although it is possible to use pthread library on non-POSIX platform like Windows, due to performance reasons HISAT2 will try to use Windows native multithreading if possible. For the support of SRA data access in HISAT2, please download and install the [NCBI-NGS] toolkit. When running `make`, specify additional variables as follow. `make USE_SRA=1 NCBI_NGS_DIR=/path/to/NCBI-NGS-directory NCBI_VDB_DIR=/path/to/NCBI-NGS-directory`, where `NCBI_NGS_DIR` and `NCBI_VDB_DIR` will be used in Makefile for -I and -L compilation options. For example, $(NCBI_NGS_DIR)/include and $(NCBI_NGS_DIR)/lib64 will be used. [Cygwin]: http://www.cygwin.com/ [MinGW]: http://www.mingw.org/ [MSYS]: http://www.mingw.org/wiki/msys [zlib]: http://cygwin.com/packages/mingw-zlib/ [pthreads]: http://sourceware.org/pthreads-win32/ [GnuWin32]: http://gnuwin32.sf.net/packages/coreutils.htm [Download]: https://sourceforge.net/projects/bowtie-bio/files/bowtie2/ [sourceforge site]: https://sourceforge.net/projects/bowtie-bio/files/bowtie2/ [source package]: http://ccb.jhu.edu/software/hisat2/downloads/hisat2-2.0.0-beta-source.zip [Xcode]: http://developer.apple.com/xcode/ [NCBI-NGS]: https://github.com/ncbi/ngs/wiki/Downloads Running HISAT2 ============= Adding to PATH -------------- By adding your new HISAT2 directory to your [PATH environment variable], you ensure that whenever you run `hisat2`, `hisat2-build` or `hisat2-inspect` from the command line, you will get the version you just installed without having to specify the entire path. This is recommended for most users. To do this, follow your operating system's instructions for adding the directory to your [PATH]. If you would like to install HISAT2 by copying the HISAT2 executable files to an existing directory in your [PATH], make sure that you copy all the executables, including `hisat2`, `hisat2-align-s`, `hisat2-align-l`, `hisat2-build`, `hisat2-build-s`, `hisat2-build-l`, `hisat2-inspect`, `hisat2-inspect-s` and `hisat2-inspect-l`. [PATH environment variable]: http://en.wikipedia.org/wiki/PATH_(variable) [PATH]: http://en.wikipedia.org/wiki/PATH_(variable) Reporting --------- The reporting mode governs how many alignments HISAT2 looks for, and how to report them. In general, when we say that a read has an alignment, we mean that it has a [valid alignment]. When we say that a read has multiple alignments, we mean that it has multiple alignments that are valid and distinct from one another. [valid alignment]: #valid-alignments-meet-or-exceed-the-minimum-score-threshold By default, HISAT2 may soft-clip reads near their 5' and 3' ends. Users can control this behavior by setting different penalties for soft-clipping ([`--sp`]) or by disallowing soft-clipping ([`--no-softclip`]). ### Distinct alignments map a read to different places Two alignments for the same individual read are "distinct" if they map the same read to different places. Specifically, we say that two alignments are distinct if there are no alignment positions where a particular read offset is aligned opposite a particular reference offset in both alignments with the same orientation. E.g. if the first alignment is in the forward orientation and aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, and the second alignment is also in the forward orientation and also aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, they are not distinct alignments. Two alignments for the same pair are distinct if either the mate 1s in the two paired-end alignments are distinct or the mate 2s in the two alignments are distinct or both. ### Default mode: search for one or more alignments, report each HISAT2 searches for up to N distinct, primary alignments for each read, where N equals the integer specified with the `-k` parameter. Primary alignments mean alignments whose alignment score is equal or higher than any other alignments. It is possible that multiple distinct alignments have the same score. That is, if `-k 2` is specified, HISAT2 will search for at most 2 distinct alignments. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS field. See the [SAM specification] for details. HISAT2 does not "find" alignments in any specific order, so for reads that have more than N distinct, valid alignments, HISAT2 does not guarantee that the N alignments reported are the best possible in terms of alignment score. Still, this mode can be effective and fast in situations where the user cares more about whether a read aligns (or aligns a certain number of times) than where exactly it originated. [SAM specification]: http://samtools.sourceforge.net/SAM1.pdf Alignment summary ------------------ When HISAT2 finishes running, it prints messages summarizing what happened. These messages are printed to the "standard error" ("stderr") filehandle. For datasets consisting of unpaired reads, the summary might look like this: 20000 reads; of these: 20000 (100.00%) were unpaired; of these: 1247 (6.24%) aligned 0 times 18739 (93.69%) aligned exactly 1 time 14 (0.07%) aligned >1 times 93.77% overall alignment rate For datasets consisting of pairs, the summary might look like this: 10000 reads; of these: 10000 (100.00%) were paired; of these: 650 (6.50%) aligned concordantly 0 times 8823 (88.23%) aligned concordantly exactly 1 time 527 (5.27%) aligned concordantly >1 times ---- 650 pairs aligned concordantly 0 times; of these: 34 (5.23%) aligned discordantly 1 time ---- 616 pairs aligned 0 times concordantly or discordantly; of these: 1232 mates make up the pairs; of these: 660 (53.57%) aligned 0 times 571 (46.35%) aligned exactly 1 time 1 (0.08%) aligned >1 times 96.70% overall alignment rate The indentation indicates how subtotals relate to totals. Wrapper ------- The `hisat2`, `hisat2-build` and `hisat2-inspect` executables are actually wrapper scripts that call binary programs as appropriate. The wrappers shield users from having to distinguish between "small" and "large" index formats, discussed briefly in the following section. Also, the `hisat2` wrapper provides some key functionality, like the ability to handle compressed inputs, and the functionality for [`--un`], [`--al`] and related options. It is recommended that you always run the hisat2 wrappers and not run the binaries directly. Small and large indexes ----------------------- `hisat2-build` can index reference genomes of any size. For genomes less than about 4 billion nucleotides in length, `hisat2-build` builds a "small" index using 32-bit numbers in various parts of the index. When the genome is longer, `hisat2-build` builds a "large" index using 64-bit numbers. Small indexes are stored in files with the `.ht2` extension, and large indexes are stored in files with the `.ht2l` extension. The user need not worry about whether a particular index is small or large; the wrapper scripts will automatically build and use the appropriate index. Performance tuning ------------------ 1. If your computer has multiple processors/cores, use `-p` The [`-p`] option causes HISAT2 to launch a specified number of parallel search threads. Each thread runs on a different processor/core and all threads find alignments in parallel, increasing alignment throughput by approximately a multiple of the number of threads (though in practice, speedup is somewhat worse than linear). Command Line ------------ ### Setting function options Some HISAT2 options specify a function rather than an individual number or setting. In these cases the user specifies three parameters: (a) a function type `F`, (b) a constant term `B`, and (c) a coefficient `A`. The available function types are constant (`C`), linear (`L`), square-root (`S`), and natural log (`G`). The parameters are specified as `F,B,A` - that is, the function type, the constant term, and the coefficient are separated by commas with no whitespace. The constant term and coefficient may be negative and/or floating-point numbers. For example, if the function specification is `L,-0.4,-0.6`, then the function defined is: f(x) = -0.4 + -0.6 * x If the function specification is `G,1,5.4`, then the function defined is: f(x) = 1.0 + 5.4 * ln(x) See the documentation for the option in question to learn what the parameter `x` is for. For example, in the case if the [`--score-min`] option, the function `f(x)` sets the minimum alignment score necessary for an alignment to be considered valid, and `x` is the read length. ### Usage hisat2 [options]* -x {-1 -2 | -U | --sra-acc } [-S ] ### Main arguments
[`-x`]: #hisat2-options-x -x The basename of the index for the reference genome. The basename is the name of any of the index files up to but not including the final `.1.ht2` / etc. `hisat2` looks for the specified index first in the current directory, then in the directory specified in the `HISAT2_INDEXES` environment variable.
[`-1`]: #hisat2-options-1 -1 Comma-separated list of files containing mate 1s (filename usually includes `_1`), e.g. `-1 flyA_1.fq,flyB_1.fq`. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in ``. Reads may be a mix of different lengths. If `-` is specified, `hisat2` will read the mate 1s from the "standard in" or "stdin" filehandle.
[`-2`]: #hisat2-options-2 -2 Comma-separated list of files containing mate 2s (filename usually includes `_2`), e.g. `-2 flyA_2.fq,flyB_2.fq`. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in ``. Reads may be a mix of different lengths. If `-` is specified, `hisat2` will read the mate 2s from the "standard in" or "stdin" filehandle.
[`-U`]: #hisat2-options-U -U Comma-separated list of files containing unpaired reads to be aligned, e.g. `lane1.fq,lane2.fq,lane3.fq,lane4.fq`. Reads may be a mix of different lengths. If `-` is specified, `hisat2` gets the reads from the "standard in" or "stdin" filehandle.
[`--sra-acc`]: #hisat2-options-sra-acc --sra-acc Comma-separated list of SRA accession numbers, e.g. `--sra-acc SRR353653,SRR353654`. Information about read types is available at http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?sp=runinfo&acc=sra-acc&retmode=xml, where sra-acc is SRA accession number. If users run HISAT2 on a computer cluster, it is recommended to disable SRA-related caching (see the instruction at [SRA-MANUAL]). [SRA-MANUAL]: https://github.com/ncbi/sra-tools/wiki/Toolkit-Configuration
[`-S`]: #hisat2-options-S -S File to write SAM alignments to. By default, alignments are written to the "standard out" or "stdout" filehandle (i.e. the console).
### Options #### Input options
[`-q`]: #hisat2-options-q -q Reads (specified with ``, ``, ``) are FASTQ files. FASTQ files usually have extension `.fq` or `.fastq`. FASTQ is the default format. See also: [`--solexa-quals`] and [`--int-quals`].
[`--qseq`]: #hisat2-options-qseq --qseq Reads (specified with ``, ``, ``) are QSEQ files. QSEQ files usually end in `_qseq.txt`. See also: [`--solexa-quals`] and [`--int-quals`].
[`-f`]: #hisat2-options-f -f Reads (specified with ``, ``, ``) are FASTA files. FASTA files usually have extension `.fa`, `.fasta`, `.mfa`, `.fna` or similar. FASTA files do not have a way of specifying quality values, so when `-f` is set, the result is as if `--ignore-quals` is also set.
[`-r`]: #hisat2-options-r -r Reads (specified with ``, ``, ``) are files with one input sequence per line, without any other information (no read names, no qualities). When `-r` is set, the result is as if `--ignore-quals` is also set.
[`-c`]: #hisat2-options-c -c The read sequences are given on command line. I.e. ``, `` and `` are comma-separated lists of reads rather than lists of read files. There is no way to specify read names or qualities, so `-c` also implies `--ignore-quals`.
[`-s`/`--skip`]: #hisat2-options-s [`-s`]: #hisat2-options-s -s/--skip Skip (i.e. do not align) the first `` reads or pairs in the input.
[`-u`/`--qupto`]: #hisat2-options-u [`-u`]: #hisat2-options-u -u/--qupto Align the first `` reads or read pairs from the input (after the [`-s`/`--skip`] reads or pairs have been skipped), then stop. Default: no limit.
[`-5`/`--trim5`]: #hisat2-options-5 [`-5`]: #hisat2-options-5 -5/--trim5 Trim `` bases from 5' (left) end of each read before alignment (default: 0).
[`-3`/`--trim3`]: #hisat2-options-3 [`-3`]: #hisat2-options-3 -3/--trim3 Trim `` bases from 3' (right) end of each read before alignment (default: 0).
[`--phred33`]: #hisat2-options-phred33-quals --phred33 Input qualities are ASCII chars equal to the [Phred quality] plus 33. This is also called the "Phred+33" encoding, which is used by the very latest Illumina pipelines. [Phred quality]: http://en.wikipedia.org/wiki/Phred_quality_score
[`--phred64`]: #hisat2-options-phred64-quals --phred64 Input qualities are ASCII chars equal to the [Phred quality] plus 64. This is also called the "Phred+64" encoding.
[`--solexa-quals`]: #hisat2-options-solexa-quals --solexa-quals Convert input qualities from [Solexa][Phred quality] (which can be negative) to [Phred][Phred quality] (which can't). This scheme was used in older Illumina GA Pipeline versions (prior to 1.3). Default: off.
[`--int-quals`]: #hisat2-options-int-quals --int-quals Quality values are represented in the read input file as space-separated ASCII integers, e.g., `40 40 30 40`..., rather than ASCII characters, e.g., `II?I`.... Integers are treated as being on the [Phred quality] scale unless [`--solexa-quals`] is also specified. Default: off.
#### Alignment options
[`--n-ceil`]: #hisat2-options-n-ceil --n-ceil Sets a function governing the maximum number of ambiguous characters (usually `N`s and/or `.`s) allowed in a read as a function of read length. For instance, specifying `-L,0,0.15` sets the N-ceiling function `f` to `f(x) = 0 + 0.15 * x`, where x is the read length. See also: [setting function options]. Reads exceeding this ceiling are [filtered out]. Default: `L,0,0.15`. [filtered out]: #filtering
[`--ignore-quals`]: #hisat2-options-ignore-quals --ignore-quals When calculating a mismatch penalty, always consider the quality value at the mismatched position to be the highest possible, regardless of the actual value. I.e. input is treated as though all quality values are high. This is also the default behavior when the input doesn't specify quality values (e.g. in [`-f`], [`-r`], or [`-c`] modes).
[`--nofw`]: #hisat2-options-nofw --nofw/--norc If `--nofw` is specified, `hisat2` will not attempt to align unpaired reads to the forward (Watson) reference strand. If `--norc` is specified, `hisat2` will not attempt to align unpaired reads against the reverse-complement (Crick) reference strand. In paired-end mode, `--nofw` and `--norc` pertain to the fragments; i.e. specifying `--nofw` causes `hisat2` to explore only those paired-end configurations corresponding to fragments from the reverse-complement (Crick) strand. Default: both strands enabled.
#### Scoring options
[`--mp`]: #hisat2-options-mp --mp MX,MN Sets the maximum (`MX`) and minimum (`MN`) mismatch penalties, both integers. A number less than or equal to `MX` and greater than or equal to `MN` is subtracted from the alignment score for each position where a read character aligns to a reference character, the characters do not match, and neither is an `N`. If [`--ignore-quals`] is specified, the number subtracted quals `MX`. Otherwise, the number subtracted is `MN + floor( (MX-MN)(MIN(Q, 40.0)/40.0) )` where Q is the Phred quality value. Default: `MX` = 6, `MN` = 2.
[`--sp`]: #hisat2-options-sp --sp MX,MN Sets the maximum (`MX`) and minimum (`MN`) penalties for soft-clipping per base, both integers. A number less than or equal to `MX` and greater than or equal to `MN` is subtracted from the alignment score for each position. The number subtracted is `MN + floor( (MX-MN)(MIN(Q, 40.0)/40.0) )` where Q is the Phred quality value. Default: `MX` = 2, `MN` = 1.
[`--sp`]: #hisat2-options-no-softclip --no-softclip Disallow soft-clipping.
[`--np`]: #hisat2-options-np --np Sets penalty for positions where the read, reference, or both, contain an ambiguous character such as `N`. Default: 1.
[`--rdg`]: #hisat2-options-rdg --rdg , Sets the read gap open (``) and extend (``) penalties. A read gap of length N gets a penalty of `` + N * ``. Default: 5, 3.
[`--rfg`]: #hisat2-options-rfg --rfg , Sets the reference gap open (``) and extend (``) penalties. A reference gap of length N gets a penalty of `` + N * ``. Default: 5, 3.
[`--score-min`]: #hisat2-options-score-min --score-min Sets a function governing the minimum alignment score needed for an alignment to be considered "valid" (i.e. good enough to report). This is a function of read length. For instance, specifying `L,0,-0.6` sets the minimum-score function `f` to `f(x) = 0 + -0.6 * x`, where `x` is the read length. See also: [setting function options]. The default is `L,0,-0.2`.
#### Spliced alignment options
[`--pen-cansplice`]: #hisat2-options-pen-cansplice --pen-cansplice Sets the penalty for each pair of canonical splice sites (e.g. GT/AG). Default: 0.
[`--pen-noncansplice`]: #hisat2-options-pen-noncansplice --pen-noncansplice Sets the penalty for each pair of non-canonical splice sites (e.g. non-GT/AG). Default: 12.
[`--pen-canintronlen`]: #hisat2-options-pen-canintronlen --pen-canintronlen Sets the penalty for long introns with canonical splice sites so that alignments with shorter introns are preferred to those with longer ones. Default: G,-8,1
[`--pen-noncanintronlen`]: #hisat2-options-pen-noncanintronlen --pen-noncanintronlen Sets the penalty for long introns with noncanonical splice sites so that alignments with shorter introns are preferred to those with longer ones. Default: G,-8,1
[`--min-intronlen`]: #hisat2-options-min-intronlen --min-intronlen Sets minimum intron length. Default: 20
[`--max-intronlen`]: #hisat2-options-max-intronlen --max-intronlen Sets maximum intron length. Default: 500000
[`--splice-infile`]: #hisat2-options-known-splicesite-infile --known-splicesite-infile With this mode, you can provide a list of known splice sites, which HISAT2 makes use of to align reads with small anchors. You can create such a list using `python hisat2_extract_splice_sites.py genes.gtf > splicesites.txt`, where `hisat2_extract_splice_sites.py` is included in the HISAT2 package, `genes.gtf` is a gene annotation file, and `splicesites.txt` is a list of splice sites with which you provide HISAT2 in this mode. Note that it is better to use indexes built using annotated transcripts (such as genome_tran or genome_snp_tran), which works better than using this option. It has no effect to provide splice sites that are already included in the indexes.
[`--novel-splicesite-outfile`]: #hisat2-options-novel-splicesite-outfile --novel-splicesite-outfile In this mode, HISAT2 reports a list of splice sites in the file : chromosome name `` genomic position of the flanking base on the left side of an intron `` genomic position of the flanking base on the right `` strand (+, -, and .) '.' indicates an unknown strand for non-canonical splice sites.
[`--novel-splicesite-infile`]: #hisat2-options-novel-splicesite-infile --novel-splicesite-infile With this mode, you can provide a list of novel splice sites that were generated from the above option "--novel-splicesite-outfile".
[`--no-temp-splicesite`]: #hisat2-options-no-temp-splicesite --no-temp-splicesite HISAT2, by default, makes use of splice sites found by earlier reads to align later reads in the same run, in particular, reads with small anchors (<= 15 bp). The option disables this default alignment strategy.
[`--no-spliced-alignment`]: #hisat2-options-no-spliced-alignment --no-spliced-alignment Disable spliced alignment.
[`--rna-strandness`]: #hisat2-options-rna-strandness --rna-strandness Specify strand-specific information: the default is unstranded. For single-end reads, use F or R. 'F' means a read corresponds to a transcript. 'R' means a read corresponds to the reverse complemented counterpart of a transcript. For paired-end reads, use either FR or RF. With this option being used, every read alignment will have an XS attribute tag: '+' means a read belongs to a transcript on '+' strand of genome. '-' means a read belongs to a transcript on '-' strand of genome. (TopHat has a similar option, --library-type option, where fr-firststrand corresponds to R and RF; fr-secondstrand corresponds to F and FR.)
[`--tmo/--transcriptome-mapping-only`]: #hisat2-options-tmo --tmo/--transcriptome-mapping-only Report only those alignments within known transcripts.
[`--dta/--downstream-transcriptome-assembly`]: #hisat2-options-dta --dta/--downstream-transcriptome-assembly Report alignments tailored for transcript assemblers including StringTie. With this option, HISAT2 requires longer anchor lengths for de novo discovery of splice sites. This leads to fewer alignments with short-anchors, which helps transcript assemblers improve significantly in computation and memory usage.
Report alignments tailored specifically for Cufflinks. In addition to what HISAT2 does with the above option (--dta), With this option, HISAT2 looks for novel splice sites with three signals (GT/AG, GC/AG, AT/AC), but all user-provided splice sites are used irrespective of their signals. HISAT2 produces an optional field, XS:A:[+-], for every spliced alignment.
[`--avoid-pseudogene`]: #hisat2-options-avoid-pseudogene --avoid-pseudogene Try to avoid aligning reads to pseudogenes. Note this option is experimental and needs further investigation.
[`--no-templatelen-adjustment`]: #hisat2-options-no-templatelen-adjustment --no-templatelen-adjustment Disables template length adjustment for RNA-seq reads.
#### Reporting options
[`-k`]: #hisat2-options-k -k It searches for at most `` distinct, primary alignments for each read. Primary alignments mean alignments whose alignment score is equal or higher than any other alignments. The search terminates when it can't find more distinct valid alignments, or when it finds ``, whichever happens first. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS field. For reads that have more than `` distinct, valid alignments, `hisat2` does not guarantee that the `` alignments reported are the best possible in terms of alignment score. Default: 5 (HFM) or 10 (HGFM) Note: HISAT2 is not designed with large values for `-k` in mind, and when aligning reads to long, repetitive genomes large `-k` can be very, very slow.
[`--max-seeds`]: #hisat2-options-max-seeds --max-seeds HISAT2, like other aligners, uses seed-and-extend approaches. HISAT2 tries to extend seeds to full-length alignments. In HISAT2, --max-seeds is used to control the maximum number of seeds that will be extended. HISAT2 extends up to these many seeds and skips the rest of the seeds. Large values for `--max-seeds` may improve alignment sensitivity, but HISAT2 is not designed with large values for `--max-seeds` in mind, and when aligning reads to long, repetitive genomes large `--max-seeds` can be very, very slow. The default value is the maximum of 5 and the value that comes with`-k`.
[`--secondary`]: #hisat2-options-secondary --secondary Report secondary alignments.
#### Paired-end options
[`-I`/`--minins`]: #hisat2-options-I [`-I`]: #hisat2-options-I -I/--minins The minimum fragment length for valid paired-end alignments.This option is valid only with --no-spliced-alignment. E.g. if `-I 60` is specified and a paired-end alignment consists of two 20-bp alignments in the appropriate orientation with a 20-bp gap between them, that alignment is considered valid (as long as [`-X`] is also satisfied). A 19-bp gap would not be valid in that case. If trimming options [`-3`] or [`-5`] are also used, the [`-I`] constraint is applied with respect to the untrimmed mates. The larger the difference between [`-I`] and [`-X`], the slower HISAT2 will run. This is because larger differences between [`-I`] and [`-X`] require that HISAT2 scan a larger window to determine if a concordant alignment exists. For typical fragment length ranges (200 to 400 nucleotides), HISAT2 is very efficient. Default: 0 (essentially imposing no minimum)
[`-X`/`--maxins`]: #hisat2-options-X [`-X`]: #hisat2-options-X -X/--maxins The maximum fragment length for valid paired-end alignments. This option is valid only with --no-spliced-alignment. E.g. if `-X 100` is specified and a paired-end alignment consists of two 20-bp alignments in the proper orientation with a 60-bp gap between them, that alignment is considered valid (as long as [`-I`] is also satisfied). A 61-bp gap would not be valid in that case. If trimming options [`-3`] or [`-5`] are also used, the `-X` constraint is applied with respect to the untrimmed mates, not the trimmed mates. The larger the difference between [`-I`] and [`-X`], the slower HISAT2 will run. This is because larger differences between [`-I`] and [`-X`] require that HISAT2 scan a larger window to determine if a concordant alignment exists. For typical fragment length ranges (200 to 400 nucleotides), HISAT2 is very efficient. Default: 500.
[`--fr`/`--rf`/`--ff`]: #hisat2-options-fr [`--fr`]: #hisat2-options-fr [`--rf`]: #hisat2-options-fr [`--ff`]: #hisat2-options-fr --fr/--rf/--ff The upstream/downstream mate orientations for a valid paired-end alignment against the forward reference strand. E.g., if `--fr` is specified and there is a candidate paired-end alignment where mate 1 appears upstream of the reverse complement of mate 2 and the fragment length constraints ([`-I`] and [`-X`]) are met, that alignment is valid. Also, if mate 2 appears upstream of the reverse complement of mate 1 and all other constraints are met, that too is valid. `--rf` likewise requires that an upstream mate1 be reverse-complemented and a downstream mate2 be forward-oriented. ` --ff` requires both an upstream mate 1 and a downstream mate 2 to be forward-oriented. Default: `--fr` (appropriate for Illumina's Paired-end Sequencing Assay).
[`--no-mixed`]: #hisat2-options-no-mixed --no-mixed By default, when `hisat2` cannot find a concordant or discordant alignment for a pair, it then tries to find alignments for the individual mates. This option disables that behavior.
[`--no-discordant`]: #hisat2-options-no-discordant --no-discordant By default, `hisat2` looks for discordant alignments if it cannot find any concordant alignments. A discordant alignment is an alignment where both mates align uniquely, but that does not satisfy the paired-end constraints ([`--fr`/`--rf`/`--ff`], [`-I`], [`-X`]). This option disables that behavior.
#### Output options
[`-t`/`--time`]: #hisat2-options-t [`-t`]: #hisat2-options-t -t/--time Print the wall-clock time required to load the index files and align the reads. This is printed to the "standard error" ("stderr") filehandle. Default: off.
[`--un`]: #hisat2-options-un [`--un-gz`]: #hisat2-options-un [`--un-bz2`]: #hisat2-options-un --un --un-gz --un-bz2 Write unpaired reads that fail to align to file at ``. These reads correspond to the SAM records with the FLAGS `0x4` bit set and neither the `0x40` nor `0x80` bits set. If `--un-gz` is specified, output will be gzip compressed. If `--un-bz2` is specified, output will be bzip2 compressed. Reads written in this way will appear exactly as they did in the input file, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the input.
[`--al`]: #hisat2-options-al [`--al-gz`]: #hisat2-options-al [`--al-bz2`]: #hisat2-options-al --al --al-gz --al-bz2 Write unpaired reads that align at least once to file at ``. These reads correspond to the SAM records with the FLAGS `0x4`, `0x40`, and `0x80` bits unset. If `--al-gz` is specified, output will be gzip compressed. If `--al-bz2` is specified, output will be bzip2 compressed. Reads written in this way will appear exactly as they did in the input file, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the input.
[`--un-conc`]: #hisat2-options-un-conc [`--un-conc-gz`]: #hisat2-options-un-conc [`--un-conc-bz2`]: #hisat2-options-un-conc --un-conc --un-conc-gz --un-conc-bz2 Write paired-end reads that fail to align concordantly to file(s) at ``. These reads correspond to the SAM records with the FLAGS `0x4` bit set and either the `0x40` or `0x80` bit set (depending on whether it's mate #1 or #2). `.1` and `.2` strings are added to the filename to distinguish which file contains mate #1 and mate #2. If a percent symbol, `%`, is used in ``, the percent symbol is replaced with `1` or `2` to make the per-mate filenames. Otherwise, `.1` or `.2` are added before the final dot in `` to make the per-mate filenames. Reads written in this way will appear exactly as they did in the input files, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the inputs.
[`--al-conc`]: #hisat2-options-al-conc [`--al-conc-gz`]: #hisat2-options-al-conc [`--al-conc-bz2`]: #hisat2-options-al-conc --al-conc --al-conc-gz --al-conc-bz2 Write paired-end reads that align concordantly at least once to file(s) at ``. These reads correspond to the SAM records with the FLAGS `0x4` bit unset and either the `0x40` or `0x80` bit set (depending on whether it's mate #1 or #2). `.1` and `.2` strings are added to the filename to distinguish which file contains mate #1 and mate #2. If a percent symbol, `%`, is used in ``, the percent symbol is replaced with `1` or `2` to make the per-mate filenames. Otherwise, `.1` or `.2` are added before the final dot in `` to make the per-mate filenames. Reads written in this way will appear exactly as they did in the input files, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the inputs.
[`--quiet`]: #hisat2-options-quiet --quiet Print nothing besides alignments and serious errors.
[`--summary-file`]: #hisat2-options-summary-file --summary-file Print alignment summary to this file.
[`--new-summary`]: #hisat2-options-new-summary --new-summary Print alignment summary in a new style, which is more machine-friendly.
[`--met-file`]: #hisat2-options-met-file --met-file Write `hisat2` metrics to file ``. Having alignment metric can be useful for debugging certain problems, especially performance issues. See also: [`--met`]. Default: metrics disabled.
[`--met-stderr`]: #hisat2-options-met-stderr --met-stderr Write `hisat2` metrics to the "standard error" ("stderr") filehandle. This is not mutually exclusive with [`--met-file`]. Having alignment metric can be useful for debugging certain problems, especially performance issues. See also: [`--met`]. Default: metrics disabled.
[`--met`]: #hisat2-options-met --met Write a new `hisat2` metrics record every `` seconds. Only matters if either [`--met-stderr`] or [`--met-file`] are specified. Default: 1.
#### SAM options
[`--no-unal`]: #hisat2-options-no-unal --no-unal Suppress SAM records for reads that failed to align.
[`--no-hd`]: #hisat2-options-no-hd --no-hd Suppress SAM header lines (starting with `@`).
[`--no-sq`]: #hisat2-options-no-sq --no-sq Suppress `@SQ` SAM header lines.
[`--rg-id`]: #hisat2-options-rg-id --rg-id Set the read group ID to ``. This causes the SAM `@RG` header line to be printed, with `` as the value associated with the `ID:` tag. It also causes the `RG:Z:` extra field to be attached to each SAM output record, with value set to ``.
[`--rg`]: #hisat2-options-rg --rg Add `` (usually of the form `TAG:VAL`, e.g. `SM:Pool1`) as a field on the `@RG` header line. Note: in order for the `@RG` line to appear, [`--rg-id`] must also be specified. This is because the `ID` tag is required by the [SAM Spec][SAM]. Specify `--rg` multiple times to set multiple fields. See the [SAM Spec][SAM] for details about what fields are legal.
[`--remove-chrname`]: #hisat2-remove-chrname --remove-chrname Remove 'chr' from reference names in alignment (e.g., chr18 to 18)
[`--add-chrname`]: #hisat2-options-add-chrname --add-chrname Add 'chr' to reference names in alignment (e.g., 18 to chr18)
[`--omit-sec-seq`]: #hisat2-options-omit-sec-seq --omit-sec-seq When printing secondary alignments, HISAT2 by default will write out the `SEQ` and `QUAL` strings. Specifying this option causes HISAT2 to print an asterisk in those fields instead.
#### Performance options
[`-o`/`--offrate`]: #hisat2-options-o [`-o`]: #hisat2-options-o [`--offrate`]: #hisat2-options-o -o/--offrate Override the offrate of the index with ``. If `` is greater than the offrate used to build the index, then some row markings are discarded when the index is read into memory. This reduces the memory footprint of the aligner but requires more time to calculate text offsets. `` must be greater than the value used to build the index.
[`-p`/`--threads`]: #hisat2-options-p [`-p`]: #hisat2-options-p -p/--threads NTHREADS Launch `NTHREADS` parallel search threads (default: 1). Threads will run on separate processors/cores and synchronize when parsing reads and outputting alignments. Searching for alignments is highly parallel, and speedup is close to linear. Increasing `-p` increases HISAT2's memory footprint. E.g. when aligning to a human genome index, increasing `-p` from 1 to 8 increases the memory footprint by a few hundred megabytes. This option is only available if `bowtie` is linked with the `pthreads` library (i.e. if `BOWTIE_PTHREADS=0` is not specified at build time).
[`--reorder`]: #hisat2-options-reorder --reorder Guarantees that output SAM records are printed in an order corresponding to the order of the reads in the original input file, even when [`-p`] is set greater than 1. Specifying `--reorder` and setting [`-p`] greater than 1 causes HISAT2 to run somewhat slower and use somewhat more memory then if `--reorder` were not specified. Has no effect if [`-p`] is set to 1, since output order will naturally correspond to input order in that case.
[`--mm`]: #hisat2-options-mm --mm Use memory-mapped I/O to load the index, rather than typical file I/O. Memory-mapping allows many concurrent `bowtie` processes on the same computer to share the same memory image of the index (i.e. you pay the memory overhead just once). This facilitates memory-efficient parallelization of `bowtie` in situations where using [`-p`] is not possible or not preferable.
#### Other options
[`--qc-filter`]: #hisat2-options-qc-filter --qc-filter Filter out reads for which the QSEQ filter field is non-zero. Only has an effect when read format is [`--qseq`]. Default: off.
[`--seed`]: #hisat2-options-seed --seed Use `` as the seed for pseudo-random number generator. Default: 0.
[`--non-deterministic`]: #hisat2-options-non-deterministic --non-deterministic Normally, HISAT2 re-initializes its pseudo-random generator for each read. It seeds the generator with a number derived from (a) the read name, (b) the nucleotide sequence, (c) the quality sequence, (d) the value of the [`--seed`] option. This means that if two reads are identical (same name, same nucleotides, same qualities) HISAT2 will find and report the same alignment(s) for both, even if there was ambiguity. When `--non-deterministic` is specified, HISAT2 re-initializes its pseudo-random generator for each read using the current time. This means that HISAT2 will not necessarily report the same alignment for two identical reads. This is counter-intuitive for some users, but might be more appropriate in situations where the input consists of many identical reads.
[`--version`]: #hisat2-options-version --version Print version information and quit.
-h/--help Print usage information and quit.
SAM output ---------- Following is a brief description of the [SAM] format as output by `hisat2`. For more details, see the [SAM format specification][SAM]. By default, `hisat2` prints a SAM header with `@HD`, `@SQ` and `@PG` lines. When one or more [`--rg`] arguments are specified, `hisat2` will also print an `@RG` line that includes all user-specified [`--rg`] tokens separated by tabs. Each subsequent line describes an alignment or, if the read failed to align, a read. Each line is a collection of at least 12 fields separated by tabs; from left to right, the fields are: 1. Name of read that aligned. Note that the [SAM specification] disallows whitespace in the read name. If the read name contains any whitespace characters, HISAT2 will truncate the name at the first whitespace character. This is similar to the behavior of other tools. 2. Sum of all applicable flags. Flags relevant to HISAT2 are:
1 The read is one of a pair
2 The alignment is one end of a proper paired-end alignment
4 The read has no reported alignments
8 The read is one of a pair and has no reported alignments
16 The alignment is to the reverse reference strand
32 The other mate in the paired-end alignment is aligned to the reverse reference strand
64 The read is mate 1 in a pair
128 The read is mate 2 in a pair
Thus, an unpaired read that aligns to the reverse reference strand will have flag 16. A paired-end read that aligns and is the first mate in the pair will have flag 83 (= 64 + 16 + 2 + 1). 3. Name of reference sequence where alignment occurs 4. 1-based offset into the forward reference strand where leftmost character of the alignment occurs 5. Mapping quality. Mapping quality of HISAT2 6. CIGAR string representation of alignment 7. Name of reference sequence where mate's alignment occurs. Set to `=` if the mate's reference sequence is the same as this alignment's, or `*` if there is no mate. 8. 1-based offset into the forward reference strand where leftmost character of the mate's alignment occurs. Offset is 0 if there is no mate. 9. Inferred fragment length. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if the mates did not align concordantly. However, size is non-0 if the mates aligned discordantly to the same chromosome. 10. Read sequence (reverse-complemented if aligned to the reverse strand) 11. ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the [Phred quality] scale and the encoding is ASCII-offset by 33 (ASCII char `!`), similarly to a [FASTQ] file. 12. Optional fields. Fields are tab-separated. `hisat2` outputs zero or more of these optional fields for each alignment, depending on the type of the alignment:
AS:i: Alignment score. Can be negative. Only present if SAM record is for an aligned read.
ZS:i: Alignment score for the best-scoring alignment found other than the alignment reported. Can be negative. Only present if the SAM record is for an aligned read and more than one alignment was found for the read. Note that, when the read is part of a concordantly-aligned pair, this score could be greater than [`AS:i`].
YS:i: Alignment score for opposite mate in the paired-end alignment. Only present if the SAM record is for a read that aligned as part of a paired-end alignment.
XN:i: The number of ambiguous bases in the reference covering this alignment. Only present if SAM record is for an aligned read.
XM:i: The number of mismatches in the alignment. Only present if SAM record is for an aligned read.
XO:i: The number of gap opens, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.
XG:i: The number of gap extensions, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.
NM:i: The edit distance; that is, the minimal number of one-nucleotide edits (substitutions, insertions and deletions) needed to transform the read string into the reference string. Only present if SAM record is for an aligned read.
YF:Z: String indicating reason why the read was filtered out. See also: [Filtering]. Only appears for reads that were filtered out.
YT:Z: Value of `UU` indicates the read was not part of a pair. Value of `CP` indicates the read was part of a pair and the pair aligned concordantly. Value of `DP` indicates the read was part of a pair and the pair aligned discordantly. Value of `UP` indicates the read was part of a pair but the pair failed to aligned either concordantly or discordantly.
MD:Z: A string representation of the mismatched reference bases in the alignment. See [SAM] format specification for details. Only present if SAM record is for an aligned read.
XS:A: Values of `+` and `-` indicate the read is mapped to transcripts on sense and anti-sense strands, respectively. Spliced alignments need to have this field, which is required in Cufflinks and StringTie. We can report this field for the canonical-splice site (GT/AG), but not for non-canonical splice sites. You can direct HISAT2 not to output such alignments (involving non-canonical splice sites) using "--pen-noncansplice 1000000".
NH:i: The number of mapped locations for the read or the pair.
Zs:Z: When the alignment of a read involves SNPs that are in the index, this option is used to indicate where exactly the read involves the SNPs. This optional field is similar to the above MD:Z field. For example, `Zs:Z:1|S|rs3747203,97|S|rs16990981` indicates the second base of the read corresponds to a known SNP (ID: rs3747203). 97 bases after the third base (the base after the second one), the read at 100th base involves another known SNP (ID: rs16990981). 'S' indicates a single nucleotide polymorphism. 'D' and 'I' indicate a deletion and an insertion, respectively.
[SAM format specification]: http://samtools.sf.net/SAM1.pdf [FASTQ]: http://en.wikipedia.org/wiki/FASTQ_format [`-S`/`--sam`]: #hisat2-options-S [`-m`]: #hisat2-options-m The `hisat2-build` indexer =========================== `hisat2-build` builds a HISAT2 index from a set of DNA sequences. `hisat2-build` outputs a set of 6 files with suffixes `.1.ht2`, `.2.ht2`, `.3.ht2`, `.4.ht2`, `.5.ht2`, `.6.ht2`, `.7.ht2`, and `.8.ht2`. In the case of a large index these suffixes will have a `ht2l` termination. These files together constitute the index: they are all that is needed to align reads to that reference. The original sequence FASTA files are no longer used by HISAT2 once the index is built. Use of Karkkainen's [blockwise algorithm] allows `hisat2-build` to trade off between running time and memory usage. `hisat2-build` has three options governing how it makes this trade: [`-p`/`--packed`], [`--bmax`]/[`--bmaxdivn`], and [`--dcv`]. By default, `hisat2-build` will automatically search for the settings that yield the best running time without exhausting memory. This behavior can be disabled using the [`-a`/`--noauto`] option. The indexer provides options pertaining to the "shape" of the index, e.g. [`--offrate`](#hisat2-build-options-o) governs the fraction of [Burrows-Wheeler] rows that are "marked" (i.e., the density of the suffix-array sample; see the original [FM Index] paper for details). All of these options are potentially profitable trade-offs depending on the application. They have been set to defaults that are reasonable for most cases according to our experiments. See [Performance tuning] for details. `hisat2-build` can generate either [small or large indexes](#small-and-large-indexes). The wrapper will decide which based on the length of the input genome. If the reference does not exceed 4 billion characters but a large index is preferred, the user can specify [`--large-index`] to force `hisat2-build` to build a large index instead. The HISAT2 index is based on the [FM Index] of Ferragina and Manzini, which in turn is based on the [Burrows-Wheeler] transform. The algorithm used to build the index is based on the [blockwise algorithm] of Karkkainen. [Blockwise algorithm]: http://portal.acm.org/citation.cfm?id=1314852 [Burrows-Wheeler]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform [Performance tuning]: #performance-tuning Command Line ------------ Usage: hisat2-build [options]* ### Notes If you use --snp, --ss, and/or --exon, hisat2-build will need about 200GB RAM for the human genome size as index building involves a graph construction. Otherwise, you will be able to build an index on your desktop with 8GB RAM. ### Main arguments
A comma-separated list of FASTA files containing the reference sequences to be aligned to, or, if [`-c`](#hisat2-build-options-c) is specified, the sequences themselves. E.g., `` might be `chr1.fa,chr2.fa,chrX.fa,chrY.fa`, or, if [`-c`](#hisat2-build-options-c) is specified, this might be `GGTCATCCT,ACGGGTCGT,CCGTTCTATGCGGCTTA`.
The basename of the index files to write. By default, `hisat2-build` writes files named `NAME.1.ht2`, `NAME.2.ht2`, `NAME.3.ht2`, `NAME.4.ht2`, `NAME.5.ht2`, `NAME.6.ht2`, `NAME.7.ht2`, and `NAME.8.ht2` where `NAME` is ``.
### Options
-f The reference input files (specified as ``) are FASTA files (usually having extension `.fa`, `.mfa`, `.fna` or similar).
-c The reference sequences are given on the command line. I.e. `` is a comma-separated list of sequences rather than a list of FASTA files.
[`--large-index`]: #hisat2-build-options-large-index --large-index Force `hisat2-build` to build a [large index](#small-and-large-indexes), even if the reference is less than ~ 4 billion nucleotides long.
[`-a`/`--noauto`]: #hisat2-build-options-a -a/--noauto Disable the default behavior whereby `hisat2-build` automatically selects values for the [`--bmax`], [`--dcv`] and [`--packed`] parameters according to available memory. Instead, user may specify values for those parameters. If memory is exhausted during indexing, an error message will be printed; it is up to the user to try new parameters.
[`--bmax`]: #hisat2-build-options-bmax --bmax The maximum number of suffixes allowed in a block. Allowing more suffixes per block makes indexing faster, but increases peak memory usage. Setting this option overrides any previous setting for [`--bmax`], or [`--bmaxdivn`]. Default (in terms of the [`--bmaxdivn`] parameter) is [`--bmaxdivn`] 4. This is configured automatically by default; use [`-a`/`--noauto`] to configure manually.
[`--bmaxdivn`]: #hisat2-build-options-bmaxdivn --bmaxdivn The maximum number of suffixes allowed in a block, expressed as a fraction of the length of the reference. Setting this option overrides any previous setting for [`--bmax`], or [`--bmaxdivn`]. Default: [`--bmaxdivn`] 4. This is configured automatically by default; use [`-a`/`--noauto`] to configure manually.
[`--dcv`]: #hisat2-build-options-dcv --dcv Use `` as the period for the difference-cover sample. A larger period yields less memory overhead, but may make suffix sorting slower, especially if repeats are present. Must be a power of 2 no greater than 4096. Default: 1024. This is configured automatically by default; use [`-a`/`--noauto`] to configure manually.
[`--nodc`]: #hisat2-build-options-nodc --nodc Disable use of the difference-cover sample. Suffix sorting becomes quadratic-time in the worst case (where the worst case is an extremely repetitive reference). Default: off.
-r/--noref Do not build the `NAME.3.ht2` and `NAME.4.ht2` portions of the index, which contain a bitpacked version of the reference sequences and are used for paired-end alignment.
-3/--justref Build only the `NAME.3.ht2` and `NAME.4.ht2` portions of the index, which contain a bitpacked version of the reference sequences and are used for paired-end alignment.
-o/--offrate To map alignments back to positions on the reference sequences, it's necessary to annotate ("mark") some or all of the [Burrows-Wheeler] rows with their corresponding location on the genome. [`-o`/`--offrate`](#hisat2-build-options-o) governs how many rows get marked: the indexer will mark every 2^`` rows. Marking more rows makes reference-position lookups faster, but requires more memory to hold the annotations at runtime. The default is 4 (every 16th row is marked; for human genome, annotations occupy about 680 megabytes).
-t/--ftabchars The ftab is the lookup table used to calculate an initial [Burrows-Wheeler] range with respect to the first `` characters of the query. A larger `` yields a larger lookup table but faster query times. The ftab has size 4^(``+1) bytes. The default setting is 10 (ftab is 4MB).
--localoffrate This option governs how many rows get marked in a local index: the indexer will mark every 2^`` rows. Marking more rows makes reference-position lookups faster, but requires more memory to hold the annotations at runtime. The default is 3 (every 8th row is marked, this occupies about 16KB per local index).
--localftabchars The local ftab is the lookup table in a local index. The default setting is 6 (ftab is 8KB per local index).
-p Launch `NTHREADS` parallel build threads (default: 1).
--snp Provide a list of SNPs (in the HISAT2's own format) as follows (five columns). SNP ID `` snp type (single, deletion, or insertion) `` chromosome name `` zero-offset based genomic position of a SNP `` alternative base (single), the length of SNP (deletion), or insertion sequence (insertion) For example, rs58784443 single 13 18447947 T Use `hisat2_extract_snps_haplotypes_UCSC.py` (in the HISAT2 package) to extract SNPs and haplotypes from a dbSNP file (e.g. http://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/snp144Common.txt.gz). or `hisat2_extract_snps_haplotypes_VCF.py` to extract SNPs and haplotypes from a VCF file (e.g. ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/supporting/GRCh38_positions/ALL.chr22.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP_no_SVs.vcf.gz).
--haplotype Provide a list of haplotypes (in the HISAT2's own format) as follows (five columns). Haplotype ID `` chromosome name `` zero-offset based left coordinate of haplotype `` zero-offset based right coordinate of haplotype `` a comma separated list of SNP ids in the haplotype For example, ht35 13 18446877 18446945 rs12381094,rs12381056,rs192016659,rs538569910 See the above option, --snp, about how to extract haplotypes. This option is not required, but haplotype information can keep the index construction from exploding and reduce the index size substantially.
--ss Note this option should be used with the following --exon option. Provide a list of splice sites (in the HISAT2's own format) as follows (four columns). chromosome name `` zero-offset based genomic position of the flanking base on the left side of an intron `` zero-offset based genomic position of the flanking base on the right `` strand Use `hisat2_extract_splice_sites.py` (in the HISAT2 package) to extract splice sites from a GTF file.
--exon Note this option should be used with the above --ss option. Provide a list of exons (in the HISAT2's own format) as follows (three columns). chromosome name `` zero-offset based left genomic position of an exon `` zero-offset based right genomic position of an exon Use `hisat2_extract_exons.py` (in the HISAT2 package) to extract exons from a GTF file.
--seed Use `` as the seed for pseudo-random number generator.
--cutoff Index only the first `` bases of the reference sequences (cumulative across sequences) and ignore the rest.
-q/--quiet `hisat2-build` is verbose by default. With this option `hisat2-build` will print only error messages.
-h/--help Print usage information and quit.
--version Print version information and quit.
The `hisat2-inspect` index inspector ===================================== `hisat2-inspect` extracts information from a HISAT2 index about what kind of index it is and what reference sequences were used to build it. When run without any options, the tool will output a FASTA file containing the sequences of the original references (with all non-`A`/`C`/`G`/`T` characters converted to `N`s). It can also be used to extract just the reference sequence names using the [`-n`/`--names`] option or a more verbose summary using the [`-s`/`--summary`] option. Command Line ------------ Usage: hisat2-inspect [options]* ### Main arguments
The basename of the index to be inspected. The basename is name of any of the index files but with the `.X.ht2` suffix omitted. `hisat2-inspect` first looks in the current directory for the index files, then in the directory specified in the `HISAT2_INDEXES` environment variable.
### Options
-a/--across When printing FASTA output, output a newline character every `` bases (default: 60).
[`-n`/`--names`]: #hisat2-inspect-options-n -n/--names Print reference sequence names, one per line, and quit.
[`-s`/`--summary`]: #hisat2-inspect-options-s -s/--summary Print a summary that includes information about index settings, as well as the names and lengths of the input sequences. The summary has this format: Colorspace <0 or 1> SA-Sample 1 in FTab-Chars Sequence-1 Sequence-2 ... Sequence-N Fields are separated by tabs. Colorspace is always set to 0 for HISAT2.
[`--snp`]: #hisat2-inspect-options-snp --snp Print SNPs, and quit.
[`--ss`]: #hisat2-inspect-options-ss --ss Print splice sites, and quit.
[`--ss-all`]: #hisat2-inspect-options-ss-all --ss-all Print splice sites including those not in the global index, and quit.
[`--exon`]: #hisat2-inspect-options-exon --exon Print exons, and quit.
-v/--verbose Print verbose output (for debugging).
--version Print version information and quit.
-h/--help Print usage information and quit.
Getting started with HISAT2 =================================================== HISAT2 comes with some example files to get you started. The example files are not scientifically significant; these files will simply let you start running HISAT2 and downstream tools right away. First follow the manual instructions to [obtain HISAT2]. Set the `HISAT2_HOME` environment variable to point to the new HISAT2 directory containing the `hisat2`, `hisat2-build` and `hisat2-inspect` binaries. This is important, as the `HISAT2_HOME` variable is used in the commands below to refer to that directory. [obtain HISAT2]: #obtaining-hisat2 Indexing a reference genome --------------------------- To create an index for the genomic region (1 million bps from the human chromosome 22 between 20,000,000 and 20,999,999) included with HISAT2, create a new temporary directory (it doesn't matter where), change into that directory, and run: $HISAT2_HOME/hisat2-build $HISAT2_HOME/example/reference/22_20-21M.fa --snp $HISAT2_HOME/example/reference/22_20-21M.snp 22_20-21M_snp The command should print many lines of output then quit. When the command completes, the current directory will contain ten new files that all start with `22_20-21M_snp` and end with `.1.ht2`, `.2.ht2`, `.3.ht2`, `.4.ht2`, `.5.ht2`, `.6.ht2`, `.7.ht2`, and `.8.ht2`. These files constitute the index - you're done! You can use `hisat2-build` to create an index for a set of FASTA files obtained from any source, including sites such as [UCSC], [NCBI], and [Ensembl]. When indexing multiple FASTA files, specify all the files using commas to separate file names. For more details on how to create an index with `hisat2-build`, see the [manual section on index building]. You may also want to bypass this process by obtaining a pre-built index. [UCSC]: http://genome.ucsc.edu/cgi-bin/hgGateway [NCBI]: http://www.ncbi.nlm.nih.gov/sites/genome [Ensembl]: http://www.ensembl.org/ [manual section on index building]: #the-hisat2-build-indexer [using a pre-built index]: #using-a-pre-built-index Aligning example reads ---------------------- Stay in the directory created in the previous step, which now contains the `22_20-21M` index files. Next, run: $HISAT2_HOME/hisat2 -f -x $HISAT2_HOME/example/index/22_20-21M_snp -U $HISAT2_HOME/example/reads/reads_1.fa -S eg1.sam This runs the HISAT2 aligner, which aligns a set of unpaired reads to the genome region using the index generated in the previous step. The alignment results in SAM format are written to the file `eg1.sam`, and a short alignment summary is written to the console. (Actually, the summary is written to the "standard error" or "stderr" filehandle, which is typically printed to the console.) To see the first few lines of the SAM output, run: head eg1.sam You will see something like this: @HD VN:1.0 SO:unsorted @SQ SN:22:20000001-21000000 LN:1000000 @PG ID:hisat2 PN:hisat2 VN:2.0.0-beta 1 0 22:20000001-21000000 397984 255 100M * 0 0 GCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU NH:i:1 2 16 22:20000001-21000000 398131 255 100M * 0 0 ATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:80A19 YT:Z:UU NH:i:1 Zs:Z:80|S|rs576159895 3 16 22:20000001-21000000 398222 255 100M * 0 0 TGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:16A83 YT:Z:UU NH:i:1 Zs:Z:16|S|rs2629364 4 16 22:20000001-21000000 398247 255 90M200N10M * 0 0 CAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAACCGCTGCACCAGGAAGTCGATCCAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU XS:A:- NH:i:1 5 16 22:20000001-21000000 398194 255 100M * 0 0 GGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:17A26A55 YT:Z:UU NH:i:1 Zs:Z:17|S|rs576159895,26|S|rs2629364 6 0 22:20000001-21000000 398069 255 100M * 0 0 CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU NH:i:1 7 0 22:20000001-21000000 397896 255 100M * 0 0 GTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:31G68 YT:Z:UU NH:i:1 Zs:Z:31|S|rs562662261 8 0 22:20000001-21000000 398150 255 100M * 0 0 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:61A26A11 YT:Z:UU NH:i:1 Zs:Z:61|S|rs576159895,26|S|rs2629364 9 16 22:20000001-21000000 398329 255 8M200N92M * 0 0 ACCAGGAAGTCGATCCAGATGTAGTGGGGGGTCACTTCGGGGGGACAGGGTTTGGGTTGACTTGCTTCCGAGGCAGCCAGGGGGTCTGCTTCCTTTATCT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU XS:A:- NH:i:1 10 16 22:20000001-21000000 398184 255 100M * 0 0 CTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:27A26A45 YT:Z:UU NH:i:1 Zs:Z:27|S|rs576159895,26|S|rs2629364 The first few lines (beginning with `@`) are SAM header lines, and the rest of the lines are SAM alignments, one line per read or mate. See the [HISAT2 manual section on SAM output] and the [SAM specification] for details about how to interpret the SAM file format. [HISAT2 manual section on SAM output]: #sam-output Paired-end example ------------------ To align paired-end reads included with HISAT2, stay in the same directory and run: $HISAT2_HOME/hisat2 -f -x $HISAT2_HOME/example/index/22_20-21M_snp -1 $HISAT2_HOME/example/reads/reads_1.fa -2 $HISAT2_HOME/example/reads/reads_2.fa -S eg2.sam This aligns a set of paired-end reads to the reference genome, with results written to the file `eg2.sam`. Using SAMtools/BCFtools downstream ---------------------------------- [SAMtools] is a collection of tools for manipulating and analyzing SAM and BAM alignment files. [BCFtools] is a collection of tools for calling variants and manipulating VCF and BCF files, and it is typically distributed with [SAMtools]. Using these tools together allows you to get from alignments in SAM format to variant calls in VCF format. This example assumes that `samtools` and `bcftools` are installed and that the directories containing these binaries are in your [PATH environment variable]. Run the paired-end example: $HISAT2_HOME/hisat -f -x $HISAT2_HOME/example/index/22_20-21M_snp -1 $HISAT2_HOME/example/reads/reads_1.fa -2 $HISAT2_HOME/example/reads/reads_2.fa -S eg2.sam Use `samtools view` to convert the SAM file into a BAM file. BAM is a the binary format corresponding to the SAM text format. Run: samtools view -bS eg2.sam > eg2.bam Use `samtools sort` to convert the BAM file to a sorted BAM file. The following command requires samtools version 1.2 or higher. samtools sort eg2.bam -o eg2.sorted.bam We now have a sorted BAM file called `eg2.sorted.bam`. Sorted BAM is a useful format because the alignments are (a) compressed, which is convenient for long-term storage, and (b) sorted, which is convenient for variant discovery. To generate variant calls in VCF format, run: samtools mpileup -uf $HISAT2_HOME/example/reference/22_20-21M.fa eg2.sorted.bam | bcftools view -bvcg - > eg2.raw.bcf Then to view the variants, run: bcftools view eg2.raw.bcf See the official SAMtools guide to [Calling SNPs/INDELs with SAMtools/BCFtools] for more details and variations on this process. [BCFtools]: http://samtools.sourceforge.net/mpileup.shtml [Calling SNPs/INDELs with SAMtools/BCFtools]: http://samtools.sourceforge.net/mpileup.shtml hisat2-2.2.1/Makefile000066400000000000000000000362731370663763200143760ustar00rootroot00000000000000# # Copyright 2015, Daehwan Kim # # This file is part of HISAT2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT. If not, see . # # # Makefile for hisat2-align, hisat2-build, hisat2-inspect # INC = GCC_PREFIX = $(shell dirname `which gcc`) GCC_SUFFIX = CC = $(GCC_PREFIX)/gcc$(GCC_SUFFIX) CPP = $(GCC_PREFIX)/g++$(GCC_SUFFIX) CXX = $(CPP) HEADERS = $(wildcard *.h) BOWTIE_MM = 1 BOWTIE_SHARED_MEM = 0 # Detect Cygwin or MinGW WINDOWS = 0 CYGWIN = 0 MINGW = 0 ifneq (,$(findstring CYGWIN,$(shell uname))) WINDOWS = 1 CYGWIN = 1 # POSIX memory-mapped files not currently supported on Windows BOWTIE_MM = 0 BOWTIE_SHARED_MEM = 0 else ifneq (,$(findstring MINGW,$(shell uname))) WINDOWS = 1 MINGW = 1 # POSIX memory-mapped files not currently supported on Windows BOWTIE_MM = 0 BOWTIE_SHARED_MEM = 0 endif endif MACOS = 0 ifneq (,$(findstring Darwin,$(shell uname))) MACOS = 1 endif EXTRA_FLAGS += -DPOPCNT_CAPABILITY -std=c++11 INC += -I. -I third_party MM_DEF = ifeq (1,$(BOWTIE_MM)) MM_DEF = -DBOWTIE_MM endif SHMEM_DEF = ifeq (1,$(BOWTIE_SHARED_MEM)) SHMEM_DEF = -DBOWTIE_SHARED_MEM endif PTHREAD_PKG = PTHREAD_LIB = ifeq (1,$(MINGW)) PTHREAD_LIB = else PTHREAD_LIB = -lpthread endif SEARCH_LIBS = BUILD_LIBS = INSPECT_LIBS = ifeq (1,$(MINGW)) BUILD_LIBS = INSPECT_LIBS = endif USE_SRA = 0 SRA_DEF = SRA_LIB = SERACH_INC = ifeq (1,$(USE_SRA)) SRA_DEF = -DUSE_SRA SRA_LIB = -lncbi-ngs-c++-static -lngs-c++-static -lncbi-vdb-static -ldl SEARCH_INC += -I$(NCBI_NGS_DIR)/include -I$(NCBI_VDB_DIR)/include SEARCH_LIBS += -L$(NCBI_NGS_DIR)/lib64 -L$(NCBI_VDB_DIR)/lib64 endif LIBS = $(PTHREAD_LIB) SHARED_CPPS = ccnt_lut.cpp ref_read.cpp alphabet.cpp shmem.cpp \ edit.cpp gfm.cpp \ reference.cpp ds.cpp multikey_qsort.cpp limit.cpp \ random_source.cpp tinythread.cpp SEARCH_CPPS = qual.cpp pat.cpp \ read_qseq.cpp aligner_seed_policy.cpp \ aligner_seed.cpp \ aligner_seed2.cpp \ aligner_sw.cpp \ aligner_sw_driver.cpp aligner_cache.cpp \ aligner_result.cpp ref_coord.cpp mask.cpp \ pe.cpp aln_sink.cpp dp_framer.cpp \ scoring.cpp presets.cpp unique.cpp \ simple_func.cpp \ random_util.cpp \ aligner_bt.cpp sse_util.cpp \ aligner_swsse.cpp outq.cpp \ aligner_swsse_loc_i16.cpp \ aligner_swsse_ee_i16.cpp \ aligner_swsse_loc_u8.cpp \ aligner_swsse_ee_u8.cpp \ aligner_driver.cpp \ splice_site.cpp BUILD_CPPS = diff_sample.cpp REPEAT_CPPS = \ mask.cpp \ qual.cpp \ aligner_bt.cpp \ scoring.cpp \ simple_func.cpp \ dp_framer.cpp \ aligner_result.cpp \ aligner_sw_driver.cpp \ aligner_sw.cpp \ aligner_swsse_ee_i16.cpp \ aligner_swsse_ee_u8.cpp \ aligner_swsse_loc_i16.cpp \ aligner_swsse_loc_u8.cpp \ aligner_swsse.cpp \ bit_packed_array.cpp \ repeat_builder.cpp HISAT2_CPPS_MAIN = $(SEARCH_CPPS) hisat2_main.cpp HISAT2_BUILD_CPPS_MAIN = $(BUILD_CPPS) hisat2_build_main.cpp HISAT2_REPEAT_CPPS_MAIN = $(REPEAT_CPPS) $(BUILD_CPPS) hisat2_repeat_main.cpp SEARCH_FRAGMENTS = $(wildcard search_*_phase*.c) VERSION = $(shell cat VERSION) # Convert BITS=?? to a -m flag BITS=32 ifeq (x86_64,$(shell uname -m)) BITS=64 endif # msys will always be 32 bit so look at the cpu arch instead. ifneq (,$(findstring AMD64,$(PROCESSOR_ARCHITEW6432))) ifeq (1,$(MINGW)) BITS=64 endif endif BITS_FLAG = ifeq (32,$(BITS)) BITS_FLAG = -m32 endif ifeq (64,$(BITS)) BITS_FLAG = -m64 endif SSE_FLAG=-msse2 DEBUG_FLAGS = -O0 -g3 $(BITS_FLAG) $(SSE_FLAG) DEBUG_DEFS = -DCOMPILER_OPTIONS="\"$(DEBUG_FLAGS) $(EXTRA_FLAGS)\"" RELEASE_FLAGS = -O3 $(BITS_FLAG) $(SSE_FLAG) -funroll-loops -g3 RELEASE_DEFS = -DCOMPILER_OPTIONS="\"$(RELEASE_FLAGS) $(EXTRA_FLAGS)\"" NOASSERT_FLAGS = -DNDEBUG FILE_FLAGS = -D_LARGEFILE_SOURCE -D_FILE_OFFSET_BITS=64 -D_GNU_SOURCE ifeq (1,$(USE_SRA)) ifeq (1, $(MACOS)) SRA_LIB += -stdlib=libc++ DEBUG_FLAGS += -mmacosx-version-min=10.10 RELEASE_FLAGS += -mmacosx-version-min=10.10 endif endif HISAT2_BIN_LIST = hisat2-build-s \ hisat2-build-l \ hisat2-align-s \ hisat2-align-l \ hisat2-inspect-s \ hisat2-inspect-l \ hisat2-repeat HISAT2_BIN_LIST_AUX = hisat2-build-s-debug \ hisat2-build-l-debug \ hisat2-align-s-debug \ hisat2-align-l-debug \ hisat2-inspect-s-debug \ hisat2-inspect-l-debug \ hisat2-repeat-debug HT2LIB_DIR = hisat2lib HT2LIB_SRCS = $(SHARED_CPPS) \ $(HT2LIB_DIR)/ht2_init.cpp \ $(HT2LIB_DIR)/ht2_repeat.cpp \ $(HT2LIB_DIR)/ht2_index.cpp HT2LIB_OBJS = $(HT2LIB_SRCS:.cpp=.o) HT2LIB_DEBUG_OBJS = $(addprefix .ht2lib-obj-debug/,$(HT2LIB_OBJS)) HT2LIB_RELEASE_OBJS = $(addprefix .ht2lib-obj-release/,$(HT2LIB_OBJS)) HT2LIB_SHARED_DEBUG_OBJS = $(addprefix .ht2lib-obj-debug-shared/,$(HT2LIB_OBJS)) HT2LIB_SHARED_RELEASE_OBJS = $(addprefix .ht2lib-obj-release-shared/,$(HT2LIB_OBJS)) HT2LIB_PKG_SRC = \ $(HT2LIB_DIR)/ht2_init.cpp \ $(HT2LIB_DIR)/ht2_repeat.cpp \ $(HT2LIB_DIR)/ht2_index.cpp \ $(HT2LIB_DIR)/ht2.h \ $(HT2LIB_DIR)/ht2_handle.h \ $(HT2LIB_DIR)/java_jni/Makefile \ $(HT2LIB_DIR)/java_jni/ht2module.c \ $(HT2LIB_DIR)/java_jni/HT2Module.java \ $(HT2LIB_DIR)/java_jni/HT2ModuleExample.java \ $(HT2LIB_DIR)/pymodule/Makefile \ $(HT2LIB_DIR)/pymodule/ht2module.c \ $(HT2LIB_DIR)/pymodule/setup.py \ $(HT2LIB_DIR)/pymodule/ht2example.py GENERAL_LIST = $(wildcard scripts/*.sh) \ $(wildcard scripts/*.pl) \ $(wildcard *.py) \ $(wildcard example/index/*.ht2) \ $(wildcard example/reads/*.fa) \ example/reference/22_20-21M.fa \ example/reference/22_20-21M.snp \ $(PTHREAD_PKG) \ hisat2 \ hisat2-build \ hisat2-inspect \ AUTHORS \ LICENSE \ NEWS \ MANUAL \ MANUAL.markdown \ TUTORIAL \ VERSION ifeq (1,$(WINDOWS)) HISAT2_BIN_LIST := $(HISAT2_BIN_LIST) hisat2.bat hisat2-build.bat hisat2-inspect.bat endif # This is helpful on Windows under MinGW/MSYS, where Make might go for # the Windows FIND tool instead. FIND=$(shell which find) SRC_PKG_LIST = $(wildcard *.h) \ $(wildcard *.hh) \ $(wildcard *.c) \ $(wildcard *.cpp) \ $(HT2LIB_PKG_SRC) \ Makefile \ CMakeLists.txt \ $(GENERAL_LIST) BIN_PKG_LIST = $(GENERAL_LIST) .PHONY: all allall both both-debug all: $(HISAT2_BIN_LIST) allall: $(HISAT2_BIN_LIST) $(HISAT2_BIN_LIST_AUX) both: hisat2-align-s hisat2-align-l hisat2-build-s hisat2-build-l both-debug: hisat2-align-s-debug hisat2-align-l-debug hisat2-build-s-debug hisat2-build-l-debug repeat: hisat2-repeat repeat-debug: hisat2-repeat-debug DEFS=-fno-strict-aliasing \ -DHISAT2_VERSION="\"`cat VERSION`\"" \ -DBUILD_HOST="\"`hostname`\"" \ -DBUILD_TIME="\"`date`\"" \ -DCOMPILER_VERSION="\"`$(CXX) -v 2>&1 | tail -1`\"" \ $(FILE_FLAGS) \ $(PREF_DEF) \ $(MM_DEF) \ $(SHMEM_DEF) # # hisat-bp targets # hisat-bp-bin: hisat_bp.cpp $(SEARCH_CPPS) $(SHARED_CPPS) $(HEADERS) $(SEARCH_FRAGMENTS) $(CXX) $(RELEASE_FLAGS) $(RELEASE_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 $(NOASSERT_FLAGS) -Wall \ $(INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT_CPPS_MAIN) \ $(LIBS) $(SEARCH_LIBS) hisat-bp-bin-debug: hisat_bp.cpp $(SEARCH_CPPS) $(SHARED_CPPS) $(HEADERS) $(SEARCH_FRAGMENTS) $(CXX) $(DEBUG_FLAGS) \ $(DEBUG_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 -Wall \ $(INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT_CPPS_MAIN) \ $(LIBS) $(SEARCH_LIBS) # # hisat2-repeat targets # hisat2-repeat: hisat2_repeat.cpp $(REPEAT_CPPS) $(SHARED_CPPS) $(HEADERS) $(CXX) $(RELEASE_FLAGS) $(RELEASE_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 -DBOWTIE_64BIT_INDEX $(NOASSERT_FLAGS) -Wall \ $(INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT2_REPEAT_CPPS_MAIN) \ $(LIBS) $(BUILD_LIBS) hisat2-repeat-debug: hisat2_repeat.cpp $(REPEAT_CPPS) $(SHARED_CPPS) $(HEADERS) $(CXX) $(DEBUG_FLAGS) $(DEBUG_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 -DBOWTIE_64BIT_INDEX -Wall \ $(INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT2_REPEAT_CPPS_MAIN) \ $(LIBS) $(BUILD_LIBS) # # hisat2-build targets # hisat2-build-s: hisat2_build.cpp $(SHARED_CPPS) $(HEADERS) $(CXX) $(RELEASE_FLAGS) $(RELEASE_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 $(NOASSERT_FLAGS) -Wall -DMASSIVE_DATA_RLCSA \ $(INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT2_BUILD_CPPS_MAIN) \ $(LIBS) $(BUILD_LIBS) hisat2-build-l: hisat2_build.cpp $(SHARED_CPPS) $(HEADERS) $(CXX) $(RELEASE_FLAGS) $(RELEASE_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 -DBOWTIE_64BIT_INDEX $(NOASSERT_FLAGS) -Wall \ $(INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT2_BUILD_CPPS_MAIN) \ $(LIBS) $(BUILD_LIBS) hisat2-build-s-debug: hisat2_build.cpp $(SHARED_CPPS) $(HEADERS) $(CXX) $(DEBUG_FLAGS) $(DEBUG_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 -Wall -DMASSIVE_DATA_RLCSA \ $(INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT2_BUILD_CPPS_MAIN) \ $(LIBS) $(BUILD_LIBS) hisat2-build-l-debug: hisat2_build.cpp $(SHARED_CPPS) $(HEADERS) $(CXX) $(DEBUG_FLAGS) $(DEBUG_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 -DBOWTIE_64BIT_INDEX -Wall \ $(INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT2_BUILD_CPPS_MAIN) \ $(LIBS) $(BUILD_LIBS) # # hisat2 targets # hisat2-align-s: hisat2.cpp $(SEARCH_CPPS) $(SHARED_CPPS) $(HEADERS) $(SEARCH_FRAGMENTS) $(CXX) $(RELEASE_FLAGS) $(RELEASE_DEFS) $(EXTRA_FLAGS) \ $(DEFS) $(SRA_DEF) -DBOWTIE2 $(NOASSERT_FLAGS) -Wall \ $(INC) $(SEARCH_INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT2_CPPS_MAIN) \ $(LIBS) $(SRA_LIB) $(SEARCH_LIBS) hisat2-align-l: hisat2.cpp $(SEARCH_CPPS) $(SHARED_CPPS) $(HEADERS) $(SEARCH_FRAGMENTS) $(CXX) $(RELEASE_FLAGS) $(RELEASE_DEFS) $(EXTRA_FLAGS) \ $(DEFS) $(SRA_DEF) -DBOWTIE2 -DBOWTIE_64BIT_INDEX $(NOASSERT_FLAGS) -Wall \ $(INC) $(SEARCH_INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT2_CPPS_MAIN) \ $(LIBS) $(SRA_LIB) $(SEARCH_LIBS) hisat2-align-s-debug: hisat2.cpp $(SEARCH_CPPS) $(SHARED_CPPS) $(HEADERS) $(SEARCH_FRAGMENTS) $(CXX) $(DEBUG_FLAGS) \ $(DEBUG_DEFS) $(EXTRA_FLAGS) \ $(DEFS) $(SRA_DEF) -DBOWTIE2 -Wall \ $(INC) $(SEARCH_INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT2_CPPS_MAIN) \ $(LIBS) $(SRA_LIB) $(SEARCH_LIBS) hisat2-align-l-debug: hisat2.cpp $(SEARCH_CPPS) $(SHARED_CPPS) $(HEADERS) $(SEARCH_FRAGMENTS) $(CXX) $(DEBUG_FLAGS) \ $(DEBUG_DEFS) $(EXTRA_FLAGS) \ $(DEFS) $(SRA_DEF) -DBOWTIE2 -DBOWTIE_64BIT_INDEX -Wall \ $(INC) $(SEARCH_INC) \ -o $@ $< \ $(SHARED_CPPS) $(HISAT2_CPPS_MAIN) \ $(LIBS) $(SRA_LIB) $(SEARCH_LIBS) # # hisat2-inspect targets # hisat2-inspect-s: hisat2_inspect.cpp $(HEADERS) $(SHARED_CPPS) $(CXX) $(RELEASE_FLAGS) \ $(RELEASE_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 -DHISAT2_INSPECT_MAIN -Wall \ $(INC) -I . \ -o $@ $< \ $(SHARED_CPPS) \ $(LIBS) $(INSPECT_LIBS) hisat2-inspect-l: hisat2_inspect.cpp $(HEADERS) $(SHARED_CPPS) $(CXX) $(RELEASE_FLAGS) \ $(RELEASE_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 -DBOWTIE_64BIT_INDEX -DHISAT2_INSPECT_MAIN -Wall \ $(INC) -I . \ -o $@ $< \ $(SHARED_CPPS) \ $(LIBS) $(INSPECT_LIBS) hisat2-inspect-s-debug: hisat2_inspect.cpp $(HEADERS) $(SHARED_CPPS) $(CXX) $(DEBUG_FLAGS) \ $(DEBUG_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 -DHISAT2_INSPECT_MAIN -Wall \ $(INC) -I . \ -o $@ $< \ $(SHARED_CPPS) \ $(LIBS) $(INSPECT_LIBS) hisat2-inspect-l-debug: hisat2_inspect.cpp $(HEADERS) $(SHARED_CPPS) $(CXX) $(DEBUG_FLAGS) \ $(DEBUG_DEFS) $(EXTRA_FLAGS) \ $(DEFS) -DBOWTIE2 -DBOWTIE_64BIT_INDEX -DHISAT2_INSPECT_MAIN -Wall \ $(INC) -I . \ -o $@ $< \ $(SHARED_CPPS) \ $(LIBS) $(INSPECT_LIBS) # # HT2LIB targets # ht2lib: libhisat2lib-debug.a libhisat2lib.a libhisat2lib-debug.so libhisat2lib.so libhisat2lib-debug.a: $(HT2LIB_DEBUG_OBJS) ar rc $@ $(HT2LIB_DEBUG_OBJS) libhisat2lib.a: $(HT2LIB_RELEASE_OBJS) ar rc $@ $(HT2LIB_RELEASE_OBJS) libhisat2lib-debug.so: $(HT2LIB_SHARED_DEBUG_OBJS) $(CXX) $(DEBUG_FLAGS) $(DEBUG_DEFS) $(EXTRA_FLAGS) $(DEFS) $(SRA_DEF) -DBOWTIE2 -Wall $(INC) $(SEARCH_INC) \ -shared -o $@ $(HT2LIB_SHARED_DEBUG_OBJS) $(LIBS) $(SRA_LIB) $(SEARCH_LIBS) libhisat2lib.so: $(HT2LIB_SHARED_RELEASE_OBJS) $(CXX) $(RELEASE_FLAGS) $(RELEASE_DEFS) $(EXTRA_FLAGS) $(DEFS) $(SRA_DEF) -DBOWTIE2 $(NOASSERT_FLAGS) -Wall $(INC) $(SEARCH_INC)\ -shared -o $@ $(HT2LIB_SHARED_RELEASE_OBJS) $(LIBS) $(SRA_LIB) $(SEARCH_LIBS) .ht2lib-obj-debug/%.o: %.cpp @mkdir -p $(dir $@)/$(dir $<) $(CXX) -fPIC $(DEBUG_FLAGS) $(DEBUG_DEFS) $(EXTRA_FLAGS) $(DEFS) $(SRA_DEF) -DBOWTIE2 -Wall $(INC) $(SEARCH_INC) \ -c -o $@ $< .ht2lib-obj-release/%.o: %.cpp @mkdir -p $(dir $@)/$(dir $<) $(CXX) -fPIC $(RELEASE_FLAGS) $(RELEASE_DEFS) $(EXTRA_FLAGS) $(DEFS) $(SRA_DEF) -DBOWTIE2 $(NOASSERT_FLAGS) -Wall $(INC) $(SEARCH_INC) \ -c -o $@ $< .ht2lib-obj-debug-shared/%.o: %.cpp @mkdir -p $(dir $@)/$(dir $<) $(CXX) -fPIC $(DEBUG_FLAGS) $(DEBUG_DEFS) $(EXTRA_FLAGS) $(DEFS) $(SRA_DEF) -DBOWTIE2 -Wall $(INC) $(SEARCH_INC) \ -c -o $@ $< .ht2lib-obj-release-shared/%.o: %.cpp @mkdir -p $(dir $@)/$(dir $<) $(CXX) -fPIC $(RELEASE_FLAGS) $(RELEASE_DEFS) $(EXTRA_FLAGS) $(DEFS) $(SRA_DEF) -DBOWTIE2 $(NOASSERT_FLAGS) -Wall $(INC) $(SEARCH_INC) \ -c -o $@ $< # # repeatexp # repeatexp: g++ -o repeatexp repeatexp.cpp -I hisat2lib libhisat2lib.a hisat2: ; hisat2.bat: echo "@echo off" > hisat2.bat echo "perl %~dp0/hisat2 %*" >> hisat2.bat hisat2-build.bat: echo "@echo off" > hisat2-build.bat echo "python %~dp0/hisat2-build %*" >> hisat2-build.bat hisat2-inspect.bat: echo "@echo off" > hisat2-inspect.bat echo "python %~dp0/hisat2-inspect %*" >> hisat2-inspect.bat .PHONY: hisat2-src hisat2-src: $(SRC_PKG_LIST) chmod a+x scripts/*.sh scripts/*.pl mkdir .src.tmp mkdir .src.tmp/hisat2-$(VERSION) zip tmp.zip $(SRC_PKG_LIST) mv tmp.zip .src.tmp/hisat2-$(VERSION) cd .src.tmp/hisat2-$(VERSION) ; unzip tmp.zip ; rm -f tmp.zip cd .src.tmp ; zip -r hisat2-$(VERSION)-source.zip hisat2-$(VERSION) cp .src.tmp/hisat2-$(VERSION)-source.zip . rm -rf .src.tmp .PHONY: hisat2-bin hisat2-bin: $(BIN_PKG_LIST) $(HISAT2_BIN_LIST) $(HISAT2_BIN_LIST_AUX) chmod a+x scripts/*.sh scripts/*.pl rm -rf .bin.tmp mkdir .bin.tmp mkdir .bin.tmp/hisat2-$(VERSION) if [ -f hisat2.exe ] ; then \ zip tmp.zip $(BIN_PKG_LIST) $(addsuffix .exe,$(HISAT2_BIN_LIST) $(HISAT2_BIN_LIST_AUX)) ; \ else \ zip tmp.zip $(BIN_PKG_LIST) $(HISAT2_BIN_LIST) $(HISAT2_BIN_LIST_AUX) ; \ fi mv tmp.zip .bin.tmp/hisat2-$(VERSION) cd .bin.tmp/hisat2-$(VERSION) ; unzip tmp.zip ; rm -f tmp.zip cd .bin.tmp ; zip -r hisat2-$(VERSION)-$(BITS).zip hisat2-$(VERSION) cp .bin.tmp/hisat2-$(VERSION)-$(BITS).zip . rm -rf .bin.tmp .PHONY: doc doc: doc/manual.inc.html MANUAL doc/manual.inc.html: MANUAL.markdown pandoc -T "HISAT2 Manual" -o $@ \ --from markdown --to HTML --toc $^ perl -i -ne \ '$$w=0 if m|^|;print if $$w;$$w=1 if m|^|;' $@ MANUAL: MANUAL.markdown perl doc/strip_markdown.pl < $^ > $@ .PHONY: clean clean: rm -f $(HISAT2_BIN_LIST) $(HISAT2_BIN_LIST_AUX) \ $(addsuffix .exe,$(HISAT2_BIN_LIST) $(HISAT2_BIN_LIST_AUX)) \ hisat2-src.zip hisat2-bin.zip rm -f core.* .tmp.head rm -rf *.dSYM rm -rf .ht2lib-obj* rm -f libhisat2lib*.a libhisat2lib*.so .PHONY: push-doc push-doc: doc/manual.inc.html scp doc/*.*html doc/indexes.txt salz-dmz:/ccb/salz7-data/www/ccb.jhu.edu/html/software/hisat2/ hisat2-2.2.1/NEWS000066400000000000000000000011101370663763200134130ustar00rootroot00000000000000HISAT 2 NEWS ============= HISAT 2 is now available for download from the project website, http://bowtie-bio.sf.net/bowtie2. 2.0.0-beta is the first version released to the public and 2.0.7 is the latest version. HISAT 2 is licensed under the GPLv3 license. See `LICENSE' file for details. Version Release History ======================= Version 2.0.0-beta - August XX, 2015 * Improved multithreading support so that Bowtie 2 now uses native Windows threads when compiled on Windows and uses a faster mutex. Threading performance should improve on all platforms. hisat2-2.2.1/README.md000066400000000000000000000312441370663763200142060ustar00rootroot00000000000000# Graph-Based Genome Alignment and Genotyping with HISAT2 and HISAT-genotype ## Contact [Daehwan Kim](https://kim-lab.org) (infphilo@gmail.com) and [Chanhee Park](https://www.linkedin.com/in/chanhee-park-97677297/) (parkchanhee@gmail.com) ## Abstract Rapid advances in next-generation sequencing technologies have dramatically changed our ability to perform genome-scale analyses. The human reference genome used for most genomic analyses represents only a small number of individuals, limiting its usefulness for genotyping. We designed a novel method, HISAT2, for representing and searching an expanded model of the human reference genome, in which a large catalogue of known genomic variants and haplotypes is incorporated into the data structure used for searching and alignment. This strategy for representing a population of genomes, along with a fast and memory-efficient search algorithm, enables more detailed and accurate variant analyses than previous methods. We demonstrate two initial applications of HISAT2: HLA typing, a critical need in human organ transplantation, and DNA fingerprinting, widely used in forensics. These applications are part of HISAT-genotype, with performance not only surpassing earlier computational methods, but matching or exceeding the accuracy of laboratory-based assays. ![](HISAT2-genotype.png) For more information, see the following websites: * [HISAT2 website](http://ccb.jhu.edu/software/hisat2) * [HISAT-genotype website](http://ccb.jhu.edu/software/hisat-genotype) ## HISAT2 HISAT2 is a fast and sensitive alignment program for mapping next-generation sequencing reads (whole-genome, transcriptome, and exome sequencing data) to a population of human genomes (as well as to a single reference genome). Based on an extension of BWT for a graph [1], we designed and implemented a graph FM index (GFM), an original approach and its first implementation to the best of our knowledge. In addition to using one global GFM index that represents general population, HISAT2 uses a large set of small GFM indexes that collectively cover the whole genome (each index representing a genomic region of 56 Kbp, with 55,000 indexes needed to cover human population). These small indexes (called local indexes) combined with several alignment strategies enable effective alignment of sequencing reads. This new indexing scheme is called Hierarchical Graph FM index (HGFM). We have developed HISAT2 based on the HISAT [2] and Bowtie 2 [3] implementations. See the [HISAT2 website](http://ccb.jhu.edu/software/hisat2/index.shtml) for more information. A few notes: 1) HISAT2's index (HGFM) size for the human reference genome and 12.3 million common SNPs is 6.2GB. The SNPs consist of 11 million single nucleotide polymorphisms, 728,000 deletions, and 555,000 insertions. Insertions and deletions used in this index are small (usually <20bp). We plan to incorporate structural variations (SV) into this index. 2) The memory footprint of HISAT2 is relatively low, 6.7GB. 3) The runtime of HISAT2 is estimated to be slightly slower than HISAT (30–100% slower for some data sets). 4) HISAT2 provides greater accuracy for alignment of reads containing SNPs. 5) We released a first (beta) version of HISAT2 in September 8, 2015. ## License [GPL-3.0](LICENSE) # For reviwers, follow the instructions below to reproduce some of the results in the manuscript. ## Code A specific version of [HISAT2 and HISAT-genotype](http://github.com/infphilo/hisat2) at GitHub is used (a branch name: hisat2_v2.2.0_beta). ## Initial setup HISAT-genotype requires a 64-bit computer running either Linux or Mac OS X and at least 8 GB of RAM (16 GB of RAM is preferred). All the commands used should be run from the Unix shell prompt within a terminal window and are prefixed with a '$' character. We refer to hisat-genotype-top as our top directory where all of our programs are located. hisat-genotype-top is a place holder that can be changed to another name according to user preference. Run the following commands to install HISAT2 and HISAT-genotype. $ git clone https://github.com/infphilo/hisat2 hisat-genotype-top $ cd hisat-genotype-top hisat-genotype-top$ git checkout hisat2_v2.2.0_beta hisat-genotype-top$ make hisat2-align-s hisat2-build-s hisat2-inspect-s To make the binaries built above and other python scripts available everywhere, add the hisat-genotype-top directory to the PATH environment variable (e.g. ~/.bashrc) export PATH=hisat-genotype-top:hisat-genotype-top/hisatgenotype_scripts:$PATH export PYTHONPATH=hisat-genotype-top/hisatgenotype_modules:$PYTHONPATH To reflect the change, run the following command: $ source ~/.bashrc Download real reads, simulated reads, and HISAT2 indexes, then move them into appropriate directories: hisat-genotype-top$ cd evaluation hisat-genotype-top/evaluation$ wget ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/hisat2_20181025.tar.gz hisat-genotype-top/evaluation$ tar xvzf hisat2_20181025.tar.gz hisat-genotype-top/evaluation$ mkdir aligners aligners/bin; cd aligners/bin; ln -s ../../../hisat2* .; cd ../.. hisat-genotype-top/evaluation$ mv hisat2/* . hisat-genotype-top/evaluation$ cd simulation; ./init.py; cd ../real; ./init.py; cd .. ## Run HISAT2 on the following simulated and real data sets. ### 10 million simulated read pairs with SNPs and with sequencing errors hisat-genotype-top/evaluation$ cd simulation/10M_DNA_mismatch_snp_reads_genome hisat-genotype-top/evaluation/simulation/10M_DNA_mismatch_snp_reads_genome$ ./calculate_read_cost.py --aligner-list hisat2 --paired-end --fresh ### 10 million simulated read pairs with SNPs and without sequencing errors hisat-genotype-top/evaluation$ cd simulation/10M_DNA_snp_reads_genome hisat-genotype-top/evaluation/simulation/10M_DNA_snp_reads_genome$ ./calculate_read_cost.py --aligner-list hisat2 --paired-end --fresh ### 10 million simulated read pairs without SNPs and with sequencing errors hisat-genotype-top/evaluation$ cd simulation/10M_DNA_mismatch_reads_genome hisat-genotype-top/evaluation/simulation/10M_DNA_mismatch_reads_genome$ ./calculate_read_cost.py --aligner-list hisat2 --paired-end --fresh ### 10 million simulated read pairs without SNPs and without sequencing errors hisat-genotype-top/evaluation$ cd simulation/10M_DNA_reads_genome hisat-genotype-top/evaluation/simulation/10M_DNA_reads_genome$ ./calculate_read_cost.py --aligner-list hisat2 --paired-end --fresh ### 10 million real read pairs hisat-genotype-top/evaluation$ cd real/DNA/10M hisat-genotype-top/evaluation/real/DNA/10M$ ./calculate_read_cost.py --aligner-list hisat2 --paired-end --fresh ### Interpreting output Example alignment output for simulated reads aligned: 1000000, multi aligned: 2654390 correctly mapped: 999963 (100.00%) uniquely and correctly mapped: 967631 (96.76%) 54694 reads per sec (all) Memory Usage: 86MB The above lines show that 1,000,000 read pairs are aligned and the total number of alignments is 2,654,390. 999,963 pairs (100.00%) are correctly aligned (e.g. one of the alignments is correct). 967,631 (96.76%) pairs are uniquely and correctly aligned. HISAT2 aligns 54,594 reads with a peak memory usage of 86 MB of RAM. Each run is expected to take up to several hours mostly due to the comparison of HISAT2’s reported alignments and true alignments and the expansion of repeat alignments. ## Details on HISAT-genotype run for HLA typing and assembly To create a directory where we perform our analysis for HLA typing and assembly, which here is referred to as hla-analysis but can be changed by the user, execute the following command. hisat-genotype-top/evaluation$ mkdir hla-analysis The current directory can be changed to hla-analysis as follows: hisat-genotype-top/evaluation$ cd hla-analysis Additional program requirements: SAMtools (version 1.3 or later) ### Downloading a Graph Reference and Index The graph reference we are going to build incorporates variants of numerous HLA alleles into the linear reference using a graph. The graph reference also includes some known variants of other regions of the genome (e.g. common small variants). To copy the graph reference, type: hisat-genotype-top/evaluation/hla-analysis$ mv ../hisat2-genotype/* . ### Typing and Assembly Since whole genome sequencing (WGS) data includes reads that are from the whole genome, the first step is to extract the reads that belong to the HLA genes by aligning them to the graph reference with HISAT2. We provide these extracted reads in hisat-genotype-top/evaluation/hla-analysis/ILMN_20181025. HISAT-genotype performs both HLA typing and assembly as follows. You can perform HLA typing and assembly for HLA-A gene on sequencing reads from the genome NA12892 (Illumina's HiSeq 2000 platform). hisat-genotype-top/evaluation/hla-analysis$ hisatgenotype_locus.py --base hla --locus-list A --assembly -1 ILMN_20181025/NA12892.hla.extracted.1.fq.gz -2 ILMN_20181025/NA12892.hla.extracted.2.fq.gz ### DNA Fingerprinting This function can be performed with the same commands used for “Typing and Assembly” and just replacing --base hla with --base codis. ### Interpreting Output Typing Output Number of reads aligned: 1507 1 A*02:01:01:02L (count: 571) 2 A*02:01:31 (count: 557) 3 A*02:20:02 (count: 557) 4 A*02:29 (count: 557) 5 A*02:321N (count: 556) 6 A*02:372 (count: 556) 7 A*02:610:02 (count: 556) 8 A*02:249 (count: 555) 9 A*02:479 (count: 555) 10 A*02:11:01 (count: 554) The above lines show the top ten alleles that the most number of reads are mapped to or compatible with. For example, the allele first ranked, A\*02:01:01:02L, is compatible with 571 reads. This raw estimate based on the number of reads should not be used to determine the two true alleles because the alleles that resemble both but are not true alleles often tend to be compatible with more reads than either of the true alleles. Thus, we apply a statistical model to identify the two true alleles as described in the main text. Abundance of alleles 1 ranked A*02:01:01:01 (abundance: 54.32%) 2 ranked A*11:01:01:01 (abundance: 45.20%) 3 ranked A*24:33 (abundance: 0.48%) The above rankings show the top three alleles that are most abundant in the sample. Normally, the top two alleles in this estimate (e.g. A\*02:01:01:01 and A\*11:01:01:01) are considered as the two alleles that best match a given sequencing data. Additional tutorials and details are available at the HISAT-genotype website: https://ccb.jhu.edu/hisat-genotype ## Data The Data directory (`/data`) contains all input files for reproducing some of our results such as from the evaluation of HISAT2 and other programs using both simulated and real reads, from typing and assembling HLA genes of Illumina Platinum Genomes using HISAT-genotype, and from building a HISAT2 graph index. * **Simulated read pairs** | Type | Number of pairs | Path | | - | - | - | | SNPs and sequencing errors included | 10,000,000 | hisat-genotype-top/evaluation/reads/simulation/10M_DNA_mismatch_snp_reads_genome | | SNPs included | 10,000,000 | hisat-genotype-top/evaluation/reads/simulation/10M_DNA_snp_reads_genome | | Sequencing errors included | 10,000,000 | hisat-genotype-top/evaluation/reads/simulation/10M_DNA_mismatch_reads_genome | | No SNPs nor sequencing errors included | 10,000,000 | hisat-genotype-top/evaluation/reads/simulation/10M_DNA_reads_genome | Each directory comes with a true alignment file in SAM format so that users know where the reads were generated in the human reference genome. * **Real read pairs** | Number of read pairs | Path | | - | - | | 10,000,000 | hisat-genotype-top/evaluation/reads/real/DNA | * **Human reference genome, SNPs, haplotypes, and HISAT2's indexes** | Type | Path | | - | - | | GRCh38 reference | hisat-genotype-top/evaluation/data/genome.fa | | SNPs | hisat-genotype-top/evaluation/data/genome.snp | | Haplotypes | hisat-genotype-top/evaluation/data/genome.haplotype | | HISAT2's prebuilt graph index for comparison with other aligners | hisat-genotype-top/evaluation/indexes/HISAT2/genome.[1-8].ht2 | | HISAT2's prebuilt graph index for genotyping | hisat-genotype-top/evaluation/hla-analysis/genotype_genome.[1-8].ht2 | ## References [1] Sirén J, Välimäki N, Mäkinen V (2014) Indexing graphs for path queries with applications in genome research. IEEE/ACM Transactions on Computational Biology and Bioinformatics 11: 375–388. doi: 10.1109/tcbb.2013.2297101 [2] Kim D, Langmead B, and Salzberg SL HISAT: a fast spliced aligner with low memory requirements, Nature methods, 2015 [3] Langmead B, Salzberg SL: Fast gapped-read alignment with Bowtie 2. Nat Methods 2012, 9:357-359 hisat2-2.2.1/TUTORIAL000066400000000000000000000003121370663763200141050ustar00rootroot00000000000000See section toward end of MANUAL entited "Getting started with HISAT2". Or, for tutorial for latest HISAT2 version, visit: https://ccb.jhu.edu/software/hisat2/manual.shtml#getting-started-with-hisat2 hisat2-2.2.1/VERSION000066400000000000000000000000061370663763200137670ustar00rootroot000000000000002.2.1 hisat2-2.2.1/_config.yml000066400000000000000000000000401370663763200150440ustar00rootroot00000000000000theme: jekyll-theme-time-machinehisat2-2.2.1/aligner_bt.cpp000066400000000000000000001513331370663763200155430ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "aligner_bt.h" #include "mask.h" using namespace std; #define CHECK_ROW_COL(rowc, colc) \ if(rowc >= 0 && colc >= 0) { \ if(!sawcell_[colc].insert(rowc)) { \ /* was already in there */ \ abort = true; \ return; \ } \ assert(local || prob_.cper_->debugCell(rowc, colc, hefc)); \ } /** * Fill in a triangle of the DP table and backtrace from the given cell to * a cell in the previous checkpoint, or to the terminal cell. */ void BtBranchTracer::triangleFill( int64_t rw, // row of cell to backtrace from int64_t cl, // column of cell to backtrace from int hef, // cell to backtrace from is H (0), E (1), or F (2) TAlScore targ, // score of cell to backtrace from TAlScore targ_final, // score of alignment we're looking for RandomSource& rnd, // pseudo-random generator int64_t& row_new, // out: row we ended up in after backtrace int64_t& col_new, // out: column we ended up in after backtrace int& hef_new, // out: H/E/F after backtrace TAlScore& targ_new, // out: score up to cell we ended up in bool& done, // out: finished tracing out an alignment? bool& abort) // out: aborted b/c cell was seen before? { assert_geq(rw, 0); assert_geq(cl, 0); assert_range(0, 2, hef); assert_lt(rw, (int64_t)prob_.qrylen_); assert_lt(cl, (int64_t)prob_.reflen_); assert(prob_.usecp_ && prob_.fill_); int64_t row = rw, col = cl; const int64_t colmin = 0; const int64_t rowmin = 0; const int64_t colmax = prob_.reflen_ - 1; const int64_t rowmax = prob_.qrylen_ - 1; assert_leq(prob_.reflen_, (TRefOff)sawcell_.size()); assert_leq(col, (int64_t)prob_.cper_->hicol()); assert_geq(col, (int64_t)prob_.cper_->locol()); assert_geq(prob_.cper_->per(), 2); size_t mod = (row + col) & prob_.cper_->lomask(); assert_lt(mod, prob_.cper_->per()); // Allocate room for diags size_t depth = mod+1; assert_leq(depth, prob_.cper_->per()); size_t breadth = depth; tri_.resize(depth); // Allocate room for each diag for(size_t i = 0; i < depth; i++) { tri_[i].resize(breadth - i); } bool upperleft = false; size_t off = (row + col) >> prob_.cper_->perpow2(); if(off == 0) { upperleft = true; } else { off--; } const TAlScore sc_rdo = prob_.sc_->readGapOpen(); const TAlScore sc_rde = prob_.sc_->readGapExtend(); const TAlScore sc_rfo = prob_.sc_->refGapOpen(); const TAlScore sc_rfe = prob_.sc_->refGapExtend(); const bool local = !prob_.sc_->monotone; int64_t row_lo = row - (int64_t)mod; const CpQuad *prev2 = NULL, *prev1 = NULL; if(!upperleft) { // Read-only pointer to cells in diagonal -2. Start one row above the // target row. prev2 = prob_.cper_->qdiag1sPtr() + (off * prob_.cper_->nrow() + row_lo - 1); // Read-only pointer to cells in diagonal -1. Start one row above the // target row prev1 = prob_.cper_->qdiag2sPtr() + (off * prob_.cper_->nrow() + row_lo - 1); #ifndef NDEBUG if(row >= (int64_t)mod) { size_t rowc = row - mod, colc = col; if(rowc > 0 && prob_.cper_->isCheckpointed(rowc-1, colc)) { TAlScore al = prev1[0].sc[0]; if(al == MIN_I16) al = MIN_I64; assert_eq(prob_.cper_->scoreTriangle(rowc-1, colc, 0), al); } if(rowc > 0 && colc > 0 && prob_.cper_->isCheckpointed(rowc-1, colc-1)) { TAlScore al = prev2[0].sc[0]; if(al == MIN_I16) al = MIN_I64; assert_eq(prob_.cper_->scoreTriangle(rowc-1, colc-1, 0), al); } } #endif } // Pointer to cells in current diagonal // For each diagonal we need to fill in for(size_t i = 0; i < depth; i++) { CpQuad * cur = tri_[i].ptr(); CpQuad * curc = cur; size_t doff = mod - i; // # diagonals we are away from target diag //assert_geq(row, (int64_t)doff); int64_t rowc = row - doff; int64_t colc = col; size_t neval = 0; // # cells evaluated in this diag ASSERT_ONLY(const CpQuad *last = NULL); // Fill this diagonal from upper right to lower left for(size_t j = 0; j < breadth; j++) { if(rowc >= rowmin && rowc <= rowmax && colc >= colmin && colc <= colmax) { neval++; int64_t fromend = prob_.qrylen_ - rowc - 1; bool allowGaps = fromend >= prob_.sc_->gapbar && rowc >= prob_.sc_->gapbar; // Fill this cell // Some things we might want to calculate about this cell up front: // 1. How many matches are possible from this cell to the cell in // row, col, in case this allows us to prune // Get character from read int qc = prob_.qry_[rowc]; // Get quality value from read int qq = prob_.qual_[rowc]; assert_geq(qq, 33); // Get character from reference int rc = prob_.ref_[colc]; assert_range(0, 16, rc); int16_t sc_diag = prob_.sc_->score(qc, rc, qq - 33); int16_t sc_h_up = MIN_I16; int16_t sc_f_up = MIN_I16; int16_t sc_h_lf = MIN_I16; int16_t sc_e_lf = MIN_I16; if(allowGaps) { if(rowc > 0) { assert(local || prev1[j+0].sc[2] < 0); if(prev1[j+0].sc[0] > MIN_I16) { sc_h_up = prev1[j+0].sc[0] - sc_rfo; if(local) sc_h_up = max(sc_h_up, 0); } if(prev1[j+0].sc[2] > MIN_I16) { sc_f_up = prev1[j+0].sc[2] - sc_rfe; if(local) sc_f_up = max(sc_f_up, 0); } #ifndef NDEBUG TAlScore hup = prev1[j+0].sc[0]; TAlScore fup = prev1[j+0].sc[2]; if(hup == MIN_I16) hup = MIN_I64; if(fup == MIN_I16) fup = MIN_I64; if(local) { hup = max(hup, 0); fup = max(fup, 0); } if(prob_.cper_->isCheckpointed(rowc-1, colc)) { assert_eq(hup, prob_.cper_->scoreTriangle(rowc-1, colc, 0)); assert_eq(fup, prob_.cper_->scoreTriangle(rowc-1, colc, 2)); } #endif } if(colc > 0) { assert(local || prev1[j+1].sc[1] < 0); if(prev1[j+1].sc[0] > MIN_I16) { sc_h_lf = prev1[j+1].sc[0] - sc_rdo; if(local) sc_h_lf = max(sc_h_lf, 0); } if(prev1[j+1].sc[1] > MIN_I16) { sc_e_lf = prev1[j+1].sc[1] - sc_rde; if(local) sc_e_lf = max(sc_e_lf, 0); } #ifndef NDEBUG TAlScore hlf = prev1[j+1].sc[0]; TAlScore elf = prev1[j+1].sc[1]; if(hlf == MIN_I16) hlf = MIN_I64; if(elf == MIN_I16) elf = MIN_I64; if(local) { hlf = max(hlf, 0); elf = max(elf, 0); } if(prob_.cper_->isCheckpointed(rowc, colc-1)) { assert_eq(hlf, prob_.cper_->scoreTriangle(rowc, colc-1, 0)); assert_eq(elf, prob_.cper_->scoreTriangle(rowc, colc-1, 1)); } #endif } } assert(rowc <= 1 || colc <= 0 || prev2 != NULL); int16_t sc_h_dg = ((rowc > 0 && colc > 0) ? prev2[j+0].sc[0] : 0); if(colc == 0 && rowc > 0 && !local) { sc_h_dg = MIN_I16; } if(sc_h_dg > MIN_I16) { sc_h_dg += sc_diag; } if(local) sc_h_dg = max(sc_h_dg, 0); // cerr << sc_diag << " " << sc_h_dg << " " << sc_h_up << " " << sc_f_up << " " << sc_h_lf << " " << sc_e_lf << endl; int mask = 0; // Calculate best ways into H, E, F cells starting with H. // Mask bits: // H: 1=diag, 2=hhoriz, 4=ehoriz, 8=hvert, 16=fvert // E: 32=hhoriz, 64=ehoriz // F: 128=hvert, 256=fvert int16_t sc_best = sc_h_dg; if(sc_h_dg > MIN_I64) { mask = 1; } if(colc > 0 && sc_h_lf >= sc_best && sc_h_lf > MIN_I64) { if(sc_h_lf > sc_best) mask = 0; mask |= 2; sc_best = sc_h_lf; } if(colc > 0 && sc_e_lf >= sc_best && sc_e_lf > MIN_I64) { if(sc_e_lf > sc_best) mask = 0; mask |= 4; sc_best = sc_e_lf; } if(rowc > 0 && sc_h_up >= sc_best && sc_h_up > MIN_I64) { if(sc_h_up > sc_best) mask = 0; mask |= 8; sc_best = sc_h_up; } if(rowc > 0 && sc_f_up >= sc_best && sc_f_up > MIN_I64) { if(sc_f_up > sc_best) mask = 0; mask |= 16; sc_best = sc_f_up; } // Calculate best way into E cell int16_t sc_e_best = sc_h_lf; if(colc > 0) { if(sc_h_lf >= sc_e_lf && sc_h_lf > MIN_I64) { if(sc_h_lf == sc_e_lf) { mask |= 64; } mask |= 32; } else if(sc_e_lf > MIN_I64) { sc_e_best = sc_e_lf; mask |= 64; } } if(sc_e_best > sc_best) { sc_best = sc_e_best; mask &= ~31; // don't go diagonal } // Calculate best way into F cell int16_t sc_f_best = sc_h_up; if(rowc > 0) { if(sc_h_up >= sc_f_up && sc_h_up > MIN_I64) { if(sc_h_up == sc_f_up) { mask |= 256; } mask |= 128; } else if(sc_f_up > MIN_I64) { sc_f_best = sc_f_up; mask |= 256; } } if(sc_f_best > sc_best) { sc_best = sc_f_best; mask &= ~127; // don't go horizontal or diagonal } // Install results in cur assert(!prob_.sc_->monotone || sc_best <= 0); assert(!prob_.sc_->monotone || sc_e_best <= 0); assert(!prob_.sc_->monotone || sc_f_best <= 0); curc->sc[0] = sc_best; assert( local || sc_e_best < 0); assert( local || sc_f_best < 0); assert(!local || sc_e_best >= 0 || sc_e_best == MIN_I16); assert(!local || sc_f_best >= 0 || sc_f_best == MIN_I16); curc->sc[1] = sc_e_best; curc->sc[2] = sc_f_best; curc->sc[3] = mask; // cerr << curc->sc[0] << " " << curc->sc[1] << " " << curc->sc[2] << " " << curc->sc[3] << endl; ASSERT_ONLY(last = curc); #ifndef NDEBUG if(prob_.cper_->isCheckpointed(rowc, colc)) { if(local) { sc_e_best = max(sc_e_best, 0); sc_f_best = max(sc_f_best, 0); } TAlScore sc_best64 = sc_best; if(sc_best == MIN_I16) sc_best64 = MIN_I64; TAlScore sc_e_best64 = sc_e_best; if(sc_e_best == MIN_I16) sc_e_best64 = MIN_I64; TAlScore sc_f_best64 = sc_f_best; if(sc_f_best == MIN_I16) sc_f_best64 = MIN_I64; assert_eq(prob_.cper_->scoreTriangle(rowc, colc, 0), sc_best64); assert_eq(prob_.cper_->scoreTriangle(rowc, colc, 1), sc_e_best64); assert_eq(prob_.cper_->scoreTriangle(rowc, colc, 2), sc_f_best64); } #endif } // Update row, col assert_lt(rowc, (int64_t)prob_.qrylen_); rowc++; colc--; curc++; } // for(size_t j = 0; j < breadth; j++) if(i == depth-1) { // Final iteration assert(last != NULL); assert_eq(1, neval); assert_neq(0, last->sc[3]); assert_eq(targ, last->sc[hef]); } else { breadth--; prev2 = prev1 + 1; prev1 = cur; } } // for(size_t i = 0; i < depth; i++) // // Now backtrack through the triangle. Abort as soon as we enter a cell // that was visited by a previous backtrace. // int64_t rowc = row, colc = col; size_t curid; int hefc = hef; if(bs_.empty()) { // Start an initial branch CHECK_ROW_COL(rowc, colc); curid = bs_.alloc(); assert_eq(0, curid); Edit e; bs_[curid].init( prob_, 0, // parent ID 0, // penalty 0, // score_en rowc, // row colc, // col e, // edit 0, // hef true, // I am the root false); // don't try to extend with exact matches bs_[curid].len_ = 0; } else { curid = bs_.size()-1; } size_t idx_orig = (row + col) >> prob_.cper_->perpow2(); while(true) { // What depth are we? size_t mod = (rowc + colc) & prob_.cper_->lomask(); assert_lt(mod, prob_.cper_->per()); CpQuad * cur = tri_[mod].ptr(); int64_t row_off = rowc - row_lo - mod; assert(!local || cur[row_off].sc[0] > 0); assert_geq(row_off, 0); int mask = cur[row_off].sc[3]; assert_gt(mask, 0); int sel = -1; // Select what type of move to make, which depends on whether we're // currently in H, E, F: if(hefc == 0) { if( (mask & 1) != 0) { // diagonal sel = 0; } else if((mask & 8) != 0) { // up to H sel = 3; } else if((mask & 16) != 0) { // up to F sel = 4; } else if((mask & 2) != 0) { // left to H sel = 1; } else if((mask & 4) != 0) { // left to E sel = 2; } } else if(hefc == 1) { if( (mask & 32) != 0) { // left to H sel = 5; } else if((mask & 64) != 0) { // left to E sel = 6; } } else { assert_eq(2, hefc); if( (mask & 128) != 0) { // up to H sel = 7; } else if((mask & 256) != 0) { // up to F sel = 8; } } assert_geq(sel, 0); // Get character from read int qc = prob_.qry_[rowc], qq = prob_.qual_[rowc]; // Get character from reference int rc = prob_.ref_[colc]; assert_range(0, 16, rc); // Now that we know what type of move to make, make it, updating our // row and column and moving updating the branch. if(sel == 0) { assert_geq(rowc, 0); assert_geq(colc, 0); TAlScore scd = prob_.sc_->score(qc, rc, qq - 33); if((rc & (1 << qc)) == 0) { // Mismatch size_t id = curid; // Check if the previous branch was the initial (bottommost) // branch with no matches. If so, the mismatch should be added // to the initial branch, instead of starting a new branch. bool empty = (bs_[curid].len_ == 0 && curid == 0); if(!empty) { id = bs_.alloc(); } Edit e((int)rowc, mask2dna[rc], "ACGTN"[qc], EDIT_TYPE_MM); assert_lt(scd, 0); TAlScore score_en = bs_[curid].score_st_ + scd; bs_[id].init( prob_, curid, // parent ID -scd, // penalty score_en, // score_en rowc, // row colc, // col e, // edit hefc, // hef empty, // root? false); // don't try to extend with exact matches //assert(!local || bs_[id].score_st_ >= 0); curid = id; } else { // Match bs_[curid].score_st_ += prob_.sc_->match(); bs_[curid].len_++; assert_leq((int64_t)bs_[curid].len_, bs_[curid].row_ + 1); } rowc--; colc--; assert(local || bs_[curid].score_st_ >= targ_final); hefc = 0; } else if((sel >= 1 && sel <= 2) || (sel >= 5 && sel <= 6)) { assert_gt(colc, 0); // Read gap size_t id = bs_.alloc(); Edit e((int)rowc+1, mask2dna[rc], '-', EDIT_TYPE_READ_GAP); TAlScore gapp = prob_.sc_->readGapOpen(); if(bs_[curid].len_ == 0 && bs_[curid].e_.inited() && bs_[curid].e_.isReadGap()) { gapp = prob_.sc_->readGapExtend(); } TAlScore score_en = bs_[curid].score_st_ - gapp; bs_[id].init( prob_, curid, // parent ID gapp, // penalty score_en, // score_en rowc, // row colc-1, // col e, // edit hefc, // hef false, // root? false); // don't try to extend with exact matches colc--; curid = id; assert( local || bs_[curid].score_st_ >= targ_final); //assert(!local || bs_[curid].score_st_ >= 0); if(sel == 1 || sel == 5) { hefc = 0; } else { hefc = 1; } } else { assert_gt(rowc, 0); // Reference gap size_t id = bs_.alloc(); Edit e((int)rowc, '-', "ACGTN"[qc], EDIT_TYPE_REF_GAP); TAlScore gapp = prob_.sc_->refGapOpen(); if(bs_[curid].len_ == 0 && bs_[curid].e_.inited() && bs_[curid].e_.isRefGap()) { gapp = prob_.sc_->refGapExtend(); } TAlScore score_en = bs_[curid].score_st_ - gapp; bs_[id].init( prob_, curid, // parent ID gapp, // penalty score_en, // score_en rowc-1, // row colc, // col e, // edit hefc, // hef false, // root? false); // don't try to extend with exact matches rowc--; curid = id; //assert(!local || bs_[curid].score_st_ >= 0); if(sel == 3 || sel == 7) { hefc = 0; } else { hefc = 2; } } CHECK_ROW_COL(rowc, colc); size_t mod_new = (rowc + colc) & prob_.cper_->lomask(); size_t idx = (rowc + colc) >> prob_.cper_->perpow2(); assert_lt(mod_new, prob_.cper_->per()); int64_t row_off_new = rowc - row_lo - mod_new; CpQuad * cur_new = NULL; if(colc >= 0 && rowc >= 0 && idx == idx_orig) { cur_new = tri_[mod_new].ptr(); } bool hit_new_tri = (idx < idx_orig && colc >= 0 && rowc >= 0); // Check whether we made it to the top row or to a cell with score 0 if(colc < 0 || rowc < 0 || (cur_new != NULL && (local && cur_new[row_off_new].sc[0] == 0))) { done = true; assert(bs_[curid].isSolution(prob_)); addSolution(curid); #ifndef NDEBUG // A check to see if any two adjacent branches in the backtrace // overlap. If they do, the whole alignment will be filtered out // in trySolution(...) size_t cur = curid; if(!bs_[cur].root_) { size_t next = bs_[cur].parentId_; while(!bs_[next].root_) { assert_neq(cur, next); if(bs_[next].len_ != 0 || bs_[cur].len_ == 0) { assert(!bs_[cur].overlap(prob_, bs_[next])); } cur = next; next = bs_[cur].parentId_; } } #endif return; } if(hit_new_tri) { assert(rowc < 0 || colc < 0 || prob_.cper_->isCheckpointed(rowc, colc)); row_new = rowc; col_new = colc; hef_new = hefc; done = false; if(rowc < 0 || colc < 0) { assert(local); targ_new = 0; } else { targ_new = prob_.cper_->scoreTriangle(rowc, colc, hefc); } if(local && targ_new == 0) { done = true; assert(bs_[curid].isSolution(prob_)); addSolution(curid); } assert((row_new >= 0 && col_new >= 0) || done); return; } } assert(false); } #ifndef NDEBUG #define DEBUG_CHECK(ss, row, col, hef) { \ if(prob_.cper_->debug() && row >= 0 && col >= 0) { \ TAlScore s = ss; \ if(s == MIN_I16) s = MIN_I64; \ if(local && s < 0) s = 0; \ TAlScore deb = prob_.cper_->debugCell(row, col, hef); \ if(local && deb < 0) deb = 0; \ assert_eq(s, deb); \ } \ } #else #define DEBUG_CHECK(ss, row, col, hef) #endif /** * Fill in a square of the DP table and backtrace from the given cell to * a cell in the previous checkpoint, or to the terminal cell. */ void BtBranchTracer::squareFill( int64_t rw, // row of cell to backtrace from int64_t cl, // column of cell to backtrace from int hef, // cell to backtrace from is H (0), E (1), or F (2) TAlScore targ, // score of cell to backtrace from TAlScore targ_final, // score of alignment we're looking for RandomSource& rnd, // pseudo-random generator int64_t& row_new, // out: row we ended up in after backtrace int64_t& col_new, // out: column we ended up in after backtrace int& hef_new, // out: H/E/F after backtrace TAlScore& targ_new, // out: score up to cell we ended up in bool& done, // out: finished tracing out an alignment? bool& abort) // out: aborted b/c cell was seen before? { assert_geq(rw, 0); assert_geq(cl, 0); assert_range(0, 2, hef); assert_lt(rw, (int64_t)prob_.qrylen_); assert_lt(cl, (int64_t)prob_.reflen_); assert(prob_.usecp_ && prob_.fill_); const bool is8_ = prob_.cper_->is8_; int64_t row = rw, col = cl; assert_leq(prob_.reflen_, (TRefOff)sawcell_.size()); assert_leq(col, (int64_t)prob_.cper_->hicol()); assert_geq(col, (int64_t)prob_.cper_->locol()); assert_geq(prob_.cper_->per(), 2); size_t xmod = col & prob_.cper_->lomask(); size_t ymod = row & prob_.cper_->lomask(); size_t xdiv = col >> prob_.cper_->perpow2(); size_t ydiv = row >> prob_.cper_->perpow2(); size_t sq_ncol = xmod+1, sq_nrow = ymod+1; sq_.resize(sq_ncol * sq_nrow); bool upper = ydiv == 0; bool left = xdiv == 0; const TAlScore sc_rdo = prob_.sc_->readGapOpen(); const TAlScore sc_rde = prob_.sc_->readGapExtend(); const TAlScore sc_rfo = prob_.sc_->refGapOpen(); const TAlScore sc_rfe = prob_.sc_->refGapExtend(); const bool local = !prob_.sc_->monotone; const CpQuad *qup = NULL; const __m128i *qlf = NULL; size_t per = prob_.cper_->per_; ASSERT_ONLY(size_t nrow = prob_.cper_->nrow()); size_t ncol = prob_.cper_->ncol(); assert_eq(prob_.qrylen_, nrow); assert_eq(prob_.reflen_, (TRefOff)ncol); size_t niter = prob_.cper_->niter_; if(!upper) { qup = prob_.cper_->qrows_.ptr() + (ncol * (ydiv-1)) + xdiv * per; } if(!left) { // Set up the column pointers to point to the first __m128i word in the // relevant column size_t off = (niter << 2) * (xdiv-1); qlf = prob_.cper_->qcols_.ptr() + off; } size_t xedge = xdiv * per; // absolute offset of leftmost cell in square size_t yedge = ydiv * per; // absolute offset of topmost cell in square size_t xi = xedge, yi = yedge; // iterators for columns, rows size_t ii = 0; // iterator into packed square // Iterate over rows, then over columns size_t m128mod = yi % prob_.cper_->niter_; size_t m128div = yi / prob_.cper_->niter_; int16_t sc_h_dg_lastrow = MIN_I16; for(size_t i = 0; i <= ymod; i++, yi++) { assert_lt(yi, nrow); xi = xedge; // Handling for first column is done outside the loop size_t fromend = prob_.qrylen_ - yi - 1; bool allowGaps = fromend >= (size_t)prob_.sc_->gapbar && yi >= (size_t)prob_.sc_->gapbar; // Get character, quality from read int qc = prob_.qry_[yi], qq = prob_.qual_[yi]; assert_geq(qq, 33); int16_t sc_h_lf_last = MIN_I16; int16_t sc_e_lf_last = MIN_I16; for(size_t j = 0; j <= xmod; j++, xi++) { assert_lt(xi, ncol); // Get character from reference int rc = prob_.ref_[xi]; assert_range(0, 16, rc); int16_t sc_diag = prob_.sc_->score(qc, rc, qq - 33); int16_t sc_h_up = MIN_I16, sc_f_up = MIN_I16, sc_h_lf = MIN_I16, sc_e_lf = MIN_I16, sc_h_dg = MIN_I16; int16_t sc_h_up_c = MIN_I16, sc_f_up_c = MIN_I16, sc_h_lf_c = MIN_I16, sc_e_lf_c = MIN_I16, sc_h_dg_c = MIN_I16; if(yi == 0) { // If I'm in the first first row or column set it to 0 sc_h_dg = 0; } else if(xi == 0) { // Do nothing; leave it at min if(local) { sc_h_dg = 0; } } else if(i == 0 && j == 0) { // Otherwise, if I'm in the upper-left square corner, I can get // it from the checkpoint sc_h_dg = qup[-1].sc[0]; } else if(j == 0) { // Otherwise, if I'm in the leftmost cell of this row, I can // get it from sc_h_lf in first column of previous row sc_h_dg = sc_h_dg_lastrow; } else { // Otherwise, I can get it from qup sc_h_dg = qup[j-1].sc[0]; } if(yi > 0 && xi > 0) DEBUG_CHECK(sc_h_dg, yi-1, xi-1, 2); // If we're in the leftmost column, calculate sc_h_lf regardless of // allowGaps. if(j == 0 && xi > 0) { // Get values for left neighbors from the checkpoint if(is8_) { size_t vecoff = (m128mod << 6) + m128div; sc_e_lf = ((uint8_t*)(qlf + 0))[vecoff]; sc_h_lf = ((uint8_t*)(qlf + 2))[vecoff]; if(local) { // No adjustment } else { if(sc_h_lf == 0) sc_h_lf = MIN_I16; else sc_h_lf -= 0xff; if(sc_e_lf == 0) sc_e_lf = MIN_I16; else sc_e_lf -= 0xff; } } else { size_t vecoff = (m128mod << 5) + m128div; sc_e_lf = ((int16_t*)(qlf + 0))[vecoff]; sc_h_lf = ((int16_t*)(qlf + 2))[vecoff]; if(local) { sc_h_lf += 0x8000; assert_geq(sc_h_lf, 0); sc_e_lf += 0x8000; assert_geq(sc_e_lf, 0); } else { if(sc_h_lf != MIN_I16) sc_h_lf -= 0x7fff; if(sc_e_lf != MIN_I16) sc_e_lf -= 0x7fff; } } DEBUG_CHECK(sc_e_lf, yi, xi-1, 0); DEBUG_CHECK(sc_h_lf, yi, xi-1, 2); sc_h_dg_lastrow = sc_h_lf; } if(allowGaps) { if(j == 0 /* at left edge */ && xi > 0 /* not extreme */) { sc_h_lf_c = sc_h_lf; sc_e_lf_c = sc_e_lf; if(sc_h_lf_c != MIN_I16) sc_h_lf_c -= sc_rdo; if(sc_e_lf_c != MIN_I16) sc_e_lf_c -= sc_rde; assert_leq(sc_h_lf_c, prob_.cper_->perf_); assert_leq(sc_e_lf_c, prob_.cper_->perf_); } else if(xi > 0) { // Get values for left neighbors from the previous iteration if(sc_h_lf_last != MIN_I16) { sc_h_lf = sc_h_lf_last; sc_h_lf_c = sc_h_lf - sc_rdo; } if(sc_e_lf_last != MIN_I16) { sc_e_lf = sc_e_lf_last; sc_e_lf_c = sc_e_lf - sc_rde; } } if(yi > 0 /* not extreme */) { // Get column values assert(qup != NULL); assert(local || qup[j].sc[2] < 0); if(qup[j].sc[0] > MIN_I16) { DEBUG_CHECK(qup[j].sc[0], yi-1, xi, 2); sc_h_up = qup[j].sc[0]; sc_h_up_c = sc_h_up - sc_rfo; } if(qup[j].sc[2] > MIN_I16) { DEBUG_CHECK(qup[j].sc[2], yi-1, xi, 1); sc_f_up = qup[j].sc[2]; sc_f_up_c = sc_f_up - sc_rfe; } } if(local) { sc_h_up_c = max(sc_h_up_c, 0); sc_f_up_c = max(sc_f_up_c, 0); sc_h_lf_c = max(sc_h_lf_c, 0); sc_e_lf_c = max(sc_e_lf_c, 0); } } if(sc_h_dg > MIN_I16) { sc_h_dg_c = sc_h_dg + sc_diag; } if(local) sc_h_dg_c = max(sc_h_dg_c, 0); int mask = 0; // Calculate best ways into H, E, F cells starting with H. // Mask bits: // H: 1=diag, 2=hhoriz, 4=ehoriz, 8=hvert, 16=fvert // E: 32=hhoriz, 64=ehoriz // F: 128=hvert, 256=fvert int16_t sc_best = sc_h_dg_c; if(sc_h_dg_c > MIN_I64) { mask = 1; } if(xi > 0 && sc_h_lf_c >= sc_best && sc_h_lf_c > MIN_I64) { if(sc_h_lf_c > sc_best) mask = 0; mask |= 2; sc_best = sc_h_lf_c; } if(xi > 0 && sc_e_lf_c >= sc_best && sc_e_lf_c > MIN_I64) { if(sc_e_lf_c > sc_best) mask = 0; mask |= 4; sc_best = sc_e_lf_c; } if(yi > 0 && sc_h_up_c >= sc_best && sc_h_up_c > MIN_I64) { if(sc_h_up_c > sc_best) mask = 0; mask |= 8; sc_best = sc_h_up_c; } if(yi > 0 && sc_f_up_c >= sc_best && sc_f_up_c > MIN_I64) { if(sc_f_up_c > sc_best) mask = 0; mask |= 16; sc_best = sc_f_up_c; } // Calculate best way into E cell int16_t sc_e_best = sc_h_lf_c; if(xi > 0) { if(sc_h_lf_c >= sc_e_lf_c && sc_h_lf_c > MIN_I64) { if(sc_h_lf_c == sc_e_lf_c) { mask |= 64; } mask |= 32; } else if(sc_e_lf_c > MIN_I64) { sc_e_best = sc_e_lf_c; mask |= 64; } } if(sc_e_best > sc_best) { sc_best = sc_e_best; mask &= ~31; // don't go diagonal } // Calculate best way into F cell int16_t sc_f_best = sc_h_up_c; if(yi > 0) { if(sc_h_up_c >= sc_f_up_c && sc_h_up_c > MIN_I64) { if(sc_h_up_c == sc_f_up_c) { mask |= 256; } mask |= 128; } else if(sc_f_up_c > MIN_I64) { sc_f_best = sc_f_up_c; mask |= 256; } } if(sc_f_best > sc_best) { sc_best = sc_f_best; mask &= ~127; // don't go horizontal or diagonal } // Install results in cur assert( local || sc_best <= 0); sq_[ii+j].sc[0] = sc_best; assert( local || sc_e_best < 0); assert( local || sc_f_best < 0); assert(!local || sc_e_best >= 0 || sc_e_best == MIN_I16); assert(!local || sc_f_best >= 0 || sc_f_best == MIN_I16); sq_[ii+j].sc[1] = sc_e_best; sq_[ii+j].sc[2] = sc_f_best; sq_[ii+j].sc[3] = mask; DEBUG_CHECK(sq_[ii+j].sc[0], yi, xi, 2); // H DEBUG_CHECK(sq_[ii+j].sc[1], yi, xi, 0); // E DEBUG_CHECK(sq_[ii+j].sc[2], yi, xi, 1); // F // Update sc_h_lf_last, sc_e_lf_last sc_h_lf_last = sc_best; sc_e_lf_last = sc_e_best; } // Update m128mod, m128div m128mod++; if(m128mod == prob_.cper_->niter_) { m128mod = 0; m128div++; } // update qup ii += sq_ncol; // dimensions of sq_ qup = sq_.ptr() + sq_ncol * i; } assert_eq(targ, sq_[ymod * sq_ncol + xmod].sc[hef]); // // Now backtrack through the triangle. Abort as soon as we enter a cell // that was visited by a previous backtrace. // int64_t rowc = row, colc = col; size_t curid; int hefc = hef; if(bs_.empty()) { // Start an initial branch CHECK_ROW_COL(rowc, colc); curid = bs_.alloc(); assert_eq(0, curid); Edit e; bs_[curid].init( prob_, 0, // parent ID 0, // penalty 0, // score_en rowc, // row colc, // col e, // edit 0, // hef true, // root? false); // don't try to extend with exact matches bs_[curid].len_ = 0; } else { curid = bs_.size()-1; } size_t ymodTimesNcol = ymod * sq_ncol; while(true) { // What depth are we? assert_eq(ymodTimesNcol, ymod * sq_ncol); CpQuad * cur = sq_.ptr() + ymodTimesNcol + xmod; int mask = cur->sc[3]; assert_gt(mask, 0); int sel = -1; // Select what type of move to make, which depends on whether we're // currently in H, E, F: if(hefc == 0) { if( (mask & 1) != 0) { // diagonal sel = 0; } else if((mask & 8) != 0) { // up to H sel = 3; } else if((mask & 16) != 0) { // up to F sel = 4; } else if((mask & 2) != 0) { // left to H sel = 1; } else if((mask & 4) != 0) { // left to E sel = 2; } } else if(hefc == 1) { if( (mask & 32) != 0) { // left to H sel = 5; } else if((mask & 64) != 0) { // left to E sel = 6; } } else { assert_eq(2, hefc); if( (mask & 128) != 0) { // up to H sel = 7; } else if((mask & 256) != 0) { // up to F sel = 8; } } assert_geq(sel, 0); // Get character from read int qc = prob_.qry_[rowc], qq = prob_.qual_[rowc]; // Get character from reference int rc = prob_.ref_[colc]; assert_range(0, 16, rc); bool xexit = false, yexit = false; // Now that we know what type of move to make, make it, updating our // row and column and moving updating the branch. if(sel == 0) { assert_geq(rowc, 0); assert_geq(colc, 0); TAlScore scd = prob_.sc_->score(qc, rc, qq - 33); if((rc & (1 << qc)) == 0) { // Mismatch size_t id = curid; // Check if the previous branch was the initial (bottommost) // branch with no matches. If so, the mismatch should be added // to the initial branch, instead of starting a new branch. bool empty = (bs_[curid].len_ == 0 && curid == 0); if(!empty) { id = bs_.alloc(); } Edit e((int)rowc, mask2dna[rc], "ACGTN"[qc], EDIT_TYPE_MM); assert_lt(scd, 0); TAlScore score_en = bs_[curid].score_st_ + scd; bs_[id].init( prob_, curid, // parent ID -scd, // penalty score_en, // score_en rowc, // row colc, // col e, // edit hefc, // hef empty, // root? false); // don't try to extend with exact matches curid = id; //assert(!local || bs_[curid].score_st_ >= 0); } else { // Match bs_[curid].score_st_ += prob_.sc_->match(); bs_[curid].len_++; assert_leq((int64_t)bs_[curid].len_, bs_[curid].row_ + 1); } if(xmod == 0) xexit = true; if(ymod == 0) yexit = true; rowc--; ymod--; ymodTimesNcol -= sq_ncol; colc--; xmod--; assert(local || bs_[curid].score_st_ >= targ_final); hefc = 0; } else if((sel >= 1 && sel <= 2) || (sel >= 5 && sel <= 6)) { assert_gt(colc, 0); // Read gap size_t id = bs_.alloc(); Edit e((int)rowc+1, mask2dna[rc], '-', EDIT_TYPE_READ_GAP); TAlScore gapp = prob_.sc_->readGapOpen(); if(bs_[curid].len_ == 0 && bs_[curid].e_.inited() && bs_[curid].e_.isReadGap()) { gapp = prob_.sc_->readGapExtend(); } //assert(!local || bs_[curid].score_st_ >= gapp); TAlScore score_en = bs_[curid].score_st_ - gapp; bs_[id].init( prob_, curid, // parent ID gapp, // penalty score_en, // score_en rowc, // row colc-1, // col e, // edit hefc, // hef false, // root? false); // don't try to extend with exact matches if(xmod == 0) xexit = true; colc--; xmod--; curid = id; assert( local || bs_[curid].score_st_ >= targ_final); //assert(!local || bs_[curid].score_st_ >= 0); if(sel == 1 || sel == 5) { hefc = 0; } else { hefc = 1; } } else { assert_gt(rowc, 0); // Reference gap size_t id = bs_.alloc(); Edit e((int)rowc, '-', "ACGTN"[qc], EDIT_TYPE_REF_GAP); TAlScore gapp = prob_.sc_->refGapOpen(); if(bs_[curid].len_ == 0 && bs_[curid].e_.inited() && bs_[curid].e_.isRefGap()) { gapp = prob_.sc_->refGapExtend(); } //assert(!local || bs_[curid].score_st_ >= gapp); TAlScore score_en = bs_[curid].score_st_ - gapp; bs_[id].init( prob_, curid, // parent ID gapp, // penalty score_en, // score_en rowc-1, // row colc, // col e, // edit hefc, // hef false, // root? false); // don't try to extend with exact matches if(ymod == 0) yexit = true; rowc--; ymod--; ymodTimesNcol -= sq_ncol; curid = id; assert( local || bs_[curid].score_st_ >= targ_final); //assert(!local || bs_[curid].score_st_ >= 0); if(sel == 3 || sel == 7) { hefc = 0; } else { hefc = 2; } } CHECK_ROW_COL(rowc, colc); CpQuad * cur_new = NULL; if(!xexit && !yexit) { cur_new = sq_.ptr() + ymodTimesNcol + xmod; } // Check whether we made it to the top row or to a cell with score 0 if(colc < 0 || rowc < 0 || (cur_new != NULL && local && cur_new->sc[0] == 0)) { done = true; assert(bs_[curid].isSolution(prob_)); addSolution(curid); #ifndef NDEBUG // A check to see if any two adjacent branches in the backtrace // overlap. If they do, the whole alignment will be filtered out // in trySolution(...) size_t cur = curid; if(!bs_[cur].root_) { size_t next = bs_[cur].parentId_; while(!bs_[next].root_) { assert_neq(cur, next); if(bs_[next].len_ != 0 || bs_[cur].len_ == 0) { assert(!bs_[cur].overlap(prob_, bs_[next])); } cur = next; next = bs_[cur].parentId_; } } #endif return; } assert(!xexit || hefc == 0 || hefc == 1); assert(!yexit || hefc == 0 || hefc == 2); if(xexit || yexit) { //assert(rowc < 0 || colc < 0 || prob_.cper_->isCheckpointed(rowc, colc)); row_new = rowc; col_new = colc; hef_new = hefc; done = false; if(rowc < 0 || colc < 0) { assert(local); targ_new = 0; } else { // TODO: Don't use scoreSquare targ_new = prob_.cper_->scoreSquare(rowc, colc, hefc); assert(local || targ_new >= targ); assert(local || targ_new >= targ_final); } if(local && targ_new == 0) { assert_eq(0, hefc); done = true; assert(bs_[curid].isSolution(prob_)); addSolution(curid); } assert((row_new >= 0 && col_new >= 0) || done); return; } } assert(false); } /** * Caller gives us score_en, row and col. We figure out score_st and len_ * by comparing characters from the strings. * * If this branch comes after a mismatch, (row, col) describe the cell that the * mismatch occurs in. len_ is initially set to 1, and the next cell we test * is the next cell up and to the left (row-1, col-1). * * If this branch comes after a read gap, (row, col) describe the leftmost cell * involved in the gap. len_ is initially set to 0, and the next cell we test * is the current cell (row, col). * * If this branch comes after a reference gap, (row, col) describe the upper * cell involved in the gap. len_ is initially set to 0, and the next cell we * test is the current cell (row, col). */ void BtBranch::init( const BtBranchProblem& prob, size_t parentId, TAlScore penalty, TAlScore score_en, int64_t row, int64_t col, Edit e, int hef, bool root, bool extend) { score_en_ = score_en; penalty_ = penalty; score_st_ = score_en_; row_ = row; col_ = col; parentId_ = parentId; e_ = e; root_ = root; assert(!root_ || parentId == 0); assert_lt(row, (int64_t)prob.qrylen_); assert_lt(col, (int64_t)prob.reflen_); // First match to check is diagonally above and to the left of the cell // where the edit occurs int64_t rowc = row; int64_t colc = col; len_ = 0; if(e.inited() && e.isMismatch()) { rowc--; colc--; len_ = 1; } int64_t match = prob.sc_->match(); bool cp = prob.usecp_; size_t iters = 0; curtailed_ = false; if(extend) { while(rowc >= 0 && colc >= 0) { int rfm = prob.ref_[colc]; assert_range(0, 16, rfm); int rdc = prob.qry_[rowc]; bool matches = (rfm & (1 << rdc)) != 0; if(!matches) { // What's the mismatch penalty? break; } // Get score from checkpointer score_st_ += match; if(cp && rowc - 1 >= 0 && colc - 1 >= 0 && prob.cper_->isCheckpointed(rowc - 1, colc - 1)) { // Possibly prune int16_t cpsc; cpsc = prob.cper_->scoreTriangle(rowc - 1, colc - 1, hef); if(cpsc + score_st_ < prob.targ_) { curtailed_ = true; break; } } iters++; rowc--; colc--; } } assert_geq(rowc, -1); assert_geq(colc, -1); len_ = (int64_t)row - rowc; assert_leq((int64_t)len_, row_+1); assert_leq((int64_t)len_, col_+1); assert_leq((int64_t)score_st_, (int64_t)prob.qrylen_ * match); } /** * Given a potential branch to add to the queue, see if we can follow the * branch a little further first. If it's still valid, or if we reach a * choice between valid outgoing paths, go ahead and add it to the queue. */ void BtBranchTracer::examineBranch( int64_t row, int64_t col, const Edit& e, TAlScore pen, // penalty associated with edit TAlScore sc, size_t parentId) { size_t id = bs_.alloc(); bs_[id].init(prob_, parentId, pen, sc, row, col, e, 0, false, true); if(bs_[id].isSolution(prob_)) { assert(bs_[id].isValid(prob_)); addSolution(id); } else { // Check if this branch is legit if(bs_[id].isValid(prob_)) { add(id); } else { bs_.pop(); } } } /** * Take all possible ways of leaving the given branch and add them to the * branch queue. */ void BtBranchTracer::addOffshoots(size_t bid) { BtBranch& b = bs_[bid]; TAlScore sc = b.score_en_; int64_t match = prob_.sc_->match(); int64_t scoreFloor = prob_.sc_->monotone ? MIN_I64 : 0; bool cp = prob_.usecp_; // Are there are any checkpoints? ASSERT_ONLY(TAlScore perfectScore = prob_.sc_->perfectScore(prob_.qrylen_)); assert_leq(prob_.targ_, perfectScore); // For each cell in the branch for(size_t i = 0 ; i < b.len_; i++) { assert_leq((int64_t)i, b.row_+1); assert_leq((int64_t)i, b.col_+1); int64_t row = b.row_ - i, col = b.col_ - i; int64_t bonusLeft = (row + 1) * match; int64_t fromend = prob_.qrylen_ - row - 1; bool allowGaps = fromend >= prob_.sc_->gapbar && row >= prob_.sc_->gapbar; if(allowGaps && row >= 0 && col >= 0) { if(col > 0) { // Try a read gap - it's either an extension or an open bool extend = b.e_.inited() && b.e_.isReadGap() && i == 0; TAlScore rdgapPen = extend ? prob_.sc_->readGapExtend() : prob_.sc_->readGapOpen(); bool prune = false; assert_gt(rdgapPen, 0); if(cp && prob_.cper_->isCheckpointed(row, col - 1)) { // Possibly prune int16_t cpsc = (int16_t)prob_.cper_->scoreTriangle(row, col - 1, 0); assert_leq(cpsc, perfectScore); assert_geq(prob_.sc_->readGapOpen(), prob_.sc_->readGapExtend()); TAlScore bonus = prob_.sc_->readGapOpen() - prob_.sc_->readGapExtend(); assert_geq(bonus, 0); if(cpsc + bonus + sc - rdgapPen < prob_.targ_) { prune = true; } } if(prune) { if(extend) { nrdexPrune_++; } else { nrdopPrune_++; } } else if(sc - rdgapPen >= scoreFloor && sc - rdgapPen + bonusLeft >= prob_.targ_) { // Yes, we can introduce a read gap here Edit e((int)row + 1, mask2dna[(int)prob_.ref_[col]], '-', EDIT_TYPE_READ_GAP); assert(e.isReadGap()); examineBranch(row, col - 1, e, rdgapPen, sc - rdgapPen, bid); if(extend) { nrdex_++; } else { nrdop_++; } } } if(row > 0) { // Try a reference gap - it's either an extension or an open bool extend = b.e_.inited() && b.e_.isRefGap() && i == 0; TAlScore rfgapPen = (b.e_.inited() && b.e_.isRefGap()) ? prob_.sc_->refGapExtend() : prob_.sc_->refGapOpen(); bool prune = false; assert_gt(rfgapPen, 0); if(cp && prob_.cper_->isCheckpointed(row - 1, col)) { // Possibly prune int16_t cpsc = (int16_t)prob_.cper_->scoreTriangle(row - 1, col, 0); assert_leq(cpsc, perfectScore); assert_geq(prob_.sc_->refGapOpen(), prob_.sc_->refGapExtend()); TAlScore bonus = prob_.sc_->refGapOpen() - prob_.sc_->refGapExtend(); assert_geq(bonus, 0); if(cpsc + bonus + sc - rfgapPen < prob_.targ_) { prune = true; } } if(prune) { if(extend) { nrfexPrune_++; } else { nrfopPrune_++; } } else if(sc - rfgapPen >= scoreFloor && sc - rfgapPen + bonusLeft >= prob_.targ_) { // Yes, we can introduce a ref gap here Edit e((int)row, '-', "ACGTN"[(int)prob_.qry_[row]], EDIT_TYPE_REF_GAP); assert(e.isRefGap()); examineBranch(row - 1, col, e, rfgapPen, sc - rfgapPen, bid); if(extend) { nrfex_++; } else { nrfop_++; } } } } // If we're at the top of the branch but not yet at the top of // the DP table, a mismatch branch is also possible. if(i == b.len_ && !b.curtailed_ && row >= 0 && col >= 0) { int rfm = prob_.ref_[col]; assert_lt(row, (int64_t)prob_.qrylen_); int rdc = prob_.qry_[row]; int rdq = prob_.qual_[row]; int scdiff = prob_.sc_->score(rdc, rfm, rdq - 33); assert_lt(scdiff, 0); // at end of branch, so can't match bool prune = false; if(cp && row > 0 && col > 0 && prob_.cper_->isCheckpointed(row - 1, col - 1)) { // Possibly prune int16_t cpsc = prob_.cper_->scoreTriangle(row - 1, col - 1, 0); assert_leq(cpsc, perfectScore); assert_leq(cpsc + scdiff + sc, perfectScore); if(cpsc + scdiff + sc < prob_.targ_) { prune = true; } } if(prune) { nmm_++; } else { // Yes, we can introduce a mismatch here if(sc + scdiff >= scoreFloor && sc + scdiff + bonusLeft >= prob_.targ_) { Edit e((int)row, mask2dna[rfm], "ACGTN"[rdc], EDIT_TYPE_MM); bool nmm = (mask2dna[rfm] == 'N' || rdc > 4); assert_neq(e.chr, e.qchr); assert_lt(scdiff, 0); examineBranch(row - 1, col - 1, e, -scdiff, sc + scdiff, bid); if(nmm) { nnmm_++; } else { nmm_++; } } } } sc += match; } } /** * Sort unsorted branches, merge them with master sorted list. */ void BtBranchTracer::flushUnsorted() { if(unsorted_.empty()) { return; } unsorted_.sort(); unsorted_.reverse(); #ifndef NDEBUG for(size_t i = 1; i < unsorted_.size(); i++) { assert_leq(bs_[unsorted_[i].second].score_st_, bs_[unsorted_[i-1].second].score_st_); } #endif EList *src2 = sortedSel_ ? &sorted1_ : &sorted2_; EList *dest = sortedSel_ ? &sorted2_ : &sorted1_; // Merge src1 and src2 into dest dest->clear(); size_t cur1 = 0, cur2 = cur_; while(cur1 < unsorted_.size() || cur2 < src2->size()) { // Take from 1 or 2 next? bool take1 = true; if(cur1 == unsorted_.size()) { take1 = false; } else if(cur2 == src2->size()) { take1 = true; } else { assert_neq(unsorted_[cur1].second, (*src2)[cur2]); take1 = bs_[unsorted_[cur1].second] < bs_[(*src2)[cur2]]; } if(take1) { dest->push_back(unsorted_[cur1++].second); // Take from list 1 } else { dest->push_back((*src2)[cur2++]); // Take from list 2 } } assert_eq(cur1, unsorted_.size()); assert_eq(cur2, src2->size()); sortedSel_ = !sortedSel_; cur_ = 0; unsorted_.clear(); } /** * Try all the solutions accumulated so far. Solutions might be rejected * if they, for instance, overlap a previous solution, have too many Ns, * fail to overlap a core diagonal, etc. */ bool BtBranchTracer::trySolutions( bool lookForOlap, SwResult& res, size_t& off, size_t& nrej, RandomSource& rnd, bool& success) { if(solutions_.size() > 0) { for(size_t i = 0; i < solutions_.size(); i++) { int ret = trySolution(solutions_[i], lookForOlap, res, off, nrej, rnd); if(ret == BT_FOUND) { success = true; return true; // there were solutions and one was good } } solutions_.clear(); success = false; return true; // there were solutions but none were good } return false; // there were no solutions to check } /** * Given the id of a branch that completes a successful backtrace, turn the * chain of branches into */ int BtBranchTracer::trySolution( size_t id, bool lookForOlap, SwResult& res, size_t& off, size_t& nrej, RandomSource& rnd) { AlnScore score; BtBranch *br = &bs_[id]; // 'br' corresponds to the leftmost edit in a right-to-left // chain of edits. EList& ned = res.alres.ned(); const BtBranch *cur = br, *prev = NULL; size_t ns = 0, nrefns = 0; size_t ngap = 0; while(true) { if(cur->e_.inited()) { if(cur->e_.isMismatch()) { if(cur->e_.qchr == 'N' || cur->e_.chr == 'N') { ns++; } } else if(cur->e_.isGap()) { ngap++; } if(cur->e_.chr == 'N') { nrefns++; } ned.push_back(cur->e_); } if(cur->root_) { break; } cur = &bs_[cur->parentId_]; } if(ns > prob_.nceil_) { // Alignment has too many Ns in it! res.reset(); assert(res.alres.ned().empty()); nrej++; return BT_REJECTED_N; } // Update 'seenPaths_' cur = br; bool rejSeen = false; // set =true if we overlap prev path bool rejCore = true; // set =true if we don't touch core diag while(true) { // Consider row, col, len, then do something int64_t row = cur->row_, col = cur->col_; assert_lt(row, (int64_t)prob_.qrylen_); size_t fromend = prob_.qrylen_ - row - 1; size_t diag = fromend + col; // Calculate the diagonal within the *trimmed* rectangle, // i.e. the rectangle we dealt with in align, gather and // backtrack. int64_t diagi = col - row; // Now adjust to the diagonal within the *untrimmed* // rectangle by adding on the amount trimmed from the left. diagi += prob_.rect_->triml; assert_lt(diag, seenPaths_.size()); // Does it overlap a core diagonal? if(diagi >= 0) { size_t diag = (size_t)diagi; if(diag >= prob_.rect_->corel && diag <= prob_.rect_->corer) { // Yes it does - it's OK rejCore = false; } } if(lookForOlap) { int64_t newlo, newhi; if(cur->len_ == 0) { if(prev != NULL && prev->len_ > 0) { // If there's a gap at the base of a non-0 length branch, the // gap will appear to overlap the branch if we give it length 1. newhi = newlo = 0; } else { // Read or ref gap with no matches coming off of it newlo = row; newhi = row + 1; } } else { // Diagonal with matches newlo = row - (cur->len_ - 1); newhi = row + 1; } assert_geq(newlo, 0); assert_geq(newhi, 0); // Does the diagonal cover cells? if(newhi > newlo) { // Check whether there is any overlap with previously traversed // cells bool added = false; const size_t sz = seenPaths_[diag].size(); for(size_t i = 0; i < sz; i++) { // Does the new interval overlap this already-seen // interval? Also of interest: does it abut this // already-seen interval? If so, we should merge them. size_t lo = seenPaths_[diag][i].first; size_t hi = seenPaths_[diag][i].second; assert_lt(lo, hi); size_t lo_sm = newlo, hi_sm = newhi; if(hi - lo < hi_sm - lo_sm) { swap(lo, lo_sm); swap(hi, hi_sm); } if((lo <= lo_sm && hi > lo_sm) || (lo < hi_sm && hi >= hi_sm)) { // One or both of the shorter interval's end points // are contained in the longer interval - so they // overlap. rejSeen = true; // Merge them into one longer interval seenPaths_[diag][i].first = min(lo, lo_sm); seenPaths_[diag][i].second = max(hi, hi_sm); #ifndef NDEBUG for(int64_t ii = seenPaths_[diag][i].first; ii < (int64_t)seenPaths_[diag][i].second; ii++) { //cerr << "trySolution rejected (" << ii << ", " << (ii + col - row) << ")" << endl; } #endif added = true; break; } else if(hi == lo_sm || lo == hi_sm) { // Merge them into one longer interval seenPaths_[diag][i].first = min(lo, lo_sm); seenPaths_[diag][i].second = max(hi, hi_sm); #ifndef NDEBUG for(int64_t ii = seenPaths_[diag][i].first; ii < (int64_t)seenPaths_[diag][i].second; ii++) { //cerr << "trySolution rejected (" << ii << ", " << (ii + col - row) << ")" << endl; } #endif added = true; // Keep going in case it overlaps one of the other // intervals } } if(!added) { seenPaths_[diag].push_back(make_pair(newlo, newhi)); } } } // After the merging that may have occurred above, it's no // longer guarnateed that all the overlapping intervals in // the list have been merged. That's OK though. We'll // still get correct answers to overlap queries. if(cur->root_) { assert_eq(0, cur->parentId_); break; } prev = cur; cur = &bs_[cur->parentId_]; } // while(cur->e_.inited()) if(rejSeen) { res.reset(); assert(res.alres.ned().empty()); nrej++; return BT_NOT_FOUND; } if(rejCore) { res.reset(); assert(res.alres.ned().empty()); nrej++; return BT_REJECTED_CORE_DIAG; } off = br->leftmostCol(); score.score_ = prob_.targ_; score.ns_ = ns; score.gaps_ = ngap; res.alres.setScore(score); res.alres.setRefNs(nrefns); size_t trimBeg = br->uppermostRow(); size_t trimEnd = prob_.qrylen_ - prob_.row_ - 1; assert_leq(trimBeg, prob_.qrylen_); assert_leq(trimEnd, prob_.qrylen_); TRefOff refoff = off + prob_.refoff_ + prob_.rect_->refl; res.alres.setShape( prob_.refid_, // ref id refoff, // 0-based ref offset prob_.treflen(), // ref length prob_.fw_, // aligned to Watson? prob_.qrylen_, // read length 0, // read id true, // pretrim soft? 0, // pretrim 5' end 0, // pretrim 3' end true, // alignment trim soft? prob_.fw_ ? trimBeg : trimEnd, // alignment trim 5' end prob_.fw_ ? trimEnd : trimBeg); // alignment trim 3' end return BT_FOUND; } /** * Get the next valid alignment given a backtrace problem. Return false * if there is no valid solution. Use a backtracking search to find the * solution. This can be very slow. */ bool BtBranchTracer::nextAlignmentBacktrace( size_t maxiter, SwResult& res, size_t& off, size_t& nrej, size_t& niter, RandomSource& rnd) { assert(!empty() || !emptySolution()); assert(prob_.inited()); // There's a subtle case where we might fail to backtracing in // local-alignment mode. The basic fact to remember is that when we're // backtracing from the highest-scoring cell in the table, we're guaranteed // to be able to backtrace without ever dipping below 0. But if we're // backtracing from a cell other than the highest-scoring cell in the // table, we might dip below 0. Dipping below 0 implies that there's a // shorted local alignment with a better score. In which case, it's // perfectly fair for us to abandon any path that dips below the floor, and // this might result in the queue becoming empty before we finish. bool result = false; niter = 0; while(!empty()) { if(trySolutions(true, res, off, nrej, rnd, result)) { return result; } if(niter++ >= maxiter) { break; } size_t brid = best(rnd); // put best branch in 'br' assert(!seen_.contains(brid)); ASSERT_ONLY(seen_.insert(brid)); #if 0 BtBranch *br = &bs_[brid]; cerr << brid << ": targ:" << prob_.targ_ << ", sc:" << br->score_st_ << ", row:" << br->uppermostRow() << ", nmm:" << nmm_ << ", nnmm:" << nnmm_ << ", nrdop:" << nrdop_ << ", nrfop:" << nrfop_ << ", nrdex:" << nrdex_ << ", nrfex:" << nrfex_ << ", nrdop_pr: " << nrdopPrune_ << ", nrfop_pr: " << nrfopPrune_ << ", nrdex_pr: " << nrdexPrune_ << ", nrfex_pr: " << nrfexPrune_ << endl; #endif addOffshoots(brid); } if(trySolutions(true, res, off, nrej, rnd, result)) { return result; } return false; } /** * Get the next valid alignment given a backtrace problem. Return false * if there is no valid solution. Use a triangle-fill backtrace to find * the solution. This is usually fast (it's O(m + n)). */ bool BtBranchTracer::nextAlignmentFill( size_t maxiter, SwResult& res, size_t& off, size_t& nrej, size_t& niter, RandomSource& rnd) { assert(prob_.inited()); assert(!emptySolution()); bool result = false; if(trySolutions(false, res, off, nrej, rnd, result)) { return result; } return false; } /** * Get the next valid alignment given the backtrace problem. Return false * if there is no valid solution, e.g., if */ bool BtBranchTracer::nextAlignment( size_t maxiter, SwResult& res, size_t& off, size_t& nrej, size_t& niter, RandomSource& rnd) { if(prob_.fill_) { return nextAlignmentFill( maxiter, res, off, nrej, niter, rnd); } else { return nextAlignmentBacktrace( maxiter, res, off, nrej, niter, rnd); } } #ifdef MAIN_ALIGNER_BT #include int main(int argc, char **argv) { size_t off = 0; RandomSource rnd(77); BtBranchTracer tr; Scoring sc = Scoring::base1(); SwResult res; tr.init( "ACGTACGT", // in: read sequence "IIIIIIII", // in: quality sequence 8, // in: read sequence length "ACGTACGT", // in: reference sequence 8, // in: reference sequence length 0, // in: reference id 0, // in: reference offset true, // in: orientation sc, // in: scoring scheme 0, // in: N ceiling 8, // in: alignment score 7, // start in this row 7, // start in this column rnd); // random gen, to choose among equal paths size_t nrej = 0; tr.nextAlignment( res, off, nrej, rnd); } #endif /*def MAIN_ALIGNER_BT*/ hisat2-2.2.1/aligner_bt.h000066400000000000000000000743221370663763200152120ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_BT_H_ #define ALIGNER_BT_H_ #include #include #include "aligner_sw_common.h" #include "aligner_result.h" #include "scoring.h" #include "edit.h" #include "limit.h" #include "dp_framer.h" #include "sse_util.h" /* Say we've filled in a DP matrix in a cost-only manner, not saving the scores * for each of the cells. At the end, we obtain a list of candidate cells and * we'd like to backtrace from them. The per-cell scores are gone, but we have * to re-create the correct path somehow. Hopefully we can do this without * recreating most or al of the score matrix, since this takes too much memory. * * Approach 1: Naively refill the matrix. * * Just refill the matrix, perhaps backwards starting from the backtrace cell. * Since this involves recreating all or most of the score matrix, this is not * a good approach. * * Approach 2: Naive backtracking. * * Conduct a search through the space of possible backtraces, rooted at the * candidate cell. To speed things along, we can prioritize paths that have a * high score and that align more characters from the read. * * The approach is simple, but it's neither fast nor memory-efficient in * general. * * Approach 3: Refilling with checkpoints. * * Refill the matrix "backwards" starting from the candidate cell, but use * checkpoints to ensure that only a series of relatively small triangles or * rectangles need to be refilled. The checkpoints must include elements from * the H, E and F matrices; not just H. After each refill, we backtrace * through the refilled area, then discard/reuse the fill memory. I call each * such fill/backtrace a mini-fill/backtrace. * * If there's only one path to be found, then this is O(m+n). But what if * there are many? And what if we would like to avoid paths that overlap in * one or more cells? There are two ways we can make this more efficient: * * 1. Remember the re-calculated E/F/H values and try to retrieve them * 2. Keep a record of cells that have already been traversed * * Legend: * * 1: Candidate cell * 2: Final cell from first mini-fill/backtrace * 3: Final cell from second mini-fill/backtrace (third not shown) * +: Checkpointed cell * *: Cell filled from first or second mini-fill/backtrace * -: Unfilled cell * * ---++--------++--------++---- * --++--------++*-------++----- * -++--(etc)-++**------++------ * ++--------+3***-----++------- * +--------++****----++-------- * --------++*****---++--------+ * -------++******--++--------++ * ------++*******-++*-------++- * -----++********++**------++-- * ----++********2+***-----++--- * ---++--------++****----++---- * --++--------++*****---++----- * -++--------++*****1--++------ * ++--------++--------++------- * * Approach 4: Backtracking with checkpoints. * * Conduct a search through the space of possible backtraces, rooted at the * candidate cell. Use "checkpoints" to prune. That is, when a backtrace * moves through a cell with a checkpointed score, consider the score * accumulated so far and the cell's saved score; abort if those two scores * add to something less than a valid score. Note we're only checkpointing H * in this case (possibly; see "subtle point"), not E or F. * * Subtle point: checkpoint scores are a result of moving forward through * the matrix whereas backtracking scores result from moving backward. This * matters becuase the two paths that meet up at a cell might have both * factored in a gap open penalty for the same gap, in which case we will * underestimate the overall score and prune a good path. Here are two ideas * for how to resolve this: * * Idea 1: when we combine the forward and backward scores to find an overall * score, and our backtrack procedure *just* made a horizontal or vertical * move, add in a "bonus" equal to the gap open penalty of the appropraite * type (read gap open for horizontal, ref gap open for vertical). This might * overcompensate, since * * Idea 2: keep the E and F values for the checkpoints around, in addition to * the H values. When it comes time to combine the score from the forward * and backward paths, we consider the last move we made in the backward * backtrace. If it's a read gap (horizontal move), then we calculate the * overall score as: * * max(Score-backward + H-forward, Score-backward + E-forward + read-open) * * If it's a reference gap (vertical move), then we calculate the overall * score as: * * max(Score-backward + H-forward, Score-backward + F-forward + ref-open) * * What does it mean to abort a backtrack? If we're starting a new branch * and there is a checkpoing in the bottommost cell of the branch, and the * overall score is less than the target, then we can simply ignore the * branch. If the checkpoint occurs in the middle of a string of matches, we * need to curtail the branch such that it doesn't include the checkpointed * cell and we won't ever try to enter the checkpointed cell, e.g., on a * mismatch. * * Approaches 3 and 4 seem reasonable, and could be combined. For simplicity, * we implement only approach 4 for now. * * Checkpoint information is propagated from the fill process to the backtracer * via a */ enum { BT_NOT_FOUND = 1, // could not obtain the backtrace because it // overlapped a previous solution BT_FOUND, // obtained a valid backtrace BT_REJECTED_N, // backtrace rejected because it had too many Ns BT_REJECTED_CORE_DIAG // backtrace rejected because it failed to overlap a // core diagonal }; /** * Parameters for a matrix of potential backtrace problems to solve. * Encapsulates information about: * * The problem given a particular reference substring: * * - The query string (nucleotides and qualities) * - The reference substring (incl. orientation, offset into overall sequence) * - Checkpoints (i.e. values of matrix cells) * - Scoring scheme and other thresholds * * The problem given a particular reference substring AND a particular row and * column from which to backtrace: * * - The row and column * - The target score */ class BtBranchProblem { public: /** * Create new uninitialized problem. */ BtBranchProblem() { reset(); } /** * Initialize a new problem. */ void initRef( const char *qry, // query string (along rows) const char *qual, // query quality string (along rows) size_t qrylen, // query string (along rows) length const char *ref, // reference string (along columns) TRefOff reflen, // in-rectangle reference string length TRefOff treflen,// total reference string length TRefId refid, // reference id TRefOff refoff, // reference offset bool fw, // orientation of problem const DPRect* rect, // dynamic programming rectangle filled out const Checkpointer* cper, // checkpointer const Scoring *sc, // scoring scheme size_t nceil) // max # Ns allowed in alignment { qry_ = qry; qual_ = qual; qrylen_ = qrylen; ref_ = ref; reflen_ = reflen; treflen_ = treflen; refid_ = refid; refoff_ = refoff; fw_ = fw; rect_ = rect; cper_ = cper; sc_ = sc; nceil_ = nceil; } /** * Initialize a new problem. */ void initBt( size_t row, // row size_t col, // column bool fill, // use a filling rather than a backtracking strategy bool usecp, // use checkpoints to short-circuit while backtracking TAlScore targ) // target score { row_ = row; col_ = col; targ_ = targ; fill_ = fill; usecp_ = usecp; if(fill) { assert(usecp_); } } /** * Reset to uninitialized state. */ void reset() { qry_ = qual_ = ref_ = NULL; cper_ = NULL; rect_ = NULL; sc_ = NULL; qrylen_ = reflen_ = treflen_ = refid_ = refoff_ = row_ = col_ = targ_ = nceil_ = 0; fill_ = fw_ = usecp_ = false; } /** * Return true iff the BtBranchProblem has been initialized. */ bool inited() const { return qry_ != NULL; } #ifndef NDEBUG /** * Sanity-check the problem. */ bool repOk() const { assert_gt(qrylen_, 0); assert_gt(reflen_, 0); assert_gt(treflen_, 0); assert_lt(row_, qrylen_); assert_lt((TRefOff)col_, reflen_); return true; } #endif size_t reflen() const { return reflen_; } size_t treflen() const { return treflen_; } protected: const char *qry_; // query string (along rows) const char *qual_; // query quality string (along rows) size_t qrylen_; // query string (along rows) length const char *ref_; // reference string (along columns) TRefOff reflen_; // in-rectangle reference string length TRefOff treflen_;// total reference string length TRefId refid_; // reference id TRefOff refoff_; // reference offset bool fw_; // orientation of problem const DPRect* rect_; // dynamic programming rectangle filled out size_t row_; // starting row size_t col_; // starting column TAlScore targ_; // target score const Checkpointer *cper_; // checkpointer bool fill_; // use mini-fills bool usecp_; // use checkpointing? const Scoring *sc_; // scoring scheme size_t nceil_; // max # Ns allowed in alignment friend class BtBranch; friend class BtBranchQ; friend class BtBranchTracer; }; /** * Encapsulates a "branch" which is a diagonal of cells (possibly of length 0) * in the matrix where all the cells are matches. These stretches are linked * together by edits to form a full backtrace path through the matrix. Lengths * are measured w/r/t to the number of rows traversed by the path, so a branch * that represents a read gap extension could have length = 0. * * At the end of the day, the full backtrace path is represented as a list of * BtBranch's where each BtBranch represents a stretch of matching cells (and * up to one mismatching cell at its bottom extreme) ending in an edit (or in * the bottommost row, in which case the edit is uninitialized). Each * BtBranch's row and col fields indicate the bottommost cell involved in the * diagonal stretch of matches, and the len_ field indicates the length of the * stretch of matches. Note that the edits themselves also correspond to * movement through the matrix. * * A related issue is how we record which cells have been visited so that we * never report a pair of paths both traversing the same (row, col) of the * overall DP matrix. This gets a little tricky because we have to take into * account the cells covered by *edits* in addition to the cells covered by the * stretches of matches. For instance: imagine a mismatch. That takes up a * cell of the DP matrix, but it may or may not be preceded by a string of * matches. It's hard to imagine how to represent this unless we let the * mismatch "count toward" the len_ of the branch and let (row, col) refer to * the cell where the mismatch occurs. * * We need BtBranches to "live forever" so that we can make some BtBranches * parents of others using parent pointers. For this reason, BtBranch's are * stored in an EFactory object in the BtBranchTracer class. */ class BtBranch { public: BtBranch() { reset(); } BtBranch( const BtBranchProblem& prob, size_t parentId, TAlScore penalty, TAlScore score_en, int64_t row, int64_t col, Edit e, int hef, bool root, bool extend) { init(prob, parentId, penalty, score_en, row, col, e, hef, root, extend); } /** * Reset to uninitialized state. */ void reset() { parentId_ = 0; score_st_ = score_en_ = len_ = row_ = col_ = 0; curtailed_ = false; e_.reset(); } /** * Caller gives us score_en, row and col. We figure out score_st and len_ * by comparing characters from the strings. */ void init( const BtBranchProblem& prob, size_t parentId, TAlScore penalty, TAlScore score_en, int64_t row, int64_t col, Edit e, int hef, bool root, bool extend); /** * Return true iff this branch ends in a solution to the backtrace problem. */ bool isSolution(const BtBranchProblem& prob) const { const bool end2end = prob.sc_->monotone; return score_st_ == prob.targ_ && (!end2end || endsInFirstRow()); } /** * Return true iff this branch could potentially lead to a valid alignment. */ bool isValid(const BtBranchProblem& prob) const { int64_t scoreFloor = prob.sc_->monotone ? MIN_I64 : 0; if(score_st_ < scoreFloor) { // Dipped below the score floor return false; } if(isSolution(prob)) { // It's a solution, so it's also valid return true; } if((int64_t)len_ > row_) { // Went all the way to the top row //assert_leq(score_st_, prob.targ_); return score_st_ == prob.targ_; } else { int64_t match = prob.sc_->match(); int64_t bonusLeft = (row_ + 1 - len_) * match; return score_st_ + bonusLeft >= prob.targ_; } } /** * Return true iff this branch overlaps with the given branch. */ bool overlap(const BtBranchProblem& prob, const BtBranch& bt) const { // Calculate this branch's diagonal assert_lt(row_, (int64_t)prob.qrylen_); size_t fromend = prob.qrylen_ - row_ - 1; size_t diag = fromend + col_; int64_t lo = 0, hi = row_ + 1; if(len_ == 0) { lo = row_; } else { lo = row_ - (len_ - 1); } // Calculate other branch's diagonal assert_lt(bt.row_, (int64_t)prob.qrylen_); size_t ofromend = prob.qrylen_ - bt.row_ - 1; size_t odiag = ofromend + bt.col_; if(diag != odiag) { return false; } int64_t olo = 0, ohi = bt.row_ + 1; if(bt.len_ == 0) { olo = bt.row_; } else { olo = bt.row_ - (bt.len_ - 1); } int64_t losm = olo, hism = ohi; if(hi - lo < ohi - olo) { swap(lo, losm); swap(hi, hism); } if((lo <= losm && hi > losm) || (lo < hism && hi >= hism)) { return true; } return false; } /** * Return true iff this branch is higher priority than the branch 'o'. */ bool operator<(const BtBranch& o) const { // Prioritize uppermost above score if(uppermostRow() != o.uppermostRow()) { return uppermostRow() < o.uppermostRow(); } if(score_st_ != o.score_st_) return score_st_ > o.score_st_; if(row_ != o.row_) return row_ < o.row_; if(col_ != o.col_) return col_ > o.col_; if(parentId_ != o.parentId_) return parentId_ > o.parentId_; assert(false); return false; } /** * Return true iff the topmost cell involved in this branch is in the top * row. */ bool endsInFirstRow() const { assert_leq((int64_t)len_, row_ + 1); return (int64_t)len_ == row_+1; } /** * Return the uppermost row covered by this branch. */ size_t uppermostRow() const { assert_geq(row_ + 1, (int64_t)len_); return row_ + 1 - (int64_t)len_; } /** * Return the leftmost column covered by this branch. */ size_t leftmostCol() const { assert_geq(col_ + 1, (int64_t)len_); return col_ + 1 - (int64_t)len_; } #ifndef NDEBUG /** * Sanity-check this BtBranch. */ bool repOk() const { assert(root_ || e_.inited()); assert_gt(len_, 0); assert_geq(col_ + 1, (int64_t)len_); assert_geq(row_ + 1, (int64_t)len_); return true; } #endif protected: // ID of the parent branch. size_t parentId_; // Penalty associated with the edit at the bottom of this branch (0 if // there is no edit) TAlScore penalty_; // Score at the beginning of the branch TAlScore score_st_; // Score at the end of the branch (taking the edit into account) TAlScore score_en_; // Length of the branch. That is, the total number of diagonal cells // involved in all the matches and in the edit (if any). Should always be // > 0. size_t len_; // The row of the final (bottommost) cell in the branch. This might be the // bottommost match if the branch has no associated edit. Otherwise, it's // the cell occupied by the edit. int64_t row_; // The column of the final (bottommost) cell in the branch. int64_t col_; // The edit at the bottom of the branch. If this is the bottommost branch // in the alignment and it does not end in an edit, then this remains // uninitialized. Edit e_; // True iff this is the bottommost branch in the alignment. We can't just // use row_ to tell us this because local alignments don't necessarily end // in the last row. bool root_; bool curtailed_; // true -> pruned at a checkpoint where we otherwise // would have had a match friend class BtBranchQ; friend class BtBranchTracer; }; /** * Instantiate and solve best-first branch-based backtraces. */ class BtBranchTracer { public: explicit BtBranchTracer() : prob_(), bs_(), seenPaths_(DP_CAT), sawcell_(DP_CAT), doTri_() { } /** * Add a branch to the queue. */ void add(size_t id) { assert(!bs_[id].isSolution(prob_)); unsorted_.push_back(make_pair(bs_[id].score_st_, id)); } /** * Add a branch to the list of solutions. */ void addSolution(size_t id) { assert(bs_[id].isSolution(prob_)); solutions_.push_back(id); } /** * Given a potential branch to add to the queue, see if we can follow the * branch a little further first. If it's still valid, or if we reach a * choice between valid outgoing paths, go ahead and add it to the queue. */ void examineBranch( int64_t row, int64_t col, const Edit& e, TAlScore pen, TAlScore sc, size_t parentId); /** * Take all possible ways of leaving the given branch and add them to the * branch queue. */ void addOffshoots(size_t bid); /** * Get the best branch and remove it from the priority queue. */ size_t best(RandomSource& rnd) { assert(!empty()); flushUnsorted(); assert_gt(sortedSel_ ? sorted1_.size() : sorted2_.size(), cur_); // Perhaps shuffle everyone who's tied for first? size_t id = sortedSel_ ? sorted1_[cur_] : sorted2_[cur_]; cur_++; return id; } /** * Return true iff there are no branches left to try. */ bool empty() const { return size() == 0; } /** * Return the size, i.e. the total number of branches contained. */ size_t size() const { return unsorted_.size() + (sortedSel_ ? sorted1_.size() : sorted2_.size()) - cur_; } /** * Return true iff there are no solutions left to try. */ bool emptySolution() const { return sizeSolution() == 0; } /** * Return the size of the solution set so far. */ size_t sizeSolution() const { return solutions_.size(); } /** * Sort unsorted branches, merge them with master sorted list. */ void flushUnsorted(); #ifndef NDEBUG /** * Sanity-check the queue. */ bool repOk() const { assert_lt(cur_, (sortedSel_ ? sorted1_.size() : sorted2_.size())); return true; } #endif /** * Initialize the tracer with respect to a new read. This involves * resetting all the state relating to the set of cells already visited */ void initRef( const char* rd, // in: read sequence const char* qu, // in: quality sequence size_t rdlen, // in: read sequence length const char* rf, // in: reference sequence size_t rflen, // in: in-rectangle reference sequence length TRefOff trflen, // in: total reference sequence length TRefId refid, // in: reference id TRefOff refoff, // in: reference offset bool fw, // in: orientation const DPRect *rect, // in: DP rectangle const Checkpointer *cper, // in: checkpointer const Scoring& sc, // in: scoring scheme size_t nceil) // in: N ceiling { prob_.initRef(rd, qu, rdlen, rf, rflen, trflen, refid, refoff, fw, rect, cper, &sc, nceil); const size_t ndiag = rflen + rdlen - 1; seenPaths_.resize(ndiag); for(size_t i = 0; i < ndiag; i++) { seenPaths_[i].clear(); } // clear each of the per-column sets if(sawcell_.size() < rflen) { size_t isz = sawcell_.size(); sawcell_.resize(rflen); for(size_t i = isz; i < rflen; i++) { sawcell_[i].setCat(DP_CAT); } } for(size_t i = 0; i < rflen; i++) { sawcell_[i].setCat(DP_CAT); sawcell_[i].clear(); // clear the set } } /** * Initialize with a new backtrace. */ void initBt( TAlScore escore, // in: alignment score size_t row, // in: start in this row size_t col, // in: start in this column bool fill, // in: use mini-filling? bool usecp, // in: use checkpointing? bool doTri, // in: triangle-shaped mini-fills? RandomSource& rnd) // in: random gen, to choose among equal paths { prob_.initBt(row, col, fill, usecp, escore); Edit e; e.reset(); unsorted_.clear(); solutions_.clear(); sorted1_.clear(); sorted2_.clear(); cur_ = 0; nmm_ = 0; // number of mismatches attempted nnmm_ = 0; // number of mismatches involving N attempted nrdop_ = 0; // number of read gap opens attempted nrfop_ = 0; // number of ref gap opens attempted nrdex_ = 0; // number of read gap extensions attempted nrfex_ = 0; // number of ref gap extensions attempted nmmPrune_ = 0; // number of mismatches attempted nnmmPrune_ = 0; // number of mismatches involving N attempted nrdopPrune_ = 0; // number of read gap opens attempted nrfopPrune_ = 0; // number of ref gap opens attempted nrdexPrune_ = 0; // number of read gap extensions attempted nrfexPrune_ = 0; // number of ref gap extensions attempted row_ = row; col_ = col; doTri_ = doTri; bs_.clear(); if(!prob_.fill_) { size_t id = bs_.alloc(); bs_[id].init( prob_, 0, // parent id 0, // penalty 0, // starting score row, // row col, // column e, 0, true, // this is the root true); // this should be extend with exact matches if(bs_[id].isSolution(prob_)) { addSolution(id); } else { add(id); } } else { int64_t row = row_, col = col_; TAlScore targsc = prob_.targ_; int hef = 0; bool done = false, abort = false; size_t depth = 0; while(!done && !abort) { // Accumulate edits as we go. We can do this by adding // BtBranches to the bs_ structure. Each step of the backtrace // either involves an edit (thereby starting a new branch) or // extends the previous branch by one more position. // // Note: if the BtBranches are in line, then trySolution can be // used to populate the SwResult and check for various // situations where we might reject the alignment (i.e. due to // a cell having been visited previously). if(doTri_) { triangleFill( row, // row of cell to backtrace from col, // column of cell to backtrace from hef, // cell to bt from: H (0), E (1), or F (2) targsc, // score of cell to backtrace from prob_.targ_, // score of alignment we're looking for rnd, // pseudo-random generator row, // out: row we ended up in after bt col, // out: column we ended up in after bt hef, // out: H/E/F after backtrace targsc, // out: score up to cell we ended up in done, // out: finished tracing out an alignment? abort); // out: aborted b/c cell was seen before? } else { squareFill( row, // row of cell to backtrace from col, // column of cell to backtrace from hef, // cell to bt from: H (0), E (1), or F (2) targsc, // score of cell to backtrace from prob_.targ_, // score of alignment we're looking for rnd, // pseudo-random generator row, // out: row we ended up in after bt col, // out: column we ended up in after bt hef, // out: H/E/F after backtrace targsc, // out: score up to cell we ended up in done, // out: finished tracing out an alignment? abort); // out: aborted b/c cell was seen before? } if(depth >= ndep_.size()) { ndep_.resize(depth+1); ndep_[depth] = 1; } else { ndep_[depth]++; } depth++; assert((row >= 0 && col >= 0) || done); } } ASSERT_ONLY(seen_.clear()); } /** * Get the next valid alignment given the backtrace problem. Return false * if there is no valid solution, e.g., if */ bool nextAlignment( size_t maxiter, SwResult& res, size_t& off, size_t& nrej, size_t& niter, RandomSource& rnd); /** * Return true iff this tracer has been initialized */ bool inited() const { return prob_.inited(); } /** * Return true iff the mini-fills are triangle-shaped. */ bool doTri() const { return doTri_; } /** * Fill in a triangle of the DP table and backtrace from the given cell to * a cell in the previous checkpoint, or to the terminal cell. */ void triangleFill( int64_t rw, // row of cell to backtrace from int64_t cl, // column of cell to backtrace from int hef, // cell to backtrace from is H (0), E (1), or F (2) TAlScore targ, // score of cell to backtrace from TAlScore targ_final, // score of alignment we're looking for RandomSource& rnd, // pseudo-random generator int64_t& row_new, // out: row we ended up in after backtrace int64_t& col_new, // out: column we ended up in after backtrace int& hef_new, // out: H/E/F after backtrace TAlScore& targ_new, // out: score up to cell we ended up in bool& done, // out: finished tracing out an alignment? bool& abort); // out: aborted b/c cell was seen before? /** * Fill in a square of the DP table and backtrace from the given cell to * a cell in the previous checkpoint, or to the terminal cell. */ void squareFill( int64_t rw, // row of cell to backtrace from int64_t cl, // column of cell to backtrace from int hef, // cell to backtrace from is H (0), E (1), or F (2) TAlScore targ, // score of cell to backtrace from TAlScore targ_final, // score of alignment we're looking for RandomSource& rnd, // pseudo-random generator int64_t& row_new, // out: row we ended up in after backtrace int64_t& col_new, // out: column we ended up in after backtrace int& hef_new, // out: H/E/F after backtrace TAlScore& targ_new, // out: score up to cell we ended up in bool& done, // out: finished tracing out an alignment? bool& abort); // out: aborted b/c cell was seen before? protected: /** * Get the next valid alignment given a backtrace problem. Return false * if there is no valid solution. Use a backtracking search to find the * solution. This can be very slow. */ bool nextAlignmentBacktrace( size_t maxiter, SwResult& res, size_t& off, size_t& nrej, size_t& niter, RandomSource& rnd); /** * Get the next valid alignment given a backtrace problem. Return false * if there is no valid solution. Use a triangle-fill backtrace to find * the solution. This is usually fast (it's O(m + n)). */ bool nextAlignmentFill( size_t maxiter, SwResult& res, size_t& off, size_t& nrej, size_t& niter, RandomSource& rnd); /** * Try all the solutions accumulated so far. Solutions might be rejected * if they, for instance, overlap a previous solution, have too many Ns, * fail to overlap a core diagonal, etc. */ bool trySolutions( bool lookForOlap, SwResult& res, size_t& off, size_t& nrej, RandomSource& rnd, bool& success); /** * See if a given solution branch works as a solution (i.e. doesn't overlap * another one, have too many Ns, fail to overlap a core diagonal, etc.) */ int trySolution( size_t id, bool lookForOlap, SwResult& res, size_t& off, size_t& nrej, RandomSource& rnd); BtBranchProblem prob_; // problem configuration EFactory bs_; // global BtBranch factory // already reported alignments going through these diagonal segments ELList > seenPaths_; ELSet sawcell_; // cells already backtraced through EList > unsorted_; // unsorted list of as-yet-unflished BtBranches EList sorted1_; // list of BtBranch, sorted by score EList sorted2_; // list of BtBranch, sorted by score EList solutions_; // list of solution branches bool sortedSel_; // true -> 1, false -> 2 size_t cur_; // cursor into sorted list to start from size_t nmm_; // number of mismatches attempted size_t nnmm_; // number of mismatches involving N attempted size_t nrdop_; // number of read gap opens attempted size_t nrfop_; // number of ref gap opens attempted size_t nrdex_; // number of read gap extensions attempted size_t nrfex_; // number of ref gap extensions attempted size_t nmmPrune_; // size_t nnmmPrune_; // size_t nrdopPrune_; // size_t nrfopPrune_; // size_t nrdexPrune_; // size_t nrfexPrune_; // size_t row_; // row size_t col_; // column bool doTri_; // true -> fill in triangles; false -> squares EList sq_; // square to fill when doing mini-fills ELList tri_; // triangle to fill when doing mini-fills EList ndep_; // # triangles mini-filled at various depths #ifndef NDEBUG ESet seen_; // seedn branch ids; should never see same twice #endif }; #endif /*ndef ALIGNER_BT_H_*/ hisat2-2.2.1/aligner_cache.cpp000066400000000000000000000102131370663763200161700ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "aligner_cache.h" #include "tinythread.h" #ifdef ALIGNER_CACHE_MAIN #include #include #include #include "random_source.h" using namespace std; enum { ARG_TESTS = 256 }; static const char *short_opts = "vCt"; static struct option long_opts[] = { {(char*)"verbose", no_argument, 0, 'v'}, {(char*)"tests", no_argument, 0, ARG_TESTS}, }; static void printUsage(ostream& os) { os << "Usage: sawhi-cache [options]*" << endl; os << "Options:" << endl; os << " --tests run unit tests" << endl; os << " -v/--verbose talkative mode" << endl; } int gVerbose = 0; static void add( RedBlack& t, Pool& p, const char *dna) { QKey qk; qk.init(BTDnaString(dna, true)); t.add(p, qk, NULL); } /** * Small tests for the AlignmentCache. */ static void aligner_cache_tests() { RedBlack rb(1024); Pool p(64 * 1024, 1024); // Small test add(rb, p, "ACGTCGATCGT"); add(rb, p, "ACATCGATCGT"); add(rb, p, "ACGACGATCGT"); add(rb, p, "ACGTAGATCGT"); add(rb, p, "ACGTCAATCGT"); add(rb, p, "ACGTCGCTCGT"); add(rb, p, "ACGTCGAACGT"); assert_eq(7, rb.size()); rb.clear(); p.clear(); // Another small test add(rb, p, "ACGTCGATCGT"); add(rb, p, "CCGTCGATCGT"); add(rb, p, "TCGTCGATCGT"); add(rb, p, "GCGTCGATCGT"); add(rb, p, "AAGTCGATCGT"); assert_eq(5, rb.size()); rb.clear(); p.clear(); // Regression test (attempt to make it smaller) add(rb, p, "CCTA"); add(rb, p, "AGAA"); add(rb, p, "TCTA"); add(rb, p, "GATC"); add(rb, p, "CTGC"); add(rb, p, "TTGC"); add(rb, p, "GCCG"); add(rb, p, "GGAT"); rb.clear(); p.clear(); // Regression test add(rb, p, "CCTA"); add(rb, p, "AGAA"); add(rb, p, "TCTA"); add(rb, p, "GATC"); add(rb, p, "CTGC"); add(rb, p, "CATC"); add(rb, p, "CAAA"); add(rb, p, "CTAT"); add(rb, p, "CTCA"); add(rb, p, "TTGC"); add(rb, p, "GCCG"); add(rb, p, "GGAT"); assert_eq(12, rb.size()); rb.clear(); p.clear(); // Larger random test EList strs; char buf[5]; for(int i = 0; i < 4; i++) { for(int j = 0; j < 4; j++) { for(int k = 0; k < 4; k++) { for(int m = 0; m < 4; m++) { buf[0] = "ACGT"[i]; buf[1] = "ACGT"[j]; buf[2] = "ACGT"[k]; buf[3] = "ACGT"[m]; buf[4] = '\0'; strs.push_back(BTDnaString(buf, true)); } } } } // Add all of the 4-mers in several different random orders RandomSource rand; for(uint32_t runs = 0; runs < 100; runs++) { rb.clear(); p.clear(); assert_eq(0, rb.size()); rand.init(runs); EList used; used.resize(256); for(int i = 0; i < 256; i++) used[i] = false; for(int i = 0; i < 256; i++) { int r = rand.nextU32() % (256-i); int unused = 0; bool added = false; for(int j = 0; j < 256; j++) { if(!used[j] && unused == r) { used[j] = true; QKey qk; qk.init(strs[j]); rb.add(p, qk, NULL); added = true; break; } if(!used[j]) unused++; } assert(added); } } } /** * A way of feeding simply tests to the seed alignment infrastructure. */ int main(int argc, char **argv) { int option_index = 0; int next_option; do { next_option = getopt_long(argc, argv, short_opts, long_opts, &option_index); switch (next_option) { case 'v': gVerbose = true; break; case ARG_TESTS: aligner_cache_tests(); return 0; case -1: break; default: { cerr << "Unknown option: " << (char)next_option << endl; printUsage(cerr); exit(1); } } } while(next_option != -1); } #endif hisat2-2.2.1/aligner_cache.h000066400000000000000000000644701370663763200156530ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_CACHE_H_ #define ALIGNER_CACHE_H_ /** * CACHEING * * By caching the results of some alignment sub-problems, we hope to * enable a "fast path" for read alignment whereby answers are mostly * looked up rather than calculated from scratch. This is particularly * effective when the input is sorted or otherwise grouped in a way * that brings together reads with (at least some) seed sequences in * common. * * But the cache is also where results are held, regardless of whether * the results are maintained & re-used across reads. * * The cache consists of two linked potions: * * 1. A multimap from seed strings (i.e. read substrings) to reference strings * that are within some edit distance (roughly speaking). This is the "seed * multimap". * * Key: Read substring (2-bit-per-base encoded + length) * Value: Set of reference substrings (i.e. keys into the suffix * array multimap). * * 2. A multimap from reference strings to the corresponding elements of the * suffix array. Elements are filled in with reference-offset info as it's * calculated. This is the "suffix array multimap" * * Key: Reference substring (2-bit-per-base encoded + length) * Value: (a) top from BWT, (b) length of range, (c) offset of first * range element in * * For both multimaps, we use a combo Red-Black tree and EList. The payload in * the Red-Black tree nodes points to a range in the EList. */ #include #include "ds.h" #include "read.h" #include "threading.h" #include "mem_ids.h" #include "simple_func.h" #include "btypes.h" #define CACHE_PAGE_SZ (16 * 1024) typedef PListSlice TSlice; /** * Key for the query multimap: the read substring and its length. */ struct QKey { /** * Initialize invalid QKey. */ QKey() { reset(); } /** * Initialize QKey from DNA string. */ QKey(const BTDnaString& s ASSERT_ONLY(, BTDnaString& tmp)) { init(s ASSERT_ONLY(, tmp)); } /** * Initialize QKey from DNA string. Rightmost character is placed in the * least significant bitpair. */ bool init( const BTDnaString& s ASSERT_ONLY(, BTDnaString& tmp)) { seq = 0; len = (uint32_t)s.length(); ASSERT_ONLY(tmp.clear()); if(len > 32) { len = 0xffffffff; return false; // wasn't cacheable } else { // Rightmost char of 's' goes in the least significant bitpair for(size_t i = 0; i < 32 && i < s.length(); i++) { int c = (int)s.get(i); assert_range(0, 4, c); if(c == 4) { len = 0xffffffff; return false; } seq = (seq << 2) | s.get(i); } ASSERT_ONLY(toString(tmp)); assert(sstr_eq(tmp, s)); assert_leq(len, 32); return true; // was cacheable } } /** * Convert this key to a DNA string. */ void toString(BTDnaString& s) { s.resize(len); uint64_t sq = seq; for(int i = (len)-1; i >= 0; i--) { s.set((uint32_t)(sq & 3), i); sq >>= 2; } } /** * Return true iff the read substring is cacheable. */ bool cacheable() const { return len != 0xffffffff; } /** * Reset to uninitialized state. */ void reset() { seq = 0; len = 0xffffffff; } /** * True -> my key is less than the given key. */ bool operator<(const QKey& o) const { return seq < o.seq || (seq == o.seq && len < o.len); } /** * True -> my key is greater than the given key. */ bool operator>(const QKey& o) const { return !(*this < o || *this == o); } /** * True -> my key is equal to the given key. */ bool operator==(const QKey& o) const { return seq == o.seq && len == o.len; } /** * True -> my key is not equal to the given key. */ bool operator!=(const QKey& o) const { return !(*this == o); } #ifndef NDEBUG /** * Check that this is a valid, initialized QKey. */ bool repOk() const { return len != 0xffffffff; } #endif uint64_t seq; // sequence uint32_t len; // length of sequence }; template class AlignmentCache; /** * Payload for the query multimap: a range of elements in the reference * string list. */ template class QVal { public: QVal() { reset(); } /** * Return the offset of the first reference substring in the qlist. */ index_t offset() const { return i_; } /** * Return the number of reference substrings associated with a read * substring. */ index_t numRanges() const { assert(valid()); return rangen_; } /** * Return the number of elements associated with all associated * reference substrings. */ index_t numElts() const { assert(valid()); return eltn_; } /** * Return true iff the read substring is not associated with any * reference substrings. */ bool empty() const { assert(valid()); return numRanges() == 0; } /** * Return true iff the QVal is valid. */ bool valid() const { return rangen_ != (index_t)OFF_MASK; } /** * Reset to invalid state. */ void reset() { i_ = 0; rangen_ = eltn_ = (index_t)OFF_MASK; } /** * Initialize Qval. */ void init(index_t i, index_t ranges, index_t elts) { i_ = i; rangen_ = ranges; eltn_ = elts; } /** * Tally another range with given number of elements. */ void addRange(index_t numElts) { rangen_++; eltn_ += numElts; } #ifndef NDEBUG /** * Check that this QVal is internally consistent and consistent * with the contents of the given cache. */ bool repOk(const AlignmentCache& ac) const; #endif protected: index_t i_; // idx of first elt in qlist index_t rangen_; // # ranges (= # associated reference substrings) index_t eltn_; // # elements (total) }; /** * Key for the suffix array multimap: the reference substring and its * length. Same as QKey so I typedef it. */ typedef QKey SAKey; /** * Payload for the suffix array multimap: (a) the top element of the * range in BWT, (b) the offset of the first elt in the salist, (c) * length of the range. */ template struct SAVal { SAVal() : topf(), topb(), i(), len(OFF_MASK) { } /** * Return true iff the SAVal is valid. */ bool valid() { return len != (index_t)OFF_MASK; } #ifndef NDEBUG /** * Check that this SAVal is internally consistent and consistent * with the contents of the given cache. */ bool repOk(const AlignmentCache& ac) const; #endif /** * Initialize the SAVal. */ void init( index_t tf, index_t tb, index_t ii, index_t ln) { topf = tf; topb = tb; i = ii; len = ln; } index_t topf; // top in BWT index_t topb; // top in BWT' index_t i; // idx of first elt in salist index_t len; // length of range }; /** * One data structure that encapsulates all of the cached information * associated with a particular reference substring. This is useful * for summarizing what info should be added to the cache for a partial * alignment. */ template class SATuple { public: SATuple() { reset(); }; SATuple(SAKey k, index_t tf, index_t tb, TSlice o) { init(k, tf, tb, o); } void init(SAKey k, index_t tf, index_t tb, TSlice o) { key = k; topf = tf; topb = tb; offs = o; } /** * Initialize this SATuple from a subrange of the SATuple 'src'. */ void init(const SATuple& src, index_t first, index_t last) { assert_neq((index_t)OFF_MASK, src.topb); key = src.key; topf = (index_t)(src.topf + first); topb = (index_t)OFF_MASK; // unknown! offs.init(src.offs, first, last); } #ifndef NDEBUG /** * Check that this SATuple is internally consistent and that its * PListSlice is consistent with its backing PList. */ bool repOk() const { assert(offs.repOk()); return true; } #endif /** * Function for ordering SATuples. This is used when prioritizing which to * explore first when extending seed hits into full alignments. Smaller * ranges get higher priority and we use 'top' to break ties, though any * way of breaking a tie would be fine. */ bool operator<(const SATuple& o) const { if(offs.size() < o.offs.size()) { return true; } if(offs.size() > o.offs.size()) { return false; } return topf < o.topf; } bool operator>(const SATuple& o) const { if(offs.size() < o.offs.size()) { return false; } if(offs.size() > o.offs.size()) { return true; } return topf > o.topf; } bool operator==(const SATuple& o) const { return key == o.key && topf == o.topf && topb == o.topb && offs == o.offs; } void reset() { topf = topb = (index_t)OFF_MASK; offs.reset(); } /** * Set the length to be at most the original length. */ void setLength(index_t nlen) { assert_leq(nlen, offs.size()); offs.setLength(nlen); } /** * Return the number of times this reference substring occurs in the * reference, which is also the size of the 'offs' TSlice. */ index_t size() const { return (index_t)offs.size(); } // bot/length of SA range equals offs.size() SAKey key; // sequence key index_t topf; // top in BWT index index_t topb; // top in BWT' index TSlice offs; // offsets }; /** * Encapsulate the data structures and routines that constitute a * particular cache, i.e., a particular stratum of the cache system, * which might comprise many strata. * * Each thread has a "current-read" AlignmentCache which is used to * build and store subproblem results as alignment is performed. When * we're finished with a read, we might copy the cached results for * that read (and perhaps a bundle of other recently-aligned reads) to * a higher-level "across-read" cache. Higher-level caches may or may * not be shared among threads. * * A cache consists chiefly of two multimaps, each implemented as a * Red-Black tree map backed by an EList. A 'version' counter is * incremented every time the cache is cleared. */ template class AlignmentCache { typedef RedBlackNode > QNode; typedef RedBlackNode > SANode; typedef PList TQList; typedef PList TSAList; public: AlignmentCache( uint64_t bytes, bool shared) : pool_(bytes, CACHE_PAGE_SZ, CA_CAT), qmap_(CACHE_PAGE_SZ, CA_CAT), qlist_(CA_CAT), samap_(CACHE_PAGE_SZ, CA_CAT), salist_(CA_CAT), shared_(shared), mutex_m(), version_(0) { } /** * Given a QVal, populate the given EList of SATuples with records * describing all of the cached information about the QVal's * reference substrings. */ template void queryQval( const QVal& qv, EList, S>& satups, index_t& nrange, index_t& nelt, bool getLock = true) { ThreadSafe ts(lockPtr(), shared_ && getLock); assert(qv.repOk(*this)); const index_t refi = qv.offset(); const index_t reff = refi + qv.numRanges(); // For each reference sequence sufficiently similar to the // query sequence in the QKey... for(index_t i = refi; i < reff; i++) { // Get corresponding SAKey, containing similar reference // sequence & length SAKey sak = qlist_.get(i); // Shouldn't have identical keys in qlist_ assert(i == refi || qlist_.get(i) != qlist_.get(i-1)); // Get corresponding SANode SANode *n = samap_.lookup(sak); assert(n != NULL); const SAVal& sav = n->payload; assert(sav.repOk(*this)); if(sav.len > 0) { nrange++; satups.expand(); satups.back().init(sak, sav.topf, sav.topb, TSlice(salist_, sav.i, sav.len)); nelt += sav.len; #ifndef NDEBUG // Shouldn't add consecutive identical entries too satups if(i > refi) { const SATuple b1 = satups.back(); const SATuple b2 = satups[satups.size()-2]; assert(b1.key != b2.key || b1.topf != b2.topf || b1.offs != b2.offs); } #endif } } } /** * Return true iff the cache has no entries in it. */ bool empty() const { bool ret = qmap_.empty(); assert(!ret || qlist_.empty()); assert(!ret || samap_.empty()); assert(!ret || salist_.empty()); return ret; } /** * Add a new query key ('qk'), usually a 2-bit encoded substring of * the read) as the key in a new Red-Black node in the qmap and * return a pointer to the node's QVal. * * The expectation is that the caller is about to set about finding * associated reference substrings, and that there will be future * calls to addOnTheFly to add associations to reference substrings * found. */ QVal* add( const QKey& qk, bool *added, bool getLock = true) { ThreadSafe ts(lockPtr(), shared_ && getLock); assert(qk.cacheable()); QNode *n = qmap_.add(pool(), qk, added); return (n != NULL ? &n->payload : NULL); } /** * Add a new association between a read sequnce ('seq') and a * reference sequence ('') */ bool addOnTheFly( QVal& qv, // qval that points to the range of reference substrings const SAKey& sak, // the key holding the reference substring index_t topf, // top range elt in BWT index index_t botf, // bottom range elt in BWT index index_t topb, // top range elt in BWT' index index_t botb, // bottom range elt in BWT' index bool getLock = true); /** * Clear the cache, i.e. turn it over. All HitGens referring to * ranges in this cache will become invalid and the corresponding * reads will have to be re-aligned. */ void clear(bool getLock = true) { ThreadSafe ts(lockPtr(), shared_ && getLock); pool_.clear(); qmap_.clear(); qlist_.clear(); samap_.clear(); salist_.clear(); version_++; } /** * Return the number of keys in the query multimap. */ index_t qNumKeys() const { return (index_t)qmap_.size(); } /** * Return the number of keys in the suffix array multimap. */ index_t saNumKeys() const { return (index_t)samap_.size(); } /** * Return the number of elements in the reference substring list. */ index_t qSize() const { return (index_t)qlist_.size(); } /** * Return the number of elements in the SA range list. */ index_t saSize() const { return (index_t)salist_.size(); } /** * Return the pool. */ Pool& pool() { return pool_; } /** * Return the lock object. */ MUTEX_T& lock() { return mutex_m; } /** * Return a const pointer to the lock object. This allows us to * write const member functions that grab the lock. */ MUTEX_T* lockPtr() const { return const_cast(&mutex_m); } /** * Return true iff this cache is shared among threads. */ bool shared() const { return shared_; } /** * Return the current "version" of the cache, i.e. the total number * of times it has turned over since its creation. */ uint32_t version() const { return version_; } protected: Pool pool_; // dispenses memory pages RedBlack > qmap_; // map from query substrings to reference substrings TQList qlist_; // list of reference substrings RedBlack > samap_; // map from reference substrings to SA ranges TSAList salist_; // list of SA ranges bool shared_; // true -> this cache is global MUTEX_T mutex_m; // mutex used for syncronization in case the the cache is shared. uint32_t version_; // cache version }; /** * Interface used to query and update a pair of caches: one thread- * local and unsynchronized, another shared and synchronized. One or * both can be NULL. */ template class AlignmentCacheIface { public: AlignmentCacheIface( AlignmentCache *current, AlignmentCache *local, AlignmentCache *shared) : qk_(), qv_(NULL), cacheable_(false), rangen_(0), eltsn_(0), current_(current), local_(local), shared_(shared) { assert(current_ != NULL); } #if 0 /** * Query the relevant set of caches, looking for a QVal to go with * the provided QKey. If the QVal is found in a cache other than * the current-read cache, it is copied into the current-read cache * first and the QVal pointer for the current-read cache is * returned. This function never returns a pointer from any cache * other than the current-read cache. If the QVal could not be * found in any cache OR if the QVal was found in a cache other * than the current-read cache but could not be copied into the * current-read cache, NULL is returned. */ QVal* queryCopy(const QKey& qk, bool getLock = true) { assert(qk.cacheable()); AlignmentCache* caches[3] = { current_, local_, shared_ }; for(int i = 0; i < 3; i++) { if(caches[i] == NULL) continue; QVal* qv = caches[i]->query(qk, getLock); if(qv != NULL) { if(i == 0) return qv; if(!current_->copy(qk, *qv, *caches[i], getLock)) { // Exhausted memory in the current cache while // attempting to copy in the qk return NULL; } QVal* curqv = current_->query(qk, getLock); assert(curqv != NULL); return curqv; } } return NULL; } /** * Query the relevant set of caches, looking for a QVal to go with * the provided QKey. If a QVal is found and which is non-NULL, * *which is set to 0 if the qval was found in the current-read * cache, 1 if it was found in the local across-read cache, and 2 * if it was found in the shared across-read cache. */ inline QVal* query( const QKey& qk, AlignmentCache** which, bool getLock = true) { assert(qk.cacheable()); AlignmentCache* caches[3] = { current_, local_, shared_ }; for(int i = 0; i < 3; i++) { if(caches[i] == NULL) continue; QVal* qv = caches[i]->query(qk, getLock); if(qv != NULL) { if(which != NULL) *which = caches[i]; return qv; } } return NULL; } #endif /** * This function is called whenever we start to align a new read or * read substring. We make key for it and store the key in qk_. * If the sequence is uncacheable, we don't actually add it to the * map but the corresponding reference substrings are still added * to the qlist_. * * Returns: * -1 if out of memory * 0 if key was found in cache * 1 if key was not found in cache (and there's enough memory to * add a new key) */ int beginAlign( const BTDnaString& seq, const BTString& qual, QVal& qv, // out: filled in if we find it in the cache bool getLock = true) { assert(repOk()); qk_.init(seq ASSERT_ONLY(, tmpdnastr_)); //if(qk_.cacheable() && (qv_ = current_->query(qk_, getLock)) != NULL) { // // qv_ holds the answer // assert(qv_->valid()); // qv = *qv_; // resetRead(); // return 1; // found in cache //} else if(qk_.cacheable()) { // Make a QNode for this key and possibly add the QNode to the // Red-Black map; but if 'seq' isn't cacheable, just create the // QNode (without adding it to the map). qv_ = current_->add(qk_, &cacheable_, getLock); } else { qv_ = &qvbuf_; } if(qv_ == NULL) { resetRead(); return -1; // Not in memory } qv_->reset(); return 0; // Need to search for it } ASSERT_ONLY(BTDnaString tmpdnastr_); /** * Called when is finished aligning a read (and so is finished * adding associated reference strings). Returns a copy of the * final QVal object and resets the alignment state of the * current-read cache. * * Also, if the alignment is cacheable, it commits it to the next * cache up in the cache hierarchy. */ QVal finishAlign(bool getLock = true) { if(!qv_->valid()) { qv_->init(0, 0, 0); } // Copy this pointer because we're about to reset the qv_ field // to NULL QVal* qv = qv_; // Commit the contents of the current-read cache to the next // cache up in the hierarchy. // If qk is cacheable, then it must be in the cache #if 0 if(qk_.cacheable()) { AlignmentCache* caches[3] = { current_, local_, shared_ }; ASSERT_ONLY(AlignmentCache* which); ASSERT_ONLY(QVal* qv2 = query(qk_, &which, true)); assert(qv2 == qv); assert(which == current_); for(int i = 1; i < 3; i++) { if(caches[i] != NULL) { // Copy this key/value pair to the to the higher // level cache and, if its memory is exhausted, // clear the cache and try again. caches[i]->clearCopy(qk_, *qv_, *current_, getLock); break; } } } #endif // Reset the state in this iface in preparation for the next // alignment. resetRead(); assert(repOk()); return *qv; } /** * A call to this member indicates that the caller has finished * with the last read (if any) and is ready to work on the next. * This gives the cache a chance to reset some of its state if * necessary. */ void nextRead() { current_->clear(); resetRead(); assert(!aligning()); } /** * Return true iff we're in the middle of aligning a sequence. */ bool aligning() const { return qv_ != NULL; } /** * Clears both the local and shared caches. */ void clear() { if(current_ != NULL) current_->clear(); if(local_ != NULL) local_->clear(); if(shared_ != NULL) shared_->clear(); } /** * Add an alignment to the running list of alignments being * compiled for the current read in the local cache. */ bool addOnTheFly( const BTDnaString& rfseq, // reference sequence close to read seq index_t topf, // top in BWT index index_t botf, // bot in BWT index index_t topb, // top in BWT' index index_t botb, // bot in BWT' index bool getLock = true) // true -> lock is not held by caller { assert(aligning()); assert(repOk()); ASSERT_ONLY(BTDnaString tmp); SAKey sak(rfseq ASSERT_ONLY(, tmp)); //assert(sak.cacheable()); if(current_->addOnTheFly((*qv_), sak, topf, botf, topb, botb, getLock)) { rangen_++; eltsn_ += (botf-topf); return true; } return false; } /** * Given a QVal, populate the given EList of SATuples with records * describing all of the cached information about the QVal's * reference substrings. */ template void queryQval( const QVal& qv, EList, S>& satups, index_t& nrange, index_t& nelt, bool getLock = true) { current_->queryQval(qv, satups, nrange, nelt, getLock); } /** * Return a pointer to the current-read cache object. */ const AlignmentCache* currentCache() const { return current_; } index_t curNumRanges() const { return rangen_; } index_t curNumElts() const { return eltsn_; } #ifndef NDEBUG /** * Check that AlignmentCacheIface is internally consistent. */ bool repOk() const { assert(current_ != NULL); assert_geq(eltsn_, rangen_); if(qv_ == NULL) { assert_eq(0, rangen_); assert_eq(0, eltsn_); } return true; } #endif /** * Return the alignment cache for the current read. */ const AlignmentCache& current() { return *current_; } protected: /** * Reset fields encoding info about the in-process read. */ void resetRead() { cacheable_ = false; rangen_ = eltsn_ = 0; qv_ = NULL; } QKey qk_; // key representation for current read substring QVal *qv_; // pointer to value representation for current read substring QVal qvbuf_; // buffer for when key is uncacheable but we need a qv bool cacheable_; // true iff the read substring currently being aligned is cacheable index_t rangen_; // number of ranges since last alignment job began index_t eltsn_; // number of elements since last alignment job began AlignmentCache *current_; // cache dedicated to the current read AlignmentCache *local_; // local, unsynchronized cache AlignmentCache *shared_; // shared, synchronized cache }; #ifndef NDEBUG /** * Check that this QVal is internally consistent and consistent * with the contents of the given cache. */ template bool QVal::repOk(const AlignmentCache& ac) const { if(rangen_ > 0) { assert_lt(i_, ac.qSize()); assert_leq(i_ + rangen_, ac.qSize()); } assert_geq(eltn_, rangen_); return true; } #endif #ifndef NDEBUG /** * Check that this SAVal is internally consistent and consistent * with the contents of the given cache. */ template bool SAVal::repOk(const AlignmentCache& ac) const { assert(len == 0 || i < ac.saSize()); assert_leq(i + len, ac.saSize()); return true; } #endif /** * Add a new association between a read sequnce ('seq') and a * reference sequence ('') */ template bool AlignmentCache::addOnTheFly( QVal& qv, // qval that points to the range of reference substrings const SAKey& sak, // the key holding the reference substring index_t topf, // top range elt in BWT index index_t botf, // bottom range elt in BWT index index_t topb, // top range elt in BWT' index index_t botb, // bottom range elt in BWT' index bool getLock) { ThreadSafe ts(lockPtr(), shared_ && getLock); bool added = true; // If this is the first reference sequence we're associating with // the query sequence, initialize the QVal. if(!qv.valid()) { qv.init((index_t)qlist_.size(), 0, 0); } qv.addRange(botf-topf); // update tally for # ranges and # elts if(!qlist_.add(pool(), sak)) { return false; // Exhausted pool memory } #ifndef NDEBUG for(index_t i = qv.offset(); i < qlist_.size(); i++) { if(i > qv.offset()) { assert(qlist_.get(i) != qlist_.get(i-1)); } } #endif assert_eq(qv.offset() + qv.numRanges(), qlist_.size()); SANode *s = samap_.add(pool(), sak, &added); if(s == NULL) { return false; // Exhausted pool memory } assert(s->key.repOk()); if(added) { s->payload.i = (index_t)salist_.size(); s->payload.len = botf - topf; s->payload.topf = topf; s->payload.topb = topb; for(size_t j = 0; j < (botf-topf); j++) { if(!salist_.add(pool(), (index_t)0xffffffff)) { // Change the payload's len field s->payload.len = (uint32_t)j; return false; // Exhausted pool memory } } assert(s->payload.repOk(*this)); } // Now that we know all allocations have succeeded, we can do a few final // updates return true; } #endif /*ALIGNER_CACHE_H_*/ hisat2-2.2.1/aligner_driver.cpp000066400000000000000000000044001370663763200164210ustar00rootroot00000000000000/* * Copyright 2012, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "aligner_driver.h" void AlignerDriverRootSelector::select( const Read& q, const Read* qo, bool nofw, bool norc, EList& confs, EList& roots) { // Calculate interval length for both mates int interval = rootIval_.f((double)q.length()); if(qo != NULL) { // Boost interval length by 20% for paired-end reads interval = (int)(interval * 1.2 + 0.5); } float pri = 0.0f; for(int fwi = 0; fwi < 2; fwi++) { bool fw = (fwi == 0); if((fw && nofw) || (!fw && norc)) { continue; } // Put down left-to-right roots w/r/t forward and reverse-complement reads { bool first = true; size_t i = 0; while(first || (i + landing_ <= q.length())) { confs.expand(); confs.back().cons.init(landing_, consExp_); roots.expand(); roots.back().init( i, // offset from 5' end true, // left-to-right? fw, // fw? q.length(), // query length pri); // root priority i += interval; first = false; } } // Put down right-to-left roots w/r/t forward and reverse-complement reads { bool first = true; size_t i = 0; while(first || (i + landing_ <= q.length())) { confs.expand(); confs.back().cons.init(landing_, consExp_); roots.expand(); roots.back().init( q.length() - i - 1, // offset from 5' end false, // left-to-right? fw, // fw? q.length(), // query length pri); // root priority i += interval; first = false; } } } } hisat2-2.2.1/aligner_driver.h000066400000000000000000000203621370663763200160730ustar00rootroot00000000000000/* * Copyright 2012, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /* * aligner_driver.h * * REDUNDANT SEED HITS * * We say that two seed hits are redundant if they trigger identical * seed-extend dynamic programming problems. Put another way, they both lie on * the same diagonal of the overall read/reference dynamic programming matrix. * Detecting redundant seed hits is simple when the seed hits are ungapped. We * do this after offset resolution but before the offset is converted to genome * coordinates (see uses of the seenDiags1_/seenDiags2_ fields for examples). * * REDUNDANT ALIGNMENTS * * In an unpaired context, we say that two alignments are redundant if they * share any cells in the global DP table. Roughly speaking, this is like * saying that two alignments are redundant if any read character aligns to the * same reference character (same reference sequence, same strand, same offset) * in both alignments. * * In a paired-end context, we say that two paired-end alignments are redundant * if the mate #1s are redundant and the mate #2s are redundant. * * How do we enforce this? In the unpaired context, this is relatively simple: * the cells from each alignment are checked against a set containing all cells * from all previous alignments. Given a new alignment, for each cell in the * new alignment we check whether it is in the set. If there is any overlap, * the new alignment is rejected as redundant. Otherwise, the new alignment is * accepted and its cells are added to the set. * * Enforcement in a paired context is a little trickier. Consider the * following approaches: * * 1. Skip anchors that are redundant with any previous anchor or opposite * alignment. This is sufficient to ensure no two concordant alignments * found are redundant. * * 2. Same as scheme 1, but with a "transitive closure" scheme for finding all * concordant pairs in the vicinity of an anchor. Consider the AB/AC * scenario from the previous paragraph. If B is the anchor alignment, we * will find AB but not AC. But under this scheme, once we find AB we then * let B be a new anchor and immediately look for its opposites. Likewise, * if we find any opposite, we make them anchors and continue searching. We * don't stop searching until every opposite is used as an anchor. * * 3. Skip anchors that are redundant with any previous anchor alignment (but * allow anchors that are redundant with previous opposite alignments). * This isn't sufficient to avoid redundant concordant alignments. To avoid * redundant concordants, we need an additional procedure that checks each * new concordant alignment one-by-one against a list of previous concordant * alignments to see if it is redundant. * * We take approach 1. */ #ifndef ALIGNER_DRIVER_H_ #define ALIGNER_DRIVER_H_ #include "aligner_seed2.h" #include "simple_func.h" #include "aln_sink.h" /** * Concrete subclass of DescentRootSelector. Puts a root every 'ival' chars, * where 'ival' is determined by user-specified parameters. A root is filtered * out if the end of the read is less than 'landing' positions away, in the * direction of the search. */ class AlignerDriverRootSelector : public DescentRootSelector { public: AlignerDriverRootSelector( double consExp, const SimpleFunc& rootIval, size_t landing) { consExp_ = consExp; rootIval_ = rootIval; landing_ = landing; } virtual ~AlignerDriverRootSelector() { } virtual void select( const Read& q, // read that we're selecting roots for const Read* qo, // opposite mate, if applicable bool nofw, // don't add roots for fw read bool norc, // don't add roots for rc read EList& confs, // put DescentConfigs here EList& roots); // put DescentRoot here protected: double consExp_; SimpleFunc rootIval_; size_t landing_; }; /** * Return values from extendSeeds and extendSeedsPaired. */ enum { // Candidates were examined exhaustively ALDRIVER_EXHAUSTED_CANDIDATES = 1, // The policy does not need us to look any further ALDRIVER_POLICY_FULFILLED, // We stopped because we ran up against a limit on how much work we should // do for one set of seed ranges, e.g. the limit on number of consecutive // unproductive DP extensions ALDRIVER_EXCEEDED_LIMIT }; /** * This class is the glue between a DescentDriver and the dynamic programming * implementations in Bowtie 2. The DescentDriver is used to find some very * high-scoring alignments, but is additionally used to rank partial alignments * so that they can be extended using dynamic programming. */ template class AlignerDriver { public: AlignerDriver( double consExp, const SimpleFunc& rootIval, size_t landing, bool veryVerbose, const SimpleFunc& totsz, const SimpleFunc& totfmops) : sel_(consExp, rootIval, landing), alsel_(), dr1_(veryVerbose), dr2_(veryVerbose) { totsz_ = totsz; totfmops_ = totfmops; } /** * Initialize driver with respect to a new read or pair. */ void initRead( const Read& q1, bool nofw, bool norc, TAlScore minsc, TAlScore maxpen, const Read* q2) { dr1_.initRead(q1, nofw, norc, minsc, maxpen, q2, &sel_); red1_.init(q1.length()); paired_ = false; if(q2 != NULL) { dr2_.initRead(*q2, nofw, norc, minsc, maxpen, &q1, &sel_); red2_.init(q2->length()); paired_ = true; } else { dr2_.reset(); } size_t totsz = totsz_.f(q1.length()); size_t totfmops = totfmops_.f(q1.length()); stop_.init( totsz, 0, true, totfmops); } /** * Start the driver. The driver will begin by conducting a best-first, * index-assisted search through the space of possible full and partial * alignments. This search may be followed up with a dynamic programming * extension step, taking a prioritized set of partial SA ranges found * during the search and extending each with DP. The process might also be * iterated, with the search being occasioanally halted so that DPs can be * tried, then restarted, etc. */ int go( const Scoring& sc, const GFM& gfmFw, const GFM& gfmBw, const BitPairReference& ref, DescentMetrics& met, WalkMetrics& wlm, PerReadMetrics& prm, RandomSource& rnd, AlnSinkWrap& sink); /** * Reset state of all DescentDrivers. */ void reset() { dr1_.reset(); dr2_.reset(); red1_.reset(); red2_.reset(); } protected: AlignerDriverRootSelector sel_; // selects where roots should go DescentAlignmentSelector alsel_; // one selector can deal with >1 drivers DescentDriver dr1_; // driver for mate 1/unpaired reads DescentDriver dr2_; // driver for paired-end reads DescentStoppingConditions stop_; // when to pause index-assisted BFS bool paired_; // current read is paired? SimpleFunc totsz_; // memory limit on best-first search data SimpleFunc totfmops_; // max # FM ops for best-first search // For detecting redundant alignments RedundantAlns red1_; // database of cells used for mate 1 alignments RedundantAlns red2_; // database of cells used for mate 2 alignments // For AlnRes::matchesRef ASSERT_ONLY(SStringExpandable raw_refbuf_); ASSERT_ONLY(SStringExpandable raw_destU32_); ASSERT_ONLY(EList raw_matches_); ASSERT_ONLY(BTDnaString tmp_rf_); ASSERT_ONLY(BTDnaString tmp_rdseq_); ASSERT_ONLY(BTString tmp_qseq_); ASSERT_ONLY(EList tmp_reflens_); ASSERT_ONLY(EList tmp_refoffs_); }; #endif /* defined(ALIGNER_DRIVER_H_) */ hisat2-2.2.1/aligner_metrics.h000066400000000000000000000257621370663763200162570ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_METRICS_H_ #define ALIGNER_METRICS_H_ #include #include #include "alphabet.h" #include "timer.h" #include "sstring.h" using namespace std; /** * Borrowed from http://www.johndcook.com/standard_deviation.html, * which in turn is borrowed from Knuth. */ class RunningStat { public: RunningStat() : m_n(0), m_tot(0.0) { } void clear() { m_n = 0; m_tot = 0.0; } void push(float x) { m_n++; m_tot += x; // See Knuth TAOCP vol 2, 3rd edition, page 232 if (m_n == 1) { m_oldM = m_newM = x; m_oldS = 0.0; } else { m_newM = m_oldM + (x - m_oldM)/m_n; m_newS = m_oldS + (x - m_oldM)*(x - m_newM); // set up for next iteration m_oldM = m_newM; m_oldS = m_newS; } } int num() const { return m_n; } double tot() const { return m_tot; } double mean() const { return (m_n > 0) ? m_newM : 0.0; } double variance() const { return ( (m_n > 1) ? m_newS/(m_n - 1) : 0.0 ); } double stddev() const { return sqrt(variance()); } private: int m_n; double m_tot; double m_oldM, m_newM, m_oldS, m_newS; }; /** * Encapsulates a set of metrics that we would like an aligner to keep * track of, so that we can possibly use it to diagnose performance * issues. */ class AlignerMetrics { public: AlignerMetrics() : curBacktracks_(0), curBwtOps_(0), first_(true), curIsLowEntropy_(false), curIsHomoPoly_(false), curHadRanges_(false), curNumNs_(0), reads_(0), homoReads_(0), lowEntReads_(0), hiEntReads_(0), alignedReads_(0), unalignedReads_(0), threeOrMoreNReads_(0), lessThanThreeNRreads_(0), bwtOpsPerRead_(), backtracksPerRead_(), bwtOpsPerHomoRead_(), backtracksPerHomoRead_(), bwtOpsPerLoEntRead_(), backtracksPerLoEntRead_(), bwtOpsPerHiEntRead_(), backtracksPerHiEntRead_(), bwtOpsPerAlignedRead_(), backtracksPerAlignedRead_(), bwtOpsPerUnalignedRead_(), backtracksPerUnalignedRead_(), bwtOpsPer0nRead_(), backtracksPer0nRead_(), bwtOpsPer1nRead_(), backtracksPer1nRead_(), bwtOpsPer2nRead_(), backtracksPer2nRead_(), bwtOpsPer3orMoreNRead_(), backtracksPer3orMoreNRead_(), timer_(cout, "", false) { } void printSummary() { if(!first_) { finishRead(); } cout << "AlignerMetrics:" << endl; cout << " # Reads: " << reads_ << endl; float hopct = (reads_ > 0) ? (((float)homoReads_)/((float)reads_)) : (0.0f); hopct *= 100.0f; cout << " % homo-polymeric: " << (hopct) << endl; float lopct = (reads_ > 0) ? ((float)lowEntReads_/(float)(reads_)) : (0.0f); lopct *= 100.0f; cout << " % low-entropy: " << (lopct) << endl; float unpct = (reads_ > 0) ? ((float)unalignedReads_/(float)(reads_)) : (0.0f); unpct *= 100.0f; cout << " % unaligned: " << (unpct) << endl; float npct = (reads_ > 0) ? ((float)threeOrMoreNReads_/(float)(reads_)) : (0.0f); npct *= 100.0f; cout << " % with 3 or more Ns: " << (npct) << endl; cout << endl; cout << " Total BWT ops: avg: " << bwtOpsPerRead_.mean() << ", stddev: " << bwtOpsPerRead_.stddev() << endl; cout << " Total Backtracks: avg: " << backtracksPerRead_.mean() << ", stddev: " << backtracksPerRead_.stddev() << endl; time_t elapsed = timer_.elapsed(); cout << " BWT ops per second: " << (bwtOpsPerRead_.tot()/elapsed) << endl; cout << " Backtracks per second: " << (backtracksPerRead_.tot()/elapsed) << endl; cout << endl; cout << " Homo-poly:" << endl; cout << " BWT ops: avg: " << bwtOpsPerHomoRead_.mean() << ", stddev: " << bwtOpsPerHomoRead_.stddev() << endl; cout << " Backtracks: avg: " << backtracksPerHomoRead_.mean() << ", stddev: " << backtracksPerHomoRead_.stddev() << endl; cout << " Low-entropy:" << endl; cout << " BWT ops: avg: " << bwtOpsPerLoEntRead_.mean() << ", stddev: " << bwtOpsPerLoEntRead_.stddev() << endl; cout << " Backtracks: avg: " << backtracksPerLoEntRead_.mean() << ", stddev: " << backtracksPerLoEntRead_.stddev() << endl; cout << " High-entropy:" << endl; cout << " BWT ops: avg: " << bwtOpsPerHiEntRead_.mean() << ", stddev: " << bwtOpsPerHiEntRead_.stddev() << endl; cout << " Backtracks: avg: " << backtracksPerHiEntRead_.mean() << ", stddev: " << backtracksPerHiEntRead_.stddev() << endl; cout << endl; cout << " Unaligned:" << endl; cout << " BWT ops: avg: " << bwtOpsPerUnalignedRead_.mean() << ", stddev: " << bwtOpsPerUnalignedRead_.stddev() << endl; cout << " Backtracks: avg: " << backtracksPerUnalignedRead_.mean() << ", stddev: " << backtracksPerUnalignedRead_.stddev() << endl; cout << " Aligned:" << endl; cout << " BWT ops: avg: " << bwtOpsPerAlignedRead_.mean() << ", stddev: " << bwtOpsPerAlignedRead_.stddev() << endl; cout << " Backtracks: avg: " << backtracksPerAlignedRead_.mean() << ", stddev: " << backtracksPerAlignedRead_.stddev() << endl; cout << endl; cout << " 0 Ns:" << endl; cout << " BWT ops: avg: " << bwtOpsPer0nRead_.mean() << ", stddev: " << bwtOpsPer0nRead_.stddev() << endl; cout << " Backtracks: avg: " << backtracksPer0nRead_.mean() << ", stddev: " << backtracksPer0nRead_.stddev() << endl; cout << " 1 N:" << endl; cout << " BWT ops: avg: " << bwtOpsPer1nRead_.mean() << ", stddev: " << bwtOpsPer1nRead_.stddev() << endl; cout << " Backtracks: avg: " << backtracksPer1nRead_.mean() << ", stddev: " << backtracksPer1nRead_.stddev() << endl; cout << " 2 Ns:" << endl; cout << " BWT ops: avg: " << bwtOpsPer2nRead_.mean() << ", stddev: " << bwtOpsPer2nRead_.stddev() << endl; cout << " Backtracks: avg: " << backtracksPer2nRead_.mean() << ", stddev: " << backtracksPer2nRead_.stddev() << endl; cout << " >2 Ns:" << endl; cout << " BWT ops: avg: " << bwtOpsPer3orMoreNRead_.mean() << ", stddev: " << bwtOpsPer3orMoreNRead_.stddev() << endl; cout << " Backtracks: avg: " << backtracksPer3orMoreNRead_.mean() << ", stddev: " << backtracksPer3orMoreNRead_.stddev() << endl; cout << endl; } /** * */ void nextRead(const BTDnaString& read) { if(!first_) { finishRead(); } first_ = false; //float ent = entropyDna5(read); float ent = 0.0f; curIsLowEntropy_ = (ent < 0.75f); curIsHomoPoly_ = (ent < 0.001f); curHadRanges_ = false; curBwtOps_ = 0; curBacktracks_ = 0; // Count Ns curNumNs_ = 0; const size_t len = read.length(); for(size_t i = 0; i < len; i++) { if((int)read[i] == 4) curNumNs_++; } } /** * */ void setReadHasRange() { curHadRanges_ = true; } /** * Commit the running statistics for this read to */ void finishRead() { reads_++; if(curIsHomoPoly_) homoReads_++; else if(curIsLowEntropy_) lowEntReads_++; else hiEntReads_++; if(curHadRanges_) alignedReads_++; else unalignedReads_++; bwtOpsPerRead_.push((float)curBwtOps_); backtracksPerRead_.push((float)curBacktracks_); // Drill down by entropy if(curIsHomoPoly_) { bwtOpsPerHomoRead_.push((float)curBwtOps_); backtracksPerHomoRead_.push((float)curBacktracks_); } else if(curIsLowEntropy_) { bwtOpsPerLoEntRead_.push((float)curBwtOps_); backtracksPerLoEntRead_.push((float)curBacktracks_); } else { bwtOpsPerHiEntRead_.push((float)curBwtOps_); backtracksPerHiEntRead_.push((float)curBacktracks_); } // Drill down by whether it aligned if(curHadRanges_) { bwtOpsPerAlignedRead_.push((float)curBwtOps_); backtracksPerAlignedRead_.push((float)curBacktracks_); } else { bwtOpsPerUnalignedRead_.push((float)curBwtOps_); backtracksPerUnalignedRead_.push((float)curBacktracks_); } if(curNumNs_ == 0) { lessThanThreeNRreads_++; bwtOpsPer0nRead_.push((float)curBwtOps_); backtracksPer0nRead_.push((float)curBacktracks_); } else if(curNumNs_ == 1) { lessThanThreeNRreads_++; bwtOpsPer1nRead_.push((float)curBwtOps_); backtracksPer1nRead_.push((float)curBacktracks_); } else if(curNumNs_ == 2) { lessThanThreeNRreads_++; bwtOpsPer2nRead_.push((float)curBwtOps_); backtracksPer2nRead_.push((float)curBacktracks_); } else { threeOrMoreNReads_++; bwtOpsPer3orMoreNRead_.push((float)curBwtOps_); backtracksPer3orMoreNRead_.push((float)curBacktracks_); } } // Running-total of the number of backtracks and BWT ops for the // current read uint32_t curBacktracks_; uint32_t curBwtOps_; protected: bool first_; // true iff the current read is low entropy bool curIsLowEntropy_; // true if current read is all 1 char (or very close) bool curIsHomoPoly_; // true iff the current read has had one or more ranges reported bool curHadRanges_; // number of Ns in current read int curNumNs_; // # reads uint32_t reads_; // # homo-poly reads uint32_t homoReads_; // # low-entropy reads uint32_t lowEntReads_; // # high-entropy reads uint32_t hiEntReads_; // # reads with alignments uint32_t alignedReads_; // # reads without alignments uint32_t unalignedReads_; // # reads with 3 or more Ns uint32_t threeOrMoreNReads_; // # reads with < 3 Ns uint32_t lessThanThreeNRreads_; // Distribution of BWT operations per read RunningStat bwtOpsPerRead_; RunningStat backtracksPerRead_; // Distribution of BWT operations per homo-poly read RunningStat bwtOpsPerHomoRead_; RunningStat backtracksPerHomoRead_; // Distribution of BWT operations per low-entropy read RunningStat bwtOpsPerLoEntRead_; RunningStat backtracksPerLoEntRead_; // Distribution of BWT operations per high-entropy read RunningStat bwtOpsPerHiEntRead_; RunningStat backtracksPerHiEntRead_; // Distribution of BWT operations per read that "aligned" (for // which a range was arrived at - range may not have necessarily // lead to an alignment) RunningStat bwtOpsPerAlignedRead_; RunningStat backtracksPerAlignedRead_; // Distribution of BWT operations per read that didn't align RunningStat bwtOpsPerUnalignedRead_; RunningStat backtracksPerUnalignedRead_; // Distribution of BWT operations/backtracks per read with no Ns RunningStat bwtOpsPer0nRead_; RunningStat backtracksPer0nRead_; // Distribution of BWT operations/backtracks per read with one N RunningStat bwtOpsPer1nRead_; RunningStat backtracksPer1nRead_; // Distribution of BWT operations/backtracks per read with two Ns RunningStat bwtOpsPer2nRead_; RunningStat backtracksPer2nRead_; // Distribution of BWT operations/backtracks per read with three or // more Ns RunningStat bwtOpsPer3orMoreNRead_; RunningStat backtracksPer3orMoreNRead_; Timer timer_; }; #endif /* ALIGNER_METRICS_H_ */ hisat2-2.2.1/aligner_report.h000066400000000000000000000017561370663763200161210ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_REPORT_H_ #define ALIGNER_REPORT_H_ #include "aligner_cache.h" class Reporter { public: /** * */ bool report(const AlignmentCacheIface& cache, const QVal& qv) { return true; // don't retry } }; #endif /*ALIGNER_REPORT_H_*/ hisat2-2.2.1/aligner_result.cpp000066400000000000000000001573761370663763200164710ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include "reference.h" #include "aligner_result.h" #include "read.h" #include "edit.h" #include "sstring.h" #include "ds.h" #include "util.h" #include "alphabet.h" using namespace std; /** * Clear all contents. */ void AlnRes::reset() { if(ned_ != NULL) { assert(aed_ != NULL); ned_->clear(); aed_->clear(); } score_.invalidate(); refcoord_.reset(); refival_.reset(); shapeSet_ = false; rdlen_ = 0; rdid_ = 0; reflen_ = 0; rdrows_ = 0; rdextent_ = 0; rdexrows_ = 0; rfextent_ = 0; refns_ = 0; type_ = ALN_RES_TYPE_UNPAIRED; fraglen_ = -1; trimSoft_ = false; trim5p_ = 0; trim3p_ = 0; pretrimSoft_ = true; pretrim5p_ = 0; pretrim3p_ = 0; seedmms_ = 0; // number of mismatches allowed in seed seedlen_ = 0; // length of seed seedival_ = 0; // interval between seeds minsc_ = 0; // minimum score nuc5p_ = 0; nuc3p_ = 0; fraglenSet_ = false; num_spliced_ = 0; assert(!refcoord_.inited()); assert(!refival_.inited()); } /** * Set the upstream-most reference offset involved in the alignment, and * the extent of the alignment (w/r/t the reference) */ void AlnRes::setShape( TRefId id, // id of reference aligned to TRefOff off, // offset of first aligned char into ref seq TRefOff reflen, // length of reference sequence aligned to bool fw, // aligned to Watson strand? size_t rdlen, // length of read after hard trimming, before soft TReadId rdid, // read ID bool pretrimSoft, // whether trimming prior to alignment was soft size_t pretrim5p, // # poss trimmed form 5p end before alignment size_t pretrim3p, // # poss trimmed form 3p end before alignment bool trimSoft, // whether local-alignment trimming was soft size_t trim5p, // # poss trimmed form 5p end during alignment size_t trim3p) // # poss trimmed form 3p end during alignment { rdlen_ = rdlen; rdid_ = rdid; rdrows_ = rdlen; refcoord_.init(id, off, fw); pretrimSoft_ = pretrimSoft; pretrim5p_ = pretrim5p; pretrim3p_ = pretrim3p; trimSoft_ = trimSoft; trim5p_ = trim5p; trim3p_ = trim3p; // Propagate trimming to the edits. We assume that the pos fields of the // edits are set w/r/t to the rows of the dynamic programming table, and // haven't taken trimming into account yet. // // TODO: The division of labor between the aligner and the AlnRes is not // clean. Perhaps the trimming and *all* of its side-effects should be // handled by the aligner. // daehwan - check this out - this doesn't seem to work with SAWHI // size_t trimBeg = fw ? trim5p : trim3p; size_t trimBeg = trim5p; if(trimBeg > 0) { for(size_t i = 0; i < ned_->size(); i++) { // Shift by trim5p, since edits are w/r/t 5p end assert_geq((*ned_)[i].pos, trimBeg); (*ned_)[i].pos -= (uint32_t)trimBeg; } } // Length after all soft trimming and any hard trimming that occurred // during alignment rdextent_ = rdlen; if(pretrimSoft_) { rdextent_ -= (pretrim5p + pretrim3p); // soft trim } rdextent_ -= (trim5p + trim3p); // soft or hard trim from alignment assert_gt(rdextent_, 0); rdexrows_ = rdextent_; calcRefExtent(); refival_.init(id, off, fw, rfextent_); reflen_ = reflen; shapeSet_ = true; } /** * Initialize new AlnRes. */ void AlnRes::init( size_t rdlen, // # chars after hard trimming TReadId rdid, // read ID AlnScore score, // alignment score const EList* ned, // nucleotide edits size_t ned_i, // first position to copy size_t ned_n, // # positions to copy const EList* aed, // ambiguous base resolutions size_t aed_i, // first position to copy size_t aed_n, // # positions to copy Coord refcoord, // leftmost ref pos of 1st al char TRefOff reflen, // length of ref aligned to LinkedEList >* raw_edits, int seedmms, // # seed mms allowed int seedlen, // seed length int seedival, // space between seeds int64_t minsc, // minimum score for valid aln int nuc5p, int nuc3p, bool pretrimSoft, size_t pretrim5p, // trimming prior to alignment size_t pretrim3p, // trimming prior to alignment bool trimSoft, size_t trim5p, // trimming from alignment size_t trim3p, // trimming from alignment bool repeat) // repeat { assert(raw_edits != NULL); assert(raw_edits_ == NULL || raw_edits_ == raw_edits); raw_edits_ = raw_edits; if(ned_ != NULL) { assert(aed_ != NULL); ned_->clear(); aed_->clear(); } else if(raw_edits_ != NULL) { assert(aed_ == NULL); assert(ned_node_ == NULL && aed_node_ == NULL); ned_node_ = raw_edits_->new_node(); aed_node_ = raw_edits_->new_node(); assert(ned_node_ != NULL && aed_node_ != NULL); ned_ = &(ned_node_->payload); aed_ = &(aed_node_->payload); } rdlen_ = rdlen; rdid_ = rdid; rdrows_ = rdlen; score_ = score; ned_->clear(); aed_->clear(); if(ned != NULL) { for(size_t i = ned_i; i < ned_i + ned_n; i++) { ned_->push_back((*ned)[i]); } } if(aed != NULL) { for(size_t i = aed_i; i < aed_i + aed_n; i++) { aed_->push_back((*aed)[i]); } } refcoord_ = refcoord; reflen_ = reflen; seedmms_ = seedmms; seedlen_ = seedlen; seedival_ = seedival; minsc_ = minsc; nuc5p_ = nuc5p; nuc3p_ = nuc3p; pretrimSoft_ = pretrimSoft; pretrim5p_ = pretrim5p; pretrim3p_ = pretrim3p; trimSoft_ = trimSoft; trim5p_ = trim5p; trim3p_ = trim3p; repeat_ = repeat; rdextent_ = rdlen; // # read characters after any hard trimming if(pretrimSoft) { rdextent_ -= (pretrim5p + pretrim3p); } if(trimSoft) { rdextent_ -= (trim5p + trim3p); } rdexrows_ = rdextent_; calcRefExtent(); setShape( refcoord.ref(), // id of reference aligned to refcoord.off(), // offset of first aligned char into ref seq reflen, // length of reference sequence aligned to refcoord.fw(), // aligned to Watson strand? rdlen, // length of read after hard trimming, before soft rdid, // read ID pretrimSoft, // whether trimming prior to alignment was soft pretrim5p, // # poss trimmed form 5p end before alignment pretrim3p, // # poss trimmed form 3p end before alignment trimSoft, // whether local-alignment trimming was soft trim5p, // # poss trimmed form 5p end during alignment trim3p); // # poss trimmed form 3p end during alignment shapeSet_ = true; num_spliced_ = 0; for(size_t i = 0; i < ned_->size(); i++) { if((*ned_)[i].type == EDIT_TYPE_SPL) { num_spliced_++; } } } /** * Clip given number of characters from the Watson-upstream end of the * alignment. */ void AlnRes::clipLeft(size_t rd_amt, size_t rf_amt) { assert_geq(rd_amt, 0); assert_geq(rf_amt, 0); assert_leq(rd_amt, rdexrows_); assert_leq(rf_amt, rfextent_); assert(trimSoft_); if(fw()) { trim5p_ += rd_amt; Edit::clipLo(*ned_, rdexrows_, rd_amt); Edit::clipLo(*aed_, rdexrows_, rd_amt); } else { trim3p_ += rd_amt; Edit::clipHi(*ned_, rdexrows_, rd_amt); Edit::clipHi(*aed_, rdexrows_, rd_amt); } rdexrows_ -= rd_amt; rdextent_ -= rd_amt; rfextent_ -= rf_amt; refcoord_.adjustOff(rf_amt); refival_.adjustOff(rf_amt); // Adjust refns_? } /** * Clip given number of characters from the Watson-downstream end of the * alignment. */ void AlnRes::clipRight(size_t rd_amt, size_t rf_amt) { assert_geq(rd_amt, 0); assert_geq(rf_amt, 0); assert_leq(rd_amt, rdexrows_); assert_leq(rf_amt, rfextent_); assert(trimSoft_); if(fw()) { trim3p_ += rd_amt; Edit::clipHi(*ned_, rdexrows_, rd_amt); Edit::clipHi(*aed_, rdexrows_, rd_amt); } else { trim5p_ += rd_amt; Edit::clipLo(*ned_, rdexrows_, rd_amt); Edit::clipLo(*aed_, rdexrows_, rd_amt); } rdexrows_ -= rd_amt; rdextent_ -= rd_amt; rfextent_ -= rf_amt; // Adjust refns_? } /** * Clip away portions of the alignment that are outside the given bounds. * Clipping is soft if soft == true, hard otherwise. Assuming for now that * there isn't any other clipping. * * Note that all clipping is expressed in terms of read positions. So if there * are reference gaps in the overhanging portion, we must */ void AlnRes::clipOutside(bool soft, TRefOff refi, TRefOff reff) { // Overhang on LHS TRefOff left = refcoord_.off(); if(left < refi) { size_t rf_amt = (size_t)(refi - left); size_t rf_i = rf_amt; size_t nedsz = ned_->size(); if(!fw()) { Edit::invertPoss(*ned_, rdexrows_, false); } for(size_t i = 0; i < nedsz; i++) { assert_lt((*ned_)[i].pos, rdexrows_); if((*ned_)[i].pos > rf_i) break; if((*ned_)[i].isRefGap()) rf_i++; } if(!fw()) { Edit::invertPoss(*ned_, rdexrows_, false); } clipLeft(rf_i, rf_amt); } // Overhang on RHS TRefOff right = refcoord_.off() + refNucExtent(); if(right > reff) { size_t rf_amt = (size_t)(right - reff); size_t rf_i = rf_amt; size_t nedsz = ned_->size(); if(fw()) { Edit::invertPoss(*ned_, rdexrows_, false); } for(size_t i = 0; i < nedsz; i++) { assert_lt((*ned_)[i].pos, rdexrows_); if((*ned_)[i].pos > rf_i) break; if((*ned_)[i].isRefGap()) rf_i++; } if(fw()) { Edit::invertPoss(*ned_, rdexrows_, false); } clipRight(rf_i, rf_amt); } } /** * Return true iff this AlnRes and the given AlnRes overlap. Two AlnRess * overlap if they share a cell in the overall dynamic programming table: * i.e. if there exists a read position s.t. that position in both reads * matches up with the same reference character. E.g., the following * alignments (drawn schematically as paths through a dynamic programming * table) are redundant: * * a b a b * \ \ \ \ * \ \ \ \ * \ \ \ \ * ---\ \ \ * \ ---\--- * ---\ \ \ * \ \ \ \ * \ \ \ \ * \ \ \ \ * a b a b * * We iterate over each read position that hasn't been hard-trimmed, but * only overlaps at positions that have also not been soft-trimmed are * considered. */ bool AlnRes::overlap(AlnRes& res) { if(fw() != res.fw() || refid() != res.refid()) { // Must be same reference and same strand in order to overlap return false; } TRefOff my_left = refoff(); // my leftmost aligned char TRefOff other_left = res.refoff(); // other leftmost aligned char TRefOff my_right = my_left + refExtent(); TRefOff other_right = other_left + res.refExtent(); if(my_right < other_left || other_right < my_left) { // The rectangular hulls of the two alignments don't overlap, so // they can't overlap at any cell return false; } // Reference and strand are the same and hulls overlap. Now go read // position by read position testing if any align identically with the // reference. // Edits are ordered and indexed from 5' to 3' to start with. We // reorder them to go from left to right along the Watson strand. if(!fw()) { invertEdits(); } if(!res.fw()) { res.invertEdits(); } size_t nedidx = 0, onedidx = 0; bool olap = false; // For each row, going left to right along Watson reference strand... for(size_t i = 0; i < rdexrows_; i++) { size_t diff = 1; // amount to shift to right for next round size_t odiff = 1; // amount to shift to right for next round // Unless there are insertions before the next position, we say // that there is one cell in this row involved in the alignment my_right = my_left + 1; other_right = other_left + 1; while(nedidx < ned_->size() && (*ned_)[nedidx].pos == i) { if((*ned_)[nedidx].isRefGap()) { // Next my_left will be in same column as this round diff = 0; } nedidx++; } while(onedidx < res.ned_->size() && (*res.ned_)[onedidx].pos == i) { if((*res.ned_)[onedidx].isRefGap()) { // Next my_left will be in same column as this round odiff = 0; } onedidx++; } if(i < rdexrows_ - 1) { // See how many inserts there are before the next read // character size_t nedidx_next = nedidx; size_t onedidx_next = onedidx; while(nedidx_next < ned_->size() && (*ned_)[nedidx_next].pos == i+1) { if((*ned_)[nedidx_next].isReadGap()) { my_right++; } nedidx_next++; } while(onedidx_next < res.ned_->size() && (*res.ned_)[onedidx_next].pos == i+1) { if((*res.ned_)[onedidx_next].isReadGap()) { other_right++; } onedidx_next++; } } // Contained? olap = (my_left >= other_left && my_right <= other_right) || (other_left >= my_left && other_right <= my_right); // Overlapping but not contained? if(!olap) { olap = (my_left <= other_left && my_right > other_left) || (other_left <= my_left && other_right > my_left); } if(olap) { break; } // How to do adjust my_left and my_right my_left = my_right + diff - 1; other_left = other_right + odiff - 1; } if(!fw()) { invertEdits(); } if(!res.fw()) { res.invertEdits(); } return olap; } #ifndef NDEBUG /** * Assuming this AlnRes is an alignment for 'rd', check that the alignment and * 'rd' are compatible with the corresponding reference sequence. */ bool AlnRes::matchesRef( const Read& rd, const BitPairReference& ref, BTDnaString& rf, BTDnaString& rdseq, BTString& qseq, SStringExpandable& raw_refbuf, SStringExpandable& destU32, EList& matches, SStringExpandable& raw_refbuf2, EList& reflens, EList& refoffs) { assert(!empty()); assert(repOk()); assert(refcoord_.inited()); size_t rdlen = rd.length(); bool fw = refcoord_.fw(); if(!fw) { assert_lt(trim3p_, rdlen); Edit::invertPoss(const_cast&>(*ned_), rdlen - trim5p_ - trim3p_, false); } size_t refallen = 0; reflens.clear(); refoffs.clear(); int64_t reflen = 0; int64_t refoff = refcoord_.off(); refoffs.push_back((uint32_t)refoff); size_t eidx = 0; assert_lt(trim5p_ + trim3p_, rdlen); for(size_t i = 0; i < rdlen - trim5p_ - trim3p_; i++, reflen++, refoff++) { while(eidx < ned_->size() && (*ned_)[eidx].pos == i) { if((*ned_)[eidx].isReadGap()) { reflen++; refoff++; } else if((*ned_)[eidx].isRefGap()) { reflen--; refoff--; } if((*ned_)[eidx].isSpliced()) { assert_gt(reflen, 0); refallen += (uint32_t)reflen; reflens.push_back((uint32_t)reflen); reflen = 0; refoff += (*ned_)[eidx].splLen; assert_gt(refoff, 0); refoffs.push_back((uint32_t)refoff); } eidx++; } } assert_gt(reflen, 0); refallen += (uint32_t)reflen; reflens.push_back((uint32_t)reflen); assert_gt(reflens.size(), 0); assert_gt(refoffs.size(), 0); assert_eq(reflens.size(), refoffs.size()); if(!fw) { assert_lt(trim3p_, rdlen); Edit::invertPoss(const_cast&>(*ned_), rdlen - trim5p_ - trim3p_, false); } // Adjust reference string length according to edits #ifndef NDEBUG if(reflens.size() == 1) { assert_eq(refallen, refNucExtent()); } #endif assert_geq(refcoord_.ref(), 0); int nsOnLeft = 0; if(refcoord_.off() < 0) { nsOnLeft = -((int)refcoord_.off()); } raw_refbuf.resize(refallen); raw_refbuf.clear(); raw_refbuf2.clear(); for(size_t i = 0; i < reflens.size(); i++) { assert_gt(reflens[i], 0); #ifndef NDEBUG if(i > 0) { assert_gt(refoffs[i], refoffs[i-1]); } #endif raw_refbuf2.resize(reflens[i] + 16); raw_refbuf2.clear(); int off = ref.getStretch( reinterpret_cast(raw_refbuf2.wbuf()), (size_t)refcoord_.ref(), (size_t)max(refoffs[i], 0), reflens[i], destU32); assert_leq(off, 16); raw_refbuf.append(raw_refbuf2.wbuf() + off, reflens[i]); } char *refbuf = raw_refbuf.wbuf(); size_t trim5 = 0, trim3 = 0; if(trimSoft_) { trim5 += trim5p_; trim3 += trim3p_; } if(pretrimSoft_) { trim5 += pretrim5p_; trim3 += pretrim3p_; } rf.clear(); rdseq.clear(); rdseq = rd.patFw; if(!fw) { rdseq.reverseComp(false); } assert_eq(rdrows_, rdseq.length()); // rdseq is the nucleotide sequence from upstream to downstream on the // Watson strand. ned_ are the nucleotide edits from upstream to // downstream. rf contains the reference characters. assert(Edit::repOk(*ned_, rdseq, fw, trim5, trim3)); Edit::toRef(rdseq, *ned_, rf, fw, trim5, trim3); assert_eq(refallen, rf.length()); matches.clear(); bool matchesOverall = true; matches.resize(refallen); matches.fill(true); for(size_t i = 0; i < refallen; i++) { if((int)i < nsOnLeft) { if((int)rf[i] != 4) { matches[i] = false; matchesOverall = false; } } else { if((int)rf[i] != (int)refbuf[i-nsOnLeft]) { matches[i] = false; matchesOverall = false; } } } if(!matchesOverall) { // Print a friendly message showing the difference between the // reference sequence obtained with Edit::toRef and the actual // reference sequence cerr << endl; Edit::printQAlignNoCheck( cerr, " ", rdseq, *ned_); cerr << " "; for(size_t i = 0; i < refallen; i++) { cerr << (matches[i] ? " " : "*"); } cerr << endl; cerr << " "; for(size_t i = 0; i < refallen-nsOnLeft; i++) { cerr << "ACGTN"[(int)refbuf[i]]; } cerr << endl; Edit::printQAlign( cerr, " ", rdseq, *ned_); cerr << endl; } return matchesOverall; } #endif /*ndef NDEBUG*/ #define COPY_BUF() { \ char *bufc = buf; \ while(*bufc != '\0') { \ *occ = *bufc; \ occ++; \ bufc++; \ } \ } /** * Initialized the stacked alignment with respect to a read string, a list of * edits (expressed left-to-right), and integers indicating how much hard and * soft trimming has occurred on either end of the read. * * s: read sequence * ed: all relevant edits, including ambiguous nucleotides * trimLS: # bases soft-trimmed from LHS * trimLH: # bases hard-trimmed from LHS * trimRS: # bases soft-trimmed from RHS * trimRH: # bases hard-trimmed from RHS */ void StackedAln::init( const BTDnaString& s, const EList& ed, size_t trimLS, size_t trimLH, size_t trimRS, size_t trimRH) { trimLS_ = trimLS; trimLH_ = trimLH; trimRS_ = trimRS; trimRH_ = trimRH; ASSERT_ONLY(size_t ln_postsoft = s.length() - trimLS - trimRS); stackRef_.clear(); stackRel_.clear(); stackSNP_.clear(); stackRead_.clear(); size_t rdoff = trimLS; for(size_t i = 0; i < ed.size(); i++) { assert_lt(ed[i].pos, ln_postsoft); size_t pos = ed[i].pos + trimLS; while(rdoff < pos) { int c = s[rdoff++]; assert_range(0, 4, c); stackRef_.push_back("ACGTN"[c]); stackRel_.push_back('='); stackSNP_.push_back(false); stackRead_.push_back("ACGTN"[c]); } if(ed[i].isMismatch()) { int c = s[rdoff++]; assert_range(0, 4, c); assert_eq(c, asc2dna[(int)ed[i].qchr]); assert_neq(c, asc2dna[(int)ed[i].chr]); stackRef_.push_back(ed[i].chr); stackRel_.push_back('X'); stackSNP_.push_back(ed[i].snpID != (uint32_t)INDEX_MAX); stackRead_.push_back("ACGTN"[c]); } else if(ed[i].isRefGap()) { int c = s[rdoff++]; assert_range(0, 4, c); assert_eq(c, asc2dna[(int)ed[i].qchr]); stackRef_.push_back('-'); stackRel_.push_back('I'); stackSNP_.push_back(ed[i].snpID != (uint32_t)INDEX_MAX); stackRead_.push_back("ACGTN"[c]); } else if(ed[i].isReadGap()) { stackRef_.push_back(ed[i].chr); stackRel_.push_back('D'); stackSNP_.push_back(ed[i].snpID != (uint32_t)INDEX_MAX); stackRead_.push_back('-'); } else if(ed[i].isSpliced()) { stackRef_.push_back('N'); stackRel_.push_back('N'); stackSNP_.push_back(false); stackRead_.push_back('N'); assert_gt(ed[i].splLen, 0); stackSkip_.push_back(ed[i].splLen); } } while(rdoff < s.length() - trimRS) { int c = s[rdoff++]; assert_range(0, 4, c); stackRef_.push_back("ACGTN"[c]); stackRel_.push_back('='); stackSNP_.push_back(false); stackRead_.push_back("ACGTN"[c]); } inited_ = true; } /** * Left-align all the gaps. If this changes the alignment and the CIGAR or * MD:Z strings have already been calculated, this renders them invalid. * * We left-align gaps with in the following way: for each gap, we check * whether the character opposite the rightmost gap character is the same * as the character opposite the character just to the left of the gap. If * this is the case, we can slide the gap to the left and make the * rightmost position previously covered by the gap into a non-gap. * * This scheme allows us to push the gap past a mismatch. BWA does seem to * allow this. It's not clear that Bowtie 2 should, since moving the * mismatch could cause a mismatch with one base quality to be replaced * with a mismatch with a different base quality. */ void StackedAln::leftAlign(bool pastMms) { assert(inited_); bool changed = false; size_t ln = stackRef_.size(); // Scan left-to-right for(size_t i = 0; i < ln; i++) { int rel = stackRel_[i]; if(rel != '=' && rel != 'X' && rel != 'N') { // Neither a match nor a mismatch - must be a gap assert(rel == 'I' || rel == 'D'); if(stackSNP_[i]) continue; size_t glen = 1; // Scan further right to measure length of gap for(size_t j = i+1; j < ln; j++) { if(rel != (int)stackRel_[j]) break; glen++; } // We've identified a gap of type 'rel' (D = deletion or read // gap, I = insertion or ref gap) with length 'glen'. Now we // can try to slide it to the left repeatedly. size_t l = i - 1; size_t r = l + glen; EList& gp = ((rel == 'I') ? stackRef_ : stackRead_); const EList& ngp = ((rel == 'I') ? stackRead_ : stackRef_); while(l > 0 && ngp[l] == ngp[r]) { if(stackRel_[l] == 'I' || stackRel_[l] == 'D') break; assert(stackRel_[l] == '=' || stackRel_[l] == 'X' || stackRel_[l] == 'N'); assert(stackRel_[r] == 'D' || stackRel_[r] == 'I'); if(!pastMms && (stackRel_[l] == 'X' || stackRel_[l] == 'N')) { break; } swap(gp[l], gp[r]); swap(stackRel_[l], stackRel_[r]); assert_neq('-', gp[r]); assert_eq('-', gp[l]); l--; r--; changed = true; } i += (glen-1); } } if(changed) { cigCalc_ = mdzCalc_ = false; } } /** * Build the CIGAR list, if it hasn't already built. Returns true iff it * was built for the first time. */ bool StackedAln::buildCigar(bool xeq) { assert(inited_); if(cigCalc_) { return false; // already done } cigOp_.clear(); cigRun_.clear(); if(trimLS_ > 0) { cigOp_.push_back('S'); cigRun_.push_back(trimLS_); } size_t numSkips = 0; size_t ln = stackRef_.size(); for(size_t i = 0; i < ln; i++) { char op = stackRel_[i]; if(!xeq && (op == 'X' || op == '=')) { op = 'M'; } size_t run; if(op != 'N') { run = 1; for(; i + run < ln; run++) { char op2 = stackRel_[i + run]; if(!xeq && (op2 == 'X' || op2 == '=')) { op2 = 'M'; } if(op2 != op) { break; } } i += (run-1); } else { assert_lt(numSkips, stackSkip_.size()); run = stackSkip_[numSkips]; numSkips++; } cigOp_.push_back(op); cigRun_.push_back(run); } if(trimRS_ > 0) { cigOp_.push_back('S'); cigRun_.push_back(trimRS_); } cigCalc_ = true; return true; } /** * Build the CIGAR list, if it hasn't already built. Returns true iff it * was built for the first time. */ bool StackedAln::buildMdz() { assert(inited_); if(mdzCalc_) { return false; // already done } mdzOp_.clear(); mdzChr_.clear(); mdzRun_.clear(); size_t ln = stackRef_.size(); for(size_t i = 0; i < ln; i++) { char op = stackRel_[i]; if(op == '=') { size_t run = 1; size_t ninserts = 0; size_t nskips = 0; // Skip over matches and insertions (ref gaps) for(; i+run < ln; run++) { if(stackRel_[i + run] == '=') { // do nothing } else if(stackRel_[i + run] == 'I') { ninserts++; } else if(stackRel_[i + run] == 'N') { nskips++; } else { break; } } i += (run - 1); mdzOp_.push_back('='); // = X or G mdzChr_.push_back('-'); mdzRun_.push_back(run - ninserts - nskips); } else if(op == 'X') { assert_neq(stackRef_[i], stackRead_[i]); mdzOp_.push_back('X'); // = X or G mdzChr_.push_back(stackRef_[i]); mdzRun_.push_back(1); } else if(op == 'D') { assert_neq('-', stackRef_[i]); mdzOp_.push_back('G'); // = X or G mdzChr_.push_back(stackRef_[i]); mdzRun_.push_back(1); } } mdzCalc_ = true; return true; } /** * Write a CIGAR representation of the alignment to the given string and/or * char buffer. */ void StackedAln::writeCigar( BTString* o, // if non-NULL, string to append to char* occ) const // if non-NULL, character string to append to { const EList& op = cigOp_; const EList& run = cigRun_; assert_eq(op.size(), run.size()); if(o != NULL || occ != NULL) { char buf[128]; ASSERT_ONLY(bool printed = false); for(size_t i = 0; i < op.size(); i++) { size_t r = run[i]; if(r > 0) { itoa10(r, buf); ASSERT_ONLY(printed = true); if(o != NULL) { o->append(buf); o->append(op[i]); } if(occ != NULL) { COPY_BUF(); *occ = op[i]; occ++; } } } assert(printed); if(occ != NULL) { *occ = '\0'; } } } /** * Write an MD:Z representation of the alignment to the given string and/or * char buffer. */ void StackedAln::writeMdz(BTString* o, char* occ) const { char buf[128]; bool mm_last = false; bool rdgap_last = false; bool first_print = true; const EList& op = mdzOp_; const EList& ch = mdzChr_; const EList& run = mdzRun_; for(size_t i = 0; i < op.size(); i++) { size_t r = run[i]; if(r > 0) { if(op[i] == '=') { // Write run length itoa10(r, buf); if(o != NULL) { o->append(buf); } if(occ != NULL) { COPY_BUF(); } first_print = false; mm_last = false; rdgap_last = false; } else if(op[i] == 'X') { if(o != NULL) { if(rdgap_last || mm_last || first_print) { o->append('0'); } o->append(ch[i]); } if(occ != NULL) { if(rdgap_last || mm_last || first_print) { *occ = '0'; occ++; } *occ = ch[i]; occ++; } first_print = false; mm_last = true; rdgap_last = false; } else if(op[i] == 'G') { if(o != NULL) { if(mm_last || first_print) { o->append('0'); } if(!rdgap_last) { o->append('^'); } o->append(ch[i]); } if(occ != NULL) { if(mm_last || first_print) { *occ = '0'; occ++; } if(!rdgap_last) { *occ = '^'; occ++; } *occ = ch[i]; occ++; } first_print = false; mm_last = false; rdgap_last = true; } } // if r > 0 } // for loop over ops if(mm_last || rdgap_last) { if(o != NULL) { o->append('0'); } if(occ != NULL) { *occ = '0'; occ++; } } if(occ != NULL) { *occ = '\0'; } } /** * Print the sequence for the read that aligned using A, C, G and * T. This will simply print the read sequence (or its reverse * complement). */ void AlnRes::printSeq( const Read& rd, // read const BTDnaString* dns, // already-decoded nucleotides BTString& o) const // buffer to write to { assert(!rd.patFw.empty()); ASSERT_ONLY(size_t written = 0); // Print decoded nucleotides assert(dns != NULL); size_t len = dns->length(); size_t st = 0; size_t en = len; for(size_t i = st; i < en; i++) { int c = dns->get(i); assert_range(0, 3, c); o.append("ACGT"[c]); ASSERT_ONLY(written++); } #ifndef NDEBUG for(size_t i = 0; i < ned_->size(); i++) { if((*ned_)[i].isReadGap()) { assert_leq((*ned_)[i].pos, dns->length()); } else { assert_lt((*ned_)[i].pos, dns->length()); } } #endif } /** * Print the quality string for the read that aligned. This will simply print * the read qualities (or their reverse). */ void AlnRes::printQuals( const Read& rd, // read const BTString* dqs, // already-decoded qualities BTString& o) const // output stream to write to { assert(dqs != NULL); size_t len = dqs->length(); // Print decoded qualities from upstream to downstream Watson for(size_t i = 1; i < len-1; i++) { o.append(dqs->get(i)); } } /** * Add all of the cells involved in the given alignment to the database. */ void RedundantAlns::add(const AlnRes& res) { assert(!cells_.empty()); TRefOff left = res.refoff(), right; const size_t len = res.readExtentRows(); if(!res.fw()) { const_cast(res).invertEdits(); } const EList& ned = res.ned(); size_t nedidx = 0; assert_leq(len, cells_.size()); // For each row... for(size_t i = 0; i < len; i++) { size_t diff = 1; // amount to shift to right for next round right = left + 1; while(nedidx < ned.size() && ned[nedidx].pos == i) { if(ned[nedidx].isRefGap()) { // Next my_left will be in same column as this round diff = 0; } nedidx++; } if(i < len - 1) { // See how many inserts there are before the next read // character size_t nedidx_next = nedidx; while(nedidx_next < ned.size() && ned[nedidx_next].pos == i+1) { if(ned[nedidx_next].isReadGap()) { right++; } nedidx_next++; } } for(TRefOff j = left; j < right; j++) { // Add to db RedundantCell c(res.refid(), res.fw(), j, i); ASSERT_ONLY(bool ret =) cells_[i].insert(c); assert(ret); } left = right + diff - 1; } if(!res.fw()) { const_cast(res).invertEdits(); } } /** * Return true iff the given alignment has at least one cell that overlaps * one of the cells in the database. */ bool RedundantAlns::overlap(const AlnRes& res) { assert(!cells_.empty()); TRefOff left = res.refoff(), right; const size_t len = res.readExtentRows(); if(!res.fw()) { const_cast(res).invertEdits(); } const EList& ned = res.ned(); size_t nedidx = 0; // For each row... bool olap = false; assert_leq(len, cells_.size()); for(size_t i = 0; i < len; i++) { size_t diff = 1; // amount to shift to right for next round right = left + 1; while(nedidx < ned.size() && ned[nedidx].pos == i) { if(ned[nedidx].isRefGap()) { // Next my_left will be in same column as this round diff = 0; } nedidx++; } if(i < len - 1) { // See how many inserts there are before the next read // character size_t nedidx_next = nedidx; while(nedidx_next < ned.size() && ned[nedidx_next].pos == i+1) { if(ned[nedidx_next].isReadGap()) { right++; } nedidx_next++; } } for(TRefOff j = left; j < right; j++) { // Add to db RedundantCell c(res.refid(), res.fw(), j, i); if(cells_[i].contains(c)) { olap = true; break; } } if(olap) { break; } left = right + diff - 1; } if(!res.fw()) { const_cast(res).invertEdits(); } return olap; } /** * Given all the paired and unpaired results involving mates #1 and #2, * calculate best and second-best scores for both mates. These are * used for future MAPQ calculations. */ void AlnSetSumm::init( const Read* rd1, const Read* rd2, const EList* rs1, const EList* rs2, const EList* rs1u, const EList* rs2u, bool exhausted1, bool exhausted2, TRefId orefid, TRefOff orefoff, bool repeat) { assert(rd1 != NULL || rd2 != NULL); assert((rs1 == NULL) == (rs2 == NULL)); AlnScore best[2], secbest[2], bestPaired, secbestPaired; size_t szs[2]; best[0].invalidate(); secbest[0].invalidate(); best[1].invalidate(); secbest[1].invalidate(); bestPaired.invalidate(); secbestPaired.invalidate(); bool paired = (rs1 != NULL && rs2 != NULL); szs[0] = szs[1] = 0; TNumAlns numAlns1 = 0, numAlns2 = 0, numAlnsPaired = 0; if(paired) { // Paired alignments assert_eq(rs1->size(), rs2->size()); szs[0] = szs[1] = rs1->size(); assert_gt(szs[0], 0); numAlnsPaired = szs[0]; for(size_t i = 0; i < rs1->size(); i++) { AlnScore sc = (*rs1)[i].score() + (*rs2)[i].score(); if(sc > bestPaired) { secbestPaired = bestPaired; bestPaired = sc; assert(VALID_AL_SCORE(bestPaired)); } else if(sc > secbestPaired) { secbestPaired = sc; assert(VALID_AL_SCORE(bestPaired)); assert(VALID_AL_SCORE(secbestPaired)); } } } for(int j = 0; j < 2; j++) { const EList* rs = (j == 0 ? rs1u : rs2u); if(rs == NULL) { continue; } assert(rs != NULL); szs[j] = rs->size(); if(j == 0) { numAlns1 = szs[j]; } else { numAlns2 = szs[j]; } //assert_gt(szs[j], 0); for(size_t i = 0; i < rs->size(); i++) { AlnScore sc = (*rs)[i].score(); if(sc > best[j]) { secbest[j] = best[j]; best[j] = sc; assert(VALID_AL_SCORE(best[j])); } else if(sc > secbest[j]) { secbest[j] = sc; assert(VALID_AL_SCORE(best[j])); assert(VALID_AL_SCORE(secbest[j])); } } } if(szs[0] > 0 || szs[1] > 0) { init( best[0], secbest[0], best[1], secbest[1], bestPaired, secbestPaired, (szs[0] == 0) ? 0 : (szs[0] - 1), (szs[1] == 0) ? 0 : (szs[1] - 1), paired, exhausted1, exhausted2, orefid, orefoff, repeat, numAlns1, numAlns2, numAlnsPaired); } else { reset(); orefid_ = orefid; orefoff_ = orefoff; repeat_ = repeat; } } /** * Print out string representation of YF:i flag for indicating whether and * why the mate was filtered. */ bool AlnFlags::printYF(BTString& o, bool first) const { const char *flag = ""; if (!lenfilt_) flag = "LN"; else if(!nfilt_ ) flag = "NS"; else if(!scfilt_ ) flag = "SC"; else if(!qcfilt_ ) flag = "QC"; if(*flag > 0) { if(!first) o.append('\t'); o.append("YF:Z:"); o.append(flag); return false; } return true; } /** * Print out string representation of YM:i flag for indicating with the * mate per se aligned repetitively. */ void AlnFlags::printYM(BTString& o) const { o.append("YM:i:"); o.append(maxed() ? '1' : '0'); } /** * Print out string representation of YM:i flag for indicating with the * pair containing the mate aligned repetitively. */ void AlnFlags::printYP(BTString& o) const { o.append("YP:i:"); o.append(maxedPair() ? '1' : '0'); } /** * Print out string representation of these flags. */ void AlnFlags::printYT(BTString& o) const { o.append("YT:Z:"); if(alignedConcordant()) { o.append("CP"); } else if(alignedDiscordant()) { o.append("DP"); } else if(alignedUnpairedMate()) { o.append("UP"); } else if(alignedUnpaired()) { o.append("UU"); } else { throw 1; } } #ifdef ALIGNER_RESULT_MAIN #include "mem_ids.h" int main() { EList op; EList ch; EList run; { // On top of each other, same length cerr << "Test case 1, simple overlap 1 ... "; AlnRes res1; res1.init( 10, AlnScore(), NULL, NULL, NULL, Coord(0, 0, true), false); AlnRes res2; res2.init( 10, AlnScore(), NULL, NULL, NULL, Coord(0, 0, true), false); assert(res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "10M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "10=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "10M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "10=")); char buf3[1024]; res1.printMD(false, false, op, ch, run, NULL, buf3); assert_eq(0, strcmp(buf3, "10")); res1.printMD(false, true, op, ch, run, NULL, buf3); assert_eq(0, strcmp(buf3, "8")); char buf4[1024]; res2.printMD(false, false, op, ch, run, NULL, buf4); assert_eq(0, strcmp(buf4, "10")); res2.printMD(false, true, op, ch, run, NULL, buf4); assert_eq(0, strcmp(buf4, "8")); cerr << "PASSED" << endl; } { // On top of each other, different lengths cerr << "Test case 2, simple overlap 2 ... "; AlnRes res1; res1.init( 10, AlnScore(), NULL, NULL, NULL, Coord(0, 0, true), false); AlnRes res2; res2.init( 11, AlnScore(), NULL, NULL, NULL, Coord(0, 0, true), false); assert(res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(11); ra.add(res1); assert(ra.overlap(res1)); assert(ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "10M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "10=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "11M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "11=")); char buf3[1024]; res1.printMD(false, false, op, ch, run, NULL, buf3); assert_eq(0, strcmp(buf3, "10")); res1.printMD(false, true, op, ch, run, NULL, buf3); assert_eq(0, strcmp(buf3, "8")); char buf4[1024]; res2.printMD(false, false, op, ch, run, NULL, buf4); assert_eq(0, strcmp(buf4, "11")); res2.printMD(false, true, op, ch, run, NULL, buf4); assert_eq(0, strcmp(buf4, "9")); cerr << "PASSED" << endl; } { // Different references cerr << "Test case 3, simple overlap 3 ... "; AlnRes res1; res1.init( 10, AlnScore(), NULL, NULL, NULL, Coord(0, 1, true), false); AlnRes res2; res2.init( 11, AlnScore(), NULL, NULL, NULL, Coord(0, 0, true), false); assert(!res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(11); ra.add(res1); assert(ra.overlap(res1)); assert(!ra.overlap(res2)); cerr << "PASSED" << endl; } { // Different references cerr << "Test case 4, simple overlap 4 ... "; AlnRes res1; res1.init( 10, AlnScore(), NULL, NULL, NULL, Coord(0, 0, true), false); AlnRes res2; res2.init( 10, AlnScore(), NULL, NULL, NULL, Coord(1, 0, true), false); assert(!res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(!ra.overlap(res2)); cerr << "PASSED" << endl; } { // Different strands cerr << "Test case 5, simple overlap 5 ... "; AlnRes res1; res1.init( 10, AlnScore(), NULL, NULL, NULL, Coord(0, 0, true), false); AlnRes res2; res2.init( 10, AlnScore(), NULL, NULL, NULL, Coord(0, 0, false), false); assert(!res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(!ra.overlap(res2)); cerr << "PASSED" << endl; } { // Different strands cerr << "Test case 6, simple overlap 6 ... "; EList ned1(RES_CAT); ned1.expand(); // 1 step to the right in the middle of the alignment ned1.back().init(5, 'A' /*chr*/, '-' /*qchr*/, EDIT_TYPE_READ_GAP); AlnRes res1; res1.init( 10, AlnScore(), &ned1, NULL, NULL, Coord(0, 5, false), false); AlnRes res2; res2.init( 10, AlnScore(), NULL, NULL, NULL, Coord(0, 6, false), false); assert(res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5M1D5M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5=1D5=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "10M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "10=")); char buf3[1024]; res1.printMD(false, false, op, ch, run, NULL, buf3); assert_eq(0, strcmp(buf3, "5^A5")); res1.printMD(false, true, op, ch, run, NULL, buf3); assert_eq(0, strcmp(buf3, "4^A4")); char buf4[1024]; res2.printMD(false, false, op, ch, run, NULL, buf4); assert_eq(0, strcmp(buf4, "10")); res2.printMD(false, true, op, ch, run, NULL, buf4); assert_eq(0, strcmp(buf4, "8")); cerr << "PASSED" << endl; } { // Different strands cerr << "Test case 7, simple overlap 7 ... "; EList ned1(RES_CAT); // 3 steps to the right in the middle of the alignment ned1.push_back(Edit(5, 'A', '-', EDIT_TYPE_READ_GAP)); ned1.push_back(Edit(5, 'C', '-', EDIT_TYPE_READ_GAP)); ned1.push_back(Edit(5, 'G', '-', EDIT_TYPE_READ_GAP)); AlnRes res1; res1.init( 10, AlnScore(), &ned1, NULL, NULL, Coord(0, 5, false), false); AlnRes res2; res2.init( 10, AlnScore(), NULL, NULL, NULL, Coord(0, 6, false), false); assert(res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5M3D5M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5=3D5=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "10M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "10=")); char buf3[1024]; res1.printMD(false, false, op, ch, run, NULL, buf3); assert_eq(0, strcmp(buf3, "5^GCA5")); res1.printMD(false, true, op, ch, run, NULL, buf3); assert_eq(0, strcmp(buf3, "4^GCA4")); char buf4[1024]; res2.printMD(false, false, op, ch, run, NULL, buf4); assert_eq(0, strcmp(buf4, "10")); res2.printMD(false, true, op, ch, run, NULL, buf4); assert_eq(0, strcmp(buf4, "8")); cerr << "PASSED" << endl; } { // Both with horizontal movements; overlap cerr << "Test case 8, simple overlap 8 ... "; EList ned1(RES_CAT); // 2 steps to the right in the middle of the alignment ned1.push_back(Edit(5, 'A', '-', EDIT_TYPE_READ_GAP)); ned1.push_back(Edit(5, 'C', '-', EDIT_TYPE_READ_GAP)); AlnRes res1; res1.init( 10, AlnScore(), &ned1, NULL, NULL, Coord(0, 5, false), false); EList ned2(RES_CAT); // 2 steps to the right in the middle of the alignment ned2.push_back(Edit(5, 'A', '-', EDIT_TYPE_READ_GAP)); ned2.push_back(Edit(5, 'C', '-', EDIT_TYPE_READ_GAP)); AlnRes res2; res2.init( 10, AlnScore(), &ned2, NULL, NULL, Coord(0, 6, false), false); assert(res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5M2D5M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5=2D5=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "5M2D5M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "5=2D5=")); cerr << "PASSED" << endl; } { // Both with horizontal movements; no overlap cerr << "Test case 9, simple overlap 9 ... "; EList ned1(RES_CAT); // 2 steps to the right in the middle of the alignment ned1.push_back(Edit(6, 'A', '-', EDIT_TYPE_READ_GAP)); ned1.push_back(Edit(6, 'C', '-', EDIT_TYPE_READ_GAP)); AlnRes res1; res1.init( 10, AlnScore(), &ned1, NULL, NULL, Coord(0, 5, true), false); EList ned2(RES_CAT); // 2 steps to the right in the middle of the alignment ned2.push_back(Edit(5, 'A', '-', EDIT_TYPE_READ_GAP)); ned2.push_back(Edit(5, 'C', '-', EDIT_TYPE_READ_GAP)); AlnRes res2; res2.init( 10, AlnScore(), &ned2, NULL, NULL, Coord(0, 6, true), false); assert(!res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(!ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "6M2D4M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "6=2D4=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "5M2D5M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "5=2D5=")); cerr << "PASSED" << endl; } { // Both with horizontal movements; no overlap. Reverse strand. cerr << "Test case 10, simple overlap 10 ... "; EList ned1(RES_CAT); // 2 steps to the right in the middle of the alignment ned1.push_back(Edit(5, 'A', '-', EDIT_TYPE_READ_GAP)); ned1.push_back(Edit(5, 'C', '-', EDIT_TYPE_READ_GAP)); AlnRes res1; res1.init( 10, AlnScore(), &ned1, NULL, NULL, Coord(0, 5, false), false); EList ned2(RES_CAT); // 2 steps to the right in the middle of the alignment ned2.push_back(Edit(6, 'A', '-', EDIT_TYPE_READ_GAP)); ned2.push_back(Edit(6, 'C', '-', EDIT_TYPE_READ_GAP)); AlnRes res2; res2.init( 10, AlnScore(), &ned2, NULL, NULL, Coord(0, 6, false), false); assert(!res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(!ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5M2D5M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5=2D5=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "4M2D6M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "4=2D6=")); cerr << "PASSED" << endl; } { // Both with vertical movements; no overlap cerr << "Test case 11, simple overlap 11 ... "; EList ned1(RES_CAT); // 2 steps to the right in the middle of the alignment ned1.push_back(Edit(5, '-', 'A', EDIT_TYPE_REF_GAP)); ned1.push_back(Edit(6, '-', 'C', EDIT_TYPE_REF_GAP)); AlnRes res1; res1.init( 10, AlnScore(), &ned1, NULL, NULL, Coord(0, 5, true), false); EList ned2(RES_CAT); // 2 steps to the right in the middle of the alignment ned2.push_back(Edit(6, '-', 'A', EDIT_TYPE_REF_GAP)); ned2.push_back(Edit(7, '-', 'C', EDIT_TYPE_REF_GAP)); AlnRes res2; res2.init( 10, AlnScore(), &ned2, NULL, NULL, Coord(0, 6, true), false); assert(!res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(!ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5M2I3M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5=2I3=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "6M2I2M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "6=2I2=")); cerr << "PASSED" << endl; } { // Both with vertical movements; no overlap cerr << "Test case 12, simple overlap 12 ... "; EList ned1(RES_CAT); // 2 steps to the right in the middle of the alignment ned1.push_back(Edit(5, '-', 'A', EDIT_TYPE_REF_GAP)); ned1.push_back(Edit(6, '-', 'C', EDIT_TYPE_REF_GAP)); AlnRes res1; res1.init( 10, AlnScore(), &ned1, NULL, NULL, Coord(0, 5, true), false); EList ned2(RES_CAT); // 2 steps to the right in the middle of the alignment ned2.push_back(Edit(5, '-', 'A', EDIT_TYPE_REF_GAP)); ned2.push_back(Edit(6, '-', 'C', EDIT_TYPE_REF_GAP)); AlnRes res2; res2.init( 10, AlnScore(), &ned2, NULL, NULL, Coord(0, 6, true), false); assert(!res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(!ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5M2I3M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5=2I3=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "5M2I3M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "5=2I3=")); cerr << "PASSED" << endl; } { // Both with vertical movements; overlap cerr << "Test case 13, simple overlap 13 ... "; EList ned1(RES_CAT); // 2 steps to the right in the middle of the alignment ned1.push_back(Edit(5, '-', 'A', EDIT_TYPE_REF_GAP)); ned1.push_back(Edit(6, '-', 'C', EDIT_TYPE_REF_GAP)); AlnRes res1; res1.init( 10, AlnScore(), &ned1, NULL, NULL, Coord(0, 5, true), false); EList ned2(RES_CAT); // 2 steps to the right in the middle of the alignment ned2.push_back(Edit(4, '-', 'A', EDIT_TYPE_REF_GAP)); ned2.push_back(Edit(5, '-', 'C', EDIT_TYPE_REF_GAP)); AlnRes res2; res2.init( 10, AlnScore(), &ned2, NULL, NULL, Coord(0, 6, true), false); assert(res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5M2I3M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5=2I3=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "4M2I4M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "4=2I4=")); cerr << "PASSED" << endl; } { // Not even close cerr << "Test case 14, simple overlap 14 ... "; EList ned1(RES_CAT); // 2 steps to the right in the middle of the alignment ned1.push_back(Edit(5, '-', 'A', EDIT_TYPE_REF_GAP)); ned1.push_back(Edit(6, '-', 'C', EDIT_TYPE_REF_GAP)); AlnRes res1; res1.init( 10, AlnScore(), &ned1, NULL, NULL, Coord(0, 5, true), false); EList ned2(RES_CAT); // 2 steps to the right in the middle of the alignment ned2.push_back(Edit(4, '-', 'A', EDIT_TYPE_REF_GAP)); ned2.push_back(Edit(5, '-', 'C', EDIT_TYPE_REF_GAP)); AlnRes res2; res2.init( 10, AlnScore(), &ned2, NULL, NULL, Coord(0, 400, true), false); assert(!res1.overlap(res2)); // Try again, but using the redundant-alignment database RedundantAlns ra; ra.reset(); ra.init(10); ra.add(res1); assert(ra.overlap(res1)); assert(!ra.overlap(res2)); char buf1[1024]; res1.printCigar(false, false, false, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5M2I3M")); res1.printCigar(false, false, true, op, run, NULL, buf1); assert_eq(0, strcmp(buf1, "5=2I3=")); char buf2[1024]; res2.printCigar(false, false, false, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "4M2I4M")); res2.printCigar(false, false, true, op, run, NULL, buf2); assert_eq(0, strcmp(buf2, "4=2I4=")); cerr << "PASSED" << endl; } { cerr << "Test case 15, CIGAR string with mismatches ... "; EList ned(RES_CAT); // 2 steps to the right in the middle of the alignment ned.push_back(Edit(0, 'C', 'A', EDIT_TYPE_MM)); ned.push_back(Edit(4, '-', 'C', EDIT_TYPE_REF_GAP)); ned.push_back(Edit(6, '-', 'C', EDIT_TYPE_REF_GAP)); ned.push_back(Edit(7, '-', 'C', EDIT_TYPE_REF_GAP)); ned.push_back(Edit(9, '-', 'A', EDIT_TYPE_READ_GAP)); ned.push_back(Edit(9, '-', 'A', EDIT_TYPE_READ_GAP)); ned.push_back(Edit(9, '-', 'A', EDIT_TYPE_READ_GAP)); ned.push_back(Edit(9, '-', 'A', EDIT_TYPE_READ_GAP)); ned.push_back(Edit(10, '-', 'A', EDIT_TYPE_MM)); AlnRes res; res.init( 11, AlnScore(), &ned, NULL, NULL, Coord(0, 44, true), false); char buf[1024]; res.printCigar(false, false, false, op, run, NULL, buf); assert_eq(0, strcmp(buf, "4M1I1M2I1M4D2M")); res.printCigar(false, false, true, op, run, NULL, buf); assert_eq(0, strcmp(buf, "1X3=1I1=2I1=4D1=1X")); cerr << "PASSED" << endl; } { cerr << "Test case 17, Overhang ... "; EList ned(RES_CAT); // 2 steps to the right in the middle of the alignment ned.push_back(Edit(0, 'N', 'A', EDIT_TYPE_MM)); ned.push_back(Edit(5, 'C', 'A', EDIT_TYPE_MM)); AlnRes res; res.init( 10, AlnScore(), &ned, NULL, NULL, Coord(0, -1, true), false); char buf[1024]; res.printCigar(false, false, false, op, run, NULL, buf); assert_eq(0, strcmp(buf, "10M")); res.printCigar(false, false, true, op, run, NULL, buf); assert_eq(0, strcmp(buf, "1X4=1X4=")); res.printMD(false, false, op, ch, run, NULL, buf); assert_eq(0, strcmp(buf, "0N4C4")); #if 0 AlnRes res2(res); // Now soft-clip away the overhang res2.clipOutside( true, // soft clip 0, // ref begins 40); // ref ends (excl) res2.printCigar(false, false, false, op, run, NULL, buf); assert_eq(0, strcmp(buf, "1S9M")); res2.printCigar(false, false, true, op, run, NULL, buf); assert_eq(0, strcmp(buf, "4=1X4=")); res2.printMD(false, false, op, ch, run, NULL, buf); assert_eq(0, strcmp(buf, "4C4")); AlnRes res3 = res; // Now hard-clip away the overhang res3.clipOutside( false, // hard clip 0, // ref begins 40); // ref ends (excl) res3.printCigar(false, false, false, op, run, NULL, buf); assert_eq(0, strcmp(buf, "9M")); res3.printCigar(false, false, true, op, run, NULL, buf); assert_eq(0, strcmp(buf, "4=1X4=")); res3.printMD(false, false, op, ch, run, NULL, buf); assert_eq(0, strcmp(buf, "4C4")); #endif cerr << "PASSED" << endl; } } #endif /*def ALIGNER_RESULT_MAIN*/ hisat2-2.2.1/aligner_result.h000066400000000000000000002014121370663763200161130ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_RESULT_H_ #define ALIGNER_RESULT_H_ #include #include #include "mem_ids.h" #include "ref_coord.h" #include "read.h" #include "filebuf.h" #include "ds.h" #include "edit.h" #include "limit.h" #include "splice_site.h" typedef int64_t TAlScore; #define VALID_AL_SCORE(x) ((x).score_ > MIN_I64) #define VALID_SCORE(x) ((x) > MIN_I64) #define INVALIDATE_SCORE(x) ((x) = MIN_I64) /** * A generic score object for an alignment. Used for accounting during * SW and elsewhere. Encapsulates the score, the number of N positions * and the number gaps in the alignment. * * The scale for 'score' is such that a perfect alignment score is 0 * and a score with non-zero penalty is less than 0. So differences * between scores work as expected, but interpreting an individual * score (larger is better) as a penalty (smaller is better) requires * taking the absolute value. */ class AlnScore { public: /** * Gapped scores are invalid until proven valid. */ inline AlnScore() { reset(); invalidate(); assert(!valid()); } /** * Gapped scores are invalid until proven valid. */ inline AlnScore( TAlScore score, TAlScore ns, TAlScore gaps, bool repeat = false, TAlScore splicescore = 0, bool knownTranscripts = false, bool nearSpliceSites = false, int leftTrim = 0, int rightTrim = 0) { score_ = score; ns_ = ns; gaps_ = gaps; repeat_ = repeat; splicescore_ = splicescore; knownTranscripts_ = knownTranscripts; nearSpliceSites_ = nearSpliceSites; leftTrim_ = leftTrim; rightTrim_ = rightTrim; hisat2_score_ = calculate_hisat2_score(); assert(valid()); } /** * Reset the score. */ void reset() { score_ = hisat2_score_ = ns_ = gaps_ = 0; repeat_ = false; splicescore_ = 0; knownTranscripts_ = false; nearSpliceSites_ = false; leftTrim_ = 0; rightTrim_ = 0; } /** * Return an invalid SwScore. */ inline static AlnScore INVALID() { AlnScore s; s.invalidate(); assert(!s.valid()); return s; } /** * Return true iff this score has a valid value. */ inline bool valid() const { return score_ != MIN_I64; } /** * Make this score invalid (and therefore <= all other scores). */ inline void invalidate() { score_ = MIN_I64; assert(!valid()); } /** * Increment the number of gaps. If currently invalid, this makes * the score valid with gaps == 1. */ inline void incNs(int nceil) { if(++ns_ > nceil) { invalidate(); } assert_lt(ns_, 0x7fffffff); } /** * Return true iff this score is > score o. * Note: An "invalid" score is <= all other scores. */ inline bool operator>(const AlnScore& o) const { if(!VALID_AL_SCORE(o)) { if(!VALID_AL_SCORE(*this)) { // both invalid return false; } else { // I'm valid, other is invalid return true; } } else if(!VALID_AL_SCORE(*this)) { // I'm invalid, other is valid return false; } return score_ > o.score_ || (score_ == o.score_ && hisat2_score_ > o.hisat2_score_); } /** * Scores are equal iff they're bitwise equal. */ inline AlnScore& operator=(const AlnScore& o) { // Profiling shows many cache misses on following lines gaps_ = o.gaps_; ns_ = o.ns_; score_ = o.score_; repeat_ = o.repeat_; hisat2_score_ = o.hisat2_score_; splicescore_ = o.splicescore_; knownTranscripts_ = o.knownTranscripts_; nearSpliceSites_ = o.nearSpliceSites_; leftTrim_ = o.leftTrim_; rightTrim_ = o.rightTrim_; assert_lt(ns_, 0x7fffffff); return *this; } /** * Scores are equal iff they're bitwise equal. */ inline bool operator==(const AlnScore& o) const { // Profiling shows cache misses on following line return VALID_AL_SCORE(*this) && VALID_AL_SCORE(o) && score_ == o.score_ && hisat2_score_ == o.hisat2_score_; } /** * Return true iff the two scores are unequal. */ inline bool operator!=(const AlnScore& o) const { return !(*this == o); } /** * Return true iff this score is >= score o. */ inline bool operator>=(const AlnScore& o) const { if(!VALID_AL_SCORE(o)) { if(!VALID_AL_SCORE(*this)) { // both invalid return false; } else { // I'm valid, other is invalid return true; } } else if(!VALID_AL_SCORE(*this)) { // I'm invalid, other is valid return false; } return score_ > o.score_ || (score_ == o.score_ && hisat2_score_ >= o.hisat2_score_); } /** * Return true iff this score is < score o. */ inline bool operator<(const AlnScore& o) const { return !operator>=(o); } /** * Return true iff this score is <= score o. */ inline bool operator<=(const AlnScore& o) const { return !operator>(o); } /** * Calculate difference between two SwScores. */ inline AlnScore operator-(const AlnScore& o) const { if(!VALID_AL_SCORE(*this)) return *this; AlnScore s; s.gaps_ = gaps_ - o.gaps_; s.ns_ = ns_; s.score_ = score_ - o.score_; s.splicescore_ = splicescore_ - o.splicescore_; assert_lt(s.ns_, 0x7fffffff); return s; } /** * Calculate sum of two SwScores. */ inline AlnScore operator+(const AlnScore& o) const { if(!VALID_AL_SCORE(*this)) return *this; AlnScore s; s.gaps_ = gaps_ + o.gaps_; s.ns_ = ns_; s.score_ = score_ + o.score_; s.repeat_ = repeat_ | o.repeat_; s.splicescore_ = splicescore_ + o.splicescore_; s.hisat2_score_ = hisat2_score_ + o.hisat2_score_; s.knownTranscripts_ = knownTranscripts_ | o.knownTranscripts_; s.nearSpliceSites_ = nearSpliceSites_ | o.nearSpliceSites_; s.leftTrim_ = leftTrim_ + o.leftTrim_; s.rightTrim_ = rightTrim_ + o.rightTrim_; assert_lt(s.ns_, 0x7fffffff); return s; } /** * Add given SwScore into this one. */ inline AlnScore operator+=(const AlnScore& o) { if(VALID_AL_SCORE(*this)) { gaps_ += o.gaps_; score_ += o.score_; repeat_ |= o.repeat_; splicescore_ += o.splicescore_; hisat2_score_ += o.hisat2_score_; knownTranscripts_ |= o.knownTranscripts_; nearSpliceSites_ |= o.nearSpliceSites_; leftTrim_ += o.leftTrim_; rightTrim_ += o.rightTrim_; } return (*this); } /** * Subtract given SwScore from this one. */ inline AlnScore operator-=(const AlnScore& o) { if(VALID_AL_SCORE(*this)) { gaps_ -= o.gaps_; score_ -= o.score_; // splicescore_ -= o.splicescore_; } return (*this); } /** * Calculate difference between two SwScores. */ inline AlnScore operator-(int o) const { return (*this) + -o; } /** * Calculate sum of a SwScore and an integer. */ inline AlnScore operator+(int o) const { if(!VALID_AL_SCORE(*this)) return *this; AlnScore s; s.gaps_ = gaps_; s.ns_ = ns_; s.score_ = score_ + o; // s.splicescore_ = splicescore_; assert_lt(s.ns_, 0x7fffffff); return s; } TAlScore score() const { return score_; } TAlScore hisat2_score() const { return hisat2_score_; } TAlScore penalty() const { return -score_; } TAlScore gaps() const { return gaps_; } TAlScore ns() const { return ns_; } bool repeat() const { return repeat_;} TAlScore splicescore() const { return splicescore_; } bool knownTranscripts() const { return knownTranscripts_; } bool nearSpliceSites() const { return nearSpliceSites_; } bool trimed() const { return leftTrim_ > 0 || rightTrim_ > 0; } TAlScore calculate_hisat2_score() const { // TAlScore 32 bits used for score_ TAlScore score = score_; if(score > MAX_I32) score = MAX_I32; else if(score < MIN_I32) score = MIN_I32; // Next 4 bits for repeat score TAlScore repeat_score = 0; if(repeat_) repeat_score = 1; // Next 4 bits for alignments against transcripts TAlScore transcript_score = 0; if(knownTranscripts_) transcript_score = 2; else if(nearSpliceSites_) transcript_score = 1; // Next 8 bits for splice site score TAlScore splicescore = splicescore_ / 100; if(splicescore > MAX_U8) splicescore = 0; else splicescore = MAX_U8 - splicescore; // Remaining 16 bits (rightmost 16 bits) for sum of left and right trim lengths TAlScore trim = leftTrim_ + rightTrim_; if(trim > MAX_U16) trim = 0; else trim = MAX_U16 - trim; return (score << 32) | (repeat_score << 28) | (transcript_score << 24) | (splicescore << 16) | trim; } // Score accumulated so far (penalties are subtracted starting at 0) TAlScore score_; // HISAT2 score, which is used internally to distinguish the alignments of RNA-seq reads TAlScore hisat2_score_; // Ns accumulated so far. An N opposite a non-gap counts as 1 N // (even if it's N-to-N) TAlScore ns_; // # gaps encountered so far, unless that number exceeds the // target, in which case the score becomes invalid and therefore <= // all other scores TAlScore gaps_; bool repeat_; // splice scores TAlScore splicescore_; // mapped to known transcripts? bool knownTranscripts_; // continuous alignment near (known) splice sites? bool nearSpliceSites_; int leftTrim_; int rightTrim_; }; enum { // This alignment is one of a pair of alignments that form a concordant // alignment for a read ALN_FLAG_PAIR_CONCORD_MATE1 = 1, ALN_FLAG_PAIR_CONCORD_MATE2, // This alignment is one of a pair of alignments that form a discordant // alignment for a read ALN_FLAG_PAIR_DISCORD_MATE1, ALN_FLAG_PAIR_DISCORD_MATE2, // This is an unpaired alignment but the read in question is a pair; // usually, this happens because the read had no reportable paired-end // alignments ALN_FLAG_PAIR_UNPAIRED_MATE1, ALN_FLAG_PAIR_UNPAIRED_MATE2, // This is an unpaired alignment of an unpaired read ALN_FLAG_PAIR_UNPAIRED }; /** * Encapsulates some general information about an alignment that doesn't belong * in AlnRes. Specifically: * * 1. Whether the alignment is paired * 2. If it's paried, whether it's concordant or discordant * 3. Whether this alignment was found after the paired-end categories were * maxed out * 4. Whether the relevant unpaired category was maxed out */ class AlnFlags { public: AlnFlags() { init( ALN_FLAG_PAIR_UNPAIRED, false, // canMax false, // maxed false, // maxedPair false, // nfilt false, // scfilt false, // lenfilt false, // qcfilt false, // mixedMode false, // primary false, // oppAligned false); // oppFw } AlnFlags( int pairing, bool canMax, bool maxed, bool maxedPair, bool nfilt, bool scfilt, bool lenfilt, bool qcfilt, bool mixedMode, bool primary, bool oppAligned, // opposite mate aligned? bool oppFw) // opposite mate aligned forward? { init(pairing, canMax, maxed, maxedPair, nfilt, scfilt, lenfilt, qcfilt, mixedMode, primary, oppAligned, oppFw); } /** * Initialize given values for all settings. */ void init( int pairing, bool canMax, bool maxed, bool maxedPair, bool nfilt, bool scfilt, bool lenfilt, bool qcfilt, bool mixedMode, bool primary, bool oppAligned, bool oppFw) { assert_gt(pairing, 0); assert_leq(pairing, ALN_FLAG_PAIR_UNPAIRED); pairing_ = pairing; canMax_ = canMax; maxed_ = maxed; maxedPair_ = maxedPair; nfilt_ = nfilt; scfilt_ = scfilt; lenfilt_ = lenfilt; qcfilt_ = qcfilt; mixedMode_ = mixedMode; primary_ = primary; oppAligned_ = oppAligned; } /** * Return true iff this alignment is from a paired-end read. */ bool partOfPair() const { assert_gt(pairing_, 0); return pairing_ < ALN_FLAG_PAIR_UNPAIRED; } #ifndef NDEBUG /** * Check that the flags are internally consistent. */ bool repOk() const { assert(partOfPair() || !maxedPair_); return true; } #endif /** * Print out string representation of YF:i flag for indicating whether and * why the mate was filtered. */ bool printYF(BTString& o, bool first) const; /** * Print out string representation of YM:i flag for indicating with the * mate per se aligned repetitively. */ void printYM(BTString& o) const; /** * Print out string representation of YM:i flag for indicating with the * pair containing the mate aligned repetitively. */ void printYP(BTString& o) const; /** * Print out string representation of these flags. */ void printYT(BTString& o) const; inline int pairing() const { return pairing_; } inline bool maxed() const { return maxed_; } inline bool maxedPair() const { return maxedPair_; } /** * Return true iff the alignment is not the primary alignment; i.e. not the * first reported alignment for the fragment. */ inline bool isPrimary() const { return primary_; } /** * Set the primary flag. */ void setPrimary(bool primary) { primary_ = primary; } /** * Return whether both paired and unpaired alignments are considered for * pairs & their constituent mates */ inline bool isMixedMode() const { return mixedMode_; } /** * Return true iff the alignment params are such that it's possible for a * read to be suppressed for being repetitive. */ inline bool canMax() const { return canMax_; } /** * Return true iff the alignment was filtered out. */ bool filtered() const { return !nfilt_ || !scfilt_ || !lenfilt_ || !qcfilt_; } /** * Return true iff the read is mate #1 of a pair, regardless of whether it * aligned as a pair. */ bool readMate1() const { return pairing_ == ALN_FLAG_PAIR_CONCORD_MATE1 || pairing_ == ALN_FLAG_PAIR_DISCORD_MATE1 || pairing_ == ALN_FLAG_PAIR_UNPAIRED_MATE1; } /** * Return true iff the read is mate #2 of a pair, regardless of whether it * aligned as a pair. */ bool readMate2() const { return pairing_ == ALN_FLAG_PAIR_CONCORD_MATE2 || pairing_ == ALN_FLAG_PAIR_DISCORD_MATE2 || pairing_ == ALN_FLAG_PAIR_UNPAIRED_MATE2; } /** * Return true iff the read aligned as either mate of a concordant pair. */ bool alignedConcordant() const { return pairing_ == ALN_FLAG_PAIR_CONCORD_MATE1 || pairing_ == ALN_FLAG_PAIR_CONCORD_MATE2; } /** * Return true iff the read aligned as either mate of a discordant pair. */ bool alignedDiscordant() const { return pairing_ == ALN_FLAG_PAIR_DISCORD_MATE1 || pairing_ == ALN_FLAG_PAIR_DISCORD_MATE2; } /** * Return true iff the read aligned as either mate of a pair, concordant or * discordant. */ bool alignedPaired() const { return alignedConcordant() || alignedDiscordant(); } /** * Return true iff the read aligned as an unpaired read. */ bool alignedUnpaired() const { return pairing_ == ALN_FLAG_PAIR_UNPAIRED; } /** * Return true iff the read aligned as an unpaired mate from a paired read. */ bool alignedUnpairedMate() const { return pairing_ == ALN_FLAG_PAIR_UNPAIRED_MATE1 || pairing_ == ALN_FLAG_PAIR_UNPAIRED_MATE2; } bool mateAligned() const { return oppAligned_; } protected: // See ALN_FLAG_PAIR_* above int pairing_; // True iff the alignment params are such that it's possible for a read to // be suppressed for being repetitive bool canMax_; // This alignment is sampled from among many alignments that, taken // together, cause this mate to align non-uniquely bool maxed_; // The paired-end read of which this mate is part has repetitive concordant // alignments bool maxedPair_; bool nfilt_; // read/mate filtered b/c proportion of Ns exceeded ceil bool scfilt_; // read/mate filtered b/c length can't provide min score bool lenfilt_; // read/mate filtered b/c less than or equal to seed mms bool qcfilt_; // read/mate filtered by upstream qc // Whether both paired and unpaired alignments are considered for pairs & // their constituent mates bool mixedMode_; // The read is the primary read bool primary_; // True iff the opposite mate aligned bool oppAligned_; }; static inline ostream& operator<<(ostream& os, const AlnScore& o) { os << o.score(); return os; } // Forward declaration class BitPairReference; // A given AlnRes can be one of these three types enum { ALN_RES_TYPE_UNPAIRED = 1, // unpaired alignment ALN_RES_TYPE_UNPAIRED_MATE1, // mate #1 in pair, aligned unpaired ALN_RES_TYPE_UNPAIRED_MATE2, // mate #2 in pair, aligned unpaired ALN_RES_TYPE_MATE1, // mate #1 in paired-end alignment ALN_RES_TYPE_MATE2 // mate #2 in paired-end alignment }; /** * Seed alignment summary */ struct SeedAlSumm { SeedAlSumm() { reset(); } void reset() { nonzTot = nonzFw = nonzRc = 0; nrangeTot = nrangeFw = nrangeRc = 0; neltTot = neltFw = neltRc = 0; minNonzRangeFw = minNonzRangeRc = 0; maxNonzRangeFw = maxNonzRangeRc = 0; minNonzEltFw = minNonzEltRc = 0; maxNonzEltFw = maxNonzEltRc = 0; } size_t nonzTot; size_t nonzFw; size_t nonzRc; size_t nrangeTot; size_t nrangeFw; size_t nrangeRc; size_t neltTot; size_t neltFw; size_t neltRc; size_t minNonzRangeFw; size_t minNonzRangeRc; size_t maxNonzRangeFw; size_t maxNonzRangeRc; size_t minNonzEltFw; size_t minNonzEltRc; size_t maxNonzEltFw; size_t maxNonzEltRc; }; /** * Encapsulates a stacked alignment, a nice intermediate format for alignments * from which to left-align gaps, print CIGAR strings, and print MD:Z strings. */ class StackedAln { public: StackedAln() : stackRef_(RES_CAT), stackRel_(RES_CAT), stackSNP_(RES_CAT), stackRead_(RES_CAT), stackSkip_(RES_CAT), cigOp_(RES_CAT), cigRun_(RES_CAT), mdzOp_(RES_CAT), mdzChr_(RES_CAT), mdzRun_(RES_CAT) { reset(); } /** * Reset to an uninitialized state. */ void reset() { inited_ = false; trimLS_ = trimLH_ = trimRS_ = trimRH_ = 0; stackRef_.clear(); stackRel_.clear(); stackSNP_.clear(); stackRead_.clear(); stackSkip_.clear(); cigDistMm_ = cigCalc_ = false; cigOp_.clear(); cigRun_.clear(); mdzCalc_ = false; mdzOp_.clear(); mdzChr_.clear(); mdzRun_.clear(); } /** * Return true iff the stacked alignment has been initialized. */ bool inited() const { return inited_; } /** * Initialized the stacked alignment with respect to a read string, a list of * edits (expressed left-to-right), and integers indicating how much hard and * soft trimming has occurred on either end of the read. * * s: read sequence * ed: all relevant edits, including ambiguous nucleotides * trimLS: # bases soft-trimmed from LHS * trimLH: # bases hard-trimmed from LHS * trimRS: # bases soft-trimmed from RHS * trimRH: # bases hard-trimmed from RHS */ void init( const BTDnaString& s, const EList& ed, size_t trimLS, size_t trimLH, size_t trimRS, size_t trimRH); /** * Left-align all the gaps. If this changes the alignment and the CIGAR or * MD:Z strings have already been calculated, this renders them invalid. * * We left-align gaps with in the following way: for each gap, we check * whether the character opposite the rightmost gap character is the same * as the character opposite the character just to the left of the gap. If * this is the case, we can slide the gap to the left and make the * rightmost position previously covered by the gap into a non-gap. * * This scheme allows us to push the gap past a mismatch. BWA does seem to * allow this. It's not clear that Bowtie 2 should, since moving the * mismatch could cause a mismatch with one base quality to be replaced * with a mismatch with a different base quality. */ void leftAlign(bool pastMms); /** * Build the CIGAR list, if it hasn't already built. Returns true iff it * was built for the first time. */ bool buildCigar(bool xeq); /** * Build the MD:Z list, if it hasn't already built. Returns true iff it * was built for the first time. */ bool buildMdz(); /** * Write a CIGAR representation of the alignment to the given string and/or * char buffer. */ void writeCigar(BTString* o, char* oc) const; /** * Write an MD:Z representation of the alignment to the given string and/or * char buffer. */ void writeMdz(BTString* o, char* oc) const; /** * Check internal consistency. */ #ifndef NDEBUG bool repOk() const { if(inited_) { assert_eq(stackRef_.size(), stackRead_.size()); assert_eq(stackRef_.size(), stackRel_.size()); } return true; } #endif protected: bool inited_; // true iff stacked alignment is initialized size_t trimLS_; // amount soft-trimmed from the LHS size_t trimLH_; // amount hard-trimmed from the LHS size_t trimRS_; // amount soft-trimmed from the RHS size_t trimRH_; // amount hard-trimmed from the RHS EList stackRef_; // reference characters EList stackRel_; // bars relating reference to read characters EList stackSNP_; // known SNP? EList stackRead_; // read characters EList stackSkip_; bool cigDistMm_; // distinguish between =/X, rather than just M bool cigCalc_; // whether we've calculated CIGAR ops/runs EList cigOp_; // CIGAR operations EList cigRun_; // CIGAR run lengths bool mdzCalc_; // whether we've calculated MD:Z ops/runs EList mdzOp_; // MD:Z operations EList mdzChr_; // MD:Z operations EList mdzRun_; // MD:Z run lengths }; /** * Encapsulates an alignment result. The result comprises: * * 1. All the nucleotide edits for both mates ('ned'). * 2. All "edits" where an ambiguous reference char is resolved to an * unambiguous char ('aed'). * 3. The score for the alginment, including summary information about the * number of gaps and Ns involved. * 4. The reference id, strand, and 0-based offset of the leftmost character * involved in the alignment. * 5. Information about trimming prior to alignment and whether it was hard or * soft. * 6. Information about trimming during alignment and whether it was hard or * soft. Local-alignment trimming is usually soft when aligning nucleotide * reads. * * Note that the AlnRes, together with the Read and an AlnSetSumm (*and* the * opposite mate's AlnRes and Read in the case of a paired-end alignment), * should contain enough information to print an entire alignment record. * * TRIMMING * * Accounting for trimming is tricky. Trimming affects: * * 1. The values of the trim* and pretrim* fields. * 2. The offsets of the Edits in the ELists. * 3. The read extent, if the trimming is soft. * 4. The read extent and the read sequence and length, if trimming is hard. * * Handling 1. is not too difficult. 2., 3., and 4. are handled in setShape(). */ class AlnRes { public: AlnRes() : // ned_(RES_CAT), // aed_(RES_CAT) ned_(NULL), aed_(NULL), ned_node_(NULL), aed_node_(NULL), raw_edits_(NULL) { reset(); } AlnRes(const AlnRes& other) : ned_(NULL), aed_(NULL), ned_node_(NULL), aed_node_(NULL), raw_edits_(NULL) { shapeSet_ = other.shapeSet_; rdlen_ = other.rdlen_; rdid_ = other.rdid_; rdrows_ = other.rdrows_; score_ = other.score_; oscore_ = other.oscore_; refcoord_ = other.refcoord_; reflen_ = other.reflen_; refival_ = other.refival_; rdextent_ = other.rdextent_; rdexrows_ = other.rdexrows_; rfextent_ = other.rfextent_; seedmms_ = other.seedmms_; seedlen_ = other.seedlen_; minsc_ = other.minsc_; nuc5p_ = other.nuc5p_; nuc3p_ = other.nuc3p_; refns_ = other.refns_; type_ = other.type_; fraglenSet_ = other.fraglenSet_; fraglen_ = other.fraglen_; pretrimSoft_ = other.pretrimSoft_; pretrim5p_ = other.pretrim5p_; pretrim3p_ = other.pretrim3p_; trimSoft_ = other.trimSoft_; trim5p_ = other.trim5p_; trim3p_ = other.trim3p_; repeat_ = other.repeat_; num_spliced_ = other.num_spliced_; raw_edits_ = other.raw_edits_; if(raw_edits_ != NULL) { assert(ned_ == NULL && aed_ == NULL); assert(ned_node_ == NULL && aed_node_ == NULL); ned_node_ = raw_edits_->new_node(); aed_node_ = raw_edits_->new_node(); assert(ned_node_ != NULL && aed_node_ != NULL); ned_ = &(ned_node_->payload); aed_ = &(aed_node_->payload); assert(other.ned_ != NULL && other.aed_ != NULL); *ned_ = *(other.ned_); *aed_ = *(other.aed_); } } AlnRes& operator=(const AlnRes& other) { if(this == &other) return *this; shapeSet_ = other.shapeSet_; rdlen_ = other.rdlen_; rdid_ = other.rdid_; rdrows_ = other.rdrows_; score_ = other.score_; oscore_ = other.oscore_; refcoord_ = other.refcoord_; reflen_ = other.reflen_; refival_ = other.refival_; rdextent_ = other.rdextent_; rdexrows_ = other.rdexrows_; rfextent_ = other.rfextent_; seedmms_ = other.seedmms_; seedlen_ = other.seedlen_; minsc_ = other.minsc_; nuc5p_ = other.nuc5p_; nuc3p_ = other.nuc3p_; refns_ = other.refns_; type_ = other.type_; fraglenSet_ = other.fraglenSet_; fraglen_ = other.fraglen_; pretrimSoft_ = other.pretrimSoft_; pretrim5p_ = other.pretrim5p_; pretrim3p_ = other.pretrim3p_; trimSoft_ = other.trimSoft_; trim5p_ = other.trim5p_; trim3p_ = other.trim3p_; repeat_ = other.repeat_; num_spliced_ = other.num_spliced_; assert(raw_edits_ == NULL || raw_edits_ == other.raw_edits_); raw_edits_ = other.raw_edits_; if(ned_ != NULL) { assert(aed_ != NULL); ned_->clear(); aed_->clear(); } else if(raw_edits_ != NULL) { assert(aed_ == NULL); assert(ned_node_ == NULL && aed_node_ == NULL); ned_node_ = raw_edits_->new_node(); aed_node_ = raw_edits_->new_node(); assert(ned_node_ != NULL && aed_node_ != NULL); ned_ = &(ned_node_->payload); aed_ = &(aed_node_->payload); } if(other.ned_ != NULL) { assert(other.aed_ != NULL); *ned_ = *(other.ned_); *aed_ = *(other.aed_); } return *this; } ~AlnRes() { #ifndef NDEBUG if(ned_node_ == NULL || aed_node_ == NULL) { assert(ned_node_ == NULL && aed_node_ == NULL); assert(ned_ == NULL && aed_ == NULL); assert(raw_edits_ == NULL); } else { assert(ned_node_ != NULL && aed_node_ != NULL); assert(ned_ != NULL && aed_ != NULL); assert(raw_edits_ != NULL); } #endif if(ned_ != NULL) { ned_->clear(); aed_->clear(); raw_edits_->delete_node(ned_node_); raw_edits_->delete_node(aed_node_); ned_ = aed_ = NULL; ned_node_ = aed_node_ = NULL; raw_edits_ = NULL; } } /* DK - temporary implementation */ void init_raw_edits(LinkedEList >* raw_edits) { if(raw_edits == NULL) return; raw_edits_ = raw_edits; assert(ned_ == NULL && aed_ == NULL); assert(ned_node_ == NULL && aed_node_ == NULL); ned_node_ = raw_edits_->new_node(); aed_node_ = raw_edits_->new_node(); assert(ned_node_ != NULL && aed_node_ != NULL); ned_ = &(ned_node_->payload); aed_ = &(aed_node_->payload); } /** * Clear all contents. */ void reset(); /** * Reverse all edit lists. */ void reverseEdits() { (*ned_).reverse(); (*aed_).reverse(); } /** * Invert positions of edits so that they're with respect to the other end * of the alignment. The assumption is that the .pos fields of the edits * in the ned_/aed_/ced_ structures are offsets with respect to the first * aligned character (i.e. after all trimming). */ void invertEdits() { assert(shapeSet_); assert_gt(rdlen_, 0); assert_gt(rdrows_, 0); Edit::invertPoss(*ned_, rdexrows_, false); Edit::invertPoss(*aed_, rdexrows_, false); } /** * Return true iff no result has been installed. */ bool empty() const { if(!VALID_AL_SCORE(score_)) { assert(ned_ == NULL || ned_->empty()); assert(aed_ == NULL || aed_->empty()); assert(!refcoord_.inited()); assert(!refival_.inited()); return true; } else { return false; } } /** * Return the identifier for the reference that the alignment * occurred in. */ inline TRefId refid() const { assert(shapeSet_); return refcoord_.ref(); } /** * Return the orientation that the alignment occurred in. */ inline int orient() const { assert(shapeSet_); return refcoord_.orient(); } /** * Return the 0-based offset of the alignment into the reference * sequence it aligned to. */ inline TRefOff refoff() const { assert(shapeSet_); return refcoord_.off(); } /** * Set arguments to coordinates for the upstream-most and downstream-most * reference positions involved in the alignment. */ inline void getCoords( Coord& st, // out: install starting coordinate here Coord& en, // out: install ending coordinate here Coord& st2, Coord& en2) const { assert(shapeSet_); st.init(refcoord_); en.init(refcoord_); en.adjustOff(refExtent() - 1); Coord right = refcoord_right(); st2.init(right); st2.adjustOff(1 - refExtent()); en2.init(right); } /** * Set arguments to coordinates for the upstream-most and downstream-most * reference positions covered by the read taking any read trimming into * account. I.e. if the upstream-most offset involved in an alignment is * 40 but the read was hard-trimmed by 5 on that end, the inferred * upstream-most covered position is 35. */ inline void getExtendedCoords( Coord& st, // out: install starting coordinate here Coord& en, // out: install ending coordinate here Coord& st2, Coord& en2) const { getCoords(st, en, st2, en2); // Take trimming into account int64_t trim_st = (fw() ? trim5p_ : trim3p_); int64_t trim_en = (fw() ? trim3p_ : trim5p_); trim_st += (fw() ? pretrim5p_ : pretrim3p_); trim_en += (fw() ? pretrim3p_ : pretrim5p_); st.adjustOff(-trim_st); en.adjustOff( trim_en); st2.adjustOff(-trim_st); en2.adjustOff( trim_en); } /** * Set the upstream-most reference offset involved in the alignment, and * the extent of the alignment (w/r/t the reference) */ void setShape( TRefId id, // id of reference aligned to TRefOff off, // offset of first aligned char into ref seq TRefOff reflen, // length of reference sequence aligned to bool fw, // aligned to Watson strand? size_t rdlen, // length of read after hard trimming, before soft TReadId rdid, // read ID bool pretrimSoft, // whether trimming prior to alignment was soft size_t pretrim5p, // # poss trimmed form 5p end before alignment size_t pretrim3p, // # poss trimmed form 3p end before alignment bool trimSoft, // whether local-alignment trimming was soft size_t trim5p, // # poss trimmed form 5p end during alignment size_t trim3p); // # poss trimmed form 3p end during alignment /** * Return true iff the reference chars involved in this alignment result * are entirely within with given bounds. */ bool within( TRefId id, TRefOff off, bool fw, size_t extent) const { if(refcoord_.ref() == id && refcoord_.off() >= off && refcoord_.off() + refExtent() <= off + extent && refcoord_.fw() == fw) { return true; } return false; } /** * Set alignment score for this alignment. */ void setScore(AlnScore score) { score_ = score; } /** * Set the upstream-most and downstream-most nucleotides. */ void setNucs(bool fw, int nup, int ndn) { nuc5p_ = fw ? nup : ndn; nuc3p_ = fw ? ndn : nup; } /** * Return the 0-based offset of the leftmost reference position involved in * the alignment. */ const Coord& refcoord() const { return refcoord_; } /** * Return the 0-based offset of the leftmost reference position involved in * the alignment. */ const Interval& refival() const { return refival_; } /** * Return the 0-based offset of the leftmost reference position involved in * the alignment. */ Coord& refcoord() { return refcoord_; } /** * Return the 0-based offset of the rightmost reference position involved in * the alignment. */ Coord refcoord_right() const { Coord coord_right = refcoord_; TRefOff right = coord_right.off() + rfextent_ - 1; for(size_t i = 0; i < ned_->size(); i++) { const Edit& ed = (*ned_)[i]; if(ed.type == EDIT_TYPE_SPL) { right += ed.splLen; } } coord_right.setOff(right); return coord_right; } /** * Return true if this alignment is to the Watson strand. */ inline bool fw() const { return refcoord_.fw(); } AlnScore score() const { return score_; } AlnScore oscore() const { return oscore_; } EList& ned() { return *ned_; } EList& aed() { return *aed_; } const EList& ned() const { return *ned_; } const EList& aed() const { return *aed_; } size_t readExtent() const { return rdextent_; } size_t readExtentRows() const { return rdexrows_; } size_t readLength() const { return rdlen_; } TReadId readID() const { return rdid_; } bool spliced() const { return num_spliced_ > 0; } size_t num_spliced() const { return num_spliced_; } uint8_t spliced_whichsense_transcript() const { uint8_t whichsense = SPL_UNKNOWN; if(spliced()) { for(size_t i = 0; i < ned_->size(); i++) { const Edit& ed = (*ned_)[i]; if(ed.type != EDIT_TYPE_SPL) continue; if(whichsense == SPL_UNKNOWN) { whichsense = ed.splDir; } else if(ed.splDir != SPL_UNKNOWN) { assert_neq(whichsense, SPL_UNKNOWN); if(whichsense == SPL_FW || whichsense == SPL_SEMI_FW) { if(ed.splDir != SPL_FW && ed.splDir != SPL_SEMI_FW) { whichsense = SPL_UNKNOWN; break; } } if(whichsense == SPL_RC || whichsense == SPL_SEMI_RC) { if(ed.splDir != SPL_RC && ed.splDir != SPL_SEMI_RC) { whichsense = SPL_UNKNOWN; break; } } } } } return whichsense; } /** * Return the number of reference nucleotides involved in the alignment * (i.e. the number of characters in the inclusive range from the first * matched-up ref char to the last). */ size_t refExtent() const { return rfextent_; } /** * Return length of reference sequence aligned to. */ TRefOff reflen() const { return reflen_; } /** * Return the number of reference nucleotides in the alignment (i.e. the * number of characters in the inclusive range from the first matched-up * ref char to the last). */ size_t refNucExtent() const { return rfextent_; } /** * Print the sequence for the read that aligned using A, C, G and * T. This will simply print the read sequence (or its reverse * complement). */ void printSeq( const Read& rd, const BTDnaString* dns, BTString& o) const; /** * Print the quality string for the read that aligned. This will * simply print the read qualities (or their reverse). */ void printQuals( const Read& rd, const BTString* dqs, BTString& o) const; /** * Print a stacked alignment with the reference on top, query on bottom, * and lines connecting matched-up positions. */ void printStacked( const Read& rd, std::ostream& o) const { printStacked(refcoord_.fw() ? rd.patFw : rd.patRc, o); } /** * Print a stacked alignment with the reference on bottom, query on top, * and lines connecting matched-up positions. */ void printStacked( const BTDnaString& seq, std::ostream& o) const { Edit::printQAlign(o, seq, *ned_); // Print reference offset below reference string o << "^" << std::endl; o << "(" << refcoord_.ref() << "," << refcoord_.off() << ")" << std::endl; } #ifndef NDEBUG /** * Check that alignment score is internally consistent. */ bool repOk() const { assert(refcoord_.repOk()); if(shapeSet_) { assert_lt(refoff(), reflen_); } assert(refival_.repOk()); assert(VALID_AL_SCORE(score_) || ned_ == NULL || ned_->empty()); assert(VALID_AL_SCORE(score_) || aed_ == NULL || aed_->empty()); assert(empty() || refcoord_.inited()); assert(empty() || refival_.inited()); assert_geq(rdexrows_, rdextent_); assert(empty() || rdextent_ > 0); assert(empty() || rfextent_ > 0); return true; } /** * Check that alignment score is internally consistent. */ bool repOk(const Read& rd) const { assert(Edit::repOk(*ned_, refcoord_.fw() ? rd.patFw : rd.patRc, refcoord_.fw(), trimmed5p(true), trimmed3p(true))); return repOk(); } #endif #ifndef NDEBUG /** * Assuming this AlnRes is an alignment for 'rd', check that the * alignment and 'rd' are compatible with the corresponding * reference sequence. */ bool matchesRef( const Read& rd, const BitPairReference& ref, BTDnaString& rf, BTDnaString& rdseq, BTString& qseq, SStringExpandable& raw_refbuf, SStringExpandable& destU32, EList& matches, SStringExpandable& raw_refbuf2, EList& reflens, EList& refoffs); #endif /** * Set information about the alignment parameters that led to this * alignment. */ void setParams( int seedmms, int seedlen, int seedival, int64_t minsc) { seedmms_ = seedmms; seedlen_ = seedlen; seedival_ = seedival; minsc_ = minsc; } // Accessors for alignment parameters int seedmms() const { return seedmms_; } int seedlen() const { return seedlen_; } int seedival() const { return seedival_; } int64_t minScore() const { return minsc_; } /** * Is the ith row from the 5' end of the DP table one of the ones * soft-trimmed away by local alignment? */ inline bool trimmedRow5p(size_t i) const { return i < trim5p_ || rdrows_ - i - 1 < trim3p_; } /** * Is the ith character from the 5' end of read sequence one of the ones * soft-trimmed away by local alignment? */ inline bool trimmedPos5p(size_t i) const { return i < trim5p_ || rdlen_ - i - 1 < trim3p_; } /** * Is the ith row from the 5' end of the DP table one of the ones that * survived local-alignment soft trimming? */ inline bool alignedRow5p(size_t i) const { return !trimmedRow5p(i); } /** * Is the ith character from the 5' end of the read sequence one of the * ones that survived local-alignment soft trimming? */ inline bool alignedPos5p(size_t i) const { return !trimmedPos5p(i); } /** * Return true iff this AlnRes and the given AlnRes overlap. Two AlnRess * overlap if they share a cell in the overall dynamic programming table: * i.e. if there exists a read position s.t. that position in both reads * matches up with the same reference character. E.g., the following * alignments (drawn schematically as paths through a dynamic programming * table) are redundant: * * a b a b * \ \ \ \ * \ \ \ \ * \ \ \ \ * ---\ \ \ * \ ---\--- * ---\ \ \ * \ \ \ \ * \ \ \ \ * \ \ \ \ * a b b a * * We iterate over each read position that hasn't been hard-trimmed, but * only overlaps at positions that have also not been soft-trimmed are * considered. */ bool overlap(AlnRes& res); /** * Return true iff this read was unpaired to begin with. */ inline bool readUnpaired() const { assert_gt(type_, 0); return type_ == ALN_RES_TYPE_UNPAIRED; } /** * Return true iff this alignment aligned in an unpaired fashion; not part * of a concordant or discordant pair. */ inline bool alignedUnpaired() const { assert_gt(type_, 0); return type_ == ALN_RES_TYPE_UNPAIRED || type_ == ALN_RES_TYPE_UNPAIRED_MATE1 || type_ == ALN_RES_TYPE_UNPAIRED_MATE2; } /** * Return true iff this alignment aligned as mate #1 or mate #2 in a pair, * either concordant or discordant. */ inline bool alignedPaired() const { assert_gt(type_, 0); return type_ == ALN_RES_TYPE_MATE1 || type_ == ALN_RES_TYPE_MATE2; } /** * Return true iff this read started as mate #1 in a pair. */ inline bool readMate1() const { assert_gt(type_, 0); return type_ == ALN_RES_TYPE_MATE1 || type_ == ALN_RES_TYPE_UNPAIRED_MATE1; } /** * Return true iff this read aligned as mate #1 in a concordant or * discordant pair. */ inline bool alignedMate1() const { assert_gt(type_, 0); return type_ == ALN_RES_TYPE_MATE1; } /** * Return true iff this alignment aligned as mate #2 in a pair, either * concordant or discordant. */ inline bool readMate2() const { assert_gt(type_, 0); return type_ == ALN_RES_TYPE_MATE2 || type_ == ALN_RES_TYPE_UNPAIRED_MATE2; } /** * Return true iff this read aligned as mate #2 in a concordant or * discordant pair. */ inline bool alignedMate2() const { assert_gt(type_, 0); return type_ == ALN_RES_TYPE_MATE2; } /** * Return true iff fragment length is set. */ bool isFraglenSet() const { return fraglenSet_; } /** * Set whether this alignment is unpaired, or is mate #1 or mate #2 in a * paired-end alignment. */ void setMateParams( int type, const AlnRes* omate, // alignment result for the opposite mate const AlnFlags& flags, // flags for this mate const SpliceSiteDB* ssdb = NULL, // splice sites uint64_t threads_rids_mindist = 0, EList* spliceSites = NULL) { assert_gt(type, 0); type_ = type; fraglen_ = 0; if(omate != NULL) { oscore_ = omate->score_; // When should we calculate a fragment length here? There are a // couple reasonable ideas: // 1. When mates align concordantly // 2. When both mates align to the same reference string // BWA seems to do 2., so that's what we'll do here. bool sameChr = true; if((sameChr && refcoord_.ref() == omate->refcoord_.ref()) || flags.alignedConcordant()) { setFragmentLength(*omate, ssdb, threads_rids_mindist, spliceSites); } else { assert(!isFraglenSet()); } } } /** * Assuming this alignment and the given alignment are at the extreme ends * of a fragment, return the length of the fragment. We take all clipping, * both hard and soft, into account here. Any clipping that occurred * earlier and isn't accounted for within Bowtie2 should be accounted for * by the user in how they set the maximum and minimum fragment length * settings. */ int64_t setFragmentLength(const AlnRes& omate, const SpliceSiteDB* ssdb = NULL, // splice sites uint64_t threads_rids_mindist = 0, EList* spliceSites = NULL) { Coord st, en, st2, en2; Coord ost, oen, ost2, oen2; assert_eq(refid(), omate.refid()); getExtendedCoords(st, en, st2, en2); omate.getExtendedCoords(ost, oen, ost2, oen2); bool imUpstream = false; if(st.off() < ost.off()) { imUpstream = true; } else if(st.off() == ost.off()) { if(st.fw() && ost.fw() && readMate1()) { imUpstream = true; } else if(st.fw() && !ost.fw()) { imUpstream = true; } else { imUpstream = false; } } else { imUpstream = false; } TRefOff up, dn, up_right, dn_left; if(imUpstream) { up = std::min(st2.off(), ost.off()); up_right = std::min(en2.off(), oen.off()); dn_left = std::max(st2.off(), ost.off()); dn = std::max(en2.off(), oen.off()); } else { up = std::min(st.off(), ost2.off()); up_right = std::min(en.off(), oen2.off()); dn_left = std::max(st.off(), ost2.off()); dn = std::max(en.off(), oen2.off()); } assert_geq(dn, up); TRefOff intron_len = 0; if(ssdb != NULL && !repeat() && up_right + 100 < dn_left) { assert(spliceSites != NULL); if(spliceSites->size() == 0) { ssdb->getRightSpliceSites(refid(), up_right, dn_left - up_right, *spliceSites); } for(size_t si = 0; si < spliceSites->size(); si++) { const SpliceSite& ss = (*spliceSites)[si]; if(!ss._fromfile && ss._readid + threads_rids_mindist > rdid_) continue; if(ss.left() <= up || ss.right() >= dn) continue; TRefOff tmp_intron_len = ss.intron_len(); if(intron_len < tmp_intron_len) { intron_len = tmp_intron_len; } } } fraglen_ = 1 + dn - up; assert_geq(fraglen_, intron_len); fraglen_ -= intron_len; if(!imUpstream) { fraglen_ = -fraglen_; } fraglenSet_ = true; return fraglen_; } /** * Return fragment length inferred by a paired-end alignment, or -1 if the * alignment is not part of a pair. */ int64_t fragmentLength() const { assert_gt(type_, 0); assert(fraglenSet_); return fraglen_; } /** * Initialize new AlnRes. */ void init( size_t rdlen, // # chars after hard trimming TReadId rdid, // read ID AlnScore score, // alignment score const EList* ned, // nucleotide edits size_t ned_i, // first position to copy size_t ned_n, // # positions to copy const EList* aed, // ambiguous base resolutions size_t aed_i, // first position to copy size_t aed_n, // # positions to copy Coord refcoord, // leftmost ref pos of 1st al char TRefOff reflen, // length of the reference LinkedEList >* raw_edits, int seedmms = -1,// # seed mms allowed int seedlen = -1,// seed length int seedival = -1,// space between seeds int64_t minsc = -1,// minimum score for valid aln int nuc5p = -1,// int nuc3p = -1, bool pretrimSoft = false, size_t pretrim5p = 0, // trimming prior to alignment size_t pretrim3p = 0, // trimming prior to alignment bool trimSoft = true, size_t trim5p = 0, // trimming from alignment size_t trim3p = 0, // trimming from alignment bool repeat = false); // repeat /** * Return number of bases trimmed from the 5' end. Argument determines * whether we're counting hard- or soft-trimmed bases. */ size_t trimmed5p(bool soft) const { size_t trim = 0; if(pretrimSoft_ == soft) trim += pretrim5p_; if(trimSoft_ == soft) trim += trim5p_; return trim; } /** * Return number of bases trimmed from the 3' end. Argument determines * whether we're counting hard- or soft-trimmed bases. */ size_t trimmed3p(bool soft) const { size_t trim = 0; if(pretrimSoft_ == soft) trim += pretrim3p_; if(trimSoft_ == soft) trim += trim3p_; return trim; } /** * Return number of bases trimmed from the left end. Argument determines * whether we're counting hard- or soft-trimmed bases. */ size_t trimmedLeft(bool soft) const { return fw() ? trimmed5p(soft) : trimmed3p(soft); } /** * Return number of bases trimmed from the right end. Argument determines * whether we're counting hard- or soft-trimmed bases. */ size_t trimmedRight(bool soft) const { return fw() ? trimmed3p(soft) : trimmed5p(soft); } bool repeat() const { return repeat_; } /** * Set the number of reference Ns covered by the alignment. */ void setRefNs(size_t refns) { refns_ = refns; } /** * Return the number of reference Ns covered by the alignment. */ size_t refNs() const { return refns_; } /** * Clip away portions of the alignment that are outside the given bounds. * Clipping is soft if soft == true, hard otherwise. */ void clipOutside(bool soft, TRefOff refi, TRefOff reff); /** * Soft trim bases from the LHS of the alignment. */ void clipLeft(size_t rd_amt, size_t rf_amt); /** * Soft trim bases from the RHS of the alignment. */ void clipRight(size_t rd_amt, size_t rf_amt); /** * In debug mode, we put a copy of the decoded nucleotide sequence here. */ ASSERT_ONLY(BTDnaString drd); /** * Return true iff this AlnRes should come before the given AlnRes in a * prioritized list of results. */ bool operator<(const AlnRes& o) const { return score_ > o.score_; } bool operator==(const AlnRes& o) const { return shapeSet_ == o.shapeSet_ && rdlen_ == o.rdlen_ && rdid_ == o.rdid_ && rdrows_ == o.rdrows_ && score_ == o.score_ && //oscore_ == o.oscore_ && *ned_ == *(o.ned_) && *aed_ == *(o.aed_) && refcoord_ == o.refcoord_ && reflen_ == o.reflen_ && refival_ == o.refival_ && rdextent_ == o.rdextent_ && rdexrows_ == o.rdexrows_ && rfextent_ == o.rfextent_ && seedmms_ == o.seedmms_ && seedlen_ == o.seedlen_ && seedival_ == o.seedival_ && minsc_ == o.minsc_ && nuc5p_ == o.nuc5p_ && nuc3p_ == o.nuc3p_ && refns_ == o.refns_ && type_ == o.type_ && fraglen_ == o.fraglen_ && pretrimSoft_ == o.pretrimSoft_ && pretrim5p_ == o.pretrim5p_ && pretrim3p_ == o.pretrim3p_ && trimSoft_ == o.trimSoft_ && trim5p_ == o.trim5p_ && trim3p_ == o.trim3p_ && repeat_ == o.repeat_ && num_spliced_ == o.num_spliced_; } /** * Initialize a StackedAln (stacked alignment) object w/r/t this alignment. */ void initStacked(const Read& rd, StackedAln& st) const { size_t trimLS = trimmed5p(true); size_t trimLH = trimmed5p(false); size_t trimRS = trimmed3p(true); size_t trimRH = trimmed3p(false); size_t len_trimmed = rd.length() - trimLS - trimRS; if(!fw()) { Edit::invertPoss(const_cast&>(*ned_), len_trimmed, false); swap(trimLS, trimRS); swap(trimLH, trimRH); } st.init( fw() ? rd.patFw : rd.patRc, *ned_, trimLS, trimLH, trimRS, trimRH); if(!fw()) { Edit::invertPoss(const_cast&>(*ned_), len_trimmed, false); } } protected: /** * Given that rdextent_ and ned_ are already set, calculate rfextent_. */ void calcRefExtent() { assert_gt(rdextent_, 0); rfextent_ = rdextent_; for(size_t i = 0; i < ned_->size(); i++) { if((*ned_)[i].isRefGap()) rfextent_--; if((*ned_)[i].isReadGap()) rfextent_++; } } bool shapeSet_; // true iff setShape() has been called size_t rdlen_; // length of the original read TReadId rdid_; // read id size_t rdrows_; // # rows in alignment problem AlnScore score_; // best SW score found AlnScore oscore_; // score of opposite mate EList* ned_; // base edits EList* aed_; // ambiguous base resolutions Coord refcoord_; // ref coordinates (seq idx, offset, orient) TRefOff reflen_; // reference length Interval refival_; // ref interval (coord + length) size_t rdextent_; // number of read chars involved in alignment size_t rdexrows_; // number of read rows involved in alignment size_t rfextent_; // number of ref chars involved in alignment int seedmms_; // number of mismatches allowed in seed int seedlen_; // length of seed int seedival_; // interval between seeds int64_t minsc_; // minimum score int nuc5p_; // 5'-most decoded base; clipped if excluding end int nuc3p_; // 3'-most decoded base; clipped if excluding end size_t refns_; // # of reference Ns overlapped int type_; // unpaired or mate #1 or mate #2? bool fraglenSet_; // true iff a fragment length has been inferred int64_t fraglen_; // inferred fragment length // A tricky aspect of trimming is that we have to decide what the units are: // read positions, reference positions??? We choose read positions here. // In other words, if an alignment overhangs the end of the reference and // part of the overhanging portion is a reference gap, we have to make sure // the trim amount reflects the number of *read characters* to trim // including the character opposite the reference gap. // Nucleotide-sequence trimming bool pretrimSoft_; // trimming prior to alignment is soft? size_t pretrim5p_; // # bases trimmed from 5p end prior to alignment size_t pretrim3p_; // # bases trimmed from 3p end prior to alignment bool trimSoft_; // trimming by local alignment is soft? size_t trim5p_; // # bases trimmed from 5p end by local alignment size_t trim3p_; // # bases trimmed from 3p end by local alignment bool repeat_; // repeat? size_t num_spliced_; LinkedEListNode >* ned_node_; LinkedEListNode >* aed_node_; LinkedEList >* raw_edits_; }; /** * Unique ID for a cell in the overall DP table. This is a helpful concept * because of our definition of "redundnant". Two alignments are redundant iff * they have at least one cell in common in the overall DP table. */ struct RedundantCell { RedundantCell() { rfid = 0; fw = true; rfoff = 0; rdoff = 0; } RedundantCell( TRefId rfid_, bool fw_, TRefOff rfoff_, size_t rdoff_) { init(rfid_, fw_, rfoff_, rdoff_); } void init( TRefId rfid_, bool fw_, TRefOff rfoff_, size_t rdoff_) { rfid = rfid_; fw = fw_; rfoff = rfoff_; rdoff = rdoff_; } /** * Return true iff this RedundantCell is less than the given RedundantCell. */ inline bool operator<(const RedundantCell& c) const { if(rfid < c.rfid) return true; if(rfid > c.rfid) return false; if(!fw && c.fw) return true; if( fw && !c.fw) return false; if(rfoff < c.rfoff) return true; if(rfoff > c.rfoff) return false; return rdoff < c.rdoff; } /** * Return true iff this RedundantCell is greater than the given * RedundantCell. */ inline bool operator>(const RedundantCell& c) const { if(rfid > c.rfid) return true; if(rfid < c.rfid) return false; if( fw && !c.fw) return true; if(!fw && c.fw) return false; if(rfoff > c.rfoff) return true; if(rfoff < c.rfoff) return false; return rdoff > c.rdoff; } /** * Return true iff this RedundantCell is equal to the given RedundantCell. */ inline bool operator==(const RedundantCell& c) const { return rfid == c.rfid && fw == c.fw && rfoff == c.rfoff && rdoff == c.rdoff; } TRefId rfid; // reference id bool fw; // orientation TRefOff rfoff; // column size_t rdoff; // row }; /** * Encapsulates data structures and routines allowing client to determine * whether one alignment is redundant (has a DP cell in common with) with a set * of others. * * Adding cells to and checking cell against this data structure can get rather * slow when there are many alignments in play. Dividing the burden over * read-position bins helps some. */ class RedundantAlns { public: RedundantAlns(int cat = DP_CAT) : cells_(cat) { } /** * Empty the cell database. */ void reset() { cells_.clear(); } /** * Initialize and set the list of sets to equal the read length. */ void init(size_t npos) { cells_.resize(npos); for(size_t i = 0; i < npos; i++) { cells_[i].clear(); } } /** * Add all of the cells involved in the given alignment to the database. */ void add(const AlnRes& res); /** * Return true iff the given alignment has at least one cell that overlaps * one of the cells in the database. */ bool overlap(const AlnRes& res); protected: EList > cells_; }; typedef uint64_t TNumAlns; /** * Encapsulates a concise summary of a set of alignment results for a * given pair or mate. Referring to the fields of this object should * provide enough information to print output records for the read. */ class AlnSetSumm { public: AlnSetSumm() { reset(); } /** * Given an unpaired read (in either rd1 or rd2) or a read pair * (mate 1 in rd1, mate 2 in rd2). */ explicit AlnSetSumm( const Read* rd1, const Read* rd2, const EList* rs1, const EList* rs2, const EList* rs1u, const EList* rs2u, bool exhausted1, bool exhausted2, TRefId orefid, TRefOff orefoff, bool repeat) { init(rd1, rd2, rs1, rs2, rs1u, rs2u, exhausted1, exhausted2, orefid, orefoff, repeat); } explicit AlnSetSumm( AlnScore best1, AlnScore secbest1, AlnScore best2, AlnScore secbest2, AlnScore bestPaired, AlnScore secbestPaired, TNumAlns other1, TNumAlns other2, bool paired, bool exhausted1, bool exhausted2, TRefId orefid, TRefOff orefoff, bool repeat, TNumAlns numAlns1, TNumAlns numAlns2, TNumAlns numAlnsPaired) { init( best1, secbest1, best2, secbest2, bestPaired, secbestPaired, other1, other2, paired, exhausted1, exhausted2, orefid, orefoff, repeat, numAlns1, numAlns2, numAlnsPaired); } /** * Set to uninitialized state. */ void reset() { best1_.invalidate(); secbest1_.invalidate(); best2_.invalidate(); secbest2_.invalidate(); bestPaired_.invalidate(); secbestPaired_.invalidate(); other1_ = other2_ = 0; paired_ = false; exhausted1_ = exhausted2_ = false; orefid_ = -1; orefoff_ = -1; repeat_ = false; numAlns1_ = numAlns2_= numAlnsPaired_ = 0; } void init( const Read* rd1, const Read* rd2, const EList* rs1, const EList* rs2, const EList* rs1u, const EList* rs2u, bool exhausted1, bool exhausted2, TRefId orefid, TRefOff orefoff, bool repeat); /** * Initialize given fields. See constructor for how fields are set. */ void init( AlnScore best1, AlnScore secbest1, AlnScore best2, AlnScore secbest2, AlnScore bestPaired, AlnScore secbestPaired, TNumAlns other1, TNumAlns other2, bool paired, bool exhausted1, bool exhausted2, TRefId orefid, TRefOff orefoff, bool repeat, TNumAlns numAlns1, TNumAlns numAlns2, TNumAlns numAlnsPaired) { best1_ = best1; secbest1_ = secbest1; best2_ = best2; secbest2_ = secbest2; bestPaired_ = bestPaired; secbestPaired_ = secbestPaired; other1_ = other1; other2_ = other2; paired_ = paired; exhausted1_ = exhausted1; exhausted2_ = exhausted2; orefid_ = orefid; orefoff_ = orefoff; repeat_ = repeat; numAlns1_ = numAlns1; numAlns2_ = numAlns2; numAlnsPaired_ = numAlnsPaired; assert(repOk()); } /** * Return true iff there is at least a best alignment */ bool empty() const { assert(repOk()); return !VALID_AL_SCORE(best1_); } #ifndef NDEBUG /** * Check that the summary is internally consistent. */ bool repOk() const { assert(other1_ == 0 || VALID_AL_SCORE(secbest1_)); assert(other1_ != 0 || !VALID_AL_SCORE(secbest1_)); assert(other2_ == 0 || VALID_AL_SCORE(secbest2_)); assert(other2_ != 0 || !VALID_AL_SCORE(secbest2_)); return true; } #endif AlnScore best1() const { return best1_; } AlnScore secbest1() const { return secbest1_; } AlnScore best2() const { return best2_; } AlnScore secbest2() const { return secbest2_; } AlnScore bestPaired() const { return bestPaired_; } AlnScore secbestPaired() const { return secbestPaired_; } TNumAlns other1() const { return other1_; } TNumAlns other2() const { return other2_; } bool paired() const { return paired_; } bool exhausted1() const { return exhausted1_; } bool exhausted2() const { return exhausted2_; } TRefId orefid() const { return orefid_; } TRefOff orefoff() const { return orefoff_; } bool repeat() const { return repeat_; } TNumAlns numAlns1() const { return numAlns1_; } TNumAlns numAlns2() const { return numAlns2_; } TNumAlns numAlnsPaired() const { return numAlnsPaired_; } void numAlns1(TNumAlns numAlns1) { numAlns1_ = numAlns1; } void numAlns2(TNumAlns numAlns2) { numAlns2_ = numAlns2; } void numAlnsPaired(TNumAlns numAlnsPaired) { numAlnsPaired_ = numAlnsPaired; } /** * */ AlnScore best(bool mate1) const { return mate1 ? best1_ : best2_; } bool exhausted(bool mate1) const { return mate1 ? exhausted1_ : exhausted2_; } /** * Return the second-best score for the specified mate. If the alignment * is paired and the specified mate aligns uniquely, return an invalid * second-best score. This allows us to treat mates separately, so that * repetitive paired-end alignments don't trump potentially unique unpaired * alignments. */ AlnScore secbestMate(bool mate1) const { return mate1 ? secbest1_ : secbest2_; } /** * Return the second-best score for the specified mate. If the alignment * is paired and the specified mate aligns uniquely, return an invalid * second-best score. This allows us to treat mates separately, so that * repetitive paired-end alignments don't trump potentially unique unpaired * alignments. */ AlnScore secbest(bool mate1) const { if(paired_) { if(mate1) { //if(!secbest1_.valid()) { return secbest1_; //} } else { //if(!secbest2_.valid()) { return secbest2_; //} } //return secbestPaired_; } else { return mate1 ? secbest1_ : secbest2_; } } protected: AlnScore bestPaired_; // best full-alignment score found for this read AlnScore secbestPaired_; // second-best AlnScore best1_; // best full-alignment score found for this read AlnScore secbest1_; // second-best AlnScore best2_; // best full-alignment score found for this read AlnScore secbest2_; // second-best TNumAlns other1_; // # more alignments within N points of second-best TNumAlns other2_; // # more alignments within N points of second-best bool paired_; // results are paired bool exhausted1_; // searched exhaustively for mate 1 alignments? bool exhausted2_; // searched exhaustively for mate 2 alignments? TRefId orefid_; TRefOff orefoff_; bool repeat_; TNumAlns numAlns1_; // number of alignments for mate 1 as singleton or discordantly mapped TNumAlns numAlns2_; // number of alignments for mate 2 as singleton or discordantly mapped TNumAlns numAlnsPaired_; // number of concordant pair alignments }; #endif hisat2-2.2.1/aligner_seed.cpp000066400000000000000000000401361370663763200160540ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "aligner_cache.h" #include "aligner_seed.h" #include "search_globals.h" #include "gfm.h" using namespace std; /** * Construct a constraint with no edits of any kind allowed. */ Constraint Constraint::exact() { Constraint c; c.edits = c.mms = c.ins = c.dels = c.penalty = 0; return c; } /** * Construct a constraint where the only constraint is a total * penalty constraint. */ Constraint Constraint::penaltyBased(int pen) { Constraint c; c.penalty = pen; return c; } /** * Construct a constraint where the only constraint is a total * penalty constraint related to the length of the read. */ Constraint Constraint::penaltyFuncBased(const SimpleFunc& f) { Constraint c; c.penFunc = f; return c; } /** * Construct a constraint where the only constraint is a total * penalty constraint. */ Constraint Constraint::mmBased(int mms) { Constraint c; c.mms = mms; c.edits = c.dels = c.ins = 0; return c; } /** * Construct a constraint where the only constraint is a total * penalty constraint. */ Constraint Constraint::editBased(int edits) { Constraint c; c.edits = edits; c.dels = c.ins = c.mms = 0; return c; } // // Some static methods for constructing some standard SeedPolicies // /** * Given a read, depth and orientation, extract a seed data structure * from the read and fill in the steps & zones arrays. The Seed * contains the sequence and quality values. */ bool Seed::instantiate( const Read& read, const BTDnaString& seq, // seed read sequence const BTString& qual, // seed quality sequence const Scoring& pens, int depth, int seedoffidx, int seedtypeidx, bool fw, InstantiatedSeed& is) const { assert(overall != NULL); int seedlen = len; if((int)read.length() < seedlen) { // Shrink seed length to fit read if necessary seedlen = (int)read.length(); } assert_gt(seedlen, 0); is.steps.resize(seedlen); is.zones.resize(seedlen); // Fill in 'steps' and 'zones' // // The 'steps' list indicates which read character should be // incorporated at each step of the search process. Often we will // simply proceed from one end to the other, in which case the // 'steps' list is ascending or descending. In some cases (e.g. // the 2mm case), we might want to switch directions at least once // during the search, in which case 'steps' will jump in the // middle. When an element of the 'steps' list is negative, this // indicates that the next // // The 'zones' list indicates which zone constraint is active at // each step. Each element of the 'zones' list is a pair; the // first pair element indicates the applicable zone when // considering either mismatch or delete (ref gap) events, while // the second pair element indicates the applicable zone when // considering insertion (read gap) events. When either pair // element is a negative number, that indicates that we are about // to leave the zone for good, at which point we may need to // evaluate whether we have reached the zone's budget. // switch(type) { case SEED_TYPE_EXACT: { for(int k = 0; k < seedlen; k++) { is.steps[k] = -(seedlen - k); // Zone 0 all the way is.zones[k].first = is.zones[k].second = 0; } break; } case SEED_TYPE_LEFT_TO_RIGHT: { for(int k = 0; k < seedlen; k++) { is.steps[k] = k+1; // Zone 0 from 0 up to ceil(len/2), then 1 is.zones[k].first = is.zones[k].second = ((k < (seedlen+1)/2) ? 0 : 1); } // Zone 1 ends at the RHS is.zones[seedlen-1].first = is.zones[seedlen-1].second = -1; break; } case SEED_TYPE_RIGHT_TO_LEFT: { for(int k = 0; k < seedlen; k++) { is.steps[k] = -(seedlen - k); // Zone 0 from 0 up to floor(len/2), then 1 is.zones[k].first = ((k < seedlen/2) ? 0 : 1); // Inserts: Zone 0 from 0 up to ceil(len/2)-1, then 1 is.zones[k].second = ((k < (seedlen+1)/2+1) ? 0 : 1); } is.zones[seedlen-1].first = is.zones[seedlen-1].second = -1; break; } case SEED_TYPE_INSIDE_OUT: { // Zone 0 from ceil(N/4) up to N-floor(N/4) int step = 0; for(int k = (seedlen+3)/4; k < seedlen - (seedlen/4); k++) { is.zones[step].first = is.zones[step].second = 0; is.steps[step++] = k+1; } // Zone 1 from N-floor(N/4) up for(int k = seedlen - (seedlen/4); k < seedlen; k++) { is.zones[step].first = is.zones[step].second = 1; is.steps[step++] = k+1; } // No Zone 1 if seedlen is short (like 2) //assert_eq(1, is.zones[step-1].first); is.zones[step-1].first = is.zones[step-1].second = -1; // Zone 2 from ((seedlen+3)/4)-1 down to 0 for(int k = ((seedlen+3)/4)-1; k >= 0; k--) { is.zones[step].first = is.zones[step].second = 2; is.steps[step++] = -(k+1); } assert_eq(2, is.zones[step-1].first); is.zones[step-1].first = is.zones[step-1].second = -2; assert_eq(seedlen, step); break; } default: throw 1; } // Instantiate constraints for(int i = 0; i < 3; i++) { is.cons[i] = zones[i]; is.cons[i].instantiate(read.length()); } is.overall = *overall; is.overall.instantiate(read.length()); // Take a sweep through the seed sequence. Consider where the Ns // occur and how zones are laid out. Calculate the maximum number // of positions we can jump over initially (e.g. with the ftab) and // perhaps set this function's return value to false, indicating // that the arrangements of Ns prevents the seed from aligning. bool streak = true; is.maxjump = 0; bool ret = true; bool ltr = (is.steps[0] > 0); // true -> left-to-right for(size_t i = 0; i < is.steps.size(); i++) { assert_neq(0, is.steps[i]); int off = is.steps[i]; off = abs(off)-1; Constraint& cons = is.cons[abs(is.zones[i].first)]; int c = seq[off]; assert_range(0, 4, c); int q = qual[off]; if(ltr != (is.steps[i] > 0) || // changed direction is.zones[i].first != 0 || // changed zone is.zones[i].second != 0) // changed zone { streak = false; } if(c == 4) { // Induced mismatch if(cons.canN(q, pens)) { cons.chargeN(q, pens); } else { // Seed disqualified due to arrangement of Ns return false; } } if(streak) is.maxjump++; } is.seedoff = depth; is.seedoffidx = seedoffidx; is.fw = fw; is.s = *this; return ret; } /** * Return a set consisting of 1 seed encapsulating an exact matching * strategy. */ void Seed::zeroMmSeeds(int ln, EList& pols, Constraint& oall) { oall.init(); // Seed policy 1: left-to-right search pols.expand(); pols.back().len = ln; pols.back().type = SEED_TYPE_EXACT; pols.back().zones[0] = Constraint::exact(); pols.back().zones[1] = Constraint::exact(); pols.back().zones[2] = Constraint::exact(); // not used pols.back().overall = &oall; } /** * Return a set of 2 seeds encapsulating a half-and-half 1mm strategy. */ void Seed::oneMmSeeds(int ln, EList& pols, Constraint& oall) { oall.init(); // Seed policy 1: left-to-right search pols.expand(); pols.back().len = ln; pols.back().type = SEED_TYPE_LEFT_TO_RIGHT; pols.back().zones[0] = Constraint::exact(); pols.back().zones[1] = Constraint::mmBased(1); pols.back().zones[2] = Constraint::exact(); // not used pols.back().overall = &oall; // Seed policy 2: right-to-left search pols.expand(); pols.back().len = ln; pols.back().type = SEED_TYPE_RIGHT_TO_LEFT; pols.back().zones[0] = Constraint::exact(); pols.back().zones[1] = Constraint::mmBased(1); pols.back().zones[1].mmsCeil = 0; pols.back().zones[2] = Constraint::exact(); // not used pols.back().overall = &oall; } /** * Return a set of 3 seeds encapsulating search roots for: * * 1. Starting from the left-hand side and searching toward the * right-hand side allowing 2 mismatches in the right half. * 2. Starting from the right-hand side and searching toward the * left-hand side allowing 2 mismatches in the left half. * 3. Starting (effectively) from the center and searching out toward * both the left and right-hand sides, allowing one mismatch on * either side. * * This is not exhaustive. There are 2 mismatch cases mised; if you * imagine the seed as divided into four successive quarters A, B, C * and D, the cases we miss are when mismatches occur in A and C or B * and D. */ void Seed::twoMmSeeds(int ln, EList& pols, Constraint& oall) { oall.init(); // Seed policy 1: left-to-right search pols.expand(); pols.back().len = ln; pols.back().type = SEED_TYPE_LEFT_TO_RIGHT; pols.back().zones[0] = Constraint::exact(); pols.back().zones[1] = Constraint::mmBased(2); pols.back().zones[2] = Constraint::exact(); // not used pols.back().overall = &oall; // Seed policy 2: right-to-left search pols.expand(); pols.back().len = ln; pols.back().type = SEED_TYPE_RIGHT_TO_LEFT; pols.back().zones[0] = Constraint::exact(); pols.back().zones[1] = Constraint::mmBased(2); pols.back().zones[1].mmsCeil = 1; // Must have used at least 1 mismatch pols.back().zones[2] = Constraint::exact(); // not used pols.back().overall = &oall; // Seed policy 3: inside-out search pols.expand(); pols.back().len = ln; pols.back().type = SEED_TYPE_INSIDE_OUT; pols.back().zones[0] = Constraint::exact(); pols.back().zones[1] = Constraint::mmBased(1); pols.back().zones[1].mmsCeil = 0; // Must have used at least 1 mismatch pols.back().zones[2] = Constraint::mmBased(1); pols.back().zones[2].mmsCeil = 0; // Must have used at least 1 mismatch pols.back().overall = &oall; } /** * Types of actions that can be taken by the SeedAligner. */ enum { SA_ACTION_TYPE_RESET = 1, SA_ACTION_TYPE_SEARCH_SEED, // 2 SA_ACTION_TYPE_FTAB, // 3 SA_ACTION_TYPE_FCHR, // 4 SA_ACTION_TYPE_MATCH, // 5 SA_ACTION_TYPE_EDIT // 6 }; #define MIN(x, y) ((x < y) ? x : y) #ifdef ALIGNER_SEED_MAIN #include #include /** * Parse an int out of optarg and enforce that it be at least 'lower'; * if it is less than 'lower', than output the given error message and * exit with an error and a usage message. */ static int parseInt(const char *errmsg, const char *arg) { long l; char *endPtr = NULL; l = strtol(arg, &endPtr, 10); if (endPtr != NULL) { return (int32_t)l; } cerr << errmsg << endl; throw 1; return -1; } enum { ARG_NOFW = 256, ARG_NORC, ARG_MM, ARG_SHMEM, ARG_TESTS, ARG_RANDOM_TESTS, ARG_SEED }; static const char *short_opts = "vCt"; static struct option long_opts[] = { {(char*)"verbose", no_argument, 0, 'v'}, {(char*)"color", no_argument, 0, 'C'}, {(char*)"timing", no_argument, 0, 't'}, {(char*)"nofw", no_argument, 0, ARG_NOFW}, {(char*)"norc", no_argument, 0, ARG_NORC}, {(char*)"mm", no_argument, 0, ARG_MM}, {(char*)"shmem", no_argument, 0, ARG_SHMEM}, {(char*)"tests", no_argument, 0, ARG_TESTS}, {(char*)"random", required_argument, 0, ARG_RANDOM_TESTS}, {(char*)"seed", required_argument, 0, ARG_SEED}, }; static void printUsage(ostream& os) { os << "Usage: ac [options]* " << endl; os << "Options:" << endl; os << " --mm memory-mapped mode" << endl; os << " --shmem shared memory mode" << endl; os << " --nofw don't align forward-oriented read" << endl; os << " --norc don't align reverse-complemented read" << endl; os << " -t/--timing show timing information" << endl; os << " -C/--color colorspace mode" << endl; os << " -v/--verbose talkative mode" << endl; } bool gNorc = false; bool gNofw = false; bool gColor = false; int gVerbose = 0; int gGapBarrier = 1; bool gColorExEnds = true; int gSnpPhred = 30; bool gReportOverhangs = true; extern void aligner_seed_tests(); extern void aligner_random_seed_tests( int num_tests, uint32_t qslo, uint32_t qshi, bool color, uint32_t seed); /** * A way of feeding simply tests to the seed alignment infrastructure. */ int main(int argc, char **argv) { bool useMm = false; bool useShmem = false; bool mmSweep = false; bool noRefNames = false; bool sanity = false; bool timing = false; int option_index = 0; int seed = 777; int next_option; do { next_option = getopt_long( argc, argv, short_opts, long_opts, &option_index); switch (next_option) { case 'v': gVerbose = true; break; case 'C': gColor = true; break; case 't': timing = true; break; case ARG_NOFW: gNofw = true; break; case ARG_NORC: gNorc = true; break; case ARG_MM: useMm = true; break; case ARG_SHMEM: useShmem = true; break; case ARG_SEED: seed = parseInt("", optarg); break; case ARG_TESTS: { aligner_seed_tests(); aligner_random_seed_tests( 100, // num references 100, // queries per reference lo 400, // queries per reference hi false, // true -> generate colorspace reference/reads 18); // pseudo-random seed return 0; } case ARG_RANDOM_TESTS: { seed = parseInt("", optarg); aligner_random_seed_tests( 100, // num references 100, // queries per reference lo 400, // queries per reference hi false, // true -> generate colorspace reference/reads seed); // pseudo-random seed return 0; } case -1: break; default: { cerr << "Unknown option: " << (char)next_option << endl; printUsage(cerr); exit(1); } } } while(next_option != -1); char *reffn; if(optind >= argc) { cerr << "No reference; quitting..." << endl; return 1; } reffn = argv[optind++]; if(optind >= argc) { cerr << "No reads; quitting..." << endl; return 1; } string gfmBase(reffn); BitPairReference ref( gfmBase, // base path gColor, // whether we expect it to be colorspace sanity, // whether to sanity-check reference as it's loaded NULL, // fasta files to sanity check reference against NULL, // another way of specifying original sequences false, // true -> infiles (2 args ago) contains raw seqs useMm, // use memory mapping to load index? useShmem, // use shared memory (not memory mapping) mmSweep, // touch all the pages after memory-mapping the index gVerbose, // verbose gVerbose); // verbose but just for startup messages Timer *t = new Timer(cerr, "Time loading fw index: ", timing); GFM gfmFw( gfmBase, 0, // don't need entireReverse for fw index true, // index is for the forward direction -1, // offrate (irrelevant) useMm, // whether to use memory-mapped files useShmem, // whether to use shared memory mmSweep, // sweep memory-mapped files !noRefNames, // load names? false, // load SA sample? true, // load ftab? true, // load rstarts? NULL, // reference map, or NULL if none is needed gVerbose, // whether to be talkative gVerbose, // talkative during initialization false, // handle memory exceptions, don't pass them up sanity); delete t; t = new Timer(cerr, "Time loading bw index: ", timing); GFM gfmBw( gfmBase + ".rev", 1, // need entireReverse false, // index is for the backward direction -1, // offrate (irrelevant) useMm, // whether to use memory-mapped files useShmem, // whether to use shared memory mmSweep, // sweep memory-mapped files !noRefNames, // load names? false, // load SA sample? true, // load ftab? false, // load rstarts? NULL, // reference map, or NULL if none is needed gVerbose, // whether to be talkative gVerbose, // talkative during initialization false, // handle memory exceptions, don't pass them up sanity); delete t; for(int i = optind; i < argc; i++) { } } #endif hisat2-2.2.1/aligner_seed.h000066400000000000000000002477671370663763200155440ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_SEED_H_ #define ALIGNER_SEED_H_ #include #include #include #include "qual.h" #include "ds.h" #include "sstring.h" #include "alphabet.h" #include "edit.h" #include "read.h" // Threading is necessary to synchronize the classes that dump // intermediate alignment results to files. Otherwise, all data herein // is constant and shared, or per-thread. #include "threading.h" #include "aligner_result.h" #include "aligner_cache.h" #include "scoring.h" #include "mem_ids.h" #include "simple_func.h" #include "btypes.h" /** * A constraint to apply to an alignment zone, or to an overall * alignment. * * The constraint can put both caps and ceilings on the number and * types of edits allowed. */ struct Constraint { Constraint() { init(); } /** * Initialize Constraint to be fully permissive. */ void init() { edits = mms = ins = dels = penalty = editsCeil = mmsCeil = insCeil = delsCeil = penaltyCeil = MAX_I; penFunc.reset(); instantiated = false; } /** * Return true iff penalities and constraints prevent us from * adding any edits. */ bool mustMatch() { assert(instantiated); return (mms == 0 && edits == 0) || penalty == 0 || (mms == 0 && dels == 0 && ins == 0); } /** * Return true iff a mismatch of the given quality is permitted. */ bool canMismatch(int q, const Scoring& cm) { assert(instantiated); return (mms > 0 || edits > 0) && penalty >= cm.mm(q); } /** * Return true iff a mismatch of the given quality is permitted. */ bool canN(int q, const Scoring& cm) { assert(instantiated); return (mms > 0 || edits > 0) && penalty >= cm.n(q); } /** * Return true iff a mismatch of *any* quality (even qual=1) is * permitted. */ bool canMismatch() { assert(instantiated); return (mms > 0 || edits > 0) && penalty > 0; } /** * Return true iff a mismatch of *any* quality (even qual=1) is * permitted. */ bool canN() { assert(instantiated); return (mms > 0 || edits > 0); } /** * Return true iff a deletion of the given extension (0=open, 1=1st * extension, etc) is permitted. */ bool canDelete(int ex, const Scoring& cm) { assert(instantiated); return (dels > 0 && edits > 0) && penalty >= cm.del(ex); } /** * Return true iff a deletion of any extension is permitted. */ bool canDelete() { assert(instantiated); return (dels > 0 || edits > 0) && penalty > 0; } /** * Return true iff an insertion of the given extension (0=open, * 1=1st extension, etc) is permitted. */ bool canInsert(int ex, const Scoring& cm) { assert(instantiated); return (ins > 0 || edits > 0) && penalty >= cm.ins(ex); } /** * Return true iff an insertion of any extension is permitted. */ bool canInsert() { assert(instantiated); return (ins > 0 || edits > 0) && penalty > 0; } /** * Return true iff a gap of any extension is permitted */ bool canGap() { assert(instantiated); return ((ins > 0 || dels > 0) || edits > 0) && penalty > 0; } /** * Charge a mismatch of the given quality. */ void chargeMismatch(int q, const Scoring& cm) { assert(instantiated); if(mms == 0) { assert_gt(edits, 0); edits--; } else mms--; penalty -= cm.mm(q); assert_geq(mms, 0); assert_geq(edits, 0); assert_geq(penalty, 0); } /** * Charge an N mismatch of the given quality. */ void chargeN(int q, const Scoring& cm) { assert(instantiated); if(mms == 0) { assert_gt(edits, 0); edits--; } else mms--; penalty -= cm.n(q); assert_geq(mms, 0); assert_geq(edits, 0); assert_geq(penalty, 0); } /** * Charge a deletion of the given extension. */ void chargeDelete(int ex, const Scoring& cm) { assert(instantiated); dels--; edits--; penalty -= cm.del(ex); assert_geq(dels, 0); assert_geq(edits, 0); assert_geq(penalty, 0); } /** * Charge an insertion of the given extension. */ void chargeInsert(int ex, const Scoring& cm) { assert(instantiated); ins--; edits--; penalty -= cm.ins(ex); assert_geq(ins, 0); assert_geq(edits, 0); assert_geq(penalty, 0); } /** * Once the constrained area is completely explored, call this * function to check whether there were *at least* as many * dissimilarities as required by the constraint. Bounds like this * are helpful to resolve instances where two search roots would * otherwise overlap in what alignments they can find. */ bool acceptable() { assert(instantiated); return edits <= editsCeil && mms <= mmsCeil && ins <= insCeil && dels <= delsCeil && penalty <= penaltyCeil; } /** * Instantiate a constraint w/r/t the read length and the constant * and linear coefficients for the penalty function. */ static int instantiate(size_t rdlen, const SimpleFunc& func) { return func.f((double)rdlen); } /** * Instantiate this constraint w/r/t the read length. */ void instantiate(size_t rdlen) { assert(!instantiated); if(penFunc.initialized()) { penalty = Constraint::instantiate(rdlen, penFunc); } instantiated = true; } int edits; // # edits permitted int mms; // # mismatches permitted int ins; // # insertions permitted int dels; // # deletions permitted int penalty; // penalty total permitted int editsCeil; // <= this many edits can be left at the end int mmsCeil; // <= this many mismatches can be left at the end int insCeil; // <= this many inserts can be left at the end int delsCeil; // <= this many deletions can be left at the end int penaltyCeil;// <= this much leftover penalty can be left at the end SimpleFunc penFunc;// penalty function; function of read len bool instantiated; // whether constraint is instantiated w/r/t read len // // Some static methods for constructing some standard Constraints // /** * Construct a constraint with no edits of any kind allowed. */ static Constraint exact(); /** * Construct a constraint where the only constraint is a total * penalty constraint. */ static Constraint penaltyBased(int pen); /** * Construct a constraint where the only constraint is a total * penalty constraint related to the length of the read. */ static Constraint penaltyFuncBased(const SimpleFunc& func); /** * Construct a constraint where the only constraint is a total * penalty constraint. */ static Constraint mmBased(int mms); /** * Construct a constraint where the only constraint is a total * penalty constraint. */ static Constraint editBased(int edits); }; /** * We divide seed search strategies into three categories: * * 1. A left-to-right search where the left half of the read is * constrained to match exactly and the right half is subject to * some looser constraint (e.g. 1mm or 2mm) * 2. Same as 1, but going right to left with the exact matching half * on the right. * 3. Inside-out search where the center half of the read is * constrained to match exactly, and the extreme quarters of the * read are subject to a looser constraint. */ enum { SEED_TYPE_EXACT = 1, SEED_TYPE_LEFT_TO_RIGHT, SEED_TYPE_RIGHT_TO_LEFT, SEED_TYPE_INSIDE_OUT }; struct InstantiatedSeed; /** * Policy dictating how to size and arrange seeds along the length of * the read, and what constraints to force on the zones of the seed. * We assume that seeds are plopped down at regular intervals from the * 5' to 3' ends, with the first seed flush to the 5' end. * * If the read is shorter than a single seed, one seed is used and it * is shrunk to accommodate the read. */ struct Seed { int len; // length of a seed int type; // dictates anchor portion, direction of search Constraint *overall; // for the overall alignment Seed() { init(0, 0, NULL); } /** * Construct and initialize this seed with given length and type. */ Seed(int ln, int ty, Constraint* oc) { init(ln, ty, oc); } /** * Initialize this seed with given length and type. */ void init(int ln, int ty, Constraint* oc) { len = ln; type = ty; overall = oc; } // If the seed is split into halves, we just use zones[0] and // zones[1]; 0 is the near half and 1 is the far half. If the seed // is split into thirds (i.e. inside-out) then 0 is the center, 1 // is the far portion on the left, and 2 is the far portion on the // right. Constraint zones[3]; /** * Once the constrained seed is completely explored, call this * function to check whether there were *at least* as many * dissimilarities as required by all constraints. Bounds like this * are helpful to resolve instances where two search roots would * otherwise overlap in what alignments they can find. */ bool acceptable() { assert(overall != NULL); return zones[0].acceptable() && zones[1].acceptable() && zones[2].acceptable() && overall->acceptable(); } /** * Given a read, depth and orientation, extract a seed data structure * from the read and fill in the steps & zones arrays. The Seed * contains the sequence and quality values. */ bool instantiate( const Read& read, const BTDnaString& seq, // already-extracted seed sequence const BTString& qual, // already-extracted seed quality sequence const Scoring& pens, int depth, int seedoffidx, int seedtypeidx, bool fw, InstantiatedSeed& si) const; /** * Return a list of Seed objects encapsulating */ static void mmSeeds( int mms, int ln, EList& pols, Constraint& oall) { if(mms == 0) { zeroMmSeeds(ln, pols, oall); } else if(mms == 1) { oneMmSeeds(ln, pols, oall); } else if(mms == 2) { twoMmSeeds(ln, pols, oall); } else throw 1; } static void zeroMmSeeds(int ln, EList&, Constraint&); static void oneMmSeeds (int ln, EList&, Constraint&); static void twoMmSeeds (int ln, EList&, Constraint&); }; /** * An instantiated seed is a seed (perhaps modified to fit the read) * plus all data needed to conduct a search of the seed. */ struct InstantiatedSeed { InstantiatedSeed() : steps(AL_CAT), zones(AL_CAT) { } // Steps map. There are as many steps as there are positions in // the seed. The map is a helpful abstraction because we sometimes // visit seed positions in an irregular order (e.g. inside-out // search). EList steps; // Zones map. For each step, records what constraint to charge an // edit to. The first entry in each pair gives the constraint for // non-insert edits and the second entry in each pair gives the // constraint for insert edits. If the value stored is negative, // this indicates that the zone is "closed out" after this // position, so zone acceptility should be checked. EList > zones; // Nucleotide sequence covering the seed, extracted from read BTDnaString *seq; // Quality sequence covering the seed, extracted from read BTString *qual; // Initial constraints governing zones 0, 1, 2. We precalculate // the effect of Ns on these. Constraint cons[3]; // Overall constraint, tailored to the read length. Constraint overall; // Maximum number of positions that the aligner may advance before // its first step. This lets the aligner know whether it can use // the ftab or not. int maxjump; // Offset of seed from 5' end of read int seedoff; // Id for seed offset; ids are such that the smallest index is the // closest to the 5' end and consecutive ids are adjacent (i.e. // there are no intervening offsets with seeds) int seedoffidx; // Type of seed (left-to-right, etc) int seedtypeidx; // Seed comes from forward-oriented read? bool fw; // Filtered out due to the pattern of Ns present. If true, this // seed should be ignored by searchAllSeeds(). bool nfiltered; // Seed this was instantiated from Seed s; #ifndef NDEBUG /** * Check that InstantiatedSeed is internally consistent. */ bool repOk() const { return true; } #endif }; /** * Simple struct for holding a end-to-end alignments for the read with at most * 2 edits. */ template struct EEHit { EEHit() { reset(); } void reset() { top = bot = 0; fw = false; e1.reset(); e2.reset(); score = MIN_I64; } void init( index_t top_, index_t bot_, const Edit* e1_, const Edit* e2_, bool fw_, int64_t score_) { top = top_; bot = bot_; if(e1_ != NULL) { e1 = *e1_; } else { e1.reset(); } if(e2_ != NULL) { e2 = *e2_; } else { e2.reset(); } fw = fw_; score = score_; } /** * Return number of mismatches in the alignment. */ int mms() const { if (e2.inited()) return 2; else if(e1.inited()) return 1; else return 0; } /** * Return the number of Ns involved in the alignment. */ int ns() const { int ns = 0; if(e1.inited() && e1.hasN()) { ns++; if(e2.inited() && e2.hasN()) { ns++; } } return ns; } /** * Return the number of Ns involved in the alignment. */ int refns() const { int ns = 0; if(e1.inited() && e1.chr == 'N') { ns++; if(e2.inited() && e2.chr == 'N') { ns++; } } return ns; } /** * Return true iff there is no hit. */ bool empty() const { return bot <= top; } /** * Higher score = higher priority. */ bool operator<(const EEHit& o) const { return score > o.score; } /** * Return the size of the alignments SA range.s */ index_t size() const { return bot - top; } #ifndef NDEBUG /** * Check that hit is sane w/r/t read. */ bool repOk(const Read& rd) const { assert_gt(bot, top); if(e1.inited()) { assert_lt(e1.pos, rd.length()); if(e2.inited()) { assert_lt(e2.pos, rd.length()); } } return true; } #endif index_t top; index_t bot; Edit e1; Edit e2; bool fw; int64_t score; }; /** * Data structure for holding all of the seed hits associated with a read. All * the seed hits for a given read are encapsulated in a single QVal object. A * QVal refers to a range of values in the qlist, where each qlist value is a * BW range and a slot to hold the hit's suffix array offset. QVals are kept * in two lists (hitsFw_ and hitsRc_), one for seeds on the forward read strand, * one for seeds on the reverse read strand. The list is indexed by read * offset index (e.g. 0=closest-to-5', 1=second-closest, etc). * * An assumption behind this data structure is that all the seeds are found * first, then downstream analyses try to extend them. In between finding the * seed hits and extending them, the sort() member function is called, which * ranks QVals according to the order they should be extended. Right now the * policy is that QVals with fewer elements (hits) should be tried first. */ template class SeedResults { public: SeedResults() : seqFw_(AL_CAT), seqRc_(AL_CAT), qualFw_(AL_CAT), qualRc_(AL_CAT), hitsFw_(AL_CAT), hitsRc_(AL_CAT), isFw_(AL_CAT), isRc_(AL_CAT), sortedFw_(AL_CAT), sortedRc_(AL_CAT), offIdx2off_(AL_CAT), rankOffs_(AL_CAT), rankFws_(AL_CAT), mm1Hit_(AL_CAT) { clear(); } /** * Set the current read. */ void nextRead(const Read& read) { read_ = &read; } /** * Set the appropriate element of either hitsFw_ or hitsRc_ to the given * QVal. A QVal encapsulates all the BW ranges for reference substrings * that are within some distance of the seed string. */ void add( const QVal& qv, // range of ranges in cache const AlignmentCache& ac, // cache index_t seedIdx, // seed index (from 5' end) bool seedFw) // whether seed is from forward read { assert(qv.repOk(ac)); assert(repOk(&ac)); assert_lt(seedIdx, hitsFw_.size()); assert_gt(numOffs_, 0); // if this fails, probably failed to call reset if(qv.empty()) return; if(seedFw) { assert(!hitsFw_[seedIdx].valid()); hitsFw_[seedIdx] = qv; numEltsFw_ += qv.numElts(); numRangesFw_ += qv.numRanges(); if(qv.numRanges() > 0) nonzFw_++; } else { assert(!hitsRc_[seedIdx].valid()); hitsRc_[seedIdx] = qv; numEltsRc_ += qv.numElts(); numRangesRc_ += qv.numRanges(); if(qv.numRanges() > 0) nonzRc_++; } numElts_ += qv.numElts(); numRanges_ += qv.numRanges(); if(qv.numRanges() > 0) { nonzTot_++; } assert(repOk(&ac)); } /** * Clear buffered seed hits and state. Set the number of seed * offsets and the read. */ void reset( const Read& read, const EList& offIdx2off, size_t numOffs) { assert_gt(numOffs, 0); clearSeeds(); numOffs_ = numOffs; seqFw_.resize(numOffs_); seqRc_.resize(numOffs_); qualFw_.resize(numOffs_); qualRc_.resize(numOffs_); hitsFw_.resize(numOffs_); hitsRc_.resize(numOffs_); isFw_.resize(numOffs_); isRc_.resize(numOffs_); sortedFw_.resize(numOffs_); sortedRc_.resize(numOffs_); offIdx2off_ = offIdx2off; for(size_t i = 0; i < numOffs_; i++) { sortedFw_[i] = sortedRc_[i] = false; hitsFw_[i].reset(); hitsRc_[i].reset(); isFw_[i].clear(); isRc_[i].clear(); } read_ = &read; sorted_ = false; } /** * Clear seed-hit state. */ void clearSeeds() { sortedFw_.clear(); sortedRc_.clear(); rankOffs_.clear(); rankFws_.clear(); offIdx2off_.clear(); hitsFw_.clear(); hitsRc_.clear(); isFw_.clear(); isRc_.clear(); seqFw_.clear(); seqRc_.clear(); nonzTot_ = 0; nonzFw_ = 0; nonzRc_ = 0; numOffs_ = 0; numRanges_ = 0; numElts_ = 0; numRangesFw_ = 0; numEltsFw_ = 0; numRangesRc_ = 0; numEltsRc_ = 0; } /** * Clear seed-hit state and end-to-end alignment state. */ void clear() { clearSeeds(); read_ = NULL; exactFwHit_.reset(); exactRcHit_.reset(); mm1Hit_.clear(); mm1Sorted_ = false; mm1Elt_ = 0; assert(empty()); } /** * Extract key summaries from this SeedResults and put into 'ssum'. */ void toSeedAlSumm(SeedAlSumm& ssum) const { // Number of positions with at least 1 range ssum.nonzTot = nonzTot_; ssum.nonzFw = nonzFw_; ssum.nonzRc = nonzRc_; // Number of ranges ssum.nrangeTot = numRanges_; ssum.nrangeFw = numRangesFw_; ssum.nrangeRc = numRangesRc_; // Number of elements ssum.neltTot = numElts_; ssum.neltFw = numEltsFw_; ssum.neltRc = numEltsRc_; // Other summaries ssum.maxNonzRangeFw = ssum.minNonzRangeFw = 0; ssum.maxNonzRangeRc = ssum.minNonzRangeRc = 0; ssum.maxNonzEltFw = ssum.minNonzEltFw = 0; ssum.maxNonzEltRc = ssum.minNonzEltRc = 0; for(size_t i = 0; i < numOffs_; i++) { if(hitsFw_[i].valid()) { if(ssum.minNonzEltFw == 0 || hitsFw_[i].numElts() < ssum.minNonzEltFw) { ssum.minNonzEltFw = hitsFw_[i].numElts(); } if(ssum.maxNonzEltFw == 0 || hitsFw_[i].numElts() > ssum.maxNonzEltFw) { ssum.maxNonzEltFw = hitsFw_[i].numElts(); } if(ssum.minNonzRangeFw == 0 || hitsFw_[i].numRanges() < ssum.minNonzRangeFw) { ssum.minNonzRangeFw = hitsFw_[i].numRanges(); } if(ssum.maxNonzRangeFw == 0 || hitsFw_[i].numRanges() > ssum.maxNonzRangeFw) { ssum.maxNonzRangeFw = hitsFw_[i].numRanges(); } } if(hitsRc_[i].valid()) { if(ssum.minNonzEltRc == 0 || hitsRc_[i].numElts() < ssum.minNonzEltRc) { ssum.minNonzEltRc = hitsRc_[i].numElts(); } if(ssum.maxNonzEltRc == 0 || hitsRc_[i].numElts() > ssum.maxNonzEltRc) { ssum.maxNonzEltRc = hitsRc_[i].numElts(); } if(ssum.minNonzRangeRc == 0 || hitsRc_[i].numRanges() < ssum.minNonzRangeRc) { ssum.minNonzRangeRc = hitsRc_[i].numRanges(); } if(ssum.maxNonzRangeRc == 0 || hitsRc_[i].numRanges() > ssum.maxNonzRangeRc) { ssum.maxNonzRangeRc = hitsRc_[i].numRanges(); } } } } /** * Return average number of hits per seed. */ float averageHitsPerSeed() const { return (float)numElts_ / (float)nonzTot_; } /** * Return median of all the non-zero per-seed # hits */ float medianHitsPerSeed() const { EList& median = const_cast&>(tmpMedian_); median.clear(); for(size_t i = 0; i < numOffs_; i++) { if(hitsFw_[i].valid() && hitsFw_[i].numElts() > 0) { median.push_back(hitsFw_[i].numElts()); } if(hitsRc_[i].valid() && hitsRc_[i].numElts() > 0) { median.push_back(hitsRc_[i].numElts()); } } if(tmpMedian_.empty()) { return 0.0f; } median.sort(); float med1 = (float)median[tmpMedian_.size() >> 1]; float med2 = med1; if((median.size() & 1) == 0) { med2 = (float)median[(tmpMedian_.size() >> 1) - 1]; } return med1 + med2 * 0.5f; } /** * Return a number that's meant to quantify how hopeful we are that this * set of seed hits will lead to good alignments. */ double uniquenessFactor() const { double result = 0.0; for(size_t i = 0; i < numOffs_; i++) { if(hitsFw_[i].valid()) { size_t nelt = hitsFw_[i].numElts(); result += (1.0 / (double)(nelt * nelt)); } if(hitsRc_[i].valid()) { size_t nelt = hitsRc_[i].numElts(); result += (1.0 / (double)(nelt * nelt)); } } return result; } /** * Return the number of ranges being held. */ index_t numRanges() const { return numRanges_; } /** * Return the number of elements being held. */ index_t numElts() const { return numElts_; } /** * Return the number of ranges being held for seeds on the forward * read strand. */ index_t numRangesFw() const { return numRangesFw_; } /** * Return the number of elements being held for seeds on the * forward read strand. */ index_t numEltsFw() const { return numEltsFw_; } /** * Return the number of ranges being held for seeds on the * reverse-complement read strand. */ index_t numRangesRc() const { return numRangesRc_; } /** * Return the number of elements being held for seeds on the * reverse-complement read strand. */ index_t numEltsRc() const { return numEltsRc_; } /** * Given an offset index, return the offset that has that index. */ index_t idx2off(size_t off) const { return offIdx2off_[off]; } /** * Return true iff there are 0 hits being held. */ bool empty() const { return numRanges() == 0; } /** * Get the QVal representing all the reference hits for the given * orientation and seed offset index. */ const QVal& hitsAtOffIdx(bool fw, size_t seedoffidx) const { assert_lt(seedoffidx, numOffs_); assert(repOk(NULL)); return fw ? hitsFw_[seedoffidx] : hitsRc_[seedoffidx]; } /** * Get the Instantiated seeds for the given orientation and offset. */ EList& instantiatedSeeds(bool fw, size_t seedoffidx) { assert_lt(seedoffidx, numOffs_); assert(repOk(NULL)); return fw ? isFw_[seedoffidx] : isRc_[seedoffidx]; } /** * Return the number of different seed offsets possible. */ index_t numOffs() const { return numOffs_; } /** * Return the read from which seeds were extracted, aligned. */ const Read& read() const { return *read_; } #ifndef NDEBUG /** * Check that this SeedResults is internally consistent. */ bool repOk( const AlignmentCache* ac, bool requireInited = false) const { if(requireInited) { assert(read_ != NULL); } if(numOffs_ > 0) { assert_eq(numOffs_, hitsFw_.size()); assert_eq(numOffs_, hitsRc_.size()); assert_leq(numRanges_, numElts_); assert_leq(nonzTot_, numRanges_); size_t nonzs = 0; for(int fw = 0; fw <= 1; fw++) { const EList >& rrs = (fw ? hitsFw_ : hitsRc_); for(size_t i = 0; i < numOffs_; i++) { if(rrs[i].valid()) { if(rrs[i].numRanges() > 0) nonzs++; if(ac != NULL) { assert(rrs[i].repOk(*ac)); } } } } assert_eq(nonzs, nonzTot_); assert(!sorted_ || nonzTot_ == rankFws_.size()); assert(!sorted_ || nonzTot_ == rankOffs_.size()); } return true; } #endif /** * Populate rankOffs_ and rankFws_ with the list of QVals that need to be * examined for this SeedResults, in order. The order is ascending by * number of elements, so QVals with fewer elements (i.e. seed sequences * that are more unique) will be tried first and QVals with more elements * (i.e. seed sequences */ void rankSeedHits(RandomSource& rnd) { while(rankOffs_.size() < nonzTot_) { index_t minsz = (index_t)0xffffffff; index_t minidx = 0; bool minfw = true; // Rank seed-hit positions in ascending order by number of elements // in all BW ranges bool rb = rnd.nextBool(); assert(rb == 0 || rb == 1); for(int fwi = 0; fwi <= 1; fwi++) { bool fw = (fwi == (rb ? 1 : 0)); EList >& rrs = (fw ? hitsFw_ : hitsRc_); EList& sorted = (fw ? sortedFw_ : sortedRc_); index_t i = (rnd.nextU32() % (index_t)numOffs_); for(index_t ii = 0; ii < numOffs_; ii++) { if(rrs[i].valid() && // valid QVal rrs[i].numElts() > 0 && // non-empty !sorted[i] && // not already sorted rrs[i].numElts() < minsz) // least elts so far? { minsz = rrs[i].numElts(); minidx = i; minfw = (fw == 1); } if((++i) == numOffs_) { i = 0; } } } assert_neq((index_t)0xffffffff, minsz); if(minfw) { sortedFw_[minidx] = true; } else { sortedRc_[minidx] = true; } rankOffs_.push_back(minidx); rankFws_.push_back(minfw); } assert_eq(rankOffs_.size(), rankFws_.size()); sorted_ = true; } /** * Return the number of orientation/offsets into the read that have * at least one seed hit. */ size_t nonzeroOffsets() const { assert(!sorted_ || nonzTot_ == rankFws_.size()); assert(!sorted_ || nonzTot_ == rankOffs_.size()); return nonzTot_; } /** * Return true iff all seeds hit for forward read. */ bool allFwSeedsHit() const { return nonzFw_ == numOffs(); } /** * Return true iff all seeds hit for revcomp read. */ bool allRcSeedsHit() const { return nonzRc_ == numOffs(); } /** * Return the minimum number of edits that an end-to-end alignment of the * fw read could have. Uses knowledge of how many seeds have exact hits * and how the seeds overlap. */ index_t fewestEditsEE(bool fw, int seedlen, int per) const { assert_gt(seedlen, 0); assert_gt(per, 0); index_t nonz = fw ? nonzFw_ : nonzRc_; if(nonz < numOffs()) { int maxdepth = (seedlen + per - 1) / per; int missing = (int)(numOffs() - nonz); return (missing + maxdepth - 1) / maxdepth; } else { // Exact hit is possible (not guaranteed) return 0; } } /** * Return the number of offsets into the forward read that have at * least one seed hit. */ index_t nonzeroOffsetsFw() const { return nonzFw_; } /** * Return the number of offsets into the reverse-complement read * that have at least one seed hit. */ index_t nonzeroOffsetsRc() const { return nonzRc_; } /** * Return a QVal of seed hits of the given rank 'r'. 'offidx' gets the id * of the offset from 5' from which it was extracted (0 for the 5-most * offset, 1 for the next closes to 5', etc). 'off' gets the offset from * the 5' end. 'fw' gets true iff the seed was extracted from the forward * read. */ const QVal& hitsByRank( index_t r, // in index_t& offidx, // out index_t& off, // out bool& fw, // out index_t& seedlen) // out { assert(sorted_); assert_lt(r, nonzTot_); if(rankFws_[r]) { fw = true; offidx = rankOffs_[r]; assert_lt(offidx, offIdx2off_.size()); off = offIdx2off_[offidx]; seedlen = (index_t)seqFw_[rankOffs_[r]].length(); return hitsFw_[rankOffs_[r]]; } else { fw = false; offidx = rankOffs_[r]; assert_lt(offidx, offIdx2off_.size()); off = offIdx2off_[offidx]; seedlen = (index_t)seqRc_[rankOffs_[r]].length(); return hitsRc_[rankOffs_[r]]; } } /** * Return an EList of seed hits of the given rank. */ const BTDnaString& seqByRank(index_t r) { assert(sorted_); assert_lt(r, nonzTot_); return rankFws_[r] ? seqFw_[rankOffs_[r]] : seqRc_[rankOffs_[r]]; } /** * Return an EList of seed hits of the given rank. */ const BTString& qualByRank(index_t r) { assert(sorted_); assert_lt(r, nonzTot_); return rankFws_[r] ? qualFw_[rankOffs_[r]] : qualRc_[rankOffs_[r]]; } /** * Return the list of extracted seed sequences for seeds on either * the forward or reverse strand. */ EList& seqs(bool fw) { return fw ? seqFw_ : seqRc_; } /** * Return the list of extracted quality sequences for seeds on * either the forward or reverse strand. */ EList& quals(bool fw) { return fw ? qualFw_ : qualRc_; } /** * Return exact end-to-end alignment of fw read. */ EEHit exactFwEEHit() const { return exactFwHit_; } /** * Return exact end-to-end alignment of rc read. */ EEHit exactRcEEHit() const { return exactRcHit_; } /** * Return const ref to list of 1-mismatch end-to-end alignments. */ const EList >& mm1EEHits() const { return mm1Hit_; } /** * Sort the end-to-end 1-mismatch alignments, prioritizing by score (higher * score = higher priority). */ void sort1mmEe(RandomSource& rnd) { assert(!mm1Sorted_); mm1Hit_.sort(); size_t streak = 0; for(size_t i = 1; i < mm1Hit_.size(); i++) { if(mm1Hit_[i].score == mm1Hit_[i-1].score) { if(streak == 0) { streak = 1; } streak++; } else { if(streak > 1) { assert_geq(i, streak); mm1Hit_.shufflePortion(i-streak, streak, rnd); } streak = 0; } } if(streak > 1) { mm1Hit_.shufflePortion(mm1Hit_.size() - streak, streak, rnd); } mm1Sorted_ = true; } /** * Add an end-to-end 1-mismatch alignment. */ void add1mmEe( index_t top, index_t bot, const Edit* e1, const Edit* e2, bool fw, int64_t score) { mm1Hit_.expand(); mm1Hit_.back().init(top, bot, e1, e2, fw, score); mm1Elt_ += (bot - top); } /** * Add an end-to-end exact alignment. */ void addExactEeFw( index_t top, index_t bot, const Edit* e1, const Edit* e2, bool fw, int64_t score) { exactFwHit_.init(top, bot, e1, e2, fw, score); } /** * Add an end-to-end exact alignment. */ void addExactEeRc( index_t top, index_t bot, const Edit* e1, const Edit* e2, bool fw, int64_t score) { exactRcHit_.init(top, bot, e1, e2, fw, score); } /** * Clear out the end-to-end exact alignments. */ void clearExactE2eHits() { exactFwHit_.reset(); exactRcHit_.reset(); } /** * Clear out the end-to-end 1-mismatch alignments. */ void clear1mmE2eHits() { mm1Hit_.clear(); // 1-mismatch end-to-end hits mm1Elt_ = 0; // number of 1-mismatch hit rows mm1Sorted_ = false; // true iff we've sorted the mm1Hit_ list } /** * Return the number of distinct exact and 1-mismatch end-to-end hits * found. */ index_t numE2eHits() const { return (index_t)(exactFwHit_.size() + exactRcHit_.size() + mm1Elt_); } /** * Return the number of distinct exact end-to-end hits found. */ index_t numExactE2eHits() const { return (index_t)(exactFwHit_.size() + exactRcHit_.size()); } /** * Return the number of distinct 1-mismatch end-to-end hits found. */ index_t num1mmE2eHits() const { return mm1Elt_; } /** * Return the length of the read that yielded the seed hits. */ index_t readLength() const { assert(read_ != NULL); return read_->length(); } protected: // As seed hits and edits are added they're sorted into these // containers EList seqFw_; // seqs for seeds from forward read EList seqRc_; // seqs for seeds from revcomp read EList qualFw_; // quals for seeds from forward read EList qualRc_; // quals for seeds from revcomp read EList > hitsFw_; // hits for forward read EList > hitsRc_; // hits for revcomp read EList > isFw_; // hits for forward read EList > isRc_; // hits for revcomp read EList sortedFw_; // true iff fw QVal was sorted/ranked EList sortedRc_; // true iff rc QVal was sorted/ranked index_t nonzTot_; // # offsets with non-zero size index_t nonzFw_; // # offsets into fw read with non-0 size index_t nonzRc_; // # offsets into rc read with non-0 size index_t numRanges_; // # ranges added index_t numElts_; // # elements added index_t numRangesFw_; // # ranges added for fw seeds index_t numEltsFw_; // # elements added for fw seeds index_t numRangesRc_; // # ranges added for rc seeds index_t numEltsRc_; // # elements added for rc seeds EList offIdx2off_;// map from offset indexes to offsets from 5' end // When the sort routine is called, the seed hits collected so far // are sorted into another set of containers that allow easy access // to hits from the lowest-ranked offset (the one with the fewest // BW elements) to the greatest-ranked offset. Offsets with 0 hits // are ignored. EList rankOffs_; // sorted offests of seeds to try EList rankFws_; // sorted orientations assoc. with rankOffs_ bool sorted_; // true if sort() called since last reset // These fields set once per read index_t numOffs_; // # different seed offsets possible const Read* read_; // read from which seeds were extracted EEHit exactFwHit_; // end-to-end exact hit for fw read EEHit exactRcHit_; // end-to-end exact hit for rc read EList > mm1Hit_; // 1-mismatch end-to-end hits index_t mm1Elt_; // number of 1-mismatch hit rows bool mm1Sorted_; // true iff we've sorted the mm1Hit_ list EList tmpMedian_; // temporary storage for calculating median }; // Forward decl template class Ebwt; template struct SideLocus; /** * Encapsulates a sumamry of what the searchAllSeeds aligner did. */ struct SeedSearchMetrics { SeedSearchMetrics() : mutex_m() { reset(); } /** * Merge this metrics object with the given object, i.e., sum each * category. This is the only safe way to update a * SeedSearchMetrics object shread by multiple threads. */ void merge(const SeedSearchMetrics& m, bool getLock = false) { ThreadSafe ts(&mutex_m, getLock); seedsearch += m.seedsearch; possearch += m.possearch; intrahit += m.intrahit; interhit += m.interhit; filteredseed += m.filteredseed; ooms += m.ooms; bwops += m.bwops; bweds += m.bweds; bestmin0 += m.bestmin0; bestmin1 += m.bestmin1; bestmin2 += m.bestmin2; } /** * Set all counters to 0. */ void reset() { seedsearch = possearch = intrahit = interhit = filteredseed = ooms = bwops = bweds = bestmin0 = bestmin1 = bestmin2 = 0; } uint64_t seedsearch; // # times we executed strategy in InstantiatedSeed uint64_t possearch; // # offsets where aligner executed >= 1 strategy uint64_t intrahit; // # offsets where current-read cache gave answer uint64_t interhit; // # offsets where across-read cache gave answer uint64_t filteredseed; // # seed instantiations skipped due to Ns uint64_t ooms; // out-of-memory errors uint64_t bwops; // Burrows-Wheeler operations uint64_t bweds; // Burrows-Wheeler edits uint64_t bestmin0; // # times the best min # edits was 0 uint64_t bestmin1; // # times the best min # edits was 1 uint64_t bestmin2; // # times the best min # edits was 2 MUTEX_T mutex_m; }; /** * Given an index and a seeding scheme, searches for seed hits. */ template class SeedAligner { public: /** * Initialize with index. */ SeedAligner() : edits_(AL_CAT), offIdx2off_(AL_CAT) { } /** * Given a read and a few coordinates that describe a substring of the * read (or its reverse complement), fill in 'seq' and 'qual' objects * with the seed sequence and qualities. */ void instantiateSeq( const Read& read, // input read BTDnaString& seq, // output sequence BTString& qual, // output qualities int len, // seed length int depth, // seed's 0-based offset from 5' end bool fw) const; // seed's orientation /** * Iterate through the seeds that cover the read and initiate a * search for each seed. */ std::pair instantiateSeeds( const EList& seeds, // search seeds index_t off, // offset into read to start extracting int per, // interval between seeds const Read& read, // read to align const Scoring& pens, // scoring scheme bool nofw, // don't align forward read bool norc, // don't align revcomp read AlignmentCacheIface& cache, // holds some seed hits from previous reads SeedResults& sr, // holds all the seed hits SeedSearchMetrics& met); // metrics /** * Iterate through the seeds that cover the read and initiate a * search for each seed. */ void searchAllSeeds( const EList& seeds, // search seeds const Ebwt* ebwtFw, // BWT index const Ebwt* ebwtBw, // BWT' index const Read& read, // read to align const Scoring& pens, // scoring scheme AlignmentCacheIface& cache, // local seed alignment cache SeedResults& hits, // holds all the seed hits SeedSearchMetrics& met, // metrics PerReadMetrics& prm); // per-read metrics /** * Sanity-check a partial alignment produced during oneMmSearch. */ bool sanityPartial( const Ebwt* ebwtFw, // BWT index const Ebwt* ebwtBw, // BWT' index const BTDnaString& seq, index_t dep, index_t len, bool do1mm, index_t topfw, index_t botfw, index_t topbw, index_t botbw); /** * Do an exact-matching sweet to establish a lower bound on number of edits * and to find exact alignments. */ size_t exactSweep( const Ebwt& ebwt, // BWT index const Read& read, // read to align const Scoring& sc, // scoring scheme bool nofw, // don't align forward read bool norc, // don't align revcomp read size_t mineMax, // don't care about edit bounds > this size_t& mineFw, // minimum # edits for forward read size_t& mineRc, // minimum # edits for revcomp read bool repex, // report 0mm hits? SeedResults& hits, // holds all the seed hits (and exact hit) SeedSearchMetrics& met); // metrics /** * Search for end-to-end alignments with up to 1 mismatch. */ bool oneMmSearch( const Ebwt* ebwtFw, // BWT index const Ebwt* ebwtBw, // BWT' index const Read& read, // read to align const Scoring& sc, // scoring int64_t minsc, // minimum score bool nofw, // don't align forward read bool norc, // don't align revcomp read bool local, // 1mm hits must be legal local alignments bool repex, // report 0mm hits? bool rep1mm, // report 1mm hits? SeedResults& hits, // holds all the seed hits (and exact hit) SeedSearchMetrics& met); // metrics protected: /** * Report a seed hit found by searchSeedBi(), but first try to extend it out in * either direction as far as possible without hitting any edits. This will * allow us to prioritize the seed hits better later on. Call reportHit() when * we're done, which actually adds the hit to the cache. Returns result from * calling reportHit(). */ bool extendAndReportHit( index_t topf, // top in BWT index_t botf, // bot in BWT index_t topb, // top in BWT' index_t botb, // bot in BWT' index_t len, // length of hit DoublyLinkedList *prevEdit); // previous edit /** * Report a seed hit found by searchSeedBi() by adding it to the cache. Return * false if the hit could not be reported because of, e.g., cache exhaustion. */ bool reportHit( index_t topf, // top in BWT index_t botf, // bot in BWT index_t topb, // top in BWT' index_t botb, // bot in BWT' index_t len, // length of hit DoublyLinkedList *prevEdit); // previous edit /** * Given an instantiated seed (in s_ and other fields), search */ bool searchSeedBi(); /** * Main, recursive implementation of the seed search. */ bool searchSeedBi( int step, // depth into steps_[] array int depth, // recursion depth index_t topf, // top in BWT index_t botf, // bot in BWT index_t topb, // top in BWT' index_t botb, // bot in BWT' SideLocus tloc, // locus for top (perhaps unititialized) SideLocus bloc, // locus for bot (perhaps unititialized) Constraint c0, // constraints to enforce in seed zone 0 Constraint c1, // constraints to enforce in seed zone 1 Constraint c2, // constraints to enforce in seed zone 2 Constraint overall, // overall constraints DoublyLinkedList *prevEdit); // previous edit /** * Get tloc and bloc ready for the next step. */ inline void nextLocsBi( SideLocus& tloc, // top locus SideLocus& bloc, // bot locus index_t topf, // top in BWT index_t botf, // bot in BWT index_t topb, // top in BWT' index_t botb, // bot in BWT' int step); // step to get ready for // Following are set in searchAllSeeds then used by searchSeed() // and other protected members. const Ebwt* ebwtFw_; // forward index (BWT) const Ebwt* ebwtBw_; // backward/mirror index (BWT') const Scoring* sc_; // scoring scheme const InstantiatedSeed* s_; // current instantiated seed const Read* read_; // read whose seeds are currently being aligned // The following are set just before a call to searchSeedBi() const BTDnaString* seq_; // sequence of current seed const BTString* qual_; // quality string for current seed index_t off_; // offset of seed currently being searched bool fw_; // orientation of seed currently being searched EList edits_; // temporary place to sort edits AlignmentCacheIface *ca_; // local alignment cache for seed alignments EList offIdx2off_; // offset idx to read offset map, set up instantiateSeeds() uint64_t bwops_; // Burrows-Wheeler operations uint64_t bwedits_; // Burrows-Wheeler edits BTDnaString tmprfdnastr_; // used in reportHit ASSERT_ONLY(ESet hits_); // Ref hits so far for seed being aligned BTDnaString tmpdnastr_; }; #define INIT_LOCS(top, bot, tloc, bloc, e) { \ if(bot - top == 1) { \ tloc.initFromRow(top, (e).eh(), (e).ebwt()); \ bloc.invalidate(); \ } else { \ SideLocus::initFromTopBot(top, bot, (e).eh(), (e).ebwt(), tloc, bloc); \ assert(bloc.valid()); \ } \ } #define SANITY_CHECK_4TUP(t, b, tp, bp) { \ ASSERT_ONLY(index_t tot = (b[0]-t[0])+(b[1]-t[1])+(b[2]-t[2])+(b[3]-t[3])); \ ASSERT_ONLY(index_t totp = (bp[0]-tp[0])+(bp[1]-tp[1])+(bp[2]-tp[2])+(bp[3]-tp[3])); \ assert_eq(tot, totp); \ } /** * Given a read and a few coordinates that describe a substring of the read (or * its reverse complement), fill in 'seq' and 'qual' objects with the seed * sequence and qualities. * * The seq field is filled with the sequence as it would align to the Watson * reference strand. I.e. if fw is false, then the sequence that appears in * 'seq' is the reverse complement of the raw read substring. */ template void SeedAligner::instantiateSeq( const Read& read, // input read BTDnaString& seq, // output sequence BTString& qual, // output qualities int len, // seed length int depth, // seed's 0-based offset from 5' end bool fw) const // seed's orientation { // Fill in 'seq' and 'qual' int seedlen = len; if((int)read.length() < seedlen) seedlen = (int)read.length(); seq.resize(len); qual.resize(len); // If fw is false, we take characters starting at the 3' end of the // reverse complement of the read. for(int i = 0; i < len; i++) { seq.set(read.patFw.windowGetDna(i, fw, read.color, depth, len), i); qual.set(read.qual.windowGet(i, fw, depth, len), i); } } /** * We assume that all seeds are the same length. * * For each seed, instantiate the seed, retracting if necessary. */ template pair SeedAligner::instantiateSeeds( const EList& seeds, // search seeds index_t off, // offset into read to start extracting int per, // interval between seeds const Read& read, // read to align const Scoring& pens, // scoring scheme bool nofw, // don't align forward read bool norc, // don't align revcomp read AlignmentCacheIface& cache,// holds some seed hits from previous reads SeedResults& sr, // holds all the seed hits SeedSearchMetrics& met) // metrics { assert(!seeds.empty()); assert_gt(read.length(), 0); // Check whether read has too many Ns offIdx2off_.clear(); int len = seeds[0].len; // assume they're all the same length #ifndef NDEBUG for(size_t i = 1; i < seeds.size(); i++) { assert_eq(len, seeds[i].len); } #endif // Calc # seeds within read interval int nseeds = 1; if((int)read.length() - (int)off > len) { nseeds += ((int)read.length() - (int)off - len) / per; } for(int i = 0; i < nseeds; i++) { offIdx2off_.push_back(per * i + (int)off); } pair ret; ret.first = 0; // # seeds that require alignment ret.second = 0; // # seeds that hit in cache with non-empty results sr.reset(read, offIdx2off_, nseeds); assert(sr.repOk(&cache.current(), true)); // require that SeedResult be initialized // For each seed position for(int fwi = 0; fwi < 2; fwi++) { bool fw = (fwi == 0); if((fw && nofw) || (!fw && norc)) { // Skip this orientation b/c user specified --nofw or --norc continue; } // For each seed position for(int i = 0; i < nseeds; i++) { int depth = i * per + (int)off; int seedlen = seeds[0].len; // Extract the seed sequence at this offset // If fw == true, we extract the characters from i*per to // i*(per-1) (exclusive). If fw == false, instantiateSeq( read, sr.seqs(fw)[i], sr.quals(fw)[i], std::min((int)seedlen, (int)read.length()), depth, fw); //QKey qk(sr.seqs(fw)[i] ASSERT_ONLY(, tmpdnastr_)); // For each search strategy EList& iss = sr.instantiatedSeeds(fw, i); for(int j = 0; j < (int)seeds.size(); j++) { iss.expand(); assert_eq(seedlen, seeds[j].len); InstantiatedSeed* is = &iss.back(); if(seeds[j].instantiate( read, sr.seqs(fw)[i], sr.quals(fw)[i], pens, depth, i, j, fw, *is)) { // Can we fill this seed hit in from the cache? ret.first++; } else { // Seed may fail to instantiate if there are Ns // that prevent it from matching met.filteredseed++; iss.pop_back(); } } } } return ret; } /** * We assume that all seeds are the same length. * * For each seed: * * 1. Instantiate all seeds, retracting them if necessary. * 2. Calculate zone boundaries for each seed */ template void SeedAligner::searchAllSeeds( const EList& seeds, // search seeds const Ebwt* ebwtFw, // BWT index const Ebwt* ebwtBw, // BWT' index const Read& read, // read to align const Scoring& pens, // scoring scheme AlignmentCacheIface& cache, // local cache for seed alignments SeedResults& sr, // holds all the seed hits SeedSearchMetrics& met, // metrics PerReadMetrics& prm) // per-read metrics { assert(!seeds.empty()); assert(ebwtFw != NULL); assert(ebwtFw->isInMemory()); assert(sr.repOk(&cache.current())); ebwtFw_ = ebwtFw; ebwtBw_ = ebwtBw; sc_ = &pens; read_ = &read; ca_ = &cache; bwops_ = bwedits_ = 0; uint64_t possearches = 0, seedsearches = 0, intrahits = 0, interhits = 0, ooms = 0; // For each instantiated seed for(int i = 0; i < (int)sr.numOffs(); i++) { size_t off = sr.idx2off(i); for(int fwi = 0; fwi < 2; fwi++) { bool fw = (fwi == 0); assert(sr.repOk(&cache.current())); EList& iss = sr.instantiatedSeeds(fw, i); if(iss.empty()) { // Cache hit in an across-read cache continue; } QVal qv; seq_ = &sr.seqs(fw)[i]; // seed sequence qual_ = &sr.quals(fw)[i]; // seed qualities off_ = off; // seed offset (from 5') fw_ = fw; // seed orientation // Tell the cache that we've started aligning, so the cache can // expect a series of on-the-fly updates int ret = cache.beginAlign(*seq_, *qual_, qv); ASSERT_ONLY(hits_.clear()); if(ret == -1) { // Out of memory when we tried to add key to map ooms++; continue; } bool abort = false; if(ret == 0) { // Not already in cache assert(cache.aligning()); possearches++; for(size_t j = 0; j < iss.size(); j++) { // Set seq_ and qual_ appropriately, using the seed sequences // and qualities already installed in SeedResults assert_eq(fw, iss[j].fw); assert_eq(i, (int)iss[j].seedoffidx); s_ = &iss[j]; // Do the search with respect to seq_, qual_ and s_. if(!searchSeedBi()) { // Memory exhausted during search ooms++; abort = true; break; } seedsearches++; assert(cache.aligning()); } if(!abort) { qv = cache.finishAlign(); } } else { // Already in cache assert_eq(1, ret); assert(qv.valid()); intrahits++; } assert(abort || !cache.aligning()); if(qv.valid()) { sr.add( qv, // range of ranges in cache cache.current(), // cache i, // seed index (from 5' end) fw); // whether seed is from forward read } } } prm.nSeedRanges = sr.numRanges(); prm.nSeedElts = sr.numElts(); prm.nSeedRangesFw = sr.numRangesFw(); prm.nSeedRangesRc = sr.numRangesRc(); prm.nSeedEltsFw = sr.numEltsFw(); prm.nSeedEltsRc = sr.numEltsRc(); prm.seedMedian = (uint64_t)(sr.medianHitsPerSeed() + 0.5); prm.seedMean = (uint64_t)sr.averageHitsPerSeed(); prm.nSdFmops += bwops_; met.seedsearch += seedsearches; met.possearch += possearches; met.intrahit += intrahits; met.interhit += interhits; met.ooms += ooms; met.bwops += bwops_; met.bweds += bwedits_; } template bool SeedAligner::sanityPartial( const Ebwt* ebwtFw, // BWT index const Ebwt* ebwtBw, // BWT' index const BTDnaString& seq, index_t dep, index_t len, bool do1mm, index_t topfw, index_t botfw, index_t topbw, index_t botbw) { tmpdnastr_.clear(); for(size_t i = dep; i < len; i++) { tmpdnastr_.append(seq[i]); } index_t top_fw = 0, bot_fw = 0; ebwtFw->contains(tmpdnastr_, &top_fw, &bot_fw); assert_eq(top_fw, topfw); assert_eq(bot_fw, botfw); if(do1mm && ebwtBw != NULL) { tmpdnastr_.reverse(); index_t top_bw = 0, bot_bw = 0; ebwtBw->contains(tmpdnastr_, &top_bw, &bot_bw); assert_eq(top_bw, topbw); assert_eq(bot_bw, botbw); } return true; } /** * Sweep right-to-left and left-to-right using exact matching. Remember all * the SA ranges encountered along the way. Report exact matches if there are * any. Calculate a lower bound on the number of edits in an end-to-end * alignment. */ template size_t SeedAligner::exactSweep( const Ebwt& ebwt, // BWT index const Read& read, // read to align const Scoring& sc, // scoring scheme bool nofw, // don't align forward read bool norc, // don't align revcomp read size_t mineMax, // don't care about edit bounds > this size_t& mineFw, // minimum # edits for forward read size_t& mineRc, // minimum # edits for revcomp read bool repex, // report 0mm hits? SeedResults& hits, // holds all the seed hits (and exact hit) SeedSearchMetrics& met) // metrics { assert_gt(mineMax, 0); index_t top = 0, bot = 0; SideLocus tloc, bloc; const size_t len = read.length(); size_t nelt = 0; for(int fwi = 0; fwi < 2; fwi++) { bool fw = (fwi == 0); if( fw && nofw) continue; if(!fw && norc) continue; const BTDnaString& seq = fw ? read.patFw : read.patRc; assert(!seq.empty()); int ftabLen = ebwt.eh().ftabChars(); size_t dep = 0; size_t nedit = 0; bool done = false; while(dep < len && !done) { top = bot = 0; size_t left = len - dep; assert_gt(left, 0); bool doFtab = ftabLen > 1 && left >= (size_t)ftabLen; if(doFtab) { // Does N interfere with use of Ftab? for(size_t i = 0; i < (size_t)ftabLen; i++) { int c = seq[len-dep-1-i]; if(c > 3) { doFtab = false; break; } } } if(doFtab) { // Use ftab ebwt.ftabLoHi(seq, len - dep - ftabLen, false, top, bot); dep += (size_t)ftabLen; } else { // Use fchr int c = seq[len-dep-1]; if(c < 4) { top = ebwt.fchr()[c]; bot = ebwt.fchr()[c+1]; } dep++; } if(bot <= top) { nedit++; if(nedit >= mineMax) { if(fw) { mineFw = nedit; } else { mineRc = nedit; } break; } continue; } INIT_LOCS(top, bot, tloc, bloc, ebwt); // Keep going while(dep < len) { int c = seq[len-dep-1]; if(c > 3) { top = bot = 0; } else { if(bloc.valid()) { bwops_ += 2; top = ebwt.mapLF(tloc, c); bot = ebwt.mapLF(bloc, c); } else { bwops_++; top = ebwt.mapLF1(top, tloc, c); if(top == (index_t)OFF_MASK) { top = bot = 0; } else { bot = top+1; } } } if(bot <= top) { nedit++; if(nedit >= mineMax) { if(fw) { mineFw = nedit; } else { mineRc = nedit; } done = true; } break; } INIT_LOCS(top, bot, tloc, bloc, ebwt); dep++; } if(done) { break; } if(dep == len) { // Set the minimum # edits if(fw) { mineFw = nedit; } else { mineRc = nedit; } // Done if(nedit == 0 && bot > top) { if(repex) { // This is an exact hit int64_t score = len * sc.match(); if(fw) { hits.addExactEeFw(top, bot, NULL, NULL, fw, score); assert(ebwt.contains(seq, NULL, NULL)); } else { hits.addExactEeRc(top, bot, NULL, NULL, fw, score); assert(ebwt.contains(seq, NULL, NULL)); } } nelt += (bot - top); } break; } dep++; } } return nelt; } /** * Search for end-to-end exact hit for read. Return true iff one is found. */ template bool SeedAligner::oneMmSearch( const Ebwt* ebwtFw, // BWT index const Ebwt* ebwtBw, // BWT' index const Read& read, // read to align const Scoring& sc, // scoring int64_t minsc, // minimum score bool nofw, // don't align forward read bool norc, // don't align revcomp read bool local, // 1mm hits must be legal local alignments bool repex, // report 0mm hits? bool rep1mm, // report 1mm hits? SeedResults& hits, // holds all the seed hits (and exact hit) SeedSearchMetrics& met) // metrics { assert(!rep1mm || ebwtBw != NULL); const size_t len = read.length(); int nceil = sc.nCeil.f((double)len); size_t ns = read.ns(); if(ns > 1) { // Can't align this with <= 1 mismatches return false; } else if(ns == 1 && !rep1mm) { // Can't align this with 0 mismatches return false; } assert_geq(len, 2); assert(!rep1mm || ebwtBw->eh().ftabChars() == ebwtFw->eh().ftabChars()); #ifndef NDEBUG if(ebwtBw != NULL) { for(int i = 0; i < 4; i++) { assert_eq(ebwtBw->fchr()[i], ebwtFw->fchr()[i]); } } #endif size_t halfFw = len >> 1; size_t halfBw = len >> 1; if((len & 1) != 0) { halfBw++; } assert_geq(halfFw, 1); assert_geq(halfBw, 1); SideLocus tloc, bloc; index_t t[4], b[4]; // dest BW ranges for BWT t[0] = t[1] = t[2] = t[3] = 0; b[0] = b[1] = b[2] = b[3] = 0; index_t tp[4], bp[4]; // dest BW ranges for BWT' tp[0] = tp[1] = tp[2] = tp[3] = 0; bp[0] = bp[1] = bp[2] = bp[3] = 0; index_t top = 0, bot = 0, topp = 0, botp = 0; // Align fw read / rc read bool results = false; for(int fwi = 0; fwi < 2; fwi++) { bool fw = (fwi == 0); if( fw && nofw) continue; if(!fw && norc) continue; // Align going right-to-left, left-to-right int lim = rep1mm ? 2 : 1; for(int ebwtfwi = 0; ebwtfwi < lim; ebwtfwi++) { bool ebwtfw = (ebwtfwi == 0); const Ebwt* ebwt = (ebwtfw ? ebwtFw : ebwtBw); const Ebwt* ebwtp = (ebwtfw ? ebwtBw : ebwtFw); assert(rep1mm || ebwt->fw()); const BTDnaString& seq = (fw ? (ebwtfw ? read.patFw : read.patFwRev) : (ebwtfw ? read.patRc : read.patRcRev)); assert(!seq.empty()); const BTString& qual = (fw ? (ebwtfw ? read.qual : read.qualRev) : (ebwtfw ? read.qualRev : read.qual)); int ftabLen = ebwt->eh().ftabChars(); size_t nea = ebwtfw ? halfFw : halfBw; // Check if there's an N in the near portion bool skip = false; for(size_t dep = 0; dep < nea; dep++) { if(seq[len-dep-1] > 3) { skip = true; break; } } if(skip) { continue; } size_t dep = 0; // Align near half if(ftabLen > 1 && (size_t)ftabLen <= nea) { // Use ftab to jump partway into near half bool rev = !ebwtfw; ebwt->ftabLoHi(seq, len - ftabLen, rev, top, bot); if(rep1mm) { ebwtp->ftabLoHi(seq, len - ftabLen, rev, topp, botp); assert_eq(bot - top, botp - topp); } if(bot - top == 0) { continue; } int c = seq[len - ftabLen]; t[c] = top; b[c] = bot; tp[c] = topp; bp[c] = botp; dep = ftabLen; // initialize tloc, bloc?? } else { // Use fchr to jump in by 1 pos int c = seq[len-1]; assert_range(0, 3, c); top = topp = tp[c] = ebwt->fchr()[c]; bot = botp = bp[c] = ebwt->fchr()[c+1]; if(bot - top == 0) { continue; } dep = 1; // initialize tloc, bloc?? } INIT_LOCS(top, bot, tloc, bloc, *ebwt); assert(sanityPartial(ebwt, ebwtp, seq, len-dep, len, rep1mm, top, bot, topp, botp)); bool do_continue = false; for(; dep < nea; dep++) { assert_lt(dep, len); int rdc = seq[len - dep - 1]; tp[0] = tp[1] = tp[2] = tp[3] = topp; bp[0] = bp[1] = bp[2] = bp[3] = botp; if(bloc.valid()) { bwops_++; t[0] = t[1] = t[2] = t[3] = b[0] = b[1] = b[2] = b[3] = 0; ebwt->mapBiLFEx(tloc, bloc, t, b, tp, bp); SANITY_CHECK_4TUP(t, b, tp, bp); top = t[rdc]; bot = b[rdc]; if(bot <= top) { do_continue = true; break; } topp = tp[rdc]; botp = bp[rdc]; assert(!rep1mm || bot - top == botp - topp); } else { assert_eq(bot, top+1); assert(!rep1mm || botp == topp+1); bwops_++; top = ebwt->mapLF1(top, tloc, rdc); if(top == (index_t)OFF_MASK) { do_continue = true; break; } bot = top + 1; t[rdc] = top; b[rdc] = bot; tp[rdc] = topp; bp[rdc] = botp; assert(!rep1mm || b[rdc] - t[rdc] == bp[rdc] - tp[rdc]); // topp/botp stay the same } INIT_LOCS(top, bot, tloc, bloc, *ebwt); assert(sanityPartial(ebwt, ebwtp, seq, len - dep - 1, len, rep1mm, top, bot, topp, botp)); } if(do_continue) { continue; } // Align far half for(; dep < len; dep++) { int rdc = seq[len-dep-1]; int quc = qual[len-dep-1]; if(rdc > 3 && nceil == 0) { break; } tp[0] = tp[1] = tp[2] = tp[3] = topp; bp[0] = bp[1] = bp[2] = bp[3] = botp; int clo = 0, chi = 3; bool match = true; if(bloc.valid()) { bwops_++; t[0] = t[1] = t[2] = t[3] = b[0] = b[1] = b[2] = b[3] = 0; ebwt->mapBiLFEx(tloc, bloc, t, b, tp, bp); SANITY_CHECK_4TUP(t, b, tp, bp); match = rdc < 4; top = t[rdc]; bot = b[rdc]; topp = tp[rdc]; botp = bp[rdc]; } else { assert_eq(bot, top+1); assert(!rep1mm || botp == topp+1); bwops_++; clo = ebwt->mapLF1(top, tloc); match = (clo == rdc); assert_range(-1, 3, clo); if(clo < 0) { break; // Hit the $ } else { t[clo] = top; b[clo] = bot = top + 1; } bp[clo] = botp; tp[clo] = topp; assert(!rep1mm || bot - top == botp - topp); assert(!rep1mm || b[clo] - t[clo] == bp[clo] - tp[clo]); chi = clo; } //assert(sanityPartial(ebwt, ebwtp, seq, len - dep - 1, len, rep1mm, top, bot, topp, botp)); if(rep1mm && (ns == 0 || rdc > 3)) { for(int j = clo; j <= chi; j++) { if(j == rdc || b[j] == t[j]) { // Either matches read or isn't a possibility continue; } // Potential mismatch - next, try size_t depm = dep + 1; index_t topm = t[j], botm = b[j]; index_t topmp = tp[j], botmp = bp[j]; assert_eq(botm - topm, botmp - topmp); index_t tm[4], bm[4]; // dest BW ranges for BWT tm[0] = t[0]; tm[1] = t[1]; tm[2] = t[2]; tm[3] = t[3]; bm[0] = b[0]; bm[1] = t[1]; bm[2] = b[2]; bm[3] = t[3]; index_t tmp[4], bmp[4]; // dest BW ranges for BWT' tmp[0] = tp[0]; tmp[1] = tp[1]; tmp[2] = tp[2]; tmp[3] = tp[3]; bmp[0] = bp[0]; bmp[1] = tp[1]; bmp[2] = bp[2]; bmp[3] = tp[3]; SideLocus tlocm, blocm; INIT_LOCS(topm, botm, tlocm, blocm, *ebwt); for(; depm < len; depm++) { int rdcm = seq[len - depm - 1]; tmp[0] = tmp[1] = tmp[2] = tmp[3] = topmp; bmp[0] = bmp[1] = bmp[2] = bmp[3] = botmp; if(blocm.valid()) { bwops_++; tm[0] = tm[1] = tm[2] = tm[3] = bm[0] = bm[1] = bm[2] = bm[3] = 0; ebwt->mapBiLFEx(tlocm, blocm, tm, bm, tmp, bmp); SANITY_CHECK_4TUP(tm, bm, tmp, bmp); topm = tm[rdcm]; botm = bm[rdcm]; topmp = tmp[rdcm]; botmp = bmp[rdcm]; if(botm <= topm) { break; } } else { assert_eq(botm, topm+1); assert_eq(botmp, topmp+1); bwops_++; topm = ebwt->mapLF1(topm, tlocm, rdcm); if(topm == (index_t)0xffffffff) { break; } botm = topm + 1; // topp/botp stay the same } INIT_LOCS(topm, botm, tlocm, blocm, *ebwt); } if(depm == len) { // Success; this is a 1MM hit size_t off5p = dep; // offset from 5' end of read size_t offstr = dep; // offset into patFw/patRc if(fw == ebwtfw) { off5p = len - off5p - 1; } if(!ebwtfw) { offstr = len - offstr - 1; } Edit e((uint32_t)off5p, j, rdc, EDIT_TYPE_MM, false); results = true; int64_t score = (len - 1) * sc.match(); // In --local mode, need to double-check that // end-to-end alignment doesn't violate local // alignment principles. Specifically, it // shouldn't to or below 0 anywhere in the middle. int pen = sc.score(rdc, (int)(1 << j), quc - 33); score += pen; bool valid = true; if(local) { int64_t locscore_fw = 0, locscore_bw = 0; for(size_t i = 0; i < len; i++) { if(i == dep) { if(locscore_fw + pen <= 0) { valid = false; break; } locscore_fw += pen; } else { locscore_fw += sc.match(); } if(len-i-1 == dep) { if(locscore_bw + pen <= 0) { valid = false; break; } locscore_bw += pen; } else { locscore_bw += sc.match(); } } } if(valid) { valid = score >= minsc; } if(valid) { #ifndef NDEBUG BTDnaString& rf = tmprfdnastr_; rf.clear(); edits_.clear(); edits_.push_back(e); if(!fw) Edit::invertPoss(edits_, len, false); Edit::toRef(fw ? read.patFw : read.patRc, edits_, rf); if(!fw) Edit::invertPoss(edits_, len, false); assert_eq(len, rf.length()); for(size_t i = 0; i < len; i++) { assert_lt((int)rf[i], 4); } ASSERT_ONLY(index_t toptmp = 0); ASSERT_ONLY(index_t bottmp = 0); assert(ebwtFw->contains(rf, &toptmp, &bottmp)); #endif index_t toprep = ebwtfw ? topm : topmp; index_t botrep = ebwtfw ? botm : botmp; assert_eq(toprep, toptmp); assert_eq(botrep, bottmp); hits.add1mmEe(toprep, botrep, &e, NULL, fw, score); } } } } if(bot > top && match) { assert_lt(rdc, 4); if(dep == len-1) { // Success; this is an exact hit if(ebwtfw && repex) { if(fw) { results = true; int64_t score = len * sc.match(); hits.addExactEeFw( ebwtfw ? top : topp, ebwtfw ? bot : botp, NULL, NULL, fw, score); assert(ebwtFw->contains(seq, NULL, NULL)); } else { results = true; int64_t score = len * sc.match(); hits.addExactEeRc( ebwtfw ? top : topp, ebwtfw ? bot : botp, NULL, NULL, fw, score); assert(ebwtFw->contains(seq, NULL, NULL)); } } break; // End of far loop } else { INIT_LOCS(top, bot, tloc, bloc, *ebwt); assert(sanityPartial(ebwt, ebwtp, seq, len - dep - 1, len, rep1mm, top, bot, topp, botp)); } } else { break; // End of far loop } } // for(; dep < len; dep++) } // for(int ebwtfw = 0; ebwtfw < 2; ebwtfw++) } // for(int fw = 0; fw < 2; fw++) return results; } /** * Wrapper for initial invcation of searchSeed. */ template bool SeedAligner::searchSeedBi() { return searchSeedBi( 0, 0, 0, 0, 0, 0, SideLocus(), SideLocus(), s_->cons[0], s_->cons[1], s_->cons[2], s_->overall, NULL); } /** * Get tloc, bloc ready for the next step. If the new range is under * the ceiling. */ template inline void SeedAligner::nextLocsBi( SideLocus& tloc, // top locus SideLocus& bloc, // bot locus index_t topf, // top in BWT index_t botf, // bot in BWT index_t topb, // top in BWT' index_t botb, // bot in BWT' int step // step to get ready for #if 0 , const SABWOffTrack* prevOt, // previous tracker SABWOffTrack& ot // current tracker #endif ) { assert_gt(botf, 0); assert(ebwtBw_ == NULL || botb > 0); assert_geq(step, 0); // next step can't be first one assert(ebwtBw_ == NULL || botf-topf == botb-topb); if(step == (int)s_->steps.size()) return; // no more steps! // Which direction are we going in next? if(s_->steps[step] > 0) { // Left to right; use BWT' if(botb - topb == 1) { // Already down to 1 row; just init top locus tloc.initFromRow(topb, ebwtBw_->eh(), ebwtBw_->ebwt()); bloc.invalidate(); } else { SideLocus::initFromTopBot( topb, botb, ebwtBw_->eh(), ebwtBw_->ebwt(), tloc, bloc); assert(bloc.valid()); } } else { // Right to left; use BWT if(botf - topf == 1) { // Already down to 1 row; just init top locus tloc.initFromRow(topf, ebwtFw_->eh(), ebwtFw_->ebwt()); bloc.invalidate(); } else { SideLocus::initFromTopBot( topf, botf, ebwtFw_->eh(), ebwtFw_->ebwt(), tloc, bloc); assert(bloc.valid()); } } // Check if we should update the tracker with this refinement #if 0 if(botf-topf <= BW_OFF_TRACK_CEIL) { if(ot.size() == 0 && prevOt != NULL && prevOt->size() > 0) { // Inherit state from the predecessor ot = *prevOt; } bool ltr = s_->steps[step-1] > 0; int adj = abs(s_->steps[step-1])-1; const Ebwt* ebwt = ltr ? ebwtBw_ : ebwtFw_; ot.update( ltr ? topb : topf, // top ltr ? botb : botf, // bot adj, // adj (to be subtracted from offset) ebwt->offs(), // offs array ebwt->eh().offRate(), // offrate (sample = every 1 << offrate elts) NULL // dead ); assert_gt(ot.size(), 0); } #endif assert(botf - topf == 1 || bloc.valid()); assert(botf - topf > 1 || !bloc.valid()); } /** * Report a seed hit found by searchSeedBi(), but first try to extend it out in * either direction as far as possible without hitting any edits. This will * allow us to prioritize the seed hits better later on. Call reportHit() when * we're done, which actually adds the hit to the cache. Returns result from * calling reportHit(). */ template bool SeedAligner::extendAndReportHit( index_t topf, // top in BWT index_t botf, // bot in BWT index_t topb, // top in BWT' index_t botb, // bot in BWT' index_t len, // length of hit DoublyLinkedList *prevEdit) // previous edit { index_t nlex = 0, nrex = 0; index_t t[4], b[4]; index_t tp[4], bp[4]; SideLocus tloc, bloc; if(off_ > 0) { const Ebwt *ebwt = ebwtFw_; assert(ebwt != NULL); // Extend left using forward index const BTDnaString& seq = fw_ ? read_->patFw : read_->patRc; // See what we get by extending index_t top = topf, bot = botf; t[0] = t[1] = t[2] = t[3] = 0; b[0] = b[1] = b[2] = b[3] = 0; tp[0] = tp[1] = tp[2] = tp[3] = topb; bp[0] = bp[1] = bp[2] = bp[3] = botb; SideLocus tloc, bloc; INIT_LOCS(top, bot, tloc, bloc, *ebwt); for(size_t ii = off_; ii > 0; ii--) { size_t i = ii-1; // Get char from read int rdc = seq.get(i); // See what we get by extending if(bloc.valid()) { bwops_++; t[0] = t[1] = t[2] = t[3] = b[0] = b[1] = b[2] = b[3] = 0; ebwt->mapBiLFEx(tloc, bloc, t, b, tp, bp); SANITY_CHECK_4TUP(t, b, tp, bp); int nonz = -1; bool abort = false; for(int j = 0; j < 4; j++) { if(b[i] > t[i]) { if(nonz >= 0) { abort = true; break; } nonz = j; top = t[i]; bot = b[i]; } } if(abort || nonz != rdc) { break; } } else { assert_eq(bot, top+1); bwops_++; int c = ebwt->mapLF1(top, tloc); if(c != rdc) { break; } bot = top + 1; } if(++nlex == 255) { break; } INIT_LOCS(top, bot, tloc, bloc, *ebwt); } } size_t rdlen = read_->length(); size_t nright = rdlen - off_ - len; if(nright > 0 && ebwtBw_ != NULL) { const Ebwt *ebwt = ebwtBw_; assert(ebwt != NULL); // Extend right using backward index const BTDnaString& seq = fw_ ? read_->patFw : read_->patRc; // See what we get by extending index_t top = topb, bot = botb; t[0] = t[1] = t[2] = t[3] = 0; b[0] = b[1] = b[2] = b[3] = 0; tp[0] = tp[1] = tp[2] = tp[3] = topb; bp[0] = bp[1] = bp[2] = bp[3] = botb; INIT_LOCS(top, bot, tloc, bloc, *ebwt); for(size_t i = off_ + len; i < rdlen; i++) { // Get char from read int rdc = seq.get(i); // See what we get by extending if(bloc.valid()) { bwops_++; t[0] = t[1] = t[2] = t[3] = b[0] = b[1] = b[2] = b[3] = 0; ebwt->mapBiLFEx(tloc, bloc, t, b, tp, bp); SANITY_CHECK_4TUP(t, b, tp, bp); int nonz = -1; bool abort = false; for(int j = 0; j < 4; j++) { if(b[i] > t[i]) { if(nonz >= 0) { abort = true; break; } nonz = j; top = t[i]; bot = b[i]; } } if(abort || nonz != rdc) { break; } } else { assert_eq(bot, top+1); bwops_++; int c = ebwt->mapLF1(top, tloc); if(c != rdc) { break; } bot = top + 1; } if(++nrex == 255) { break; } INIT_LOCS(top, bot, tloc, bloc, *ebwt); } } assert_lt(nlex, rdlen); assert_leq(nlex, off_); assert_lt(nrex, rdlen); return reportHit(topf, botf, topb, botb, len, prevEdit); } /** * Report a seed hit found by searchSeedBi() by adding it to the cache. Return * false if the hit could not be reported because of, e.g., cache exhaustion. */ template bool SeedAligner::reportHit( index_t topf, // top in BWT index_t botf, // bot in BWT index_t topb, // top in BWT' index_t botb, // bot in BWT' index_t len, // length of hit DoublyLinkedList *prevEdit) // previous edit { // Add information about the seed hit to AlignmentCache. This // information eventually makes its way back to the SeedResults // object when we call finishAlign(...). BTDnaString& rf = tmprfdnastr_; rf.clear(); edits_.clear(); if(prevEdit != NULL) { prevEdit->toList(edits_); Edit::sort(edits_); assert(Edit::repOk(edits_, *seq_)); Edit::toRef(*seq_, edits_, rf); } else { rf = *seq_; } // Sanity check: shouldn't add the same hit twice. If this // happens, it may be because our zone Constraints are not set up // properly and erroneously return true from acceptable() when they // should return false in some cases. assert_eq(hits_.size(), ca_->curNumRanges()); assert(hits_.insert(rf)); if(!ca_->addOnTheFly(rf, topf, botf, topb, botb)) { return false; } assert_eq(hits_.size(), ca_->curNumRanges()); #ifndef NDEBUG // Sanity check that the topf/botf and topb/botb ranges really // correspond to the reference sequence aligned to { BTDnaString rfr; index_t tpf, btf, tpb, btb; tpf = btf = tpb = btb = 0; assert(ebwtFw_->contains(rf, &tpf, &btf)); if(ebwtBw_ != NULL) { rfr = rf; rfr.reverse(); assert(ebwtBw_->contains(rfr, &tpb, &btb)); assert_eq(tpf, topf); assert_eq(btf, botf); assert_eq(tpb, topb); assert_eq(btb, botb); } } #endif return true; } /** * Given a seed, search. Assumes zone 0 = no backtracking. * * Return a list of Seed hits. * 1. Edits * 2. Bidirectional BWT range(s) on either end */ template bool SeedAligner::searchSeedBi( int step, // depth into steps_[] array int depth, // recursion depth index_t topf, // top in BWT index_t botf, // bot in BWT index_t topb, // top in BWT' index_t botb, // bot in BWT' SideLocus tloc, // locus for top (perhaps unititialized) SideLocus bloc, // locus for bot (perhaps unititialized) Constraint c0, // constraints to enforce in seed zone 0 Constraint c1, // constraints to enforce in seed zone 1 Constraint c2, // constraints to enforce in seed zone 2 Constraint overall, // overall constraints to enforce DoublyLinkedList *prevEdit // previous edit #if 0 , const SABWOffTrack* prevOt // prev off tracker (if tracking started) #endif ) { assert(s_ != NULL); const InstantiatedSeed& s = *s_; assert_gt(s.steps.size(), 0); assert(ebwtBw_ == NULL || ebwtBw_->eh().ftabChars() == ebwtFw_->eh().ftabChars()); #ifndef NDEBUG for(int i = 0; i < 4; i++) { assert(ebwtBw_ == NULL || ebwtBw_->fchr()[i] == ebwtFw_->fchr()[i]); } #endif if(step == (int)s.steps.size()) { // Finished aligning seed assert(c0.acceptable()); assert(c1.acceptable()); assert(c2.acceptable()); if(!reportHit(topf, botf, topb, botb, seq_->length(), prevEdit)) { return false; // Memory exhausted } return true; } #ifndef NDEBUG if(depth > 0) { assert(botf - topf == 1 || bloc.valid()); assert(botf - topf > 1 || !bloc.valid()); } #endif int off; index_t tp[4], bp[4]; // dest BW ranges for "prime" index if(step == 0) { // Just starting assert(prevEdit == NULL); assert(!tloc.valid()); assert(!bloc.valid()); off = s.steps[0]; bool ltr = off > 0; off = abs(off)-1; // Check whether/how far we can jump using ftab or fchr int ftabLen = ebwtFw_->eh().ftabChars(); if(ftabLen > 1 && ftabLen <= s.maxjump) { if(!ltr) { assert_geq(off+1, ftabLen-1); off = off - ftabLen + 1; } ebwtFw_->ftabLoHi(*seq_, off, false, topf, botf); #ifdef NDEBUG if(botf - topf == 0) return true; #endif #ifdef NDEBUG if(ebwtBw_ != NULL) { topb = ebwtBw_->ftabHi(*seq_, off); botb = topb + (botf-topf); } #else if(ebwtBw_ != NULL) { ebwtBw_->ftabLoHi(*seq_, off, false, topb, botb); assert_eq(botf-topf, botb-topb); } if(botf - topf == 0) return true; #endif step += ftabLen; } else if(s.maxjump > 0) { // Use fchr int c = (*seq_)[off]; assert_range(0, 3, c); topf = topb = ebwtFw_->fchr()[c]; botf = botb = ebwtFw_->fchr()[c+1]; if(botf - topf == 0) return true; step++; } else { assert_eq(0, s.maxjump); topf = topb = 0; botf = botb = ebwtFw_->fchr()[4]; } if(step == (int)s.steps.size()) { // Finished aligning seed assert(c0.acceptable()); assert(c1.acceptable()); assert(c2.acceptable()); if(!reportHit(topf, botf, topb, botb, seq_->length(), prevEdit)) { return false; // Memory exhausted } return true; } nextLocsBi(tloc, bloc, topf, botf, topb, botb, step); assert(tloc.valid()); } else assert(prevEdit != NULL); assert(tloc.valid()); assert(botf - topf == 1 || bloc.valid()); assert(botf - topf > 1 || !bloc.valid()); assert_geq(step, 0); index_t t[4], b[4]; // dest BW ranges Constraint* zones[3] = { &c0, &c1, &c2 }; ASSERT_ONLY(index_t lasttot = botf - topf); for(int i = step; i < (int)s.steps.size(); i++) { assert_gt(botf, topf); assert(botf - topf == 1 || bloc.valid()); assert(botf - topf > 1 || !bloc.valid()); assert(ebwtBw_ == NULL || botf-topf == botb-topb); assert(tloc.valid()); off = s.steps[i]; bool ltr = off > 0; const Ebwt* ebwt = ltr ? ebwtBw_ : ebwtFw_; assert(ebwt != NULL); if(ltr) { tp[0] = tp[1] = tp[2] = tp[3] = topf; bp[0] = bp[1] = bp[2] = bp[3] = botf; } else { tp[0] = tp[1] = tp[2] = tp[3] = topb; bp[0] = bp[1] = bp[2] = bp[3] = botb; } t[0] = t[1] = t[2] = t[3] = b[0] = b[1] = b[2] = b[3] = 0; if(bloc.valid()) { // Range delimited by tloc/bloc has size >1. If size == 1, // we use a simpler query (see if(!bloc.valid()) blocks below) bwops_++; ebwt->mapBiLFEx(tloc, bloc, t, b, tp, bp); ASSERT_ONLY(index_t tot = (b[0]-t[0])+(b[1]-t[1])+(b[2]-t[2])+(b[3]-t[3])); ASSERT_ONLY(index_t totp = (bp[0]-tp[0])+(bp[1]-tp[1])+(bp[2]-tp[2])+(bp[3]-tp[3])); assert_eq(tot, totp); assert_leq(tot, lasttot); ASSERT_ONLY(lasttot = tot); } index_t *tf = ltr ? tp : t, *tb = ltr ? t : tp; index_t *bf = ltr ? bp : b, *bb = ltr ? b : bp; off = abs(off)-1; // bool leaveZone = s.zones[i].first < 0; //bool leaveZoneIns = zones_[i].second < 0; Constraint& cons = *zones[abs(s.zones[i].first)]; Constraint& insCons = *zones[abs(s.zones[i].second)]; int c = (*seq_)[off]; assert_range(0, 4, c); int q = (*qual_)[off]; // Is it legal for us to advance on characters other than 'c'? if(!(cons.mustMatch() && !overall.mustMatch()) || c == 4) { // There may be legal edits bool bail = false; if(!bloc.valid()) { // Range delimited by tloc/bloc has size 1 index_t ntop = ltr ? topb : topf; bwops_++; int cc = ebwt->mapLF1(ntop, tloc); assert_range(-1, 3, cc); if(cc < 0) bail = true; else { t[cc] = ntop; b[cc] = ntop+1; } } if(!bail) { if((cons.canMismatch(q, *sc_) && overall.canMismatch(q, *sc_)) || c == 4) { Constraint oldCons = cons, oldOvCons = overall; SideLocus oldTloc = tloc, oldBloc = bloc; if(c != 4) { cons.chargeMismatch(q, *sc_); overall.chargeMismatch(q, *sc_); } // Can leave the zone as-is if(!leaveZone || (cons.acceptable() && overall.acceptable())) { for(int j = 0; j < 4; j++) { if(j == c || b[j] == t[j]) continue; // Potential mismatch nextLocsBi(tloc, bloc, tf[j], bf[j], tb[j], bb[j], i+1); int loff = off; if(!ltr) loff = (int)(s.steps.size() - loff - 1); assert(prevEdit == NULL || prevEdit->next == NULL); Edit edit(off, j, c, EDIT_TYPE_MM, false); DoublyLinkedList editl; editl.payload = edit; if(prevEdit != NULL) { prevEdit->next = &editl; editl.prev = prevEdit; } assert(editl.next == NULL); bwedits_++; if(!searchSeedBi( i+1, // depth into steps_[] array depth+1, // recursion depth tf[j], // top in BWT bf[j], // bot in BWT tb[j], // top in BWT' bb[j], // bot in BWT' tloc, // locus for top (perhaps unititialized) bloc, // locus for bot (perhaps unititialized) c0, // constraints to enforce in seed zone 0 c1, // constraints to enforce in seed zone 1 c2, // constraints to enforce in seed zone 2 overall, // overall constraints to enforce &editl)) // latest edit { return false; } if(prevEdit != NULL) prevEdit->next = NULL; } } else { // Not enough edits to make this path // non-redundant with other seeds } cons = oldCons; overall = oldOvCons; tloc = oldTloc; bloc = oldBloc; } if(cons.canGap() && overall.canGap()) { throw 1; // TODO int delEx = 0; if(cons.canDelete(delEx, *sc_) && overall.canDelete(delEx, *sc_)) { // Try delete } int insEx = 0; if(insCons.canInsert(insEx, *sc_) && overall.canInsert(insEx, *sc_)) { // Try insert } } } // if(!bail) } if(c == 4) { return true; // couldn't handle the N } if(leaveZone && (!cons.acceptable() || !overall.acceptable())) { // Not enough edits to make this path non-redundant with // other seeds return true; } if(!bloc.valid()) { assert(ebwtBw_ == NULL || bp[c] == tp[c]+1); // Range delimited by tloc/bloc has size 1 index_t top = ltr ? topb : topf; bwops_++; t[c] = ebwt->mapLF1(top, tloc, c); if(t[c] == (index_t)OFF_MASK) { return true; } assert_geq(t[c], ebwt->fchr()[c]); assert_lt(t[c], ebwt->fchr()[c+1]); b[c] = t[c]+1; assert_gt(b[c], 0); } assert(ebwtBw_ == NULL || bf[c]-tf[c] == bb[c]-tb[c]); assert_leq(bf[c]-tf[c], lasttot); ASSERT_ONLY(lasttot = bf[c]-tf[c]); if(b[c] == t[c]) { return true; } topf = tf[c]; botf = bf[c]; topb = tb[c]; botb = bb[c]; if(i+1 == (int)s.steps.size()) { // Finished aligning seed assert(c0.acceptable()); assert(c1.acceptable()); assert(c2.acceptable()); if(!reportHit(topf, botf, topb, botb, seq_->length(), prevEdit)) { return false; // Memory exhausted } return true; } nextLocsBi(tloc, bloc, tf[c], bf[c], tb[c], bb[c], i+1); } return true; } #endif /*ALIGNER_SEED_H_*/ hisat2-2.2.1/aligner_seed2.cpp000066400000000000000000001212631370663763200161370ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include #include "aligner_seed2.h" #include "assert_helpers.h" #include "gfm.h" #ifdef ALIGNER_SEED2_MAIN #include #include "sstring.h" using namespace std; /** * A way of feeding simply tests to the seed alignment infrastructure. */ int main(int argc, char **argv) { EList strs; // GCTATATAGCGCGCTCGCATCATTTTGTGT strs.push_back(string("CATGTCAGCTATATAGCGCGCTCGCATCATTTTGTGTGTAAACCA" "NNNNNNNNNN" "CATGTCAGCTATATAGCGCGCTCGCATCATTTTGTGTGTAAACCA")); // GCTATATAGCGCGCTTGCATCATTTTGTGT // ^ bool packed = false; int color = 0; pair gfms = GFM::fromStrings >( strs, packed, REF_READ_REVERSE, Ebwt::default_bigEndian, Ebwt::default_lineRate, Ebwt::default_offRate, Ebwt::default_ftabChars, ".aligner_seed2.cpp.tmp", Ebwt::default_useBlockwise, Ebwt::default_bmax, Ebwt::default_bmaxMultSqrt, Ebwt::default_bmaxDivN, Ebwt::default_dcv, Ebwt::default_seed, false, // verbose false, // autoMem false); // sanity gfms.first->loadIntoMemory (-1, true, true, true, true, false); gfms.second->loadIntoMemory(1, true, true, true, true, false); int testnum = 0; // Query is longer than ftab and matches exactly twice for(int rc = 0; rc < 2; rc++) { for(int i = 0; i < 2; i++) { cerr << "Test " << (++testnum) << endl; cerr << " Query with length greater than ftab" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; // Set up the read BTDnaString seq ("GCTATATAGCGCGCTCGCATCATTTTGTGT", true); BTString qual("ABCDEFGHIabcdefghiABCDEFGHIabc"); if(rc) { seq.reverseComp(); qual.reverse(); } dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; conf.cons.init(GFM::default_ftabChars, 1.0); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig (i == 0) ? 0 : (seq.length() - 1), // 5' offset into read of root (i == 0) ? true : false, // left-to-right? rc == 0, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert_eq(2, dr.sink().nelt()); } } // Query has length euqal to ftab and matches exactly twice for(int i = 0; i < 2; i++) { cerr << "Test " << (++testnum) << endl; cerr << " Query with length equal to ftab" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; // Set up the read BTDnaString seq ("GCTATATAGC", true); BTString qual("ABCDEFGHIa"); dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; conf.cons.init(GFM::default_ftabChars, 1.0); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig (i == 0) ? 0 : (seq.length() - 1), // 5' offset into read of root (i == 0) ? true : false, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert_eq(2, dr.sink().nelt()); } // Query has length less than ftab length and matches exactly twice for(int i = 0; i < 2; i++) { cerr << "Test " << (++testnum) << endl; cerr << " Query with length less than ftab" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; // Set up the read BTDnaString seq ("GCTATATAG", true); BTString qual("ABCDEFGHI"); dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; conf.cons.init(GFM::default_ftabChars, 1.0); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig (i == 0) ? 0 : (seq.length() - 1), // 5' offset into read of root (i == 0) ? true : false, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert_eq(2, dr.sink().nelt()); } // Search root is in the middle of the read, requiring a bounce for(int i = 0; i < 2; i++) { cerr << "Test " << (++testnum) << endl; cerr << " Search root in middle of read" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; // Set up the read // 012345678901234567890123456789 BTDnaString seq ("GCTATATAGCGCGCTCGCATCATTTTGTGT", true); BTString qual("ABCDEFGHIabcdefghiABCDEFGHIabc"); TIndexOffU top, bot; top = bot = 0; bool ret = gfms.first->contains("GCGCTCGCATCATTTTGTGT", &top, &bot); cerr << ret << ", " << top << ", " << bot << endl; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; conf.cons.init(GFM::default_ftabChars, 1.0); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig (i == 0) ? 10 : (seq.length() - 1 - 10), // 5' offset into read of root (i == 0) ? true : false, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert_eq(2, dr.sink().nelt()); } delete gfms.first; delete gfms.second; strs.clear(); strs.push_back(string("CATGTCAGCTATATAGCGCGCTCGCATCATTTTGTGTGTAAACCA" "NNNNNNNNNN" "CATGTCAGCTATATAGCG")); gfms = GFM::fromStrings >( strs, packed, REF_READ_REVERSE, GFM::default_bigEndian, GFM::default_lineRate, GFM::default_offRate, GFM::default_ftabChars, ".aligner_seed2.cpp.tmp", GFM::default_useBlockwise, GFM::default_bmax, GfM::default_bmaxMultSqrt, GFM::default_bmaxDivN, GFM::default_dcv, GFM::default_seed, false, // verbose false, // autoMem false); // sanity gfms.first->loadIntoMemory (-1, true, true, true, true, false); gfms.second->loadIntoMemory(1, true, true, true, true, false); // Query is longer than ftab and matches exactly once. One search root for // forward read. { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { BTDnaString seq ("GCTATATAGCGCGCTCGCATCATTTTGTGT", true); BTString qual("ABCDEFGHIabcdefghiABCDEFGHIabc"); for(size_t j = 0; j < seq.length(); j++) { cerr << "Test " << (++testnum) << endl; cerr << " Query with length greater than ftab and matches exactly once" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; // Set up the read dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; conf.cons.init(GFM::default_ftabChars, 1.0); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); assert_eq(1, dr.sink().nelt()); } } } // Query is longer than ftab and its reverse complement matches exactly // once. Search roots on forward and reverse-comp reads. { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { BTDnaString seq ("GCTATATAGCGCGCTCGCATCATTTTGTGT", true); BTString qual("ABCDEFGHIabcdefghiABCDEFGHIabc"); for(size_t j = 0; j < seq.length(); j++) { cerr << "Test " << (++testnum) << endl; cerr << " Query with length greater than ftab and reverse complement matches exactly once" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; // Set up the read dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; conf.cons.init(GFM::default_ftabChars, 1.0); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? true, // forward? 0.0f); // root priority dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? false, // forward? 1.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); assert_eq(1, dr.sink().nelt()); } } } // Query is longer than ftab and matches exactly once with one mismatch { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { // Set up the read // Ref: CATGTCAGCTATATAGCGCGCTCGCATCATTTTGTGTGTAAACCA // |||||||||||||||||||||||||||||| BTDnaString orig("GCTATATAGCGCGCTCGCATCATTTTGTGT", true); // 012345678901234567890123456789 BTString qual("ABCDEFGHIabcdefghiABCDEFGHIabc"); for(size_t k = 0; k < orig.length(); k++) { BTDnaString seq = orig; seq.set(seq[k] ^ 3, k); for(size_t j = 0; j < seq.length(); j++) { // Assume left-to-right size_t beg = j; size_t end = j + GFM::default_ftabChars; // Mismatch penalty is 3, so we have to skip starting // points that are within 2 from the mismatch if((i > 0 && j > 0) || j == seq.length()-1) { // Right-to-left if(beg < GFM::default_ftabChars) { beg = 0; } else { beg -= GFM::default_ftabChars; } end -= GFM::default_ftabChars; } size_t kk = k; //if(rc) { // kk = seq.length() - k - 1; //} if(beg <= kk && end > kk) { continue; } if((j > kk) ? (j - kk <= 2) : (kk - j <= 2)) { continue; } cerr << "Test " << (++testnum) << endl; cerr << " Query with length greater than ftab and matches exactly once with 1mm" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(0, 1.0); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); cerr << dr.sink()[0].topf << ", " << dr.sink()[0].botf << endl; assert_eq(1, dr.sink().nelt()); last_topf = dr.sink()[0].topf; last_botf = dr.sink()[0].botf; } } } } // Query is longer than ftab and matches exactly once with one N mismatch { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { // Set up the read // Ref: CATGTCAGCTATATAGCGCGCTCGCATCATTTTGTGTGTAAACCA // |||||||||||||||||||||||||||||| BTDnaString orig("GCTATATAGCGCGCTCGCATCATTTTGTGT", true); // 012345678901234567890123456789 BTString qual("ABCDEFGHIabcdefghiABCDEFGHIabc"); for(size_t k = 0; k < orig.length(); k++) { BTDnaString seq = orig; seq.set(4, k); for(size_t j = 0; j < seq.length(); j++) { // Assume left-to-right size_t beg = j; size_t end = j + GFM::default_ftabChars; // Mismatch penalty is 3, so we have to skip starting // points that are within 2 from the mismatch if((i > 0 && j > 0) || j == seq.length()-1) { // Right-to-left if(beg < GFM::default_ftabChars) { beg = 0; } else { beg -= GFM::default_ftabChars; } end -= GFM::default_ftabChars; } if(beg <= k && end > k) { continue; } if((j > k) ? (j - k <= 2) : (k - j <= 2)) { continue; } cerr << "Test " << (++testnum) << endl; cerr << " Query with length greater than ftab and matches exactly once with 1mm" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(0, 1.0); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert_eq(sc.n(40), dr.sink()[0].pen); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); cerr << dr.sink()[0].topf << ", " << dr.sink()[0].botf << endl; assert_eq(1, dr.sink().nelt()); last_topf = dr.sink()[0].topf; last_botf = dr.sink()[0].botf; } } } } // Throw a bunch of queries with a bunch of Ns in and try to force an assert { RandomSource rnd(79); for(int i = 0; i < 2; i++) { // Set up the read // Ref: CATGTCAGCTATATAGCGCGCTCGCATCATTTTGTGTGTAAACCA // |||||||||||||||||||||||||||||| BTDnaString orig("GCTATATAGCGCGCTCGCATCATTTTGTGT", true); // 012345678901234567890123456789 BTString qual("ABCDEFGHIabcdefghiABCDEFGHIabc"); if(i == 1) { orig.reverseComp(); qual.reverse(); } for(size_t trials = 0; trials < 100; trials++) { BTDnaString seq = orig; size_t ns = 10; for(size_t k = 0; k < ns; k++) { size_t pos = rnd.nextU32() % seq.length(); seq.set(4, pos); } cerr << "Test " << (++testnum) << endl; cerr << " Query with a bunch of Ns" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(GFM::default_ftabChars, 1.0); conf.expol = DESC_EX_NONE; // Set up the search roots for(size_t k = 0; k < ns; k++) { size_t j = rnd.nextU32() % seq.length(); bool ltr = (rnd.nextU2() == 0) ? true : false; bool fw = (rnd.nextU2() == 0) ? true : false; dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root ltr, // left-to-right? fw, // forward? 0.0f); // root priority } // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); } } } // Query is longer than ftab and matches exactly once with one mismatch { RandomSource rnd(77); size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { // Set up the read // Ref: CATGTCAGCTATATAGCGCGCTCGCATCATTTTGTGTGTAAACCA // |||||||||||||||||||||||||||||| BTDnaString orig("GCTATATAGCGCGCTCGCATCATTTTGTGT", true); // 012345678901234567890123456789 BTString qual("ABCDEFGHIabcdefghiABCDEFGHIabc"); // revcomp: ACACAAAATGATGCGAGCGCGCTATATAGC // revqual: cbaIHGFEDCBAihgfedcbaIHGFEDCBA bool fwi = (i == 0); if(!fwi) { orig.reverseComp(); } for(size_t k = 0; k < orig.length(); k++) { BTDnaString seq = orig; seq.set(seq[k] ^ 3, k); cerr << "Test " << (++testnum) << endl; cerr << " Query with length greater than ftab and matches exactly once with 1mm. Many search roots." << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(0, 1.0); conf.expol = DESC_EX_NONE; // Set up several random search roots bool onegood = false; for(size_t y = 0; y < 10; y++) { size_t j = rnd.nextU32() % seq.length(); bool ltr = (rnd.nextU2() == 0) ? true : false; bool fw = (rnd.nextU2() == 0) ? true : false; dr.addRoot( conf, // DescentConfig (TReadOff)j, // 5' offset into read of root ltr, // left-to-right? fw, // forward? (float)((float)y * 1.0f)); // root priority // Assume left-to-right size_t beg = j; size_t end = j + GFM::default_ftabChars; // Mismatch penalty is 3, so we have to skip starting // points that are within 2 from the mismatch if(!ltr) { // Right-to-left if(beg < GFM::default_ftabChars) { beg = 0; } else { beg -= GFM::default_ftabChars; } end -= GFM::default_ftabChars; } bool good = true; if(fw != fwi) { good = false; } if(beg <= k && end > k) { good = false; } if((j > k) ? (j - k <= 2) : (k - j <= 2)) { good = false; } if(good) { onegood = true; } } if(!onegood) { continue; } // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); cerr << dr.sink()[0].topf << ", " << dr.sink()[0].botf << endl; assert_eq(1, dr.sink().nelt()); last_topf = dr.sink()[0].topf; last_botf = dr.sink()[0].botf; } } } // Query is longer than ftab and matches exactly once with one read gap { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { for(int k = 0; k < 2; k++) { // Set up the read // GCTATATAGCGCGCCTGCATCATTTTGTGT // Ref: CATGTCAGCTATATAGCGCGCTCGCATCATTTTGTGTGTAAACCA // |||||||||||||||/////////////// BTDnaString seq ("GCTATATAGCGCGCTGCATCATTTTGTGT", true); // 01234567890123456789012345678 // 87654321098765432109876543210 BTString qual("ABCDEFGHIabcdefghiABCDEFGHIab"); if(k == 1) { seq.reverseComp(); qual.reverse(); } assert_eq(seq.length(), qual.length()); // js iterate over offsets from 5' end for the search root for(size_t j = 0; j < seq.length(); j++) { // Assume left-to-right size_t beg = j; if(k == 1) { beg = seq.length() - beg - 1; } size_t end = beg + GFM::default_ftabChars; // Mismatch penalty is 3, so we have to skip starting // points that are within 2 from the mismatch if((i > 0 && j > 0) || j == seq.length()-1) { // Right-to-left if(beg < GFM::default_ftabChars) { beg = 0; } else { beg -= GFM::default_ftabChars; } end -= GFM::default_ftabChars; } assert_geq(end, beg); if(beg <= 15 && end >= 15) { continue; } cerr << "Test " << (++testnum) << endl; cerr << " Query matches once with a read gap of length 1" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; Read q("test", seq.toZBuf(), qual.toZBuf()); assert(q.repOk()); dr.initRead(q, -30, 30); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(0, 0.5); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? k == 0, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert_eq(sc.readGapOpen() + 0 * sc.readGapExtend(), dr.sink()[0].pen); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); cerr << dr.sink()[0].topf << ", " << dr.sink()[0].botf << endl; assert_eq(1, dr.sink().nelt()); last_topf = dr.sink()[0].topf; last_botf = dr.sink()[0].botf; } }} } // Query is longer than ftab and matches exactly once with one read gap of // length 3 { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { for(int k = 0; k < 2; k++) { // Set up the read // GCTATATAGCGCGCGCTCATCATTTTGTGT // Ref: CATGTCAGCTATATAGCGCGCTCGCATCATTTTGTGTGTAAACCA // |||||||||||||| ||||||||||||| BTDnaString seq ("GCTATATAGCGCGC" "CATCATTTTGTGT", true); // 01234567890123 4567890123456 // 65432109876543 2109876543210 BTString qual("ABCDEFGHIabcde" "fghiABCDEFGHI"); if(k == 1) { seq.reverseComp(); qual.reverse(); } for(size_t j = 0; j < seq.length(); j++) { // Assume left-to-right size_t beg = j; if(k == 1) { beg = seq.length() - beg - 1; } size_t end = beg + GFM::default_ftabChars; // Mismatch penalty is 3, so we have to skip starting // points that are within 2 from the mismatch if((i > 0 && j > 0) || j == seq.length()-1) { // Right-to-left if(beg < GFM::default_ftabChars) { beg = 0; } else { beg -= GFM::default_ftabChars; } end -= GFM::default_ftabChars; } if(beg <= 14 && end >= 14) { continue; } cerr << "Test " << (++testnum) << endl; cerr << " Query matches once with a read gap of length 3" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(0, 0.2); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? k == 0, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); // Need to adjust the mismatch penalty up to avoid alignments // with lots of mismatches. sc.setMmPen(COST_MODEL_CONSTANT, 6, 6); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert_eq(sc.readGapOpen() + 2 * sc.readGapExtend(), dr.sink()[0].pen); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); cerr << dr.sink()[0].topf << ", " << dr.sink()[0].botf << endl; assert_eq(1, dr.sink().nelt()); last_topf = dr.sink()[0].topf; last_botf = dr.sink()[0].botf; } }} } // Query is longer than ftab and matches exactly once with one reference gap { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { // Set up the read // Ref: CATGTCAGCTATATAGCGCGC" "TCGCATCATTTTGTGTGTAAACCA // |||||||||||||| |||||||||||||||| BTDnaString seq ("GCTATATAGCGCGCA""TCGCATCATTTTGTGT", true); // 012345678901234 5678901234567890 BTString qual("ABCDEFGHIabcdef""ghiABCDEFGHIabcd"); for(size_t j = 0; j < seq.length(); j++) { // Assume left-to-right size_t beg = j; size_t end = j + GFM::default_ftabChars; // Mismatch penalty is 3, so we have to skip starting // points that are within 2 from the mismatch if((i > 0 && j > 0) || j == seq.length()-1) { // Right-to-left if(beg < GFM::default_ftabChars) { beg = 0; } else { beg -= GFM::default_ftabChars; } end -= GFM::default_ftabChars; } if(beg <= 14 && end >= 14) { continue; } cerr << "Test " << (++testnum) << endl; cerr << " Query matches once with a reference gap of length 1" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(1, 0.5); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); // Need to adjust the mismatch penalty up to avoid alignments // with lots of mismatches. sc.setMmPen(COST_MODEL_CONSTANT, 6, 6); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert_eq(sc.refGapOpen() + 0 * sc.refGapExtend(), dr.sink()[0].pen); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); cerr << dr.sink()[0].topf << ", " << dr.sink()[0].botf << endl; assert_eq(1, dr.sink().nelt()); last_topf = dr.sink()[0].topf; last_botf = dr.sink()[0].botf; } } } // Query is longer than ftab and matches exactly once with one reference gap { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { // Set up the read // Ref: CATGTCAGCTATATAGCGCGC" "TCGCATCATTTTGTGTGTAAACCA // |||||||||||||| |||||||||||||||| BTDnaString seq ("GCTATATAGCGCGCATG""TCGCATCATTTTGTGT", true); // 01234567890123456 7890123456789012 BTString qual("ABCDEFGHIabcdefgh""iABCDEFGHIabcdef"); for(size_t j = 0; j < seq.length(); j++) { // Assume left-to-right size_t beg = j; size_t end = j + GFM::default_ftabChars; // Mismatch penalty is 3, so we have to skip starting // points that are within 2 from the mismatch if((i > 0 && j > 0) || j == seq.length()-1) { // Right-to-left if(beg < GFM::default_ftabChars) { beg = 0; } else { beg -= GFM::default_ftabChars; } end -= GFM::default_ftabChars; } if(beg <= 14 && end >= 14) { continue; } if(beg <= 15 && end >= 15) { continue; } if(beg <= 16 && end >= 16) { continue; } cerr << "Test " << (++testnum) << endl; cerr << " Query matches once with a reference gap of length 1" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -30, 30); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(1, 0.25); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); // Need to adjust the mismatch penalty up to avoid alignments // with lots of mismatches. sc.setMmPen(COST_MODEL_CONSTANT, 6, 6); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert_eq(sc.refGapOpen() + 2 * sc.refGapExtend(), dr.sink()[0].pen); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); cerr << dr.sink()[0].topf << ", " << dr.sink()[0].botf << endl; assert_eq(1, dr.sink().nelt()); last_topf = dr.sink()[0].topf; last_botf = dr.sink()[0].botf; } } } // Query is longer than ftab and matches exactly once with one read gap, // one ref gap, and one mismatch { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { // Set up the read // Ref: CATGTCAGCT ATATAGCGCGCT CGCATCATTTTGTGTGTAAACCA // |||||||||| |||||||||||| |||||| ||||||||||||| BTDnaString seq ("CATGTCAGCT""GATATAGCGCGCT" "GCATCAATTTGTGTGTAAAC", true); // 0123456789 0123456789012 34567890123456789012 BTString qual("ABCDEFGHIa""bcdefghiACDEF" "GHIabcdefghijkABCDEF"); for(size_t j = 0; j < seq.length(); j++) { // Assume left-to-right size_t beg = j; size_t end = j + GFM::default_ftabChars; // Mismatch penalty is 3, so we have to skip starting // points that are within 2 from the mismatch if((i > 0 && j > 0) || j == seq.length()-1) { // Right-to-left if(beg < GFM::default_ftabChars) { beg = 0; } else { beg -= GFM::default_ftabChars; } end -= GFM::default_ftabChars; } if(beg <= 10 && end >= 10) { continue; } if(beg <= 22 && end >= 22) { continue; } if(beg <= 30 && end >= 30) { continue; } cerr << "Test " << (++testnum) << endl; cerr << " Query matches once with a read gap of length 1" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -50, 50); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(1, 0.5); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(1, dr.sink().nrange()); assert_eq(sc.readGapOpen() + sc.refGapOpen() + sc.mm((int)'d' - 33), dr.sink()[0].pen); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); cerr << dr.sink()[0].topf << ", " << dr.sink()[0].botf << endl; assert_eq(1, dr.sink().nelt()); last_topf = dr.sink()[0].topf; last_botf = dr.sink()[0].botf; } } } delete gfms.first; delete gfms.second; // Ref CATGTCAGCT-ATATAGCGCGCTCGCATCATTTTGTGTGTAAAC // |||||||||| |||||||||||| |||||| ||||||||||||| // Rd CATGTCAGCTGATATAGCGCGCT-GCATCAATTTGTGTGTAAAC strs.clear(); strs.push_back(string("CATGTCAGCTATATAGCGCGCTCGCATCATTTTGTGTGTAAAC" "NNNNNNNNNN" "CATGTCAGCTGATATAGCGCGCTCGCATCATTTTGTGTGTAAAC" // same but without first ref gap "N" "CATGTCAGCTATATAGCGCGCTGCATCATTTTGTGTGTAAAC" // same but without first read gap "N" "CATGTCAGCTATATAGCGCGCTCGCATCAATTTGTGTGTAAAC" // same but without first mismatch "N" "CATGTCAGCTGATATAGCGCGCTGCATCAATTTGTGTGTAAAC" // Exact match for read )); gfms = GFM::fromStrings >( strs, packed, REF_READ_REVERSE, GFM::default_bigEndian, GFM::default_lineRate, GFM::default_offRate, GFM::default_ftabChars, ".aligner_seed2.cpp.tmp", GFM::default_useBlockwise, GFM::default_bmax, GFM::default_bmaxMultSqrt, GFM::default_bmaxDivN, GFM::default_dcv, GFM::default_seed, false, // verbose false, // autoMem false); // sanity gfms.first->loadIntoMemory (color, -1, true, true, true, true, false); gfms.second->loadIntoMemory(color, 1, true, true, true, true, false); // Query is longer than ftab and matches exactly once with one read gap, // one ref gap, and one mismatch { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { // Set up the read // Ref: CATGTCAGCT ATATAGCGCGCT CGCATCATTTTGTGTGTAAACCA // |||||||||| |||||||||||| |||||| ||||||||||||| BTDnaString seq ("CATGTCAGCT""GATATAGCGCGCT" "GCATCAATTTGTGTGTAAAC", true); // 0123456789 0123456789012 34567890123456789012 BTString qual("ABCDEFGHIa""bcdefghiACDEF" "GHIabcdefghijkABCDEF"); for(size_t j = 0; j < seq.length(); j++) { // Assume left-to-right size_t beg = j; size_t end = j + GFM::default_ftabChars; // Mismatch penalty is 3, so we have to skip starting // points that are within 2 from the mismatch if((i > 0 && j > 0) || j == seq.length()-1) { // Right-to-left if(beg < GFM::default_ftabChars) { beg = 0; } else { beg -= GFM::default_ftabChars; } end -= GFM::default_ftabChars; } if(beg <= 10 && end >= 10) { continue; } if(beg <= 22 && end >= 22) { continue; } if(beg <= 30 && end >= 30) { continue; } cerr << "Test " << (++testnum) << endl; cerr << " Query matches once with a read gap of length 1" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -50, 50); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(1, 0.5); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(5, dr.sink().nrange()); assert_eq(0, dr.sink()[0].pen); assert_eq(min(sc.readGapOpen(), sc.refGapOpen()) + sc.mm((int)'d' - 33), dr.sink()[1].pen); assert_eq(max(sc.readGapOpen(), sc.refGapOpen()) + sc.mm((int)'d' - 33), dr.sink()[2].pen); assert_eq(sc.readGapOpen() + sc.refGapOpen(), dr.sink()[3].pen); assert_eq(sc.readGapOpen() + sc.refGapOpen() + sc.mm((int)'d' - 33), dr.sink()[4].pen); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); cerr << dr.sink()[0].topf << ", " << dr.sink()[0].botf << endl; assert_eq(5, dr.sink().nelt()); last_topf = dr.sink()[0].topf; last_botf = dr.sink()[0].botf; } } } // Query is longer than ftab and matches exactly once with one read gap, // one ref gap, one mismatch, and one N { size_t last_topf = std::numeric_limits::max(); size_t last_botf = std::numeric_limits::max(); for(int i = 0; i < 2; i++) { // Set up the read // Ref: CATGTCAGCT ATATAGCGCGCT CGCATCATTTTGTGTGTAAACCA // |||||||||| |||||||||||| |||||| |||||| |||||| BTDnaString seq ("CATGTCAGCT""GATATAGCGCGCT" "GCATCAATTTGTGNGTAAAC", true); // 0123456789 0123456789012 34567890123456789012 BTString qual("ABCDEFGHIa""bcdefghiACDEF" "GHIabcdefghijkABCDEF"); for(size_t j = 0; j < seq.length(); j++) { // Assume left-to-right size_t beg = j; size_t end = j + GFM::default_ftabChars; // Mismatch penalty is 3, so we have to skip starting // points that are within 2 from the mismatch if((i > 0 && j > 0) || j == seq.length()-1) { // Right-to-left if(beg < GFM::default_ftabChars) { beg = 0; } else { beg -= GFM::default_ftabChars; } end -= GFM::default_ftabChars; } if(beg <= 10 && end >= 10) { continue; } if(beg <= 22 && end >= 22) { continue; } if(beg <= 30 && end >= 30) { continue; } if(beg <= 36 && end >= 36) { continue; } cerr << "Test " << (++testnum) << endl; cerr << " Query matches with various patterns of gaps, mismatches and Ns" << endl; DescentMetrics mets; PerReadMetrics prm; DescentDriver dr; dr.initRead(Read("test", seq.toZBuf(), qual.toZBuf()), -50, 50); // Set up the DescentConfig DescentConfig conf; // Changed conf.cons.init(1, 0.5); conf.expol = DESC_EX_NONE; // Set up the search roots dr.addRoot( conf, // DescentConfig j, // 5' offset into read of root i == 0, // left-to-right? true, // forward? 0.0f); // root priority // Do the search Scoring sc = Scoring::base1(); sc.setNPen(COST_MODEL_CONSTANT, 1); dr.go(sc, *gfms.first, *gfms.second, mets, prm); // Confirm that an exact-matching alignment was found assert_eq(5, dr.sink().nrange()); assert_eq(sc.n(40), dr.sink()[0].pen); assert_eq(sc.n(40) + min(sc.readGapOpen(), sc.refGapOpen()) + sc.mm((int)'d' - 33), dr.sink()[1].pen); assert_eq(sc.n(40) + max(sc.readGapOpen(), sc.refGapOpen()) + sc.mm((int)'d' - 33), dr.sink()[2].pen); assert_eq(sc.n(40) + sc.readGapOpen() + sc.refGapOpen(), dr.sink()[3].pen); assert_eq(sc.n(40) + sc.readGapOpen() + sc.refGapOpen() + sc.mm((int)'d' - 33), dr.sink()[4].pen); assert(last_topf == std::numeric_limits::max() || last_topf == dr.sink()[0].topf); assert(last_botf == std::numeric_limits::max() || last_botf == dr.sink()[0].botf); cerr << dr.sink()[0].topf << ", " << dr.sink()[0].botf << endl; assert_eq(5, dr.sink().nelt()); last_topf = dr.sink()[0].topf; last_botf = dr.sink()[0].botf; } } } delete gfms.first; delete gfms.second; cerr << "DONE" << endl; } #endif hisat2-2.2.1/aligner_seed2.h000066400000000000000000003772121370663763200156130ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_SEED2_H_ #define ALIGNER_SEED2_H_ /** * The user of the DescentDriver class specifies a collection of search roots. * Logic for picking these search roots is located elsewhere, not in this * module. The search roots are annotated with a priority score, which * * The heap is a min-heap over pairs, where the first element of each pair is * the score associated with a descent and the second element of each pair is * the descent ID. * * Weeding out redundant descents is key; otherwise we end up reporting slight * variations on the same alignment repeatedly, including variations with poor * scores. What criteria do we use to determine whether two paths are * redundant? * * Here's an example where the same set of read characters have been aligned in * all three cases: * * Alignment 1 (sc = 0): * Rd: GCTATATAGCGCGCTCGCATCATTTTGTGT * |||||||||||||||||||||||||||||| * Rf: GCTATATAGCGCGCTCGCATCATTTTGTGT * * Alignment 2 (sc = -22): * Rd: GCTATATAGCGCGCTCGCATCATTTTGTGT * ||||||||||||||||||||||| | ||| * Rf: GCTATATAGCGCGCTCGCATCAT--TTTGT * * Alignment 3 (sc = -22): * Rd: GCTATATAGCGCGCTCGCATCATT--TTGTGT * |||||||||||||||||||||||| ||||| * Rf: GCTATATAGCGCGCTCGCATCATTTTGTGTGT * * Rf from aln 1: GCTATATAGCGCGCTCGCATCATTTTGTGT * Rf from aln 2: GCTATATAGCGCGCTCGCATCATTTTGT * Rf from aln 3: GCTATATAGCGCGCTCGCATCATTTTGTGTGT * * Are alignments 2 and 3 redundant with alignment 1? We can't totally say * without knowing the associated SA ranges. Take alignments 1 and 2. Either * the SA ranges are the same or the SA range for 2 contains the SA range for * 1. If they're the same, then alignment 2 is redundant with alignment 1. * Otherwise, *some* of the elements in the SA range for alignment 2 are not * redundant. * * In that example, the same read characters are aligned in all three * alignments. Is it possible and profitable to consider scenarios where an * alignment might be redundant with another alignment * * Another question is *when* do we try to detect the redundancy? Before we * try to extend through the matches, or after. After is easier, but less work * has been avoided. * * What data structure do we query to determine whether there's redundancy? * The situation is harder when we try to detect overlaps between SA ranges * rather than identical SA ranges. Maybe: read intervals -> intersection tree -> penalties. * * 1. If we're introducing a gap and we could have introduced it deeper in the * descent with the same effect w/r/t homopolymer length. * 2. If we have Descent A with penalty B and Descent a with penalty b, and A * aligns read characters [X, Y] to SA range [Z, W], and B aligns read * characters [x, y] to SA range [z, w], then A is redundant with B if * [x, y] is within [X, Y]. * * Found an alignment with total penalty = 3 * GCAATATAGCGCGCTCGCATCATTTTGTGT * || ||||||||||||||||||||||||||| * GCTATATAGCGCGCTCGCATCATTTTGTGT * * Found an alignment with total penalty = 27 * gCAATATAGCGCGCTCGCATCATTTTGTGT * | |||||||||||||||||||||||| * TATA-TAGCGCGCTCGCATCATTTTGTGT */ #include #include #include #include #include "assert_helpers.h" #include "random_util.h" #include "aligner_result.h" #include "gfm.h" #include "simple_func.h" #include "scoring.h" #include "edit.h" #include "read.h" #include "ds.h" #include "group_walk.h" #include "btypes.h" typedef size_t TReadOff; typedef int64_t TScore; typedef float TRootPri; typedef size_t TDescentId; typedef size_t TRootId; /** * enum encapsulating a few different policies for how we might extend descents * in the direction opposite from their primary direction. */ enum { // Never extened in the direction opposite from the primary. Just go in // the primary direction until the bounce. DESC_EX_NONE = 1, // When we're finished extending out the matches for a descent, try to // extend in the opposite direction in a way that extends all branches // simultaneously. The Descent.nex_ field contains the number of positions // we were able to extend through in this way. DESC_EX_FROM_1ST_BRANCH = 2, // Each time we add an edge to the summary, extend it in the opposite // direction. The DescentEdge.nex field contains the number of positions // we were able to extend through, and this in turn gets propagated to // Descent.nex_ if and when we branch from the DescentEdge. DESC_EX_EACH_EDGE = 3 }; /** * Counters to keep track of how much work is being done. */ struct DescentMetrics { DescentMetrics() { reset(); } void reset() { bwops = bwops_1 = bwops_bi = recalc = branch = branch_mm = branch_del = branch_ins = heap_max = descent_max = descentpos_max = nex = 0; } uint64_t bwops; // # FM Index opbs uint64_t bwops_1; // # LF1 FM Index opbs uint64_t bwops_bi; // # BiEx FM Index opbs uint64_t recalc; // # times outgoing edge summary was recalculated uint64_t branch; // # times we descended from another descent uint64_t branch_mm; // # times branch was on a mismatch uint64_t branch_del; // # times branch was on a deletion uint64_t branch_ins; // # times branch was on a insertion uint64_t heap_max; // maximum size of Descent heap uint64_t descent_max; // maximum size of Descent factory uint64_t descentpos_max; // maximum size of DescentPos factory uint64_t nex; // # extensions }; /** * Priority used to rank which descent we should branch from next. Right now, * priority is governed by a 4-tuple. From higher to lower priority: * * 1. Penalty accumulated so far * 2. Depth into the search space, including extensions * 3. Width of the SA range (i.e. uniqueness) * 4. Root priority */ struct DescentPriority { DescentPriority() { reset(); } DescentPriority( TScore pen_, size_t depth_, TIndexOffU width_, float rootpri_) { pen = pen_; depth = depth_; width = width_; rootpri = rootpri_; } /** * Initialize new DescentPriority. */ void init(TScore pen_, size_t depth_, TIndexOffU width_, float rootpri_) { pen = pen_; depth = depth_; width = width_; rootpri = rootpri_; } /** * Reset to uninitialized state. */ void reset() { width = 0; } /** * Return true iff DescentPriority is initialized. */ bool inited() const { return width > 0; } /** * Return true iff this priority is prior to given priority. */ bool operator<(const DescentPriority& o) const { assert(inited()); assert(o.inited()); // 1st priority: penalty accumulated so far if(pen < o.pen) return true; if(pen > o.pen) return false; // 2nd priority: depth into the search space, including extensions if(depth > o.depth) return true; if(depth < o.depth) return false; // 3rd priority: width of the SA range (i.e. uniqueness) if(width < o.width) return true; if(width > o.width) return false; // 4th priority: root priority if(rootpri > o.rootpri) return true; return false; } /** * Return true iff this priority is prior to or equal to given priority. */ bool operator<=(const DescentPriority& o) const { assert(inited()); assert(o.inited()); // 1st priority: penalty accumulated so far if(pen < o.pen) return true; if(pen > o.pen) return false; // 2nd priority: depth into the search space, including extensions if(depth > o.depth) return true; if(depth < o.depth) return false; // 3rd priority: width of the SA range (i.e. uniqueness) if(width < o.depth) return true; if(width > o.width) return false; // 4th priority: root priority if(rootpri > o.rootpri) return true; return true; } /** * Return true iff this priority is prior to or equal to given priority. */ bool operator==(const DescentPriority& o) const { assert(inited()); assert(o.inited()); return pen == o.pen && depth == o.depth && width == o.width && rootpri == o.rootpri; } TScore pen; // total penalty accumulated so far size_t depth; // depth from root of descent TIndexOffU width; // width of the SA range float rootpri; // priority of the root }; static inline std::ostream& operator<<( std::ostream& os, const DescentPriority& o) { os << "[" << o.pen << ", " << o.depth << ", " << o.width << ", " << o.rootpri << "]"; return os; } static inline std::ostream& operator<<( std::ostream& os, const std::pair& o) { os << "{[" << o.first.pen << ", " << o.first.depth << ", " << o.first.width << ", " << o.first.rootpri << "], " << o.second << "}"; return os; } typedef std::pair TDescentPair; /** * Encapsulates the constraints limiting which outgoing edges are permitted. * Specifically, we constrain the total penalty accumulated so far so that some * outgoing edges will exceed the limit and be pruned. The limit is set * according to our "depth" into the search, as measured by the number of read * characters aligned so far. We divide the depth domain into two pieces, a * piece close to the root, where the penty is constrained to be 0, and the * remainder, where the maximum penalty is an interpolation between 0 and the * maximum penalty */ struct DescentConstraints { DescentConstraints() { reset(); } /** * Initialize with new constraint function. */ DescentConstraints(size_t nzero, double exp) { init(nzero, exp); } /** * Initialize with given function. */ void init(size_t nzero_, double exp_) { nzero = nzero_ > 0 ? nzero_ : 1; exp = exp_; #ifndef NDEBUG for(size_t i = 1; i < nzero_ + 5; i++) { assert_geq(get(i, nzero_ + 10, 100), get(i-1, nzero_ + 10, 100)); } #endif } /** * Reset to uninitialized state. */ void reset() { nzero = 0; exp = -1.0f; } /** * Return true iff the DescentConstraints has been initialized. */ bool inited() const { return exp >= 0.0f; } /** * Get the maximum penalty total for depth 'off'. */ inline TScore get(TReadOff off, TReadOff rdlen, TAlScore maxpen) const { if(off < nzero || nzero >= rdlen) { return 0; } double frac = (double)(off - nzero) / (rdlen - nzero); if(fabs(exp - 1.0f) > 0.00001) { if(fabs(exp - 2.0f) < 0.00001) { frac *= frac; } else { frac = pow(frac, exp); } } return (TAlScore)(frac * maxpen + 0.5f); } size_t nzero; double exp; }; /** * Encapsulates settings governing how we descent. */ struct DescentConfig { DescentConfig() { reset(); } /** * Reset the DescentConfig to an uninitialized state. */ void reset() { expol = 0; } /** * Return true iff this DescentConfig is initialized. */ bool inited() const { return expol != 0; } DescentConstraints cons; // constraints int expol; // extend policy }; /** * Encapsulates the state of a Descent that allows us to determine whether it * is redundant with another Descent. Two Descents are redundant if: * * 1. Both are aligning the same read orientation (fw or rc) * 2. Both are growing the alignment in the same direction (left-to-right or * right-to-left) * 3. They have aligned exactly the same read characters (which are always * consecutive in the read) * 4. The corresponding reference strings are identical */ struct DescentRedundancyKey { DescentRedundancyKey() { reset(); } DescentRedundancyKey( TReadOff al5pf_, size_t rflen_, TIndexOffU topf_, TIndexOffU botf_) { init(al5pf_, rflen_, topf_, botf_); } void reset() { al5pf = 0; rflen = 0; topf = botf = 0; } bool inited() const { return rflen > 0; } void init( TReadOff al5pf_, size_t rflen_, TIndexOffU topf_, TIndexOffU botf_) { al5pf = al5pf_; rflen = rflen_; topf = topf_; botf = botf_; } bool operator==(const DescentRedundancyKey& o) const { return al5pf == o.al5pf && rflen == o.rflen && topf == o.topf && botf == o.botf; } bool operator<(const DescentRedundancyKey& o) const { if(al5pf < o.al5pf) return true; if(al5pf > o.al5pf) return false; if(rflen < o.rflen) return true; if(rflen > o.rflen) return false; if(topf < o.topf) return true; if(topf > o.topf) return false; return botf < o.botf; } TReadOff al5pf; // 3'-most aligned char, as offset from 5' end size_t rflen; // number of reference characters involved in alignment TIndexOffU topf; // top w/r/t forward index TIndexOffU botf; // bot w/r/t forward index }; /** * Map from pairs to top, bot, penalty triples. */ class DescentRedundancyChecker { public: DescentRedundancyChecker() { reset(); } void clear() { reset(); } /** * Reset to uninitialized state. */ void reset() { bits_.reset(); inited_ = false; totsz_ = 0; // total size totcap_ = 0; // total capacity } const static int NPARTS = 8; const static int PART_MASK = 7; const static int NBITS = (1 << 16); /** * Initialize using given read length. */ void init(TReadOff rdlen) { reset(); // daehwan - for debugging purposes #if 0 bits_.resize(NBITS); maplist_fl_.resize(NPARTS); maplist_fr_.resize(NPARTS); maplist_rl_.resize(NPARTS); maplist_rr_.resize(NPARTS); for(int i = 0; i < NPARTS; i++) { maplist_fl_[i].resize(rdlen); maplist_fr_[i].resize(rdlen); maplist_rl_[i].resize(rdlen); maplist_rr_[i].resize(rdlen); totcap_ += maplist_fl_[i].totalCapacityBytes(); totcap_ += maplist_fr_[i].totalCapacityBytes(); totcap_ += maplist_rl_[i].totalCapacityBytes(); totcap_ += maplist_rr_[i].totalCapacityBytes(); for(size_t j = 0; j < rdlen; j++) { maplist_fl_[i][j].clear(); maplist_fr_[i][j].clear(); maplist_rl_[i][j].clear(); maplist_rr_[i][j].clear(); totcap_ += maplist_fl_[i][j].totalCapacityBytes(); totcap_ += maplist_fr_[i][j].totalCapacityBytes(); totcap_ += maplist_rl_[i][j].totalCapacityBytes(); totcap_ += maplist_rr_[i][j].totalCapacityBytes(); } } #endif inited_ = true; } /** * Return true iff the checker is initialized. */ bool inited() const { return inited_; } /** * Check if this partial alignment is redundant with one that we've already * explored. */ bool check( bool fw, bool l2r, TReadOff al5pi, TReadOff al5pf, size_t rflen, TIndexOffU topf, TIndexOffU botf, TScore pen) { // daehwan - for debugging purposes return true; assert(inited_); assert(topf > 0 || botf > 0); DescentRedundancyKey k(al5pf, rflen, topf, botf); size_t i = std::numeric_limits::max(); size_t mask = topf & PART_MASK; EMap& map = (fw ? (l2r ? maplist_fl_[mask][al5pi] : maplist_fr_[mask][al5pi]) : (l2r ? maplist_rl_[mask][al5pi] : maplist_rr_[mask][al5pi])); size_t key = (topf & 255) | ((botf & 255) << 8); if(bits_.test(key) && map.containsEx(k, i)) { // Already contains the key assert_lt(i, map.size()); assert_geq(pen, map[i].second); return false; } assert(!map.containsEx(k, i)); size_t oldsz = map.totalSizeBytes(); size_t oldcap = map.totalCapacityBytes(); map.insert(make_pair(k, pen)); bits_.set(key); totsz_ += (map.totalSizeBytes() - oldsz); totcap_ += (map.totalCapacityBytes() - oldcap); return true; } /** * Check if this partial alignment is redundant with one that we've already * explored using the Bw index SA range. */ bool contains( bool fw, bool l2r, TReadOff al5pi, TReadOff al5pf, size_t rflen, TIndexOffU topf, TIndexOffU botf, TScore pen) { // daehwan - for debugging purposes return false; assert(inited_); size_t key = (topf & 255) | ((botf & 255) << 8); if(!bits_.test(key)) { return false; } DescentRedundancyKey k(al5pf, rflen, topf, botf); size_t mask = topf & PART_MASK; EMap& map = (fw ? (l2r ? maplist_fl_[mask][al5pi] : maplist_fr_[mask][al5pi]) : (l2r ? maplist_rl_[mask][al5pi] : maplist_rr_[mask][al5pi])); return map.contains(k); } /** * Return the total size of the redundancy map. */ size_t totalSizeBytes() const { return totsz_; } /** * Return the total capacity of the redundancy map. */ size_t totalCapacityBytes() const { return totcap_; } protected: bool inited_; // initialized? size_t totsz_; // total size size_t totcap_; // total capacity // List of maps. Each entry is a map for all the DescentRedundancyKeys // with al5pi equal to the offset into the list. ELList, NPARTS, 100> maplist_fl_; // fw, l2r ELList, NPARTS, 100> maplist_rl_; // !fw, l2r ELList, NPARTS, 100> maplist_fr_; // fw, !l2r ELList, NPARTS, 100> maplist_rr_; // !fw, !l2r EBitList<128> bits_; }; /** * A search root. Consists of an offset from the 5' end read and flags * indicating (a) whether we're initially heading left-to-right or * right-to-left, and (b) whether we're examining the read or its reverse * complement. * * A root also comes with a priority ("pri") score indicating how promising it * is as a root. Promising roots have long stretches of high-quality, * non-repetitive nucleotides in the first several ply of the search tree. * Also, roots beginning at the 5' end of the read may receive a higher * priority. */ struct DescentRoot { DescentRoot() { reset(); } DescentRoot(size_t off5p_, bool l2r_, bool fw_, size_t len, float pri_) { init(off5p_, l2r_, fw_, len, pri_); } /** * Reset this DescentRoot to uninitialized state. */ void reset() { off5p = std::numeric_limits::max(); } /** * Return true iff this DescentRoot is uninitialized. */ bool inited() const { return off5p == std::numeric_limits::max(); } /** * Initialize a new descent root. */ void init(size_t off5p_, bool l2r_, bool fw_, size_t len, float pri_) { off5p = off5p_; l2r = l2r_; fw = fw_; pri = pri_; assert_lt(off5p, len); } TReadOff off5p; // root origin offset, expressed as offset from 5' end bool l2r; // true -> move in left-to-right direction bool fw; // true -> work with forward read, false -> revcomp float pri; // priority of seed }; /** * Set of flags indicating outgoing edges we've tried from a DescentPos. */ struct DescentPosFlags { DescentPosFlags() { reset(); } /** * Set all flags to 1, indicating all outgoing edges are yet to be * explored. */ void reset() { mm_a = mm_c = mm_g = mm_t = rdg_a = rdg_c = rdg_g = rdg_t = rfg = 1; reserved = 0; } /** * Return true iff all outgoing edges have already been explored. */ bool exhausted() const { return ((uint16_t*)this)[0] == 0; } /** * Return false iff the specified mismatch has already been explored. */ bool mmExplore(int c) { assert_range(0, 3, c); if(c == 0) { return mm_a; } else if(c == 1) { return mm_c; } else if(c == 2) { return mm_g; } else { return mm_t; } } /** * Try to explore a mismatch. Return false iff it has already been * explored. */ bool mmSet(int c) { assert_range(0, 3, c); if(c == 0) { bool ret = mm_a; mm_a = 0; return ret; } else if(c == 1) { bool ret = mm_c; mm_c = 0; return ret; } else if(c == 2) { bool ret = mm_g; mm_g = 0; return ret; } else { bool ret = mm_t; mm_t = 0; return ret; } } /** * Return false iff specified read gap has already been explored. */ bool rdgExplore(int c) { assert_range(0, 3, c); if(c == 0) { return rdg_a; } else if(c == 1) { return rdg_c; } else if(c == 2) { return rdg_g; } else { return rdg_t; } } /** * Try to explore a read gap. Return false iff it has already been * explored. */ bool rdgSet(int c) { assert_range(0, 3, c); if(c == 0) { bool ret = rdg_a; rdg_a = 0; return ret; } else if(c == 1) { bool ret = rdg_c; rdg_c = 0; return ret; } else if(c == 2) { bool ret = rdg_g; rdg_g = 0; return ret; } else { bool ret = rdg_t; rdg_t = 0; return ret; } } /** * Return false iff the reference gap has already been explored. */ bool rfgExplore() { return rfg; } /** * Try to explore a reference gap. Return false iff it has already been * explored. */ bool rfgSet() { bool ret = rfg; rfg = 0; return ret; } uint16_t mm_a : 1; uint16_t mm_c : 1; uint16_t mm_g : 1; uint16_t mm_t : 1; uint16_t rdg_a : 1; uint16_t rdg_c : 1; uint16_t rdg_g : 1; uint16_t rdg_t : 1; uint16_t rfg : 1; uint16_t reserved : 7; }; /** * FM Index state associated with a single position in a descent. For both the * forward and backward indexes, it stores the four SA ranges corresponding to * the four nucleotides. */ struct DescentPos { /** * Reset all tops and bots to 0. */ void reset() { topf[0] = topf[1] = topf[2] = topf[3] = 0; botf[0] = botf[1] = botf[2] = botf[3] = 0; topb[0] = topb[1] = topb[2] = topb[3] = 0; botb[0] = botb[1] = botb[2] = botb[3] = 0; c = -1; flags.reset(); } /** * Return true iff DescentPos has been initialized. */ bool inited() const { return c >= 0; } #ifndef NDEBUG /** * Check that DescentPos is internally consistent. */ bool repOk() const { assert_range(0, 3, (int)c); return true; } #endif TIndexOffU topf[4]; // SA range top indexes in fw index TIndexOffU botf[4]; // SA range bottom indexes (exclusive) in fw index TIndexOffU topb[4]; // SA range top indexes in bw index TIndexOffU botb[4]; // SA range bottom indexes (exclusive) in bw index char c; // read char that would yield match DescentPosFlags flags; // flags }; /** * Encapsulates an edge outgoing from a descent. */ struct DescentEdge { DescentEdge() { reset(); } DescentEdge( Edit e_, TReadOff off5p_, DescentPriority pri_, size_t posFlag_, TReadOff nex_ #ifndef NDEBUG , size_t d_, TIndexOffU topf_, TIndexOffU botf_, TIndexOffU topb_, TIndexOffU botb_ #endif ) { init(e_, off5p_, pri_, posFlag_ #ifndef NDEBUG , d_, topf_, botf_, topb_, botb_ #endif ); } /** * Return true iff edge is initialized. */ bool inited() const { return e.inited(); } /** * Reset to uninitialized state. */ void reset() { e.reset(); } /** * Initialize DescentEdge given 5' offset, nucleotide, and priority. */ void init( Edit e_, TReadOff off5p_, DescentPriority pri_, size_t posFlag_ #ifndef NDEBUG , size_t d_, TIndexOffU topf_, TIndexOffU botf_, TIndexOffU topb_, TIndexOffU botb_ #endif ) { e = e_; off5p = off5p_; pri = pri_; posFlag = posFlag_; #ifndef NDEBUG d = d_; topf = topf_; botf = botf_; topb = topb_; botb = botb_; #endif } /** * Update flags to show this edge as visited. */ void updateFlags(EFactory& pf) { if(inited()) { if(e.isReadGap()) { assert_neq('-', e.chr); pf[posFlag].flags.rdgSet(asc2dna[e.chr]); } else if(e.isRefGap()) { pf[posFlag].flags.rfgSet(); } else { assert_neq('-', e.chr); pf[posFlag].flags.mmSet(asc2dna[e.chr]); } } } /** * Return true iff this edge has higher priority than the given edge. */ bool operator<(const DescentEdge& o) const { if(inited() && !o.inited()) { return true; } else if(!inited()) { return false; } return pri < o.pri; } DescentPriority pri; // priority of the edge //TReadOff nex; // # extends possible from this edge size_t posFlag; // depth of DescentPos where flag should be set #ifndef NDEBUG // This can be recreated by looking at the edit, the paren't descent's // len_, al5pi_, al5pf_. I have it here so we can sanity check. size_t d; TIndexOffU topf, botf, topb, botb; #endif Edit e; TReadOff off5p; }; /** * Encapsulates an incomplete summary of the outgoing edges from a descent. We * don't try to store information about all outgoing edges, because doing so * will generally be wasteful. We'll typically only try a handful of them per * descent. */ class DescentOutgoing { public: /** * Return the best edge and rotate in preparation for next call. */ DescentEdge rotate() { DescentEdge tmp = best1; assert(!(best2 < tmp)); best1 = best2; assert(!(best3 < best2)); best2 = best3; assert(!(best4 < best3)); best3 = best4; assert(!(best5 < best4)); best4 = best5; best5.reset(); return tmp; } /** * Given a potental outgoing edge, place it where it belongs in the running * list of best 5 outgoing edges from this descent. */ void update(DescentEdge e) { if(!best1.inited()) { best1 = e; } else if(e < best1) { best5 = best4; best4 = best3; best3 = best2; best2 = best1; best1 = e; } else if(!best2.inited()) { best2 = e; } else if(e < best2) { best5 = best4; best4 = best3; best3 = best2; best2 = e; } else if(!best3.inited()) { best3 = e; } else if(e < best3) { best5 = best4; best4 = best3; best3 = e; } else if(!best4.inited()) { best4 = e; } else if(e < best4) { best5 = best4; best4 = e; } else if(!best5.inited() || e < best5) { best5 = e; } } /** * Clear all the outgoing edges stored here. */ void clear() { best1.reset(); best2.reset(); best3.reset(); best4.reset(); best5.reset(); } /** * Return true iff there are no outgoing edges currently represented in * this summary. There may still be outgoing edges, they just haven't * been added to the summary. */ bool empty() const { return !best1.inited(); } /** * Return the DescentPriority of the best outgoing edge. */ DescentPriority bestPri() const { assert(!empty()); return best1.pri; } DescentEdge best1; // best DescentEdge best2; // 2nd-best DescentEdge best3; // 3rd-best DescentEdge best4; // 4th-best DescentEdge best5; // 5th-best }; template class DescentAlignmentSink; /** * Encapsulates a descent through a search tree, along a path of matches. * Descents that are part of the same alignment form a chain. Two aligments * adjacent in the chain are connected either by an edit, or by a switch in * direction. Because a descent might have a different direction from the * DescentRoot it ultimately came from, it has its own 'l2r' field, which might * differ from the root's. */ template class Descent { public: Descent() { reset(); } /** * Initialize a new descent branching from the given descent via the given * edit. Return false if the Descent has no outgoing edges (and can * therefore have its memory freed), true otherwise. */ bool init( const Read& q, // query TRootId rid, // root id const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score TAlScore maxpen, // maximum penalty TReadOff al5pi, // offset from 5' of 1st aligned char TReadOff al5pf, // offset from 5' of last aligned char TIndexOffU topf, // SA range top in FW index TIndexOffU botf, // SA range bottom in FW index TIndexOffU topb, // SA range top in BW index TIndexOffU botb, // SA range bottom in BW index bool l2r, // direction this descent will go in size_t descid, // my ID TDescentId parent, // parent ID TScore pen, // total penalties so far const Edit& e, // edit for incoming edge const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index DescentRedundancyChecker& re, // redundancy checker EFactory& df, // Descent factory EFactory& pf, // DescentPos factory const EList& rs, // roots const EList& cs, // configs EHeap& heap, // heap DescentAlignmentSink& alsink, // alignment sink DescentMetrics& met, // metrics PerReadMetrics& prm); // per-read metrics /** * Initialize a new descent beginning at the given root. Return false if * the Descent has no outgoing edges (and can therefore have its memory * freed), true otherwise. */ bool init( const Read& q, // query TRootId rid, // root id const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score TAlScore maxpen, // maximum penalty size_t descid, // id of this Descent const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index DescentRedundancyChecker& re, // redundancy checker EFactory& df, // Descent factory EFactory& pf, // DescentPos factory const EList& rs, // roots const EList& cs, // configs EHeap& heap, // heap DescentAlignmentSink& alsink, // alignment sink DescentMetrics& met, // metrics PerReadMetrics& prm); // per-read metrics /** * Return true iff this Descent has been initialized. */ bool inited() const { return descid_ != std::numeric_limits::max(); } /** * Reset to uninitialized state. */ void reset() { lastRecalc_ = true; descid_ = std::numeric_limits::max(); } /** * Return true iff this Descent is a search root. */ bool root() const { return parent_ == std::numeric_limits::max(); } /** * Return the edit. */ const Edit& edit() const { return edit_; } /** * Return id of parent. */ TDescentId parent() const { return parent_; } /** * Take the best outgoing edge and follow it. */ void followBestOutgoing( const Read& q, // read const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score TAlScore maxpen, // maximum penalty DescentRedundancyChecker& re, // redundancy checker EFactory& df, // factory with Descent EFactory& pf, // factory with DescentPoss const EList& rs, // roots const EList& cs, // configs EHeap& heap, // heap of descents DescentAlignmentSink& alsink, // alignment sink DescentMetrics& met, // metrics PerReadMetrics& prm); // per-read metrics /** * Return true iff no outgoing edges from this descent remain unexplored. */ bool empty() const { return lastRecalc_ && out_.empty(); } #ifndef NDEBUG /** * Return true iff the Descent is internally consistent. */ bool repOk(const Read *q) const { // A non-root can have an uninitialized edit_ if it is from a bounce //assert( root() || edit_.inited()); assert(!root() || !edit_.inited()); assert_eq(botf_ - topf_, botb_ - topb_); if(q != NULL) { assert_leq(len_, q->length()); } return true; } #endif size_t al5pi() const { return al5pi_; } size_t al5pf() const { return al5pf_; } bool l2r() const { return l2r_; } /** * Print a stacked representation of this descent and all its parents. Assumes that */ void print( std::ostream* os, const char *prefix, const Read& q, size_t trimLf, size_t trimRg, bool fw, const EList& edits, size_t ei, size_t en, BTDnaString& rf) const; /** * Collect all the edits */ void collectEdits( EList& edits, const Edit *e, EFactory& df) { // Take just the portion of the read that has aligned up until this // point size_t nuninited = 0; size_t ei = edits.size(); size_t en = 0; if(e != NULL && e->inited()) { edits.push_back(*e); en++; } size_t cur = descid_; while(cur != std::numeric_limits::max()) { if(!df[cur].edit().inited()) { nuninited++; assert_leq(nuninited, 2); } else { edits.push_back(df[cur].edit()); en++; } cur = df[cur].parent(); } // Sort just the edits we just added edits.sortPortion(ei, en); } protected: /** * */ bool bounce( const Read& q, // query string TIndexOffU topf, // SA range top in fw index TIndexOffU botf, // SA range bottom in fw index TIndexOffU topb, // SA range top in bw index TIndexOffU botb, // SA range bottom in bw index const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score TAlScore maxpen, // maximum penalty DescentRedundancyChecker& re, // redundancy checker EFactory& df, // factory with Descent EFactory& pf, // factory with DescentPoss const EList& rs, // roots const EList& cs, // configs EHeap& heap, // heap of descents DescentAlignmentSink& alsink, // alignment sink DescentMetrics& met, // metrics PerReadMetrics& prm); // per-read metrics /** * Given the forward and backward indexes, and given topf/botf/topb/botb, * get tloc, bloc ready for the next step. */ void nextLocsBi( const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index SideLocus& tloc, // top locus SideLocus& bloc, // bot locus index_t topf, // top in BWT index_t botf, // bot in BWT index_t topb, // top in BWT' index_t botb); // bot in BWT' /** * Advance this descent by following read matches as far as possible. */ bool followMatches( const Read& q, // query string const Scoring& sc, // scoring scheme const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index DescentRedundancyChecker& re, // redundancy checker EFactory& df, // Descent factory EFactory& pf, // DescentPos factory const EList& rs, // roots const EList& cs, // configs EHeap& heap, // heap DescentAlignmentSink& alsink, // alignment sink DescentMetrics& met, // metrics PerReadMetrics& prm, // per-read metrics bool& branches, // out: true -> there are > 0 ways to branch bool& hitEnd, // out: true -> hit read end with non-empty range bool& done, // out: true -> we made a full alignment TReadOff& off5p_i, // out: initial 5' offset TIndexOffU& topf_bounce, // out: top of SA range for fw idx for bounce TIndexOffU& botf_bounce, // out: bot of SA range for fw idx for bounce TIndexOffU& topb_bounce, // out: top of SA range for bw idx for bounce TIndexOffU& botb_bounce); // out: bot of SA range for bw idx for bounce /** * Recalculate our summary of the outgoing edges from this descent. When * deciding what outgoing edges are legal, we abide by constraints. * Typically, they limit the total of the penalties accumulated so far, as * a function of distance from the search root. E.g. a constraint might * disallow any gaps or mismatches within 20 ply of the search root, then * allow 1 mismatch within 30 ply, then allow up to 1 mismatch or 1 gap * within 40 ply, etc. */ size_t recalcOutgoing( const Read& q, // query string const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score TAlScore maxpen, // maximum penalty DescentRedundancyChecker& re, // redundancy checker EFactory& pf, // factory with DescentPoss const EList& rs, // roots const EList& cs, // configs PerReadMetrics& prm); // per-read metrics TRootId rid_; // root id TReadOff al5pi_; // lo offset from 5' end of aligned read char TReadOff al5pf_; // hi offset from 5' end of aligned read char bool l2r_; // left-to-right? int gapadd_; // net ref characters additional TReadOff off5p_i_; // offset we started out at for this descent TIndexOffU topf_, botf_; // incoming SA range w/r/t forward index TIndexOffU topb_, botb_; // incoming SA range w/r/t forward index size_t descid_; // ID of this descent TDescentId parent_; // ID of parent descent TScore pen_; // total penalties accumulated so far size_t posid_; // ID of 1st elt of the DescentPos factory w/ // descent pos info for this descent size_t len_; // length of stretch of matches DescentOutgoing out_; // summary of outgoing edges Edit edit_; // edit joining this descent with parent bool lastRecalc_; // set by recalcOutgoing if out edges empty }; /** * An alignment result from a Descent. */ struct DescentAlignment { DescentAlignment() { reset(); } /** * Reset DescentAlignment to be uninitialized. */ void reset() { topf = botf = 0; pen = 0; fw = false; ei = en = 0; } /** * Initialize this DescentAlignment. */ void init( TScore pen_, bool fw_, TIndexOffU topf_, TIndexOffU botf_, size_t ei_, size_t en_) { assert_gt(botf_, topf_); pen = pen_; fw = fw_; topf = topf_; botf = botf_; ei = ei_; en = en_; } /** * Return true iff DescentAlignment is initialized. */ bool inited() const { return botf > topf; } /** * Return true iff the alignment is perfect (has no edits) */ bool perfect() const { return pen == 0; } /** * Return the number of elements in this range. */ size_t size() const { return botf - topf; } TScore pen; // score bool fw; // forward or revcomp aligned? TIndexOffU topf; // top in forward index TIndexOffU botf; // bot in forward index size_t ei; // First edit in DescentAlignmentSink::edits_ involved in aln size_t en; // # edits in DescentAlignmentSink::edits_ involved in aln }; /** * A partial alignment result from a Descent where the reference offset has * been resolved. */ struct DescentPartialResolvedAlignment { DescentPartialResolvedAlignment() { reset(); } /** * Reset DescentAlignment to be uninitialized. */ void reset() { topf = botf = 0; pen = 0; fw = false; ei = en = 0; refcoord.reset(); } /** * Initialize this DescentAlignment. */ void init( TScore pen_, bool fw_, TIndexOffU topf_, TIndexOffU botf_, size_t ei_, size_t en_, const Coord& refcoord_) { assert_gt(botf_, topf_); pen = pen_; fw = fw_; topf = topf_; botf = botf_; ei = ei_; en = en_; refcoord = refcoord_; } /** * Return true iff DescentAlignment is initialized. */ bool inited() const { return botf > topf; } /** * Return the number of elements in this range. */ size_t size() const { return botf - topf; } TScore pen; // score bool fw; // forward or revcomp aligned? TIndexOffU topf; // top in forward index TIndexOffU botf; // bot in forward index size_t ei; // First edit in DescentAlignmentSink::edits_ involved in aln size_t en; // # edits in DescentAlignmentSink::edits_ involved in aln Coord refcoord; // reference coord of leftmost ref char involved }; /** * Class that accepts alignments found during descent and maintains the state * required to dispense them to consumers in an appropriate order. * * As for order in which they are dispensed, in order to maintain uniform * distribution over equal-scoring alignments, a good policy may be not to * dispense alignments at a given score stratum until *all* alignments at that * stratum have been accumulated (i.e. until our best-first search has moved on * to a worse stratum). This also has the advantage that, for each alignment, * we can also report the number of other alignments in that cost stratum. * * A lazier alternative is to assume that the order in which alignments in a * given stratum arrive is already pseudo-random, which frees us from having to * wait until the entire stratum has been explored. But there is reason to * think that this order is not truly pseudo-random, since our root placement * and root priorities will tend to first lead us to alignments with certain * patterns of edits. */ template class DescentAlignmentSink { public: /** * If this is the final descent in a complete end-to-end alignment, report * the alignment. */ bool reportAlignment( const Read& q, // query string const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index TIndexOffU topf, // SA range top in forward index TIndexOffU botf, // SA range bottom in forward index TIndexOffU topb, // SA range top in backward index TIndexOffU botb, // SA range bottom in backward index TDescentId id, // id of leaf Descent TRootId rid, // id of search root const Edit& e, // final edit, if needed TScore pen, // total penalty EFactory >& df, // factory with Descent EFactory& pf, // factory with DescentPoss const EList& rs, // roots const EList& cs); // configs /** * Reset to uninitialized state. */ void reset() { edits_.clear(); als_.clear(); lhs_.clear(); rhs_.clear(); nelt_ = 0; bestPen_ = worstPen_ = std::numeric_limits::max(); } /** * Return the total size occupued by the Descent driver and all its * constituent parts. */ size_t totalSizeBytes() const { return edits_.totalSizeBytes() + als_.totalSizeBytes() + lhs_.totalSizeBytes() + rhs_.totalSizeBytes() + sizeof(size_t); } /** * Return the total capacity of the Descent driver and all its constituent * parts. */ size_t totalCapacityBytes() const { return edits_.totalCapacityBytes() + als_.totalCapacityBytes() + lhs_.totalCapacityBytes() + rhs_.totalCapacityBytes() + sizeof(size_t); } /** * Return the number of SA ranges involved in hits. */ size_t nrange() const { return als_.size(); } /** * Return the number of SA elements involved in hits. */ size_t nelt() const { return nelt_; } /** * The caller provides 'i', which is an offset of a particular element in * one of the SA ranges in the current stratum. This function returns, in * 'al' and 'off', information about the element in terms of the range it's * part of and its offset into that range. */ void elt(size_t i, DescentAlignment& al, size_t& ri, size_t& off) const { assert_lt(i, nelt()); for(size_t j = 0; j < als_.size(); j++) { if(i < als_[j].size()) { al = als_[j]; ri = j; off = i; return; } i -= als_[j].size(); } assert(false); } /** * Get a particular alignment. */ const DescentAlignment& operator[](size_t i) const { return als_[i]; } /** * Return true iff (a) we found an alignment since the sink was initialized * or since the last time advanceStratum() was called, and (b) the penalty * associated with the current-best task on the heap ('best') is worse * (higher) than the penalty associated with the alignments found most * recently (worstPen_). */ bool stratumDone(TAlScore bestPen) const { if(nelt_ > 0 && bestPen > worstPen_) { return true; } return false; } /** * The alignment consumer calls this to indicate that they are done with * all the alignments in the current best non-empty stratum. We can * therefore mark all those alignments as "reported" and start collecting * results for the next stratum. */ void advanceStratum() { assert_gt(nelt_, 0); edits_.clear(); als_.clear(); // Don't reset lhs_ or rhs_ nelt_ = 0; bestPen_ = worstPen_ = std::numeric_limits::max(); } #ifndef NDEBUG /** * Check that alignment sink is internally consistent. */ bool repOk() const { assert_geq(nelt_, als_.size()); for(size_t i = 1; i < als_.size(); i++) { assert_geq(als_[i].pen, als_[i-1].pen); } assert(bestPen_ == std::numeric_limits::max() || worstPen_ >= bestPen_); return true; } #endif TAlScore bestPenalty() const { return bestPen_; } TAlScore worstPenalty() const { return worstPen_; } size_t editsSize() const { return edits_.size(); } size_t alsSize() const { return als_.size(); } size_t lhsSize() const { return lhs_.size(); } size_t rhsSize() const { return rhs_.size(); } const EList& edits() const { return edits_; } protected: EList edits_; EList als_; ESet > lhs_; ESet > rhs_; size_t nelt_; TAlScore bestPen_; // best (smallest) penalty among as-yet-unreported alns TAlScore worstPen_; // worst (greatest) penalty among as-yet-unreported alns #ifndef NDEBUG BTDnaString tmprfdnastr_; #endif }; /** * Class that aggregates partial alignments taken from a snapshot of the * DescentDriver heap. */ class DescentPartialResolvedAlignmentSink { public: /** * Reset to uninitialized state. */ void reset() { edits_.clear(); als_.clear(); nelt_ = 0; bestPen_ = worstPen_ = std::numeric_limits::max(); } /** * Return the total size occupued by the Descent driver and all its * constituent parts. */ size_t totalSizeBytes() const { return edits_.totalSizeBytes() + als_.totalSizeBytes() + sizeof(size_t); } /** * Return the total capacity of the Descent driver and all its constituent * parts. */ size_t totalCapacityBytes() const { return edits_.totalCapacityBytes() + als_.totalCapacityBytes() + sizeof(size_t); } /** * Return the number of SA ranges involved in hits. */ size_t nrange() const { return als_.size(); } /** * Return the number of SA elements involved in hits. */ size_t nelt() const { return nelt_; } /** * The caller provides 'i', which is an offset of a particular element in * one of the SA ranges in the current stratum. This function returns, in * 'al' and 'off', information about the element in terms of the range it's * part of and its offset into that range. */ void elt(size_t i, DescentPartialResolvedAlignment& al, size_t& ri, size_t& off) const { assert_lt(i, nelt()); for(size_t j = 0; j < als_.size(); j++) { if(i < als_[j].size()) { al = als_[j]; ri = j; off = i; return; } i -= als_[j].size(); } assert(false); } /** * Get a particular alignment. */ const DescentPartialResolvedAlignment& operator[](size_t i) const { return als_[i]; } /** * Return true iff (a) we found an alignment since the sink was initialized * or since the last time advanceStratum() was called, and (b) the penalty * associated with the current-best task on the heap ('best') is worse * (higher) than the penalty associated with the alignments found most * recently (worstPen_). */ bool stratumDone(TAlScore bestPen) const { if(nelt_ > 0 && bestPen > worstPen_) { return true; } return false; } /** * The alignment consumer calls this to indicate that they are done with * all the alignments in the current best non-empty stratum. We can * therefore mark all those alignments as "reported" and start collecting * results for the next stratum. */ void advanceStratum() { assert_gt(nelt_, 0); edits_.clear(); als_.clear(); nelt_ = 0; bestPen_ = worstPen_ = std::numeric_limits::max(); } #ifndef NDEBUG /** * Check that partial alignment sink is internally consistent. */ bool repOk() const { assert_geq(nelt_, als_.size()); //for(size_t i = 1; i < als_.size(); i++) { // assert_geq(als_[i].pen, als_[i-1].pen); //} assert(bestPen_ == std::numeric_limits::max() || worstPen_ >= bestPen_); return true; } #endif TAlScore bestPenalty() const { return bestPen_; } TAlScore worstPenalty() const { return worstPen_; } size_t editsSize() const { return edits_.size(); } size_t alsSize() const { return als_.size(); } const EList& edits() const { return edits_; } protected: EList edits_; EList als_; size_t nelt_; TAlScore bestPen_; // best (smallest) penalty among as-yet-unreported alns TAlScore worstPen_; // worst (greatest) penalty among as-yet-unreported alns }; /** * Abstract parent for classes that select descent roots and descent * configurations given information about the read. */ class DescentRootSelector { public: virtual ~DescentRootSelector() { } virtual void select( const Read& q, // read that we're selecting roots for const Read* qo, // opposite mate, if applicable bool nofw, // don't add roots for fw read bool norc, // don't add roots for rc read EList& confs, // put DescentConfigs here EList& roots) = 0; // put DescentRoot here }; /** * Encapsulates a set of conditions governing when the DescentDriver should * stop. */ struct DescentStoppingConditions { DescentStoppingConditions() { reset(); } DescentStoppingConditions( size_t totsz_, size_t nfound_, bool stra_, size_t nbwop_) { init(totsz_, nfound_, stra_, nbwop_); } /** * Reset to uninitialized state. */ void reset() { totsz = nfound = nbwop = std::numeric_limits::max(); stra = false; assert(!inited()); } /** * Initialize this DescentStoppingConditions. */ void init( size_t totsz_, size_t nfound_, bool stra_, size_t nbwop_) { totsz = totsz_; nfound = nfound_; stra = stra_; nbwop = nbwop_; assert(inited()); } /** * Return true iff this instance is initialized. */ bool inited() const { return totsz != std::numeric_limits::max(); } size_t totsz; // total size of all the expandable data structures in bytes size_t nfound; // # alignments found bool stra; // stop after each non-empty stratum size_t nbwop; // # Burrows-Wheeler (rank) operations performed }; enum { DESCENT_DRIVER_ALN = 1, DESCENT_DRIVER_STRATA = 2, DESCENT_DRIVER_MEM = 4, DESCENT_DRIVER_BWOPS = 8, DESCENT_DRIVER_DONE = 16 }; /** * Class responsible for advancing all the descents. The initial descents may * emanate from several different locations in the read. Note that descents * may become redundant with each other, and should then be eliminated. */ template class DescentDriver { public: DescentDriver(bool veryVerbose) : veryVerbose_(veryVerbose) { reset(); } /** * Initialize driver with respect to a new read. If a DescentRootSelector * is specified, then it is used to obtain roots as well. */ void initRead( const Read& q, bool nofw, bool norc, TAlScore minsc, TAlScore maxpen, const Read* qu = NULL, DescentRootSelector *sel = NULL) { reset(); q_ = q; minsc_ = minsc; maxpen_ = maxpen; if(sel != NULL) { sel->select(q_, qu, nofw, norc, confs_, roots_); } re_.init(q.length()); } /** * Add a new search root, which might (a) prefer to move in a left-to-right * direction, and might (b) be with respect to the read or its reverse * complement. */ void addRoot( const DescentConfig& conf, TReadOff off, bool l2r, bool fw, float pri) { confs_.push_back(conf); assert_lt(off, q_.length()); if(l2r && off == q_.length()-1) { l2r = !l2r; } else if(!l2r && off == 0) { l2r = !l2r; } roots_.push_back(DescentRoot(off, l2r, fw, q_.length(), pri)); } /** * Clear out the DescentRoots currently configured. */ void clearRoots() { confs_.clear(); roots_.clear(); } /** * Clear the Descent driver so that we're ready to re-start seed alignment * for the current read. */ void resetRead() { df_.clear(); // clear Descents assert_leq(df_.totalSizeBytes(), 100); pf_.clear(); // clear DescentPoss assert_leq(pf_.totalSizeBytes(), 100); heap_.clear(); // clear Heap assert_leq(heap_.totalSizeBytes(), 100); roots_.clear(); // clear roots assert_leq(roots_.totalSizeBytes(), 100); confs_.clear(); // clear confs assert_leq(confs_.totalSizeBytes(), 100); alsink_.reset(); // clear alignment sink assert_leq(alsink_.totalSizeBytes(), 100); re_.reset(); assert_leq(re_.totalSizeBytes(), 100); rootsInited_ = 0; // haven't yet created initial descents curPen_ = 0; // } /** * Clear the Descent driver so that we're ready to re-start seed alignment * for the current read. */ void reset() { resetRead(); } /** * Perform seed alignment. */ void go( const Scoring& sc, // scoring scheme const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index DescentMetrics& met, // metrics PerReadMetrics& prm); // per-read metrics /** * Perform seed alignment until some stopping condition is satisfied. */ int advance( const DescentStoppingConditions& stopc, // stopping conditions const Scoring& sc, // scoring scheme const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index DescentMetrics& met, // metrics PerReadMetrics& prm); // per-read metrics #ifndef NDEBUG /** * Return true iff this DescentDriver is well formed. Throw an assertion * otherwise. */ bool repOk() const { return true; } #endif /** * Return the number of end-to-end alignments reported. */ size_t numAlignments() const { return alsink_.nelt(); } /** * Return the associated DescentAlignmentSink object. */ const DescentAlignmentSink& sink() const { return alsink_; } /** * Return the associated DescentAlignmentSink object. */ DescentAlignmentSink& sink() { return alsink_; } /** * Return the total size occupued by the Descent driver and all its * constituent parts. */ size_t totalSizeBytes() const { return df_.totalSizeBytes() + pf_.totalSizeBytes() + heap_.totalSizeBytes() + roots_.totalSizeBytes() + confs_.totalSizeBytes() + alsink_.totalSizeBytes() + re_.totalSizeBytes(); } /** * Return the total capacity of the Descent driver and all its constituent * parts. */ size_t totalCapacityBytes() const { return df_.totalCapacityBytes() + pf_.totalCapacityBytes() + heap_.totalCapacityBytes() + roots_.totalCapacityBytes() + confs_.totalCapacityBytes() + alsink_.totalCapacityBytes() + re_.totalCapacityBytes(); } /** * Return a const ref to the query. */ const Read& query() const { return q_; } /** * Return the minimum score that must be achieved by an alignment in order * for it to be considered "valid". */ TAlScore minScore() const { return minsc_; } protected: Read q_; // query nucleotide and quality strings TAlScore minsc_; // minimum score TAlScore maxpen_; // maximum penalty EFactory > df_; // factory holding all the Descents, which // must be referred to by ID EFactory pf_; // factory holding all the DescentPoss, which // must be referred to by ID EList roots_; // search roots EList confs_; // configuration params for each root size_t rootsInited_; // # initial Descents already created EHeap heap_; // priority queue of Descents DescentAlignmentSink alsink_; // alignment sink DescentRedundancyChecker re_; // redundancy checker TAlScore curPen_; // current penalty bool veryVerbose_; // print lots of partial alignments EList tmpedit_; BTDnaString tmprfdnastr_; }; /** * Selects alignments to report from a complete non-empty stratum of * alignments stored in the DescentAlignmentSink. */ template class DescentAlignmentSelector { public: DescentAlignmentSelector() : gwstate_(GW_CAT) { reset(); } /** * Initialize a new selector w/r/t a DescentAlignmentSink holding a * non-empty alignment stratum. */ void init( const Read& q, const DescentAlignmentSink& sink, const GFM& gfmFw, // forward Bowtie index for walking left const BitPairReference& ref, // bitpair-encoded reference RandomSource& rnd, // pseudo-random generator for sampling rows WalkMetrics& met) { // We're going to sample from space of *alignments*, not ranges. So // when we extract a sample, we'll have to do a little extra work to // convert it to a coordinate. rnd_.init( sink.nelt(), // # elements to choose from true); // without replacement offs_.resize(sink.nelt()); offs_.fill(std::numeric_limits::max()); sas_.resize(sink.nrange()); gws_.resize(sink.nrange()); size_t ei = 0; for(size_t i = 0; i < sas_.size(); i++) { size_t en = sink[i].botf - sink[i].topf; sas_[i].init(sink[i].topf, q.length(), EListSlice(offs_, ei, en)); gws_[i].init(gfmFw, ref, sas_[i], rnd, met); ei += en; } } /** * Reset the selector. */ void reset() { rnd_.reset(); } /** * Return true iff the selector is currently initialized. */ bool inited() const { return rnd_.size() > 0; } /** * Get next alignment and convert it to an AlnRes. */ bool next( const DescentDriver& dr, const GFM& gfmFw, // forward Bowtie index for walking left const BitPairReference& ref, // bitpair-encoded reference RandomSource& rnd, AlnRes& rs, WalkMetrics& met, PerReadMetrics& prm) { // Sample one alignment randomly from pool of remaining alignments size_t ri = (size_t)rnd_.next(rnd); size_t off = 0; DescentAlignment al; size_t rangei = 0; // Convert random alignment index into a coordinate dr.sink().elt(ri, al, rangei, off); assert_lt(off, al.size()); Coord refcoord; WalkResult wr; TIndexOffU tidx = 0, toff = 0, tlen = 0; gws_[rangei].advanceElement( (TIndexOffU)off, gfmFw, // forward Bowtie index for walking left ref, // bitpair-encoded reference sas_[rangei], // SA range with offsets gwstate_, // GroupWalk state; scratch space wr, // put the result here met, // metrics prm); // per-read metrics assert_neq(OFF_MASK, wr.toff); bool straddled = false; gfmFw.joinedToTextOff( wr.elt.len, wr.toff, tidx, toff, tlen, true, // reject straddlers? straddled); // straddled? if(tidx == OFF_MASK) { // The seed hit straddled a reference boundary so the seed // hit isn't valid return false; } // Coordinate of the seed hit w/r/t the pasted reference string refcoord.init(tidx, (int64_t)toff, dr.sink()[rangei].fw); const EList& edits = dr.sink().edits(); size_t ns = 0, ngap = 0, nrefn = 0; for(size_t i = al.ei; i < al.ei + al.en; i++) { if(edits[i].qchr == 'N' || edits[i].chr == 'N') ns++; if(edits[i].chr == 'N') nrefn++; if(edits[i].isGap()) ngap++; } AlnScore asc( -dr.sink().bestPenalty(), // numeric score ns, // # Ns ngap); // # gaps rs.init( dr.query().length(), // # chars after hard trimming asc, // alignment score &dr.sink().edits(), // nucleotide edits array al.ei, // nucleotide edits first pos al.en, // nucleotide edits last pos NULL, // ambig base array 0, // ambig base first pos 0, // ambig base last pos refcoord, // coord of leftmost aligned char in ref tlen, // length of reference aligned to -1, // # seed mms allowed -1, // seed length -1, // seed interval dr.minScore(), // minimum score for valid alignment -1, // nuc5p (for colorspace) -1, // nuc3p (for colorspace) false, // soft pre-trimming? 0, // 5p pre-trimming 0, // 3p pre-trimming false, // soft trimming? 0, // 5p trimming 0); // 3p trimming rs.setRefNs(nrefn); return true; } /** * Return true iff all elements have been reported. */ bool done() const { return rnd_.done(); } /** * Return the total size occupued by the Descent driver and all its * constituent parts. */ size_t totalSizeBytes() const { return rnd_.totalSizeBytes() + offs_.totalSizeBytes() + sas_.totalSizeBytes() + gws_.totalSizeBytes(); } /** * Return the total capacity of the Descent driver and all its constituent * parts. */ size_t totalCapacityBytes() const { return rnd_.totalCapacityBytes() + offs_.totalCapacityBytes() + sas_.totalCapacityBytes() + gws_.totalCapacityBytes(); } protected: Random1toN rnd_; EList offs_; EList, index_t> > sas_; EList, 16> > gws_; GroupWalkState gwstate_; }; /** * Selects and prioritizes partial alignments from the heap of the * DescentDriver. We assume that the heap is no longer changing (i.e. that the * DescentDriver is done). Usually, the user will then attempt to extend the * partial alignments into full alignments. This can happen incrementally; * that is, the user might ask for the partial alignments one "batch" at a * time, and the selector will only do as much work is necessary to supply each * requesteded batch. * * The actual work done here includes: (a) scanning the heap for high-priority * partial alignments, (b) setting up the rnd_, offs_, sas_, gws_, and gwstate_ * fields and resolving offsets of partial alignments, (c) packaging and * delivering batches of results to the caller. * * How to prioritize partial alignments? One idea is to use the same * penalty-based prioritization used in the heap. This has pros: (a) maintains * the guarantee that we're visiting alignments in best-to-worst order in * end-to-end alignment mode, (b) the heap is already prioritized this way, so * it's easier for us to compile high-priority partial alignments. But the con * is that it doesn't take depth into account, which could mean that we're * extending a lot of very short partial alignments first. * * A problem we should keep in mind is that some */ template class DescentPartialAlignmentSelector { public: DescentPartialAlignmentSelector() : gwstate_(GW_CAT) { reset(); } /** * Initialize a new selector w/r/t a read, index and heap of partial * alignments. */ void init( const Read& q, // read const EHeap& heap, // the heap w/ the partial alns TAlScore depthBonus, // use depth when prioritizing size_t nbatch, // # of alignments in a batch const GFM& gfmFw, // forward Bowtie index for walk-left const BitPairReference& ref, // bitpair-encoded reference RandomSource& rnd, // pseudo-randoms for sampling rows WalkMetrics& met) // metrics re: offset resolution { // Make our internal heap if(depthBonus > 0) { heap_.clear(); for(size_t i = 0; i < heap.size(); i++) { TDescentPair p = heap[i]; p.first.pen += depthBonus * p.first.depth; heap_.insert(p); } } else { heap_ = heap; } #if 0 // We're going to sample from space of *alignments*, not ranges. So // when we extract a sample, we'll have to do a little extra work to // convert it to a coordinate. rnd_.init( sink.nelt(), // # elements to choose from true); // without replacement offs_.resize(sink.nelt()); offs_.fill(std::numeric_limits::max()); sas_.resize(sink.nrange()); gws_.resize(sink.nrange()); size_t ei = 0; for(size_t i = 0; i < sas_.size(); i++) { size_t en = sink[i].botf - sink[i].topf; sas_[i].init(sink[i].topf, q.length(), EListSlice(offs_, ei, en)); gws_[i].init(gfmFw, ref, sas_[i], rnd, met); ei += en; } #endif } /** * */ void compileBatch() { } /** * Reset the selector. */ void reset() { heap_.clear(); } /** * Return true iff the selector is currently initialized. */ bool inited() const { return !heap_.empty(); } /** * Get next alignment and convert it to an AlnRes. */ bool next( const DescentDriver& dr, const GFM& gfmFw, // forward Bowtie index for walking left const BitPairReference& ref, // bitpair-encoded reference RandomSource& rnd, AlnRes& rs, WalkMetrics& met, PerReadMetrics& prm) { // Sample one alignment randomly from pool of remaining alignments size_t ri = (size_t)rnd_.next(rnd); size_t off = 0; DescentAlignment al; size_t rangei = 0; // Convert random alignment index into a coordinate dr.sink().elt(ri, al, rangei, off); assert_lt(off, al.size()); Coord refcoord; WalkResult wr; uint32_t tidx = 0, toff = 0, tlen = 0; gws_[rangei].advanceElement( (uint32_t)off, gfmFw, // forward Bowtie index for walking left ref, // bitpair-encoded reference sas_[rangei], // SA range with offsets gwstate_, // GroupWalk state; scratch space wr, // put the result here met, // metrics prm); // per-read metrics assert_neq(0xffffffff, wr.toff); bool straddled = false; gfmFw.joinedToTextOff( wr.elt.len, wr.toff, tidx, toff, tlen, true, // reject straddlers? straddled); // straddled? if(tidx == 0xffffffff) { // The seed hit straddled a reference boundary so the seed // hit isn't valid return false; } // Coordinate of the seed hit w/r/t the pasted reference string refcoord.init(tidx, (int64_t)toff, dr.sink()[rangei].fw); const EList& edits = dr.sink().edits(); size_t ns = 0, ngap = 0, nrefn = 0; for(size_t i = al.ei; i < al.ei + al.en; i++) { if(edits[i].qchr == 'N' || edits[i].chr == 'N') ns++; if(edits[i].chr == 'N') nrefn++; if(edits[i].isGap()) ngap++; } return true; } /** * Return true iff all elements have been reported. */ bool done() const { return rnd_.done(); } /** * Return the total size occupued by the Descent driver and all its * constituent parts. */ size_t totalSizeBytes() const { return heap_.totalSizeBytes() + rnd_.totalSizeBytes() + offs_.totalSizeBytes() + sas_.totalSizeBytes() + gws_.totalSizeBytes(); } /** * Return the total capacity of the Descent driver and all its constituent * parts. */ size_t totalCapacityBytes() const { return heap_.totalCapacityBytes() + rnd_.totalCapacityBytes() + offs_.totalCapacityBytes() + sas_.totalCapacityBytes() + gws_.totalCapacityBytes(); } protected: // This class's working heap. This might simply be a copy of the original // heap, or it might be re-prioritized in some way. EHeap heap_; Random1toN rnd_; EList offs_; EList, index_t> > sas_; EList, 16> > gws_; GroupWalkState gwstate_; }; /** * Drive the process of descending from all search roots. */ template void DescentDriver::go( const Scoring& sc, // scoring scheme const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index DescentMetrics& met, // metrics PerReadMetrics& prm) // per-read metrics { assert(q_.repOk()); // Convert DescentRoots to the initial Descents for(size_t i = 0; i < roots_.size(); i++) { size_t dfsz = df_.size(); size_t pfsz = pf_.size(); TDescentId id = df_.alloc(); Edit e_null; assert(!e_null.inited()); bool succ = df_[id].init( q_, // read i, // root and conf id sc, // scoring scheme minsc_, // minimum score maxpen_, // maximum penalty id, // new Descent's id gfmFw, // forward index gfmBw, // mirror index re_, // redundancy checker df_, // Descent factory pf_, // DescentPos factory roots_, // DescentRoots confs_, // DescentConfs heap_, // heap alsink_, // alignment sink met, // metrics prm); // per-read metrics if(veryVerbose_) { bool fw = roots_[i].fw; tmpedit_.clear(); df_[id].print( &cerr, "", q_, 0, 0, fw, tmpedit_, 0, tmpedit_.size(), tmprfdnastr_); } if(!succ) { // Reclaim memory we had used for this descent and its DescentPos info df_.resize(dfsz); pf_.resize(pfsz); } } // Advance until some stopping condition bool stop = heap_.empty(); while(!stop) { // Pop off the highest-priority descent. Note that some outgoing edges // might have since been explored, which could reduce the priority of // the descent once we . TDescentPair p = heap_.pop(); df_.alloc(); df_.pop(); df_[p.second].followBestOutgoing( q_, // read gfmFw, // index over text gfmBw, // index over reverse text sc, // scoring scheme minsc_, // minimum score maxpen_, // maximum penalty re_, // redundancy checker df_, // Descent factory pf_, // DescentPos factory roots_, // confs_, // heap_, // priority queue for Descents alsink_, // alignment sink met, // metrics prm); // per-read metrics stop = heap_.empty(); } } /** * Perform seed alignment until some stopping condition is satisfied. */ template int DescentDriver::advance( const DescentStoppingConditions& stopc, // stopping conditions const Scoring& sc, // scoring scheme const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index DescentMetrics& met, // metrics PerReadMetrics& prm) // per-read metrics { size_t nbwop_i = met.bwops; while(rootsInited_ < roots_.size()) { size_t dfsz = df_.size(); size_t pfsz = pf_.size(); TDescentId id = df_.alloc(); Edit e_null; assert(!e_null.inited()); bool succ = df_[id].init( q_, // query rootsInited_, // root and conf id sc, // scoring scheme minsc_, // minimum score maxpen_, // maximum penalty id, // new Descent's id gfmFw, // forward index gfmBw, // mirror index re_, // redundancy checker df_, // Descent factory pf_, // DescentPos factory roots_, // DescentRoots confs_, // DescentConfs heap_, // heap alsink_, // alignment sink met, // metrics prm); // per-read metrics if(!succ) { // Reclaim memory we had used for this descent and its DescentPos info df_.resize(dfsz); pf_.resize(pfsz); } rootsInited_++; TAlScore best = std::numeric_limits::max(); if(!heap_.empty()) { best = heap_.top().first.pen; } if(stopc.nfound > 0 && alsink_.nelt() > stopc.nfound) { return DESCENT_DRIVER_ALN; } if(alsink_.stratumDone(best)) { return DESCENT_DRIVER_STRATA; } if(stopc.nbwop > 0 && (met.bwops - nbwop_i) > stopc.nbwop) { return DESCENT_DRIVER_BWOPS; } if(stopc.totsz > 0 && totalSizeBytes() > stopc.totsz) { return DESCENT_DRIVER_MEM; } } // Advance until some stopping condition bool stop = heap_.empty(); while(!stop) { // Pop off the highest-priority descent. Note that some outgoing edges // might have since been explored, which could reduce the priority of // the descent once we . TDescentPair p = heap_.pop(); df_.alloc(); df_.pop(); df_[p.second].followBestOutgoing( q_, gfmFw, gfmBw, sc, minsc_, // minimum score maxpen_, // maximum penalty re_, // redundancy checker df_, // Descent factory pf_, // DescentPos factory roots_, confs_, heap_, alsink_, met, prm); // per-read metrics TAlScore best = std::numeric_limits::max(); if(!heap_.empty()) { best = heap_.top().first.pen; } if(stopc.nfound > 0 && alsink_.nelt() > stopc.nfound) { return DESCENT_DRIVER_ALN; } if(alsink_.stratumDone(best)) { return DESCENT_DRIVER_STRATA; } if(stopc.nbwop > 0 && (met.bwops - nbwop_i) > stopc.nbwop) { return DESCENT_DRIVER_BWOPS; } if(stopc.totsz > 0 && totalSizeBytes() > stopc.totsz) { return DESCENT_DRIVER_MEM; } stop = heap_.empty(); } return DESCENT_DRIVER_DONE; } /** * If this is the final descent in a complete end-to-end alignment, report * the alignment. */ template bool DescentAlignmentSink::reportAlignment( const Read& q, // query string const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index TIndexOffU topf, // SA range top in forward index TIndexOffU botf, // SA range bottom in forward index TIndexOffU topb, // SA range top in backward index TIndexOffU botb, // SA range bottom in backward index TDescentId id, // id of leaf Descent TRootId rid, // id of search root const Edit& e, // final edit, if needed TScore pen, // total penalty EFactory >& df, // factory with Descent EFactory& pf, // factory with DescentPoss const EList& rs, // roots const EList& cs) // configs { TDescentId cur = id; ASSERT_ONLY(const Descent& desc = df[id]); const bool fw = rs[rid].fw; ASSERT_ONLY(size_t len = q.length()); assert(q.repOk()); assert_lt(desc.al5pf(), len); // Adjust al5pi and al5pf to take the final edit into account (if // there is one) // Check if this is redundant with a previous reported alignment Triple lhs(topf, botf, 0); Triple rhs(topb, botb, q.length()-1); if(!lhs_.insert(lhs)) { rhs_.insert(rhs); return false; // Already there } if(!rhs_.insert(rhs)) { return false; // Already there } size_t ei = edits_.size(); df[cur].collectEdits(edits_, &e, df); size_t en = edits_.size() - ei; #ifndef NDEBUG { for(size_t i = 1; i < en; i++) { assert_geq(edits_[ei+i].pos, edits_[ei+i-1].pos); } // Now figure out how much we refrained from aligning on either // side. size_t trimLf = 0; size_t trimRg = 0; BTDnaString& rf = tmprfdnastr_; rf.clear(); if(!fw) { // Edit offsets are w/r/t 5' end, but desc.print wants them w/r/t // the *left* end of the read sequence that aligned Edit::invertPoss(edits_, len, ei, en, true); } desc.print(NULL, "", q, trimLf, trimRg, fw, edits_, ei, en, rf); if(!fw) { // Invert them back to how they were before Edit::invertPoss(edits_, len, ei, en, true); } ASSERT_ONLY(TIndexOffU toptmp = 0); ASSERT_ONLY(TIndexOffU bottmp = 0); // Check that the edited string occurs in the reference if(!gfmFw.contains(rf, &toptmp, &bottmp)) { std::cerr << rf << std::endl; assert(false); } } #endif als_.expand(); als_.back().init(pen, fw, topf, botf, ei, en); nelt_ += (botf - topf); if(bestPen_ == std::numeric_limits::max() || pen < bestPen_) { bestPen_ = pen; } if(worstPen_ == std::numeric_limits::max() || pen > worstPen_) { worstPen_ = pen; } return true; } /** * Initialize a new descent branching from the given descent via the given * edit. Return false if the Descent has no outgoing edges (and can * therefore have its memory freed), true otherwise. */ template bool Descent::init( const Read& q, // query TRootId rid, // root id const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score TAlScore maxpen, // maximum penalty TReadOff al5pi, // offset from 5' of 1st aligned char TReadOff al5pf, // offset from 5' of last aligned char TIndexOffU topf, // SA range top in FW index TIndexOffU botf, // SA range bottom in FW index TIndexOffU topb, // SA range top in BW index TIndexOffU botb, // SA range bottom in BW index bool l2r, // direction this descent will go in size_t descid, // my ID TDescentId parent, // parent ID TScore pen, // total penalties so far const Edit& e, // edit for incoming edge const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index DescentRedundancyChecker& re, // redundancy checker EFactory& df, // Descent factory EFactory& pf, // DescentPos factory const EList& rs, // roots const EList& cs, // configs EHeap& heap, // heap DescentAlignmentSink& alsink, // alignment sink DescentMetrics& met, // metrics PerReadMetrics& prm) // per-read metrics { assert(q.repOk()); rid_ = rid; al5pi_ = al5pi; al5pf_ = al5pf; l2r_ = l2r; topf_ = topf; botf_ = botf; topb_ = topb; botb_ = botb; descid_ = descid; parent_ = parent; pen_ = pen; posid_ = std::numeric_limits::max(); len_ = 0; out_.clear(); edit_ = e; lastRecalc_ = true; gapadd_ = df[parent].gapadd_; if(e.inited()) { if(e.isReadGap()) { gapadd_++; } else if(e.isRefGap()) { gapadd_--; } } bool branches = false, hitEnd = false, done = false; TIndexOffU topf_new = 0, botf_new = 0, topb_new = 0, botb_new = 0; off5p_i_ = 0; #ifndef NDEBUG size_t depth = al5pf_ - al5pi_ + 1; TAlScore maxpend = cs[rid_].cons.get(depth, q.length(), maxpen); assert_geq(maxpend, pen_); // can't have already exceeded max penalty #endif bool matchSucc = followMatches( q, sc, gfmFw, gfmBw, re, df, pf, rs, cs, heap, alsink, met, prm, branches, hitEnd, done, off5p_i_, topf_new, botf_new, topb_new, botb_new); bool bounceSucc = false; if(matchSucc && hitEnd && !done) { assert(topf_new > 0 || botf_new > 0); bounceSucc = bounce( q, topf_new, botf_new, topb_new, botb_new, gfmFw, gfmBw, sc, minsc, // minimum score maxpen, // maximum penalty re, df, pf, rs, cs, heap, alsink, met, // descent metrics prm); // per-read metrics } if(matchSucc) { // Calculate info about outgoing edges recalcOutgoing(q, sc, minsc, maxpen, re, pf, rs, cs, prm); if(!empty()) { heap.insert(make_pair(out_.bestPri(), descid)); // Add to heap } } return !empty() || bounceSucc; } /** * Initialize a new descent beginning at the given root. Return false if * the Descent has no outgoing edges (and can therefore have its memory * freed), true otherwise. */ template bool Descent::init( const Read& q, // query TRootId rid, // root id const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score TAlScore maxpen, // maximum penalty size_t descid, // id of this Descent const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index DescentRedundancyChecker& re, // redundancy checker EFactory >& df, // Descent factory EFactory& pf, // DescentPos factory const EList& rs, // roots const EList& cs, // configs EHeap& heap, // heap DescentAlignmentSink& alsink, // alignment sink DescentMetrics& met, // metrics PerReadMetrics& prm) // per-read metrics { rid_ = rid; al5pi_ = rs[rid].off5p; al5pf_ = rs[rid].off5p; assert_lt(al5pi_, q.length()); assert_lt(al5pf_, q.length()); l2r_ = rs[rid].l2r; topf_ = botf_ = topb_ = botb_ = 0; descid_ = descid; parent_ = std::numeric_limits::max(); pen_ = 0; posid_ = std::numeric_limits::max(); len_ = 0; out_.clear(); edit_.reset(); lastRecalc_ = true; gapadd_ = 0; bool branches = false, hitEnd = false, done = false; TIndexOffU topf_new = 0, botf_new = 0, topb_new = 0, botb_new = 0; off5p_i_ = 0; bool matchSucc = followMatches( q, sc, gfmFw, gfmBw, re, df, pf, rs, cs, heap, alsink, met, prm, branches, hitEnd, done, off5p_i_, topf_new, botf_new, topb_new, botb_new); bool bounceSucc = false; if(matchSucc && hitEnd && !done) { assert(topf_new > 0 || botf_new > 0); bounceSucc = bounce( q, topf_new, botf_new, topb_new, botb_new, gfmFw, gfmBw, sc, minsc, // minimum score maxpen, // maximum penalty re, df, pf, rs, cs, heap, alsink, met, // descent metrics prm); // per-read metrics } // Calculate info about outgoing edges assert(empty()); if(matchSucc) { recalcOutgoing(q, sc, minsc, maxpen, re, pf, rs, cs, prm); if(!empty()) { heap.insert(make_pair(out_.bestPri(), descid)); // Add to heap } } return !empty() || bounceSucc; } /** * Recalculate our summary of the outgoing edges from this descent. When * deciding what outgoing edges are legal, we abide by constraints. * Typically, they limit the total of the penalties accumulated so far, as * a function of distance from the search root. E.g. a constraint might * disallow any gaps or mismatches within 20 ply of the search root, then * allow 1 mismatch within 30 ply, then allow up to 1 mismatch or 1 gap * within 40 ply, etc. * * Return the total number of valid outgoing edges found. * * TODO: Eliminate outgoing gap edges that are redundant with others owing to * the DNA sequence and the fact that we don't care to distinguish among * "equivalent" homopolymer extensinos and retractions. */ template size_t Descent::recalcOutgoing( const Read& q, // query string const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score TAlScore maxpen, // maximum penalty DescentRedundancyChecker& re, // redundancy checker EFactory& pf, // factory with DescentPoss const EList& rs, // roots const EList& cs, // configs PerReadMetrics& prm) // per-read metrics { assert_eq(botf_ - topf_, botb_ - topb_); assert(out_.empty()); assert(repOk(&q)); // Get initial 5' and 3' offsets bool fw = rs[rid_].fw; float rootpri = rs[rid_].pri; bool toward3p = (l2r_ == fw); size_t off5p = off5p_i_; assert_geq(al5pf_, al5pi_); size_t off3p = q.length() - off5p - 1; // By "depth" we essentially mean the number of characters already aligned size_t depth, extrai = 0, extraf = 0; size_t cur5pi = al5pi_, cur5pf = al5pf_; if(toward3p) { // Toward 3' cur5pf = off5p; depth = off5p - al5pi_; // Failed to match out to the end? if(al5pf_ < q.length() - 1) { extraf = 1; // extra } } else { // Toward 5' cur5pi = off5p; depth = al5pf_ - off5p; if(al5pi_ > 0) { extrai = 1; } } // Get gap penalties TScore pen_rdg_ex = sc.readGapExtend(), pen_rfg_ex = sc.refGapExtend(); TScore pen_rdg_op = sc.readGapOpen(), pen_rfg_op = sc.refGapOpen(); // Top and bot in the direction of the descent TIndexOffU top = l2r_ ? topb_ : topf_; TIndexOffU bot = l2r_ ? botb_ : botf_; // Top and bot in the opposite direction TIndexOffU topp = l2r_ ? topf_ : topb_; TIndexOffU botp = l2r_ ? botf_ : botb_; assert_eq(botp - topp, bot - top); DescentEdge edge; size_t nout = 0; // Enumerate all outgoing edges, starting at the root and going out size_t d = posid_; // At first glance, we might think we should be bounded by al5pi_ and // al5pf_, but those delimit the positions that matched between reference // and read. If we hit a position that failed to match as part of // followMatches, then we also want to evaluate ways of leaving that // position, which adds one more position to viist. while(off5p >= al5pi_ - extrai && off5p <= al5pf_ + extraf) { assert_lt(off5p, q.length()); assert_lt(off3p, q.length()); TScore maxpend = cs[rid_].cons.get(depth, q.length(), maxpen); assert(depth > 0 || maxpend == 0); assert_geq(maxpend, pen_); // can't have already exceeded max penalty TScore diff = maxpend - pen_; // room we have left // Get pointer to SA ranges in the direction of descent const TIndexOffU *t = l2r_ ? pf[d].topb : pf[d].topf; const TIndexOffU *b = l2r_ ? pf[d].botb : pf[d].botf; const TIndexOffU *tp = l2r_ ? pf[d].topf : pf[d].topb; const TIndexOffU *bp = l2r_ ? pf[d].botf : pf[d].botb; assert_eq(pf[d].botf - pf[d].topf, pf[d].botb - pf[d].topb); // What are the read char / quality? std::pair p = q.get(off5p, fw); int c = p.first; assert_range(0, 4, c); // Only entertain edits if there is at least one type of edit left and // there is some penalty budget left if(!pf[d].flags.exhausted() && diff > 0) { // What would the penalty be if we mismatched at this position? // This includes the case where the mismatch is for an N in the // read. int qq = p.second; assert_geq(qq, 0); TScore pen_mm = sc.mm(c, qq); if(pen_mm <= diff) { for(int j = 0; j < 4; j++) { if(j == c) continue; // Match, not mismatch if(b[j] <= t[j]) { continue; // No outgoing edge with this nucleotide } if(!pf[d].flags.mmExplore(j)) { continue; // Already been explored } TIndexOffU topf = pf[d].topf[j], botf = pf[d].botf[j]; ASSERT_ONLY(TIndexOffU topb = pf[d].topb[j], botb = pf[d].botb[j]); if(re.contains(fw, l2r_, cur5pi, cur5pf, cur5pf - cur5pi + 1 + gapadd_, topf, botf, pen_ + pen_mm)) { prm.nRedSkip++; continue; // Redundant with a path already explored } prm.nRedFail++; TIndexOffU width = b[j] - t[j]; Edit edit((uint32_t)off5p, (int)("ACGTN"[j]), (int)("ACGTN"[c]), EDIT_TYPE_MM); DescentPriority pri(pen_ + pen_mm, depth, width, rootpri); assert(topf != 0 || botf != 0); assert(topb != 0 || botb != 0); assert_eq(botb - topb, botf - topf); edge.init(edit, off5p, pri, d #ifndef NDEBUG , d, topf, botf, topb, botb #endif ); out_.update(edge); nout++; } } bool gapsAllowed = (off5p >= (size_t)sc.gapbar && off3p >= (size_t)sc.gapbar); if(gapsAllowed) { assert_gt(depth, 0); // An easy redundancy check is: if all ways of proceeding are // matches, then there's no need to entertain gaps here. // Shifting the gap one position further downstream is // guarnteed not to be worse. size_t totwidth = (b[0] - t[0]) + (b[1] - t[1]) + (b[2] - t[2]) + (b[3] - t[3]); assert(c > 3 || b[c] - t[c] <= totwidth); bool allmatch = c < 4 && (totwidth == (b[c] - t[c])); bool rdex = false, rfex = false; size_t cur5pi_i = cur5pi, cur5pf_i = cur5pf; if(toward3p) { cur5pf_i--; } else { cur5pi_i++; } if(off5p == off5p_i_ && edit_.inited()) { // If we're at the root of the descent, and the descent // branched on a gap, then this could be scored as an // extension of that gap. if(pen_rdg_ex <= diff && edit_.isReadGap()) { // Extension of a read gap rdex = true; for(int j = 0; j < 4; j++) { if(b[j] <= t[j]) { continue; // No outgoing edge with this nucleotide } if(!pf[d].flags.rdgExplore(j)) { continue; // Already been explored } TIndexOffU topf = pf[d].topf[j], botf = pf[d].botf[j]; ASSERT_ONLY(TIndexOffU topb = pf[d].topb[j], botb = pf[d].botb[j]); assert(topf != 0 || botf != 0); assert(topb != 0 || botb != 0); if(re.contains(fw, l2r_, cur5pi_i, cur5pf_i, cur5pf - cur5pi + 1 + gapadd_, topf, botf, pen_ + pen_rdg_ex)) { prm.nRedSkip++; continue; // Redundant with a path already explored } prm.nRedFail++; TIndexOffU width = b[j] - t[j]; // off5p holds the offset from the 5' of the next // character we were trying to align when we decided to // introduce a read gap (before that character). If we // were walking toward the 5' end, we need to increment // by 1. uint32_t off = (uint32_t)off5p + (toward3p ? 0 : 1); Edit edit(off, (int)("ACGTN"[j]), '-', EDIT_TYPE_READ_GAP); assert(edit.pos2 != std::numeric_limits::max()); edit.pos2 = edit_.pos2 + (toward3p ? 1 : -1); DescentPriority pri(pen_ + pen_rdg_ex, depth, width, rootpri); assert(topf != 0 || botf != 0); assert(topb != 0 || botb != 0); assert_eq(botb - topb, botf - topf); edge.init(edit, off5p, pri, d #ifndef NDEBUG , d, topf, botf, topb, botb #endif ); out_.update(edge); nout++; } } if(pen_rfg_ex <= diff && edit_.isRefGap()) { // Extension of a reference gap rfex = true; if(pf[d].flags.rfgExplore()) { TIndexOffU topf = l2r_ ? topp : top; TIndexOffU botf = l2r_ ? botp : bot; ASSERT_ONLY(TIndexOffU topb = l2r_ ? top : topp); ASSERT_ONLY(TIndexOffU botb = l2r_ ? bot : botp); assert(topf != 0 || botf != 0); assert(topb != 0 || botb != 0); size_t nrefal = cur5pf - cur5pi + gapadd_; if(!re.contains(fw, l2r_, cur5pi, cur5pf, nrefal, topf, botf, pen_ + pen_rfg_ex)) { TIndexOffU width = bot - top; Edit edit((uint32_t)off5p, '-', (int)("ACGTN"[c]), EDIT_TYPE_REF_GAP); DescentPriority pri(pen_ + pen_rfg_ex, depth, width, rootpri); assert(topf != 0 || botf != 0); assert(topb != 0 || botb != 0); edge.init(edit, off5p, pri, d #ifndef NDEBUG // It's a little unclear what the depth ought to be. // Is it the depth we were at when we did the ref // gap? I.e. the depth of the flags where rfgExplore() // returned true? Or is it the depth where we can // retrieve the appropriate top/bot? We make it the // latter, might wrap around, indicating we should get // top/bot from the descent's topf_, ... fields. , (d == posid_) ? std::numeric_limits::max() : (d - 1), topf, botf, topb, botb #endif ); out_.update(edge); nout++; prm.nRedFail++; } else { prm.nRedSkip++; } } } } if(!allmatch && pen_rdg_op <= diff && !rdex) { // Opening a new read gap for(int j = 0; j < 4; j++) { if(b[j] <= t[j]) { continue; // No outgoing edge with this nucleotide } if(!pf[d].flags.rdgExplore(j)) { continue; // Already been explored } TIndexOffU topf = pf[d].topf[j], botf = pf[d].botf[j]; ASSERT_ONLY(TIndexOffU topb = pf[d].topb[j], botb = pf[d].botb[j]); assert(topf != 0 || botf != 0); assert(topb != 0 || botb != 0); if(re.contains(fw, l2r_, cur5pi_i, cur5pf_i, cur5pf - cur5pi + 1 + gapadd_, topf, botf, pen_ + pen_rdg_op)) { prm.nRedSkip++; continue; // Redundant with a path already explored } prm.nRedFail++; TIndexOffU width = b[j] - t[j]; // off5p holds the offset from the 5' of the next // character we were trying to align when we decided to // introduce a read gap (before that character). If we // were walking toward the 5' end, we need to increment // by 1. uint32_t off = (uint32_t)off5p + (toward3p ? 0 : 1); Edit edit(off, (int)("ACGTN"[j]), '-', EDIT_TYPE_READ_GAP); assert(edit.pos2 != std::numeric_limits::max()); DescentPriority pri(pen_ + pen_rdg_op, depth, width, rootpri); assert(topf != 0 || botf != 0); assert(topb != 0 || botb != 0); assert_eq(botb - topb, botf - topf); edge.init(edit, off5p, pri, d #ifndef NDEBUG , d, topf, botf, topb, botb #endif ); out_.update(edge); nout++; } } if(!allmatch && pen_rfg_op <= diff && !rfex) { // Opening a new reference gap if(pf[d].flags.rfgExplore()) { TIndexOffU topf = l2r_ ? topp : top; TIndexOffU botf = l2r_ ? botp : bot; ASSERT_ONLY(TIndexOffU topb = l2r_ ? top : topp); ASSERT_ONLY(TIndexOffU botb = l2r_ ? bot : botp); assert(topf != 0 || botf != 0); assert(topb != 0 || botb != 0); size_t nrefal = cur5pf - cur5pi + gapadd_; if(!re.contains(fw, l2r_, cur5pi, cur5pf, nrefal, topf, botf, pen_ + pen_rfg_op)) { TIndexOffU width = bot - top; Edit edit((uint32_t)off5p, '-', (int)("ACGTN"[c]), EDIT_TYPE_REF_GAP); DescentPriority pri(pen_ + pen_rfg_op, depth, width, rootpri); assert(topf != 0 || botf != 0); assert(topb != 0 || botb != 0); edge.init(edit, off5p, pri, d #ifndef NDEBUG // It's a little unclear what the depth ought to be. // Is it the depth we were at when we did the ref // gap? I.e. the depth of the flags where rfgExplore() // returned true? Or is it the depth where we can // retrieve the appropriate top/bot? We make it the // latter, might wrap around, indicating we should get // top/bot from the descent's topf_, ... fields. , (d == posid_) ? std::numeric_limits::max() : (d - 1), topf, botf, topb, botb #endif ); out_.update(edge); nout++; prm.nRedFail++; } else { prm.nRedSkip++; } } } } } // Update off5p, off3p, depth d++; depth++; assert_leq(depth, al5pf_ - al5pi_ + 2); if(toward3p) { if(off3p == 0) { break; } off5p++; off3p--; cur5pf++; } else { if(off5p == 0) { break; } off3p++; off5p--; cur5pi--; } // Update top and bot if(off5p >= al5pi_ - extrai && off5p <= al5pf_ + extraf) { assert_range(0, 3, c); top = t[c]; topp = tp[c]; bot = b[c]; botp = bp[c]; assert_eq(bot-top, botp-topp); } } lastRecalc_ = (nout <= 5); out_.best1.updateFlags(pf); out_.best2.updateFlags(pf); out_.best3.updateFlags(pf); out_.best4.updateFlags(pf); out_.best5.updateFlags(pf); return nout; } template void Descent::print( std::ostream *os, const char *prefix, const Read& q, size_t trimLf, size_t trimRg, bool fw, const EList& edits, size_t ei, size_t en, BTDnaString& rf) const { const BTDnaString& read = fw ? q.patFw : q.patRc; size_t eidx = ei; if(os != NULL) { *os << prefix; } // Print read for(size_t i = 0; i < read.length(); i++) { if(i < trimLf || i >= read.length() - trimRg) { if(os != NULL) { *os << (char)tolower(read.toChar(i)); } continue; } bool del = false, mm = false; while(eidx < ei + en && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { if(os != NULL) { *os << '-'; } } else if(edits[eidx].isRefGap()) { del = true; assert_eq((int)edits[eidx].qchr, read.toChar(i)); if(os != NULL) { *os << read.toChar(i); } } else { mm = true; assert(edits[eidx].isMismatch()); assert_eq((int)edits[eidx].qchr, read.toChar(i)); if(os != NULL) { *os << (char)edits[eidx].qchr; } } eidx++; } if(!del && !mm) { // Print read character if(os != NULL) { *os << read.toChar(i); } } } if(os != NULL) { *os << endl; *os << prefix; } eidx = ei; // Print match bars for(size_t i = 0; i < read.length(); i++) { if(i < trimLf || i >= read.length() - trimRg) { if(os != NULL) { *os << ' '; } continue; } bool del = false, mm = false; while(eidx < ei + en && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { if(os != NULL) { *os << ' '; } } else if(edits[eidx].isRefGap()) { del = true; if(os != NULL) { *os << ' '; } } else { mm = true; assert(edits[eidx].isMismatch()); if(os != NULL) { *os << ' '; } } eidx++; } if(!del && !mm && os != NULL) { *os << '|'; } } if(os != NULL) { *os << endl; *os << prefix; } eidx = ei; // Print reference for(size_t i = 0; i < read.length(); i++) { if(i < trimLf || i >= read.length() - trimRg) { if(os != NULL) { *os << ' '; } continue; } bool del = false, mm = false; while(eidx < ei + en && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { rf.appendChar((char)edits[eidx].chr); if(os != NULL) { *os << (char)edits[eidx].chr; } } else if(edits[eidx].isRefGap()) { del = true; if(os != NULL) { *os << '-'; } } else { mm = true; assert(edits[eidx].isMismatch()); rf.appendChar((char)edits[eidx].chr); if(os != NULL) { *os << (char)edits[eidx].chr; } } eidx++; } if(!del && !mm) { rf.append(read[i]); if(os != NULL) { *os << read.toChar(i); } } } if(os != NULL) { *os << endl; } } /** * Create a new Descent */ template bool Descent::bounce( const Read& q, // query string TIndexOffU topf, // SA range top in fw index TIndexOffU botf, // SA range bottom in fw index TIndexOffU topb, // SA range top in bw index TIndexOffU botb, // SA range bottom in bw index const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score TAlScore maxpen, // maximum penalty DescentRedundancyChecker& re, // redundancy checker EFactory >& df, // factory with Descent EFactory& pf, // factory with DescentPoss const EList& rs, // roots const EList& cs, // configs EHeap& heap, // heap of descents DescentAlignmentSink& alsink, // alignment sink DescentMetrics& met, // metrics PerReadMetrics& prm) // per-read metrics { assert_gt(botf, topf); assert(al5pi_ == 0 || al5pf_ == q.length()-1); assert(!(al5pi_ == 0 && al5pf_ == q.length()-1)); size_t dfsz = df.size(); size_t pfsz = pf.size(); TDescentId id = df.alloc(); Edit e_null; assert(!e_null.inited()); // Follow matches bool succ = df[id].init( q, // query rid_, // root id sc, // scoring scheme minsc, // minimum score maxpen, // maximum penalty al5pi_, // new near-5' extreme al5pf_, // new far-5' extreme topf, // SA range top in FW index botf, // SA range bottom in FW index topb, // SA range top in BW index botb, // SA range bottom in BW index !l2r_, // direction this descent will go in; opposite from parent id, // my ID descid_, // parent ID pen_, // total penalties so far - same as parent e_null, // edit for incoming edge; uninitialized if bounced gfmFw, // forward index gfmBw, // mirror index re, // redundancy checker df, // Descent factory pf, // DescentPos factory rs, // DescentRoot list cs, // DescentConfig list heap, // heap alsink, // alignment sink met, // metrics prm); // per-read metrics if(!succ) { // Reclaim memory we had used for this descent and its DescentPos info df.resize(dfsz); pf.resize(pfsz); } return succ; } /** * Take the best outgoing edge and place it in the heap. When deciding what * outgoing edges exist, abide by constraints in DescentConfig. These * constraints limit total penalty accumulated so far versus distance from * search root. E.g. a constraint might disallow any gaps or mismatches within * 20 ply of the root, then allow 1 mismatch within 30 ply, 1 mismatch or 1 gap * within 40 ply, etc. */ template void Descent::followBestOutgoing( const Read& q, // query string const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score TAlScore maxpen, // maximum penalty DescentRedundancyChecker& re, // redundancy checker EFactory >& df, // factory with Descent EFactory& pf, // factory with DescentPoss const EList& rs, // roots const EList& cs, // configs EHeap& heap, // heap of descents DescentAlignmentSink& alsink, // alignment sink DescentMetrics& met, // metrics PerReadMetrics& prm) // per-read metrics { // We assume this descent has been popped off the heap. We'll re-add it if // it hasn't been exhausted. assert(q.repOk()); assert(!empty()); assert(!out_.empty()); while(!out_.empty()) { DescentPriority best = out_.bestPri(); DescentEdge e = out_.rotate(); TReadOff al5pi_new = al5pi_, al5pf_new = al5pf_; bool fw = rs[rid_].fw; bool toward3p = (l2r_ == fw); TReadOff edoff = e.off5p; // 5' offset of edit assert_leq(edoff, al5pf_ + 1); assert_geq(edoff + 1, al5pi_); if(out_.empty()) { if(!lastRecalc_) { // This might allocate new Descents recalcOutgoing(q, sc, minsc, maxpen, re, pf, rs, cs, prm); if(empty()) { // Could happen, since some outgoing edges may have become // redundant in the meantime. break; } } else { assert(empty()); } } TReadOff doff; // edit's offset into this descent int chr = asc2dna[e.e.chr]; // hitEnd is set to true iff this edit pushes us to the extreme 5' or 3' // end of the alignment bool hitEnd = false; // done is set to true iff this edit aligns the only remaining character of // the read bool done = false; if(toward3p) { // The 3' extreme of the new Descent is further in (away from the 3' // end) than the parent's. al5pf_new = doff = edoff; if(e.e.isReadGap()) { // We didn't actually consume the read character at 'edoff', so // retract al5pf_new by one position. This doesn't effect the // "depth" (doff) of the SA range we took, though. assert_gt(al5pf_new, 0); al5pf_new--; } assert_lt(al5pf_new, q.length()); hitEnd = (al5pf_new == q.length() - 1); done = (hitEnd && al5pi_new == 0); assert_geq(doff, off5p_i_); doff = doff - off5p_i_; assert_leq(doff, len_); } else { // The 5' extreme of the new Descent is further in (away from the 5' // end) than the parent's. al5pi_new = doff = edoff; if(e.e.isReadGap()) { // We didn't actually consume the read character at 'edoff', so // move al5pi_new closer to the 3' end by one position. This // doesn't effect the "depth" (doff) of the SA range we took, // though. al5pi_new++; } hitEnd = (al5pi_new == 0); done = (hitEnd && al5pf_new == q.length() - 1); assert_geq(off5p_i_, doff); doff = off5p_i_ - doff; assert_leq(doff, len_); } // Check if this is redundant with an already-explored path bool l2r = l2r_; // gets overridden if we bounce if(!done && hitEnd) { // Alignment finsihed extending in one direction l2r = !l2r; } size_t dfsz = df.size(); size_t pfsz = pf.size(); TIndexOffU topf, botf, topb, botb; size_t d = posid_ + doff; if(e.e.isRefGap()) { d--; // might underflow if(doff == 0) { topf = topf_; botf = botf_; topb = topb_; botb = botb_; d = std::numeric_limits::max(); assert_eq(botf-topf, botb-topb); } else { assert_gt(al5pf_new, 0); assert_gt(d, 0); chr = pf[d].c; assert(pf[d].inited()); assert_range(0, 3, chr); topf = pf[d].topf[chr]; botf = pf[d].botf[chr]; topb = pf[d].topb[chr]; botb = pf[d].botb[chr]; assert_eq(botf-topf, botb-topb); } } else { // A read gap or a mismatch assert(pf[d].inited()); topf = pf[d].topf[chr]; botf = pf[d].botf[chr]; topb = pf[d].topb[chr]; botb = pf[d].botb[chr]; assert_eq(botf-topf, botb-topb); } assert_eq(d, e.d); assert_eq(topf, e.topf); assert_eq(botf, e.botf); assert_eq(topb, e.topb); assert_eq(botb, e.botb); if(done) { // Aligned the entire read end-to-end. Presumably there's no need to // create a new Descent object. We just report the alignment. alsink.reportAlignment( q, // query gfmFw, // forward index gfmBw, // backward index topf, // top of SA range in forward index botf, // bottom of SA range in forward index topb, // top of SA range in backward index botb, // bottom of SA range in backward index descid_, // Descent at the leaf rid_, // root id e.e, // extra edit, if necessary best.pen, // penalty df, // factory with Descent pf, // factory with DescentPoss rs, // roots cs); // configs assert(alsink.repOk()); return; } assert(al5pi_new != 0 || al5pf_new != q.length() - 1); TDescentId id = df.alloc(); bool succ = df[id].init( q, // query rid_, // root id sc, // scoring scheme minsc, // minimum score maxpen, // maximum penalty al5pi_new, // new near-5' extreme al5pf_new, // new far-5' extreme topf, // SA range top in FW index botf, // SA range bottom in FW index topb, // SA range top in BW index botb, // SA range bottom in BW index l2r, // direction this descent will go in id, // my ID descid_, // parent ID best.pen, // total penalties so far e.e, // edit for incoming edge; uninitialized if bounced gfmFw, // forward index gfmBw, // mirror index re, // redundancy checker df, // Descent factory pf, // DescentPos factory rs, // DescentRoot list cs, // DescentConfig list heap, // heap alsink, // alignment sink met, // metrics prm); // per-read metrics if(!succ) { // Reclaim memory we had used for this descent and its DescentPos info df.resize(dfsz); pf.resize(pfsz); } break; } if(!empty()) { // Re-insert this Descent with its new priority heap.insert(make_pair(out_.bestPri(), descid_)); } } /** * Given the forward and backward indexes, and given topf/botf/topb/botb, get * tloc, bloc ready for the next step. */ template void Descent::nextLocsBi( const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index SideLocus& tloc, // top locus SideLocus& bloc, // bot locus index_t topf, // top in BWT index_t botf, // bot in BWT index_t topb, // top in BWT' index_t botb) // bot in BWT' { assert_gt(botf, 0); // Which direction are we going in next? if(l2r_) { // Left to right; use BWT' if(botb - topb == 1) { // Already down to 1 row; just init top locus tloc.initFromRow(topb, gfmBw.gh(), gfmBw.gfm()); bloc.invalidate(); } else { SideLocus::initFromTopBot( topb, botb, gfmBw.gh(), gfmBw.gfm(), tloc, bloc); assert(bloc.valid()); } } else { // Right to left; use BWT if(botf - topf == 1) { // Already down to 1 row; just init top locus tloc.initFromRow(topf, gfmFw.gh(), gfmFw.gfm()); bloc.invalidate(); } else { SideLocus::initFromTopBot( topf, botf, gfmFw.gh(), gfmFw.gfm(), tloc, bloc); assert(bloc.valid()); } } // Check if we should update the tracker with this refinement assert(botf - topf == 1 || bloc.valid()); assert(botf - topf > 1 || !bloc.valid()); } /** * Advance this descent by following read matches as far as possible. * * This routine doesn't have to consider the whole gamut of constraints on * which outgoing edges can be followed. If it is a root descent, it does have * to know how deep the no-edit constraint goes, though, so we can decide * whether using the ftab would potentially jump over relevant branch points. * Apart from that, though, we simply proceed as far as it can go by matching * characters in the query, irrespective of the constraints. * recalcOutgoing(...) and followBestOutgoing(...) do have to consider these * constraints, though. * * Conceptually, as we make descending steps, we have: * 1. Before each step, a single range indicating how we departed the previous * step * 2. As part of each step, a quad of ranges indicating what range would result * if we proceeded on an a, c, g ot t * * Return true iff it is possible to branch from this descent. If we haven't * exceeded the no-branch depth. */ template bool Descent::followMatches( const Read& q, // query string const Scoring& sc, // scoring scheme const GFM& gfmFw, // forward index const GFM& gfmBw, // mirror index DescentRedundancyChecker& re, // redundancy checker EFactory >& df, // Descent factory EFactory& pf, // DescentPos factory const EList& rs, // roots const EList& cs, // configs EHeap& heap, // heap DescentAlignmentSink& alsink, // alignment sink DescentMetrics& met, // metrics PerReadMetrics& prm, // per-read metrics bool& branches, // out: true -> there are > 0 ways to branch bool& hitEnd, // out: true -> hit read end with non-empty range bool& done, // out: true -> we made a full alignment TReadOff& off5p_i, // out: initial 5' offset TIndexOffU& topf_bounce, // out: top of SA range for fw idx for bounce TIndexOffU& botf_bounce, // out: bot of SA range for fw idx for bounce TIndexOffU& topb_bounce, // out: top of SA range for bw idx for bounce TIndexOffU& botb_bounce) // out: bot of SA range for bw idx for bounce { // TODO: make these full-fledged parameters size_t nobranchDepth = 20; bool stopOnN = true; assert(q.repOk()); assert(repOk(&q)); assert_eq(gfmFw.eh().ftabChars(), gfmBw.gh().ftabChars()); #ifndef NDEBUG for(int i = 0; i < 4; i++) { assert_eq(gfmFw.fchr()[i], gfmBw.fchr()[i]); } #endif SideLocus tloc, bloc; TIndexOffU topf = topf_, botf = botf_, topb = topb_, botb = botb_; bool fw = rs[rid_].fw; bool toward3p; size_t off5p; assert_lt(al5pi_, q.length()); assert_lt(al5pf_, q.length()); while(true) { toward3p = (l2r_ == fw); assert_geq(al5pf_, al5pi_); assert(al5pi_ != 0 || al5pf_ != q.length() - 1); if(toward3p) { if(al5pf_ == q.length()-1) { l2r_ = !l2r_; continue; } if(al5pi_ == al5pf_ && root()) { off5p = off5p_i = al5pi_; } else { off5p = off5p_i = (al5pf_ + 1); } } else { if(al5pi_ == 0) { l2r_ = !l2r_; continue; } assert_gt(al5pi_, 0); if(al5pi_ == al5pf_ && root()) { off5p = off5p_i = al5pi_; } else { off5p = off5p_i = (al5pi_ - 1); } } break; } size_t off3p = q.length() - off5p - 1; assert_lt(off5p, q.length()); assert_lt(off3p, q.length()); bool firstPos = true; assert_eq(0, len_); // Number of times pf.alloc() is called. So we can sanity check it. size_t nalloc = 0; // Set to true as soon as we encounter a branch point along this descent. branches = false; // hitEnd is set to true iff this edit pushes us to the extreme 5' or 3' // end of the alignment hitEnd = false; // done is set to true iff this edit aligns the only remaining character of // the read done = false; if(root()) { assert_eq(al5pi_, al5pf_); // Check whether/how far we can jump using ftab int ftabLen = gfmFw.gh().ftabChars(); bool ftabFits = true; if(toward3p && ftabLen + off5p > q.length()) { ftabFits = false; } else if(!toward3p && off5p < (size_t)ftabLen) { ftabFits = false; } bool useFtab = ftabLen > 1 && (size_t)ftabLen <= nobranchDepth && ftabFits; bool ftabFailed = false; if(useFtab) { prm.nFtabs++; // Forward index: right-to-left size_t off_r2l = fw ? off5p : q.length() - off5p - 1; if(l2r_) { // } else { assert_geq((int)off_r2l, ftabLen - 1); off_r2l -= (ftabLen - 1); } bool ret = gfmFw.ftabLoHi(fw ? q.patFw : q.patRc, off_r2l, false, // reverse topf, botf); if(!ret) { // Encountered an N or something else that made it impossible // to use the ftab ftabFailed = true; } else { if(botf - topf == 0) { return false; } int c_r2l = fw ? q.patFw[off_r2l] : q.patRc[off_r2l]; // Backward index: left-to-right size_t off_l2r = fw ? off5p : q.length() - off5p - 1; if(l2r_) { // } else { assert_geq((int)off_l2r, ftabLen - 1); off_l2r -= (ftabLen - 1); } ASSERT_ONLY(bool ret2 = ) gfmBw.ftabLoHi(fw ? q.patFw : q.patRc, off_l2r, false, // don't reverse topb, botb); assert(ret == ret2); int c_l2r = fw ? q.patFw[off_l2r + ftabLen - 1] : q.patRc[off_l2r + ftabLen - 1]; assert_eq(botf - topf, botb - topb); if(toward3p) { assert_geq((int)off3p, ftabLen - 1); off5p += ftabLen; off3p -= ftabLen; } else { assert_geq((int)off5p, ftabLen - 1); off5p -= ftabLen; off3p += ftabLen; } len_ += ftabLen; if(toward3p) { // By convention, al5pf_ and al5pi_ start out equal, so we only // advance al5pf_ by ftabLen - 1 (not ftabLen) al5pf_ += (ftabLen - 1); // -1 accounts for inclusive al5pf_ if(al5pf_ == q.length() - 1) { hitEnd = true; done = (al5pi_ == 0); } } else { // By convention, al5pf_ and al5pi_ start out equal, so we only // advance al5pi_ by ftabLen - 1 (not ftabLen) al5pi_ -= (ftabLen - 1); if(al5pi_ == 0) { hitEnd = true; done = (al5pf_ == q.length()-1); } } // Allocate DescentPos data structures and leave them empty. We // jumped over them by doing our lookup in the ftab, so we have no // info about outgoing edges from them, besides the matching // outgoing edge from the last pos which is in topf/botf and // topb/botb. size_t id = 0; if(firstPos) { posid_ = pf.alloc(); pf[posid_].reset(); firstPos = false; for(int i = 1; i < ftabLen; i++) { id = pf.alloc(); pf[id].reset(); } } else { for(int i = 0; i < ftabLen; i++) { id = pf.alloc(); pf[id].reset(); } } assert_eq(botf-topf, botb-topb); pf[id].c = l2r_ ? c_l2r : c_r2l; pf[id].topf[l2r_ ? c_l2r : c_r2l] = topf; pf[id].botf[l2r_ ? c_l2r : c_r2l] = botf; pf[id].topb[l2r_ ? c_l2r : c_r2l] = topb; pf[id].botb[l2r_ ? c_l2r : c_r2l] = botb; assert(pf[id].inited()); nalloc += ftabLen; } } if(!useFtab || ftabFailed) { // Can't use ftab, use fchr instead int rdc = q.getc(off5p, fw); // If rdc is N, that's pretty bad! That means we placed a root // right on an N. The only thing we can reasonably do is to pick a // nucleotide at random and proceed. if(rdc > 3) { return false; } assert_range(0, 3, rdc); topf = topb = gfmFw.fchr()[rdc]; botf = botb = gfmFw.fchr()[rdc+1]; if(botf - topf == 0) { return false; } if(toward3p) { off5p++; off3p--; } else { off5p--; off3p++; } len_++; if(toward3p) { if(al5pf_ == q.length()-1) { hitEnd = true; done = (al5pi_ == 0); } } else { if(al5pi_ == 0) { hitEnd = true; done = (al5pf_ == q.length()-1); } } // Allocate DescentPos data structure. We could fill it with the // four ranges from fchr if we wanted to, but that will never be // relevant. size_t id = 0; if(firstPos) { posid_ = id = pf.alloc(); firstPos = false; } else { id = pf.alloc(); } assert_eq(botf-topf, botb-topb); pf[id].c = rdc; pf[id].topf[rdc] = topf; pf[id].botf[rdc] = botf; pf[id].topb[rdc] = topb; pf[id].botb[rdc] = botb; assert(pf[id].inited()); nalloc++; } assert_gt(botf, topf); assert_eq(botf - topf, botb - topb); // Check if this is redundant with an already-explored path if(!re.check(fw, l2r_, al5pi_, al5pf_, al5pf_ - al5pi_ + 1 + gapadd_, topf, botf, pen_)) { prm.nRedSkip++; return false; } prm.nRedFail++; // not pruned by redundancy list prm.nRedIns++; // inserted into redundancy list } if(done) { Edit eempty; alsink.reportAlignment( q, // query gfmFw, // forward index gfmBw, // backward index topf, // top of SA range in forward index botf, // bottom of SA range in forward index topb, // top of SA range in backward index botb, // bottom of SA range in backward index descid_, // Descent at the leaf rid_, // root id eempty, // extra edit, if necessary pen_, // penalty df, // factory with Descent pf, // factory with DescentPoss rs, // roots cs); // configs assert(alsink.repOk()); return true; } else if(hitEnd) { assert(botf > 0 || topf > 0); assert_gt(botf, topf); topf_bounce = topf; botf_bounce = botf; topb_bounce = topb; botb_bounce = botb; return true; // Bounced } // We just advanced either ftabLen characters, or 1 character, // depending on whether we used ftab or fchr. nextLocsBi(gfmFw, gfmBw, tloc, bloc, topf, botf, topb, botb); assert(tloc.valid()); assert(botf - topf == 1 || bloc.valid()); assert(botf - topf > 1 || !bloc.valid()); TIndexOffU t[4], b[4]; // dest BW ranges TIndexOffU tp[4], bp[4]; // dest BW ranges for "prime" index ASSERT_ONLY(TIndexOff lasttot = botf - topf); bool fail = false; while(!fail && !hitEnd) { assert(!done); int rdc = q.getc(off5p, fw); int rdq = q.getq(off5p); assert_range(0, 4, rdc); assert_gt(botf, topf); assert(botf - topf == 1 || bloc.valid()); assert(botf - topf > 1 || !bloc.valid()); assert(tloc.valid()); TIndexOffU width = botf - topf; bool ltr = l2r_; const GFM& gfm = ltr ? gfmBw : gfmFw; t[0] = t[1] = t[2] = t[3] = b[0] = b[1] = b[2] = b[3] = 0; int only = -1; // if we only get 1 non-empty range, this is the char size_t nopts = 1; if(bloc.valid()) { // Set up initial values for the primes if(ltr) { tp[0] = tp[1] = tp[2] = tp[3] = topf; bp[0] = bp[1] = bp[2] = bp[3] = botf; } else { tp[0] = tp[1] = tp[2] = tp[3] = topb; bp[0] = bp[1] = bp[2] = bp[3] = botb; } // Range delimited by tloc/bloc has size >1. If size == 1, // we use a simpler query (see if(!bloc.valid()) blocks below) met.bwops++; met.bwops_bi++; prm.nSdFmops++; if(prm.doFmString) { prm.fmString.add(false, pen_, 1); } gfm.mapBiLFEx(tloc, bloc, t, b, tp, bp); // t, b, tp and bp now filled ASSERT_ONLY(TIndexOffU tot = (b[0]-t[0])+(b[1]-t[1])+(b[2]-t[2])+(b[3]-t[3])); ASSERT_ONLY(TIndexOffU totp = (bp[0]-tp[0])+(bp[1]-tp[1])+(bp[2]-tp[2])+(bp[3]-tp[3])); assert_eq(tot, totp); assert_leq(tot, lasttot); ASSERT_ONLY(lasttot = tot); fail = (rdc > 3 || b[rdc] <= t[rdc]); size_t nopts = 0; if(b[0] > t[0]) { nopts++; only = 0; } if(b[1] > t[1]) { nopts++; only = 1; } if(b[2] > t[2]) { nopts++; only = 2; } if(b[3] > t[3]) { nopts++; only = 3; } if(!fail && b[rdc] - t[rdc] < width) { branches = true; } } else { tp[0] = tp[1] = tp[2] = tp[3] = bp[0] = bp[1] = bp[2] = bp[3] = 0; // Range delimited by tloc/bloc has size 1 TIndexOffU ntop = ltr ? topb : topf; met.bwops++; met.bwops_1++; prm.nSdFmops++; if(prm.doFmString) { prm.fmString.add(false, pen_, 1); } int cc = gfm.mapLF1(ntop, tloc); assert_range(-1, 3, cc); fail = (cc != rdc); if(fail) { branches = true; } if(cc >= 0) { only = cc; t[cc] = ntop; b[cc] = ntop+1; tp[cc] = ltr ? topf : topb; bp[cc] = ltr ? botf : botb; } } // Now figure out what to do with our N. int origRdc = rdc; if(rdc == 4) { fail = true; } else { topf = ltr ? tp[rdc] : t[rdc]; botf = ltr ? bp[rdc] : b[rdc]; topb = ltr ? t[rdc] : tp[rdc]; botb = ltr ? b[rdc] : bp[rdc]; assert_eq(botf - topf, botb - topb); } // The trouble with !stopOnN is that we don't have a way to store the N // edits. There could be several per Descent. if(rdc == 4 && !stopOnN && nopts == 1) { fail = false; rdc = only; int pen = sc.n(rdq); assert_gt(pen, 0); pen_ += pen; } assert_range(0, 4, origRdc); assert_range(0, 4, rdc); // If 'fail' is true, we failed to align this read character. We still // install the SA ranges into the DescentPos and increment len_ in this // case. // Convert t, tp, b, bp info tf, bf, tb, bb TIndexOffU *tf = ltr ? tp : t; TIndexOffU *bf = ltr ? bp : b; TIndexOffU *tb = ltr ? t : tp; TIndexOffU *bb = ltr ? b : bp; // Allocate DescentPos data structure. if(firstPos) { posid_ = pf.alloc(); firstPos = false; } else { pf.alloc(); } nalloc++; pf[posid_ + len_].reset(); pf[posid_ + len_].c = origRdc; for(size_t i = 0; i < 4; i++) { pf[posid_ + len_].topf[i] = tf[i]; pf[posid_ + len_].botf[i] = bf[i]; pf[posid_ + len_].topb[i] = tb[i]; pf[posid_ + len_].botb[i] = bb[i]; assert_eq(pf[posid_ + len_].botf[i] - pf[posid_ + len_].topf[i], pf[posid_ + len_].botb[i] - pf[posid_ + len_].topb[i]); } if(!fail) { // Check if this is redundant with an already-explored path size_t al5pf = al5pf_, al5pi = al5pi_; if(toward3p) { al5pf++; } else { al5pi--; } fail = !re.check(fw, l2r_, al5pi, al5pf, al5pf - al5pi + 1 + gapadd_, topf, botf, pen_); if(fail) { prm.nRedSkip++; } else { prm.nRedFail++; // not pruned by redundancy list prm.nRedIns++; // inserted into redundancy list } } if(!fail) { len_++; if(toward3p) { al5pf_++; off5p++; off3p--; if(al5pf_ == q.length() - 1) { hitEnd = true; done = (al5pi_ == 0); } } else { assert_gt(al5pi_, 0); al5pi_--; off5p--; off3p++; if(al5pi_ == 0) { hitEnd = true; done = (al5pf_ == q.length() - 1); } } } if(!fail && !hitEnd) { nextLocsBi(gfmFw, gfmBw, tloc, bloc, tf[rdc], bf[rdc], tb[rdc], bb[rdc]); } } assert_geq(al5pf_, al5pi_); assert(!root() || al5pf_ - al5pi_ + 1 == nalloc || al5pf_ - al5pi_ + 2 == nalloc); assert_geq(pf.size(), nalloc); if(done) { Edit eempty; alsink.reportAlignment( q, // query gfmFw, // forward index gfmBw, // backward index topf, // top of SA range in forward index botf, // bottom of SA range in forward index topb, // top of SA range in backward index botb, // bottom of SA range in backward index descid_, // Descent at the leaf rid_, // root id eempty, // extra edit, if necessary pen_, // penalty df, // factory with Descent pf, // factory with DescentPoss rs, // roots cs); // configs assert(alsink.repOk()); return true; } else if(hitEnd) { assert(botf > 0 || topf > 0); assert_gt(botf, topf); topf_bounce = topf; botf_bounce = botf; topb_bounce = topb; botb_bounce = botb; return true; // Bounced } assert(repOk(&q)); assert(!hitEnd || topf_bounce > 0 || botf_bounce > 0); return true; } #endif hisat2-2.2.1/aligner_seed_policy.cpp000066400000000000000000000731451370663763200174410ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include #include #include #include "ds.h" #include "aligner_seed_policy.h" #include "mem_ids.h" using namespace std; static int parseFuncType(const std::string& otype) { string type = otype; if(type == "C" || type == "Constant") { return SIMPLE_FUNC_CONST; } else if(type == "L" || type == "Linear") { return SIMPLE_FUNC_LINEAR; } else if(type == "S" || type == "Sqrt") { return SIMPLE_FUNC_SQRT; } else if(type == "G" || type == "Log") { return SIMPLE_FUNC_LOG; } std::cerr << "Error: Bad function type '" << otype.c_str() << "'. Should be C (constant), L (linear), " << "S (square root) or G (natural log)." << std::endl; throw 1; } #define PARSE_FUNC(fv) { \ if(ctoks.size() >= 1) { \ fv.setType(parseFuncType(ctoks[0])); \ } \ if(ctoks.size() >= 2) { \ double co; \ istringstream tmpss(ctoks[1]); \ tmpss >> co; \ fv.setConst(co); \ } \ if(ctoks.size() >= 3) { \ double ce; \ istringstream tmpss(ctoks[2]); \ tmpss >> ce; \ fv.setCoeff(ce); \ } \ if(ctoks.size() >= 4) { \ double mn; \ istringstream tmpss(ctoks[3]); \ tmpss >> mn; \ fv.setMin(mn); \ } \ if(ctoks.size() >= 5) { \ double mx; \ istringstream tmpss(ctoks[4]); \ tmpss >> mx; \ fv.setMin(mx); \ } \ } /** * Parse alignment policy when provided in this format: * =;=;=... * * And label=value possibilities are: * * Bonus for a match * ----------------- * * MA=xx (default: MA=0, or MA=2 if --local is set) * * xx = Each position where equal read and reference characters match up * in the alignment contriubtes this amount to the total score. * * Penalty for a mismatch * ---------------------- * * MMP={Cxx|Q|RQ} (default: MMP=C6) * * Cxx = Each mismatch costs xx. If MMP=Cxx is specified, quality * values are ignored when assessing penalities for mismatches. * Q = Each mismatch incurs a penalty equal to the mismatched base's * value. * R = Each mismatch incurs a penalty equal to the mismatched base's * rounded quality value. Qualities are rounded off to the * nearest 10, and qualities greater than 30 are rounded to 30. * * Penalty for position with N (in either read or reference) * --------------------------------------------------------- * * NP={Cxx|Q|RQ} (default: NP=C1) * * Cxx = Each alignment position with an N in either the read or the * reference costs xx. If NP=Cxx is specified, quality values are * ignored when assessing penalities for Ns. * Q = Each alignment position with an N in either the read or the * reference incurs a penalty equal to the read base's quality * value. * R = Each alignment position with an N in either the read or the * reference incurs a penalty equal to the read base's rounded * quality value. Qualities are rounded off to the nearest 10, * and qualities greater than 30 are rounded to 30. * * Penalty for a read gap * ---------------------- * * RDG=xx,yy (default: RDG=5,3) * * xx = Read gap open penalty. * yy = Read gap extension penalty. * * Total cost incurred by a read gap = xx + (yy * gap length) * * Penalty for a reference gap * --------------------------- * * RFG=xx,yy (default: RFG=5,3) * * xx = Reference gap open penalty. * yy = Reference gap extension penalty. * * Total cost incurred by a reference gap = xx + (yy * gap length) * * Minimum score for valid alignment * --------------------------------- * * MIN=xx,yy (defaults: MIN=-0.6,-0.6, or MIN=0.0,0.66 if --local is set) * * xx,yy = For a read of length N, the total score must be at least * xx + (read length * yy) for the alignment to be valid. The * total score is the sum of all negative penalties (from * mismatches and gaps) and all positive bonuses. The minimum * can be negative (and is by default in global alignment mode). * * Score floor for local alignment * ------------------------------- * * FL=xx,yy (defaults: FL=-Infinity,0.0, or FL=0.0,0.0 if --local is set) * * xx,yy = If a cell in the dynamic programming table has a score less * than xx + (read length * yy), then no valid alignment can go * through it. Defaults are highly recommended. * * N ceiling * --------- * * NCEIL=xx,yy (default: NCEIL=0.0,0.15) * * xx,yy = For a read of length N, the number of alignment * positions with an N in either the read or the * reference cannot exceed * ceiling = xx + (read length * yy). If the ceiling is * exceeded, the alignment is considered invalid. * * Seeds * ----- * * SEED=mm,len,ival (default: SEED=0,22) * * mm = Maximum number of mismatches allowed within a seed. * Must be >= 0 and <= 2. Note that 2-mismatch mode is * not fully sensitive; i.e. some 2-mismatch seed * alignments may be missed. * len = Length of seed. * ival = Interval between seeds. If not specified, seed * interval is determined by IVAL. * * Seed interval * ------------- * * IVAL={L|S|C},xx,yy (default: IVAL=S,1.0,0.0) * * L = let interval between seeds be a linear function of the * read length. xx and yy are the constant and linear * coefficients respectively. In other words, the interval * equals a * len + b, where len is the read length. * Intervals less than 1 are rounded up to 1. * S = let interval between seeds be a function of the sqaure * root of the read length. xx and yy are the * coefficients. In other words, the interval equals * a * sqrt(len) + b, where len is the read length. * Intervals less than 1 are rounded up to 1. * C = Like S but uses cube root of length instead of square * root. * * Example 1: * * SEED=1,10,5 and read sequence is TGCTATCGTACGATCGTAC: * * The following seeds are extracted from the forward * representation of the read and aligned to the reference * allowing up to 1 mismatch: * * Read: TGCTATCGTACGATCGTACA * * Seed 1+: TGCTATCGTA * Seed 2+: TCGTACGATC * Seed 3+: CGATCGTACA * * ...and the following are extracted from the reverse-complement * representation of the read and align to the reference allowing * up to 1 mismatch: * * Seed 1-: TACGATAGCA * Seed 2-: GATCGTACGA * Seed 3-: TGTACGATCG * * Example 2: * * SEED=1,20,20 and read sequence is TGCTATCGTACGATC. The seed * length is 20 but the read is only 15 characters long. In this * case, Bowtie2 automatically shrinks the seed length to be equal * to the read length. * * Read: TGCTATCGTACGATC * * Seed 1+: TGCTATCGTACGATC * Seed 1-: GATCGTACGATAGCA * * Example 3: * * SEED=1,10,10 and read sequence is TGCTATCGTACGATC. Only one seed * fits on the read; a second seed would overhang the end of the read * by 5 positions. In this case, Bowtie2 extracts one seed. * * Read: TGCTATCGTACGATC * * Seed 1+: TGCTATCGTA * Seed 1-: TACGATAGCA */ void SeedAlignmentPolicy::parseString( const std::string& s, bool local, bool noisyHpolymer, bool ignoreQuals, int& bonusMatchType, int& bonusMatch, int& penMmcType, int& penMmcMax, int& penMmcMin, int& penScMax, int& penScMin, int& penNType, int& penN, int& penRdExConst, int& penRfExConst, int& penRdExLinear, int& penRfExLinear, SimpleFunc& costMin, SimpleFunc& nCeil, bool& nCatPair, int& multiseedMms, int& multiseedLen, SimpleFunc& multiseedIval, size_t& failStreak, size_t& seedRounds, SimpleFunc* penCanIntronLen, SimpleFunc* penNoncanIntronLen) { bonusMatchType = local ? DEFAULT_MATCH_BONUS_TYPE_LOCAL : DEFAULT_MATCH_BONUS_TYPE; bonusMatch = local ? DEFAULT_MATCH_BONUS_LOCAL : DEFAULT_MATCH_BONUS; penMmcType = ignoreQuals ? DEFAULT_MM_PENALTY_TYPE_IGNORE_QUALS : DEFAULT_MM_PENALTY_TYPE; penMmcMax = DEFAULT_MM_PENALTY_MAX; penMmcMin = DEFAULT_MM_PENALTY_MIN; penNType = DEFAULT_N_PENALTY_TYPE; penN = DEFAULT_N_PENALTY; penScMax = DEFAULT_SC_PENALTY_MAX; penScMin = DEFAULT_SC_PENALTY_MIN; const double DMAX = std::numeric_limits::max(); costMin.init( local ? SIMPLE_FUNC_LOG : SIMPLE_FUNC_LINEAR, local ? DEFAULT_MIN_CONST_LOCAL : 0.0f, local ? DEFAULT_MIN_LINEAR_LOCAL : -0.2f); nCeil.init( SIMPLE_FUNC_LINEAR, 0.0f, DMAX, DEFAULT_N_CEIL_CONST, DEFAULT_N_CEIL_LINEAR); multiseedIval.init( DEFAULT_IVAL, 1.0f, DMAX, DEFAULT_IVAL_B, DEFAULT_IVAL_A); nCatPair = DEFAULT_N_CAT_PAIR; if(!noisyHpolymer) { penRdExConst = DEFAULT_READ_GAP_CONST; penRdExLinear = DEFAULT_READ_GAP_LINEAR; penRfExConst = DEFAULT_REF_GAP_CONST; penRfExLinear = DEFAULT_REF_GAP_LINEAR; } else { penRdExConst = DEFAULT_READ_GAP_CONST_BADHPOLY; penRdExLinear = DEFAULT_READ_GAP_LINEAR_BADHPOLY; penRfExConst = DEFAULT_REF_GAP_CONST_BADHPOLY; penRfExLinear = DEFAULT_REF_GAP_LINEAR_BADHPOLY; } multiseedMms = DEFAULT_SEEDMMS; multiseedLen = DEFAULT_SEEDLEN; EList toks(MISC_CAT); string tok; istringstream ss(s); int setting = 0; // Get each ;-separated token while(getline(ss, tok, ';')) { setting++; EList etoks(MISC_CAT); string etok; // Divide into tokens on either side of = istringstream ess(tok); while(getline(ess, etok, '=')) { etoks.push_back(etok); } // Must be exactly 1 = if(etoks.size() != 2) { cerr << "Error parsing alignment policy setting " << setting << "; must be bisected by = sign" << endl << "Policy: " << s.c_str() << endl; assert(false); throw 1; } // LHS is tag, RHS value string tag = etoks[0], val = etoks[1]; // Separate value into comma-separated tokens EList ctoks(MISC_CAT); string ctok; istringstream css(val); while(getline(css, ctok, ',')) { ctoks.push_back(ctok); } if(ctoks.size() == 0) { cerr << "Error parsing alignment policy setting " << setting << "; RHS must have at least 1 token" << endl << "Policy: " << s.c_str() << endl; assert(false); throw 1; } for(size_t i = 0; i < ctoks.size(); i++) { if(ctoks[i].length() == 0) { cerr << "Error parsing alignment policy setting " << setting << "; token " << i+1 << " on RHS had length=0" << endl << "Policy: " << s.c_str() << endl; assert(false); throw 1; } } // Bonus for a match // MA=xx (default: MA=0, or MA=10 if --local is set) if(tag == "MA") { if(ctoks.size() != 1) { cerr << "Error parsing alignment policy setting " << setting << "; RHS must have 1 token" << endl << "Policy: " << s.c_str() << endl; assert(false); throw 1; } string tmp = ctoks[0]; istringstream tmpss(tmp); tmpss >> bonusMatch; } // Scoring for mismatches // MMP={Cxx|Q|RQ} // Cxx = constant, where constant is integer xx // Qxx = equal to quality, scaled // R = equal to maq-rounded quality value (rounded to nearest // 10, can't be greater than 30) else if(tag == "MMP") { if(ctoks.size() > 3) { cerr << "Error parsing alignment policy setting " << "'" << tag.c_str() << "'" << "; RHS must have at most 3 tokens" << endl << "Policy: '" << s.c_str() << "'" << endl; assert(false); throw 1; } if(ctoks[0][0] == 'C') { string tmp = ctoks[0].substr(1); // Parse constant penalty istringstream tmpss(tmp); tmpss >> penMmcMax; penMmcMin = penMmcMax; // Parse constant penalty penMmcType = COST_MODEL_CONSTANT; } else if(ctoks[0][0] == 'Q') { if(ctoks.size() >= 2) { string tmp = ctoks[1]; istringstream tmpss(tmp); tmpss >> penMmcMax; } else { penMmcMax = DEFAULT_MM_PENALTY_MAX; } if(ctoks.size() >= 3) { string tmp = ctoks[2]; istringstream tmpss(tmp); tmpss >> penMmcMin; } else { penMmcMin = DEFAULT_MM_PENALTY_MIN; } if(penMmcMin > penMmcMax) { cerr << "Error: Maximum mismatch penalty (" << penMmcMax << ") is less than minimum penalty (" << penMmcMin << endl; throw 1; } // Set type to =quality penMmcType = COST_MODEL_QUAL; } else if(ctoks[0][0] == 'R') { // Set type to=Maq-quality penMmcType = COST_MODEL_ROUNDED_QUAL; } else { cerr << "Error parsing alignment policy setting " << "'" << tag.c_str() << "'" << "; RHS must start with C, Q or R" << endl << "Policy: '" << s.c_str() << "'" << endl; assert(false); throw 1; } } else if(tag == "SCP") { if(ctoks.size() > 3) { cerr << "Error parsing alignment policy setting " << "'" << tag.c_str() << "'" << "; SCP must have at most 3 tokens" << endl << "Policy: '" << s.c_str() << "'" << endl; assert(false); throw 1; } istringstream tmpMax(ctoks[1]); tmpMax >> penScMax; istringstream tmpMin(ctoks[1]); tmpMin >> penScMin; if(penScMin > penScMax) { cerr << "max (" << penScMax << ") should be >= min (" << penScMin << ")" << endl; assert(false); throw 1; } if(penScMin < 1) { cerr << "min (" << penScMin << ") should be greater than 0" << endl; assert(false); throw 1; } } // Scoring for mismatches where read char=N // NP={Cxx|Q|RQ} // Cxx = constant, where constant is integer xx // Q = equal to quality // R = equal to maq-rounded quality value (rounded to nearest // 10, can't be greater than 30) else if(tag == "NP") { if(ctoks.size() != 1) { cerr << "Error parsing alignment policy setting " << "'" << tag.c_str() << "'" << "; RHS must have 1 token" << endl << "Policy: '" << s.c_str() << "'" << endl; assert(false); throw 1; } if(ctoks[0][0] == 'C') { string tmp = ctoks[0].substr(1); // Parse constant penalty istringstream tmpss(tmp); tmpss >> penN; // Parse constant penalty penNType = COST_MODEL_CONSTANT; } else if(ctoks[0][0] == 'Q') { // Set type to =quality penNType = COST_MODEL_QUAL; } else if(ctoks[0][0] == 'R') { // Set type to=Maq-quality penNType = COST_MODEL_ROUNDED_QUAL; } else { cerr << "Error parsing alignment policy setting " << "'" << tag.c_str() << "'" << "; RHS must start with C, Q or R" << endl << "Policy: '" << s.c_str() << "'" << endl; assert(false); throw 1; } } // Scoring for read gaps // RDG=xx,yy,zz // xx = read gap open penalty // yy = read gap extension penalty constant coefficient // (defaults to open penalty) // zz = read gap extension penalty linear coefficient // (defaults to 0) else if(tag == "RDG") { if(ctoks.size() >= 1) { istringstream tmpss(ctoks[0]); tmpss >> penRdExConst; } else { penRdExConst = noisyHpolymer ? DEFAULT_READ_GAP_CONST_BADHPOLY : DEFAULT_READ_GAP_CONST; } if(ctoks.size() >= 2) { istringstream tmpss(ctoks[1]); tmpss >> penRdExLinear; } else { penRdExLinear = noisyHpolymer ? DEFAULT_READ_GAP_LINEAR_BADHPOLY : DEFAULT_READ_GAP_LINEAR; } } // Scoring for reference gaps // RFG=xx,yy,zz // xx = ref gap open penalty // yy = ref gap extension penalty constant coefficient // (defaults to open penalty) // zz = ref gap extension penalty linear coefficient // (defaults to 0) else if(tag == "RFG") { if(ctoks.size() >= 1) { istringstream tmpss(ctoks[0]); tmpss >> penRfExConst; } else { penRfExConst = noisyHpolymer ? DEFAULT_REF_GAP_CONST_BADHPOLY : DEFAULT_REF_GAP_CONST; } if(ctoks.size() >= 2) { istringstream tmpss(ctoks[1]); tmpss >> penRfExLinear; } else { penRfExLinear = noisyHpolymer ? DEFAULT_REF_GAP_LINEAR_BADHPOLY : DEFAULT_REF_GAP_LINEAR; } } // Minimum score as a function of read length // MIN=xx,yy // xx = constant coefficient // yy = linear coefficient else if(tag == "MIN") { PARSE_FUNC(costMin); } // Per-read N ceiling as a function of read length // NCEIL=xx,yy // xx = N ceiling constant coefficient // yy = N ceiling linear coefficient (set to 0 if unspecified) else if(tag == "NCEIL") { PARSE_FUNC(nCeil); } /* * Seeds * ----- * * SEED=mm,len,ival (default: SEED=0,22) * * mm = Maximum number of mismatches allowed within a seed. * Must be >= 0 and <= 2. Note that 2-mismatch mode is * not fully sensitive; i.e. some 2-mismatch seed * alignments may be missed. * len = Length of seed. * ival = Interval between seeds. If not specified, seed * interval is determined by IVAL. */ else if(tag == "SEED") { if(ctoks.size() > 2) { cerr << "Error parsing alignment policy setting " << "'" << tag.c_str() << "'; RHS must have 1 or 2 tokens, " << "had " << ctoks.size() << ". " << "Policy: '" << s.c_str() << "'" << endl; assert(false); throw 1; } if(ctoks.size() >= 1) { istringstream tmpss(ctoks[0]); tmpss >> multiseedMms; if(multiseedMms > 1) { cerr << "Error: -N was set to " << multiseedMms << ", but cannot be set greater than 1" << endl; throw 1; } if(multiseedMms < 0) { cerr << "Error: -N was set to a number less than 0 (" << multiseedMms << ")" << endl; throw 1; } } if(ctoks.size() >= 2) { istringstream tmpss(ctoks[1]); tmpss >> multiseedLen; } else { multiseedLen = DEFAULT_SEEDLEN; } } else if(tag == "SEEDLEN") { if(ctoks.size() > 1) { cerr << "Error parsing alignment policy setting " << "'" << tag.c_str() << "'; RHS must have 1 token, " << "had " << ctoks.size() << ". " << "Policy: '" << s.c_str() << "'" << endl; assert(false); throw 1; } if(ctoks.size() >= 1) { istringstream tmpss(ctoks[0]); tmpss >> multiseedLen; } } else if(tag == "DPS") { if(ctoks.size() > 1) { cerr << "Error parsing alignment policy setting " << "'" << tag.c_str() << "'; RHS must have 1 token, " << "had " << ctoks.size() << ". " << "Policy: '" << s.c_str() << "'" << endl; assert(false); throw 1; } if(ctoks.size() >= 1) { istringstream tmpss(ctoks[0]); tmpss >> failStreak; } } else if(tag == "ROUNDS") { if(ctoks.size() > 1) { cerr << "Error parsing alignment policy setting " << "'" << tag.c_str() << "'; RHS must have 1 token, " << "had " << ctoks.size() << ". " << "Policy: '" << s.c_str() << "'" << endl; assert(false); throw 1; } if(ctoks.size() >= 1) { istringstream tmpss(ctoks[0]); tmpss >> seedRounds; } } /* * Seed interval * ------------- * * IVAL={L|S|C},a,b (default: IVAL=S,1.0,0.0) * * L = let interval between seeds be a linear function of the * read length. xx and yy are the constant and linear * coefficients respectively. In other words, the interval * equals a * len + b, where len is the read length. * Intervals less than 1 are rounded up to 1. * S = let interval between seeds be a function of the sqaure * root of the read length. xx and yy are the * coefficients. In other words, the interval equals * a * sqrt(len) + b, where len is the read length. * Intervals less than 1 are rounded up to 1. * C = Like S but uses cube root of length instead of square * root. */ else if(tag == "IVAL") { PARSE_FUNC(multiseedIval); } else if(tag == "CANINTRONLEN") { assert(penCanIntronLen != NULL); PARSE_FUNC((*penCanIntronLen)); } else if(tag == "NONCANINTRONLEN") { assert(penNoncanIntronLen != NULL); PARSE_FUNC((*penNoncanIntronLen)); } else { // Unknown tag cerr << "Unexpected alignment policy setting " << "'" << tag.c_str() << "'" << endl << "Policy: '" << s.c_str() << "'" << endl; assert(false); throw 1; } } } #ifdef ALIGNER_SEED_POLICY_MAIN int main() { int bonusMatchType; int bonusMatch; int penMmcType; int penMmc; int penScMax; int penScMin; int penNType; int penN; int penRdExConst; int penRfExConst; int penRdExLinear; int penRfExLinear; SimpleFunc costMin; SimpleFunc costFloor; SimpleFunc nCeil; bool nCatPair; int multiseedMms; int multiseedLen; SimpleFunc msIval; SimpleFunc posfrac; SimpleFunc rowmult; uint32_t mhits; { cout << "Case 1: Defaults 1 ... "; const char *pol = ""; SeedAlignmentPolicy::parseString( string(pol), false, // --local? false, // noisy homopolymers a la 454? false, // ignore qualities? bonusMatchType, bonusMatch, penMmcType, penMmc, penScMax, penScMin, penNType, penN, penRdExConst, penRfExConst, penRdExLinear, penRfExLinear, costMin, costFloor, nCeil, nCatPair, multiseedMms, multiseedLen, msIval, mhits); assert_eq(DEFAULT_MATCH_BONUS_TYPE, bonusMatchType); assert_eq(DEFAULT_MATCH_BONUS, bonusMatch); assert_eq(DEFAULT_MM_PENALTY_TYPE, penMmcType); assert_eq(DEFAULT_MM_PENALTY_MAX, penMmcMax); assert_eq(DEFAULT_MM_PENALTY_MIN, penMmcMin); assert_eq(DEFAULT_N_PENALTY_TYPE, penNType); assert_eq(DEFAULT_N_PENALTY, penN); assert_eq(DEFAULT_MIN_CONST, costMin.getConst()); assert_eq(DEFAULT_MIN_LINEAR, costMin.getCoeff()); assert_eq(DEFAULT_FLOOR_CONST, costFloor.getConst()); assert_eq(DEFAULT_FLOOR_LINEAR, costFloor.getCoeff()); assert_eq(DEFAULT_N_CEIL_CONST, nCeil.getConst()); assert_eq(DEFAULT_N_CAT_PAIR, nCatPair); assert_eq(DEFAULT_READ_GAP_CONST, penRdExConst); assert_eq(DEFAULT_READ_GAP_LINEAR, penRdExLinear); assert_eq(DEFAULT_REF_GAP_CONST, penRfExConst); assert_eq(DEFAULT_REF_GAP_LINEAR, penRfExLinear); assert_eq(DEFAULT_SEEDMMS, multiseedMms); assert_eq(DEFAULT_SEEDLEN, multiseedLen); assert_eq(DEFAULT_IVAL, msIval.getType()); assert_eq(DEFAULT_IVAL_A, msIval.getCoeff()); assert_eq(DEFAULT_IVAL_B, msIval.getConst()); cout << "PASSED" << endl; } { cout << "Case 2: Defaults 2 ... "; const char *pol = ""; SeedAlignmentPolicy::parseString( string(pol), false, // --local? true, // noisy homopolymers a la 454? false, // ignore qualities? bonusMatchType, bonusMatch, penMmcType, penMmc, penNType, penN, penRdExConst, penRfExConst, penRdExLinear, penRfExLinear, costMin, costFloor, nCeil, nCatPair, multiseedMms, multiseedLen, msIval, mhits); assert_eq(DEFAULT_MATCH_BONUS_TYPE, bonusMatchType); assert_eq(DEFAULT_MATCH_BONUS, bonusMatch); assert_eq(DEFAULT_MM_PENALTY_TYPE, penMmcType); assert_eq(DEFAULT_MM_PENALTY_MAX, penMmc); assert_eq(DEFAULT_MM_PENALTY_MIN, penMmc); assert_eq(DEFAULT_N_PENALTY_TYPE, penNType); assert_eq(DEFAULT_N_PENALTY, penN); assert_eq(DEFAULT_MIN_CONST, costMin.getConst()); assert_eq(DEFAULT_MIN_LINEAR, costMin.getCoeff()); assert_eq(DEFAULT_FLOOR_CONST, costFloor.getConst()); assert_eq(DEFAULT_FLOOR_LINEAR, costFloor.getCoeff()); assert_eq(DEFAULT_N_CEIL_CONST, nCeil.getConst()); assert_eq(DEFAULT_N_CAT_PAIR, nCatPair); assert_eq(DEFAULT_READ_GAP_CONST_BADHPOLY, penRdExConst); assert_eq(DEFAULT_READ_GAP_LINEAR_BADHPOLY, penRdExLinear); assert_eq(DEFAULT_REF_GAP_CONST_BADHPOLY, penRfExConst); assert_eq(DEFAULT_REF_GAP_LINEAR_BADHPOLY, penRfExLinear); assert_eq(DEFAULT_SEEDMMS, multiseedMms); assert_eq(DEFAULT_SEEDLEN, multiseedLen); assert_eq(DEFAULT_IVAL, msIval.getType()); assert_eq(DEFAULT_IVAL_A, msIval.getCoeff()); assert_eq(DEFAULT_IVAL_B, msIval.getConst()); cout << "PASSED" << endl; } { cout << "Case 3: Defaults 3 ... "; const char *pol = ""; SeedAlignmentPolicy::parseString( string(pol), true, // --local? false, // noisy homopolymers a la 454? false, // ignore qualities? bonusMatchType, bonusMatch, penMmcType, penMmc, penNType, penN, penRdExConst, penRfExConst, penRdExLinear, penRfExLinear, costMin, costFloor, nCeil, nCatPair, multiseedMms, multiseedLen, msIval, mhits); assert_eq(DEFAULT_MATCH_BONUS_TYPE_LOCAL, bonusMatchType); assert_eq(DEFAULT_MATCH_BONUS_LOCAL, bonusMatch); assert_eq(DEFAULT_MM_PENALTY_TYPE, penMmcType); assert_eq(DEFAULT_MM_PENALTY_MAX, penMmcMax); assert_eq(DEFAULT_MM_PENALTY_MIN, penMmcMin); assert_eq(DEFAULT_N_PENALTY_TYPE, penNType); assert_eq(DEFAULT_N_PENALTY, penN); assert_eq(DEFAULT_MIN_CONST_LOCAL, costMin.getConst()); assert_eq(DEFAULT_MIN_LINEAR_LOCAL, costMin.getCoeff()); assert_eq(DEFAULT_FLOOR_CONST_LOCAL, costFloor.getConst()); assert_eq(DEFAULT_FLOOR_LINEAR_LOCAL, costFloor.getCoeff()); assert_eq(DEFAULT_N_CEIL_CONST, nCeil.getConst()); assert_eq(DEFAULT_N_CEIL_LINEAR, nCeil.getCoeff()); assert_eq(DEFAULT_N_CAT_PAIR, nCatPair); assert_eq(DEFAULT_READ_GAP_CONST, penRdExConst); assert_eq(DEFAULT_READ_GAP_LINEAR, penRdExLinear); assert_eq(DEFAULT_REF_GAP_CONST, penRfExConst); assert_eq(DEFAULT_REF_GAP_LINEAR, penRfExLinear); assert_eq(DEFAULT_SEEDMMS, multiseedMms); assert_eq(DEFAULT_SEEDLEN, multiseedLen); assert_eq(DEFAULT_IVAL, msIval.getType()); assert_eq(DEFAULT_IVAL_A, msIval.getCoeff()); assert_eq(DEFAULT_IVAL_B, msIval.getConst()); cout << "PASSED" << endl; } { cout << "Case 4: Simple string 1 ... "; const char *pol = "MMP=C44;MA=4;RFG=24,12;FL=C,8;RDG=2;NP=C4;MIN=C,7"; SeedAlignmentPolicy::parseString( string(pol), true, // --local? false, // noisy homopolymers a la 454? false, // ignore qualities? bonusMatchType, bonusMatch, penMmcType, penMmc, penNType, penN, penRdExConst, penRfExConst, penRdExLinear, penRfExLinear, costMin, costFloor, nCeil, nCatPair, multiseedMms, multiseedLen, msIval, mhits); assert_eq(COST_MODEL_CONSTANT, bonusMatchType); assert_eq(4, bonusMatch); assert_eq(COST_MODEL_CONSTANT, penMmcType); assert_eq(44, penMmc); assert_eq(COST_MODEL_CONSTANT, penNType); assert_eq(4.0f, penN); assert_eq(7, costMin.getConst()); assert_eq(DEFAULT_MIN_LINEAR_LOCAL, costMin.getCoeff()); assert_eq(8, costFloor.getConst()); assert_eq(DEFAULT_FLOOR_LINEAR_LOCAL, costFloor.getCoeff()); assert_eq(DEFAULT_N_CEIL_CONST, nCeil.getConst()); assert_eq(DEFAULT_N_CEIL_LINEAR, nCeil.getCoeff()); assert_eq(DEFAULT_N_CAT_PAIR, nCatPair); assert_eq(2.0f, penRdExConst); assert_eq(DEFAULT_READ_GAP_LINEAR, penRdExLinear); assert_eq(24.0f, penRfExConst); assert_eq(12.0f, penRfExLinear); assert_eq(DEFAULT_SEEDMMS, multiseedMms); assert_eq(DEFAULT_SEEDLEN, multiseedLen); assert_eq(DEFAULT_IVAL, msIval.getType()); assert_eq(DEFAULT_IVAL_A, msIval.getCoeff()); assert_eq(DEFAULT_IVAL_B, msIval.getConst()); cout << "PASSED" << endl; } } #endif /*def ALIGNER_SEED_POLICY_MAIN*/ hisat2-2.2.1/aligner_seed_policy.h000066400000000000000000000202771370663763200171040ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_SEED_POLICY_H_ #define ALIGNER_SEED_POLICY_H_ #include "scoring.h" #include "simple_func.h" #define DEFAULT_SEEDMMS 0 #define DEFAULT_SEEDLEN 22 #define DEFAULT_IVAL SIMPLE_FUNC_SQRT #define DEFAULT_IVAL_A 1.15f #define DEFAULT_IVAL_B 0.0f #define DEFAULT_UNGAPPED_HITS 6 /** * Encapsulates the set of all parameters that affect what the * SeedAligner does with reads. */ class SeedAlignmentPolicy { public: /** * Parse alignment policy when provided in this format: * =;=;=... * * And label=value possibilities are: * * Bonus for a match * ----------------- * * MA=xx (default: MA=0, or MA=2 if --local is set) * * xx = Each position where equal read and reference characters match up * in the alignment contriubtes this amount to the total score. * * Penalty for a mismatch * ---------------------- * * MMP={Cxx|Q|RQ} (default: MMP=C6) * * Cxx = Each mismatch costs xx. If MMP=Cxx is specified, quality * values are ignored when assessing penalities for mismatches. * Q = Each mismatch incurs a penalty equal to the mismatched base's * value. * R = Each mismatch incurs a penalty equal to the mismatched base's * rounded quality value. Qualities are rounded off to the * nearest 10, and qualities greater than 30 are rounded to 30. * * Penalty for position with N (in either read or reference) * --------------------------------------------------------- * * NP={Cxx|Q|RQ} (default: NP=C1) * * Cxx = Each alignment position with an N in either the read or the * reference costs xx. If NP=Cxx is specified, quality values are * ignored when assessing penalities for Ns. * Q = Each alignment position with an N in either the read or the * reference incurs a penalty equal to the read base's quality * value. * R = Each alignment position with an N in either the read or the * reference incurs a penalty equal to the read base's rounded * quality value. Qualities are rounded off to the nearest 10, * and qualities greater than 30 are rounded to 30. * * Penalty for a read gap * ---------------------- * * RDG=xx,yy (default: RDG=5,3) * * xx = Read gap open penalty. * yy = Read gap extension penalty. * * Total cost incurred by a read gap = xx + (yy * gap length) * * Penalty for a reference gap * --------------------------- * * RFG=xx,yy (default: RFG=5,3) * * xx = Reference gap open penalty. * yy = Reference gap extension penalty. * * Total cost incurred by a reference gap = xx + (yy * gap length) * * Minimum score for valid alignment * --------------------------------- * * MIN=xx,yy (defaults: MIN=-0.6,-0.6, or MIN=0.0,0.66 if --local is set) * * xx,yy = For a read of length N, the total score must be at least * xx + (read length * yy) for the alignment to be valid. The * total score is the sum of all negative penalties (from * mismatches and gaps) and all positive bonuses. The minimum * can be negative (and is by default in global alignment mode). * * N ceiling * --------- * * NCEIL=xx,yy (default: NCEIL=0.0,0.15) * * xx,yy = For a read of length N, the number of alignment * positions with an N in either the read or the * reference cannot exceed * ceiling = xx + (read length * yy). If the ceiling is * exceeded, the alignment is considered invalid. * * Seeds * ----- * * SEED=mm,len,ival (default: SEED=0,22) * * mm = Maximum number of mismatches allowed within a seed. * Must be >= 0 and <= 2. Note that 2-mismatch mode is * not fully sensitive; i.e. some 2-mismatch seed * alignments may be missed. * len = Length of seed. * ival = Interval between seeds. If not specified, seed * interval is determined by IVAL. * * Seed interval * ------------- * * IVAL={L|S|C},xx,yy (default: IVAL=S,1.0,0.0) * * L = let interval between seeds be a linear function of the * read length. xx and yy are the constant and linear * coefficients respectively. In other words, the interval * equals a * len + b, where len is the read length. * Intervals less than 1 are rounded up to 1. * S = let interval between seeds be a function of the sqaure * root of the read length. xx and yy are the * coefficients. In other words, the interval equals * a * sqrt(len) + b, where len is the read length. * Intervals less than 1 are rounded up to 1. * C = Like S but uses cube root of length instead of square * root. * * Example 1: * * SEED=1,10,5 and read sequence is TGCTATCGTACGATCGTAC: * * The following seeds are extracted from the forward * representation of the read and aligned to the reference * allowing up to 1 mismatch: * * Read: TGCTATCGTACGATCGTACA * * Seed 1+: TGCTATCGTA * Seed 2+: TCGTACGATC * Seed 3+: CGATCGTACA * * ...and the following are extracted from the reverse-complement * representation of the read and align to the reference allowing * up to 1 mismatch: * * Seed 1-: TACGATAGCA * Seed 2-: GATCGTACGA * Seed 3-: TGTACGATCG * * Example 2: * * SEED=1,20,20 and read sequence is TGCTATCGTACGATC. The seed * length is 20 but the read is only 15 characters long. In this * case, Bowtie2 automatically shrinks the seed length to be equal * to the read length. * * Read: TGCTATCGTACGATC * * Seed 1+: TGCTATCGTACGATC * Seed 1-: GATCGTACGATAGCA * * Example 3: * * SEED=1,10,10 and read sequence is TGCTATCGTACGATC. Only one seed * fits on the read; a second seed would overhang the end of the read * by 5 positions. In this case, Bowtie2 extracts one seed. * * Read: TGCTATCGTACGATC * * Seed 1+: TGCTATCGTA * Seed 1-: TACGATAGCA */ static void parseString( const std::string& s, bool local, bool noisyHpolymer, bool ignoreQuals, int& bonusMatchType, int& bonusMatch, int& penMmcType, int& penMmcMax, int& penMmcMin, int& penScMax, int& penScMin, int& penNType, int& penN, int& penRdExConst, int& penRfExConst, int& penRdExLinear, int& penRfExLinear, SimpleFunc& costMin, SimpleFunc& nCeil, bool& nCatPair, int& multiseedMms, int& multiseedLen, SimpleFunc& multiseedIval, size_t& failStreak, size_t& seedRounds, SimpleFunc* penCanIntronLen = NULL, SimpleFunc* penNoncanIntronLen = NULL); }; #endif /*ndef ALIGNER_SEED_POLICY_H_*/ hisat2-2.2.1/aligner_sw.cpp000066400000000000000000003127421370663763200155720ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include // -- BTL remove -- //#include //#include // -- -- #include "aligner_sw.h" #include "aligner_result.h" #include "search_globals.h" #include "scoring.h" #include "mask.h" /** * Initialize with a new read. */ void SwAligner::initRead( const BTDnaString& rdfw, // forward read sequence const BTDnaString& rdrc, // revcomp read sequence const BTString& qufw, // forward read qualities const BTString& qurc, // reverse read qualities size_t rdi, // offset of first read char to align size_t rdf, // offset of last read char to align const Scoring& sc) // scoring scheme { assert_gt(rdf, rdi); int nceil = sc.nCeil.f((double)rdfw.length()); rdfw_ = &rdfw; // read sequence rdrc_ = &rdrc; // read sequence qufw_ = &qufw; // read qualities qurc_ = &qurc; // read qualities rdi_ = rdi; // offset of first read char to align rdf_ = rdf; // offset of last read char to align sc_ = ≻ // scoring scheme nceil_ = nceil; // max # Ns allowed in ref portion of aln readSse16_ = false; // true -> sse16 from now on for this read initedRead_ = true; #ifndef NO_SSE sseU8fwBuilt_ = false; // built fw query profile, 8-bit score sseU8rcBuilt_ = false; // built rc query profile, 8-bit score sseI16fwBuilt_ = false; // built fw query profile, 16-bit score sseI16rcBuilt_ = false; // built rc query profile, 16-bit score #endif } /** * Initialize with a new alignment problem. */ void SwAligner::initRef( bool fw, // whether to forward or revcomp read is aligning TRefId refidx, // id of reference aligned against const DPRect& rect, // DP rectangle char *rf, // reference sequence size_t rfi, // offset of first reference char to align to size_t rff, // offset of last reference char to align to TRefOff reflen, // length of reference sequence const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score bool enable8, // use 8-bit SSE if possible? size_t cminlen, // minimum length for using checkpointing scheme size_t cpow2, // interval b/t checkpointed diags; 1 << this bool doTri, // triangular mini-fills? bool extend) // is this a seed extension? { size_t readGaps = sc.maxReadGaps(minsc, rdfw_->length()); size_t refGaps = sc.maxRefGaps(minsc, rdfw_->length()); assert_geq(readGaps, 0); assert_geq(refGaps, 0); assert_gt(rff, rfi); rdgap_ = readGaps; // max # gaps in read rfgap_ = refGaps; // max # gaps in reference state_ = STATE_INITED; fw_ = fw; // orientation rd_ = fw ? rdfw_ : rdrc_; // read sequence qu_ = fw ? qufw_ : qurc_; // quality sequence refidx_ = refidx; // id of reference aligned against rf_ = rf; // reference sequence rfi_ = rfi; // offset of first reference char to align to rff_ = rff; // offset of last reference char to align to reflen_ = reflen; // length of entire reference sequence rect_ = ▭ // DP rectangle minsc_ = minsc; // minimum score cural_ = 0; // idx of next alignment to give out initedRef_ = true; // indicate we've initialized the ref portion enable8_ = enable8; // use 8-bit SSE if possible? extend_ = extend; // true iff this is a seed extension cperMinlen_ = cminlen; // reads shorter than this won't use checkpointer cperPerPow2_ = cpow2; // interval b/t checkpointed diags; 1 << this cperEf_ = true; // whether to checkpoint H, E, and F cperTri_ = doTri; // triangular mini-fills? bter_.initRef( fw_ ? rdfw_->buf() : // in: read sequence rdrc_->buf(), fw_ ? qufw_->buf() : // in: quality sequence qurc_->buf(), // daehwan // rd_->length(), // in: read sequence length rdf_ - rdi_, rf_ + rfi_, // in: reference sequence rff_ - rfi_, // in: in-rectangle reference sequence length reflen, // in: total reference sequence length refidx_, // in: reference id rfi_, // in: reference offset fw_, // in: orientation rect_, // in: DP rectangle &cper_, // in: checkpointer *sc_, // in: scoring scheme nceil_); // in: N ceiling } /** * Given a read, an alignment orientation, a range of characters in a referece * sequence, and a bit-encoded version of the reference, set up and execute the * corresponding dynamic programming problem. * * The caller has already narrowed down the relevant portion of the reference * using, e.g., the location of a seed hit, or the range of possible fragment * lengths if we're searching for the opposite mate in a pair. */ void SwAligner::initRef( bool fw, // whether to forward or revcomp read is aligning TRefId refidx, // reference aligned against const DPRect& rect, // DP rectangle const BitPairReference& refs, // Reference strings TRefOff reflen, // length of reference sequence const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score bool enable8, // use 8-bit SSE if possible? size_t cminlen, // minimum length for using checkpointing scheme size_t cpow2, // interval b/t checkpointed diags; 1 << this bool doTri, // triangular mini-fills? bool extend, // true iff this is a seed extension size_t upto, // count the number of Ns up to this offset size_t& nsUpto) // output: the number of Ns up to 'upto' { TRefOff rfi = rect.refl; TRefOff rff = rect.refr + 1; assert_gt(rff, rfi); // Capture an extra reference character outside the rectangle so that we // can check matches in the next column over to the right rff++; // rflen = full length of the reference substring to consider, including // overhang off the boundaries of the reference sequence const size_t rflen = (size_t)(rff - rfi); // Figure the number of Ns we're going to add to either side size_t leftNs = (rfi >= 0 ? 0 : (size_t)std::abs(static_cast(rfi))); leftNs = min(leftNs, rflen); size_t rightNs = (rff <= (TRefOff)reflen ? 0 : (size_t)std::abs(static_cast(rff - reflen))); rightNs = min(rightNs, rflen); // rflenInner = length of just the portion that doesn't overhang ref ends assert_geq(rflen, leftNs + rightNs); const size_t rflenInner = rflen - (leftNs + rightNs); #ifndef NDEBUG bool haveRfbuf2 = false; EList rfbuf2(rflen); // This is really slow, so only do it some of the time if((rand() % 10) == 0) { TRefOff rfii = rfi; for(size_t i = 0; i < rflen; i++) { if(rfii < 0 || (TRefOff)rfii >= reflen) { rfbuf2.push_back(4); } else { rfbuf2.push_back(refs.getBase(refidx, (uint32_t)rfii)); } rfii++; } haveRfbuf2 = true; } #endif // rfbuf_ = uint32_t list large enough to accommodate both the reference // sequence and any Ns we might add to either side. rfwbuf_.resize((rflen + 16) / 4); int offset = refs.getStretch( rfwbuf_.ptr(), // buffer to store words in refidx, // which reference (rfi < 0) ? 0 : (size_t)rfi, // starting offset (can't be < 0) rflenInner // length to grab (exclude overhang) ASSERT_ONLY(, tmp_destU32_));// for BitPairReference::getStretch() assert_leq(offset, 16); rf_ = (char*)rfwbuf_.ptr() + offset; // Shift ref chars away from 0 so we can stick Ns at the beginning if(leftNs > 0) { // Slide everyone down for(size_t i = rflenInner; i > 0; i--) { rf_[i+leftNs-1] = rf_[i-1]; } // Add Ns for(size_t i = 0; i < leftNs; i++) { rf_[i] = 4; } } if(rightNs > 0) { // Add Ns to the end for(size_t i = 0; i < rightNs; i++) { rf_[i + leftNs + rflenInner] = 4; } } #ifndef NDEBUG // Sanity check reference characters for(size_t i = 0; i < rflen; i++) { assert(!haveRfbuf2 || rf_[i] == rfbuf2[i]); assert_range(0, 4, (int)rf_[i]); } #endif // Count Ns and convert reference characters into A/C/G/T masks. Ambiguous // nucleotides (IUPAC codes) have more than one mask bit set. If a // reference scanner was provided, use it to opportunistically resolve seed // hits. nsUpto = 0; for(size_t i = 0; i < rflen; i++) { // rf_[i] gets mask version of refence char, with N=16 if(i < upto && rf_[i] > 3) { nsUpto++; } rf_[i] = (1 << rf_[i]); } // Correct for having captured an extra reference character rff--; initRef( fw, // whether to forward or revcomp read is aligning refidx, // id of reference aligned against rect, // DP rectangle rf_, // reference sequence, wrapped up in BTString object 0, // use the whole thing (size_t)(rff - rfi), // ditto reflen, // reference length sc, // scoring scheme minsc, // minimum score enable8, // use 8-bit SSE if possible? cminlen, // minimum length for using checkpointing scheme cpow2, // interval b/t checkpointed diags; 1 << this doTri, // triangular mini-fills? extend); // true iff this is a seed extension } /** * Given a read, an alignment orientation, a range of characters in a referece * sequence, and a bit-encoded version of the reference, set up and execute the * corresponding ungapped alignment problem. There can only be one solution. * * The caller has already narrowed down the relevant portion of the reference * using, e.g., the location of a seed hit, or the range of possible fragment * lengths if we're searching for the opposite mate in a pair. */ int SwAligner::ungappedAlign( const BTDnaString& rd, // read sequence (could be RC) const BTString& qu, // qual sequence (could be rev) const Coord& coord, // coordinate aligned to const BitPairReference& refs, // Reference strings size_t reflen, // length of reference sequence const Scoring& sc, // scoring scheme bool ohang, // allow overhang? TAlScore minsc, // minimum score SwResult& res) // put alignment result here { const size_t len = rd.length(); int nceil = sc.nCeil.f((double)len); int ns = 0; TRefOff rfi = coord.off(); TRefOff rff = rfi + (TRefOff)len; TRefId refidx = coord.ref(); assert_gt(rff, rfi); // Figure the number of Ns we're going to add to either side size_t leftNs = 0; if(rfi < 0) { if(ohang) { leftNs = (size_t)(-rfi); } else { return 0; } } size_t rightNs = 0; if(rff > (TRefOff)reflen) { if(ohang) { rightNs = (size_t)(rff - (TRefOff)reflen); } else { return 0; } } if((leftNs + rightNs) > (size_t)nceil) { return 0; } // rflenInner = length of just the portion that doesn't overhang ref ends assert_geq(len, leftNs + rightNs); const size_t rflenInner = len - (leftNs + rightNs); #ifndef NDEBUG bool haveRfbuf2 = false; EList rfbuf2(len); // This is really slow, so only do it some of the time if((rand() % 10) == 0) { TRefOff rfii = rfi; for(size_t i = 0; i < len; i++) { if(rfii < 0 || (size_t)rfii >= reflen) { rfbuf2.push_back(4); } else { rfbuf2.push_back(refs.getBase(refidx, (uint32_t)rfii)); } rfii++; } haveRfbuf2 = true; } #endif // rfbuf_ = uint32_t list large enough to accommodate both the reference // sequence and any Ns we might add to either side. rfwbuf_.resize((len + 16) / 4); int offset = refs.getStretch( rfwbuf_.ptr(), // buffer to store words in refidx, // which reference (rfi < 0) ? 0 : (size_t)rfi, // starting offset (can't be < 0) rflenInner // length to grab (exclude overhang) ASSERT_ONLY(, tmp_destU32_));// for BitPairReference::getStretch() assert_leq(offset, 16); rf_ = (char*)rfwbuf_.ptr() + offset; // Shift ref chars away from 0 so we can stick Ns at the beginning if(leftNs > 0) { // Slide everyone down for(size_t i = rflenInner; i > 0; i--) { rf_[i+leftNs-1] = rf_[i-1]; } // Add Ns for(size_t i = 0; i < leftNs; i++) { rf_[i] = 4; } } if(rightNs > 0) { // Add Ns to the end for(size_t i = 0; i < rightNs; i++) { rf_[i + leftNs + rflenInner] = 4; } } #ifndef NDEBUG // Sanity check reference characters for(size_t i = 0; i < len; i++) { assert(!haveRfbuf2 || rf_[i] == rfbuf2[i]); assert_range(0, 4, (int)rf_[i]); } #endif // Count Ns and convert reference characters into A/C/G/T masks. Ambiguous // nucleotides (IUPAC codes) have more than one mask bit set. If a // reference scanner was provided, use it to opportunistically resolve seed // hits. TAlScore score = 0; res.alres.reset(); size_t rowi = 0; size_t rowf = len-1; if(sc.monotone) { for(size_t i = 0; i < len; i++) { // rf_[i] gets mask version of refence char, with N=16 assert_geq(qu[i], 33); score += sc.score(rd[i], (int)(1 << rf_[i]), qu[i] - 33, ns); assert_leq(score, 0); if(score < minsc || ns > nceil) { // Fell below threshold return 0; } } // Got a result! Fill in the rest of the result object. } else { // Definitely ways to short-circuit this. E.g. if diff between cur // score and minsc can't be met by matches. TAlScore floorsc = 0; TAlScore scoreMax = floorsc; size_t lastfloor = 0; rowi = MAX_SIZE_T; size_t sols = 0; for(size_t i = 0; i < len; i++) { score += sc.score(rd[i], (int)(1 << rf_[i]), qu[i] - 33, ns); if(score >= minsc && score >= scoreMax) { scoreMax = score; rowf = i; if(rowi != lastfloor) { rowi = lastfloor; sols++; } } if(score <= floorsc) { score = floorsc; lastfloor = i+1; } } if(ns > nceil || scoreMax < minsc) { // Too many Ns return 0; } if(sols > 1) { // >1 distinct solution in this diag; defer to DP aligner return -1; } score = scoreMax; // Got a result! Fill in the rest of the result object. } // Now fill in the edits res.alres.setScore(AlnScore(score, ns, 0)); assert_geq(rowf, rowi); EList& ned = res.alres.ned(); size_t refns = 0; ASSERT_ONLY(BTDnaString refstr); for(size_t i = rowi; i <= rowf; i++) { ASSERT_ONLY(refstr.append((int)rf_[i])); if(rf_[i] > 3 || rd[i] != rf_[i]) { // Add edit Edit e((int)i, mask2dna[1 << (int)rf_[i]], "ACGTN"[(int)rd[i]], EDIT_TYPE_MM); ned.push_back(e); if(rf_[i] > 3) { refns++; } } } assert(Edit::repOk(ned, rd)); bool fw = coord.fw(); assert_leq(rowf, len-1); size_t trimEnd = (len-1) - rowf; res.alres.setShape( coord.ref(), // ref id coord.off()+rowi, // 0-based ref offset reflen, // length of reference sequence aligned to fw, // aligned to Watson? len, // read length 0, // read ID true, // pretrim soft? 0, // pretrim 5' end 0, // pretrim 3' end true, // alignment trim soft? fw ? rowi : trimEnd, // alignment trim 5' end fw ? trimEnd : rowi); // alignment trim 3' end res.alres.setRefNs(refns); assert(res.repOk()); #ifndef NDEBUG BTDnaString editstr; Edit::toRef(rd, ned, editstr, true, rowi, trimEnd); if(refstr != editstr) { cerr << "Decoded nucleotides and edits don't match reference:" << endl; cerr << " score: " << res.alres.score().score() << endl; cerr << " edits: "; Edit::print(cerr, ned); cerr << endl; cerr << " decoded nucs: " << rd << endl; cerr << " edited nucs: " << editstr << endl; cerr << " reference nucs: " << refstr << endl; assert(0); } #endif if(!fw) { // All edits are currently w/r/t upstream end; if read aligned to Crick // strand, invert them to be w/r/t 5' end instead. res.alres.invertEdits(); } return 1; } /** * Align read 'rd' to reference using read & reference information given * last time init() was called. */ bool SwAligner::align( RandomSource& rnd, // source of pseudo-randoms TAlScore& best) // best alignment score observed in DP matrix { assert(initedRef() && initedRead()); assert_eq(STATE_INITED, state_); state_ = STATE_ALIGNED; // Reset solutions lists btncand_.clear(); btncanddone_.clear(); btncanddoneSucc_ = btncanddoneFail_ = 0; best = std::numeric_limits::min(); sse8succ_ = sse16succ_ = false; int flag = 0; size_t rdlen = rdf_ - rdi_; bool checkpointed = rdlen >= cperMinlen_; bool gathered = false; // Did gathering happen along with alignment? if(sc_->monotone) { // End-to-end if(enable8_ && !readSse16_ && minsc_ >= -254) { // 8-bit end-to-end if(checkpointed) { best = alignGatherEE8(flag, false); if(flag == 0) { gathered = true; } } else { best = alignNucleotidesEnd2EndSseU8(flag, false); #ifndef NDEBUG int flagtmp = 0; TAlScore besttmp = alignGatherEE8(flagtmp, true); // debug assert_eq(flagtmp, flag); assert_eq(besttmp, best); #endif } sse8succ_ = (flag == 0); #ifndef NDEBUG { int flag2 = 0; TAlScore best2 = alignNucleotidesEnd2EndSseI16(flag2, true); { int flagtmp = 0; TAlScore besttmp = alignGatherEE16(flagtmp, true); assert_eq(flagtmp, flag2); assert(flag2 != 0 || best2 == besttmp); } assert(flag < 0 || best == best2); sse16succ_ = (flag2 == 0); } #endif /*ndef NDEBUG*/ } else { // 16-bit end-to-end if(checkpointed) { best = alignGatherEE16(flag, false); if(flag == 0) { gathered = true; } } else { best = alignNucleotidesEnd2EndSseI16(flag, false); #ifndef NDEBUG int flagtmp = 0; TAlScore besttmp = alignGatherEE16(flagtmp, true); assert_eq(flagtmp, flag); assert_eq(besttmp, best); #endif } sse16succ_ = (flag == 0); } } else { // Local flag = -2; if(enable8_ && !readSse16_) { // 8-bit local if(checkpointed) { best = alignGatherLoc8(flag, false); if(flag == 0) { gathered = true; } } else { best = alignNucleotidesLocalSseU8(flag, false); #ifndef NDEBUG int flagtmp = 0; TAlScore besttmp = alignGatherLoc8(flagtmp, true); assert_eq(flag, flagtmp); assert_eq(best, besttmp); #endif } } if(flag == -2) { // 16-bit local flag = 0; if(checkpointed) { best = alignNucleotidesLocalSseI16(flag, false); best = alignGatherLoc16(flag, false); if(flag == 0) { gathered = true; } } else { best = alignNucleotidesLocalSseI16(flag, false); #ifndef NDEBUG int flagtmp = 0; TAlScore besttmp = alignGatherLoc16(flagtmp, true); assert_eq(flag, flagtmp); assert_eq(best, besttmp); #endif } sse16succ_ = (flag == 0); } else { sse8succ_ = (flag == 0); #ifndef NDEBUG int flag2 = 0; TAlScore best2 = alignNucleotidesLocalSseI16(flag2, true); { int flagtmp = 0; TAlScore besttmp = alignGatherLoc16(flagtmp, true); assert_eq(flag2, flagtmp); assert(flag2 != 0 || best2 == besttmp); } assert(flag2 < 0 || best == best2); sse16succ_ = (flag2 == 0); #endif /*ndef NDEBUG*/ } } #ifndef NDEBUG if(!checkpointed && (rand() & 15) == 0 && sse8succ_ && sse16succ_) { SSEData& d8 = fw_ ? sseU8fw_ : sseU8rc_; SSEData& d16 = fw_ ? sseI16fw_ : sseI16rc_; assert_eq(d8.mat_.nrow(), d16.mat_.nrow()); assert_eq(d8.mat_.ncol(), d16.mat_.ncol()); for(size_t i = 0; i < d8.mat_.nrow(); i++) { for(size_t j = 0; j < colstop_; j++) { int h8 = d8.mat_.helt(i, j); int h16 = d16.mat_.helt(i, j); int e8 = d8.mat_.eelt(i, j); int e16 = d16.mat_.eelt(i, j); int f8 = d8.mat_.felt(i, j); int f16 = d16.mat_.felt(i, j); TAlScore h8s = (sc_->monotone ? (h8 - 0xff ) : h8); TAlScore h16s = (sc_->monotone ? (h16 - 0x7fff) : (h16 + 0x8000)); TAlScore e8s = (sc_->monotone ? (e8 - 0xff ) : e8); TAlScore e16s = (sc_->monotone ? (e16 - 0x7fff) : (e16 + 0x8000)); TAlScore f8s = (sc_->monotone ? (f8 - 0xff ) : f8); TAlScore f16s = (sc_->monotone ? (f16 - 0x7fff) : (f16 + 0x8000)); if(h8s < minsc_) { h8s = minsc_ - 1; } if(h16s < minsc_) { h16s = minsc_ - 1; } if(e8s < minsc_) { e8s = minsc_ - 1; } if(e16s < minsc_) { e16s = minsc_ - 1; } if(f8s < minsc_) { f8s = minsc_ - 1; } if(f16s < minsc_) { f16s = minsc_ - 1; } if((h8 != 0 || (int16_t)h16 != (int16_t)0x8000) && h8 > 0) { assert_eq(h8s, h16s); } if((e8 != 0 || (int16_t)e16 != (int16_t)0x8000) && e8 > 0) { assert_eq(e8s, e16s); } if((f8 != 0 || (int16_t)f16 != (int16_t)0x8000) && f8 > 0) { assert_eq(f8s, f16s); } } } } #endif assert(repOk()); cural_ = 0; if(best == MIN_I64 || best < minsc_) { return false; } if(!gathered) { // Look for solutions using SSE matrix assert(sse8succ_ || sse16succ_); if(sc_->monotone) { if(sse8succ_) { gatherCellsNucleotidesEnd2EndSseU8(best); #ifndef NDEBUG if(sse16succ_) { cand_tmp_ = btncand_; gatherCellsNucleotidesEnd2EndSseI16(best); cand_tmp_.sort(); btncand_.sort(); assert(cand_tmp_ == btncand_); } #endif /*ndef NDEBUG*/ } else { gatherCellsNucleotidesEnd2EndSseI16(best); } } else { if(sse8succ_) { gatherCellsNucleotidesLocalSseU8(best); #ifndef NDEBUG if(sse16succ_) { cand_tmp_ = btncand_; gatherCellsNucleotidesLocalSseI16(best); cand_tmp_.sort(); btncand_.sort(); assert(cand_tmp_ == btncand_); } #endif /*ndef NDEBUG*/ } else { gatherCellsNucleotidesLocalSseI16(best); } } } if(!btncand_.empty()) { btncand_.sort(); } return !btncand_.empty(); } /** * Populate the given SwResult with information about the "next best" * alignment if there is one. If there isn't one, false is returned. Note * that false might be returned even though a call to done() would have * returned false. */ bool SwAligner::nextAlignment( SwResult& res, TAlScore minsc, RandomSource& rnd) { assert(initedRead() && initedRef()); assert_eq(STATE_ALIGNED, state_); assert(repOk()); if(done()) { res.reset(); return false; } assert(!done()); size_t off = 0, nbts = 0; assert_lt(cural_, btncand_.size()); assert(res.repOk()); // For each candidate cell that we should try to backtrack from... const size_t candsz = btncand_.size(); size_t SQ = dpRows() >> 4; if(SQ == 0) SQ = 1; size_t rdlen = rdf_ - rdi_; bool checkpointed = rdlen >= cperMinlen_; while(cural_ < candsz) { // Doing 'continue' anywhere in here simply causes us to move on to the // next candidate if(btncand_[cural_].score < minsc) { btncand_[cural_].fate = BT_CAND_FATE_FILT_SCORE; nbtfiltsc_++; cural_++; continue; } nbts = 0; assert(sse8succ_ || sse16succ_); size_t row = btncand_[cural_].row; size_t col = btncand_[cural_].col; assert_lt(row, dpRows()); assert_lt((TRefOff)col, rff_-rfi_); if(sse16succ_) { SSEData& d = fw_ ? sseI16fw_ : sseI16rc_; if(!checkpointed && d.mat_.reset_[row] && d.mat_.reportedThrough(row, col)) { // Skipping this candidate because a previous candidate already // moved through this cell btncand_[cural_].fate = BT_CAND_FATE_FILT_START; //cerr << " skipped becuase starting cell was covered" << endl; nbtfiltst_++; cural_++; continue; } } else if(sse8succ_) { SSEData& d = fw_ ? sseU8fw_ : sseU8rc_; if(!checkpointed && d.mat_.reset_[row] && d.mat_.reportedThrough(row, col)) { // Skipping this candidate because a previous candidate already // moved through this cell btncand_[cural_].fate = BT_CAND_FATE_FILT_START; //cerr << " skipped becuase starting cell was covered" << endl; nbtfiltst_++; cural_++; continue; } } if(sc_->monotone) { bool ret = false; if(sse8succ_) { uint32_t reseed = rnd.nextU32() + 1; rnd.init(reseed); res.reset(); if(checkpointed) { size_t maxiter = MAX_SIZE_T; size_t niter = 0; ret = backtrace( btncand_[cural_].score, // in: expected score true, // in: use mini-fill? true, // in: use checkpoints? res, // out: store results (edits and scores) here off, // out: store diagonal projection of origin row, // start in this rectangle row col, // start in this rectangle column maxiter, // max # extensions to try niter, // # extensions tried rnd); // random gen, to choose among equal paths } else { ret = backtraceNucleotidesEnd2EndSseU8( btncand_[cural_].score, // in: expected score res, // out: store results (edits and scores) here off, // out: store diagonal projection of origin nbts, // out: # backtracks row, // start in this rectangle row col, // start in this rectangle column rnd); // random gen, to choose among equal paths } #ifndef NDEBUG // if(...) statement here should check not whether the primary // alignment was checkpointed, but whether a checkpointed // alignment was done at all. if(!checkpointed) { SwResult res2; res2.alres = res.alres; res2.alres.reset(); size_t maxiter2 = MAX_SIZE_T; size_t niter2 = 0; bool ret2 = backtrace( btncand_[cural_].score, // in: expected score true, // in: use mini-fill? true, // in: use checkpoints? res2, // out: store results (edits and scores) here off, // out: store diagonal projection of origin row, // start in this rectangle row col, // start in this rectangle column maxiter2, // max # extensions to try niter2, // # extensions tried rnd); // random gen, to choose among equal paths // After the first alignment, there's no guarantee we'll // get the same answer from both backtrackers because of // differences in how they handle marking cells as // reported-through. assert(cural_ > 0 || !ret || ret == ret2); assert(cural_ > 0 || !ret || res.alres == res2.alres); } if(sse16succ_ && !checkpointed) { SwResult res2; res2.alres = res.alres; res2.alres.reset(); size_t off2, nbts2 = 0; rnd.init(reseed); bool ret2 = backtraceNucleotidesEnd2EndSseI16( btncand_[cural_].score, // in: expected score res2, // out: store results (edits and scores) here off2, // out: store diagonal projection of origin nbts2, // out: # backtracks row, // start in this rectangle row col, // start in this rectangle column rnd); // random gen, to choose among equal paths assert_eq(ret, ret2); assert_eq(nbts, nbts2); assert(!ret || res2.alres.score() == res.alres.score()); #if 0 if(!checkpointed && (rand() & 15) == 0) { // Check that same cells are reported through SSEData& d8 = fw_ ? sseU8fw_ : sseU8rc_; SSEData& d16 = fw_ ? sseI16fw_ : sseI16rc_; for(size_t i = d8.mat_.nrow(); i > 0; i--) { for(size_t j = 0; j < d8.mat_.ncol(); j++) { assert_eq(d8.mat_.reportedThrough(i-1, j), d16.mat_.reportedThrough(i-1, j)); } } } #endif } #endif rnd.init(reseed+1); // debug/release pseudo-randoms in lock step } else if(sse16succ_) { uint32_t reseed = rnd.nextU32() + 1; res.reset(); if(checkpointed) { size_t maxiter = MAX_SIZE_T; size_t niter = 0; ret = backtrace( btncand_[cural_].score, // in: expected score true, // in: use mini-fill? true, // in: use checkpoints? res, // out: store results (edits and scores) here off, // out: store diagonal projection of origin row, // start in this rectangle row col, // start in this rectangle column maxiter, // max # extensions to try niter, // # extensions tried rnd); // random gen, to choose among equal paths } else { ret = backtraceNucleotidesEnd2EndSseI16( btncand_[cural_].score, // in: expected score res, // out: store results (edits and scores) here off, // out: store diagonal projection of origin nbts, // out: # backtracks row, // start in this rectangle row col, // start in this rectangle column rnd); // random gen, to choose among equal paths } #ifndef NDEBUG // if(...) statement here should check not whether the primary // alignment was checkpointed, but whether a checkpointed // alignment was done at all. if(!checkpointed) { SwResult res2; size_t maxiter2 = MAX_SIZE_T; size_t niter2 = 0; bool ret2 = backtrace( btncand_[cural_].score, // in: expected score true, // in: use mini-fill? true, // in: use checkpoints? res2, // out: store results (edits and scores) here off, // out: store diagonal projection of origin row, // start in this rectangle row col, // start in this rectangle column maxiter2, // max # extensions to try niter2, // # extensions tried rnd); // random gen, to choose among equal paths // After the first alignment, there's no guarantee we'll // get the same answer from both backtrackers because of // differences in how they handle marking cells as // reported-through. assert(cural_ > 0 || !ret || ret == ret2); assert(cural_ > 0 || !ret || res.alres == res2.alres); } #endif rnd.init(reseed); // debug/release pseudo-randoms in lock step } if(ret) { btncand_[cural_].fate = BT_CAND_FATE_SUCCEEDED; break; } else { btncand_[cural_].fate = BT_CAND_FATE_FAILED; } } else { // Local alignment // Check if this solution is "dominated" by a prior one. // Domination is a heuristic designed to eliminate the vast // majority of valid-but-redundant candidates lying in the // "penumbra" of a high-scoring alignment. bool dom = false; { size_t donesz = btncanddone_.size(); const size_t col = btncand_[cural_].col; const size_t row = btncand_[cural_].row; for(size_t i = 0; i < donesz; i++) { assert_gt(btncanddone_[i].fate, 0); size_t colhi = col, rowhi = row; size_t rowlo = btncanddone_[i].row; size_t collo = btncanddone_[i].col; if(colhi < collo) swap(colhi, collo); if(rowhi < rowlo) swap(rowhi, rowlo); if(colhi - collo <= SQ && rowhi - rowlo <= SQ) { // Skipping this candidate because it's "dominated" by // a previous candidate dom = true; break; } } } if(dom) { btncand_[cural_].fate = BT_CAND_FATE_FILT_DOMINATED; nbtfiltdo_++; cural_++; continue; } bool ret = false; if(sse8succ_) { uint32_t reseed = rnd.nextU32() + 1; res.reset(); rnd.init(reseed); if(checkpointed) { size_t maxiter = MAX_SIZE_T; size_t niter = 0; ret = backtrace( btncand_[cural_].score, // in: expected score true, // in: use mini-fill? true, // in: use checkpoints? res, // out: store results (edits and scores) here off, // out: store diagonal projection of origin row, // start in this rectangle row col, // start in this rectangle column maxiter, // max # extensions to try niter, // # extensions tried rnd); // random gen, to choose among equal paths } else { ret = backtraceNucleotidesLocalSseU8( btncand_[cural_].score, // in: expected score res, // out: store results (edits and scores) here off, // out: store diagonal projection of origin nbts, // out: # backtracks row, // start in this rectangle row col, // start in this rectangle column rnd); // random gen, to choose among equal paths } #ifndef NDEBUG // if(...) statement here should check not whether the primary // alignment was checkpointed, but whether a checkpointed // alignment was done at all. if(!checkpointed) { SwResult res2; size_t maxiter2 = MAX_SIZE_T; size_t niter2 = 0; bool ret2 = backtrace( btncand_[cural_].score, // in: expected score true, // in: use mini-fill? true, // in: use checkpoints? res2, // out: store results (edits and scores) here off, // out: store diagonal projection of origin row, // start in this rectangle row col, // start in this rectangle column maxiter2, // max # extensions to try niter2, // # extensions tried rnd); // random gen, to choose among equal paths // After the first alignment, there's no guarantee we'll // get the same answer from both backtrackers because of // differences in how they handle marking cells as // reported-through. assert(cural_ > 0 || !ret || ret == ret2); assert(cural_ > 0 || !ret || res.alres == res2.alres); } if(!checkpointed && sse16succ_) { SwResult res2; size_t off2, nbts2 = 0; rnd.init(reseed); // same b/t backtrace calls bool ret2 = backtraceNucleotidesLocalSseI16( btncand_[cural_].score, // in: expected score res2, // out: store results (edits and scores) here off2, // out: store diagonal projection of origin nbts2, // out: # backtracks row, // start in this rectangle row col, // start in this rectangle column rnd); // random gen, to choose among equal paths assert_eq(ret, ret2); assert_eq(nbts, nbts2); assert(!ret || res2.alres.score() == res.alres.score()); #if 0 if(!checkpointed && (rand() & 15) == 0) { // Check that same cells are reported through SSEData& d8 = fw_ ? sseU8fw_ : sseU8rc_; SSEData& d16 = fw_ ? sseI16fw_ : sseI16rc_; for(size_t i = d8.mat_.nrow(); i > 0; i--) { for(size_t j = 0; j < d8.mat_.ncol(); j++) { assert_eq(d8.mat_.reportedThrough(i-1, j), d16.mat_.reportedThrough(i-1, j)); } } } #endif } #endif rnd.init(reseed+1); // debug/release pseudo-randoms in lock step } else if(sse16succ_) { uint32_t reseed = rnd.nextU32() + 1; res.reset(); if(checkpointed) { size_t maxiter = MAX_SIZE_T; size_t niter = 0; ret = backtrace( btncand_[cural_].score, // in: expected score true, // in: use mini-fill? true, // in: use checkpoints? res, // out: store results (edits and scores) here off, // out: store diagonal projection of origin row, // start in this rectangle row col, // start in this rectangle column maxiter, // max # extensions to try niter, // # extensions tried rnd); // random gen, to choose among equal paths } else { ret = backtraceNucleotidesLocalSseI16( btncand_[cural_].score, // in: expected score res, // out: store results (edits and scores) here off, // out: store diagonal projection of origin nbts, // out: # backtracks row, // start in this rectangle row col, // start in this rectangle column rnd); // random gen, to choose among equal paths } #ifndef NDEBUG // if(...) statement here should check not whether the primary // alignment was checkpointed, but whether a checkpointed // alignment was done at all. if(!checkpointed) { SwResult res2; size_t maxiter2 = MAX_SIZE_T; size_t niter2 = 0; bool ret2 = backtrace( btncand_[cural_].score, // in: expected score true, // in: use mini-fill? true, // in: use checkpoints? res2, // out: store results (edits and scores) here off, // out: store diagonal projection of origin row, // start in this rectangle row col, // start in this rectangle column maxiter2, // max # extensions to try niter2, // # extensions tried rnd); // random gen, to choose among equal paths // After the first alignment, there's no guarantee we'll // get the same answer from both backtrackers because of // differences in how they handle marking cells as // reported-through. assert(cural_ > 0 || !ret || ret == ret2); assert(cural_ > 0 || !ret || res.alres == res2.alres); } #endif rnd.init(reseed); // same b/t backtrace calls } if(ret) { btncand_[cural_].fate = BT_CAND_FATE_SUCCEEDED; btncanddone_.push_back(btncand_[cural_]); btncanddoneSucc_++; assert(res.repOk()); break; } else { btncand_[cural_].fate = BT_CAND_FATE_FAILED; btncanddone_.push_back(btncand_[cural_]); btncanddoneFail_++; } } cural_++; } // while(cural_ < btncand_.size()) if(cural_ == btncand_.size()) { assert(res.repOk()); return false; } assert(!res.alres.empty()); assert(res.repOk()); if(!fw_) { // All edits are currently w/r/t upstream end; if read aligned // to Crick strand, we need to invert them so that they're // w/r/t the read's 5' end instead. res.alres.invertEdits(); } cural_++; assert(res.repOk()); return true; } #ifdef MAIN_ALIGNER_SW #include #include #include #include "scoring.h" #include "aligner_seed_policy.h" int gGapBarrier; int gSnpPhred; static int bonusMatchType; // how to reward matches static int bonusMatch; // constant if match bonus is a constant static int penMmcType; // how to penalize mismatches static int penMmc; // constant if mm pelanty is a constant static int penNType; // how to penalize Ns in the read static int penN; // constant if N pelanty is a constant static bool nPairCat; // true -> concatenate mates before N filter static int penRdExConst; // constant coeff for cost of gap in read static int penRfExConst; // constant coeff for cost of gap in ref static int penRdExLinear; // linear coeff for cost of gap in read static int penRfExLinear; // linear coeff for cost of gap in ref static float costMinConst; // constant coeff for min score w/r/t read len static float costMinLinear; // linear coeff for min score w/r/t read len static float costFloorConst; // constant coeff for score floor w/r/t read len static float costFloorLinear;// linear coeff for score floor w/r/t read len static float nCeilConst; // constant coeff for N ceiling w/r/t read len static float nCeilLinear; // linear coeff for N ceiling w/r/t read len static bool nCatPair; // concat mates before applying N filter? static int multiseedMms; // mismatches permitted in a multiseed seed static int multiseedLen; // length of multiseed seeds static int multiseedIvalType; static float multiseedIvalA; static float multiseedIvalB; static float posmin; static float posfrac; static float rowmult; enum { ARG_TESTS = 256 }; static const char *short_opts = "s:m:r:d:i:"; static struct option long_opts[] = { {(char*)"snppen", required_argument, 0, 's'}, {(char*)"misspen", required_argument, 0, 'm'}, {(char*)"seed", required_argument, 0, 'r'}, {(char*)"align-policy", no_argument, 0, 'A'}, {(char*)"test", no_argument, 0, ARG_TESTS}, }; static void printUsage(ostream& os) { os << "Usage: aligner_sw [options]*" << endl; os << "Options:" << endl; os << " -s/--snppen penalty incurred by SNP; used for decoding" << endl; os << " -m/--misspen quality to use for read chars" << endl; os << " -r/-seed seed for pseudo-random generator" << endl; } /** * Parse a T from a string 's' */ template T parse(const char *s) { T tmp; stringstream ss(s); ss >> tmp; return tmp; } static EList stbuf, enbuf; static BTDnaString btread; static BTString btqual; static BTString btref; static BTString btref2; static BTDnaString readrc; static BTString qualrc; /** * Helper function for running a case consisting of a read (sequence * and quality), a reference string, and an offset that anchors the 0th * character of the read to a reference position. */ static void doTestCase( SwAligner& al, const BTDnaString& read, const BTString& qual, const BTString& refin, TRefOff off, EList *en, const Scoring& sc, TAlScore minsc, SwResult& res, bool nsInclusive, bool filterns, uint32_t seed) { RandomSource rnd(seed); btref2 = refin; assert_eq(read.length(), qual.length()); size_t nrow = read.length(); TRefOff rfi, rff; // Calculate the largest possible number of read and reference gaps given // 'minsc' and 'pens' size_t maxgaps; size_t padi, padf; { int readGaps = sc.maxReadGaps(minsc, read.length()); int refGaps = sc.maxRefGaps(minsc, read.length()); assert_geq(readGaps, 0); assert_geq(refGaps, 0); int maxGaps = max(readGaps, refGaps); padi = 2 * maxGaps; padf = maxGaps; maxgaps = (size_t)maxGaps; } size_t nceil = (size_t)sc.nCeil.f((double)read.length()); size_t width = 1 + padi + padf; rfi = off; off = 0; // Pad the beginning of the reference with Ns if necessary if(rfi < padi) { size_t beginpad = (size_t)(padi - rfi); for(size_t i = 0; i < beginpad; i++) { btref2.insert('N', 0); off--; } rfi = 0; } else { rfi -= padi; } assert_geq(rfi, 0); // Pad the end of the reference with Ns if necessary while(rfi + nrow + padi + padf > btref2.length()) { btref2.append('N'); } rff = rfi + nrow + padi + padf; // Convert reference string to masks for(size_t i = 0; i < btref2.length(); i++) { if(toupper(btref2[i]) == 'N' && !nsInclusive) { btref2.set(16, i); } else { int num = 0; int alts[] = {4, 4, 4, 4}; decodeNuc(toupper(btref2[i]), num, alts); assert_leq(num, 4); assert_gt(num, 0); btref2.set(0, i); for(int j = 0; j < num; j++) { btref2.set(btref2[i] | (1 << alts[j]), i); } } } bool fw = true; uint32_t refidx = 0; size_t solwidth = width; if(maxgaps >= solwidth) { solwidth = 0; } else { solwidth -= maxgaps; } if(en == NULL) { enbuf.resize(solwidth); enbuf.fill(true); en = &enbuf; } assert_geq(rfi, 0); assert_gt(rff, rfi); readrc = read; qualrc = qual; al.initRead( read, // read sequence readrc, qual, // read qualities qualrc, 0, // offset of first character within 'read' to consider read.length(), // offset of last char (exclusive) in 'read' to consider floorsc); // local-alignment score floor al.initRef( fw, // 'read' is forward version of read? refidx, // id of reference aligned to off, // offset of upstream ref char aligned against btref2.wbuf(), // reference sequence (masks) rfi, // offset of first char in 'ref' to consider rff, // offset of last char (exclusive) in 'ref' to consider width, // # bands to do (width of parallelogram) solwidth, // # rightmost cols where solns can end sc, // scoring scheme minsc, // minimum score for valid alignment maxgaps, // max of max # read gaps, ref gaps 0, // amount to truncate on left-hand side en); // mask indicating which columns we can end in if(filterns) { al.filter((int)nceil); } al.align(rnd); } /** * Another interface for running a case. */ static void doTestCase2( SwAligner& al, const char *read, const char *qual, const char *refin, TRefOff off, const Scoring& sc, float costMinConst, float costMinLinear, SwResult& res, bool nsInclusive = false, bool filterns = false, uint32_t seed = 0) { btread.install(read, true); TAlScore minsc = (TAlScore)(Scoring::linearFunc( btread.length(), costMinConst, costMinLinear)); TAlScore floorsc = (TAlScore)(Scoring::linearFunc( btread.length(), costFloorConst, costFloorLinear)); btqual.install(qual); btref.install(refin); doTestCase( al, btread, btqual, btref, off, NULL, sc, minsc, floorsc, res, nsInclusive, filterns, seed ); } /** * Another interface for running a case. */ static void doTestCase3( SwAligner& al, const char *read, const char *qual, const char *refin, TRefOff off, Scoring& sc, float costMinConst, float costMinLinear, float nCeilConst, float nCeilLinear, SwResult& res, bool nsInclusive = false, bool filterns = false, uint32_t seed = 0) { btread.install(read, true); // Calculate the penalty ceiling for the read TAlScore minsc = (TAlScore)(Scoring::linearFunc( btread.length(), costMinConst, costMinLinear)); TAlScore floorsc = (TAlScore)(Scoring::linearFunc( btread.length(), costFloorConst, costFloorLinear)); btqual.install(qual); btref.install(refin); sc.nCeil.setType(SIMPLE_FUNC_LINEAR); sc.nCeil.setConst(costMinConst); sc.nCeil.setCoeff(costMinLinear); doTestCase( al, btread, btqual, btref, off, NULL, sc, minsc, floorsc, res, nsInclusive, filterns, seed ); } /** * Another interface for running a case. Like doTestCase3 but caller specifies * st_ and en_ lists. */ static void doTestCase4( SwAligner& al, const char *read, const char *qual, const char *refin, TRefOff off, EList& en, Scoring& sc, float costMinConst, float costMinLinear, float nCeilConst, float nCeilLinear, SwResult& res, bool nsInclusive = false, bool filterns = false, uint32_t seed = 0) { btread.install(read, true); // Calculate the penalty ceiling for the read TAlScore minsc = (TAlScore)(Scoring::linearFunc( btread.length(), costMinConst, costMinLinear)); TAlScore floorsc = (TAlScore)(Scoring::linearFunc( btread.length(), costFloorConst, costFloorLinear)); btqual.install(qual); btref.install(refin); sc.nCeil.setType(SIMPLE_FUNC_LINEAR); sc.nCeil.setConst(costMinConst); sc.nCeil.setCoeff(costMinLinear); doTestCase( al, btread, btqual, btref, off, &en, sc, minsc, floorsc, res, nsInclusive, filterns, seed ); } /** * Do a set of unit tests. */ static void doTests() { bonusMatchType = DEFAULT_MATCH_BONUS_TYPE; bonusMatch = DEFAULT_MATCH_BONUS; penMmcType = DEFAULT_MM_PENALTY_TYPE; penMmc = DEFAULT_MM_PENALTY; penSnp = DEFAULT_SNP_PENALTY; penNType = DEFAULT_N_PENALTY_TYPE; penN = DEFAULT_N_PENALTY; nPairCat = DEFAULT_N_CAT_PAIR; penRdExConst = DEFAULT_READ_GAP_CONST; penRfExConst = DEFAULT_REF_GAP_CONST; penRdExLinear = DEFAULT_READ_GAP_LINEAR; penRfExLinear = DEFAULT_REF_GAP_LINEAR; costMinConst = DEFAULT_MIN_CONST; costMinLinear = DEFAULT_MIN_LINEAR; costFloorConst = DEFAULT_FLOOR_CONST; costFloorLinear = DEFAULT_FLOOR_LINEAR; nCeilConst = 1.0f; // constant factor in N ceil w/r/t read len nCeilLinear = 0.1f; // coeff of linear term in N ceil w/r/t read len multiseedMms = DEFAULT_SEEDMMS; multiseedLen = DEFAULT_SEEDLEN; // Set up penalities Scoring sc( bonusMatch, penMmcType, // how to penalize mismatches 30, // constant if mm pelanty is a constant 30, // penalty for decoded SNP costMinConst, // constant factor in N ceiling w/r/t read length costMinLinear, // coeff of linear term in N ceiling w/r/t read length costFloorConst, // constant factor in N ceiling w/r/t read length costFloorLinear, // coeff of linear term in N ceiling w/r/t read length nCeilConst, // constant factor in N ceiling w/r/t read length nCeilLinear, // coeff of linear term in N ceiling w/r/t read length penNType, // how to penalize Ns in the read penN, // constant if N pelanty is a constant nPairCat, // true -> concatenate mates before N filtering 25, // constant coeff for cost of gap in read 25, // constant coeff for cost of gap in ref 15, // linear coeff for cost of gap in read 15, // linear coeff for cost of gap in ref 1, // # rows at top/bot can only be entered diagonally -1, // min row idx to backtrace from; -1 = no limit false // sort results first by row then by score? ); // Set up alternative penalities Scoring sc2( bonusMatch, COST_MODEL_QUAL, // how to penalize mismatches 30, // constant if mm pelanty is a constant 30, // penalty for decoded SNP costMinConst, // constant factor in N ceiling w/r/t read length costMinLinear, // coeff of linear term in N ceiling w/r/t read length costFloorConst, // constant factor in N ceiling w/r/t read length costFloorLinear, // coeff of linear term in N ceiling w/r/t read length 1.0f, // constant factor in N ceiling w/r/t read length 1.0f, // coeff of linear term in N ceiling w/r/t read length penNType, // how to penalize Ns in the read penN, // constant if N pelanty is a constant nPairCat, // true -> concatenate mates before N filtering 25, // constant coeff for cost of gap in read 25, // constant coeff for cost of gap in ref 15, // linear coeff for cost of gap in read 15, // linear coeff for cost of gap in ref 1, // # rows at top/bot can only be entered diagonally -1, // min row idx to backtrace from; -1 = no limit false // sort results first by row then by score? ); SwResult res; // // Basic nucleotide-space tests // cerr << "Running tests..." << endl; int tests = 1; bool nIncl = false; bool nfilter = false; SwAligner al; RandomSource rnd(73); for(int i = 0; i < 3; i++) { cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", exact)..."; sc.rdGapConst = 40; sc.rfGapConst = 40; sc.rdGapLinear = 15; sc.rfGapLinear = 15; // A C G T A C G T // H E F H E F H E F H E F H E F H E F H E F H E F // A 0 lo lo -30 lo lo -30 lo lo -30 lo lo 0 lo lo -30 lo lo-30 lo lo-30 lo lo // C -30 lo -55 0 -85 -85 -55 -55 -85 // G -30 lo -70 -55 -85 -55 0 -100-100 // T -30 lo -85 -60 -85 -70 -55-100 -55 // A 0 lo -85 -55 -55 -85 -70 -70 -70 // C -30 lo -55 0 -85-100 -55 -55 -85 // G -30 lo -70 -55 -85 -55 0 -100-100 // T -30 lo -85 -60 -85 -70 -55-100 -55 doTestCase2( al, "ACGTACGT", // read "IIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 0); assert_eq(res.alres.score().ns(), 0); assert(res.alres.ned().empty()); assert(res.alres.aed().empty()); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1mm allowed by minsc)..."; sc.setMmPen(COST_MODEL_CONSTANT, 30); //sc.setMatchBonus(10); doTestCase2( al, "ACGTTCGT", // read "IIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), -30); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1mm allowed by minsc, check qual 1)..."; doTestCase2( al, "ACGTTCGT", // read "ABCDEFGH", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc2, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); size_t lo, hi; if(i == 0) { lo = 0; hi = 1; } else if(i == 1) { lo = 1; hi = 2; } else { lo = 2; hi = 3; } for(size_t j = lo; j < hi; j++) { al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(j*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), -36); assert_eq(res.alres.score().ns(), 0); res.reset(); } al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1mm allowed by minsc, check qual 2)..."; doTestCase2( al, "ACGAACGT", // read "ABCDEFGH", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc2, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), -35); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); assert(res.empty()); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1mm allowed by minsc, check qual )..."; assert(res.empty()); doTestCase2( al, "TCGTACGT", // read "ABCDEFGH", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc2, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), -32); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); assert(res.empty()); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1mm at the beginning, allowed by minsc)..."; doTestCase2( al, "CCGTACGT", // read "IIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), -30); assert_eq(res.alres.score().ns(), 0); assert_eq(1, res.alres.ned().size()); assert_eq(0, res.alres.aed().size()); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1 n in read, allowed)..."; doTestCase3( al, "ACGTNCGT", // read "IIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling 1.0f, // allow 1 N 0.0f, // allow 1 N res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), -1); assert_eq(res.alres.score().ns(), 1); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 2 n in read, allowed)..."; doTestCase3( al, "ACGNNCGT", // read "IIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling 2.0f, // const coeff for N ceiling 0.0f, // linear coeff for N ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), -2); assert_eq(res.alres.score().ns(), 2); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 2 n in read, 1 at beginning, allowed)..."; doTestCase2( al, "NCGTNCGT", // read "IIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), -2); assert_eq(res.alres.score().ns(), 2); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1 n in ref, allowed)..."; doTestCase2( al, "ACGTACGT", // read "IIIIIIII", // qual "ACGTNCGTACGTANGT", // ref in i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), -1); assert_eq(res.alres.score().ns(), 1); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1mm disallowed by minsc)..."; doTestCase2( al, "ACGTTCGT", // read "IIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -10.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); // Read gap with equal read and ref gap penalties cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", read gap allowed by minsc)..."; assert(res.empty()); sc.rfGapConst = 25; sc.rdGapConst = 25; sc.rfGapLinear = 15; sc.rdGapLinear = 15; doTestCase2( al, "ACGTCGT", // read "IIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -40); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", read gap disallowed by minsc)..."; sc.rfGapConst = 25; sc.rdGapConst = 25; sc.rfGapLinear = 15; sc.rdGapLinear = 15; doTestCase2( al, "ACGTCGT", // read "IIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); res.reset(); cerr << "PASSED" << endl; // Ref gap with equal read and ref gap penalties cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", ref gap allowed by minsc)..."; doTestCase2( al, "ACGTAACGT", // read "IIIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -40); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", read gap disallowed by gap barrier)..."; sc.rfGapConst = 25; sc.rdGapConst = 25; sc.rfGapLinear = 15; sc.rdGapLinear = 15; sc.gapbar = 4; doTestCase2( al, "ACGTCGT", // read "IIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns sc.gapbar = 1; al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); res.reset(); cerr << "PASSED" << endl; // Ref gap with equal read and ref gap penalties cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", ref gap allowed by minsc, gapbar=3)..."; sc.gapbar = 3; doTestCase2( al, "ACGTAACGT", // read "IIIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns sc.gapbar = 1; assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -40); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; // Ref gap with equal read and ref gap penalties cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", ref gap allowed by minsc, gapbar=4)..."; sc.gapbar = 4; doTestCase2( al, "ACGTAACGT", // read "IIIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns sc.gapbar = 1; assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -40); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", ref gap disallowed by minsc)..."; doTestCase2( al, "ACGTAACGT", // read "IIIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); assert(al.done()); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", ref gap disallowed by gap barrier)..."; sc.gapbar = 5; doTestCase2( al, "ACGTAACGT", // read "IIIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns sc.gapbar = 1; al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); assert(al.done()); cerr << "PASSED" << endl; // Read gap with one read gap and zero ref gaps allowed cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1 read gap, ref gaps disallowed by minsc)..."; sc.rfGapConst = 35; sc.rdGapConst = 25; sc.rfGapLinear = 20; sc.rdGapLinear = 10; doTestCase2( al, "ACGTCGT", // read "IIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -35); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", gaps disallowed by minsc)..."; sc.rfGapConst = 25; sc.rdGapConst = 25; sc.rfGapLinear = 10; sc.rdGapLinear = 10; doTestCase2( al, "ACGTCGT", // read "IIIIIII", // qual "ACGTACGTACGTACGT", // ref i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); assert(res.empty()); cerr << "PASSED" << endl; // Ref gap with one ref gap and zero read gaps allowed sc.rfGapConst = 25; sc.rdGapConst = 35; sc.rfGapLinear = 12; sc.rdGapLinear = 22; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1 ref gap, read gaps disallowed by minsc)..."; assert(res.empty()); doTestCase2( al, "ACGTAACGT", "IIIIIIIII", "ACGTACGTACGTACGT", i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -37); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", gaps disallowed by minsc)..."; doTestCase2( al, "ACGTAACGT", "IIIIIIIII", "ACGTACGTACGTACGT", i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); cerr << "PASSED" << endl; // Read gap with one read gap and two ref gaps allowed sc.rfGapConst = 20; sc.rdGapConst = 25; sc.rfGapLinear = 10; sc.rdGapLinear = 15; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1 read gap, 2 ref gaps allowed by minsc)..."; doTestCase2( al, "ACGTCGT", "IIIIIII", "ACGTACGTACGTACGT", i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -40); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", gaps disallowed by minsc)..."; doTestCase2( al, "ACGTCGT", "IIIIIII", "ACGTACGTACGTACGT", i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); cerr << "PASSED" << endl; // Ref gap with one ref gap and two read gaps allowed sc.rfGapConst = 25; sc.rdGapConst = 11; // if this were 10, we'd have ties sc.rfGapLinear = 15; sc.rdGapLinear = 10; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1 ref gap, 2 read gaps allowed by minsc)..."; doTestCase2( al, "ACGTAACGT", "IIIIIIIII", "ACGTACGTACGTACGT", i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -40); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", gaps disallowed by minsc)..."; doTestCase2( al, "ACGTAACGT", "IIIIIIIII", "ACGTACGTACGTACGT", i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(res.empty()); res.reset(); assert(al.done()); cerr << "PASSED" << endl; // Read gap with two read gaps and two ref gaps allowed sc.rfGapConst = 15; sc.rdGapConst = 15; sc.rfGapLinear = 10; sc.rdGapLinear = 10; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 2 ref gaps, 2 read gaps allowed by minsc)..."; doTestCase3( al, "ACGTCGT", "IIIIIII", "ACGTACGTACGTACGT", i*4, // off sc, // scoring scheme -40.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling 1.0, // const coeff for N ceiling 0.0, // linear coeff for N ceiling res, // result nIncl, // Ns inclusive (not mismatches) true); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); if(!res.empty()) { //al.printResultStacked(res, cerr); cerr << endl; } assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -25); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); // The following alignment is possible when i == 2: // ACGTACGTACGTACGTN // A x // C x // G x // T x // C x // G x // T x assert(i == 2 || res.empty()); res.reset(); cerr << "PASSED" << endl; sc.rfGapConst = 10; sc.rdGapConst = 10; sc.rfGapLinear = 10; sc.rdGapLinear = 10; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1 ref gap, 1 read gap allowed by minsc)..."; doTestCase2( al, "ACGTCGT", "IIIIIII", "ACGTACGTACGTACGT", i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -20); assert_eq(res.alres.score().ns(), 0); res.reset(); cerr << "PASSED" << endl; // Ref gap with two ref gaps and zero read gaps allowed sc.rfGapConst = 15; sc.rdGapConst = 15; sc.rfGapLinear = 5; sc.rdGapLinear = 5; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 2 ref gaps, 2 read gaps allowed by minsc)..."; // Careful: it might be possible for the read to align with overhang // instead of with a gap doTestCase3( al, "ACGTAACGT", "IIIIIIIII", "ACGTACGTACGTACGT", i*4, // off sc, // scoring scheme -35.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling 1.0f, // needed to avoid overhang alignments 0.0f, // needed to avoid overhang alignments res, // result nIncl, // Ns inclusive (not mismatches) true); // filter Ns if(i == 0) { lo = 0; hi = 1; } else if(i == 1) { lo = 1; hi = 2; } else { lo = 2; hi = 3; } for(size_t j = lo; j < hi; j++) { al.nextAlignment(res, rnd); assert(!res.empty()); //al.printResultStacked(res, cerr); cerr << endl; assert(res.alres.refoff() == 0 || res.alres.refoff() == 4 || res.alres.refoff() == 8); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -20); assert_eq(res.alres.score().ns(), 0); res.reset(); } al.nextAlignment(res, rnd); //assert(res.empty()); //res.reset(); cerr << "PASSED" << endl; sc.rfGapConst = 25; sc.rdGapConst = 25; sc.rfGapLinear = 4; sc.rdGapLinear = 4; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1 ref gap, 1 read gap allowed by minsc)..."; doTestCase2( al, "ACGTAACGT", "IIIIIIIII", "ACGTACGTACGTACGT", i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 1); assert_eq(res.alres.score().score(), -29); assert_eq(res.alres.score().ns(), 0); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", short read)..."; doTestCase2( al, "A", "I", "AAAAAAAAAAAA", i*4, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 0); assert_eq(res.alres.score().ns(), 0); res.reset(); cerr << "PASSED" << endl; if(i == 0) { cerr << " Test " << tests++ << " (nuc space, offset 0, short read & ref)..."; doTestCase2( al, "A", "I", "A", 0, // off sc, // scoring scheme -30.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 0); assert_eq(res.alres.score().ns(), 0); res.reset(); cerr << "PASSED" << endl; } cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", short read, many allowed gaps)..."; doTestCase2( al, "A", "I", "AAAAAAAAAAAA", i*4, // off sc, // scoring scheme -150.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 0); assert_eq(res.alres.score().ns(), 0); res.reset(); cerr << "PASSED" << endl; if(i == 0) { cerr << " Test " << tests++ << " (nuc space, offset 0, short read & ref, " << "many allowed gaps)..."; doTestCase2( al, "A", "I", "A", 0, // off sc, // scoring scheme -150.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 0); assert_eq(res.alres.score().ns(), 0); res.reset(); cerr << "PASSED" << endl; } } // A test case where a valid alignment with a worse score should be // accepted over a valid alignment with a better score but too many // Ns cerr << " Test " << tests++ << " (N ceiling 1)..."; sc.mmcostType = COST_MODEL_CONSTANT; sc.mmcost = 10; sc.snp = 30; sc.nCeilConst = 0.0f; sc.nCeilLinear = 0.0f; sc.rfGapConst = 10; sc.rdGapLinear = 10; sc.rfGapConst = 10; sc.rfGapLinear = 10; sc.setNPen(COST_MODEL_CONSTANT, 2); sc.gapbar = 1; // No Ns allowed, so this hit should be filtered doTestCase2( al, "ACGTACGT", // read seq "IIIIIIII", // read quals "NCGTACGT", // ref seq 0, // offset sc, // scoring scheme -25.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result false, // ns are in inclusive true, // nfilter 0); al.nextAlignment(res, rnd); assert(res.empty()); cerr << "PASSED" << endl; res.reset(); // 1 N allowed, so this hit should stand cerr << " Test " << tests++ << " (N ceiling 2)..."; doTestCase3( al, "ACGTACGT", // read seq "IIIIIIII", // read quals "NCGTACGT", // ref seq 0, // offset sc, // scoring scheme -25.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling 1.0f, // constant coefficient for # Ns allowed 0.0f, // linear coefficient for # Ns allowed res, // result false, // ns are in inclusive false, // nfilter - NOTE: FILTER OFF 0); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(0, res.alres.score().gaps()); assert_eq(-2, res.alres.score().score()); assert_eq(1, res.alres.score().ns()); cerr << "PASSED" << endl; res.reset(); // 1 N allowed, but we set st_ such that this hit should not stand for(size_t i = 0; i < 2; i++) { cerr << " Test " << tests++ << " (N ceiling 2 with st_ override)..."; EList en; en.resize(3); en.fill(true); if(i == 1) { en[1] = false; } sc.rfGapConst = 10; sc.rdGapLinear = 10; sc.rfGapConst = 10; sc.rfGapLinear = 10; doTestCase4( al, "ACGTACGT", // read seq "IIIIIIII", // read quals "NCGTACGT", // ref seq 0, // offset en, // rectangle columns where solution can end sc, // scoring scheme -25.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling 1.0f, // constant coefficient for # Ns allowed 0.0f, // linear coefficient for # Ns allowed res, // result false, // ns are in inclusive false, // nfilter - NOTE: FILTER OFF 0); al.nextAlignment(res, rnd); if(i > 0) { assert(res.empty()); } else { assert(!res.empty()); } cerr << "PASSED" << endl; res.reset(); } // No Ns allowed, so this hit should be filtered cerr << " Test " << tests++ << " (N ceiling 3)..."; sc.nCeilConst = 1.0f; sc.nCeilLinear = 0.0f; doTestCase2( al, "ACGTACGT", // read seq "IIIIIIII", // read quals "NCGTACGT", // ref seq 0, // offset sc, // scoring scheme -25.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result false, // ns are in inclusive true, // nfilter - NOTE: FILTER ON 0); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(0, res.alres.score().gaps()); assert_eq(-2, res.alres.score().score()); assert_eq(1, res.alres.score().ns()); cerr << "PASSED" << endl; res.reset(); // No Ns allowed, so this hit should be filtered cerr << " Test " << tests++ << " (redundant alignment elimination 1)..."; sc.nCeilConst = 1.0f; sc.nCeilLinear = 0.0f; sc.rfGapConst = 25; sc.rdGapLinear = 15; sc.rfGapConst = 25; sc.rfGapLinear = 15; doTestCase2( al, // 1 2 3 4 // 01234567890123456789012345678901234567890123456 "AGGCTATGCCTCTGACGCGATATCGGCGCCCACTTCAGAGCTAACCG", "IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII", "TTTTTTTTAGGCTATGCCTCTGACGCGATATCGGCGCCCACTTCAGAGCTAACCGTTTTTTT", // 01234567890123456789012345678901234567890123456789012345678901 // 1 2 3 4 5 6 8, // offset sc, // scoring scheme -25.0f, // const coeff for cost ceiling -5.0f, // linear coeff for cost ceiling res, // result false, // ns are in inclusive true, // nfilter - NOTE: FILTER ON 0); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(8, res.alres.refoff()); assert_eq(47, res.alres.refExtent()); assert_eq(0, res.alres.score().gaps()); assert_eq(0, res.alres.score().score()); assert_eq(0, res.alres.score().ns()); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); cerr << "PASSED" << endl; res.reset(); } /** * Do a set of unit tests for local alignment. */ static void doLocalTests() { bonusMatchType = DEFAULT_MATCH_BONUS_TYPE; bonusMatch = DEFAULT_MATCH_BONUS_LOCAL; penMmcType = DEFAULT_MM_PENALTY_TYPE; penMmc = DEFAULT_MM_PENALTY; penSnp = DEFAULT_SNP_PENALTY; penNType = DEFAULT_N_PENALTY_TYPE; penN = DEFAULT_N_PENALTY; nPairCat = DEFAULT_N_CAT_PAIR; penRdExConst = DEFAULT_READ_GAP_CONST; penRfExConst = DEFAULT_REF_GAP_CONST; penRdExLinear = DEFAULT_READ_GAP_LINEAR; penRfExLinear = DEFAULT_REF_GAP_LINEAR; costMinConst = DEFAULT_MIN_CONST_LOCAL; costMinLinear = DEFAULT_MIN_LINEAR_LOCAL; costFloorConst = DEFAULT_FLOOR_CONST_LOCAL; costFloorLinear = DEFAULT_FLOOR_LINEAR_LOCAL; nCeilConst = 1.0f; // constant factor in N ceil w/r/t read len nCeilLinear = 0.1f; // coeff of linear term in N ceil w/r/t read len multiseedMms = DEFAULT_SEEDMMS; multiseedLen = DEFAULT_SEEDLEN; // Set up penalities Scoring sc( 10, penMmcType, // how to penalize mismatches 30, // constant if mm pelanty is a constant penSnp, // penalty for decoded SNP costMinConst, // constant factor in N ceiling w/r/t read length costMinLinear, // coeff of linear term in N ceiling w/r/t read length costFloorConst, // constant factor in N ceiling w/r/t read length costFloorLinear, // coeff of linear term in N ceiling w/r/t read length nCeilConst, // constant factor in N ceiling w/r/t read length nCeilLinear, // coeff of linear term in N ceiling w/r/t read length penNType, // how to penalize Ns in the read penN, // constant if N pelanty is a constant nPairCat, // true -> concatenate mates before N filtering 25, // constant coeff for cost of gap in read 25, // constant coeff for cost of gap in ref 15, // linear coeff for cost of gap in read 15, // linear coeff for cost of gap in ref 1, // # rows at top/bot can only be entered diagonally -1, // min row idx to backtrace from; -1 = no limit false // sort results first by row then by score? ); SwResult res; // // Basic nucleotide-space tests // cerr << "Running local tests..." << endl; int tests = 1; bool nIncl = false; bool nfilter = false; SwAligner al; RandomSource rnd(73); for(int i = 0; i < 3; i++) { cerr << " Test " << tests++ << " (short nuc space, offset " << (i*4) << ", exact)..."; sc.rdGapConst = 40; sc.rfGapConst = 40; doTestCase2( al, "ACGT", // read "IIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme 0.0f, // const coeff for cost ceiling 8.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(4, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 40); assert_eq(res.alres.score().ns(), 0); assert(res.alres.ned().empty()); assert(res.alres.aed().empty()); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); res.reset(); cerr << "PASSED" << endl; // 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 // A C G T A C G T A C G T A C G T // 0 C // 1 C x // 2 G x // 3 T x cerr << " Test " << tests++ << " (short nuc space, offset " << (i*4) << ", 1mm)..."; sc.rdGapConst = 40; sc.rfGapConst = 40; doTestCase2( al, "CCGT", // read "IIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme 0.0f, // const coeff for cost ceiling 7.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4+1, res.alres.refoff()); assert_eq(3, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 30); assert_eq(res.alres.score().ns(), 0); assert(res.alres.ned().empty()); assert(res.alres.aed().empty()); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (short nuc space, offset " << (i*4) << ", 1mm)..."; sc.rdGapConst = 40; sc.rfGapConst = 40; doTestCase2( al, "ACGA", // read "IIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme 0.0f, // const coeff for cost ceiling 7.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(3, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 30); assert_eq(res.alres.score().ns(), 0); assert(res.alres.ned().empty()); assert(res.alres.aed().empty()); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); res.reset(); cerr << "PASSED" << endl; if(i == 0) { cerr << " Test " << tests++ << " (short nuc space, offset " << (i*4) << ", 1mm, match bonus=20)..."; sc.rdGapConst = 40; sc.rfGapConst = 40; sc.setMatchBonus(20); doTestCase2( al, "TTGT", // read "IIII", // qual "TTGA", // ref in i*4, // off sc, // scoring scheme 25.0f, // const coeff for cost ceiling 0.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(3, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 60); assert_eq(res.alres.score().ns(), 0); assert(res.alres.ned().empty()); assert(res.alres.aed().empty()); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); res.reset(); sc.setMatchBonus(10); cerr << "PASSED" << endl; } cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", exact)..."; sc.rdGapConst = 40; sc.rfGapConst = 40; doTestCase2( al, "ACGTACGT", // read "IIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme 0.0f, // const coeff for cost ceiling 8.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 80); assert_eq(res.alres.score().ns(), 0); assert(res.alres.ned().empty()); assert(res.alres.aed().empty()); res.reset(); al.nextAlignment(res, rnd); assert(res.empty()); assert(al.done()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (long nuc space, offset " << (i*8) << ", exact)..."; sc.rdGapConst = 40; sc.rfGapConst = 40; doTestCase2( al, "ACGTACGTACGTACGTACGTA", // read "IIIIIIIIIIIIIIIIIIIII", // qual "ACGTACGTACGTACGTACGTACGTACGTACGTACGTA", // ref in // ACGTACGTACGTACGTACGT // ACGTACGTACGTACGTACGT // ACGTACGTACGTACGTACGT i*8, // off sc, // scoring scheme 0.0f, // const coeff for cost ceiling 8.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*8, res.alres.refoff()); assert_eq(21, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 210); assert_eq(res.alres.score().ns(), 0); assert(res.alres.ned().empty()); assert(res.alres.aed().empty()); res.reset(); al.nextAlignment(res, rnd); //assert(res.empty()); //assert(al.done()); res.reset(); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (nuc space, offset " << (i*4) << ", 1mm allowed by minsc)..."; doTestCase2( al, "ACGTTCGT", // read "IIIIIIII", // qual "ACGTACGTACGTACGT", // ref in i*4, // off sc, // scoring scheme 0.0f, // const coeff for cost ceiling 5.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*4, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 40); assert_eq(res.alres.score().ns(), 0); res.reset(); al.nextAlignment(res, rnd); //assert(res.empty()); //assert(al.done()); cerr << "PASSED" << endl; cerr << " Test " << tests++ << " (long nuc space, offset " << (i*8) << ", 6mm allowed by minsc)..."; sc.rdGapConst = 50; sc.rfGapConst = 50; sc.rdGapLinear = 45; sc.rfGapLinear = 45; doTestCase2( al, "ACGTACGATGCATCGTACGTA", // read "IIIIIIIIIIIIIIIIIIIII", // qual "ACGTACGTACGTACGTACGTACGTACGTACGTACGTA", // ref in // ACGTACGTACGTACGTACGT // ACGTACGTACGTACGTACGT // ACGTACGTACGTACGTACGT i*8, // off sc, // scoring scheme 0.0f, // const coeff for cost ceiling 1.0f, // linear coeff for cost ceiling res, // result nIncl, // Ns inclusive (not mismatches) nfilter); // filter Ns assert(!al.done()); al.nextAlignment(res, rnd); assert(!res.empty()); assert_eq(i*8 + 13, res.alres.refoff()); assert_eq(8, res.alres.refExtent()); assert_eq(res.alres.score().gaps(), 0); assert_eq(res.alres.score().score(), 80); assert_eq(res.alres.score().ns(), 0); assert(res.alres.ned().empty()); assert(res.alres.aed().empty()); res.reset(); al.nextAlignment(res, rnd); res.reset(); cerr << "PASSED" << endl; } } int main(int argc, char **argv) { int option_index = 0; int next_option; unsigned seed = 0; gGapBarrier = 1; gSnpPhred = 30; bool nsInclusive = false; bool nfilter = false; bonusMatchType = DEFAULT_MATCH_BONUS_TYPE; bonusMatch = DEFAULT_MATCH_BONUS; penMmcType = DEFAULT_MM_PENALTY_TYPE; penMmc = DEFAULT_MM_PENALTY; penSnp = DEFAULT_SNP_PENALTY; penNType = DEFAULT_N_PENALTY_TYPE; penN = DEFAULT_N_PENALTY; penRdExConst = DEFAULT_READ_GAP_CONST; penRfExConst = DEFAULT_REF_GAP_CONST; penRdExLinear = DEFAULT_READ_GAP_LINEAR; penRfExLinear = DEFAULT_REF_GAP_LINEAR; costMinConst = DEFAULT_MIN_CONST; costMinLinear = DEFAULT_MIN_LINEAR; costFloorConst = DEFAULT_FLOOR_CONST; costFloorLinear = DEFAULT_FLOOR_LINEAR; nCeilConst = 1.0f; // constant factor in N ceiling w/r/t read length nCeilLinear = 1.0f; // coeff of linear term in N ceiling w/r/t read length nCatPair = false; multiseedMms = DEFAULT_SEEDMMS; multiseedLen = DEFAULT_SEEDLEN; multiseedIvalType = DEFAULT_IVAL; multiseedIvalA = DEFAULT_IVAL_A; multiseedIvalB = DEFAULT_IVAL_B; mhits = 1; do { next_option = getopt_long(argc, argv, short_opts, long_opts, &option_index); switch (next_option) { case 's': gSnpPhred = parse(optarg); break; case 'r': seed = parse(optarg); break; case ARG_TESTS: { doTests(); cout << "PASSED end-to-ends" << endl; doLocalTests(); cout << "PASSED locals" << endl; return 0; } case 'A': { bool localAlign = false; bool noisyHpolymer = false; bool ignoreQuals = false; SeedAlignmentPolicy::parseString( optarg, localAlign, noisyHpolymer, ignoreQuals, bonusMatchType, bonusMatch, penMmcType, penMmc, penNType, penN, penRdExConst, penRfExConst, penRdExLinear, penRfExLinear, costMinConst, costMinLinear, costFloorConst, costFloorLinear, nCeilConst, nCeilLinear, nCatPair, multiseedMms, multiseedLen, multiseedIvalType, multiseedIvalA, multiseedIvalB, posmin); break; } case -1: break; default: { cerr << "Unknown option: " << (char)next_option << endl; printUsage(cerr); exit(1); } } } while(next_option != -1); srand(seed); if(argc - optind < 4) { cerr << "Need at least 4 arguments" << endl; printUsage(cerr); exit(1); } BTDnaString read; BTString ref, qual; // Get read read.installChars(argv[optind]); // Get qualities qual.install(argv[optind+1]); assert_eq(read.length(), qual.length()); // Get reference ref.install(argv[optind+2]); // Get reference offset size_t off = parse(argv[optind+3]); // Set up penalities Scoring sc( false, // local alignment? false, // bad homopolymer? bonusMatchType, bonusMatch, penMmcType, // how to penalize mismatches penMmc, // constant if mm pelanty is a constant costMinConst, costMinLinear, costFloorConst, costFloorLinear, nCeilConst, // N ceiling constant coefficient nCeilLinear, // N ceiling linear coefficient penNType, // how to penalize Ns in the read penN, // constant if N pelanty is a constant nCatPair, // true -> concatenate mates before N filtering penRdExConst, // constant cost of extending a gap in the read penRfExConst, // constant cost of extending a gap in the reference penRdExLinear, // coeff of linear term for cost of gap extension in read penRfExLinear // coeff of linear term for cost of gap extension in ref ); // Calculate the penalty ceiling for the read TAlScore minsc = Scoring::linearFunc( read.length(), costMinConst, costMinLinear); TAlScore floorsc = Scoring::linearFunc( read.length(), costFloorConst, costFloorLinear); SwResult res; SwAligner al; doTestCase( al, read, qual, ref, off, NULL, sc, minsc, res, nsInclusive, nfilter, seed); } #endif /*MAIN_ALIGNER_SW*/ hisat2-2.2.1/aligner_sw.h000066400000000000000000000615741370663763200152430ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /* * aligner_sw.h * * Classes and routines for solving dynamic programming problems in aid of read * alignment. Goals include the ability to handle: * * - Both read alignment, where the query must align end-to-end, and local * alignment, where we seek a high-scoring alignment that need not involve * the entire query. * - Situations where: (a) we've found a seed hit and are trying to extend it * into a larger hit, (b) we've found an alignment for one mate of a pair and * are trying to find a nearby alignment for the other mate, (c) we're * aligning against an entire reference sequence. * - Caller-specified indicators for what columns of the dynamic programming * matrix we are allowed to start in or end in. * * TODO: * * - A slicker way to filter out alignments that violate a ceiling placed on * the number of Ns permitted in the reference portion of the alignment. * Right now we accomplish this by masking out ending columns that correspond * to *ungapped* alignments with too many Ns. This results in false * positives and false negatives for gapped alignments. The margin of error * (# of Ns by which we might miscount) is bounded by the number of gaps. */ /** * |-maxgaps-| * ***********oooooooooooooooooooooo - * ***********ooooooooooooooooooooo | * ***********oooooooooooooooooooo | * ***********ooooooooooooooooooo | * ***********oooooooooooooooooo | * ***********ooooooooooooooooo read len * ***********oooooooooooooooo | * ***********ooooooooooooooo | * ***********oooooooooooooo | * ***********ooooooooooooo | * ***********oooooooooooo - * |-maxgaps-| * |-readlen-| * |-------skip--------| */ #ifndef ALIGNER_SW_H_ #define ALIGNER_SW_H_ #define INLINE_CUPS #include #include #include #include "threading.h" #include #include "aligner_sw_common.h" #include "aligner_sw_nuc.h" #include "ds.h" #include "aligner_seed.h" #include "reference.h" #include "random_source.h" #include "mem_ids.h" #include "aligner_result.h" #include "mask.h" #include "dp_framer.h" #include "aligner_swsse.h" #include "aligner_bt.h" #define QUAL2(d, f) sc_->mm((int)(*rd_)[rdi_ + d], \ (int) rf_ [rfi_ + f], \ (int)(*qu_)[rdi_ + d] - 33) #define QUAL(d) sc_->mm((int)(*rd_)[rdi_ + d], \ (int)(*qu_)[rdi_ + d] - 33) #define N_SNP_PEN(c) (((int)rf_[rfi_ + c] > 15) ? sc_->n(30) : sc_->penSnp) /** * SwAligner * ========= * * Ensapsulates facilities for alignment using dynamic programming. Handles * alignment of nucleotide reads against known reference nucleotides. * * The class is stateful. First the user must call init() to initialize the * object with details regarding the dynamic programming problem to be solved. * Next, the user calls align() to fill the dynamic programming matrix and * calculate summaries describing the solutions. Finally the user calls * nextAlignment(...), perhaps repeatedly, to populate the SwResult object with * the next result. Results are dispensend in best-to-worst, left-to-right * order. * * The class expects the read string, quality string, and reference string * provided by the caller live at least until the user is finished aligning and * obtaining alignments from this object. * * There is a design tradeoff between hiding/exposing details of the genome and * its strands to the SwAligner. In a sense, a better design is to hide * details such as the id of the reference sequence aligned to, or whether * we're aligning the read in its original forward orientation or its reverse * complement. But this means that any alignment results returned by SwAligner * have to be extended to include those details before they're useful to the * caller. We opt for messy but expedient - the reference id and orientation * of the read are given to SwAligner, remembered, and used to populate * SwResults. * * LOCAL VS GLOBAL * * The dynamic programming aligner supports both local and global alignment, * and one option in between. To implement global alignment, the aligner (a) * allows negative scores (i.e. doesn't necessarily clamp them up to 0), (b) * checks in rows other than the last row for acceptable solutions, and (c) * optionally adds a bonus to the score for matches. * * For global alignment, we: * * (a) Allow negative scores * (b) Check only in the last row * (c) Either add a bonus for matches or not (doesn't matter) * * For local alignment, we: * * (a) Clamp scores to 0 * (b) Check in any row for a sufficiently high score * (c) Add a bonus for matches * * An in-between solution is to allow alignments to be curtailed on the * right-hand side if a better score can be achieved thereby, but not on the * left. For this, we: * * (a) Allow negative scores * (b) Check in any row for a sufficiently high score * (c) Either add a bonus for matches or not (doesn't matter) * * REDUNDANT ALIGNMENTS * * When are two alignments distinct and when are they redundant (not distinct)? * At one extreme, we might say the best alignment from any given dynamic * programming problem is redundant with all other alignments from that # problem. At the other extreme, we might say that any two alignments with * distinct starting points and edits are distinct. The former is probably too * conservative for mate-finding DP problems. The latter is certainly too * permissive, since two alignments that differ only in how gaps are arranged * should not be considered distinct. * * Some in-between solutions are: * * (a) If two alignments share an end point on either end, they are redundant. * Otherwise, they are distinct. * (b) If two alignments share *both* end points, they are redundant. * (c) If two alignments share any cells in the DP table, they are redundant. * (d) 2 alignments are redundant if either end within N poss of each other * (e) Like (d) but both instead of either * (f, g) Like d, e, but where N is tied to maxgaps somehow * * Why not (a)? One reason is that it's possible for two alignments to have * different start & end positions but share many cells. Consider alignments 1 * and 2 below; their end-points are labeled. * * 1 2 * \ \ * -\ * \ * \ * \ * -\ * \ \ * 1 2 * * 1 and 2 are distinct according to (a) but they share many cells in common. * * Why not (f, g)? It fixes the problem with (a) above by forcing the * alignments to be spread so far that they can't possibly share diagonal cells * in common */ class SwAligner { typedef std::pair SizeTPair; // States that the aligner can be in enum { STATE_UNINIT, // init() hasn't been called yet STATE_INITED, // init() has been called, but not align() STATE_ALIGNED, // align() has been called }; const static size_t ALPHA_SIZE = 5; public: explicit SwAligner() : sseU8fw_(DP_CAT), sseU8rc_(DP_CAT), sseI16fw_(DP_CAT), sseI16rc_(DP_CAT), state_(STATE_UNINIT), initedRead_(false), readSse16_(false), initedRef_(false), rfwbuf_(DP_CAT), btnstack_(DP_CAT), btcells_(DP_CAT), btdiag_(), btncand_(DP_CAT), btncanddone_(DP_CAT), btncanddoneSucc_(0), btncanddoneFail_(0), cper_(), cperMinlen_(), cperPerPow2_(), cperEf_(), cperTri_(), colstop_(0), lastsolcol_(0), cural_(0) ASSERT_ONLY(, cand_tmp_(DP_CAT)) { } /** * Prepare the dynamic programming driver with a new read and a new scoring * scheme. */ void initRead( const BTDnaString& rdfw, // read sequence for fw read const BTDnaString& rdrc, // read sequence for rc read const BTString& qufw, // read qualities for fw read const BTString& qurc, // read qualities for rc read size_t rdi, // offset of first read char to align size_t rdf, // offset of last read char to align const Scoring& sc); // scoring scheme /** * Initialize with a new alignment problem. */ void initRef( bool fw, // whether to forward or revcomp read is aligning TRefId refidx, // id of reference aligned against const DPRect& rect, // DP rectangle char *rf, // reference sequence size_t rfi, // offset of first reference char to align to size_t rff, // offset of last reference char to align to TRefOff reflen, // length of reference sequence const Scoring& sc, // scoring scheme TAlScore minsc, // minimum score bool enable8, // use 8-bit SSE if possible? size_t cminlen, // minimum length for using checkpointing scheme size_t cpow2, // interval b/t checkpointed diags; 1 << this bool doTri, // triangular mini-fills? bool extend); // true iff this is a seed extension /** * Given a read, an alignment orientation, a range of characters in a * referece sequence, and a bit-encoded version of the reference, * execute the corresponding dynamic programming problem. * * Here we expect that the caller has already narrowed down the relevant * portion of the reference (e.g. using a seed hit) and all we do is * banded dynamic programming in the vicinity of that portion. This is not * the function to call if we are trying to solve the whole alignment * problem with dynamic programming (that is TODO). * * Returns true if an alignment was found, false otherwise. */ void initRef( bool fw, // whether to forward or revcomp read aligned TRefId refidx, // reference aligned against const DPRect& rect, // DP rectangle const BitPairReference& refs, // Reference strings TRefOff reflen, // length of reference sequence const Scoring& sc, // scoring scheme TAlScore minsc, // minimum alignment score bool enable8, // use 8-bit SSE if possible? size_t cminlen, // minimum length for using checkpointing scheme size_t cpow2, // interval b/t checkpointed diags; 1 << this bool doTri, // triangular mini-fills? bool extend, // true iff this is a seed extension size_t upto, // count the number of Ns up to this offset size_t& nsUpto); // output: the number of Ns up to 'upto' /** * Given a read, an alignment orientation, a range of characters in a * referece sequence, and a bit-encoded version of the reference, set up * and execute the corresponding ungapped alignment problem. There can * only be one solution. * * The caller has already narrowed down the relevant portion of the * reference using, e.g., the location of a seed hit, or the range of * possible fragment lengths if we're searching for the opposite mate in a * pair. */ int ungappedAlign( const BTDnaString& rd, // read sequence (could be RC) const BTString& qu, // qual sequence (could be rev) const Coord& coord, // coordinate aligned to const BitPairReference& refs, // Reference strings size_t reflen, // length of reference sequence const Scoring& sc, // scoring scheme bool ohang, // allow overhang? TAlScore minsc, // minimum score SwResult& res); // put alignment result here /** * Align read 'rd' to reference using read & reference information given * last time init() was called. Uses dynamic programming. */ bool align(RandomSource& rnd, TAlScore& best); /** * Populate the given SwResult with information about the "next best" * alignment if there is one. If there isn't one, false is returned. Note * that false might be returned even though a call to done() would have * returned false. */ bool nextAlignment( SwResult& res, TAlScore minsc, RandomSource& rnd); /** * Print out an alignment result as an ASCII DP table. */ void printResultStacked( const SwResult& res, std::ostream& os) { res.alres.printStacked(*rd_, os); } /** * Return true iff there are no more solution cells to backtace from. * Note that this may return false in situations where there are actually * no more solutions, but that hasn't been discovered yet. */ bool done() const { assert(initedRead() && initedRef()); return cural_ == btncand_.size(); } /** * Return true iff this SwAligner has been initialized with a read to align. */ inline bool initedRef() const { return initedRef_; } /** * Return true iff this SwAligner has been initialized with a reference to * align against. */ inline bool initedRead() const { return initedRead_; } /** * Reset, signaling that we're done with this dynamic programming problem * and won't be asking for any more alignments. */ inline void reset() { initedRef_ = initedRead_ = false; } #ifndef NDEBUG /** * Check that aligner is internally consistent. */ bool repOk() const { assert_gt(dpRows(), 0); // Check btncand_ for(size_t i = 0; i < btncand_.size(); i++) { assert(btncand_[i].repOk()); assert_geq(btncand_[i].score, minsc_); } return true; } #endif /** * Return the number of alignments given out so far by nextAlignment(). */ size_t numAlignmentsReported() const { return cural_; } /** * Merge tallies in the counters related to filling the DP table. */ void merge( SSEMetrics& sseU8ExtendMet, SSEMetrics& sseU8MateMet, SSEMetrics& sseI16ExtendMet, SSEMetrics& sseI16MateMet, uint64_t& nbtfiltst, uint64_t& nbtfiltsc, uint64_t& nbtfiltdo) { sseU8ExtendMet.merge(sseU8ExtendMet_); sseU8MateMet.merge(sseU8MateMet_); sseI16ExtendMet.merge(sseI16ExtendMet_); sseI16MateMet.merge(sseI16MateMet_); nbtfiltst += nbtfiltst_; nbtfiltsc += nbtfiltsc_; nbtfiltdo += nbtfiltdo_; } /** * Reset all the counters related to filling in the DP table to 0. */ void resetCounters() { sseU8ExtendMet_.reset(); sseU8MateMet_.reset(); sseI16ExtendMet_.reset(); sseI16MateMet_.reset(); nbtfiltst_ = nbtfiltsc_ = nbtfiltdo_ = 0; } /** * Return the size of the DP problem. */ size_t size() const { return dpRows() * (rff_ - rfi_); } protected: /** * Return the number of rows that will be in the dynamic programming table. */ inline size_t dpRows() const { assert(initedRead_); return rdf_ - rdi_; } /** * Align nucleotides from read 'rd' to the reference string 'rf' using * vector instructions. Return the score of the best alignment found, or * the minimum integer if an alignment could not be found. Flag is set to * 0 if an alignment is found, -1 if no valid alignment is found, or -2 if * the score saturated at any point during alignment. */ TAlScore alignNucleotidesEnd2EndSseU8( // unsigned 8-bit elements int& flag, bool debug); TAlScore alignNucleotidesLocalSseU8( // unsigned 8-bit elements int& flag, bool debug); TAlScore alignNucleotidesEnd2EndSseI16( // signed 16-bit elements int& flag, bool debug); TAlScore alignNucleotidesLocalSseI16( // signed 16-bit elements int& flag, bool debug); /** * Aligns by filling a dynamic programming matrix with the SSE-accelerated, * banded DP approach of Farrar. As it goes, it determines which cells we * might backtrace from and tallies the best (highest-scoring) N backtrace * candidate cells per diagonal. Also returns the alignment score of the best * alignment in the matrix. * * This routine does *not* maintain a matrix holding the entire matrix worth of * scores, nor does it maintain any other dense O(mn) data structure, as this * would quickly exhaust memory for queries longer than about 10,000 kb. * Instead, in the fill stage it maintains two columns worth of scores at a * time (current/previous, or right/left) - these take O(m) space. When * finished with the current column, it determines which cells from the * previous column, if any, are candidates we might backtrace from to find a * full alignment. A candidate cell has a score that rises above the threshold * and isn't improved upon by a match in the next column. The best N * candidates per diagonal are stored in a O(m + n) data structure. */ TAlScore alignGatherEE8( // unsigned 8-bit elements int& flag, bool debug); TAlScore alignGatherLoc8( // unsigned 8-bit elements int& flag, bool debug); TAlScore alignGatherEE16( // signed 16-bit elements int& flag, bool debug); TAlScore alignGatherLoc16( // signed 16-bit elements int& flag, bool debug); /** * Build query profile look up tables for the read. The query profile look * up table is organized as a 1D array indexed by [i][j] where i is the * reference character in the current DP column (0=A, 1=C, etc), and j is * the segment of the query we're currently working on. */ void buildQueryProfileEnd2EndSseU8(bool fw); void buildQueryProfileLocalSseU8(bool fw); /** * Build query profile look up tables for the read. The query profile look * up table is organized as a 1D array indexed by [i][j] where i is the * reference character in the current DP column (0=A, 1=C, etc), and j is * the segment of the query we're currently working on. */ void buildQueryProfileEnd2EndSseI16(bool fw); void buildQueryProfileLocalSseI16(bool fw); bool gatherCellsNucleotidesLocalSseU8(TAlScore best); bool gatherCellsNucleotidesEnd2EndSseU8(TAlScore best); bool gatherCellsNucleotidesLocalSseI16(TAlScore best); bool gatherCellsNucleotidesEnd2EndSseI16(TAlScore best); bool backtraceNucleotidesLocalSseU8( TAlScore escore, // in: expected score SwResult& res, // out: store results (edits and scores) here size_t& off, // out: store diagonal projection of origin size_t& nbts, // out: # backtracks size_t row, // start in this rectangle row size_t col, // start in this rectangle column RandomSource& rand); // random gen, to choose among equal paths bool backtraceNucleotidesLocalSseI16( TAlScore escore, // in: expected score SwResult& res, // out: store results (edits and scores) here size_t& off, // out: store diagonal projection of origin size_t& nbts, // out: # backtracks size_t row, // start in this rectangle row size_t col, // start in this rectangle column RandomSource& rand); // random gen, to choose among equal paths bool backtraceNucleotidesEnd2EndSseU8( TAlScore escore, // in: expected score SwResult& res, // out: store results (edits and scores) here size_t& off, // out: store diagonal projection of origin size_t& nbts, // out: # backtracks size_t row, // start in this rectangle row size_t col, // start in this rectangle column RandomSource& rand); // random gen, to choose among equal paths bool backtraceNucleotidesEnd2EndSseI16( TAlScore escore, // in: expected score SwResult& res, // out: store results (edits and scores) here size_t& off, // out: store diagonal projection of origin size_t& nbts, // out: # backtracks size_t row, // start in this rectangle row size_t col, // start in this rectangle column RandomSource& rand); // random gen, to choose among equal paths bool backtrace( TAlScore escore, // in: expected score bool fill, // in: use mini-fill? bool usecp, // in: use checkpoints? SwResult& res, // out: store results (edits and scores) here size_t& off, // out: store diagonal projection of origin size_t row, // start in this rectangle row size_t col, // start in this rectangle column size_t maxiter,// max # extensions to try size_t& niter, // # extensions tried RandomSource& rnd) // random gen, to choose among equal paths { bter_.initBt( escore, // in: alignment score row, // in: start in this row col, // in: start in this column fill, // in: use mini-fill? usecp, // in: use checkpoints? cperTri_, // in: triangle-shaped mini-fills? rnd); // in: random gen, to choose among equal paths assert(bter_.inited()); size_t nrej = 0; if(bter_.emptySolution()) { return false; } else { return bter_.nextAlignment(maxiter, res, off, nrej, niter, rnd); } } const BTDnaString *rd_; // read sequence const BTString *qu_; // read qualities const BTDnaString *rdfw_; // read sequence for fw read const BTDnaString *rdrc_; // read sequence for rc read const BTString *qufw_; // read qualities for fw read const BTString *qurc_; // read qualities for rc read TReadOff rdi_; // offset of first read char to align TReadOff rdf_; // offset of last read char to align bool fw_; // true iff read sequence is original fw read TRefId refidx_; // id of reference aligned against TRefOff reflen_; // length of entire reference sequence const DPRect* rect_; // DP rectangle char *rf_; // reference sequence TRefOff rfi_; // offset of first ref char to align to TRefOff rff_; // offset of last ref char to align to (excl) size_t rdgap_; // max # gaps in read size_t rfgap_; // max # gaps in reference bool enable8_;// enable 8-bit sse bool extend_; // true iff this is a seed-extend problem const Scoring *sc_; // penalties for edit types TAlScore minsc_; // penalty ceiling for valid alignments int nceil_; // max # Ns allowed in ref portion of aln bool sse8succ_; // whether 8-bit worked bool sse16succ_; // whether 16-bit worked SSEData sseU8fw_; // buf for fw query, 8-bit score SSEData sseU8rc_; // buf for rc query, 8-bit score SSEData sseI16fw_; // buf for fw query, 16-bit score SSEData sseI16rc_; // buf for rc query, 16-bit score bool sseU8fwBuilt_; // built fw query profile, 8-bit score bool sseU8rcBuilt_; // built rc query profile, 8-bit score bool sseI16fwBuilt_; // built fw query profile, 16-bit score bool sseI16rcBuilt_; // built rc query profile, 16-bit score SSEMetrics sseU8ExtendMet_; SSEMetrics sseU8MateMet_; SSEMetrics sseI16ExtendMet_; SSEMetrics sseI16MateMet_; int state_; // state bool initedRead_; // true iff initialized with initRead bool readSse16_; // true -> sse16 from now on for read bool initedRef_; // true iff initialized with initRef EList rfwbuf_; // buffer for wordized ref stretches EList btnstack_; // backtrace stack for nucleotides EList btcells_; // cells involved in current backtrace NBest btdiag_; // per-diagonal backtrace candidates EList btncand_; // cells we might backtrace from EList btncanddone_; // candidates that we investigated size_t btncanddoneSucc_; // # investigated and succeeded size_t btncanddoneFail_; // # investigated and failed BtBranchTracer bter_; // backtracer Checkpointer cper_; // structure for saving checkpoint cells size_t cperMinlen_; // minimum length for using checkpointer size_t cperPerPow2_; // checkpoint every 1 << perpow2 diags (& next) bool cperEf_; // store E and F in addition to H? bool cperTri_; // checkpoint for triangular mini-fills? size_t colstop_; // bailed on DP loop after this many cols size_t lastsolcol_; // last DP col with valid cell size_t cural_; // index of next alignment to be given uint64_t nbtfiltst_; // # candidates filtered b/c starting cell was seen uint64_t nbtfiltsc_; // # candidates filtered b/c score uninteresting uint64_t nbtfiltdo_; // # candidates filtered b/c dominated by other cell ASSERT_ONLY(SStringExpandable tmp_destU32_); ASSERT_ONLY(BTDnaString tmp_editstr_, tmp_refstr_); ASSERT_ONLY(EList cand_tmp_); }; #endif /*ALIGNER_SW_H_*/ hisat2-2.2.1/aligner_sw_common.h000066400000000000000000000210361370663763200166000ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_SW_COMMON_H_ #define ALIGNER_SW_COMMON_H_ #include "aligner_result.h" /** * Encapsulates the result of a dynamic programming alignment, including * colorspace alignments. In our case, the result is a combination of: * * 1. All the nucleotide edits * 2. All the "edits" where an ambiguous reference char is resolved to * an unambiguous char. * 3. All the color edits (if applicable) * 4. All the color miscalls (if applicable). This is a subset of 3. * 5. The score of the best alginment * 6. The score of the second-best alignment * * Having scores for the best and second-best alignments gives us an * idea of where gaps may make reassembly beneficial. */ struct SwResult { SwResult() : alres(), sws(0), swcups(0), swrows(0), swskiprows(0), swskip(0), swsucc(0), swfail(0), swbts(0) { } /** * Clear all contents. */ void reset() { sws = swcups = swrows = swskiprows = swskip = swsucc = swfail = swbts = 0; alres.reset(); } /** * Reverse all edit lists. */ void reverse() { alres.reverseEdits(); } /** * Return true iff no result has been installed. */ bool empty() const { return alres.empty(); } #ifndef NDEBUG /** * Check that result is internally consistent. */ bool repOk() const { assert(alres.repOk()); return true; } /** * Check that result is internally consistent w/r/t read. */ bool repOk(const Read& rd) const { assert(alres.repOk(rd)); return true; } #endif AlnRes alres; uint64_t sws; // # DP problems solved uint64_t swcups; // # DP cell updates uint64_t swrows; // # DP row updates uint64_t swskiprows; // # skipped DP row updates (b/c no valid alignments can go thru row) uint64_t swskip; // # DP problems skipped by sse filter uint64_t swsucc; // # DP problems resulting in alignment uint64_t swfail; // # DP problems not resulting in alignment uint64_t swbts; // # DP backtrace steps int nup; // upstream decoded nucleotide; for colorspace reads int ndn; // downstream decoded nucleotide; for colorspace reads }; /** * Encapsulates counters that measure how much work has been done by * the dynamic programming driver and aligner. */ struct SwMetrics { SwMetrics() : mutex_m() { reset(); } void reset() { sws = swcups = swrows = swskiprows = swskip = swsucc = swfail = swbts = sws10 = sws5 = sws3 = rshit = ungapsucc = ungapfail = ungapnodec = 0; exatts = exranges = exrows = exsucc = exooms = 0; mm1atts = mm1ranges = mm1rows = mm1succ = mm1ooms = 0; sdatts = sdranges = sdrows = sdsucc = sdooms = 0; } void init( uint64_t sws_, uint64_t sws10_, uint64_t sws5_, uint64_t sws3_, uint64_t swcups_, uint64_t swrows_, uint64_t swskiprows_, uint64_t swskip_, uint64_t swsucc_, uint64_t swfail_, uint64_t swbts_, uint64_t rshit_, uint64_t ungapsucc_, uint64_t ungapfail_, uint64_t ungapnodec_, uint64_t exatts_, uint64_t exranges_, uint64_t exrows_, uint64_t exsucc_, uint64_t exooms_, uint64_t mm1atts_, uint64_t mm1ranges_, uint64_t mm1rows_, uint64_t mm1succ_, uint64_t mm1ooms_, uint64_t sdatts_, uint64_t sdranges_, uint64_t sdrows_, uint64_t sdsucc_, uint64_t sdooms_) { sws = sws_; sws10 = sws10_; sws5 = sws5_; sws3 = sws3_; swcups = swcups_; swrows = swrows_; swskiprows = swskiprows_; swskip = swskip_; swsucc = swsucc_; swfail = swfail_; swbts = swbts_; ungapsucc = ungapsucc_; ungapfail = ungapfail_; ungapnodec = ungapnodec_; // Exact end-to-end attempts exatts = exatts_; exranges = exranges_; exrows = exrows_; exsucc = exsucc_; exooms = exooms_; // 1-mismatch end-to-end attempts mm1atts = mm1atts_; mm1ranges = mm1ranges_; mm1rows = mm1rows_; mm1succ = mm1succ_; mm1ooms = mm1ooms_; // Seed attempts sdatts = sdatts_; sdranges = sdranges_; sdrows = sdrows_; sdsucc = sdsucc_; sdooms = sdooms_; } /** * Merge (add) the counters in the given SwResult object into this * SwMetrics object. */ void update(const SwResult& r) { sws += r.sws; swcups += r.swcups; swrows += r.swrows; swskiprows += r.swskiprows; swskip += r.swskip; swsucc += r.swsucc; swfail += r.swfail; swbts += r.swbts; } /** * Merge (add) the counters in the given SwMetrics object into this * object. This is the only safe way to update a SwMetrics shared * by multiple threads. */ void merge(const SwMetrics& r, bool getLock = false) { ThreadSafe ts(&mutex_m, getLock); sws += r.sws; sws10 += r.sws10; sws5 += r.sws5; sws3 += r.sws3; swcups += r.swcups; swrows += r.swrows; swskiprows += r.swskiprows; swskip += r.swskip; swsucc += r.swsucc; swfail += r.swfail; swbts += r.swbts; rshit += r.rshit; ungapsucc += r.ungapsucc; ungapfail += r.ungapfail; ungapnodec += r.ungapnodec; exatts += r.exatts; exranges += r.exranges; exrows += r.exrows; exsucc += r.exsucc; exooms += r.exooms; mm1atts += r.mm1atts; mm1ranges += r.mm1ranges; mm1rows += r.mm1rows; mm1succ += r.mm1succ; mm1ooms += r.mm1ooms; sdatts += r.sdatts; sdranges += r.sdranges; sdrows += r.sdrows; sdsucc += r.sdsucc; sdooms += r.sdooms; } void tallyGappedDp(size_t readGaps, size_t refGaps) { size_t mx = max(readGaps, refGaps); if(mx < 10) sws10++; if(mx < 5) sws5++; if(mx < 3) sws3++; } uint64_t sws; // # DP problems solved uint64_t sws10; // # DP problems solved where max gaps < 10 uint64_t sws5; // # DP problems solved where max gaps < 5 uint64_t sws3; // # DP problems solved where max gaps < 3 uint64_t swcups; // # DP cell updates uint64_t swrows; // # DP row updates uint64_t swskiprows; // # skipped DP rows (b/c no valid alns go thru row) uint64_t swskip; // # DP problems skipped by sse filter uint64_t swsucc; // # DP problems resulting in alignment uint64_t swfail; // # DP problems not resulting in alignment uint64_t swbts; // # DP backtrace steps uint64_t rshit; // # DP problems avoided b/c seed hit was redundant uint64_t ungapsucc; // # DP problems avoided b/c seed hit was redundant uint64_t ungapfail; // # DP problems avoided b/c seed hit was redundant uint64_t ungapnodec; // # DP problems avoided b/c seed hit was redundant uint64_t exatts; // total # attempts at exact-hit end-to-end aln uint64_t exranges; // total # ranges returned by exact-hit queries uint64_t exrows; // total # rows returned by exact-hit queries uint64_t exsucc; // exact-hit yielded non-empty result uint64_t exooms; // exact-hit offset memory exhausted uint64_t mm1atts; // total # attempts at 1mm end-to-end aln uint64_t mm1ranges; // total # ranges returned by 1mm-hit queries uint64_t mm1rows; // total # rows returned by 1mm-hit queries uint64_t mm1succ; // 1mm-hit yielded non-empty result uint64_t mm1ooms; // 1mm-hit offset memory exhausted uint64_t sdatts; // total # attempts to find seed alignments uint64_t sdranges; // total # seed-alignment ranges found uint64_t sdrows; // total # seed-alignment rows found uint64_t sdsucc; // # times seed alignment yielded >= 1 hit uint64_t sdooms; // # times an OOM occurred during seed alignment MUTEX_T mutex_m; }; // The various ways that one might backtrack from a later cell (either oall, // rdgap or rfgap) to an earlier cell enum { SW_BT_OALL_DIAG, // from oall cell to oall cell SW_BT_OALL_REF_OPEN, // from oall cell to oall cell SW_BT_OALL_READ_OPEN, // from oall cell to oall cell SW_BT_RDGAP_EXTEND, // from rdgap cell to rdgap cell SW_BT_RFGAP_EXTEND // from rfgap cell to rfgap cell }; #endif /*def ALIGNER_SW_COMMON_H_*/ hisat2-2.2.1/aligner_sw_driver.cpp000066400000000000000000000013401370663763200171320ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ hisat2-2.2.1/aligner_sw_driver.h000066400000000000000000003236331370663763200166130ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /* * aligner_sw_driver.h * * REDUNDANT SEED HITS * * We say that two seed hits are redundant if they trigger identical * seed-extend dynamic programming problems. Put another way, they both lie on * the same diagonal of the overall read/reference dynamic programming matrix. * Detecting redundant seed hits is simple when the seed hits are ungapped. We * do this after offset resolution but before the offset is converted to genome * coordinates (see uses of the seenDiags1_/seenDiags2_ fields for examples). * * REDUNDANT ALIGNMENTS * * In an unpaired context, we say that two alignments are redundant if they * share any cells in the global DP table. Roughly speaking, this is like * saying that two alignments are redundant if any read character aligns to the * same reference character (same reference sequence, same strand, same offset) * in both alignments. * * In a paired-end context, we say that two paired-end alignments are redundant * if the mate #1s are redundant and the mate #2s are redundant. * * How do we enforce this? In the unpaired context, this is relatively simple: * the cells from each alignment are checked against a set containing all cells * from all previous alignments. Given a new alignment, for each cell in the * new alignment we check whether it is in the set. If there is any overlap, * the new alignment is rejected as redundant. Otherwise, the new alignment is * accepted and its cells are added to the set. * * Enforcement in a paired context is a little trickier. Consider the * following approaches: * * 1. Skip anchors that are redundant with any previous anchor or opposite * alignment. This is sufficient to ensure no two concordant alignments * found are redundant. * * 2. Same as scheme 1, but with a "transitive closure" scheme for finding all * concordant pairs in the vicinity of an anchor. Consider the AB/AC * scenario from the previous paragraph. If B is the anchor alignment, we * will find AB but not AC. But under this scheme, once we find AB we then * let B be a new anchor and immediately look for its opposites. Likewise, * if we find any opposite, we make them anchors and continue searching. We * don't stop searching until every opposite is used as an anchor. * * 3. Skip anchors that are redundant with any previous anchor alignment (but * allow anchors that are redundant with previous opposite alignments). * This isn't sufficient to avoid redundant concordant alignments. To avoid * redundant concordants, we need an additional procedure that checks each * new concordant alignment one-by-one against a list of previous concordant * alignments to see if it is redundant. * * We take approach 1. */ #ifndef ALIGNER_SW_DRIVER_H_ #define ALIGNER_SW_DRIVER_H_ #include // -- BTL remove -- #include #include // -- -- #include #include "ds.h" #include "aligner_seed.h" #include "aligner_sw.h" #include "aligner_cache.h" #include "reference.h" #include "group_walk.h" #include "gfm.h" #include "mem_ids.h" #include "aln_sink.h" #include "pe.h" #include "ival_list.h" #include "simple_func.h" #include "random_util.h" #include "dp_framer.h" using namespace std; template struct SeedPos { SeedPos() : fw(false), offidx(0), rdoff(0), seedlen(0) { } SeedPos( bool fw_, index_t offidx_, index_t rdoff_, index_t seedlen_) { init(fw_, offidx_, rdoff_, seedlen_); } void init( bool fw_, index_t offidx_, index_t rdoff_, index_t seedlen_) { fw = fw_; offidx = offidx_; rdoff = rdoff_; seedlen = seedlen_; } bool operator<(const SeedPos& o) const { if(offidx < o.offidx) return true; if(offidx > o.offidx) return false; if(rdoff < o.rdoff) return true; if(rdoff > o.rdoff) return false; if(seedlen < o.seedlen) return true; if(seedlen > o.seedlen) return false; if(fw && !o.fw) return true; if(!fw && o.fw) return false; return false; } bool operator>(const SeedPos& o) const { if(offidx < o.offidx) return false; if(offidx > o.offidx) return true; if(rdoff < o.rdoff) return false; if(rdoff > o.rdoff) return true; if(seedlen < o.seedlen) return false; if(seedlen > o.seedlen) return true; if(fw && !o.fw) return false; if(!fw && o.fw) return true; return false; } bool operator==(const SeedPos& o) const { return fw == o.fw && offidx == o.offidx && rdoff == o.rdoff && seedlen == o.seedlen; } bool fw; index_t offidx; index_t rdoff; index_t seedlen; }; /** * An SATuple along with the associated seed position. */ template struct SATupleAndPos { SATuple sat; // result for this seed hit SeedPos pos; // seed position that yielded the range this was taken from index_t origSz; // size of range this was taken from index_t nlex; // # position we can extend seed hit to left w/o edit index_t nrex; // # position we can extend seed hit to right w/o edit bool operator<(const SATupleAndPos& o) const { if(sat < o.sat) return true; if(sat > o.sat) return false; return pos < o.pos; } bool operator==(const SATupleAndPos& o) const { return sat == o.sat && pos == o.pos; } }; /** * Encapsulates the weighted random sampling scheme we want to use to pick * which seed hit range to sample a row from. */ template class RowSampler { public: RowSampler(int cat = 0) : elim_(cat), masses_(cat) { mass_ = 0.0f; } /** * Initialze sampler with respect to a range of elements in a list of * SATupleAndPos's. */ void init( const EList, 16>& salist, index_t sai, index_t saf, bool lensq, // whether to square the numerator, which = extended length bool szsq) // whether to square denominator, which = { assert_gt(saf, sai); elim_.resize(saf - sai); elim_.fill(false); // Initialize mass mass_ = 0.0f; masses_.resize(saf - sai); for(index_t i = sai; i < saf; i++) { index_t len = salist[i].nlex + salist[i].nrex + 1; // + salist[i].sat.key.len; double num = (double)len; if(lensq) { num *= num; } double denom = (double)salist[i].sat.size(); if(szsq) { denom *= denom; } masses_[i - sai] = num / denom; mass_ += masses_[i - sai]; } } /** * Caller is indicating that the bin at index i is exhausted and we should * exclude it from our sampling from now on. */ void finishedRange(index_t i) { assert_lt(i, masses_.size()); elim_[i] = true; mass_ -= masses_[i]; } /** * Sample randomly from the mass. */ size_t next(RandomSource& rnd) { // Throw the dart double rd = rnd.nextFloat() * mass_; double mass_sofar = 0.0f; size_t sz = masses_.size(); size_t last_unelim = std::numeric_limits::max(); for(size_t i = 0; i < sz; i++) { if(!elim_[i]) { last_unelim = i; mass_sofar += masses_[i]; if(rd < mass_sofar) { // This is the one we hit return i; } } } assert_neq(std::numeric_limits::max(), last_unelim); return last_unelim; } protected: double mass_; // total probability mass to throw darts at EList elim_; // whether the range is eliminated EList masses_; // mass of each range }; /** * Return values from extendSeeds and extendSeedsPaired. */ enum { // All end-to-end and seed hits were examined // The policy does not need us to look any further EXTEND_EXHAUSTED_CANDIDATES = 1, EXTEND_POLICY_FULFILLED, // We stopped because we reached a point where the only remaining // alignments of interest have perfect scores, but we already investigated // perfect alignments EXTEND_PERFECT_SCORE, // We stopped because we ran up against a limit on how much work we should // do for one set of seed ranges, e.g. the limit on number of consecutive // unproductive DP extensions EXTEND_EXCEEDED_SOFT_LIMIT, // We stopped because we ran up against a limit on how much work we should // do for overall before giving up on a mate EXTEND_EXCEEDED_HARD_LIMIT }; /** * Data structure encapsulating a range that's been extended out in two * directions. */ struct ExtendRange { void init(size_t off_, size_t len_, size_t sz_) { off = off_; len = len_; sz = sz_; } size_t off; // offset of extended region size_t len; // length between extremes of extended region size_t sz; // # of elements in SA range }; template class SwDriver { typedef PList TSAList; public: SwDriver(size_t bytes) : satups_(DP_CAT), gws_(DP_CAT), seenDiags1_(DP_CAT), seenDiags2_(DP_CAT), redAnchor_(DP_CAT), redMate1_(DP_CAT), redMate2_(DP_CAT), pool_(bytes, CACHE_PAGE_SZ, DP_CAT), salistEe_(DP_CAT), gwstate_(GW_CAT) { } /** * Given a collection of SeedHits for a single read, extend seed alignments * into full alignments. Where possible, try to avoid redundant offset * lookups and dynamic programming problems. Optionally report alignments * to a AlnSinkWrap object as they are discovered. * * If 'reportImmediately' is true, returns true iff a call to * mhs->report() returned true (indicating that the reporting * policy is satisfied and we can stop). Otherwise, returns false. */ int extendSeeds( Read& rd, // read to align bool mate1, // true iff rd is mate #1 SeedResults& sh, // seed hits to extend into full alignments const GFM& gfmFw, // BWT const GFM* gfmBw, // BWT' const BitPairReference& ref, // Reference strings SwAligner& swa, // dynamic programming aligner const Scoring& sc, // scoring scheme int seedmms, // # mismatches allowed in seed int seedlen, // length of seed int seedival, // interval between seeds TAlScore& minsc, // minimum score for anchor int nceil, // maximum # Ns permitted in ref portion size_t maxhalf, // maximum width on one side of DP table bool doUngapped, // do ungapped alignment size_t maxIters, // stop after this many seed-extend loop iters size_t maxUg, // max # ungapped extends size_t maxDp, // max # DPs size_t maxUgStreak, // stop after streak of this many ungap fails size_t maxDpStreak, // stop after streak of this many dp fails bool doExtend, // do seed extension bool enable8, // use 8-bit SSE where possible size_t cminlen, // use checkpointer if read longer than this size_t cpow2, // interval between diagonals to checkpoint bool doTri, // triangular mini-fills int tighten, // -M score tightening mode AlignmentCacheIface& ca, // alignment cache for seed hits RandomSource& rnd, // pseudo-random source WalkMetrics& wlm, // group walk left metrics SwMetrics& swmSeed, // DP metrics for seed-extend PerReadMetrics& prm, // per-read metrics AlnSinkWrap* mhs, // HitSink for multiseed-style aligner bool reportImmediately, // whether to report hits immediately to mhs bool& exhaustive); /** * Given a collection of SeedHits for a read pair, extend seed * alignments into full alignments and then look for the opposite * mate using dynamic programming. Where possible, try to avoid * redundant offset lookups. Optionally report alignments to a * AlnSinkWrap object as they are discovered. * * If 'reportImmediately' is true, returns true iff a call to * mhs->report() returned true (indicating that the reporting * policy is satisfied and we can stop). Otherwise, returns false. */ int extendSeedsPaired( Read& rd, // mate to align as anchor Read& ord, // mate to align as opposite bool anchor1, // true iff anchor mate is mate1 bool oppFilt, // true iff opposite mate was filtered out SeedResults& sh, // seed hits for anchor const GFM& gfmFw, // BWT const GFM* gfmBw, // BWT' const BitPairReference& ref, // Reference strings SwAligner& swa, // dyn programming aligner for anchor SwAligner& swao, // dyn programming aligner for opposite const Scoring& sc, // scoring scheme const PairedEndPolicy& pepol,// paired-end policy int seedmms, // # mismatches allowed in seed int seedlen, // length of seed int seedival, // interval between seeds TAlScore& minsc, // minimum score for anchor TAlScore& ominsc, // minimum score for opposite int nceil, // max # Ns permitted in ref for anchor int onceil, // max # Ns permitted in ref for opposite bool nofw, // don't align forward read bool norc, // don't align revcomp read size_t maxhalf, // maximum width on one side of DP table bool doUngapped, // do ungapped alignment size_t maxIters, // stop after this many seed-extend loop iters size_t maxUg, // max # ungapped extends size_t maxDp, // max # DPs size_t maxEeStreak, // stop after streak of this many end-to-end fails size_t maxUgStreak, // stop after streak of this many ungap fails size_t maxDpStreak, // stop after streak of this many dp fails size_t maxMateStreak, // stop seed range after N mate-find fails bool doExtend, // do seed extension bool enable8, // use 8-bit SSE where possible size_t cminlen, // use checkpointer if read longer than this size_t cpow2, // interval between diagonals to checkpoint bool doTri, // triangular mini-fills int tighten, // -M score tightening mode AlignmentCacheIface& cs, // alignment cache for seed hits RandomSource& rnd, // pseudo-random source WalkMetrics& wlm, // group walk left metrics SwMetrics& swmSeed, // DP metrics for seed-extend SwMetrics& swmMate, // DP metrics for mate finidng PerReadMetrics& prm, // per-read metrics for anchor AlnSinkWrap* msink, // AlnSink wrapper for multiseed-style aligner bool swMateImmediately, // whether to look for mate immediately bool reportImmediately, // whether to report hits immediately to msink bool discord, // look for discordant alignments? bool mixed, // look for unpaired as well as paired alns? bool& exhaustive); /** * Prepare for a new read. */ void nextRead(bool paired, size_t mate1len, size_t mate2len) { redAnchor_.reset(); seenDiags1_.reset(); seenDiags2_.reset(); seedExRangeFw_[0].clear(); // mate 1 fw seedExRangeFw_[1].clear(); // mate 2 fw seedExRangeRc_[0].clear(); // mate 1 rc seedExRangeRc_[1].clear(); // mate 2 rc size_t maxlen = mate1len; if(paired) { redMate1_.reset(); redMate1_.init(mate1len); redMate2_.reset(); redMate2_.init(mate2len); if(mate2len > maxlen) { maxlen = mate2len; } } redAnchor_.init(maxlen); } protected: bool eeSaTups( const Read& rd, // read SeedResults& sh, // seed hits to extend into full alignments const GFM& gfm, // BWT const BitPairReference& ref, // Reference strings RandomSource& rnd, // pseudo-random generator WalkMetrics& wlm, // group walk left metrics SwMetrics& swmSeed, // metrics for seed extensions index_t& nelt_out, // out: # elements total index_t maxelts, // max # elts to report bool all); // report all hits? void extend( const Read& rd, // read const GFM& gfmFw, // Forward Bowtie index const GFM* gfmBw, // Backward Bowtie index index_t topf, // top in fw index index_t botf, // bot in fw index index_t topb, // top in bw index index_t botb, // bot in bw index bool fw, // seed orientation index_t off, // seed offset from 5' end index_t len, // seed length PerReadMetrics& prm, // per-read metrics index_t& nlex, // # positions we can extend to left w/o edit index_t& nrex); // # positions we can extend to right w/o edit void prioritizeSATups( const Read& rd, // read SeedResults& sh, // seed hits to extend into full alignments const GFM& gfmFw, // BWT const GFM* gfmBw, // BWT' const BitPairReference& ref, // Reference strings int seedmms, // # seed mismatches allowed index_t maxelt, // max elts we'll consider bool doExtend, // extend out seeds bool lensq, // square extended length bool szsq, // square SA range size index_t nsm, // if range as <= nsm elts, it's "small" AlignmentCacheIface& ca, // alignment cache for seed hits RandomSource& rnd, // pseudo-random generator WalkMetrics& wlm, // group walk left metrics PerReadMetrics& prm, // per-read metrics index_t& nelt_out, // out: # elements total bool all); // report all hits? Random1toN rand_; // random number generators EList rands_; // random number generators EList rands2_; // random number generators EList, 16> eehits_; // holds end-to-end hits EList, 16> satpos_; // holds SATuple, SeedPos pairs EList, 16> satpos2_; // holds SATuple, SeedPos pairs EList, 16> satups_; // holds SATuples to explore elements from EList > gws_; // list of GroupWalks; no particular order EList mateStreaks_; // mate-find fail streaks RowSampler rowsamp_; // row sampler // Ranges that we've extended through when extending seed hits EList seedExRangeFw_[2]; EList seedExRangeRc_[2]; // Data structures encapsulating the diagonals that have already been used // to seed alignment for mate 1 and mate 2. EIvalMergeListBinned seenDiags1_; EIvalMergeListBinned seenDiags2_; // For weeding out redundant alignments RedundantAlns redAnchor_; // database of cells used for anchor alignments RedundantAlns redMate1_; // database of cells used for mate 1 alignments RedundantAlns redMate2_; // database of cells used for mate 2 alignments // For holding results for anchor (res_) and opposite (ores_) mates SwResult resGap_; // temp holder for alignment result SwResult oresGap_; // temp holder for alignment result, opp mate SwResult resUngap_; // temp holder for ungapped alignment result SwResult oresUngap_; // temp holder for ungap. aln. opp mate SwResult resEe_; // temp holder for ungapped alignment result SwResult oresEe_; // temp holder for ungap. aln. opp mate Pool pool_; // memory pages for salistExact_ TSAList salistEe_; // PList for offsets for end-to-end hits GroupWalkState gwstate_; // some per-thread state shared by all GroupWalks // For AlnRes::matchesRef: ASSERT_ONLY(SStringExpandable raw_refbuf_); ASSERT_ONLY(SStringExpandable raw_destU32_); ASSERT_ONLY(EList raw_matches_); ASSERT_ONLY(BTDnaString tmp_rf_); ASSERT_ONLY(BTDnaString tmp_rdseq_); ASSERT_ONLY(BTString tmp_qseq_); ASSERT_ONLY(EList tmp_reflens_); ASSERT_ONLY(EList tmp_refoffs_); }; #define TIMER_START() \ struct timeval tv_i, tv_f; \ struct timezone tz_i, tz_f; \ size_t total_usecs; \ gettimeofday(&tv_i, &tz_i) #define IF_TIMER_END() \ gettimeofday(&tv_f, &tz_f); \ total_usecs = \ (tv_f.tv_sec - tv_i.tv_sec) * 1000000 + (tv_f.tv_usec - tv_i.tv_usec); \ if(total_usecs > 300000) /* * aligner_sw_driver.cpp * * Routines that drive the alignment process given a collection of seed hits. * This is generally done in a few stages: extendSeeds visits the set of * seed-hit BW elements in some order; for each element visited it resolves its * reference offset; once the reference offset is known, bounds for a dynamic * programming subproblem are established; if these bounds are distinct from * the bounds we've already tried, we solve the dynamic programming subproblem * and report the hit; if the AlnSinkWrap indicates that we can stop, we * return, otherwise we continue on to the next BW element. */ /** * Given end-to-end alignment results stored in the SeedResults structure, set * up all of our state for resolving and keeping track of reference offsets for * hits. Order the list of ranges to examine such that all exact end-to-end * alignments are examined before any 1mm end-to-end alignments. * * Note: there might be a lot of hits and a lot of wide ranges to look for * here. We use 'maxelt'. */ template bool SwDriver::eeSaTups( const Read& rd, // read SeedResults& sh, // seed hits to extend into full alignments const GFM& gfm, // BWT const BitPairReference& ref, // Reference strings RandomSource& rnd, // pseudo-random generator WalkMetrics& wlm, // group walk left metrics SwMetrics& swmSeed, // metrics for seed extensions index_t& nelt_out, // out: # elements total index_t maxelt, // max elts we'll consider bool all) // report all hits? { assert_eq(0, nelt_out); gws_.clear(); rands_.clear(); satpos_.clear(); eehits_.clear(); // First, count up the total number of satpos_, rands_, eehits_, and gws_ // we're going to tuse index_t nobj = 0; if(!sh.exactFwEEHit().empty()) nobj++; if(!sh.exactRcEEHit().empty()) nobj++; nobj += sh.mm1EEHits().size(); nobj = min(nobj, maxelt); gws_.ensure(nobj); rands_.ensure(nobj); satpos_.ensure(nobj); eehits_.ensure(nobj); index_t tot = sh.exactFwEEHit().size() + sh.exactRcEEHit().size(); bool succ = false; bool firstEe = true; bool done = false; if(tot > 0) { bool fwFirst = true; // Pick fw / rc to go first in a weighted random fashion #ifdef BOWTIE_64BIT_INDEX index_t rn64 = rnd.nextU64(); index_t rn = rn64 % (uint64_t)tot; #else index_t rn32 = rnd.nextU32(); index_t rn = rn32 % (uint32_t)tot; #endif if(rn >= sh.exactFwEEHit().size()) { fwFirst = false; } for(int fwi = 0; fwi < 2 && !done; fwi++) { bool fw = ((fwi == 0) == fwFirst); EEHit hit = fw ? sh.exactFwEEHit() : sh.exactRcEEHit(); if(hit.empty()) { continue; } assert(hit.fw == fw); if(hit.bot > hit.top) { // Possibly adjust bot and width if we would have exceeded maxelt index_t tops[2] = { hit.top, 0 }; index_t bots[2] = { hit.bot, 0 }; index_t width = hit.bot - hit.top; if(nelt_out + width > maxelt) { index_t trim = (index_t)((nelt_out + width) - maxelt); #ifdef BOWTIE_64BIT_INDEX index_t rn = rnd.nextU64() % width; #else index_t rn = rnd.nextU32() % width; #endif index_t newwidth = width - trim; if(hit.top + rn + newwidth > hit.bot) { // Two pieces tops[0] = hit.top + rn; bots[0] = hit.bot; tops[1] = hit.top; bots[1] = hit.top + newwidth - (bots[0] - tops[0]); } else { // One piece tops[0] = hit.top + rn; bots[0] = tops[0] + newwidth; } assert_leq(bots[0], hit.bot); assert_leq(bots[1], hit.bot); assert_geq(bots[0], tops[0]); assert_geq(bots[1], tops[1]); assert_eq(newwidth, (bots[0] - tops[0]) + (bots[1] - tops[1])); } for(int i = 0; i < 2 && !done; i++) { if(bots[i] <= tops[i]) break; index_t width = bots[i] - tops[i]; index_t top = tops[i]; // Clear list where resolved offsets are stored swmSeed.exranges++; swmSeed.exrows += width; if(!succ) { swmSeed.exsucc++; succ = true; } if(firstEe) { salistEe_.clear(); pool_.clear(); firstEe = false; } // We have to be careful not to allocate excessive amounts of memory here TSlice o(salistEe_, (index_t)salistEe_.size(), width); for(index_t i = 0; i < width; i++) { if(!salistEe_.add(pool_, (index_t)OFF_MASK)) { swmSeed.exooms++; return false; } } assert(!done); eehits_.push_back(hit); satpos_.expand(); satpos_.back().sat.init(SAKey(), top, (index_t)OFF_MASK, o); satpos_.back().sat.key.seq = MAX_U64; satpos_.back().sat.key.len = (index_t)rd.length(); satpos_.back().pos.init(fw, 0, 0, (index_t)rd.length()); satpos_.back().origSz = width; rands_.expand(); rands_.back().init(width, all); gws_.expand(); SARangeWithOffs sa; sa.topf = satpos_.back().sat.topf; sa.len = satpos_.back().sat.key.len; sa.offs = satpos_.back().sat.offs; gws_.back().init( gfm, // forward Bowtie index ref, // reference sequences sa, // SATuple rnd, // pseudo-random generator wlm); // metrics assert(gws_.back().repOk(sa)); nelt_out += width; if(nelt_out >= maxelt) { done = true; } } } } } succ = false; if(!done && !sh.mm1EEHits().empty()) { sh.sort1mmEe(rnd); index_t sz = sh.mm1EEHits().size(); for(index_t i = 0; i < sz && !done; i++) { EEHit hit = sh.mm1EEHits()[i]; assert(hit.repOk(rd)); assert(!hit.empty()); // Possibly adjust bot and width if we would have exceeded maxelt index_t tops[2] = { hit.top, 0 }; index_t bots[2] = { hit.bot, 0 }; index_t width = hit.bot - hit.top; if(nelt_out + width > maxelt) { index_t trim = (index_t)((nelt_out + width) - maxelt); #ifdef BOWTIE_64BIT_INDEX index_t rn = rnd.nextU64() % width; #else index_t rn = rnd.nextU32() % width; #endif index_t newwidth = width - trim; if(hit.top + rn + newwidth > hit.bot) { // Two pieces tops[0] = hit.top + rn; bots[0] = hit.bot; tops[1] = hit.top; bots[1] = hit.top + newwidth - (bots[0] - tops[0]); } else { // One piece tops[0] = hit.top + rn; bots[0] = tops[0] + newwidth; } assert_leq(bots[0], hit.bot); assert_leq(bots[1], hit.bot); assert_geq(bots[0], tops[0]); assert_geq(bots[1], tops[1]); assert_eq(newwidth, (bots[0] - tops[0]) + (bots[1] - tops[1])); } for(int i = 0; i < 2 && !done; i++) { if(bots[i] <= tops[i]) break; index_t width = bots[i] - tops[i]; index_t top = tops[i]; // Clear list where resolved offsets are stored swmSeed.mm1ranges++; swmSeed.mm1rows += width; if(!succ) { swmSeed.mm1succ++; succ = true; } if(firstEe) { salistEe_.clear(); pool_.clear(); firstEe = false; } TSlice o(salistEe_, (index_t)salistEe_.size(), width); for(size_t i = 0; i < width; i++) { if(!salistEe_.add(pool_, (index_t)OFF_MASK)) { swmSeed.mm1ooms++; return false; } } eehits_.push_back(hit); satpos_.expand(); satpos_.back().sat.init(SAKey(), top, (index_t)OFF_MASK, o); satpos_.back().sat.key.seq = MAX_U64; satpos_.back().sat.key.len = (index_t)rd.length(); satpos_.back().pos.init(hit.fw, 0, 0, (index_t)rd.length()); satpos_.back().origSz = width; rands_.expand(); rands_.back().init(width, all); gws_.expand(); SARangeWithOffs sa; sa.topf = satpos_.back().sat.topf; sa.len = satpos_.back().sat.key.len; sa.offs = satpos_.back().sat.offs; gws_.back().init( gfm, // forward Bowtie index ref, // reference sequences sa, // SATuple rnd, // pseudo-random generator wlm); // metrics assert(gws_.back().repOk(sa)); nelt_out += width; if(nelt_out >= maxelt) { done = true; } } } } return true; } /** * Extend a seed hit out on either side. Requires that we know the seed hit's * offset into the read and orientation. Also requires that we know top/bot * for the seed hit in both the forward and (if we want to extend to the right) * reverse index. */ template void SwDriver::extend( const Read& rd, // read const GFM& gfmFw, // Forward Bowtie index const GFM* gfmBw, // Backward Bowtie index index_t topf, // top in fw index index_t botf, // bot in fw index index_t topb, // top in bw index index_t botb, // bot in bw index bool fw, // seed orientation index_t off, // seed offset from 5' end index_t len, // seed length PerReadMetrics& prm, // per-read metrics index_t& nlex, // # positions we can extend to left w/o edit index_t& nrex) // # positions we can extend to right w/o edit { index_t t[4], b[4]; index_t tp[4], bp[4]; SideLocus tloc, bloc; index_t rdlen = (index_t)rd.length(); index_t lim = fw ? off : rdlen - len - off; // We're about to add onto the beginning, so reverse it #ifndef NDEBUG if(false) { // TODO: This will sometimes fail even when the extension is legitimate // This is because contains() comes in from one extreme or the other, // whereas we started from the inside and worked outwards. This // affects which Ns are OK and which are not OK. // Have to do both because whether we can get through an N depends on // which direction we're coming in bool fwContains = gfmFw.contains(tmp_rdseq_); tmp_rdseq_.reverse(); bool bwContains = gfmBw != NULL && gfmBw->contains(tmp_rdseq_); tmp_rdseq_.reverse(); assert(fwContains || bwContains); } #endif ASSERT_ONLY(tmp_rdseq_.reverse()); if(lim > 0) { const GFM *gfm = &gfmFw; assert(gfm != NULL); // Extend left using forward index const BTDnaString& seq = fw ? rd.patFw : rd.patRc; // See what we get by extending index_t top = topf, bot = botf; t[0] = t[1] = t[2] = t[3] = 0; b[0] = b[1] = b[2] = b[3] = 0; tp[0] = tp[1] = tp[2] = tp[3] = topb; bp[0] = bp[1] = bp[2] = bp[3] = botb; SideLocus tloc, bloc; INIT_LOCS(top, bot, tloc, bloc, *gfm); for(index_t ii = 0; ii < lim; ii++) { // Starting to left of seed (mapBiLFEx(tloc, bloc, t, b, tp, bp); SANITY_CHECK_4TUP(t, b, tp, bp); int nonz = -1; bool abort = false; size_t origSz = bot - top; for(int j = 0; j < 4; j++) { if(b[j] > t[j]) { if(nonz >= 0) { abort = true; break; } nonz = j; top = t[j]; bot = b[j]; } } assert_leq(bot - top, origSz); if(abort || (nonz != rdc && rdc <= 3) || bot - top < origSz) { break; } } else { assert_eq(bot, top+1); prm.nSdFmops++; int c = gfm->mapLF1(top, tloc); if(c != rdc && rdc <= 3) { break; } bot = top + 1; } ASSERT_ONLY(tmp_rdseq_.append(rdc)); if(++nlex == 255) { break; } INIT_LOCS(top, bot, tloc, bloc, *gfm); } } // We're about to add onto the end, so re-reverse ASSERT_ONLY(tmp_rdseq_.reverse()); lim = fw ? rdlen - len - off : off; if(lim > 0 && gfmBw != NULL) { const GFM *gfm = gfmBw; assert(gfm != NULL); // Extend right using backward index const BTDnaString& seq = fw ? rd.patFw : rd.patRc; // See what we get by extending index_t top = topb, bot = botb; t[0] = t[1] = t[2] = t[3] = 0; b[0] = b[1] = b[2] = b[3] = 0; tp[0] = tp[1] = tp[2] = tp[3] = topf; bp[0] = bp[1] = bp[2] = bp[3] = botf; INIT_LOCS(top, bot, tloc, bloc, *gfm); for(index_t ii = 0; ii < lim; ii++) { // Starting to right of seed (mapBiLFEx(tloc, bloc, t, b, tp, bp); SANITY_CHECK_4TUP(t, b, tp, bp); int nonz = -1; bool abort = false; size_t origSz = bot - top; for(int j = 0; j < 4; j++) { if(b[j] > t[j]) { if(nonz >= 0) { abort = true; break; } nonz = j; top = t[j]; bot = b[j]; } } assert_leq(bot - top, origSz); if(abort || (nonz != rdc && rdc <= 3) || bot - top < origSz) { break; } } else { assert_eq(bot, top+1); prm.nSdFmops++; int c = gfm->mapLF1(top, tloc); if(c != rdc && rdc <= 3) { break; } bot = top + 1; } ASSERT_ONLY(tmp_rdseq_.append(rdc)); if(++nrex == 255) { break; } INIT_LOCS(top, bot, tloc, bloc, *gfm); } } #ifndef NDEBUG if(false) { // TODO: This will sometimes fail even when the extension is legitimate // This is because contains() comes in from one extreme or the other, // whereas we started from the inside and worked outwards. This // affects which Ns are OK and which are not OK. // Have to do both because whether we can get through an N depends on // which direction we're coming in bool fwContains = gfmFw.contains(tmp_rdseq_); tmp_rdseq_.reverse(); bool bwContains = gfmBw != NULL && gfmBw->contains(tmp_rdseq_); tmp_rdseq_.reverse(); assert(fwContains || bwContains); } #endif assert_lt(nlex, rdlen); assert_lt(nrex, rdlen); return; } /** * Given seed results, set up all of our state for resolving and keeping * track of reference offsets for hits. */ template void SwDriver::prioritizeSATups( const Read& read, // read SeedResults& sh, // seed hits to extend into full alignments const GFM& gfmFw, // BWT const GFM* gfmBw, // BWT const BitPairReference& ref, // Reference strings int seedmms, // # mismatches allowed in seed index_t maxelt, // max elts we'll consider bool doExtend, // do extension of seed hits? bool lensq, // square length in weight calculation bool szsq, // square range size in weight calculation index_t nsm, // if range as <= nsm elts, it's "small" AlignmentCacheIface& ca, // alignment cache for seed hits RandomSource& rnd, // pseudo-random generator WalkMetrics& wlm, // group walk left metrics PerReadMetrics& prm, // per-read metrics index_t& nelt_out, // out: # elements total bool all) // report all hits? { const index_t nonz = sh.nonzeroOffsets(); // non-zero positions const int matei = (read.mate <= 1 ? 0 : 1); satups_.clear(); gws_.clear(); rands_.clear(); rands2_.clear(); satpos_.clear(); satpos2_.clear(); index_t nrange = 0, nelt = 0, nsmall = 0, nsmall_elts = 0; bool keepWhole = false; EList, 16>& satpos = keepWhole ? satpos_ : satpos2_; for(index_t i = 0; i < nonz; i++) { bool fw = true; index_t offidx = 0, rdoff = 0, seedlen = 0; QVal qv = sh.hitsByRank(i, offidx, rdoff, fw, seedlen); assert(qv.valid()); assert(!qv.empty()); assert(qv.repOk(ca.current())); ca.queryQval(qv, satups_, nrange, nelt); for(size_t j = 0; j < satups_.size(); j++) { const index_t sz = satups_[j].size(); // Check whether this hit occurs inside the extended boundaries of // another hit we already processed for this read. if(seedmms == 0) { // See if we're covered by a previous extended seed hit EList& range = fw ? seedExRangeFw_[matei] : seedExRangeRc_[matei]; bool skip = false; for(index_t k = 0; k < range.size(); k++) { index_t p5 = range[k].off; index_t len = range[k].len; if(p5 <= rdoff && p5 + len >= (rdoff + seedlen)) { if(sz <= range[k].sz) { skip = true; break; } } } if(skip) { assert_gt(nrange, 0); nrange--; assert_geq(nelt, sz); nelt -= sz; continue; // Skip this seed } } satpos.expand(); satpos.back().sat = satups_[j]; satpos.back().origSz = sz; satpos.back().pos.init(fw, offidx, rdoff, seedlen); if(sz <= nsm) { nsmall++; nsmall_elts += sz; } satpos.back().nlex = satpos.back().nrex = 0; #ifndef NDEBUG tmp_rdseq_.clear(); uint64_t key = satpos.back().sat.key.seq; for(size_t k = 0; k < seedlen; k++) { int c = (int)(key & 3); tmp_rdseq_.append(c); key >>= 2; } tmp_rdseq_.reverse(); #endif index_t nlex = 0, nrex = 0; if(doExtend) { extend( read, gfmFw, gfmBw, satpos.back().sat.topf, (index_t)(satpos.back().sat.topf + sz), satpos.back().sat.topb, (index_t)(satpos.back().sat.topb + sz), fw, rdoff, seedlen, prm, nlex, nrex); } satpos.back().nlex = nlex; satpos.back().nrex = nrex; if(seedmms == 0 && (nlex > 0 || nrex > 0)) { assert_geq(rdoff, (fw ? nlex : nrex)); index_t p5 = rdoff - (fw ? nlex : nrex); EList& range = fw ? seedExRangeFw_[matei] : seedExRangeRc_[matei]; range.expand(); range.back().off = p5; range.back().len = seedlen + nlex + nrex; range.back().sz = sz; } } satups_.clear(); } assert_leq(nsmall, nrange); nelt_out = nelt; // return the total number of elements assert_eq(nrange, satpos.size()); satpos.sort(); if(keepWhole) { gws_.ensure(nrange); rands_.ensure(nrange); for(index_t i = 0; i < nrange; i++) { gws_.expand(); SARangeWithOffs sa; sa.topf = satpos_.back().sat.topf; sa.len = satpos_.back().sat.key.len; sa.offs = satpos_.back().sat.offs; gws_.back().init( gfmFw, // forward Bowtie index ref, // reference sequences sa, // SA tuples: ref hit, salist range rnd, // pseudo-random generator wlm); // metrics assert(gws_.back().initialized()); rands_.expand(); rands_.back().init(satpos_[i].sat.size(), all); } return; } // Resize satups_ list so that ranges having elements that we might // possibly explore are present satpos_.ensure(min(maxelt, nelt)); gws_.ensure(min(maxelt, nelt)); rands_.ensure(min(maxelt, nelt)); rands2_.ensure(min(maxelt, nelt)); size_t nlarge_elts = nelt - nsmall_elts; if(maxelt < nelt) { size_t diff = nelt - maxelt; if(diff >= nlarge_elts) { nlarge_elts = 0; } else { nlarge_elts -= diff; } } index_t nelt_added = 0; // Now we have a collection of ranges in satpos2_. Now we want to decide // how we explore elements from them. The basic idea is that: for very // small guys, where "very small" means that the size of the range is less // than or equal to the parameter 'nsz', we explore them in their entirety // right away. For the rest, we want to select in a way that is (a) // random, and (b) weighted toward examining elements from the smaller // ranges more frequently (and first). // // 1. do the smalls for(index_t j = 0; j < nsmall && nelt_added < maxelt; j++) { satpos_.expand(); satpos_.back() = satpos2_[j]; gws_.expand(); SARangeWithOffs sa; sa.topf = satpos_.back().sat.topf; sa.len = satpos_.back().sat.key.len; sa.offs = satpos_.back().sat.offs; gws_.back().init( gfmFw, // forward Bowtie index ref, // reference sequences sa, // SA tuples: ref hit, salist range rnd, // pseudo-random generator wlm); // metrics assert(gws_.back().initialized()); rands_.expand(); rands_.back().init(satpos_.back().sat.size(), all); nelt_added += satpos_.back().sat.size(); #ifndef NDEBUG for(size_t k = 0; k < satpos_.size()-1; k++) { assert(!(satpos_[k] == satpos_.back())); } #endif } if(nelt_added >= maxelt || nsmall == satpos2_.size()) { nelt_out = nelt_added; return; } // 2. do the non-smalls // Initialize the row sampler rowsamp_.init(satpos2_, nsmall, satpos2_.size(), lensq, szsq); // Initialize the random choosers rands2_.resize(satpos2_.size()); for(index_t j = 0; j < satpos2_.size(); j++) { rands2_[j].reset(); } while(nelt_added < maxelt && nelt_added < nelt) { // Pick a non-small range to sample from index_t ri = rowsamp_.next(rnd) + nsmall; assert_geq(ri, nsmall); assert_lt(ri, satpos2_.size()); // Initialize random element chooser for that range if(!rands2_[ri].inited()) { rands2_[ri].init(satpos2_[ri].sat.size(), all); assert(!rands2_[ri].done()); } assert(!rands2_[ri].done()); // Choose an element from the range uint32_t r = rands2_[ri].next(rnd); if(rands2_[ri].done()) { // Tell the row sampler this range is done rowsamp_.finishedRange(ri - nsmall); } // Add the element to the satpos_ list SATuple sat; TSlice o; o.init(satpos2_[ri].sat.offs, r, r+1); sat.init(satpos2_[ri].sat.key, (index_t)(satpos2_[ri].sat.topf + r), (index_t)OFF_MASK, o); satpos_.expand(); satpos_.back().sat = sat; satpos_.back().origSz = satpos2_[ri].origSz; satpos_.back().pos = satpos2_[ri].pos; // Initialize GroupWalk object gws_.expand(); SARangeWithOffs sa; sa.topf = sat.topf; sa.len = sat.key.len; sa.offs = sat.offs; gws_.back().init( gfmFw, // forward Bowtie index ref, // reference sequences sa, // SA tuples: ref hit, salist range rnd, // pseudo-random generator wlm); // metrics assert(gws_.back().initialized()); // Initialize random selector rands_.expand(); rands_.back().init(1, all); nelt_added++; } nelt_out = nelt_added; return; } enum { FOUND_NONE = 0, FOUND_EE, FOUND_UNGAPPED, }; /** * Given a collection of SeedHits for a single read, extend seed alignments * into full alignments. Where possible, try to avoid redundant offset lookups * and dynamic programming wherever possible. Optionally report alignments to * a AlnSinkWrap object as they are discovered. * * If 'reportImmediately' is true, returns true iff a call to msink->report() * returned true (indicating that the reporting policy is satisfied and we can * stop). Otherwise, returns false. */ template int SwDriver::extendSeeds( Read& rd, // read to align bool mate1, // true iff rd is mate #1 SeedResults& sh, // seed hits to extend into full alignments const GFM& gfmFw, // BWT const GFM* gfmBw, // BWT' const BitPairReference& ref, // Reference strings SwAligner& swa, // dynamic programming aligner const Scoring& sc, // scoring scheme int seedmms, // # mismatches allowed in seed int seedlen, // length of seed int seedival, // interval between seeds TAlScore& minsc, // minimum score for anchor int nceil, // maximum # Ns permitted in reference portion size_t maxhalf, // max width in either direction for DP tables bool doUngapped, // do ungapped alignment size_t maxIters, // stop after this many seed-extend loop iters size_t maxUg, // stop after this many ungaps size_t maxDp, // stop after this many dps size_t maxUgStreak, // stop after streak of this many ungap fails size_t maxDpStreak, // stop after streak of this many dp fails bool doExtend, // do seed extension bool enable8, // use 8-bit SSE where possible size_t cminlen, // use checkpointer if read longer than this size_t cpow2, // interval between diagonals to checkpoint bool doTri, // triangular mini-fills? int tighten, // -M score tightening mode AlignmentCacheIface& ca, // alignment cache for seed hits RandomSource& rnd, // pseudo-random source WalkMetrics& wlm, // group walk left metrics SwMetrics& swmSeed, // DP metrics for seed-extend PerReadMetrics& prm, // per-read metrics AlnSinkWrap* msink, // AlnSink wrapper for multiseed-style aligner bool reportImmediately, // whether to report hits immediately to msink bool& exhaustive) // set to true iff we searched all seeds exhaustively { bool all = msink->allHits(); // typedef std::pair UPair; assert(!reportImmediately || msink != NULL); assert(!reportImmediately || !msink->maxed()); assert_geq(nceil, 0); assert_leq((size_t)nceil, rd.length()); // Calculate the largest possible number of read and reference gaps const index_t rdlen = (index_t)rd.length(); TAlScore perfectScore = sc.perfectScore(rdlen); DynProgFramer dpframe(!gReportOverhangs); swa.reset(); // Initialize a set of GroupWalks, one for each seed. Also, intialize the // accompanying lists of reference seed hits (satups*) const index_t nsm = 5; const index_t nonz = sh.nonzeroOffsets(); // non-zero positions index_t eeHits = sh.numE2eHits(); bool eeMode = eeHits > 0; bool firstEe = true; bool firstExtend = true; // Reset all the counters related to streaks prm.nEeFail = 0; prm.nUgFail = 0; prm.nDpFail = 0; index_t nelt = 0, neltLeft = 0; index_t rows = rdlen; index_t eltsDone = 0; // cerr << "===" << endl; while(true) { if(eeMode) { if(firstEe) { firstEe = false; eeMode = eeSaTups( rd, // read sh, // seed hits to extend into full alignments gfmFw, // BWT ref, // Reference strings rnd, // pseudo-random generator wlm, // group walk left metrics swmSeed, // seed-extend metrics nelt, // out: # elements total maxIters, // max # to report all); // report all hits? assert_eq(gws_.size(), rands_.size()); assert_eq(gws_.size(), satpos_.size()); } else { eeMode = false; } } if(!eeMode) { if(nonz == 0) { return EXTEND_EXHAUSTED_CANDIDATES; // No seed hits! Bail. } if(minsc == perfectScore) { return EXTEND_PERFECT_SCORE; // Already found all perfect hits! } if(firstExtend) { nelt = 0; prioritizeSATups( rd, // read sh, // seed hits to extend into full alignments gfmFw, // BWT gfmBw, // BWT' ref, // Reference strings seedmms, // # seed mismatches allowed maxIters, // max rows to consider per position doExtend, // extend out seeds true, // square extended length true, // square SA range size nsm, // smallness threshold ca, // alignment cache for seed hits rnd, // pseudo-random generator wlm, // group walk left metrics prm, // per-read metrics nelt, // out: # elements total all); // report all hits? assert_eq(gws_.size(), rands_.size()); assert_eq(gws_.size(), satpos_.size()); neltLeft = nelt; firstExtend = false; } if(neltLeft == 0) { // Finished examining gapped candidates break; } } for(size_t i = 0; i < gws_.size(); i++) { if(eeMode && eehits_[i].score < minsc) { return EXTEND_PERFECT_SCORE; } bool is_small = satpos_[i].sat.size() < nsm; bool fw = satpos_[i].pos.fw; index_t rdoff = satpos_[i].pos.rdoff; index_t seedhitlen = satpos_[i].pos.seedlen; if(!fw) { // 'rdoff' and 'offidx' are with respect to the 5' end of // the read. Here we convert rdoff to be with respect to // the upstream (3') end of ther read. rdoff = (index_t)(rdlen - rdoff - seedhitlen); } bool first = true; // If the range is small, investigate all elements now. If the // range is large, just investigate one and move on - we might come // back to this range later. index_t riter = 0; while(!rands_[i].done() && (first || is_small || eeMode)) { assert(!gws_[i].done()); riter++; if(minsc == perfectScore) { if(!eeMode || eehits_[i].score < perfectScore) { return EXTEND_PERFECT_SCORE; } } else if(eeMode && eehits_[i].score < minsc) { break; } if(prm.nExDps >= maxDp || prm.nMateDps >= maxDp) { return EXTEND_EXCEEDED_HARD_LIMIT; } if(prm.nExUgs >= maxUg || prm.nMateUgs >= maxUg) { return EXTEND_EXCEEDED_HARD_LIMIT; } if(prm.nExIters >= maxIters) { return EXTEND_EXCEEDED_HARD_LIMIT; } prm.nExIters++; first = false; // Resolve next element offset WalkResult wr; uint32_t elt = rands_[i].next(rnd); //cerr << "elt=" << elt << endl; SARangeWithOffs sa; sa.topf = satpos_[i].sat.topf; sa.len = satpos_[i].sat.key.len; sa.offs = satpos_[i].sat.offs; gws_[i].advanceElement((index_t)elt, gfmFw, ref, sa, gwstate_, wr, wlm, prm); eltsDone++; if(!eeMode) { assert_gt(neltLeft, 0); neltLeft--; } assert_neq((index_t)OFF_MASK, wr.toff); index_t tidx = 0, toff = 0, tlen = 0; bool straddled = false; gfmFw.joinedToTextOff( wr.elt.len, wr.toff, tidx, toff, tlen, eeMode, // reject straddlers? straddled); // did it straddle? if(tidx == (index_t)OFF_MASK) { // The seed hit straddled a reference boundary so the seed hit // isn't valid continue; } #ifndef NDEBUG if(!eeMode && !straddled) { // Check that seed hit matches reference uint64_t key = satpos_[i].sat.key.seq; for(index_t k = 0; k < wr.elt.len; k++) { int c = ref.getBase(tidx, toff + wr.elt.len - k - 1); assert_leq(c, 3); int ck = (int)(key & 3); key >>= 2; assert_eq(c, ck); } } #endif // Find offset of alignment's upstream base assuming net gaps=0 // between beginning of read and beginning of seed hit int64_t refoff = (int64_t)toff - rdoff; // Coordinate of the seed hit w/r/t the pasted reference string Coord refcoord(tidx, refoff, fw); if(seenDiags1_.locusPresent(refcoord)) { // Already handled alignments seeded on this diagonal prm.nRedundants++; swmSeed.rshit++; continue; } // Now that we have a seed hit, there are many issues to solve // before we have a completely framed dynamic programming problem. // They include: // // 1. Setting reference offsets on either side of the seed hit, // accounting for where the seed occurs in the read // 2. Adjusting the width of the banded dynamic programming problem // and adjusting reference bounds to allow for gaps in the // alignment // 3. Accounting for the edges of the reference, which can impact // the width of the DP problem and reference bounds. // 4. Perhaps filtering the problem down to a smaller problem based // on what DPs we've already solved for this read // // We do #1 here, since it is simple and we have all the seed-hit // information here. #2 and #3 are handled in the DynProgFramer. int readGaps = 0, refGaps = 0; bool ungapped = false; if(!eeMode) { readGaps = sc.maxReadGaps(minsc, rdlen); refGaps = sc.maxRefGaps(minsc, rdlen); ungapped = (readGaps == 0 && refGaps == 0); } int state = FOUND_NONE; bool found = false; if(eeMode) { resEe_.reset(); resEe_.alres.reset(); const EEHit& h = eehits_[i]; assert_leq(h.score, perfectScore); resEe_.alres.setScore(AlnScore(h.score, h.ns(), 0)); resEe_.alres.setShape( refcoord.ref(), // ref id refcoord.off(), // 0-based ref offset tlen, // length of reference fw, // aligned to Watson? rdlen, // read length true, // pretrim soft? 0, // pretrim 5' end 0, // pretrim 3' end true, // alignment trim soft? 0, // alignment trim 5' end 0); // alignment trim 3' end resEe_.alres.setRefNs(h.refns()); if(h.mms() > 0) { assert_eq(1, h.mms()); assert_lt(h.e1.pos, rd.length()); resEe_.alres.ned().push_back(h.e1); } assert(resEe_.repOk(rd)); state = FOUND_EE; found = true; Interval refival(refcoord, 1); seenDiags1_.add(refival); } else if(doUngapped && ungapped) { resUngap_.reset(); int al = swa.ungappedAlign( fw ? rd.patFw : rd.patRc, fw ? rd.qual : rd.qualRev, refcoord, ref, tlen, sc, gReportOverhangs, minsc, resUngap_); Interval refival(refcoord, 1); seenDiags1_.add(refival); prm.nExUgs++; if(al == 0) { prm.nExUgFails++; prm.nUgFail++; if(prm.nUgFail >= maxUgStreak) { return EXTEND_EXCEEDED_SOFT_LIMIT; } swmSeed.ungapfail++; continue; } else if(al == -1) { prm.nExUgFails++; prm.nUgFail++; // count this as failure if(prm.nUgFail >= maxUgStreak) { return EXTEND_EXCEEDED_SOFT_LIMIT; } swmSeed.ungapnodec++; } else { prm.nExUgSuccs++; prm.nUgLastSucc = prm.nExUgs-1; if(prm.nUgFail > prm.nUgFailStreak) { prm.nUgFailStreak = prm.nUgFail; } prm.nUgFail = 0; found = true; state = FOUND_UNGAPPED; swmSeed.ungapsucc++; } } int64_t pastedRefoff = (int64_t)wr.toff - rdoff; DPRect rect; if(state == FOUND_NONE) { found = dpframe.frameSeedExtensionRect( refoff, // ref offset implied by seed hit assuming no gaps rows, // length of read sequence used in DP table tlen, // length of reference readGaps, // max # of read gaps permitted in opp mate alignment refGaps, // max # of ref gaps permitted in opp mate alignment (size_t)nceil, // # Ns permitted maxhalf, // max width in either direction rect); // DP rectangle assert(rect.repOk()); // Add the seed diagonal at least seenDiags1_.add(Interval(refcoord, 1)); if(!found) { continue; } } int64_t leftShift = refoff - rect.refl; size_t nwindow = 0; if(toff >= rect.refl) { nwindow = (size_t)(toff - rect.refl); } // NOTE: We might be taking off more than we should because the // pasted string omits non-A/C/G/T characters, but we included them // when calculating leftShift. We'll account for this later. pastedRefoff -= leftShift; size_t nsInLeftShift = 0; if(state == FOUND_NONE) { if(!swa.initedRead()) { // Initialize the aligner with a new read swa.initRead( rd.patFw, // fw version of query rd.patRc, // rc version of query rd.qual, // fw version of qualities rd.qualRev,// rc version of qualities 0, // off of first char in 'rd' to consider rdlen, // off of last char (excl) in 'rd' to consider sc); // scoring scheme } swa.initRef( fw, // whether to align forward or revcomp read tidx, // reference aligned against rect, // DP rectangle ref, // Reference strings tlen, // length of reference sequence sc, // scoring scheme minsc, // minimum score permitted enable8, // use 8-bit SSE if possible? cminlen, // minimum length for using checkpointing scheme cpow2, // interval b/t checkpointed diags; 1 << this doTri, // triangular mini-fills? true, // this is a seed extension - not finding a mate nwindow, nsInLeftShift); // Because of how we framed the problem, we can say that we've // exhaustively scored the seed diagonal as well as maxgaps // diagonals on either side Interval refival(tidx, 0, fw, 0); rect.initIval(refival); seenDiags1_.add(refival); // Now fill the dynamic programming matrix and return true iff // there is at least one valid alignment TAlScore bestCell = std::numeric_limits::min(); found = swa.align(rnd, bestCell); swmSeed.tallyGappedDp(readGaps, refGaps); prm.nExDps++; if(!found) { prm.nExDpFails++; prm.nDpFail++; if(prm.nDpFail >= maxDpStreak) { return EXTEND_EXCEEDED_SOFT_LIMIT; } if(bestCell > std::numeric_limits::min() && bestCell > prm.bestLtMinscMate1) { prm.bestLtMinscMate1 = bestCell; } continue; // Look for more anchor alignments } else { prm.nExDpSuccs++; prm.nDpLastSucc = prm.nExDps-1; if(prm.nDpFail > prm.nDpFailStreak) { prm.nDpFailStreak = prm.nDpFail; } prm.nDpFail = 0; } } bool firstInner = true; while(true) { assert(found); SwResult *res = NULL; if(state == FOUND_EE) { if(!firstInner) { break; } res = &resEe_; } else if(state == FOUND_UNGAPPED) { if(!firstInner) { break; } res = &resUngap_; } else { resGap_.reset(); assert(resGap_.empty()); if(swa.done()) { break; } swa.nextAlignment(resGap_, minsc, rnd); found = !resGap_.empty(); if(!found) { break; } res = &resGap_; } assert(res != NULL); firstInner = false; assert(res->alres.matchesRef( rd, ref, tmp_rf_, tmp_rdseq_, tmp_qseq_, raw_refbuf_, raw_destU32_, raw_matches_, tmp_reflens_, tmp_refoffs_)); Interval refival(tidx, 0, fw, tlen); assert_gt(res->alres.refExtent(), 0); if(gReportOverhangs && !refival.containsIgnoreOrient(res->alres.refival())) { res->alres.clipOutside(true, 0, tlen); if(res->alres.refExtent() == 0) { continue; } } assert(gReportOverhangs || refival.containsIgnoreOrient(res->alres.refival())); // Did the alignment fall entirely outside the reference? if(!refival.overlapsIgnoreOrient(res->alres.refival())) { continue; } // Is this alignment redundant with one we've seen previously? if(redAnchor_.overlap(res->alres)) { // Redundant with an alignment we found already continue; } redAnchor_.add(res->alres); // Annotate the AlnRes object with some key parameters // that were used to obtain the alignment. res->alres.setParams( seedmms, // # mismatches allowed in seed seedlen, // length of seed seedival, // interval between seeds minsc); // minimum score for valid alignment if(reportImmediately) { assert(msink != NULL); assert(res->repOk()); // Check that alignment accurately reflects the // reference characters aligned to assert(res->alres.matchesRef( rd, ref, tmp_rf_, tmp_rdseq_, tmp_qseq_, raw_refbuf_, raw_destU32_, raw_matches_, tmp_reflens_, tmp_refoffs_)); // Report an unpaired alignment assert(!msink->maxed()); if(msink->report( 0, mate1 ? &res->alres : NULL, mate1 ? NULL : &res->alres)) { // Short-circuited because a limit, e.g. -k, -m or // -M, was exceeded return EXTEND_POLICY_FULFILLED; } if(tighten > 0 && msink->Mmode() && msink->hasSecondBestUnp1()) { if(tighten == 1) { if(msink->bestUnp1() >= minsc) { minsc = msink->bestUnp1(); if(minsc < perfectScore && msink->bestUnp1() == msink->secondBestUnp1()) { minsc++; } } } else if(tighten == 2) { if(msink->secondBestUnp1() >= minsc) { minsc = msink->secondBestUnp1(); if(minsc < perfectScore) { minsc++; } } } else { TAlScore diff = msink->bestUnp1() - msink->secondBestUnp1(); TAlScore bot = msink->secondBestUnp1() + ((diff*3)/4); if(bot >= minsc) { minsc = bot; if(minsc < perfectScore) { minsc++; } } } assert_leq(minsc, perfectScore); } } } // At this point we know that we aren't bailing, and will // continue to resolve seed hits. } // while(!gws_[i].done()) } } // Short-circuited because a limit, e.g. -k, -m or -M, was exceeded return EXTEND_EXHAUSTED_CANDIDATES; } /** * Given a collection of SeedHits for both mates in a read pair, extend seed * alignments into full alignments and then look for the opposite mate using * dynamic programming. Where possible, try to avoid redundant offset lookups. * Optionally report alignments to a AlnSinkWrap object as they are discovered. * * If 'reportImmediately' is true, returns true iff a call to * msink->report() returned true (indicating that the reporting * policy is satisfied and we can stop). Otherwise, returns false. * * REDUNDANT SEED HITS * * See notes at top of aligner_sw_driver.h. * * REDUNDANT ALIGNMENTS * * See notes at top of aligner_sw_driver.h. * * MIXING PAIRED AND UNPAIRED ALIGNMENTS * * There are distinct paired-end alignment modes for the cases where (a) the * user does or does not want to see unpaired alignments for individual mates * when there are no reportable paired-end alignments involving both mates, and * (b) the user does or does not want to see discordant paired-end alignments. * The modes have implications for this function and for the AlnSinkWrap, since * it affects when we're "done." Also, whether the user has asked us to report * discordant alignments affects whether and how much searching for unpaired * alignments we must do (i.e. if there are no paired-end alignments, we must * at least do -m 1 for both mates). * * Mode 1: Just concordant paired-end. Print only concordant paired-end * alignments. As soon as any limits (-k/-m/-M) are reached, stop. * * Mode 2: Concordant and discordant paired-end. If -k/-m/-M limits are * reached for paired-end alignments, stop. Otherwise, if no paired-end * alignments are found, align both mates in an unpaired -m 1 fashion. If * there is exactly one unpaired alignment for each mate, report the * combination as a discordant alignment. * * Mode 3: Concordant paired-end if possible, otherwise unpaired. If -k/-M * limit is reached for paired-end alignmnts, stop. If -m limit is reached for * paired-end alignments or no paired-end alignments are found, align both * mates in an unpaired fashion. All the same settings governing validity and * reportability in paired-end mode apply here too (-k/-m/-M/etc). * * Mode 4: Concordant or discordant paired-end if possible, otherwise unpaired. * If -k/-M limit is reached for paired-end alignmnts, stop. If -m limit is * reached for paired-end alignments or no paired-end alignments are found, * align both mates in an unpaired fashion. If the -m limit was reached, there * is no need to search for a discordant alignment, and unapired alignment can * proceed as in Mode 3. If no paired-end alignments were found, then unpaired * alignment proceeds as in Mode 3 but with this caveat: alignment must be at * least as thorough as dictated by -m 1 up until the point where * * Print paired-end alignments when there are reportable paired-end * alignments, otherwise report reportable unpaired alignments. If -k limit is * reached for paired-end alignments, stop. If -m/-M limit is reached for * paired-end alignments, stop searching for paired-end alignments and look * only for unpaired alignments. If searching only for unpaired alignments, * respect -k/-m/-M limits separately for both mates. * * The return value from the AlnSinkWrap's report member function must be * specific enough to distinguish between: * * 1. Stop searching for paired-end alignments * 2. Stop searching for alignments for unpaired alignments for mate #1 * 3. Stop searching for alignments for unpaired alignments for mate #2 * 4. Stop searching for any alignments * * Note that in Mode 2, options affecting validity and reportability of * alignments apply . E.g. if -m 1 is specified * * WORKFLOW * * Our general approach to finding paired and unpaired alignments here * is as follows: * * - For mate in mate1, mate2: * - For each seed hit in mate: * - Try to extend it into a full alignment; if we can't, continue * to the next seed hit * - Look for alignment for opposite mate; if we can't find one, * - * - * */ template int SwDriver::extendSeedsPaired( Read& rd, // mate to align as anchor Read& ord, // mate to align as opposite bool anchor1, // true iff anchor mate is mate1 bool oppFilt, // true iff opposite mate was filtered out SeedResults& sh, // seed hits for anchor const GFM& gfmFw, // BWT const GFM* gfmBw, // BWT' const BitPairReference& ref, // Reference strings SwAligner& swa, // dynamic programming aligner for anchor SwAligner& oswa, // dynamic programming aligner for opposite const Scoring& sc, // scoring scheme const PairedEndPolicy& pepol,// paired-end policy int seedmms, // # mismatches allowed in seed int seedlen, // length of seed int seedival, // interval between seeds TAlScore& minsc, // minimum score for valid anchor aln TAlScore& ominsc, // minimum score for valid opposite aln int nceil, // max # Ns permitted in ref for anchor int onceil, // max # Ns permitted in ref for opposite bool nofw, // don't align forward read bool norc, // don't align revcomp read size_t maxhalf, // max width in either direction for DP tables bool doUngapped, // do ungapped alignment size_t maxIters, // stop after this many seed-extend loop iters size_t maxUg, // stop after this many ungaps size_t maxDp, // stop after this many dps size_t maxEeStreak, // stop after streak of this many end-to-end fails size_t maxUgStreak, // stop after streak of this many ungap fails size_t maxDpStreak, // stop after streak of this many dp fails size_t maxMateStreak, // stop seed range after N mate-find fails bool doExtend, // do seed extension bool enable8, // use 8-bit SSE where possible size_t cminlen, // use checkpointer if read longer than this size_t cpow2, // interval between diagonals to checkpoint bool doTri, // triangular mini-fills? int tighten, // -M score tightening mode AlignmentCacheIface& ca, // alignment cache for seed hits RandomSource& rnd, // pseudo-random source WalkMetrics& wlm, // group walk left metrics SwMetrics& swmSeed, // DP metrics for seed-extend SwMetrics& swmMate, // DP metrics for mate finidng PerReadMetrics& prm, // per-read metrics AlnSinkWrap* msink, // AlnSink wrapper for multiseed-style aligner bool swMateImmediately, // whether to look for mate immediately bool reportImmediately, // whether to report hits immediately to msink bool discord, // look for discordant alignments? bool mixed, // look for unpaired as well as paired alns? bool& exhaustive) { bool all = msink->allHits(); // typedef std::pair U32Pair; assert(!reportImmediately || msink != NULL); assert(!reportImmediately || !msink->maxed()); assert(!msink->state().doneWithMate(anchor1)); assert_geq(nceil, 0); assert_geq(onceil, 0); assert_leq((size_t)nceil, rd.length()); assert_leq((size_t)onceil, ord.length()); const index_t rdlen = rd.length(); const index_t ordlen = ord.length(); const TAlScore perfectScore = sc.perfectScore(rdlen); const TAlScore operfectScore = sc.perfectScore(ordlen); assert_leq(minsc, perfectScore); assert(oppFilt || ominsc <= operfectScore); TAlScore bestPairScore = perfectScore + operfectScore; if(tighten > 0 && msink->Mmode() && msink->hasSecondBestPair()) { // Paired-end alignments should have at least this score from now TAlScore ps; if(tighten == 1) { ps = msink->bestPair(); } else if(tighten == 2) { ps = msink->secondBestPair(); } else { TAlScore diff = msink->bestPair() - msink->secondBestPair(); ps = msink->secondBestPair() + (diff * 3)/4; } if(tighten == 1 && ps < bestPairScore && msink->bestPair() == msink->secondBestPair()) { ps++; } if(tighten >= 2 && ps < bestPairScore) { ps++; } // Anchor mate must have score at least 'ps' minus the best possible // score for the opposite mate. TAlScore nc = ps - operfectScore; if(nc > minsc) { minsc = nc; } assert_leq(minsc, perfectScore); } DynProgFramer dpframe(!gReportOverhangs); swa.reset(); oswa.reset(); // Initialize a set of GroupWalks, one for each seed. Also, intialize the // accompanying lists of reference seed hits (satups*) const index_t nsm = 5; const index_t nonz = sh.nonzeroOffsets(); // non-zero positions index_t eeHits = sh.numE2eHits(); bool eeMode = eeHits > 0; bool firstEe = true; bool firstExtend = true; // Reset all the counters related to streaks prm.nEeFail = 0; prm.nUgFail = 0; prm.nDpFail = 0; index_t nelt = 0, neltLeft = 0; const index_t rows = rdlen; const index_t orows = ordlen; index_t eltsDone = 0; while(true) { if(eeMode) { if(firstEe) { firstEe = false; eeMode = eeSaTups( rd, // read sh, // seed hits to extend into full alignments gfmFw, // BWT ref, // Reference strings rnd, // pseudo-random generator wlm, // group walk left metrics swmSeed, // seed-extend metrics nelt, // out: # elements total maxIters, // max elts to report all); // report all hits assert_eq(gws_.size(), rands_.size()); assert_eq(gws_.size(), satpos_.size()); neltLeft = nelt; // Initialize list that contains the mate-finding failure // streak for each range mateStreaks_.resize(gws_.size()); mateStreaks_.fill(0); } else { eeMode = false; } } if(!eeMode) { if(nonz == 0) { // No seed hits! Bail. return EXTEND_EXHAUSTED_CANDIDATES; } if(msink->Mmode() && minsc == perfectScore) { // Already found all perfect hits! return EXTEND_PERFECT_SCORE; } if(firstExtend) { nelt = 0; prioritizeSATups( rd, // read sh, // seed hits to extend into full alignments gfmFw, // BWT gfmBw, // BWT' ref, // Reference strings seedmms, // # seed mismatches allowed maxIters, // max rows to consider per position doExtend, // extend out seeds true, // square extended length true, // square SA range size nsm, // smallness threshold ca, // alignment cache for seed hits rnd, // pseudo-random generator wlm, // group walk left metrics prm, // per-read metrics nelt, // out: # elements total all); // report all hits? assert_eq(gws_.size(), rands_.size()); assert_eq(gws_.size(), satpos_.size()); neltLeft = nelt; firstExtend = false; mateStreaks_.resize(gws_.size()); mateStreaks_.fill(0); } if(neltLeft == 0) { // Finished examining gapped candidates break; } } for(index_t i = 0; i < gws_.size(); i++) { if(eeMode && eehits_[i].score < minsc) { return EXTEND_PERFECT_SCORE; } bool is_small = satpos_[i].sat.size() < nsm; bool fw = satpos_[i].pos.fw; index_t rdoff = satpos_[i].pos.rdoff; index_t seedhitlen = satpos_[i].pos.seedlen; if(!fw) { // 'rdoff' and 'offidx' are with respect to the 5' end of // the read. Here we convert rdoff to be with respect to // the upstream (3') end of ther read. rdoff = (index_t)(rdlen - rdoff - seedhitlen); } bool first = true; // If the range is small, investigate all elements now. If the // range is large, just investigate one and move on - we might come // back to this range later. while(!rands_[i].done() && (first || is_small || eeMode)) { if(minsc == perfectScore) { if(!eeMode || eehits_[i].score < perfectScore) { return EXTEND_PERFECT_SCORE; } } else if(eeMode && eehits_[i].score < minsc) { break; } if(prm.nExDps >= maxDp || prm.nMateDps >= maxDp) { return EXTEND_EXCEEDED_HARD_LIMIT; } if(prm.nExUgs >= maxUg || prm.nMateUgs >= maxUg) { return EXTEND_EXCEEDED_HARD_LIMIT; } if(prm.nExIters >= maxIters) { return EXTEND_EXCEEDED_HARD_LIMIT; } if(eeMode && prm.nEeFail >= maxEeStreak) { return EXTEND_EXCEEDED_SOFT_LIMIT; } if(!eeMode && prm.nDpFail >= maxDpStreak) { return EXTEND_EXCEEDED_SOFT_LIMIT; } if(!eeMode && prm.nUgFail >= maxUgStreak) { return EXTEND_EXCEEDED_SOFT_LIMIT; } if(mateStreaks_[i] >= maxMateStreak) { // Don't try this seed range anymore rands_[i].setDone(); assert(rands_[i].done()); break; } prm.nExIters++; first = false; assert(!gws_[i].done()); // Resolve next element offset WalkResult wr; uint32_t elt = rands_[i].next(rnd); SARangeWithOffs sa; sa.topf = satpos_[i].sat.topf; sa.len = satpos_[i].sat.key.len; sa.offs = satpos_[i].sat.offs; gws_[i].advanceElement((index_t)elt, gfmFw, ref, sa, gwstate_, wr, wlm, prm); eltsDone++; assert_gt(neltLeft, 0); neltLeft--; assert_neq((index_t)OFF_MASK, wr.toff); index_t tidx = 0, toff = 0, tlen = 0; bool straddled = false; gfmFw.joinedToTextOff( wr.elt.len, wr.toff, tidx, toff, tlen, eeMode, // reject straddlers? straddled); // straddled? if(tidx == (index_t)OFF_MASK) { // The seed hit straddled a reference boundary so the seed hit // isn't valid continue; } #ifndef NDEBUG if(!eeMode && !straddled) { // Check that seed hit matches reference uint64_t key = satpos_[i].sat.key.seq; for(index_t k = 0; k < wr.elt.len; k++) { int c = ref.getBase(tidx, toff + wr.elt.len - k - 1); assert_leq(c, 3); int ck = (int)(key & 3); key >>= 2; assert_eq(c, ck); } } #endif // Find offset of alignment's upstream base assuming net gaps=0 // between beginning of read and beginning of seed hit int64_t refoff = (int64_t)toff - rdoff; EIvalMergeListBinned& seenDiags = anchor1 ? seenDiags1_ : seenDiags2_; // Coordinate of the seed hit w/r/t the pasted reference string Coord refcoord(tidx, refoff, fw); if(seenDiags.locusPresent(refcoord)) { // Already handled alignments seeded on this diagonal prm.nRedundants++; swmSeed.rshit++; continue; } // Now that we have a seed hit, there are many issues to solve // before we have a completely framed dynamic programming problem. // They include: // // 1. Setting reference offsets on either side of the seed hit, // accounting for where the seed occurs in the read // 2. Adjusting the width of the banded dynamic programming problem // and adjusting reference bounds to allow for gaps in the // alignment // 3. Accounting for the edges of the reference, which can impact // the width of the DP problem and reference bounds. // 4. Perhaps filtering the problem down to a smaller problem based // on what DPs we've already solved for this read // // We do #1 here, since it is simple and we have all the seed-hit // information here. #2 and #3 are handled in the DynProgFramer. int readGaps = 0, refGaps = 0; bool ungapped = false; if(!eeMode) { readGaps = sc.maxReadGaps(minsc, rdlen); refGaps = sc.maxRefGaps(minsc, rdlen); ungapped = (readGaps == 0 && refGaps == 0); } int state = FOUND_NONE; bool found = false; // In unpaired mode, a seed extension is successful if it // results in a full alignment that meets the minimum score // threshold. In paired-end mode, a seed extension is // successful if it results in a *full paired-end* alignment // that meets the minimum score threshold. if(eeMode) { resEe_.reset(); resEe_.alres.reset(); const EEHit& h = eehits_[i]; assert_leq(h.score, perfectScore); resEe_.alres.setScore(AlnScore(h.score, h.ns(), 0)); resEe_.alres.setShape( refcoord.ref(), // ref id refcoord.off(), // 0-based ref offset tlen, // reference length fw, // aligned to Watson? rdlen, // read length true, // pretrim soft? 0, // pretrim 5' end 0, // pretrim 3' end true, // alignment trim soft? 0, // alignment trim 5' end 0); // alignment trim 3' end resEe_.alres.setRefNs(h.refns()); if(h.mms() > 0) { assert_eq(1, h.mms()); assert_lt(h.e1.pos, rd.length()); resEe_.alres.ned().push_back(h.e1); } assert(resEe_.repOk(rd)); state = FOUND_EE; found = true; Interval refival(refcoord, 1); seenDiags.add(refival); prm.nExEes++; prm.nEeFail++; // say it's failed until proven successful prm.nExEeFails++; } else if(doUngapped && ungapped) { resUngap_.reset(); int al = swa.ungappedAlign( fw ? rd.patFw : rd.patRc, fw ? rd.qual : rd.qualRev, refcoord, ref, tlen, sc, gReportOverhangs, minsc, // minimum resUngap_); Interval refival(refcoord, 1); seenDiags.add(refival); prm.nExUgs++; prm.nUgFail++; // say it's failed until proven successful prm.nExUgFails++; if(al == 0) { swmSeed.ungapfail++; continue; } else if(al == -1) { swmSeed.ungapnodec++; } else { found = true; state = FOUND_UNGAPPED; swmSeed.ungapsucc++; } } int64_t pastedRefoff = (int64_t)wr.toff - rdoff; DPRect rect; if(state == FOUND_NONE) { found = dpframe.frameSeedExtensionRect( refoff, // ref offset implied by seed hit assuming no gaps rows, // length of read sequence used in DP table tlen, // length of reference readGaps, // max # of read gaps permitted in opp mate alignment refGaps, // max # of ref gaps permitted in opp mate alignment (size_t)nceil, // # Ns permitted maxhalf, // max width in either direction rect); // DP rectangle assert(rect.repOk()); // Add the seed diagonal at least seenDiags.add(Interval(refcoord, 1)); if(!found) { continue; } } int64_t leftShift = refoff - rect.refl; size_t nwindow = 0; if(toff >= rect.refl) { nwindow = (size_t)(toff - rect.refl); } // NOTE: We might be taking off more than we should because the // pasted string omits non-A/C/G/T characters, but we included them // when calculating leftShift. We'll account for this later. pastedRefoff -= leftShift; size_t nsInLeftShift = 0; if(state == FOUND_NONE) { if(!swa.initedRead()) { // Initialize the aligner with a new read swa.initRead( rd.patFw, // fw version of query rd.patRc, // rc version of query rd.qual, // fw version of qualities rd.qualRev,// rc version of qualities 0, // off of first char in 'rd' to consider rdlen, // off of last char (excl) in 'rd' to consider sc); // scoring scheme } swa.initRef( fw, // whether to align forward or revcomp read tidx, // reference aligned against rect, // DP rectangle ref, // Reference strings tlen, // length of reference sequence sc, // scoring scheme minsc, // minimum score permitted enable8, // use 8-bit SSE if possible? cminlen, // minimum length for using checkpointing scheme cpow2, // interval b/t checkpointed diags; 1 << this doTri, // triangular mini-fills? true, // this is a seed extension - not finding a mate nwindow, nsInLeftShift); // Because of how we framed the problem, we can say that we've // exhaustively scored the seed diagonal as well as maxgaps // diagonals on either side Interval refival(tidx, 0, fw, 0); rect.initIval(refival); seenDiags.add(refival); // Now fill the dynamic programming matrix and return true iff // there is at least one valid alignment TAlScore bestCell = std::numeric_limits::min(); found = swa.align(rnd, bestCell); swmSeed.tallyGappedDp(readGaps, refGaps); prm.nExDps++; prm.nDpFail++; // failed until proven successful prm.nExDpFails++; // failed until proven successful if(!found) { TAlScore bestLast = anchor1 ? prm.bestLtMinscMate1 : prm.bestLtMinscMate2; if(bestCell > std::numeric_limits::min() && bestCell > bestLast) { if(anchor1) { prm.bestLtMinscMate1 = bestCell; } else { prm.bestLtMinscMate2 = bestCell; } } continue; // Look for more anchor alignments } } bool firstInner = true; bool foundConcordant = false; while(true) { assert(found); SwResult *res = NULL; if(state == FOUND_EE) { if(!firstInner) { break; } res = &resEe_; assert(res->repOk(rd)); } else if(state == FOUND_UNGAPPED) { if(!firstInner) { break; } res = &resUngap_; assert(res->repOk(rd)); } else { resGap_.reset(); assert(resGap_.empty()); if(swa.done()) { break; } swa.nextAlignment(resGap_, minsc, rnd); found = !resGap_.empty(); if(!found) { break; } res = &resGap_; assert(res->repOk(rd)); } // TODO: If we're just taking anchor alignments out of the // same rectangle, aren't we getting very similar // rectangles for the opposite mate each time? Seems like // we could save some work by detecting this. assert(res != NULL); firstInner = false; assert(res->alres.matchesRef( rd, ref, tmp_rf_, tmp_rdseq_, tmp_qseq_, raw_refbuf_, raw_destU32_, raw_matches_, tmp_reflens_, tmp_refoffs_)); Interval refival(tidx, 0, fw, tlen); assert_gt(res->alres.refExtent(), 0); if(gReportOverhangs && !refival.containsIgnoreOrient(res->alres.refival())) { res->alres.clipOutside(true, 0, tlen); if(res->alres.refExtent() == 0) { continue; } } assert(gReportOverhangs || refival.containsIgnoreOrient(res->alres.refival())); // Did the alignment fall entirely outside the reference? if(!refival.overlapsIgnoreOrient(res->alres.refival())) { continue; } // Is this alignment redundant with one we've seen previously? if(redAnchor_.overlap(res->alres)) { continue; } redAnchor_.add(res->alres); // Annotate the AlnRes object with some key parameters // that were used to obtain the alignment. res->alres.setParams( seedmms, // # mismatches allowed in seed seedlen, // length of seed seedival, // interval between seeds minsc); // minimum score for valid alignment bool foundMate = false; TRefOff off = res->alres.refoff(); if( msink->state().doneWithMate(!anchor1) && !msink->state().doneWithMate( anchor1)) { // We're done with the opposite mate but not with the // anchor mate; don't try to mate up the anchor. swMateImmediately = false; } if(found && swMateImmediately) { assert(!msink->state().doneWithMate(!anchor1)); bool oleft = false, ofw = false; int64_t oll = 0, olr = 0, orl = 0, orr = 0; assert(!msink->state().done()); foundMate = !oppFilt; TAlScore ominsc_cur = ominsc; //bool oungapped = false; int oreadGaps = 0, orefGaps = 0; //int oungappedAlign = -1; // defer if(foundMate) { // Adjust ominsc given the alignment score of the // anchor mate ominsc_cur = ominsc; if(tighten > 0 && msink->Mmode() && msink->hasSecondBestPair()) { // Paired-end alignments should have at least this score from now TAlScore ps; if(tighten == 1) { ps = msink->bestPair(); } else if(tighten == 2) { ps = msink->secondBestPair(); } else { TAlScore diff = msink->bestPair() - msink->secondBestPair(); ps = msink->secondBestPair() + (diff * 3)/4; } if(tighten == 1 && ps < bestPairScore && msink->bestPair() == msink->secondBestPair()) { ps++; } if(tighten >= 2 && ps < bestPairScore) { ps++; } // Anchor mate must have score at least 'ps' minus the best possible // score for the opposite mate. TAlScore nc = ps - res->alres.score().score(); if(nc > ominsc_cur) { ominsc_cur = nc; assert_leq(ominsc_cur, operfectScore); } } oreadGaps = sc.maxReadGaps(ominsc_cur, ordlen); orefGaps = sc.maxRefGaps (ominsc_cur, ordlen); //oungapped = (oreadGaps == 0 && orefGaps == 0); // TODO: Something lighter-weight than DP to scan // for other mate?? //if(oungapped) { // oresUngap_.reset(); // oungappedAlign = oswa.ungappedAlign( // ofw ? ord.patFw : ord.patRc, // ofw ? ord.qual : ord.qualRev, // orefcoord, // ref, // otlen, // sc, // gReportOverhangs, // ominsc_cur, // 0, // oresUngap_); //} foundMate = pepol.otherMate( anchor1, // anchor mate is mate #1? fw, // anchor aligned to Watson? off, // offset of anchor mate orows + oreadGaps, // max # columns spanned by alignment tlen, // reference length anchor1 ? rd.length() : ord.length(), // mate 1 len anchor1 ? ord.length() : rd.length(), // mate 2 len oleft, // out: look left for opposite mate? oll, olr, orl, orr, ofw); } DPRect orect; if(foundMate) { foundMate = dpframe.frameFindMateRect( !oleft, // true iff anchor alignment is to the left oll, // leftmost Watson off for LHS of opp aln olr, // rightmost Watson off for LHS of opp aln orl, // leftmost Watson off for RHS of opp aln orr, // rightmost Watson off for RHS of opp aln orows, // length of opposite mate tlen, // length of reference sequence aligned to oreadGaps, // max # of read gaps in opp mate aln orefGaps, // max # of ref gaps in opp mate aln (size_t)onceil, // max # Ns on opp mate maxhalf, // max width in either direction orect); // DP rectangle assert(!foundMate || orect.refr >= orect.refl); } if(foundMate) { oresGap_.reset(); assert(oresGap_.empty()); if(!oswa.initedRead()) { oswa.initRead( ord.patFw, // read to align ord.patRc, // qualities ord.qual, // read to align ord.qualRev,// qualities 0, // off of first char to consider ordlen, // off of last char (ex) to consider sc); // scoring scheme } // Given the boundaries defined by refi and reff, initilize // the SwAligner with the dynamic programming problem that // aligns the read to this reference stretch. size_t onsInLeftShift = 0; assert_geq(orect.refr, orect.refl); oswa.initRef( ofw, // align forward or revcomp read? tidx, // reference aligned against orect, // DP rectangle ref, // Reference strings tlen, // length of reference sequence sc, // scoring scheme ominsc_cur,// min score for valid alignments enable8, // use 8-bit SSE if possible? cminlen, // minimum length for using checkpointing scheme cpow2, // interval b/t checkpointed diags; 1 << this doTri, // triangular mini-fills? false, // this is finding a mate - not seed ext 0, // nwindow? onsInLeftShift); // TODO: Can't we add some diagonals to the // opposite mate's seenDiags when we fill in the // opposite mate's DP? Or can we? We might want // to use this again as an anchor - will that still // happen? Also, isn't there a problem with // consistency of the minimum score? Minimum score // here depends in part on the score of the anchor // alignment here, but it won't when the current // opposite becomes the anchor. // Because of how we framed the problem, we can say // that we've exhaustively explored the "core" // diagonals //Interval orefival(tidx, 0, ofw, 0); //orect.initIval(orefival); //oseenDiags.add(orefival); // Now fill the dynamic programming matrix, return true // iff there is at least one valid alignment TAlScore bestCell = std::numeric_limits::min(); foundMate = oswa.align(rnd, bestCell); prm.nMateDps++; swmMate.tallyGappedDp(oreadGaps, orefGaps); if(!foundMate) { TAlScore bestLast = anchor1 ? prm.bestLtMinscMate2 : prm.bestLtMinscMate1; if(bestCell > std::numeric_limits::min() && bestCell > bestLast) { if(anchor1) { prm.bestLtMinscMate2 = bestCell; } else { prm.bestLtMinscMate1 = bestCell; } } } } bool didAnchor = false; do { oresGap_.reset(); assert(oresGap_.empty()); if(foundMate && oswa.done()) { foundMate = false; } else if(foundMate) { oswa.nextAlignment(oresGap_, ominsc_cur, rnd); foundMate = !oresGap_.empty(); assert(!foundMate || oresGap_.alres.matchesRef( ord, ref, tmp_rf_, tmp_rdseq_, tmp_qseq_, raw_refbuf_, raw_destU32_, raw_matches_, tmp_reflens_, tmp_refoffs_)); } if(foundMate) { // Redundant with one we've seen previously? if(!redAnchor_.overlap(oresGap_.alres)) { redAnchor_.add(oresGap_.alres); } assert_eq(ofw, oresGap_.alres.fw()); // Annotate the AlnRes object with some key parameters // that were used to obtain the alignment. oresGap_.alres.setParams( seedmms, // # mismatches allowed in seed seedlen, // length of seed seedival, // interval between seeds ominsc); // minimum score for valid alignment assert_gt(oresGap_.alres.refExtent(), 0); if(gReportOverhangs && !refival.containsIgnoreOrient(oresGap_.alres.refival())) { oresGap_.alres.clipOutside(true, 0, tlen); foundMate = oresGap_.alres.refExtent() > 0; } if(foundMate && ((!gReportOverhangs && !refival.containsIgnoreOrient(oresGap_.alres.refival())) || !refival.overlapsIgnoreOrient(oresGap_.alres.refival()))) { foundMate = false; } } ASSERT_ONLY(TRefId refid); TRefOff off1, off2; size_t len1, len2; bool fw1, fw2; int pairCl = PE_ALS_DISCORD; if(foundMate) { ASSERT_ONLY(refid =) res->alres.refid(); assert_eq(refid, oresGap_.alres.refid()); off1 = anchor1 ? off : oresGap_.alres.refoff(); off2 = anchor1 ? oresGap_.alres.refoff() : off; len1 = anchor1 ? res->alres.refExtent() : oresGap_.alres.refExtent(); len2 = anchor1 ? oresGap_.alres.refExtent() : res->alres.refExtent(); fw1 = anchor1 ? res->alres.fw() : oresGap_.alres.fw(); fw2 = anchor1 ? oresGap_.alres.fw() : res->alres.fw(); // Check that final mate alignments are consistent with // paired-end fragment constraints pairCl = pepol.peClassifyPair( off1, len1, fw1, off2, len2, fw2); // Instead of trying //foundMate = pairCl != PE_ALS_DISCORD; } if(msink->state().doneConcordant()) { foundMate = false; } if(reportImmediately) { if(foundMate) { // Report pair to the AlnSinkWrap assert(!msink->state().doneConcordant()); assert(msink != NULL); assert(res->repOk()); assert(oresGap_.repOk()); // Report an unpaired alignment assert(!msink->maxed()); assert(!msink->state().done()); bool doneUnpaired = false; //if(mixed || discord) { // Report alignment for mate #1 as an // unpaired alignment. if(!anchor1 || !didAnchor) { if(anchor1) { didAnchor = true; } const AlnRes& r1 = anchor1 ? res->alres : oresGap_.alres; if(!redMate1_.overlap(r1)) { redMate1_.add(r1); if(msink->report(0, &r1, NULL)) { doneUnpaired = true; // Short-circuited } } } // Report alignment for mate #2 as an // unpaired alignment. if(anchor1 || !didAnchor) { if(!anchor1) { didAnchor = true; } const AlnRes& r2 = anchor1 ? oresGap_.alres : res->alres; if(!redMate2_.overlap(r2)) { redMate2_.add(r2); if(msink->report(0, NULL, &r2)) { doneUnpaired = true; // Short-circuited } } } //} // if(mixed || discord) bool donePaired = false; if(pairCl != PE_ALS_DISCORD) { foundConcordant = true; if(msink->report( 0, anchor1 ? &res->alres : &oresGap_.alres, anchor1 ? &oresGap_.alres : &res->alres)) { // Short-circuited because a limit, e.g. // -k, -m or -M, was exceeded donePaired = true; } else { if(tighten > 0 && msink->Mmode() && msink->hasSecondBestPair()) { // Paired-end alignments should have at least this score from now TAlScore ps; if(tighten == 1) { ps = msink->bestPair(); } else if(tighten == 2) { ps = msink->secondBestPair(); } else { TAlScore diff = msink->bestPair() - msink->secondBestPair(); ps = msink->secondBestPair() + (diff * 3)/4; } if(tighten == 1 && ps < bestPairScore && msink->bestPair() == msink->secondBestPair()) { ps++; } if(tighten >= 2 && ps < bestPairScore) { ps++; } // Anchor mate must have score at least 'ps' minus the best possible // score for the opposite mate. TAlScore nc = ps - operfectScore; if(nc > minsc) { minsc = nc; assert_leq(minsc, perfectScore); if(minsc > res->alres.score().score()) { // We're done with this anchor break; } } assert_leq(minsc, perfectScore); } } } // if(pairCl != PE_ALS_DISCORD) if(donePaired || doneUnpaired) { return EXTEND_POLICY_FULFILLED; } if(msink->state().doneWithMate(anchor1)) { // We're now done with the mate that we're // currently using as our anchor. We're not // with the read overall. return EXTEND_POLICY_FULFILLED; } } else if((mixed || discord) && !didAnchor) { didAnchor = true; // Report unpaired hit for anchor assert(msink != NULL); assert(res->repOk()); // Check that alignment accurately reflects the // reference characters aligned to assert(res->alres.matchesRef( rd, ref, tmp_rf_, tmp_rdseq_, tmp_qseq_, raw_refbuf_, raw_destU32_, raw_matches_, tmp_reflens_, tmp_refoffs_)); // Report an unpaired alignment assert(!msink->maxed()); assert(!msink->state().done()); // Report alignment for mate #1 as an // unpaired alignment. if(!msink->state().doneUnpaired(anchor1)) { const AlnRes& r = res->alres; RedundantAlns& red = anchor1 ? redMate1_ : redMate2_; const AlnRes* r1 = anchor1 ? &res->alres : NULL; const AlnRes* r2 = anchor1 ? NULL : &res->alres; if(!red.overlap(r)) { red.add(r); if(msink->report(0, r1, r2)) { return EXTEND_POLICY_FULFILLED; // Short-circuited } } } if(msink->state().doneWithMate(anchor1)) { // Done with mate, but not read overall return EXTEND_POLICY_FULFILLED; } } } } while(!oresGap_.empty()); } // if(found && swMateImmediately) else if(found) { assert(!msink->state().doneWithMate(anchor1)); // We found an anchor alignment but did not attempt to find // an alignment for the opposite mate (probably because // we're done with it) if(reportImmediately && (mixed || discord)) { // Report unpaired hit for anchor assert(msink != NULL); assert(res->repOk()); // Check that alignment accurately reflects the // reference characters aligned to assert(res->alres.matchesRef( rd, ref, tmp_rf_, tmp_rdseq_, tmp_qseq_, raw_refbuf_, raw_destU32_, raw_matches_, tmp_reflens_, tmp_refoffs_)); // Report an unpaired alignment assert(!msink->maxed()); assert(!msink->state().done()); // Report alignment for mate #1 as an // unpaired alignment. if(!msink->state().doneUnpaired(anchor1)) { const AlnRes& r = res->alres; RedundantAlns& red = anchor1 ? redMate1_ : redMate2_; const AlnRes* r1 = anchor1 ? &res->alres : NULL; const AlnRes* r2 = anchor1 ? NULL : &res->alres; if(!red.overlap(r)) { red.add(r); if(msink->report(0, r1, r2)) { return EXTEND_POLICY_FULFILLED; // Short-circuited } } } if(msink->state().doneWithMate(anchor1)) { // Done with mate, but not read overall return EXTEND_POLICY_FULFILLED; } } } } // while(true) if(foundConcordant) { prm.nMateDpSuccs++; mateStreaks_[i] = 0; // Register this as a success. Now we need to // make the streak variables reflect the // success. if(state == FOUND_UNGAPPED) { assert_gt(prm.nUgFail, 0); assert_gt(prm.nExUgFails, 0); prm.nExUgFails--; prm.nExUgSuccs++; prm.nUgLastSucc = prm.nExUgs-1; if(prm.nUgFail > prm.nUgFailStreak) { prm.nUgFailStreak = prm.nUgFail; } prm.nUgFail = 0; } else if(state == FOUND_EE) { assert_gt(prm.nEeFail, 0); assert_gt(prm.nExEeFails, 0); prm.nExEeFails--; prm.nExEeSuccs++; prm.nEeLastSucc = prm.nExEes-1; if(prm.nEeFail > prm.nEeFailStreak) { prm.nEeFailStreak = prm.nEeFail; } prm.nEeFail = 0; } else { assert_gt(prm.nDpFail, 0); assert_gt(prm.nExDpFails, 0); prm.nExDpFails--; prm.nExDpSuccs++; prm.nDpLastSucc = prm.nExDps-1; if(prm.nDpFail > prm.nDpFailStreak) { prm.nDpFailStreak = prm.nDpFail; } prm.nDpFail = 0; } } else { prm.nMateDpFails++; mateStreaks_[i]++; } // At this point we know that we aren't bailing, and will continue to resolve seed hits. } // while(!gw.done()) } // for(size_t i = 0; i < gws_.size(); i++) } return EXTEND_EXHAUSTED_CANDIDATES; } #endif /*ALIGNER_SW_DRIVER_H_*/ hisat2-2.2.1/aligner_sw_nuc.h000066400000000000000000000160551370663763200161020ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_SW_NUC_H_ #define ALIGNER_SW_NUC_H_ #include #include "aligner_sw_common.h" #include "aligner_result.h" /** * Encapsulates a backtrace stack frame. Includes enough information that we * can "pop" back up to this frame and choose to make a different backtracking * decision. The information included is: * * 1. The mask at the decision point. When we first move through the mask and * when we backtrack to it, we're careful to mask out the bit corresponding * to the path we're taking. When we move through it after removing the * last bit from the mask, we're careful to pop it from the stack. * 2. The sizes of the edit lists. When we backtrack, we resize the lists back * down to these sizes to get rid of any edits introduced since the branch * point. */ struct DpNucFrame { /** * Initialize a new DpNucFrame stack frame. */ void init( size_t nedsz_, size_t aedsz_, size_t celsz_, size_t row_, size_t col_, size_t gaps_, size_t readGaps_, size_t refGaps_, AlnScore score_, int ct_) { nedsz = nedsz_; aedsz = aedsz_; celsz = celsz_; row = row_; col = col_; gaps = gaps_; readGaps = readGaps_; refGaps = refGaps_; score = score_; ct = ct_; } size_t nedsz; // size of the nucleotide edit list at branch (before // adding the branch edit) size_t aedsz; // size of ambiguous nucleotide edit list at branch size_t celsz; // size of cell-traversed list at branch size_t row; // row of cell where branch occurred size_t col; // column of cell where branch occurred size_t gaps; // number of gaps before branch occurred size_t readGaps; // number of read gaps before branch occurred size_t refGaps; // number of ref gaps before branch occurred AlnScore score; // score where branch occurred int ct; // table type (oall, rdgap or rfgap) }; enum { BT_CAND_FATE_SUCCEEDED = 1, BT_CAND_FATE_FAILED, BT_CAND_FATE_FILT_START, // skipped b/c starting cell already explored BT_CAND_FATE_FILT_DOMINATED, // skipped b/c it was dominated BT_CAND_FATE_FILT_SCORE // skipped b/c score not interesting anymore }; /** * Encapsulates a cell that we might want to backtrace from. */ struct DpBtCandidate { DpBtCandidate() { reset(); } DpBtCandidate(size_t row_, size_t col_, TAlScore score_) { init(row_, col_, score_); } void reset() { init(0, 0, 0); } void init(size_t row_, size_t col_, TAlScore score_) { row = row_; col = col_; score = score_; // 0 = invalid; this should be set later according to what happens // before / during the backtrace fate = 0; } /** * Return true iff this candidate is (heuristically) dominated by the given * candidate. We say that candidate A dominates candidate B if (a) B is * somewhere in the N x N square that extends up and to the left of A, * where N is an arbitrary number like 20, and (b) B's score is <= than * A's. */ inline bool dominatedBy(const DpBtCandidate& o) { const size_t SQ = 40; size_t rowhi = row; size_t rowlo = o.row; if(rowhi < rowlo) swap(rowhi, rowlo); size_t colhi = col; size_t collo = o.col; if(colhi < collo) swap(colhi, collo); return (colhi - collo) <= SQ && (rowhi - rowlo) <= SQ; } /** * Return true if this candidate is "greater than" (should be considered * later than) the given candidate. */ bool operator>(const DpBtCandidate& o) const { if(score < o.score) return true; if(score > o.score) return false; if(row < o.row ) return true; if(row > o.row ) return false; if(col < o.col ) return true; if(col > o.col ) return false; return false; } /** * Return true if this candidate is "less than" (should be considered * sooner than) the given candidate. */ bool operator<(const DpBtCandidate& o) const { if(score > o.score) return true; if(score < o.score) return false; if(row > o.row ) return true; if(row < o.row ) return false; if(col > o.col ) return true; if(col < o.col ) return false; return false; } /** * Return true if this candidate equals the given candidate. */ bool operator==(const DpBtCandidate& o) const { return row == o.row && col == o.col && score == o.score; } bool operator>=(const DpBtCandidate& o) const { return !((*this) < o); } bool operator<=(const DpBtCandidate& o) const { return !((*this) > o); } #ifndef NDEBUG /** * Check internal consistency. */ bool repOk() const { assert(VALID_SCORE(score)); return true; } #endif size_t row; // cell row size_t col; // cell column w/r/t LHS of rectangle TAlScore score; // score fo alignment int fate; // flag indicating whether we succeeded, failed, skipped }; template class NBest { public: NBest() { nelt_ = nbest_ = n_ = 0; } bool inited() const { return nelt_ > 0; } void init(size_t nelt, size_t nbest) { nelt_ = nelt; nbest_ = nbest; elts_.resize(nelt * nbest); ncur_.resize(nelt); ncur_.fill(0); n_ = 0; } /** * Add a new result to bin 'elt'. Where it gets prioritized in the list of * results in that bin depends on the result of operator>. */ bool add(size_t elt, const T& o) { assert_lt(elt, nelt_); const size_t ncur = ncur_[elt]; assert_leq(ncur, nbest_); n_++; for(size_t i = 0; i < nbest_ && i <= ncur; i++) { if(o > elts_[nbest_ * elt + i] || i >= ncur) { // Insert it here // Move everyone from here on down by one slot for(int j = (int)ncur; j > (int)i; j--) { if(j < (int)nbest_) { elts_[nbest_ * elt + j] = elts_[nbest_ * elt + j - 1]; } } elts_[nbest_ * elt + i] = o; if(ncur < nbest_) { ncur_[elt]++; } return true; } } return false; } /** * Return true iff there are no solutions. */ bool empty() const { return n_ == 0; } /** * Dump all the items in our payload into the given EList. */ template void dump(TList& l) const { if(empty()) return; for(size_t i = 0; i < nelt_; i++) { assert_leq(ncur_[i], nbest_); for(size_t j = 0; j < ncur_[i]; j++) { l.push_back(elts_[i * nbest_ + j]); } } } protected: size_t nelt_; size_t nbest_; EList elts_; EList ncur_; size_t n_; // total # results added }; #endif /*def ALIGNER_SW_NUC_H_*/ hisat2-2.2.1/aligner_swsse.cpp000066400000000000000000000051541370663763200163010ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include "aligner_sw_common.h" #include "aligner_swsse.h" /** * Given a number of rows (nrow), a number of columns (ncol), and the * number of words to fit inside a single __m128i vector, initialize the * matrix buffer to accomodate the needed configuration of vectors. */ void SSEMatrix::init( size_t nrow, size_t ncol, size_t wperv) { nrow_ = nrow; ncol_ = ncol; wperv_ = wperv; nvecPerCol_ = (nrow + (wperv-1)) / wperv; // The +1 is so that we don't have to special-case the final column; // instead, we just write off the end of the useful part of the table // with pvEStore. try { matbuf_.resizeNoCopy((ncol+1) * nvecPerCell_ * nvecPerCol_); } catch(exception& e) { cerr << "Tried to allocate DP matrix with " << (ncol+1) << " columns, " << nvecPerCol_ << " vectors per column, and and " << nvecPerCell_ << " vectors per cell" << endl; throw e; } assert(wperv_ == 8 || wperv_ == 16); vecshift_ = (wperv_ == 8) ? 3 : 4; nvecrow_ = (nrow + (wperv_-1)) >> vecshift_; nveccol_ = ncol; colstride_ = nvecPerCol_ * nvecPerCell_; rowstride_ = nvecPerCell_; inited_ = true; } /** * Initialize the matrix of masks and backtracking flags. */ void SSEMatrix::initMasks() { assert_gt(nrow_, 0); assert_gt(ncol_, 0); masks_.resize(nrow_); reset_.resizeNoCopy(nrow_); reset_.fill(false); } /** * Given a row, col and matrix (i.e. E, F or H), return the corresponding * element. */ int SSEMatrix::eltSlow(size_t row, size_t col, size_t mat) const { assert_lt(row, nrow_); assert_lt(col, ncol_); assert_leq(mat, 3); // Move to beginning of column/row size_t rowelt = row / nvecrow_; size_t rowvec = row % nvecrow_; size_t eltvec = (col * colstride_) + (rowvec * rowstride_) + mat; if(wperv_ == 16) { return (int)((uint8_t*)(matbuf_.ptr() + eltvec))[rowelt]; } else { assert_eq(8, wperv_); return (int)((int16_t*)(matbuf_.ptr() + eltvec))[rowelt]; } } hisat2-2.2.1/aligner_swsse.h000066400000000000000000000354551370663763200157550ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALIGNER_SWSSE_H_ #define ALIGNER_SWSSE_H_ #include "ds.h" #include "mem_ids.h" #include "random_source.h" #include "scoring.h" #include "mask.h" #include "sse_util.h" #include struct SSEMetrics { SSEMetrics():mutex_m() { reset(); } void clear() { reset(); } void reset() { dp = dpsat = dpfail = dpsucc = col = cell = inner = fixup = gathsol = bt = btfail = btsucc = btcell = corerej = nrej = 0; } void merge(const SSEMetrics& o, bool getLock = false) { ThreadSafe ts(&mutex_m, getLock); dp += o.dp; dpsat += o.dpsat; dpfail += o.dpfail; dpsucc += o.dpsucc; col += o.col; cell += o.cell; inner += o.inner; fixup += o.fixup; gathsol += o.gathsol; bt += o.bt; btfail += o.btfail; btsucc += o.btsucc; btcell += o.btcell; corerej += o.corerej; nrej += o.nrej; } uint64_t dp; // DPs tried uint64_t dpsat; // DPs saturated uint64_t dpfail; // DPs failed uint64_t dpsucc; // DPs succeeded uint64_t col; // DP columns uint64_t cell; // DP cells uint64_t inner; // DP inner loop iters uint64_t fixup; // DP fixup loop iters uint64_t gathsol; // DP gather solution cells found uint64_t bt; // DP backtraces uint64_t btfail; // DP backtraces failed uint64_t btsucc; // DP backtraces succeeded uint64_t btcell; // DP backtrace cells traversed uint64_t corerej; // DP backtrace core rejections uint64_t nrej; // DP backtrace N rejections MUTEX_T mutex_m; }; /** * Encapsulates matrix information calculated by the SSE aligner. * * Matrix memory is laid out as follows: * * - Elements (individual cell scores) are packed into __m128i vectors * - Vectors are packed into quartets, quartet elements correspond to: a vector * from E, one from F, one from H, and one that's "reserved" * - Quartets are packed into columns, where the number of quartets is * determined by the number of query characters divided by the number of * elements per vector * * Regarding the "reserved" element of the vector quartet: we use it for two * things. First, we use the first column of reserved vectors to stage the * initial column of H vectors. Second, we use the "reserved" vectors during * the backtrace procedure to store information about (a) which cells have been * traversed, (b) whether the cell is "terminal" (in local mode), etc. */ struct SSEMatrix { // Each matrix element is a quartet of vectors. These constants are used // to identify members of the quartet. const static size_t E = 0; const static size_t F = 1; const static size_t H = 2; const static size_t TMP = 3; SSEMatrix(int cat = 0) : nvecPerCell_(4), matbuf_(cat) { } /** * Return a pointer to the matrix buffer. */ inline __m128i *ptr() { assert(inited_); return matbuf_.ptr(); } /** * Return a pointer to the E vector at the given row and column. Note: * here row refers to rows of vectors, not rows of elements. */ inline __m128i* evec(size_t row, size_t col) { assert_lt(row, nvecrow_); assert_lt(col, nveccol_); size_t elt = row * rowstride() + col * colstride() + E; assert_lt(elt, matbuf_.size()); return ptr() + elt; } /** * Like evec, but it's allowed to ask for a pointer to one column after the * final one. */ inline __m128i* evecUnsafe(size_t row, size_t col) { assert_lt(row, nvecrow_); assert_leq(col, nveccol_); size_t elt = row * rowstride() + col * colstride() + E; assert_lt(elt, matbuf_.size()); return ptr() + elt; } /** * Return a pointer to the F vector at the given row and column. Note: * here row refers to rows of vectors, not rows of elements. */ inline __m128i* fvec(size_t row, size_t col) { assert_lt(row, nvecrow_); assert_lt(col, nveccol_); size_t elt = row * rowstride() + col * colstride() + F; assert_lt(elt, matbuf_.size()); return ptr() + elt; } /** * Return a pointer to the H vector at the given row and column. Note: * here row refers to rows of vectors, not rows of elements. */ inline __m128i* hvec(size_t row, size_t col) { assert_lt(row, nvecrow_); assert_lt(col, nveccol_); size_t elt = row * rowstride() + col * colstride() + H; assert_lt(elt, matbuf_.size()); return ptr() + elt; } /** * Return a pointer to the TMP vector at the given row and column. Note: * here row refers to rows of vectors, not rows of elements. */ inline __m128i* tmpvec(size_t row, size_t col) { assert_lt(row, nvecrow_); assert_lt(col, nveccol_); size_t elt = row * rowstride() + col * colstride() + TMP; assert_lt(elt, matbuf_.size()); return ptr() + elt; } /** * Like tmpvec, but it's allowed to ask for a pointer to one column after * the final one. */ inline __m128i* tmpvecUnsafe(size_t row, size_t col) { assert_lt(row, nvecrow_); assert_leq(col, nveccol_); size_t elt = row * rowstride() + col * colstride() + TMP; assert_lt(elt, matbuf_.size()); return ptr() + elt; } /** * Given a number of rows (nrow), a number of columns (ncol), and the * number of words to fit inside a single __m128i vector, initialize the * matrix buffer to accomodate the needed configuration of vectors. */ void init( size_t nrow, size_t ncol, size_t wperv); /** * Return the number of __m128i's you need to skip over to get from one * cell to the cell one column over from it. */ inline size_t colstride() const { return colstride_; } /** * Return the number of __m128i's you need to skip over to get from one * cell to the cell one row down from it. */ inline size_t rowstride() const { return rowstride_; } /** * Given a row, col and matrix (i.e. E, F or H), return the corresponding * element. */ int eltSlow(size_t row, size_t col, size_t mat) const; /** * Given a row, col and matrix (i.e. E, F or H), return the corresponding * element. */ inline int elt(size_t row, size_t col, size_t mat) const { assert(inited_); assert_lt(row, nrow_); assert_lt(col, ncol_); assert_lt(mat, 3); // Move to beginning of column/row size_t rowelt = row / nvecrow_; size_t rowvec = row % nvecrow_; size_t eltvec = (col * colstride_) + (rowvec * rowstride_) + mat; assert_lt(eltvec, matbuf_.size()); if(wperv_ == 16) { return (int)((uint8_t*)(matbuf_.ptr() + eltvec))[rowelt]; } else { assert_eq(8, wperv_); return (int)((int16_t*)(matbuf_.ptr() + eltvec))[rowelt]; } } /** * Return the element in the E matrix at element row, col. */ inline int eelt(size_t row, size_t col) const { return elt(row, col, E); } /** * Return the element in the F matrix at element row, col. */ inline int felt(size_t row, size_t col) const { return elt(row, col, F); } /** * Return the element in the H matrix at element row, col. */ inline int helt(size_t row, size_t col) const { return elt(row, col, H); } /** * Return true iff the given cell has its reportedThru bit set. */ inline bool reportedThrough( size_t row, // current row size_t col) const // current column { return (masks_[row][col] & (1 << 0)) != 0; } /** * Set the given cell's reportedThru bit. */ inline void setReportedThrough( size_t row, // current row size_t col) // current column { masks_[row][col] |= (1 << 0); } /** * Return true iff the H mask has been set with a previous call to hMaskSet. */ bool isHMaskSet( size_t row, // current row size_t col) const; // current column /** * Set the given cell's H mask. This is the mask of remaining legal ways to * backtrack from the H cell at this coordinate. It's 5 bits long and has * offset=2 into the 16-bit field. */ void hMaskSet( size_t row, // current row size_t col, // current column int mask); /** * Return true iff the E mask has been set with a previous call to eMaskSet. */ bool isEMaskSet( size_t row, // current row size_t col) const; // current column /** * Set the given cell's E mask. This is the mask of remaining legal ways to * backtrack from the E cell at this coordinate. It's 2 bits long and has * offset=8 into the 16-bit field. */ void eMaskSet( size_t row, // current row size_t col, // current column int mask); /** * Return true iff the F mask has been set with a previous call to fMaskSet. */ bool isFMaskSet( size_t row, // current row size_t col) const; // current column /** * Set the given cell's F mask. This is the mask of remaining legal ways to * backtrack from the F cell at this coordinate. It's 2 bits long and has * offset=11 into the 16-bit field. */ void fMaskSet( size_t row, // current row size_t col, // current column int mask); /** * Analyze a cell in the SSE-filled dynamic programming matrix. Determine & * memorize ways that we can backtrack from the cell. If there is at least one * way to backtrack, select one at random and return the selection. * * There are a few subtleties to keep in mind regarding which cells can be at * the end of a backtrace. First of all: cells from which we can backtrack * should not be at the end of a backtrace. But have to distinguish between * cells whose masks eventually become 0 (we shouldn't end at those), from * those whose masks were 0 all along (we can end at those). */ void analyzeCell( size_t row, // current row size_t col, // current column size_t ct, // current cell type: E/F/H int refc, int readc, int readq, const Scoring& sc, // scoring scheme int64_t offsetsc, // offset to add to each score RandomSource& rand, // rand gen for choosing among equal options bool& empty, // out: =true iff no way to backtrace int& cur, // out: =type of transition bool& branch, // out: =true iff we chose among >1 options bool& canMoveThru, // out: =true iff ... bool& reportedThru); // out: =true iff ... /** * Initialize the matrix of masks and backtracking flags. */ void initMasks(); /** * Return the number of rows in the dynamic programming matrix. */ size_t nrow() const { return nrow_; } /** * Return the number of columns in the dynamic programming matrix. */ size_t ncol() const { return ncol_; } /** * Prepare a row so we can use it to store masks. */ void resetRow(size_t i) { assert(!reset_[i]); masks_[i].resizeNoCopy(ncol_); masks_[i].fillZero(); reset_[i] = true; } bool inited_; // initialized? size_t nrow_; // # rows size_t ncol_; // # columns size_t nvecrow_; // # vector rows (<= nrow_) size_t nveccol_; // # vector columns (<= ncol_) size_t wperv_; // # words per vector size_t vecshift_; // # bits to shift to divide by words per vec size_t nvecPerCol_; // # vectors per column size_t nvecPerCell_; // # vectors per matrix cell (4) size_t colstride_; // # vectors b/t adjacent cells in same row size_t rowstride_; // # vectors b/t adjacent cells in same col EList_m128i matbuf_; // buffer for holding vectors ELList masks_; // buffer for masks/backtracking flags EList reset_; // true iff row in masks_ has been reset }; /** * All the data associated with the query profile and other data needed for SSE * alignment of a query. */ struct SSEData { SSEData(int cat = 0) : profbuf_(cat), mat_(cat) { } EList_m128i profbuf_; // buffer for query profile & temp vecs EList_m128i vecbuf_; // buffer for 2 column vectors (not using mat_) size_t qprofStride_; // stride for query profile size_t gbarStride_; // gap barrier for query profile SSEMatrix mat_; // SSE matrix for holding all E, F, H vectors size_t maxPen_; // biggest penalty of all size_t maxBonus_; // biggest bonus of all size_t lastIter_; // which 128-bit striped word has final row? size_t lastWord_; // which word within 128-word has final row? int bias_; // all scores shifted up by this for unsigned }; /** * Return true iff the H mask has been set with a previous call to hMaskSet. */ inline bool SSEMatrix::isHMaskSet( size_t row, // current row size_t col) const // current column { return (masks_[row][col] & (1 << 1)) != 0; } /** * Set the given cell's H mask. This is the mask of remaining legal ways to * backtrack from the H cell at this coordinate. It's 5 bits long and has * offset=2 into the 16-bit field. */ inline void SSEMatrix::hMaskSet( size_t row, // current row size_t col, // current column int mask) { assert_lt(mask, 32); masks_[row][col] &= ~(31 << 1); masks_[row][col] |= (1 << 1 | mask << 2); } /** * Return true iff the E mask has been set with a previous call to eMaskSet. */ inline bool SSEMatrix::isEMaskSet( size_t row, // current row size_t col) const // current column { return (masks_[row][col] & (1 << 7)) != 0; } /** * Set the given cell's E mask. This is the mask of remaining legal ways to * backtrack from the E cell at this coordinate. It's 2 bits long and has * offset=8 into the 16-bit field. */ inline void SSEMatrix::eMaskSet( size_t row, // current row size_t col, // current column int mask) { assert_lt(mask, 4); masks_[row][col] &= ~(7 << 7); masks_[row][col] |= (1 << 7 | mask << 8); } /** * Return true iff the F mask has been set with a previous call to fMaskSet. */ inline bool SSEMatrix::isFMaskSet( size_t row, // current row size_t col) const // current column { return (masks_[row][col] & (1 << 10)) != 0; } /** * Set the given cell's F mask. This is the mask of remaining legal ways to * backtrack from the F cell at this coordinate. It's 2 bits long and has * offset=11 into the 16-bit field. */ inline void SSEMatrix::fMaskSet( size_t row, // current row size_t col, // current column int mask) { assert_lt(mask, 4); masks_[row][col] &= ~(7 << 10); masks_[row][col] |= (1 << 10 | mask << 11); } #define ROWSTRIDE_2COL 4 #define ROWSTRIDE 4 #endif /*ndef ALIGNER_SWSSE_H_*/ hisat2-2.2.1/aligner_swsse_ee_i16.cpp000066400000000000000000001742031370663763200174330ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /** * aligner_sw_sse.cpp * * Versions of key alignment functions that use vector instructions to * accelerate dynamic programming. Based chiefly on the striped Smith-Waterman * paper and implementation by Michael Farrar. See: * * Farrar M. Striped Smith-Waterman speeds database searches six times over * other SIMD implementations. Bioinformatics. 2007 Jan 15;23(2):156-61. * http://sites.google.com/site/farrarmichael/smith-waterman * * While the paper describes an implementation of Smith-Waterman, we extend it * do end-to-end read alignment as well as local alignment. The change * required for this is minor: we simply let vmax be the maximum element in the * score domain rather than the minimum. * * The vectorized dynamic programming implementation lacks some features that * make it hard to adapt to solving the entire dynamic-programming alignment * problem. For instance: * * - It doesn't respect gap barriers on either end of the read * - It just gives a maximum; not enough information to backtrace without * redoing some alignment * - It's a little difficult to handle st_ and en_, especially st_. * - The query profile mechanism makes handling of ambiguous reference bases a * little tricky (16 cols in query profile lookup table instead of 5) * * Given the drawbacks, it is tempting to use SSE dynamic programming as a * filter rather than as an aligner per se. Here are a few ideas for how it * can be extended to handle more of the alignment problem: * * - Save calculated scores to a big array as we go. We return to this array * to find and backtrace from good solutions. */ #include #include "aligner_sw.h" static const size_t NBYTES_PER_REG = 16; static const size_t NWORDS_PER_REG = 8; static const size_t NBITS_PER_WORD = 16; static const size_t NBYTES_PER_WORD = 2; // In 16-bit end-to-end mode, we have the option of using signed saturated // arithmetic. Because we have signed arithmetic, there's no need to add/subtract // bias when building an applying the query profile. The lowest value we can // use is 0x8000, and the greatest is 0x7fff. typedef int16_t TCScore; /** * Build query profile look up tables for the read. The query profile look * up table is organized as a 1D array indexed by [i][j] where i is the * reference character in the current DP column (0=A, 1=C, etc), and j is * the segment of the query we're currently working on. */ void SwAligner::buildQueryProfileEnd2EndSseI16(bool fw) { bool& done = fw ? sseI16fwBuilt_ : sseI16rcBuilt_; if(done) { return; } done = true; const BTDnaString* rd = fw ? rdfw_ : rdrc_; const BTString* qu = fw ? qufw_ : qurc_; // daehwan - allows to align a portion of a read, not the whole // const size_t len = rd->length(); const size_t len = dpRows(); const size_t seglen = (len + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // How many __m128i's are needed size_t n128s = 64 + // slack bytes, for alignment? (seglen * ALPHA_SIZE) // query profile data * 2; // & gap barrier data assert_gt(n128s, 0); SSEData& d = fw ? sseI16fw_ : sseI16rc_; d.profbuf_.resizeNoCopy(n128s); assert(!d.profbuf_.empty()); d.maxPen_ = d.maxBonus_ = 0; d.lastIter_ = d.lastWord_ = 0; d.qprofStride_ = d.gbarStride_ = 2; d.bias_ = 0; // no bias when words are signed // For each reference character A, C, G, T, N ... for(size_t refc = 0; refc < ALPHA_SIZE; refc++) { // For each segment ... for(size_t i = 0; i < seglen; i++) { size_t j = i; int16_t *qprofWords = reinterpret_cast(d.profbuf_.ptr() + (refc * seglen * 2) + (i * 2)); int16_t *gbarWords = reinterpret_cast(d.profbuf_.ptr() + (refc * seglen * 2) + (i * 2) + 1); // For each sub-word (byte) ... for(size_t k = 0; k < NWORDS_PER_REG; k++) { int sc = 0; *gbarWords = 0; if(j < len) { int readc = (*rd)[j]; int readq = (*qu)[j]; sc = sc_->score(readc, (int)(1 << refc), readq - 33); size_t j_from_end = len - j - 1; if(j < (size_t)sc_->gapbar || j_from_end < (size_t)sc_->gapbar) { // Inside the gap barrier *gbarWords = 0x8000; // add this twice } } if(refc == 0 && j == len-1) { // Remember which 128-bit word and which smaller word has // the final row d.lastIter_ = i; d.lastWord_ = k; } if(sc < 0) { if((size_t)(-sc) > d.maxPen_) { d.maxPen_ = (size_t)(-sc); } } else { if((size_t)sc > d.maxBonus_) { d.maxBonus_ = (size_t)sc; } } *qprofWords = (int16_t)sc; gbarWords++; qprofWords++; j += seglen; // update offset into query } } } } #ifndef NDEBUG /** * Return true iff the cell has sane E/F/H values w/r/t its predecessors. */ static bool cellOkEnd2EndI16( SSEData& d, size_t row, size_t col, int refc, int readc, int readq, const Scoring& sc) // scoring scheme { TCScore floorsc = 0x8000; TCScore ceilsc = MAX_I64; TAlScore offsetsc = -0x7fff; TAlScore sc_h_cur = (TAlScore)d.mat_.helt(row, col); TAlScore sc_e_cur = (TAlScore)d.mat_.eelt(row, col); TAlScore sc_f_cur = (TAlScore)d.mat_.felt(row, col); if(sc_h_cur > floorsc) { sc_h_cur += offsetsc; } if(sc_e_cur > floorsc) { sc_e_cur += offsetsc; } if(sc_f_cur > floorsc) { sc_f_cur += offsetsc; } bool gapsAllowed = true; size_t rowFromEnd = d.mat_.nrow() - row - 1; if(row < (size_t)sc.gapbar || rowFromEnd < (size_t)sc.gapbar) { gapsAllowed = false; } bool e_left_trans = false, h_left_trans = false; bool f_up_trans = false, h_up_trans = false; bool h_diag_trans = false; if(gapsAllowed) { TAlScore sc_h_left = floorsc; TAlScore sc_e_left = floorsc; TAlScore sc_h_up = floorsc; TAlScore sc_f_up = floorsc; if(col > 0 && sc_e_cur > floorsc && sc_e_cur <= ceilsc) { sc_h_left = d.mat_.helt(row, col-1) + offsetsc; sc_e_left = d.mat_.eelt(row, col-1) + offsetsc; e_left_trans = (sc_e_left > floorsc && sc_e_cur == sc_e_left - sc.readGapExtend()); h_left_trans = (sc_h_left > floorsc && sc_e_cur == sc_h_left - sc.readGapOpen()); assert(e_left_trans || h_left_trans); } if(row > 0 && sc_f_cur > floorsc && sc_f_cur <= ceilsc) { sc_h_up = d.mat_.helt(row-1, col) + offsetsc; sc_f_up = d.mat_.felt(row-1, col) + offsetsc; f_up_trans = (sc_f_up > floorsc && sc_f_cur == sc_f_up - sc.refGapExtend()); h_up_trans = (sc_h_up > floorsc && sc_f_cur == sc_h_up - sc.refGapOpen()); assert(f_up_trans || h_up_trans); } } else { assert_geq(floorsc, sc_e_cur); assert_geq(floorsc, sc_f_cur); } if(col > 0 && row > 0 && sc_h_cur > floorsc && sc_h_cur <= ceilsc) { TAlScore sc_h_upleft = d.mat_.helt(row-1, col-1) + offsetsc; TAlScore sc_diag = sc.score(readc, (int)refc, readq - 33); h_diag_trans = sc_h_cur == sc_h_upleft + sc_diag; } assert( sc_h_cur <= floorsc || e_left_trans || h_left_trans || f_up_trans || h_up_trans || h_diag_trans || sc_h_cur > ceilsc || row == 0 || col == 0); return true; } #endif /*ndef NDEBUG*/ #ifdef NDEBUG #define assert_all_eq0(x) #define assert_all_gt(x, y) #define assert_all_gt_lo(x) #define assert_all_lt(x, y) #define assert_all_lt_hi(x) #else #define assert_all_eq0(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_xor_si128(z, z); \ tmp = _mm_cmpeq_epi16(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_gt(x, y) { \ __m128i tmp = _mm_cmpgt_epi16(x, y); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_gt_lo(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_xor_si128(z, z); \ tmp = _mm_cmpgt_epi16(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_lt(x, y) { \ __m128i tmp = _mm_cmplt_epi16(x, y); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_leq(x, y) { \ __m128i tmp = _mm_cmpgt_epi16(x, y); \ assert_eq(0x0000, _mm_movemask_epi8(tmp)); \ } #define assert_all_lt_hi(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_cmpeq_epi16(z, z); \ z = _mm_srli_epi16(z, 1); \ tmp = _mm_cmplt_epi16(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #endif /** * Aligns by filling a dynamic programming matrix with the SSE-accelerated, * banded DP approach of Farrar. As it goes, it determines which cells we * might backtrace from and tallies the best (highest-scoring) N backtrace * candidate cells per diagonal. Also returns the alignment score of the best * alignment in the matrix. * * This routine does *not* maintain a matrix holding the entire matrix worth of * scores, nor does it maintain any other dense O(mn) data structure, as this * would quickly exhaust memory for queries longer than about 10,000 kb. * Instead, in the fill stage it maintains two columns worth of scores at a * time (current/previous, or right/left) - these take O(m) space. When * finished with the current column, it determines which cells from the * previous column, if any, are candidates we might backtrace from to find a * full alignment. A candidate cell has a score that rises above the threshold * and isn't improved upon by a match in the next column. The best N * candidates per diagonal are stored in a O(m + n) data structure. */ TAlScore SwAligner::alignGatherEE16(int& flag, bool debug) { assert_leq(rdf_, rd_->length()); assert_leq(rdf_, qu_->length()); assert_lt(rfi_, rff_); assert_lt(rdi_, rdf_); assert_eq(rd_->length(), qu_->length()); assert_geq(sc_->gapbar, 1); assert(repOk()); #ifndef NDEBUG for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert_range(0, 16, (int)rf_[i]); } #endif SSEData& d = fw_ ? sseI16fw_ : sseI16rc_; SSEMetrics& met = extend_ ? sseI16ExtendMet_ : sseI16MateMet_; if(!debug) met.dp++; buildQueryProfileEnd2EndSseI16(fw_); assert(!d.profbuf_.empty()); assert_eq(0, d.maxBonus_); size_t iter = (dpRows() + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // iter = segLen // Now set up the score vectors. We just need two columns worth, which // we'll call "left" and "right". d.vecbuf_.resize(4 * 2 * iter); d.vecbuf_.zero(); __m128i *vbuf_l = d.vecbuf_.ptr(); __m128i *vbuf_r = d.vecbuf_.ptr() + (4 * iter); // This is the data structure that holds candidate cells per diagonal. const size_t ndiags = rff_ - rfi_ + dpRows() - 1; if(!debug) { btdiag_.init(ndiags, 2); } // Data structure that holds checkpointed anti-diagonals TAlScore perfectScore = sc_->perfectScore(dpRows()); bool checkpoint = true; bool cpdebug = false; #ifndef NDEBUG cpdebug = dpRows() < 1000; #endif cper_.init( dpRows(), // # rows rff_ - rfi_, // # columns cperPerPow2_, // checkpoint every 1 << perpow2 diags (& next) perfectScore, // perfect score (for sanity checks) false, // matrix cells have 8-bit scores? cperTri_, // triangular mini-fills? false, // alignment is local? cpdebug); // save all cells for debugging? // Many thanks to Michael Farrar for releasing his striped Smith-Waterman // implementation: // // http://sites.google.com/site/farrarmichael/smith-waterman // // Much of the implmentation below is adapted from Michael's code. // Set all elts to reference gap open penalty __m128i rfgapo = _mm_setzero_si128(); __m128i rfgape = _mm_setzero_si128(); __m128i rdgapo = _mm_setzero_si128(); __m128i rdgape = _mm_setzero_si128(); __m128i vlo = _mm_setzero_si128(); __m128i vhi = _mm_setzero_si128(); __m128i vhilsw = _mm_setzero_si128(); __m128i vlolsw = _mm_setzero_si128(); __m128i ve = _mm_setzero_si128(); __m128i vf = _mm_setzero_si128(); __m128i vh = _mm_setzero_si128(); __m128i vhd = _mm_setzero_si128(); __m128i vhdtmp = _mm_setzero_si128(); __m128i vtmp = _mm_setzero_si128(); assert_gt(sc_->refGapOpen(), 0); assert_leq(sc_->refGapOpen(), MAX_I16); rfgapo = _mm_insert_epi16(rfgapo, sc_->refGapOpen(), 0); rfgapo = _mm_shufflelo_epi16(rfgapo, 0); rfgapo = _mm_shuffle_epi32(rfgapo, 0); // Set all elts to reference gap extension penalty assert_gt(sc_->refGapExtend(), 0); assert_leq(sc_->refGapExtend(), MAX_I16); assert_leq(sc_->refGapExtend(), sc_->refGapOpen()); rfgape = _mm_insert_epi16(rfgape, sc_->refGapExtend(), 0); rfgape = _mm_shufflelo_epi16(rfgape, 0); rfgape = _mm_shuffle_epi32(rfgape, 0); // Set all elts to read gap open penalty assert_gt(sc_->readGapOpen(), 0); assert_leq(sc_->readGapOpen(), MAX_I16); rdgapo = _mm_insert_epi16(rdgapo, sc_->readGapOpen(), 0); rdgapo = _mm_shufflelo_epi16(rdgapo, 0); rdgapo = _mm_shuffle_epi32(rdgapo, 0); // Set all elts to read gap extension penalty assert_gt(sc_->readGapExtend(), 0); assert_leq(sc_->readGapExtend(), MAX_I16); assert_leq(sc_->readGapExtend(), sc_->readGapOpen()); rdgape = _mm_insert_epi16(rdgape, sc_->readGapExtend(), 0); rdgape = _mm_shufflelo_epi16(rdgape, 0); rdgape = _mm_shuffle_epi32(rdgape, 0); // Set all elts to 0x8000 (min value for signed 16-bit) vlo = _mm_cmpeq_epi16(vlo, vlo); // all elts = 0xffff vlo = _mm_slli_epi16(vlo, NBITS_PER_WORD-1); // all elts = 0x8000 // Set all elts to 0x7fff (max value for signed 16-bit) vhi = _mm_cmpeq_epi16(vhi, vhi); // all elts = 0xffff vhi = _mm_srli_epi16(vhi, 1); // all elts = 0x7fff // vlolsw: topmost (least sig) word set to 0x8000, all other words=0 vlolsw = _mm_shuffle_epi32(vlo, 0); vlolsw = _mm_srli_si128(vlolsw, NBYTES_PER_REG - NBYTES_PER_WORD); // vhilsw: topmost (least sig) word set to 0x7fff, all other words=0 vhilsw = _mm_shuffle_epi32(vhi, 0); vhilsw = _mm_srli_si128(vhilsw, NBYTES_PER_REG - NBYTES_PER_WORD); // Points to a long vector of __m128i where each element is a block of // contiguous cells in the E, F or H matrix. If the index % 3 == 0, then // the block of cells is from the E matrix. If index % 3 == 1, they're // from the F matrix. If index % 3 == 2, then they're from the H matrix. // Blocks of cells are organized in the same interleaved manner as they are // calculated by the Farrar algorithm. const __m128i *pvScore; // points into the query profile const size_t colstride = ROWSTRIDE_2COL * iter; // Initialize the H and E vectors in the first matrix column __m128i *pvELeft = vbuf_l + 0; __m128i *pvERight = vbuf_r + 0; /* __m128i *pvFLeft = vbuf_l + 1; */ __m128i *pvFRight = vbuf_r + 1; __m128i *pvHLeft = vbuf_l + 2; __m128i *pvHRight = vbuf_r + 2; // Maximum score in final row bool found = false; TCScore lrmax = MIN_I16; for(size_t i = 0; i < iter; i++) { _mm_store_si128(pvERight, vlo); pvERight += ROWSTRIDE_2COL; // Could initialize Hs to high or low. If high, cells in the lower // triangle will have somewhat more legitiate scores, but still won't // be exhaustively scored. _mm_store_si128(pvHRight, vlo); pvHRight += ROWSTRIDE_2COL; } assert_gt(sc_->gapbar, 0); size_t nfixup = 0; // Fill in the table as usual but instead of using the same gap-penalty // vector for each iteration of the inner loop, load words out of a // pre-calculated gap vector parallel to the query profile. The pre- // calculated gap vectors enforce the gap barrier constraint by making it // infinitely costly to introduce a gap in barrier rows. // // AND use a separate loop to fill in the first row of the table, enforcing // the st_ constraints in the process. This is awkward because it // separates the processing of the first row from the others and might make // it difficult to use the first-row results in the next row, but it might // be the simplest and least disruptive way to deal with the st_ constraint. for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { // Swap left and right; vbuf_l is the vector on the left, which we // generally load from, and vbuf_r is the vector on the right, which we // generally store to. swap(vbuf_l, vbuf_r); pvELeft = vbuf_l + 0; pvERight = vbuf_r + 0; /* pvFLeft = vbuf_l + 1; */ pvFRight = vbuf_r + 1; pvHLeft = vbuf_l + 2; pvHRight = vbuf_r + 2; // Fetch the appropriate query profile. Note that elements of rf_ must // be numbers, not masks. const int refc = (int)rf_[i]; // Fetch the appropriate query profile size_t off = (size_t)firsts5[refc] * iter * 2; pvScore = d.profbuf_.ptr() + off; // even elts = query profile, odd = gap barrier // Set all cells to low value vf = _mm_cmpeq_epi16(vf, vf); vf = _mm_slli_epi16(vf, NBITS_PER_WORD-1); vf = _mm_or_si128(vf, vlolsw); // Load H vector from the final row of the previous column vh = _mm_load_si128(pvHLeft + colstride - ROWSTRIDE_2COL); // Shift 2 bytes down so that topmost (least sig) cell gets 0 vh = _mm_slli_si128(vh, NBYTES_PER_WORD); // Fill topmost (least sig) cell with high value vh = _mm_or_si128(vh, vhilsw); // For each character in the reference text: size_t j; for(j = 0; j < iter; j++) { // Load cells from E, calculated previously ve = _mm_load_si128(pvELeft); vhd = _mm_load_si128(pvHLeft); assert_all_lt(ve, vhi); pvELeft += ROWSTRIDE_2COL; // Store cells in F, calculated previously vf = _mm_adds_epi16(vf, pvScore[1]); // veto some ref gap extensions vf = _mm_adds_epi16(vf, pvScore[1]); // veto some ref gap extensions _mm_store_si128(pvFRight, vf); pvFRight += ROWSTRIDE_2COL; // Factor in query profile (matches and mismatches) vh = _mm_adds_epi16(vh, pvScore[0]); // Update H, factoring in E and F vh = _mm_max_epi16(vh, vf); // Update vE value vhdtmp = vhd; vhd = _mm_subs_epi16(vhd, rdgapo); vhd = _mm_adds_epi16(vhd, pvScore[1]); // veto some read gap opens vhd = _mm_adds_epi16(vhd, pvScore[1]); // veto some read gap opens ve = _mm_subs_epi16(ve, rdgape); ve = _mm_max_epi16(ve, vhd); vh = _mm_max_epi16(vh, ve); // Save the new vH values _mm_store_si128(pvHRight, vh); pvHRight += ROWSTRIDE_2COL; vtmp = vh; assert_all_lt(ve, vhi); // Load the next h value vh = vhdtmp; pvHLeft += ROWSTRIDE_2COL; // Save E values _mm_store_si128(pvERight, ve); pvERight += ROWSTRIDE_2COL; // Update vf value vtmp = _mm_subs_epi16(vtmp, rfgapo); vf = _mm_subs_epi16(vf, rfgape); assert_all_lt(vf, vhi); vf = _mm_max_epi16(vf, vtmp); pvScore += 2; // move on to next query profile / gap veto } // pvHStore, pvELoad, pvEStore have all rolled over to the next column pvFRight -= colstride; // reset to start of column vtmp = _mm_load_si128(pvFRight); pvHRight -= colstride; // reset to start of column vh = _mm_load_si128(pvHRight); pvScore = d.profbuf_.ptr() + off + 1; // reset veto vector // vf from last row gets shifted down by one to overlay the first row // rfgape has already been subtracted from it. vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_or_si128(vf, vlolsw); vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epi16(vtmp, vf); vtmp = _mm_cmpgt_epi16(vf, vtmp); int cmp = _mm_movemask_epi8(vtmp); // If any element of vtmp is greater than H - gap-open... j = 0; while(cmp != 0x0000) { // Store this vf _mm_store_si128(pvFRight, vf); pvFRight += ROWSTRIDE_2COL; // Update vh w/r/t new vf vh = _mm_max_epi16(vh, vf); // Save vH values _mm_store_si128(pvHRight, vh); pvHRight += ROWSTRIDE_2COL; pvScore += 2; assert_lt(j, iter); if(++j == iter) { pvFRight -= colstride; vtmp = _mm_load_si128(pvFRight); // load next vf ASAP pvHRight -= colstride; vh = _mm_load_si128(pvHRight); // load next vh ASAP pvScore = d.profbuf_.ptr() + off + 1; j = 0; vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_or_si128(vf, vlolsw); } else { vtmp = _mm_load_si128(pvFRight); // load next vf ASAP vh = _mm_load_si128(pvHRight); // load next vh ASAP } // Update F with another gap extension vf = _mm_subs_epi16(vf, rfgape); vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epi16(vtmp, vf); vtmp = _mm_cmpgt_epi16(vf, vtmp); cmp = _mm_movemask_epi8(vtmp); nfixup++; } // Check in the last row for the maximum so far __m128i *vtmp = vbuf_r + 2 /* H */ + (d.lastIter_ * ROWSTRIDE_2COL); // Note: we may not want to extract from the final row TCScore lr = ((TCScore*)(vtmp))[d.lastWord_]; found = true; if(lr > lrmax) { lrmax = lr; } // Now we'd like to know whether the bottommost element of the right // column is a candidate we might backtrace from. First question is: // did it exceed the minimum score threshold? TAlScore score = (TAlScore)(lr - 0x7fff); if(lr == MIN_I16) { score = MIN_I64; } if(!debug && score >= minsc_) { DpBtCandidate cand(dpRows() - 1, i - rfi_, score); btdiag_.add(i - rfi_, cand); } // Save some elements to checkpoints if(checkpoint) { __m128i *pvE = vbuf_r + 0; __m128i *pvF = vbuf_r + 1; __m128i *pvH = vbuf_r + 2; size_t coli = i - rfi_; if(coli < cper_.locol_) cper_.locol_ = coli; if(coli > cper_.hicol_) cper_.hicol_ = coli; if(cperTri_) { size_t rc_mod = coli & cper_.lomask_; assert_lt(rc_mod, cper_.per_); int64_t row = -(int64_t)rc_mod-1; int64_t row_mod = row; int64_t row_div = 0; size_t idx = coli >> cper_.perpow2_; size_t idxrow = idx * cper_.nrow_; assert_eq(4, ROWSTRIDE_2COL); bool done = false; while(true) { row += (cper_.per_ - 2); row_mod += (cper_.per_ - 2); for(size_t j = 0; j < 2; j++) { row++; row_mod++; if(row >= 0 && (size_t)row < cper_.nrow_) { // Update row divided by iter_ and mod iter_ while(row_mod >= (int64_t)iter) { row_mod -= (int64_t)iter; row_div++; } size_t delt = idxrow + row; size_t vecoff = (row_mod << 5) + row_div; assert_lt(row_div, 8); int16_t h_sc = ((int16_t*)pvH)[vecoff]; int16_t e_sc = ((int16_t*)pvE)[vecoff]; int16_t f_sc = ((int16_t*)pvF)[vecoff]; if(h_sc != MIN_I16) h_sc -= 0x7fff; if(e_sc != MIN_I16) e_sc -= 0x7fff; if(f_sc != MIN_I16) f_sc -= 0x7fff; assert_leq(h_sc, cper_.perf_); assert_leq(e_sc, cper_.perf_); assert_leq(f_sc, cper_.perf_); CpQuad *qdiags = ((j == 0) ? cper_.qdiag1s_.ptr() : cper_.qdiag2s_.ptr()); qdiags[delt].sc[0] = h_sc; qdiags[delt].sc[1] = e_sc; qdiags[delt].sc[2] = f_sc; } // if(row >= 0 && row < nrow_) else if(row >= 0 && (size_t)row >= cper_.nrow_) { done = true; break; } } // end of loop over anti-diags if(done) { break; } idx++; idxrow += cper_.nrow_; } } else { // If this is the first column, take this opportunity to // pre-calculate the coordinates of the elements we're going to // checkpoint. if(coli == 0) { size_t cpi = cper_.per_-1; size_t cpimod = cper_.per_-1; size_t cpidiv = 0; cper_.commitMap_.clear(); while(cpi < cper_.nrow_) { while(cpimod >= iter) { cpimod -= iter; cpidiv++; } size_t vecoff = (cpimod << 5) + cpidiv; cper_.commitMap_.push_back(vecoff); cpi += cper_.per_; cpimod += cper_.per_; } } // Save all the rows size_t rowoff = 0; size_t sz = cper_.commitMap_.size(); for(size_t i = 0; i < sz; i++, rowoff += cper_.ncol_) { size_t vecoff = cper_.commitMap_[i]; int16_t h_sc = ((int16_t*)pvH)[vecoff]; int16_t e_sc = ((int16_t*)pvE)[vecoff]; int16_t f_sc = ((int16_t*)pvF)[vecoff]; if(h_sc != MIN_I16) h_sc -= 0x7fff; if(e_sc != MIN_I16) e_sc -= 0x7fff; if(f_sc != MIN_I16) f_sc -= 0x7fff; assert_leq(h_sc, cper_.perf_); assert_leq(e_sc, cper_.perf_); assert_leq(f_sc, cper_.perf_); CpQuad& dst = cper_.qrows_[rowoff + coli]; dst.sc[0] = h_sc; dst.sc[1] = e_sc; dst.sc[2] = f_sc; } // Is this a column we'd like to checkpoint? if((coli & cper_.lomask_) == cper_.lomask_) { // Save the column using memcpys assert_gt(coli, 0); size_t wordspercol = cper_.niter_ * ROWSTRIDE_2COL; size_t coloff = (coli >> cper_.perpow2_) * wordspercol; __m128i *dst = cper_.qcols_.ptr() + coloff; memcpy(dst, vbuf_r, sizeof(__m128i) * wordspercol); } } if(cper_.debug_) { // Save the column using memcpys size_t wordspercol = cper_.niter_ * ROWSTRIDE_2COL; size_t coloff = coli * wordspercol; __m128i *dst = cper_.qcolsD_.ptr() + coloff; memcpy(dst, vbuf_r, sizeof(__m128i) * wordspercol); } } } // Update metrics if(!debug) { size_t ninner = (rff_ - rfi_) * iter; met.col += (rff_ - rfi_); // DP columns met.cell += (ninner * NWORDS_PER_REG); // DP cells met.inner += ninner; // DP inner loop iters met.fixup += nfixup; // DP fixup loop iters } flag = 0; // Did we find a solution? TAlScore score = MIN_I64; if(!found) { flag = -1; // no if(!debug) met.dpfail++; return MIN_I64; } else { score = (TAlScore)(lrmax - 0x7fff); if(score < minsc_) { flag = -1; // no if(!debug) met.dpfail++; return score; } } // Could we have saturated? if(lrmax == MIN_I16) { flag = -2; // yes if(!debug) met.dpsat++; return MIN_I64; } // Now take all the backtrace candidates in the btdaig_ structure and // dump them into the btncand_ array. They'll be sorted later. if(!debug) { btdiag_.dump(btncand_); assert(!btncand_.empty()); } // Return largest score if(!debug) met.dpsucc++; return score; } /** * Solve the current alignment problem using SSE instructions that operate on 8 * signed 16-bit values packed into a single 128-bit register. */ TAlScore SwAligner::alignNucleotidesEnd2EndSseI16(int& flag, bool debug) { assert_leq(rdf_, rd_->length()); assert_leq(rdf_, qu_->length()); assert_lt(rfi_, rff_); assert_lt(rdi_, rdf_); assert_eq(rd_->length(), qu_->length()); assert_geq(sc_->gapbar, 1); assert(repOk()); #ifndef NDEBUG for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert_range(0, 16, (int)rf_[i]); } #endif SSEData& d = fw_ ? sseI16fw_ : sseI16rc_; SSEMetrics& met = extend_ ? sseI16ExtendMet_ : sseI16MateMet_; if(!debug) met.dp++; buildQueryProfileEnd2EndSseI16(fw_); assert(!d.profbuf_.empty()); assert_eq(0, d.maxBonus_); size_t iter = (dpRows() + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // iter = segLen // Many thanks to Michael Farrar for releasing his striped Smith-Waterman // implementation: // // http://sites.google.com/site/farrarmichael/smith-waterman // // Much of the implmentation below is adapted from Michael's code. // Set all elts to reference gap open penalty __m128i rfgapo = _mm_setzero_si128(); __m128i rfgape = _mm_setzero_si128(); __m128i rdgapo = _mm_setzero_si128(); __m128i rdgape = _mm_setzero_si128(); __m128i vlo = _mm_setzero_si128(); __m128i vhi = _mm_setzero_si128(); __m128i vhilsw = _mm_setzero_si128(); __m128i vlolsw = _mm_setzero_si128(); __m128i ve = _mm_setzero_si128(); __m128i vf = _mm_setzero_si128(); __m128i vh = _mm_setzero_si128(); #if 0 __m128i vhd = _mm_setzero_si128(); __m128i vhdtmp = _mm_setzero_si128(); #endif __m128i vtmp = _mm_setzero_si128(); assert_gt(sc_->refGapOpen(), 0); assert_leq(sc_->refGapOpen(), MAX_I16); rfgapo = _mm_insert_epi16(rfgapo, sc_->refGapOpen(), 0); rfgapo = _mm_shufflelo_epi16(rfgapo, 0); rfgapo = _mm_shuffle_epi32(rfgapo, 0); // Set all elts to reference gap extension penalty assert_gt(sc_->refGapExtend(), 0); assert_leq(sc_->refGapExtend(), MAX_I16); assert_leq(sc_->refGapExtend(), sc_->refGapOpen()); rfgape = _mm_insert_epi16(rfgape, sc_->refGapExtend(), 0); rfgape = _mm_shufflelo_epi16(rfgape, 0); rfgape = _mm_shuffle_epi32(rfgape, 0); // Set all elts to read gap open penalty assert_gt(sc_->readGapOpen(), 0); assert_leq(sc_->readGapOpen(), MAX_I16); rdgapo = _mm_insert_epi16(rdgapo, sc_->readGapOpen(), 0); rdgapo = _mm_shufflelo_epi16(rdgapo, 0); rdgapo = _mm_shuffle_epi32(rdgapo, 0); // Set all elts to read gap extension penalty assert_gt(sc_->readGapExtend(), 0); assert_leq(sc_->readGapExtend(), MAX_I16); assert_leq(sc_->readGapExtend(), sc_->readGapOpen()); rdgape = _mm_insert_epi16(rdgape, sc_->readGapExtend(), 0); rdgape = _mm_shufflelo_epi16(rdgape, 0); rdgape = _mm_shuffle_epi32(rdgape, 0); // Set all elts to 0x8000 (min value for signed 16-bit) vlo = _mm_cmpeq_epi16(vlo, vlo); // all elts = 0xffff vlo = _mm_slli_epi16(vlo, NBITS_PER_WORD-1); // all elts = 0x8000 // Set all elts to 0x7fff (max value for signed 16-bit) vhi = _mm_cmpeq_epi16(vhi, vhi); // all elts = 0xffff vhi = _mm_srli_epi16(vhi, 1); // all elts = 0x7fff // vlolsw: topmost (least sig) word set to 0x8000, all other words=0 vlolsw = _mm_shuffle_epi32(vlo, 0); vlolsw = _mm_srli_si128(vlolsw, NBYTES_PER_REG - NBYTES_PER_WORD); // vhilsw: topmost (least sig) word set to 0x7fff, all other words=0 vhilsw = _mm_shuffle_epi32(vhi, 0); vhilsw = _mm_srli_si128(vhilsw, NBYTES_PER_REG - NBYTES_PER_WORD); // Points to a long vector of __m128i where each element is a block of // contiguous cells in the E, F or H matrix. If the index % 3 == 0, then // the block of cells is from the E matrix. If index % 3 == 1, they're // from the F matrix. If index % 3 == 2, then they're from the H matrix. // Blocks of cells are organized in the same interleaved manner as they are // calculated by the Farrar algorithm. const __m128i *pvScore; // points into the query profile d.mat_.init(dpRows(), rff_ - rfi_, NWORDS_PER_REG); const size_t colstride = d.mat_.colstride(); assert_eq(ROWSTRIDE, colstride / iter); // Initialize the H and E vectors in the first matrix column __m128i *pvHTmp = d.mat_.tmpvec(0, 0); __m128i *pvETmp = d.mat_.evec(0, 0); // Maximum score in final row bool found = false; TCScore lrmax = MIN_I16; for(size_t i = 0; i < iter; i++) { _mm_store_si128(pvETmp, vlo); // Could initialize Hs to high or low. If high, cells in the lower // triangle will have somewhat more legitiate scores, but still won't // be exhaustively scored. _mm_store_si128(pvHTmp, vlo); pvETmp += ROWSTRIDE; pvHTmp += ROWSTRIDE; } // These are swapped just before the innermost loop __m128i *pvHStore = d.mat_.hvec(0, 0); __m128i *pvHLoad = d.mat_.tmpvec(0, 0); __m128i *pvELoad = d.mat_.evec(0, 0); __m128i *pvEStore = d.mat_.evecUnsafe(0, 1); __m128i *pvFStore = d.mat_.fvec(0, 0); __m128i *pvFTmp = NULL; assert_gt(sc_->gapbar, 0); size_t nfixup = 0; // Fill in the table as usual but instead of using the same gap-penalty // vector for each iteration of the inner loop, load words out of a // pre-calculated gap vector parallel to the query profile. The pre- // calculated gap vectors enforce the gap barrier constraint by making it // infinitely costly to introduce a gap in barrier rows. // // AND use a separate loop to fill in the first row of the table, enforcing // the st_ constraints in the process. This is awkward because it // separates the processing of the first row from the others and might make // it difficult to use the first-row results in the next row, but it might // be the simplest and least disruptive way to deal with the st_ constraint. colstop_ = rff_ - 1; lastsolcol_ = 0; for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert(pvFStore == d.mat_.fvec(0, i - rfi_)); assert(pvHStore == d.mat_.hvec(0, i - rfi_)); // Fetch the appropriate query profile. Note that elements of rf_ must // be numbers, not masks. const int refc = (int)rf_[i]; size_t off = (size_t)firsts5[refc] * iter * 2; pvScore = d.profbuf_.ptr() + off; // even elts = query profile, odd = gap barrier // Set all cells to low value vf = _mm_cmpeq_epi16(vf, vf); vf = _mm_slli_epi16(vf, NBITS_PER_WORD-1); vf = _mm_or_si128(vf, vlolsw); // Load H vector from the final row of the previous column vh = _mm_load_si128(pvHLoad + colstride - ROWSTRIDE); // Shift 2 bytes down so that topmost (least sig) cell gets 0 vh = _mm_slli_si128(vh, NBYTES_PER_WORD); // Fill topmost (least sig) cell with high value vh = _mm_or_si128(vh, vhilsw); // For each character in the reference text: size_t j; for(j = 0; j < iter; j++) { // Load cells from E, calculated previously ve = _mm_load_si128(pvELoad); #if 0 vhd = _mm_load_si128(pvHLoad); #endif assert_all_lt(ve, vhi); pvELoad += ROWSTRIDE; // Store cells in F, calculated previously vf = _mm_adds_epi16(vf, pvScore[1]); // veto some ref gap extensions vf = _mm_adds_epi16(vf, pvScore[1]); // veto some ref gap extensions _mm_store_si128(pvFStore, vf); pvFStore += ROWSTRIDE; // Factor in query profile (matches and mismatches) vh = _mm_adds_epi16(vh, pvScore[0]); // Update H, factoring in E and F vh = _mm_max_epi16(vh, ve); vh = _mm_max_epi16(vh, vf); // Save the new vH values _mm_store_si128(pvHStore, vh); pvHStore += ROWSTRIDE; // Update vE value vtmp = vh; #if 0 vhdtmp = vhd; vhd = _mm_subs_epi16(vhd, rdgapo); vhd = _mm_adds_epi16(vhd, pvScore[1]); // veto some read gap opens vhd = _mm_adds_epi16(vhd, pvScore[1]); // veto some read gap opens ve = _mm_subs_epi16(ve, rdgape); ve = _mm_max_epi16(ve, vhd); #else vh = _mm_subs_epi16(vh, rdgapo); vh = _mm_adds_epi16(vh, pvScore[1]); // veto some read gap opens vh = _mm_adds_epi16(vh, pvScore[1]); // veto some read gap opens ve = _mm_subs_epi16(ve, rdgape); ve = _mm_max_epi16(ve, vh); #endif assert_all_lt(ve, vhi); // Load the next h value #if 0 vh = vhdtmp; #else vh = _mm_load_si128(pvHLoad); #endif pvHLoad += ROWSTRIDE; // Save E values _mm_store_si128(pvEStore, ve); pvEStore += ROWSTRIDE; // Update vf value vtmp = _mm_subs_epi16(vtmp, rfgapo); vf = _mm_subs_epi16(vf, rfgape); assert_all_lt(vf, vhi); vf = _mm_max_epi16(vf, vtmp); pvScore += 2; // move on to next query profile / gap veto } // pvHStore, pvELoad, pvEStore have all rolled over to the next column pvFTmp = pvFStore; pvFStore -= colstride; // reset to start of column vtmp = _mm_load_si128(pvFStore); pvHStore -= colstride; // reset to start of column vh = _mm_load_si128(pvHStore); #if 0 #else pvEStore -= colstride; // reset to start of column ve = _mm_load_si128(pvEStore); #endif pvHLoad = pvHStore; // new pvHLoad = pvHStore pvScore = d.profbuf_.ptr() + off + 1; // reset veto vector // vf from last row gets shifted down by one to overlay the first row // rfgape has already been subtracted from it. vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_or_si128(vf, vlolsw); vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epi16(vtmp, vf); vtmp = _mm_cmpgt_epi16(vf, vtmp); int cmp = _mm_movemask_epi8(vtmp); // If any element of vtmp is greater than H - gap-open... j = 0; while(cmp != 0x0000) { // Store this vf _mm_store_si128(pvFStore, vf); pvFStore += ROWSTRIDE; // Update vh w/r/t new vf vh = _mm_max_epi16(vh, vf); // Save vH values _mm_store_si128(pvHStore, vh); pvHStore += ROWSTRIDE; // Update E in case it can be improved using our new vh #if 0 #else vh = _mm_subs_epi16(vh, rdgapo); vh = _mm_adds_epi16(vh, *pvScore); // veto some read gap opens vh = _mm_adds_epi16(vh, *pvScore); // veto some read gap opens ve = _mm_max_epi16(ve, vh); _mm_store_si128(pvEStore, ve); pvEStore += ROWSTRIDE; #endif pvScore += 2; assert_lt(j, iter); if(++j == iter) { pvFStore -= colstride; vtmp = _mm_load_si128(pvFStore); // load next vf ASAP pvHStore -= colstride; vh = _mm_load_si128(pvHStore); // load next vh ASAP #if 0 #else pvEStore -= colstride; ve = _mm_load_si128(pvEStore); // load next ve ASAP #endif pvScore = d.profbuf_.ptr() + off + 1; j = 0; vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_or_si128(vf, vlolsw); } else { vtmp = _mm_load_si128(pvFStore); // load next vf ASAP vh = _mm_load_si128(pvHStore); // load next vh ASAP #if 0 #else ve = _mm_load_si128(pvEStore); // load next vh ASAP #endif } // Update F with another gap extension vf = _mm_subs_epi16(vf, rfgape); vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epi16(vtmp, vf); vtmp = _mm_cmpgt_epi16(vf, vtmp); cmp = _mm_movemask_epi8(vtmp); nfixup++; } #ifndef NDEBUG if((rand() & 15) == 0) { // This is a work-intensive sanity check; each time we finish filling // a column, we check that each H, E, and F is sensible. for(size_t k = 0; k < dpRows(); k++) { assert(cellOkEnd2EndI16( d, k, // row i - rfi_, // col refc, // reference mask (int)(*rd_)[rdi_+k], // read char (int)(*qu_)[rdi_+k], // read quality *sc_)); // scoring scheme } } #endif __m128i *vtmp = d.mat_.hvec(d.lastIter_, i-rfi_); // Note: we may not want to extract from the final row TCScore lr = ((TCScore*)(vtmp))[d.lastWord_]; found = true; if(lr > lrmax) { lrmax = lr; } // pvELoad and pvHLoad are already where they need to be // Adjust the load and store vectors here. pvHStore = pvHLoad + colstride; pvEStore = pvELoad + colstride; pvFStore = pvFTmp; } // Update metrics if(!debug) { size_t ninner = (rff_ - rfi_) * iter; met.col += (rff_ - rfi_); // DP columns met.cell += (ninner * NWORDS_PER_REG); // DP cells met.inner += ninner; // DP inner loop iters met.fixup += nfixup; // DP fixup loop iters } flag = 0; // Did we find a solution? TAlScore score = MIN_I64; if(!found) { flag = -1; // no if(!debug) met.dpfail++; return MIN_I64; } else { score = (TAlScore)(lrmax - 0x7fff); if(score < minsc_) { flag = -1; // no if(!debug) met.dpfail++; return score; } } // Could we have saturated? if(lrmax == MIN_I16) { flag = -2; // yes if(!debug) met.dpsat++; return MIN_I64; } // Return largest score if(!debug) met.dpsucc++; return score; } /** * Given a filled-in DP table, populate the btncand_ list with candidate cells * that might be at the ends of valid alignments. No need to do this unless * the maximum score returned by the align*() func is >= the minimum. * * Only cells that are exhaustively scored are candidates. Those are the * cells inside the shape made of o's in this: * * |-maxgaps-| * ********************************* - * ******************************** | * ******************************* | * ****************************** | * ***************************** | * **************************** read len * *************************** | * ************************** | * ************************* | * ************************ | * ***********oooooooooooo - * |-maxgaps-| * |-readlen-| * |-------skip--------| * * And it's possible for the shape to be truncated on the left and right sides. * * */ bool SwAligner::gatherCellsNucleotidesEnd2EndSseI16(TAlScore best) { // What's the minimum number of rows that can possibly be spanned by an // alignment that meets the minimum score requirement? assert(sse16succ_); const size_t ncol = rff_ - rfi_; const size_t nrow = dpRows(); assert_gt(nrow, 0); btncand_.clear(); btncanddone_.clear(); SSEData& d = fw_ ? sseI16fw_ : sseI16rc_; SSEMetrics& met = extend_ ? sseI16ExtendMet_ : sseI16MateMet_; assert(!d.profbuf_.empty()); const size_t colstride = d.mat_.colstride(); ASSERT_ONLY(bool sawbest = false); __m128i *pvH = d.mat_.hvec(d.lastIter_, 0); for(size_t j = 0; j < ncol; j++) { TAlScore sc = (TAlScore)(((TCScore*)pvH)[d.lastWord_] - 0x7fff); assert_leq(sc, best); ASSERT_ONLY(sawbest = (sawbest || sc == best)); if(sc >= minsc_) { // Yes, this is legit met.gathsol++; btncand_.expand(); btncand_.back().init(nrow-1, j, sc); } pvH += colstride; } assert(sawbest); if(!btncand_.empty()) { d.mat_.initMasks(); } return !btncand_.empty(); } #define MOVE_VEC_PTR_UP(vec, rowvec, rowelt) { \ if(rowvec == 0) { \ rowvec += d.mat_.nvecrow_; \ vec += d.mat_.colstride_; \ rowelt--; \ } \ rowvec--; \ vec -= ROWSTRIDE; \ } #define MOVE_VEC_PTR_LEFT(vec, rowvec, rowelt) { vec -= d.mat_.colstride_; } #define MOVE_VEC_PTR_UPLEFT(vec, rowvec, rowelt) { \ MOVE_VEC_PTR_UP(vec, rowvec, rowelt); \ MOVE_VEC_PTR_LEFT(vec, rowvec, rowelt); \ } #define MOVE_ALL_LEFT() { \ MOVE_VEC_PTR_LEFT(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_LEFT(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_LEFT(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_LEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define MOVE_ALL_UP() { \ MOVE_VEC_PTR_UP(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_UP(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_UP(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_UP(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define MOVE_ALL_UPLEFT() { \ MOVE_VEC_PTR_UPLEFT(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_UPLEFT(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_UPLEFT(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_UPLEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define NEW_ROW_COL(row, col) { \ rowelt = row / d.mat_.nvecrow_; \ rowvec = row % d.mat_.nvecrow_; \ eltvec = (col * d.mat_.colstride_) + (rowvec * ROWSTRIDE); \ cur_vec = d.mat_.matbuf_.ptr() + eltvec; \ left_vec = cur_vec; \ left_rowelt = rowelt; \ left_rowvec = rowvec; \ MOVE_VEC_PTR_LEFT(left_vec, left_rowvec, left_rowelt); \ up_vec = cur_vec; \ up_rowelt = rowelt; \ up_rowvec = rowvec; \ MOVE_VEC_PTR_UP(up_vec, up_rowvec, up_rowelt); \ upleft_vec = up_vec; \ upleft_rowelt = up_rowelt; \ upleft_rowvec = up_rowvec; \ MOVE_VEC_PTR_LEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } /** * Given the dynamic programming table and a cell, trace backwards from the * cell and install the edits and score/penalty in the appropriate fields * of res. The RandomSource is used to break ties among equally good ways * of tracing back. * * Whenever we enter a cell, we check whether the read/ref coordinates of * that cell correspond to a cell we traversed constructing a previous * alignment. If so, we backtrack to the last decision point, mask out the * path that led to the previously observed cell, and continue along a * different path; or, if there are no more paths to try, we give up. * * If an alignment is found, 'off' is set to the alignment's upstream-most * reference character's offset into the chromosome and true is returned. * Otherwise, false is returned. */ bool SwAligner::backtraceNucleotidesEnd2EndSseI16( TAlScore escore, // in: expected score SwResult& res, // out: store results (edits and scores) here size_t& off, // out: store diagonal projection of origin size_t& nbts, // out: # backtracks size_t row, // start in this row size_t col, // start in this column RandomSource& rnd) // random gen, to choose among equal paths { assert_lt(row, dpRows()); assert_lt(col, (size_t)(rff_ - rfi_)); SSEData& d = fw_ ? sseI16fw_ : sseI16rc_; SSEMetrics& met = extend_ ? sseI16ExtendMet_ : sseI16MateMet_; met.bt++; assert(!d.profbuf_.empty()); assert_lt(row, rd_->length()); btnstack_.clear(); // empty the backtrack stack btcells_.clear(); // empty the cells-so-far list AlnScore score; score.score_ = 0; score.gaps_ = score.ns_ = 0; size_t origCol = col; size_t gaps = 0, readGaps = 0, refGaps = 0; res.alres.reset(); EList& ned = res.alres.ned(); assert(ned.empty()); assert_gt(dpRows(), row); size_t trimEnd = dpRows() - row - 1; size_t trimBeg = 0; size_t ct = SSEMatrix::H; // cell type // Row and col in terms of where they fall in the SSE vector matrix size_t rowelt, rowvec, eltvec; size_t left_rowelt, up_rowelt, upleft_rowelt; size_t left_rowvec, up_rowvec, upleft_rowvec; __m128i *cur_vec, *left_vec, *up_vec, *upleft_vec; NEW_ROW_COL(row, col); while((int)row >= 0) { met.btcell++; nbts++; int readc = (*rd_)[rdi_ + row]; int refm = (int)rf_[rfi_ + col]; int readq = (*qu_)[row]; assert_leq(col, origCol); // Get score in this cell bool empty = false, reportedThru, canMoveThru, branch = false; int cur = SSEMatrix::H; if(!d.mat_.reset_[row]) { d.mat_.resetRow(row); } reportedThru = d.mat_.reportedThrough(row, col); canMoveThru = true; if(reportedThru) { canMoveThru = false; } else { empty = false; if(row > 0) { assert_gt(row, 0); size_t rowFromEnd = d.mat_.nrow() - row - 1; bool gapsAllowed = true; if(row < (size_t)sc_->gapbar || rowFromEnd < (size_t)sc_->gapbar) { gapsAllowed = false; } const TAlScore floorsc = MIN_I64; const int offsetsc = -0x7fff; // Move to beginning of column/row if(ct == SSEMatrix::E) { // AKA rdgap assert_gt(col, 0); TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::E))[rowelt] + offsetsc; assert(gapsAllowed); // Currently in the E matrix; incoming transition must come from the // left. It's either a gap open from the H matrix or a gap extend from // the E matrix. // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; // Get H score of cell to the left TAlScore sc_h_left = ((TCScore*)(left_vec + SSEMatrix::H))[left_rowelt] + offsetsc; if(sc_h_left > floorsc && sc_h_left - sc_->readGapOpen() == sc_cur) { mask |= (1 << 0); } // Get E score of cell to the left TAlScore sc_e_left = ((TCScore*)(left_vec + SSEMatrix::E))[left_rowelt] + offsetsc; if(sc_e_left > floorsc && sc_e_left - sc_->readGapExtend() == sc_cur) { mask |= (1 << 1); } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isEMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 8) & 3; } if(mask == 3) { #if 1 // Pick H -> E cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 2); // might choose E later #else if(rnd.nextU2()) { // Pick H -> E cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 2); // might choose E later } else { // Pick E -> E cell cur = SW_BT_RDGAP_EXTEND; d.mat_.eMaskSet(row, col, 1); // might choose H later } #endif branch = true; } else if(mask == 2) { // I chose the E cell cur = SW_BT_RDGAP_EXTEND; d.mat_.eMaskSet(row, col, 0); // done } else if(mask == 1) { // I chose the H cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 0); // done } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } assert(!empty || !canMoveThru); } else if(ct == SSEMatrix::F) { // AKA rfgap assert_gt(row, 0); assert(gapsAllowed); TAlScore sc_h_up = ((TCScore*)(up_vec + SSEMatrix::H))[up_rowelt] + offsetsc; TAlScore sc_f_up = ((TCScore*)(up_vec + SSEMatrix::F))[up_rowelt] + offsetsc; TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::F))[rowelt] + offsetsc; // Currently in the F matrix; incoming transition must come from above. // It's either a gap open from the H matrix or a gap extend from the F // matrix. // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; // Get H score of cell above if(sc_h_up > floorsc && sc_h_up - sc_->refGapOpen() == sc_cur) { mask |= (1 << 0); } // Get F score of cell above if(sc_f_up > floorsc && sc_f_up - sc_->refGapExtend() == sc_cur) { mask |= (1 << 1); } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isFMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 11) & 3; } if(mask == 3) { #if 1 // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 2); // might choose E later #else if(rnd.nextU2()) { // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 2); // might choose E later } else { // I chose the F cell cur = SW_BT_RFGAP_EXTEND; d.mat_.fMaskSet(row, col, 1); // might choose E later } #endif branch = true; } else if(mask == 2) { // I chose the F cell cur = SW_BT_RFGAP_EXTEND; d.mat_.fMaskSet(row, col, 0); // done } else if(mask == 1) { // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 0); // done } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } assert(!empty || !canMoveThru); } else { assert_eq(SSEMatrix::H, ct); TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::H))[rowelt] + offsetsc; TAlScore sc_f_up = ((TCScore*)(up_vec + SSEMatrix::F))[up_rowelt] + offsetsc; TAlScore sc_h_up = ((TCScore*)(up_vec + SSEMatrix::H))[up_rowelt] + offsetsc; TAlScore sc_h_left = col > 0 ? (((TCScore*)(left_vec + SSEMatrix::H))[left_rowelt] + offsetsc) : floorsc; TAlScore sc_e_left = col > 0 ? (((TCScore*)(left_vec + SSEMatrix::E))[left_rowelt] + offsetsc) : floorsc; TAlScore sc_h_upleft = col > 0 ? (((TCScore*)(upleft_vec + SSEMatrix::H))[upleft_rowelt] + offsetsc) : floorsc; TAlScore sc_diag = sc_->score(readc, refm, readq - 33); // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; if(gapsAllowed) { if(sc_h_up > floorsc && sc_cur == sc_h_up - sc_->refGapOpen()) { mask |= (1 << 0); } if(sc_h_left > floorsc && sc_cur == sc_h_left - sc_->readGapOpen()) { mask |= (1 << 1); } if(sc_f_up > floorsc && sc_cur == sc_f_up - sc_->refGapExtend()) { mask |= (1 << 2); } if(sc_e_left > floorsc && sc_cur == sc_e_left - sc_->readGapExtend()) { mask |= (1 << 3); } } if(sc_h_upleft > floorsc && sc_cur == sc_h_upleft + sc_diag) { mask |= (1 << 4); } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isHMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 2) & 31; } assert(gapsAllowed || mask == (1 << 4) || mask == 0); int opts = alts5[mask]; int select = -1; if(opts == 1) { select = firsts5[mask]; assert_geq(mask, 0); d.mat_.hMaskSet(row, col, 0); } else if(opts > 1) { #if 1 if( (mask & 16) != 0) { select = 4; // H diag } else if((mask & 1) != 0) { select = 0; // H up } else if((mask & 4) != 0) { select = 2; // F up } else if((mask & 2) != 0) { select = 1; // H left } else if((mask & 8) != 0) { select = 3; // E left } #else select = randFromMask(rnd, mask); #endif assert_geq(mask, 0); mask &= ~(1 << select); assert(gapsAllowed || mask == (1 << 4) || mask == 0); d.mat_.hMaskSet(row, col, mask); branch = true; } else { /* No way to backtrack! */ } if(select != -1) { if(select == 4) { cur = SW_BT_OALL_DIAG; } else if(select == 0) { cur = SW_BT_OALL_REF_OPEN; } else if(select == 1) { cur = SW_BT_OALL_READ_OPEN; } else if(select == 2) { cur = SW_BT_RFGAP_EXTEND; } else { assert_eq(3, select) cur = SW_BT_RDGAP_EXTEND; } } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } } assert(!empty || !canMoveThru || ct == SSEMatrix::H); } } d.mat_.setReportedThrough(row, col); assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); // Cell was involved in a previously-reported alignment? if(!canMoveThru) { if(!btnstack_.empty()) { // Remove all the cells from list back to and including the // cell where the branch occurred btcells_.resize(btnstack_.back().celsz); // Pop record off the top of the stack ned.resize(btnstack_.back().nedsz); //aed.resize(btnstack_.back().aedsz); row = btnstack_.back().row; col = btnstack_.back().col; gaps = btnstack_.back().gaps; readGaps = btnstack_.back().readGaps; refGaps = btnstack_.back().refGaps; score = btnstack_.back().score; ct = btnstack_.back().ct; btnstack_.pop_back(); assert(!sc_->monotone || score.score() >= escore); NEW_ROW_COL(row, col); continue; } else { // No branch points to revisit; just give up res.reset(); met.btfail++; // DP backtraces failed return false; } } assert(!reportedThru); assert(!sc_->monotone || score.score() >= minsc_); if(empty || row == 0) { assert_eq(SSEMatrix::H, ct); btcells_.expand(); btcells_.back().first = row; btcells_.back().second = col; // This cell is at the end of a legitimate alignment trimBeg = row; assert_eq(btcells_.size(), dpRows() - trimBeg - trimEnd + readGaps); break; } if(branch) { // Add a frame to the backtrack stack btnstack_.expand(); btnstack_.back().init( ned.size(), 0, // aed.size() btcells_.size(), row, col, gaps, readGaps, refGaps, score, (int)ct); } btcells_.expand(); btcells_.back().first = row; btcells_.back().second = col; switch(cur) { // Move up and to the left. If the reference nucleotide in the // source row mismatches the read nucleotide, penalize // it and add a nucleotide mismatch. case SW_BT_OALL_DIAG: { assert_gt(row, 0); assert_gt(col, 0); // Check for color mismatch int readC = (*rd_)[row]; int refNmask = (int)rf_[rfi_+col]; assert_gt(refNmask, 0); int m = matchesEx(readC, refNmask); ct = SSEMatrix::H; if(m != 1) { Edit e( (int)row, mask2dna[refNmask], "ACGTN"[readC], EDIT_TYPE_MM); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); int pen = QUAL2(row, col); score.score_ -= pen; assert(!sc_->monotone || score.score() >= escore); } else { // Reward a match int64_t bonus = sc_->match(30); score.score_ += bonus; assert(!sc_->monotone || score.score() >= escore); } if(m == -1) { score.ns_++; } row--; col--; MOVE_ALL_UPLEFT(); assert(VALID_AL_SCORE(score)); break; } // Move up. Add an edit encoding the ref gap. case SW_BT_OALL_REF_OPEN: { assert_gt(row, 0); Edit e( (int)row, '-', "ACGTN"[(int)(*rd_)[row]], EDIT_TYPE_REF_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); row--; ct = SSEMatrix::H; int pen = sc_->refGapOpen(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; refGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_UP(); break; } // Move up. Add an edit encoding the ref gap. case SW_BT_RFGAP_EXTEND: { assert_gt(row, 1); Edit e( (int)row, '-', "ACGTN"[(int)(*rd_)[row]], EDIT_TYPE_REF_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); row--; ct = SSEMatrix::F; int pen = sc_->refGapExtend(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; refGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_UP(); break; } case SW_BT_OALL_READ_OPEN: { assert_gt(col, 0); Edit e( (int)row+1, mask2dna[(int)rf_[rfi_+col]], '-', EDIT_TYPE_READ_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); col--; ct = SSEMatrix::H; int pen = sc_->readGapOpen(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; readGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_LEFT(); break; } case SW_BT_RDGAP_EXTEND: { assert_gt(col, 1); Edit e( (int)row+1, mask2dna[(int)rf_[rfi_+col]], '-', EDIT_TYPE_READ_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); col--; ct = SSEMatrix::E; int pen = sc_->readGapExtend(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; readGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_LEFT(); break; } default: throw 1; } } // while((int)row > 0) assert_eq(0, trimBeg); assert_eq(0, trimEnd); assert_geq(col, 0); assert_eq(SSEMatrix::H, ct); // The number of cells in the backtracs should equal the number of read // bases after trimming plus the number of gaps assert_eq(btcells_.size(), dpRows() - trimBeg - trimEnd + readGaps); // Check whether we went through a core diagonal and set 'reported' flag on // each cell bool overlappedCoreDiag = false; for(size_t i = 0; i < btcells_.size(); i++) { size_t rw = btcells_[i].first; size_t cl = btcells_[i].second; // Calculate the diagonal within the *trimmed* rectangle, i.e. the // rectangle we dealt with in align, gather and backtrack. int64_t diagi = cl - rw; // Now adjust to the diagonal within the *untrimmed* rectangle by // adding on the amount trimmed from the left. diagi += rect_->triml; if(diagi >= 0) { size_t diag = (size_t)diagi; if(diag >= rect_->corel && diag <= rect_->corer) { overlappedCoreDiag = true; break; } } assert(d.mat_.reportedThrough(rw, cl)); } if(!overlappedCoreDiag) { // Must overlap a core diagonal. Otherwise, we run the risk of // reporting an alignment that overlaps (and trumps) a higher-scoring // alignment that lies partially outside the dynamic programming // rectangle. res.reset(); met.corerej++; return false; } int readC = (*rd_)[rdi_+row]; // get last char in read int refNmask = (int)rf_[rfi_+col]; // get last ref char ref involved in aln assert_gt(refNmask, 0); int m = matchesEx(readC, refNmask); if(m != 1) { Edit e((int)row, mask2dna[refNmask], "ACGTN"[readC], EDIT_TYPE_MM); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); score.score_ -= QUAL2(row, col); assert_geq(score.score(), minsc_); } else { score.score_ += sc_->match(30); } if(m == -1) { score.ns_++; } if(score.ns_ > nceil_) { // Alignment has too many Ns in it! res.reset(); met.nrej++; return false; } res.reverse(); assert(Edit::repOk(ned, (*rd_))); assert_eq(score.score(), escore); assert_leq(gaps, rdgap_ + rfgap_); off = col; assert_lt(col + (size_t)rfi_, (size_t)rff_); score.gaps_ = gaps; res.alres.setScore(score); res.alres.setShape( refidx_, // ref id off + rfi_ + rect_->refl, // 0-based ref offset reflen_, // reference length fw_, // aligned to Watson? rdf_ - rdi_, // read length 0, // read ID true, // pretrim soft? 0, // pretrim 5' end 0, // pretrim 3' end true, // alignment trim soft? fw_ ? trimBeg : trimEnd, // alignment trim 5' end fw_ ? trimEnd : trimBeg); // alignment trim 3' end size_t refns = 0; for(size_t i = col; i <= origCol; i++) { if((int)rf_[rfi_+i] > 15) { refns++; } } res.alres.setRefNs(refns); assert(Edit::repOk(ned, (*rd_), true, trimBeg, trimEnd)); assert(res.repOk()); #ifndef NDEBUG size_t gapsCheck = 0; for(size_t i = 0; i < ned.size(); i++) { if(ned[i].isGap()) gapsCheck++; } assert_eq(gaps, gapsCheck); BTDnaString refstr; for(size_t i = col; i <= origCol; i++) { refstr.append(firsts5[(int)rf_[rfi_+i]]); } BTDnaString editstr; // daehwan // Edit::toRef((*rd_), ned, editstr, true, trimBeg, trimEnd); Edit::toRef((*rd_), ned, editstr, true, trimBeg + rdi_, trimEnd + (rd_->length() - rdf_)); if(refstr != editstr) { cerr << "Decoded nucleotides and edits don't match reference:" << endl; cerr << " score: " << score.score() << " (" << gaps << " gaps)" << endl; cerr << " edits: "; Edit::print(cerr, ned); cerr << endl; cerr << " decoded nucs: " << (*rd_) << endl; cerr << " edited nucs: " << editstr << endl; cerr << " reference nucs: " << refstr << endl; assert(0); } #endif met.btsucc++; // DP backtraces succeeded return true; } hisat2-2.2.1/aligner_swsse_ee_u8.cpp000066400000000000000000001723721370663763200173750ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /** * aligner_sw_sse.cpp * * Versions of key alignment functions that use vector instructions to * accelerate dynamic programming. Based chiefly on the striped Smith-Waterman * paper and implementation by Michael Farrar. See: * * Farrar M. Striped Smith-Waterman speeds database searches six times over * other SIMD implementations. Bioinformatics. 2007 Jan 15;23(2):156-61. * http://sites.google.com/site/farrarmichael/smith-waterman * * While the paper describes an implementation of Smith-Waterman, we extend it * do end-to-end read alignment as well as local alignment. The change * required for this is minor: we simply let vmax be the maximum element in the * score domain rather than the minimum. * * The vectorized dynamic programming implementation lacks some features that * make it hard to adapt to solving the entire dynamic-programming alignment * problem. For instance: * * - It doesn't respect gap barriers on either end of the read * - It just gives a maximum; not enough information to backtrace without * redoing some alignment * - It's a little difficult to handle st_ and en_, especially st_. * - The query profile mechanism makes handling of ambiguous reference bases a * little tricky (16 cols in query profile lookup table instead of 5) * * Given the drawbacks, it is tempting to use SSE dynamic programming as a * filter rather than as an aligner per se. Here are a few ideas for how it * can be extended to handle more of the alignment problem: * * - Save calculated scores to a big array as we go. We return to this array * to find and backtrace from good solutions. */ #include #include "aligner_sw.h" static const size_t NBYTES_PER_REG = 16; static const size_t NWORDS_PER_REG = 16; // static const size_t NBITS_PER_WORD = 8; static const size_t NBYTES_PER_WORD = 1; // In end-to-end mode, we start high (255) and go low (0). Factoring in // a query profile involves unsigned saturating subtraction, so all the // query profile elements should be expressed as a positive penalty rather // than a negative score. typedef uint8_t TCScore; /** * Build query profile look up tables for the read. The query profile look * up table is organized as a 1D array indexed by [i][j] where i is the * reference character in the current DP column (0=A, 1=C, etc), and j is * the segment of the query we're currently working on. */ void SwAligner::buildQueryProfileEnd2EndSseU8(bool fw) { bool& done = fw ? sseU8fwBuilt_ : sseU8rcBuilt_; if(done) { return; } done = true; const BTDnaString* rd = fw ? rdfw_ : rdrc_; const BTString* qu = fw ? qufw_ : qurc_; // daehwan - allows to align a portion of a read, not the whole. // const size_t len = rd->length(); const size_t len = dpRows(); const size_t seglen = (len + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // How many __m128i's are needed size_t n128s = 64 + // slack bytes, for alignment? (seglen * ALPHA_SIZE) // query profile data * 2; // & gap barrier data assert_gt(n128s, 0); SSEData& d = fw ? sseU8fw_ : sseU8rc_; d.profbuf_.resizeNoCopy(n128s); assert(!d.profbuf_.empty()); d.maxPen_ = d.maxBonus_ = 0; d.lastIter_ = d.lastWord_ = 0; d.qprofStride_ = d.gbarStride_ = 2; d.bias_ = 0; // no bias needed for end-to-end alignment; just use subtraction // For each reference character A, C, G, T, N ... for(size_t refc = 0; refc < ALPHA_SIZE; refc++) { // For each segment ... for(size_t i = 0; i < seglen; i++) { size_t j = i; uint8_t *qprofWords = reinterpret_cast(d.profbuf_.ptr() + (refc * seglen * 2) + (i * 2)); uint8_t *gbarWords = reinterpret_cast(d.profbuf_.ptr() + (refc * seglen * 2) + (i * 2) + 1); // For each sub-word (byte) ... for(size_t k = 0; k < NWORDS_PER_REG; k++) { int sc = 0; *gbarWords = 0; if(j < len) { int readc = (*rd)[j]; int readq = (*qu)[j]; sc = sc_->score(readc, (int)(1 << refc), readq - 33); // Make score positive, to fit in an unsigned sc = -sc; assert_range(0, 255, sc); size_t j_from_end = len - j - 1; if(j < (size_t)sc_->gapbar || j_from_end < (size_t)sc_->gapbar) { // Inside the gap barrier *gbarWords = 0xff; } } if(refc == 0 && j == len-1) { // Remember which 128-bit word and which smaller word has // the final row d.lastIter_ = i; d.lastWord_ = k; } if((size_t)sc > d.maxPen_) { d.maxPen_ = (size_t)sc; } *qprofWords = (uint8_t)sc; gbarWords++; qprofWords++; j += seglen; // update offset into query } } } } #ifndef NDEBUG /** * Return true iff the cell has sane E/F/H values w/r/t its predecessors. */ static bool cellOkEnd2EndU8( SSEData& d, size_t row, size_t col, int refc, int readc, int readq, const Scoring& sc) // scoring scheme { TCScore floorsc = 0; TAlScore ceilsc = MAX_I64; TAlScore offsetsc = -0xff; TAlScore sc_h_cur = (TAlScore)d.mat_.helt(row, col); TAlScore sc_e_cur = (TAlScore)d.mat_.eelt(row, col); TAlScore sc_f_cur = (TAlScore)d.mat_.felt(row, col); if(sc_h_cur > floorsc) { sc_h_cur += offsetsc; } if(sc_e_cur > floorsc) { sc_e_cur += offsetsc; } if(sc_f_cur > floorsc) { sc_f_cur += offsetsc; } bool gapsAllowed = true; size_t rowFromEnd = d.mat_.nrow() - row - 1; if(row < (size_t)sc.gapbar || rowFromEnd < (size_t)sc.gapbar) { gapsAllowed = false; } bool e_left_trans = false, h_left_trans = false; bool f_up_trans = false, h_up_trans = false; bool h_diag_trans = false; if(gapsAllowed) { TAlScore sc_h_left = floorsc; TAlScore sc_e_left = floorsc; TAlScore sc_h_up = floorsc; TAlScore sc_f_up = floorsc; if(col > 0 && sc_e_cur > floorsc && sc_e_cur <= ceilsc) { sc_h_left = d.mat_.helt(row, col-1) + offsetsc; sc_e_left = d.mat_.eelt(row, col-1) + offsetsc; e_left_trans = (sc_e_left > floorsc && sc_e_cur == sc_e_left - sc.readGapExtend()); h_left_trans = (sc_h_left > floorsc && sc_e_cur == sc_h_left - sc.readGapOpen()); assert(e_left_trans || h_left_trans); // Check that we couldn't have got a better E score assert_geq(sc_e_cur, sc_e_left - sc.readGapExtend()); assert_geq(sc_e_cur, sc_h_left - sc.readGapOpen()); } if(row > 0 && sc_f_cur > floorsc && sc_f_cur <= ceilsc) { sc_h_up = d.mat_.helt(row-1, col) + offsetsc; sc_f_up = d.mat_.felt(row-1, col) + offsetsc; f_up_trans = (sc_f_up > floorsc && sc_f_cur == sc_f_up - sc.refGapExtend()); h_up_trans = (sc_h_up > floorsc && sc_f_cur == sc_h_up - sc.refGapOpen()); assert(f_up_trans || h_up_trans); // Check that we couldn't have got a better F score assert_geq(sc_f_cur, sc_f_up - sc.refGapExtend()); assert_geq(sc_f_cur, sc_h_up - sc.refGapOpen()); } } else { assert_geq(floorsc, sc_e_cur); assert_geq(floorsc, sc_f_cur); } if(col > 0 && row > 0 && sc_h_cur > floorsc && sc_h_cur <= ceilsc) { TAlScore sc_h_upleft = d.mat_.helt(row-1, col-1) + offsetsc; TAlScore sc_diag = sc.score(readc, (int)refc, readq - 33); h_diag_trans = sc_h_cur == sc_h_upleft + sc_diag; } assert( sc_h_cur <= floorsc || e_left_trans || h_left_trans || f_up_trans || h_up_trans || h_diag_trans || sc_h_cur > ceilsc || row == 0 || col == 0); return true; } #endif /*ndef NDEBUG*/ #ifdef NDEBUG #define assert_all_eq0(x) #define assert_all_gt(x, y) #define assert_all_gt_lo(x) #define assert_all_lt(x, y) #define assert_all_lt_hi(x) #else #define assert_all_eq0(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_xor_si128(z, z); \ tmp = _mm_cmpeq_epi16(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_gt(x, y) { \ __m128i tmp = _mm_cmpgt_epu8(x, y); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_gt_lo(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_xor_si128(z, z); \ tmp = _mm_cmpgt_epu8(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_lt(x, y) { \ __m128i z = _mm_setzero_si128(); \ z = _mm_xor_si128(z, z); \ __m128i tmp = _mm_subs_epu8(y, x); \ tmp = _mm_cmpeq_epi16(tmp, z); \ assert_eq(0x0000, _mm_movemask_epi8(tmp)); \ } #define assert_all_lt_hi(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_cmpeq_epu8(z, z); \ z = _mm_srli_epu8(z, 1); \ tmp = _mm_cmplt_epu8(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #endif /** * Aligns by filling a dynamic programming matrix with the SSE-accelerated, * banded DP approach of Farrar. As it goes, it determines which cells we * might backtrace from and tallies the best (highest-scoring) N backtrace * candidate cells per diagonal. Also returns the alignment score of the best * alignment in the matrix. * * This routine does *not* maintain a matrix holding the entire matrix worth of * scores, nor does it maintain any other dense O(mn) data structure, as this * would quickly exhaust memory for queries longer than about 10,000 kb. * Instead, in the fill stage it maintains two columns worth of scores at a * time (current/previous, or right/left) - these take O(m) space. When * finished with the current column, it determines which cells from the * previous column, if any, are candidates we might backtrace from to find a * full alignment. A candidate cell has a score that rises above the threshold * and isn't improved upon by a match in the next column. The best N * candidates per diagonal are stored in a O(m + n) data structure. */ TAlScore SwAligner::alignGatherEE8(int& flag, bool debug) { assert_leq(rdf_, rd_->length()); assert_leq(rdf_, qu_->length()); assert_lt(rfi_, rff_); assert_lt(rdi_, rdf_); assert_eq(rd_->length(), qu_->length()); assert_geq(sc_->gapbar, 1); assert(repOk()); #ifndef NDEBUG for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert_range(0, 16, (int)rf_[i]); } #endif SSEData& d = fw_ ? sseU8fw_ : sseU8rc_; SSEMetrics& met = extend_ ? sseU8ExtendMet_ : sseU8MateMet_; if(!debug) met.dp++; buildQueryProfileEnd2EndSseU8(fw_); assert(!d.profbuf_.empty()); assert_eq(0, d.maxBonus_); size_t iter = (dpRows() + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // iter = segLen int dup; // Now set up the score vectors. We just need two columns worth, which // we'll call "left" and "right". d.vecbuf_.resize(4 * 2 * iter); d.vecbuf_.zero(); __m128i *vbuf_l = d.vecbuf_.ptr(); __m128i *vbuf_r = d.vecbuf_.ptr() + (4 * iter); // This is the data structure that holds candidate cells per diagonal. const size_t ndiags = rff_ - rfi_ + dpRows() - 1; if(!debug) { btdiag_.init(ndiags, 2); } // Data structure that holds checkpointed anti-diagonals TAlScore perfectScore = sc_->perfectScore(dpRows()); bool checkpoint = true; bool cpdebug = false; #ifndef NDEBUG cpdebug = dpRows() < 1000; #endif cper_.init( dpRows(), // # rows rff_ - rfi_, // # columns cperPerPow2_, // checkpoint every 1 << perpow2 diags (& next) perfectScore, // perfect score (for sanity checks) true, // matrix cells have 8-bit scores? cperTri_, // triangular mini-fills? false, // alignment is local? cpdebug); // save all cells for debugging? // Many thanks to Michael Farrar for releasing his striped Smith-Waterman // implementation: // // http://sites.google.com/site/farrarmichael/smith-waterman // // Much of the implmentation below is adapted from Michael's code. // Set all elts to reference gap open penalty __m128i rfgapo = _mm_setzero_si128(); __m128i rfgape = _mm_setzero_si128(); __m128i rdgapo = _mm_setzero_si128(); __m128i rdgape = _mm_setzero_si128(); __m128i vlo = _mm_setzero_si128(); __m128i vhi = _mm_setzero_si128(); __m128i ve = _mm_setzero_si128(); __m128i vf = _mm_setzero_si128(); __m128i vh = _mm_setzero_si128(); __m128i vhd = _mm_setzero_si128(); __m128i vhdtmp = _mm_setzero_si128(); __m128i vtmp = _mm_setzero_si128(); __m128i vzero = _mm_setzero_si128(); __m128i vhilsw = _mm_setzero_si128(); assert_gt(sc_->refGapOpen(), 0); assert_leq(sc_->refGapOpen(), MAX_U8); dup = (sc_->refGapOpen() << 8) | (sc_->refGapOpen() & 0x00ff); rfgapo = _mm_insert_epi16(rfgapo, dup, 0); rfgapo = _mm_shufflelo_epi16(rfgapo, 0); rfgapo = _mm_shuffle_epi32(rfgapo, 0); // Set all elts to reference gap extension penalty assert_gt(sc_->refGapExtend(), 0); assert_leq(sc_->refGapExtend(), MAX_U8); assert_leq(sc_->refGapExtend(), sc_->refGapOpen()); dup = (sc_->refGapExtend() << 8) | (sc_->refGapExtend() & 0x00ff); rfgape = _mm_insert_epi16(rfgape, dup, 0); rfgape = _mm_shufflelo_epi16(rfgape, 0); rfgape = _mm_shuffle_epi32(rfgape, 0); // Set all elts to read gap open penalty assert_gt(sc_->readGapOpen(), 0); assert_leq(sc_->readGapOpen(), MAX_U8); dup = (sc_->readGapOpen() << 8) | (sc_->readGapOpen() & 0x00ff); rdgapo = _mm_insert_epi16(rdgapo, dup, 0); rdgapo = _mm_shufflelo_epi16(rdgapo, 0); rdgapo = _mm_shuffle_epi32(rdgapo, 0); // Set all elts to read gap extension penalty assert_gt(sc_->readGapExtend(), 0); assert_leq(sc_->readGapExtend(), MAX_U8); assert_leq(sc_->readGapExtend(), sc_->readGapOpen()); dup = (sc_->readGapExtend() << 8) | (sc_->readGapExtend() & 0x00ff); rdgape = _mm_insert_epi16(rdgape, dup, 0); rdgape = _mm_shufflelo_epi16(rdgape, 0); rdgape = _mm_shuffle_epi32(rdgape, 0); vhi = _mm_cmpeq_epi16(vhi, vhi); // all elts = 0xffff vlo = _mm_xor_si128(vlo, vlo); // all elts = 0 // vhilsw: topmost (least sig) word set to 0x7fff, all other words=0 vhilsw = _mm_shuffle_epi32(vhi, 0); vhilsw = _mm_srli_si128(vhilsw, NBYTES_PER_REG - NBYTES_PER_WORD); // Points to a long vector of __m128i where each element is a block of // contiguous cells in the E, F or H matrix. If the index % 3 == 0, then // the block of cells is from the E matrix. If index % 3 == 1, they're // from the F matrix. If index % 3 == 2, then they're from the H matrix. // Blocks of cells are organized in the same interleaved manner as they are // calculated by the Farrar algorithm. const __m128i *pvScore; // points into the query profile const size_t colstride = ROWSTRIDE_2COL * iter; // Initialize the H and E vectors in the first matrix column __m128i *pvELeft = vbuf_l + 0; __m128i *pvERight = vbuf_r + 0; /* __m128i *pvFLeft = vbuf_l + 1; */ __m128i *pvFRight = vbuf_r + 1; __m128i *pvHLeft = vbuf_l + 2; __m128i *pvHRight = vbuf_r + 2; // Maximum score in final row bool found = false; TCScore lrmax = MIN_U8; for(size_t i = 0; i < iter; i++) { _mm_store_si128(pvERight, vlo); pvERight += ROWSTRIDE_2COL; // Could initialize Hs to high or low. If high, cells in the lower // triangle will have somewhat more legitiate scores, but still won't // be exhaustively scored. _mm_store_si128(pvHRight, vlo); pvHRight += ROWSTRIDE_2COL; } assert_gt(sc_->gapbar, 0); size_t nfixup = 0; // Fill in the table as usual but instead of using the same gap-penalty // vector for each iteration of the inner loop, load words out of a // pre-calculated gap vector parallel to the query profile. The pre- // calculated gap vectors enforce the gap barrier constraint by making it // infinitely costly to introduce a gap in barrier rows. // // AND use a separate loop to fill in the first row of the table, enforcing // the st_ constraints in the process. This is awkward because it // separates the processing of the first row from the others and might make // it difficult to use the first-row results in the next row, but it might // be the simplest and least disruptive way to deal with the st_ constraint. for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { // Swap left and right; vbuf_l is the vector on the left, which we // generally load from, and vbuf_r is the vector on the right, which we // generally store to. swap(vbuf_l, vbuf_r); pvELeft = vbuf_l + 0; pvERight = vbuf_r + 0; /* pvFLeft = vbuf_l + 1; */ pvFRight = vbuf_r + 1; pvHLeft = vbuf_l + 2; pvHRight = vbuf_r + 2; // Fetch the appropriate query profile. Note that elements of rf_ must // be numbers, not masks. const int refc = (int)rf_[i]; // Fetch the appropriate query profile size_t off = (size_t)firsts5[refc] * iter * 2; pvScore = d.profbuf_.ptr() + off; // even elts = query profile, odd = gap barrier // Set all cells to low value vf = _mm_xor_si128(vf, vf); // Load H vector from the final row of the previous column vh = _mm_load_si128(pvHLeft + colstride - ROWSTRIDE_2COL); // Shift 2 bytes down so that topmost (least sig) cell gets 0 vh = _mm_slli_si128(vh, NBYTES_PER_WORD); // Fill topmost (least sig) cell with high value vh = _mm_or_si128(vh, vhilsw); // For each character in the reference text: size_t j; for(j = 0; j < iter; j++) { // Load cells from E, calculated previously ve = _mm_load_si128(pvELeft); vhd = _mm_load_si128(pvHLeft); assert_all_lt(ve, vhi); pvELeft += ROWSTRIDE_2COL; // Store cells in F, calculated previously vf = _mm_subs_epu8(vf, pvScore[1]); // veto some ref gap extensions _mm_store_si128(pvFRight, vf); pvFRight += ROWSTRIDE_2COL; // Factor in query profile (matches and mismatches) vh = _mm_subs_epu8(vh, pvScore[0]); // Update H, factoring in E and F vh = _mm_max_epu8(vh, vf); // Update vE value vhdtmp = vhd; vhd = _mm_subs_epu8(vhd, rdgapo); vhd = _mm_subs_epu8(vhd, pvScore[1]); // veto some read gap opens ve = _mm_subs_epu8(ve, rdgape); ve = _mm_max_epu8(ve, vhd); vh = _mm_max_epu8(vh, ve); // Save the new vH values _mm_store_si128(pvHRight, vh); pvHRight += ROWSTRIDE_2COL; vtmp = vh; assert_all_lt(ve, vhi); // Load the next h value vh = vhdtmp; pvHLeft += ROWSTRIDE_2COL; // Save E values _mm_store_si128(pvERight, ve); pvERight += ROWSTRIDE_2COL; // Update vf value vtmp = _mm_subs_epu8(vtmp, rfgapo); vf = _mm_subs_epu8(vf, rfgape); assert_all_lt(vf, vhi); vf = _mm_max_epu8(vf, vtmp); pvScore += 2; // move on to next query profile / gap veto } // pvHStore, pvELoad, pvEStore have all rolled over to the next column pvFRight -= colstride; // reset to start of column vtmp = _mm_load_si128(pvFRight); pvHRight -= colstride; // reset to start of column vh = _mm_load_si128(pvHRight); pvScore = d.profbuf_.ptr() + off + 1; // reset veto vector // vf from last row gets shifted down by one to overlay the first row // rfgape has already been subtracted from it. vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_subs_epu8(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epu8(vtmp, vf); vtmp = _mm_subs_epu8(vf, vtmp); vtmp = _mm_cmpeq_epi8(vtmp, vzero); int cmp = _mm_movemask_epi8(vtmp); // If any element of vtmp is greater than H - gap-open... j = 0; while(cmp != 0xffff) { // Store this vf _mm_store_si128(pvFRight, vf); pvFRight += ROWSTRIDE_2COL; // Update vh w/r/t new vf vh = _mm_max_epu8(vh, vf); // Save vH values _mm_store_si128(pvHRight, vh); pvHRight += ROWSTRIDE_2COL; pvScore += 2; assert_lt(j, iter); if(++j == iter) { pvFRight -= colstride; vtmp = _mm_load_si128(pvFRight); // load next vf ASAP pvHRight -= colstride; vh = _mm_load_si128(pvHRight); // load next vh ASAP pvScore = d.profbuf_.ptr() + off + 1; j = 0; vf = _mm_slli_si128(vf, NBYTES_PER_WORD); } else { vtmp = _mm_load_si128(pvFRight); // load next vf ASAP vh = _mm_load_si128(pvHRight); // load next vh ASAP } // Update F with another gap extension vf = _mm_subs_epu8(vf, rfgape); vf = _mm_subs_epu8(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epu8(vtmp, vf); vtmp = _mm_subs_epu8(vf, vtmp); vtmp = _mm_cmpeq_epi8(vtmp, vzero); cmp = _mm_movemask_epi8(vtmp); nfixup++; } // Check in the last row for the maximum so far __m128i *vtmp = vbuf_r + 2 /* H */ + (d.lastIter_ * ROWSTRIDE_2COL); // Note: we may not want to extract from the final row TCScore lr = ((TCScore*)(vtmp))[d.lastWord_]; found = true; if(lr > lrmax) { lrmax = lr; } // Now we'd like to know whether the bottommost element of the right // column is a candidate we might backtrace from. First question is: // did it exceed the minimum score threshold? TAlScore score = (TAlScore)(lr - 0xff); if(lr == MIN_U8) { score = MIN_I64; } if(!debug && score >= minsc_) { DpBtCandidate cand(dpRows() - 1, i - rfi_, score); btdiag_.add(i - rfi_, cand); } // Save some elements to checkpoints if(checkpoint) { __m128i *pvE = vbuf_r + 0; __m128i *pvF = vbuf_r + 1; __m128i *pvH = vbuf_r + 2; size_t coli = i - rfi_; if(coli < cper_.locol_) cper_.locol_ = coli; if(coli > cper_.hicol_) cper_.hicol_ = coli; if(cperTri_) { size_t rc_mod = coli & cper_.lomask_; assert_lt(rc_mod, cper_.per_); int64_t row = -(int64_t)rc_mod-1; int64_t row_mod = row; int64_t row_div = 0; size_t idx = coli >> cper_.perpow2_; size_t idxrow = idx * cper_.nrow_; assert_eq(4, ROWSTRIDE_2COL); bool done = false; while(true) { row += (cper_.per_ - 2); row_mod += (cper_.per_ - 2); for(size_t j = 0; j < 2; j++) { row++; row_mod++; if(row >= 0 && (size_t)row < cper_.nrow_) { // Update row divided by iter_ and mod iter_ while(row_mod >= (int64_t)iter) { row_mod -= (int64_t)iter; row_div++; } size_t delt = idxrow + row; size_t vecoff = (row_mod << 6) + row_div; assert_lt(row_div, 16); int16_t h_sc = ((uint8_t*)pvH)[vecoff]; int16_t e_sc = ((uint8_t*)pvE)[vecoff]; int16_t f_sc = ((uint8_t*)pvF)[vecoff]; if(h_sc == 0) h_sc = MIN_I16; else h_sc -= 0xff; if(e_sc == 0) e_sc = MIN_I16; else e_sc -= 0xff; if(f_sc == 0) f_sc = MIN_I16; else f_sc -= 0xff; assert_leq(h_sc, cper_.perf_); assert_leq(e_sc, cper_.perf_); assert_leq(f_sc, cper_.perf_); CpQuad *qdiags = ((j == 0) ? cper_.qdiag1s_.ptr() : cper_.qdiag2s_.ptr()); qdiags[delt].sc[0] = h_sc; qdiags[delt].sc[1] = e_sc; qdiags[delt].sc[2] = f_sc; } // if(row >= 0 && row < nrow_) else if(row >= 0 && (size_t)row >= cper_.nrow_) { done = true; break; } } // end of loop over anti-diags if(done) { break; } idx++; idxrow += cper_.nrow_; } } else { // If this is the first column, take this opportunity to // pre-calculate the coordinates of the elements we're going to // checkpoint. if(coli == 0) { size_t cpi = cper_.per_-1; size_t cpimod = cper_.per_-1; size_t cpidiv = 0; cper_.commitMap_.clear(); while(cpi < cper_.nrow_) { while(cpimod >= iter) { cpimod -= iter; cpidiv++; } size_t vecoff = (cpimod << 6) + cpidiv; cper_.commitMap_.push_back(vecoff); cpi += cper_.per_; cpimod += cper_.per_; } } // Save all the rows size_t rowoff = 0; size_t sz = cper_.commitMap_.size(); for(size_t i = 0; i < sz; i++, rowoff += cper_.ncol_) { size_t vecoff = cper_.commitMap_[i]; int16_t h_sc = ((uint8_t*)pvH)[vecoff]; //int16_t e_sc = ((uint8_t*)pvE)[vecoff]; int16_t f_sc = ((uint8_t*)pvF)[vecoff]; if(h_sc == 0) h_sc = MIN_I16; else h_sc -= 0xff; //if(e_sc == 0) e_sc = MIN_I16; //else e_sc -= 0xff; if(f_sc == 0) f_sc = MIN_I16; else f_sc -= 0xff; assert_leq(h_sc, cper_.perf_); //assert_leq(e_sc, cper_.perf_); assert_leq(f_sc, cper_.perf_); CpQuad& dst = cper_.qrows_[rowoff + coli]; dst.sc[0] = h_sc; //dst.sc[1] = e_sc; dst.sc[2] = f_sc; } // Is this a column we'd like to checkpoint? if((coli & cper_.lomask_) == cper_.lomask_) { // Save the column using memcpys assert_gt(coli, 0); size_t wordspercol = cper_.niter_ * ROWSTRIDE_2COL; size_t coloff = (coli >> cper_.perpow2_) * wordspercol; __m128i *dst = cper_.qcols_.ptr() + coloff; memcpy(dst, vbuf_r, sizeof(__m128i) * wordspercol); } } if(cper_.debug_) { // Save the column using memcpys size_t wordspercol = cper_.niter_ * ROWSTRIDE_2COL; size_t coloff = coli * wordspercol; __m128i *dst = cper_.qcolsD_.ptr() + coloff; memcpy(dst, vbuf_r, sizeof(__m128i) * wordspercol); } } } // Update metrics if(!debug) { size_t ninner = (rff_ - rfi_) * iter; met.col += (rff_ - rfi_); // DP columns met.cell += (ninner * NWORDS_PER_REG); // DP cells met.inner += ninner; // DP inner loop iters met.fixup += nfixup; // DP fixup loop iters } flag = 0; // Did we find a solution? TAlScore score = MIN_I64; if(!found) { flag = -1; // no if(!debug) met.dpfail++; return MIN_I64; } else { score = (TAlScore)(lrmax - 0xff); if(score < minsc_) { flag = -1; // no if(!debug) met.dpfail++; return score; } } // Could we have saturated? if(lrmax == MIN_U8) { flag = -2; // yes if(!debug) met.dpsat++; return MIN_I64; } // Now take all the backtrace candidates in the btdaig_ structure and // dump them into the btncand_ array. They'll be sorted later. if(!debug) { btdiag_.dump(btncand_); assert(!btncand_.empty()); } // Return largest score if(!debug) met.dpsucc++; return score; } /** * Solve the current alignment problem using SSE instructions that operate on 16 * unsigned 8-bit values packed into a single 128-bit register. */ TAlScore SwAligner::alignNucleotidesEnd2EndSseU8(int& flag, bool debug) { assert_leq(rdf_, rd_->length()); assert_leq(rdf_, qu_->length()); assert_lt(rfi_, rff_); assert_lt(rdi_, rdf_); assert_eq(rd_->length(), qu_->length()); assert_geq(sc_->gapbar, 1); assert(repOk()); #ifndef NDEBUG for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert_range(0, 16, (int)rf_[i]); } #endif SSEData& d = fw_ ? sseU8fw_ : sseU8rc_; SSEMetrics& met = extend_ ? sseU8ExtendMet_ : sseU8MateMet_; if(!debug) met.dp++; buildQueryProfileEnd2EndSseU8(fw_); assert(!d.profbuf_.empty()); assert_eq(0, d.maxBonus_); size_t iter = (dpRows() + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // iter = segLen int dup; // Many thanks to Michael Farrar for releasing his striped Smith-Waterman // implementation: // // http://sites.google.com/site/farrarmichael/smith-waterman // // Much of the implmentation below is adapted from Michael's code. // Set all elts to reference gap open penalty __m128i rfgapo = _mm_setzero_si128(); __m128i rfgape = _mm_setzero_si128(); __m128i rdgapo = _mm_setzero_si128(); __m128i rdgape = _mm_setzero_si128(); __m128i vlo = _mm_setzero_si128(); __m128i vhi = _mm_setzero_si128(); __m128i ve = _mm_setzero_si128(); __m128i vf = _mm_setzero_si128(); __m128i vh = _mm_setzero_si128(); #if 0 __m128i vhd = _mm_setzero_si128(); __m128i vhdtmp = _mm_setzero_si128(); #endif __m128i vtmp = _mm_setzero_si128(); __m128i vzero = _mm_setzero_si128(); __m128i vhilsw = _mm_setzero_si128(); assert_gt(sc_->refGapOpen(), 0); assert_leq(sc_->refGapOpen(), MAX_U8); dup = (sc_->refGapOpen() << 8) | (sc_->refGapOpen() & 0x00ff); rfgapo = _mm_insert_epi16(rfgapo, dup, 0); rfgapo = _mm_shufflelo_epi16(rfgapo, 0); rfgapo = _mm_shuffle_epi32(rfgapo, 0); // Set all elts to reference gap extension penalty assert_gt(sc_->refGapExtend(), 0); assert_leq(sc_->refGapExtend(), MAX_U8); assert_leq(sc_->refGapExtend(), sc_->refGapOpen()); dup = (sc_->refGapExtend() << 8) | (sc_->refGapExtend() & 0x00ff); rfgape = _mm_insert_epi16(rfgape, dup, 0); rfgape = _mm_shufflelo_epi16(rfgape, 0); rfgape = _mm_shuffle_epi32(rfgape, 0); // Set all elts to read gap open penalty assert_gt(sc_->readGapOpen(), 0); assert_leq(sc_->readGapOpen(), MAX_U8); dup = (sc_->readGapOpen() << 8) | (sc_->readGapOpen() & 0x00ff); rdgapo = _mm_insert_epi16(rdgapo, dup, 0); rdgapo = _mm_shufflelo_epi16(rdgapo, 0); rdgapo = _mm_shuffle_epi32(rdgapo, 0); // Set all elts to read gap extension penalty assert_gt(sc_->readGapExtend(), 0); assert_leq(sc_->readGapExtend(), MAX_U8); assert_leq(sc_->readGapExtend(), sc_->readGapOpen()); dup = (sc_->readGapExtend() << 8) | (sc_->readGapExtend() & 0x00ff); rdgape = _mm_insert_epi16(rdgape, dup, 0); rdgape = _mm_shufflelo_epi16(rdgape, 0); rdgape = _mm_shuffle_epi32(rdgape, 0); vhi = _mm_cmpeq_epi16(vhi, vhi); // all elts = 0xffff vlo = _mm_xor_si128(vlo, vlo); // all elts = 0 // vhilsw: topmost (least sig) word set to 0x7fff, all other words=0 vhilsw = _mm_shuffle_epi32(vhi, 0); vhilsw = _mm_srli_si128(vhilsw, NBYTES_PER_REG - NBYTES_PER_WORD); // Points to a long vector of __m128i where each element is a block of // contiguous cells in the E, F or H matrix. If the index % 3 == 0, then // the block of cells is from the E matrix. If index % 3 == 1, they're // from the F matrix. If index % 3 == 2, then they're from the H matrix. // Blocks of cells are organized in the same interleaved manner as they are // calculated by the Farrar algorithm. const __m128i *pvScore; // points into the query profile d.mat_.init(dpRows(), rff_ - rfi_, NWORDS_PER_REG); const size_t colstride = d.mat_.colstride(); //const size_t rowstride = d.mat_.rowstride(); assert_eq(ROWSTRIDE, colstride / iter); // Initialize the H and E vectors in the first matrix column __m128i *pvHTmp = d.mat_.tmpvec(0, 0); __m128i *pvETmp = d.mat_.evec(0, 0); // Maximum score in final row bool found = false; TCScore lrmax = MIN_U8; for(size_t i = 0; i < iter; i++) { _mm_store_si128(pvETmp, vlo); _mm_store_si128(pvHTmp, vlo); // start high in end-to-end mode pvETmp += ROWSTRIDE; pvHTmp += ROWSTRIDE; } // These are swapped just before the innermost loop __m128i *pvHStore = d.mat_.hvec(0, 0); __m128i *pvHLoad = d.mat_.tmpvec(0, 0); __m128i *pvELoad = d.mat_.evec(0, 0); __m128i *pvEStore = d.mat_.evecUnsafe(0, 1); __m128i *pvFStore = d.mat_.fvec(0, 0); __m128i *pvFTmp = NULL; assert_gt(sc_->gapbar, 0); size_t nfixup = 0; // Fill in the table as usual but instead of using the same gap-penalty // vector for each iteration of the inner loop, load words out of a // pre-calculated gap vector parallel to the query profile. The pre- // calculated gap vectors enforce the gap barrier constraint by making it // infinitely costly to introduce a gap in barrier rows. // // AND use a separate loop to fill in the first row of the table, enforcing // the st_ constraints in the process. This is awkward because it // separates the processing of the first row from the others and might make // it difficult to use the first-row results in the next row, but it might // be the simplest and least disruptive way to deal with the st_ constraint. colstop_ = rff_ - 1; lastsolcol_ = 0; for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert(pvFStore == d.mat_.fvec(0, i - rfi_)); assert(pvHStore == d.mat_.hvec(0, i - rfi_)); // Fetch the appropriate query profile. Note that elements of rf_ must // be numbers, not masks. const int refc = (int)rf_[i]; size_t off = (size_t)firsts5[refc] * iter * 2; pvScore = d.profbuf_.ptr() + off; // even elts = query profile, odd = gap barrier // Set all cells to low value vf = _mm_xor_si128(vf, vf); // Load H vector from the final row of the previous column vh = _mm_load_si128(pvHLoad + colstride - ROWSTRIDE); // Shift 2 bytes down so that topmost (least sig) cell gets 0 vh = _mm_slli_si128(vh, NBYTES_PER_WORD); // Fill topmost (least sig) cell with high value vh = _mm_or_si128(vh, vhilsw); // For each character in the reference text: size_t j; for(j = 0; j < iter; j++) { // Load cells from E, calculated previously ve = _mm_load_si128(pvELoad); #if 0 vhd = _mm_load_si128(pvHLoad); #endif assert_all_lt(ve, vhi); pvELoad += ROWSTRIDE; // Store cells in F, calculated previously vf = _mm_subs_epu8(vf, pvScore[1]); // veto some ref gap extensions _mm_store_si128(pvFStore, vf); pvFStore += ROWSTRIDE; // Factor in query profile (matches and mismatches) vh = _mm_subs_epu8(vh, pvScore[0]); // Update H, factoring in E and F vh = _mm_max_epu8(vh, ve); vh = _mm_max_epu8(vh, vf); // Save the new vH values _mm_store_si128(pvHStore, vh); pvHStore += ROWSTRIDE; // Update vE value vtmp = vh; #if 0 vhdtmp = vhd; vhd = _mm_subs_epu8(vhd, rdgapo); vhd = _mm_subs_epu8(vhd, pvScore[1]); // veto some read gap opens ve = _mm_subs_epu8(ve, rdgape); ve = _mm_max_epu8(ve, vhd); #else vh = _mm_subs_epu8(vh, rdgapo); vh = _mm_subs_epu8(vh, pvScore[1]); // veto some read gap opens ve = _mm_subs_epu8(ve, rdgape); ve = _mm_max_epu8(ve, vh); #endif assert_all_lt(ve, vhi); // Load the next h value #if 0 vh = vhdtmp; #else vh = _mm_load_si128(pvHLoad); #endif pvHLoad += ROWSTRIDE; // Save E values _mm_store_si128(pvEStore, ve); pvEStore += ROWSTRIDE; // Update vf value vtmp = _mm_subs_epu8(vtmp, rfgapo); vf = _mm_subs_epu8(vf, rfgape); assert_all_lt(vf, vhi); vf = _mm_max_epu8(vf, vtmp); pvScore += 2; // move on to next query profile / gap veto } // pvHStore, pvELoad, pvEStore have all rolled over to the next column pvFTmp = pvFStore; pvFStore -= colstride; // reset to start of column vtmp = _mm_load_si128(pvFStore); pvHStore -= colstride; // reset to start of column vh = _mm_load_si128(pvHStore); #if 0 #else pvEStore -= colstride; // reset to start of column ve = _mm_load_si128(pvEStore); #endif pvHLoad = pvHStore; // new pvHLoad = pvHStore pvScore = d.profbuf_.ptr() + off + 1; // reset veto vector // vf from last row gets shifted down by one to overlay the first row // rfgape has already been subtracted from it. vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_subs_epu8(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epu8(vtmp, vf); vtmp = _mm_subs_epu8(vf, vtmp); vtmp = _mm_cmpeq_epi8(vtmp, vzero); int cmp = _mm_movemask_epi8(vtmp); // If any element of vtmp is greater than H - gap-open... j = 0; while(cmp != 0xffff) { // Store this vf _mm_store_si128(pvFStore, vf); pvFStore += ROWSTRIDE; // Update vh w/r/t new vf vh = _mm_max_epu8(vh, vf); // Save vH values _mm_store_si128(pvHStore, vh); pvHStore += ROWSTRIDE; // Update E in case it can be improved using our new vh #if 0 #else vh = _mm_subs_epu8(vh, rdgapo); vh = _mm_subs_epu8(vh, *pvScore); // veto some read gap opens ve = _mm_max_epu8(ve, vh); _mm_store_si128(pvEStore, ve); pvEStore += ROWSTRIDE; #endif pvScore += 2; assert_lt(j, iter); if(++j == iter) { pvFStore -= colstride; vtmp = _mm_load_si128(pvFStore); // load next vf ASAP pvHStore -= colstride; vh = _mm_load_si128(pvHStore); // load next vh ASAP #if 0 #else pvEStore -= colstride; ve = _mm_load_si128(pvEStore); // load next ve ASAP #endif pvScore = d.profbuf_.ptr() + off + 1; j = 0; vf = _mm_slli_si128(vf, NBYTES_PER_WORD); } else { vtmp = _mm_load_si128(pvFStore); // load next vf ASAP vh = _mm_load_si128(pvHStore); // load next vh ASAP #if 0 #else ve = _mm_load_si128(pvEStore); // load next vh ASAP #endif } // Update F with another gap extension vf = _mm_subs_epu8(vf, rfgape); vf = _mm_subs_epu8(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epu8(vtmp, vf); vtmp = _mm_subs_epu8(vf, vtmp); vtmp = _mm_cmpeq_epi8(vtmp, vzero); cmp = _mm_movemask_epi8(vtmp); nfixup++; } #ifndef NDEBUG if(true && (rand() & 15) == 0) { // This is a work-intensive sanity check; each time we finish filling // a column, we check that each H, E, and F is sensible. for(size_t k = 0; k < dpRows(); k++) { assert(cellOkEnd2EndU8( d, k, // row i - rfi_, // col refc, // reference mask (int)(*rd_)[rdi_+k], // read char (int)(*qu_)[rdi_+k], // read quality *sc_)); // scoring scheme } } #endif __m128i *vtmp = d.mat_.hvec(d.lastIter_, i-rfi_); // Note: we may not want to extract from the final row TCScore lr = ((TCScore*)(vtmp))[d.lastWord_]; found = true; if(lr > lrmax) { lrmax = lr; } // pvELoad and pvHLoad are already where they need to be // Adjust the load and store vectors here. pvHStore = pvHLoad + colstride; pvEStore = pvELoad + colstride; pvFStore = pvFTmp; } // Update metrics if(!debug) { size_t ninner = (rff_ - rfi_) * iter; met.col += (rff_ - rfi_); // DP columns met.cell += (ninner * NWORDS_PER_REG); // DP cells met.inner += ninner; // DP inner loop iters met.fixup += nfixup; // DP fixup loop iters } flag = 0; // Did we find a solution? TAlScore score = MIN_I64; if(!found) { flag = -1; // no if(!debug) met.dpfail++; return MIN_I64; } else { score = (TAlScore)(lrmax - 0xff); if(score < minsc_) { flag = -1; // no if(!debug) met.dpfail++; return score; } } // Could we have saturated? if(lrmax == MIN_U8) { flag = -2; // yes if(!debug) met.dpsat++; return MIN_I64; } // Return largest score if(!debug) met.dpsucc++; return score; } /** * Given a filled-in DP table, populate the btncand_ list with candidate cells * that might be at the ends of valid alignments. No need to do this unless * the maximum score returned by the align*() func is >= the minimum. * * Only cells that are exhaustively scored are candidates. Those are the * cells inside the shape made of o's in this: * * |-maxgaps-| * ********************************* - * ******************************** | * ******************************* | * ****************************** | * ***************************** | * **************************** read len * *************************** | * ************************** | * ************************* | * ************************ | * ***********oooooooooooo - * |-maxgaps-| * |-readlen-| * |-------skip--------| * * And it's possible for the shape to be truncated on the left and right sides. * * */ bool SwAligner::gatherCellsNucleotidesEnd2EndSseU8(TAlScore best) { // What's the minimum number of rows that can possibly be spanned by an // alignment that meets the minimum score requirement? assert(sse8succ_); const size_t ncol = rff_ - rfi_; const size_t nrow = dpRows(); assert_gt(nrow, 0); btncand_.clear(); btncanddone_.clear(); SSEData& d = fw_ ? sseU8fw_ : sseU8rc_; SSEMetrics& met = extend_ ? sseU8ExtendMet_ : sseU8MateMet_; assert(!d.profbuf_.empty()); const size_t colstride = d.mat_.colstride(); ASSERT_ONLY(bool sawbest = false); __m128i *pvH = d.mat_.hvec(d.lastIter_, 0); for(size_t j = 0; j < ncol; j++) { TAlScore sc = (TAlScore)(((TCScore*)pvH)[d.lastWord_] - 0xff); assert_leq(sc, best); ASSERT_ONLY(sawbest = (sawbest || sc == best)); if(sc >= minsc_) { // Yes, this is legit met.gathsol++; btncand_.expand(); btncand_.back().init(nrow-1, j, sc); } pvH += colstride; } assert(sawbest); if(!btncand_.empty()) { d.mat_.initMasks(); } return !btncand_.empty(); } #define MOVE_VEC_PTR_UP(vec, rowvec, rowelt) { \ if(rowvec == 0) { \ rowvec += d.mat_.nvecrow_; \ vec += d.mat_.colstride_; \ rowelt--; \ } \ rowvec--; \ vec -= ROWSTRIDE; \ } #define MOVE_VEC_PTR_LEFT(vec, rowvec, rowelt) { vec -= d.mat_.colstride_; } #define MOVE_VEC_PTR_UPLEFT(vec, rowvec, rowelt) { \ MOVE_VEC_PTR_UP(vec, rowvec, rowelt); \ MOVE_VEC_PTR_LEFT(vec, rowvec, rowelt); \ } #define MOVE_ALL_LEFT() { \ MOVE_VEC_PTR_LEFT(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_LEFT(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_LEFT(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_LEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define MOVE_ALL_UP() { \ MOVE_VEC_PTR_UP(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_UP(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_UP(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_UP(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define MOVE_ALL_UPLEFT() { \ MOVE_VEC_PTR_UPLEFT(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_UPLEFT(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_UPLEFT(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_UPLEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define NEW_ROW_COL(row, col) { \ rowelt = row / d.mat_.nvecrow_; \ rowvec = row % d.mat_.nvecrow_; \ eltvec = (col * d.mat_.colstride_) + (rowvec * ROWSTRIDE); \ cur_vec = d.mat_.matbuf_.ptr() + eltvec; \ left_vec = cur_vec; \ left_rowelt = rowelt; \ left_rowvec = rowvec; \ MOVE_VEC_PTR_LEFT(left_vec, left_rowvec, left_rowelt); \ up_vec = cur_vec; \ up_rowelt = rowelt; \ up_rowvec = rowvec; \ MOVE_VEC_PTR_UP(up_vec, up_rowvec, up_rowelt); \ upleft_vec = up_vec; \ upleft_rowelt = up_rowelt; \ upleft_rowvec = up_rowvec; \ MOVE_VEC_PTR_LEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } /** * Given the dynamic programming table and a cell, trace backwards from the * cell and install the edits and score/penalty in the appropriate fields * of res. The RandomSource is used to break ties among equally good ways * of tracing back. * * Whenever we enter a cell, we check whether the read/ref coordinates of * that cell correspond to a cell we traversed constructing a previous * alignment. If so, we backtrack to the last decision point, mask out the * path that led to the previously observed cell, and continue along a * different path; or, if there are no more paths to try, we give up. * * If an alignment is found, 'off' is set to the alignment's upstream-most * reference character's offset into the chromosome and true is returned. * Otherwise, false is returned. */ bool SwAligner::backtraceNucleotidesEnd2EndSseU8( TAlScore escore, // in: expected score SwResult& res, // out: store results (edits and scores) here size_t& off, // out: store diagonal projection of origin size_t& nbts, // out: # backtracks size_t row, // start in this row size_t col, // start in this column RandomSource& rnd) // random gen, to choose among equal paths { assert_lt(row, dpRows()); assert_lt(col, (size_t)(rff_ - rfi_)); SSEData& d = fw_ ? sseU8fw_ : sseU8rc_; SSEMetrics& met = extend_ ? sseU8ExtendMet_ : sseU8MateMet_; met.bt++; assert(!d.profbuf_.empty()); assert_lt(row, rd_->length()); btnstack_.clear(); // empty the backtrack stack btcells_.clear(); // empty the cells-so-far list AlnScore score; score.score_ = 0; score.gaps_ = score.ns_ = 0; size_t origCol = col; size_t gaps = 0, readGaps = 0, refGaps = 0; res.alres.reset(); EList& ned = res.alres.ned(); assert(ned.empty()); assert_gt(dpRows(), row); size_t trimEnd = dpRows() - row - 1; size_t trimBeg = 0; size_t ct = SSEMatrix::H; // cell type // Row and col in terms of where they fall in the SSE vector matrix size_t rowelt, rowvec, eltvec; size_t left_rowelt, up_rowelt, upleft_rowelt; size_t left_rowvec, up_rowvec, upleft_rowvec; __m128i *cur_vec, *left_vec, *up_vec, *upleft_vec; NEW_ROW_COL(row, col); while((int)row >= 0) { met.btcell++; nbts++; int readc = (*rd_)[rdi_ + row]; int refm = (int)rf_[rfi_ + col]; int readq = (*qu_)[row]; assert_leq(col, origCol); // Get score in this cell bool empty = false, reportedThru, canMoveThru, branch = false; int cur = SSEMatrix::H; if(!d.mat_.reset_[row]) { d.mat_.resetRow(row); } reportedThru = d.mat_.reportedThrough(row, col); canMoveThru = true; if(reportedThru) { canMoveThru = false; } else { empty = false; if(row > 0) { assert_gt(row, 0); size_t rowFromEnd = d.mat_.nrow() - row - 1; bool gapsAllowed = true; if(row < (size_t)sc_->gapbar || rowFromEnd < (size_t)sc_->gapbar) { gapsAllowed = false; } const TAlScore floorsc = MIN_I64; const int offsetsc = -0xff; // Move to beginning of column/row if(ct == SSEMatrix::E) { // AKA rdgap assert_gt(col, 0); TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::E))[rowelt] + offsetsc; assert(gapsAllowed); // Currently in the E matrix; incoming transition must come from the // left. It's either a gap open from the H matrix or a gap extend from // the E matrix. // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; // Get H score of cell to the left TAlScore sc_h_left = ((TCScore*)(left_vec + SSEMatrix::H))[left_rowelt] + offsetsc; if(sc_h_left > floorsc && sc_h_left - sc_->readGapOpen() == sc_cur) { mask |= (1 << 0); } // Get E score of cell to the left TAlScore sc_e_left = ((TCScore*)(left_vec + SSEMatrix::E))[left_rowelt] + offsetsc; if(sc_e_left > floorsc && sc_e_left - sc_->readGapExtend() == sc_cur) { mask |= (1 << 1); } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isEMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 8) & 3; } if(mask == 3) { #if 1 // Pick H -> E cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 2); // might choose E later #else if(rnd.nextU2()) { // Pick H -> E cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 2); // might choose E later } else { // Pick E -> E cell cur = SW_BT_RDGAP_EXTEND; d.mat_.eMaskSet(row, col, 1); // might choose H later } #endif branch = true; } else if(mask == 2) { // I chose the E cell cur = SW_BT_RDGAP_EXTEND; d.mat_.eMaskSet(row, col, 0); // done } else if(mask == 1) { // I chose the H cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 0); // done } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } assert(!empty || !canMoveThru); } else if(ct == SSEMatrix::F) { // AKA rfgap assert_gt(row, 0); assert(gapsAllowed); TAlScore sc_h_up = ((TCScore*)(up_vec + SSEMatrix::H))[up_rowelt] + offsetsc; TAlScore sc_f_up = ((TCScore*)(up_vec + SSEMatrix::F))[up_rowelt] + offsetsc; TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::F))[rowelt] + offsetsc; // Currently in the F matrix; incoming transition must come from above. // It's either a gap open from the H matrix or a gap extend from the F // matrix. // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; // Get H score of cell above if(sc_h_up > floorsc && sc_h_up - sc_->refGapOpen() == sc_cur) { mask |= (1 << 0); } // Get F score of cell above if(sc_f_up > floorsc && sc_f_up - sc_->refGapExtend() == sc_cur) { mask |= (1 << 1); } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isFMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 11) & 3; } if(mask == 3) { #if 1 // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 2); // might choose E later #else if(rnd.nextU2()) { // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 2); // might choose E later } else { // I chose the F cell cur = SW_BT_RFGAP_EXTEND; d.mat_.fMaskSet(row, col, 1); // might choose E later } #endif branch = true; } else if(mask == 2) { // I chose the F cell cur = SW_BT_RFGAP_EXTEND; d.mat_.fMaskSet(row, col, 0); // done } else if(mask == 1) { // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 0); // done } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } assert(!empty || !canMoveThru); } else { assert_eq(SSEMatrix::H, ct); TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::H))[rowelt] + offsetsc; TAlScore sc_f_up = ((TCScore*)(up_vec + SSEMatrix::F))[up_rowelt] + offsetsc; TAlScore sc_h_up = ((TCScore*)(up_vec + SSEMatrix::H))[up_rowelt] + offsetsc; TAlScore sc_h_left = col > 0 ? (((TCScore*)(left_vec + SSEMatrix::H))[left_rowelt] + offsetsc) : floorsc; TAlScore sc_e_left = col > 0 ? (((TCScore*)(left_vec + SSEMatrix::E))[left_rowelt] + offsetsc) : floorsc; TAlScore sc_h_upleft = col > 0 ? (((TCScore*)(upleft_vec + SSEMatrix::H))[upleft_rowelt] + offsetsc) : floorsc; TAlScore sc_diag = sc_->score(readc, refm, readq - 33); // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; if(gapsAllowed) { if(sc_h_up > floorsc && sc_cur == sc_h_up - sc_->refGapOpen()) { mask |= (1 << 0); } if(sc_h_left > floorsc && sc_cur == sc_h_left - sc_->readGapOpen()) { mask |= (1 << 1); } if(sc_f_up > floorsc && sc_cur == sc_f_up - sc_->refGapExtend()) { mask |= (1 << 2); } if(sc_e_left > floorsc && sc_cur == sc_e_left - sc_->readGapExtend()) { mask |= (1 << 3); } } if(sc_h_upleft > floorsc && sc_cur == sc_h_upleft + sc_diag) { mask |= (1 << 4); } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isHMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 2) & 31; } assert(gapsAllowed || mask == (1 << 4) || mask == 0); int opts = alts5[mask]; int select = -1; if(opts == 1) { select = firsts5[mask]; assert_geq(mask, 0); d.mat_.hMaskSet(row, col, 0); } else if(opts > 1) { #if 1 if( (mask & 16) != 0) { select = 4; // H diag } else if((mask & 1) != 0) { select = 0; // H up } else if((mask & 4) != 0) { select = 2; // F up } else if((mask & 2) != 0) { select = 1; // H left } else if((mask & 8) != 0) { select = 3; // E left } #else select = randFromMask(rnd, mask); #endif assert_geq(mask, 0); mask &= ~(1 << select); assert(gapsAllowed || mask == (1 << 4) || mask == 0); d.mat_.hMaskSet(row, col, mask); branch = true; } else { /* No way to backtrack! */ } if(select != -1) { if(select == 4) { cur = SW_BT_OALL_DIAG; } else if(select == 0) { cur = SW_BT_OALL_REF_OPEN; } else if(select == 1) { cur = SW_BT_OALL_READ_OPEN; } else if(select == 2) { cur = SW_BT_RFGAP_EXTEND; } else { assert_eq(3, select) cur = SW_BT_RDGAP_EXTEND; } } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } } assert(!empty || !canMoveThru || ct == SSEMatrix::H); } } //cerr << "reportedThrough rejected (" << row << ", " << col << ")" << endl; d.mat_.setReportedThrough(row, col); assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); // Cell was involved in a previously-reported alignment? if(!canMoveThru) { if(!btnstack_.empty()) { // Remove all the cells from list back to and including the // cell where the branch occurred btcells_.resize(btnstack_.back().celsz); // Pop record off the top of the stack ned.resize(btnstack_.back().nedsz); //aed.resize(btnstack_.back().aedsz); row = btnstack_.back().row; col = btnstack_.back().col; gaps = btnstack_.back().gaps; readGaps = btnstack_.back().readGaps; refGaps = btnstack_.back().refGaps; score = btnstack_.back().score; ct = btnstack_.back().ct; btnstack_.pop_back(); assert(!sc_->monotone || score.score() >= escore); NEW_ROW_COL(row, col); continue; } else { // No branch points to revisit; just give up res.reset(); met.btfail++; // DP backtraces failed return false; } } assert(!reportedThru); assert(!sc_->monotone || score.score() >= minsc_); if(empty || row == 0) { assert_eq(SSEMatrix::H, ct); btcells_.expand(); btcells_.back().first = row; btcells_.back().second = col; // This cell is at the end of a legitimate alignment trimBeg = row; assert_eq(0, trimBeg); assert_eq(btcells_.size(), dpRows() - trimBeg - trimEnd + readGaps); break; } if(branch) { // Add a frame to the backtrack stack btnstack_.expand(); btnstack_.back().init( ned.size(), 0, // aed.size() btcells_.size(), row, col, gaps, readGaps, refGaps, score, (int)ct); } btcells_.expand(); btcells_.back().first = row; btcells_.back().second = col; switch(cur) { // Move up and to the left. If the reference nucleotide in the // source row mismatches the read nucleotide, penalize // it and add a nucleotide mismatch. case SW_BT_OALL_DIAG: { assert_gt(row, 0); assert_gt(col, 0); // Check for color mismatch int readC = (*rd_)[row]; int refNmask = (int)rf_[rfi_+col]; assert_gt(refNmask, 0); int m = matchesEx(readC, refNmask); ct = SSEMatrix::H; if(m != 1) { Edit e( (int)row, mask2dna[refNmask], "ACGTN"[readC], EDIT_TYPE_MM); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); int pen = QUAL2(row, col); score.score_ -= pen; assert(!sc_->monotone || score.score() >= escore); } else { // Reward a match int64_t bonus = sc_->match(30); score.score_ += bonus; assert(!sc_->monotone || score.score() >= escore); } if(m == -1) { score.ns_++; } row--; col--; MOVE_ALL_UPLEFT(); assert(VALID_AL_SCORE(score)); break; } // Move up. Add an edit encoding the ref gap. case SW_BT_OALL_REF_OPEN: { assert_gt(row, 0); Edit e( (int)row, '-', "ACGTN"[(int)(*rd_)[row]], EDIT_TYPE_REF_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); row--; ct = SSEMatrix::H; int pen = sc_->refGapOpen(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; refGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_UP(); break; } // Move up. Add an edit encoding the ref gap. case SW_BT_RFGAP_EXTEND: { assert_gt(row, 1); Edit e( (int)row, '-', "ACGTN"[(int)(*rd_)[row]], EDIT_TYPE_REF_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); row--; ct = SSEMatrix::F; int pen = sc_->refGapExtend(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; refGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_UP(); break; } case SW_BT_OALL_READ_OPEN: { assert_gt(col, 0); Edit e( (int)row+1, mask2dna[(int)rf_[rfi_+col]], '-', EDIT_TYPE_READ_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); col--; ct = SSEMatrix::H; int pen = sc_->readGapOpen(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; readGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_LEFT(); break; } case SW_BT_RDGAP_EXTEND: { assert_gt(col, 1); Edit e( (int)row+1, mask2dna[(int)rf_[rfi_+col]], '-', EDIT_TYPE_READ_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); col--; ct = SSEMatrix::E; int pen = sc_->readGapExtend(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; readGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_LEFT(); break; } default: throw 1; } } // while((int)row > 0) assert_eq(0, trimBeg); assert_eq(0, trimEnd); assert_geq(col, 0); assert_eq(SSEMatrix::H, ct); // The number of cells in the backtracs should equal the number of read // bases after trimming plus the number of gaps assert_eq(btcells_.size(), dpRows() - trimBeg - trimEnd + readGaps); // Check whether we went through a core diagonal and set 'reported' flag on // each cell bool overlappedCoreDiag = false; for(size_t i = 0; i < btcells_.size(); i++) { size_t rw = btcells_[i].first; size_t cl = btcells_[i].second; // Calculate the diagonal within the *trimmed* rectangle, i.e. the // rectangle we dealt with in align, gather and backtrack. int64_t diagi = cl - rw; // Now adjust to the diagonal within the *untrimmed* rectangle by // adding on the amount trimmed from the left. diagi += rect_->triml; if(diagi >= 0) { size_t diag = (size_t)diagi; if(diag >= rect_->corel && diag <= rect_->corer) { overlappedCoreDiag = true; break; } } #ifndef NDEBUG //assert(!d.mat_.reportedThrough(rw, cl)); //d.mat_.setReportedThrough(rw, cl); assert(d.mat_.reportedThrough(rw, cl)); #endif } if(!overlappedCoreDiag) { // Must overlap a core diagonal. Otherwise, we run the risk of // reporting an alignment that overlaps (and trumps) a higher-scoring // alignment that lies partially outside the dynamic programming // rectangle. res.reset(); met.corerej++; return false; } int readC = (*rd_)[rdi_+row]; // get last char in read int refNmask = (int)rf_[rfi_+col]; // get last ref char ref involved in aln assert_gt(refNmask, 0); int m = matchesEx(readC, refNmask); if(m != 1) { Edit e((int)row, mask2dna[refNmask], "ACGTN"[readC], EDIT_TYPE_MM); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); score.score_ -= QUAL2(row, col); assert_geq(score.score(), minsc_); } else { score.score_ += sc_->match(30); } if(m == -1) { score.ns_++; } if(score.ns_ > nceil_) { // Alignment has too many Ns in it! res.reset(); met.nrej++; return false; } res.reverse(); assert(Edit::repOk(ned, (*rd_))); assert_eq(score.score(), escore); assert_leq(gaps, rdgap_ + rfgap_); off = col; assert_lt(col + (size_t)rfi_, (size_t)rff_); score.gaps_ = gaps; res.alres.setScore(score); res.alres.setShape( refidx_, // ref id off + rfi_ + rect_->refl, // 0-based ref offset reflen_, // length of entire reference fw_, // aligned to Watson? rdf_ - rdi_, // read length 0, // read ID true, // pretrim soft? 0, // pretrim 5' end 0, // pretrim 3' end true, // alignment trim soft? fw_ ? trimBeg : trimEnd, // alignment trim 5' end fw_ ? trimEnd : trimBeg); // alignment trim 3' end size_t refns = 0; for(size_t i = col; i <= origCol; i++) { if((int)rf_[rfi_+i] > 15) { refns++; } } res.alres.setRefNs(refns); assert(Edit::repOk(ned, (*rd_), true, trimBeg, trimEnd)); assert(res.repOk()); #ifndef NDEBUG size_t gapsCheck = 0; for(size_t i = 0; i < ned.size(); i++) { if(ned[i].isGap()) gapsCheck++; } assert_eq(gaps, gapsCheck); BTDnaString refstr; for(size_t i = col; i <= origCol; i++) { refstr.append(firsts5[(int)rf_[rfi_+i]]); } BTDnaString editstr; // daehwan // Edit::toRef((*rd_), ned, editstr, true, trimBeg, trimEnd); Edit::toRef((*rd_), ned, editstr, true, trimBeg + rdi_, trimEnd + (rd_->length() - rdf_)); if(refstr != editstr) { cerr << "Decoded nucleotides and edits don't match reference:" << endl; cerr << " score: " << score.score() << " (" << gaps << " gaps)" << endl; cerr << " edits: "; Edit::print(cerr, ned); cerr << endl; cerr << " decoded nucs: " << (*rd_) << endl; cerr << " edited nucs: " << editstr << endl; cerr << " reference nucs: " << refstr << endl; assert(0); } #endif met.btsucc++; // DP backtraces succeeded return true; } hisat2-2.2.1/aligner_swsse_loc_i16.cpp000066400000000000000000002250441370663763200176170ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /** * aligner_sw_sse.cpp * * Versions of key alignment functions that use vector instructions to * accelerate dynamic programming. Based chiefly on the striped Smith-Waterman * paper and implementation by Michael Farrar. See: * * Farrar M. Striped Smith-Waterman speeds database searches six times over * other SIMD implementations. Bioinformatics. 2007 Jan 15;23(2):156-61. * http://sites.google.com/site/farrarmichael/smith-waterman * * While the paper describes an implementation of Smith-Waterman, we extend it * do end-to-end read alignment as well as local alignment. The change * required for this is minor: we simply let vmax be the maximum element in the * score domain rather than the minimum. * * The vectorized dynamic programming implementation lacks some features that * make it hard to adapt to solving the entire dynamic-programming alignment * problem. For instance: * * - It doesn't respect gap barriers on either end of the read * - It just gives a maximum; not enough information to backtrace without * redoing some alignment * - It's a little difficult to handle st_ and en_, especially st_. * - The query profile mechanism makes handling of ambiguous reference bases a * little tricky (16 cols in query profile lookup table instead of 5) * * Given the drawbacks, it is tempting to use SSE dynamic programming as a * filter rather than as an aligner per se. Here are a few ideas for how it * can be extended to handle more of the alignment problem: * * - Save calculated scores to a big array as we go. We return to this array * to find and backtrace from good solutions. */ #include #include "aligner_sw.h" static const size_t NBYTES_PER_REG = 16; static const size_t NWORDS_PER_REG = 8; static const size_t NBITS_PER_WORD = 16; static const size_t NBYTES_PER_WORD = 2; // In 16-bit local mode, we have the option of using signed saturated // arithmetic. Because we have signed arithmetic, there's no need to // add/subtract bias when building an applying the query profile. The lowest // value we can use is 0x8000, greatest is 0x7fff. typedef int16_t TCScore; /** * Build query profile look up tables for the read. The query profile look * up table is organized as a 1D array indexed by [i][j] where i is the * reference character in the current DP column (0=A, 1=C, etc), and j is * the segment of the query we're currently working on. */ void SwAligner::buildQueryProfileLocalSseI16(bool fw) { bool& done = fw ? sseI16fwBuilt_ : sseI16rcBuilt_; if(done) { return; } done = true; const BTDnaString* rd = fw ? rdfw_ : rdrc_; const BTString* qu = fw ? qufw_ : qurc_; const size_t len = rd->length(); const size_t seglen = (len + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // How many __m128i's are needed size_t n128s = 64 + // slack bytes, for alignment? (seglen * ALPHA_SIZE) // query profile data * 2; // & gap barrier data assert_gt(n128s, 0); SSEData& d = fw ? sseI16fw_ : sseI16rc_; d.profbuf_.resizeNoCopy(n128s); assert(!d.profbuf_.empty()); d.maxPen_ = d.maxBonus_ = 0; d.lastIter_ = d.lastWord_ = 0; d.qprofStride_ = d.gbarStride_ = 2; d.bias_ = 0; // no bias when words are signed // For each reference character A, C, G, T, N ... for(size_t refc = 0; refc < ALPHA_SIZE; refc++) { // For each segment ... for(size_t i = 0; i < seglen; i++) { size_t j = i; int16_t *qprofWords = reinterpret_cast(d.profbuf_.ptr() + (refc * seglen * 2) + (i * 2)); int16_t *gbarWords = reinterpret_cast(d.profbuf_.ptr() + (refc * seglen * 2) + (i * 2) + 1); // For each sub-word (byte) ... for(size_t k = 0; k < NWORDS_PER_REG; k++) { int sc = 0; *gbarWords = 0; if(j < len) { int readc = (*rd)[j]; int readq = (*qu)[j]; sc = sc_->score(readc, (int)(1 << refc), readq - 33); size_t j_from_end = len - j - 1; if(j < (size_t)sc_->gapbar || j_from_end < (size_t)sc_->gapbar) { // Inside the gap barrier *gbarWords = 0x8000; // add this twice } } if(refc == 0 && j == len-1) { // Remember which 128-bit word and which smaller word has // the final row d.lastIter_ = i; d.lastWord_ = k; } if(sc < 0) { if((size_t)(-sc) > d.maxPen_) { d.maxPen_ = (size_t)(-sc); } } else { if((size_t)sc > d.maxBonus_) { d.maxBonus_ = (size_t)sc; } } *qprofWords = (int16_t)sc; gbarWords++; qprofWords++; j += seglen; // update offset into query } } } } #ifndef NDEBUG /** * Return true iff the cell has sane E/F/H values w/r/t its predecessors. */ static bool cellOkLocalI16( SSEData& d, size_t row, size_t col, int refc, int readc, int readq, const Scoring& sc) // scoring scheme { TCScore floorsc = MIN_I16; TCScore ceilsc = MIN_I16-1; TAlScore offsetsc = 0x8000; TAlScore sc_h_cur = (TAlScore)d.mat_.helt(row, col); TAlScore sc_e_cur = (TAlScore)d.mat_.eelt(row, col); TAlScore sc_f_cur = (TAlScore)d.mat_.felt(row, col); if(sc_h_cur > floorsc) { sc_h_cur += offsetsc; } if(sc_e_cur > floorsc) { sc_e_cur += offsetsc; } if(sc_f_cur > floorsc) { sc_f_cur += offsetsc; } bool gapsAllowed = true; size_t rowFromEnd = d.mat_.nrow() - row - 1; if(row < (size_t)sc.gapbar || rowFromEnd < (size_t)sc.gapbar) { gapsAllowed = false; } bool e_left_trans = false, h_left_trans = false; bool f_up_trans = false, h_up_trans = false; bool h_diag_trans = false; if(gapsAllowed) { TAlScore sc_h_left = floorsc; TAlScore sc_e_left = floorsc; TAlScore sc_h_up = floorsc; TAlScore sc_f_up = floorsc; if(col > 0 && sc_e_cur > floorsc && sc_e_cur <= ceilsc) { sc_h_left = d.mat_.helt(row, col-1) + offsetsc; sc_e_left = d.mat_.eelt(row, col-1) + offsetsc; e_left_trans = (sc_e_left > floorsc && sc_e_cur == sc_e_left - sc.readGapExtend()); h_left_trans = (sc_h_left > floorsc && sc_e_cur == sc_h_left - sc.readGapOpen()); assert(e_left_trans || h_left_trans); } if(row > 0 && sc_f_cur > floorsc && sc_f_cur <= ceilsc) { sc_h_up = d.mat_.helt(row-1, col) + offsetsc; sc_f_up = d.mat_.felt(row-1, col) + offsetsc; f_up_trans = (sc_f_up > floorsc && sc_f_cur == sc_f_up - sc.refGapExtend()); h_up_trans = (sc_h_up > floorsc && sc_f_cur == sc_h_up - sc.refGapOpen()); assert(f_up_trans || h_up_trans); } } else { assert_geq(floorsc, sc_e_cur); assert_geq(floorsc, sc_f_cur); } if(col > 0 && row > 0 && sc_h_cur > floorsc && sc_h_cur <= ceilsc) { TAlScore sc_h_upleft = d.mat_.helt(row-1, col-1) + offsetsc; TAlScore sc_diag = sc.score(readc, (int)refc, readq - 33); h_diag_trans = sc_h_cur == sc_h_upleft + sc_diag; } assert( sc_h_cur <= floorsc || e_left_trans || h_left_trans || f_up_trans || h_up_trans || h_diag_trans || sc_h_cur > ceilsc || row == 0 || col == 0); return true; } #endif /*ndef NDEBUG*/ #ifdef NDEBUG #define assert_all_eq0(x) #define assert_all_gt(x, y) #define assert_all_gt_lo(x) #define assert_all_lt(x, y) #define assert_all_lt_hi(x) #else #define assert_all_eq0(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_xor_si128(z, z); \ tmp = _mm_cmpeq_epi16(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_gt(x, y) { \ __m128i tmp = _mm_cmpgt_epi16(x, y); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_gt_lo(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_xor_si128(z, z); \ tmp = _mm_cmpgt_epi16(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_lt(x, y) { \ __m128i tmp = _mm_cmplt_epi16(x, y); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_leq(x, y) { \ __m128i tmp = _mm_cmpgt_epi16(x, y); \ assert_eq(0x0000, _mm_movemask_epi8(tmp)); \ } #define assert_all_lt_hi(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_cmpeq_epi16(z, z); \ z = _mm_srli_epi16(z, 1); \ tmp = _mm_cmplt_epi16(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #endif /** * Aligns by filling a dynamic programming matrix with the SSE-accelerated, * banded DP approach of Farrar. As it goes, it determines which cells we * might backtrace from and tallies the best (highest-scoring) N backtrace * candidate cells per diagonal. Also returns the alignment score of the best * alignment in the matrix. * * This routine does *not* maintain a matrix holding the entire matrix worth of * scores, nor does it maintain any other dense O(mn) data structure, as this * would quickly exhaust memory for queries longer than about 10,000 kb. * Instead, in the fill stage it maintains two columns worth of scores at a * time (current/previous, or right/left) - these take O(m) space. When * finished with the current column, it determines which cells from the * previous column, if any, are candidates we might backtrace from to find a * full alignment. A candidate cell has a score that rises above the threshold * and isn't improved upon by a match in the next column. The best N * candidates per diagonal are stored in a O(m + n) data structure. */ TAlScore SwAligner::alignGatherLoc16(int& flag, bool debug) { assert_leq(rdf_, rd_->length()); assert_leq(rdf_, qu_->length()); assert_lt(rfi_, rff_); assert_lt(rdi_, rdf_); assert_eq(rd_->length(), qu_->length()); assert_geq(sc_->gapbar, 1); assert_gt(minsc_, 0); assert_leq(minsc_, MAX_I16); assert(repOk()); #ifndef NDEBUG for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert_range(0, 16, (int)rf_[i]); } #endif SSEData& d = fw_ ? sseI16fw_ : sseI16rc_; SSEMetrics& met = extend_ ? sseI16ExtendMet_ : sseI16MateMet_; if(!debug) met.dp++; buildQueryProfileLocalSseI16(fw_); assert(!d.profbuf_.empty()); assert_gt(d.maxBonus_, 0); size_t iter = (dpRows() + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // iter = segLen // Now set up the score vectors. We just need two columns worth, which // we'll call "left" and "right". d.vecbuf_.resize(ROWSTRIDE_2COL * iter * 2); d.vecbuf_.zero(); __m128i *vbuf_l = d.vecbuf_.ptr(); __m128i *vbuf_r = d.vecbuf_.ptr() + (ROWSTRIDE_2COL * iter); // This is the data structure that holds candidate cells per diagonal. const size_t ndiags = rff_ - rfi_ + dpRows() - 1; if(!debug) { btdiag_.init(ndiags, 2); } // Data structure that holds checkpointed anti-diagonals TAlScore perfectScore = sc_->perfectScore(dpRows()); bool checkpoint = true; bool cpdebug = false; #ifndef NDEBUG cpdebug = dpRows() < 1000; #endif cper_.init( dpRows(), // # rows rff_ - rfi_, // # columns cperPerPow2_, // checkpoint every 1 << perpow2 diags (& next) perfectScore, // perfect score (for sanity checks) false, // matrix cells have 8-bit scores? cperTri_, // triangular mini-fills? true, // alignment is local? cpdebug); // save all cells for debugging? // Many thanks to Michael Farrar for releasing his striped Smith-Waterman // implementation: // // http://sites.google.com/site/farrarmichael/smith-waterman // // Much of the implmentation below is adapted from Michael's code. // Set all elts to reference gap open penalty __m128i rfgapo = _mm_setzero_si128(); __m128i rfgape = _mm_setzero_si128(); __m128i rdgapo = _mm_setzero_si128(); __m128i rdgape = _mm_setzero_si128(); __m128i vlo = _mm_setzero_si128(); __m128i vhi = _mm_setzero_si128(); __m128i vlolsw = _mm_setzero_si128(); __m128i vmax = _mm_setzero_si128(); __m128i vcolmax = _mm_setzero_si128(); __m128i vmaxtmp = _mm_setzero_si128(); __m128i ve = _mm_setzero_si128(); __m128i vf = _mm_setzero_si128(); __m128i vh = _mm_setzero_si128(); __m128i vhd = _mm_setzero_si128(); __m128i vhdtmp = _mm_setzero_si128(); __m128i vtmp = _mm_setzero_si128(); __m128i vzero = _mm_setzero_si128(); __m128i vminsc = _mm_setzero_si128(); assert_gt(sc_->refGapOpen(), 0); assert_leq(sc_->refGapOpen(), MAX_I16); rfgapo = _mm_insert_epi16(rfgapo, sc_->refGapOpen(), 0); rfgapo = _mm_shufflelo_epi16(rfgapo, 0); rfgapo = _mm_shuffle_epi32(rfgapo, 0); // Set all elts to reference gap extension penalty assert_gt(sc_->refGapExtend(), 0); assert_leq(sc_->refGapExtend(), MAX_I16); assert_leq(sc_->refGapExtend(), sc_->refGapOpen()); rfgape = _mm_insert_epi16(rfgape, sc_->refGapExtend(), 0); rfgape = _mm_shufflelo_epi16(rfgape, 0); rfgape = _mm_shuffle_epi32(rfgape, 0); // Set all elts to read gap open penalty assert_gt(sc_->readGapOpen(), 0); assert_leq(sc_->readGapOpen(), MAX_I16); rdgapo = _mm_insert_epi16(rdgapo, sc_->readGapOpen(), 0); rdgapo = _mm_shufflelo_epi16(rdgapo, 0); rdgapo = _mm_shuffle_epi32(rdgapo, 0); // Set all elts to read gap extension penalty assert_gt(sc_->readGapExtend(), 0); assert_leq(sc_->readGapExtend(), MAX_I16); assert_leq(sc_->readGapExtend(), sc_->readGapOpen()); rdgape = _mm_insert_epi16(rdgape, sc_->readGapExtend(), 0); rdgape = _mm_shufflelo_epi16(rdgape, 0); rdgape = _mm_shuffle_epi32(rdgape, 0); // Set all elts to minimum score threshold. Actually, to 1 less than the // threshold so we can use gt instead of geq. vminsc = _mm_insert_epi16(vminsc, (int)minsc_-1, 0); vminsc = _mm_shufflelo_epi16(vminsc, 0); vminsc = _mm_shuffle_epi32(vminsc, 0); // Set all elts to 0x8000 (min value for signed 16-bit) vlo = _mm_cmpeq_epi16(vlo, vlo); // all elts = 0xffff vlo = _mm_slli_epi16(vlo, NBITS_PER_WORD-1); // all elts = 0x8000 // Set all elts to 0x7fff (max value for signed 16-bit) vhi = _mm_cmpeq_epi16(vhi, vhi); // all elts = 0xffff vhi = _mm_srli_epi16(vhi, 1); // all elts = 0x7fff // Set all elts to 0x8000 (min value for signed 16-bit) vmax = vlo; // vlolsw: topmost (least sig) word set to 0x8000, all other words=0 vlolsw = _mm_shuffle_epi32(vlo, 0); vlolsw = _mm_srli_si128(vlolsw, NBYTES_PER_REG - NBYTES_PER_WORD); // Points to a long vector of __m128i where each element is a block of // contiguous cells in the E, F or H matrix. If the index % 3 == 0, then // the block of cells is from the E matrix. If index % 3 == 1, they're // from the F matrix. If index % 3 == 2, then they're from the H matrix. // Blocks of cells are organized in the same interleaved manner as they are // calculated by the Farrar algorithm. const __m128i *pvScore; // points into the query profile const size_t colstride = ROWSTRIDE_2COL * iter; // Initialize the H and E vectors in the first matrix column __m128i *pvELeft = vbuf_l + 0; __m128i *pvERight = vbuf_r + 0; //__m128i *pvFLeft = vbuf_l + 1; __m128i *pvFRight = vbuf_r + 1; __m128i *pvHLeft = vbuf_l + 2; __m128i *pvHRight = vbuf_r + 2; for(size_t i = 0; i < iter; i++) { // start low in local mode _mm_store_si128(pvERight, vlo); pvERight += ROWSTRIDE_2COL; _mm_store_si128(pvHRight, vlo); pvHRight += ROWSTRIDE_2COL; // Note: right and left are going to be swapped as soon as we enter // the outer loop below } assert_gt(sc_->gapbar, 0); size_t nfixup = 0; TAlScore matchsc = sc_->match(30); TAlScore leftmax = MIN_I64; // Fill in the table as usual but instead of using the same gap-penalty // vector for each iteration of the inner loop, load words out of a // pre-calculated gap vector parallel to the query profile. The pre- // calculated gap vectors enforce the gap barrier constraint by making it // infinitely costly to introduce a gap in barrier rows. // // AND use a separate loop to fill in the first row of the table, enforcing // the st_ constraints in the process. This is awkward because it // separates the processing of the first row from the others and might make // it difficult to use the first-row results in the next row, but it might // be the simplest and least disruptive way to deal with the st_ constraint. size_t off = MAX_SIZE_T, lastoff; bool bailed = false; for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { // Swap left and right; vbuf_l is the vector on the left, which we // generally load from, and vbuf_r is the vector on the right, which we // generally store to. swap(vbuf_l, vbuf_r); pvELeft = vbuf_l + 0; pvERight = vbuf_r + 0; /* pvFLeft = vbuf_l + 1; */ pvFRight = vbuf_r + 1; pvHLeft = vbuf_l + 2; pvHRight = vbuf_r + 2; // Fetch this column's reference mask const int refm = (int)rf_[i]; // Fetch the appropriate query profile lastoff = off; off = (size_t)firsts5[refm] * iter * 2; pvScore = d.profbuf_.ptr() + off; // even elts = query profile, odd = gap barrier // Load H vector from the final row of the previous column. // ??? perhaps we should calculate the next iter's F instead of the // current iter's? The way we currently do it, seems like it will // almost always require at least one fixup loop iter (to recalculate // this topmost F). vh = _mm_load_si128(pvHLeft + colstride - ROWSTRIDE_2COL); // Set all F cells to low value vf = _mm_cmpeq_epi16(vf, vf); vf = _mm_slli_epi16(vf, NBITS_PER_WORD-1); vf = _mm_or_si128(vf, vlolsw); // vf now contains the vertical contribution // Store cells in F, calculated previously // No need to veto ref gap extensions, they're all 0x8000s _mm_store_si128(pvFRight, vf); pvFRight += ROWSTRIDE_2COL; // Shift down so that topmost (least sig) cell gets 0 vh = _mm_slli_si128(vh, NBYTES_PER_WORD); // Fill topmost (least sig) cell with low value vh = _mm_or_si128(vh, vlolsw); // We pull out one loop iteration to make it easier to veto values in the top row // Load cells from E, calculated previously ve = _mm_load_si128(pvELeft); vhd = _mm_load_si128(pvHLeft); assert_all_lt(ve, vhi); pvELeft += ROWSTRIDE_2COL; // ve now contains the horizontal contribution // Factor in query profile (matches and mismatches) vh = _mm_adds_epi16(vh, pvScore[0]); // vh now contains the diagonal contribution // Update vE value vhdtmp = vhd; vhd = _mm_subs_epi16(vhd, rdgapo); vhd = _mm_adds_epi16(vhd, pvScore[1]); // veto some read gap opens vhd = _mm_adds_epi16(vhd, pvScore[1]); // veto some read gap opens ve = _mm_subs_epi16(ve, rdgape); ve = _mm_max_epi16(ve, vhd); // Update H, factoring in E and F vh = _mm_max_epi16(vh, ve); // F won't change anything! vf = vh; // Update highest score so far vcolmax = vh; // Save the new vH values _mm_store_si128(pvHRight, vh); assert_all_lt(ve, vhi); vh = vhdtmp; assert_all_lt(ve, vhi); pvHRight += ROWSTRIDE_2COL; pvHLeft += ROWSTRIDE_2COL; // Save E values _mm_store_si128(pvERight, ve); pvERight += ROWSTRIDE_2COL; // Update vf value vf = _mm_subs_epi16(vf, rfgapo); assert_all_lt(vf, vhi); pvScore += 2; // move on to next query profile // For each character in the reference text: size_t j; for(j = 1; j < iter; j++) { // Load cells from E, calculated previously ve = _mm_load_si128(pvELeft); vhd = _mm_load_si128(pvHLeft); assert_all_lt(ve, vhi); pvELeft += ROWSTRIDE_2COL; // Store cells in F, calculated previously vf = _mm_adds_epi16(vf, pvScore[1]); // veto some ref gap extensions vf = _mm_adds_epi16(vf, pvScore[1]); // veto some ref gap extensions _mm_store_si128(pvFRight, vf); pvFRight += ROWSTRIDE_2COL; // Factor in query profile (matches and mismatches) vh = _mm_adds_epi16(vh, pvScore[0]); vh = _mm_max_epi16(vh, vf); // Update vE value vhdtmp = vhd; vhd = _mm_subs_epi16(vhd, rdgapo); vhd = _mm_adds_epi16(vhd, pvScore[1]); // veto some read gap opens vhd = _mm_adds_epi16(vhd, pvScore[1]); // veto some read gap opens ve = _mm_subs_epi16(ve, rdgape); ve = _mm_max_epi16(ve, vhd); vh = _mm_max_epi16(vh, ve); vtmp = vh; // Update highest score encountered this far vcolmax = _mm_max_epi16(vcolmax, vh); // Save the new vH values _mm_store_si128(pvHRight, vh); vh = vhdtmp; assert_all_lt(ve, vhi); pvHRight += ROWSTRIDE_2COL; pvHLeft += ROWSTRIDE_2COL; // Save E values _mm_store_si128(pvERight, ve); pvERight += ROWSTRIDE_2COL; // Update vf value vtmp = _mm_subs_epi16(vtmp, rfgapo); vf = _mm_subs_epi16(vf, rfgape); assert_all_lt(vf, vhi); vf = _mm_max_epi16(vf, vtmp); pvScore += 2; // move on to next query profile / gap veto } // pvHStore, pvELoad, pvEStore have all rolled over to the next column pvFRight -= colstride; // reset to start of column vtmp = _mm_load_si128(pvFRight); pvHRight -= colstride; // reset to start of column vh = _mm_load_si128(pvHRight); pvScore = d.profbuf_.ptr() + off + 1; // reset veto vector // vf from last row gets shifted down by one to overlay the first row // rfgape has already been subtracted from it. vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_or_si128(vf, vlolsw); vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epi16(vtmp, vf); vtmp = _mm_cmpgt_epi16(vf, vtmp); int cmp = _mm_movemask_epi8(vtmp); // If any element of vtmp is greater than H - gap-open... j = 0; while(cmp != 0x0000) { // Store this vf _mm_store_si128(pvFRight, vf); pvFRight += ROWSTRIDE_2COL; // Update vh w/r/t new vf vh = _mm_max_epi16(vh, vf); // Save vH values _mm_store_si128(pvHRight, vh); pvHRight += ROWSTRIDE_2COL; // Update highest score encountered so far. vcolmax = _mm_max_epi16(vcolmax, vh); pvScore += 2; assert_lt(j, iter); if(++j == iter) { pvFRight -= colstride; vtmp = _mm_load_si128(pvFRight); // load next vf ASAP pvHRight -= colstride; vh = _mm_load_si128(pvHRight); // load next vh ASAP pvScore = d.profbuf_.ptr() + off + 1; j = 0; vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_or_si128(vf, vlolsw); } else { vtmp = _mm_load_si128(pvFRight); // load next vf ASAP vh = _mm_load_si128(pvHRight); // load next vh ASAP } // Update F with another gap extension vf = _mm_subs_epi16(vf, rfgape); vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epi16(vtmp, vf); vtmp = _mm_cmpgt_epi16(vf, vtmp); cmp = _mm_movemask_epi8(vtmp); nfixup++; } // Now we'd like to know exactly which cells in the left column are // candidates we might backtrace from. First question is: did *any* // elements in the column exceed the minimum score threshold? if(!debug && leftmax >= minsc_) { // Yes. Next question is: which cells are candidates? We have to // allow matches in the right column to override matches above and // to the left in the left column. assert_gt(i - rfi_, 0); pvHLeft = vbuf_l + 2; assert_lt(lastoff, MAX_SIZE_T); pvScore = d.profbuf_.ptr() + lastoff; // even elts = query profile, odd = gap barrier for(size_t k = 0; k < iter; k++) { vh = _mm_load_si128(pvHLeft); vtmp = _mm_cmpgt_epi16(pvScore[0], vzero); int cmp = _mm_movemask_epi8(vtmp); if(cmp != 0) { // At least one candidate in this mask. Now iterate // through vm/vh to evaluate individual cells. for(size_t m = 0; m < NWORDS_PER_REG; m++) { size_t row = k + m * iter; if(row >= dpRows()) { break; } TAlScore sc = (TAlScore)(((TCScore *)&vh)[m] + 0x8000); if(sc >= minsc_) { if(((TCScore *)&vtmp)[m] != 0) { // Add to data structure holding all candidates size_t col = i - rfi_ - 1; // -1 b/c prev col size_t frombot = dpRows() - row - 1; DpBtCandidate cand(row, col, sc); btdiag_.add(frombot + col, cand); } } } } pvHLeft += ROWSTRIDE_2COL; pvScore += 2; } } // Save some elements to checkpoints if(checkpoint) { __m128i *pvE = vbuf_r + 0; __m128i *pvF = vbuf_r + 1; __m128i *pvH = vbuf_r + 2; size_t coli = i - rfi_; if(coli < cper_.locol_) cper_.locol_ = coli; if(coli > cper_.hicol_) cper_.hicol_ = coli; if(cperTri_) { size_t rc_mod = coli & cper_.lomask_; assert_lt(rc_mod, cper_.per_); int64_t row = -(int64_t)rc_mod-1; int64_t row_mod = row; int64_t row_div = 0; size_t idx = coli >> cper_.perpow2_; size_t idxrow = idx * cper_.nrow_; assert_eq(4, ROWSTRIDE_2COL); bool done = false; while(true) { row += (cper_.per_ - 2); row_mod += (cper_.per_ - 2); for(size_t j = 0; j < 2; j++) { row++; row_mod++; if(row >= 0 && (size_t)row < cper_.nrow_) { // Update row divided by iter_ and mod iter_ while(row_mod >= (int64_t)iter) { row_mod -= (int64_t)iter; row_div++; } size_t delt = idxrow + row; size_t vecoff = (row_mod << 5) + row_div; assert_lt(row_div, 8); int16_t h_sc = ((int16_t*)pvH)[vecoff]; int16_t e_sc = ((int16_t*)pvE)[vecoff]; int16_t f_sc = ((int16_t*)pvF)[vecoff]; h_sc += 0x8000; assert_geq(h_sc, 0); e_sc += 0x8000; assert_geq(e_sc, 0); f_sc += 0x8000; assert_geq(f_sc, 0); assert_leq(h_sc, cper_.perf_); assert_leq(e_sc, cper_.perf_); assert_leq(f_sc, cper_.perf_); CpQuad *qdiags = ((j == 0) ? cper_.qdiag1s_.ptr() : cper_.qdiag2s_.ptr()); qdiags[delt].sc[0] = h_sc; qdiags[delt].sc[1] = e_sc; qdiags[delt].sc[2] = f_sc; } // if(row >= 0 && row < nrow_) else if(row >= 0 && (size_t)row >= cper_.nrow_) { done = true; break; } } // end of loop over anti-diags if(done) { break; } idx++; idxrow += cper_.nrow_; } } else { // If this is the first column, take this opportunity to // pre-calculate the coordinates of the elements we're going to // checkpoint. if(coli == 0) { size_t cpi = cper_.per_-1; size_t cpimod = cper_.per_-1; size_t cpidiv = 0; cper_.commitMap_.clear(); while(cpi < cper_.nrow_) { while(cpimod >= iter) { cpimod -= iter; cpidiv++; } size_t vecoff = (cpimod << 5) + cpidiv; cper_.commitMap_.push_back(vecoff); cpi += cper_.per_; cpimod += cper_.per_; } } // Save all the rows size_t rowoff = 0; size_t sz = cper_.commitMap_.size(); for(size_t i = 0; i < sz; i++, rowoff += cper_.ncol_) { size_t vecoff = cper_.commitMap_[i]; int16_t h_sc = ((int16_t*)pvH)[vecoff]; //int16_t e_sc = ((int16_t*)pvE)[vecoff]; int16_t f_sc = ((int16_t*)pvF)[vecoff]; h_sc += 0x8000; assert_geq(h_sc, 0); //e_sc += 0x8000; assert_geq(e_sc, 0); f_sc += 0x8000; assert_geq(f_sc, 0); assert_leq(h_sc, cper_.perf_); //assert_leq(e_sc, cper_.perf_); assert_leq(f_sc, cper_.perf_); CpQuad& dst = cper_.qrows_[rowoff + coli]; dst.sc[0] = h_sc; //dst.sc[1] = e_sc; dst.sc[2] = f_sc; } // Is this a column we'd like to checkpoint? if((coli & cper_.lomask_) == cper_.lomask_) { // Save the column using memcpys assert_gt(coli, 0); size_t wordspercol = cper_.niter_ * ROWSTRIDE_2COL; size_t coloff = (coli >> cper_.perpow2_) * wordspercol; __m128i *dst = cper_.qcols_.ptr() + coloff; memcpy(dst, vbuf_r, sizeof(__m128i) * wordspercol); } } if(cper_.debug_) { // Save the column using memcpys size_t wordspercol = cper_.niter_ * ROWSTRIDE_2COL; size_t coloff = coli * wordspercol; __m128i *dst = cper_.qcolsD_.ptr() + coloff; memcpy(dst, vbuf_r, sizeof(__m128i) * wordspercol); } } vmax = _mm_max_epi16(vmax, vcolmax); { // Get single largest score in this column vmaxtmp = vcolmax; vtmp = _mm_srli_si128(vmaxtmp, 8); vmaxtmp = _mm_max_epi16(vmaxtmp, vtmp); vtmp = _mm_srli_si128(vmaxtmp, 4); vmaxtmp = _mm_max_epi16(vmaxtmp, vtmp); vtmp = _mm_srli_si128(vmaxtmp, 2); vmaxtmp = _mm_max_epi16(vmaxtmp, vtmp); int16_t ret = _mm_extract_epi16(vmaxtmp, 0); TAlScore score = (TAlScore)(ret + 0x8000); if(ret == MIN_I16) { score = MIN_I64; } if(score < minsc_) { size_t ncolleft = rff_ - i - 1; if(max(score, 0) + (TAlScore)ncolleft * matchsc < minsc_) { // Bail! There can't possibly be a valid alignment that // passes through this column. bailed = true; break; } } leftmax = score; } } lastoff = off; // Now we'd like to know exactly which cells in the *rightmost* column are // candidates we might backtrace from. Did *any* elements exceed the // minimum score threshold? if(!debug && !bailed && leftmax >= minsc_) { // Yes. Next question is: which cells are candidates? We have to // allow matches in the right column to override matches above and // to the left in the left column. pvHLeft = vbuf_r + 2; assert_lt(lastoff, MAX_SIZE_T); pvScore = d.profbuf_.ptr() + lastoff; // even elts = query profile, odd = gap barrier for(size_t k = 0; k < iter; k++) { vh = _mm_load_si128(pvHLeft); vtmp = _mm_cmpgt_epi16(pvScore[0], vzero); int cmp = _mm_movemask_epi8(vtmp); if(cmp != 0) { // At least one candidate in this mask. Now iterate // through vm/vh to evaluate individual cells. for(size_t m = 0; m < NWORDS_PER_REG; m++) { size_t row = k + m * iter; if(row >= dpRows()) { break; } TAlScore sc = (TAlScore)(((TCScore *)&vh)[m] + 0x8000); if(sc >= minsc_) { if(((TCScore *)&vtmp)[m] != 0) { // Add to data structure holding all candidates size_t col = rff_ - rfi_ - 1; // -1 b/c prev col size_t frombot = dpRows() - row - 1; DpBtCandidate cand(row, col, sc); btdiag_.add(frombot + col, cand); } } } } pvHLeft += ROWSTRIDE_2COL; pvScore += 2; } } // Find largest score in vmax vtmp = _mm_srli_si128(vmax, 8); vmax = _mm_max_epi16(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 4); vmax = _mm_max_epi16(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 2); vmax = _mm_max_epi16(vmax, vtmp); int16_t ret = _mm_extract_epi16(vmax, 0); // Update metrics if(!debug) { size_t ninner = (rff_ - rfi_) * iter; met.col += (rff_ - rfi_); // DP columns met.cell += (ninner * NWORDS_PER_REG); // DP cells met.inner += ninner; // DP inner loop iters met.fixup += nfixup; // DP fixup loop iters } flag = 0; // Did we find a solution? TAlScore score = MIN_I64; if(ret == MIN_I16) { flag = -1; // no if(!debug) met.dpfail++; return MIN_I64; } else { score = (TAlScore)(ret + 0x8000); if(score < minsc_) { flag = -1; // no if(!debug) met.dpfail++; return score; } } // Could we have saturated? if(ret == MAX_I16) { flag = -2; // yes if(!debug) met.dpsat++; return MIN_I64; } // Now take all the backtrace candidates in the btdaig_ structure and // dump them into the btncand_ array. They'll be sorted later. if(!debug) { btdiag_.dump(btncand_); assert(!btncand_.empty()); } // Return largest score if(!debug) met.dpsucc++; return score; } /** * Solve the current alignment problem using SSE instructions that operate on 8 * signed 16-bit values packed into a single 128-bit register. */ TAlScore SwAligner::alignNucleotidesLocalSseI16(int& flag, bool debug) { assert_leq(rdf_, rd_->length()); assert_leq(rdf_, qu_->length()); assert_lt(rfi_, rff_); assert_lt(rdi_, rdf_); assert_eq(rd_->length(), qu_->length()); assert_geq(sc_->gapbar, 1); assert(repOk()); #ifndef NDEBUG for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert_range(0, 16, (int)rf_[i]); } #endif SSEData& d = fw_ ? sseI16fw_ : sseI16rc_; SSEMetrics& met = extend_ ? sseI16ExtendMet_ : sseI16MateMet_; if(!debug) met.dp++; buildQueryProfileLocalSseI16(fw_); assert(!d.profbuf_.empty()); assert_gt(d.maxBonus_, 0); size_t iter = (dpRows() + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // iter = segLen // Many thanks to Michael Farrar for releasing his striped Smith-Waterman // implementation: // // http://sites.google.com/site/farrarmichael/smith-waterman // // Much of the implmentation below is adapted from Michael's code. // Set all elts to reference gap open penalty __m128i rfgapo = _mm_setzero_si128(); __m128i rfgape = _mm_setzero_si128(); __m128i rdgapo = _mm_setzero_si128(); __m128i rdgape = _mm_setzero_si128(); __m128i vlo = _mm_setzero_si128(); __m128i vhi = _mm_setzero_si128(); __m128i vlolsw = _mm_setzero_si128(); __m128i vmax = _mm_setzero_si128(); __m128i vcolmax = _mm_setzero_si128(); __m128i vmaxtmp = _mm_setzero_si128(); __m128i ve = _mm_setzero_si128(); __m128i vf = _mm_setzero_si128(); __m128i vh = _mm_setzero_si128(); __m128i vtmp = _mm_setzero_si128(); assert_gt(sc_->refGapOpen(), 0); assert_leq(sc_->refGapOpen(), MAX_I16); rfgapo = _mm_insert_epi16(rfgapo, sc_->refGapOpen(), 0); rfgapo = _mm_shufflelo_epi16(rfgapo, 0); rfgapo = _mm_shuffle_epi32(rfgapo, 0); // Set all elts to reference gap extension penalty assert_gt(sc_->refGapExtend(), 0); assert_leq(sc_->refGapExtend(), MAX_I16); assert_leq(sc_->refGapExtend(), sc_->refGapOpen()); rfgape = _mm_insert_epi16(rfgape, sc_->refGapExtend(), 0); rfgape = _mm_shufflelo_epi16(rfgape, 0); rfgape = _mm_shuffle_epi32(rfgape, 0); // Set all elts to read gap open penalty assert_gt(sc_->readGapOpen(), 0); assert_leq(sc_->readGapOpen(), MAX_I16); rdgapo = _mm_insert_epi16(rdgapo, sc_->readGapOpen(), 0); rdgapo = _mm_shufflelo_epi16(rdgapo, 0); rdgapo = _mm_shuffle_epi32(rdgapo, 0); // Set all elts to read gap extension penalty assert_gt(sc_->readGapExtend(), 0); assert_leq(sc_->readGapExtend(), MAX_I16); assert_leq(sc_->readGapExtend(), sc_->readGapOpen()); rdgape = _mm_insert_epi16(rdgape, sc_->readGapExtend(), 0); rdgape = _mm_shufflelo_epi16(rdgape, 0); rdgape = _mm_shuffle_epi32(rdgape, 0); // Set all elts to 0x8000 (min value for signed 16-bit) vlo = _mm_cmpeq_epi16(vlo, vlo); // all elts = 0xffff vlo = _mm_slli_epi16(vlo, NBITS_PER_WORD-1); // all elts = 0x8000 // Set all elts to 0x7fff (max value for signed 16-bit) vhi = _mm_cmpeq_epi16(vhi, vhi); // all elts = 0xffff vhi = _mm_srli_epi16(vhi, 1); // all elts = 0x7fff // Set all elts to 0x8000 (min value for signed 16-bit) vmax = vlo; // vlolsw: topmost (least sig) word set to 0x8000, all other words=0 vlolsw = _mm_shuffle_epi32(vlo, 0); vlolsw = _mm_srli_si128(vlolsw, NBYTES_PER_REG - NBYTES_PER_WORD); // Points to a long vector of __m128i where each element is a block of // contiguous cells in the E, F or H matrix. If the index % 3 == 0, then // the block of cells is from the E matrix. If index % 3 == 1, they're // from the F matrix. If index % 3 == 2, then they're from the H matrix. // Blocks of cells are organized in the same interleaved manner as they are // calculated by the Farrar algorithm. const __m128i *pvScore; // points into the query profile d.mat_.init(dpRows(), rff_ - rfi_, NWORDS_PER_REG); const size_t colstride = d.mat_.colstride(); //const size_t rowstride = d.mat_.rowstride(); assert_eq(ROWSTRIDE, colstride / iter); // Initialize the H and E vectors in the first matrix column __m128i *pvHTmp = d.mat_.tmpvec(0, 0); __m128i *pvETmp = d.mat_.evec(0, 0); for(size_t i = 0; i < iter; i++) { _mm_store_si128(pvETmp, vlo); _mm_store_si128(pvHTmp, vlo); // start low in local mode pvETmp += ROWSTRIDE; pvHTmp += ROWSTRIDE; } // These are swapped just before the innermost loop __m128i *pvHStore = d.mat_.hvec(0, 0); __m128i *pvHLoad = d.mat_.tmpvec(0, 0); __m128i *pvELoad = d.mat_.evec(0, 0); __m128i *pvEStore = d.mat_.evecUnsafe(0, 1); __m128i *pvFStore = d.mat_.fvec(0, 0); __m128i *pvFTmp = NULL; assert_gt(sc_->gapbar, 0); size_t nfixup = 0; TAlScore matchsc = sc_->match(30); // Fill in the table as usual but instead of using the same gap-penalty // vector for each iteration of the inner loop, load words out of a // pre-calculated gap vector parallel to the query profile. The pre- // calculated gap vectors enforce the gap barrier constraint by making it // infinitely costly to introduce a gap in barrier rows. // // AND use a separate loop to fill in the first row of the table, enforcing // the st_ constraints in the process. This is awkward because it // separates the processing of the first row from the others and might make // it difficult to use the first-row results in the next row, but it might // be the simplest and least disruptive way to deal with the st_ constraint. colstop_ = rff_ - rfi_; lastsolcol_ = 0; for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert(pvFStore == d.mat_.fvec(0, i - rfi_)); assert(pvHStore == d.mat_.hvec(0, i - rfi_)); // Fetch this column's reference mask const int refm = (int)rf_[i]; // Fetch the appropriate query profile size_t off = (size_t)firsts5[refm] * iter * 2; pvScore = d.profbuf_.ptr() + off; // even elts = query profile, odd = gap barrier // Load H vector from the final row of the previous column vh = _mm_load_si128(pvHLoad + colstride - ROWSTRIDE); // Set all F cells to low value vf = _mm_cmpeq_epi16(vf, vf); vf = _mm_slli_epi16(vf, NBITS_PER_WORD-1); vf = _mm_or_si128(vf, vlolsw); // vf now contains the vertical contribution // Store cells in F, calculated previously // No need to veto ref gap extensions, they're all 0x8000s _mm_store_si128(pvFStore, vf); pvFStore += ROWSTRIDE; // Shift down so that topmost (least sig) cell gets 0 vh = _mm_slli_si128(vh, NBYTES_PER_WORD); // Fill topmost (least sig) cell with low value vh = _mm_or_si128(vh, vlolsw); // We pull out one loop iteration to make it easier to veto values in the top row // Load cells from E, calculated previously ve = _mm_load_si128(pvELoad); assert_all_lt(ve, vhi); pvELoad += ROWSTRIDE; // ve now contains the horizontal contribution // Factor in query profile (matches and mismatches) vh = _mm_adds_epi16(vh, pvScore[0]); // vh now contains the diagonal contribution // Update H, factoring in E and F vtmp = _mm_max_epi16(vh, ve); // F won't change anything! vh = vtmp; // Update highest score so far vcolmax = vlo; vcolmax = _mm_max_epi16(vcolmax, vh); // Save the new vH values _mm_store_si128(pvHStore, vh); pvHStore += ROWSTRIDE; // Update vE value vf = vh; vh = _mm_subs_epi16(vh, rdgapo); vh = _mm_adds_epi16(vh, pvScore[1]); // veto some read gap opens vh = _mm_adds_epi16(vh, pvScore[1]); // veto some read gap opens ve = _mm_subs_epi16(ve, rdgape); ve = _mm_max_epi16(ve, vh); assert_all_lt(ve, vhi); // Load the next h value vh = _mm_load_si128(pvHLoad); pvHLoad += ROWSTRIDE; // Save E values _mm_store_si128(pvEStore, ve); pvEStore += ROWSTRIDE; // Update vf value vf = _mm_subs_epi16(vf, rfgapo); assert_all_lt(vf, vhi); pvScore += 2; // move on to next query profile // For each character in the reference text: size_t j; for(j = 1; j < iter; j++) { // Load cells from E, calculated previously ve = _mm_load_si128(pvELoad); assert_all_lt(ve, vhi); pvELoad += ROWSTRIDE; // Store cells in F, calculated previously vf = _mm_adds_epi16(vf, pvScore[1]); // veto some ref gap extensions vf = _mm_adds_epi16(vf, pvScore[1]); // veto some ref gap extensions _mm_store_si128(pvFStore, vf); pvFStore += ROWSTRIDE; // Factor in query profile (matches and mismatches) vh = _mm_adds_epi16(vh, pvScore[0]); // Update H, factoring in E and F vh = _mm_max_epi16(vh, ve); vh = _mm_max_epi16(vh, vf); // Update highest score encountered this far vcolmax = _mm_max_epi16(vcolmax, vh); // Save the new vH values _mm_store_si128(pvHStore, vh); pvHStore += ROWSTRIDE; // Update vE value vtmp = vh; vh = _mm_subs_epi16(vh, rdgapo); vh = _mm_adds_epi16(vh, pvScore[1]); // veto some read gap opens vh = _mm_adds_epi16(vh, pvScore[1]); // veto some read gap opens ve = _mm_subs_epi16(ve, rdgape); ve = _mm_max_epi16(ve, vh); assert_all_lt(ve, vhi); // Load the next h value vh = _mm_load_si128(pvHLoad); pvHLoad += ROWSTRIDE; // Save E values _mm_store_si128(pvEStore, ve); pvEStore += ROWSTRIDE; // Update vf value vtmp = _mm_subs_epi16(vtmp, rfgapo); vf = _mm_subs_epi16(vf, rfgape); assert_all_lt(vf, vhi); vf = _mm_max_epi16(vf, vtmp); pvScore += 2; // move on to next query profile / gap veto } // pvHStore, pvELoad, pvEStore have all rolled over to the next column pvFTmp = pvFStore; pvFStore -= colstride; // reset to start of column vtmp = _mm_load_si128(pvFStore); pvHStore -= colstride; // reset to start of column vh = _mm_load_si128(pvHStore); pvEStore -= colstride; // reset to start of column ve = _mm_load_si128(pvEStore); pvHLoad = pvHStore; // new pvHLoad = pvHStore pvScore = d.profbuf_.ptr() + off + 1; // reset veto vector // vf from last row gets shifted down by one to overlay the first row // rfgape has already been subtracted from it. vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_or_si128(vf, vlolsw); vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epi16(vtmp, vf); vtmp = _mm_cmpgt_epi16(vf, vtmp); int cmp = _mm_movemask_epi8(vtmp); // If any element of vtmp is greater than H - gap-open... j = 0; while(cmp != 0x0000) { // Store this vf _mm_store_si128(pvFStore, vf); pvFStore += ROWSTRIDE; // Update vh w/r/t new vf vh = _mm_max_epi16(vh, vf); // Save vH values _mm_store_si128(pvHStore, vh); pvHStore += ROWSTRIDE; // Update highest score encountered this far vcolmax = _mm_max_epi16(vcolmax, vh); // Update E in case it can be improved using our new vh vh = _mm_subs_epi16(vh, rdgapo); vh = _mm_adds_epi16(vh, *pvScore); // veto some read gap opens vh = _mm_adds_epi16(vh, *pvScore); // veto some read gap opens ve = _mm_max_epi16(ve, vh); _mm_store_si128(pvEStore, ve); pvEStore += ROWSTRIDE; pvScore += 2; assert_lt(j, iter); if(++j == iter) { pvFStore -= colstride; vtmp = _mm_load_si128(pvFStore); // load next vf ASAP pvHStore -= colstride; vh = _mm_load_si128(pvHStore); // load next vh ASAP pvEStore -= colstride; ve = _mm_load_si128(pvEStore); // load next ve ASAP pvScore = d.profbuf_.ptr() + off + 1; j = 0; vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_or_si128(vf, vlolsw); } else { vtmp = _mm_load_si128(pvFStore); // load next vf ASAP vh = _mm_load_si128(pvHStore); // load next vh ASAP ve = _mm_load_si128(pvEStore); // load next vh ASAP } // Update F with another gap extension vf = _mm_subs_epi16(vf, rfgape); vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_adds_epi16(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epi16(vtmp, vf); vtmp = _mm_cmpgt_epi16(vf, vtmp); cmp = _mm_movemask_epi8(vtmp); nfixup++; } #ifndef NDEBUG if((rand() & 15) == 0) { // This is a work-intensive sanity check; each time we finish filling // a column, we check that each H, E, and F is sensible. for(size_t k = 0; k < dpRows(); k++) { assert(cellOkLocalI16( d, k, // row i - rfi_, // col refm, // reference mask (int)(*rd_)[rdi_+k], // read char (int)(*qu_)[rdi_+k], // read quality *sc_)); // scoring scheme } } #endif // Store column maximum vector in first element of tmp vmax = _mm_max_epi16(vmax, vcolmax); _mm_store_si128(d.mat_.tmpvec(0, i - rfi_), vcolmax); { // Get single largest score in this column vmaxtmp = vcolmax; vtmp = _mm_srli_si128(vmaxtmp, 8); vmaxtmp = _mm_max_epi16(vmaxtmp, vtmp); vtmp = _mm_srli_si128(vmaxtmp, 4); vmaxtmp = _mm_max_epi16(vmaxtmp, vtmp); vtmp = _mm_srli_si128(vmaxtmp, 2); vmaxtmp = _mm_max_epi16(vmaxtmp, vtmp); int16_t ret = _mm_extract_epi16(vmaxtmp, 0); TAlScore score = (TAlScore)(ret + 0x8000); if(score < minsc_) { size_t ncolleft = rff_ - i - 1; if(score + (TAlScore)ncolleft * matchsc < minsc_) { // Bail! We're guaranteed not to see a valid alignment in // the rest of the matrix colstop_ = (i+1) - rfi_; break; } } else { lastsolcol_ = i - rfi_; } } // pvELoad and pvHLoad are already where they need to be // Adjust the load and store vectors here. pvHStore = pvHLoad + colstride; pvEStore = pvELoad + colstride; pvFStore = pvFTmp; } // Find largest score in vmax vtmp = _mm_srli_si128(vmax, 8); vmax = _mm_max_epi16(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 4); vmax = _mm_max_epi16(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 2); vmax = _mm_max_epi16(vmax, vtmp); int16_t ret = _mm_extract_epi16(vmax, 0); // Update metrics if(!debug) { size_t ninner = (rff_ - rfi_) * iter; met.col += (rff_ - rfi_); // DP columns met.cell += (ninner * NWORDS_PER_REG); // DP cells met.inner += ninner; // DP inner loop iters met.fixup += nfixup; // DP fixup loop iters } flag = 0; // Did we find a solution? TAlScore score = MIN_I64; if(ret == MIN_I16) { flag = -1; // no if(!debug) met.dpfail++; return MIN_I64; } else { score = (TAlScore)(ret + 0x8000); if(score < minsc_) { flag = -1; // no if(!debug) met.dpfail++; return score; } } // Could we have saturated? if(ret == MAX_I16) { flag = -2; // yes if(!debug) met.dpsat++; return MIN_I64; } // Return largest score if(!debug) met.dpsucc++; return score; } /** * Given a filled-in DP table, populate the btncand_ list with candidate cells * that might be at the ends of valid alignments. No need to do this unless * the maximum score returned by the align*() func is >= the minimum. * * We needn't consider cells that have no chance of reaching any of the core * diagonals. These are the cells that are more than 'maxgaps' cells away from * a core diagonal. * * We need to be careful to consider that the rectangle might be truncated on * one or both ends. * * The seed extend case looks like this: * * |Rectangle| 0: seed diagonal * **OO0oo---- o: "RHS gap" diagonals * -**OO0oo--- O: "LHS gap" diagonals * --**OO0oo-- *: "LHS extra" diagonals * ---**OO0oo- -: cells that can't possibly be involved in a valid * ----**OO0oo alignment that overlaps one of the core diagonals * * The anchor-to-left case looks like this: * * |Anchor| | ---- Rectangle ---- | * o---------OO0000000000000oo------ 0: mate diagonal (also core diags!) * -o---------OO0000000000000oo----- o: "RHS gap" diagonals * --o---------OO0000000000000oo---- O: "LHS gap" diagonals * ---oo--------OO0000000000000oo--- *: "LHS extra" diagonals * -----o--------OO0000000000000oo-- -: cells that can't possibly be * ------o--------OO0000000000000oo- involved in a valid alignment that * -------o--------OO0000000000000oo overlaps one of the core diagonals * XXXXXXXXXXXXX * | RHS Range | * ^ ^ * rl rr * * The anchor-to-right case looks like this: * * ll lr * v v * | LHS Range | * XXXXXXXXXXXXX |Anchor| * OO0000000000000oo--------o-------- 0: mate diagonal (also core diags!) * -OO0000000000000oo--------o------- o: "RHS gap" diagonals * --OO0000000000000oo--------o------ O: "LHS gap" diagonals * ---OO0000000000000oo--------oo---- *: "LHS extra" diagonals * ----OO0000000000000oo---------o--- -: cells that can't possibly be * -----OO0000000000000oo---------o-- involved in a valid alignment that * ------OO0000000000000oo---------o- overlaps one of the core diagonals * | ---- Rectangle ---- | */ bool SwAligner::gatherCellsNucleotidesLocalSseI16(TAlScore best) { // What's the minimum number of rows that can possibly be spanned by an // alignment that meets the minimum score requirement? assert(sse16succ_); size_t bonus = (size_t)sc_->match(30); const size_t ncol = lastsolcol_ + 1; const size_t nrow = dpRows(); assert_gt(nrow, 0); btncand_.clear(); btncanddone_.clear(); SSEData& d = fw_ ? sseI16fw_ : sseI16rc_; SSEMetrics& met = extend_ ? sseI16ExtendMet_ : sseI16MateMet_; assert(!d.profbuf_.empty()); //const size_t rowstride = d.mat_.rowstride(); //const size_t colstride = d.mat_.colstride(); size_t iter = (dpRows() + (NWORDS_PER_REG - 1)) / NWORDS_PER_REG; assert_gt(iter, 0); assert_geq(minsc_, 0); assert_gt(bonus, 0); size_t minrow = (size_t)(((minsc_ + bonus - 1) / bonus) - 1); for(size_t j = 0; j < ncol; j++) { // Establish the range of rows where a backtrace from the cell in this // row/col is close enough to one of the core diagonals that it could // conceivably count size_t nrow_lo = MIN_SIZE_T; size_t nrow_hi = nrow; // First, check if there is a cell in this column with a score // above the score threshold __m128i vmax = *d.mat_.tmpvec(0, j); __m128i vtmp = _mm_srli_si128(vmax, 8); vmax = _mm_max_epi16(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 4); vmax = _mm_max_epi16(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 2); vmax = _mm_max_epi16(vmax, vtmp); TAlScore score = (TAlScore)((int16_t)_mm_extract_epi16(vmax, 0) + 0x8000); assert_geq(score, 0); #ifndef NDEBUG { // Start in upper vector row and move down TAlScore max = 0; vmax = *d.mat_.tmpvec(0, j); __m128i *pvH = d.mat_.hvec(0, j); for(size_t i = 0; i < iter; i++) { for(size_t k = 0; k < NWORDS_PER_REG; k++) { TAlScore sc = (TAlScore)(((TCScore*)pvH)[k] + 0x8000); TAlScore scm = (TAlScore)(((TCScore*)&vmax)[k] + 0x8000); assert_leq(sc, scm); if(sc > max) { max = sc; } } pvH += ROWSTRIDE; } assert_eq(max, score); } #endif if(score < minsc_) { // Scores in column aren't good enough continue; } // Get pointer to first cell in column to examine: __m128i *pvHorig = d.mat_.hvec(0, j); __m128i *pvH = pvHorig; // Get pointer to the vector in the following column that corresponds // to the cells diagonally down and to the right from the cells in pvH __m128i *pvHSucc = (j < ncol-1) ? d.mat_.hvec(0, j+1) : NULL; // Start in upper vector row and move down for(size_t i = 0; i < iter; i++) { if(pvHSucc != NULL) { pvHSucc += ROWSTRIDE; if(i == iter-1) { pvHSucc = d.mat_.hvec(0, j+1); } } // Which elements of this vector are exhaustively scored? size_t rdoff = i; for(size_t k = 0; k < NWORDS_PER_REG; k++) { // Is this row, col one that we can potential backtrace from? // I.e. are we close enough to a core diagonal? if(rdoff >= nrow_lo && rdoff < nrow_hi) { // This cell has been exhaustively scored if(rdoff >= minrow) { // ... and it could potentially score high enough TAlScore sc = (TAlScore)(((TCScore*)pvH)[k] + 0x8000); assert_leq(sc, best); if(sc >= minsc_) { // This is a potential solution bool matchSucc = false; int readc = (*rd_)[rdoff]; int refc = rf_[j + rfi_]; bool match = ((refc & (1 << readc)) != 0); if(rdoff < dpRows()-1) { int readcSucc = (*rd_)[rdoff+1]; int refcSucc = rf_[j + rfi_ + 1]; assert_range(0, 16, refcSucc); matchSucc = ((refcSucc & (1 << readcSucc)) != 0); } if(match && !matchSucc) { // Yes, this is legit met.gathsol++; btncand_.expand(); btncand_.back().init(rdoff, j, sc); } } } } else { // Already saw every element in the vector that's been // exhaustively scored break; } rdoff += iter; } pvH += ROWSTRIDE; } } if(!btncand_.empty()) { d.mat_.initMasks(); } return !btncand_.empty(); } #define MOVE_VEC_PTR_UP(vec, rowvec, rowelt) { \ if(rowvec == 0) { \ rowvec += d.mat_.nvecrow_; \ vec += d.mat_.colstride_; \ rowelt--; \ } \ rowvec--; \ vec -= ROWSTRIDE; \ } #define MOVE_VEC_PTR_LEFT(vec, rowvec, rowelt) { vec -= d.mat_.colstride_; } #define MOVE_VEC_PTR_UPLEFT(vec, rowvec, rowelt) { \ MOVE_VEC_PTR_UP(vec, rowvec, rowelt); \ MOVE_VEC_PTR_LEFT(vec, rowvec, rowelt); \ } #define MOVE_ALL_LEFT() { \ MOVE_VEC_PTR_LEFT(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_LEFT(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_LEFT(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_LEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define MOVE_ALL_UP() { \ MOVE_VEC_PTR_UP(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_UP(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_UP(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_UP(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define MOVE_ALL_UPLEFT() { \ MOVE_VEC_PTR_UPLEFT(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_UPLEFT(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_UPLEFT(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_UPLEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define NEW_ROW_COL(row, col) { \ rowelt = row / d.mat_.nvecrow_; \ rowvec = row % d.mat_.nvecrow_; \ eltvec = (col * d.mat_.colstride_) + (rowvec * ROWSTRIDE); \ cur_vec = d.mat_.matbuf_.ptr() + eltvec; \ left_vec = cur_vec; \ left_rowelt = rowelt; \ left_rowvec = rowvec; \ MOVE_VEC_PTR_LEFT(left_vec, left_rowvec, left_rowelt); \ up_vec = cur_vec; \ up_rowelt = rowelt; \ up_rowvec = rowvec; \ MOVE_VEC_PTR_UP(up_vec, up_rowvec, up_rowelt); \ upleft_vec = up_vec; \ upleft_rowelt = up_rowelt; \ upleft_rowvec = up_rowvec; \ MOVE_VEC_PTR_LEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } /** * Given the dynamic programming table and a cell, trace backwards from the * cell and install the edits and score/penalty in the appropriate fields of * res. The RandomSource is used to break ties among equally good ways of * tracing back. * * Whenever we enter a cell, we check if its read/ref coordinates correspond to * a cell we traversed constructing a previous alignment. If so, we backtrack * to the last decision point, mask out the path that led to the previously * observed cell, and continue along a different path. If there are no more * paths to try, we stop. * * If an alignment is found, 'off' is set to the alignment's upstream-most * reference character's offset and true is returned. Otherwise, false is * returned. * * In local alignment mode, this method is liable to be slow, especially for * long reads. This is chiefly because if there is one valid solution * (especially if it is pretty high scoring), then many, many paths shooting * off that solution's path will also have valid solutions. */ bool SwAligner::backtraceNucleotidesLocalSseI16( TAlScore escore, // in: expected score SwResult& res, // out: store results (edits and scores) here size_t& off, // out: store diagonal projection of origin size_t& nbts, // out: # backtracks size_t row, // start in this row size_t col, // start in this column RandomSource& rnd) // random gen, to choose among equal paths { assert_lt(row, dpRows()); assert_lt(col, (size_t)(rff_ - rfi_)); SSEData& d = fw_ ? sseI16fw_ : sseI16rc_; SSEMetrics& met = extend_ ? sseI16ExtendMet_ : sseI16MateMet_; met.bt++; assert(!d.profbuf_.empty()); assert_lt(row, rd_->length()); btnstack_.clear(); // empty the backtrack stack btcells_.clear(); // empty the cells-so-far list AlnScore score; score.score_ = score.gaps_ = score.ns_ = 0; size_t origCol = col; size_t gaps = 0, readGaps = 0, refGaps = 0; res.alres.reset(); EList& ned = res.alres.ned(); assert(ned.empty()); assert_gt(dpRows(), row); size_t trimEnd = dpRows() - row - 1; size_t trimBeg = 0; size_t ct = SSEMatrix::H; // cell type // Row and col in terms of where they fall in the SSE vector matrix size_t rowelt, rowvec, eltvec; size_t left_rowelt, up_rowelt, upleft_rowelt; size_t left_rowvec, up_rowvec, upleft_rowvec; __m128i *cur_vec, *left_vec, *up_vec, *upleft_vec; const size_t gbar = sc_->gapbar; NEW_ROW_COL(row, col); // If 'backEliminate' is true, then every time we visit a cell, we remove // edges into the cell. We do this to avoid some of the thrashing around // that occurs when there are lots of valid candidates in the same DP // problem. //const bool backEliminate = true; while((int)row >= 0) { // TODO: As soon as we enter a cell, set it as being reported through, // *and* mark all cells that point into this cell as being reported // through. This will save us from having to consider quite so many // candidates. met.btcell++; nbts++; int readc = (*rd_)[rdi_ + row]; int refm = (int)rf_[rfi_ + col]; int readq = (*qu_)[row]; assert_leq(col, origCol); // Get score in this cell bool empty = false, reportedThru, canMoveThru, branch = false; int cur = SSEMatrix::H; if(!d.mat_.reset_[row]) { d.mat_.resetRow(row); } reportedThru = d.mat_.reportedThrough(row, col); canMoveThru = true; if(reportedThru) { canMoveThru = false; } else { empty = false; if(row > 0) { size_t rowFromEnd = d.mat_.nrow() - row - 1; bool gapsAllowed = !(row < gbar || rowFromEnd < gbar); const int floorsc = 0; const int offsetsc = 0x8000; // Move to beginning of column/row if(ct == SSEMatrix::E) { // AKA rdgap assert_gt(col, 0); TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::E))[rowelt] + offsetsc; assert(gapsAllowed); // Currently in the E matrix; incoming transition must come from the // left. It's either a gap open from the H matrix or a gap extend from // the E matrix. // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; // Get H score of cell to the left TAlScore sc_h_left = ((TCScore*)(left_vec + SSEMatrix::H))[left_rowelt] + offsetsc; if(sc_h_left > floorsc && sc_h_left - sc_->readGapOpen() == sc_cur) { mask |= (1 << 0); // horiz H -> E move possible } // Get E score of cell to the left TAlScore sc_e_left = ((TCScore*)(left_vec + SSEMatrix::E))[left_rowelt] + offsetsc; if(sc_e_left > floorsc && sc_e_left - sc_->readGapExtend() == sc_cur) { mask |= (1 << 1); // horiz E -> E move possible } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isEMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 8) & 3; } if(mask == 3) { // Horiz H -> E or horiz E -> E moves possible #if 1 // Pick H -> E cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 2); // might choose E later #else if(rnd.nextU2()) { // Pick H -> E cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 2); // might choose E later } else { // Pick E -> E cell cur = SW_BT_RDGAP_EXTEND; d.mat_.eMaskSet(row, col, 1); // might choose H later } #endif branch = true; } else if(mask == 2) { // Only horiz E -> E move possible, pick it cur = SW_BT_RDGAP_EXTEND; d.mat_.eMaskSet(row, col, 0); // done } else if(mask == 1) { // I chose the H cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 0); // done } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } if(!branch) { // Is this where we can eliminate some incoming paths as well? } assert(!empty || !canMoveThru); } else if(ct == SSEMatrix::F) { // AKA rfgap assert_gt(row, 0); assert(gapsAllowed); TAlScore sc_h_up = ((TCScore*)(up_vec + SSEMatrix::H))[up_rowelt] + offsetsc; TAlScore sc_f_up = ((TCScore*)(up_vec + SSEMatrix::F))[up_rowelt] + offsetsc; TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::F))[rowelt] + offsetsc; // Currently in the F matrix; incoming transition must come from above. // It's either a gap open from the H matrix or a gap extend from the F // matrix. // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; // Get H score of cell above if(sc_h_up > floorsc && sc_h_up - sc_->refGapOpen() == sc_cur) { mask |= (1 << 0); } // Get F score of cell above if(sc_f_up > floorsc && sc_f_up - sc_->refGapExtend() == sc_cur) { mask |= (1 << 1); } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isFMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 11) & 3; } if(mask == 3) { #if 1 // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 2); // might choose E later #else if(rnd.nextU2()) { // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 2); // might choose E later } else { // I chose the F cell cur = SW_BT_RFGAP_EXTEND; d.mat_.fMaskSet(row, col, 1); // might choose E later } #endif branch = true; } else if(mask == 2) { // I chose the F cell cur = SW_BT_RFGAP_EXTEND; d.mat_.fMaskSet(row, col, 0); // done } else if(mask == 1) { // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 0); // done } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } assert(!empty || !canMoveThru); } else { assert_eq(SSEMatrix::H, ct); TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::H))[rowelt] + offsetsc; TAlScore sc_f_up = ((TCScore*)(up_vec + SSEMatrix::F))[up_rowelt] + offsetsc; TAlScore sc_h_up = ((TCScore*)(up_vec + SSEMatrix::H))[up_rowelt] + offsetsc; TAlScore sc_h_left = col > 0 ? (((TCScore*)(left_vec + SSEMatrix::H))[left_rowelt] + offsetsc) : floorsc; TAlScore sc_e_left = col > 0 ? (((TCScore*)(left_vec + SSEMatrix::E))[left_rowelt] + offsetsc) : floorsc; TAlScore sc_h_upleft = col > 0 ? (((TCScore*)(upleft_vec + SSEMatrix::H))[upleft_rowelt] + offsetsc) : floorsc; TAlScore sc_diag = sc_->score(readc, refm, readq - 33); // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; if(gapsAllowed) { if(sc_h_up > floorsc && sc_cur == sc_h_up - sc_->refGapOpen()) { mask |= (1 << 0); } if(sc_h_left > floorsc && sc_cur == sc_h_left - sc_->readGapOpen()) { mask |= (1 << 1); } if(sc_f_up > floorsc && sc_cur == sc_f_up - sc_->refGapExtend()) { mask |= (1 << 2); } if(sc_e_left > floorsc && sc_cur == sc_e_left - sc_->readGapExtend()) { mask |= (1 << 3); } } if(sc_h_upleft > floorsc && sc_cur == sc_h_upleft + sc_diag) { mask |= (1 << 4); // diagonal is } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isHMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 2) & 31; } assert(gapsAllowed || mask == (1 << 4) || mask == 0); int opts = alts5[mask]; int select = -1; if(opts == 1) { select = firsts5[mask]; assert_geq(mask, 0); d.mat_.hMaskSet(row, col, 0); } else if(opts > 1) { #if 1 if( (mask & 16) != 0) { select = 4; // H diag } else if((mask & 1) != 0) { select = 0; // H up } else if((mask & 4) != 0) { select = 2; // F up } else if((mask & 2) != 0) { select = 1; // H left } else if((mask & 8) != 0) { select = 3; // E left } #else select = randFromMask(rnd, mask); #endif assert_geq(mask, 0); mask &= ~(1 << select); assert(gapsAllowed || mask == (1 << 4) || mask == 0); d.mat_.hMaskSet(row, col, mask); branch = true; } else { /* No way to backtrack! */ } if(select != -1) { if(select == 4) { cur = SW_BT_OALL_DIAG; } else if(select == 0) { cur = SW_BT_OALL_REF_OPEN; } else if(select == 1) { cur = SW_BT_OALL_READ_OPEN; } else if(select == 2) { cur = SW_BT_RFGAP_EXTEND; } else { assert_eq(3, select) cur = SW_BT_RDGAP_EXTEND; } } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } } assert(!empty || !canMoveThru || ct == SSEMatrix::H); } // if(row > 0) } // else clause of if(reportedThru) if(!reportedThru) { d.mat_.setReportedThrough(row, col); } assert(d.mat_.reportedThrough(row, col)); //if(backEliminate && row < d.mat_.nrow()-1) { // // Possibly pick off neighbors below and to the right if the // // neighbor's only way of backtracking is through this cell. //} assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); // Cell was involved in a previously-reported alignment? if(!canMoveThru) { if(!btnstack_.empty()) { // Remove all the cells from list back to and including the // cell where the branch occurred btcells_.resize(btnstack_.back().celsz); // Pop record off the top of the stack ned.resize(btnstack_.back().nedsz); //aed.resize(btnstack_.back().aedsz); row = btnstack_.back().row; col = btnstack_.back().col; gaps = btnstack_.back().gaps; readGaps = btnstack_.back().readGaps; refGaps = btnstack_.back().refGaps; score = btnstack_.back().score; ct = btnstack_.back().ct; btnstack_.pop_back(); assert(!sc_->monotone || score.score() >= escore); NEW_ROW_COL(row, col); continue; } else { // No branch points to revisit; just give up res.reset(); met.btfail++; // DP backtraces failed return false; } } assert(!reportedThru); assert(!sc_->monotone || score.score() >= minsc_); if(empty || row == 0) { assert_eq(SSEMatrix::H, ct); btcells_.expand(); btcells_.back().first = row; btcells_.back().second = col; // This cell is at the end of a legitimate alignment trimBeg = row; assert_eq(btcells_.size(), dpRows() - trimBeg - trimEnd + readGaps); break; } if(branch) { // Add a frame to the backtrack stack btnstack_.expand(); btnstack_.back().init( ned.size(), 0, // aed.size() btcells_.size(), row, col, gaps, readGaps, refGaps, score, (int)ct); } btcells_.expand(); btcells_.back().first = row; btcells_.back().second = col; switch(cur) { // Move up and to the left. If the reference nucleotide in the // source row mismatches the read nucleotide, penalize // it and add a nucleotide mismatch. case SW_BT_OALL_DIAG: { assert_gt(row, 0); assert_gt(col, 0); int readC = (*rd_)[row]; int refNmask = (int)rf_[rfi_+col]; assert_gt(refNmask, 0); int m = matchesEx(readC, refNmask); ct = SSEMatrix::H; if(m != 1) { Edit e( (int)row, mask2dna[refNmask], "ACGTN"[readC], EDIT_TYPE_MM); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); int pen = QUAL2(row, col); score.score_ -= pen; assert(!sc_->monotone || score.score() >= escore); } else { // Reward a match int64_t bonus = sc_->match(30); score.score_ += bonus; assert(!sc_->monotone || score.score() >= escore); } if(m == -1) { score.ns_++; } row--; col--; MOVE_ALL_UPLEFT(); assert(VALID_AL_SCORE(score)); break; } // Move up. Add an edit encoding the ref gap. case SW_BT_OALL_REF_OPEN: { assert_gt(row, 0); Edit e( (int)row, '-', "ACGTN"[(int)(*rd_)[row]], EDIT_TYPE_REF_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); row--; ct = SSEMatrix::H; int pen = sc_->refGapOpen(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; refGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_UP(); break; } // Move up. Add an edit encoding the ref gap. case SW_BT_RFGAP_EXTEND: { assert_gt(row, 1); Edit e( (int)row, '-', "ACGTN"[(int)(*rd_)[row]], EDIT_TYPE_REF_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); row--; ct = SSEMatrix::F; int pen = sc_->refGapExtend(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; refGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_UP(); break; } case SW_BT_OALL_READ_OPEN: { assert_gt(col, 0); Edit e( (int)row+1, mask2dna[(int)rf_[rfi_+col]], '-', EDIT_TYPE_READ_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); col--; ct = SSEMatrix::H; int pen = sc_->readGapOpen(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; readGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_LEFT(); break; } case SW_BT_RDGAP_EXTEND: { assert_gt(col, 1); Edit e( (int)row+1, mask2dna[(int)rf_[rfi_+col]], '-', EDIT_TYPE_READ_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); col--; ct = SSEMatrix::E; int pen = sc_->readGapExtend(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; readGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_LEFT(); break; } default: throw 1; } } // while((int)row > 0) assert_geq(col, 0); assert_eq(SSEMatrix::H, ct); // The number of cells in the backtracs should equal the number of read // bases after trimming plus the number of gaps assert_eq(btcells_.size(), dpRows() - trimBeg - trimEnd + readGaps); // Check whether we went through a core diagonal and set 'reported' flag on // each cell bool overlappedCoreDiag = false; for(size_t i = 0; i < btcells_.size(); i++) { size_t rw = btcells_[i].first; size_t cl = btcells_[i].second; // Calculate the diagonal within the *trimmed* rectangle, i.e. the // rectangle we dealt with in align, gather and backtrack. int64_t diagi = cl - rw; // Now adjust to the diagonal within the *untrimmed* rectangle by // adding on the amount trimmed from the left. diagi += rect_->triml; if(diagi >= 0) { size_t diag = (size_t)diagi; if(diag >= rect_->corel && diag <= rect_->corer) { overlappedCoreDiag = true; break; } } #ifndef NDEBUG //assert(!d.mat_.reportedThrough(rw, cl)); //d.mat_.setReportedThrough(rw, cl); assert(d.mat_.reportedThrough(rw, cl)); #endif } if(!overlappedCoreDiag) { // Must overlap a core diagonal. Otherwise, we run the risk of // reporting an alignment that overlaps (and trumps) a higher-scoring // alignment that lies partially outside the dynamic programming // rectangle. res.reset(); met.corerej++; return false; } int readC = (*rd_)[rdi_+row]; // get last char in read int refNmask = (int)rf_[rfi_+col]; // get last ref char ref involved in aln assert_gt(refNmask, 0); int m = matchesEx(readC, refNmask); if(m != 1) { Edit e((int)row, mask2dna[refNmask], "ACGTN"[readC], EDIT_TYPE_MM); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); score.score_ -= QUAL2(row, col); assert_geq(score.score(), minsc_); } else { score.score_ += sc_->match(30); } if(m == -1) { score.ns_++; } if(score.ns_ > nceil_) { // Alignment has too many Ns in it! res.reset(); met.nrej++; return false; } res.reverse(); assert(Edit::repOk(ned, (*rd_))); assert_eq(score.score(), escore); assert_leq(gaps, rdgap_ + rfgap_); off = col; assert_lt(col + (size_t)rfi_, (size_t)rff_); score.gaps_ = gaps; res.alres.setScore(score); res.alres.setShape( refidx_, // ref id off + rfi_ + rect_->refl, // 0-based ref offset reflen_, // reference length fw_, // aligned to Watson? rdf_ - rdi_, // read length 0, // read ID true, // pretrim soft? 0, // pretrim 5' end 0, // pretrim 3' end true, // alignment trim soft? fw_ ? trimBeg : trimEnd, // alignment trim 5' end fw_ ? trimEnd : trimBeg); // alignment trim 3' end size_t refns = 0; for(size_t i = col; i <= origCol; i++) { if((int)rf_[rfi_+i] > 15) { refns++; } } res.alres.setRefNs(refns); assert(Edit::repOk(ned, (*rd_), true, trimBeg, trimEnd)); assert(res.repOk()); #ifndef NDEBUG size_t gapsCheck = 0; for(size_t i = 0; i < ned.size(); i++) { if(ned[i].isGap()) gapsCheck++; } assert_eq(gaps, gapsCheck); BTDnaString refstr; for(size_t i = col; i <= origCol; i++) { refstr.append(firsts5[(int)rf_[rfi_+i]]); } BTDnaString editstr; Edit::toRef((*rd_), ned, editstr, true, trimBeg, trimEnd); if(refstr != editstr) { cerr << "Decoded nucleotides and edits don't match reference:" << endl; cerr << " score: " << score.score() << " (" << gaps << " gaps)" << endl; cerr << " edits: "; Edit::print(cerr, ned); cerr << endl; cerr << " decoded nucs: " << (*rd_) << endl; cerr << " edited nucs: " << editstr << endl; cerr << " reference nucs: " << refstr << endl; assert(0); } #endif met.btsucc++; // DP backtraces succeeded return true; } hisat2-2.2.1/aligner_swsse_loc_u8.cpp000066400000000000000000002225331370663763200175540ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /** * aligner_sw_sse.cpp * * Versions of key alignment functions that use vector instructions to * accelerate dynamic programming. Based chiefly on the striped Smith-Waterman * paper and implementation by Michael Farrar. See: * * Farrar M. Striped Smith-Waterman speeds database searches six times over * other SIMD implementations. Bioinformatics. 2007 Jan 15;23(2):156-61. * http://sites.google.com/site/farrarmichael/smith-waterman * * While the paper describes an implementation of Smith-Waterman, we extend it * do end-to-end read alignment as well as local alignment. The change * required for this is minor: we simply let vmax be the maximum element in the * score domain rather than the minimum. * * The vectorized dynamic programming implementation lacks some features that * make it hard to adapt to solving the entire dynamic-programming alignment * problem. For instance: * * - It doesn't respect gap barriers on either end of the read * - It just gives a maximum; not enough information to backtrace without * redoing some alignment * - It's a little difficult to handle st_ and en_, especially st_. * - The query profile mechanism makes handling of ambiguous reference bases a * little tricky (16 cols in query profile lookup table instead of 5) * * Given the drawbacks, it is tempting to use SSE dynamic programming as a * filter rather than as an aligner per se. Here are a few ideas for how it * can be extended to handle more of the alignment problem: * * - Save calculated scores to a big array as we go. We return to this array * to find and backtrace from good solutions. */ #include #include "aligner_sw.h" // static const size_t NBYTES_PER_REG = 16; static const size_t NWORDS_PER_REG = 16; // static const size_t NBITS_PER_WORD = 8; static const size_t NBYTES_PER_WORD = 1; // In local mode, we start low (0) and go high (255). Factoring in a query // profile involves unsigned saturating addition. All query profile elements // should be expressed as a positive number; this is done by adding -min // where min is the smallest (negative) score in the query profile. typedef uint8_t TCScore; /** * Build query profile look up tables for the read. The query profile look * up table is organized as a 1D array indexed by [i][j] where i is the * reference character in the current DP column (0=A, 1=C, etc), and j is * the segment of the query we're currently working on. */ void SwAligner::buildQueryProfileLocalSseU8(bool fw) { bool& done = fw ? sseU8fwBuilt_ : sseU8rcBuilt_; if(done) { return; } done = true; const BTDnaString* rd = fw ? rdfw_ : rdrc_; const BTString* qu = fw ? qufw_ : qurc_; const size_t len = rd->length(); const size_t seglen = (len + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // How many __m128i's are needed size_t n128s = 64 + // slack bytes, for alignment? (seglen * ALPHA_SIZE) // query profile data * 2; // & gap barrier data assert_gt(n128s, 0); SSEData& d = fw ? sseU8fw_ : sseU8rc_; d.profbuf_.resizeNoCopy(n128s); assert(!d.profbuf_.empty()); d.maxPen_ = d.maxBonus_ = 0; d.lastIter_ = d.lastWord_ = 0; d.qprofStride_ = d.gbarStride_ = 2; d.bias_ = 0; // Calculate bias for(size_t refc = 0; refc < ALPHA_SIZE; refc++) { for(size_t i = 0; i < len; i++) { int readc = (*rd)[i]; int readq = (*qu)[i]; int sc = sc_->score(readc, (int)(1 << refc), readq - 33); if(sc < 0 && sc < d.bias_) { d.bias_ = sc; } } } assert_leq(d.bias_, 0); d.bias_ = -d.bias_; // For each reference character A, C, G, T, N ... for(size_t refc = 0; refc < ALPHA_SIZE; refc++) { // For each segment ... for(size_t i = 0; i < seglen; i++) { size_t j = i; uint8_t *qprofWords = reinterpret_cast(d.profbuf_.ptr() + (refc * seglen * 2) + (i * 2)); uint8_t *gbarWords = reinterpret_cast(d.profbuf_.ptr() + (refc * seglen * 2) + (i * 2) + 1); // For each sub-word (byte) ... for(size_t k = 0; k < NWORDS_PER_REG; k++) { int sc = 0; *gbarWords = 0; if(j < len) { int readc = (*rd)[j]; int readq = (*qu)[j]; sc = sc_->score(readc, (int)(1 << refc), readq - 33); assert_range(0, 255, sc + d.bias_); size_t j_from_end = len - j - 1; if(j < (size_t)sc_->gapbar || j_from_end < (size_t)sc_->gapbar) { // Inside the gap barrier *gbarWords = 0xff; } } if(refc == 0 && j == len-1) { // Remember which 128-bit word and which smaller word has // the final row d.lastIter_ = i; d.lastWord_ = k; } if(sc < 0) { if((size_t)(-sc) > d.maxPen_) { d.maxPen_ = (size_t)(-sc); } } else { if((size_t)sc > d.maxBonus_) { d.maxBonus_ = (size_t)sc; } } *qprofWords = (uint8_t)(sc + d.bias_); gbarWords++; qprofWords++; j += seglen; // update offset into query } } } } #ifndef NDEBUG /** * Return true iff the cell has sane E/F/H values w/r/t its predecessors. */ static bool cellOkLocalU8( SSEData& d, size_t row, size_t col, int refc, int readc, int readq, const Scoring& sc) // scoring scheme { TCScore floorsc = 0; TCScore ceilsc = 255 - d.bias_ - 1; TAlScore offsetsc = 0; TAlScore sc_h_cur = (TAlScore)d.mat_.helt(row, col); TAlScore sc_e_cur = (TAlScore)d.mat_.eelt(row, col); TAlScore sc_f_cur = (TAlScore)d.mat_.felt(row, col); if(sc_h_cur > floorsc) { sc_h_cur += offsetsc; } if(sc_e_cur > floorsc) { sc_e_cur += offsetsc; } if(sc_f_cur > floorsc) { sc_f_cur += offsetsc; } bool gapsAllowed = true; size_t rowFromEnd = d.mat_.nrow() - row - 1; if(row < (size_t)sc.gapbar || rowFromEnd < (size_t)sc.gapbar) { gapsAllowed = false; } bool e_left_trans = false, h_left_trans = false; bool f_up_trans = false, h_up_trans = false; bool h_diag_trans = false; if(gapsAllowed) { TAlScore sc_h_left = floorsc; TAlScore sc_e_left = floorsc; TAlScore sc_h_up = floorsc; TAlScore sc_f_up = floorsc; if(col > 0 && sc_e_cur > floorsc && sc_e_cur <= ceilsc) { sc_h_left = d.mat_.helt(row, col-1) + offsetsc; sc_e_left = d.mat_.eelt(row, col-1) + offsetsc; e_left_trans = (sc_e_left > floorsc && sc_e_cur == sc_e_left - sc.readGapExtend()); h_left_trans = (sc_h_left > floorsc && sc_e_cur == sc_h_left - sc.readGapOpen()); assert(e_left_trans || h_left_trans); } if(row > 0 && sc_f_cur > floorsc && sc_f_cur <= ceilsc) { sc_h_up = d.mat_.helt(row-1, col) + offsetsc; sc_f_up = d.mat_.felt(row-1, col) + offsetsc; f_up_trans = (sc_f_up > floorsc && sc_f_cur == sc_f_up - sc.refGapExtend()); h_up_trans = (sc_h_up > floorsc && sc_f_cur == sc_h_up - sc.refGapOpen()); assert(f_up_trans || h_up_trans); } } else { assert_geq(floorsc, sc_e_cur); assert_geq(floorsc, sc_f_cur); } if(col > 0 && row > 0 && sc_h_cur > floorsc && sc_h_cur <= ceilsc) { TAlScore sc_h_upleft = d.mat_.helt(row-1, col-1) + offsetsc; TAlScore sc_diag = sc.score(readc, (int)refc, readq - 33); h_diag_trans = sc_h_cur == sc_h_upleft + sc_diag; } assert( sc_h_cur <= floorsc || e_left_trans || h_left_trans || f_up_trans || h_up_trans || h_diag_trans || sc_h_cur > ceilsc || row == 0 || col == 0); return true; } #endif /*ndef NDEBUG*/ #ifdef NDEBUG #define assert_all_eq0(x) #define assert_all_gt(x, y) #define assert_all_gt_lo(x) #define assert_all_lt(x, y) #define assert_all_lt_hi(x) #else #define assert_all_eq0(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_xor_si128(z, z); \ tmp = _mm_cmpeq_epi16(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_gt(x, y) { \ __m128i tmp = _mm_cmpgt_epu8(x, y); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_gt_lo(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_xor_si128(z, z); \ tmp = _mm_cmpgt_epu8(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #define assert_all_lt(x, y) { \ __m128i z = _mm_setzero_si128(); \ z = _mm_xor_si128(z, z); \ __m128i tmp = _mm_subs_epu8(y, x); \ tmp = _mm_cmpeq_epi16(tmp, z); \ assert_eq(0x0000, _mm_movemask_epi8(tmp)); \ } #define assert_all_lt_hi(x) { \ __m128i z = _mm_setzero_si128(); \ __m128i tmp = _mm_setzero_si128(); \ z = _mm_cmpeq_epu8(z, z); \ z = _mm_srli_epu8(z, 1); \ tmp = _mm_cmplt_epu8(x, z); \ assert_eq(0xffff, _mm_movemask_epi8(tmp)); \ } #endif /** * Aligns by filling a dynamic programming matrix with the SSE-accelerated, * banded DP approach of Farrar. As it goes, it determines which cells we * might backtrace from and tallies the best (highest-scoring) N backtrace * candidate cells per diagonal. Also returns the alignment score of the best * alignment in the matrix. * * This routine does *not* maintain a matrix holding the entire matrix worth of * scores, nor does it maintain any other dense O(mn) data structure, as this * would quickly exhaust memory for queries longer than about 10,000 kb. * Instead, in the fill stage it maintains two columns worth of scores at a * time (current/previous, or right/left) - these take O(m) space. When * finished with the current column, it determines which cells from the * previous column, if any, are candidates we might backtrace from to find a * full alignment. A candidate cell has a score that rises above the threshold * and isn't improved upon by a match in the next column. The best N * candidates per diagonal are stored in a O(m + n) data structure. */ TAlScore SwAligner::alignGatherLoc8(int& flag, bool debug) { assert_leq(rdf_, rd_->length()); assert_leq(rdf_, qu_->length()); assert_lt(rfi_, rff_); assert_lt(rdi_, rdf_); assert_eq(rd_->length(), qu_->length()); assert_geq(sc_->gapbar, 1); assert_gt(minsc_, 0); assert(repOk()); #ifndef NDEBUG for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert_range(0, 16, (int)rf_[i]); } #endif SSEData& d = fw_ ? sseU8fw_ : sseU8rc_; SSEMetrics& met = extend_ ? sseU8ExtendMet_ : sseU8MateMet_; if(!debug) met.dp++; buildQueryProfileLocalSseU8(fw_); assert(!d.profbuf_.empty()); assert_gt(d.bias_, 0); assert_lt(d.bias_, 127); assert_gt(d.maxBonus_, 0); size_t iter = (dpRows() + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // iter = segLen // Now set up the score vectors. We just need two columns worth, which // we'll call "left" and "right". d.vecbuf_.resize(ROWSTRIDE_2COL * iter * 2); d.vecbuf_.zero(); __m128i *vbuf_l = d.vecbuf_.ptr(); __m128i *vbuf_r = d.vecbuf_.ptr() + (ROWSTRIDE_2COL * iter); // This is the data structure that holds candidate cells per diagonal. const size_t ndiags = rff_ - rfi_ + dpRows() - 1; if(!debug) { btdiag_.init(ndiags, 2); } // Data structure that holds checkpointed anti-diagonals TAlScore perfectScore = sc_->perfectScore(dpRows()); bool checkpoint = true; bool cpdebug = false; #ifndef NDEBUG cpdebug = dpRows() < 1000; #endif cper_.init( dpRows(), // # rows rff_ - rfi_, // # columns cperPerPow2_, // checkpoint every 1 << perpow2 diags (& next) perfectScore, // perfect score (for sanity checks) true, // matrix cells have 8-bit scores? cperTri_, // triangular mini-fills? true, // alignment is local? cpdebug); // save all cells for debugging? // Many thanks to Michael Farrar for releasing his striped Smith-Waterman // implementation: // // http://sites.google.com/site/farrarmichael/smith-waterman // // Much of the implmentation below is adapted from Michael's code. // Set all elts to reference gap open penalty __m128i rfgapo = _mm_setzero_si128(); __m128i rfgape = _mm_setzero_si128(); __m128i rdgapo = _mm_setzero_si128(); __m128i rdgape = _mm_setzero_si128(); __m128i vlo = _mm_setzero_si128(); __m128i vhi = _mm_setzero_si128(); __m128i vmax = _mm_setzero_si128(); __m128i vcolmax = _mm_setzero_si128(); __m128i vmaxtmp = _mm_setzero_si128(); __m128i ve = _mm_setzero_si128(); __m128i vf = _mm_setzero_si128(); __m128i vh = _mm_setzero_si128(); __m128i vhd = _mm_setzero_si128(); __m128i vhdtmp = _mm_setzero_si128(); __m128i vtmp = _mm_setzero_si128(); __m128i vzero = _mm_setzero_si128(); __m128i vbias = _mm_setzero_si128(); __m128i vbiasm1 = _mm_setzero_si128(); __m128i vminsc = _mm_setzero_si128(); int dup; assert_gt(sc_->refGapOpen(), 0); assert_leq(sc_->refGapOpen(), MAX_U8); dup = (sc_->refGapOpen() << 8) | (sc_->refGapOpen() & 0x00ff); rfgapo = _mm_insert_epi16(rfgapo, dup, 0); rfgapo = _mm_shufflelo_epi16(rfgapo, 0); rfgapo = _mm_shuffle_epi32(rfgapo, 0); // Set all elts to reference gap extension penalty assert_gt(sc_->refGapExtend(), 0); assert_leq(sc_->refGapExtend(), MAX_U8); assert_leq(sc_->refGapExtend(), sc_->refGapOpen()); dup = (sc_->refGapExtend() << 8) | (sc_->refGapExtend() & 0x00ff); rfgape = _mm_insert_epi16(rfgape, dup, 0); rfgape = _mm_shufflelo_epi16(rfgape, 0); rfgape = _mm_shuffle_epi32(rfgape, 0); // Set all elts to read gap open penalty assert_gt(sc_->readGapOpen(), 0); assert_leq(sc_->readGapOpen(), MAX_U8); dup = (sc_->readGapOpen() << 8) | (sc_->readGapOpen() & 0x00ff); rdgapo = _mm_insert_epi16(rdgapo, dup, 0); rdgapo = _mm_shufflelo_epi16(rdgapo, 0); rdgapo = _mm_shuffle_epi32(rdgapo, 0); // Set all elts to read gap extension penalty assert_gt(sc_->readGapExtend(), 0); assert_leq(sc_->readGapExtend(), MAX_U8); assert_leq(sc_->readGapExtend(), sc_->readGapOpen()); dup = (sc_->readGapExtend() << 8) | (sc_->readGapExtend() & 0x00ff); rdgape = _mm_insert_epi16(rdgape, dup, 0); rdgape = _mm_shufflelo_epi16(rdgape, 0); rdgape = _mm_shuffle_epi32(rdgape, 0); // Set all elts to minimum score threshold. Actually, to 1 less than the // threshold so we can use gt instead of geq. dup = (((int)minsc_ - 1) << 8) | (((int)minsc_ - 1) & 0x00ff); vminsc = _mm_insert_epi16(vminsc, dup, 0); vminsc = _mm_shufflelo_epi16(vminsc, 0); vminsc = _mm_shuffle_epi32(vminsc, 0); dup = ((d.bias_ - 1) << 8) | ((d.bias_ - 1) & 0x00ff); vbiasm1 = _mm_insert_epi16(vbiasm1, dup, 0); vbiasm1 = _mm_shufflelo_epi16(vbiasm1, 0); vbiasm1 = _mm_shuffle_epi32(vbiasm1, 0); vhi = _mm_cmpeq_epi16(vhi, vhi); // all elts = 0xffff vlo = _mm_xor_si128(vlo, vlo); // all elts = 0 vmax = vlo; // Make a vector of bias offsets dup = (d.bias_ << 8) | (d.bias_ & 0x00ff); vbias = _mm_insert_epi16(vbias, dup, 0); vbias = _mm_shufflelo_epi16(vbias, 0); vbias = _mm_shuffle_epi32(vbias, 0); // Points to a long vector of __m128i where each element is a block of // contiguous cells in the E, F or H matrix. If the index % 3 == 0, then // the block of cells is from the E matrix. If index % 3 == 1, they're // from the F matrix. If index % 3 == 2, then they're from the H matrix. // Blocks of cells are organized in the same interleaved manner as they are // calculated by the Farrar algorithm. const __m128i *pvScore; // points into the query profile const size_t colstride = ROWSTRIDE_2COL * iter; // Initialize the H and E vectors in the first matrix column __m128i *pvELeft = vbuf_l + 0; __m128i *pvERight = vbuf_r + 0; /* __m128i *pvFLeft = vbuf_l + 1; */ __m128i *pvFRight = vbuf_r + 1; __m128i *pvHLeft = vbuf_l + 2; __m128i *pvHRight = vbuf_r + 2; for(size_t i = 0; i < iter; i++) { // start low in local mode _mm_store_si128(pvERight, vlo); pvERight += ROWSTRIDE_2COL; _mm_store_si128(pvHRight, vlo); pvHRight += ROWSTRIDE_2COL; } assert_gt(sc_->gapbar, 0); size_t nfixup = 0; TAlScore matchsc = sc_->match(30); TAlScore leftmax = MIN_I64; // Fill in the table as usual but instead of using the same gap-penalty // vector for each iteration of the inner loop, load words out of a // pre-calculated gap vector parallel to the query profile. The pre- // calculated gap vectors enforce the gap barrier constraint by making it // infinitely costly to introduce a gap in barrier rows. // // AND use a separate loop to fill in the first row of the table, enforcing // the st_ constraints in the process. This is awkward because it // separates the processing of the first row from the others and might make // it difficult to use the first-row results in the next row, but it might // be the simplest and least disruptive way to deal with the st_ constraint. size_t off = MAX_SIZE_T, lastoff; bool bailed = false; for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { // Swap left and right; vbuf_l is the vector on the left, which we // generally load from, and vbuf_r is the vector on the right, which we // generally store to. swap(vbuf_l, vbuf_r); pvELeft = vbuf_l + 0; pvERight = vbuf_r + 0; /* pvFLeft = vbuf_l + 1; */ pvFRight = vbuf_r + 1; pvHLeft = vbuf_l + 2; pvHRight = vbuf_r + 2; // Fetch this column's reference mask const int refm = (int)rf_[i]; // Fetch the appropriate query profile lastoff = off; off = (size_t)firsts5[refm] * iter * 2; pvScore = d.profbuf_.ptr() + off; // even elts = query profile, odd = gap barrier // Load H vector from the final row of the previous column. // ??? perhaps we should calculate the next iter's F instead of the // current iter's? The way we currently do it, seems like it will // almost always require at least one fixup loop iter (to recalculate // this topmost F). vh = _mm_load_si128(pvHLeft + colstride - ROWSTRIDE_2COL); // Set all cells to low value vf = _mm_xor_si128(vf, vf); // vf now contains the vertical contribution // Store cells in F, calculated previously // No need to veto ref gap extensions, they're all 0x00s _mm_store_si128(pvFRight, vf); pvFRight += ROWSTRIDE_2COL; // Shift down so that topmost (least sig) cell gets 0 vh = _mm_slli_si128(vh, NBYTES_PER_WORD); // We pull out one loop iteration to make it easier to veto values in the top row // Load cells from E, calculated previously ve = _mm_load_si128(pvELeft); vhd = _mm_load_si128(pvHLeft); assert_all_lt(ve, vhi); pvELeft += ROWSTRIDE_2COL; // ve now contains the horizontal contribution // Factor in query profile (matches and mismatches) vh = _mm_adds_epu8(vh, pvScore[0]); vh = _mm_subs_epu8(vh, vbias); // vh now contains the diagonal contribution vhdtmp = vhd; vhd = _mm_subs_epu8(vhd, rdgapo); vhd = _mm_subs_epu8(vhd, pvScore[1]); // veto some read gap opens ve = _mm_subs_epu8(ve, rdgape); ve = _mm_max_epu8(ve, vhd); vh = _mm_max_epu8(vh, ve); vf = vh; // Update highest score so far vcolmax = vh; // Save the new vH values _mm_store_si128(pvHRight, vh); vh = vhdtmp; assert_all_lt(ve, vhi); pvHRight += ROWSTRIDE_2COL; pvHLeft += ROWSTRIDE_2COL; // Save E values _mm_store_si128(pvERight, ve); pvERight += ROWSTRIDE_2COL; // Update vf value vf = _mm_subs_epu8(vf, rfgapo); assert_all_lt(vf, vhi); pvScore += 2; // move on to next query profile // For each character in the reference text: size_t j; for(j = 1; j < iter; j++) { // Load cells from E, calculated previously ve = _mm_load_si128(pvELeft); vhd = _mm_load_si128(pvHLeft); assert_all_lt(ve, vhi); pvELeft += ROWSTRIDE_2COL; // Store cells in F, calculated previously vf = _mm_subs_epu8(vf, pvScore[1]); // veto some ref gap extensions _mm_store_si128(pvFRight, vf); pvFRight += ROWSTRIDE_2COL; // Factor in query profile (matches and mismatches) vh = _mm_adds_epu8(vh, pvScore[0]); vh = _mm_subs_epu8(vh, vbias); // Update H, factoring in E and F vh = _mm_max_epu8(vh, vf); vhdtmp = vhd; vhd = _mm_subs_epu8(vhd, rdgapo); vhd = _mm_subs_epu8(vhd, pvScore[1]); // veto some read gap opens ve = _mm_subs_epu8(ve, rdgape); ve = _mm_max_epu8(ve, vhd); vh = _mm_max_epu8(vh, ve); vtmp = vh; // Update highest score encountered this far vcolmax = _mm_max_epu8(vcolmax, vh); // Save the new vH values _mm_store_si128(pvHRight, vh); vh = vhdtmp; assert_all_lt(ve, vhi); pvHRight += ROWSTRIDE_2COL; pvHLeft += ROWSTRIDE_2COL; // Save E values _mm_store_si128(pvERight, ve); pvERight += ROWSTRIDE_2COL; // Update vf value vtmp = _mm_subs_epu8(vtmp, rfgapo); vf = _mm_subs_epu8(vf, rfgape); assert_all_lt(vf, vhi); vf = _mm_max_epu8(vf, vtmp); pvScore += 2; // move on to next query profile / gap veto } // pvHStore, pvELoad, pvEStore have all rolled over to the next column pvFRight -= colstride; // reset to start of column vtmp = _mm_load_si128(pvFRight); pvHRight -= colstride; // reset to start of column vh = _mm_load_si128(pvHRight); pvScore = d.profbuf_.ptr() + off + 1; // reset veto vector // vf from last row gets shifted down by one to overlay the first row // rfgape has already been subtracted from it. vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_subs_epu8(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epu8(vtmp, vf); // TODO: We're testing whether F changed. Can't we just assume that F // did change and instead check whether H changed? Might save us from // entering the fixup loop. vtmp = _mm_subs_epu8(vf, vtmp); vtmp = _mm_cmpeq_epi8(vtmp, vzero); int cmp = _mm_movemask_epi8(vtmp); // If any element of vtmp is greater than H - gap-open... j = 0; while(cmp != 0xffff) { // Store this vf _mm_store_si128(pvFRight, vf); pvFRight += ROWSTRIDE_2COL; // Update vh w/r/t new vf vh = _mm_max_epu8(vh, vf); // Save vH values _mm_store_si128(pvHRight, vh); pvHRight += ROWSTRIDE_2COL; // Update highest score encountered so far. vcolmax = _mm_max_epu8(vcolmax, vh); pvScore += 2; assert_lt(j, iter); if(++j == iter) { pvFRight -= colstride; vtmp = _mm_load_si128(pvFRight); // load next vf ASAP pvHRight -= colstride; vh = _mm_load_si128(pvHRight); // load next vh ASAP pvScore = d.profbuf_.ptr() + off + 1; j = 0; vf = _mm_slli_si128(vf, NBYTES_PER_WORD); } else { vtmp = _mm_load_si128(pvFRight); // load next vf ASAP vh = _mm_load_si128(pvHRight); // load next vh ASAP } // Update F with another gap extension vf = _mm_subs_epu8(vf, rfgape); vf = _mm_subs_epu8(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epu8(vtmp, vf); vtmp = _mm_subs_epu8(vf, vtmp); vtmp = _mm_cmpeq_epi8(vtmp, vzero); cmp = _mm_movemask_epi8(vtmp); nfixup++; } // Now we'd like to know exactly which cells in the left column are // candidates we might backtrace from. First question is: did *any* // elements in the column exceed the minimum score threshold? if(!debug && leftmax >= minsc_) { // Yes. Next question is: which cells are candidates? We have to // allow matches in the right column to override matches above and // to the left in the left column. assert_gt(i - rfi_, 0); pvHLeft = vbuf_l + 2; assert_lt(lastoff, MAX_SIZE_T); pvScore = d.profbuf_.ptr() + lastoff; // even elts = query profile, odd = gap barrier for(size_t k = 0; k < iter; k++) { vh = _mm_load_si128(pvHLeft); vtmp = _mm_cmpgt_epi8(pvScore[0], vbiasm1); int cmp = _mm_movemask_epi8(vtmp); if(cmp != 0xffff) { // At least one candidate in this mask. Now iterate // through vm/vh to evaluate individual cells. for(size_t m = 0; m < NWORDS_PER_REG; m++) { size_t row = k + m * iter; if(row >= dpRows()) { break; } if(((TCScore *)&vtmp)[m] > 0 && ((TCScore *)&vh)[m] >= minsc_) { TCScore sc = ((TCScore *)&vh)[m]; assert_geq(sc, minsc_); // Add to data structure holding all candidates size_t col = i - rfi_ - 1; // -1 b/c prev col size_t frombot = dpRows() - row - 1; DpBtCandidate cand(row, col, sc); btdiag_.add(frombot + col, cand); } } } pvHLeft += ROWSTRIDE_2COL; pvScore += 2; } } // Save some elements to checkpoints if(checkpoint) { __m128i *pvE = vbuf_r + 0; __m128i *pvF = vbuf_r + 1; __m128i *pvH = vbuf_r + 2; size_t coli = i - rfi_; if(coli < cper_.locol_) cper_.locol_ = coli; if(coli > cper_.hicol_) cper_.hicol_ = coli; if(cperTri_) { // Checkpoint for triangular mini-fills size_t rc_mod = coli & cper_.lomask_; assert_lt(rc_mod, cper_.per_); int64_t row = -(int64_t)rc_mod-1; int64_t row_mod = row; int64_t row_div = 0; size_t idx = coli >> cper_.perpow2_; size_t idxrow = idx * cper_.nrow_; assert_eq(4, ROWSTRIDE_2COL); bool done = false; while(true) { row += (cper_.per_ - 2); row_mod += (cper_.per_ - 2); for(size_t j = 0; j < 2; j++) { row++; row_mod++; if(row >= 0 && (size_t)row < cper_.nrow_) { // Update row divided by iter_ and mod iter_ while(row_mod >= (int64_t)iter) { row_mod -= (int64_t)iter; row_div++; } size_t delt = idxrow + row; size_t vecoff = (row_mod << 6) + row_div; assert_lt(row_div, 16); int16_t h_sc = ((uint8_t*)pvH)[vecoff]; int16_t e_sc = ((uint8_t*)pvE)[vecoff]; int16_t f_sc = ((uint8_t*)pvF)[vecoff]; assert_leq(h_sc, cper_.perf_); assert_leq(e_sc, cper_.perf_); assert_leq(f_sc, cper_.perf_); CpQuad *qdiags = ((j == 0) ? cper_.qdiag1s_.ptr() : cper_.qdiag2s_.ptr()); qdiags[delt].sc[0] = h_sc; qdiags[delt].sc[1] = e_sc; qdiags[delt].sc[2] = f_sc; } // if(row >= 0 && row < nrow_) else if(row >= 0 && (size_t)row >= cper_.nrow_) { done = true; break; } } // for(size_t j = 0; j < 2; j++) if(done) { break; } idx++; idxrow += cper_.nrow_; } // while(true) } else { // Checkpoint for square mini-fills // If this is the first column, take this opportunity to // pre-calculate the coordinates of the elements we're going to // checkpoint. if(coli == 0) { size_t cpi = cper_.per_-1; size_t cpimod = cper_.per_-1; size_t cpidiv = 0; cper_.commitMap_.clear(); while(cpi < cper_.nrow_) { while(cpimod >= iter) { cpimod -= iter; cpidiv++; } size_t vecoff = (cpimod << 6) + cpidiv; cper_.commitMap_.push_back(vecoff); cpi += cper_.per_; cpimod += cper_.per_; } } // Save all the rows size_t rowoff = 0; size_t sz = cper_.commitMap_.size(); for(size_t i = 0; i < sz; i++, rowoff += cper_.ncol_) { size_t vecoff = cper_.commitMap_[i]; int16_t h_sc = ((uint8_t*)pvH)[vecoff]; //int16_t e_sc = ((uint8_t*)pvE)[vecoff]; int16_t f_sc = ((uint8_t*)pvF)[vecoff]; assert_leq(h_sc, cper_.perf_); //assert_leq(e_sc, cper_.perf_); assert_leq(f_sc, cper_.perf_); CpQuad& dst = cper_.qrows_[rowoff + coli]; dst.sc[0] = h_sc; //dst.sc[1] = e_sc; dst.sc[2] = f_sc; } // Is this a column we'd like to checkpoint? if((coli & cper_.lomask_) == cper_.lomask_) { // Save the column using memcpys assert_gt(coli, 0); size_t wordspercol = cper_.niter_ * ROWSTRIDE_2COL; size_t coloff = (coli >> cper_.perpow2_) * wordspercol; __m128i *dst = cper_.qcols_.ptr() + coloff; memcpy(dst, vbuf_r, sizeof(__m128i) * wordspercol); } } if(cper_.debug_) { // Save the column using memcpys size_t wordspercol = cper_.niter_ * ROWSTRIDE_2COL; size_t coloff = coli * wordspercol; __m128i *dst = cper_.qcolsD_.ptr() + coloff; memcpy(dst, vbuf_r, sizeof(__m128i) * wordspercol); } } // Store column maximum vector in first element of tmp vmax = _mm_max_epu8(vmax, vcolmax); { // Get single largest score in this column vmaxtmp = vcolmax; vtmp = _mm_srli_si128(vmaxtmp, 8); vmaxtmp = _mm_max_epu8(vmaxtmp, vtmp); vtmp = _mm_srli_si128(vmaxtmp, 4); vmaxtmp = _mm_max_epu8(vmaxtmp, vtmp); vtmp = _mm_srli_si128(vmaxtmp, 2); vmaxtmp = _mm_max_epu8(vmaxtmp, vtmp); vtmp = _mm_srli_si128(vmaxtmp, 1); vmaxtmp = _mm_max_epu8(vmaxtmp, vtmp); int score = _mm_extract_epi16(vmaxtmp, 0); score = score & 0x00ff; // Could we have saturated? if(score + d.bias_ >= 255) { flag = -2; // yes if(!debug) met.dpsat++; return MIN_I64; } if(score < minsc_) { size_t ncolleft = rff_ - i - 1; if(score + (TAlScore)ncolleft * matchsc < minsc_) { // Bail! There can't possibly be a valid alignment that // passes through this column. bailed = true; break; } } leftmax = score; } } lastoff = off; // Now we'd like to know exactly which cells in the *rightmost* column are // candidates we might backtrace from. Did *any* elements exceed the // minimum score threshold? if(!debug && !bailed && leftmax >= minsc_) { // Yes. Next question is: which cells are candidates? We have to // allow matches in the right column to override matches above and // to the left in the left column. pvHLeft = vbuf_r + 2; assert_lt(lastoff, MAX_SIZE_T); pvScore = d.profbuf_.ptr() + lastoff; // even elts = query profile, odd = gap barrier for(size_t k = 0; k < iter; k++) { vh = _mm_load_si128(pvHLeft); vtmp = _mm_cmpgt_epi8(pvScore[0], vbiasm1); int cmp = _mm_movemask_epi8(vtmp); if(cmp != 0xffff) { // At least one candidate in this mask. Now iterate // through vm/vh to evaluate individual cells. for(size_t m = 0; m < NWORDS_PER_REG; m++) { size_t row = k + m * iter; if(row >= dpRows()) { break; } if(((TCScore *)&vtmp)[m] > 0 && ((TCScore *)&vh)[m] >= minsc_) { TCScore sc = ((TCScore *)&vh)[m]; assert_geq(sc, minsc_); // Add to data structure holding all candidates size_t col = rff_ - rfi_ - 1; // -1 b/c prev col size_t frombot = dpRows() - row - 1; DpBtCandidate cand(row, col, sc); btdiag_.add(frombot + col, cand); } } } pvHLeft += ROWSTRIDE_2COL; pvScore += 2; } } // Find largest score in vmax vtmp = _mm_srli_si128(vmax, 8); vmax = _mm_max_epu8(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 4); vmax = _mm_max_epu8(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 2); vmax = _mm_max_epu8(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 1); vmax = _mm_max_epu8(vmax, vtmp); // Update metrics if(!debug) { size_t ninner = (rff_ - rfi_) * iter; met.col += (rff_ - rfi_); // DP columns met.cell += (ninner * NWORDS_PER_REG); // DP cells met.inner += ninner; // DP inner loop iters met.fixup += nfixup; // DP fixup loop iters } int score = _mm_extract_epi16(vmax, 0); score = score & 0x00ff; flag = 0; // Could we have saturated? if(score + d.bias_ >= 255) { flag = -2; // yes if(!debug) met.dpsat++; return MIN_I64; } // Did we find a solution? if(score == MIN_U8 || score < minsc_) { flag = -1; // no if(!debug) met.dpfail++; return (TAlScore)score; } // Now take all the backtrace candidates in the btdaig_ structure and // dump them into the btncand_ array. They'll be sorted later. if(!debug) { assert(!btdiag_.empty()); btdiag_.dump(btncand_); assert(!btncand_.empty()); } // Return largest score if(!debug) met.dpsucc++; return (TAlScore)score; } /** * Solve the current alignment problem using SSE instructions that operate on 16 * unsigned 8-bit values packed into a single 128-bit register. */ TAlScore SwAligner::alignNucleotidesLocalSseU8(int& flag, bool debug) { assert_leq(rdf_, rd_->length()); assert_leq(rdf_, qu_->length()); assert_lt(rfi_, rff_); assert_lt(rdi_, rdf_); assert_eq(rd_->length(), qu_->length()); assert_geq(sc_->gapbar, 1); assert(repOk()); #ifndef NDEBUG for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert_range(0, 16, (int)rf_[i]); } #endif SSEData& d = fw_ ? sseU8fw_ : sseU8rc_; SSEMetrics& met = extend_ ? sseU8ExtendMet_ : sseU8MateMet_; if(!debug) met.dp++; buildQueryProfileLocalSseU8(fw_); assert(!d.profbuf_.empty()); assert_geq(d.bias_, 0); assert_gt(d.maxBonus_, 0); size_t iter = (dpRows() + (NWORDS_PER_REG-1)) / NWORDS_PER_REG; // iter = segLen int dup; // Many thanks to Michael Farrar for releasing his striped Smith-Waterman // implementation: // // http://sites.google.com/site/farrarmichael/smith-waterman // // Much of the implmentation below is adapted from Michael's code. // Set all elts to reference gap open penalty __m128i rfgapo = _mm_setzero_si128(); __m128i rfgape = _mm_setzero_si128(); __m128i rdgapo = _mm_setzero_si128(); __m128i rdgape = _mm_setzero_si128(); __m128i vlo = _mm_setzero_si128(); __m128i vhi = _mm_setzero_si128(); __m128i vmax = _mm_setzero_si128(); __m128i vcolmax = _mm_setzero_si128(); __m128i vmaxtmp = _mm_setzero_si128(); __m128i ve = _mm_setzero_si128(); __m128i vf = _mm_setzero_si128(); __m128i vh = _mm_setzero_si128(); __m128i vtmp = _mm_setzero_si128(); __m128i vzero = _mm_setzero_si128(); __m128i vbias = _mm_setzero_si128(); assert_gt(sc_->refGapOpen(), 0); assert_leq(sc_->refGapOpen(), MAX_U8); dup = (sc_->refGapOpen() << 8) | (sc_->refGapOpen() & 0x00ff); rfgapo = _mm_insert_epi16(rfgapo, dup, 0); rfgapo = _mm_shufflelo_epi16(rfgapo, 0); rfgapo = _mm_shuffle_epi32(rfgapo, 0); // Set all elts to reference gap extension penalty assert_gt(sc_->refGapExtend(), 0); assert_leq(sc_->refGapExtend(), MAX_U8); assert_leq(sc_->refGapExtend(), sc_->refGapOpen()); dup = (sc_->refGapExtend() << 8) | (sc_->refGapExtend() & 0x00ff); rfgape = _mm_insert_epi16(rfgape, dup, 0); rfgape = _mm_shufflelo_epi16(rfgape, 0); rfgape = _mm_shuffle_epi32(rfgape, 0); // Set all elts to read gap open penalty assert_gt(sc_->readGapOpen(), 0); assert_leq(sc_->readGapOpen(), MAX_U8); dup = (sc_->readGapOpen() << 8) | (sc_->readGapOpen() & 0x00ff); rdgapo = _mm_insert_epi16(rdgapo, dup, 0); rdgapo = _mm_shufflelo_epi16(rdgapo, 0); rdgapo = _mm_shuffle_epi32(rdgapo, 0); // Set all elts to read gap extension penalty assert_gt(sc_->readGapExtend(), 0); assert_leq(sc_->readGapExtend(), MAX_U8); assert_leq(sc_->readGapExtend(), sc_->readGapOpen()); dup = (sc_->readGapExtend() << 8) | (sc_->readGapExtend() & 0x00ff); rdgape = _mm_insert_epi16(rdgape, dup, 0); rdgape = _mm_shufflelo_epi16(rdgape, 0); rdgape = _mm_shuffle_epi32(rdgape, 0); vhi = _mm_cmpeq_epi16(vhi, vhi); // all elts = 0xffff vlo = _mm_xor_si128(vlo, vlo); // all elts = 0 vmax = vlo; // Make a vector of bias offsets dup = (d.bias_ << 8) | (d.bias_ & 0x00ff); vbias = _mm_insert_epi16(vbias, dup, 0); vbias = _mm_shufflelo_epi16(vbias, 0); vbias = _mm_shuffle_epi32(vbias, 0); // Points to a long vector of __m128i where each element is a block of // contiguous cells in the E, F or H matrix. If the index % 3 == 0, then // the block of cells is from the E matrix. If index % 3 == 1, they're // from the F matrix. If index % 3 == 2, then they're from the H matrix. // Blocks of cells are organized in the same interleaved manner as they are // calculated by the Farrar algorithm. const __m128i *pvScore; // points into the query profile d.mat_.init(dpRows(), rff_ - rfi_, NWORDS_PER_REG); const size_t colstride = d.mat_.colstride(); //const size_t rowstride = d.mat_.rowstride(); assert_eq(ROWSTRIDE, colstride / iter); // Initialize the H and E vectors in the first matrix column __m128i *pvHTmp = d.mat_.tmpvec(0, 0); __m128i *pvETmp = d.mat_.evec(0, 0); for(size_t i = 0; i < iter; i++) { _mm_store_si128(pvETmp, vlo); _mm_store_si128(pvHTmp, vlo); // start low in local mode pvETmp += ROWSTRIDE; pvHTmp += ROWSTRIDE; } // These are swapped just before the innermost loop __m128i *pvHStore = d.mat_.hvec(0, 0); __m128i *pvHLoad = d.mat_.tmpvec(0, 0); __m128i *pvELoad = d.mat_.evec(0, 0); __m128i *pvEStore = d.mat_.evecUnsafe(0, 1); __m128i *pvFStore = d.mat_.fvec(0, 0); __m128i *pvFTmp = NULL; assert_gt(sc_->gapbar, 0); size_t nfixup = 0; TAlScore matchsc = sc_->match(30); // Fill in the table as usual but instead of using the same gap-penalty // vector for each iteration of the inner loop, load words out of a // pre-calculated gap vector parallel to the query profile. The pre- // calculated gap vectors enforce the gap barrier constraint by making it // infinitely costly to introduce a gap in barrier rows. // // AND use a separate loop to fill in the first row of the table, enforcing // the st_ constraints in the process. This is awkward because it // separates the processing of the first row from the others and might make // it difficult to use the first-row results in the next row, but it might // be the simplest and least disruptive way to deal with the st_ constraint. colstop_ = rff_ - rfi_; lastsolcol_ = 0; for(size_t i = (size_t)rfi_; i < (size_t)rff_; i++) { assert(pvFStore == d.mat_.fvec(0, i - rfi_)); assert(pvHStore == d.mat_.hvec(0, i - rfi_)); // Fetch this column's reference mask const int refm = (int)rf_[i]; // Fetch the appropriate query profile size_t off = (size_t)firsts5[refm] * iter * 2; pvScore = d.profbuf_.ptr() + off; // even elts = query profile, odd = gap barrier // Load H vector from the final row of the previous column vh = _mm_load_si128(pvHLoad + colstride - ROWSTRIDE); // Set all cells to low value vf = _mm_xor_si128(vf, vf); // Store cells in F, calculated previously // No need to veto ref gap extensions, they're all 0x00s _mm_store_si128(pvFStore, vf); pvFStore += ROWSTRIDE; // Shift down so that topmost (least sig) cell gets 0 vh = _mm_slli_si128(vh, NBYTES_PER_WORD); // We pull out one loop iteration to make it easier to veto values in the top row // Load cells from E, calculated previously ve = _mm_load_si128(pvELoad); assert_all_lt(ve, vhi); pvELoad += ROWSTRIDE; // Factor in query profile (matches and mismatches) vh = _mm_adds_epu8(vh, pvScore[0]); vh = _mm_subs_epu8(vh, vbias); // Update H, factoring in E and F vh = _mm_max_epu8(vh, ve); vh = _mm_max_epu8(vh, vf); // Update highest score so far vcolmax = _mm_xor_si128(vcolmax, vcolmax); vcolmax = _mm_max_epu8(vcolmax, vh); // Save the new vH values _mm_store_si128(pvHStore, vh); pvHStore += ROWSTRIDE; // Update vE value vf = vh; vh = _mm_subs_epu8(vh, rdgapo); vh = _mm_subs_epu8(vh, pvScore[1]); // veto some read gap opens ve = _mm_subs_epu8(ve, rdgape); ve = _mm_max_epu8(ve, vh); assert_all_lt(ve, vhi); // Load the next h value vh = _mm_load_si128(pvHLoad); pvHLoad += ROWSTRIDE; // Save E values _mm_store_si128(pvEStore, ve); pvEStore += ROWSTRIDE; // Update vf value vf = _mm_subs_epu8(vf, rfgapo); assert_all_lt(vf, vhi); pvScore += 2; // move on to next query profile // For each character in the reference text: size_t j; for(j = 1; j < iter; j++) { // Load cells from E, calculated previously ve = _mm_load_si128(pvELoad); assert_all_lt(ve, vhi); pvELoad += ROWSTRIDE; // Store cells in F, calculated previously vf = _mm_subs_epu8(vf, pvScore[1]); // veto some ref gap extensions _mm_store_si128(pvFStore, vf); pvFStore += ROWSTRIDE; // Factor in query profile (matches and mismatches) vh = _mm_adds_epu8(vh, pvScore[0]); vh = _mm_subs_epu8(vh, vbias); // Update H, factoring in E and F vh = _mm_max_epu8(vh, ve); vh = _mm_max_epu8(vh, vf); // Update highest score encountered this far vcolmax = _mm_max_epu8(vcolmax, vh); // Save the new vH values _mm_store_si128(pvHStore, vh); pvHStore += ROWSTRIDE; // Update vE value vtmp = vh; vh = _mm_subs_epu8(vh, rdgapo); vh = _mm_subs_epu8(vh, pvScore[1]); // veto some read gap opens ve = _mm_subs_epu8(ve, rdgape); ve = _mm_max_epu8(ve, vh); assert_all_lt(ve, vhi); // Load the next h value vh = _mm_load_si128(pvHLoad); pvHLoad += ROWSTRIDE; // Save E values _mm_store_si128(pvEStore, ve); pvEStore += ROWSTRIDE; // Update vf value vtmp = _mm_subs_epu8(vtmp, rfgapo); vf = _mm_subs_epu8(vf, rfgape); assert_all_lt(vf, vhi); vf = _mm_max_epu8(vf, vtmp); pvScore += 2; // move on to next query profile / gap veto } // pvHStore, pvELoad, pvEStore have all rolled over to the next column pvFTmp = pvFStore; pvFStore -= colstride; // reset to start of column vtmp = _mm_load_si128(pvFStore); pvHStore -= colstride; // reset to start of column vh = _mm_load_si128(pvHStore); pvEStore -= colstride; // reset to start of column ve = _mm_load_si128(pvEStore); pvHLoad = pvHStore; // new pvHLoad = pvHStore pvScore = d.profbuf_.ptr() + off + 1; // reset veto vector // vf from last row gets shifted down by one to overlay the first row // rfgape has already been subtracted from it. vf = _mm_slli_si128(vf, NBYTES_PER_WORD); vf = _mm_subs_epu8(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epu8(vtmp, vf); vtmp = _mm_subs_epu8(vf, vtmp); vtmp = _mm_cmpeq_epi8(vtmp, vzero); int cmp = _mm_movemask_epi8(vtmp); // If any element of vtmp is greater than H - gap-open... j = 0; while(cmp != 0xffff) { // Store this vf _mm_store_si128(pvFStore, vf); pvFStore += ROWSTRIDE; // Update vh w/r/t new vf vh = _mm_max_epu8(vh, vf); // Save vH values _mm_store_si128(pvHStore, vh); pvHStore += ROWSTRIDE; // Update highest score encountered this far vcolmax = _mm_max_epu8(vcolmax, vh); // Update E in case it can be improved using our new vh vh = _mm_subs_epu8(vh, rdgapo); vh = _mm_subs_epu8(vh, *pvScore); // veto some read gap opens ve = _mm_max_epu8(ve, vh); _mm_store_si128(pvEStore, ve); pvEStore += ROWSTRIDE; pvScore += 2; assert_lt(j, iter); if(++j == iter) { pvFStore -= colstride; vtmp = _mm_load_si128(pvFStore); // load next vf ASAP pvHStore -= colstride; vh = _mm_load_si128(pvHStore); // load next vh ASAP pvEStore -= colstride; ve = _mm_load_si128(pvEStore); // load next ve ASAP pvScore = d.profbuf_.ptr() + off + 1; j = 0; vf = _mm_slli_si128(vf, NBYTES_PER_WORD); } else { vtmp = _mm_load_si128(pvFStore); // load next vf ASAP vh = _mm_load_si128(pvHStore); // load next vh ASAP ve = _mm_load_si128(pvEStore); // load next vh ASAP } // Update F with another gap extension vf = _mm_subs_epu8(vf, rfgape); vf = _mm_subs_epu8(vf, *pvScore); // veto some ref gap extensions vf = _mm_max_epu8(vtmp, vf); vtmp = _mm_subs_epu8(vf, vtmp); vtmp = _mm_cmpeq_epi8(vtmp, vzero); cmp = _mm_movemask_epi8(vtmp); nfixup++; } #ifndef NDEBUG if((rand() & 15) == 0) { // This is a work-intensive sanity check; each time we finish filling // a column, we check that each H, E, and F is sensible. for(size_t k = 0; k < dpRows(); k++) { assert(cellOkLocalU8( d, k, // row i - rfi_, // col refm, // reference mask (int)(*rd_)[rdi_+k], // read char (int)(*qu_)[rdi_+k], // read quality *sc_)); // scoring scheme } } #endif // Store column maximum vector in first element of tmp vmax = _mm_max_epu8(vmax, vcolmax); _mm_store_si128(d.mat_.tmpvec(0, i - rfi_), vcolmax); { // Get single largest score in this column vmaxtmp = vcolmax; vtmp = _mm_srli_si128(vmaxtmp, 8); vmaxtmp = _mm_max_epu8(vmaxtmp, vtmp); vtmp = _mm_srli_si128(vmaxtmp, 4); vmaxtmp = _mm_max_epu8(vmaxtmp, vtmp); vtmp = _mm_srli_si128(vmaxtmp, 2); vmaxtmp = _mm_max_epu8(vmaxtmp, vtmp); vtmp = _mm_srli_si128(vmaxtmp, 1); vmaxtmp = _mm_max_epu8(vmaxtmp, vtmp); int score = _mm_extract_epi16(vmaxtmp, 0); score = score & 0x00ff; // Could we have saturated? if(score + d.bias_ >= 255) { flag = -2; // yes if(!debug) met.dpsat++; return MIN_I64; } if(score < minsc_) { size_t ncolleft = rff_ - i - 1; if(score + (TAlScore)ncolleft * matchsc < minsc_) { // Bail! We're guaranteed not to see a valid alignment in // the rest of the matrix colstop_ = (i+1) - rfi_; break; } } else { lastsolcol_ = i - rfi_; } } // pvELoad and pvHLoad are already where they need to be // Adjust the load and store vectors here. pvHStore = pvHLoad + colstride; pvEStore = pvELoad + colstride; pvFStore = pvFTmp; } // Find largest score in vmax vtmp = _mm_srli_si128(vmax, 8); vmax = _mm_max_epu8(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 4); vmax = _mm_max_epu8(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 2); vmax = _mm_max_epu8(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 1); vmax = _mm_max_epu8(vmax, vtmp); // Update metrics if(!debug) { size_t ninner = (rff_ - rfi_) * iter; met.col += (rff_ - rfi_); // DP columns met.cell += (ninner * NWORDS_PER_REG); // DP cells met.inner += ninner; // DP inner loop iters met.fixup += nfixup; // DP fixup loop iters } int score = _mm_extract_epi16(vmax, 0); score = score & 0x00ff; flag = 0; // Could we have saturated? if(score + d.bias_ >= 255) { flag = -2; // yes if(!debug) met.dpsat++; return MIN_I64; } // Did we find a solution? if(score == MIN_U8 || score < minsc_) { flag = -1; // no if(!debug) met.dpfail++; return (TAlScore)score; } // Return largest score if(!debug) met.dpsucc++; return (TAlScore)score; } /** * Given a filled-in DP table, populate the btncand_ list with candidate cells * that might be at the ends of valid alignments. No need to do this unless * the maximum score returned by the align*() func is >= the minimum. * * We needn't consider cells that have no chance of reaching any of the core * diagonals. These are the cells that are more than 'maxgaps' cells away from * a core diagonal. * * We need to be careful to consider that the rectangle might be truncated on * one or both ends. * * The seed extend case looks like this: * * |Rectangle| 0: seed diagonal * **OO0oo---- o: "RHS gap" diagonals * -**OO0oo--- O: "LHS gap" diagonals * --**OO0oo-- *: "LHS extra" diagonals * ---**OO0oo- -: cells that can't possibly be involved in a valid * ----**OO0oo alignment that overlaps one of the core diagonals * * The anchor-to-left case looks like this: * * |Anchor| | ---- Rectangle ---- | * o---------OO0000000000000oo------ 0: mate diagonal (also core diags!) * -o---------OO0000000000000oo----- o: "RHS gap" diagonals * --o---------OO0000000000000oo---- O: "LHS gap" diagonals * ---oo--------OO0000000000000oo--- *: "LHS extra" diagonals * -----o--------OO0000000000000oo-- -: cells that can't possibly be * ------o--------OO0000000000000oo- involved in a valid alignment that * -------o--------OO0000000000000oo overlaps one of the core diagonals * XXXXXXXXXXXXX * | RHS Range | * ^ ^ * rl rr * * The anchor-to-right case looks like this: * * ll lr * v v * | LHS Range | * XXXXXXXXXXXXX |Anchor| * OO0000000000000oo--------o-------- 0: mate diagonal (also core diags!) * -OO0000000000000oo--------o------- o: "RHS gap" diagonals * --OO0000000000000oo--------o------ O: "LHS gap" diagonals * ---OO0000000000000oo--------oo---- *: "LHS extra" diagonals * ----OO0000000000000oo---------o--- -: cells that can't possibly be * -----OO0000000000000oo---------o-- involved in a valid alignment that * ------OO0000000000000oo---------o- overlaps one of the core diagonals * | ---- Rectangle ---- | */ bool SwAligner::gatherCellsNucleotidesLocalSseU8(TAlScore best) { // What's the minimum number of rows that can possibly be spanned by an // alignment that meets the minimum score requirement? assert(sse8succ_); size_t bonus = (size_t)sc_->match(30); const size_t ncol = lastsolcol_ + 1; const size_t nrow = dpRows(); assert_gt(nrow, 0); btncand_.clear(); btncanddone_.clear(); SSEData& d = fw_ ? sseU8fw_ : sseU8rc_; SSEMetrics& met = extend_ ? sseU8ExtendMet_ : sseU8MateMet_; assert(!d.profbuf_.empty()); //const size_t rowstride = d.mat_.rowstride(); //const size_t colstride = d.mat_.colstride(); size_t iter = (dpRows() + (NWORDS_PER_REG - 1)) / NWORDS_PER_REG; assert_gt(iter, 0); assert_geq(minsc_, 0); assert_gt(bonus, 0); size_t minrow = (size_t)(((minsc_ + bonus - 1) / bonus) - 1); for(size_t j = 0; j < ncol; j++) { // Establish the range of rows where a backtrace from the cell in this // row/col is close enough to one of the core diagonals that it could // conceivably count size_t nrow_lo = MIN_SIZE_T; size_t nrow_hi = nrow; // First, check if there is a cell in this column with a score // above the score threshold __m128i vmax = *d.mat_.tmpvec(0, j); __m128i vtmp = _mm_srli_si128(vmax, 8); vmax = _mm_max_epu8(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 4); vmax = _mm_max_epu8(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 2); vmax = _mm_max_epu8(vmax, vtmp); vtmp = _mm_srli_si128(vmax, 1); vmax = _mm_max_epu8(vmax, vtmp); int score = _mm_extract_epi16(vmax, 0); score = score & 0x00ff; #ifndef NDEBUG { // Start in upper vector row and move down TAlScore max = 0; __m128i *pvH = d.mat_.hvec(0, j); for(size_t i = 0; i < iter; i++) { for(size_t k = 0; k < NWORDS_PER_REG; k++) { TAlScore sc = (TAlScore)((TCScore*)pvH)[k]; if(sc > max) { max = sc; } } pvH += ROWSTRIDE; } assert_eq(max, score); } #endif if((TAlScore)score < minsc_) { // Scores in column aren't good enough continue; } // Get pointer to first cell in column to examine: __m128i *pvHorig = d.mat_.hvec(0, j); __m128i *pvH = pvHorig; // Get pointer to the vector in the following column that corresponds // to the cells diagonally down and to the right from the cells in pvH __m128i *pvHSucc = (j < ncol-1) ? d.mat_.hvec(0, j+1) : NULL; // Start in upper vector row and move down for(size_t i = 0; i < iter; i++) { if(pvHSucc != NULL) { pvHSucc += ROWSTRIDE; if(i == iter-1) { pvHSucc = d.mat_.hvec(0, j+1); } } // Which elements of this vector are exhaustively scored? size_t rdoff = i; for(size_t k = 0; k < NWORDS_PER_REG; k++) { // Is this row, col one that we can potential backtrace from? // I.e. are we close enough to a core diagonal? if(rdoff >= nrow_lo && rdoff < nrow_hi) { // This cell has been exhaustively scored if(rdoff >= minrow) { // ... and it could potentially score high enough TAlScore sc = (TAlScore)((TCScore*)pvH)[k]; assert_leq(sc, best); if(sc >= minsc_) { // This is a potential solution bool matchSucc = false; int readc = (*rd_)[rdoff]; int refc = rf_[j + rfi_]; bool match = ((refc & (1 << readc)) != 0); if(rdoff < dpRows()-1) { int readcSucc = (*rd_)[rdoff+1]; int refcSucc = rf_[j + rfi_ + 1]; assert_range(0, 16, refcSucc); matchSucc = ((refcSucc & (1 << readcSucc)) != 0); } if(match && !matchSucc) { // Yes, this is legit met.gathsol++; btncand_.expand(); btncand_.back().init(rdoff, j, sc); } } } } else { // Already saw every element in the vector that's been // exhaustively scored break; } rdoff += iter; } pvH += ROWSTRIDE; } } if(!btncand_.empty()) { d.mat_.initMasks(); } return !btncand_.empty(); } #define MOVE_VEC_PTR_UP(vec, rowvec, rowelt) { \ if(rowvec == 0) { \ rowvec += d.mat_.nvecrow_; \ vec += d.mat_.colstride_; \ rowelt--; \ } \ rowvec--; \ vec -= ROWSTRIDE; \ } #define MOVE_VEC_PTR_LEFT(vec, rowvec, rowelt) { vec -= d.mat_.colstride_; } #define MOVE_VEC_PTR_UPLEFT(vec, rowvec, rowelt) { \ MOVE_VEC_PTR_UP(vec, rowvec, rowelt); \ MOVE_VEC_PTR_LEFT(vec, rowvec, rowelt); \ } #define MOVE_ALL_LEFT() { \ MOVE_VEC_PTR_LEFT(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_LEFT(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_LEFT(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_LEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define MOVE_ALL_UP() { \ MOVE_VEC_PTR_UP(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_UP(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_UP(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_UP(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define MOVE_ALL_UPLEFT() { \ MOVE_VEC_PTR_UPLEFT(cur_vec, rowvec, rowelt); \ MOVE_VEC_PTR_UPLEFT(left_vec, left_rowvec, left_rowelt); \ MOVE_VEC_PTR_UPLEFT(up_vec, up_rowvec, up_rowelt); \ MOVE_VEC_PTR_UPLEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } #define NEW_ROW_COL(row, col) { \ rowelt = row / d.mat_.nvecrow_; \ rowvec = row % d.mat_.nvecrow_; \ eltvec = (col * d.mat_.colstride_) + (rowvec * ROWSTRIDE); \ cur_vec = d.mat_.matbuf_.ptr() + eltvec; \ left_vec = cur_vec; \ left_rowelt = rowelt; \ left_rowvec = rowvec; \ MOVE_VEC_PTR_LEFT(left_vec, left_rowvec, left_rowelt); \ up_vec = cur_vec; \ up_rowelt = rowelt; \ up_rowvec = rowvec; \ MOVE_VEC_PTR_UP(up_vec, up_rowvec, up_rowelt); \ upleft_vec = up_vec; \ upleft_rowelt = up_rowelt; \ upleft_rowvec = up_rowvec; \ MOVE_VEC_PTR_LEFT(upleft_vec, upleft_rowvec, upleft_rowelt); \ } /** * Given the dynamic programming table and a cell, trace backwards from the * cell and install the edits and score/penalty in the appropriate fields * of SwResult res, which contains an AlnRes. The RandomSource is used to * break ties among equally good ways of tracing back. * * Upon entering a cell, we check if the read/ref coordinates of the cell * correspond to a cell we traversed constructing a previous alignment. If so, * we backtrack to the last decision point, mask out the path that led to the * previously observed cell, and continue along a different path; or, if there * are no more paths to try, we give up. * * An alignment found is subject to a filtering step designed to remove * alignments that could spuriously trump a better alignment falling partially * outside the rectangle. * * 1 * 67890123456 0: seed diagonal * **OO0oo---- o: right-hand "gap" diagonals: band of 'maxgap' diags * -**OO0oo--- O: left-hand "gap" diagonals: band of 'maxgap' diags * --**OO0oo-- *: "extra" diagonals: additional band of 'maxgap' diags * ---**OO0oo- +: cells not in any of the above * ----**OO0oo * |-| * Gotta touch one of these diags * * Basically, the filtering step removes alignments that do not at some point * touch a cell labeled '0' or 'O' in the diagram above. * */ bool SwAligner::backtraceNucleotidesLocalSseU8( TAlScore escore, // in: expected score SwResult& res, // out: store results (edits and scores) here size_t& off, // out: store diagonal projection of origin size_t& nbts, // out: # backtracks size_t row, // start in this row size_t col, // start in this column RandomSource& rnd) // random gen, to choose among equal paths { assert_lt(row, dpRows()); assert_lt(col, (size_t)(rff_ - rfi_)); SSEData& d = fw_ ? sseU8fw_ : sseU8rc_; SSEMetrics& met = extend_ ? sseU8ExtendMet_ : sseU8MateMet_; met.bt++; assert(!d.profbuf_.empty()); assert_lt(row, rd_->length()); btnstack_.clear(); // empty the backtrack stack btcells_.clear(); // empty the cells-so-far list AlnScore score; score.score_ = 0; score.gaps_ = score.ns_ = 0; size_t origCol = col; size_t gaps = 0, readGaps = 0, refGaps = 0; res.alres.reset(); EList& ned = res.alres.ned(); assert(ned.empty()); assert_gt(dpRows(), row); size_t trimEnd = dpRows() - row - 1; size_t trimBeg = 0; size_t ct = SSEMatrix::H; // cell type // Row and col in terms of where they fall in the SSE vector matrix size_t rowelt, rowvec, eltvec; size_t left_rowelt, up_rowelt, upleft_rowelt; size_t left_rowvec, up_rowvec, upleft_rowvec; __m128i *cur_vec, *left_vec, *up_vec, *upleft_vec; NEW_ROW_COL(row, col); while((int)row >= 0) { met.btcell++; nbts++; int readc = (*rd_)[rdi_ + row]; int refm = (int)rf_[rfi_ + col]; int readq = (*qu_)[row]; assert_leq(col, origCol); // Get score in this cell bool empty = false, reportedThru, canMoveThru, branch = false; int cur = SSEMatrix::H; if(!d.mat_.reset_[row]) { d.mat_.resetRow(row); } reportedThru = d.mat_.reportedThrough(row, col); canMoveThru = true; if(reportedThru) { canMoveThru = false; } else { empty = false; if(row > 0) { assert_gt(row, 0); size_t rowFromEnd = d.mat_.nrow() - row - 1; bool gapsAllowed = true; if(row < (size_t)sc_->gapbar || rowFromEnd < (size_t)sc_->gapbar) { gapsAllowed = false; } const int floorsc = 0; const int offsetsc = 0; // Move to beginning of column/row if(ct == SSEMatrix::E) { // AKA rdgap assert_gt(col, 0); TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::E))[rowelt] + offsetsc; assert(gapsAllowed); // Currently in the E matrix; incoming transition must come from the // left. It's either a gap open from the H matrix or a gap extend from // the E matrix. // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; // Get H score of cell to the left TAlScore sc_h_left = ((TCScore*)(left_vec + SSEMatrix::H))[left_rowelt] + offsetsc; if(sc_h_left > 0 && sc_h_left - sc_->readGapOpen() == sc_cur) { mask |= (1 << 0); } // Get E score of cell to the left TAlScore sc_e_left = ((TCScore*)(left_vec + SSEMatrix::E))[left_rowelt] + offsetsc; if(sc_e_left > 0 && sc_e_left - sc_->readGapExtend() == sc_cur) { mask |= (1 << 1); } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isEMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 8) & 3; } if(mask == 3) { #if 1 // Pick H -> E cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 2); // might choose E later #else if(rnd.nextU2()) { // Pick H -> E cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 2); // might choose E later } else { // Pick E -> E cell cur = SW_BT_RDGAP_EXTEND; d.mat_.eMaskSet(row, col, 1); // might choose H later } #endif branch = true; } else if(mask == 2) { // I chose the E cell cur = SW_BT_RDGAP_EXTEND; d.mat_.eMaskSet(row, col, 0); // done } else if(mask == 1) { // I chose the H cell cur = SW_BT_OALL_READ_OPEN; d.mat_.eMaskSet(row, col, 0); // done } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } assert(!empty || !canMoveThru); } else if(ct == SSEMatrix::F) { // AKA rfgap assert_gt(row, 0); assert(gapsAllowed); TAlScore sc_h_up = ((TCScore*)(up_vec + SSEMatrix::H))[up_rowelt] + offsetsc; TAlScore sc_f_up = ((TCScore*)(up_vec + SSEMatrix::F))[up_rowelt] + offsetsc; TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::F))[rowelt] + offsetsc; // Currently in the F matrix; incoming transition must come from above. // It's either a gap open from the H matrix or a gap extend from the F // matrix. // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; // Get H score of cell above if(sc_h_up > floorsc && sc_h_up - sc_->refGapOpen() == sc_cur) { mask |= (1 << 0); } // Get F score of cell above if(sc_f_up > floorsc && sc_f_up - sc_->refGapExtend() == sc_cur) { mask |= (1 << 1); } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isFMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 11) & 3; } if(mask == 3) { #if 1 // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 2); // might choose E later #else if(rnd.nextU2()) { // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 2); // might choose E later } else { // I chose the F cell cur = SW_BT_RFGAP_EXTEND; d.mat_.fMaskSet(row, col, 1); // might choose E later } #endif branch = true; } else if(mask == 2) { // I chose the F cell cur = SW_BT_RFGAP_EXTEND; d.mat_.fMaskSet(row, col, 0); // done } else if(mask == 1) { // I chose the H cell cur = SW_BT_OALL_REF_OPEN; d.mat_.fMaskSet(row, col, 0); // done } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } assert(!empty || !canMoveThru); } else { assert_eq(SSEMatrix::H, ct); TAlScore sc_cur = ((TCScore*)(cur_vec + SSEMatrix::H))[rowelt] + offsetsc; TAlScore sc_f_up = ((TCScore*)(up_vec + SSEMatrix::F))[up_rowelt] + offsetsc; TAlScore sc_h_up = ((TCScore*)(up_vec + SSEMatrix::H))[up_rowelt] + offsetsc; TAlScore sc_h_left = col > 0 ? (((TCScore*)(left_vec + SSEMatrix::H))[left_rowelt] + offsetsc) : floorsc; TAlScore sc_e_left = col > 0 ? (((TCScore*)(left_vec + SSEMatrix::E))[left_rowelt] + offsetsc) : floorsc; TAlScore sc_h_upleft = col > 0 ? (((TCScore*)(upleft_vec + SSEMatrix::H))[upleft_rowelt] + offsetsc) : floorsc; TAlScore sc_diag = sc_->score(readc, refm, readq - 33); // TODO: save and restore origMask as well as mask int origMask = 0, mask = 0; if(gapsAllowed) { if(sc_h_up > floorsc && sc_cur == sc_h_up - sc_->refGapOpen()) { mask |= (1 << 0); } if(sc_h_left > floorsc && sc_cur == sc_h_left - sc_->readGapOpen()) { mask |= (1 << 1); } if(sc_f_up > floorsc && sc_cur == sc_f_up - sc_->refGapExtend()) { mask |= (1 << 2); } if(sc_e_left > floorsc && sc_cur == sc_e_left - sc_->readGapExtend()) { mask |= (1 << 3); } } if(sc_h_upleft > floorsc && sc_cur == sc_h_upleft + sc_diag) { mask |= (1 << 4); } origMask = mask; assert(origMask > 0 || sc_cur <= sc_->match()); if(d.mat_.isHMaskSet(row, col)) { mask = (d.mat_.masks_[row][col] >> 2) & 31; } assert(gapsAllowed || mask == (1 << 4) || mask == 0); int opts = alts5[mask]; int select = -1; if(opts == 1) { select = firsts5[mask]; assert_geq(mask, 0); d.mat_.hMaskSet(row, col, 0); } else if(opts > 1) { #if 1 if( (mask & 16) != 0) { select = 4; // H diag } else if((mask & 1) != 0) { select = 0; // H up } else if((mask & 4) != 0) { select = 2; // F up } else if((mask & 2) != 0) { select = 1; // H left } else if((mask & 8) != 0) { select = 3; // E left } #else select = randFromMask(rnd, mask); #endif assert_geq(mask, 0); mask &= ~(1 << select); assert(gapsAllowed || mask == (1 << 4) || mask == 0); d.mat_.hMaskSet(row, col, mask); branch = true; } else { /* No way to backtrack! */ } if(select != -1) { if(select == 4) { cur = SW_BT_OALL_DIAG; } else if(select == 0) { cur = SW_BT_OALL_REF_OPEN; } else if(select == 1) { cur = SW_BT_OALL_READ_OPEN; } else if(select == 2) { cur = SW_BT_RFGAP_EXTEND; } else { assert_eq(3, select) cur = SW_BT_RDGAP_EXTEND; } } else { empty = true; // It's empty, so the only question left is whether we should be // allowed in terimnate in this cell. If it's got a valid score // then we *shouldn't* be allowed to terminate here because that // means it's part of a larger alignment that was already reported. canMoveThru = (origMask == 0); } } assert(!empty || !canMoveThru || ct == SSEMatrix::H); } } d.mat_.setReportedThrough(row, col); assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); // Cell was involved in a previously-reported alignment? if(!canMoveThru) { if(!btnstack_.empty()) { // Remove all the cells from list back to and including the // cell where the branch occurred btcells_.resize(btnstack_.back().celsz); // Pop record off the top of the stack ned.resize(btnstack_.back().nedsz); //aed.resize(btnstack_.back().aedsz); row = btnstack_.back().row; col = btnstack_.back().col; gaps = btnstack_.back().gaps; readGaps = btnstack_.back().readGaps; refGaps = btnstack_.back().refGaps; score = btnstack_.back().score; ct = btnstack_.back().ct; btnstack_.pop_back(); assert(!sc_->monotone || score.score() >= escore); NEW_ROW_COL(row, col); continue; } else { // No branch points to revisit; just give up res.reset(); met.btfail++; // DP backtraces failed return false; } } assert(!reportedThru); assert(!sc_->monotone || score.score() >= minsc_); if(empty || row == 0) { assert_eq(SSEMatrix::H, ct); btcells_.expand(); btcells_.back().first = row; btcells_.back().second = col; // This cell is at the end of a legitimate alignment trimBeg = row; assert_eq(btcells_.size(), dpRows() - trimBeg - trimEnd + readGaps); break; } if(branch) { // Add a frame to the backtrack stack btnstack_.expand(); btnstack_.back().init( ned.size(), 0, // aed.size() btcells_.size(), row, col, gaps, readGaps, refGaps, score, (int)ct); } btcells_.expand(); btcells_.back().first = row; btcells_.back().second = col; switch(cur) { // Move up and to the left. If the reference nucleotide in the // source row mismatches the read nucleotide, penalize // it and add a nucleotide mismatch. case SW_BT_OALL_DIAG: { assert_gt(row, 0); assert_gt(col, 0); // Check for color mismatch int readC = (*rd_)[row]; int refNmask = (int)rf_[rfi_+col]; assert_gt(refNmask, 0); int m = matchesEx(readC, refNmask); ct = SSEMatrix::H; if(m != 1) { Edit e( (int)row, mask2dna[refNmask], "ACGTN"[readC], EDIT_TYPE_MM); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); int pen = QUAL2(row, col); score.score_ -= pen; assert(!sc_->monotone || score.score() >= escore); } else { // Reward a match int64_t bonus = sc_->match(30); score.score_ += bonus; assert(!sc_->monotone || score.score() >= escore); } if(m == -1) { score.ns_++; } row--; col--; MOVE_ALL_UPLEFT(); assert(VALID_AL_SCORE(score)); break; } // Move up. Add an edit encoding the ref gap. case SW_BT_OALL_REF_OPEN: { assert_gt(row, 0); Edit e( (int)row, '-', "ACGTN"[(int)(*rd_)[row]], EDIT_TYPE_REF_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); row--; ct = SSEMatrix::H; int pen = sc_->refGapOpen(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; refGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_UP(); break; } // Move up. Add an edit encoding the ref gap. case SW_BT_RFGAP_EXTEND: { assert_gt(row, 1); Edit e( (int)row, '-', "ACGTN"[(int)(*rd_)[row]], EDIT_TYPE_REF_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); row--; ct = SSEMatrix::F; int pen = sc_->refGapExtend(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; refGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_UP(); break; } case SW_BT_OALL_READ_OPEN: { assert_gt(col, 0); Edit e( (int)row+1, mask2dna[(int)rf_[rfi_+col]], '-', EDIT_TYPE_READ_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); col--; ct = SSEMatrix::H; int pen = sc_->readGapOpen(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; readGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_LEFT(); break; } case SW_BT_RDGAP_EXTEND: { assert_gt(col, 1); Edit e( (int)row+1, mask2dna[(int)rf_[rfi_+col]], '-', EDIT_TYPE_READ_GAP); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); assert_geq(row, (size_t)sc_->gapbar); assert_geq((int)(rdf_-rdi_-row-1), sc_->gapbar-1); col--; ct = SSEMatrix::E; int pen = sc_->readGapExtend(); score.score_ -= pen; assert(!sc_->monotone || score.score() >= minsc_); gaps++; readGaps++; assert_eq(gaps, Edit::numGaps(ned)); assert_leq(gaps, rdgap_ + rfgap_); MOVE_ALL_LEFT(); break; } default: throw 1; } } // while((int)row > 0) assert_geq(col, 0); assert_eq(SSEMatrix::H, ct); // The number of cells in the backtracs should equal the number of read // bases after trimming plus the number of gaps assert_eq(btcells_.size(), dpRows() - trimBeg - trimEnd + readGaps); // Check whether we went through a core diagonal and set 'reported' flag on // each cell bool overlappedCoreDiag = false; for(size_t i = 0; i < btcells_.size(); i++) { size_t rw = btcells_[i].first; size_t cl = btcells_[i].second; // Calculate the diagonal within the *trimmed* rectangle, i.e. the // rectangle we dealt with in align, gather and backtrack. int64_t diagi = cl - rw; // Now adjust to the diagonal within the *untrimmed* rectangle by // adding on the amount trimmed from the left. diagi += rect_->triml; if(diagi >= 0) { size_t diag = (size_t)diagi; if(diag >= rect_->corel && diag <= rect_->corer) { overlappedCoreDiag = true; break; } } #ifndef NDEBUG //assert(!d.mat_.reportedThrough(rw, cl)); //d.mat_.setReportedThrough(rw, cl); assert(d.mat_.reportedThrough(rw, cl)); #endif } if(!overlappedCoreDiag) { // Must overlap a core diagonal. Otherwise, we run the risk of // reporting an alignment that overlaps (and trumps) a higher-scoring // alignment that lies partially outside the dynamic programming // rectangle. res.reset(); met.corerej++; return false; } int readC = (*rd_)[rdi_+row]; // get last char in read int refNmask = (int)rf_[rfi_+col]; // get last ref char ref involved in aln assert_gt(refNmask, 0); int m = matchesEx(readC, refNmask); if(m != 1) { Edit e((int)row, mask2dna[refNmask], "ACGTN"[readC], EDIT_TYPE_MM); assert(e.repOk()); assert(ned.empty() || ned.back().pos >= row); ned.push_back(e); score.score_ -= QUAL2(row, col); assert_geq(score.score(), minsc_); } else { score.score_ += sc_->match(30); } if(m == -1) { score.ns_++; } if(score.ns_ > nceil_) { // Alignment has too many Ns in it! res.reset(); met.nrej++; return false; } res.reverse(); assert(Edit::repOk(ned, (*rd_))); assert_eq(score.score(), escore); assert_leq(gaps, rdgap_ + rfgap_); off = col; assert_lt(col + (size_t)rfi_, (size_t)rff_); score.gaps_ = gaps; res.alres.setScore(score); res.alres.setShape( refidx_, // ref id off + rfi_ + rect_->refl, // 0-based ref offset reflen_, // reference length fw_, // aligned to Watson? rdf_ - rdi_, // read length 0, // read ID true, // pretrim soft? 0, // pretrim 5' end 0, // pretrim 3' end true, // alignment trim soft? fw_ ? trimBeg : trimEnd, // alignment trim 5' end fw_ ? trimEnd : trimBeg); // alignment trim 3' end size_t refns = 0; for(size_t i = col; i <= origCol; i++) { if((int)rf_[rfi_+i] > 15) { refns++; } } res.alres.setRefNs(refns); assert(Edit::repOk(ned, (*rd_), true, trimBeg, trimEnd)); assert(res.repOk()); #ifndef NDEBUG size_t gapsCheck = 0; for(size_t i = 0; i < ned.size(); i++) { if(ned[i].isGap()) gapsCheck++; } assert_eq(gaps, gapsCheck); BTDnaString refstr; for(size_t i = col; i <= origCol; i++) { refstr.append(firsts5[(int)rf_[rfi_+i]]); } BTDnaString editstr; Edit::toRef((*rd_), ned, editstr, true, trimBeg, trimEnd); if(refstr != editstr) { cerr << "Decoded nucleotides and edits don't match reference:" << endl; cerr << " score: " << score.score() << " (" << gaps << " gaps)" << endl; cerr << " edits: "; Edit::print(cerr, ned); cerr << endl; cerr << " decoded nucs: " << (*rd_) << endl; cerr << " edited nucs: " << editstr << endl; cerr << " reference nucs: " << refstr << endl; assert(0); } #endif met.btsucc++; // DP backtraces succeeded return true; } hisat2-2.2.1/aln_sink.cpp000066400000000000000000000625601370663763200152360ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include #include "aln_sink.h" #include "aligner_seed.h" #include "util.h" using namespace std; /** * Initialize state machine with a new read. The state we start in depends * on whether it's paired-end or unpaired. */ void ReportingState::nextRead(bool paired) { paired_ = paired; if(paired) { state_ = CONCORDANT_PAIRS; doneConcord_ = false; doneDiscord_ = p_.discord ? false : true; doneUnpair1_ = p_.mixed ? false : true; doneUnpair2_ = p_.mixed ? false : true; exitConcord_ = ReportingState::EXIT_DID_NOT_EXIT; exitDiscord_ = p_.discord ? ReportingState::EXIT_DID_NOT_EXIT : ReportingState::EXIT_DID_NOT_ENTER; exitUnpair1_ = p_.mixed ? ReportingState::EXIT_DID_NOT_EXIT : ReportingState::EXIT_DID_NOT_ENTER; exitUnpair2_ = p_.mixed ? ReportingState::EXIT_DID_NOT_EXIT : ReportingState::EXIT_DID_NOT_ENTER; } else { // Unpaired state_ = UNPAIRED; doneConcord_ = true; doneDiscord_ = true; doneUnpair1_ = false; doneUnpair2_ = true; exitConcord_ = ReportingState::EXIT_DID_NOT_ENTER; // not relevant exitDiscord_ = ReportingState::EXIT_DID_NOT_ENTER; // not relevant exitUnpair1_ = ReportingState::EXIT_DID_NOT_EXIT; exitUnpair2_ = ReportingState::EXIT_DID_NOT_ENTER; // not relevant } doneUnpair_ = doneUnpair1_ && doneUnpair2_; done_ = false; nconcord_ = ndiscord_ = nunpair1_ = nunpair2_ = 0; nunpairRepeat1_ = nunpairRepeat2_ = 0; concordBest_ = getMinScore(); } /** * Caller uses this member function to indicate that one additional * concordant alignment has been found. */ bool ReportingState::foundConcordant(TAlScore score) { assert(paired_); assert_geq(state_, ReportingState::CONCORDANT_PAIRS); assert(!doneConcord_); if(score > concordBest_) { concordBest_ = score; nconcord_ = 0; } nconcord_++; // DK CONCORDANT - debugging purpuses // areDone(nconcord_, doneConcord_, exitConcord_); // No need to search for discordant alignments if there are one or more // concordant alignments. doneDiscord_ = true; exitDiscord_ = ReportingState::EXIT_SHORT_CIRCUIT_TRUMPED; if(doneConcord_) { // If we're finished looking for concordant alignments, do we have to // continue on to search for unpaired alignments? Only if our exit // from the concordant stage is EXIT_SHORT_CIRCUIT_M. If it's // EXIT_SHORT_CIRCUIT_k or EXIT_WITH_ALIGNMENTS, we can skip unpaired. assert_neq(ReportingState::EXIT_NO_ALIGNMENTS, exitConcord_); if(exitConcord_ != ReportingState::EXIT_SHORT_CIRCUIT_M) { if(!doneUnpair1_) { doneUnpair1_ = true; exitUnpair1_ = ReportingState::EXIT_SHORT_CIRCUIT_TRUMPED; } if(!doneUnpair2_) { doneUnpair2_ = true; exitUnpair2_ = ReportingState::EXIT_SHORT_CIRCUIT_TRUMPED; } } } updateDone(); return done(); } /** * Caller uses this member function to indicate that one additional unpaired * mate alignment has been found for the specified mate. */ bool ReportingState::foundUnpaired(bool mate1, bool repeat) { assert_gt(state_, ReportingState::NO_READ); // Note: it's not right to assert !doneUnpair1_/!doneUnpair2_ here. // Even if we're done with finding if(mate1) { nunpair1_++; if(repeat) { nunpairRepeat1_++; } // Did we just finish with this mate? if(!doneUnpair1_) { areDone(nunpair1_, doneUnpair1_, exitUnpair1_); if(doneUnpair1_) { doneUnpair_ = doneUnpair1_ && doneUnpair2_; updateDone(); } } if(nunpair1_ > 1) { doneDiscord_ = true; exitDiscord_ = ReportingState::EXIT_NO_ALIGNMENTS; } } else { nunpair2_++; if(repeat) { nunpairRepeat2_++; } // Did we just finish with this mate? if(!doneUnpair2_) { areDone(nunpair2_, doneUnpair2_, exitUnpair2_); if(doneUnpair2_) { doneUnpair_ = doneUnpair1_ && doneUnpair2_; updateDone(); } } if(nunpair2_ > 1) { doneDiscord_ = true; exitDiscord_ = ReportingState::EXIT_NO_ALIGNMENTS; } } return done(); } /** * Called to indicate that the aligner has finished searching for * alignments. This gives us a chance to finalize our state. * * TODO: Keep track of short-circuiting information. */ void ReportingState::finish() { if(!doneConcord_) { doneConcord_ = true; exitConcord_ = ((nconcord_ > 0) ? ReportingState::EXIT_WITH_ALIGNMENTS : ReportingState::EXIT_NO_ALIGNMENTS); } assert_gt(exitConcord_, EXIT_DID_NOT_EXIT); if(!doneUnpair1_) { doneUnpair1_ = true; exitUnpair1_ = ((nunpair1_ > 0) ? ReportingState::EXIT_WITH_ALIGNMENTS : ReportingState::EXIT_NO_ALIGNMENTS); } assert_gt(exitUnpair1_, EXIT_DID_NOT_EXIT); if(!doneUnpair2_) { doneUnpair2_ = true; exitUnpair2_ = ((nunpair2_ > 0) ? ReportingState::EXIT_WITH_ALIGNMENTS : ReportingState::EXIT_NO_ALIGNMENTS); } assert_gt(exitUnpair2_, EXIT_DID_NOT_EXIT); if(!doneDiscord_) { // Check if the unpaired alignments should be converted to a single // discordant paired-end alignment. assert_eq(0, ndiscord_); if(nconcord_ == 0 && nunpair1_ == 1 && nunpair2_ == 1) { convertUnpairedToDiscordant(); } doneDiscord_ = true; exitDiscord_ = ((ndiscord_ > 0) ? ReportingState::EXIT_WITH_ALIGNMENTS : ReportingState::EXIT_NO_ALIGNMENTS); } assert(!paired_ || exitDiscord_ > ReportingState::EXIT_DID_NOT_EXIT); doneUnpair_ = done_ = true; assert(done()); } /** * Populate given counters with the number of various kinds of alignments * to report for this read. Concordant alignments are preferable to (and * mutually exclusive with) discordant alignments, and paired-end * alignments are preferable to unpaired alignments. * * The caller also needs some additional information for the case where a * pair or unpaired read aligns repetitively. If the read is paired-end * and the paired-end has repetitive concordant alignments, that should be * reported, and 'pairMax' is set to true to indicate this. If the read is * paired-end, does not have any conordant alignments, but does have * repetitive alignments for one or both mates, then that should be * reported, and 'unpair1Max' and 'unpair2Max' are set accordingly. * * Note that it's possible in the case of a paired-end read for the read to * have repetitive concordant alignments, but for one mate to have a unique * unpaired alignment. */ void ReportingState::getReport( uint64_t& nconcordAln, // # concordant alignments to report uint64_t& ndiscordAln, // # discordant alignments to report uint64_t& nunpair1Aln, // # unpaired alignments for mate #1 to report uint64_t& nunpair2Aln, // # unpaired alignments for mate #2 to report uint64_t& nunpairRepeat1Aln, // # unpaired alignments for mate #1 to report uint64_t& nunpairRepeat2Aln, // # unpaired alignments for mate #2 to report bool& pairMax, // repetitive concordant alignments bool& unpair1Max, // repetitive alignments for mate #1 bool& unpair2Max) // repetitive alignments for mate #2 const { nconcordAln = ndiscordAln = nunpair1Aln = nunpair2Aln = 0; nunpairRepeat1Aln = nunpairRepeat2Aln = 0; pairMax = unpair1Max = unpair2Max = false; assert_gt(p_.khits, 0); assert_gt(p_.mhits, 0); if(paired_) { // Do we have 1 or more concordant alignments to report? if(exitConcord_ == ReportingState::EXIT_SHORT_CIRCUIT_k) { // k at random assert_geq(nconcord_, (uint64_t)p_.khits); nconcordAln = p_.khits; return; } else if(exitConcord_ == ReportingState::EXIT_SHORT_CIRCUIT_M) { assert(p_.msample); assert_gt(nconcord_, 0); pairMax = true; // repetitive concordant alignments if(p_.mixed) { unpair1Max = nunpair1_ > (uint64_t)p_.mhits; unpair2Max = nunpair2_ > (uint64_t)p_.mhits; } // Not sure if this is OK nconcordAln = 1; // 1 at random return; } else if(exitConcord_ == ReportingState::EXIT_WITH_ALIGNMENTS) { assert_gt(nconcord_, 0); // <= k at random nconcordAln = min(p_.khits, nconcord_); } assert(!p_.mhitsSet() || nconcord_ <= (uint64_t)p_.mhits+1); // Do we have a discordant alignment to report? if(exitDiscord_ == ReportingState::EXIT_WITH_ALIGNMENTS) { // Report discordant assert(p_.discord); ndiscordAln = 1; return; } } assert_neq(ReportingState::EXIT_SHORT_CIRCUIT_TRUMPED, exitUnpair1_); assert_neq(ReportingState::EXIT_SHORT_CIRCUIT_TRUMPED, exitUnpair2_); if((paired_ && !p_.mixed) || nunpair1_ + nunpair2_ == 0) { // Unpaired alignments either not reportable or non-existant return; } // Do we have 1 or more alignments for mate #1 to report? if(exitUnpair1_ == ReportingState::EXIT_SHORT_CIRCUIT_k) { // k at random assert_geq(nunpair1_, (uint64_t)p_.khits); nunpair1Aln = p_.khits; } else if(exitUnpair1_ == ReportingState::EXIT_SHORT_CIRCUIT_M) { assert(p_.msample); assert_gt(nunpair1_, 0); unpair1Max = true; // repetitive alignments for mate #1 nunpair1Aln = 1; // 1 at random } else if(exitUnpair1_ == ReportingState::EXIT_WITH_ALIGNMENTS) { assert_gt(nunpair1_, 0); // <= k at random nunpair1Aln = min(nunpair1_, (uint64_t)p_.khits); } assert(!p_.mhitsSet() || paired_ || nunpair1_ <= (uint64_t)p_.mhits+1); if(p_.repeat) nunpairRepeat1Aln = nunpairRepeat1_; // Do we have 2 or more alignments for mate #2 to report? if(exitUnpair2_ == ReportingState::EXIT_SHORT_CIRCUIT_k) { // k at random nunpair2Aln = p_.khits; } else if(exitUnpair2_ == ReportingState::EXIT_SHORT_CIRCUIT_M) { assert(p_.msample); assert_gt(nunpair2_, 0); unpair2Max = true; // repetitive alignments for mate #1 nunpair2Aln = 1; // 1 at random } else if(exitUnpair2_ == ReportingState::EXIT_WITH_ALIGNMENTS) { assert_gt(nunpair2_, 0); // <= k at random nunpair2Aln = min(nunpair2_, (uint64_t)p_.khits); } assert(!p_.mhitsSet() || paired_ || nunpair2_ <= (uint64_t)p_.mhits+1); if(p_.repeat) nunpairRepeat2Aln = nunpairRepeat2_; } /** * Given the number of alignments in a category, check whether we * short-circuited out of the category. Set the done and exit arguments to * indicate whether and how we short-circuited. */ inline void ReportingState::areDone( uint64_t cnt, // # alignments in category bool& done, // out: whether we short-circuited out of category int& exit) const // out: if done, how we short-circuited (-k? -m? etc) { assert(!done); // Have we exceeded the -k limit? assert_gt(p_.khits, 0); assert_gt(p_.mhits, 0); if(cnt >= (uint64_t)p_.khits && !p_.mhitsSet()) { done = true; exit = ReportingState::EXIT_SHORT_CIRCUIT_k; } // Have we exceeded the -m or -M limit? else if(p_.mhitsSet() && cnt > (uint64_t)p_.mhits) { done = true; assert(p_.msample); exit = ReportingState::EXIT_SHORT_CIRCUIT_M; } } #ifdef ALN_SINK_MAIN #include bool testDones( const ReportingState& st, bool done1, bool done2, bool done3, bool done4, bool done5, bool done6) { assert(st.doneConcordant() == done1); assert(st.doneDiscordant() == done2); assert(st.doneUnpaired(true) == done3); assert(st.doneUnpaired(false) == done4); assert(st.doneUnpaired() == done5); assert(st.done() == done6); assert(st.repOk()); return true; } int main(void) { cerr << "Case 1 (simple unpaired 1) ... "; { uint64_t nconcord = 0, ndiscord = 0, nunpair1 = 0, nunpair2 = 0; bool pairMax = false, unpair1Max = false, unpair2Max = false; ReportingParams rp( 2, // khits 0, // mhits 0, // pengap false, // msample false, // discord false); // mixed ReportingState st(rp); st.nextRead(false); // unpaired read assert(testDones(st, true, true, false, true, false, false)); st.foundUnpaired(true); assert(testDones(st, true, true, false, true, false, false)); st.foundUnpaired(true); assert(testDones(st, true, true, true, true, true, true)); st.finish(); assert(testDones(st, true, true, true, true, true, true)); assert_eq(0, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(2, st.numUnpaired1()); assert_eq(0, st.numUnpaired2()); assert(st.repOk()); st.getReport(nconcord, ndiscord, nunpair1, nunpair2, pairMax, unpair1Max, unpair2Max); assert_eq(0, nconcord); assert_eq(0, ndiscord); assert_eq(2, nunpair1); assert_eq(0, nunpair2); assert(!pairMax); assert(!unpair1Max); assert(!unpair2Max); } cerr << "PASSED" << endl; cerr << "Case 2 (simple unpaired 1) ... "; { uint64_t nconcord = 0, ndiscord = 0, nunpair1 = 0, nunpair2 = 0; bool pairMax = false, unpair1Max = false, unpair2Max = false; ReportingParams rp( 2, // khits 3, // mhits 0, // pengap false, // msample false, // discord false); // mixed ReportingState st(rp); st.nextRead(false); // unpaired read assert(testDones(st, true, true, false, true, false, false)); st.foundUnpaired(true); assert(testDones(st, true, true, false, true, false, false)); st.foundUnpaired(true); assert(testDones(st, true, true, false, true, false, false)); st.foundUnpaired(true); assert(testDones(st, true, true, false, true, false, false)); st.foundUnpaired(true); assert(testDones(st, true, true, true, true, true, true)); assert_eq(0, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(4, st.numUnpaired1()); assert_eq(0, st.numUnpaired2()); st.finish(); assert(testDones(st, true, true, true, true, true, true)); assert_eq(0, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(4, st.numUnpaired1()); assert_eq(0, st.numUnpaired2()); assert(st.repOk()); st.getReport(nconcord, ndiscord, nunpair1, nunpair2, pairMax, unpair1Max, unpair2Max); assert_eq(0, nconcord); assert_eq(0, ndiscord); assert_eq(0, nunpair1); assert_eq(0, nunpair2); assert(!pairMax); assert(unpair1Max); assert(!unpair2Max); } cerr << "PASSED" << endl; cerr << "Case 3 (simple paired 1) ... "; { uint64_t nconcord = 0, ndiscord = 0, nunpair1 = 0, nunpair2 = 0; bool pairMax = false, unpair1Max = false, unpair2Max = false; ReportingParams rp( 2, // khits 3, // mhits 0, // pengap false, // msample false, // discord false); // mixed ReportingState st(rp); st.nextRead(true); // unpaired read assert(testDones(st, false, true, true, true, true, false)); st.foundUnpaired(true); assert(testDones(st, false, true, true, true, true, false)); st.foundUnpaired(true); assert(testDones(st, false, true, true, true, true, false)); st.foundUnpaired(true); assert(testDones(st, false, true, true, true, true, false)); st.foundUnpaired(true); assert(testDones(st, false, true, true, true, true, false)); st.foundUnpaired(false); assert(testDones(st, false, true, true, true, true, false)); st.foundUnpaired(false); assert(testDones(st, false, true, true, true, true, false)); st.foundUnpaired(false); assert(testDones(st, false, true, true, true, true, false)); st.foundUnpaired(false); assert(testDones(st, false, true, true, true, true, false)); st.foundConcordant(); assert(testDones(st, false, true, true, true, true, false)); st.foundConcordant(); assert(testDones(st, false, true, true, true, true, false)); st.foundConcordant(); assert(testDones(st, false, true, true, true, true, false)); st.foundConcordant(); assert(testDones(st, true, true, true, true, true, true)); assert_eq(4, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(4, st.numUnpaired1()); assert_eq(4, st.numUnpaired2()); st.finish(); assert(testDones(st, true, true, true, true, true, true)); assert_eq(4, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(4, st.numUnpaired1()); assert_eq(4, st.numUnpaired2()); assert(st.repOk()); st.getReport(nconcord, ndiscord, nunpair1, nunpair2, pairMax, unpair1Max, unpair2Max); assert_eq(0, nconcord); assert_eq(0, ndiscord); assert_eq(0, nunpair1); assert_eq(0, nunpair2); assert(pairMax); assert(!unpair1Max); // because !mixed assert(!unpair2Max); // because !mixed } cerr << "PASSED" << endl; cerr << "Case 4 (simple paired 2) ... "; { uint64_t nconcord = 0, ndiscord = 0, nunpair1 = 0, nunpair2 = 0; bool pairMax = false, unpair1Max = false, unpair2Max = false; ReportingParams rp( 2, // khits 3, // mhits 0, // pengap false, // msample true, // discord true); // mixed ReportingState st(rp); st.nextRead(true); // unpaired read assert(testDones(st, false, false, false, false, false, false)); st.foundUnpaired(true); assert(testDones(st, false, false, false, false, false, false)); st.foundUnpaired(true); assert(testDones(st, false, true, false, false, false, false)); st.foundUnpaired(true); assert(testDones(st, false, true, false, false, false, false)); st.foundUnpaired(true); assert(testDones(st, false, true, true, false, false, false)); st.foundUnpaired(false); assert(testDones(st, false, true, true, false, false, false)); st.foundUnpaired(false); assert(testDones(st, false, true, true, false, false, false)); st.foundUnpaired(false); assert(testDones(st, false, true, true, false, false, false)); st.foundUnpaired(false); assert(testDones(st, false, true, true, true, true, false)); st.foundConcordant(); assert(testDones(st, false, true, true, true, true, false)); st.foundConcordant(); assert(testDones(st, false, true, true, true, true, false)); st.foundConcordant(); assert(testDones(st, false, true, true, true, true, false)); st.foundConcordant(); assert(testDones(st, true, true, true, true, true, true)); assert_eq(4, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(4, st.numUnpaired1()); assert_eq(4, st.numUnpaired2()); st.finish(); assert(testDones(st, true, true, true, true, true, true)); assert_eq(4, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(4, st.numUnpaired1()); assert_eq(4, st.numUnpaired2()); assert(st.repOk()); st.getReport(nconcord, ndiscord, nunpair1, nunpair2, pairMax, unpair1Max, unpair2Max); assert_eq(0, nconcord); assert_eq(0, ndiscord); assert_eq(0, nunpair1); assert_eq(0, nunpair2); assert(pairMax); assert(unpair1Max); assert(unpair2Max); } cerr << "PASSED" << endl; cerr << "Case 5 (potential discordant after concordant) ... "; { uint64_t nconcord = 0, ndiscord = 0, nunpair1 = 0, nunpair2 = 0; bool pairMax = false, unpair1Max = false, unpair2Max = false; ReportingParams rp( 2, // khits 3, // mhits 0, // pengap false, // msample true, // discord true); // mixed ReportingState st(rp); st.nextRead(true); assert(testDones(st, false, false, false, false, false, false)); st.foundUnpaired(true); st.foundUnpaired(false); st.foundConcordant(); assert(testDones(st, false, true, false, false, false, false)); st.finish(); assert(testDones(st, true, true, true, true, true, true)); assert_eq(1, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(1, st.numUnpaired1()); assert_eq(1, st.numUnpaired2()); assert(st.repOk()); st.getReport(nconcord, ndiscord, nunpair1, nunpair2, pairMax, unpair1Max, unpair2Max); assert_eq(1, nconcord); assert_eq(0, ndiscord); assert_eq(0, nunpair1); assert_eq(0, nunpair2); assert(!pairMax); assert(!unpair1Max); assert(!unpair2Max); } cerr << "PASSED" << endl; cerr << "Case 6 (true discordant) ... "; { uint64_t nconcord = 0, ndiscord = 0, nunpair1 = 0, nunpair2 = 0; bool pairMax = false, unpair1Max = false, unpair2Max = false; ReportingParams rp( 2, // khits 3, // mhits 0, // pengap false, // msample true, // discord true); // mixed ReportingState st(rp); st.nextRead(true); assert(testDones(st, false, false, false, false, false, false)); st.foundUnpaired(true); st.foundUnpaired(false); assert(testDones(st, false, false, false, false, false, false)); st.finish(); assert(testDones(st, true, true, true, true, true, true)); assert_eq(0, st.numConcordant()); assert_eq(1, st.numDiscordant()); assert_eq(0, st.numUnpaired1()); assert_eq(0, st.numUnpaired2()); assert(st.repOk()); st.getReport(nconcord, ndiscord, nunpair1, nunpair2, pairMax, unpair1Max, unpair2Max); assert_eq(0, nconcord); assert_eq(1, ndiscord); assert_eq(0, nunpair1); assert_eq(0, nunpair2); assert(!pairMax); assert(!unpair1Max); assert(!unpair2Max); } cerr << "PASSED" << endl; cerr << "Case 7 (unaligned pair & uniquely aligned mate, mixed-mode) ... "; { uint64_t nconcord = 0, ndiscord = 0, nunpair1 = 0, nunpair2 = 0; bool pairMax = false, unpair1Max = false, unpair2Max = false; ReportingParams rp( 1, // khits 1, // mhits 0, // pengap false, // msample true, // discord true); // mixed ReportingState st(rp); st.nextRead(true); // unpaired read // assert(st.doneConcordant() == done1); // assert(st.doneDiscordant() == done2); // assert(st.doneUnpaired(true) == done3); // assert(st.doneUnpaired(false) == done4); // assert(st.doneUnpaired() == done5); // assert(st.done() == done6); st.foundUnpaired(true); assert(testDones(st, false, false, false, false, false, false)); st.foundUnpaired(true); assert(testDones(st, false, true, true, false, false, false)); assert_eq(0, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(2, st.numUnpaired1()); assert_eq(0, st.numUnpaired2()); st.finish(); st.getReport(nconcord, ndiscord, nunpair1, nunpair2, pairMax, unpair1Max, unpair2Max); assert_eq(0, nconcord); assert_eq(0, ndiscord); assert_eq(0, nunpair1); assert_eq(0, nunpair2); assert(!pairMax); assert(unpair1Max); assert(!unpair2Max); } cerr << "PASSED" << endl; cerr << "Case 8 (unaligned pair & uniquely aligned mate, NOT mixed-mode) ... "; { uint64_t nconcord = 0, ndiscord = 0, nunpair1 = 0, nunpair2 = 0; bool pairMax = false, unpair1Max = false, unpair2Max = false; ReportingParams rp( 1, // khits 1, // mhits 0, // pengap false, // msample true, // discord false); // mixed ReportingState st(rp); st.nextRead(true); // unpaired read // assert(st.doneConcordant() == done1); // assert(st.doneDiscordant() == done2); // assert(st.doneUnpaired(true) == done3); // assert(st.doneUnpaired(false) == done4); // assert(st.doneUnpaired() == done5); // assert(st.done() == done6); st.foundUnpaired(true); assert(testDones(st, false, false, true, true, true, false)); st.foundUnpaired(true); assert(testDones(st, false, true, true, true, true, false)); assert_eq(0, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(2, st.numUnpaired1()); assert_eq(0, st.numUnpaired2()); st.finish(); st.getReport(nconcord, ndiscord, nunpair1, nunpair2, pairMax, unpair1Max, unpair2Max); assert_eq(0, nconcord); assert_eq(0, ndiscord); assert_eq(0, nunpair1); assert_eq(0, nunpair2); assert(!pairMax); assert(!unpair1Max); // not really relevant assert(!unpair2Max); // not really relevant } cerr << "PASSED" << endl; cerr << "Case 9 (repetitive pair, only one mate repetitive) ... "; { uint64_t nconcord = 0, ndiscord = 0, nunpair1 = 0, nunpair2 = 0; bool pairMax = false, unpair1Max = false, unpair2Max = false; ReportingParams rp( 1, // khits 1, // mhits 0, // pengap true, // msample true, // discord true); // mixed ReportingState st(rp); st.nextRead(true); // unpaired read // assert(st.doneConcordant() == done1); // assert(st.doneDiscordant() == done2); // assert(st.doneUnpaired(true) == done3); // assert(st.doneUnpaired(false) == done4); // assert(st.doneUnpaired() == done5); // assert(st.done() == done6); st.foundConcordant(); assert(st.repOk()); st.foundUnpaired(true); assert(st.repOk()); st.foundUnpaired(false); assert(st.repOk()); assert(testDones(st, false, true, false, false, false, false)); assert(st.repOk()); st.foundConcordant(); assert(st.repOk()); st.foundUnpaired(true); assert(st.repOk()); assert(testDones(st, true, true, true, false, false, false)); assert_eq(2, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(2, st.numUnpaired1()); assert_eq(1, st.numUnpaired2()); st.foundUnpaired(false); assert(st.repOk()); assert(testDones(st, true, true, true, true, true, true)); assert_eq(2, st.numConcordant()); assert_eq(0, st.numDiscordant()); assert_eq(2, st.numUnpaired1()); assert_eq(2, st.numUnpaired2()); st.finish(); st.getReport(nconcord, ndiscord, nunpair1, nunpair2, pairMax, unpair1Max, unpair2Max); assert_eq(1, nconcord); assert_eq(0, ndiscord); assert_eq(0, nunpair1); assert_eq(0, nunpair2); assert(pairMax); assert(unpair1Max); // not really relevant assert(unpair2Max); // not really relevant } cerr << "PASSED" << endl; } #endif /*def ALN_SINK_MAIN*/ hisat2-2.2.1/aln_sink.h000066400000000000000000003240531370663763200147010ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALN_SINK_H_ #define ALN_SINK_H_ #include #include "read.h" #include "unique.h" #include "sam.h" #include "ds.h" #include "simple_func.h" #include "outq.h" #include #include "alt.h" #include "splice_site.h" static const TAlScore getMinScore() { return std::numeric_limits::min() / 2; } // Forward decl template class SeedResults; enum { OUTPUT_SAM = 1 }; /** * Metrics summarizing the work done by the reporter and summarizing * the number of reads that align, that fail to align, and that align * non-uniquely. */ struct ReportingMetrics { ReportingMetrics():mutex_m() { reset(); } void reset() { init(0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0); } void init( uint64_t nread_, uint64_t npaired_, uint64_t nunpaired_, uint64_t nconcord_uni_, uint64_t nconcord_uni1_, uint64_t nconcord_uni2_, uint64_t nconcord_rep_, uint64_t nconcord_0_, uint64_t ndiscord_, uint64_t nunp_0_uni_, uint64_t nunp_0_uni1_, uint64_t nunp_0_uni2_, uint64_t nunp_0_rep_, uint64_t nunp_0_0_, uint64_t nunp_rep_uni_, uint64_t nunp_rep_uni1_, uint64_t nunp_rep_uni2_, uint64_t nunp_rep_rep_, uint64_t nunp_rep_0_, uint64_t nunp_uni_, uint64_t nunp_uni1_, uint64_t nunp_uni2_, uint64_t nunp_rep_, uint64_t nunp_0_, uint64_t sum_best1_, uint64_t sum_best2_, uint64_t sum_best_) { nread = nread_; npaired = npaired_; nunpaired = nunpaired_; nconcord_uni = nconcord_uni_; nconcord_uni1 = nconcord_uni1_; nconcord_uni2 = nconcord_uni2_; nconcord_rep = nconcord_rep_; nconcord_0 = nconcord_0_; ndiscord = ndiscord_; nunp_0_uni = nunp_0_uni_; nunp_0_uni1 = nunp_0_uni1_; nunp_0_uni2 = nunp_0_uni2_; nunp_0_rep = nunp_0_rep_; nunp_0_0 = nunp_0_0_; nunp_rep_uni = nunp_rep_uni_; nunp_rep_uni1 = nunp_rep_uni1_; nunp_rep_uni2 = nunp_rep_uni2_; nunp_rep_rep = nunp_rep_rep_; nunp_rep_0 = nunp_rep_0_; nunp_uni = nunp_uni_; nunp_uni1 = nunp_uni1_; nunp_uni2 = nunp_uni2_; nunp_rep = nunp_rep_; nunp_0 = nunp_0_; sum_best1 = sum_best1_; sum_best2 = sum_best2_; sum_best = sum_best_; } /** * Merge (add) the counters in the given ReportingMetrics object * into this object. This is the only safe way to update a * ReportingMetrics shared by multiple threads. */ void merge(const ReportingMetrics& met, bool getLock = false) { ThreadSafe ts(&mutex_m, getLock); nread += met.nread; npaired += met.npaired; nunpaired += met.nunpaired; nconcord_uni += met.nconcord_uni; nconcord_uni1 += met.nconcord_uni1; nconcord_uni2 += met.nconcord_uni2; nconcord_rep += met.nconcord_rep; nconcord_0 += met.nconcord_0; ndiscord += met.ndiscord; nunp_0_uni += met.nunp_0_uni; nunp_0_uni1 += met.nunp_0_uni1; nunp_0_uni2 += met.nunp_0_uni2; nunp_0_rep += met.nunp_0_rep; nunp_0_0 += met.nunp_0_0; nunp_rep_uni += met.nunp_rep_uni; nunp_rep_uni1 += met.nunp_rep_uni1; nunp_rep_uni2 += met.nunp_rep_uni2; nunp_rep_rep += met.nunp_rep_rep; nunp_rep_0 += met.nunp_rep_0; nunp_uni += met.nunp_uni; nunp_uni1 += met.nunp_uni1; nunp_uni2 += met.nunp_uni2; nunp_rep += met.nunp_rep; nunp_0 += met.nunp_0; sum_best1 += met.sum_best1; sum_best2 += met.sum_best2; sum_best += met.sum_best; } uint64_t nread; // # reads uint64_t npaired; // # pairs uint64_t nunpaired; // # unpaired reads // Paired // Concordant uint64_t nconcord_uni; // # pairs with unique concordant alns uint64_t nconcord_uni1; // # pairs with exactly 1 concordant alns uint64_t nconcord_uni2; // # pairs with >1 concordant aln, still unique uint64_t nconcord_rep; // # pairs with repetitive concordant alns uint64_t nconcord_0; // # pairs with 0 concordant alns // Discordant uint64_t ndiscord; // # pairs with 1 discordant aln // Unpaired from failed pairs uint64_t nunp_0_uni; // # unique from nconcord_0_ - ndiscord_ uint64_t nunp_0_uni1; // # pairs with exactly 1 concordant alns uint64_t nunp_0_uni2; // # pairs with >1 concordant aln, still unique uint64_t nunp_0_rep; // # repetitive from uint64_t nunp_0_0; // # with 0 alignments // Unpaired from repetitive pairs uint64_t nunp_rep_uni; // # pairs with unique concordant alns uint64_t nunp_rep_uni1; // # pairs with exactly 1 concordant alns uint64_t nunp_rep_uni2; // # pairs with >1 concordant aln, still unique uint64_t nunp_rep_rep; // # pairs with repetitive concordant alns uint64_t nunp_rep_0; // # pairs with 0 concordant alns // Unpaired uint64_t nunp_uni; // # unique from nconcord_0_ - ndiscord_ uint64_t nunp_uni1; // # pairs with exactly 1 concordant alns uint64_t nunp_uni2; // # pairs with >1 concordant aln, still unique uint64_t nunp_rep; // # repetitive from uint64_t nunp_0; // # with 0 alignments uint64_t sum_best1; // Sum of all the best alignment scores uint64_t sum_best2; // Sum of all the second-best alignment scores uint64_t sum_best; // Sum of all the best and second-best MUTEX_T mutex_m; }; // Type for expression numbers of hits typedef int64_t THitInt; /** * Parameters affecting reporting of alignments, specifically -k & -a, * -m & -M. */ struct ReportingParams { explicit ReportingParams( THitInt khits_, THitInt kseeds_, THitInt mhits_, THitInt pengap_, bool msample_, bool discord_, bool mixed_, bool secondary_, bool localAlign_, int bowtie2_dp_, bool sensitive_, bool repeat_) { init( khits_, kseeds_, mhits_, pengap_, msample_, discord_, mixed_, secondary_, localAlign_, bowtie2_dp_, sensitive_, repeat_); } void init( THitInt khits_, THitInt kseeds_, THitInt mhits_, THitInt pengap_, bool msample_, bool discord_, bool mixed_, bool secondary_, bool localAlign_, int bowtie2_dp_, bool sensitive_, bool repeat_) { khits = khits_; // -k (or high if -a) kseeds = kseeds_; mhits = ((mhits_ == 0) ? std::numeric_limits::max() : mhits_); pengap = pengap_; msample = msample_; discord = discord_; mixed = mixed_; secondary = secondary_; localAlign = localAlign_; bowtie2_dp = bowtie2_dp_; sensitive = sensitive_; repeat = repeat_; } #ifndef NDEBUG /** * Check that reporting parameters are internally consistent. */ bool repOk() const { assert_geq(khits, 1); assert_geq(mhits, 1); return true; } #endif /** * Return true iff a -m or -M limit was set by the user. */ inline bool mhitsSet() const { return mhits < std::numeric_limits::max(); } /** * Return a multiplier that indicates how many alignments we might look for * (max). We can use this to boost parameters like ROWM and POSF * appropriately. */ inline THitInt mult() const { if(mhitsSet()) { return mhits+1; } return khits; } /** * Given ROWM, POSF thresholds, boost them according to mult(). */ void boostThreshold(SimpleFunc& func) { THitInt mul = mult(); assert_gt(mul, 0); if(mul == std::numeric_limits::max()) { func.setMin(std::numeric_limits::max()); } else if(mul > 1) { func.mult(mul); } } /** * Return true iff we are reporting all hits. */ bool allHits() const { return khits == std::numeric_limits::max(); } // Number of alignments to report THitInt khits; // Number of seeds allowed to extend THitInt kseeds; // Read is non-unique if mhits-1 next-best alignments are within // pengap of the best alignment THitInt mhits, pengap; // true if -M is specified, meaning that if the -M ceiling is // exceeded, we should report 'khits' alignments chosen at random // from those found bool msample; // true iff we should seek and report discordant paired-end alignments for // paired-end reads. bool discord; // true iff we should seek and report unpaired mate alignments when there // are paired-end alignments for a paired-end read, or if the number of // paired-end alignments exceeds the -m ceiling. bool mixed; // true iff we allow secondary alignments to be output (secondary alignments // have lower scores) bool secondary; // true iff we allow local alignment (not implemented yet) bool localAlign; // true iff we allow dynamic alignment int bowtie2_dp; // true iff we allow sensitive alignment bool sensitive; // true iff we output alignments to repeat sequences bool repeat; }; /** * A state machine keeping track of the number and type of alignments found so * far. Its purpose is to inform the caller as to what stage the alignment is * in and what categories of alignment are still of interest. This information * should allow the caller to short-circuit some alignment work. Another * purpose is to tell the AlnSinkWrap how many and what type of alignment to * report. * * TODO: This class does not keep accurate information about what * short-circuiting took place. If a read is identical to a previous read, * there should be a way to query this object to determine what work, if any, * has to be re-done for the new read. */ class ReportingState { public: enum { NO_READ = 1, // haven't got a read yet CONCORDANT_PAIRS, // looking for concordant pairs DISCORDANT_PAIRS, // looking for discordant pairs UNPAIRED, // looking for unpaired DONE // finished looking }; // Flags for different ways we can finish out a category of potential // alignments. enum { EXIT_DID_NOT_EXIT = 1, // haven't finished EXIT_DID_NOT_ENTER, // never tried search EXIT_SHORT_CIRCUIT_k, // -k exceeded EXIT_SHORT_CIRCUIT_M, // -M exceeded EXIT_SHORT_CIRCUIT_TRUMPED, // made irrelevant EXIT_CONVERTED_TO_DISCORDANT, // unpair became discord EXIT_NO_ALIGNMENTS, // none found EXIT_WITH_ALIGNMENTS // some found }; ReportingState(const ReportingParams& p) : p_(p) { reset(); } /** * Set all state to uninitialized defaults. */ void reset() { state_ = ReportingState::NO_READ; paired_ = false; nconcord_ = 0; ndiscord_ = 0; nunpair1_ = 0; nunpair2_ = 0; nunpairRepeat1_ = 0; nunpairRepeat2_ = 0; doneConcord_ = false; doneDiscord_ = false; doneUnpair_ = false; doneUnpair1_ = false; doneUnpair2_ = false; exitConcord_ = ReportingState::EXIT_DID_NOT_ENTER; exitDiscord_ = ReportingState::EXIT_DID_NOT_ENTER; exitUnpair1_ = ReportingState::EXIT_DID_NOT_ENTER; exitUnpair2_ = ReportingState::EXIT_DID_NOT_ENTER; concordBest_ = getMinScore(); done_ = false; } /** * Return true iff this ReportingState has been initialized with a call to * nextRead() since the last time reset() was called. */ bool inited() const { return state_ != ReportingState::NO_READ; } /** * Initialize state machine with a new read. The state we start in depends * on whether it's paired-end or unpaired. */ void nextRead(bool paired); /** * Caller uses this member function to indicate that one additional * concordant alignment has been found. */ bool foundConcordant(TAlScore score); /** * Caller uses this member function to indicate that one additional * discordant alignment has been found. */ bool foundUnpaired(bool mate1, bool repeat = false); /** * Called to indicate that the aligner has finished searching for * alignments. This gives us a chance to finalize our state. * * TODO: Keep track of short-circuiting information. */ void finish(); /** * Populate given counters with the number of various kinds of alignments * to report for this read. Concordant alignments are preferable to (and * mutually exclusive with) discordant alignments, and paired-end * alignments are preferable to unpaired alignments. * * The caller also needs some additional information for the case where a * pair or unpaired read aligns repetitively. If the read is paired-end * and the paired-end has repetitive concordant alignments, that should be * reported, and 'pairMax' is set to true to indicate this. If the read is * paired-end, does not have any conordant alignments, but does have * repetitive alignments for one or both mates, then that should be * reported, and 'unpair1Max' and 'unpair2Max' are set accordingly. * * Note that it's possible in the case of a paired-end read for the read to * have repetitive concordant alignments, but for one mate to have a unique * unpaired alignment. */ void getReport( uint64_t& nconcordAln, // # concordant alignments to report uint64_t& ndiscordAln, // # discordant alignments to report uint64_t& nunpair1Aln, // # unpaired alignments for mate #1 to report uint64_t& nunpair2Aln, // # unpaired alignments for mate #2 to report uint64_t& nunpairRepeat1Aln, // # unpaired alignments for mate #1 to report uint64_t& nunpairRepeat2Aln, // # unpaired alignments for mate #2 to report bool& pairMax, // repetitive concordant alignments bool& unpair1Max, // repetitive alignments for mate #1 bool& unpair2Max) // repetitive alignments for mate #2 const; /** * Return an integer representing the alignment state we're in. */ inline int state() const { return state_; } /** * If false, there's no need to solve any more dynamic programming problems * for finding opposite mates. */ inline bool doneConcordant() const { return doneConcord_; } /** * If false, there's no need to seek any more discordant alignment. */ inline bool doneDiscordant() const { return doneDiscord_; } /** * If false, there's no need to seek any more unpaired alignments for the * specified mate. Note: this doesn't necessarily mean we can stop looking * for alignments for the mate, since this might be necessary for finding * concordant and discordant alignments. */ inline bool doneUnpaired(bool mate1) const { return mate1 ? doneUnpair1_ : doneUnpair2_; } /** * If false, no further consideration of the given mate is necessary. It's * not needed for *any* class of alignment: concordant, discordant or * unpaired. */ inline bool doneWithMate(bool mate1) const { bool doneUnpair = mate1 ? doneUnpair1_ : doneUnpair2_; uint64_t nun = mate1 ? nunpair1_ : nunpair2_; if(!doneUnpair || !doneConcord_) { return false; // still needed for future concordant/unpaired alns } if(!doneDiscord_ && nun == 0) { return false; // still needed for future discordant alignments } return true; // done } /** * Return true iff there's no need to seek any more unpaired alignments. */ inline bool doneUnpaired() const { return doneUnpair_; } /** * Return true iff all alignment stages have been exited. */ inline bool done() const { return done_; } inline uint64_t numConcordant() const { return nconcord_; } inline uint64_t numDiscordant() const { return ndiscord_; } inline uint64_t numUnpaired1() const { return nunpair1_; } inline uint64_t numUnpaired2() const { return nunpair2_; } inline uint64_t numUnpairedRepeat1() const { return nunpairRepeat1_; } inline uint64_t numUnpairedRepeat2() const { return nunpairRepeat2_; } inline int exitConcordant() const { return exitConcord_; } inline int exitDiscordant() const { return exitDiscord_; } inline int exitUnpaired1() const { return exitUnpair1_; } inline int exitUnpaired2() const { return exitUnpair2_; } inline int64_t concordBest() const { return concordBest_; } #ifndef NDEBUG /** * Check that ReportingState is internally consistent. */ bool repOk() const { assert(p_.discord || doneDiscord_); assert(p_.mixed || !paired_ || doneUnpair_); assert(doneUnpair_ || !doneUnpair1_ || !doneUnpair2_); if(p_.mhitsSet()) { assert_leq(numConcordant(), (uint64_t)p_.mhits+1); assert_leq(numDiscordant(), (uint64_t)p_.mhits+1); assert(paired_ || numUnpaired1() <= (uint64_t)p_.mhits+1); assert(paired_ || numUnpaired2() <= (uint64_t)p_.mhits+1); } assert(done() || !doneWithMate(true) || !doneWithMate(false)); return true; } #endif /** * Return ReportingParams object governing this ReportingState. */ const ReportingParams& params() const { return p_; } protected: /** * Update state to reflect situation after converting two unique unpaired * alignments, one for mate 1 and one for mate 2, into a single discordant * alignment. */ void convertUnpairedToDiscordant() { assert_eq(1, numUnpaired1()); assert_eq(1, numUnpaired2()); assert_eq(0, numDiscordant()); exitUnpair1_ = exitUnpair2_ = ReportingState::EXIT_CONVERTED_TO_DISCORDANT; nunpair1_ = nunpair2_ = 0; nunpairRepeat1_ = nunpairRepeat2_ = 0; ndiscord_ = 1; assert_eq(1, numDiscordant()); } /** * Given the number of alignments in a category, check whether we * short-circuited out of the category. Set the done and exit arguments to * indicate whether and how we short-circuited. */ inline void areDone( uint64_t cnt, // # alignments in category bool& done, // out: whether we short-circuited out of category int& exit) const; // out: if done, how we short-circuited (-k? -m? etc) /** * Update done_ field to reflect whether we're totally done now. */ inline void updateDone() { doneUnpair_ = doneUnpair1_ && doneUnpair2_; done_ = doneUnpair_ && doneDiscord_ && doneConcord_; } const ReportingParams& p_; // reporting parameters int state_; // state we're currently in bool paired_; // true iff read we're currently handling is paired uint64_t nconcord_; // # concordants found so far uint64_t ndiscord_; // # discordants found so far uint64_t nunpair1_; // # unpaired alignments found so far for mate 1 uint64_t nunpair2_; // # unpaired alignments found so far for mate 2 uint64_t nunpairRepeat1_; // # unpaired repeat alignments found so far for mate 1 uint64_t nunpairRepeat2_; // # unpaired repeat alignments found so far for mate 2 bool doneConcord_; // true iff we're no longner interested in concordants bool doneDiscord_; // true iff we're no longner interested in discordants bool doneUnpair_; // no longner interested in unpaired alns bool doneUnpair1_; // no longner interested in unpaired alns for mate 1 bool doneUnpair2_; // no longner interested in unpaired alns for mate 2 int exitConcord_; // flag indicating how we exited concordant state int exitDiscord_; // flag indicating how we exited discordant state int exitUnpair1_; // flag indicating how we exited unpaired 1 state int exitUnpair2_; // flag indicating how we exited unpaired 2 state TAlScore concordBest_; // bool done_; // done with all alignments }; /** * Global hit sink for hits from the MultiSeed aligner. Encapsulates * all aspects of the MultiSeed aligner hitsink that are global to all * threads. This includes aspects relating to: * * (a) synchronized access to the output stream * (b) the policy to be enforced by the per-thread wrapper * * TODO: Implement splitting up of alignments into separate files * according to genomic coordinate. */ template class AlnSink { typedef EList StrList; public: explicit AlnSink( OutputQueue& oq, const StrList& refnames, const StrList& repnames, bool quiet, ALTDB* altdb = NULL, SpliceSiteDB* ssdb = NULL) : oq_(oq), refnames_(refnames), repnames_(repnames), quiet_(quiet), altdb_(altdb), spliceSiteDB_(ssdb) { } /** * Destroy HitSinkobject; */ virtual ~AlnSink() { } /** * Called when the AlnSink is wrapped by a new AlnSinkWrap. This helps us * keep track of whether the main lock or any of the per-stream locks will * be contended by multiple threads. */ void addWrapper() { numWrappers_++; } /** * Append a single hit to the given output stream. If * synchronization is required, append() assumes the caller has * already grabbed the appropriate lock. */ virtual void append( BTString& o, StackedAln& staln, size_t threadId, const Read *rd1, const Read *rd2, const TReadId rdid, AlnRes *rs1, AlnRes *rs2, const AlnSetSumm& summ, const SeedAlSumm& ssm1, const SeedAlSumm& ssm2, const AlnFlags* flags1, const AlnFlags* flags2, const PerReadMetrics& prm, const Mapq& mapq, const Scoring& sc, bool report2) = 0; /** * Report a given batch of hits for the given read or read pair. * Should be called just once per read pair. Assumes all the * alignments are paired, split between rs1 and rs2. * * The caller hasn't decided which alignments get reported as primary * or secondary; that's up to the routine. Because the caller might * want to know this, we use the pri1 and pri2 out arguments to * convey this. */ virtual void reportHits( BTString& o, // write to this buffer StackedAln& staln, // StackedAln to write stacked alignment size_t threadId, // which thread am I? const Read *rd1, // mate #1 const Read *rd2, // mate #2 const TReadId rdid, // read ID const EList& select1, // random subset of rd1s const EList* select2, // random subset of rd2s EList *rs1, // alignments for mate #1 EList *rs2, // alignments for mate #2 bool maxed, // true iff -m/-M exceeded const AlnSetSumm& summ, // summary const SeedAlSumm& ssm1, // seed alignment summ const SeedAlSumm& ssm2, // seed alignment summ const AlnFlags* flags1, // flags for mate #1 const AlnFlags* flags2, // flags for mate #2 const PerReadMetrics& prm, // per-read metrics const Mapq& mapq, // MAPQ generator const Scoring& sc, // scoring scheme bool getLock = true) // true iff lock held by caller { // There are a few scenarios: // 1. Read is unpaired, in which case rd2 is NULL // 2. Read is paired-end and we're reporting concordant alignments // 3. Read is paired-end and we're reporting discordant alignments // 4. Read is paired-end and we're reporting unpaired alignments for // both mates // 5. Read is paired-end and we're reporting an unpaired alignments for // just one mate or the other assert(rd1 != NULL || rd2 != NULL); assert(rs1 != NULL || rs2 != NULL); AlnFlags flagscp1, flagscp2; if(flags1 != NULL) { flagscp1 = *flags1; flags1 = &flagscp1; flagscp1.setPrimary(true); } if(flags2 != NULL) { flagscp2 = *flags2; flags2 = &flagscp2; flagscp2.setPrimary(true); } if(select2 != NULL) { // Handle case 5 assert(rd1 != NULL); assert(flags1 != NULL); assert(rd2 != NULL); assert(flags2 != NULL); assert_gt(select1.size(), 0); assert_gt(select2->size(), 0); AlnRes* r1pri = ((rs1 != NULL) ? &rs1->get(select1[0]) : NULL); AlnRes* r2pri = ((rs2 != NULL) ? &rs2->get((*select2)[0]) : NULL); append(o, staln, threadId, rd1, rd2, rdid, r1pri, r2pri, summ, ssm1, ssm2, flags1, flags2, prm, mapq, sc, true); flagscp1.setPrimary(false); flagscp2.setPrimary(false); for(size_t i = 1; i < select1.size(); i++) { AlnRes* r1 = ((rs1 != NULL) ? &rs1->get(select1[i]) : NULL); append(o, staln, threadId, rd1, rd2, rdid, r1, r2pri, summ, ssm1, ssm2, flags1, flags2, prm, mapq, sc, false); } for(size_t i = 1; i < select2->size(); i++) { AlnRes* r2 = ((rs2 != NULL) ? &rs2->get((*select2)[i]) : NULL); append(o, staln, threadId, rd2, rd1, rdid, r2, r1pri, summ, ssm2, ssm1, flags2, flags1, prm, mapq, sc, false); } } else { // Handle cases 1-4 for(size_t i = 0; i < select1.size(); i++) { AlnRes* r1 = ((rs1 != NULL) ? &rs1->get(select1[i]) : NULL); AlnRes* r2 = ((rs2 != NULL) ? &rs2->get(select1[i]) : NULL); append(o, staln, threadId, rd1, rd2, rdid, r1, r2, summ, ssm1, ssm2, flags1, flags2, prm, mapq, sc, true); if(flags1 != NULL) { flagscp1.setPrimary(false); } if(flags2 != NULL) { flagscp2.setPrimary(false); } } } } /** * Report an unaligned read. Typically we do nothing, but we might * want to print a placeholder when output is chained. */ virtual void reportUnaligned( BTString& o, // write to this string StackedAln& staln, // StackedAln to write stacked alignment size_t threadId, // which thread am I? const Read *rd1, // mate #1 const Read *rd2, // mate #2 const TReadId rdid, // read ID const AlnSetSumm& summ, // summary const SeedAlSumm& ssm1, // seed alignment summary const SeedAlSumm& ssm2, // seed alignment summary const AlnFlags* flags1, // flags for mate #1 const AlnFlags* flags2, // flags for mate #2 const PerReadMetrics& prm, // per-read metrics const Mapq& mapq, // MAPQ calculator const Scoring& sc, // scoring scheme bool report2, // report alns for both mates? bool getLock = true) // true iff lock held by caller { append(o, staln, threadId, rd1, rd2, rdid, NULL, NULL, summ, ssm1, ssm2, flags1, flags2, prm, mapq, sc, report2); } /** * Print summary of how many reads aligned, failed to align and aligned * repetitively. Write it to stderr. Optionally write Hadoop counter * updates. */ void printAlSumm( ostream& out, const ReportingMetrics& met, size_t repThresh, // threshold for uniqueness, or max if no thresh bool discord, // looked for discordant alignments bool mixed, // looked for unpaired alignments where paired failed? bool newSummary, // alignment summary in a new style bool hadoopOut); // output Hadoop counters? /** * Called when all alignments are complete. It is assumed that no * synchronization is necessary. */ void finish( ostream& out, size_t repThresh, bool discord, bool mixed, bool newSummary, bool hadoopOut) { // Close output streams if(!quiet_) { printAlSumm( out, met_, repThresh, discord, mixed, newSummary, hadoopOut); } } #ifndef NDEBUG /** * Check that hit sink is internally consistent. */ bool repOk() const { return true; } #endif // // Related to reporting seed hits // /** * Given a Read and associated, filled-in SeedResults objects, * print a record summarizing the seed hits. */ void reportSeedSummary( BTString& o, const Read& rd, TReadId rdid, size_t threadId, const SeedResults& rs, bool getLock = true); /** * Given a Read, print an empty record (all 0s). */ void reportEmptySeedSummary( BTString& o, const Read& rd, TReadId rdid, size_t threadId, bool getLock = true); /** * Append a batch of unresolved seed alignment results (i.e. seed * alignments where all we know is the reference sequence aligned * to and its SA range, not where it falls in the reference * sequence) to the given output stream in Bowtie's seed-alignment * verbose-mode format. */ virtual void appendSeedSummary( BTString& o, const Read& rd, const TReadId rdid, size_t seedsTried, size_t nonzero, size_t ranges, size_t elts, size_t seedsTriedFw, size_t nonzeroFw, size_t rangesFw, size_t eltsFw, size_t seedsTriedRc, size_t nonzeroRc, size_t rangesRc, size_t eltsRc); /** * Merge given metrics in with ours by summing all individual metrics. */ void mergeMetrics(const ReportingMetrics& met, bool getLock = true) { met_.merge(met, getLock); } /** * Return mutable reference to the shared OutputQueue. */ OutputQueue& outq() { return oq_; } protected: OutputQueue& oq_; // output queue int numWrappers_; // # threads owning a wrapper for this HitSink const StrList& refnames_; // reference names const StrList& repnames_; // repeat names bool quiet_; // true -> don't print alignment stats at the end ReportingMetrics met_; // global repository of reporting metrics ALTDB* altdb_; SpliceSiteDB* spliceSiteDB_; // }; /** * Per-thread hit sink "wrapper" for the MultiSeed aligner. Encapsulates * aspects of the MultiSeed aligner hit sink that are per-thread. This * includes aspects relating to: * * (a) Enforcement of the reporting policy * (b) Tallying of results * (c) Storing of results for the previous read in case this allows us to * short-circuit some work for the next read (i.e. if it's identical) * * PHASED ALIGNMENT ASSUMPTION * * We make some assumptions about how alignment proceeds when we try to * short-circuit work for identical reads. Specifically, we assume that for * each read the aligner proceeds in a series of stages (or perhaps just one * stage). In each stage, the aligner either: * * (a) Finds no alignments, or * (b) Finds some alignments and short circuits out of the stage with some * random reporting involved (e.g. in -k and/or -M modes), or * (c) Finds all of the alignments in the stage * * In the event of (a), the aligner proceeds to the next stage and keeps * trying; we can skip the stage entirely for the next read if it's identical. * In the event of (b), or (c), the aligner stops and does not proceed to * further stages. In the event of (b1), if the next read is identical we * would like to tell the aligner to start again at the beginning of the stage * that was short-circuited. * * In any event, the rs1_/rs2_/rs1u_/rs2u_ fields contain the alignments found * in the last alignment stage attempted. * * HANDLING REPORTING LIMITS * * The user can specify reporting limits, like -k (specifies number of * alignments to report out of those found) and -M (specifies a ceiling s.t. if * there are more alignments than the ceiling, read is called repetitive and * best found is reported). Enforcing these limits is straightforward for * unpaired alignments: if a new alignment causes us to exceed the -M ceiling, * we can stop looking. * * The case where both paired-end and unpaired alignments are possible is * trickier. Once we have a number of unpaired alignments that exceeds the * ceiling, we can stop looking *for unpaired alignments* - but we can't * necessarily stop looking for paired-end alignments, since there may yet be * more to find. However, if the input read is not a pair, then we can stop at * this point. If the input read is a pair and we have a number of paired * aligments that exceeds the -M ceiling, we can stop looking. * * CONCORDANT & DISCORDANT, PAIRED & UNPAIRED * * A note on paired-end alignment: Clearly, if an input read is * paired-end and we find either concordant or discordant paired-end * alignments for the read, then we would like to tally and report * those alignments as such (and not as groups of 2 unpaired * alignments). And if we fail to find any paired-end alignments, but * we do find some unpaired alignments for one mate or the other, then * we should clearly tally and report those alignments as unpaired * alignments (if the user so desires). * * The situation is murkier when there are no paired-end alignments, * but there are unpaired alignments for *both* mates. In this case, * we might want to pick out zero or more pairs of mates and classify * those pairs as discordant paired-end alignments. And we might want * to classify the remaining alignments as unpaired. But how do we * pick which pairs if any to call discordant? * * Because the most obvious use for discordant pairs is for identifying * large-scale variation, like rearrangements or large indels, we would * usually like to be conservative about what we call a discordant * alignment. If there's a good chance that one or the other of the * two mates has a good alignment to another place on the genome, this * compromises the evidence for the large-scale variant. For this * reason, Bowtie 2's policy is: if there are no paired-end alignments * and there is *exactly one alignment each* for both mates, then the * two alignments are paired and treated as a discordant paired-end * alignment. Otherwise, all alignments are treated as unpaired * alignments. * * When both paired and unpaired alignments are discovered by the * aligner, only the paired alignments are reported by default. This * is sensible considering relative likelihoods: if a good paired-end * alignment is found, it is much more likely that the placement of * the two mates implied by that paired alignment is correct than any * placement implied by an unpaired alignment. * * */ template class AlnSinkWrap { public: AlnSinkWrap( AlnSink& g, // AlnSink being wrapped const ReportingParams& rp, // Parameters governing reporting Mapq& mapq, // Mapq calculator size_t threadId, // Thread ID bool secondary = false, // Secondary alignments const SpliceSiteDB* ssdb = NULL, // splice sites uint64_t threads_rids_mindist = 0) : // synchronization g_(g), rp_(rp), threadid_(threadId), mapq_(mapq), secondary_(secondary), ssdb_(ssdb), threads_rids_mindist_(threads_rids_mindist), init_(false), maxed1_(false), // read is pair and we maxed out mate 1 unp alns maxed2_(false), // read is pair and we maxed out mate 2 unp alns maxedOverall_(false), // alignments found so far exceed -m/-M ceiling bestPair_(getMinScore()), best2Pair_(getMinScore()), bestUnp1_(getMinScore()), best2Unp1_(getMinScore()), bestUnp2_(getMinScore()), best2Unp2_(getMinScore()), bestUnpRepeat1_(getMinScore()), best2UnpRepeat1_(getMinScore()), bestUnpRepeat2_(getMinScore()), best2UnpRepeat2_(getMinScore()), bestSplicedPair_(0), best2SplicedPair_(0), bestSplicedUnp1_(0), best2SplicedUnp1_(0), bestSplicedUnp2_(0), best2SplicedUnp2_(0), rd1_(NULL), // mate 1 rd2_(NULL), // mate 2 rdid_(std::numeric_limits::max()), // read id rs1_(), // mate 1 alignments for paired-end alignments rs2_(), // mate 2 alignments for paired-end alignments rs1u_(), // mate 1 unpaired alignments rs2u_(), // mate 2 unpaired alignments select1_(), // for selecting random subsets for mate 1 select2_(), // for selecting random subsets for mate 2 st_(rp) // reporting state - what's left to do? { assert(rp_.repOk()); } /** * Initialize the wrapper with a new read pair and return an * integer >= -1 indicating which stage the aligner should start * at. If -1 is returned, the aligner can skip the read entirely. * at. If . Checks if the new read pair is identical to the * previous pair. If it is, then we return the id of the first * stage to run. */ int nextRead( // One of the other of rd1, rd2 will = NULL if read is unpaired const Read* rd1, // new mate #1 const Read* rd2, // new mate #2 TReadId rdid, // read ID for new pair bool qualitiesMatter);// aln policy distinguishes b/t quals? /** * Inform global, shared AlnSink object that we're finished with * this read. The global AlnSink is responsible for updating * counters, creating the output record, and delivering the record * to the appropriate output stream. */ void finishRead( const SeedResults *sr1, // seed alignment results for mate 1 const SeedResults *sr2, // seed alignment results for mate 2 bool exhaust1, // mate 1 exhausted? bool exhaust2, // mate 2 exhausted? bool nfilt1, // mate 1 N-filtered? bool nfilt2, // mate 2 N-filtered? bool scfilt1, // mate 1 score-filtered? bool scfilt2, // mate 2 score-filtered? bool lenfilt1, // mate 1 length-filtered? bool lenfilt2, // mate 2 length-filtered? bool qcfilt1, // mate 1 qc-filtered? bool qcfilt2, // mate 2 qc-filtered? bool sortByScore, // prioritize alignments by score RandomSource& rnd, // pseudo-random generator ReportingMetrics& met, // reporting metrics const PerReadMetrics& prm, // per-read metrics const Scoring& sc, // scoring scheme bool suppressSeedSummary = true, bool suppressAlignments = false, bool templateLenAdjustment = true); /** * Called by the aligner when a new unpaired or paired alignment is * discovered in the given stage. This function checks whether the * addition of this alignment causes the reporting policy to be * violated (by meeting or exceeding the limits set by -k, -m, -M), * in which case true is returned immediately and the aligner is * short circuited. Otherwise, the alignment is tallied and false * is returned. */ bool report( int stage, const AlnRes* rs1, const AlnRes* rs2, bool alignMate = false); #ifndef NDEBUG /** * Check that hit sink wrapper is internally consistent. */ bool repOk() const { assert_eq(rs2_.size(), rs1_.size()); if(rp_.mhitsSet()) { assert_gt(rp_.mhits, 0); assert_leq((int)rs1_.size(), rp_.mhits+1); assert_leq((int)rs2_.size(), rp_.mhits+1); assert(readIsPair() || (int)rs1u_.size() <= rp_.mhits+1); assert(readIsPair() || (int)rs2u_.size() <= rp_.mhits+1); } if(init_) { assert(rd1_ != NULL); assert_neq(std::numeric_limits::max(), rdid_); } //assert_eq(st_.numConcordant() + st_.numDiscordant(), rs1_.size()); //assert_eq(st_.numUnpaired1(), rs1u_.size()); //assert_eq(st_.numUnpaired2(), rs2u_.size()); assert(st_.repOk()); return true; } #endif /** * Return true iff no alignments have been reported to this wrapper * since the last call to nextRead(). */ bool empty() const { return rs1_.empty() && rs1u_.empty() && rs2u_.empty(); } /** * Return true iff we have already encountered a number of alignments that * exceeds the -m/-M ceiling. TODO: how does this distinguish between * pairs and mates? */ bool maxed() const { return maxedOverall_; } /** * Return true if the current read is paired. */ bool readIsPair() const { return rd1_ != NULL && rd2_ != NULL; } /** * Return true iff nextRead() has been called since the last time * finishRead() was called. */ bool inited() const { return init_; } /** * Return a const ref to the ReportingState object associated with the * AlnSinkWrap. */ const ReportingState& state() const { return st_; } const ReportingParams& reportingParams() { return rp_;} /** * Return true iff we're in -M mode. */ bool Mmode() const { return rp_.mhitsSet(); } /** * Return true iff the policy is to report all hits. */ bool allHits() const { return rp_.allHits(); } /** * Return true iff at least two alignments have been reported so far for an * unpaired read or mate 1. */ bool hasSecondBestUnp1() const { return best2Unp1_ != getMinScore(); } /** * Return true iff at least two alignments have been reported so far for * mate 2. */ bool hasSecondBestUnp2() const { return best2Unp2_ != getMinScore(); } /** * Return true iff at least two paired-end alignments have been reported so * far. */ bool hasSecondBestPair() const { return best2Pair_ != getMinScore(); } /** * Get best score observed so far for an unpaired read or mate 1. */ TAlScore bestUnp1() const { return bestUnp1_; } /** * Get second-best score observed so far for an unpaired read or mate 1. */ TAlScore secondBestUnp1() const { return best2Unp1_; } /** * Get best score observed so far for mate 2. */ TAlScore bestUnp2() const { return bestUnp2_; } /** * Get second-best score observed so far for mate 2. */ TAlScore secondBestUnp2() const { return best2Unp2_; } /** * Get best score observed so far for an unpaired read or mate 1. */ TAlScore bestUnpRepeat1() const { return bestUnpRepeat1_; } /** * Get second-best score observed so far for an unpaired read or mate 1. */ TAlScore secondBestUnpRepeat1() const { return best2UnpRepeat1_; } /** * Get best score observed so far for mate 2. */ TAlScore bestUnpRepeat2() const { return bestUnpRepeat2_; } /** * Get second-best score observed so far for mate 2. */ TAlScore secondBestUnpRepeat2() const { return best2UnpRepeat2_; } /** * Get best score observed so far for paired-end read. */ TAlScore bestPair() const { return bestPair_; } /** * Get second-best score observed so far for paired-end read. */ TAlScore secondBestPair() const { return best2Pair_; } index_t bestSplicedPair() const { return bestSplicedPair_; } index_t best2SplicedPair() const { return best2SplicedPair_; } index_t bestSplicedUnp1() const { return bestSplicedUnp1_; } index_t best2SplicedUnp1() const { return best2SplicedUnp1_; } index_t bestSplicedUnp2() const { return bestSplicedUnp2_; } index_t best2SplicedUnp2() const { return best2SplicedUnp2_; } bool secondary() const { return secondary_; } /** * */ void getUnp1(const EList*& rs) const { rs = &rs1u_; } void getUnp2(const EList*& rs) const { rs = &rs2u_; } void getPair(const EList*& rs1, const EList*& rs2) const { rs1 = &rs1_; rs2 = &rs2_; } index_t numUnp1() const { return rs1u_.size(); } index_t numUnp2() const { return rs2u_.size(); } index_t numPair() const { assert_eq(rs1_.size(), rs2_.size()); return rs1_.size(); } pair numBestUnp(index_t rdi) const { index_t numGenome = 0, numRepeat = 0; TAlScore maxScore = getMinScore(); const EList& rs = (rdi == 0 ? rs1u_ : rs2u_); for(size_t i = 0; i < rs.size(); i++) { TAlScore curScore = rs[i].score().score(); if(curScore > maxScore) { numGenome = numRepeat = 0; maxScore = curScore; } if(curScore >= maxScore) { if(rs[i].repeat()) { numRepeat++; } else { numGenome++; } } } return pair(numGenome, numRepeat); } pair numBestPair() const { index_t numGenome = 0, numRepeat = 0; TAlScore maxScore = getMinScore(); assert_eq(rs1_.size(), rs2_.size()); for(size_t i = 0; i < rs1_.size(); i++) { TAlScore curScore = rs1_[i].score().score() + rs2_[i].score().score(); if(curScore > maxScore) { numGenome = numRepeat = 0; maxScore = curScore; } if(curScore >= maxScore) { if(rs1_[i].repeat() || rs2_[i].repeat()) { assert(rs1_[i].repeat() && rs2_[i].repeat()); numRepeat++; } else { numGenome++; } } } return pair(numGenome, numRepeat); } protected: /** * Return true iff the read in rd1/rd2 matches the last read handled, which * should still be in rd1_/rd2_. */ bool sameRead( const Read* rd1, const Read* rd2, bool qualitiesMatter); /** * If there is a configuration of unpaired alignments that fits our * criteria for there being one or more discordant alignments, then * shift the discordant alignments over to the rs1_/rs2_ lists, clear the * rs1u_/rs2u_ lists and return true. Otherwise, return false. */ bool prepareDiscordants(); /** * Given that rs is already populated with alignments, consider the * alignment policy and make random selections where necessary. E.g. if we * found 10 alignments and the policy is -k 2 -m 20, select 2 alignments at * random. We "select" an alignment by setting the parallel entry in the * 'select' list to true. */ size_t selectAlnsToReport( const EList& rs, // alignments to select from uint64_t num, // number of alignments to select EList& select, // list to put results in RandomSource& rnd) const; /** * rs1 (possibly together with rs2 if reads are paired) are populated with * alignments. Here we prioritize them according to alignment score, and * some randomness to break ties. Priorities are returned in the 'select' * list. */ size_t selectByScore( const EList* rs1, // alignments to select from (mate 1) const EList* rs2, // alignments to select from (mate 2, or NULL) uint64_t num, // number of alignments to select EList& select, // prioritized list to put results in RandomSource& rnd) const; AlnSink& g_; // global alignment sink ReportingParams rp_; // reporting parameters: khits, mhits etc size_t threadid_; // thread ID Mapq& mapq_; // mapq calculator bool secondary_; // allow for secondary alignments const SpliceSiteDB* ssdb_; // splice sites uint64_t threads_rids_mindist_; // synchronization bool init_; // whether we're initialized w/ read pair bool maxed1_; // true iff # unpaired mate-1 alns reported so far exceeded -m/-M bool maxed2_; // true iff # unpaired mate-2 alns reported so far exceeded -m/-M bool maxedOverall_; // true iff # paired-end alns reported so far exceeded -m/-M TAlScore bestPair_; // greatest score so far for paired-end TAlScore best2Pair_; // second-greatest score so far for paired-end TAlScore bestUnp1_; // greatest score so far for unpaired/mate1 TAlScore best2Unp1_; // second-greatest score so far for unpaired/mate1 TAlScore bestUnp2_; // greatest score so far for mate 2 TAlScore best2Unp2_; // second-greatest score so far for mate 2 TAlScore bestUnpRepeat1_; // greatest score so far for repeat unpaired/mate1 TAlScore best2UnpRepeat1_; // second-greatest score so far for repeat unpaired/mate1 TAlScore bestUnpRepeat2_; // greatest score so far for repeat mate 2 TAlScore best2UnpRepeat2_; // second-greatest score so far for repeat mate 2 index_t bestSplicedPair_; index_t best2SplicedPair_; index_t bestSplicedUnp1_; index_t best2SplicedUnp1_; index_t bestSplicedUnp2_; index_t best2SplicedUnp2_; const Read* rd1_; // mate #1 const Read* rd2_; // mate #2 TReadId rdid_; // read ID (potentially used for ordering) EList rs1_; // paired alignments for mate #1 EList rs2_; // paired alignments for mate #2 EList rs1u_; // unpaired alignments for mate #1 EList rs2u_; // unpaired alignments for mate #2 EList select1_; // parallel to rs1_/rs2_ - which to report EList select2_; // parallel to rs1_/rs2_ - which to report ReportingState st_; // reporting state - what's left to do? EList > selectBuf_; BTString obuf_; StackedAln staln_; EList spliceSites_; }; /** * An AlnSink concrete subclass for printing SAM alignments. The user might * want to customize SAM output in various ways. We encapsulate all these * customizations, and some of the key printing routines, in the SamConfig * class in sam.h/sam.cpp. */ template class AlnSinkSam : public AlnSink { typedef EList StrList; public: AlnSinkSam( OutputQueue& oq, // output queue const SamConfig& samc, // settings & routines for SAM output const StrList& refnames, // reference names const StrList& repnames, // repeat names bool quiet, // don't print alignment summary at end ALTDB* altdb = NULL, SpliceSiteDB* ssdb = NULL) : AlnSink( oq, refnames, repnames, quiet, altdb, ssdb), samc_(samc) { } virtual ~AlnSinkSam() { } /** * Append a single alignment result, which might be paired or * unpaired, to the given output stream in Bowtie's verbose-mode * format. If the alignment is paired-end, print mate1's alignment * then mate2's alignment. */ virtual void append( BTString& o, // write output to this string StackedAln& staln, // StackedAln to write stacked alignment size_t threadId, // which thread am I? const Read* rd1, // mate #1 const Read* rd2, // mate #2 const TReadId rdid, // read ID AlnRes* rs1, // alignments for mate #1 AlnRes* rs2, // alignments for mate #2 const AlnSetSumm& summ, // summary const SeedAlSumm& ssm1, // seed alignment summary const SeedAlSumm& ssm2, // seed alignment summary const AlnFlags* flags1, // flags for mate #1 const AlnFlags* flags2, // flags for mate #2 const PerReadMetrics& prm, // per-read metrics const Mapq& mapq, // MAPQ calculator const Scoring& sc, // scoring scheme bool report2) // report alns for both mates { assert(rd1 != NULL || rd2 != NULL); if(rd1 != NULL) { assert(flags1 != NULL); appendMate(o, staln, *rd1, rd2, rdid, rs1, rs2, summ, ssm1, ssm2, *flags1, prm, mapq, sc); if(rs1 != NULL && rs1->spliced() && this->spliceSiteDB_ != NULL) { this->spliceSiteDB_->addSpliceSite(*rd1, *rs1); } } if(rd2 != NULL && report2) { assert(flags2 != NULL); appendMate(o, staln, *rd2, rd1, rdid, rs2, rs1, summ, ssm2, ssm1, *flags2, prm, mapq, sc); if(rs2 != NULL && rs2->spliced() && this->spliceSiteDB_ != NULL) { this->spliceSiteDB_->addSpliceSite(*rd2, *rs2); } } } protected: /** * Append a single per-mate alignment result to the given output * stream. If the alignment is part of a pair, information about * the opposite mate and its alignment are given in rdo/rso. */ void appendMate( BTString& o, StackedAln& staln, const Read& rd, const Read* rdo, const TReadId rdid, AlnRes* rs, AlnRes* rso, const AlnSetSumm& summ, const SeedAlSumm& ssm, const SeedAlSumm& ssmo, const AlnFlags& flags, const PerReadMetrics& prm, // per-read metrics const Mapq& mapq, // MAPQ calculator const Scoring& sc); // scoring scheme const SamConfig& samc_; // settings & routines for SAM output BTDnaString dseq_; // buffer for decoded read sequence BTString dqual_; // buffer for decoded quality sequence }; static inline std::ostream& printPct( std::ostream& os, uint64_t num, uint64_t denom) { double pct = 0.0f; if(denom != 0) { pct = 100.0 * (double)num / (double)denom; } os << fixed << setprecision(2) << pct << '%'; return os; } /** * Print a friendly summary of: * * 1. How many reads were aligned and had one or more alignments * reported * 2. How many reads exceeded the -m or -M ceiling and therefore had * their alignments suppressed or sampled * 3. How many reads failed to align entirely * * Optionally print a series of Hadoop streaming-style counter updates * with similar information. */ template void AlnSink::printAlSumm( ostream& out, const ReportingMetrics& met, size_t repThresh, // threshold for uniqueness, or max if no thresh bool discord, // looked for discordant alignments bool mixed, // looked for unpaired alignments where paired failed? bool newSummary, // alignment summary in a new style bool hadoopOut) // output Hadoop counters? { // NOTE: there's a filtering step at the very beginning, so everything // being reported here is post filtering bool canRep = repThresh != MAX_SIZE_T; if(hadoopOut) { out << "reporter:counter:HISAT2,Reads processed," << met.nread << endl; } uint64_t totread = met.nread; uint64_t totpair = met.npaired; uint64_t totunpair = met.nunpaired; uint64_t tot_al_cand = totunpair + totpair*2; uint64_t tot_al = (met.nconcord_uni + met.nconcord_rep) * 2 + (met.ndiscord) * 2 + met.nunp_0_uni + met.nunp_0_rep + met.nunp_uni + met.nunp_rep; assert_leq(tot_al, tot_al_cand); if(newSummary) { out << "HISAT2 summary stats:" << endl; if(totpair > 0) { uint64_t ncondiscord_0 = met.nconcord_0 - met.ndiscord; out << "\tTotal pairs: " << totpair << endl; out << "\t\tAligned concordantly or discordantly 0 time: " << ncondiscord_0 << " ("; printPct(out, ncondiscord_0, met.npaired); out << ")" << endl; out << "\t\tAligned concordantly 1 time: " << met.nconcord_uni1 << " ("; printPct(out, met.nconcord_uni1, met.npaired); out << ")" << endl; out << "\t\tAligned concordantly >1 times: " << met.nconcord_uni2 << " ("; printPct(out, met.nconcord_uni2, met.npaired); out << ")" << endl; out << "\t\tAligned discordantly 1 time: " << met.ndiscord << " ("; printPct(out, met.ndiscord, met.npaired); out << ")" << endl; out << "\tTotal unpaired reads: " << ncondiscord_0 * 2 << endl; out << "\t\tAligned 0 time: " << met.nunp_0_0 << " ("; printPct(out, met.nunp_0_0, ncondiscord_0 * 2); out << ")" << endl; out << "\t\tAligned 1 time: " << met.nunp_0_uni1 << " ("; printPct(out, met.nunp_0_uni1, ncondiscord_0 * 2); out << ")" << endl; out << "\t\tAligned >1 times: " << met.nunp_0_uni2 << " ("; printPct(out, met.nunp_0_uni2, ncondiscord_0 * 2); out << ")" << endl; } else { out << "\tTotal reads: " << totread << endl; out << "\t\tAligned 0 time: " << met.nunp_0 << " ("; printPct(out, met.nunp_0, met.nunpaired); out << ")" << endl; out << "\t\tAligned 1 time: " << met.nunp_uni1 << " ("; printPct(out, met.nunp_uni1, met.nunpaired); out << ")" << endl; out << "\t\tAligned >1 times: " << met.nunp_uni2 << " ("; printPct(out, met.nunp_uni2, met.nunpaired); out << ")" << endl; } out << "\tOverall alignment rate: "; printPct(out, tot_al, tot_al_cand); out << endl; } else { if(totread > 0) { out << "" << totread << " reads; of these:" << endl; } else { assert_eq(0, met.npaired); assert_eq(0, met.nunpaired); out << "" << totread << " reads" << endl; } if(totpair > 0) { // Paired output out << " " << totpair << " ("; printPct(out, totpair, totread); out << ") were paired; of these:" << endl; // Concordants out << " " << met.nconcord_0 << " ("; printPct(out, met.nconcord_0, met.npaired); out << ") aligned concordantly 0 times" << endl; if(canRep) { // Print the number that aligned concordantly exactly once assert_eq(met.nconcord_uni, met.nconcord_uni1+met.nconcord_uni2); out << " " << met.nconcord_uni1 << " ("; printPct(out, met.nconcord_uni1, met.npaired); out << ") aligned concordantly exactly 1 time" << endl; // Print the number that aligned concordantly more than once but // fewer times than the limit out << " " << met.nconcord_uni2+met.nconcord_rep << " ("; printPct(out, met.nconcord_uni2+met.nconcord_rep, met.npaired); out << ") aligned concordantly >1 times" << endl; } else { // Print the number that aligned concordantly exactly once assert_eq(met.nconcord_uni, met.nconcord_uni1+met.nconcord_uni2); out << " " << met.nconcord_uni1 << " ("; printPct(out, met.nconcord_uni1, met.npaired); out << ") aligned concordantly exactly 1 time" << endl; // Print the number that aligned concordantly more than once out << " " << met.nconcord_uni2 << " ("; printPct(out, met.nconcord_uni2, met.npaired); out << ") aligned concordantly >1 times" << endl; } if(discord) { // TODO: what about discoardant and on separate chromosomes? // Bring out the unaligned pair total so we can subtract discordants out << " ----" << endl; out << " " << met.nconcord_0 << " pairs aligned concordantly 0 times; of these:" << endl; // Discordants out << " " << met.ndiscord << " ("; printPct(out, met.ndiscord, met.nconcord_0); out << ") aligned discordantly 1 time" << endl; } uint64_t ncondiscord_0 = met.nconcord_0 - met.ndiscord; if(mixed) { // Bring out the unaligned pair total so we can subtract discordants out << " ----" << endl; out << " " << ncondiscord_0 << " pairs aligned 0 times concordantly or discordantly; of these:" << endl; out << " " << (ncondiscord_0 * 2) << " mates make up the pairs; of these:" << endl; out << " " << met.nunp_0_0 << " " << "("; printPct(out, met.nunp_0_0, ncondiscord_0 * 2); out << ") aligned 0 times" << endl; if(canRep) { // Print the number that aligned exactly once assert_eq(met.nunp_0_uni, met.nunp_0_uni1+met.nunp_0_uni2); out << " " << met.nunp_0_uni1 << " ("; printPct(out, met.nunp_0_uni1, ncondiscord_0 * 2); out << ") aligned exactly 1 time" << endl; // Print the number that aligned more than once but fewer times // than the limit out << " " << met.nunp_0_uni2+met.nunp_0_rep << " ("; printPct(out, met.nunp_0_uni2+met.nunp_0_rep, ncondiscord_0 * 2); out << ") aligned >1 times" << endl; } else { // Print the number that aligned exactly once assert_eq(met.nunp_0_uni, met.nunp_0_uni1+met.nunp_0_uni2); out << " " << met.nunp_0_uni1 << " ("; printPct(out, met.nunp_0_uni1, ncondiscord_0 * 2); out << ") aligned exactly 1 time" << endl; // Print the number that aligned more than once but fewer times // than the limit out << " " << met.nunp_0_uni2 << " ("; printPct(out, met.nunp_0_uni2, ncondiscord_0 * 2); out << ") aligned >1 times" << endl; } } } if(totunpair > 0) { // Unpaired output out << " " << totunpair << " ("; printPct(out, totunpair, totread); out << ") were unpaired; of these:" << endl; out << " " << met.nunp_0 << " ("; printPct(out, met.nunp_0, met.nunpaired); out << ") aligned 0 times" << endl; if(hadoopOut) { out << "reporter:counter:HISAT 2,Unpaired reads with 0 alignments," << met.nunpaired << endl; } if(canRep) { // Print the number that aligned exactly once assert_eq(met.nunp_uni, met.nunp_uni1+met.nunp_uni2); out << " " << met.nunp_uni1 << " ("; printPct(out, met.nunp_uni1, met.nunpaired); out << ") aligned exactly 1 time" << endl; // Print the number that aligned more than once but fewer times // than the limit out << " " << met.nunp_uni2+met.nunp_rep << " ("; printPct(out, met.nunp_uni2+met.nunp_rep, met.nunpaired); out << ") aligned >1 times" << endl; } else { // Print the number that aligned exactly once assert_eq(met.nunp_uni, met.nunp_uni1+met.nunp_uni2); out << " " << met.nunp_uni1 << " ("; printPct(out, met.nunp_uni1, met.nunpaired); out << ") aligned exactly 1 time" << endl; // Print the number that aligned more than once out << " " << met.nunp_uni2 << " ("; printPct(out, met.nunp_uni2, met.nunpaired); out << ") aligned >1 times" << endl; } } printPct(out, tot_al, tot_al_cand); out << " overall alignment rate" << endl; } } /** * Return true iff the read in rd1/rd2 matches the last read handled, which * should still be in rd1_/rd2_. */ template bool AlnSinkWrap::sameRead( // One of the other of rd1, rd2 will = NULL if read is unpaired const Read* rd1, // new mate #1 const Read* rd2, // new mate #2 bool qualitiesMatter) // aln policy distinguishes b/t quals? { bool same = false; if(rd1_ != NULL || rd2_ != NULL) { // This is not the first time the sink was initialized with // a read. Check if new read/pair is identical to previous // read/pair if((rd1_ == NULL) == (rd1 == NULL) && (rd2_ == NULL) == (rd2 == NULL)) { bool m1same = (rd1 == NULL && rd1_ == NULL); if(!m1same) { assert(rd1 != NULL); assert(rd1_ != NULL); m1same = Read::same( rd1->patFw, // new seq rd1->qual, // new quals rd1_->patFw, // old seq rd1_->qual, // old quals qualitiesMatter); } if(m1same) { bool m2same = (rd2 == NULL && rd2_ == NULL); if(!m2same) { m2same = Read::same( rd2->patFw, // new seq rd2->qual, // new quals rd2_->patFw, // old seq rd2_->qual, // old quals qualitiesMatter); } same = m2same; } } } return same; } /** * Initialize the wrapper with a new read pair and return an integer >= -1 * indicating which stage the aligner should start at. If -1 is returned, the * aligner can skip the read entirely. Checks if the new read pair is * identical to the previous pair. If it is, then we return the id of the * first stage to run. */ template int AlnSinkWrap::nextRead( // One of the other of rd1, rd2 will = NULL if read is unpaired const Read* rd1, // new mate #1 const Read* rd2, // new mate #2 TReadId rdid, // read ID for new pair bool qualitiesMatter) // aln policy distinguishes b/t quals? { assert(!init_); assert(rd1 != NULL || rd2 != NULL); init_ = true; // Keep copy of new read, so that we can compare it with the // next one if(rd1 != NULL) { rd1_ = rd1; } else rd1_ = NULL; if(rd2 != NULL) { rd2_ = rd2; } else rd2_ = NULL; rdid_ = rdid; // Caller must now align the read maxed1_ = false; maxed2_ = false; maxedOverall_ = false; bestPair_ = best2Pair_ = bestUnp1_ = best2Unp1_ = bestUnp2_ = best2Unp2_ = std::numeric_limits::min(); bestUnpRepeat1_ = best2UnpRepeat1_ = bestUnpRepeat2_ = best2UnpRepeat2_ = std::numeric_limits::min(); bestSplicedPair_ = best2SplicedPair_ = bestSplicedUnp1_ = best2SplicedUnp1_ = bestSplicedUnp2_ = best2SplicedUnp2_ = 0; rs1_.clear(); // clear out paired-end alignments rs2_.clear(); // clear out paired-end alignments rs1u_.clear(); // clear out unpaired alignments for mate #1 rs2u_.clear(); // clear out unpaired alignments for mate #2 st_.nextRead(readIsPair()); // reset state assert(empty()); assert(!maxed()); // Start from the first stage return 0; } /** * Inform global, shared AlnSink object that we're finished with this read. * The global AlnSink is responsible for updating counters, creating the output * record, and delivering the record to the appropriate output stream. * * What gets reported for a paired-end alignment? * * 1. If there are reportable concordant alignments, report those and stop * 2. If there are reportable discordant alignments, report those and stop * 3. If unpaired alignments can be reported: * 3a. Report # * Update metrics. Only ambiguity is: what if a pair aligns repetitively and * one of its mates aligns uniquely? * * uint64_t al; // # mates w/ >= 1 reported alignment * uint64_t unal; // # mates w/ 0 alignments * uint64_t max; // # mates withheld for exceeding -M/-m ceiling * uint64_t al_concord; // # pairs w/ >= 1 concordant alignment * uint64_t al_discord; // # pairs w/ >= 1 discordant alignment * uint64_t max_concord; // # pairs maxed out * uint64_t unal_pair; // # pairs where neither mate aligned */ template void AlnSinkWrap::finishRead( const SeedResults *sr1, // seed alignment results for mate 1 const SeedResults *sr2, // seed alignment results for mate 2 bool exhaust1, // mate 1 exhausted? bool exhaust2, // mate 2 exhausted? bool nfilt1, // mate 1 N-filtered? bool nfilt2, // mate 2 N-filtered? bool scfilt1, // mate 1 score-filtered? bool scfilt2, // mate 2 score-filtered? bool lenfilt1, // mate 1 length-filtered? bool lenfilt2, // mate 2 length-filtered? bool qcfilt1, // mate 1 qc-filtered? bool qcfilt2, // mate 2 qc-filtered? bool sortByScore, // prioritize alignments by score RandomSource& rnd, // pseudo-random generator ReportingMetrics& met, // reporting metrics const PerReadMetrics& prm, // per-read metrics const Scoring& sc, // scoring scheme bool suppressSeedSummary, // = true bool suppressAlignments, // = false bool templateLenAdjustment) // = true { obuf_.clear(); OutputQueueMark qqm(g_.outq(), obuf_, rdid_, threadid_); assert(init_); if(!suppressSeedSummary) { if(sr1 != NULL) { assert(rd1_ != NULL); // Mate exists and has non-empty SeedResults g_.reportSeedSummary(obuf_, *rd1_, rdid_, threadid_, *sr1, true); } else if(rd1_ != NULL) { // Mate exists but has NULL SeedResults g_.reportEmptySeedSummary(obuf_, *rd1_, rdid_, true); } if(sr2 != NULL) { assert(rd2_ != NULL); // Mate exists and has non-empty SeedResults g_.reportSeedSummary(obuf_, *rd2_, rdid_, threadid_, *sr2, true); } else if(rd2_ != NULL) { // Mate exists but has NULL SeedResults g_.reportEmptySeedSummary(obuf_, *rd2_, rdid_, true); } } if(!suppressAlignments) { // Ask the ReportingState what to report st_.finish(); uint64_t nconcord = 0, ndiscord = 0, nunpair1 = 0, nunpair2 = 0; uint64_t nunpairRepeat1 = 0, nunpairRepeat2 = 0; bool pairMax = false, unpair1Max = false, unpair2Max = false; st_.getReport( nconcord, ndiscord, nunpair1, nunpair2, nunpairRepeat1, nunpairRepeat2, pairMax, unpair1Max, unpair2Max); assert_leq(nconcord, rs1_.size()); assert_leq(nunpair1, rs1u_.size()); assert_leq(nunpair2, rs2u_.size()); assert_leq(ndiscord, 1); assert_gt(rp_.khits, 0); assert_gt(rp_.mhits, 0); assert(!pairMax || rs1_.size() >= (uint64_t)rp_.mhits); assert(!unpair1Max || rs1u_.size() >= (uint64_t)rp_.mhits); assert(!unpair2Max || rs2u_.size() >= (uint64_t)rp_.mhits); met.nread++; if(readIsPair()) { met.npaired++; } else { met.nunpaired++; } // Report concordant paired-end alignments if possible if(nconcord > 0) { AlnSetSumm concordSumm( rd1_, rd2_, &rs1_, &rs2_, &rs1u_, &rs2u_, exhaust1, exhaust2, -1, -1, false); // Possibly select a random subset size_t off; if(sortByScore) { // Sort by score then pick from low to high off = selectByScore(&rs1_, &rs2_, nconcord, select1_, rnd); } else { // Select subset randomly off = selectAlnsToReport(rs1_, nconcord, select1_, rnd); } concordSumm.numAlnsPaired(select1_.size()); assert_lt(off, rs1_.size()); const AlnRes *rs1 = &rs1_[off]; const AlnRes *rs2 = &rs2_[off]; AlnFlags flags1( ALN_FLAG_PAIR_CONCORD_MATE1, st_.params().mhitsSet(), unpair1Max, pairMax, nfilt1, scfilt1, lenfilt1, qcfilt1, st_.params().mixed, true, // primary true, // opp aligned rs2->fw()); // opp fw AlnFlags flags2( ALN_FLAG_PAIR_CONCORD_MATE2, st_.params().mhitsSet(), unpair2Max, pairMax, nfilt2, scfilt2, lenfilt2, qcfilt2, st_.params().mixed, false, // primary true, // opp aligned rs1->fw()); // opp fw // Issue: we only set the flags once, but some of the flags might // vary from pair to pair among the pairs we're reporting. For // instance, whether a given mate aligns to the forward strand. SeedAlSumm ssm1, ssm2; if(sr1 != NULL && sr2 != NULL) { sr1->toSeedAlSumm(ssm1); sr2->toSeedAlSumm(ssm2); } for(size_t i = 0; i < rs1_.size(); i++) { spliceSites_.clear(); if(templateLenAdjustment) { rs1_[i].setMateParams(ALN_RES_TYPE_MATE1, &rs2_[i], flags1, ssdb_, threads_rids_mindist_, &spliceSites_); rs2_[i].setMateParams(ALN_RES_TYPE_MATE2, &rs1_[i], flags2, ssdb_, threads_rids_mindist_, &spliceSites_); } else { rs1_[i].setMateParams(ALN_RES_TYPE_MATE1, &rs2_[i], flags1); rs2_[i].setMateParams(ALN_RES_TYPE_MATE2, &rs1_[i], flags2); } assert_eq(abs(rs1_[i].fragmentLength()), abs(rs2_[i].fragmentLength())); } assert(!select1_.empty()); g_.reportHits( obuf_, staln_, threadid_, rd1_, rd2_, rdid_, select1_, NULL, &rs1_, &rs2_, pairMax, concordSumm, ssm1, ssm2, &flags1, &flags2, prm, mapq_, sc); if(pairMax) { met.nconcord_rep++; } else { met.nconcord_uni++; assert(!rs1_.empty()); if(select1_.size() == 1) { met.nconcord_uni1++; } else { met.nconcord_uni2++; } } init_ = false; //g_.outq().finishRead(obuf_, rdid_, threadid_); return; } // Report concordant paired-end alignments if possible else if(ndiscord > 0) { ASSERT_ONLY(bool ret =) prepareDiscordants(); assert(ret); assert_eq(1, rs1_.size()); assert_eq(1, rs2_.size()); AlnSetSumm discordSumm( rd1_, rd2_, &rs1_, &rs2_, &rs1u_, &rs2u_, exhaust1, exhaust2, -1, -1, false); const AlnRes *rs1 = &rs1_[0]; const AlnRes *rs2 = &rs2_[0]; AlnFlags flags1( ALN_FLAG_PAIR_DISCORD_MATE1, st_.params().mhitsSet(), false, pairMax, nfilt1, scfilt1, lenfilt1, qcfilt1, st_.params().mixed, true, // primary true, // opp aligned rs2->fw()); // opp fw AlnFlags flags2( ALN_FLAG_PAIR_DISCORD_MATE2, st_.params().mhitsSet(), false, pairMax, nfilt2, scfilt2, lenfilt2, qcfilt2, st_.params().mixed, false, // primary true, // opp aligned rs1->fw()); // opp fw SeedAlSumm ssm1, ssm2; if(sr1 != NULL) sr1->toSeedAlSumm(ssm1); if(sr2 != NULL) sr2->toSeedAlSumm(ssm2); for(size_t i = 0; i < rs1_.size(); i++) { rs1_[i].setMateParams(ALN_RES_TYPE_MATE1, &rs2_[i], flags1); rs2_[i].setMateParams(ALN_RES_TYPE_MATE2, &rs1_[i], flags2); assert(rs1_[i].isFraglenSet() == rs2_[i].isFraglenSet()); assert(!rs1_[i].isFraglenSet() || abs(rs1_[i].fragmentLength()) == abs(rs2_[i].fragmentLength())); } ASSERT_ONLY(size_t off); if(sortByScore) { // Sort by score then pick from low to high ASSERT_ONLY(off =) selectByScore(&rs1_, &rs2_, ndiscord, select1_, rnd); } else { // Select subset randomly ASSERT_ONLY(off =) selectAlnsToReport(rs1_, ndiscord, select1_, rnd); } assert_eq(0, off); assert(!select1_.empty()); g_.reportHits( obuf_, staln_, threadid_, rd1_, rd2_, rdid_, select1_, NULL, &rs1_, &rs2_, pairMax, discordSumm, ssm1, ssm2, &flags1, &flags2, prm, mapq_, sc); met.nconcord_0++; met.ndiscord++; init_ = false; //g_.outq().finishRead(obuf_, rdid_, threadid_); return; } // If we're at this point, at least one mate failed to align. // BTL: That's not true. It could be that there are no concordant // alignments but both mates have unpaired alignments, with one of // the mates having more than one. //assert(nunpair1 == 0 || nunpair2 == 0); assert(!pairMax); const AlnRes *repRs1 = NULL, *repRs2 = NULL; AlnSetSumm summ1, summ2; AlnFlags flags1, flags2; TRefId refid = -1; TRefOff refoff = -1; bool repeat = false; bool rep1 = rd1_ != NULL && nunpair1 > 0; bool rep2 = rd2_ != NULL && nunpair2 > 0; // This is the preliminary if statement for mate 1 - here we're // gathering some preliminary information, making it possible to call // g_.reportHits(...) with information about both mates potentially if(rep1) { // Mate 1 aligned at least once if(rep2) { summ1.init( rd1_, rd2_, NULL, NULL, &rs1u_, &rs2u_, exhaust1, exhaust2, -1, -1, false); } else { summ1.init( rd1_, NULL, NULL, NULL, &rs1u_, NULL, exhaust1, exhaust2, -1, -1, false); } size_t off; if(sortByScore) { // Sort by score then pick from low to high off = selectByScore(&rs1u_, NULL, nunpair1, select1_, rnd); } else { // Select subset randomly off = selectAlnsToReport(rs1u_, nunpair1, select1_, rnd); } summ1.numAlns1(select1_.size()); summ2.numAlns1(select1_.size()); repRs1 = &rs1u_[off]; } else if(rd1_ != NULL) { // Mate 1 failed to align - don't do anything yet. First we want // to collect information on mate 2 in case that factors into the // summary assert(!unpair1Max); } if(rep2) { if(rep1) { summ2.init( rd1_, rd2_, NULL, NULL, &rs1u_, &rs2u_, exhaust1, exhaust2, -1, -1, false); } else { summ2.init( NULL, rd2_, NULL, NULL, NULL, &rs2u_, exhaust1, exhaust2, -1, -1, false); } size_t off; if(sortByScore) { // Sort by score then pick from low to high off = selectByScore(&rs2u_, NULL, nunpair2, select2_, rnd); } else { // Select subset randomly off = selectAlnsToReport(rs2u_, nunpair2, select2_, rnd); } repRs2 = &rs2u_[off]; summ1.numAlns2(select2_.size()); summ2.numAlns2(select2_.size()); } else if(rd2_ != NULL) { // Mate 2 failed to align - don't do anything yet. First we want // to collect information on mate 1 in case that factors into the // summary assert(!unpair2Max); } // Update counters given that one mate didn't align if(readIsPair()) { met.nconcord_0++; } if(rd1_ != NULL) { if(nunpair1 > 0) { // Update counters if(readIsPair()) { if(unpair1Max) met.nunp_0_rep++; else { met.nunp_0_uni++; assert(!rs1u_.empty()); if(select1_.size() == 1) { met.nunp_0_uni1++; } else { met.nunp_0_uni2++; } } } else { if(unpair1Max) met.nunp_rep++; else { met.nunp_uni++; assert(!rs1u_.empty()); if(select1_.size() == 1) { met.nunp_uni1++; } else { met.nunp_uni2++; } } } } else if(unpair1Max) { // Update counters if(readIsPair()) met.nunp_0_rep++; else met.nunp_rep++; } else { // Update counters if(readIsPair()) met.nunp_0_0++; else met.nunp_0++; } } if(rd2_ != NULL) { if(nunpair2 > 0) { // Update counters if(readIsPair()) { if(unpair2Max) met.nunp_0_rep++; else { assert(!rs2u_.empty()); met.nunp_0_uni++; if(select2_.size() == 1) { met.nunp_0_uni1++; } else { met.nunp_0_uni2++; } } } else { if(unpair2Max) met.nunp_rep++; else { assert(!rs2u_.empty()); met.nunp_uni++; if(select2_.size() == 1) { met.nunp_uni1++; } else { met.nunp_uni2++; } } } } else if(unpair2Max) { // Update counters if(readIsPair()) met.nunp_0_rep++; else met.nunp_rep++; } else { // Update counters if(readIsPair()) met.nunp_0_0++; else met.nunp_0++; } } // Now set up flags if(rep1) { // Initialize flags. Note: We want to have information about how // the other mate aligned (if it did) at this point flags1.init( readIsPair() ? ALN_FLAG_PAIR_UNPAIRED_MATE1 : ALN_FLAG_PAIR_UNPAIRED, st_.params().mhitsSet(), unpair1Max, pairMax, nfilt1, scfilt1, lenfilt1, qcfilt1, st_.params().mixed, true, // primary repRs2 != NULL, // opp aligned repRs2 == NULL || repRs2->fw()); // opp fw for(size_t i = 0; i < rs1u_.size(); i++) { rs1u_[i].setMateParams(ALN_RES_TYPE_UNPAIRED_MATE1, NULL, flags1); } } if(rep2) { // Initialize flags. Note: We want to have information about how // the other mate aligned (if it did) at this point flags2.init( readIsPair() ? ALN_FLAG_PAIR_UNPAIRED_MATE2 : ALN_FLAG_PAIR_UNPAIRED, st_.params().mhitsSet(), unpair2Max, pairMax, nfilt2, scfilt2, lenfilt2, qcfilt2, st_.params().mixed, true, // primary repRs1 != NULL, // opp aligned repRs1 == NULL || repRs1->fw()); // opp fw for(size_t i = 0; i < rs2u_.size(); i++) { rs2u_[i].setMateParams(ALN_RES_TYPE_UNPAIRED_MATE2, NULL, flags2); } } // Now report mate 1 if(rep1) { SeedAlSumm ssm1, ssm2; if(sr1 != NULL) sr1->toSeedAlSumm(ssm1); if(sr2 != NULL) sr2->toSeedAlSumm(ssm2); assert(!select1_.empty()); g_.reportHits( obuf_, staln_, threadid_, rd1_, repRs2 != NULL ? rd2_ : NULL, rdid_, select1_, repRs2 != NULL ? &select2_ : NULL, &rs1u_, repRs2 != NULL ? &rs2u_ : NULL, unpair1Max, summ1, ssm1, ssm2, &flags1, repRs2 != NULL ? &flags2 : NULL, prm, mapq_, sc); assert_lt(select1_[0], rs1u_.size()); refid = rs1u_[select1_[0]].refid(); refoff = rs1u_[select1_[0]].refoff(); repeat = rs1u_[select1_[0]].repeat(); } // Now report mate 2 if(rep2 && !rep1) { SeedAlSumm ssm1, ssm2; if(sr1 != NULL) sr1->toSeedAlSumm(ssm1); if(sr2 != NULL) sr2->toSeedAlSumm(ssm2); assert(!select2_.empty()); g_.reportHits( obuf_, staln_, threadid_, rd2_, repRs1 != NULL ? rd1_ : NULL, rdid_, select2_, repRs1 != NULL ? &select1_ : NULL, &rs2u_, repRs1 != NULL ? &rs1u_ : NULL, unpair2Max, summ2, ssm1, ssm2, &flags2, repRs1 != NULL ? &flags1 : NULL, prm, mapq_, sc); assert_lt(select2_[0], rs2u_.size()); refid = rs2u_[select2_[0]].refid(); refoff = rs2u_[select2_[0]].refoff(); repeat = rs2u_[select2_[0]].repeat(); } if(rd1_ != NULL && nunpair1 == 0) { if(nunpair2 > 0) { assert_neq(-1, refid); summ1.init( rd1_, NULL, NULL, NULL, NULL, NULL, exhaust1, exhaust2, refid, refoff, repeat); } else { summ1.init( rd1_, NULL, NULL, NULL, NULL, NULL, exhaust1, exhaust2, -1, -1, false); } SeedAlSumm ssm1, ssm2; if(sr1 != NULL) sr1->toSeedAlSumm(ssm1); if(sr2 != NULL) sr2->toSeedAlSumm(ssm2); flags1.init( readIsPair() ? ALN_FLAG_PAIR_UNPAIRED_MATE1 : ALN_FLAG_PAIR_UNPAIRED, st_.params().mhitsSet(), false, false, nfilt1, scfilt1, lenfilt1, qcfilt1, st_.params().mixed, true, // primary repRs2 != NULL, // opp aligned (repRs2 != NULL) ? repRs2->fw() : false); // opp fw g_.reportUnaligned( obuf_, // string to write output to staln_, threadid_, rd1_, // read 1 NULL, // read 2 rdid_, // read id summ1, // summ ssm1, // ssm2, &flags1, // flags 1 NULL, // flags 2 prm, // per-read metrics mapq_, // MAPQ calculator sc, // scoring scheme true); // get lock? } if(rd2_ != NULL && nunpair2 == 0) { if(nunpair1 > 0) { assert_neq(-1, refid); summ2.init( NULL, rd2_, NULL, NULL, NULL, NULL, exhaust1, exhaust2, refid, refoff, repeat); } else { summ2.init( NULL, rd2_, NULL, NULL, NULL, NULL, exhaust1, exhaust2, -1, -1, false); } SeedAlSumm ssm1, ssm2; if(sr1 != NULL) sr1->toSeedAlSumm(ssm1); if(sr2 != NULL) sr2->toSeedAlSumm(ssm2); flags2.init( readIsPair() ? ALN_FLAG_PAIR_UNPAIRED_MATE2 : ALN_FLAG_PAIR_UNPAIRED, st_.params().mhitsSet(), false, false, nfilt2, scfilt2, lenfilt2, qcfilt2, st_.params().mixed, true, // primary repRs1 != NULL, // opp aligned (repRs1 != NULL) ? repRs1->fw() : false); // opp fw g_.reportUnaligned( obuf_, // string to write output to staln_, threadid_, rd2_, // read 1 NULL, // read 2 rdid_, // read id summ2, // summ ssm1, ssm2, &flags2, // flags 1 NULL, // flags 2 prm, // per-read metrics mapq_, // MAPQ calculator sc, // scoring scheme true); // get lock? } } // if(suppress alignments) init_ = false; return; } /** * Called by the aligner when a new unpaired or paired alignment is * discovered in the given stage. This function checks whether the * addition of this alignment causes the reporting policy to be * violated (by meeting or exceeding the limits set by -k, -m, -M), * in which case true is returned immediately and the aligner is * short circuited. Otherwise, the alignment is tallied and false * is returned. */ template bool AlnSinkWrap::report( int stage, const AlnRes* rs1, const AlnRes* rs2, bool alignMate) { assert(init_); assert(rs1 != NULL || rs2 != NULL); assert(rs1 == NULL || !rs1->empty()); assert(rs2 == NULL || !rs2->empty()); assert(rs1 == NULL || rs1->repOk()); assert(rs2 == NULL || rs2->repOk()); bool paired = (rs1 != NULL && rs2 != NULL); bool one = (rs1 != NULL); const AlnRes* rsa = one ? rs1 : rs2; const AlnRes* rsb = one ? rs2 : rs1; // Tally overall alignment score TAlScore score = rsa->score().score(); if(rsb != NULL) score += rsb->score().score(); index_t num_spliced = (index_t)rsa->num_spliced(); if(rsb != NULL) num_spliced += (index_t)rsb->num_spliced(); if(paired) { assert(readIsPair()); st_.foundConcordant(score); rs1_.push_back(*rs1); rs2_.push_back(*rs2); } else { st_.foundUnpaired(one, rsa->repeat()); if(one) { rs1u_.push_back(*rs1); } else { rs2u_.push_back(*rs2); } } // Update best score so far if(paired) { if(score > bestPair_) { best2Pair_ = bestPair_; bestPair_ = score; best2SplicedPair_ = bestSplicedPair_; bestSplicedPair_ = num_spliced; } else if(score > best2Pair_) { best2Pair_ = score; best2SplicedPair_ = num_spliced; } } else { if(one) { if(score > bestUnp1_) { best2Unp1_ = bestUnp1_; bestUnp1_ = score; best2SplicedUnp1_ = bestSplicedUnp1_; bestSplicedUnp1_ = num_spliced; } else if(score > best2Unp1_) { best2Unp1_ = score; best2SplicedUnp1_ = num_spliced; } if(rs1->repeat()) { if(score > bestUnpRepeat1_) { best2UnpRepeat1_ = bestUnpRepeat1_; bestUnpRepeat1_ = score; } else if(score > best2UnpRepeat1_) { best2UnpRepeat1_ = score; } } } else { if(score > bestUnp2_) { best2Unp2_ = bestUnp2_; bestUnp2_ = score; best2SplicedUnp2_ = bestSplicedUnp2_; bestSplicedUnp2_ = num_spliced; } else if(score > best2Unp2_) { best2Unp2_ = score; best2SplicedUnp1_ = num_spliced; } if(rs2->repeat()) { if(score > bestUnpRepeat2_) { best2UnpRepeat2_ = bestUnpRepeat2_; bestUnpRepeat2_ = score; } else if(score > best2UnpRepeat2_) { best2UnpRepeat2_ = score; } } } } return st_.done(); } /** * If there is a configuration of unpaired alignments that fits our * criteria for there being one or more discordant alignments, then * shift the discordant alignments over to the rs1_/rs2_ lists, clear the * rs1u_/rs2u_ lists and return true. Otherwise, return false. */ template bool AlnSinkWrap::prepareDiscordants() { if(rs1u_.size() == 1 && rs2u_.size() == 1) { assert(rs1_.empty()); assert(rs2_.empty()); rs1_.push_back(rs1u_[0]); rs2_.push_back(rs2u_[0]); return true; } return false; } /** * rs1 (possibly together with rs2 if reads are paired) are populated with * alignments. Here we prioritize them according to alignment score, and * some randomness to break ties. Priorities are returned in the 'select' * list. */ template size_t AlnSinkWrap::selectByScore( const EList* rs1, // alignments to select from (mate 1) const EList* rs2, // alignments to select from (mate 2, or NULL) uint64_t num, // number of alignments to select EList& select, // prioritized list to put results in RandomSource& rnd) const { assert(init_); assert(repOk()); assert_gt(num, 0); assert(rs1 != NULL); size_t sz = rs1->size(); // sz = # alignments found assert_leq(num, sz); if(sz < num) { num = sz; } // num = # to select if(sz < 1) { return 0; } select.resize((size_t)num); // Use 'selectBuf_' as a temporary list for sorting purposes EList >& buf = const_cast >& >(selectBuf_); buf.resize(sz); // Sort by score. If reads are pairs, sort by sum of mate scores. for(size_t i = 0; i < sz; i++) { buf[i].first = (*rs1)[i].score().hisat2_score(); if(rs2 != NULL) { buf[i].first += (*rs2)[i].score().hisat2_score(); } buf[i].second = i; // original offset } buf.sort(); buf.reverse(); // sort in descending order by score // Randomize streaks of alignments that are equal by score size_t streak = 0; for(size_t i = 1; i < buf.size(); i++) { if(buf[i].first == buf[i-1].first) { if(streak == 0) { streak = 1; } streak++; } else { if(streak > 1) { assert_geq(i, streak); buf.shufflePortion(i-streak, streak, rnd); } streak = 0; } } if(streak > 1) { buf.shufflePortion(buf.size() - streak, streak, rnd); } select.clear(); for(size_t i = 0; i < buf.size(); i++) { index_t add = buf[i].second; if(i >= num && !(*rs1)[add].repeat()) { assert(rs2 == NULL || !(*rs2)[add].repeat()); break; } select.push_back(add); } if(!secondary_) { assert_geq(buf.size(), select.size()); for(size_t i = 0; i + 1 < select.size(); i++) { if(buf[i].first != buf[i+1].first) { select.resize(i+1); break; } } } // Returns index of the representative alignment, but in 'select' also // returns the indexes of the next best selected alignments in order by // score. return selectBuf_[0].second; } /** * Given that rs is already populated with alignments, consider the * alignment policy and make random selections where necessary. E.g. if we * found 10 alignments and the policy is -k 2 -m 20, select 2 alignments at * random. We "select" an alignment by setting the parallel entry in the * 'select' list to true. * * Return the "representative" alignment. This is simply the first one * selected. That will also be what SAM calls the "primary" alignment. */ template size_t AlnSinkWrap::selectAlnsToReport( const EList& rs, // alignments to select from uint64_t num, // number of alignments to select EList& select, // list to put results in RandomSource& rnd) const { assert(init_); assert(repOk()); assert_gt(num, 0); size_t sz = rs.size(); if(sz < num) { num = sz; } if(sz < 1) { return 0; } select.resize((size_t)num); if(sz == 1) { assert_eq(1, num); select[0] = 0; return 0; } // Select a random offset into the list of alignments uint32_t off = rnd.nextU32() % (uint32_t)sz; uint32_t offOrig = off; // Now take elements starting at that offset, wrapping around to 0 if // necessary. Leave the rest. for(size_t i = 0; i < num; i++) { select[i] = off; off++; if(off == sz) { off = 0; } } return offOrig; } #define NOT_SUPPRESSED !suppress_[field++] #define BEGIN_FIELD { \ if(firstfield) firstfield = false; \ else o.append('\t'); \ } #define WRITE_TAB { \ if(firstfield) firstfield = false; \ else o.append('\t'); \ } #define WRITE_NUM(o, x) { \ itoa10(x, buf); \ o.append(buf); \ } /** * Print a seed summary to the first output stream in the outs_ list. */ template void AlnSink::reportSeedSummary( BTString& o, const Read& rd, TReadId rdid, size_t threadId, const SeedResults& rs, bool getLock) { appendSeedSummary( o, // string to write to rd, // read rdid, // read id rs.numOffs()*2, // # seeds tried rs.nonzeroOffsets(), // # seeds with non-empty results rs.numRanges(), // # ranges for all seed hits rs.numElts(), // # elements for all seed hits rs.numOffs(), // # seeds tried from fw read rs.nonzeroOffsetsFw(), // # seeds with non-empty results from fw read rs.numRangesFw(), // # ranges for seed hits from fw read rs.numEltsFw(), // # elements for seed hits from fw read rs.numOffs(), // # seeds tried from rc read rs.nonzeroOffsetsRc(), // # seeds with non-empty results from fw read rs.numRangesRc(), // # ranges for seed hits from fw read rs.numEltsRc()); // # elements for seed hits from fw read } /** * Print an empty seed summary to the first output stream in the outs_ list. */ template void AlnSink::reportEmptySeedSummary( BTString& o, const Read& rd, TReadId rdid, size_t threadId, bool getLock) { appendSeedSummary( o, // string to append to rd, // read rdid, // read id 0, // # seeds tried 0, // # seeds with non-empty results 0, // # ranges for all seed hits 0, // # elements for all seed hits 0, // # seeds tried from fw read 0, // # seeds with non-empty results from fw read 0, // # ranges for seed hits from fw read 0, // # elements for seed hits from fw read 0, // # seeds tried from rc read 0, // # seeds with non-empty results from fw read 0, // # ranges for seed hits from fw read 0); // # elements for seed hits from fw read } /** * Print the given string. If ws = true, print only up to and not * including the first space or tab. Useful for printing reference * names. */ template static inline void printUptoWs( BTString& s, const T& str, bool chopws) { size_t len = str.length(); for(size_t i = 0; i < len; i++) { if(!chopws || (str[i] != ' ' && str[i] != '\t')) { s.append(str[i]); } else { break; } } } /** * Append a batch of unresolved seed alignment summary results (i.e. * seed alignments where all we know is the reference sequence aligned * to and its SA range, not where it falls in the reference * sequence) to the given output stream in Bowtie's seed-sumamry * verbose-mode format. * * The seed summary format is: * * - One line per read * - A typical line consists of a set of tab-delimited fields: * * 1. Read name * 2. Total number of seeds extracted from the read * 3. Total number of seeds that aligned to the reference at * least once (always <= field 2) * 4. Total number of distinct BW ranges found in all seed hits * (always >= field 3) * 5. Total number of distinct BW elements found in all seed * hits (always >= field 4) * 6-9.: Like 2-5. but just for seeds extracted from the * forward representation of the read * 10-13.: Like 2-5. but just for seeds extracted from the * reverse-complement representation of the read * * Note that fields 6 and 10 should add to field 2, 7 and 11 * should add to 3, etc. * * - Lines for reads that are filtered out for any reason (e.g. too * many Ns) have columns 2 through 13 set to 0. */ template void AlnSink::appendSeedSummary( BTString& o, const Read& rd, const TReadId rdid, size_t seedsTried, size_t nonzero, size_t ranges, size_t elts, size_t seedsTriedFw, size_t nonzeroFw, size_t rangesFw, size_t eltsFw, size_t seedsTriedRc, size_t nonzeroRc, size_t rangesRc, size_t eltsRc) { char buf[1024]; bool firstfield = true; // // Read name // BEGIN_FIELD; printUptoWs(o, rd.name, true); // // Total number of seeds tried // BEGIN_FIELD; WRITE_NUM(o, seedsTried); // // Total number of seeds tried where at least one range was found. // BEGIN_FIELD; WRITE_NUM(o, nonzero); // // Total number of ranges found // BEGIN_FIELD; WRITE_NUM(o, ranges); // // Total number of elements found // BEGIN_FIELD; WRITE_NUM(o, elts); // // The same four numbers, but only for seeds extracted from the // forward read representation. // BEGIN_FIELD; WRITE_NUM(o, seedsTriedFw); BEGIN_FIELD; WRITE_NUM(o, nonzeroFw); BEGIN_FIELD; WRITE_NUM(o, rangesFw); BEGIN_FIELD; WRITE_NUM(o, eltsFw); // // The same four numbers, but only for seeds extracted from the // reverse complement read representation. // BEGIN_FIELD; WRITE_NUM(o, seedsTriedRc); BEGIN_FIELD; WRITE_NUM(o, nonzeroRc); BEGIN_FIELD; WRITE_NUM(o, rangesRc); BEGIN_FIELD; WRITE_NUM(o, eltsRc); o.append('\n'); } /** * Append a single hit to the given output stream in Bowtie's * verbose-mode format. */ template void AlnSinkSam::appendMate( BTString& o, // append to this string StackedAln& staln, // store stacked alignment struct here const Read& rd, const Read* rdo, const TReadId rdid, AlnRes* rs, AlnRes* rso, const AlnSetSumm& summ, const SeedAlSumm& ssm, const SeedAlSumm& ssmo, const AlnFlags& flags, const PerReadMetrics& prm, const Mapq& mapqCalc, const Scoring& sc) { if(rs == NULL && samc_.omitUnalignedReads()) { return; } char buf[1024]; char mapqInps[1024]; if(rs != NULL) { staln.reset(); rs->initStacked(rd, staln); staln.leftAlign(false /* not past MMs */); } int offAdj = 0; // QNAME samc_.printReadName(o, rd.name, flags.partOfPair()); o.append('\t'); // FLAG int fl = 0; if(flags.partOfPair()) { fl |= SAM_FLAG_PAIRED; if(flags.alignedConcordant()) { fl |= SAM_FLAG_MAPPED_PAIRED; } if(!flags.mateAligned()) { // Other fragment is unmapped fl |= SAM_FLAG_MATE_UNMAPPED; } fl |= (flags.readMate1() ? SAM_FLAG_FIRST_IN_PAIR : SAM_FLAG_SECOND_IN_PAIR); if(flags.mateAligned() && rso != NULL) { if(!rso->fw()) { fl |= SAM_FLAG_MATE_STRAND; } } } if(!flags.isPrimary()) { fl |= SAM_FLAG_NOT_PRIMARY; } if(rs != NULL && !rs->fw()) { fl |= SAM_FLAG_QUERY_STRAND; } if(rs == NULL) { // Failed to align fl |= SAM_FLAG_UNMAPPED; } itoa10(fl, buf); o.append(buf); o.append('\t'); // RNAME if(rs != NULL) { samc_.printRefNameFromIndex(o, (size_t)rs->refid(), rs->repeat()); o.append('\t'); } else { if(summ.orefid() != -1) { // Opposite mate aligned but this one didn't - print the opposite // mate's RNAME and POS as is customary assert(flags.partOfPair()); samc_.printRefNameFromIndex(o, (size_t)summ.orefid(), summ.repeat()); } else { // No alignment o.append('*'); } o.append('\t'); } // POS // Note: POS is *after* soft clipping. I.e. POS points to the // upstream-most character *involved in the clipped alignment*. if(rs != NULL) { itoa10(rs->refoff()+1+offAdj, buf); o.append(buf); o.append('\t'); } else { if(summ.orefid() != -1) { // Opposite mate aligned but this one didn't - print the opposite // mate's RNAME and POS as is customary assert(flags.partOfPair()); itoa10(summ.orefoff()+1+offAdj, buf); o.append(buf); } else { // No alignment o.append('0'); } o.append('\t'); } // MAPQ mapqInps[0] = '\0'; if(rs != NULL) { itoa10(mapqCalc.mapq( summ, flags, rd.mate < 2, rd.length(), rdo == NULL ? 0 : rdo->length(), mapqInps), buf); o.append(buf); o.append('\t'); } else { // No alignment o.append("0\t"); } // CIGAR if(rs != NULL) { staln.buildCigar(false); staln.writeCigar(&o, NULL); o.append('\t'); } else { // No alignment o.append("*\t"); } // RNEXT if(rs != NULL && flags.partOfPair()) { if(rso != NULL && (rs->refid() != rso->refid() || rs->repeat() != rso->repeat())) { samc_.printRefNameFromIndex(o, (size_t)rso->refid(), rso->repeat()); o.append('\t'); } else { o.append("=\t"); } } else if(summ.orefid() != -1) { // The convention if this mate fails to align but the other doesn't is // to copy the mate's details into here o.append("=\t"); } else { o.append("*\t"); } // PNEXT if(rs != NULL && flags.partOfPair()) { if(rso != NULL) { itoa10(rso->refoff()+1, buf); o.append(buf); o.append('\t'); } else { // The convenstion is that if this mate aligns but the opposite // doesn't, we print this mate's offset here itoa10(rs->refoff()+1, buf); o.append(buf); o.append('\t'); } } else if(summ.orefid() != -1) { // The convention if this mate fails to align but the other doesn't is // to copy the mate's details into here itoa10(summ.orefoff()+1, buf); o.append(buf); o.append('\t'); } else { o.append("0\t"); } // ISIZE if(rs != NULL && rs->isFraglenSet()) { itoa10(rs->fragmentLength(), buf); o.append(buf); o.append('\t'); } else { // No fragment o.append("0\t"); } // SEQ if(!flags.isPrimary() && samc_.omitSecondarySeqQual()) { o.append('*'); } else { // Print the read if(rd.patFw.length() == 0) { o.append('*'); } else { if(rs == NULL || rs->fw()) { o.append(rd.patFw.toZBuf()); } else { o.append(rd.patRc.toZBuf()); } } } o.append('\t'); // QUAL if(!flags.isPrimary() && samc_.omitSecondarySeqQual()) { o.append('*'); } else { // Print the quals if(rd.qual.length() == 0) { o.append('*'); } else { if(rs == NULL || rs->fw()) { o.append(rd.qual.toZBuf()); } else { o.append(rd.qualRev.toZBuf()); } } } o.append('\t'); // // Optional fields // if(rs != NULL) { samc_.printAlignedOptFlags( o, // output buffer true, // first opt flag printed is first overall? rd, // read *rs, // individual alignment result staln, // stacked alignment flags, // alignment flags summ, // summary of alignments for this read ssm, // seed alignment summary prm, // per-read metrics sc, // scoring scheme mapqInps, // inputs to MAPQ calculation this->altdb_); } else { samc_.printEmptyOptFlags( o, // output buffer true, // first opt flag printed is first overall? rd, // read flags, // alignment flags summ, // summary of alignments for this read ssm, // seed alignment summary prm, // per-read metrics sc); // scoring scheme } o.append('\n'); } #endif /*ndef ALN_SINK_H_*/ hisat2-2.2.1/alphabet.cpp000066400000000000000000000455701370663763200152220ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include #include #include "alphabet.h" using namespace std; /** * Mapping from ASCII characters to DNA categories: * * 0 = invalid - error * 1 = DNA * 2 = IUPAC (ambiguous DNA) * 3 = not an error, but unmatchable; alignments containing this * character are invalid */ uint8_t asc2dnacat[] = { /* 0 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 16 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 32 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 3, 0, 0, /* - */ /* 48 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 64 */ 0, 1, 2, 1, 2, 0, 0, 1, 2, 0, 0, 2, 0, 2, 2, 0, /* A B C D G H K M N */ /* 80 */ 0, 0, 2, 2, 1, 0, 2, 2, 2, 2, 0, 0, 0, 0, 0, 0, /* R S T V W X Y */ /* 96 */ 0, 1, 2, 1, 2, 0, 0, 1, 2, 0, 0, 2, 0, 2, 2, 0, /* a b c d g h k m n */ /* 112 */ 0, 0, 2, 2, 1, 0, 2, 2, 2, 2, 0, 0, 0, 0, 0, 0, /* r s t v w x y */ /* 128 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 144 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 160 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 176 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 192 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 208 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 224 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 240 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, }; // 5-bit pop count int mask2popcnt[] = { 0, 1, 1, 2, 1, 2, 2, 3, 1, 2, 2, 3, 2, 3, 3, 4, 1, 2, 2, 3, 2, 3, 3, 4, 2, 3, 3, 4, 3, 4, 4, 5 }; /** * Mapping from masks to ASCII characters for ambiguous nucleotides. */ char mask2dna[] = { '?', // 0 'A', // 1 'C', // 2 'M', // 3 'G', // 4 'R', // 5 'S', // 6 'V', // 7 'T', // 8 'W', // 9 'Y', // 10 'H', // 11 'K', // 12 'D', // 13 'B', // 14 'N', // 15 (inclusive N) 'N' // 16 (exclusive N) }; /** * Mapping from ASCII characters for ambiguous nucleotides into masks: */ uint8_t asc2dnamask[] = { /* 0 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 16 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 32 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 48 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 64 */ 0, 1,14, 2,13, 0, 0, 4,11, 0, 0,12, 0, 3,15, 0, /* A B C D G H K M N */ /* 80 */ 0, 0, 5, 6, 8, 0, 7, 9, 0,10, 0, 0, 0, 0, 0, 0, /* R S T V W Y */ /* 96 */ 0, 1,14, 2,13, 0, 0, 4,11, 0, 0,12, 0, 3,15, 0, /* a b c d g h k m n */ /* 112 */ 0, 0, 5, 6, 8, 0, 7, 9, 0,10, 0, 0, 0, 0, 0, 0, /* r s t v w y */ /* 128 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 144 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 160 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 176 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 192 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 208 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 224 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 240 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, }; /** * Convert a pair of DNA masks to a color mask * * */ uint8_t dnamasks2colormask[16][16] = { /* 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 */ /* 0 */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 }, /* 1 */ { 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 }, /* 2 */ { 0, 2, 1, 3, 8, 10, 9, 11, 4, 6, 5, 7, 12, 14, 13, 15 }, /* 3 */ { 0, 3, 3, 3, 12, 15, 15, 15, 12, 15, 15, 15, 12, 15, 15, 15 }, /* 4 */ { 0, 4, 8, 12, 1, 5, 9, 13, 2, 6, 10, 14, 3, 7, 11, 15 }, /* 5 */ { 0, 5, 10, 15, 5, 5, 15, 15, 10, 15, 10, 15, 15, 15, 15, 15 }, /* 6 */ { 0, 6, 9, 15, 9, 15, 9, 15, 6, 6, 15, 15, 15, 15, 15, 15 }, /* 7 */ { 0, 7, 11, 15, 13, 15, 15, 15, 14, 15, 15, 15, 15, 15, 15, 15 }, /* 8 */ { 0, 8, 4, 12, 2, 10, 6, 14, 1, 9, 5, 13, 3, 11, 7, 15 }, /* 9 */ { 0, 9, 6, 15, 6, 15, 6, 15, 9, 9, 15, 15, 15, 15, 15, 15 }, /* 10 */ { 0, 10, 5, 15, 10, 10, 15, 15, 5, 15, 5, 15, 15, 15, 15, 15 }, /* 11 */ { 0, 11, 7, 15, 14, 15, 15, 15, 13, 15, 15, 15, 15, 15, 15, 15 }, /* 12 */ { 0, 12, 12, 12, 3, 15, 15, 15, 3, 15, 15, 15, 3, 15, 15, 15 }, /* 13 */ { 0, 13, 14, 15, 7, 15, 15, 15, 11, 15, 15, 15, 15, 15, 15, 15 }, /* 14 */ { 0, 14, 13, 15, 11, 15, 15, 15, 7, 15, 15, 15, 15, 15, 15, 15 }, /* 15 */ { 0, 15, 15, 15, 15, 15, 15, 15, 15, 15, 15, 15, 15, 15, 15, 15 } }; /** * Mapping from ASCII characters for ambiguous nucleotides into masks: */ char asc2dnacomp[] = { /* 0 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 16 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 32 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,'-', 0, 0, /* 48 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 64 */ 0,'T','V','G','H', 0, 0,'C','D', 0, 0,'M', 0,'K','N', 0, /* A B C D G H K M N */ /* 80 */ 0, 0,'Y','S','A', 0,'B','W', 0,'R', 0, 0, 0, 0, 0, 0, /* R S T V W Y */ /* 96 */ 0,'T','V','G','H', 0, 0,'C','D', 0, 0,'M', 0,'K','N', 0, /* a b c d g h k m n */ /* 112 */ 0, 0,'Y','S','A', 0,'B','W', 0,'R', 0, 0, 0, 0, 0, 0, /* r s t v w y */ /* 128 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 144 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 160 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 176 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 192 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 208 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 224 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 240 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 }; /** * Mapping from ASCII characters for ambiguous nucleotides into masks: */ char col2dna[] = { /* 0 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 16 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 32 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,'-','N', 0, /* - . */ /* 48 */'A','C','G','T','N', 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 0 1 2 3 4 */ /* 64 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 80 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 96 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 112 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 128 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 144 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 160 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 176 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 192 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 208 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 224 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 240 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 }; /** * Mapping from ASCII characters for ambiguous nucleotides into masks: */ char dna2col[] = { /* 0 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 16 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 32 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,'-', 0, 0, /* 48 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 64 */ 0,'0', 0,'1', 0, 0, 0,'2', 0, 0, 0, 0, 0, 0,'4', 0, /* A C G N */ /* 80 */ 0, 0, 0, 0,'3', 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* T */ /* 92 */ 0,'0', 0,'1', 0, 0, 0,'2', 0, 0, 0, 0, 0, 0,'4', 0, /* a c g n */ /* 112 */ 0, 0, 0, 0,'3', 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* t */ /* 128 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 144 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 160 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 176 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 192 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 208 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 224 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 240 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 }; /** * Mapping from ASCII characters for ambiguous nucleotides into masks: */ const char* dna2colstr[] = { /* 0 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", /* 16 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", /* 32 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "-", "?", "?", /* 48 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", /* 64 */ "?", "0","1|2|3","1","0|2|3","?", "?", "2","0|1|3","?", "?", "2|3", "?", "0|1", ".", "?", /* A B C D G H K M N */ /* 80 */ "?", "?", "0|2","1|2", "3", "?","0|1|2","0|3","?", "1|3", "?", "?", "?", "?", "?", "?", /* R S T V W Y */ /* 92 */ "?", "?","1|2|3","1","0|2|3","?", "?", "2","0|1|3","?", "?", "2|3", "?", "0|1", ".", "?", /* a b c d g h k m n */ /* 112 */ "?", "0", "0|2","1|2", "3", "?","0|1|2","0|3","?", "1|3", "?", "?", "?", "?", "?", "?", /* r s t v w y */ /* 128 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", /* 144 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", /* 160 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", /* 176 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", /* 192 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", /* 208 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", /* 224 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", /* 240 */ "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?", "?" }; /** * Mapping from ASCII characters to color categories: * * 0 = invalid - error * 1 = valid color * 2 = IUPAC (ambiguous DNA) - there is no such thing for colors to my * knowledge * 3 = not an error, but unmatchable; alignments containing this * character are invalid */ uint8_t asc2colcat[] = { /* 0 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 16 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 32 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 3, 3, 0, /* - . */ /* 48 */ 1, 1, 1, 1, 3, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 0 1 2 3 4 */ /* 64 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 80 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 96 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 112 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 128 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 144 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 160 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 176 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 192 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 208 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 224 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 240 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, }; /** * Set the category for all IUPAC codes. By default they're in * category 2 (IUPAC), but sometimes we'd like to put them in category * 3 (unmatchable), for example. */ void setIupacsCat(uint8_t cat) { assert(cat < 4); asc2dnacat[(int)'B'] = asc2dnacat[(int)'b'] = asc2dnacat[(int)'D'] = asc2dnacat[(int)'d'] = asc2dnacat[(int)'H'] = asc2dnacat[(int)'h'] = asc2dnacat[(int)'K'] = asc2dnacat[(int)'k'] = asc2dnacat[(int)'M'] = asc2dnacat[(int)'m'] = asc2dnacat[(int)'N'] = asc2dnacat[(int)'n'] = asc2dnacat[(int)'R'] = asc2dnacat[(int)'r'] = asc2dnacat[(int)'S'] = asc2dnacat[(int)'s'] = asc2dnacat[(int)'V'] = asc2dnacat[(int)'v'] = asc2dnacat[(int)'W'] = asc2dnacat[(int)'w'] = asc2dnacat[(int)'X'] = asc2dnacat[(int)'x'] = asc2dnacat[(int)'Y'] = asc2dnacat[(int)'y'] = cat; } /// For converting from ASCII to the Dna5 code where A=0, C=1, G=2, /// T=3, N=4 uint8_t asc2dna[] = { /* 0 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 16 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 32 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 48 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 64 */ 0, 0, 0, 1, 0, 0, 0, 2, 0, 0, 0, 0, 0, 0, 4, 0, /* A C G N */ /* 80 */ 0, 0, 0, 0, 3, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* T */ /* 96 */ 0, 0, 0, 1, 0, 0, 0, 2, 0, 0, 0, 0, 0, 0, 4, 0, /* a c g n */ /* 112 */ 0, 0, 0, 0, 3, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* t */ /* 128 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 144 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 160 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 176 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 192 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 208 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 224 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 240 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, }; /// Convert an ascii char representing a base or a color to a 2-bit /// code: 0=A,0; 1=C,1; 2=G,2; 3=T,3; 4=N,. uint8_t asc2dnaOrCol[] = { /* 0 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 16 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 32 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 4, 4, 0, /* - . */ /* 48 */ 0, 1, 2, 3, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 0 1 2 3 */ /* 64 */ 0, 0, 0, 1, 0, 0, 0, 2, 0, 0, 0, 0, 0, 0, 4, 0, /* A C G N */ /* 80 */ 0, 0, 0, 0, 3, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* T */ /* 96 */ 0, 0, 0, 1, 0, 0, 0, 2, 0, 0, 0, 0, 0, 0, 4, 0, /* a c g n */ /* 112 */ 0, 0, 0, 0, 3, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* t */ /* 128 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 144 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 160 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 176 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 192 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 208 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 224 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 240 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, }; /// For converting from ASCII to the Dna5 code where A=0, C=1, G=2, /// T=3, N=4 uint8_t asc2col[] = { /* 0 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 16 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 32 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 4, 4, 0, /* - . */ /* 48 */ 0, 1, 2, 3, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 0 1 2 3 */ /* 64 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 80 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 96 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 112 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 128 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 144 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 160 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 176 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 192 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 208 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 224 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, /* 240 */ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, }; /** * Convert a nucleotide and a color to the paired nucleotide. Indexed * first by nucleotide then by color. Note that this is exactly the * same as the dinuc2color array. */ uint8_t nuccol2nuc[5][5] = { /* B G O R . */ /* A */ {0, 1, 2, 3, 4}, /* C */ {1, 0, 3, 2, 4}, /* G */ {2, 3, 0, 1, 4}, /* T */ {3, 2, 1, 0, 4}, /* N */ {4, 4, 4, 4, 4} }; /** * Convert a pair of nucleotides to a color. */ uint8_t dinuc2color[5][5] = { /* A */ {0, 1, 2, 3, 4}, /* C */ {1, 0, 3, 2, 4}, /* G */ {2, 3, 0, 1, 4}, /* T */ {3, 2, 1, 0, 4}, /* N */ {4, 4, 4, 4, 4} }; /// Convert bit encoded DNA char to its complement int dnacomp[5] = { 3, 2, 1, 0, 4 }; const char *iupacs = "!ACMGRSVTWYHKDBN!acmgrsvtwyhkdbn"; char mask2iupac[16] = { -1, 'A', // 0001 'C', // 0010 'M', // 0011 'G', // 0100 'R', // 0101 'S', // 0110 'V', // 0111 'T', // 1000 'W', // 1001 'Y', // 1010 'H', // 1011 'K', // 1100 'D', // 1101 'B', // 1110 'N', // 1111 }; int maskcomp[16] = { 0, // 0000 (!) -> 0000 (!) 8, // 0001 (A) -> 1000 (T) 4, // 0010 (C) -> 0100 (G) 12, // 0011 (M) -> 1100 (K) 2, // 0100 (G) -> 0010 (C) 10, // 0101 (R) -> 1010 (Y) 6, // 0110 (S) -> 0110 (S) 14, // 0111 (V) -> 1110 (B) 1, // 1000 (T) -> 0001 (A) 9, // 1001 (W) -> 1001 (W) 5, // 1010 (Y) -> 0101 (R) 13, // 1011 (H) -> 1101 (D) 3, // 1100 (K) -> 0011 (M) 11, // 1101 (D) -> 1011 (H) 7, // 1110 (B) -> 0111 (V) 15, // 1111 (N) -> 1111 (N) }; hisat2-2.2.1/alphabet.h000066400000000000000000000127631370663763200146650ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ALPHABETS_H_ #define ALPHABETS_H_ #include #include #include #include #include "assert_helpers.h" using namespace std; /// Convert an ascii char to a DNA category. Categories are: /// 0 -> invalid /// 1 -> unambiguous a, c, g or t /// 2 -> ambiguous /// 3 -> unmatchable extern uint8_t asc2dnacat[]; /// Convert masks to ambiguous nucleotides extern char mask2dna[]; /// Convert ambiguous ASCII nuceleotide to mask extern uint8_t asc2dnamask[]; /// Convert mask to # of alternative in the mask extern int mask2popcnt[]; /// Convert an ascii char to a 2-bit base: 0=A, 1=C, 2=G, 3=T, 4=N extern uint8_t asc2dna[]; /// Convert an ascii char representing a base or a color to a 2-bit /// code: 0=A,0; 1=C,1; 2=G,2; 3=T,3; 4=N,. extern uint8_t asc2dnaOrCol[]; /// Convert a pair of DNA masks to a color mask extern uint8_t dnamasks2colormask[16][16]; /// Convert an ascii char to a color category. Categories are: /// 0 -> invalid /// 1 -> unambiguous 0, 1, 2 or 3 /// 2 -> ambiguous (not applicable for colors) /// 3 -> unmatchable extern uint8_t asc2colcat[]; /// Convert an ascii char to a 2-bit base: 0=A, 1=C, 2=G, 3=T, 4=N extern uint8_t asc2col[]; /// Convert an ascii char to its DNA complement, including IUPACs extern char asc2dnacomp[]; /// Convert a pair of 2-bit (and 4=N) encoded DNA bases to a color extern uint8_t dinuc2color[5][5]; /// Convert a 2-bit nucleotide (and 4=N) and a color to the /// corresponding 2-bit nucleotide extern uint8_t nuccol2nuc[5][5]; /// Convert a 4-bit mask into an IUPAC code extern char mask2iupac[16]; /// Convert an ascii color to an ascii dna char extern char col2dna[]; /// Convert an ascii dna to a color char extern char dna2col[]; /// Convert an ascii dna to a color char extern const char* dna2colstr[]; /// Convert bit encoded DNA char to its complement extern int dnacomp[5]; /// String of all DNA and IUPAC characters extern const char *iupacs; /// Map from masks to their reverse-complement masks extern int maskcomp[16]; /** * Return true iff c is a Dna character. */ static inline bool isDna(char c) { return asc2dnacat[(int)c] > 0; } /** * Return true iff c is a color character. */ static inline bool isColor(char c) { return asc2colcat[(int)c] > 0; } /** * Return true iff c is an ambiguous Dna character. */ static inline bool isAmbigNuc(char c) { return asc2dnacat[(int)c] == 2; } /** * Return true iff c is an ambiguous color character. */ static inline bool isAmbigColor(char c) { return asc2colcat[(int)c] == 2; } /** * Return true iff c is an ambiguous character. */ static inline bool isAmbig(char c, bool color) { return (color ? asc2colcat[(int)c] : asc2dnacat[(int)c]) == 2; } /** * Return true iff c is an unambiguous DNA character. */ static inline bool isUnambigNuc(char c) { return asc2dnacat[(int)c] == 1; } /** * Return the DNA complement of the given ASCII char. */ static inline char comp(char c) { switch(c) { case 'a': return 't'; case 'A': return 'T'; case 'c': return 'g'; case 'C': return 'G'; case 'g': return 'c'; case 'G': return 'C'; case 't': return 'a'; case 'T': return 'A'; default: return c; } } /** * Return the reverse complement of a bit-encoded nucleotide. */ static inline int compDna(int c) { assert_leq(c, 4); return dnacomp[c]; } /** * Return true iff c is an unambiguous Dna character. */ static inline bool isUnambigDna(char c) { return asc2dnacat[(int)c] == 1; } /** * Return true iff c is an unambiguous color character (0,1,2,3). */ static inline bool isUnambigColor(char c) { return asc2colcat[(int)c] == 1; } /// Convert a pair of 2-bit (and 4=N) encoded DNA bases to a color extern uint8_t dinuc2color[5][5]; /** * Decode a not-necessarily-ambiguous nucleotide. */ static inline void decodeNuc(char c , int& num, int *alts) { switch(c) { case 'A': alts[0] = 0; num = 1; break; case 'C': alts[0] = 1; num = 1; break; case 'G': alts[0] = 2; num = 1; break; case 'T': alts[0] = 3; num = 1; break; case 'M': alts[0] = 0; alts[1] = 1; num = 2; break; case 'R': alts[0] = 0; alts[1] = 2; num = 2; break; case 'W': alts[0] = 0; alts[1] = 3; num = 2; break; case 'S': alts[0] = 1; alts[1] = 2; num = 2; break; case 'Y': alts[0] = 1; alts[1] = 3; num = 2; break; case 'K': alts[0] = 2; alts[1] = 3; num = 2; break; case 'V': alts[0] = 0; alts[1] = 1; alts[2] = 2; num = 3; break; case 'H': alts[0] = 0; alts[1] = 1; alts[2] = 3; num = 3; break; case 'D': alts[0] = 0; alts[1] = 2; alts[2] = 3; num = 3; break; case 'B': alts[0] = 1; alts[1] = 2; alts[2] = 3; num = 3; break; case 'N': alts[0] = 0; alts[1] = 1; alts[2] = 2; alts[3] = 3; num = 4; break; default: { std::cerr << "Bad IUPAC code: " << c << ", (int: " << (int)c << ")" << std::endl; throw std::runtime_error(""); } } } extern void setIupacsCat(uint8_t cat); #endif /*ALPHABETS_H_*/ hisat2-2.2.1/alt.h000066400000000000000000000204111370663763200136520ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef ALT_H_ #define ALT_H_ #include #include #include #include "assert_helpers.h" #include "word_io.h" #include "mem_ids.h" using namespace std; enum ALT_TYPE { ALT_NONE = 0, ALT_SNP_SGL, // single nucleotide substitution ALT_SNP_INS, // small insertion wrt reference genome ALT_SNP_DEL, // small deletion wrt reference genome ALT_SNP_ALT, // alternative sequence (to be implemented ...) ALT_SPLICESITE, ALT_EXON }; template struct ALT { ALT() { reset(); } void reset() { type = ALT_NONE; pos = len = 0; seq = 0; } ALT_TYPE type; union { index_t pos; index_t left; }; union { index_t len; index_t right; }; union { uint64_t seq; // used to store 32 bp, but it can be used to store a pointer to EList struct { union { bool fw; bool reversed; }; bool excluded; }; }; public: // in order to support a sequence longer than 32 bp bool snp() const { return type == ALT_SNP_SGL || type == ALT_SNP_DEL || type == ALT_SNP_INS; } bool splicesite() const { return type == ALT_SPLICESITE; } bool mismatch() const { return type == ALT_SNP_SGL; } bool gap() const { return type == ALT_SNP_DEL || type == ALT_SNP_INS || type == ALT_SPLICESITE; } bool deletion() const { return type == ALT_SNP_DEL; } bool insertion() const { return type == ALT_SNP_INS; } bool exon() const { return type == ALT_EXON; } bool operator< (const ALT& o) const { if(pos != o.pos) return pos < o.pos; if(type != o.type) { if(type == ALT_NONE || o.type == ALT_NONE) { return type == ALT_NONE; } if(type == ALT_SNP_INS) return true; else if(o.type == ALT_SNP_INS) return false; return type < o.type; } if(len != o.len) return len < o.len; if(seq != o.seq) return seq < o.seq; return false; } bool compatibleWith(const ALT& o) const { if(pos == o.pos) return false; // sort the two SNPs const ALT& a = (pos < o.pos ? *this : o); const ALT& b = (pos < o.pos ? o : *this); if(a.snp()) { if(a.type == ALT_SNP_DEL || a.type == ALT_SNP_INS) { if(b.pos <= a.pos + a.len) { return false; } } } else if(a.splicesite()) { if(b.pos <= a.right + 2) { return false; } } else { assert(false); } return true; } bool isSame(const ALT& o) const { if(type != o.type) return false; if(type == ALT_SNP_SGL) { return pos == o.pos && seq == o.seq; } else if(type == ALT_SNP_DEL || type == ALT_SNP_INS || type == ALT_SPLICESITE) { if(type == ALT_SNP_INS) { if(seq != o.seq) return false; } if(reversed == o.reversed) { return pos == o.pos && len == o.len; } else { if(reversed) { return pos - len + 1 == o.pos && len == o.len; } else { return pos == o.pos - o.len + 1 && len == o.len; } } } else { assert(false); } return true; } #ifndef NDEBUG bool repOk() const { if(type == ALT_SNP_SGL) { if(len != 1) { assert(false); return false; } if(seq > 3) { assert(false); return false; } } else if(type == ALT_SNP_DEL) { if(len <= 0) { assert(false); return false; } if(seq != 0) { assert(false); return false; } } else if(type == ALT_SNP_INS) { if(len <= 0) { assert(false); return false; } } else if(type == ALT_SPLICESITE) { assert_lt(left, right); assert_leq(fw, 1); }else { assert(false); return false; } return true; } #endif bool write(ofstream& f_out, bool bigEndian) const { writeIndex(f_out, pos, bigEndian); writeU32(f_out, type, bigEndian); writeIndex(f_out, len, bigEndian); writeIndex(f_out, seq, bigEndian); return true; } bool read(ifstream& f_in, bool bigEndian) { pos = readIndex(f_in, bigEndian); type = (ALT_TYPE)readU32(f_in, bigEndian); assert_neq(type, ALT_SNP_ALT); len = readIndex(f_in, bigEndian); seq = readIndex(f_in, bigEndian); return true; } }; template struct Haplotype { Haplotype() { reset(); } void reset() { left = right = 0; alts.clear(); } index_t left; index_t right; EList alts; bool operator< (const Haplotype& o) const { if(left != o.left) return left < o.left; if(right != o.right) return right < o.right; return false; } bool write(ofstream& f_out, bool bigEndian) const { writeIndex(f_out, left, bigEndian); writeIndex(f_out, right, bigEndian); writeIndex(f_out, alts.size(), bigEndian); for(index_t i = 0; i < alts.size(); i++) { writeIndex(f_out, alts[i], bigEndian); } return true; } bool read(ifstream& f_in, bool bigEndian) { left = readIndex(f_in, bigEndian); right = readIndex(f_in, bigEndian); assert_leq(left, right); index_t num_alts = readIndex(f_in, bigEndian); alts.resizeExact(num_alts); alts.clear(); for(index_t i = 0; i < num_alts; i++) { alts.push_back(readIndex(f_in, bigEndian)); } return true; } }; template class ALTDB { public: ALTDB() : _snp(false), _ss(false), _exon(false) {} virtual ~ALTDB() {} bool hasSNPs() const { return _snp; } bool hasSpliceSites() const { return _ss; } bool hasExons() const { return _exon; } void setSNPs(bool snp) { _snp = snp; } void setSpliceSites(bool ss) { _ss = ss; } void setExons(bool exon) { _exon = exon; } EList >& alts() { return _alts; } EList& altnames() { return _altnames; } EList >& haplotypes() { return _haplotypes; } EList& haplotype_maxrights() { return _haplotype_maxrights; } const EList >& alts() const { return _alts; } const EList& altnames() const { return _altnames; } const EList >& haplotypes() const { return _haplotypes; } const EList& haplotype_maxrights() const { return _haplotype_maxrights; } private: bool _snp; bool _ss; bool _exon; EList > _alts; EList _altnames; EList > _haplotypes; EList _haplotype_maxrights; }; #endif /*ifndef ALT_H_*/ hisat2-2.2.1/assert_helpers.h000066400000000000000000000225211370663763200161210ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ASSERT_HELPERS_H_ #define ASSERT_HELPERS_H_ #include #include #include #include /** * Assertion for release-enabled assertions */ class ReleaseAssertException : public std::runtime_error { public: ReleaseAssertException(const std::string& msg = "") : std::runtime_error(msg) {} }; /** * Macros for release-enabled assertions, and helper macros to make * all assertion error messages more helpful. */ #ifndef NDEBUG #define ASSERT_ONLY(...) __VA_ARGS__ #else #define ASSERT_ONLY(...) #endif #define rt_assert(b) \ if(!(b)) { \ std::cout << "rt_assert at " << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(); \ } #define rt_assert_msg(b,msg) \ if(!(b)) { \ std::cout << msg << " at " << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(msg); \ } #define rt_assert_eq(ex,ac) \ if(!((ex) == (ac))) { \ std::cout << "rt_assert_eq: expected (" << (ex) << ", 0x" << std::hex << (ex) << std::dec << ") got (" << (ac) << ", 0x" << std::hex << (ac) << std::dec << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(); \ } #define rt_assert_eq_msg(ex,ac,msg) \ if(!((ex) == (ac))) { \ std::cout << "rt_assert_eq: " << msg << ": (" << (ex) << ", 0x" << std::hex << (ex) << std::dec << ") got (" << (ac) << ", 0x" << std::hex << (ac) << std::dec << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(msg); \ } #ifndef NDEBUG #define assert_eq(ex,ac) \ if(!((ex) == (ac))) { \ std::cout << "assert_eq: expected (" << (ex) << ", 0x" << std::hex << (ex) << std::dec << ") got (" << (ac) << ", 0x" << std::hex << (ac) << std::dec << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #define assert_eq_msg(ex,ac,msg) \ if(!((ex) == (ac))) { \ std::cout << "assert_eq: " << msg << ": (" << (ex) << ", 0x" << std::hex << (ex) << std::dec << ") got (" << (ac) << ", 0x" << std::hex << (ac) << std::dec << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #else #define assert_eq(ex,ac) #define assert_eq_msg(ex,ac,msg) #endif #define rt_assert_neq(ex,ac) \ if(!((ex) != (ac))) { \ std::cout << "rt_assert_neq: expected not (" << (ex) << ", 0x" << std::hex << (ex) << std::dec << ") got (" << (ac) << ", 0x" << std::hex << (ac) << std::dec << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(); \ } #define rt_assert_neq_msg(ex,ac,msg) \ if(!((ex) != (ac))) { \ std::cout << "rt_assert_neq: " << msg << ": (" << (ex) << ", 0x" << std::hex << (ex) << std::dec << ") got (" << (ac) << ", 0x" << std::hex << (ac) << std::dec << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(msg); \ } #ifndef NDEBUG #define assert_neq(ex,ac) \ if(!((ex) != (ac))) { \ std::cout << "assert_neq: expected not (" << (ex) << ", 0x" << std::hex << (ex) << std::dec << ") got (" << (ac) << ", 0x" << std::hex << (ac) << std::dec << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #define assert_neq_msg(ex,ac,msg) \ if(!((ex) != (ac))) { \ std::cout << "assert_neq: " << msg << ": (" << (ex) << ", 0x" << std::hex << (ex) << std::dec << ") got (" << (ac) << ", 0x" << std::hex << (ac) << std::dec << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #else #define assert_neq(ex,ac) #define assert_neq_msg(ex,ac,msg) #endif #define rt_assert_gt(a,b) \ if(!((a) > (b))) { \ std::cout << "rt_assert_gt: expected (" << (a) << ") > (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(); \ } #define rt_assert_gt_msg(a,b,msg) \ if(!((a) > (b))) { \ std::cout << "rt_assert_gt: " << msg << ": (" << (a) << ") > (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(msg); \ } #ifndef NDEBUG #define assert_gt(a,b) \ if(!((a) > (b))) { \ std::cout << "assert_gt: expected (" << (a) << ") > (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #define assert_gt_msg(a,b,msg) \ if(!((a) > (b))) { \ std::cout << "assert_gt: " << msg << ": (" << (a) << ") > (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #else #define assert_gt(a,b) #define assert_gt_msg(a,b,msg) #endif #define rt_assert_geq(a,b) \ if(!((a) >= (b))) { \ std::cout << "rt_assert_geq: expected (" << (a) << ") >= (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(); \ } #define rt_assert_geq_msg(a,b,msg) \ if(!((a) >= (b))) { \ std::cout << "rt_assert_geq: " << msg << ": (" << (a) << ") >= (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(msg); \ } #ifndef NDEBUG #define assert_geq(a,b) \ if(!((a) >= (b))) { \ std::cout << "assert_geq: expected (" << (a) << ") >= (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #define assert_geq_msg(a,b,msg) \ if(!((a) >= (b))) { \ std::cout << "assert_geq: " << msg << ": (" << (a) << ") >= (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #else #define assert_geq(a,b) #define assert_geq_msg(a,b,msg) #endif #define rt_assert_lt(a,b) \ if(!(a < b)) { \ std::cout << "rt_assert_lt: expected (" << a << ") < (" << b << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(); \ } #define rt_assert_lt_msg(a,b,msg) \ if(!(a < b)) { \ std::cout << "rt_assert_lt: " << msg << ": (" << a << ") < (" << b << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(msg); \ } #ifndef NDEBUG #define assert_lt(a,b) \ if(!(a < b)) { \ std::cout << "assert_lt: expected (" << a << ") < (" << b << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #define assert_lt_msg(a,b,msg) \ if(!(a < b)) { \ std::cout << "assert_lt: " << msg << ": (" << a << ") < (" << b << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #else #define assert_lt(a,b) #define assert_lt_msg(a,b,msg) #endif #define rt_assert_leq(a,b) \ if(!((a) <= (b))) { \ std::cout << "rt_assert_leq: expected (" << (a) << ") <= (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(); \ } #define rt_assert_leq_msg(a,b,msg) \ if(!((a) <= (b))) { \ std::cout << "rt_assert_leq: " << msg << ": (" << (a) << ") <= (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ throw ReleaseAssertException(msg); \ } #ifndef NDEBUG #define assert_leq(a,b) \ if(!((a) <= (b))) { \ std::cout << "assert_leq: expected (" << (a) << ") <= (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #define assert_leq_msg(a,b,msg) \ if(!((a) <= (b))) { \ std::cout << "assert_leq: " << msg << ": (" << (a) << ") <= (" << (b) << ")" << std::endl; \ std::cout << __FILE__ << ":" << __LINE__ << std::endl; \ assert(0); \ } #else #define assert_leq(a,b) #define assert_leq_msg(a,b,msg) #endif #ifndef NDEBUG #define assert_in(c, s) assert_in2(c, s, __FILE__, __LINE__) static inline void assert_in2(char c, const char *str, const char *file, int line) { const char *s = str; while(*s != '\0') { if(c == *s) return; s++; } std::cout << "assert_in: (" << c << ") not in (" << str << ")" << std::endl; std::cout << file << ":" << line << std::endl; assert(0); } #else #define assert_in(c, s) #endif #ifndef NDEBUG #define assert_range(b, e, v) assert_range_helper(b, e, v, __FILE__, __LINE__) template inline static void assert_range_helper(const T& begin, const T& end, const T& val, const char *file, int line) { if(val < begin || val > end) { std::cout << "assert_range: (" << val << ") not in [" << begin << ", " << end << "]" << std::endl; std::cout << file << ":" << line << std::endl; assert(0); } } #else #define assert_range(b, e, v) #endif #endif /*ASSERT_HELPERS_H_*/ hisat2-2.2.1/banded.cpp000066400000000000000000000014671370663763200146540ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include "banded.h" #ifdef MAIN_BANDED int main(void) { } #endif hisat2-2.2.1/banded.h000066400000000000000000000023351370663763200143140ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef BANDED_H_ #define BANDED_H_ #include "sse_util.h" /** * Use SSE instructions to quickly find stretches with lots of matches, then * resolve alignments. */ class BandedSseAligner { public: void init( int *q, // query, maskized size_t qi, // query start size_t qf, // query end int *r, // reference, maskized size_t ri, // reference start size_t rf) // reference end { } void nextAlignment() { } protected: EList_m128i mat_; }; #endif hisat2-2.2.1/binary_sa_search.h000066400000000000000000000067171370663763200164030ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef BINARY_SA_SEARCH_H_ #define BINARY_SA_SEARCH_H_ #include #include #include #include "alphabet.h" #include "assert_helpers.h" #include "ds.h" #include "btypes.h" /** * Do a binary search using the suffix of 'host' beginning at offset * 'qry' as the query and 'sa' as an already-lexicographically-sorted * list of suffixes of host. 'sa' may be all suffixes of host or just * a subset. Returns the index in sa of the smallest suffix of host * that is larger than qry, or length(sa) if all suffixes of host are * less than qry. * * We use the Manber and Myers optimization of maintaining a pair of * counters for the longest lcp observed so far on the left- and right- * hand sides and using the min of the two as a way of skipping over * characters at the beginning of a new round. * * Returns maximum value if the query suffix matches an element of sa. */ template inline TIndexOffU binarySASearch( const TStr& host, TIndexOffU qry, const EList& sa) { TIndexOffU lLcp = 0, rLcp = 0; // greatest observed LCPs on left and right TIndexOffU l = 0, r = (TIndexOffU)sa.size()+1; // binary-search window TIndexOffU hostLen = (TIndexOffU)host.length(); while(true) { assert_gt(r, l); TIndexOffU m = (l+r) >> 1; if(m == l) { // Binary-search window has closed: we have an answer if(m > 0 && sa[m-1] == qry) { return std::numeric_limits::max(); // qry matches } assert_leq(m, sa.size()); return m; // Return index of right-hand suffix } assert_gt(m, 0); TIndexOffU suf = sa[m-1]; if(suf == qry) { return std::numeric_limits::max(); // query matches an elt of sa } TIndexOffU lcp = min(lLcp, rLcp); #ifndef NDEBUG if(sstr_suf_upto_neq(host, qry, host, suf, lcp)) { assert(0); } #endif // Keep advancing lcp, but stop when query mismatches host or // when the counter falls off either the query or the suffix while(suf+lcp < hostLen && qry+lcp < hostLen && host[suf+lcp] == host[qry+lcp]) { lcp++; } // Fell off the end of either the query or the sa elt? bool fell = (suf+lcp == hostLen || qry+lcp == hostLen); if((fell && qry+lcp == hostLen) || (!fell && host[suf+lcp] < host[qry+lcp])) { // Query is greater than sa elt l = m; // update left bound lLcp = max(lLcp, lcp); // update left lcp } else if((fell && suf+lcp == hostLen) || (!fell && host[suf+lcp] > host[qry+lcp])) { // Query is less than sa elt r = m; // update right bound rLcp = max(rLcp, lcp); // update right lcp } else { assert(false); // Must be one or the other! } } // Shouldn't get here assert(false); return std::numeric_limits::max(); } #endif /*BINARY_SA_SEARCH_H_*/ hisat2-2.2.1/bit_packed_array.cpp000066400000000000000000000162031370663763200167140ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include #include #include "timer.h" #include "aligner_sw.h" #include "aligner_result.h" #include "scoring.h" #include "sstring.h" #include "bit_packed_array.h" TIndexOffU BitPackedArray::get(size_t index) const { assert_lt(index, cur_); pair addr = indexToAddress(index); uint64_t *block = blocks_[addr.first]; pair pos = columnToPosition(addr.second); TIndexOffU val = getItem(block, pos.first, pos.second); return val; } #define write_fp(x) fp.write((const char *)&(x), sizeof((x))) void BitPackedArray::writeFile(ofstream &fp) { size_t sz = 0; write_fp(item_bit_size_); write_fp(elm_bit_size_); write_fp(items_per_block_bit_); write_fp(items_per_block_bit_mask_); write_fp(items_per_block_); write_fp(cur_); write_fp(sz_); write_fp(block_size_); // number of blocks sz = blocks_.size(); write_fp(sz); for(size_t i = 0; i < sz; i++) { fp.write((const char *)blocks_[i], block_size_); } } void BitPackedArray::writeFile(const char *filename) { ofstream fp(filename, std::ofstream::binary); writeFile(fp); fp.close(); } void BitPackedArray::writeFile(const string &filename) { writeFile(filename.c_str()); } #define read_fp(x) fp.read((char *)&(x), sizeof((x))) void BitPackedArray::readFile(ifstream &fp) { size_t val_sz = 0; read_fp(val_sz); init_by_log2(val_sz); //rt_assert_eq(val_sz, item_bit_size_); read_fp(val_sz); rt_assert_eq(val_sz, elm_bit_size_); read_fp(val_sz); rt_assert_eq(val_sz, items_per_block_bit_); read_fp(val_sz); rt_assert_eq(val_sz, items_per_block_bit_mask_); read_fp(val_sz); rt_assert_eq(val_sz, items_per_block_); // skip cur_ size_t prev_cnt = 0; read_fp(prev_cnt); cur_ = 0; // skip sz_ size_t prev_sz = 0; read_fp(prev_sz); sz_ = 0; // block_size_ read_fp(val_sz); rt_assert_eq(val_sz, block_size_); // alloc blocks allocItems(prev_cnt); rt_assert_eq(prev_sz, sz_); // number of blocks read_fp(val_sz); rt_assert_eq(val_sz, blocks_.size()); for(size_t i = 0; i < blocks_.size(); i++) { fp.read((char *)blocks_[i], block_size_); } cur_ = prev_cnt; } void BitPackedArray::readFile(const char *filename) { ifstream fp(filename, std::ifstream::binary); readFile(fp); fp.close(); } void BitPackedArray::readFile(const string &filename) { readFile(filename.c_str()); } void BitPackedArray::put(size_t index, TIndexOffU val) { assert_lt(index, cur_); pair addr = indexToAddress(index); uint64_t *block = blocks_[addr.first]; pair pos = columnToPosition(addr.second); setItem(block, pos.first, pos.second, val); } void BitPackedArray::pushBack(TIndexOffU val) { if(cur_ == sz_) { allocItems(items_per_block_); } put(cur_++, val); assert_leq(cur_, sz_); } TIndexOffU BitPackedArray::getItem(uint64_t *block, size_t idx, size_t offset) const { size_t remains = item_bit_size_; TIndexOffU val = 0; while(remains > 0) { size_t bits = min(elm_bit_size_ - offset, remains); uint64_t mask = bitToMask(bits); // get value from block TIndexOffU t = (block[idx] >> offset) & mask; val = val | (t << (item_bit_size_ - remains)); remains -= bits; offset = 0; idx++; } return val; } void BitPackedArray::setItem(uint64_t *block, size_t idx, size_t offset, TIndexOffU val) { size_t remains = item_bit_size_; while(remains > 0) { size_t bits = min(elm_bit_size_ - offset, remains); uint64_t mask = bitToMask(bits); uint64_t dest_mask = mask << offset; // get 'bits' lsb from val uint64_t t = val & mask; val >>= bits; // save 't' to block[idx] t <<= offset; block[idx] &= ~(dest_mask); // clear block[idx] |= t; idx++; remains -= bits; offset = 0; } } pair BitPackedArray::indexToAddress(size_t index) const { pair addr; addr.first = index >> items_per_block_bit_; addr.second = index & items_per_block_bit_mask_; return addr; } pair BitPackedArray::columnToPosition(size_t col) const { pair pos; pos.first = (col * item_bit_size_) / elm_bit_size_; pos.second = (col * item_bit_size_) % elm_bit_size_; return pos; } void BitPackedArray::expand(size_t count) { if((cur_ + count) > sz_) { allocItems(count); } cur_ += count; assert_leq(cur_, sz_); } void BitPackedArray::allocSize(size_t sz) { size_t num_block = (sz * sizeof(uint64_t) + block_size_ - 1) / block_size_; for(size_t i = 0; i < num_block; i++) { uint64_t *ptr = new uint64_t[block_size_]; blocks_.push_back(ptr); sz_ += items_per_block_; } } void BitPackedArray::allocItems(size_t count) { size_t sz = (count * item_bit_size_ + elm_bit_size_ - 1) / elm_bit_size_; allocSize(sz); } void BitPackedArray::init_by_log2(size_t ceil_log2) { item_bit_size_ = ceil_log2; elm_bit_size_ = sizeof(uint64_t) * 8; items_per_block_bit_ = 20; // 1M items_per_block_ = 1ULL << (items_per_block_bit_); items_per_block_bit_mask_ = items_per_block_ - 1; block_size_ = (items_per_block_ * item_bit_size_ + elm_bit_size_ - 1) / elm_bit_size_ * sizeof(uint64_t); cur_ = 0; sz_ = 0; } void BitPackedArray::init(size_t max_value) { init_by_log2((size_t)ceil(log2(max_value))); } void BitPackedArray::dump() const { cerr << "item_bit_size_: " << item_bit_size_ << endl; cerr << "block_size_: " << block_size_ << endl; cerr << "items_per_block_: " << items_per_block_ << endl; cerr << "cur_: " << cur_ << endl; cerr << "sz_: " << sz_ << endl; cerr << "number of blocks: " << blocks_.size() << endl; } size_t BitPackedArray::getMemUsage() const { size_t tot = blocks_.size() * block_size_; tot += blocks_.totalCapacityBytes(); return tot; } BitPackedArray::~BitPackedArray() { for(size_t i = 0; i < blocks_.size(); i++) { uint64_t *ptr = blocks_[i]; delete [] ptr; } } void BitPackedArray::reset() { cur_ = 0; sz_ = 0; for(size_t i = 0; i < blocks_.size(); i++) { uint64_t *ptr = blocks_[i]; delete [] ptr; } blocks_.clear(); } hisat2-2.2.1/bit_packed_array.h000066400000000000000000000054351370663763200163660ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef __HISAT2_BIT_PACKED_ARRAY_H #define __HISAT2_BIT_PACKED_ARRAY_H #include #include #include #include #include "assert_helpers.h" #include "word_io.h" #include "mem_ids.h" #include "ds.h" using namespace std; class BitPackedArray { public: BitPackedArray () {} ~BitPackedArray(); /** * Return true iff there are no items * @return */ inline bool empty() const { return cur_ == 0; } inline size_t size() const { return cur_; } TIndexOffU get(size_t idx) const; inline TIndexOffU operator[](size_t i) const { return get(i); } void pushBack(TIndexOffU val); void init(size_t max_value); void reset(); void writeFile(const char *filename); void writeFile(const string& filename); void writeFile(ofstream &fp); void readFile(const char *filename); void readFile(const string& filename); void readFile(ifstream &fp); void dump() const; size_t getMemUsage() const; private: void init_by_log2(size_t ceil_log2); void put(size_t index, TIndexOffU val); inline uint64_t bitToMask(size_t bit) const { return (uint64_t) ((1ULL << bit) - 1); } TIndexOffU getItem(uint64_t *block, size_t idx, size_t offset) const; void setItem(uint64_t *block, size_t idx, size_t offset, TIndexOffU val); pair indexToAddress(size_t index) const; pair columnToPosition(size_t col) const; void expand(size_t count = 1); void allocSize(size_t sz); void allocItems(size_t count); private: size_t item_bit_size_; // item bit size(e.g. 33bit) size_t elm_bit_size_; // 64bit size_t items_per_block_bit_; size_t items_per_block_bit_mask_; size_t items_per_block_; // number of items in block size_t cur_; // current item count size_t sz_; // maximum item count size_t block_size_; // block size in byte // List of packed array EList blocks_; }; #endif //__HISAT2_BIT_PACKED_ARRAY_H hisat2-2.2.1/bitpack.h000066400000000000000000000042411370663763200145120ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef BITPACK_H_ #define BITPACK_H_ #include #include "assert_helpers.h" /** * Routines for marshalling 2-bit values into and out of 8-bit or * 32-bit hosts */ static inline void pack_2b_in_8b(const int two, uint8_t& eight, const int off) { assert_lt(two, 4); assert_lt(off, 4); eight |= (two << (off*2)); } static inline int unpack_2b_from_8b(const uint8_t eight, const int off) { assert_lt(off, 4); return ((eight >> (off*2)) & 0x3); } static inline void pack_2b_in_32b(const int two, uint32_t& thirty2, const int off) { assert_lt(two, 4); assert_lt(off, 16); thirty2 |= (two << (off*2)); } static inline int unpack_2b_from_32b(const uint32_t thirty2, const int off) { assert_lt(off, 16); return ((thirty2 >> (off*2)) & 0x3); } /** * Routines for marshalling 1-bit values into and out of 8-bit or * 32-bit hosts */ static inline void pack_1b_in_8b(const int one, uint8_t& eight, const int off) { assert_lt(one, 2); assert_lt(off, 8); eight |= (one << off); } static inline int unpack_1b_from_8b(const uint8_t eight, const int off) { assert_lt(off, 2); return ((eight >> off) & 0x1); } static inline void pack_1b_in_32b(const int one, uint32_t& thirty2, const int off) { assert_lt(one, 2); assert_lt(off, 32); thirty2 |= (one << off); } static inline int unpack_1b_from_32b(const uint32_t thirty2, const int off) { assert_lt(off, 32); return ((thirty2 >> off) & 0x1); } #endif /*BITPACK_H_*/ hisat2-2.2.1/blockwise_sa.h000066400000000000000000001167611370663763200155550ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef BLOCKWISE_SA_H_ #define BLOCKWISE_SA_H_ #include #include #include #include #include #include "assert_helpers.h" #include "diff_sample.h" #include "multikey_qsort.h" #include "random_source.h" #include "binary_sa_search.h" #include "zbox.h" #include "alphabet.h" #include "timer.h" #include "ds.h" #include "mem_ids.h" #include "word_io.h" using namespace std; // Helpers for printing verbose messages #ifndef VMSG_NL #define VMSG_NL(...) \ if(this->verbose()) { \ stringstream tmp; \ tmp << __VA_ARGS__ << endl; \ this->verbose(tmp.str()); \ } #endif #ifndef VMSG #define VMSG(...) \ if(this->verbose()) { \ stringstream tmp; \ tmp << __VA_ARGS__; \ this->verbose(tmp.str()); \ } #endif /** * Abstract parent class for blockwise suffix-array building schemes. */ template class BlockwiseSA { public: BlockwiseSA(const TStr& __text, TIndexOffU __bucketSz, bool __sanityCheck = false, bool __passMemExc = false, bool __verbose = false, ostream& __logger = cout) : _text(__text), _bucketSz(max(__bucketSz, 2u)), _sanityCheck(__sanityCheck), _passMemExc(__passMemExc), _verbose(__verbose), _itrBucket(EBWTB_CAT), _itrBucketPos(OFF_MASK), _itrPushedBackSuffix(OFF_MASK), _logger(__logger) { } virtual ~BlockwiseSA() { } /** * Get the next suffix; compute the next bucket if necessary. */ virtual TIndexOffU nextSuffix() = 0; /** * Return true iff the next call to nextSuffix will succeed. */ bool hasMoreSuffixes() { if(_itrPushedBackSuffix != OFF_MASK) return true; try { _itrPushedBackSuffix = nextSuffix(); } catch(out_of_range& e) { assert_eq(OFF_MASK, _itrPushedBackSuffix); return false; } return true; } /** * Reset the suffix iterator so that the next call to nextSuffix() * returns the lexicographically-first suffix. */ void resetSuffixItr() { _itrBucket.clear(); _itrBucketPos = OFF_MASK; _itrPushedBackSuffix = OFF_MASK; reset(); assert(suffixItrIsReset()); } /** * Returns true iff the next call to nextSuffix() returns the * lexicographically-first suffix. */ bool suffixItrIsReset() { return _itrBucket.size() == 0 && _itrBucketPos == OFF_MASK && _itrPushedBackSuffix == OFF_MASK && isReset(); } const TStr& text() const { return _text; } TIndexOffU bucketSz() const { return _bucketSz; } bool sanityCheck() const { return _sanityCheck; } bool verbose() const { return _verbose; } ostream& log() const { return _logger; } size_t size() const { return _text.length()+1; } protected: /// Reset back to the first block virtual void reset() = 0; /// Return true iff reset to the first block virtual bool isReset() = 0; /** * Grab the next block of sorted suffixes. The block is guaranteed * to have at most _bucketSz elements. */ virtual void nextBlock(int cur_block, int tid = 0) = 0; /// Return true iff more blocks are available virtual bool hasMoreBlocks() const = 0; /// Optionally output a verbose message void verbose(const string& s) const { if(this->verbose()) { this->log() << s.c_str(); this->log().flush(); } } const TStr& _text; /// original string const TIndexOffU _bucketSz; /// target maximum bucket size const bool _sanityCheck; /// whether to perform sanity checks const bool _passMemExc; /// true -> pass on memory exceptions const bool _verbose; /// be talkative EList _itrBucket; /// current bucket TIndexOffU _itrBucketPos;/// offset into current bucket TIndexOffU _itrPushedBackSuffix; /// temporary slot for lookahead ostream& _logger; /// write log messages here }; /** * Abstract parent class for a blockwise suffix array builder that * always doles out blocks in lexicographical order. */ template class InorderBlockwiseSA : public BlockwiseSA { public: InorderBlockwiseSA(const TStr& __text, TIndexOffU __bucketSz, bool __sanityCheck = false, bool __passMemExc = false, bool __verbose = false, ostream& __logger = cout) : BlockwiseSA(__text, __bucketSz, __sanityCheck, __passMemExc, __verbose, __logger) { } }; /** * Build the SA a block at a time according to the scheme outlined in * Karkkainen's "Fast BWT" paper. */ template class KarkkainenBlockwiseSA : public InorderBlockwiseSA { public: typedef DifferenceCoverSample TDC; KarkkainenBlockwiseSA(const TStr& __text, TIndexOffU __bucketSz, int __nthreads, uint32_t __dcV, uint32_t __seed = 0, bool __sanityCheck = false, bool __passMemExc = false, bool __verbose = false, string base_fname = "", ostream& __logger = cout) : InorderBlockwiseSA(__text, __bucketSz, __sanityCheck, __passMemExc, __verbose, __logger), _sampleSuffs(EBWTB_CAT), _nthreads(__nthreads), _itrBucketIdx(0), _cur(0), _dcV(__dcV), _dc(EBWTB_CAT), _built(false), _base_fname(base_fname), _bigEndian(currentlyBigEndian()) { _randomSrc.init(__seed); reset(); } ~KarkkainenBlockwiseSA() { if(_threads.size() > 0) { for (size_t tid = 0; tid < _threads.size(); tid++) { _threads[tid]->join(); delete _threads[tid]; } } } /** * Allocate an amount of memory that simulates the peak memory * usage of the DifferenceCoverSample with the given text and v. * Throws bad_alloc if it's not going to fit in memory. Returns * the approximate number of bytes the Cover takes at all times. */ static size_t simulateAllocs(const TStr& text, TIndexOffU bucketSz) { size_t len = text.length(); // _sampleSuffs and _itrBucket are in memory at the peak size_t bsz = bucketSz; size_t sssz = len / max(bucketSz-1, 1); AutoArray tmp(bsz + sssz + (1024 * 1024 /*out of caution*/), EBWT_CAT); return bsz; } static void nextBlock_Worker(void *vp) { pair param = *(pair*)vp; KarkkainenBlockwiseSA* sa = param.first; int tid = param.second; while(true) { size_t cur = 0; { ThreadSafe ts(&sa->_mutex, sa->_nthreads > 1); cur = sa->_cur; if(cur > sa->_sampleSuffs.size()) break; sa->_cur++; } sa->nextBlock((int)cur, tid); // Write suffixes into a file std::ostringstream number; number << cur; const string fname = sa->_base_fname + "." + number.str() + ".sa"; ofstream sa_file(fname.c_str(), ios::binary); if(!sa_file.good()) { cerr << "Could not open file for writing a bucket: \"" << fname << "\"" << endl; throw 1; } const EList& bucket = sa->_itrBuckets[tid]; writeIndex(sa_file, (TIndexOffU)bucket.size(), sa->_bigEndian); for(size_t i = 0; i < bucket.size(); i++) { writeIndex(sa_file, bucket[i], sa->_bigEndian); } sa_file.close(); sa->_itrBuckets[tid].clear(); sa->_done[cur] = true; } } /** * Get the next suffix; compute the next bucket if necessary. */ virtual TIndexOffU nextSuffix() { // Launch threads if not if(this->_nthreads > 1) { if(_threads.size() == 0) { _done.resize(_sampleSuffs.size() + 1); _done.fill(false); _itrBuckets.resize(this->_nthreads); for(int tid = 0; tid < this->_nthreads; tid++) { _tparams.expand(); _tparams.back().first = this; _tparams.back().second = tid; _threads.push_back(new tthread::thread(nextBlock_Worker, (void*)&_tparams.back())); } assert_eq(_threads.size(), (size_t)this->_nthreads); } } if(this->_itrPushedBackSuffix != OFF_MASK) { TIndexOffU tmp = this->_itrPushedBackSuffix; this->_itrPushedBackSuffix = OFF_MASK; return tmp; } while(this->_itrBucketPos >= this->_itrBucket.size() || this->_itrBucket.size() == 0) { if(!hasMoreBlocks()) { throw out_of_range("No more suffixes"); } if(this->_nthreads == 1) { nextBlock((int)_cur); _cur++; } else { while(!_done[this->_itrBucketIdx]) { #if defined(_TTHREAD_WIN32_) Sleep(1); #elif defined(_TTHREAD_POSIX_) const static timespec ts = {0, 1000000}; // 1 millisecond nanosleep(&ts, NULL); #endif } // Read suffixes from a file std::ostringstream number; number << this->_itrBucketIdx; const string fname = _base_fname + "." + number.str() + ".sa"; ifstream sa_file(fname.c_str(), ios::binary); if(!sa_file.good()) { cerr << "Could not open file for reading a bucket: \"" << fname << "\"" << endl; throw 1; } size_t numSAs = readIndex(sa_file, _bigEndian); this->_itrBucket.resizeExact(numSAs); for(size_t i = 0; i < numSAs; i++) { this->_itrBucket[i] = readIndex(sa_file, _bigEndian); } sa_file.close(); std::remove(fname.c_str()); } this->_itrBucketIdx++; this->_itrBucketPos = 0; } return this->_itrBucket[this->_itrBucketPos++]; } /// Defined in blockwise_sa.cpp virtual void nextBlock(int cur_block, int tid = 0); /// Defined in blockwise_sa.cpp virtual void qsort(EList& bucket); /// Return true iff more blocks are available virtual bool hasMoreBlocks() const { return this->_itrBucketIdx <= _sampleSuffs.size(); } /// Return the difference-cover period uint32_t dcV() const { return _dcV; } protected: /** * Initialize the state of the blockwise suffix sort. If the * difference cover sample and the sample set have not yet been * built, build them. Then reset the block cursor to point to * the first block. */ virtual void reset() { if(!_built) { build(); } assert(_built); _cur = 0; } /// Return true iff we're about to dole out the first bucket virtual bool isReset() { return _cur == 0; } private: /** * Calculate the difference-cover sample and sample suffixes. */ void build() { // Calculate difference-cover sample assert(_dc.get() == NULL); if(_dcV != 0) { _dc.init(new TDC(this->text(), _dcV, this->verbose(), this->sanityCheck())); _dc.get()->build(this->_nthreads); } // Calculate sample suffixes if(this->bucketSz() <= this->text().length()) { VMSG_NL("Building samples"); buildSamples(); } else { VMSG_NL("Skipping building samples since text length " << this->text().length() << " is less than bucket size: " << this->bucketSz()); } _built = true; } /** * Calculate the lcp between two suffixes using the difference * cover as a tie-breaker. If the tie-breaker is employed, then * the calculated lcp may be an underestimate. * * Defined in blockwise_sa.cpp */ inline bool tieBreakingLcp(TIndexOffU aOff, TIndexOffU bOff, TIndexOffU& lcp, bool& lcpIsSoft); /** * Compare two suffixes using the difference-cover sample. */ inline bool suffixCmp(TIndexOffU cmp, TIndexOffU i, int64_t& j, int64_t& k, bool& kSoft, const EList& z); void buildSamples(); EList _sampleSuffs; /// sample suffixes int _nthreads; /// # of threads TIndexOffU _itrBucketIdx; TIndexOffU _cur; /// offset to 1st elt of next block const uint32_t _dcV; /// difference-cover periodicity PtrWrap _dc; /// queryable difference-cover data bool _built; /// whether samples/DC have been built RandomSource _randomSrc; /// source of pseudo-randoms MUTEX_T _mutex; /// synchronization of output message string _base_fname; /// base file name for storing SA blocks bool _bigEndian; /// bigEndian? EList _threads; /// thread list EList > _tparams; ELList _itrBuckets; /// buckets EList _done; /// is a block processed? }; /** * Qsort the set of suffixes whose offsets are in 'bucket'. */ template inline void KarkkainenBlockwiseSA::qsort(EList& bucket) { const TStr& t = this->text(); TIndexOffU *s = bucket.ptr(); size_t slen = bucket.size(); TIndexOffU len = (TIndexOffU)t.length(); if(_dc.get() != NULL) { // Use the difference cover as a tie-breaker if we have it VMSG_NL(" (Using difference cover)"); // Extract the 'host' array because it's faster to work // with than the EList<> container const uint8_t *host = (const uint8_t *)t.buf(); assert(_dc.get() != NULL); mkeyQSortSufDcU8(t, host, len, s, slen, *_dc.get(), 4, this->verbose(), this->sanityCheck()); } else { VMSG_NL(" (Not using difference cover)"); // We don't have a difference cover - just do a normal // suffix sort mkeyQSortSuf(t, s, slen, 4, this->verbose(), this->sanityCheck()); } } /** * Qsort the set of suffixes whose offsets are in 'bucket'. This * specialization for packed strings does not attempt to extract and * operate directly on the host string; the fact that the string is * packed means that the array cannot be sorted directly. */ template<> inline void KarkkainenBlockwiseSA::qsort( EList& bucket) { const S2bDnaString& t = this->text(); TIndexOffU *s = bucket.ptr(); size_t slen = bucket.size(); size_t len = t.length(); if(_dc.get() != NULL) { // Use the difference cover as a tie-breaker if we have it VMSG_NL(" (Using difference cover)"); // Can't use the text's 'host' array because the backing // store for the packed string is not one-char-per-elt. mkeyQSortSufDcU8(t, t, len, s, slen, *_dc.get(), 4, this->verbose(), this->sanityCheck()); } else { VMSG_NL(" (Not using difference cover)"); // We don't have a difference cover - just do a normal // suffix sort mkeyQSortSuf(t, s, slen, 4, this->verbose(), this->sanityCheck()); } } template struct BinarySortingParam { const TStr* t; const EList* sampleSuffs; EList bucketSzs; EList bucketReps; size_t begin; size_t end; }; template static void BinarySorting_worker(void *vp) { BinarySortingParam* param = (BinarySortingParam*)vp; const TStr& t = *(param->t); size_t len = t.length(); const EList& sampleSuffs = *(param->sampleSuffs); EList& bucketSzs = param->bucketSzs; EList& bucketReps = param->bucketReps; ASSERT_ONLY(size_t numBuckets = bucketSzs.size()); size_t begin = param->begin; size_t end = param->end; // Iterate through every suffix in the text, determine which // bucket it falls into by doing a binary search across the // sorted list of samples, and increment a counter associated // with that bucket. Also, keep one representative for each // bucket so that we can split it later. We loop in ten // stretches so that we can print out a helpful progress // message. (This step can take a long time.) for(TIndexOffU i = (TIndexOffU)begin; i < end && i < len; i++) { TIndexOffU r = binarySASearch(t, i, sampleSuffs); if(r == std::numeric_limits::max()) continue; // r was one of the samples assert_lt(r, numBuckets); bucketSzs[r]++; assert_lt(bucketSzs[r], len); if(bucketReps[r] == OFF_MASK || (i & 100) == 0) { bucketReps[r] = i; // clobbers previous one, but that's OK } } } /** * Select a set of bucket-delineating sample suffixes such that no * bucket is greater than the requested upper limit. Some care is * taken to make each bucket's size close to the limit without * going over. */ template void KarkkainenBlockwiseSA::buildSamples() { const TStr& t = this->text(); TIndexOffU bsz = this->bucketSz()-1; // subtract 1 to leave room for sample size_t len = this->text().length(); // Prepare _sampleSuffs array _sampleSuffs.clear(); TIndexOffU numSamples = (TIndexOffU)((len/bsz)+1)<<1; // ~len/bsz x 2 assert_gt(numSamples, 0); VMSG_NL("Reserving space for " << numSamples << " sample suffixes"); if(this->_passMemExc) { _sampleSuffs.resizeExact(numSamples); // Randomly generate samples. Allow duplicates for now. VMSG_NL("Generating random suffixes"); for(size_t i = 0; i < numSamples; i++) { #ifdef BOWTIE_64BIT_INDEX _sampleSuffs[i] = (TIndexOffU)(_randomSrc.nextU64() % len); #else _sampleSuffs[i] = (TIndexOffU)(_randomSrc.nextU32() % len); #endif } } else { try { _sampleSuffs.resizeExact(numSamples); // Randomly generate samples. Allow duplicates for now. VMSG_NL("Generating random suffixes"); for(size_t i = 0; i < numSamples; i++) { #ifdef BOWTIE_64BIT_INDEX _sampleSuffs[i] = (TIndexOffU)(_randomSrc.nextU64() % len); #else _sampleSuffs[i] = (TIndexOffU)(_randomSrc.nextU32() % len); #endif } } catch(bad_alloc &e) { if(this->_passMemExc) { throw e; // rethrow immediately } else { cerr << "Could not allocate sample suffix container of " << (numSamples * OFF_SIZE) << " bytes." << endl << "Please try using a smaller number of blocks by specifying a larger --bmax or" << endl << "a smaller --bmaxdivn" << endl; throw 1; } } } // Remove duplicates; very important to do this before the call to // mkeyQSortSuf so that it doesn't try to calculate lexicographical // relationships between very long, identical strings, which takes // an extremely long time in general, and causes the stack to grow // linearly with the size of the input { Timer timer(cout, "QSorting sample offsets, eliminating duplicates time: ", this->verbose()); VMSG_NL("QSorting " << _sampleSuffs.size() << " sample offsets, eliminating duplicates"); _sampleSuffs.sort(); size_t sslen = _sampleSuffs.size(); for(size_t i = 0; i < sslen-1; i++) { if(_sampleSuffs[i] == _sampleSuffs[i+1]) { _sampleSuffs.erase(i--); sslen--; } } } // Multikey quicksort the samples { Timer timer(cout, " Multikey QSorting samples time: ", this->verbose()); VMSG_NL("Multikey QSorting " << _sampleSuffs.size() << " samples"); this->qsort(_sampleSuffs); } // Calculate bucket sizes VMSG_NL("Calculating bucket sizes"); int limit = 5; // Iterate until all buckets are less than while(--limit >= 0) { TIndexOffU numBuckets = (TIndexOffU)_sampleSuffs.size()+1; AutoArray threads(this->_nthreads); EList > tparams; for(int tid = 0; tid < this->_nthreads; tid++) { // Calculate bucket sizes by doing a binary search for each // suffix and noting where it lands tparams.expand(); try { // Allocate and initialize containers for holding bucket // sizes and representatives. tparams.back().bucketSzs.resizeExact(numBuckets); tparams.back().bucketReps.resizeExact(numBuckets); tparams.back().bucketSzs.fillZero(); tparams.back().bucketReps.fill(OFF_MASK); } catch(bad_alloc &e) { if(this->_passMemExc) { throw e; // rethrow immediately } else { cerr << "Could not allocate sizes, representatives (" << ((numBuckets*8)>>10) << " KB) for blocks." << endl << "Please try using a smaller number of blocks by specifying a larger --bmax or a" << endl << "smaller --bmaxdivn." << endl; throw 1; } } tparams.back().t = &t; tparams.back().sampleSuffs = &_sampleSuffs; tparams.back().begin = (tid == 0 ? 0 : len / this->_nthreads * tid); tparams.back().end = (tid + 1 == this->_nthreads ? len : len / this->_nthreads * (tid + 1)); if(this->_nthreads == 1) { BinarySorting_worker((void*)&tparams.back()); } else { threads[tid] = new tthread::thread(BinarySorting_worker, (void*)&tparams.back()); } } if(this->_nthreads > 1) { for (int tid = 0; tid < this->_nthreads; tid++) { threads[tid]->join(); } } EList& bucketSzs = tparams[0].bucketSzs; EList& bucketReps = tparams[0].bucketReps; for(int tid = 1; tid < this->_nthreads; tid++) { for(size_t j = 0; j < numBuckets; j++) { bucketSzs[j] += tparams[tid].bucketSzs[j]; if(bucketReps[j] == OFF_MASK) { bucketReps[j] = tparams[tid].bucketReps[j]; } } } // Check for large buckets and mergeable pairs of small buckets // and split/merge as necessary TIndexOff added = 0; TIndexOff merged = 0; assert_eq(bucketSzs.size(), numBuckets); assert_eq(bucketReps.size(), numBuckets); { Timer timer(cout, " Splitting and merging time: ", this->verbose()); VMSG_NL("Splitting and merging"); for(TIndexOffU i = 0; i < numBuckets; i++) { TIndexOffU mergedSz = bsz + 1; assert(bucketSzs[(size_t)i] == 0 || bucketReps[(size_t)i] != OFF_MASK); if(i < numBuckets-1) { mergedSz = bucketSzs[(size_t)i] + bucketSzs[(size_t)i+1] + 1; } // Merge? if(mergedSz <= bsz) { bucketSzs[(size_t)i+1] += (bucketSzs[(size_t)i]+1); // The following may look strange, but it's necessary // to ensure that the merged bucket has a representative bucketReps[(size_t)i+1] = _sampleSuffs[(size_t)i+added]; _sampleSuffs.erase((size_t)i+added); bucketSzs.erase((size_t)i); bucketReps.erase((size_t)i); i--; // might go to -1 but ++ will overflow back to 0 numBuckets--; merged++; assert_eq(numBuckets, _sampleSuffs.size()+1-added); assert_eq(numBuckets, bucketSzs.size()); } // Split? else if(bucketSzs[(size_t)i] > bsz) { // Add an additional sample from the bucketReps[] // set accumulated in the binarySASearch loop; this // effectively splits the bucket _sampleSuffs.insert(bucketReps[(size_t)i], (TIndexOffU)(i + (added++))); } } } if(added == 0) { //if(this->verbose()) { // cout << "Final bucket sizes:" << endl; // cout << " (begin): " << bucketSzs[0] << " (" << (int)(bsz - bucketSzs[0]) << ")" << endl; // for(uint32_t i = 1; i < numBuckets; i++) { // cout << " " << bucketSzs[i] << " (" << (int)(bsz - bucketSzs[i]) << ")" << endl; // } //} break; } // Otherwise, continue until no more buckets need to be // split VMSG_NL("Split " << added << ", merged " << merged << "; iterating..."); } // Do *not* force a do-over // if(limit == 0) { // VMSG_NL("Iterated too many times; trying again..."); // buildSamples(); // } VMSG_NL("Avg bucket size: " << ((double)(len-_sampleSuffs.size()) / (_sampleSuffs.size()+1)) << " (target: " << bsz << ")"); } /** * Do a simple LCP calculation on two strings. */ template inline static TIndexOffU suffixLcp(const T& t, TIndexOffU aOff, TIndexOffU bOff) { TIndexOffU c = 0; size_t len = t.length(); assert_leq(aOff, len); assert_leq(bOff, len); while(aOff + c < len && bOff + c < len && t[aOff + c] == t[bOff + c]) c++; return c; } /** * Calculate the lcp between two suffixes using the difference * cover as a tie-breaker. If the tie-breaker is employed, then * the calculated lcp may be an underestimate. If the tie-breaker is * employed, lcpIsSoft will be set to true (otherwise, false). */ template inline bool KarkkainenBlockwiseSA::tieBreakingLcp(TIndexOffU aOff, TIndexOffU bOff, TIndexOffU& lcp, bool& lcpIsSoft) { const TStr& t = this->text(); TIndexOffU c = 0; TIndexOffU tlen = (TIndexOffU)t.length(); assert_leq(aOff, tlen); assert_leq(bOff, tlen); assert(_dc.get() != NULL); uint32_t dcDist = _dc.get()->tieBreakOff(aOff, bOff); lcpIsSoft = false; // hard until proven soft while(c < dcDist && // we haven't hit the tie breaker c < tlen-aOff && // we haven't fallen off of LHS suffix c < tlen-bOff && // we haven't fallen off of RHS suffix t[aOff+c] == t[bOff+c]) // we haven't hit a mismatch c++; lcp = c; if(c == tlen-aOff) { // Fell off LHS (a), a is greater return false; } else if(c == tlen-bOff) { // Fell off RHS (b), b is greater return true; } else if(c == dcDist) { // Hit a tie-breaker element lcpIsSoft = true; assert_neq(dcDist, 0xffffffff); return _dc.get()->breakTie(aOff+c, bOff+c) < 0; } else { assert_neq(t[aOff+c], t[bOff+c]); return t[aOff+c] < t[bOff+c]; } } /** * Lookup a suffix LCP in the given z array; if the element is not * filled in then calculate it from scratch. */ template static TIndexOffU lookupSuffixZ( const T& t, TIndexOffU zOff, TIndexOffU off, const EList& z) { if(zOff < z.size()) { TIndexOffU ret = z[zOff]; assert_eq(ret, suffixLcp(t, off + zOff, off)); return ret; } assert_leq(off + zOff, t.length()); return suffixLcp(t, off + zOff, off); } /** * true -> i < cmp * false -> i > cmp */ template inline bool KarkkainenBlockwiseSA::suffixCmp( TIndexOffU cmp, TIndexOffU i, int64_t& j, int64_t& k, bool& kSoft, const EList& z) { const TStr& t = this->text(); TIndexOffU len = (TIndexOffU)t.length(); // i is not covered by any previous match TIndexOffU l; if((int64_t)i > k) { k = i; // so that i + lHi == kHi l = 0; // erase any previous l kSoft = false; // To be extended } // i is covered by a previous match else /* i <= k */ { assert_gt((int64_t)i, j); TIndexOffU zIdx = (TIndexOffU)(i-j); assert_leq(zIdx, len-cmp); if(zIdx < _dcV || _dc.get() == NULL) { // Go as far as the Z-box says l = lookupSuffixZ(t, zIdx, cmp, z); if(i + l > len) { l = len-i; } assert_leq(i + l, len); // Possibly to be extended } else { // But we're past the point of no-more-Z-boxes bool ret = tieBreakingLcp(i, cmp, l, kSoft); // Sanity-check tie-breaker if(this->sanityCheck()) { if(ret) assert(sstr_suf_lt(t, i, t, cmp, false)); else assert(sstr_suf_gt(t, i, t, cmp, false)); } j = i; k = i + l; if(this->sanityCheck()) { if(kSoft) { assert_leq(l, suffixLcp(t, i, cmp)); } else { assert_eq (l, suffixLcp(t, i, cmp)); } } return ret; } } // Z box extends exactly as far as previous match (or there // is neither a Z box nor a previous match) if((int64_t)(i + l) == k) { // Extend while(l < len-cmp && k < (int64_t)len && t[(size_t)(cmp+l)] == t[(size_t)k]) { k++; l++; } j = i; // update furthest-extending LHS kSoft = false; assert_eq(l, suffixLcp(t, i, cmp)); } // Z box extends further than previous match else if((int64_t)(i + l) > k) { l = (TIndexOffU)(k - i); // point to just after previous match j = i; // update furthest-extending LHS if(kSoft) { while(l < len-cmp && k < (int64_t)len && t[(size_t)(cmp+l)] == t[(size_t)k]) { k++; l++; } kSoft = false; assert_eq(l, suffixLcp(t, i, cmp)); } else assert_eq(l, suffixLcp(t, i, cmp)); } // Check that calculated lcp matches actual lcp if(this->sanityCheck()) { if(!kSoft) { // l should exactly match lcp assert_eq(l, suffixLcp(t, i, cmp)); } else { // l is an underestimate of LCP assert_leq(l, suffixLcp(t, i, cmp)); } } assert_leq(l+i, len); assert_leq(l, len-cmp); // i and cmp should not be the same suffix assert(l != len-cmp || i+l != len); // Now we're ready to do a comparison on the next char if(l+i != len && ( l == len-cmp || // departure from paper algorithm: // falling off pattern implies // pattern is *greater* in our case t[i + l] < t[cmp + l])) { // Case 2: Text suffix is less than upper sample suffix #ifndef NDEBUG if(this->sanityCheck()) { assert(sstr_suf_lt(t, i, t, cmp, false)); } #endif return true; // suffix at i is less than suffix at cmp } else { // Case 3: Text suffix is greater than upper sample suffix #ifndef NDEBUG if(this->sanityCheck()) { assert(sstr_suf_gt(t, i, t, cmp, false)); } #endif return false; // suffix at i is less than suffix at cmp } } /** * Retrieve the next block. This is the most performance-critical part * of the blockwise suffix sorting process. */ template void KarkkainenBlockwiseSA::nextBlock(int cur_block, int tid) { #ifndef NDEBUG if(this->_nthreads > 1) { assert_lt(tid, this->_itrBuckets.size()); } #endif EList& bucket = (this->_nthreads > 1 ? this->_itrBuckets[tid] : this->_itrBucket); { ThreadSafe ts(&_mutex, this->_nthreads > 1); VMSG_NL("Getting block " << (cur_block+1) << " of " << _sampleSuffs.size()+1); } assert(_built); assert_gt(_dcV, 3); assert_leq(cur_block, _sampleSuffs.size()); const TStr& t = this->text(); TIndexOffU len = (TIndexOffU)t.length(); // Set up the bucket bucket.clear(); TIndexOffU lo = OFF_MASK, hi = OFF_MASK; if(_sampleSuffs.size() == 0) { // Special case: if _sampleSuffs is 0, then multikey-quicksort // everything { ThreadSafe ts(&_mutex, this->_nthreads > 1); VMSG_NL(" No samples; assembling all-inclusive block"); } assert_eq(0, cur_block); try { if(bucket.capacity() < this->bucketSz()) { bucket.reserveExact(len+1); } bucket.resize(len); for(TIndexOffU i = 0; i < len; i++) { bucket[i] = i; } } catch(bad_alloc &e) { if(this->_passMemExc) { throw e; // rethrow immediately } else { cerr << "Could not allocate a master suffix-array block of " << ((len+1) * 4) << " bytes" << endl << "Please try using a larger number of blocks by specifying a smaller --bmax or" << endl << "a larger --bmaxdivn" << endl; throw 1; } } } else { try { { ThreadSafe ts(&_mutex, this->_nthreads > 1); VMSG_NL(" Reserving size (" << this->bucketSz() << ") for bucket " << (cur_block+1)); } // BTL: Add a +100 fudge factor; there seem to be instances // where a bucket ends up having one more elt than bucketSz() if(bucket.size() < this->bucketSz()+100) { bucket.reserveExact(this->bucketSz()+100); } } catch(bad_alloc &e) { if(this->_passMemExc) { throw e; // rethrow immediately } else { cerr << "Could not allocate a suffix-array block of " << ((this->bucketSz()+1) * 4) << " bytes" << endl; cerr << "Please try using a larger number of blocks by specifying a smaller --bmax or" << endl << "a larger --bmaxdivn" << endl; throw 1; } } // Select upper and lower bounds from _sampleSuffs[] and // calculate the Z array up to the difference-cover periodicity // for both. Be careful about first/last buckets. EList zLo(EBWTB_CAT), zHi(EBWTB_CAT); assert_geq(cur_block, 0); assert_leq((size_t)cur_block, _sampleSuffs.size()); bool first = (cur_block == 0); bool last = ((size_t)cur_block == _sampleSuffs.size()); try { // Timer timer(cout, " Calculating Z arrays time: ", this->verbose()); { ThreadSafe ts(&_mutex, this->_nthreads > 1); VMSG_NL(" Calculating Z arrays for bucket " << (cur_block+1)); } if(!last) { // Not the last bucket assert_lt(cur_block, _sampleSuffs.size()); hi = _sampleSuffs[cur_block]; zHi.resizeExact(_dcV); zHi.fillZero(); assert_eq(zHi[0], 0); calcZ(t, hi, zHi, this->verbose(), this->sanityCheck()); } if(!first) { // Not the first bucket assert_gt(cur_block, 0); assert_leq(cur_block, _sampleSuffs.size()); lo = _sampleSuffs[cur_block-1]; zLo.resizeExact(_dcV); zLo.fillZero(); assert_gt(_dcV, 3); assert_eq(zLo[0], 0); calcZ(t, lo, zLo, this->verbose(), this->sanityCheck()); } } catch(bad_alloc &e) { if(this->_passMemExc) { throw e; // rethrow immediately } else { cerr << "Could not allocate a z-array of " << (_dcV * 4) << " bytes" << endl; cerr << "Please try using a larger number of blocks by specifying a smaller --bmax or" << endl << "a larger --bmaxdivn" << endl; throw 1; } } // This is the most critical loop in the algorithm; this is where // we iterate over all suffixes in the text and pick out those that // fall into the current bucket. // // This loop is based on the SMALLERSUFFIXES function outlined on // p7 of the "Fast BWT" paper // int64_t kHi = -1, kLo = -1; int64_t jHi = -1, jLo = -1; bool kHiSoft = false, kLoSoft = false; assert_eq(0, bucket.size()); { // Timer timer(cout, " Block accumulator loop time: ", this->verbose()); { ThreadSafe ts(&_mutex, this->_nthreads > 1); VMSG_NL(" Entering block accumulator loop for bucket " << (cur_block+1) << ":"); } TIndexOffU lenDiv10 = (len + 9) / 10; for(TIndexOffU iten = 0, ten = 0; iten < len; iten += lenDiv10, ten++) { TIndexOffU itenNext = iten + lenDiv10; { ThreadSafe ts(&_mutex, this->_nthreads > 1); if(ten > 0) VMSG_NL(" bucket " << (cur_block+1) << ": " << (ten * 10) << "%"); } for(TIndexOffU i = iten; i < itenNext && i < len; i++) { assert_lt(jLo, (int64_t)i); assert_lt(jHi, (int64_t)i); // Advance the upper-bound comparison by one character if(i == hi || i == lo) continue; // equal to one of the bookends if(hi != OFF_MASK && !suffixCmp(hi, i, jHi, kHi, kHiSoft, zHi)) { continue; // not in the bucket } if(lo != OFF_MASK && suffixCmp(lo, i, jLo, kLo, kLoSoft, zLo)) { continue; // not in the bucket } // In the bucket! - add it assert_lt(i, len); try { bucket.push_back(i); } catch(bad_alloc &e) { cerr << "Could not append element to block of " << ((bucket.size()) * OFF_SIZE) << " bytes" << endl; if(this->_passMemExc) { throw e; // rethrow immediately } else { cerr << "Please try using a larger number of blocks by specifying a smaller --bmax or" << endl << "a larger --bmaxdivn" << endl; throw 1; } } // Not necessarily true; we allow overflowing buckets // since we can't guarantee that a good set of sample // suffixes can be found in a reasonable amount of time //assert_lt(bucket.size(), this->bucketSz()); } } // end loop over all suffixes of t { ThreadSafe ts(&_mutex, this->_nthreads > 1); VMSG_NL(" bucket " << (cur_block+1) << ": 100%"); } } } // end else clause of if(_sampleSuffs.size() == 0) // Sort the bucket if(bucket.size() > 0) { Timer timer(cout, " Sorting block time: ", this->verbose()); { ThreadSafe ts(&_mutex, this->_nthreads > 1); VMSG_NL(" Sorting block of length " << bucket.size() << " for bucket " << (cur_block+1)); } this->qsort(bucket); } if(hi != OFF_MASK) { // Not the final bucket; throw in the sample on the RHS bucket.push_back(hi); } else { // Final bucket; throw in $ suffix bucket.push_back(len); } { ThreadSafe ts(&_mutex, this->_nthreads > 1); VMSG_NL("Returning block of " << bucket.size() << " for bucket " << (cur_block+1)); } } #endif /*BLOCKWISE_SA_H_*/ hisat2-2.2.1/bp_aligner.h000066400000000000000000001733251370663763200152110ustar00rootroot00000000000000/* * Copyright 2014, Daehwan Kim * * This file is part of HISAT. * * HISAT is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT. If not, see . */ #ifndef BP_ALIGNER_H_ #define BP_ALIGNER_H_ #include "hi_aligner.h" /** * With a hierarchical indexing, SplicedAligner provides several alignment strategies * , which enable effective alignment of RNA-seq reads */ template class BP_Aligner : public HI_Aligner { public: /** * Initialize with index. */ BP_Aligner( const Ebwt& ebwt, const EList& refnames, MUTEX_T* mutex, uint64_t threads_rids_mindist = 0, bool no_spliced_alignment = false) : HI_Aligner(ebwt, threads_rids_mindist, no_spliced_alignment), _refnames(refnames), _mutex(mutex), _done(false) { } ~BP_Aligner() { } /** * Aligns a read or a pair * This funcion is called per read or pair */ virtual int go( const Scoring& sc, const Ebwt& ebwtFw, const Ebwt& ebwtBw, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink) { _done = false; index_t rdi; bool fw; while(this->nextBWT(sc, ebwtFw, ebwtBw, ref, rdi, fw, wlm, prm, him, rnd, sink)) { // given the partial alignment, try to extend it to full alignments this->align(sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, fw, wlm, prm, swm, him, rnd, sink); if(_done) break; } return EXTEND_POLICY_FULFILLED; } /** * Given a partial alignment of a read, try to further extend * the alignment bidirectionally using a combination of * local search, extension, and global search */ virtual void hybridSearch( const Scoring& sc, const Ebwt& ebwtFw, const Ebwt& ebwtBw, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, bool fw, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink); /** * Given a partial alignment of a read, try to further extend * the alignment bidirectionally using a combination of * local search, extension, and global search */ virtual void hybridSearch_recur( const Scoring& sc, const Ebwt& ebwtFw, const Ebwt& ebwtBw, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, const GenomeHit& hit, index_t hitoff, index_t hitlen, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink, index_t dep = 0); private: EList _refnames; MUTEX_T* _mutex; bool _done; }; /** * Given a partial alignment of a read, try to further extend * the alignment bidirectionally using a combination of * local search, extension, and global search */ template void BP_Aligner::hybridSearch( const Scoring& sc, const Ebwt& ebwtFw, const Ebwt& ebwtBw, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, bool fw, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink) { assert_lt(rdi, 2); assert(this->_rds[rdi] != NULL); him.localatts++; // before further alignment using local search, extend the partial alignments directly // by comparing with the corresponding genomic sequences // this extension is performed without any mismatches allowed for(index_t hi = 0; hi < this->_genomeHits.size(); hi++) { GenomeHit& genomeHit = this->_genomeHits[hi]; index_t leftext = (index_t)OFF_MASK, rightext = (index_t)OFF_MASK; genomeHit.extend(*(this->_rds[rdi]), ref, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, this->_minK_local, leftext, rightext); } // for the candidate alignments, examine the longest (best) one first this->_genomeHits_done.resize(this->_genomeHits.size()); this->_genomeHits_done.fill(false); for(size_t hi = 0; hi < this->_genomeHits.size(); hi++) { index_t hj = 0; for(; hj < this->_genomeHits.size(); hj++) { if(!this->_genomeHits_done[hj]) break; } if(hj >= this->_genomeHits.size()) break; for(index_t hk = hj + 1; hk < this->_genomeHits.size(); hk++) { if(this->_genomeHits_done[hk]) continue; GenomeHit& genomeHit_j = this->_genomeHits[hj]; GenomeHit& genomeHit_k = this->_genomeHits[hk]; if(genomeHit_k.hitcount() > genomeHit_j.hitcount() || (genomeHit_k.hitcount() == genomeHit_j.hitcount() && genomeHit_k.len() > genomeHit_j.len())) { hj = hk; } } // given a candidate partial alignment, extend it bidirectionally him.anchoratts++; GenomeHit& genomeHit = this->_genomeHits[hj]; hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, genomeHit, genomeHit.rdoff(), genomeHit.len(), wlm, prm, swm, him, rnd, sink); this->_genomeHits_done[hj] = true; if(_done) return; } } /** * Given a partial alignment of a read, try to further extend * the alignment bidirectionally using a combination of * local search, extension, and global search */ template void BP_Aligner::hybridSearch_recur( const Scoring& sc, const Ebwt& ebwtFw, const Ebwt& ebwtBw, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, const GenomeHit& hit, index_t hitoff, index_t hitlen, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink, index_t dep) { if(_done) return; him.localsearchrecur++; assert_lt(rdi, 2); assert(this->_rds[rdi] != NULL); const Read& rd = *(this->_rds[rdi]); index_t rdlen = rd.length(); if(hit.score() < this->_minsc[rdi]) return; // if it's already examined, just return if(hitoff == hit.rdoff() - hit.trim5() && hitlen == hit.len() + hit.trim5() + hit.trim3()) { if(this->isSearched(hit, rdi)) return; this->addSearched(hit, rdi); } // for effective use of memory allocation and deallocation if(this->_coords.size() <= dep) { this->_coords.expand(); assert_leq(this->_local_genomeHits.size(), dep); this->_local_genomeHits.expand(); assert_leq(this->_spliceSites.size(), dep); this->_spliceSites.expand(); } EList& coords = this->_coords[dep]; EList >& local_genomeHits = this->_local_genomeHits[dep]; EList& spliceSites = this->_spliceSites[dep]; // daehwan - for debugging purposes #if 0 cout << rd.name << "\t" << (hit.fw() ? "+" : "-") << "\t" << hitoff << "\t" << hitoff + hitlen << "\t" << "( " << hit.rdoff() << "\t" << hit.rdoff() + hit.len() << " )" << "\t" << hit.refoff() << "\t" << hit.getRightOff() << "\t" << hit.score() << "\t" << "dep: " << dep << "\t"; Edit::print(cout, hit.edits()); cout << endl; #endif assert_leq(hitoff + hitlen, rdlen); // if this is a full alignment, report it if(hitoff == 0 && hitlen == rdlen) { if(!this->redundant(sink, rdi, hit)) { // this->reportHit(sc, ebwtFw, ref, sink, rdi, hit); return; } } else if(hitoff > 0 && (hitoff + hitlen == rdlen || hitoff + hitoff < rdlen - hitlen)) { // extend the partial alignment in the left direction index_t fragoff = 0, fraglen = 0, left = 0; hit.getLeft(fragoff, fraglen, left); const index_t minMatchLen = this->_minK_local; // make use of a list of known or novel splice sites to further align the read if(fraglen >= minMatchLen && left >= minMatchLen && !this->_no_spliced_alignment) { spliceSites.clear(); ssdb.getLeftSpliceSites(hit.ref(), left + minMatchLen, minMatchLen * 2, spliceSites); for(size_t si = 0; si < spliceSites.size(); si++) { const SpliceSite& ss = spliceSites[si]; if(!ss._fromfile && ss._readid + this->_thread_rids_mindist > rd.rdid) continue; if(left + fraglen - 1 < ss.right()) continue; index_t frag2off = ss.left() - (ss.right() - left); if(frag2off + 1 < hitoff) continue; GenomeHit tempHit; tempHit.init(hit.fw(), 0, hitoff, 0, // trim5 0, // trim3 hit.ref(), frag2off + 1 - hitoff, this->_sharedVars); if(!tempHit.compatibleWith(hit, this->_no_spliced_alignment)) continue; int64_t minsc = this->_minsc[rdi]; bool combined = tempHit.combineWith(hit, rd, ref, ssdb, swa, swm, sc, minsc, rnd, this->_minK_local, 1, 1, false); if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); if(combined && tempHit.score() >= minsc) { assert_eq(tempHit.trim5(), 0); assert_leq(tempHit.rdoff() + tempHit.len() + tempHit.trim3(), rdlen); hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff(), tempHit.len() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, dep + 1); } } } // choose a local index based on the genomic location of the partial alignment const HierEbwt* hierEbwtFw = (const HierEbwt*)(&ebwtFw); const LocalEbwt* localEbwtFw = hierEbwtFw->getLocalEbwt(hit.ref(), hit.refoff()); assert_leq(localEbwtFw->_localOffset, hit.refoff()); bool success = false, first = true; index_t count = 0; // consider at most two local indexes index_t max_count = 2; int64_t prev_score = hit.score(); local_genomeHits.clear(); while(!success && count++ < max_count) { if(him.localindexatts >= this->max_localindexatts) return; if(first) { first = false; } else { localEbwtFw = hierEbwtFw->prevLocalEbwt(localEbwtFw); if(localEbwtFw == NULL || localEbwtFw->empty()) break; } // local index search index_t extlen = 0; local_index_t top = (local_index_t)OFF_MASK, bot = (local_index_t)OFF_MASK; index_t extoff = hitoff - 1; if(extoff > 0) extoff -= 1; if(extoff < minAnchorLen) { extoff = minAnchorLen; } index_t nelt = (index_t)OFF_MASK; index_t max_nelt = std::max(5, extlen); bool no_extension = false; bool uniqueStop; // daehwan - for debugging purposes // index_t minUniqueLen = this->_minK_local; index_t minUniqueLen = (index_t)OFF_MASK; for(; extoff < rdlen; extoff++) { extlen = 0; // daehwan - for debugging purposes // uniqueStop = true; uniqueStop = false; him.localindexatts++; nelt = this->localEbwtSearch( localEbwtFw, // BWT index NULL, // BWT index rd, // read to align sc, // scoring scheme hit.fw(), false, // searchfw, extoff, extlen, top, bot, rnd, uniqueStop, minUniqueLen); if(extoff + 1 - extlen >= hitoff) { no_extension = true; break; } if(nelt <= max_nelt) break; } assert_leq(top, bot); assert_eq(nelt, (index_t)(bot - top)); assert_leq(extlen, extoff + 1); if(nelt > 0 && nelt <= max_nelt && extlen >= minAnchorLen && !no_extension) { assert_leq(nelt, max_nelt); coords.clear(); bool straddled = false; // get genomic locations for this local search this->getGenomeCoords_local( *localEbwtFw, ref, rnd, top, bot, hit.fw(), extoff + 1 - extlen, extlen, coords, wlm, prm, him, true, // reject straddled? straddled); assert_leq(coords.size(), nelt); coords.sort(); for(int ri = coords.size() - 1; ri >= 0; ri--) { const Coord& coord = coords[ri]; GenomeHit tempHit; tempHit.init(coord.orient(), extoff + 1 - extlen, extlen, 0, // trim5 0, // trim3 coord.ref(), coord.off(), this->_sharedVars); // daehwan - for debugging purposes if(coord.ref() == hit.ref() && coord.off() > hit.refoff() && coord.off() < hit.refoff() + 64000) { index_t leftext = (index_t)OFF_MASK, rightext = (index_t)0; index_t mm = 1; tempHit.extend( rd, ref, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, this->_minK_local, leftext, rightext, mm); #if 0 cout << endl; cout << rd.rdid << "\t" << rd.name << endl; cout << "\ttype: " << 1 << endl; cout << "\t" << hit.ref() << endl; cout << "\t\ttempHit " << (tempHit.fw() ? "+" : "-") << "\t" << tempHit.refoff() << "\t" << tempHit.rdoff() << "\t" << tempHit.len() << "\t" << tempHit.score() << endl; cout << "\t\tanchHit " << (hit.fw() ? "+" : "-") << "\t" << hit.refoff() << "\t" << hit.rdoff() << "\t" << hit.len() << "\t" << hit.score() << endl; spliceSites.clear(); ssdb.getRightSpliceSites(hit.ref(), hit.refoff() - minMatchLen, minMatchLen * 2, spliceSites); for(size_t si = 0; si < spliceSites.size(); si++) { const SpliceSite& ss = spliceSites[si]; if(ss.right() > tempHit.refoff()) { index_t dist = ss.right() - 1 - (tempHit.refoff() + tempHit.len() - 1); cout << rd.rdid << "s\t\t\t" << ss.left() + 1 << "\t" << ss.right() - 1 << "\t" << (ss.fw() ? "+" : "-") << "\t" << dist << endl; } } spliceSites.clear(); ssdb.getLeftSpliceSites(tempHit.ref(), tempHit.getRightOff() + minMatchLen, minMatchLen * 2, spliceSites); for(size_t si = 0; si < spliceSites.size(); si++) { const SpliceSite& ss = spliceSites[si]; if(ss.left() < hit.refoff()) { index_t dist = hit.refoff() - (ss.left() + 1); cout << rd.rdid << "s\t\t\t" << ss.left() + 1 << "\t" << ss.right() - 1 << "\t" << (ss.fw() ? "+" : "-") << "\t" << dist << endl; } } if(tempHit.rdoff() + tempHit.len() + 1 >= hit.rdoff()) return; #else assert_lt(hit.ref(), _refnames.size()); { ThreadSafe t(const_cast(_mutex), true); cout << rd.name << "\t" << _refnames[hit.ref()] << "\t" << (tempHit.fw() ? "+" : "-") << "\t" << tempHit.refoff() << "\t" << tempHit.rdoff() << "\t" << tempHit.len() << "\t" << tempHit.score() << "\t" << (hit.fw() ? "+" : "-") << "\t" << hit.refoff() << "\t" << hit.rdoff() << "\t" << hit.len() << "\t" << hit.score() << endl; } _done = true; return; #endif } // check if the partial alignment is compatible with the new alignment using the local index if(!tempHit.compatibleWith(hit, this->_no_spliced_alignment)) { if(count == 1) continue; else break; } if(uniqueStop) { assert_eq(coords.size(), 1); index_t leftext = (index_t)OFF_MASK, rightext = (index_t)0; tempHit.extend(rd, ref, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, this->_minK_local, leftext, rightext); } // combine the partial alignment and the new alignment int64_t minsc = this->_minsc[rdi]; bool combined = tempHit.combineWith(hit, rd, ref, ssdb, swa, swm, sc, minsc, rnd, this->_minK_local); if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); if(combined && tempHit.score() >= minsc) { assert_eq(tempHit.trim5(), 0); assert_leq(tempHit.rdoff() + tempHit.len() + tempHit.trim3(), rdlen); if(tempHit.score() >= prev_score - sc.mmpMax) { // extend the new partial alignment recursively hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff(), tempHit.len() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, dep + 1); } else { local_genomeHits.push_back(tempHit); } } } } int64_t minsc = (rdi == 0 ? sink.bestUnp1() : sink.bestUnp2()); if(minsc >= prev_score - sc.mmpMax) success = true; if(!success && (count == max_count || hierEbwtFw->prevLocalEbwt(localEbwtFw) == NULL)) { for(index_t ti = 0; ti < local_genomeHits.size(); ti++) { GenomeHit& tempHit = local_genomeHits[ti]; int64_t minsc = this->_minsc[rdi]; if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); if(tempHit.score() >= minsc) { hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff(), tempHit.len() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, dep + 1); } } } } // while(!success && count++ < 2) if(!success) { if(hitoff > this->_minK) { index_t extlen = 0; index_t top = (index_t)OFF_MASK, bot = (index_t)OFF_MASK; index_t extoff = hitoff - 1; bool uniqueStop = true; // perform global search for long introns index_t nelt = this->globalEbwtSearch( ebwtFw, // BWT index rd, // read to align sc, // scoring scheme hit.fw(), extoff, extlen, top, bot, rnd, uniqueStop); if(nelt <= 5 && extlen >= this->_minK) { coords.clear(); bool straddled = false; this->getGenomeCoords( ebwtFw, ref, rnd, top, bot, hit.fw(), bot - top, extoff + 1 - extlen, extlen, coords, wlm, prm, him, true, // reject straddled? straddled); assert_leq(coords.size(), nelt); coords.sort(); for(int ri = coords.size() - 1; ri >= 0; ri--) { const Coord& coord = coords[ri]; GenomeHit tempHit; tempHit.init(coord.orient(), extoff + 1 - extlen, extlen, 0, // trim5 0, // trim3 coord.ref(), coord.off(), this->_sharedVars); if(!tempHit.compatibleWith(hit, this->_no_spliced_alignment)) continue; if(uniqueStop) { assert_eq(coords.size(), 1); index_t leftext = (index_t)OFF_MASK, rightext = (index_t)0; tempHit.extend(rd, ref, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, this->_minK_local, leftext, rightext); } int64_t minsc = this->_minsc[rdi]; bool combined = tempHit.combineWith(hit, rd, ref, ssdb, swa, swm, sc, minsc, rnd, this->_minK_local); if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); if(combined && tempHit.score() >= minsc) { assert_eq(tempHit.trim5(), 0); assert_leq(tempHit.rdoff() + tempHit.len() + tempHit.trim3(), rdlen); hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff(), tempHit.len() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, dep + 1); } } } } GenomeHit tempHit = hit; if(tempHit.rdoff() <= 5) { index_t trim5 = tempHit.rdoff(); tempHit.trim5(trim5); assert_leq(tempHit.len() + tempHit.trim5() + tempHit.trim3(), rdlen); hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, tempHit, 0, tempHit.len() + tempHit.trim5() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, dep + 1); return; } // extend the partial alignment directly comparing with the corresponding genomic sequence // with mismatches or a gap allowed int64_t minsc = this->_minsc[rdi]; assert_geq(tempHit.score(), minsc); index_t mm = (tempHit.score() - minsc) / sc.mmpMax; index_t leftext = (index_t)OFF_MASK, rightext = (index_t)0; index_t num_mismatch_allowed = 1; if(hitoff <= this->_minK_local) { num_mismatch_allowed = min(tempHit.rdoff(), mm); } him.localextatts++; tempHit.extend(rd, ref, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, this->_minK_local, leftext, rightext, num_mismatch_allowed); if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); if(tempHit.score() >= minsc && leftext >= min(this->_minK_local, hit.rdoff())) { assert_eq(tempHit.trim5(), 0); assert_leq(tempHit.rdoff() + tempHit.len() + tempHit.trim3(), rdlen); hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff(), tempHit.len() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, dep + 1); } else if(hitoff > this->_minK_local) { // skip some bases of a read index_t jumplen = hitoff > this->_minK ? this->_minK : this->_minK_local; assert_leq(hitoff, hit.rdoff()); int64_t expected_score = hit.score() - (hit.rdoff() - hitoff) / jumplen * sc.mmpMax - sc.mmpMax; if(expected_score >= minsc) { assert_lt(hitlen + jumplen, rdlen); assert_eq(hit.trim5(), 0); assert_leq(hitoff + hitlen, rdlen); hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, hit, hitoff - jumplen, hitlen + jumplen, wlm, prm, swm, him, rnd, sink, dep + 1); } } } } else { // extend the partial alignment in the right direction assert_lt(hitoff + hitlen, rdlen); index_t fragoff = 0, fraglen = 0, right = 0; hit.getRight(fragoff, fraglen, right); const index_t minMatchLen = this->_minK_local; // make use of a list of known or novel splice sites to further align the read if(fraglen >= minMatchLen && !this->_no_spliced_alignment) { spliceSites.clear(); assert_gt(fraglen, 0); ssdb.getRightSpliceSites(hit.ref(), right + fraglen - minMatchLen, minMatchLen * 2, spliceSites); for(size_t si = 0; si < spliceSites.size(); si++) { const SpliceSite& ss = spliceSites[si]; if(!ss._fromfile && ss._readid + this->_thread_rids_mindist > rd.rdid) continue; if(right > ss.left()) continue; index_t frag2off = ss.right() - ss.left() + right + fraglen - 1; GenomeHit tempHit; tempHit.init(hit.fw(), fragoff + fraglen, rdlen - fragoff - fraglen, 0, // trim5 0, // trim3 hit.ref(), frag2off, this->_sharedVars); if(!hit.compatibleWith(tempHit, this->_no_spliced_alignment)) continue; GenomeHit combinedHit = hit; int64_t minsc = this->_minsc[rdi]; bool combined = combinedHit.combineWith(tempHit, rd, ref, ssdb, swa, swm, sc, minsc, rnd, this->_minK_local, 1, 1, false); if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); if(combined && combinedHit.score() >= minsc) { assert_leq(combinedHit.trim5(), combinedHit.rdoff()); assert_eq(combinedHit.rdoff() + combinedHit.len(), rdlen); hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, combinedHit, combinedHit.rdoff() - combinedHit.trim5(), combinedHit.len() + combinedHit.trim5(), wlm, prm, swm, him, rnd, sink, dep + 1); } } } // choose a local index based on the genomic location of the partial alignment const HierEbwt* hierEbwtFw = (const HierEbwt*)(&ebwtFw); const LocalEbwt* localEbwtFw = hierEbwtFw->getLocalEbwt(hit.ref(), hit.refoff()); bool success = false, first = true; index_t count = 0; index_t max_count = 2; int64_t prev_score = hit.score(); local_genomeHits.clear(); while(!success && count++ < max_count) { if(him.localindexatts >= this->max_localindexatts) return; if(first) { first = false; } else { localEbwtFw = hierEbwtFw->nextLocalEbwt(localEbwtFw); if(localEbwtFw == NULL || localEbwtFw->empty()) break; } // local index search index_t extlen = 0; local_index_t top = (local_index_t)OFF_MASK, bot = (local_index_t)OFF_MASK; // daehwan - for debugging purposes // index_t extoff = hitoff + hitlen + this->_minK_local; index_t extoff = hitoff + hitlen + this->_minK_local * 3; if(extoff + 1 < rdlen) extoff += 1; if(extoff >= rdlen) { extoff = rdlen - 1; } index_t nelt = (index_t)OFF_MASK; index_t max_nelt = std::max(5, extlen); bool no_extension = false; bool uniqueStop; // daehwan - for debugging purposes // index_t minUniqueLen = this->_minK_local; index_t minUniqueLen = (index_t)OFF_MASK; index_t maxHitLen = max(extoff + 1 - hitoff - hitlen, this->_minK_local); for(; maxHitLen < extoff + 1 && extoff < rdlen;) { extlen = 0; uniqueStop = false; him.localindexatts++; nelt = this->localEbwtSearch( localEbwtFw, // BWT index NULL, // BWT index rd, // read to align sc, // scoring scheme hit.fw(), false, // searchfw, extoff, extlen, top, bot, rnd, uniqueStop, minUniqueLen, maxHitLen); if(extoff < hitoff + hitlen) { no_extension = true; break; } if(nelt <= max_nelt) break; if(extoff + 1 < rdlen) extoff++; else { if(extlen < maxHitLen) break; else maxHitLen++; } } assert_leq(top, bot); assert_eq(nelt, (index_t)(bot - top)); assert_leq(extlen, extoff + 1); assert_leq(extoff, rdlen); if(nelt > 0 && nelt <= max_nelt && extlen >= minAnchorLen && !no_extension) { assert_leq(nelt, max_nelt); coords.clear(); bool straddled = false; // get genomic locations for this local search this->getGenomeCoords_local( *localEbwtFw, ref, rnd, top, bot, hit.fw(), extoff + 1 - extlen, extlen, coords, wlm, prm, him, true, // reject straddled? straddled); assert_leq(coords.size(), nelt); coords.sort(); for(index_t ri = 0; ri < coords.size(); ri++) { const Coord& coord = coords[ri]; GenomeHit tempHit; tempHit.init(coord.orient(), extoff + 1 - extlen, extlen, 0, // trim5 0, // trim3 coord.ref(), coord.off(), this->_sharedVars); // daehwan - for debugging purposes if(coord.ref() == hit.ref() && coord.off() < hit.refoff() && coord.off() + 64000 > hit.refoff()) { index_t leftext = (index_t)0, rightext = (index_t)OFF_MASK; index_t mm = 1; tempHit.extend( rd, ref, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, this->_minK_local, leftext, rightext, mm); #if 0 cout << endl; cout << rd.rdid << "\t" << rd.name << endl; cout << "\ttype: " << 2 << endl; cout << "\t" << hit.ref() << endl; cout << "\t\ttempHit " << (tempHit.fw() ? "+" : "-") << "\t" << tempHit.refoff() << "\t" << tempHit.rdoff() << "\t" << tempHit.len() << "\t" << tempHit.score() << endl; cout << "\t\tanchHit " << (hit.fw() ? "+" : "-") << "\t" << hit.refoff() << "\t" << hit.rdoff() << "\t" << hit.len() << "\t" << hit.score() << endl; spliceSites.clear(); ssdb.getRightSpliceSites(tempHit.ref(), tempHit.refoff() - minMatchLen, minMatchLen * 2, spliceSites); for(size_t si = 0; si < spliceSites.size(); si++) { const SpliceSite& ss = spliceSites[si]; if(ss.right() > hit.getRightOff()) { index_t dist = ss.right() - 1 - hit.getRightOff(); cout << rd.rdid << "s\t\t\t" << ss.left() + 1 << "\t" << ss.right() - 1 << "\t" << (ss.fw() ? "+" : "-") << "\t" << dist << endl; } } spliceSites.clear(); ssdb.getLeftSpliceSites(hit.ref(), hit.getRightOff() + minMatchLen, minMatchLen * 2, spliceSites); for(size_t si = 0; si < spliceSites.size(); si++) { const SpliceSite& ss = spliceSites[si]; if(ss.left() < tempHit.refoff()) { index_t dist = tempHit.refoff() - (ss.left() + 1); cout << rd.rdid << "s\t\t\t" << ss.left() + 1 << "\t" << ss.right() - 1 << "\t" << (ss.fw() ? "+" : "-") << "\t" << dist << endl; } } if(hit.rdoff() + hit.len() - 1 <= tempHit.rdoff()) return; #else assert_lt(hit.ref(), _refnames.size()); { ThreadSafe t(const_cast(_mutex), true); cout << rd.name << "\t" << _refnames[hit.ref()] << "\t" << (hit.fw() ? "+" : "-") << "\t" << hit.refoff() << "\t" << hit.rdoff() << "\t" << hit.len() << "\t" << hit.score() << "\t" << (tempHit.fw() ? "+" : "-") << "\t" << tempHit.refoff() << "\t" << tempHit.rdoff() << "\t" << tempHit.len() << "\t" << tempHit.score() << endl; } _done = true; return; #endif } // check if the partial alignment is compatible with the new alignment using the local index if(!hit.compatibleWith(tempHit, this->_no_spliced_alignment)) { if(count == 1) continue; else break; } index_t leftext = (index_t)0, rightext = (index_t)OFF_MASK; tempHit.extend(rd, ref, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, this->_minK_local, leftext, rightext); GenomeHit combinedHit = hit; int64_t minsc = this->_minsc[rdi]; // combine the partial alignment and the new alignment bool combined = combinedHit.combineWith(tempHit, rd, ref, ssdb, swa, swm, sc, minsc, rnd, this->_minK_local); if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); if(combined && combinedHit.score() >= minsc) { assert_leq(combinedHit.trim5(), combinedHit.rdoff()); if(combinedHit.score() >= prev_score - sc.mmpMax) { // extend the new partial alignment recursively hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, combinedHit, combinedHit.rdoff() - combinedHit.trim5(), combinedHit.len() + combinedHit.trim5(), wlm, prm, swm, him, rnd, sink, dep + 1); } else { local_genomeHits.push_back(combinedHit); } } } } int64_t minsc = (rdi == 0 ? sink.bestUnp1() : sink.bestUnp2()); if(minsc >= prev_score - sc.mmpMax) success = true; if(!success && (count == max_count || hierEbwtFw->nextLocalEbwt(localEbwtFw) == NULL) ) { for(index_t ti = 0; ti < local_genomeHits.size(); ti++) { GenomeHit& tempHit = local_genomeHits[ti]; int64_t minsc = this->_minsc[rdi]; if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); if(tempHit.score() >= minsc) { hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff() - tempHit.trim5(), tempHit.len() + tempHit.trim5(), wlm, prm, swm, him, rnd, sink, dep + 1); } } } } // while(!success && count++ < 2) if(!success) { // perform global search for long introns if(hitoff + hitlen + this->_minK + 1 < rdlen) { index_t extlen = 0; index_t top = (index_t)OFF_MASK, bot = (index_t)OFF_MASK; index_t extoff = hitoff + hitlen + this->_minK + 1; bool uniqueStop = true; index_t nelt = this->globalEbwtSearch( ebwtFw, // BWT index rd, // read to align sc, // scoring scheme hit.fw(), extoff, extlen, top, bot, rnd, uniqueStop); if(nelt <= 5 && extlen >= this->_minK) { coords.clear(); bool straddled = false; this->getGenomeCoords( ebwtFw, ref, rnd, top, bot, hit.fw(), bot - top, extoff + 1 - extlen, extlen, coords, wlm, prm, him, true, // reject straddled straddled); assert_leq(coords.size(), nelt); coords.sort(); for(index_t ri = 0; ri < coords.size(); ri++) { const Coord& coord = coords[ri]; GenomeHit tempHit; tempHit.init(coord.orient(), extoff + 1 - extlen, extlen, 0, // trim5 0, // trim3 coord.ref(), coord.off(), this->_sharedVars); if(!hit.compatibleWith(tempHit, this->_no_spliced_alignment)) continue; index_t leftext = (index_t)0, rightext = (index_t)OFF_MASK; tempHit.extend(rd, ref, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, this->_minK_local, leftext, rightext); GenomeHit combinedHit = hit; int64_t minsc = this->_minsc[rdi]; bool combined = combinedHit.combineWith(tempHit, rd, ref, ssdb, swa, swm, sc, minsc, rnd, this->_minK_local); if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); if(combined && combinedHit.score() >= minsc) { assert_leq(combinedHit.trim5(), combinedHit.rdoff()); hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, combinedHit, combinedHit.rdoff() - combinedHit.trim5(), combinedHit.len() + combinedHit.trim5(), wlm, prm, swm, him, rnd, sink, dep + 1); } } } } GenomeHit tempHit = hit; assert(tempHit.trim5() == 0 || hitoff == 0); if(rdlen - hitoff - tempHit.len() - tempHit.trim5() <= 5) { index_t trim3 = rdlen - hitoff - tempHit.len() - tempHit.trim5(); tempHit.trim3(trim3); assert_leq(tempHit.trim5(), tempHit.rdoff()); assert_leq(tempHit.len() + tempHit.trim5() + tempHit.trim3(), rdlen); hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff() - tempHit.trim5(), tempHit.len() + tempHit.trim5() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, dep + 1); return; } // extend the partial alignment directly comparing with the corresponding genomic sequence // with mismatches or a gap allowed int64_t minsc = this->_minsc[rdi]; assert_geq(tempHit.score(), minsc); index_t leftext = (index_t)0, rightext = (index_t)OFF_MASK; index_t mm = (tempHit.score() - minsc) / sc.mmpMax; index_t num_mismatch_allowed = 1; if(rdlen - hitoff - hitlen <= this->_minK_local) { num_mismatch_allowed = min(rdlen - tempHit.rdoff() - tempHit.len(), mm); } him.localextatts++; tempHit.extend(rd, ref, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, this->_minK_local, leftext, rightext, num_mismatch_allowed); if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); if(tempHit.score() >= minsc && rightext >= min(this->_minK_local, rdlen - hit.len() - hit.rdoff())) { assert_eq(tempHit.trim3(), 0); assert_leq(tempHit.trim5(), tempHit.rdoff()); hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff() - tempHit.trim5(), tempHit.len() + tempHit.trim5(), wlm, prm, swm, him, rnd, sink, dep + 1); } else if(hitoff + hitlen + this->_minK_local < rdlen) { // skip some bases of a read index_t jumplen = hitoff + hitlen + this->_minK < rdlen ? this->_minK : this->_minK_local; assert_lt(hitoff + hitlen + jumplen, rdlen); assert_leq(hit.len(), hitlen); int64_t expected_score = hit.score() - (hitlen - hit.len()) / jumplen * sc.mmpMax - sc.mmpMax; if(expected_score >= minsc) { assert_eq(hit.trim3(), 0); hybridSearch_recur( sc, ebwtFw, ebwtBw, ref, swa, ssdb, rdi, hit, hitoff, hitlen + jumplen, wlm, prm, swm, him, rnd, sink, dep + 1); } } } } } #endif /*BP_ALIGNER_H_*/ hisat2-2.2.1/btypes.h000066400000000000000000000025021370663763200144010ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef BOWTIE_INDEX_TYPES_H #define BOWTIE_INDEX_TYPES_H #ifdef BOWTIE_64BIT_INDEX #define OFF_MASK 0xffffffffffffffff #define OFF_LEN_MASK 0xc000000000000000 #define LS_SIZE 0x100000000000000 #define OFF_SIZE 8 #define INDEX_MAX 0xffffffffffffffff typedef uint64_t TIndexOffU; typedef int64_t TIndexOff; #else #define OFF_MASK 0xffffffff #define OFF_LEN_MASK 0xc0000000 #define LS_SIZE 0x10000000 #define OFF_SIZE 4 #define INDEX_MAX 0xffffffff typedef uint32_t TIndexOffU; typedef int TIndexOff; #endif /* BOWTIE_64BIT_INDEX */ extern const std::string gfm_ext; #endif /* BOWTIE_INDEX_TYPES_H */ hisat2-2.2.1/ccnt_lut.cpp000066400000000000000000000037041370663763200152460ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include /* Generated by gen_lookup_tables.pl */ uint8_t cCntLUT_4[4][4][256]; uint8_t cCntLUT_4_rev[4][4][256]; uint8_t cCntBIT[8][256]; int countCnt(int by, int c, uint8_t str) { int count = 0; if(by == 0) by = 4; while(by-- > 0) { int c2 = str & 3; str >>= 2; if(c == c2) count++; } return count; } int countCnt_rev(int by, int c, uint8_t str) { int count = 0; if(by == 0) by = 4; while(by-- > 0) { int c2 = (str >> 6) & 3; str <<= 2; if(c == c2) count++; } return count; } void initializeCntLut() { for(int by = 0; by < 4; by++) { for(int c = 0; c < 4; c++) { for(int str = 0; str < 256; str++) { cCntLUT_4[by][c][str] = countCnt(by, c, str); cCntLUT_4_rev[by][c][str] = countCnt_rev(by, c, str); } } } } int countBit(int b, uint8_t str) { int count = 0; if(b == 0) b = 8; while(b-- > 0) { if(str & 0x1) count++; str >>= 1; } return count; } void initializeCntBit() { for(int b = 0; b < 8; b++) { for(int str = 0; str < 256; str++) { cCntBIT[b][str] = countBit(b, str); } } } hisat2-2.2.1/diff_sample.cpp000066400000000000000000000107601370663763200157040ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "diff_sample.h" struct sampleEntry clDCs[16]; bool clDCs_calced = false; /// have clDCs been calculated? /** * Entries 4-57 are transcribed from page 6 of Luk and Wong's paper * "Two New Quorum Based Algorithms for Distributed Mutual Exclusion", * which is also used and cited in the Burkhardt and Karkkainen's * papers on difference covers for sorting. These samples are optimal * according to Luk and Wong. * * All other entries are generated via the exhaustive algorithm in * calcExhaustiveDC(). * * The 0 is stored at the end of the sample as an end-of-list marker, * but 0 is also an element of each. * * Note that every difference cover has a 0 and a 1. Intuitively, * any optimal difference cover sample can be oriented (i.e. rotated) * such that it includes 0 and 1 as elements. * * All samples in this list have been verified to be complete covers. * * A value of 0xffffffff in the first column indicates that there is no * sample for that value of v. We do not keep samples for values of v * less than 3, since they are trivial (and the caller probably didn't * mean to ask for it). */ uint32_t dc0to64[65][10] = { {0xffffffff}, // 0 {0xffffffff}, // 1 {0xffffffff}, // 2 {1, 0}, // 3 {1, 2, 0}, // 4 {1, 2, 0}, // 5 {1, 3, 0}, // 6 {1, 3, 0}, // 7 {1, 2, 4, 0}, // 8 {1, 2, 4, 0}, // 9 {1, 2, 5, 0}, // 10 {1, 2, 5, 0}, // 11 {1, 3, 7, 0}, // 12 {1, 3, 9, 0}, // 13 {1, 2, 3, 7, 0}, // 14 {1, 2, 3, 7, 0}, // 15 {1, 2, 5, 8, 0}, // 16 {1, 2, 4, 12, 0}, // 17 {1, 2, 5, 11, 0}, // 18 {1, 2, 6, 9, 0}, // 19 {1, 2, 3, 6, 10, 0}, // 20 {1, 4, 14, 16, 0}, // 21 {1, 2, 3, 7, 11, 0}, // 22 {1, 2, 3, 7, 11, 0}, // 23 {1, 2, 3, 7, 15, 0}, // 24 {1, 2, 3, 8, 12, 0}, // 25 {1, 2, 5, 9, 15, 0}, // 26 {1, 2, 5, 13, 22, 0}, // 27 {1, 4, 15, 20, 22, 0}, // 28 {1, 2, 3, 4, 9, 14, 0}, // 29 {1, 2, 3, 4, 9, 19, 0}, // 30 {1, 3, 8, 12, 18, 0}, // 31 {1, 2, 3, 7, 11, 19, 0}, // 32 {1, 2, 3, 6, 16, 27, 0}, // 33 {1, 2, 3, 7, 12, 20, 0}, // 34 {1, 2, 3, 8, 12, 21, 0}, // 35 {1, 2, 5, 12, 14, 20, 0}, // 36 {1, 2, 4, 10, 15, 22, 0}, // 37 {1, 2, 3, 4, 8, 14, 23, 0}, // 38 {1, 2, 4, 13, 18, 33, 0}, // 39 {1, 2, 3, 4, 9, 14, 24, 0}, // 40 {1, 2, 3, 4, 9, 15, 25, 0}, // 41 {1, 2, 3, 4, 9, 15, 25, 0}, // 42 {1, 2, 3, 4, 10, 15, 26, 0}, // 43 {1, 2, 3, 6, 16, 27, 38, 0}, // 44 {1, 2, 3, 5, 12, 18, 26, 0}, // 45 {1, 2, 3, 6, 18, 25, 38, 0}, // 46 {1, 2, 3, 5, 16, 22, 40, 0}, // 47 {1, 2, 5, 9, 20, 26, 36, 0}, // 48 {1, 2, 5, 24, 33, 36, 44, 0}, // 49 {1, 3, 8, 17, 28, 32, 38, 0}, // 50 {1, 2, 5, 11, 18, 30, 38, 0}, // 51 {1, 2, 3, 4, 6, 14, 21, 30, 0}, // 52 {1, 2, 3, 4, 7, 21, 29, 44, 0}, // 53 {1, 2, 3, 4, 9, 15, 21, 31, 0}, // 54 {1, 2, 3, 4, 6, 19, 26, 47, 0}, // 55 {1, 2, 3, 4, 11, 16, 33, 39, 0}, // 56 {1, 3, 13, 32, 36, 43, 52, 0}, // 57 // Generated by calcExhaustiveDC() {1, 2, 3, 7, 21, 33, 37, 50, 0}, // 58 {1, 2, 3, 6, 13, 21, 35, 44, 0}, // 59 {1, 2, 4, 9, 15, 25, 30, 42, 0}, // 60 {1, 2, 3, 7, 15, 25, 36, 45, 0}, // 61 {1, 2, 4, 10, 32, 39, 46, 51, 0}, // 62 {1, 2, 6, 8, 20, 38, 41, 54, 0}, // 63 {1, 2, 5, 14, 16, 34, 42, 59, 0} // 64 }; hisat2-2.2.1/diff_sample.h000066400000000000000000000750151370663763200153550ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef DIFF_SAMPLE_H_ #define DIFF_SAMPLE_H_ #include #include #include "assert_helpers.h" #include "multikey_qsort.h" #include "timer.h" #include "ds.h" #include "mem_ids.h" #include "ls.h" #include "btypes.h" using namespace std; #ifndef VMSG_NL #define VMSG_NL(...) \ if(this->verbose()) { \ stringstream tmp; \ tmp << __VA_ARGS__ << endl; \ this->verbose(tmp.str()); \ } #endif #ifndef VMSG #define VMSG(...) \ if(this->verbose()) { \ stringstream tmp; \ tmp << __VA_ARGS__; \ this->verbose(tmp.str()); \ } #endif /** * Routines for calculating, sanity-checking, and dispensing difference * cover samples to clients. */ /** * */ struct sampleEntry { uint32_t maxV; uint32_t numSamples; uint32_t samples[128]; }; /// Array of Colbourn and Ling calculated difference covers up to /// r = 16 (maxV = 5953) extern struct sampleEntry clDCs[16]; extern bool clDCs_calced; /// have clDCs been calculated? /** * Check that the given difference cover 'ds' actually covers all * differences for a periodicity of v. */ template static bool dcRepOk(T v, EList& ds) { // diffs[] records all the differences observed AutoArray covered(v, EBWT_CAT); for(T i = 1; i < v; i++) { covered[i] = false; } for(T di = T(); di < ds.size(); di++) { for(T dj = di+1; dj < ds.size(); dj++) { assert_lt(ds[di], ds[dj]); T d1 = (ds[dj] - ds[di]); T d2 = (ds[di] + v - ds[dj]); assert_lt(d1, v); assert_lt(d2, v); covered[d1] = true; covered[d2] = true; } } bool ok = true; for(T i = 1; i < v; i++) { if(covered[i] == false) { ok = false; break; } } return ok; } /** * Return true iff each element of ts (with length 'limit') is greater * than the last. */ template static bool increasing(T* ts, size_t limit) { for(size_t i = 0; i < limit-1; i++) { if(ts[i+1] <= ts[i]) return false; } return true; } /** * Return true iff the given difference cover covers difference 'diff' * mod 'v'. */ template static inline bool hasDifference(T *ds, T d, T v, T diff) { // diffs[] records all the differences observed for(T di = T(); di < d; di++) { for(T dj = di+1; dj < d; dj++) { assert_lt(ds[di], ds[dj]); T d1 = (ds[dj] - ds[di]); T d2 = (ds[di] + v - ds[dj]); assert_lt(d1, v); assert_lt(d2, v); if(d1 == diff || d2 == diff) return true; } } return false; } /** * Exhaustively calculate optimal difference cover samples for v = 4, * 8, 16, 32, 64, 128, 256 and store results in p2DCs[] */ template void calcExhaustiveDC(T i, bool verbose = false, bool sanityCheck = false) { T v = i; AutoArray diffs(v, EBWT_CAT); // v is the target period T ld = (T)ceil(sqrt(v)); // ud is the upper bound on |D| T ud = v / 2; // for all possible |D|s bool ok = true; T *ds = NULL; T d; for(d = ld; d <= ud+1; d++) { // for all possible |D| samples AutoArray ds(d, EBWT_CAT); for(T j = 0; j < d; j++) { ds[j] = j; } assert(increasing(ds, d)); while(true) { // reset diffs[] for(T t = 1; t < v; t++) { diffs[t] = false; } T diffCnt = 0; // diffs[] records all the differences observed for(T di = 0; di < d; di++) { for(T dj = di+1; dj < d; dj++) { assert_lt(ds[di], ds[dj]); T d1 = (ds[dj] - ds[di]); T d2 = (ds[di] + v - ds[dj]); assert_lt(d1, v); assert_lt(d2, v); assert_gt(d1, 0); assert_gt(d2, 0); if(!diffs[d1]) { diffCnt++; diffs[d1] = true; } if(!diffs[d2]) { diffCnt++; diffs[d2] = true; } } } // Do we observe all possible differences (except 0) ok = diffCnt == v-1; if(ok) { // Yes, all differences are covered break; } else { // Advance ds // (Following is commented out because it turns out // it's slow) // Find a missing difference //uint32_t missing = 0xffffffff; //for(uint32_t t = 1; t < v; t++) { // if(diffs[t] == false) { // missing = diffs[t]; // break; // } //} //assert_neq(missing, 0xffffffff); assert(increasing(ds, d)); bool advanced = false; bool keepGoing = false; do { keepGoing = false; for(T bd = d-1; bd > 1; bd--) { T dif = (d-1)-bd; if(ds[bd] < v-1-dif) { ds[bd]++; assert_neq(0, ds[bd]); // Reset subsequent ones for(T bdi = bd+1; bdi < d; bdi++) { assert_eq(0, ds[bdi]); ds[bdi] = ds[bdi-1]+1; assert_gt(ds[bdi], ds[bdi-1]); } assert(increasing(ds, d)); // (Following is commented out because // it turns out it's slow) // See if the new DC has the missing value //if(!hasDifference(ds, d, v, missing)) { // keepGoing = true; // break; //} advanced = true; break; } else { ds[bd] = 0; // keep going } } } while(keepGoing); // No solution for this |D| if(!advanced) break; assert(increasing(ds, d)); } } // next sample assignment if(ok) { break; } } // next |D| assert(ok); cout << "Did exhaustive v=" << v << " |D|=" << d << endl; cout << " "; for(T i = 0; i < d; i++) { cout << ds[i]; if(i < d-1) cout << ","; } cout << endl; } /** * Routune for calculating the elements of clDCs up to r = 16 using the * technique of Colbourn and Ling. * * See http://citeseer.ist.psu.edu/211575.html */ template void calcColbournAndLingDCs(bool verbose = false, bool sanityCheck = false) { for(T r = 0; r < 16; r++) { T maxv = 24*r*r + 36*r + 13; // Corollary 2.3 T numsamp = 6*r + 4; clDCs[r].maxV = maxv; clDCs[r].numSamples = numsamp; memset(clDCs[r].samples, 0, 4 * 128); T i; // clDCs[r].samples[0] = 0; // Fill in the 1^r part of the B series for(i = 1; i < r+1; i++) { clDCs[r].samples[i] = clDCs[r].samples[i-1] + 1; } // Fill in the (r + 1)^1 part clDCs[r].samples[r+1] = clDCs[r].samples[r] + r + 1; // Fill in the (2r + 1)^r part for(i = r+2; i < r+2+r; i++) { clDCs[r].samples[i] = clDCs[r].samples[i-1] + 2*r + 1; } // Fill in the (4r + 3)^(2r + 1) part for(i = r+2+r; i < r+2+r+2*r+1; i++) { clDCs[r].samples[i] = clDCs[r].samples[i-1] + 4*r + 3; } // Fill in the (2r + 2)^(r + 1) part for(i = r+2+r+2*r+1; i < r+2+r+2*r+1+r+1; i++) { clDCs[r].samples[i] = clDCs[r].samples[i-1] + 2*r + 2; } // Fill in the last 1^r part for(i = r+2+r+2*r+1+r+1; i < r+2+r+2*r+1+r+1+r; i++) { clDCs[r].samples[i] = clDCs[r].samples[i-1] + 1; } assert_eq(i, numsamp); assert_lt(i, 128); if(sanityCheck) { // diffs[] records all the differences observed AutoArray diffs(maxv, EBWT_CAT); for(T i = 0; i < numsamp; i++) { for(T j = i+1; j < numsamp; j++) { T d1 = (clDCs[r].samples[j] - clDCs[r].samples[i]); T d2 = (clDCs[r].samples[i] + maxv - clDCs[r].samples[j]); assert_lt(d1, maxv); assert_lt(d2, maxv); diffs[d1] = true; diffs[d2] = true; } } // Should have observed all possible differences (except 0) for(T i = 1; i < maxv; i++) { if(diffs[i] == false) cout << r << ", " << i << endl; assert(diffs[i] == true); } } } clDCs_calced = true; } /** * A precalculated list of difference covers. */ extern uint32_t dc0to64[65][10]; /** * Get a difference cover for the requested periodicity v. */ template static EList getDiffCover( T v, bool verbose = false, bool sanityCheck = false) { assert_gt(v, 2); EList ret; ret.clear(); // Can we look it up in our hardcoded array? if(v <= 64 && dc0to64[v][0] == 0xffffffff) { if(verbose) cout << "v in hardcoded area, but hardcoded entry was all-fs" << endl; return ret; } else if(v <= 64) { ret.push_back(0); for(size_t i = 0; i < 10; i++) { if(dc0to64[v][i] == 0) break; ret.push_back(dc0to64[v][i]); } if(sanityCheck) assert(dcRepOk(v, ret)); return ret; } // Can we look it up in our calcColbournAndLingDCs array? if(!clDCs_calced) { calcColbournAndLingDCs(verbose, sanityCheck); assert(clDCs_calced); } for(size_t i = 0; i < 16; i++) { if(v <= clDCs[i].maxV) { for(size_t j = 0; j < clDCs[i].numSamples; j++) { T s = clDCs[i].samples[j]; if(s >= v) { s %= v; for(size_t k = 0; k < ret.size(); k++) { if(s == ret[k]) break; if(s < ret[k]) { ret.insert(s, k); break; } } } else { ret.push_back(s % v); } } if(sanityCheck) assert(dcRepOk(v, ret)); return ret; } } cerr << "Error: Could not find a difference cover sample for v=" << v << endl; throw 1; } /** * Calculate and return a delta map based on the given difference cover * and periodicity v. */ template static EList getDeltaMap(T v, const EList& dc) { // Declare anchor-map-related items EList amap; size_t amapEnts = 1; amap.resizeExact((size_t)v); amap.fill(0xffffffff); amap[0] = 0; // Print out difference cover (and optionally calculate // anchor map) for(size_t i = 0; i < dc.size(); i++) { for(size_t j = i+1; j < dc.size(); j++) { assert_gt(dc[j], dc[i]); T diffLeft = dc[j] - dc[i]; T diffRight = dc[i] + v - dc[j]; assert_lt(diffLeft, v); assert_lt(diffRight, v); if(amap[diffLeft] == 0xffffffff) { amap[diffLeft] = dc[i]; amapEnts++; } if(amap[diffRight] == 0xffffffff) { amap[diffRight] = dc[j]; amapEnts++; } } } return amap; } /** * Return population count (count of all bits set to 1) of i. */ template static unsigned int popCount(T i) { unsigned int cnt = 0; for(size_t j = 0; j < sizeof(T)*8; j++) { if(i & 1) cnt++; i >>= 1; } return cnt; } /** * Calculate log-base-2 of i */ template static unsigned int myLog2(T i) { assert_eq(1, popCount(i)); // must be power of 2 for(size_t j = 0; j < sizeof(T)*8; j++) { if(i & 1) return (int)j; i >>= 1; } assert(false); return 0xffffffff; } /** * */ template class DifferenceCoverSample { public: DifferenceCoverSample(const TStr& __text, uint32_t __v, bool __verbose = false, bool __sanity = false, ostream& __logger = cout) : _text(__text), _v(__v), _verbose(__verbose), _sanity(__sanity), _ds(getDiffCover(_v, _verbose, _sanity)), _dmap(getDeltaMap(_v, _ds)), _d((uint32_t)_ds.size()), _doffs(), _isaPrime(), _dInv(), _log2v(myLog2(_v)), _vmask(OFF_MASK << _log2v), _logger(__logger) { assert_gt(_d, 0); assert_eq(1, popCount(_v)); // must be power of 2 // Build map from d's to idx's _dInv.resizeExact((size_t)v()); _dInv.fill(0xffffffff); uint32_t lim = (uint32_t)_ds.size(); for(uint32_t i = 0; i < lim; i++) { _dInv[_ds[i]] = i; } } /** * Allocate an amount of memory that simulates the peak memory * usage of the DifferenceCoverSample with the given text and v. * Throws bad_alloc if it's not going to fit in memory. Returns * the approximate number of bytes the Cover takes at all times. */ static size_t simulateAllocs(const TStr& text, uint32_t v) { EList ds(getDiffCover(v, false /*verbose*/, false /*sanity*/)); size_t len = text.length(); size_t sPrimeSz = (len / v) * ds.size(); // sPrime, sPrimeOrder, _isaPrime all exist in memory at // once and that's the peak AutoArray aa(sPrimeSz * 3 + (1024 * 1024 /*out of caution*/), EBWT_CAT); return sPrimeSz * 4; // sPrime array } uint32_t v() const { return _v; } uint32_t log2v() const { return _log2v; } uint32_t vmask() const { return _vmask; } uint32_t modv(TIndexOffU i) const { return (uint32_t)(i & ~_vmask); } TIndexOffU divv(TIndexOffU i) const { return i >> _log2v; } uint32_t d() const { return _d; } bool verbose() const { return _verbose; } bool sanityCheck() const { return _sanity; } const TStr& text() const { return _text; } const EList& ds() const { return _ds; } const EList& dmap() const { return _dmap; } ostream& log() const { return _logger; } void build(int nthreads); uint32_t tieBreakOff(TIndexOffU i, TIndexOffU j) const; int64_t breakTie(TIndexOffU i, TIndexOffU j) const; bool isCovered(TIndexOffU i) const; TIndexOffU rank(TIndexOffU i) const; /** * Print out the suffix array such that every sample offset has its * rank filled in and every non-sample offset is shown as '-'. */ void print(ostream& out) { for(size_t i = 0; i < _text.length(); i++) { if(isCovered(i)) { out << rank(i); } else { out << "-"; } if(i < _text.length()-1) { out << ","; } } out << endl; } private: void doBuiltSanityCheck() const; void buildSPrime(EList& sPrime, size_t padding); bool built() const { return _isaPrime.size() > 0; } void verbose(const string& s) const { if(this->verbose()) { this->log() << s.c_str(); this->log().flush(); } } const TStr& _text; // text to sample uint32_t _v; // periodicity of sample bool _verbose; // bool _sanity; // EList _ds; // samples: idx -> d EList _dmap; // delta map uint32_t _d; // |D| - size of sample EList _doffs; // offsets into sPrime/isaPrime for each d idx EList _isaPrime; // ISA' array EList _dInv; // Map from d -> idx uint32_t _log2v; TIndexOffU _vmask; ostream& _logger; }; /** * Sanity-check the difference cover by first inverting _isaPrime then * checking that each successive suffix really is less than the next. */ template void DifferenceCoverSample::doBuiltSanityCheck() const { uint32_t v = this->v(); assert(built()); VMSG_NL(" Doing sanity check"); TIndexOffU added = 0; EList sorted; sorted.resizeExact(_isaPrime.size()); sorted.fill(OFF_MASK); for(size_t di = 0; di < this->d(); di++) { uint32_t d = _ds[di]; size_t i = 0; for(size_t doi = _doffs[di]; doi < _doffs[di+1]; doi++, i++) { assert_eq(OFF_MASK, sorted[_isaPrime[doi]]); // Maps the offset of the suffix to its rank sorted[_isaPrime[doi]] = (TIndexOffU)(v*i + d); added++; } } assert_eq(added, _isaPrime.size()); #ifndef NDEBUG for(size_t i = 0; i < sorted.size()-1; i++) { assert(sstr_suf_lt(this->text(), sorted[i], this->text(), sorted[i+1], false)); } #endif } /** * Build the s' array by sampling suffixes (suffix offsets, actually) * from t according to the difference-cover sample and pack them into * an array of machine words in the order dictated by the "mu" mapping * described in Burkhardt. * * Also builds _doffs map. */ template void DifferenceCoverSample::buildSPrime( EList& sPrime, size_t padding) { const TStr& t = this->text(); const EList& ds = this->ds(); TIndexOffU tlen = (TIndexOffU)t.length(); uint32_t v = this->v(); uint32_t d = this->d(); assert_gt(v, 2); assert_lt(d, v); // Record where each d section should begin in sPrime TIndexOffU tlenDivV = this->divv(tlen); uint32_t tlenModV = this->modv(tlen); TIndexOffU sPrimeSz = 0; assert(_doffs.empty()); _doffs.resizeExact((size_t)d+1); for(uint32_t di = 0; di < d; di++) { // mu mapping TIndexOffU sz = tlenDivV + ((ds[di] <= tlenModV) ? 1 : 0); assert_geq(sz, 0); _doffs[di] = sPrimeSz; sPrimeSz += sz; } _doffs[d] = sPrimeSz; #ifndef NDEBUG if(tlenDivV > 0) { for(size_t i = 0; i < d; i++) { assert_gt(_doffs[i+1], _doffs[i]); TIndexOffU diff = _doffs[i+1] - _doffs[i]; assert(diff == tlenDivV || diff == tlenDivV+1); } } #endif assert_eq(_doffs.size(), d+1); // Size sPrime appropriately sPrime.resizeExact((size_t)sPrimeSz + padding); sPrime.fill(OFF_MASK); // Slot suffixes from text into sPrime according to the mu // mapping; where the mapping would leave a blank, insert a 0 TIndexOffU added = 0; TIndexOffU i = 0; for(uint64_t ti = 0; ti <= tlen; ti += v) { for(uint32_t di = 0; di < d; di++) { TIndexOffU tti = (TIndexOffU)ti + (TIndexOffU)ds[di]; if(tti > tlen) break; TIndexOffU spi = _doffs[di] + i; assert_lt(spi, _doffs[di+1]); assert_leq(tti, tlen); assert_lt(spi, sPrimeSz); assert_eq(OFF_MASK, sPrime[spi]); sPrime[spi] = tti; added++; } i++; } assert_eq(added, sPrimeSz); } /** * Return true iff suffixes with offsets suf1 and suf2 out of host * string 'host' are identical up to depth 'v'. */ template static inline bool suffixSameUpTo( const TStr& host, TIndexOffU suf1, TIndexOffU suf2, TIndexOffU v) { for(TIndexOffU i = 0; i < v; i++) { bool endSuf1 = suf1+i >= host.length(); bool endSuf2 = suf2+i >= host.length(); if((endSuf1 && !endSuf2) || (!endSuf1 && endSuf2)) return false; if(endSuf1 && endSuf2) return true; if(host[suf1+i] != host[suf2+i]) return false; } return true; } template struct VSortingParam { DifferenceCoverSample* dcs; TIndexOffU* sPrimeArr; size_t sPrimeSz; TIndexOffU* sPrimeOrderArr; size_t depth; const EList* boundaries; size_t* cur; MUTEX_T* mutex; }; template static void VSorting_worker(void *vp) { VSortingParam* param = (VSortingParam*)vp; DifferenceCoverSample* dcs = param->dcs; const TStr& host = dcs->text(); const size_t hlen = host.length(); uint32_t v = dcs->v(); while(true) { size_t cur = 0; { ThreadSafe ts(param->mutex, true); cur = *(param->cur); (*param->cur)++; } if(cur >= param->boundaries->size()) return; size_t begin = (cur == 0 ? 0 : (*param->boundaries)[cur-1]); size_t end = (*param->boundaries)[cur]; assert_leq(begin, end); if(end - begin <= 1) continue; mkeyQSortSuf2( host, hlen, param->sPrimeArr, param->sPrimeSz, param->sPrimeOrderArr, 4, begin, end, param->depth, v); } } /** * Calculates a ranking of all suffixes in the sample and stores them, * packed according to the mu mapping, in _isaPrime. */ template void DifferenceCoverSample::build(int nthreads) { // Local names for relevant types VMSG_NL("Building DifferenceCoverSample"); // Local names for relevant data const TStr& t = this->text(); uint32_t v = this->v(); assert_gt(v, 2); // Build s' EList sPrime; // Need to allocate 2 extra elements at the end of the sPrime and _isaPrime // arrays. One element that's less than all others, and another that acts // as needed padding for the Larsson-Sadakane sorting code. size_t padding = 1; VMSG_NL(" Building sPrime"); buildSPrime(sPrime, padding); size_t sPrimeSz = sPrime.size() - padding; assert_gt(sPrime.size(), padding); assert_leq(sPrime.size(), t.length() + padding + 1); TIndexOffU nextRank = 0; { VMSG_NL(" Building sPrimeOrder"); EList sPrimeOrder; sPrimeOrder.resizeExact(sPrimeSz); for(TIndexOffU i = 0; i < sPrimeSz; i++) { sPrimeOrder[i] = i; } // sPrime now holds suffix-offsets for DC samples. { Timer timer(cout, " V-Sorting samples time: ", this->verbose()); VMSG_NL(" V-Sorting samples"); // Extract backing-store array from sPrime and sPrimeOrder; // the mkeyQSortSuf2 routine works on the array for maximum // efficiency TIndexOffU *sPrimeArr = (TIndexOffU*)sPrime.ptr(); assert_eq(sPrimeArr[0], sPrime[0]); assert_eq(sPrimeArr[sPrimeSz-1], sPrime[sPrimeSz-1]); TIndexOffU *sPrimeOrderArr = (TIndexOffU*)sPrimeOrder.ptr(); assert_eq(sPrimeOrderArr[0], sPrimeOrder[0]); assert_eq(sPrimeOrderArr[sPrimeSz-1], sPrimeOrder[sPrimeSz-1]); // Sort sample suffixes up to the vth character using a // multikey quicksort. Sort time is proportional to the // number of samples times v. It isn't quadratic. // sPrimeOrder is passed in as a swapping partner for // sPrimeArr, i.e., every time the multikey qsort swaps // elements in sPrime, it swaps the same elements in // sPrimeOrder too. This allows us to easily reconstruct // what the sort did. if(nthreads == 1) { mkeyQSortSuf2(t, sPrimeArr, sPrimeSz, sPrimeOrderArr, 4, this->verbose(), this->sanityCheck(), v); } else { int query_depth = 0; int tmp_nthreads = nthreads; while(tmp_nthreads > 0) { query_depth++; tmp_nthreads >>= 1; } EList boundaries; // bucket boundaries for parallelization TIndexOffU *sOrig = NULL; if(this->sanityCheck()) { sOrig = new TIndexOffU[sPrimeSz]; memcpy(sOrig, sPrimeArr, OFF_SIZE * sPrimeSz); } mkeyQSortSuf2(t, sPrimeArr, sPrimeSz, sPrimeOrderArr, 4, this->verbose(), false, query_depth, &boundaries); if(boundaries.size() > 0) { AutoArray threads(nthreads); EList > tparams; size_t cur = 0; MUTEX_T mutex; for(int tid = 0; tid < nthreads; tid++) { // Calculate bucket sizes by doing a binary search for each // suffix and noting where it lands tparams.expand(); tparams.back().dcs = this; tparams.back().sPrimeArr = sPrimeArr; tparams.back().sPrimeSz = sPrimeSz; tparams.back().sPrimeOrderArr = sPrimeOrderArr; tparams.back().depth = query_depth; tparams.back().boundaries = &boundaries; tparams.back().cur = &cur; tparams.back().mutex = &mutex; threads[tid] = new tthread::thread(VSorting_worker, (void*)&tparams.back()); } for (int tid = 0; tid < nthreads; tid++) { threads[tid]->join(); } } if(this->sanityCheck()) { sanityCheckOrderedSufs(t, t.length(), sPrimeArr, sPrimeSz, v); for(size_t i = 0; i < sPrimeSz; i++) { assert_eq(sPrimeArr[i], sOrig[sPrimeOrderArr[i]]); } delete[] sOrig; } } // Make sure sPrime and sPrimeOrder are consistent with // their respective backing-store arrays assert_eq(sPrimeArr[0], sPrime[0]); assert_eq(sPrimeArr[sPrimeSz-1], sPrime[sPrimeSz-1]); assert_eq(sPrimeOrderArr[0], sPrimeOrder[0]); assert_eq(sPrimeOrderArr[sPrimeSz-1], sPrimeOrder[sPrimeSz-1]); } // Now assign the ranking implied by the sorted sPrime/sPrimeOrder // arrays back into sPrime. VMSG_NL(" Allocating rank array"); _isaPrime.resizeExact(sPrime.size()); ASSERT_ONLY(_isaPrime.fill(OFF_MASK)); assert_gt(_isaPrime.size(), 0); { Timer timer(cout, " Ranking v-sort output time: ", this->verbose()); VMSG_NL(" Ranking v-sort output"); for(size_t i = 0; i < sPrimeSz-1; i++) { // Place the appropriate ranking _isaPrime[sPrimeOrder[i]] = nextRank; // If sPrime[i] and sPrime[i+1] are identical up to v, then we // should give the next suffix the same rank if(!suffixSameUpTo(t, sPrime[i], sPrime[i+1], v)) nextRank++; } _isaPrime[sPrimeOrder[sPrimeSz-1]] = nextRank; // finish off #ifndef NDEBUG for(size_t i = 0; i < sPrimeSz; i++) { assert_neq(OFF_MASK, _isaPrime[i]); assert_lt(_isaPrime[i], sPrimeSz); } #endif } // sPrimeOrder is destroyed // All the information we need is now in _isaPrime } _isaPrime[_isaPrime.size()-1] = (TIndexOffU)sPrimeSz; sPrime[sPrime.size()-1] = (TIndexOffU)sPrimeSz; // _isaPrime[_isaPrime.size()-1] and sPrime[sPrime.size()-1] are just // spacer for the Larsson-Sadakane routine to use { Timer timer(cout, " Invoking Larsson-Sadakane on ranks time: ", this->verbose()); VMSG_NL(" Invoking Larsson-Sadakane on ranks"); if(sPrime.size() >= LS_SIZE) { cerr << "Error; sPrime array has so many elements that it can't be converted to a signed array without overflow." << endl; throw 1; } LarssonSadakane ls; ls.suffixsort( (TIndexOff*)_isaPrime.ptr(), (TIndexOff*)sPrime.ptr(), (TIndexOff)sPrimeSz, (TIndexOff)sPrime.size(), 0); } // chop off final character of _isaPrime _isaPrime.resizeExact(sPrimeSz); for(size_t i = 0; i < _isaPrime.size(); i++) { _isaPrime[i]--; } #ifndef NDEBUG for(size_t i = 0; i < sPrimeSz-1; i++) { assert_lt(_isaPrime[i], sPrimeSz); assert(i == 0 || _isaPrime[i] != _isaPrime[i-1]); } #endif VMSG_NL(" Sanity-checking and returning"); if(this->sanityCheck()) doBuiltSanityCheck(); } /** * Return true iff index i within the text is covered by the difference * cover sample. Allow i to be off the end of the text; simplifies * logic elsewhere. */ template bool DifferenceCoverSample::isCovered(TIndexOffU i) const { assert(built()); uint32_t modi = this->modv(i); assert_lt(modi, _dInv.size()); return _dInv[modi] != 0xffffffff; } /** * Given a text offset that's covered, return its lexicographical rank * among the sample suffixes. */ template TIndexOffU DifferenceCoverSample::rank(TIndexOffU i) const { assert(built()); assert_lt(i, this->text().length()); uint32_t imodv = this->modv(i); assert_neq(0xffffffff, _dInv[imodv]); // must be in the sample TIndexOffU ioff = this->divv(i); assert_lt(ioff, _doffs[_dInv[imodv]+1] - _doffs[_dInv[imodv]]); TIndexOffU isaIIdx = _doffs[_dInv[imodv]] + ioff; assert_lt(isaIIdx, _isaPrime.size()); TIndexOffU isaPrimeI = _isaPrime[isaIIdx]; assert_leq(isaPrimeI, _isaPrime.size()); return isaPrimeI; } /** * Return: < 0 if suffix i is lexicographically less than suffix j; > 0 * if suffix j is lexicographically greater. */ template int64_t DifferenceCoverSample::breakTie(TIndexOffU i, TIndexOffU j) const { assert(built()); assert_neq(i, j); assert_lt(i, this->text().length()); assert_lt(j, this->text().length()); uint32_t imodv = this->modv(i); uint32_t jmodv = this->modv(j); assert_neq(0xffffffff, _dInv[imodv]); // must be in the sample assert_neq(0xffffffff, _dInv[jmodv]); // must be in the sample uint32_t dimodv = _dInv[imodv]; uint32_t djmodv = _dInv[jmodv]; TIndexOffU ioff = this->divv(i); TIndexOffU joff = this->divv(j); assert_lt(dimodv+1, _doffs.size()); assert_lt(djmodv+1, _doffs.size()); // assert_lt: expected (32024) < (0) assert_lt(ioff, _doffs[dimodv+1] - _doffs[dimodv]); assert_lt(joff, _doffs[djmodv+1] - _doffs[djmodv]); TIndexOffU isaIIdx = _doffs[dimodv] + ioff; TIndexOffU isaJIdx = _doffs[djmodv] + joff; assert_lt(isaIIdx, _isaPrime.size()); assert_lt(isaJIdx, _isaPrime.size()); assert_neq(isaIIdx, isaJIdx); // ranks must be unique TIndexOffU isaPrimeI = _isaPrime[isaIIdx]; TIndexOffU isaPrimeJ = _isaPrime[isaJIdx]; assert_neq(isaPrimeI, isaPrimeJ); // ranks must be unique assert_leq(isaPrimeI, _isaPrime.size()); assert_leq(isaPrimeJ, _isaPrime.size()); return (int64_t)isaPrimeI - (int64_t)isaPrimeJ; } /** * Given i, j, return the number of additional characters that need to * be compared before the difference cover can break the tie. */ template uint32_t DifferenceCoverSample::tieBreakOff(TIndexOffU i, TIndexOffU j) const { const TStr& t = this->text(); const EList& dmap = this->dmap(); assert(built()); // It's actually convenient to allow this, but we're permitted to // return nonsense in that case if(t[i] != t[j]) return 0xffffffff; //assert_eq(t[i], t[j]); // if they're unequal, there's no tie to break uint32_t v = this->v(); assert_neq(i, j); assert_lt(i, t.length()); assert_lt(j, t.length()); uint32_t imod = this->modv(i); uint32_t jmod = this->modv(j); uint32_t diffLeft = (jmod >= imod)? (jmod - imod) : (jmod + v - imod); uint32_t diffRight = (imod >= jmod)? (imod - jmod) : (imod + v - jmod); assert_lt(diffLeft, dmap.size()); assert_lt(diffRight, dmap.size()); uint32_t destLeft = dmap[diffLeft]; // offset where i needs to be uint32_t destRight = dmap[diffRight]; // offset where i needs to be assert(isCovered(destLeft)); assert(isCovered(destLeft+diffLeft)); assert(isCovered(destRight)); assert(isCovered(destRight+diffRight)); assert_lt(destLeft, v); assert_lt(destRight, v); uint32_t deltaLeft = (destLeft >= imod)? (destLeft - imod) : (destLeft + v - imod); if(deltaLeft == v) deltaLeft = 0; uint32_t deltaRight = (destRight >= jmod)? (destRight - jmod) : (destRight + v - jmod); if(deltaRight == v) deltaRight = 0; assert_lt(deltaLeft, v); assert_lt(deltaRight, v); assert(isCovered(i+deltaLeft)); assert(isCovered(j+deltaLeft)); assert(isCovered(i+deltaRight)); assert(isCovered(j+deltaRight)); return min(deltaLeft, deltaRight); } #endif /*DIFF_SAMPLE_H_*/ hisat2-2.2.1/docs/000077500000000000000000000000001370663763200136535ustar00rootroot00000000000000hisat2-2.2.1/docs/404.html000066400000000000000000000002151370663763200150460ustar00rootroot00000000000000--- layout: page title: 404 Not Found permalink: 404.html hide: true share: false --- Sorry, the requested page wasn't found on the server. hisat2-2.2.1/docs/Gemfile000066400000000000000000000001201370663763200151370ustar00rootroot00000000000000source 'https://rubygems.org' gem 'github-pages' gem 'jekyll-feed' gem 'jemoji' hisat2-2.2.1/docs/LICENSE000066400000000000000000000020671370663763200146650ustar00rootroot00000000000000The MIT License (MIT) Copyright (c) 2014 Rohan Chandra Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions: The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software. THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.hisat2-2.2.1/docs/README.md000066400000000000000000000032171370663763200151350ustar00rootroot00000000000000# jekyll-ttskch-theme A simple and customizable theme for Jekyll. > This theme was renamed from _jekyll-**qck**-theme_ to _jekyll-**tch**-theme_ at 2016.06.02. > And renamed again from _jekyll-**tch**-theme_ to _jekyll-**ttskch**-theme_ at 2016.09.23. ## Screen shot ![image](https://cloud.githubusercontent.com/assets/4360663/18776176/62611b38-81a2-11e6-875b-86a66aa8f15c.png) ## Features * A lot of Markdown features (also GitHub Flavored Markdown) * `:emoji:` ready :+1: * Easy color-scheme customization * Tags list page * Monthly Archives page * Search feature without any Jekyll plugins * `` tag feature * Anchor links for each headings * Sticky side nav * Responsive * OGP ready * Share buttons ready ## Getting started 1. [Fork me](https://github.com/ttskch/jekyll-ttskch-theme/fork) 2. Rename the repository from `jekyll-ttskch-theme` to `{username}.github.io` ([learn more](https://pages.github.com/)) 3. Modify `_config.yml` 4. Modify `_sass/base/_variables.scss` if you need to change colors or font sizes 5. Add new posts into `_posts/` :smiley: ## Demo You can see live demo at below: * https://ttskch.github.io/jekyll-ttskch-theme/ ## Thanks for using :wink: * http://ttskch.github.io * http://sitaramshelke.github.io * http://jffourmond.github.io * http://vbflash8.github.io * http://luqitao.github.io * http://harusametime.github.io * http://gitzxon.github.io * http://hutsonlu.github.io * http://k0-1.github.io * http://anthonygore.github.io * http://getjsdojo.github.io * http://georgezhuo.github.io * http://neontapir.github.io * https://sasukeh.github.io * https://blog.guilhermegarnier.com Please PR if you want to add your blog. hisat2-2.2.1/docs/_config.yml000066400000000000000000000032721370663763200160060ustar00rootroot00000000000000# # Basic settings. # url: http://DaehwanKimLab.github.io baseurl: /hisat2 title: HISAT2 description: graph-based alignment of next generation sequencing reads to a population of genomes avatar: /assets/img/ogp.png # favicon: /favicon.ico favicon: /assets/img/ogp.png # language: ja language: en # # Icons # icons: rss: true email: github: DaehwanKimLab bitbucket: twitter: facebook: google_plus: tumblr: behance: dribbble: flickr: instagram: linkedin: # full URL pinterest: reddit: soundcloud: stack_exchange: # full URL steam: wordpress: youtube: # # default for front matter # defaults: - scope: path: "" values: category: "main" # # Prettify url. # permalink: pretty # # Scripts. # google_analytics: # e.g. UA-000000-01 disqus: # # Localizations. # str_next: Next str_prev: Prev str_read_more: Read more... str_search: Search str_recent_posts: Recent posts str_show_all_posts: Show all posts # # Recent posts. # recent_posts_num: 10 # # Pagination. # paginate: 10 paginate_path: page/:num # # Social. # share_buttons: twitter: true facebook: false # needs ogp.fb.app_id hatena: false ogp: image_url: //ttskch.github.io/jekyll-ttskch-theme/assets/img/ogp.png fb: admin: # facebook admin id app_id: # facebook application id # # Plugins. # gems: - jekyll-paginate - jekyll-feed - jemoji # # Styles: see "_sass/base/_variables.scss" # # # !! Danger zone !! # include: ["_pages"] markdown: kramdown kramdown: input: GFM syntax_highlighter: rouge excerpt_separator: sass: sass_dir: _sass style: :compressed # or :expanded exclude: - Gemfile - Gemfile.lock - LICENSE - README.md - vendor hisat2-2.2.1/docs/_data/000077500000000000000000000000001370663763200147235ustar00rootroot00000000000000hisat2-2.2.1/docs/_data/collaborate.yml000066400000000000000000000005341370663763200177370ustar00rootroot00000000000000- name: Lyda Hill Department of Bioinformatics, The University of Texas Southwestern Medical Center url: https://www.utsouthwestern.edu/departments/bioinformatics logo: /assets/img/bioinformatics_utsw_logo.png - name: Center for Computational Biologoy, Johns Hopkins University url: http://ccb.jhu.edu logo: /assets/img/ccb_jhu_logo_tmp.pnghisat2-2.2.1/docs/_data/contributor.yml000066400000000000000000000004601370663763200200200ustar00rootroot00000000000000- name: Chanhee Park url: /chanhee.park/ - name: Ben Langmead url: http://www.langmead-lab.org/ - name: Yun (Leo) Zhang url: /leo.zhang/ - name: Steven Salzberg url: https://salzberg-lab.org/in-the-news/about-me/ - name: Daehwan Kim url: https://kim-lab.org/daehwan-kim-principal-investigator/ hisat2-2.2.1/docs/_data/download-binary.yml000066400000000000000000000062731370663763200205470ustar00rootroot00000000000000latest_version: 2.2.1,2.2.0,2.1.0 release: - version: 2.2.1 date: 7/24/2020 name: HISAT2 artifacts: Source: https://cloud.biohpc.swmed.edu/index.php/s/fE9QCsX3NH4QwBi/download OSX_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/an8KdGxxRdSRXjr/download Linux_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/4pMgDq4oAF9QCfA/download - version: 2.2.0 date: 2/6/2020 name: HISAT2 artifacts: Source: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-220-source/download OSX_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-220-OSX_x86_64/download Linux_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-220-Linux_x86_64/download - version: 2.1.0 date: 6/8/2017 name: HISAT2 artifacts: Source: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-210-source/download OSX_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-210-OSX_x86_64/download Linux_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-210-Linux_x86_64/download Windows: http://www.di.fc.ul.pt/~afalcao/hisat2_windows.html - version: 2.0.5 date: 11/4/2016 name: HISAT2 artifacts: Source: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-205-source/download OSX_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-205-OSX_x86_64/download Linux_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-205-Linux_x86_64/download - version: 2.0.4 date: 5/18/2016 name: HISAT2 artifacts: Source: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-204-source/download OSX_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-204-OSX_x86_64/download Linux_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-204-Linux_x86_64/download - version: 2.0.3-beta date: 3/28/2016 name: HISAT2 artifacts: Source: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-203-beta-source/download OSX_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-203-beta-OSX_x86_64/download Linux_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-203-beta-Linux_x86_64/download - version: 2.0.2-beta date: 3/17/2016 name: HISAT2 artifacts: Source: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-202-beta-source/download OSX_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-202-beta-OSX_x86_64/download Linux_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-202-beta-Linux_x86_64/download - version: 2.0.1-beta date: 11/19/2015 name: HISAT2 artifacts: Source: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-201-beta-source/download OSX_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-201-beta-OSX_x86_64/download Linux_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-201-beta-Linux_x86_64/download - version: 2.0.0-beta date: 9/8/2015 name: HISAT2 artifacts: Source: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-200-beta-source/download OSX_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-200-beta-OSX_x86_64/download Linux_x86_64: https://cloud.biohpc.swmed.edu/index.php/s/hisat2-200-beta-Linux_x86_64/download hisat2-2.2.1/docs/_data/download-index.yml000066400000000000000000000056771370663763200204010ustar00rootroot00000000000000- organism: H. sapiens data: GRCh38: genome: url: https://cloud.biohpc.swmed.edu/index.php/s/grch38/download genome_snp: url: https://cloud.biohpc.swmed.edu/index.php/s/grch38_snp/download genome_tran: url: https://cloud.biohpc.swmed.edu/index.php/s/grch38_tran/download genome_snp_tran: url: https://cloud.biohpc.swmed.edu/index.php/s/grch38_snp_tran/download genome_rep(above 2.2.0): url: https://cloud.biohpc.swmed.edu/index.php/s/grch38_rep/download genome_snp_rep(above 2.2.0): url: https://cloud.biohpc.swmed.edu/index.php/s/grch38_snp_rep/download UCSC hg38: genome: url: https://cloud.biohpc.swmed.edu/index.php/s/hg38/download genome_tran: url: https://cloud.biohpc.swmed.edu/index.php/s/hg38_tran/download GRCh37: genome: url: https://cloud.biohpc.swmed.edu/index.php/s/grch37/download genome_snp: url: https://cloud.biohpc.swmed.edu/index.php/s/grch37_snp/download genome_tran: url: https://cloud.biohpc.swmed.edu/index.php/s/grch37_tran/download genome_snp_tran: url: https://cloud.biohpc.swmed.edu/index.php/s/grch37_snp_tran/download UCSC hg19: genome: url: https://cloud.biohpc.swmed.edu/index.php/s/hg19/download - organism: M. musculus data: GRCm38: genome: url: https://cloud.biohpc.swmed.edu/index.php/s/grcm38/download genome_snp: url: https://cloud.biohpc.swmed.edu/index.php/s/grcm38_snp/download genome_tran: url: https://cloud.biohpc.swmed.edu/index.php/s/grcm38_tran/download genome_snp_tran: url: https://cloud.biohpc.swmed.edu/index.php/s/grcm38_snp_tran/download UCSC mm10: genome: url: https://cloud.biohpc.swmed.edu/index.php/s/mm10/download - organism: R. norvegicus data: UCSC rn6: genome: url: https://cloud.biohpc.swmed.edu/index.php/s/rn6/download - organism: D. melanogaster data: BDGP6: genome: url: ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/bdgp6.tar.gz genome_tran: url: ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/bdgp6_tran.tar.gz UCSC dm6: genome: url: ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/dm6.tar.gz - organism: C. elegans data: WBcel235: genome: url: ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/wbcel235.tar.gz genome_tran: url: ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/wbcel235_tran.tar.gz UCSC ce10: genome: url: ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/ce10.tar.gz - organism: S. cerevisiae data: R64-1-1: genome: url: ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/r64.tar.gz genome_tran: url: ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/r64_tran.tar.gz UCSC sacCer3: genome: url: ftp://ftp.ccb.jhu.edu/pub/infphilo/hisat2/data/sc3.tar.gz hisat2-2.2.1/docs/_includes/000077500000000000000000000000001370663763200156205ustar00rootroot00000000000000hisat2-2.2.1/docs/_includes/article-footer.html000066400000000000000000000002251370663763200214240ustar00rootroot00000000000000

hisat2-2.2.1/docs/_includes/article-header.html000066400000000000000000000036471370663763200213710ustar00rootroot00000000000000{% assign page = include.page %}

{% if include.link %} {{ page.title }} {% else %} {{ page.title }} {% endif %}

    {% assign tags_num = (page.tags | size) %} {% if tags_num > 0 %}
    • {% endif %} {% for tag in page.tags %}
  • #{{ tag }}
    • {% endfor %}
    {% if page.date %}
  • {{ page.date | date: "%Y-%m-%d" }}
    • {% endif %} {% if page.author %}
  • {{ page.author }}
    • {% if page.icons %}
    • {% include icons.html icons=page.icons %}
    • {% endif %} {% endif %}
{% if site.share_buttons and include.share != false %}
{% include share-buttons.html page=page %}
{% endif %} {% if include.eye_catch != false and page.eye_catch %}

{% endif %}
hisat2-2.2.1/docs/_includes/disqus.html000066400000000000000000000010631370663763200200160ustar00rootroot00000000000000
hisat2-2.2.1/docs/_includes/fb-root.html000066400000000000000000000006751370663763200200660ustar00rootroot00000000000000 {% if site.share_buttons.facebook %}
{% endif %} hisat2-2.2.1/docs/_includes/google-analytics.html000066400000000000000000000010031370663763200217410ustar00rootroot00000000000000 {% if site.google_analytics %} {% endif %} hisat2-2.2.1/docs/_includes/icons.html000066400000000000000000000061711370663763200176260ustar00rootroot00000000000000{% assign icons = include.icons %} {% if icons.rss %}
    • {% endif %} {% if icons.email %}
    • {% endif %} {% if icons.github %}
    • {% endif %} {% if icons.bitbucket %}
    • {% endif %} {% if icons.twitter %}
    • {% endif %} {% if icons.facebook %}
    • {% endif %} {% if icons.google_plus %}
    • {% endif %} {% if icons.tumblr %}
    • {% endif %} {% if icons.behance %}
    • {% endif %} {% if icons.dribbble %}
    • {% endif %} {% if icons.flickr %}
    • {% endif %} {% if icons.instagram %}
    • {% endif %} {% if icons.linkedin %}
    • {% endif %} {% if icons.pinterest %}
    • {% endif %} {% if icons.reddit %}
    • {% endif %} {% if icons.soundcloud %}
    • {% endif %} {% if icons.stack_exchange %}
    • {% endif %} {% if icons.steam %}
    • {% endif %} {% if icons.wordpress %}
    • {% endif %} {% if icons.youtube %}
    • {% endif %} hisat2-2.2.1/docs/_includes/page-url-resolver.html000066400000000000000000000004031370663763200220560ustar00rootroot00000000000000{% assign page = include.page %} {% if page.canonical %} {% assign url = page.canonical | prepend: site.baseurl | prepend: site.url %} {% else %} {% assign url = page.url | replace: 'index.html', '' | prepend: site.baseurl | prepend: site.url %} {% endif %} hisat2-2.2.1/docs/_includes/paginator.html000066400000000000000000000014061370663763200204730ustar00rootroot00000000000000{% if paginator.total_pages > 1 %} {% endif %} hisat2-2.2.1/docs/_includes/share-buttons.html000066400000000000000000000033571370663763200213140ustar00rootroot00000000000000{% include page-url-resolver.html page=include.page %} {% assign title = include.page.title | append: ' | ' | append: site.title %}
    {% if site.share_buttons.twitter %}
    {% endif %} {% if site.share_buttons.facebook %}
    {% endif %} {% if site.share_buttons.hatena %}
    このエントリーをはてなブックマークに追加
    {% endif %}
    hisat2-2.2.1/docs/_layouts/000077500000000000000000000000001370663763200155125ustar00rootroot00000000000000hisat2-2.2.1/docs/_layouts/default.html000066400000000000000000000173341370663763200200340ustar00rootroot00000000000000 {% capture title %}{% if page.title %}{{ page.title }} | {% endif %}{{ site.title }}{% endcapture %} {% include page-url-resolver.html page=page %} {% if page.excerpt %} {% assign description = page.excerpt | strip_html | strip_newlines | truncate: 160 %} {% else %} {% assign description = site.description %} {% endif %} {{ title }} {% if page.eye_catch %} {% assign ogp_image_url = page.eye_catch %} {% else %} {% assign ogp_image_url = site.ogp.image_url %} {% endif %}
    {% if site.avatar %} {% endif %}

    {{ site.title }}
    {{ site.description }}

    {{ content }}
    {% if page.id %} {% endif %} {% if page.permalink == '/search/' %} {% endif %} {% include fb-root.html %} {% include google-analytics.html %} hisat2-2.2.1/docs/_layouts/page.html000066400000000000000000000005041370663763200173130ustar00rootroot00000000000000--- layout: default ---
    {% include article-header.html page=page link=false share=page.share %}
    {{ content }}
    {% include article-footer.html page=page share=page.share %}
    hisat2-2.2.1/docs/_layouts/post.html000066400000000000000000000006521370663763200173700ustar00rootroot00000000000000--- layout: default ---
    {% include article-header.html page=page link=false share=page.share %}
    {{ content }}
    {% include article-footer.html page=page share=page.share %}
    {% if site.disqus %}
    {% include disqus.html %}
    {% endif %} hisat2-2.2.1/docs/_pages/000077500000000000000000000000001370663763200151115ustar00rootroot00000000000000hisat2-2.2.1/docs/_pages/about.md000066400000000000000000000015551370663763200165530ustar00rootroot00000000000000--- layout: page title: About permalink: /about/ order: 2 share: false --- **HISAT2** is a fast and sensitive alignment program for mapping next-generation sequencing reads (both DNA and RNA) to a population of human genomes as well as to a single reference genome. Based on an extension of BWT for graphs ([Sirén et al. 2014](http://dl.acm.org/citation.cfm?id=2674828)), we designed and implemented a graph FM index (GFM), an original approach and its first implementation. In addition to using one global GFM index that represents a population of human genomes, HISAT2 uses a large set of small GFM indexes that collectively cover the whole genome. These small indexes (called local indexes), combined with several alignment strategies, enable rapid and accurate alignment of sequencing reads. This new indexing scheme is called a Hierarchical Graph FM index (HGFM). hisat2-2.2.1/docs/_pages/archives-all.html000066400000000000000000000006551370663763200203570ustar00rootroot00000000000000--- layout: page title: All Posts permalink: /archives/all/ hide: true share: false ---
    {% for post in site.posts %}
    {% include article-header.html page=post link=true share=false eye_catch=false %}
    {% endfor %}
    hisat2-2.2.1/docs/_pages/archives.html000066400000000000000000000016661370663763200176140ustar00rootroot00000000000000--- layout: page title: Archives permalink: /archives/ order: 3 share: false hide: true --- {% for post in site.posts %} {% unless post.next %}

    {{ post.date | date: '%Y' }}

      {% else %} {% assign year = post.date | date: '%Y' %} {% assign next_year = post.next.date | date: '%Y' %} {% if year != next_year %}

    {{ post.date | date: '%Y' }}

      {% endif %} {% endunless %} {% assign month = post.date | date: '%m' %} {% assign next_month = post.next.date | date: '%m' %} {% if year != next_year or month != next_month %}
    • {{ post.date | date: '%Y/%m' }}
      • {% endif %} {% endfor %} {% if site.posts %}
    {% endif %} {{ site.str_show_all_posts }} hisat2-2.2.1/docs/_pages/contributors/000077500000000000000000000000001370663763200176465ustar00rootroot00000000000000hisat2-2.2.1/docs/_pages/contributors/chanheepark.md000066400000000000000000000004521370663763200224420ustar00rootroot00000000000000--- layout: page title: Chanhee Park permalink: /chanhee.park/ order: 1 share: false category: contributor --- Chanhee Park is a Scientific Software Engineer in the Kim Lab at UTSW responsible for maintaining and improving HISAT2. [Linkedin](https://www.linkedin.com/in/chanhee-park-97677297/) hisat2-2.2.1/docs/_pages/contributors/yunleozhang.md000066400000000000000000000005071370663763200225350ustar00rootroot00000000000000--- layout: page title: Yun (Leo) Zhang permalink: /leo.zhang/ order: 1 share: false category: contributor --- Yun (Leo) is a biomedical engineering graduate student at UT Southwestern Medical Center. His main research includes developing advance alignment tools. [Linkedin](https://www.linkedin.com/in/zhang-yun-a9565891/) hisat2-2.2.1/docs/_pages/download.md000066400000000000000000000033341370663763200172450ustar00rootroot00000000000000--- layout: page title: Download permalink: /download/ order: 4 share: false --- Please cite: >Kim, D., Paggi, J.M., Park, C. _et al._ Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. _Nat Biotechnol_ **37**, 907–915 (2019). - TOC {:toc} ## Index {% for item in site.data.download-index %} ### {{ item.organism }} {% for data in item.data %}
  • {{ data[0] }}
    • {% for genome in data[1] %} {% endfor %}
    {{ genome[0] }}{{ genome[1].url }}
    {% endfor %} {% endfor %} genome: HISAT2 index for reference genome_snp: HISAT2 Graph index for reference plus SNPs genome_tran: HISAT2 Graph index for reference plus transcripts genome_snp_tran: HISAT2 Graph index for reference plus SNPs and transcripts ## Binaries {: binaries } {% assign targets = site.data.download-binary.latest_version | split: "," %} {% for release in site.data.download-binary.release %} {% assign version = release['version'] %} {% if targets contains version or targets == null %} {% assign name = release['name'] %} ### Version: {{name}} {{version}} {% for artifact in release['artifacts'] %} {% assign type = artifact[0] %} {% endfor %}
    Release Date: {{release['date']}}
    {{type}}{{artifact[1]}}
    {% endif %} {% endfor %} hisat2-2.2.1/docs/_pages/hisat2.md000066400000000000000000000255241370663763200166350ustar00rootroot00000000000000--- layout: page title: Main permalink: /main/ order: 1 share: false --- **HISAT2** is a fast and sensitive alignment program for mapping next-generation sequencing reads (both DNA and RNA) to a population of human genomes as well as to a single reference genome. Based on an extension of BWT for graphs ([Sirén et al. 2014](http://dl.acm.org/citation.cfm?id=2674828)), we designed and implemented a graph FM index (GFM), an original approach and its first implementation. In addition to using one global GFM index that represents a population of human genomes, **HISAT2** uses a large set of small GFM indexes that collectively cover the whole genome. These small indexes (called local indexes), combined with several alignment strategies, enable rapid and accurate alignment of sequencing reads. This new indexing scheme is called a Hierarchical Graph FM index (HGFM). ### HISAT 2.2.1 release 7/24/2020 This patch version includes the following changes. * Python3 support * Remove the HISAT-genotype related scripts. HISAT-genotype moved to [http://daehwankimlab.github.io/hisat-genotype/](http://daehwankimlab.github.io/hisat-genotype/) * Fixed bugs related to `--read-lengths` option ### HISAT 2.2.0 release 2/6/2020 This major version update includes a new feature to handle “repeat” reads. Based on sets of 100-bp simulated and 101-bp real reads that we tested, we found that 2.6-3.4% and 1.4-1.8% of the reads were mapped to >5 locations and >100 locations, respectively. Attempting to report all alignments would likely consume a prohibitive amount of disk space. In order to address this issue, our repeat indexing and alignment approach directly aligns reads to repeat sequences, resulting in one repeat alignment per read. HISAT2 provides application programming interfaces (API) for C++, Python, and JAVA that rapidly retrieve genomic locations from repeat alignments for use in downstream analyses. Other minor bug fixes are also included as follows: * Fixed occasional sign (+ or -) issues of template lengths in SAM file * Fixed duplicate read alignments in SAM file * Skip a splice site if exon's last base or first base is ambiguous (N) ### Index files are moved to a different location. 8/30/2019 Due to a high volume of index downloads, we have moved HISAT2 index files to a different location in order to provide faster download speed. If you use wget or curl to download index files, then you may need to use the following commands to get the correct file name. * `wget --content-disposition` *download_link* * `curl -OJ` *download_link* ### [The HISAT2 paper](https://www.nature.com/articles/s41587-019-0201-4) is out in *Nature Biotechnology*. 8/2/2019 ### HISAT 2.1.0 release 6/8/2017 * This major version includes the first release of HISAT-genotype, which currently performs HLA typing, DNA fingerprinting analysis, and CYP typing on whole genome sequencing (WGS) reads. We plan to extend the system so that it can analyze not just a few genes, but a whole human genome. Please refer to [the HISAT-genotype website](https://daehwankimlab.github.io/hisat-genotype) for more details. * HISAT2 can be directly compiled and executed on Windows system using Visual Studio, thanks to [Nigel Dyer](http://www2.warwick.ac.uk/fac/sci/systemsbiology/staff/dyer/). * Implemented `--new-summary` option to output a new style of alignment summary, which is easier to parse for programming purposes. * Implemented `--summary-file` option to output alignment summary to a file in addition to the terminal (e.g. stderr). * Fixed discrepancy in HISAT2’s alignment summary. * Implemented `--no-templatelen-adjustment` option to disable automatic template length adjustment for RNA-seq reads. ### HISAT2 2.0.5 release 11/4/2016 Version 2.0.5 is a minor release with the following changes. * Due to a policy change (HTTP to HTTPS) in using SRA data (`--sra-option`), users are strongly encouraged to use this version. As of 11/9/2016, NCBI will begin a permanent redirect to HTTPS, which means the previous versions of HISAT2 no longer works with `--sra-acc` option soon. * Implemented `-I` and `-X` options for specifying minimum and maximum fragment lengths. The options are valid only when used with `--no-spliced-alignment`, which is used for the alignment of DNA-seq reads. * Fixed some cases where reads with SNPs on their 5' ends were not properly aligned. * Implemented `--no-softclip` option to disable soft-clipping. * Implemented `--max-seeds` to specify the maximum number of seeds that HISAT2 will try to extend to full-length alignments (see [the manual] for details). ### [HISAT, StringTie and Ballgown protocol](http://www.nature.com/nprot/journal/v11/n9/full/nprot.2016.095.html) published at Nature Protocols 8/11/2016 ### HISAT2 2.0.4 Windows binary available [here](http://www.di.fc.ul.pt/~afalcao/hisat2_windows.html), thanks to [Andre Osorio Falcao](http://www.di.fc.ul.pt/~afalcao/) 5/24/2016 ### HISAT2 2.0.4 release 5/18/2016 Version 2.0.4 is a minor release with the following changes. * Improved template length estimation (the 9th column of the SAM format) of RNA-seq reads by taking introns into account. * Introduced two options, `--remove-chrname` and `--add-chrname`, to remove "chr" from reference names or add "chr" to reference names in the alignment output, respectively (the 3rd column of the SAM format). * Changed the maximum of mapping quality (the 5th column of the SAM format) from 255 to 60. Note that 255 is an undefined value according to the SAM manual and some programs would not work with this value (255) properly. * Fixed NH (number of hits) in the alignment output. * HISAT2 allows indels of any length pertaining to minimum alignment score (previously, the maximum length of indels was 3 bp). * Fixed several cases that alignment goes beyond reference sequences. * Fixed reporting duplicate alignments. ### HISAT2 2.0.3-beta release 3/28/2016 Version 2.0.3-beta is a minor release with the following changes. * Fixed graph index building when using both SNPs and transcripts. As a result, genome_snp_tran indexes here on the HISAT2 website have been rebuilt. * Included some missing files needed to follow the small test example (see [the manual] for details). ### HISAT2 2.0.2-beta release 3/17/2016 **Note (3/19/2016):** this version is slightly updated to handle reporting splice sites with the correct chromosome names. Version 2.0.2-beta is a major release with the following changes. * Memory mappaped IO (`--mm` option) works now. * Building linear index can be now done using multi-threads. * Changed the minimum score for alignment in keeping with read lengths, so it's now `--score-min L,0.0,-0.2`, meaning a minimum score of -20 for 100-bp reads and -30 for 150-bp reads. * Fixed a bug that the same read was written into a file multiple times when `--un-conc` was used. * Fixed another bug that caused reads to map beyond reference sequences. * Introduced `--haplotype` option in the hisat2-build (index building), which is used with `--snp` option together to incorporate those SNP combinations present in the human population. This option also prevents graph construction from exploding due to exponential combinations of SNPs in small genomic regions. * Provided a new python script to extract SNPs and haplotypes from VCF files, hisat2_extract_snps_haplotypes_VCF.py * Changed several python script names as follows< * *extract_splice_sites.py* to *hisat2_extract_splice_sites.py* * *extract_exons.py* to *hisat2_extract_exons.py* * *extract_snps.py* to *hisat2_extract_snps_haplotypes_UCSC.py* ### HISAT2 2.0.1-beta release 11/19/2015 Version 2.0.1-beta is a maintenance release with the following changes. * Fixed a bug that caused reads to map beyond reference sequences. * Fixed a deadlock issue that happened very rarely. * Fixed a bug that led to illegal memory access when reading SNP information. * Fixed a system-specific bug related to popcount instruction. ### HISAT2 2.0.0-beta release 9/8/2015 - first release We extended the BWT/FM index to incorporate genomic differences among individuals into the reference genome, while keeping memory requirements low enough to fit the entire index onto a desktop computer. Using this novel Hierarchical Graph FM index (HGFM) approach, we built a new alignment system, HISAT2, with an index that incorporates ~12.3M common SNPs from the dbSNP database. HISAT2 provides greater alignment accuracy for reads containing SNPs. * HISAT2's index size for the human reference genome and 12.3 million common SNPs is 6.2GB (the memory footprint of HISAT2 is 6.7GB). The SNPs consist of 11 million single nucleotide polymorphisms, 728,000 deletions, and 555,000 insertions. The insertions and deletions used in this index are small (usually <20bp). * HISAT2 comes with several index types: * Hierarchical FM index (HFM) for a reference genome (index base: genome) * Hierarchical Graph FM index (HGFM) for a reference genome plus SNPs (index base: genome_snp) * Hierarchical Graph FM index (HGFM) for a reference genome plus transcripts (index base: genome_tran) * Hierarchical Graph FM index (HGFM) for a reference genome plus SNPs and transcripts (index base: genome_snp_tran) * HISAT2 is a successor to both [HISAT](http://ccb.jhu.edu/software/hisat) and [TopHat2](http://ccb.jhu.edu/software/tophat). We recommend that HISAT and TopHat2 users switch to HISAT2. * HISAT2 can be considered an enhanced version of HISAT with many improvements and bug fixes. The alignment speed and memory requirements of HISAT2 are virtually the same as those of HISAT when using the HFM index (genome). * When using graph-based indexes (HGFM), the runtime of HISAT2 is slightly slower than HISAT (30~80% additional CPU time). * HISAT2 allows for mapping reads directly against transcripts, similar to that of TopHat2 (use genome_tran or genome_snp_tran). * When reads contain SNPs, the SNP information is provided as an optional field in the SAM output of HISAT2 (e.g., **Zs:Z:1|S|rs3747203,97|S|rs16990981** - see [the manual] for details). This feature enables fast and sensitive genotyping in downstream analyses. Note that there is no alignment penalty for mismatches, insertions, and deletions if they correspond to known SNPs. * HISAT2 provides options for transcript assemblers (e.g., StringTie and Cufflinks) to work better with the alignment from HISAT2 (see options such as `--dta` and `--dta-cufflinks`). * Some slides about HISAT2 are found [here]({{ '/assets/data/HISAT2-first_release-Sept_8_2015.pdf' | prepend: site.baseurl }}) and we are preparing detailed documention. * We plan to incorporate a larger set of SNPs and structural variations (SV) into this index (e.g., long insertions/deletions, inversions, and translocations). [the manual]: {{ site.baseurl }}{% link _pages/manual.md %} ### The HISAT2 source code is available in a [public GitHub repository](https://github.com/DaehwanKimLab/hisat2) (5/30/2015). hisat2-2.2.1/docs/_pages/howto.md000066400000000000000000000043201370663763200165720ustar00rootroot00000000000000--- layout: page title: HowTo permalink: /howto/ order: 5 share: false --- ## HOWTO {: .no_toc} - TOC {:toc} ### Building indexes Depend on your purpose, you have to download reference sequence, gene annotation and SNP files. We also provides scripts to build indexes. [Download]({{ site.baseurl }}{% link _pages/download.md %}) #### Prepare data 1. Download reference ``` $ wget ftp://ftp.ensembl.org/pub/release-84/fasta/homo_sapiens/dna/Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz $ gzip -d Homo_sapiens.GRCh38.dna.primary_assembly.fa.gz $ mv Homo_sapiens.GRCh38.dna.primary_assembly.fa genome.fa ``` 1. Download GTF and make exon, splicesite file. If you want to build HFM index, you can skip this step. ``` $ wget ftp://ftp.ensembl.org/pub/release-84/gtf/homo_sapiens/Homo_sapiens.GRCh38.84.gtf.gz $ gzip -d Homo_sapiens.GRCh38.84.gtf.gz $ mv Homo_sapiens.GRCh38.84.gtf genome.gtf $ hisat2_extract_splice_sites.py genome.gtf > genome.ss $ hisat2_extract_exons.py genome.gtf > genome.exon ``` 1. Download SNP If you want to build HFM index, you can skip this step. ``` $ wget http://hgdownload.cse.ucsc.edu/goldenPath/hg38/database/snp144Common.txt.gz $ gzip -d snp144Common.txt.gz ``` Convert chromosome names of UCSC Database to Ensembl Annotation ``` $ awk 'BEGIN{OFS="\t"} {if($2 ~ /^chr/) {$2 = substr($2, 4)}; if($2 == "M") {$2 = "MT"} print}' snp144Common.txt > snp144Common.txt.ensembl ``` make SNPs and haplotype file ``` $ hisat2_extract_snps_haplotypes_UCSC.py genome.fa snp144Common.txt.ensembl genome ``` #### Build HFM index It takes about 20 minutes(depend on HW spec) to build index, and requires at least 6GB memory. ``` $ hisat2-build -p 16 genome.fa genome ``` #### Build HGFM index with SNPs ``` $ hisat2-build -p 16 --snp genome.snp --haplotype genome.haplotype genome.fa genome_snp ``` #### Build HGFM index with transcripts It takes about 1 hour(depend on HW spec) to build index, and requires at least 160GB memory. ``` $ hisat2-build -p 16 --exon genome.exon --ss genome.ss genome.fa genome_tran ``` #### Build HGFM index with SNPs and transcripts ``` $ hisat2-build -p 16 --snp genome.snp --haplotype genome.haplotype --exon genome.exon --ss genome.ss genome.fa genome_snp_tran ``` hisat2-2.2.1/docs/_pages/links.md000066400000000000000000000010331370663763200165500ustar00rootroot00000000000000--- layout: page title: Links permalink: /links/ order: 6 share: false --- * KimLab - * github - * hisat-genotype - * github for hisat-genotype - * Lyda Hill Department of Bioinformatics at UT Southwestern Medical Center - * Center for Computational Biology at Johns Hopkins University - hisat2-2.2.1/docs/_pages/manual.md000066400000000000000000002255101370663763200167150ustar00rootroot00000000000000--- layout: page title: Manual permalink: /manual/ order: 3 share: false --- Introduction ============ What is HISAT2? ----------------- HISAT2 is a fast and sensitive alignment program for mapping next-generation sequencing reads (whole-genome, transcriptome, and exome sequencing data) against the general human population (as well as against a single reference genome). Based on [GCSA] (an extension of [BWT] for a graph), we designed and implemented a graph FM index (GFM), an original approach and its first implementation to the best of our knowledge. In addition to using one global GFM index that represents general population, HISAT2 uses a large set of small GFM indexes that collectively cover the whole genome (each index representing a genomic region of 56 Kbp, with 55,000 indexes needed to cover human population). These small indexes (called local indexes) combined with several alignment strategies enable effective alignment of sequencing reads. This new indexing scheme is called Hierarchical Graph FM index (HGFM). We have developed HISAT 2 based on the [HISAT] and [Bowtie2] implementations. HISAT2 outputs alignments in [SAM] format, enabling interoperation with a large number of other tools (e.g. [SAMtools], [GATK]) that use SAM. HISAT2 is distributed under the [GPLv3 license], and it runs on the command line under Linux, Mac OS X and Windows. [HISAT2]: https://daehwankimlab.github.io/hisat2 [HISAT]: http://ccb.jhu.edu/software/hisat [Bowtie2]: http://bowtie-bio.sf.net/bowtie2 [Bowtie]: http://bowtie-bio.sf.net [Bowtie1]: http://bowtie-bio.sf.net [GCSA]: http://ieeexplore.ieee.org/xpls/abs_all.jsp?arnumber=6698337&tag=1 [Burrows-Wheeler Transform]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform [BWT]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform [FM Index]: http://en.wikipedia.org/wiki/FM-index [SAM]: http://samtools.sourceforge.net/SAM1.pdf [SAMtools]: http://samtools.sourceforge.net [GATK]: http://www.broadinstitute.org/gsa/wiki/index.php/The_Genome_Analysis_Toolkit [TopHat2]: http://ccb.jhu.edu/software/tophat [Cufflinks]: http://cufflinks.cbcb.umd.edu/ [Crossbow]: http://bowtie-bio.sf.net/crossbow [Myrna]: http://bowtie-bio.sf.net/myrna [Bowtie paper]: http://genomebiology.com/2009/10/3/R25 [GPLv3 license]: http://www.gnu.org/licenses/gpl-3.0.html Obtaining HISAT2 ================== Download HISAT2 sources and binaries from the Releases sections on the right side. Binaries are available for Intel architectures (`x86_64`) running Linux, and Mac OS X. Building from source -------------------- Building HISAT2 from source requires a GNU-like environment with GCC, GNU Make and other basics. It should be possible to build HISAT2 on most vanilla Linux installations or on a Mac installation with [Xcode] installed. HISAT2 can also be built on Windows using [Cygwin] or [MinGW] (MinGW recommended). For a MinGW build the choice of what compiler is to be used is important since this will determine if a 32 or 64 bit code can be successfully compiled using it. If there is a need to generate both 32 and 64 bit on the same machine then a multilib MinGW has to be properly installed. [MSYS], the [zlib] library, and depending on architecture [pthreads] library are also required. We are recommending a 64 bit build since it has some clear advantages in real life research problems. In order to simplify the MinGW setup it might be worth investigating popular MinGW personal builds since these are coming already prepared with most of the toolchains needed. First, download the [source package] from the Download section on the right side. Unzip the file, change to the unzipped directory, and build the HISAT2 tools by running GNU `make` (usually with the command `make`, but sometimes with `gmake`) with no arguments. If building with MinGW, run `make` from the MSYS environment. HISAT2 is using the multithreading software model in order to speed up execution times on SMP architectures where this is possible. On POSIX platforms (like linux, Mac OS, etc) it needs the pthread library. Although it is possible to use pthread library on non-POSIX platform like Windows, due to performance reasons HISAT2 will try to use Windows native multithreading if possible. For the support of SRA data access in HISAT2, please download and install the [NCBI-NGS] toolkit. When running `make`, specify additional variables as follow. `make USE_SRA=1 NCBI_NGS_DIR=/path/to/NCBI-NGS-directory NCBI_VDB_DIR=/path/to/NCBI-NGS-directory`, where `NCBI_NGS_DIR` and `NCBI_VDB_DIR` will be used in Makefile for `-I` and `-L` compilation options. For example, `$(NCBI_NGS_DIR)/include` and `$(NCBI_NGS_DIR)/lib64` will be used. [Cygwin]: http://www.cygwin.com/ [MinGW]: http://www.mingw.org/ [MSYS]: http://www.mingw.org/wiki/msys [zlib]: http://cygwin.com/packages/mingw-zlib/ [pthreads]: http://sourceware.org/pthreads-win32/ [GnuWin32]: http://gnuwin32.sf.net/packages/coreutils.htm [Download]: https://sourceforge.net/projects/bowtie-bio/files/bowtie2/ [sourceforge site]: https://sourceforge.net/projects/bowtie-bio/files/bowtie2/ [source package]: {{ site.baseurl }}{% link _pages/download.md %} [Xcode]: http://developer.apple.com/xcode/ [NCBI-NGS]: https://github.com/ncbi/ngs/wiki/Downloads Running HISAT2 ============= Adding to PATH -------------- By adding your new HISAT2 directory to your [PATH environment variable], you ensure that whenever you run `hisat2`, `hisat2-build` or `hisat2-inspect` from the command line, you will get the version you just installed without having to specify the entire path. This is recommended for most users. To do this, follow your operating system's instructions for adding the directory to your [PATH]. If you would like to install HISAT2 by copying the HISAT2 executable files to an existing directory in your [PATH], make sure that you copy all the executables, including `hisat2`, `hisat2-align-s`, `hisat2-align-l`, `hisat2-build`, `hisat2-build-s`, `hisat2-build-l`, `hisat2-inspect`, `hisat2-inspect-s` and `hisat2-inspect-l`. [PATH environment variable]: http://en.wikipedia.org/wiki/PATH_(variable) [PATH]: http://en.wikipedia.org/wiki/PATH_(variable) Reporting --------- The reporting mode governs how many alignments HISAT2 looks for, and how to report them. In general, when we say that a read has an alignment, we mean that it has a [valid alignment]. When we say that a read has multiple alignments, we mean that it has multiple alignments that are valid and distinct from one another. [valid alignment]: #valid-alignments-meet-or-exceed-the-minimum-score-threshold By default, HISAT2 may soft-clip reads near their 5' and 3' ends. Users can control this behavior by setting different penalties for soft-clipping ([`--sp`]) or by disallowing soft-clipping ([`--no-softclip`]). ### Distinct alignments map a read to different places Two alignments for the same individual read are "distinct" if they map the same read to different places. Specifically, we say that two alignments are distinct if there are no alignment positions where a particular read offset is aligned opposite a particular reference offset in both alignments with the same orientation. E.g. if the first alignment is in the forward orientation and aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, and the second alignment is also in the forward orientation and also aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, they are not distinct alignments. Two alignments for the same pair are distinct if either the mate 1s in the two paired-end alignments are distinct or the mate 2s in the two alignments are distinct or both. ### Default mode: search for one or more alignments, report each HISAT2 searches for up to N distinct, primary alignments for each read, where N equals the integer specified with the `-k` parameter. Primary alignments mean alignments whose alignment score is equal or higher than any other alignments. It is possible that multiple distinct alignments have the same score. That is, if `-k 2` is specified, HISAT2 will search for at most 2 distinct alignments. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS field. See the [SAM specification] for details. HISAT2 does not "find" alignments in any specific order, so for reads that have more than N distinct, valid alignments, HISAT2 does not guarantee that the N alignments reported are the best possible in terms of alignment score. Still, this mode can be effective and fast in situations where the user cares more about whether a read aligns (or aligns a certain number of times) than where exactly it originated. [SAM specification]: http://samtools.sourceforge.net/SAM1.pdf Alignment summary ------------------ When HISAT2 finishes running, it prints messages summarizing what happened. These messages are printed to the "standard error" ("stderr") filehandle. For datasets consisting of unpaired reads, the summary might look like this: 20000 reads; of these: 20000 (100.00%) were unpaired; of these: 1247 (6.24%) aligned 0 times 18739 (93.69%) aligned exactly 1 time 14 (0.07%) aligned >1 times 93.77% overall alignment rate For datasets consisting of pairs, the summary might look like this: 10000 reads; of these: 10000 (100.00%) were paired; of these: 650 (6.50%) aligned concordantly 0 times 8823 (88.23%) aligned concordantly exactly 1 time 527 (5.27%) aligned concordantly >1 times ---- 650 pairs aligned concordantly 0 times; of these: 34 (5.23%) aligned discordantly 1 time ---- 616 pairs aligned 0 times concordantly or discordantly; of these: 1232 mates make up the pairs; of these: 660 (53.57%) aligned 0 times 571 (46.35%) aligned exactly 1 time 1 (0.08%) aligned >1 times 96.70% overall alignment rate The indentation indicates how subtotals relate to totals. Wrapper ------- The `hisat2`, `hisat2-build` and `hisat2-inspect` executables are actually wrapper scripts that call binary programs as appropriate. The wrappers shield users from having to distinguish between "small" and "large" index formats, discussed briefly in the following section. Also, the `hisat2` wrapper provides some key functionality, like the ability to handle compressed inputs, and the functionality for [`--un`], [`--al`] and related options. It is recommended that you always run the hisat2 wrappers and not run the binaries directly. Small and large indexes ----------------------- `hisat2-build` can index reference genomes of any size. For genomes less than about 4 billion nucleotides in length, `hisat2-build` builds a "small" index using 32-bit numbers in various parts of the index. When the genome is longer, `hisat2-build` builds a "large" index using 64-bit numbers. Small indexes are stored in files with the `.ht2` extension, and large indexes are stored in files with the `.ht2l` extension. The user need not worry about whether a particular index is small or large; the wrapper scripts will automatically build and use the appropriate index. Performance tuning ------------------ 1. If your computer has multiple processors/cores, use `-p` The [`-p`] option causes HISAT2 to launch a specified number of parallel search threads. Each thread runs on a different processor/core and all threads find alignments in parallel, increasing alignment throughput by approximately a multiple of the number of threads (though in practice, speedup is somewhat worse than linear). Command Line ------------ ### Setting function options Some HISAT2 options specify a function rather than an individual number or setting. In these cases the user specifies three parameters: (a) a function type `F`, (b) a constant term `B`, and (c) a coefficient `A`. The available function types are constant (`C`), linear (`L`), square-root (`S`), and natural log (`G`). The parameters are specified as `F,B,A` - that is, the function type, the constant term, and the coefficient are separated by commas with no whitespace. The constant term and coefficient may be negative and/or floating-point numbers. For example, if the function specification is `L,-0.4,-0.6`, then the function defined is: f(x) = -0.4 + -0.6 * x If the function specification is `G,1,5.4`, then the function defined is: f(x) = 1.0 + 5.4 * ln(x) See the documentation for the option in question to learn what the parameter `x` is for. For example, in the case if the [`--score-min`] option, the function `f(x)` sets the minimum alignment score necessary for an alignment to be considered valid, and `x` is the read length. ### Usage hisat2 [options]* -x {-1 -2 | -U | --sra-acc } [-S ] ### Main arguments * `-x ` The basename of the index for the reference genome. The basename is the name of any of the index files up to but not including the final `.1.ht2` / etc. `hisat2` looks for the specified index first in the current directory, then in the directory specified in the `HISAT2_INDEXES` environment variable. {: #hisat2-options-x} [`-x`]: #hisat2-options-x * `-1 ` Comma-separated list of files containing mate 1s (filename usually includes `_1`), e.g. `-1 flyA_1.fq,flyB_1.fq`. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in ``. Reads may be a mix of different lengths. If `-` is specified, `hisat2` will read the mate 1s from the "standard in" or "stdin" filehandle. {: #hisat2-options-1} [`-1`]: #hisat2-options-1 * `-2 ` Comma-separated list of files containing mate 2s (filename usually includes `_2`), e.g. `-2 flyA_2.fq,flyB_2.fq`. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in ``. Reads may be a mix of different lengths. If `-` is specified, `hisat2` will read the mate 2s from the "standard in" or "stdin" filehandle. {: #hisat2-options-2} [`-2`]: #hisat2-options-2 * `-U ` Comma-separated list of files containing unpaired reads to be aligned, e.g. `lane1.fq,lane2.fq,lane3.fq,lane4.fq`. Reads may be a mix of different lengths. If `-` is specified, `hisat2` gets the reads from the "standard in" or "stdin" filehandle. {: #hisat2-options-U} [`-U`]: #hisat2-options-U * `--sra-acc ` Comma-separated list of SRA accession numbers, e.g. `--sra-acc SRR353653,SRR353654`. Information about read types is available at http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?sp=runinfo&acc=sra-acc&retmode=xml, where sra-acc is SRA accession number. If users run HISAT2 on a computer cluster, it is recommended to disable SRA-related caching (see the instruction at [SRA-MANUAL]). {: #hisat2-options-sra-acc} [`--sra-acc`]: #hisat2-options-sra-acc [SRA-MANUAL]: https://github.com/ncbi/sra-tools/wiki/Toolkit-Configuration * `-S ` File to write SAM alignments to. By default, alignments are written to the "standard out" or "stdout" filehandle (i.e. the console). {: #hisat2-options-S} [`-S`]: #hisat2-options-S ### Options #### Input options * `-q` Reads (specified with ``, ``, ``) are FASTQ files. FASTQ files usually have extension `.fq` or `.fastq`. FASTQ is the default format. See also: [`--solexa-quals`] and [`--int-quals`]. {: #hisat2-options-q} [`-q`]: #hisat2-options-q * `--qseq` Reads (specified with ``, ``, ``) are QSEQ files. QSEQ files usually end in `_qseq.txt`. See also: [`--solexa-quals`] and [`--int-quals`]. {: #hisat2-options-qseq} [`--qseq`]: #hisat2-options-qseq * `-f` Reads (specified with ``, ``, ``) are FASTA files. FASTA files usually have extension `.fa`, `.fasta`, `.mfa`, `.fna` or similar. FASTA files do not have a way of specifying quality values, so when `-f` is set, the result is as if `--ignore-quals` is also set. {: #hisat2-options-f} [`-f`]: #hisat2-options-f * `-r` Reads (specified with ``, ``, ``) are files with one input sequence per line, without any other information (no read names, no qualities). When `-r` is set, the result is as if `--ignore-quals` is also set. {: #hisat2-options-r} [`-r`]: #hisat2-options-r * `-c` The read sequences are given on command line. I.e. ``, `` and `` are comma-separated lists of reads rather than lists of read files. There is no way to specify read names or qualities, so `-c` also implies `--ignore-quals`. {: #hisat2-options-c} [`-c`]: #hisat2-options-c * `-s/--skip ` Skip (i.e. do not align) the first `` reads or pairs in the input. {: #hisat2-options-s} [`-s`/`--skip`]: #hisat2-options-s [`-s`]: #hisat2-options-s * `-u/--upto ` Align the first `` reads or read pairs from the input (after the [`-s`/`--skip`] reads or pairs have been skipped), then stop. Default: no limit. {: #hisat2-options-u} [`-u`/`--qupto`]: #hisat2-options-u [`-u`]: #hisat2-options-u * `-5/--trim5 ` Trim `` bases from 5' (left) end of each read before alignment (default: 0). {: #hisat2-options-5} [`-5`/`--trim5`]: #hisat2-options-5 [`-5`]: #hisat2-options-5 * `-3/--trim3 ` Trim `` bases from 3' (right) end of each read before alignment (default: 0). {: #hisat2-options-3} [`-3`/`--trim3`]: #hisat2-options-3 [`-3`]: #hisat2-options-3 * `--phred33` Input qualities are ASCII chars equal to the [Phred quality] plus 33. This is also called the "Phred+33" encoding, which is used by the very latest Illumina pipelines. {: #hisat2-options-phred33-quals} [`--phred33`]: #hisat2-options-phred33-quals [Phred quality]: http://en.wikipedia.org/wiki/Phred_quality_score * `--phred64` Input qualities are ASCII chars equal to the [Phred quality] plus 64. This is also called the "Phred+64" encoding. {: #hisat2-options-phred64-quals} [`--phred64`]: #hisat2-options-phred64-quals * `--solexa-quals` Convert input qualities from [Solexa][Phred quality] (which can be negative) to [Phred][Phred quality] (which can't). This scheme was used in older Illumina GA Pipeline versions (prior to 1.3). Default: off. {: #hisat2-options-solexa-quals} [`--solexa-quals`]: #hisat2-options-solexa-quals * `--int-quals` Quality values are represented in the read input file as space-separated ASCII integers, e.g., `40 40 30 40`..., rather than ASCII characters, e.g., `II?I`.... Integers are treated as being on the [Phred quality] scale unless [`--solexa-quals`] is also specified. Default: off. {: #hisat2-options-int-quals} [`--int-quals`]: #hisat2-options-int-quals #### Alignment options * `--n-ceil ` Sets a function governing the maximum number of ambiguous characters (usually `N`s and/or `.`s) allowed in a read as a function of read length. For instance, specifying `-L,0,0.15` sets the N-ceiling function `f` to `f(x) = 0 + 0.15 * x`, where x is the read length. See also: [setting function options]. Reads exceeding this ceiling are [filtered out]. Default: `L,0,0.15`. {: #hisat2-options-n-ceil} [`--n-ceil`]: #hisat2-options-n-ceil [filtered out]: #filtering * `--ignore-quals` When calculating a mismatch penalty, always consider the quality value at the mismatched position to be the highest possible, regardless of the actual value. I.e. input is treated as though all quality values are high. This is also the default behavior when the input doesn't specify quality values (e.g. in [`-f`], [`-r`], or [`-c`] modes). {: #hisat2-options-ignore-quals} [`--ignore-quals`]: #hisat2-options-ignore-quals * `--nofw/--norc` If `--nofw` is specified, `hisat2` will not attempt to align unpaired reads to the forward (Watson) reference strand. If `--norc` is specified, `hisat2` will not attempt to align unpaired reads against the reverse-complement (Crick) reference strand. In paired-end mode, `--nofw` and `--norc` pertain to the fragments; i.e. specifying `--nofw` causes `hisat2` to explore only those paired-end configurations corresponding to fragments from the reverse-complement (Crick) strand. Default: both strands enabled. {: #hisat2-options-nofw} [`--nofw`]: #hisat2-options-nofw #### Scoring options * `--mp MX,MN` Sets the maximum (`MX`) and minimum (`MN`) mismatch penalties, both integers. A number less than or equal to `MX` and greater than or equal to `MN` is subtracted from the alignment score for each position where a read character aligns to a reference character, the characters do not match, and neither is an `N`. If [`--ignore-quals`] is specified, the number subtracted quals `MX`. Otherwise, the number subtracted is `MN + floor( (MX-MN)(MIN(Q, 40.0)/40.0) )` where Q is the Phred quality value. Default: `MX` = 6, `MN` = 2. {: #hisat2-options-mp} [`--mp`]: #hisat2-options-mp * `--sp MX,MN` Sets the maximum (`MX`) and minimum (`MN`) penalties for soft-clipping per base, both integers. A number less than or equal to `MX` and greater than or equal to `MN` is subtracted from the alignment score for each position. The number subtracted is `MN + floor( (MX-MN)(MIN(Q, 40.0)/40.0) )` where Q is the Phred quality value. Default: `MX` = 2, `MN` = 1. {: #hisat2-options-sp} [`--sp`]: #hisat2-options-sp * `--no-softclip` Disallow soft-clipping. {: #hisat2-options-no-softclip} [`--sp`]: #hisat2-options-no-softclip * `--np ` Sets penalty for positions where the read, reference, or both, contain an ambiguous character such as `N`. Default: 1. {: #hisat2-options-np} [`--np`]: #hisat2-options-np * `--rdg ,` Sets the read gap open (``) and extend (``) penalties. A read gap of length N gets a penalty of `` + N * ``. Default: 5, 3. {: #hisat2-options-rdg} [`--rdg`]: #hisat2-options-rdg * `--rfg ,` Sets the reference gap open (``) and extend (``) penalties. A reference gap of length N gets a penalty of `` + N * ``. Default: 5, 3. {: #hisat2-options-rfg} [`--rfg`]: #hisat2-options-rfg * `--score-min ` Sets a function governing the minimum alignment score needed for an alignment to be considered "valid" (i.e. good enough to report). This is a function of read length. For instance, specifying `L,0,-0.6` sets the minimum-score function `f` to `f(x) = 0 + -0.6 * x`, where `x` is the read length. See also: [setting function options]. The default is `L,0,-0.2`. {: #hisat2-options-score-min} [`--score-min`]: #hisat2-options-score-min #### Spliced alignment options * `--pen-cansplice ` Sets the penalty for each pair of canonical splice sites (e.g. GT/AG). Default: 0. {: #hisat2-options-pen-cansplice} [`--pen-cansplice`]: #hisat2-options-pen-cansplice * `--pen-noncansplice ` Sets the penalty for each pair of non-canonical splice sites (e.g. non-GT/AG). Default: 12. {: #hisat2-options-pen-noncansplice} [`--pen-noncansplice`]: #hisat2-options-pen-noncansplice * `--pen-canintronlen ` Sets the penalty for long introns with canonical splice sites so that alignments with shorter introns are preferred to those with longer ones. Default: G,-8,1 {: #hisat2-options-pen-canintronlen} [`--pen-canintronlen`]: #hisat2-options-pen-canintronlen * `--pen-noncanintronlen ` Sets the penalty for long introns with noncanonical splice sites so that alignments with shorter introns are preferred to those with longer ones. Default: G,-8,1 {: #hisat2-options-pen-noncanintronlen} [`--pen-noncanintronlen`]: #hisat2-options-pen-noncanintronlen * `--min-intronlen ` Sets minimum intron length. Default: 20 {: #hisat2-options-min-intronlen} [`--min-intronlen`]: #hisat2-options-min-intronlen * `--max-intronlen ` Sets maximum intron length. Default: 500000 {: #hisat2-options-max-intronlen} [`--max-intronlen`]: #hisat2-options-max-intronlen * `--known-splicesite-infile ` With this mode, you can provide a list of known splice sites, which HISAT2 makes use of to align reads with small anchors. You can create such a list using `python hisat2_extract_splice_sites.py genes.gtf > splicesites.txt`, where `hisat2_extract_splice_sites.py` is included in the HISAT2 package, `genes.gtf` is a gene annotation file, and `splicesites.txt` is a list of splice sites with which you provide HISAT2 in this mode. Note that it is better to use indexes built using annotated transcripts (such as genome_tran or genome_snp_tran), which works better than using this option. It has no effect to provide splice sites that are already included in the indexes. {: #hisat2-options-known-splicesite-infile} [`--splice-infile`]: #hisat2-options-known-splicesite-infile * `--novel-splicesite-outfile ` In this mode, HISAT2 reports a list of splice sites in the file : > chromosome name `` genomic position of the flanking base on the left side of an intron `` genomic position of the flanking base on the right `` strand (+, -, and .) '.' indicates an unknown strand for non-canonical splice sites. {: #hisat2-options-novel-splicesite-outfile} [`--novel-splicesite-outfile`]: #hisat2-options-novel-splicesite-outfile * `--novel-splicesite-infile ` With this mode, you can provide a list of novel splice sites that were generated from the above option "--novel-splicesite-outfile". {: #hisat2-options-novel-splicesite-infile} [`--novel-splicesite-infile`]: #hisat2-options-novel-splicesite-infile * `--no-temp-splicesite` HISAT2, by default, makes use of splice sites found by earlier reads to align later reads in the same run, in particular, reads with small anchors (<= 15 bp). The option disables this default alignment strategy. {: #hisat2-options-no-temp-splicesite} [`--no-temp-splicesite`]: #hisat2-options-no-temp-splicesite * `--no-spliced-alignment` Disable spliced alignment. {: #hisat2-options-no-spliced-alignment} [`--no-spliced-alignment`]: #hisat2-options-no-spliced-alignment * `--rna-strandness ` Specify strand-specific information: the default is unstranded. For single-end reads, use F or R. >'F' means a read corresponds to a transcript. >'R' means a read corresponds to the reverse complemented counterpart of a transcript. For paired-end reads, use either FR or RF. With this option being used, every read alignment will have an XS attribute tag: >'+' means a read belongs to a transcript on '+' strand of genome. >'-' means a read belongs to a transcript on '-' strand of genome. (TopHat has a similar option, --library-type option, where fr-firststrand corresponds to R and RF; fr-secondstrand corresponds to F and FR.) {: #hisat2-options-rna-strandness} [`--rna-strandness`]: #hisat2-options-rna-strandness * `--tmo/--transcriptome-mapping-only` Report only those alignments within known transcripts. {: #hisat2-options-tmo} [`--tmo/--transcriptome-mapping-only`]: #hisat2-options-tmo * `--dta/--downstream-transcriptome-assembly` Report alignments tailored for transcript assemblers including StringTie. With this option, HISAT2 requires longer anchor lengths for de novo discovery of splice sites. This leads to fewer alignments with short-anchors, which helps transcript assemblers improve significantly in computation and memory usage. {: #hisat2-options-dta} [`--dta/--downstream-transcriptome-assembly`]: #hisat2-options-dta * `--dta-cufflinks` Report alignments tailored specifically for Cufflinks. In addition to what HISAT2 does with the above option (--dta), With this option, HISAT2 looks for novel splice sites with three signals (GT/AG, GC/AG, AT/AC), but all user-provided splice sites are used irrespective of their signals. HISAT2 produces an optional field, XS:A:[+-], for every spliced alignment. {: #hisat2-options-dta-cufflinks} [`--dta-cufflinks`]: #hisat2-options-dta-cufflinks * `--avoid-pseudogene` Try to avoid aligning reads to pseudogenes. Note this option is experimental and needs further investigation. {: #hisat2-options-avoid-pseudogene} [`--avoid-pseudogene`]: #hisat2-options-avoid-pseudogene * `--no-templatelen-adjustment` Disables template length adjustment for RNA-seq reads. {: #hisat2-options-no-templatelen-adjustment} [`--no-templatelen-adjustment`]: #hisat2-options-no-templatelen-adjustment #### Reporting options * `-k ` It searches for at most `` distinct, primary alignments for each read. Primary alignments mean alignments whose alignment score is equal to or higher than any other alignments. The search terminates when it cannot find more distinct valid alignments, or when it finds ``, whichever happens first. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS field. For reads that have more than `` distinct, valid alignments, **hisat2** does not guarantee that the `` alignments reported are the best possible in terms of alignment score. Default: 5 (linear index) or 10 (graph index).

    Note: HISAT2 is not designed with large values for `-k` in mind, and when aligning reads to long, repetitive genomes, large `-k` could make alignment much slower. {: #hisat2-options-k} [`-k`]: #hisat2-options-k * `--max-seeds ` HISAT2, like other aligners, uses seed-and-extend approaches. HISAT2 tries to extend seeds to full-length alignments. In HISAT2, `--max-seeds` is used to control the maximum number of seeds that will be extended. For DNA-read alignment ([`--no-spliced-alignment`]), HISAT2 extends up to these many seeds and skips the rest of the seeds. For RNA-read alignment, HISAT2 skips extending seeds and reports no alignments if the number of seeds is larger than the number specified with the option, to be compatible with previous versions of HISAT2. Large values for `--max-seeds` may improve alignment sensitivity, but HISAT2 is not designed with large values for `--max-seeds` in mind, and when aligning reads to long, repetitive genomes, large `--max-seeds` could make alignment much slower. The default value is the maximum of 5 and the value that comes with `-k` times 2. {: #hisat2-options-max-seeds} [`--max-seeds`]: #hisat2-options-max-seeds * `-a/--all` HISAT2 reports all alignments it can find. Using the option is equivalent to using both [`--max-seeds`] and [`-k`] with the maximum value that a 64-bit signed integer can represent (9,223,372,036,854,775,807). {: #hisat2-options-a} [`-a`/`--all`]: #hisat2-options-a [`-a`]: #hisat2-options-a * `--secondary` Report secondary alignments. {: #hisat2-options-secondary} [`--secondary`]: #hisat2-options-secondary #### Paired-end options * `-I/--minins ` The minimum fragment length for valid paired-end alignments.This option is valid only with [`--no-spliced-alignment`]. E.g. if `-I 60` is specified and a paired-end alignment consists of two 20-bp alignments in the appropriate orientation with a 20-bp gap between them, that alignment is considered valid (as long as [`-X`] is also satisfied). A 19-bp gap would not be valid in that case. If trimming options [`-3`] or [`-5`] are also used, the [`-I`] constraint is applied with respect to the untrimmed mates.

    The larger the difference between [`-I`] and [`-X`], the slower HISAT2 will run. This is because larger differences between [`-I`] and [`-X`] require that HISAT2 scan a larger window to determine if a concordant alignment exists. For typical fragment length ranges (200 to 400 nucleotides), HISAT2 is very efficient.

    Default: 0 (essentially imposing no minimum) {: #hisat2-options-I} [`-I`/`--minins`]: #hisat2-options-I [`-I`]: #hisat2-options-I * `-X/--maxins ` The maximum fragment length for valid paired-end alignments. This option is valid only with [`--no-spliced-alignment`]. E.g. if `-X 100` is specified and a paired-end alignment consists of two 20-bp alignments in the proper orientation with a 60-bp gap between them, that alignment is considered valid (as long as [`-I`] is also satisfied). A 61-bp gap would not be valid in that case. If trimming options [`-3`] or [`-5`] are also used, the `-X` constraint is applied with respect to the untrimmed mates, not the trimmed mates.

    The larger the difference between [`-I`] and [`-X`], the slower HISAT2 will run. This is because larger differences between [`-I`] and [`-X`] require that HISAT2 scan a larger window to determine if a concordant alignment exists. For typical fragment length ranges (200 to 400 nucleotides), HISAT2 is very efficient.

    Default: 500. {: #hisat2-options-X} [`-X`/`--maxins`]: #hisat2-options-X [`-X`]: #hisat2-options-X * `--fr/--rf/--ff` The upstream/downstream mate orientations for a valid paired-end alignment against the forward reference strand. E.g., if `--fr` is specified and there is a candidate paired-end alignment where mate 1 appears upstream of the reverse complement of mate 2 and the fragment length constraints ([`-I`] and [`-X`]) are met, that alignment is valid. Also, if mate 2 appears upstream of the reverse complement of mate 1 and all other constraints are met, that too is valid. `--rf` likewise requires that an upstream mate1 be reverse-complemented and a downstream mate2 be forward-oriented. `--ff` requires both an upstream mate 1 and a downstream mate 2 to be forward-oriented. Default: `--fr` (appropriate for Illumina's Paired-end Sequencing Assay). {: #hisat2-options-fr} [`--fr/--rf/--ff`]: #hisat2-options-fr [`--fr`]: #hisat2-options-fr [`--rf`]: #hisat2-options-fr [`--ff`]: #hisat2-options-fr * `--no-mixed` By default, when `hisat2` cannot find a concordant or discordant alignment for a pair, it then tries to find alignments for the individual mates. This option disables that behavior. {: #hisat2-options-no-mixed} [`--no-mixed`]: #hisat2-options-no-mixed * `--no-discordant` By default, `hisat2` looks for discordant alignments if it cannot find any concordant alignments. A discordant alignment is an alignment where both mates align uniquely, but that does not satisfy the paired-end constraints ([`--fr/--rf/--ff`], [`-I`], [`-X`]). This option disables that behavior. {: #hisat2-options-no-discordant} [`--no-discordant`]: #hisat2-options-no-discordant #### Output options * `-t/--time` Print the wall-clock time required to load the index files and align the reads. This is printed to the "standard error" ("stderr") filehandle. Default: off. {: #hisat2-options-t} [`-t`/`--time`]: #hisat2-options-t [`-t`]: #hisat2-options-t * `--un `, `--un-gz `, `--un-bz2 ` Write unpaired reads that fail to align to file at ``. These reads correspond to the SAM records with the FLAGS `0x4` bit set and neither the `0x40` nor `0x80` bits set. If `--un-gz` is specified, output will be gzip compressed. If `--un-bz2` is specified, output will be bzip2 compressed. Reads written in this way will appear exactly as they did in the input file, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the input. {: #hisat2-options-un} [`--un`]: #hisat2-options-un [`--un-gz`]: #hisat2-options-un [`--un-bz2`]: #hisat2-options-un * `--al `, `--al-gz `, `--al-bz2 ` Write unpaired reads that align at least once to file at ``. These reads correspond to the SAM records with the FLAGS `0x4`, `0x40`, and `0x80` bits unset. If `--al-gz` is specified, output will be gzip compressed. If `--al-bz2` is specified, output will be bzip2 compressed. Reads written in this way will appear exactly as they did in the input file, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the input. {: #hisat2-options-al} [`--al`]: #hisat2-options-al [`--al-gz`]: #hisat2-options-al [`--al-bz2`]: #hisat2-options-al * `--un-conc `, `--un-conc-gz `, `--un-conc-bz2 ` Write paired-end reads that fail to align concordantly to file(s) at ``. These reads correspond to the SAM records with the FLAGS `0x4` bit set and either the `0x40` or `0x80` bit set (depending on whether it's mate #1 or #2). `.1` and `.2` strings are added to the filename to distinguish which file contains mate #1 and mate #2. If a percent symbol, `%`, is used in ``, the percent symbol is replaced with `1` or `2` to make the per-mate filenames. Otherwise, `.1` or `.2` are added before the final dot in `` to make the per-mate filenames. Reads written in this way will appear exactly as they did in the input files, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the inputs. {: #hisat2-options-un-conc} [`--un-conc`]: #hisat2-options-un-conc [`--un-conc-gz`]: #hisat2-options-un-conc [`--un-conc-bz2`]: #hisat2-options-un-conc * `--al-conc `, `--al-conc-gz `, `--al-conc-bz2 ` Write paired-end reads that align concordantly at least once to file(s) at ``. These reads correspond to the SAM records with the FLAGS `0x4` bit unset and either the `0x40` or `0x80` bit set (depending on whether it's mate #1 or #2). `.1` and `.2` strings are added to the filename to distinguish which file contains mate #1 and mate #2. If a percent symbol, `%`, is used in ``, the percent symbol is replaced with `1` or `2` to make the per-mate filenames. Otherwise, `.1` or `.2` are added before the final dot in `` to make the per-mate filenames. Reads written in this way will appear exactly as they did in the input files, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the inputs. {: #hisat2-options-al-conc} [`--al-conc`]: #hisat2-options-al-conc [`--al-conc-gz`]: #hisat2-options-al-conc [`--al-conc-bz2`]: #hisat2-options-al-conc * `--quiet` Print nothing besides alignments and serious errors. {: #hisat2-options-quiet} [`--quiet`]: #hisat2-options-quiet * `--summary-file` Print alignment summary to this file. {: #hisat2-options-summary-file} [`--summary-file`]: #hisat2-options-summary-file * `--new-summary` Print alignment summary in a new style, which is more machine-friendly. {: #hisat2-options-new-summary} [`--new-summary`]: #hisat2-options-new-summary * `--met-file ` Write `hisat2` metrics to file ``. Having alignment metric can be useful for debugging certain problems, especially performance issues. See also: [`--met`]. Default: metrics disabled. {: #hisat2-options-met-file} [`--met-file`]: #hisat2-options-met-file * `--met-stderr` Write `hisat2` metrics to the "standard error" ("stderr") filehandle. This is not mutually exclusive with [`--met-file`]. Having alignment metric can be useful for debugging certain problems, especially performance issues. See also: [`--met`]. Default: metrics disabled. {: #hisat2-options-met-stderr} [`--met-stderr`]: #hisat2-options-met-stderr * `--met ` Write a new `hisat2` metrics record every `` seconds. Only matters if either [`--met-stderr`] or [`--met-file`] are specified. Default: 1. {: #hisat2-options-met} [`--met`]: #hisat2-options-met #### SAM options * `--no-unal` Suppress SAM records for reads that failed to align. {: #hisat2-options-no-unal} [`--no-unal`]: #hisat2-options-no-unal * `--no-hd` Suppress SAM header lines (starting with `@`). {: #hisat2-options-no-hd} [`--no-hd`]: #hisat2-options-no-hd * `--no-sq` Suppress `@SQ` SAM header lines. {: #hisat2-options-no-sq} [`--no-sq`]: #hisat2-options-no-sq * `--rg-id ` Set the read group ID to ``. This causes the SAM `@RG` header line to be printed, with `` as the value associated with the `ID:` tag. It also causes the `RG:Z:` extra field to be attached to each SAM output record, with value set to ``. {: #hisat2-options-rg-id} [`--rg-id`]: #hisat2-options-rg-id * `--rg ` Add `` (usually of the form `TAG:VAL`, e.g. `SM:Pool1`) as a field on the `@RG` header line. Note: in order for the `@RG` line to appear, [`--rg-id`] must also be specified. This is because the `ID` tag is required by the [SAM Spec][SAM]. Specify `--rg` multiple times to set multiple fields. See the [SAM Spec][SAM] for details about what fields are legal. {: #hisat2-options-rg} [`--rg`]: #hisat2-options-rg * `--remove-chrname` Remove 'chr' from reference names in alignment (e.g., chr18 to 18) {: #hisat2-remove-chrname} [`--remove-chrname`]: #hisat2-remove-chrname * `--add-chrname` Add 'chr' to reference names in alignment (e.g., 18 to chr18) {: #hisat2-options-add-chrname} [`--add-chrname`]: #hisat2-options-add-chrname * `--omit-sec-seq` When printing secondary alignments, HISAT2 by default will write out the `SEQ` and `QUAL` strings. Specifying this option causes HISAT2 to print an asterisk in those fields instead. {: #hisat2-options-omit-sec-seq} [`--omit-sec-seq`]: #hisat2-options-omit-sec-seq #### Performance options * `-o/--offrate ` Override the offrate of the index with ``. If `` is greater than the offrate used to build the index, then some row markings are discarded when the index is read into memory. This reduces the memory footprint of the aligner but requires more time to calculate text offsets. `` must be greater than the value used to build the index. {: #hisat2-options-o} [`-o`/`--offrate`]: #hisat2-options-o [`-o`]: #hisat2-options-o [`--offrate`]: #hisat2-options-o * `-p/--threads NTHREADS` Launch `NTHREADS` parallel search threads (default: 1). Threads will run on separate processors/cores and synchronize when parsing reads and outputting alignments. Searching for alignments is highly parallel, and speedup is close to linear. Increasing `-p` increases HISAT2's memory footprint. E.g. when aligning to a human genome index, increasing `-p` from 1 to 8 increases the memory footprint by a few hundred megabytes. This option is only available if HISAT2 is linked with the `pthreads` library. {: #hisat2-options-p} [`-p`/`--threads`]: #hisat2-options-p [`-p`]: #hisat2-options-p * `--reorder` Guarantees that output SAM records are printed in an order corresponding to the order of the reads in the original input file, even when [`-p`] is set greater than 1. Specifying `--reorder` and setting [`-p`] greater than 1 causes HISAT2 to run somewhat slower and use somewhat more memory then if `--reorder` were not specified. Has no effect if [`-p`] is set to 1, since output order will naturally correspond to input order in that case. {: #hisat2-options-reorder} [`--reorder`]: #hisat2-options-reorder * `--mm` Use memory-mapped I/O to load the index, rather than typical file I/O. Memory-mapping allows many concurrent `bowtie` processes on the same computer to share the same memory image of the index (i.e. you pay the memory overhead just once). This facilitates memory-efficient parallelization of `bowtie` in situations where using [`-p`] is not possible or not preferable. {: #hisat2-options-mm} [`--mm`]: #hisat2-options-mm #### Other options * `--qc-filter` Filter out reads for which the QSEQ filter field is non-zero. Only has an effect when read format is [`--qseq`]. Default: off. {: #hisat2-options-qc-filter} [`--qc-filter`]: #hisat2-options-qc-filter * `--seed ` Use `` as the seed for pseudo-random number generator. Default: 0. {: #hisat2-options-seed} [`--seed`]: #hisat2-options-seed * `--non-deterministic` Normally, HISAT2 re-initializes its pseudo-random generator for each read. It seeds the generator with a number derived from (a) the read name, (b) the nucleotide sequence, (c) the quality sequence, (d) the value of the [`--seed`] option. This means that if two reads are identical (same name, same nucleotides, same qualities) HISAT2 will find and report the same alignment(s) for both, even if there was ambiguity. When `--non-deterministic` is specified, HISAT2 re-initializes its pseudo-random generator for each read using the current time. This means that HISAT2 will not necessarily report the same alignment for two identical reads. This is counter-intuitive for some users, but might be more appropriate in situations where the input consists of many identical reads. {: #hisat2-options-non-deterministic} [`--non-deterministic`]: #hisat2-options-non-deterministic * `--version` Print version information and quit. {: #hisat2-options-version} [`--version`]: #hisat2-options-version * `-h/--help` Print usage information and quit. {: #hisat2-options-h} [`-h`]: #hisat2-options-h SAM output ---------- Following is a brief description of the [SAM] format as output by `hisat2`. For more details, see the [SAM format specification][SAM]. By default, `hisat2` prints a SAM header with `@HD`, `@SQ` and `@PG` lines. When one or more [`--rg`] arguments are specified, `hisat2` will also print an `@RG` line that includes all user-specified [`--rg`] tokens separated by tabs. Each subsequent line describes an alignment or, if the read failed to align, a read. Each line is a collection of at least 12 fields separated by tabs; from left to right, the fields are: 1. Name of read that aligned. Note that the [SAM specification] disallows whitespace in the read name. If the read name contains any whitespace characters, HISAT2 will truncate the name at the first whitespace character. This is similar to the behavior of other tools. 2. Sum of all applicable flags. Flags relevant to HISAT2 are * 1: The read is one of a pair * 2: The alignment is one end of a proper paired-end alignment * 4: The read has no reported alignments * 8: The read is one of a pair and has no reported alignments * 16: The alignment is to the reverse reference strand * 32: The other mate in the paired-end alignment is aligned to the reverse reference strand * 64: The read is mate 1 in a pair * 128: The read is mate 2 in a pair ^ Thus, an unpaired read that aligns to the reverse reference strand will have flag 16. A paired-end read that aligns and is the first mate in the pair will have flag 83 (= 64 + 16 + 2 + 1). 3. Name of reference sequence where alignment occurs 4. 1-based offset into the forward reference strand where leftmost character of the alignment occurs 5. Mapping quality. Mapping quality of HISAT2 6. CIGAR string representation of alignment 7. Name of reference sequence where mate's alignment occurs. Set to `=` if the mate's reference sequence is the same as this alignment's, or `*` if there is no mate. 8. 1-based offset into the forward reference strand where leftmost character of the mate's alignment occurs. Offset is 0 if there is no mate. 9. Inferred fragment length. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if the mates did not align concordantly. However, size is non-0 if the mates aligned discordantly to the same chromosome. 10. Read sequence (reverse-complemented if aligned to the reverse strand) 11. ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the [Phred quality] scale and the encoding is ASCII-offset by 33 (ASCII char `!`), similarly to a [FASTQ] file. 12. Optional fields. Fields are tab-separated. `hisat2` outputs zero or more of these optional fields for each alignment, depending on the type of the alignment: * {: #hisat2-opt-fields-as} `AS:i:` : Alignment score. Can be negative. Only present if SAM record is for an aligned read. * {: #hisat2-opt-fields-xs} `ZS:i:` : Alignment score for the best-scoring alignment found other than the alignment reported. Can be negative. Only present if the SAM record is for an aligned read and more than one alignment was found for the read. Note that, when the read is part of a concordantly-aligned pair, this score could be greater than [`AS:i`]. * {: #hisat2-opt-fields-ys} `YS:i:` : Alignment score for opposite mate in the paired-end alignment. Only present if the SAM record is for a read that aligned as part of a paired-end alignment. * {: #hisat2-opt-fields-xn} `XN:i:` : The number of ambiguous bases in the reference covering this alignment. Only present if SAM record is for an aligned read. * {: #hisat2-opt-fields-xm} `XM:i:` : The number of mismatches in the alignment. Only present if SAM record is for an aligned read. * {: #hisat2-opt-fields-xo} `XO:i:` : The number of gap opens, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read. * {: #hisat2-opt-fields-xg} `XG:i:` : The number of gap extensions, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read. * {: #hisat2-opt-fields-nm} `NM:i:` : The edit distance; that is, the minimal number of one-nucleotide edits (substitutions, insertions and deletions) needed to transform the read string into the reference string. Only present if SAM record is for an aligned read. * {: #hisat2-opt-fields-yf} `YF:Z:` : String indicating reason why the read was filtered out. See also: [Filtering]. Only appears for reads that were filtered out. * {: #hisat2-opt-fields-yt} `YT:Z:` : Value of `UU` indicates the read was not part of a pair. Value of `CP` indicates the read was part of a pair and the pair aligned concordantly. Value of `DP` indicates the read was part of a pair and the pair aligned discordantly. Value of `UP` indicates the read was part of a pair but the pair failed to aligned either concordantly or discordantly. * {: #hisat2-opt-fields-md} `MD:Z:` : A string representation of the mismatched reference bases in the alignment. See [SAM] format specification for details. Only present if SAM record is for an aligned read. * {: #hisat2-opt-fields-xs} `XS:A:` : Values of `+` and `-` indicate the read is mapped to transcripts on sense and anti-sense strands, respectively. Spliced alignments need to have this field, which is required in Cufflinks and StringTie. We can report this field for the canonical-splice site (GT/AG), but not for non-canonical splice sites. You can direct HISAT2 not to output such alignments (involving non-canonical splice sites) using "--pen-noncansplice 1000000". * {: #hisat2-opt-fields-nh} `NH:i:` : The number of mapped locations for the read or the pair. * {: #hisat2-opt-fields-Zs} `Zs:Z:` : When the alignment of a read involves SNPs that are in the index, this option is used to indicate where exactly the read involves the SNPs. This optional field is similar to the above MD:Z field. For example, `Zs:Z:1|S|rs3747203,97|S|rs16990981` indicates the second base of the read corresponds to a known SNP (ID: rs3747203). 97 bases after the third base (the base after the second one), the read at 100th base involves another known SNP (ID: rs16990981). 'S' indicates a single nucleotide polymorphism. 'D' and 'I' indicate a deletion and an insertion, respectively. [SAM format specification]: http://samtools.sf.net/SAM1.pdf [FASTQ]: http://en.wikipedia.org/wiki/FASTQ_format [`-S`/`--sam`]: #hisat2-options-S [`-m`]: #hisat2-options-m [`AS:i`]: #hisat2-opt-fields-as [`ZS:i`]: #hisat2-opt-fields-xs [`YS:i`]: #hisat2-opt-fields-ys [`XN:i`]: #hisat2-opt-fields-xn [`XM:i`]: #hisat2-opt-fields-xm [`XO:i`]: #hisat2-opt-fields-xo [`XG:i`]: #hisat2-opt-fields-xg [`NM:i`]: #hisat2-opt-fields-nm [`YF:Z`]: #hisat2-opt-fields-yf [`YT:Z`]: #hisat2-opt-fields-yt [`MD:Z`]: #hisat2-opt-fields-md [`XS:A`]: #hisat2-opt-fields-xs [`NH:i`]: #hisat2-opt-fields-nh [`Zs:Z`]: #hisat2-opt-fields-Zs The `hisat2-build` indexer =========================== `hisat2-build` builds a HISAT2 index from a set of DNA sequences. `hisat2-build` outputs a set of 6 files with suffixes `.1.ht2`, `.2.ht2`, `.3.ht2`, `.4.ht2`, `.5.ht2`, `.6.ht2`, `.7.ht2`, and `.8.ht2`. In the case of a large index these suffixes will have a `ht2l` termination. These files together constitute the index: they are all that is needed to align reads to that reference. The original sequence FASTA files are no longer used by HISAT2 once the index is built. Use of Karkkainen's [blockwise algorithm] allows `hisat2-build` to trade off between running time and memory usage. `hisat2-build` has three options governing how it makes this trade: [`-p`/`--packed`], [`--bmax`]/[`--bmaxdivn`], and [`--dcv`]. By default, `hisat2-build` will automatically search for the settings that yield the best running time without exhausting memory. This behavior can be disabled using the [`-a`/`--noauto`] option. The indexer provides options pertaining to the "shape" of the index, e.g. [`--offrate`](#hisat2-build-options-o) governs the fraction of [Burrows-Wheeler] rows that are "marked" (i.e., the density of the suffix-array sample; see the original [FM Index] paper for details). All of these options are potentially profitable trade-offs depending on the application. They have been set to defaults that are reasonable for most cases according to our experiments. See [Performance tuning] for details. `hisat2-build` can generate either [small or large indexes](#small-and-large-indexes). The wrapper will decide which based on the length of the input genome. If the reference does not exceed 4 billion characters but a large index is preferred, the user can specify [`--large-index`] to force `hisat2-build` to build a large index instead. The HISAT2 index is based on the [FM Index] of Ferragina and Manzini, which in turn is based on the [Burrows-Wheeler] transform. The algorithm used to build the index is based on the [blockwise algorithm] of Karkkainen. [Blockwise algorithm]: http://portal.acm.org/citation.cfm?id=1314852 [Burrows-Wheeler]: http://en.wikipedia.org/wiki/Burrows-Wheeler_transform [Performance tuning]: #performance-tuning Command Line ------------ Usage: hisat2-build [options]* ### Notes If you use --snp, --ss, and/or --exon, hisat2-build will need about 200GB RAM for the human genome size as index building involves a graph construction. Otherwise, you will be able to build an index on your desktop with 8GB RAM. ### Main arguments * `` A comma-separated list of FASTA files containing the reference sequences to be aligned to, or, if [`-c`](#hisat2-build-options-c) is specified, the sequences themselves. E.g., `` might be `chr1.fa,chr2.fa,chrX.fa,chrY.fa`, or, if [`-c`](#hisat2-build-options-c) is specified, this might be `GGTCATCCT,ACGGGTCGT,CCGTTCTATGCGGCTTA`. {: #hisat2-build-options-ref} * `` The basename of the index files to write. By default, `hisat2-build` writes files named `NAME.1.ht2`, `NAME.2.ht2`, `NAME.3.ht2`, `NAME.4.ht2`, `NAME.5.ht2`, `NAME.6.ht2`, `NAME.7.ht2`, and `NAME.8.ht2` where `NAME` is ``. {: #hisat2-build-options-base} ### Options * `-f` The reference input files (specified as ``) are FASTA files (usually having extension `.fa`, `.mfa`, `.fna` or similar). {: #hisat2-build-options-f} * `-c` The reference sequences are given on the command line. I.e. `` is a comma-separated list of sequences rather than a list of FASTA files. {: #hisat2-build-options-c} * `--large-index` Force `hisat2-build` to build a [large index](#small-and-large-indexes), even if the reference is less than ~ 4 billion nucleotides long. {: #hisat2-build-options-large-index} [`--large-index`]: #hisat2-build-options-large-index * `-a/--noauto` Disable the default behavior whereby `hisat2-build` automatically selects values for the [`--bmax`], [`--dcv`] and [`--packed`] parameters according to available memory. Instead, user may specify values for those parameters. If memory is exhausted during indexing, an error message will be printed; it is up to the user to try new parameters. {: #hisat2-build-options-a} [`-a`/`--noauto`]: #hisat2-build-options-a * `--bmax ` The maximum number of suffixes allowed in a block. Allowing more suffixes per block makes indexing faster, but increases peak memory usage. Setting this option overrides any previous setting for [`--bmax`], or [`--bmaxdivn`]. Default (in terms of the [`--bmaxdivn`] parameter) is [`--bmaxdivn`] 4. This is configured automatically by default; use [`-a`/`--noauto`] to configure manually. {: #hisat2-build-options-bmax} [`--bmax`]: #hisat2-build-options-bmax * `--bmaxdivn ` The maximum number of suffixes allowed in a block, expressed as a fraction of the length of the reference. Setting this option overrides any previous setting for [`--bmax`], or [`--bmaxdivn`]. Default: [`--bmaxdivn`] 4. This is configured automatically by default; use [`-a`/`--noauto`] to configure manually. {: #hisat2-build-options-bmaxdivn} [`--bmaxdivn`]: #hisat2-build-options-bmaxdivn * `--dcv ` Use `` as the period for the difference-cover sample. A larger period yields less memory overhead, but may make suffix sorting slower, especially if repeats are present. Must be a power of 2 no greater than 4096. Default: 1024. This is configured automatically by default; use [`-a`/`--noauto`] to configure manually. {: #hisat2-build-options-dcv} [`--dcv`]: #hisat2-build-options-dcv * `--nodc` Disable use of the difference-cover sample. Suffix sorting becomes quadratic-time in the worst case (where the worst case is an extremely repetitive reference). Default: off. {: #hisat2-build-options-nodc} [`--nodc`]: #hisat2-build-options-nodc * `-r/--noref` Do not build the `NAME.3.ht2` and `NAME.4.ht2` portions of the index, which contain a bitpacked version of the reference sequences and are used for paired-end alignment. {: #hisat2-build-options-r} * `-3/--justref` Build only the `NAME.3.ht2` and `NAME.4.ht2` portions of the index, which contain a bitpacked version of the reference sequences and are used for paired-end alignment. {: #hisat2-build-options-3} * `-o/--offrate ` To map alignments back to positions on the reference sequences, it's necessary to annotate ("mark") some or all of the [Burrows-Wheeler] rows with their corresponding location on the genome. [`-o`/`--offrate`](#hisat2-build-options-o) governs how many rows get marked: the indexer will mark every 2^`` rows. Marking more rows makes reference-position lookups faster, but requires more memory to hold the annotations at runtime. The default is 4 (every 16th row is marked; for human genome, annotations occupy about 680 megabytes). {: #hisat2-build-options-o} * `-t/--ftabchars ` The ftab is the lookup table used to calculate an initial [Burrows-Wheeler] range with respect to the first `` characters of the query. A larger `` yields a larger lookup table but faster query times. The ftab has size 4^(``+1) bytes. The default setting is 10 (ftab is 4MB). {: #hisat2-build-options-t} * `--localoffrate ` This option governs how many rows get marked in a local index: the indexer will mark every 2^`` rows. Marking more rows makes reference-position lookups faster, but requires more memory to hold the annotations at runtime. The default is 3 (every 8th row is marked, this occupies about 16KB per local index). {: #hisat2-build-options-localoffrate} * `--localftabchars ` The local ftab is the lookup table in a local index. The default setting is 6 (ftab is 8KB per local index). {: #hisat2-build-options-localftabchars} * `-p ` Launch `NTHREADS` parallel build threads (default: 1). {: #hisat2-build-options-p} * `--snp ` Provide a list of SNPs (in the HISAT2's own format) as follows (five columns). SNP ID snp type (single, deletion, or insertion) chromosome name zero-offset based genomic position of a SNP alternative base (single), the length of SNP (deletion), or insertion sequence (insertion) For example, rs58784443 single 13 18447947 T Use `hisat2_extract_snps_haplotypes_UCSC.py` (in the HISAT2 package) to extract SNPs and haplotypes from a dbSNP file (e.g. http://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/snp144Common.txt.gz). or `hisat2_extract_snps_haplotypes_VCF.py` to extract SNPs and haplotypes from a VCF file (e.g. ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/supporting/GRCh38_positions/ALL.chr22.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP_no_SVs.vcf.gz). {: #hisat2-build-options-snp} * `--haplotype ` Provide a list of haplotypes (in the HISAT2's own format) as follows (five columns). Haplotype ID chromosome name zero-offset based left coordinate of haplotype zero-offset based right coordinate of haplotype a comma separated list of SNP ids in the haplotype For example, ht35 13 18446877 18446945 rs12381094,rs12381056,rs192016659,rs538569910 See the above option, --snp, about how to extract haplotypes. This option is not required, but haplotype information can keep the index construction from exploding and reduce the index size substantially. {: #hisat2-build-options-haplotype} * `--ss ` Note this option should be used with the following [`--exon`](#hisat2-build-options-exon) option. Provide a list of splice sites (in the HISAT2's own format) as follows (four columns). chromosome name zero-offset based genomic position of the flanking base on the left side of an intron zero-offset based genomic position of the flanking base on the right strand Use `hisat2_extract_splice_sites.py` (in the HISAT2 package) to extract splice sites from a GTF file. {: #hisat2-build-options-ss} * `--exon ` Note this option should be used with the above [`--ss`](#hisat2-build-options-ss) option. Provide a list of exons (in the HISAT2's own format) as follows (three columns). chromosome name zero-offset based left genomic position of an exon zero-offset based right genomic position of an exon Use `hisat2_extract_exons.py` (in the HISAT2 package) to extract exons from a GTF file. {: #hisat2-build-options-exon} * `--seed ` Use `` as the seed for pseudo-random number generator. {: #hisat2-build-options-seed} * `--cutoff ` Index only the first `` bases of the reference sequences (cumulative across sequences) and ignore the rest. {: #hisat2-build-options-cutoff} * `-q/--quiet` `hisat2-build` is verbose by default. With this option `hisat2-build` will print only error messages. {: #hisat2-build-options-q} * `-h/--help` Print usage information and quit. {: #hisat2-build-options-h} * `--version` Print version information and quit. {: #hisat2-build-options-version} The `hisat2-inspect` index inspector ===================================== `hisat2-inspect` extracts information from a HISAT2 index about what kind of index it is and what reference sequences were used to build it. When run without any options, the tool will output a FASTA file containing the sequences of the original references (with all non-`A`/`C`/`G`/`T` characters converted to `N`s). It can also be used to extract just the reference sequence names using the [`-n`/`--names`] option or a more verbose summary using the [`-s`/`--summary`] option. Command Line ------------ Usage: hisat2-inspect [options]* ### Main arguments * `` The basename of the index to be inspected. The basename is name of any of the index files but with the `.X.ht2` suffix omitted. `hisat2-inspect` first looks in the current directory for the index files, then in the directory specified in the `HISAT2_INDEXES` environment variable. {: #hisat2-inspect-options-base} ### Options * `-a/--across ` When printing FASTA output, output a newline character every `` bases (default: 60). {: #hisat2-inspect-options-a} * `-n/--names` Print reference sequence names, one per line, and quit. {: #hisat2-inspect-options-n} [`-n`/`--names`]: #hisat2-inspect-options-n * `-s/--summary` Print a summary that includes information about index settings, as well as the names and lengths of the input sequences. The summary has this format: Colorspace <0 or 1> SA-Sample 1 in FTab-Chars Sequence-1 Sequence-2 ... Sequence-N Fields are separated by tabs. Colorspace is always set to 0 for HISAT2. {: #hisat2-inspect-options-s} [`-s`/`--summary`]: #hisat2-inspect-options-s * `--snp` Print SNPs, and quit. {: #hisat2-inspect-options-snp} [`--snp`]: #hisat2-inspect-options-snp * `--ss` Print splice sites, and quit. {: #hisat2-inspect-options-ss} [`--ss`]: #hisat2-inspect-options-ss * `--ss-all` Print splice sites including those not in the global index, and quit. {: #hisat2-inspect-options-ss-all} [`--ss-all`]: #hisat2-inspect-options-ss-all * `--exon` Print exons, and quit. {: #hisat2-inspect-options-exon} [`--exon`]: #hisat2-inspect-options-exon * `-v/--verbose` Print verbose output (for debugging). * `--version` Print version information and quit. * `-h/--help` Print usage information and quit. Getting started with HISAT2 =================================================== HISAT2 comes with some example files to get you started. The example files are not scientifically significant; these files will simply let you start running HISAT2 and downstream tools right away. First follow the manual instructions to [obtain HISAT2]. Set the `HISAT2_HOME` environment variable to point to the new HISAT2 directory containing the `hisat2`, `hisat2-build` and `hisat2-inspect` binaries. This is important, as the `HISAT2_HOME` variable is used in the commands below to refer to that directory. [obtain HISAT2]: #obtaining-hisat2 Indexing a reference genome --------------------------- To create an index for the genomic region (1 million bps from the human chromosome 22 between 20,000,000 and 20,999,999) included with HISAT2, create a new temporary directory (it doesn't matter where), change into that directory, and run: $HISAT2_HOME/hisat2-build $HISAT2_HOME/example/reference/22_20-21M.fa --snp $HISAT2_HOME/example/reference/22_20-21M.snp 22_20-21M_snp The command should print many lines of output then quit. When the command completes, the current directory will contain ten new files that all start with `22_20-21M_snp` and end with `.1.ht2`, `.2.ht2`, `.3.ht2`, `.4.ht2`, `.5.ht2`, `.6.ht2`, `.7.ht2`, and `.8.ht2`. These files constitute the index - you're done! You can use `hisat2-build` to create an index for a set of FASTA files obtained from any source, including sites such as [UCSC], [NCBI], and [Ensembl]. When indexing multiple FASTA files, specify all the files using commas to separate file names. For more details on how to create an index with `hisat2-build`, see the [manual section on index building]. You may also want to bypass this process by obtaining a pre-built index. [UCSC]: http://genome.ucsc.edu/cgi-bin/hgGateway [NCBI]: http://www.ncbi.nlm.nih.gov/sites/genome [Ensembl]: http://www.ensembl.org/ [manual section on index building]: #the-hisat2-build-indexer [using a pre-built index]: #using-a-pre-built-index Aligning example reads ---------------------- Stay in the directory created in the previous step, which now contains the `22_20-21M` index files. Next, run: $HISAT2_HOME/hisat2 -f -x $HISAT2_HOME/example/index/22_20-21M_snp -U $HISAT2_HOME/example/reads/reads_1.fa -S eg1.sam This runs the HISAT2 aligner, which aligns a set of unpaired reads to the genome region using the index generated in the previous step. The alignment results in SAM format are written to the file `eg1.sam`, and a short alignment summary is written to the console. (Actually, the summary is written to the `"standard error"` or `"stderr"` filehandle, which is typically printed to the console.) To see the first few lines of the SAM output, run: head eg1.sam You will see something like this: @HD VN:1.0 SO:unsorted @SQ SN:22:20000001-21000000 LN:1000000 @PG ID:hisat2 PN:hisat2 VN:2.0.0-beta 1 0 22:20000001-21000000 397984 255 100M * 0 0 GCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU NH:i:1 2 16 22:20000001-21000000 398131 255 100M * 0 0 ATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:80A19 YT:Z:UU NH:i:1 Zs:Z:80|S|rs576159895 3 16 22:20000001-21000000 398222 255 100M * 0 0 TGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:16A83 YT:Z:UU NH:i:1 Zs:Z:16|S|rs2629364 4 16 22:20000001-21000000 398247 255 90M200N10M * 0 0 CAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAACCGCTGCACCAGGAAGTCGATCCAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU XS:A:- NH:i:1 5 16 22:20000001-21000000 398194 255 100M * 0 0 GGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:17A26A55 YT:Z:UU NH:i:1 Zs:Z:17|S|rs576159895,26|S|rs2629364 6 0 22:20000001-21000000 398069 255 100M * 0 0 CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU NH:i:1 7 0 22:20000001-21000000 397896 255 100M * 0 0 GTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGA IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:31G68 YT:Z:UU NH:i:1 Zs:Z:31|S|rs562662261 8 0 22:20000001-21000000 398150 255 100M * 0 0 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:61A26A11 YT:Z:UU NH:i:1 Zs:Z:61|S|rs576159895,26|S|rs2629364 9 16 22:20000001-21000000 398329 255 8M200N92M * 0 0 ACCAGGAAGTCGATCCAGATGTAGTGGGGGGTCACTTCGGGGGGACAGGGTTTGGGTTGACTTGCTTCCGAGGCAGCCAGGGGGTCTGCTTCCTTTATCT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:100 YT:Z:UU XS:A:- NH:i:1 10 16 22:20000001-21000000 398184 255 100M * 0 0 CTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATC IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:27A26A45 YT:Z:UU NH:i:1 Zs:Z:27|S|rs576159895,26|S|rs2629364 The first few lines (beginning with `@`) are SAM header lines, and the rest of the lines are SAM alignments, one line per read or mate. See the [HISAT2 manual section on SAM output] and the [SAM specification] for details about how to interpret the SAM file format. [HISAT2 manual section on SAM output]: #sam-output Paired-end example ------------------ To align paired-end reads included with HISAT2, stay in the same directory and run: $HISAT2_HOME/hisat2 -f -x $HISAT2_HOME/example/index/22_20-21M_snp -1 $HISAT2_HOME/example/reads/reads_1.fa -2 $HISAT2_HOME/example/reads/reads_2.fa -S eg2.sam This aligns a set of paired-end reads to the reference genome, with results written to the file `eg2.sam`. Using SAMtools/BCFtools downstream ---------------------------------- [SAMtools] is a collection of tools for manipulating and analyzing SAM and BAM alignment files. [BCFtools] is a collection of tools for calling variants and manipulating VCF and BCF files, and it is typically distributed with [SAMtools]. Using these tools together allows you to get from alignments in SAM format to variant calls in VCF format. This example assumes that `samtools` and `bcftools` are installed and that the directories containing these binaries are in your [PATH environment variable]. Run the paired-end example: $HISAT2_HOME/hisat -f -x $HISAT2_HOME/example/index/22_20-21M_snp -1 $HISAT2_HOME/example/reads/reads_1.fa -2 $HISAT2_HOME/example/reads/reads_2.fa -S eg2.sam Use `samtools view` to convert the SAM file into a BAM file. BAM is a the binary format corresponding to the SAM text format. Run: samtools view -bS eg2.sam > eg2.bam Use `samtools sort` to convert the BAM file to a sorted BAM file. The following command requires samtools version 1.2 or higher. samtools sort eg2.bam -o eg2.sorted.bam We now have a sorted BAM file called `eg2.sorted.bam`. Sorted BAM is a useful format because the alignments are (a) compressed, which is convenient for long-term storage, and (b) sorted, which is convenient for variant discovery. To generate variant calls in VCF format, run: samtools mpileup -uf $HISAT2_HOME/example/reference/22_20-21M.fa eg2.sorted.bam | bcftools view -bvcg - > eg2.raw.bcf Then to view the variants, run: bcftools view eg2.raw.bcf See the official SAMtools guide to [Calling SNPs/INDELs with SAMtools/BCFtools] for more details and variations on this process. [BCFtools]: http://samtools.sourceforge.net/mpileup.shtml [Calling SNPs/INDELs with SAMtools/BCFtools]: http://samtools.sourceforge.net/mpileup.shtml hisat2-2.2.1/docs/_pages/search.html000066400000000000000000000011231370663763200172410ustar00rootroot00000000000000--- layout: page title: Search Results permalink: /search/ hide: true share: false ---

    hisat2-2.2.1/docs/_pages/tags.html000066400000000000000000000004001370663763200167270ustar00rootroot00000000000000--- layout: page title: Tags permalink: /tags/ order: 2 share: false hide: true --- hisat2-2.2.1/docs/_posts/000077500000000000000000000000001370663763200151625ustar00rootroot00000000000000hisat2-2.2.1/docs/_posts/2000-01-01-kim.md000066400000000000000000000005251370663763200173010ustar00rootroot00000000000000--- layout: post title: Daehwan Kim tags: daehwankim eye_catch: https://avatars0.githubusercontent.com/u/28678667?s=460&v=4 --- Daehwan Kim is an Assistant Professor at UT Southwestern and was the original designer who layed much of the ground work for HISAT-genotype. [Webpage](https://kim-lab.org/daehwan-kim-principal-investigator/) hisat2-2.2.1/docs/_posts/2000-01-02-salzberg.md000066400000000000000000000006361370663763200203360ustar00rootroot00000000000000--- layout: post title: Steven Salzberg tags: stevensalzberg eye_catch: https://avatars0.githubusercontent.com/u/28678667?s=460&v=4 --- Steven Salzberg is the Bloomberg Distinguished Professor of Biomedical Engineering, Computer Science, and Biostatistics at Johns Hopkins University, where I’m also Director of the Center for Computational Biology. [Webpage](https://salzberg-lab.org/in-the-news/about-me/) hisat2-2.2.1/docs/_posts/2000-01-03-langmead.md000066400000000000000000000004071370663763200202720ustar00rootroot00000000000000--- layout: post title: Ben Langmead tags: benlangmead eye_catch: https://avatars0.githubusercontent.com/u/28678667?s=460&v=4 --- Ben Langmead is an Associate Professor of Computer Science at Johns Hopkins University. [Webpage](http://www.langmead-lab.org/) hisat2-2.2.1/docs/_posts/2019-07-28-park.md000066400000000000000000000005271370663763200175110ustar00rootroot00000000000000--- layout: post title: Chanhee Park tags: chanheepark eye_catch: https://avatars0.githubusercontent.com/u/28678667?s=460&v=4 --- Chanhee Park is a Scientific Software Engineer in the Kim Lab at UTSW responsible for maintaining and improving HISAT2, the core of HISAT-genotype. 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R /Im85 402 0 R /Im47 326 0 R /Im119 470 0 R /Im81 394 0 R /Im124 480 0 R /Im122 476 0 R /Im134 500 0 R /Im158 548 0 R /Im126 484 0 R /Im69 370 0 R /Im108 448 0 R /Im136 504 0 R /Im95 422 0 R /Im117 466 0 R /Im157 546 0 R /Im109 450 0 R /Im52 336 0 R /Im76 384 0 R /Im110 452 0 R /Im98 428 0 R /Im106 444 0 R /Im145 522 0 R /Im79 390 0 R /Im102 436 0 R /Im64 360 0 R /Im113 458 0 R /Im111 454 0 R /Im153 538 0 R /Im141 514 0 R >> >> endobj 430 0 obj << /Length 431 0 R /Type /XObject /Subtype /Image /Width 156 /Height 172 /Interpolate true /ColorSpace 7 0 R /SMask 550 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  : endstream endobj 431 0 obj 373 endobj 414 0 obj << /Length 415 0 R /Type /XObject /Subtype /Image /Width 162 /Height 140 /Interpolate true /ColorSpace 7 0 R /SMask 552 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 415 0 obj 320 endobj 332 0 obj << /Length 333 0 R /Type /XObject /Subtype /Image /Width 282 /Height 95 /Interpolate true /ColorSpace 7 0 R /SMask 554 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` : endstream endobj 333 0 obj 374 endobj 406 0 obj << /Length 407 0 R /Type /XObject /Subtype /Image /Width 207 /Height 264 /Interpolate true /ColorSpace 7 0 R /SMask 556 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om /@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`{` endstream endobj 407 0 obj 738 endobj 416 0 obj << /Length 417 0 R /Type /XObject /Subtype /Image /Width 114 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 558 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 417 0 obj 178 endobj 334 0 obj << /Length 335 0 R /Type /XObject /Subtype /Image /Width 264 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 560 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om_@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` A endstream endobj 335 0 obj 383 endobj 438 0 obj << /Length 439 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 562 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 439 0 obj 202 endobj 398 0 obj << /Length 399 0 R /Type /XObject /Subtype /Image /Width 152 /Height 124 /Interpolate true /ColorSpace 7 0 R /SMask 564 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàS_Pa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`;0 endstream endobj 399 0 obj 270 endobj 532 0 obj << /Length 533 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 566 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 533 0 obj 196 endobj 418 0 obj << /Length 419 0 R /Type /XObject /Subtype /Image /Width 193 /Height 169 /Interpolate true /ColorSpace 7 0 R /SMask 568 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`a`~J endstream endobj 419 0 obj 450 endobj 492 0 obj << /Length 493 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 570 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 493 0 obj 196 endobj 342 0 obj << /Length 343 0 R /Type /XObject /Subtype /Image /Width 155 /Height 173 /Interpolate true /ColorSpace 7 0 R /SMask 572 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`:L endstream endobj 343 0 obj 374 endobj 518 0 obj << /Length 519 0 R /Type /XObject /Subtype /Image /Width 62 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 574 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0`w`K endstream endobj 519 0 obj 106 endobj 388 0 obj << /Length 389 0 R /Type /XObject /Subtype /Image /Width 179 /Height 189 /Interpolate true /ColorSpace 7 0 R /SMask 576 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om ?@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0p endstream endobj 389 0 obj 466 endobj 412 0 obj << /Length 413 0 R /Type /XObject /Subtype /Image /Width 114 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 578 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 413 0 obj 178 endobj 376 0 obj << /Length 377 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 580 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 377 0 obj 177 endobj 536 0 obj << /Length 537 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 582 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 537 0 obj 151 endobj 506 0 obj << /Length 507 0 R /Type /XObject /Subtype /Image /Width 117 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 584 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`ρ endstream endobj 507 0 obj 183 endobj 460 0 obj << /Length 461 0 R /Type /XObject /Subtype /Image /Width 113 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 586 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0p:0  endstream endobj 461 0 obj 177 endobj 488 0 obj << /Length 489 0 R /Type /XObject /Subtype /Image /Width 116 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 588 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 489 0 obj 183 endobj 358 0 obj << /Length 359 0 R /Type /XObject /Subtype /Image /Width 536 /Height 129 /Interpolate true /ColorSpace 7 0 R /SMask 590 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`*u endstream endobj 359 0 obj 927 endobj 496 0 obj << /Length 497 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 592 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 497 0 obj 202 endobj 338 0 obj << /Length 339 0 R /Type /XObject /Subtype /Image /Width 297 /Height 239 /Interpolate true /ColorSpace 7 0 R /SMask 594 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om ?@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0p@ endstream endobj 339 0 obj 952 endobj 486 0 obj << /Length 487 0 R /Type /XObject /Subtype /Image /Width 154 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 596 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 487 0 obj 201 endobj 498 0 obj << /Length 499 0 R /Type /XObject /Subtype /Image /Width 116 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 598 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`` endstream endobj 499 0 obj 182 endobj 516 0 obj << /Length 517 0 R /Type /XObject /Subtype /Image /Width 149 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 600 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 517 0 obj 195 endobj 432 0 obj << /Length 433 0 R /Type /XObject /Subtype /Image /Width 117 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 602 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`ρ endstream endobj 433 0 obj 183 endobj 346 0 obj << /Length 347 0 R /Type /XObject /Subtype /Image /Width 247 /Height 153 /Interpolate true /ColorSpace 7 0 R /SMask 604 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om /@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` \ endstream endobj 347 0 obj 518 endobj 526 0 obj << /Length 527 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 606 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 527 0 obj 112 endobj 328 0 obj << /Length 329 0 R /Type /XObject /Subtype /Image /Width 155 /Height 125 /Interpolate true /ColorSpace 7 0 R /SMask 608 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0p  endstream endobj 329 0 obj 277 endobj 530 0 obj << /Length 531 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 610 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 531 0 obj 112 endobj 380 0 obj << /Length 381 0 R /Type /XObject /Subtype /Image /Width 117 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 612 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 381 0 obj 184 endobj 528 0 obj << /Length 529 0 R /Type /XObject /Subtype /Image /Width 154 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 614 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 529 0 obj 201 endobj 510 0 obj << /Length 511 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 616 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 511 0 obj 177 endobj 446 0 obj << /Length 447 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 618 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 447 0 obj 202 endobj 420 0 obj << /Length 421 0 R /Type /XObject /Subtype /Image /Width 114 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 620 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`70F endstream endobj 421 0 obj 180 endobj 490 0 obj << /Length 491 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 622 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 491 0 obj 151 endobj 426 0 obj << /Length 427 0 R /Type /XObject /Subtype /Image /Width 95 /Height 65 /Interpolate true /ColorSpace 7 0 R /SMask 624 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0`H] endstream endobj 427 0 obj 104 endobj 440 0 obj << /Length 441 0 R /Type /XObject /Subtype /Image /Width 116 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 626 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 441 0 obj 183 endobj 468 0 obj << /Length 469 0 R /Type /XObject /Subtype /Image /Width 114 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 628 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`70F endstream endobj 469 0 obj 180 endobj 442 0 obj << /Length 443 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 630 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 443 0 obj 202 endobj 354 0 obj << /Length 355 0 R /Type /XObject /Subtype /Image /Width 308 /Height 128 /Interpolate true /ColorSpace 7 0 R /SMask 632 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om /@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`70 endstream endobj 355 0 obj 539 endobj 434 0 obj << /Length 435 0 R /Type /XObject /Subtype /Image /Width 149 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 634 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 435 0 obj 195 endobj 404 0 obj << /Length 405 0 R /Type /XObject /Subtype /Image /Width 114 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 636 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`70F endstream endobj 405 0 obj 180 endobj 356 0 obj << /Length 357 0 R /Type /XObject /Subtype /Image /Width 290 /Height 143 /Interpolate true /ColorSpace 7 0 R /SMask 638 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Omo@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 357 0 obj 566 endobj 410 0 obj << /Length 411 0 R /Type /XObject /Subtype /Image /Width 162 /Height 137 /Interpolate true /ColorSpace 7 0 R /SMask 640 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Oo@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`% endstream endobj 411 0 obj 314 endobj 456 0 obj << /Length 457 0 R /Type /XObject /Subtype /Image /Width 114 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 642 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`70F endstream endobj 457 0 obj 180 endobj 462 0 obj << /Length 463 0 R /Type /XObject /Subtype /Image /Width 149 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 644 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 463 0 obj 195 endobj 382 0 obj << /Length 383 0 R /Type /XObject /Subtype /Image /Width 207 /Height 109 /Interpolate true /ColorSpace 7 0 R /SMask 646 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om ?@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`x endstream endobj 383 0 obj 319 endobj 464 0 obj << /Length 465 0 R /Type /XObject /Subtype /Image /Width 113 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 648 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0p:0  endstream endobj 465 0 obj 177 endobj 340 0 obj << /Length 341 0 R /Type /XObject /Subtype /Image /Width 163 /Height 167 /Interpolate true /ColorSpace 7 0 R /SMask 650 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`? endstream endobj 341 0 obj 380 endobj 482 0 obj << /Length 483 0 R /Type /XObject /Subtype /Image /Width 156 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 652 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 483 0 obj 203 endobj 524 0 obj << /Length 525 0 R /Type /XObject /Subtype /Image /Width 156 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 654 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 525 0 obj 203 endobj 540 0 obj << /Length 541 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 656 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 541 0 obj 151 endobj 542 0 obj << /Length 543 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 658 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 543 0 obj 151 endobj 374 0 obj << /Length 375 0 R /Type /XObject /Subtype /Image /Width 149 /Height 211 /Interpolate true /ColorSpace 7 0 R /SMask 660 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`wp| endstream endobj 375 0 obj 435 endobj 508 0 obj << /Length 509 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 662 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 509 0 obj 196 endobj 366 0 obj << /Length 367 0 R /Type /XObject /Subtype /Image /Width 308 /Height 315 /Interpolate true /ColorSpace 7 0 R /SMask 664 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` q0 endstream endobj 367 0 obj 1293 endobj 478 0 obj << /Length 479 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 666 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 479 0 obj 196 endobj 512 0 obj << /Length 513 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 668 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 513 0 obj 196 endobj 424 0 obj << /Length 425 0 R /Type /XObject /Subtype /Image /Width 114 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 670 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`70F endstream endobj 425 0 obj 180 endobj 344 0 obj << /Length 345 0 R /Type /XObject /Subtype /Image /Width 305 /Height 190 /Interpolate true /ColorSpace 7 0 R /SMask 672 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`>8 endstream endobj 345 0 obj 781 endobj 502 0 obj << /Length 503 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 674 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 503 0 obj 112 endobj 396 0 obj << /Length 397 0 R /Type /XObject /Subtype /Image /Width 114 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 676 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`70F endstream endobj 397 0 obj 180 endobj 350 0 obj << /Length 351 0 R /Type /XObject /Subtype /Image /Width 423 /Height 201 /Interpolate true /ColorSpace 7 0 R /SMask 678 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 351 0 obj 1136 endobj 474 0 obj << /Length 475 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 680 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 475 0 obj 196 endobj 364 0 obj << /Length 365 0 R /Type /XObject /Subtype /Image /Width 308 /Height 84 /Interpolate true /ColorSpace 7 0 R /SMask 682 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 090/? endstream endobj 365 0 obj 361 endobj 348 0 obj << /Length 349 0 R /Type /XObject /Subtype /Image /Width 341 /Height 128 /Interpolate true /ColorSpace 7 0 R /SMask 684 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 349 0 obj 595 endobj 494 0 obj << /Length 495 0 R /Type /XObject /Subtype /Image /Width 62 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 686 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0`w`K endstream endobj 495 0 obj 106 endobj 534 0 obj << /Length 535 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 688 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 535 0 obj 177 endobj 520 0 obj << /Length 521 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 690 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 521 0 obj 202 endobj 362 0 obj << /Length 363 0 R /Type /XObject /Subtype /Image /Width 206 /Height 84 /Interpolate true /ColorSpace 7 0 R /SMask 692 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Omo@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0  endstream endobj 363 0 obj 250 endobj 386 0 obj << /Length 387 0 R /Type /XObject /Subtype /Image /Width 276 /Height 109 /Interpolate true /ColorSpace 7 0 R /SMask 694 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`m`` endstream endobj 387 0 obj 416 endobj 372 0 obj << /Length 373 0 R /Type /XObject /Subtype /Image /Width 289 /Height 89 /Interpolate true /ColorSpace 7 0 R /SMask 696 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàS_Pa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`-z endstream endobj 373 0 obj 360 endobj 378 0 obj << /Length 379 0 R /Type /XObject /Subtype /Image /Width 155 /Height 219 /Interpolate true /ColorSpace 7 0 R /SMask 698 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`r` endstream endobj 379 0 obj 467 endobj 544 0 obj << /Length 545 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 700 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 545 0 obj 151 endobj 392 0 obj << /Length 393 0 R /Type /XObject /Subtype /Image /Width 116 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 702 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 393 0 obj 183 endobj 352 0 obj << /Length 353 0 R /Type /XObject /Subtype /Image /Width 565 /Height 262 /Interpolate true /ColorSpace 7 0 R /SMask 704 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 353 0 obj 1960 endobj 368 0 obj << /Length 369 0 R /Type /XObject /Subtype /Image /Width 262 /Height 85 /Interpolate true /ColorSpace 7 0 R /SMask 706 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`q`  endstream endobj 369 0 obj 313 endobj 472 0 obj << /Length 473 0 R /Type /XObject /Subtype /Image /Width 114 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 708 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`70F endstream endobj 473 0 obj 180 endobj 400 0 obj << /Length 401 0 R /Type /XObject /Subtype /Image /Width 113 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 710 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0p:0  endstream endobj 401 0 obj 177 endobj 330 0 obj << /Length 331 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 712 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 331 0 obj 112 endobj 408 0 obj << /Length 409 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 714 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 409 0 obj 177 endobj 402 0 obj << /Length 403 0 R /Type /XObject /Subtype /Image /Width 150 /Height 146 /Interpolate true /ColorSpace 7 0 R /SMask 716 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`> endstream endobj 403 0 obj 310 endobj 326 0 obj << /Length 327 0 R /Type /XObject /Subtype /Image /Width 273 /Height 102 /Interpolate true /ColorSpace 7 0 R /SMask 718 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` Fa endstream endobj 327 0 obj 387 endobj 470 0 obj << /Length 471 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 720 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 471 0 obj 202 endobj 394 0 obj << /Length 395 0 R /Type /XObject /Subtype /Image /Width 150 /Height 211 /Interpolate true /ColorSpace 7 0 R /SMask 722 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  O( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`r endstream endobj 395 0 obj 437 endobj 480 0 obj << /Length 481 0 R /Type /XObject /Subtype /Image /Width 114 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 724 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`70F endstream endobj 481 0 obj 180 endobj 476 0 obj << /Length 477 0 R /Type /XObject /Subtype /Image /Width 114 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 726 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`70F endstream endobj 477 0 obj 180 endobj 500 0 obj << /Length 501 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 728 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 501 0 obj 202 endobj 548 0 obj << /Length 549 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 730 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 549 0 obj 151 endobj 484 0 obj << /Length 485 0 R /Type /XObject /Subtype /Image /Width 117 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 732 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 485 0 obj 184 endobj 370 0 obj << /Length 371 0 R /Type /XObject /Subtype /Image /Width 305 /Height 111 /Interpolate true /ColorSpace 7 0 R /SMask 734 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0p30 endstream endobj 371 0 obj 466 endobj 448 0 obj << /Length 449 0 R /Type /XObject /Subtype /Image /Width 117 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 736 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 449 0 obj 184 endobj 504 0 obj << /Length 505 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 738 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 505 0 obj 202 endobj 422 0 obj << /Length 423 0 R /Type /XObject /Subtype /Image /Width 226 /Height 241 /Interpolate true /ColorSpace 7 0 R /SMask 740 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0~d endstream endobj 423 0 obj 736 endobj 466 0 obj << /Length 467 0 R /Type /XObject /Subtype /Image /Width 156 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 742 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 467 0 obj 203 endobj 546 0 obj << /Length 547 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 744 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 547 0 obj 151 endobj 450 0 obj << /Length 451 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 746 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 451 0 obj 196 endobj 336 0 obj << /Length 337 0 R /Type /XObject /Subtype /Image /Width 309 /Height 265 /Interpolate true /ColorSpace 7 0 R /SMask 748 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàS_Pa 0` 0` 0` 0` 0` 0` 0` 0` 0` 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806 0 obj << /Length 807 0 R /Type /XObject /Subtype /Image /Width 594 /Height 203 /Interpolate true /ColorSpace 7 0 R /SMask 844 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` ] endstream endobj 807 0 obj 1601 endobj 812 0 obj << /Length 813 0 R /Type /XObject /Subtype /Image /Width 206 /Height 84 /Interpolate true /ColorSpace 7 0 R /SMask 846 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Omo@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0  endstream endobj 813 0 obj 250 endobj 794 0 obj << /Length 795 0 R /Type /XObject /Subtype /Image /Width 156 /Height 211 /Interpolate true /ColorSpace 7 0 R /SMask 848 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 795 0 obj 454 endobj 826 0 obj << /Length 827 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 850 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 827 0 obj 177 endobj 818 0 obj << /Length 819 0 R /Type /XObject /Subtype /Image /Width 116 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 852 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`` endstream endobj 819 0 obj 182 endobj 828 0 obj << /Length 829 0 R /Type /XObject /Subtype /Image /Width 149 /Height 145 /Interpolate true /ColorSpace 7 0 R /SMask 854 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Omo@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`90/ endstream endobj 829 0 obj 306 endobj 784 0 obj << /Length 785 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 856 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 785 0 obj 112 endobj 814 0 obj << /Length 815 0 R /Type /XObject /Subtype /Image /Width 305 /Height 111 /Interpolate true /ColorSpace 7 0 R /SMask 858 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0p30 endstream endobj 815 0 obj 466 endobj 800 0 obj << /Length 801 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 860 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 801 0 obj 177 endobj 816 0 obj << /Length 817 0 R /Type /XObject /Subtype /Image /Width 154 /Height 149 /Interpolate true /ColorSpace 7 0 R /SMask 862 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 817 0 obj 323 endobj 820 0 obj << /Length 821 0 R /Type /XObject /Subtype /Image /Width 155 /Height 225 /Interpolate true /ColorSpace 7 0 R /SMask 864 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 821 0 obj 480 endobj 834 0 obj << /Length 835 0 R /Type /XObject /Subtype /Image /Width 277 /Height 102 /Interpolate true /ColorSpace 7 0 R /SMask 866 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` xK) endstream endobj 835 0 obj 393 endobj 796 0 obj << /Length 797 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 868 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 797 0 obj 112 endobj 836 0 obj << /Length 837 0 R /Type /XObject /Subtype /Image /Width 191 /Height 194 /Interpolate true /ColorSpace 7 0 R /SMask 870 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`I endstream endobj 837 0 obj 508 endobj 838 0 obj << /Length 839 0 R /Type /XObject /Subtype /Image /Width 65 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 872 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om ?@a 0` 0` 0` 0` 0` 0` 0` 0` 0` O endstream endobj 839 0 obj 113 endobj 832 0 obj << /Length 833 0 R /Type /XObject /Subtype /Image /Width 276 /Height 109 /Interpolate true /ColorSpace 7 0 R /SMask 874 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`m`` endstream endobj 833 0 obj 416 endobj 786 0 obj << /Length 787 0 R /Type /XObject /Subtype /Image /Width 276 /Height 109 /Interpolate true /ColorSpace 7 0 R /SMask 876 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`m`` endstream endobj 787 0 obj 416 endobj 824 0 obj << /Length 825 0 R /Type /XObject /Subtype /Image /Width 149 /Height 211 /Interpolate true /ColorSpace 7 0 R /SMask 878 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`wp| endstream endobj 825 0 obj 435 endobj 790 0 obj << /Length 791 0 R /Type /XObject /Subtype /Image /Width 150 /Height 234 /Interpolate true /ColorSpace 7 0 R /SMask 880 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` c endstream endobj 791 0 obj 483 endobj 804 0 obj << /Length 805 0 R /Type /XObject /Subtype /Image /Width 65 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 882 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om ?@a 0` 0` 0` 0` 0` 0` 0` 0` 0` O endstream endobj 805 0 obj 113 endobj 788 0 obj << /Length 789 0 R /Type /XObject /Subtype /Image /Width 264 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 884 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om_@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` A endstream endobj 789 0 obj 383 endobj 798 0 obj << /Length 799 0 R /Type /XObject /Subtype /Image /Width 150 /Height 211 /Interpolate true /ColorSpace 7 0 R /SMask 886 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  O( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`r endstream endobj 799 0 obj 437 endobj 830 0 obj << /Length 831 0 R /Type /XObject /Subtype /Image /Width 113 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 888 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0p:0  endstream endobj 831 0 obj 177 endobj 782 0 obj << /Length 783 0 R /Type /XObject /Subtype /Image /Width 156 /Height 140 /Interpolate true /ColorSpace 7 0 R /SMask 890 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 783 0 obj 308 endobj 778 0 obj << /Length 779 0 R /Type /XObject /Subtype /Image /Width 150 /Height 152 /Interpolate true /ColorSpace 7 0 R /SMask 892 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 ? endstream endobj 779 0 obj 322 endobj 780 0 obj << /Length 781 0 R /Type /XObject /Subtype /Image /Width 62 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 894 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0`LJ endstream endobj 781 0 obj 109 endobj 810 0 obj << /Length 811 0 R /Type /XObject /Subtype /Image /Width 242 /Height 115 /Interpolate true /ColorSpace 7 0 R /SMask 896 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`F1 endstream endobj 811 0 obj 388 endobj 802 0 obj << /Length 803 0 R /Type /XObject /Subtype /Image /Width 156 /Height 166 /Interpolate true /ColorSpace 7 0 R /SMask 898 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`o`/ endstream endobj 803 0 obj 362 endobj 822 0 obj << /Length 823 0 R /Type /XObject /Subtype /Image /Width 117 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 900 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`ρ endstream endobj 823 0 obj 183 endobj 866 0 obj << /Length 867 0 R /Type /XObject /Subtype /Image /Width 277 /Height 102 /ColorSpace /DeviceGray /Interpolate true /BitsPerComponent 8 /Filter /FlateDecode >> stream x흋[Zo@APATDD^"TDJ'Z{jxlD?3gfI'MC@䗯' M$xƯz,3D2Ǐczr|1!HI)@}:CN_q`DZɩ))I 11>V"d<W!x)NLM{f>5^̌q].Ȉ"ce3.a/,"/B@`~?7&0"!G!h| wai5H&D<E++p Q@78&A飱{BYXof FX.\.l7Sz"D((x|^`#1TDܮ~R|X7f۫kjR-vۅ<< (lFNv}r]=b}rrbt:v:<8':wlXtu9hT@ pg-@]g+kˋ3c:<x(aDB>=hgW'|E2i9+[}PsK;>m`|CPtŦQV&I47vNI,blोQP"AL~aG(6'mh DM L:01i?X< % |p4drd0Qҟ&_k(,rwlQg]M[d6fܮ)M<  sʓI@ӻd{ݝY$//_QPhO,|OƷFwsuy= G(V#&35Lɍe2!9]P%!#!fZΥ7H\ܮɉ#-Wu[(unW'PQ+Aay F(\_]4jb~k#!\fH/"bQ`,S?^\^u =foŲ 7@y|]#NLy\K^ȅFlwNx;v/.ps%wDd$ z΀n,d>P`N[K2^B),O>~ȸZ̗=888<S[.W*Z }Pj)B#xHp 6:TZ1`pi6VrmyQ 0/>c;gh,X^$v$Stz+$Le AbdxKGrAۙ`Y qzt{Y^^YY]Fb8Hfx4hX0SI$aRK?r}Caq|9홙:~| Px)JBwJ:lON!{+Zd瞚XlB< 02:6x"rng?BOb}# P(43 dM\Pza\ L2XFF<8!8)!Ì^x (g+6Q4 '%`^>C.7WR.A$ ZE#cvsSA,gn64o}1D*wW'jb#!cH`crYod ?Jjwޮ!;k?& 9m/Y[Tr}"r}_\ k#*9Bx5vw/!D*}~r)+7E>pry| '7BER:iVn Ҕ`H/w>HoW;BE!Y 1*oqD8@8+g)u/:͝tt{+M’w $sS`TtTNE6Oo?q/ ŭ Vp9J8!GqS.o6ð wX*.~Gu'^_[-\P3eʯ 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/Im217 960 0 R /Im201 928 0 R /Im204 934 0 R /Im203 932 0 R /Im206 938 0 R /Im221 968 0 R /Im198 922 0 R /Im220 966 0 R /Im212 950 0 R /Im194 914 0 R /Im210 946 0 R /Im193 912 0 R /Im202 930 0 R /Im200 926 0 R /Im216 958 0 R /Im205 936 0 R /Im211 948 0 R /Im215 956 0 R >> >> endobj 952 0 obj << /Length 953 0 R /Type /XObject /Subtype /Image /Width 117 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 970 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 953 0 obj 184 endobj 942 0 obj << /Length 943 0 R /Type /XObject /Subtype /Image /Width 305 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 972 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om ?@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`wZ endstream endobj 943 0 obj 443 endobj 910 0 obj << /Length 911 0 R /Type /XObject /Subtype /Image /Width 248 /Height 146 /Interpolate true /ColorSpace 7 0 R /SMask 974 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  O( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`_ endstream endobj 911 0 obj 497 endobj 924 0 obj << /Length 925 0 R /Type /XObject /Subtype /Image /Width 150 /Height 234 /Interpolate true /ColorSpace 7 0 R /SMask 976 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` c endstream endobj 925 0 obj 483 endobj 964 0 obj << /Length 965 0 R /Type /XObject /Subtype /Image /Width 116 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 978 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 965 0 obj 183 endobj 962 0 obj << /Length 963 0 R /Type /XObject /Subtype /Image /Width 149 /Height 145 /Interpolate true /ColorSpace 7 0 R /SMask 980 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Omo@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`90/ endstream endobj 963 0 obj 306 endobj 916 0 obj << /Length 917 0 R /Type /XObject /Subtype /Image /Width 156 /Height 140 /Interpolate true /ColorSpace 7 0 R /SMask 982 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 917 0 obj 308 endobj 918 0 obj << /Length 919 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 984 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 919 0 obj 112 endobj 944 0 obj << /Length 945 0 R /Type /XObject /Subtype /Image /Width 318 /Height 262 /Interpolate true /ColorSpace 7 0 R /SMask 986 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`?Љ endstream endobj 945 0 obj 1113 endobj 908 0 obj << /Length 909 0 R /Type /XObject /Subtype /Image /Width 65 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 988 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om ?@a 0` 0` 0` 0` 0` 0` 0` 0` 0` O endstream endobj 909 0 obj 113 endobj 940 0 obj << /Length 941 0 R /Type /XObject /Subtype /Image /Width 662 /Height 333 /Interpolate true /ColorSpace 7 0 R /SMask 990 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`` endstream endobj 941 0 obj 2907 endobj 906 0 obj << /Length 907 0 R /Type /XObject /Subtype /Image /Width 191 /Height 194 /Interpolate true /ColorSpace 7 0 R /SMask 992 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`I endstream endobj 907 0 obj 508 endobj 954 0 obj << /Length 955 0 R /Type /XObject /Subtype /Image /Width 155 /Height 225 /Interpolate true /ColorSpace 7 0 R /SMask 994 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 955 0 obj 480 endobj 920 0 obj << /Length 921 0 R /Type /XObject /Subtype /Image /Width 276 /Height 109 /Interpolate true /ColorSpace 7 0 R /SMask 996 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`m`` endstream endobj 921 0 obj 416 endobj 960 0 obj << /Length 961 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 998 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 961 0 obj 177 endobj 928 0 obj << /Length 929 0 R /Type /XObject /Subtype /Image /Width 156 /Height 211 /Interpolate true /ColorSpace 7 0 R /SMask 1000 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 929 0 obj 454 endobj 934 0 obj << /Length 935 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1002 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 935 0 obj 177 endobj 932 0 obj << /Length 933 0 R /Type /XObject /Subtype /Image /Width 150 /Height 211 /Interpolate true /ColorSpace 7 0 R /SMask 1004 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  O( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`r endstream endobj 933 0 obj 437 endobj 938 0 obj << /Length 939 0 R /Type /XObject /Subtype /Image /Width 65 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 1006 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om ?@a 0` 0` 0` 0` 0` 0` 0` 0` 0` O endstream endobj 939 0 obj 113 endobj 968 0 obj << /Length 969 0 R /Type /XObject /Subtype /Image /Width 276 /Height 109 /Interpolate true /ColorSpace 7 0 R /SMask 1008 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`m`` endstream endobj 969 0 obj 416 endobj 922 0 obj << /Length 923 0 R /Type /XObject /Subtype /Image /Width 307 /Height 98 /Interpolate true /ColorSpace 7 0 R /SMask 1010 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`s`` endstream endobj 923 0 obj 416 endobj 966 0 obj << /Length 967 0 R /Type /XObject /Subtype /Image /Width 276 /Height 109 /Interpolate true /ColorSpace 7 0 R /SMask 1012 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`m`` endstream endobj 967 0 obj 416 endobj 950 0 obj << /Length 951 0 R /Type /XObject /Subtype /Image /Width 155 /Height 148 /Interpolate true /ColorSpace 7 0 R /SMask 1014 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`{`  endstream endobj 951 0 obj 323 endobj 914 0 obj << /Length 915 0 R /Type /XObject /Subtype /Image /Width 62 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 1016 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0`LJ endstream endobj 915 0 obj 109 endobj 946 0 obj << /Length 947 0 R /Type /XObject /Subtype /Image /Width 206 /Height 84 /Interpolate true /ColorSpace 7 0 R /SMask 1018 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Omo@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0  endstream endobj 947 0 obj 250 endobj 912 0 obj << /Length 913 0 R /Type /XObject /Subtype /Image /Width 150 /Height 177 /Interpolate true /ColorSpace 7 0 R /SMask 1020 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`ׁ71 endstream endobj 913 0 obj 371 endobj 930 0 obj << /Length 931 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1022 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 931 0 obj 112 endobj 926 0 obj << /Length 927 0 R /Type /XObject /Subtype /Image /Width 62 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 1024 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0`LJ endstream endobj 927 0 obj 109 endobj 958 0 obj << /Length 959 0 R /Type /XObject /Subtype /Image /Width 149 /Height 211 /Interpolate true /ColorSpace 7 0 R /SMask 1026 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`wp| endstream endobj 959 0 obj 435 endobj 936 0 obj << /Length 937 0 R /Type /XObject /Subtype /Image /Width 156 /Height 166 /Interpolate true /ColorSpace 7 0 R /SMask 1028 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`o`/ endstream endobj 937 0 obj 362 endobj 948 0 obj << /Length 949 0 R /Type /XObject /Subtype /Image /Width 305 /Height 111 /Interpolate true /ColorSpace 7 0 R /SMask 1030 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0p30 endstream endobj 949 0 obj 466 endobj 956 0 obj << /Length 957 0 R /Type /XObject /Subtype /Image /Width 120 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1032 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0@ endstream endobj 957 0 obj 187 endobj 1014 0 obj << /Length 1015 0 R /Type /XObject /Subtype /Image 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"reUB+HU_gB>|^cO*Fy˗"b9k wMlVky%Cb05ť !bu):BMЖ5 |485κq*$})f񓦭 Ow](|k^2S`0*S[֘, ]3zd1Lu9|q[݀qCr1FBa`\ +W0ξW2lQ%]ɔa"m2< &m4)HU3pIs[[GPrϰ'6>dP-ω0(JȚG:J!K*: U[.ώvklma 2Ek6WT eMrcoߗ^%mKrBo"YK뭶^W1fo陲+7i1ӥ!ї?~O?}~Nj}YC'~nߑ$k1gWQ H0IsRv0GMVi"}h9mN+_OV4 M=IC;R1o^73:2ӰH$H$ 9lR|DzE"I'ͥmw}$DBG^0rx56ldB~#f$<$]:n3 ݚ%$%["n }K:1,(oa?{oĔ_NBذ@D]/"{䈹G9g_DFz0CS9]Ǐ/P9#v pAL0&!eo-s8iq00اu7 ~m!||S]:1b3=p7TIތ[Q endstream endobj 1037 0 obj 7997 endobj 1034 0 obj << /Type /Page /Parent 1035 0 R /Resources 1038 0 R /Contents 1036 0 R /MediaBox [0 0 720 540] >> endobj 1038 0 obj << /ProcSet [ /PDF /Text /ImageB /ImageC /ImageI ] /ColorSpace << /Cs2 8 0 R /Cs1 7 0 R >> /Font << /TT2 10 0 R >> /XObject << /Im246 1087 0 R /Im275 1145 0 R /Im256 1107 0 R /Im264 1123 0 R /Im245 1085 0 R /Im269 1133 0 R /Im233 1061 0 R /Im223 1041 0 R /Im274 1143 0 R /Im231 1057 0 R /Im242 1079 0 R /Im222 1039 0 R /Im251 1097 0 R /Im226 1047 0 R /Im224 1043 0 R /Im268 1131 0 R /Im240 1075 0 R /Im232 1059 0 R /Im258 1111 0 R /Im250 1095 0 R /Im263 1121 0 R /Im261 1117 0 R /Im266 1127 0 R /Im244 1083 0 R /Im247 1089 0 R /Im227 1049 0 R /Im249 1093 0 R /Im234 1063 0 R /Im252 1099 0 R /Im253 1101 0 R /Im248 1091 0 R /Im241 1077 0 R /Im265 1125 0 R /Im259 1113 0 R /Im225 1045 0 R /Im271 1137 0 R /Im273 1141 0 R /Im255 1105 0 R /Im235 1065 0 R /Im228 1051 0 R /Im262 1119 0 R /Im230 1055 0 R /Im267 1129 0 R /Im270 1135 0 R /Im236 1067 0 R /Im243 1081 0 R /Im254 1103 0 R /Im239 1073 0 R /Im229 1053 0 R /Im257 1109 0 R /Im238 1071 0 R /Im272 1139 0 R /Im260 1115 0 R /Im237 1069 0 R >> >> endobj 1087 0 obj << /Length 1088 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1147 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 1088 0 obj 196 endobj 1145 0 obj << /Length 1146 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 1149 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 1146 0 obj 151 endobj 1107 0 obj << /Length 1108 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1151 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 1108 0 obj 202 endobj 1123 0 obj << /Length 1124 0 R /Type /XObject /Subtype /Image /Width 149 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1153 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 1124 0 obj 195 endobj 1085 0 obj << /Length 1086 0 R /Type /XObject /Subtype /Image /Width 62 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1155 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0`w`K endstream endobj 1086 0 obj 106 endobj 1133 0 obj << /Length 1134 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 1157 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 1134 0 obj 151 endobj 1061 0 obj << /Length 1062 0 R /Type /XObject /Subtype /Image /Width 117 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 1159 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 1062 0 obj 184 endobj 1041 0 obj << /Length 1042 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1161 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 1042 0 obj 177 endobj 1143 0 obj << /Length 1144 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 1163 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 1144 0 obj 151 endobj 1057 0 obj << /Length 1058 0 R /Type /XObject /Subtype /Image /Width 114 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1165 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 1058 0 obj 178 endobj 1079 0 obj << /Length 1080 0 R /Type /XObject /Subtype /Image /Width 149 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1167 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 1080 0 obj 195 endobj 1039 0 obj << /Length 1040 0 R /Type /XObject /Subtype /Image /Width 149 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1169 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 1040 0 obj 195 endobj 1097 0 obj << /Length 1098 0 R /Type /XObject /Subtype /Image /Width 62 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1171 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0`w`K endstream endobj 1098 0 obj 106 endobj 1047 0 obj << /Length 1048 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1173 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 1048 0 obj 202 endobj 1043 0 obj << /Length 1044 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1175 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 1044 0 obj 196 endobj 1131 0 obj << /Length 1132 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 1177 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 1132 0 obj 151 endobj 1075 0 obj << /Length 1076 0 R /Type /XObject /Subtype /Image /Width 149 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1179 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 1076 0 obj 195 endobj 1059 0 obj << /Length 1060 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1181 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 1060 0 obj 202 endobj 1111 0 obj << /Length 1112 0 R /Type /XObject /Subtype /Image /Width 154 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1183 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 1112 0 obj 201 endobj 1095 0 obj << /Length 1096 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1185 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 1096 0 obj 196 endobj 1121 0 obj << /Length 1122 0 R /Type /XObject /Subtype /Image /Width 62 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1187 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0`w`K endstream endobj 1122 0 obj 106 endobj 1117 0 obj << /Length 1118 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1189 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 1118 0 obj 112 endobj 1127 0 obj << /Length 1128 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 1191 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 1128 0 obj 151 endobj 1083 0 obj << /Length 1084 0 R /Type /XObject /Subtype /Image /Width 149 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1193 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 1084 0 obj 195 endobj 1089 0 obj << /Length 1090 0 R /Type /XObject /Subtype /Image /Width 62 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1195 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0`w`K endstream endobj 1090 0 obj 106 endobj 1049 0 obj << /Length 1050 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1197 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 1050 0 obj 177 endobj 1093 0 obj << /Length 1094 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1199 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 1094 0 obj 112 endobj 1063 0 obj << /Length 1064 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1201 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 1064 0 obj 202 endobj 1099 0 obj << /Length 1100 0 R /Type /XObject /Subtype /Image /Width 156 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1203 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 1100 0 obj 203 endobj 1101 0 obj << /Length 1102 0 R /Type /XObject /Subtype /Image /Width 65 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1205 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`~O8 endstream endobj 1102 0 obj 112 endobj 1091 0 obj << /Length 1092 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1207 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 1092 0 obj 202 endobj 1077 0 obj << /Length 1078 0 R /Type /XObject /Subtype /Image /Width 116 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1209 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`` endstream endobj 1078 0 obj 182 endobj 1125 0 obj << /Length 1126 0 R /Type /XObject /Subtype /Image /Width 62 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1211 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream xЁàSPa 0` 0` 0` 0` 0` 0` 0` 0` 0`w`K endstream endobj 1126 0 obj 106 endobj 1113 0 obj << /Length 1114 0 R /Type /XObject /Subtype /Image /Width 89 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 1213 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`m endstream endobj 1114 0 obj 146 endobj 1045 0 obj << /Length 1046 0 R /Type /XObject /Subtype /Image /Width 114 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1215 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 1046 0 obj 178 endobj 1137 0 obj << /Length 1138 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 1217 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 1138 0 obj 151 endobj 1141 0 obj << /Length 1142 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 1219 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 1142 0 obj 151 endobj 1105 0 obj << /Length 1106 0 R /Type /XObject /Subtype /Image /Width 65 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 1221 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om ?@a 0` 0` 0` 0` 0` 0` 0` 0` 0` O endstream endobj 1106 0 obj 113 endobj 1065 0 obj << /Length 1066 0 R /Type /XObject /Subtype /Image /Width 120 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 1223 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`?0 endstream endobj 1066 0 obj 188 endobj 1051 0 obj << /Length 1052 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1225 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0 endstream endobj 1052 0 obj 196 endobj 1119 0 obj << /Length 1120 0 R /Type /XObject /Subtype /Image /Width 149 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1227 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 1120 0 obj 195 endobj 1055 0 obj << /Length 1056 0 R /Type /XObject /Subtype /Image /Width 156 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1229 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`   endstream endobj 1056 0 obj 203 endobj 1129 0 obj << /Length 1130 0 R /Type /XObject /Subtype /Image /Width 29 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 1231 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om ?@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`vD endstream endobj 1130 0 obj 156 endobj 1135 0 obj << /Length 1136 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 1233 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 1136 0 obj 151 endobj 1067 0 obj << /Length 1068 0 R /Type /XObject /Subtype /Image /Width 154 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1235 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 1068 0 obj 201 endobj 1081 0 obj << /Length 1082 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1237 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 1082 0 obj 177 endobj 1103 0 obj << /Length 1104 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1239 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 1104 0 obj 202 endobj 1073 0 obj << /Length 1074 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1241 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 1074 0 obj 177 endobj 1053 0 obj << /Length 1054 0 R /Type /XObject /Subtype /Image /Width 113 /Height 104 /Interpolate true /ColorSpace 7 0 R /SMask 1243 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om7@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`  endstream endobj 1054 0 obj 177 endobj 1109 0 obj << /Length 1110 0 R /Type /XObject /Subtype /Image /Width 61 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 1245 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0`60K endstream endobj 1110 0 obj 107 endobj 1071 0 obj << /Length 1072 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1247 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 1072 0 obj 202 endobj 1139 0 obj << /Length 1140 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /Interpolate true /ColorSpace 7 0 R /SMask 1249 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om O@a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0r0 endstream endobj 1140 0 obj 151 endobj 1115 0 obj << /Length 1116 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /Interpolate true /ColorSpace 7 0 R /SMask 1251 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x1 Om @a 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0`~ endstream endobj 1116 0 obj 202 endobj 1069 0 obj << /Length 1070 0 R /Type /XObject /Subtype /Image /Width 117 /Height 105 /Interpolate true /ColorSpace 7 0 R /SMask 1253 0 R /BitsPerComponent 8 /Filter /FlateDecode >> stream x  Om( 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` 0` endstream endobj 1070 0 obj 184 endobj 1151 0 obj << /Length 1152 0 R /Type /XObject /Subtype /Image /Width 155 /Height 88 /ColorSpace /DeviceGray /Interpolate true /BitsPerComponent 8 /Filter /FlateDecode >> stream xYS@Fq@ApDDE"t24RV)íWӼ84ߛ P Tc4D?PȠY8 z42jư%nɣvݬfSxb,]fn_ QTUBDU9,^yfCi2Ѵ,D4-Jd=l/CdXT. R) y-H>n:O |Zo4aҸWg 9`c۾n'' '#\jw ׹kԊZL:щj<%]j3Dcݨt{Xf+zg2 2]fj)x6_,!OGQEHlZo ^'fQTtryff%=R`<_m~Cm=[ }Jޥ0 I»&A2)5ZxR$H&FB RjɤHh]J  -Ka$R#w)5LdRj$.ILJޥ0 I»&A2)5ZxR$H&FB Rj]}k,L" eM? 86 tnJ`l;|~L&SLƣcv rDZGB|z58E:ݞ^}_2Ck%sjZE!?5 R&\&.8mX<%JH'Cs S),ˊ%( az~u>uXtflit[dlYPzXuzbeځgA|;} endstream endobj 1152 0 obj 838 endobj 1167 0 obj << /Length 1168 0 R /Type /XObject /Subtype /Image /Width 149 /Height 88 /ColorSpace /DeviceGray /Interpolate true /BitsPerComponent 8 /Filter /FlateDecode >> stream xr@E hh0&I&lrȴ=^f\}V@u [WF"?sm0XF";EQ96*dhXs9DQ2Q9AS WF&p(E]"р=j0Z8<`춏 hD*d1Iq=9UuN;Id rRL\,'#AVdUKJz`lsqVVeIJv?03vx-8.C+t6cf6;W_d6Xg5~qQLiPhR?]ft*gtU~6J.v?`f]φJFɟCJ{8_w|ԩf#Pw u )b )b )b )b )b )b )b )b )b )b )b ٩ok7ϵ-73mЂ6TћW/j1ymu7xx 'bl:UF%4x~} endstream endobj 1168 0 obj 839 endobj 1175 0 obj << /Length 1176 0 R /Type /XObject /Subtype /Image /Width 150 /Height 88 /ColorSpace /DeviceGray /Interpolate true /BitsPerComponent 8 /Filter /FlateDecode >> stream x[O@Z  R9YDD|B%;wf y:1ఙs"A$7| fer( roL͸Yu34 ŰE^ϹXP'+j h/yy4I1WVC:N,GAE?E9xOL3I ]SD!8K+.W,W5T+=, (DPmڝtz)"GRќFSZ1>xuKXHJX`,Sj3doײF/ %|j9v +sJ4#[Ζ3z>?o=q%a#3v9}k=?S{f 3j6x.I*y .WPr qU  .WPr qU  .WPr qU  .WPr qU  .WPr qU  .WPr qU /nv~~%-uzfzB NG:K,ѠSD- Г zu013ݧdGOtJ0KV{LܬSz;0䠞4ZF^)0(|.@M7f6N.g%U;dE% ;E5UU5x\J iahZ(}g>{}x>I@= uEj- mOa7 .AϹr C~v~c endstream endobj 1176 0 obj 881 endobj 1231 0 obj << /Length 1232 0 R /Type /XObject /Subtype /Image /Width 29 /Height 348 /ColorSpace /DeviceGray /Interpolate true /BitsPerComponent 8 /Filter /FlateDecode >> stream xn@ dB@)MRs' :,GU%M.>yYߗجGn$=0#2[ R( ( Uu0twCm@y`Hu 8<(o*D8Y[/¦25i0z5UẓjIl DsZ,n脃2emA3Krqدa ^ZTϯæ.R߼~VԛßZp=F1 x ? `Ds AL0L /̄!%lDV Vޯ`g16sxqPl$j6EtލA^:V7Z„>Dz̓Hdڮ/yD4;żuBGE K2?q?-[$NbBEJ SKf:~g^R +6 pOliJݹ44}@ (_wQ#c둔:oC=x endstream endobj 1232 0 obj 544 endobj 1149 0 obj << /Length 1150 0 R /Type /XObject /Subtype /Image /Width 28 /Height 348 /ColorSpace /DeviceGray /Interpolate true /BitsPerComponent 8 /Filter /FlateDecode >> stream xis0˒`V,K WBojn~L?|pF^9$GR+%zMTRoJEGct-J aBMڢ Ym1ӐbUQd~!H;TT%iDez(!(Gr鈔A:,grb&FEU-Af6q(عԘa>oiizy}ۭ,ङ#{ X+rEz////xxxxxa|:=@_}@_}Y Ww#////^ j{x}91ϻf9Nڬ&|~T`\.8|z,Qx0)y G3NCrer~"|$Cy7q/t9=6”E q=u8D)L:cb2Y@߫? 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    Frequently Asked Questions


    hisat2-2.2.1/docs_jhu/footer.inc.html000066400000000000000000000006471370663763200174640ustar00rootroot00000000000000 hisat2-2.2.1/docs_jhu/index.html000066400000000000000000000002001370663763200165060ustar00rootroot00000000000000 HISAT2 (under construction)

    hisat2-2.2.1/docs_jhu/index.shtml000066400000000000000000000267531370663763200167160ustar00rootroot00000000000000
    HISAT2 is a fast and sensitive alignment program for mapping next-generation sequencing reads (both DNA and RNA) to a population of human genomes (as well as to a single reference genome). Based on an extension of BWT for graphs [Sirén et al. 2014], we designed and implemented a graph FM index (GFM), an original approach and its first implementation to the best of our knowledge. In addition to using one global GFM index that represents a population of human genomes, HISAT2 uses a large set of small GFM indexes that collectively cover the whole genome (each index representing a genomic region of 56 Kbp, with 55,000 indexes needed to cover the human population). These small indexes (called local indexes), combined with several alignment strategies, enable rapid and accurate alignment of sequencing reads. This new indexing scheme is called a Hierarchical Graph FM index (HGFM).
    		Open Source Software

    HISAT2 2.1.0 release 6/8/2017

    HISAT2 2.0.5 release 11/4/2016

    Version 2.0.5 is a minor release with the following changes.
    • Due to a policy change (HTTP to HTTPS) in using SRA data (`--sra-option`), users are strongly encouraged to use this version. As of 11/9/2016, NCBI will begin a permanent redirect to HTTPS, which means the previous versions of HISAT2 no longer works with `--sra-acc` option soon.
    • Implemented -I and -X options for specifying minimum and maximum fragment lengths. The options are valid only when used with --no-spliced-alignment, which is used for the alignment of DNA-seq reads.
    • Fixed some cases where reads with SNPs on their 5' ends were not properly aligned.
    • Implemented --no-softclip option to disable soft-clipping.
    • Implemented --max-seeds to specify the maximum number of seeds that HISAT2 will try to extend to full-length alignments (see the manual for details).

    HISAT, StringTie and Ballgown protocol published at Nature Protocols 8/11/2016

    HISAT2 2.0.4 Windows binary available here, thanks to Andre Osorio Falcao 5/24/2016

    HISAT2 2.0.4 release 5/18/2016

    Version 2.0.4 is a minor release with the following changes.
    • Improved template length estimation (the 9th column of the SAM format) of RNA-seq reads by taking introns into account.
    • Introduced two options, --remove-chrname and --add-chrname, to remove "chr" from reference names or add "chr" to reference names in the alignment output, respectively (the 3rd column of the SAM format).
    • Changed the maximum of mapping quality (the 5th column of the SAM format) from 255 to 60. Note that 255 is an undefined value according to the SAM manual and some programs would not work with this value (255) properly.
    • Fixed NH (number of hits) in the alignment output.
    • HISAT2 allows indels of any length pertaining to minimum alignment score (previously, the maximum length of indels was 3 bp).
    • Fixed several cases that alignment goes beyond reference sequences.
    • Fixed reporting duplicate alignments.

    HISAT2 2.0.3-beta release 3/28/2016

    Version 2.0.3-beta is a minor release with the following changes.
    • Fixed graph index building when using both SNPs and transcripts. As a result, genome_snp_tran indexes here on the HISAT2 website have been rebuilt.
    • Included some missing files needed to follow the small test example (see the manual for details).

    HISAT2 2.0.2-beta release 3/17/2016

    Note (3/19/2016): this version is slightly updated to handle reporting splice sites with the correct chromosome names.
    Version 2.0.2-beta is a major release with the following changes.
    • Memory mappaped IO (--mm option) works now.
    • Building linear index can be now done using multi-threads.
    • Changed the minimum score for alignment in keeping with read lengths, so it's now --score-min L,0.0,-0.2, meaning a minimum score of -20 for 100-bp reads and -30 for 150-bp reads.
    • Fixed a bug that the same read was written into a file multiple times when --un-conc was used.
    • Fixed another bug that caused reads to map beyond reference sequences.
    • Introduced --haplotype option in the hisat2-build (index building), which is used with --snp option together to incorporate those SNP combinations present in the human population. This option also prevents graph construction from exploding due to exponential combinations of SNPs in small genomic regions.
    • Provided a new python script to extract SNPs and haplotypes from VCF files, hisat2_extract_snps_haplotypes_VCF.py
    • Changed several python script names as follows
      1. extract_splice_sites.py to hisat2_extract_splice_sites.py
      2. extract_exons.py to hisat2_extract_exons.py
      3. extract_snps.py to hisat2_extract_snps_haplotypes_UCSC.py

    HISAT2 2.0.1-beta release 11/19/2015

    Version 2.0.1-beta is a maintenance release with the following changes.
    • Fixed a bug that caused reads to map beyond reference sequences.
    • Fixed a deadlock issue that happened very rarely.
    • Fixed a bug that led to illegal memory access when reading SNP information.
    • Fixed a system-specific bug related to popcount instruction.

    HISAT2 2.0.0-beta release 9/8/2015 - first release

    We extended the BWT/FM index to incorporate genomic differences among individuals into the reference genome, while keeping memory requirements low enough to fit the entire index onto a desktop computer. Using this novel Hierarchical Graph FM index (HGFM) approach, we built a new alignment system, HISAT2, with an index that incorporates ~12.3M common SNPs from the dbSNP database. HISAT2 provides greater alignment accuracy for reads containing SNPs.
    • HISAT2's index size for the human reference genome and 12.3 million common SNPs is 6.2GB (the memory footprint of HISAT2 is 6.7GB). The SNPs consist of 11 million single nucleotide polymorphisms, 728,000 deletions, and 555,000 insertions. The insertions and deletions used in this index are small (usually <20bp).
    • HISAT2 comes with several index types:
      1. Hierarchical FM index (HFM) for a reference genome (index base: genome)
      2. Hierarchical Graph FM index (HGFM) for a reference genome plus SNPs (index base: genome_snp)
      3. Hierarchical Graph FM index (HGFM) for a reference genome plus transcripts (index base: genome_tran)
      4. Hierarchical Graph FM index (HGFM) for a reference genome plus SNPs and transcripts (index base: genome_snp_tran)
    • HISAT2 is a successor to both HISAT and TopHat2. We recommend that HISAT and TopHat2 users switch to HISAT2.
      1. HISAT2 can be considered an enhanced version of HISAT with many improvements and bug fixes. The alignment speed and memory requirements of HISAT2 are virtually the same as those of HISAT when using the HFM index (genome).
      2. When using graph-based indexes (HGFM), the runtime of HISAT2 is slightly slower than HISAT (30~80% additional CPU time).
      3. HISAT2 allows for mapping reads directly against transcripts, similar to that of TopHat2 (use genome_tran or genome_snp_tran).
    • When reads contain SNPs, the SNP information is provided as an optional field in the SAM output of HISAT2 (e.g., Zs:Z:1|S|rs3747203,97|S|rs16990981 - see the manual for details). This feature enables fast and sensitive genotyping in downstream analyses. Note that there is no alignment penalty for mismatches, insertions, and deletions if they correspond to known SNPs.
    • HISAT2 provides options for transcript assemblers (e.g., StringTie and Cufflinks) to work better with the alignment from HISAT2 (see options such as --dta and --dta-cufflinks).
    • Some slides about HISAT2 are found here and we are preparing detailed documention.
    • We plan to incorporate a larger set of SNPs and structural variations (SV) into this index (e.g., long insertions/deletions, inversions, and translocations).

    The HISAT2 source code is available in a public GitHub repository (5/30/2015).

    hisat2-2.2.1/docs_jhu/indexes.txt000066400000000000000000000021421370663763200167200ustar00rootroot00000000000000Each of the HISAT2 indexes available here on the website comes with a shell script (e.g. make_grch38.sh) that provides instructions (or shell commands) for downloading a reference sequence, gene annotations, and SNPs, and building a HISAT2 index. You can use the script to build the same HISAT2 index we provide. HISAT2 indexes named genome_tran or genome_snp_tran use Ensembl gene annotations, which include many more transcripts than RefSeq annotations, due to the inclusion of annotations as predicted by software. The HISAT2 indexes for the human genome include only primary assembly. If you choose to include alternative sequences introduced in GRCh38 and build your own HISAT2 indexes, then please be aware that those alternative sequences are nearly identical to the primary assembly in GRCh38 and as a result some reads may map to many more locations compared to when using the primary assembly only. HISAT2 often skips those multi-mapped reads if the number of potential locations is more than the value specified by the -k option. You may want to use a higher value for the -k option to resolve the issue. hisat2-2.2.1/docs_jhu/manual.inc.html000066400000000000000000002571531370663763200174510ustar00rootroot00000000000000

    Introduction

    What is HISAT2?

    HISAT2 is a fast and sensitive alignment program for mapping next-generation sequencing reads (whole-genome, transcriptome, and exome sequencing data) against the general human population (as well as against a single reference genome). Based on GCSA (an extension of BWT for a graph), we designed and implemented a graph FM index (GFM), an original approach and its first implementation to the best of our knowledge. In addition to using one global GFM index that represents general population, HISAT2 uses a large set of small GFM indexes that collectively cover the whole genome (each index representing a genomic region of 56 Kbp, with 55,000 indexes needed to cover human population). These small indexes (called local indexes) combined with several alignment strategies enable effective alignment of sequencing reads. This new indexing scheme is called Hierarchical Graph FM index (HGFM). We have developed HISAT 2 based on the HISAT and Bowtie2 implementations. HISAT2 outputs alignments in SAM format, enabling interoperation with a large number of other tools (e.g. SAMtools, GATK) that use SAM. HISAT2 is distributed under the GPLv3 license, and it runs on the command line under Linux, Mac OS X and Windows.

    Obtaining HISAT2

    Download HISAT2 sources and binaries from the Releases sections on the right side. Binaries are available for Intel architectures (x86_64) running Linux, and Mac OS X.

    Building from source

    Building HISAT2 from source requires a GNU-like environment with GCC, GNU Make and other basics. It should be possible to build HISAT2 on most vanilla Linux installations or on a Mac installation with Xcode installed. HISAT2 can also be built on Windows using Cygwin or MinGW (MinGW recommended). For a MinGW build the choice of what compiler is to be used is important since this will determine if a 32 or 64 bit code can be successfully compiled using it. If there is a need to generate both 32 and 64 bit on the same machine then a multilib MinGW has to be properly installed. MSYS, the zlib library, and depending on architecture pthreads library are also required. We are recommending a 64 bit build since it has some clear advantages in real life research problems. In order to simplify the MinGW setup it might be worth investigating popular MinGW personal builds since these are coming already prepared with most of the toolchains needed.

    First, download the source package from the Releases section on the right side. Unzip the file, change to the unzipped directory, and build the HISAT2 tools by running GNU make (usually with the command make, but sometimes with gmake) with no arguments. If building with MinGW, run make from the MSYS environment.

    HISAT2 is using the multithreading software model in order to speed up execution times on SMP architectures where this is possible. On POSIX platforms (like linux, Mac OS, etc) it needs the pthread library. Although it is possible to use pthread library on non-POSIX platform like Windows, due to performance reasons HISAT2 will try to use Windows native multithreading if possible.

    For the support of SRA data access in HISAT2, please download and install the NCBI-NGS toolkit. When running make, specify additional variables as follow. make USE_SRA=1 NCBI_NGS_DIR=/path/to/NCBI-NGS-directory NCBI_VDB_DIR=/path/to/NCBI-NGS-directory, where NCBI_NGS_DIR and NCBI_VDB_DIR will be used in Makefile for -I and -L compilation options. For example, $(NCBI_NGS_DIR)/include and $(NCBI_NGS_DIR)/lib64 will be used.

    Running HISAT2

    Adding to PATH

    By adding your new HISAT2 directory to your PATH environment variable, you ensure that whenever you run hisat2, hisat2-build or hisat2-inspect from the command line, you will get the version you just installed without having to specify the entire path. This is recommended for most users. To do this, follow your operating system's instructions for adding the directory to your PATH.

    If you would like to install HISAT2 by copying the HISAT2 executable files to an existing directory in your PATH, make sure that you copy all the executables, including hisat2, hisat2-align-s, hisat2-align-l, hisat2-build, hisat2-build-s, hisat2-build-l, hisat2-inspect, hisat2-inspect-s and hisat2-inspect-l.

    Reporting

    The reporting mode governs how many alignments HISAT2 looks for, and how to report them.

    In general, when we say that a read has an alignment, we mean that it has a valid alignment. When we say that a read has multiple alignments, we mean that it has multiple alignments that are valid and distinct from one another.

    By default, HISAT2 may soft-clip reads near their 5' and 3' ends. Users can control this behavior by setting different penalties for soft-clipping (--sp) or by disallowing soft-clipping ([--no-softclip]).

    Distinct alignments map a read to different places

    Two alignments for the same individual read are "distinct" if they map the same read to different places. Specifically, we say that two alignments are distinct if there are no alignment positions where a particular read offset is aligned opposite a particular reference offset in both alignments with the same orientation. E.g. if the first alignment is in the forward orientation and aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, and the second alignment is also in the forward orientation and also aligns the read character at read offset 10 to the reference character at chromosome 3, offset 3,445,245, they are not distinct alignments.

    Two alignments for the same pair are distinct if either the mate 1s in the two paired-end alignments are distinct or the mate 2s in the two alignments are distinct or both.

    Default mode: search for one or more alignments, report each

    HISAT2 searches for up to N distinct, primary alignments for each read, where N equals the integer specified with the -k parameter. Primary alignments mean alignments whose alignment score is equal or higher than any other alignments. It is possible that multiple distinct alignments have the same score. That is, if -k 2 is specified, HISAT2 will search for at most 2 distinct alignments. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS field. See the SAM specification for details.

    HISAT2 does not "find" alignments in any specific order, so for reads that have more than N distinct, valid alignments, HISAT2 does not guarantee that the N alignments reported are the best possible in terms of alignment score. Still, this mode can be effective and fast in situations where the user cares more about whether a read aligns (or aligns a certain number of times) than where exactly it originated.

    Alignment summary

    When HISAT2 finishes running, it prints messages summarizing what happened. These messages are printed to the "standard error" ("stderr") filehandle. For datasets consisting of unpaired reads, the summary might look like this:

    20000 reads; of these:
  20000 (100.00%) were unpaired; of these:
    1247 (6.24%) aligned 0 times
    18739 (93.69%) aligned exactly 1 time
    14 (0.07%) aligned >1 times
93.77% overall alignment rate

    For datasets consisting of pairs, the summary might look like this:

    10000 reads; of these:
  10000 (100.00%) were paired; of these:
    650 (6.50%) aligned concordantly 0 times
    8823 (88.23%) aligned concordantly exactly 1 time
    527 (5.27%) aligned concordantly >1 times
    ----
    650 pairs aligned concordantly 0 times; of these:
      34 (5.23%) aligned discordantly 1 time
    ----
    616 pairs aligned 0 times concordantly or discordantly; of these:
      1232 mates make up the pairs; of these:
        660 (53.57%) aligned 0 times
        571 (46.35%) aligned exactly 1 time
        1 (0.08%) aligned >1 times
96.70% overall alignment rate

    The indentation indicates how subtotals relate to totals.

    Wrapper

    The hisat2, hisat2-build and hisat2-inspect executables are actually wrapper scripts that call binary programs as appropriate. The wrappers shield users from having to distinguish between "small" and "large" index formats, discussed briefly in the following section. Also, the hisat2 wrapper provides some key functionality, like the ability to handle compressed inputs, and the functionality for --un, --al and related options.

    It is recommended that you always run the hisat2 wrappers and not run the binaries directly.

    Small and large indexes

    hisat2-build can index reference genomes of any size. For genomes less than about 4 billion nucleotides in length, hisat2-build builds a "small" index using 32-bit numbers in various parts of the index. When the genome is longer, hisat2-build builds a "large" index using 64-bit numbers. Small indexes are stored in files with the .ht2 extension, and large indexes are stored in files with the .ht2l extension. The user need not worry about whether a particular index is small or large; the wrapper scripts will automatically build and use the appropriate index.

    Performance tuning

    1. If your computer has multiple processors/cores, use -p

      The -p option causes HISAT2 to launch a specified number of parallel search threads. Each thread runs on a different processor/core and all threads find alignments in parallel, increasing alignment throughput by approximately a multiple of the number of threads (though in practice, speedup is somewhat worse than linear).

    Command Line

    Setting function options

    Some HISAT2 options specify a function rather than an individual number or setting. In these cases the user specifies three parameters: (a) a function type F, (b) a constant term B, and (c) a coefficient A. The available function types are constant (C), linear (L), square-root (S), and natural log (G). The parameters are specified as F,B,A - that is, the function type, the constant term, and the coefficient are separated by commas with no whitespace. The constant term and coefficient may be negative and/or floating-point numbers.

    For example, if the function specification is L,-0.4,-0.6, then the function defined is:

    f(x) = -0.4 + -0.6 * x

    If the function specification is G,1,5.4, then the function defined is:

    f(x) = 1.0 + 5.4 * ln(x)

    See the documentation for the option in question to learn what the parameter x is for. For example, in the case if the --score-min option, the function f(x) sets the minimum alignment score necessary for an alignment to be considered valid, and x is the read length.

    Usage

    hisat2 [options]* -x <hisat2-idx> {-1 <m1> -2 <m2> | -U <r> | --sra-acc <SRA accession number>} [-S <hit>]

    Main arguments

    -x <hisat2-idx>

    The basename of the index for the reference genome. The basename is the name of any of the index files up to but not including the final .1.ht2 / etc. hisat2 looks for the specified index first in the current directory, then in the directory specified in the HISAT2_INDEXES environment variable.

    -1 <m1>

    Comma-separated list of files containing mate 1s (filename usually includes _1), e.g. -1 flyA_1.fq,flyB_1.fq. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in <m2>. Reads may be a mix of different lengths. If - is specified, hisat2 will read the mate 1s from the "standard in" or "stdin" filehandle.

    -2 <m2>

    Comma-separated list of files containing mate 2s (filename usually includes _2), e.g. -2 flyA_2.fq,flyB_2.fq. Sequences specified with this option must correspond file-for-file and read-for-read with those specified in <m1>. Reads may be a mix of different lengths. If - is specified, hisat2 will read the mate 2s from the "standard in" or "stdin" filehandle.

    -U <r>

    Comma-separated list of files containing unpaired reads to be aligned, e.g. lane1.fq,lane2.fq,lane3.fq,lane4.fq. Reads may be a mix of different lengths. If - is specified, hisat2 gets the reads from the "standard in" or "stdin" filehandle.

    --sra-acc <SRA accession number>

    Comma-separated list of SRA accession numbers, e.g. --sra-acc SRR353653,SRR353654. Information about read types is available at http://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?sp=runinfo&acc=sra-acc&retmode=xml, where sra-acc is SRA accession number. If users run HISAT2 on a computer cluster, it is recommended to disable SRA-related caching (see the instruction at SRA-MANUAL).

    -S <hit>

    File to write SAM alignments to. By default, alignments are written to the "standard out" or "stdout" filehandle (i.e. the console).

    Options

    Input options

    -q

    Reads (specified with <m1>, <m2>, <s>) are FASTQ files. FASTQ files usually have extension .fq or .fastq. FASTQ is the default format. See also: --solexa-quals and --int-quals.

    --qseq

    Reads (specified with <m1>, <m2>, <s>) are QSEQ files. QSEQ files usually end in _qseq.txt. See also: --solexa-quals and --int-quals.

    -f

    Reads (specified with <m1>, <m2>, <s>) are FASTA files. FASTA files usually have extension .fa, .fasta, .mfa, .fna or similar. FASTA files do not have a way of specifying quality values, so when -f is set, the result is as if --ignore-quals is also set.

    -r

    Reads (specified with <m1>, <m2>, <s>) are files with one input sequence per line, without any other information (no read names, no qualities). When -r is set, the result is as if --ignore-quals is also set.

    -c

    The read sequences are given on command line. I.e. <m1>, <m2> and <singles> are comma-separated lists of reads rather than lists of read files. There is no way to specify read names or qualities, so -c also implies --ignore-quals.

    -s/--skip <int>

    Skip (i.e. do not align) the first <int> reads or pairs in the input.

    -u/--qupto <int>

    Align the first <int> reads or read pairs from the input (after the -s/--skip reads or pairs have been skipped), then stop. Default: no limit.

    -5/--trim5 <int>

    Trim <int> bases from 5' (left) end of each read before alignment (default: 0).

    -3/--trim3 <int>

    Trim <int> bases from 3' (right) end of each read before alignment (default: 0).

    --phred33

    Input qualities are ASCII chars equal to the Phred quality plus 33. This is also called the "Phred+33" encoding, which is used by the very latest Illumina pipelines.

    --phred64

    Input qualities are ASCII chars equal to the Phred quality plus 64. This is also called the "Phred+64" encoding.

    --solexa-quals

    Convert input qualities from Solexa (which can be negative) to Phred (which can't). This scheme was used in older Illumina GA Pipeline versions (prior to 1.3). Default: off.

    --int-quals

    Quality values are represented in the read input file as space-separated ASCII integers, e.g., 40 40 30 40..., rather than ASCII characters, e.g., II?I.... Integers are treated as being on the Phred quality scale unless --solexa-quals is also specified. Default: off.

    Alignment options

    --n-ceil <func>

    Sets a function governing the maximum number of ambiguous characters (usually Ns and/or .s) allowed in a read as a function of read length. For instance, specifying -L,0,0.15 sets the N-ceiling function f to f(x) = 0 + 0.15 * x, where x is the read length. See also: [setting function options]. Reads exceeding this ceiling are filtered out. Default: L,0,0.15.

    --ignore-quals

    When calculating a mismatch penalty, always consider the quality value at the mismatched position to be the highest possible, regardless of the actual value. I.e. input is treated as though all quality values are high. This is also the default behavior when the input doesn't specify quality values (e.g. in -f, -r, or -c modes).

    --nofw/--norc

    If --nofw is specified, hisat2 will not attempt to align unpaired reads to the forward (Watson) reference strand. If --norc is specified, hisat2 will not attempt to align unpaired reads against the reverse-complement (Crick) reference strand. In paired-end mode, --nofw and --norc pertain to the fragments; i.e. specifying --nofw causes hisat2 to explore only those paired-end configurations corresponding to fragments from the reverse-complement (Crick) strand. Default: both strands enabled.

    Scoring options

    --mp MX,MN

    Sets the maximum (MX) and minimum (MN) mismatch penalties, both integers. A number less than or equal to MX and greater than or equal to MN is subtracted from the alignment score for each position where a read character aligns to a reference character, the characters do not match, and neither is an N. If --ignore-quals is specified, the number subtracted quals MX. Otherwise, the number subtracted is MN + floor( (MX-MN)(MIN(Q, 40.0)/40.0) ) where Q is the Phred quality value. Default: MX = 6, MN = 2.

    --sp MX,MN

    Sets the maximum (MX) and minimum (MN) penalties for soft-clipping per base, both integers. A number less than or equal to MX and greater than or equal to MN is subtracted from the alignment score for each position. The number subtracted is MN + floor( (MX-MN)(MIN(Q, 40.0)/40.0) ) where Q is the Phred quality value. Default: MX = 2, MN = 1.

    --no-softclip

    Disallow soft-clipping.

    --np <int>

    Sets penalty for positions where the read, reference, or both, contain an ambiguous character such as N. Default: 1.

    --rdg <int1>,<int2>

    Sets the read gap open (<int1>) and extend (<int2>) penalties. A read gap of length N gets a penalty of <int1> + N * <int2>. Default: 5, 3.

    --rfg <int1>,<int2>

    Sets the reference gap open (<int1>) and extend (<int2>) penalties. A reference gap of length N gets a penalty of <int1> + N * <int2>. Default: 5, 3.

    --score-min <func>

    Sets a function governing the minimum alignment score needed for an alignment to be considered "valid" (i.e. good enough to report). This is a function of read length. For instance, specifying L,0,-0.6 sets the minimum-score function f to f(x) = 0 + -0.6 * x, where x is the read length. See also: [setting function options]. The default is L,0,-0.2.

    Spliced alignment options

    --pen-cansplice <int>

    Sets the penalty for each pair of canonical splice sites (e.g. GT/AG). Default: 0.

    --pen-noncansplice <int>

    Sets the penalty for each pair of non-canonical splice sites (e.g. non-GT/AG). Default: 12.

    --pen-canintronlen <func>

    Sets the penalty for long introns with canonical splice sites so that alignments with shorter introns are preferred to those with longer ones. Default: G,-8,1

    --pen-noncanintronlen <func>

    Sets the penalty for long introns with noncanonical splice sites so that alignments with shorter introns are preferred to those with longer ones. Default: G,-8,1

    --min-intronlen <int>

    Sets minimum intron length. Default: 20

    --max-intronlen <int>

    Sets maximum intron length. Default: 500000

    --known-splicesite-infile <path>

    With this mode, you can provide a list of known splice sites, which HISAT2 makes use of to align reads with small anchors.
    You can create such a list using python hisat2_extract_splice_sites.py genes.gtf > splicesites.txt, where hisat2_extract_splice_sites.py is included in the HISAT2 package, genes.gtf is a gene annotation file, and splicesites.txt is a list of splice sites with which you provide HISAT2 in this mode. Note that it is better to use indexes built using annotated transcripts (such as genome_tran or genome_snp_tran), which works better than using this option. It has no effect to provide splice sites that are already included in the indexes.

    --novel-splicesite-outfile <path>

    In this mode, HISAT2 reports a list of splice sites in the file :
    chromosome name <tab> genomic position of the flanking base on the left side of an intron <tab> genomic position of the flanking base on the right <tab> strand (+, -, and .) '.' indicates an unknown strand for non-canonical splice sites.

    --novel-splicesite-infile <path>

    With this mode, you can provide a list of novel splice sites that were generated from the above option "--novel-splicesite-outfile".

    --no-temp-splicesite

    HISAT2, by default, makes use of splice sites found by earlier reads to align later reads in the same run, in particular, reads with small anchors (<= 15 bp).
    The option disables this default alignment strategy.

    --no-spliced-alignment

    Disable spliced alignment.

    --rna-strandness <string>

    Specify strand-specific information: the default is unstranded.
    For single-end reads, use F or R. 'F' means a read corresponds to a transcript. 'R' means a read corresponds to the reverse complemented counterpart of a transcript. For paired-end reads, use either FR or RF.
    With this option being used, every read alignment will have an XS attribute tag: '+' means a read belongs to a transcript on '+' strand of genome. '-' means a read belongs to a transcript on '-' strand of genome.

    (TopHat has a similar option, --library-type option, where fr-firststrand corresponds to R and RF; fr-secondstrand corresponds to F and FR.) 
    --tmo/--transcriptome-mapping-only

    Report only those alignments within known transcripts.

    --dta/--downstream-transcriptome-assembly

    Report alignments tailored for transcript assemblers including StringTie. With this option, HISAT2 requires longer anchor lengths for de novo discovery of splice sites. This leads to fewer alignments with short-anchors, which helps transcript assemblers improve significantly in computation and memory usage.

    Report alignments tailored specifically for Cufflinks. In addition to what HISAT2 does with the above option (--dta), With this option, HISAT2 looks for novel splice sites with three signals (GT/AG, GC/AG, AT/AC), but all user-provided splice sites are used irrespective of their signals. HISAT2 produces an optional field, XS:A:[+-], for every spliced alignment.

    --no-templatelen-adjustment

    Disables template length adjustment for RNA-seq reads.

    Reporting options

    -k <int>

    It searches for at most <int> distinct, primary alignments for each read. Primary alignments mean alignments whose alignment score is equal or higher than any other alignments. The search terminates when it can't find more distinct valid alignments, or when it finds <int>, whichever happens first. The alignment score for a paired-end alignment equals the sum of the alignment scores of the individual mates. Each reported read or pair alignment beyond the first has the SAM 'secondary' bit (which equals 256) set in its FLAGS field. For reads that have more than <int> distinct, valid alignments, hisat2 does not guarantee that the <int> alignments reported are the best possible in terms of alignment score. Default: 5 (HFM) or 10 (HGFM)

    Note: HISAT2 is not designed with large values for -k in mind, and when aligning reads to long, repetitive genomes large -k can be very, very slow.

    --max-seeds <int>

    HISAT2, like other aligners, uses seed-and-extend approaches. HISAT2 tries to extend seeds to full-length alignments. In HISAT2, --max-seeds is used to control the maximum number of seeds that will be extended. HISAT2 extends up to these many seeds and skips the rest of the seeds. Large values for --max-seeds may improve alignment sensitivity, but HISAT2 is not designed with large values for --max-seeds in mind, and when aligning reads to long, repetitive genomes large --max-seeds can be very, very slow. The default value is the maximum of 5 and the value that comes with-k.

    --secondary

    Report secondary alignments.

    Paired-end options

    -I/--minins <int>

    The minimum fragment length for valid paired-end alignments.This option is valid only with --no-spliced-alignment. E.g. if -I 60 is specified and a paired-end alignment consists of two 20-bp alignments in the appropriate orientation with a 20-bp gap between them, that alignment is considered valid (as long as -X is also satisfied). A 19-bp gap would not be valid in that case. If trimming options -3 or -5 are also used, the -I constraint is applied with respect to the untrimmed mates.

    The larger the difference between -I and -X, the slower HISAT2 will run. This is because larger differences between -I and -X require that HISAT2 scan a larger window to determine if a concordant alignment exists. For typical fragment length ranges (200 to 400 nucleotides), HISAT2 is very efficient.

    Default: 0 (essentially imposing no minimum)

    -X/--maxins <int>

    The maximum fragment length for valid paired-end alignments. This option is valid only with --no-spliced-alignment. E.g. if -X 100 is specified and a paired-end alignment consists of two 20-bp alignments in the proper orientation with a 60-bp gap between them, that alignment is considered valid (as long as -I is also satisfied). A 61-bp gap would not be valid in that case. If trimming options -3 or -5 are also used, the -X constraint is applied with respect to the untrimmed mates, not the trimmed mates.

    The larger the difference between -I and -X, the slower HISAT2 will run. This is because larger differences between -I and -X require that HISAT2 scan a larger window to determine if a concordant alignment exists. For typical fragment length ranges (200 to 400 nucleotides), HISAT2 is very efficient.

    Default: 500.

    --fr/--rf/--ff

    The upstream/downstream mate orientations for a valid paired-end alignment against the forward reference strand. E.g., if --fr is specified and there is a candidate paired-end alignment where mate 1 appears upstream of the reverse complement of mate 2 and the fragment length constraints (-I and -X) are met, that alignment is valid. Also, if mate 2 appears upstream of the reverse complement of mate 1 and all other constraints are met, that too is valid. --rf likewise requires that an upstream mate1 be reverse-complemented and a downstream mate2 be forward-oriented. --ff requires both an upstream mate 1 and a downstream mate 2 to be forward-oriented. Default: --fr (appropriate for Illumina's Paired-end Sequencing Assay).

    --no-mixed

    By default, when hisat2 cannot find a concordant or discordant alignment for a pair, it then tries to find alignments for the individual mates. This option disables that behavior.

    --no-discordant

    By default, hisat2 looks for discordant alignments if it cannot find any concordant alignments. A discordant alignment is an alignment where both mates align uniquely, but that does not satisfy the paired-end constraints (--fr/--rf/--ff, -I, -X). This option disables that behavior.

    Output options

    -t/--time

    Print the wall-clock time required to load the index files and align the reads. This is printed to the "standard error" ("stderr") filehandle. Default: off.

    --un <path>
--un-gz <path>
--un-bz2 <path>

    Write unpaired reads that fail to align to file at <path>. These reads correspond to the SAM records with the FLAGS 0x4 bit set and neither the 0x40 nor 0x80 bits set. If --un-gz is specified, output will be gzip compressed. If --un-bz2 is specified, output will be bzip2 compressed. Reads written in this way will appear exactly as they did in the input file, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the input.

    --al <path>
--al-gz <path>
--al-bz2 <path>

    Write unpaired reads that align at least once to file at <path>. These reads correspond to the SAM records with the FLAGS 0x4, 0x40, and 0x80 bits unset. If --al-gz is specified, output will be gzip compressed. If --al-bz2 is specified, output will be bzip2 compressed. Reads written in this way will appear exactly as they did in the input file, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the input.

    --un-conc <path>
--un-conc-gz <path>
--un-conc-bz2 <path>

    Write paired-end reads that fail to align concordantly to file(s) at <path>. These reads correspond to the SAM records with the FLAGS 0x4 bit set and either the 0x40 or 0x80 bit set (depending on whether it's mate #1 or #2). .1 and .2 strings are added to the filename to distinguish which file contains mate #1 and mate #2. If a percent symbol, %, is used in <path>, the percent symbol is replaced with 1 or 2 to make the per-mate filenames. Otherwise, .1 or .2 are added before the final dot in <path> to make the per-mate filenames. Reads written in this way will appear exactly as they did in the input files, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the inputs.

    --al-conc <path>
--al-conc-gz <path>
--al-conc-bz2 <path>

    Write paired-end reads that align concordantly at least once to file(s) at <path>. These reads correspond to the SAM records with the FLAGS 0x4 bit unset and either the 0x40 or 0x80 bit set (depending on whether it's mate #1 or #2). .1 and .2 strings are added to the filename to distinguish which file contains mate #1 and mate #2. If a percent symbol, %, is used in <path>, the percent symbol is replaced with 1 or 2 to make the per-mate filenames. Otherwise, .1 or .2 are added before the final dot in <path> to make the per-mate filenames. Reads written in this way will appear exactly as they did in the input files, without any modification (same sequence, same name, same quality string, same quality encoding). Reads will not necessarily appear in the same order as they did in the inputs.

    --quiet

    Print nothing besides alignments and serious errors.

    --summary-file

    Print alignment summary to this file.

    --new-summary

    Print alignment summary in a new style, which is more machine-friendly.

    --met-file <path>

    Write hisat2 metrics to file <path>. Having alignment metric can be useful for debugging certain problems, especially performance issues. See also: --met. Default: metrics disabled.

    --met-stderr

    Write hisat2 metrics to the "standard error" ("stderr") filehandle. This is not mutually exclusive with --met-file. Having alignment metric can be useful for debugging certain problems, especially performance issues. See also: --met. Default: metrics disabled.

    --met <int>

    Write a new hisat2 metrics record every <int> seconds. Only matters if either --met-stderr or --met-file are specified. Default: 1.

    SAM options

    --no-unal

    Suppress SAM records for reads that failed to align.

    --no-hd

    Suppress SAM header lines (starting with @).

    --no-sq

    Suppress @SQ SAM header lines.

    --rg-id <text>

    Set the read group ID to <text>. This causes the SAM @RG header line to be printed, with <text> as the value associated with the ID: tag. It also causes the RG:Z: extra field to be attached to each SAM output record, with value set to <text>.

    --rg <text>

    Add <text> (usually of the form TAG:VAL, e.g. SM:Pool1) as a field on the @RG header line. Note: in order for the @RG line to appear, --rg-id must also be specified. This is because the ID tag is required by the SAM Spec. Specify --rg multiple times to set multiple fields. See the SAM Spec for details about what fields are legal.

    --remove-chrname

    Remove 'chr' from reference names in alignment (e.g., chr18 to 18)

    --add-chrname

    Add 'chr' to reference names in alignment (e.g., 18 to chr18)

    --omit-sec-seq

    When printing secondary alignments, HISAT2 by default will write out the SEQ and QUAL strings. Specifying this option causes HISAT2 to print an asterisk in those fields instead.

    Performance options

    -o/--offrate <int>

    Override the offrate of the index with <int>. If <int> is greater than the offrate used to build the index, then some row markings are discarded when the index is read into memory. This reduces the memory footprint of the aligner but requires more time to calculate text offsets. <int> must be greater than the value used to build the index.

    -p/--threads NTHREADS

    Launch NTHREADS parallel search threads (default: 1). Threads will run on separate processors/cores and synchronize when parsing reads and outputting alignments. Searching for alignments is highly parallel, and speedup is close to linear. Increasing -p increases HISAT2's memory footprint. E.g. when aligning to a human genome index, increasing -p from 1 to 8 increases the memory footprint by a few hundred megabytes. This option is only available if bowtie is linked with the pthreads library (i.e. if BOWTIE_PTHREADS=0 is not specified at build time).

    --reorder

    Guarantees that output SAM records are printed in an order corresponding to the order of the reads in the original input file, even when -p is set greater than 1. Specifying --reorder and setting -p greater than 1 causes HISAT2 to run somewhat slower and use somewhat more memory then if --reorder were not specified. Has no effect if -p is set to 1, since output order will naturally correspond to input order in that case.

    --mm

    Use memory-mapped I/O to load the index, rather than typical file I/O. Memory-mapping allows many concurrent bowtie processes on the same computer to share the same memory image of the index (i.e. you pay the memory overhead just once). This facilitates memory-efficient parallelization of bowtie in situations where using -p is not possible or not preferable.

    Other options

    --qc-filter

    Filter out reads for which the QSEQ filter field is non-zero. Only has an effect when read format is --qseq. Default: off.

    --seed <int>

    Use <int> as the seed for pseudo-random number generator. Default: 0.

    --non-deterministic

    Normally, HISAT2 re-initializes its pseudo-random generator for each read. It seeds the generator with a number derived from (a) the read name, (b) the nucleotide sequence, (c) the quality sequence, (d) the value of the --seed option. This means that if two reads are identical (same name, same nucleotides, same qualities) HISAT2 will find and report the same alignment(s) for both, even if there was ambiguity. When --non-deterministic is specified, HISAT2 re-initializes its pseudo-random generator for each read using the current time. This means that HISAT2 will not necessarily report the same alignment for two identical reads. This is counter-intuitive for some users, but might be more appropriate in situations where the input consists of many identical reads.

    --version

    Print version information and quit.

    -h/--help

    Print usage information and quit.

    SAM output

    Following is a brief description of the SAM format as output by hisat2. For more details, see the SAM format specification.

    By default, hisat2 prints a SAM header with @HD, @SQ and @PG lines. When one or more --rg arguments are specified, hisat2 will also print an @RG line that includes all user-specified --rg tokens separated by tabs.

    Each subsequent line describes an alignment or, if the read failed to align, a read. Each line is a collection of at least 12 fields separated by tabs; from left to right, the fields are:

    1. Name of read that aligned.

      Note that the SAM specification disallows whitespace in the read name. If the read name contains any whitespace characters, HISAT2 will truncate the name at the first whitespace character. This is similar to the behavior of other tools.

    2. Sum of all applicable flags. Flags relevant to HISAT2 are:

      1

      The read is one of a pair

      2

      The alignment is one end of a proper paired-end alignment

      4

      The read has no reported alignments

      8

      The read is one of a pair and has no reported alignments

      16

      The alignment is to the reverse reference strand

      32

      The other mate in the paired-end alignment is aligned to the reverse reference strand

      64

      The read is mate 1 in a pair

      128

      The read is mate 2 in a pair

      Thus, an unpaired read that aligns to the reverse reference strand will have flag 16. A paired-end read that aligns and is the first mate in the pair will have flag 83 (= 64 + 16 + 2 + 1).

    3. Name of reference sequence where alignment occurs

    4. 1-based offset into the forward reference strand where leftmost character of the alignment occurs

    5. Mapping quality

    6. CIGAR string representation of alignment

    7. Name of reference sequence where mate's alignment occurs. Set to = if the mate's reference sequence is the same as this alignment's, or * if there is no mate.

    8. 1-based offset into the forward reference strand where leftmost character of the mate's alignment occurs. Offset is 0 if there is no mate.

    9. Inferred fragment length. Size is negative if the mate's alignment occurs upstream of this alignment. Size is 0 if the mates did not align concordantly. However, size is non-0 if the mates aligned discordantly to the same chromosome.

    10. Read sequence (reverse-complemented if aligned to the reverse strand)

    11. ASCII-encoded read qualities (reverse-complemented if the read aligned to the reverse strand). The encoded quality values are on the Phred quality scale and the encoding is ASCII-offset by 33 (ASCII char !), similarly to a FASTQ file.

    12. Optional fields. Fields are tab-separated. hisat2 outputs zero or more of these optional fields for each alignment, depending on the type of the alignment:

      AS:i:<N>

      Alignment score. Can be negative. Only present if SAM record is for an aligned read.

      ZS:i:<N>

      Alignment score for the best-scoring alignment found other than the alignment reported. Can be negative. Only present if the SAM record is for an aligned read and more than one alignment was found for the read. Note that, when the read is part of a concordantly-aligned pair, this score could be greater than [AS:i].

      YS:i:<N>

      Alignment score for opposite mate in the paired-end alignment. Only present if the SAM record is for a read that aligned as part of a paired-end alignment.

      XN:i:<N>

      The number of ambiguous bases in the reference covering this alignment. Only present if SAM record is for an aligned read.

      XM:i:<N>

      The number of mismatches in the alignment. Only present if SAM record is for an aligned read.

      XO:i:<N>

      The number of gap opens, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.

      XG:i:<N>

      The number of gap extensions, for both read and reference gaps, in the alignment. Only present if SAM record is for an aligned read.

      NM:i:<N>

      The edit distance; that is, the minimal number of one-nucleotide edits (substitutions, insertions and deletions) needed to transform the read string into the reference string. Only present if SAM record is for an aligned read.

      YF:Z:<S>

      String indicating reason why the read was filtered out. See also: [Filtering]. Only appears for reads that were filtered out.

      YT:Z:<S>

      Value of UU indicates the read was not part of a pair. Value of CP indicates the read was part of a pair and the pair aligned concordantly. Value of DP indicates the read was part of a pair and the pair aligned discordantly. Value of UP indicates the read was part of a pair but the pair failed to aligned either concordantly or discordantly.

      MD:Z:<S>

      A string representation of the mismatched reference bases in the alignment. See SAM format specification for details. Only present if SAM record is for an aligned read.

      XS:A:<A>

      Values of + and - indicate the read is mapped to transcripts on sense and anti-sense strands, respectively. Spliced alignments need to have this field, which is required in Cufflinks and StringTie.
      We can report this field for the canonical-splice site (GT/AG), but not for non-canonical splice sites. You can direct HISAT2 not to output such alignments (involving non-canonical splice sites) using "--pen-noncansplice 1000000".

      NH:i:<N>

      The number of mapped locations for the read or the pair.

      Zs:Z:<S>
      		 When the alignment of a read involves SNPs that are in the index, this option is used to indicate where exactly the read involves the SNPs. This optional field is similar to the above MD:Z field. For example, Zs:Z:1|S|rs3747203,97|S|rs16990981 indicates the second base of the read corresponds to a known SNP (ID: rs3747203). 97 bases after the third base (the base after the second one), the read at 100th base involves another known SNP (ID: rs16990981). 'S' indicates a single nucleotide polymorphism. 'D' and 'I' indicate a deletion and an insertion, respectively.

    The hisat2-build indexer

    hisat2-build builds a HISAT2 index from a set of DNA sequences. hisat2-build outputs a set of 6 files with suffixes .1.ht2, .2.ht2, .3.ht2, .4.ht2, .5.ht2, .6.ht2, .7.ht2, and .8.ht2. In the case of a large index these suffixes will have a ht2l termination. These files together constitute the index: they are all that is needed to align reads to that reference. The original sequence FASTA files are no longer used by HISAT2 once the index is built.

    Use of Karkkainen's blockwise algorithm allows hisat2-build to trade off between running time and memory usage. hisat2-build has three options governing how it makes this trade: [-p/--packed], --bmax/--bmaxdivn, and --dcv. By default, hisat2-build will automatically search for the settings that yield the best running time without exhausting memory. This behavior can be disabled using the -a/--noauto option.

    The indexer provides options pertaining to the "shape" of the index, e.g. --offrate governs the fraction of Burrows-Wheeler rows that are "marked" (i.e., the density of the suffix-array sample; see the original FM Index paper for details). All of these options are potentially profitable trade-offs depending on the application. They have been set to defaults that are reasonable for most cases according to our experiments. See Performance tuning for details.

    hisat2-build can generate either small or large indexes. The wrapper will decide which based on the length of the input genome. If the reference does not exceed 4 billion characters but a large index is preferred, the user can specify --large-index to force hisat2-build to build a large index instead.

    The HISAT2 index is based on the FM Index of Ferragina and Manzini, which in turn is based on the Burrows-Wheeler transform. The algorithm used to build the index is based on the blockwise algorithm of Karkkainen.

    Command Line

    Usage:

    hisat2-build [options]* <reference_in> <ht2_base>

    Notes

    If you use --snp, --ss, and/or --exon, hisat2-build will need about 200GB RAM for the human genome size as index building involves a graph construction. 
Otherwise, you will be able to build an index on your desktop with 8GB RAM.

    Main arguments

    <reference_in>

    A comma-separated list of FASTA files containing the reference sequences to be aligned to, or, if -c is specified, the sequences themselves. E.g., <reference_in> might be chr1.fa,chr2.fa,chrX.fa,chrY.fa, or, if -c is specified, this might be GGTCATCCT,ACGGGTCGT,CCGTTCTATGCGGCTTA.

    <ht2_base>

    The basename of the index files to write. By default, hisat2-build writes files named NAME.1.ht2, NAME.2.ht2, NAME.3.ht2, NAME.4.ht2, NAME.5.ht2, NAME.6.ht2, NAME.7.ht2, and NAME.8.ht2 where NAME is <ht2_base>.

    Options

    -f

    The reference input files (specified as <reference_in>) are FASTA files (usually having extension .fa, .mfa, .fna or similar).

    -c

    The reference sequences are given on the command line. I.e. <reference_in> is a comma-separated list of sequences rather than a list of FASTA files.

    --large-index

    Force hisat2-build to build a large index, even if the reference is less than ~ 4 billion nucleotides long.

    -a/--noauto

    Disable the default behavior whereby hisat2-build automatically selects values for the --bmax, --dcv and [--packed] parameters according to available memory. Instead, user may specify values for those parameters. If memory is exhausted during indexing, an error message will be printed; it is up to the user to try new parameters.

    --bmax <int>

    The maximum number of suffixes allowed in a block. Allowing more suffixes per block makes indexing faster, but increases peak memory usage. Setting this option overrides any previous setting for --bmax, or --bmaxdivn. Default (in terms of the --bmaxdivn parameter) is --bmaxdivn 4. This is configured automatically by default; use -a/--noauto to configure manually.

    --bmaxdivn <int>

    The maximum number of suffixes allowed in a block, expressed as a fraction of the length of the reference. Setting this option overrides any previous setting for --bmax, or --bmaxdivn. Default: --bmaxdivn 4. This is configured automatically by default; use -a/--noauto to configure manually.

    --dcv <int>

    Use <int> as the period for the difference-cover sample. A larger period yields less memory overhead, but may make suffix sorting slower, especially if repeats are present. Must be a power of 2 no greater than 4096. Default: 1024. This is configured automatically by default; use -a/--noauto to configure manually.

    --nodc

    Disable use of the difference-cover sample. Suffix sorting becomes quadratic-time in the worst case (where the worst case is an extremely repetitive reference). Default: off.

    -r/--noref

    Do not build the NAME.3.ht2 and NAME.4.ht2 portions of the index, which contain a bitpacked version of the reference sequences and are used for paired-end alignment.

    -3/--justref

    Build only the NAME.3.ht2 and NAME.4.ht2 portions of the index, which contain a bitpacked version of the reference sequences and are used for paired-end alignment.

    -o/--offrate <int>

    To map alignments back to positions on the reference sequences, it's necessary to annotate ("mark") some or all of the Burrows-Wheeler rows with their corresponding location on the genome. -o/--offrate governs how many rows get marked: the indexer will mark every 2^<int> rows. Marking more rows makes reference-position lookups faster, but requires more memory to hold the annotations at runtime. The default is 4 (every 16th row is marked; for human genome, annotations occupy about 680 megabytes).

    -t/--ftabchars <int>

    The ftab is the lookup table used to calculate an initial Burrows-Wheeler range with respect to the first <int> characters of the query. A larger <int> yields a larger lookup table but faster query times. The ftab has size 4^(<int>+1) bytes. The default setting is 10 (ftab is 4MB).

    --localoffrate <int>

    This option governs how many rows get marked in a local index: the indexer will mark every 2^<int> rows. Marking more rows makes reference-position lookups faster, but requires more memory to hold the annotations at runtime. The default is 3 (every 8th row is marked, this occupies about 16KB per local index).

    --localftabchars <int>

    The local ftab is the lookup table in a local index. The default setting is 6 (ftab is 8KB per local index).

    -p <int>

    Launch NTHREADS parallel build threads (default: 1).

    --snp <path>

    Provide a list of SNPs (in the HISAT2's own format) as follows (five columns).

    SNP ID <tab> snp type (single, deletion, or insertion) <tab> chromosome name <tab> zero-offset based genomic position of a SNP <tab> alternative base (single), the length of SNP (deletion), or insertion sequence (insertion)

    For example, rs58784443 single 13 18447947 T

    Use hisat2_extract_snps_haplotypes_UCSC.py (in the HISAT2 package) to extract SNPs and haplotypes from a dbSNP file (e.g. http://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/snp144Common.txt.gz). or hisat2_extract_snps_haplotypes_VCF.py to extract SNPs and haplotypes from a VCF file (e.g. ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/supporting/GRCh38_positions/ALL.chr22.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP_no_SVs.vcf.gz).

    --haplotype <path>

    Provide a list of haplotypes (in the HISAT2's own format) as follows (five columns).

    Haplotype ID <tab> chromosome name <tab> zero-offset based left coordinate of haplotype <tab> zero-offset based right coordinate of haplotype <tab> a comma separated list of SNP ids in the haplotype

    For example, ht35 13 18446877 18446945 rs12381094,rs12381056,rs192016659,rs538569910

    See the above option, --snp, about how to extract haplotypes. This option is not required, but haplotype information can keep the index construction from exploding and reduce the index size substantially.

    --ss <path>

    Note this option should be used with the following --exon option. Provide a list of splice sites (in the HISAT2's own format) as follows (four columns).

    chromosome name <tab> zero-offset based genomic position of the flanking base on the left side of an intron <tab> zero-offset based genomic position of the flanking base on the right <tab> strand

    Use hisat2_extract_splice_sites.py (in the HISAT2 package) to extract splice sites from a GTF file.

    --exon <path>

    Note this option should be used with the above --ss option. Provide a list of exons (in the HISAT2's own format) as follows (three columns).

    chromosome name <tab> zero-offset based left genomic position of an exon <tab> zero-offset based right genomic position of an exon

    Use hisat2_extract_exons.py (in the HISAT2 package) to extract exons from a GTF file.

    --seed <int>

    Use <int> as the seed for pseudo-random number generator.

    --cutoff <int>

    Index only the first <int> bases of the reference sequences (cumulative across sequences) and ignore the rest.

    -q/--quiet

    hisat2-build is verbose by default. With this option hisat2-build will print only error messages.

    -h/--help

    Print usage information and quit.

    --version

    Print version information and quit.

    The hisat2-inspect index inspector

    hisat2-inspect extracts information from a HISAT2 index about what kind of index it is and what reference sequences were used to build it. When run without any options, the tool will output a FASTA file containing the sequences of the original references (with all non-A/C/G/T characters converted to Ns). It can also be used to extract just the reference sequence names using the -n/--names option or a more verbose summary using the -s/--summary option.

    Command Line

    Usage:

    hisat2-inspect [options]* <ht2_base>

    Main arguments

    <ht2_base>

    The basename of the index to be inspected. The basename is name of any of the index files but with the .X.ht2 suffix omitted. hisat2-inspect first looks in the current directory for the index files, then in the directory specified in the HISAT2_INDEXES environment variable.

    Options

    -a/--across <int>

    When printing FASTA output, output a newline character every <int> bases (default: 60).

    -n/--names

    Print reference sequence names, one per line, and quit.

    -s/--summary

    Print a summary that includes information about index settings, as well as the names and lengths of the input sequences. The summary has this format:

    Colorspace  <0 or 1>
SA-Sample   1 in <sample>
FTab-Chars  <chars>
Sequence-1  <name>  <len>
Sequence-2  <name>  <len>
...
Sequence-N  <name>  <len>

    Fields are separated by tabs. Colorspace is always set to 0 for HISAT2.

    --snp

    Print SNPs, and quit.

    --ss

    Print splice sites, and quit.

    --ss-all

    Print splice sites including those not in the global index, and quit.

    --exon

    Print exons, and quit.

    -v/--verbose

    Print verbose output (for debugging).

    --version

    Print version information and quit.

    -h/--help

    Print usage information and quit.

    Getting started with HISAT2

    HISAT2 comes with some example files to get you started. The example files are not scientifically significant; these files will simply let you start running HISAT2 and downstream tools right away.

    First follow the manual instructions to obtain HISAT2. Set the HISAT2_HOME environment variable to point to the new HISAT2 directory containing the hisat2, hisat2-build and hisat2-inspect binaries. This is important, as the HISAT2_HOME variable is used in the commands below to refer to that directory.

    Indexing a reference genome

    To create an index for the genomic region (1 million bps from the human chromosome 22 between 20,000,000 and 20,999,999) included with HISAT2, create a new temporary directory (it doesn't matter where), change into that directory, and run:

    $HISAT2_HOME/hisat2-build $HISAT2_HOME/example/reference/22_20-21M.fa --snp $HISAT2_HOME/example/reference/22_20-21M.snp 22_20-21M_snp

    The command should print many lines of output then quit. When the command completes, the current directory will contain ten new files that all start with 22_20-21M_snp and end with .1.ht2, .2.ht2, .3.ht2, .4.ht2, .5.ht2, .6.ht2, .7.ht2, and .8.ht2. These files constitute the index - you're done!

    You can use hisat2-build to create an index for a set of FASTA files obtained from any source, including sites such as UCSC, NCBI, and Ensembl. When indexing multiple FASTA files, specify all the files using commas to separate file names. For more details on how to create an index with hisat2-build, see the manual section on index building. You may also want to bypass this process by obtaining a pre-built index.

    Aligning example reads

    Stay in the directory created in the previous step, which now contains the 22_20-21M index files. Next, run:

    $HISAT2_HOME/hisat2 -f -x $HISAT2_HOME/example/index/22_20-21M_snp -U $HISAT2_HOME/example/reads/reads_1.fa -S eg1.sam

    This runs the HISAT2 aligner, which aligns a set of unpaired reads to the genome region using the index generated in the previous step. The alignment results in SAM format are written to the file eg1.sam, and a short alignment summary is written to the console. (Actually, the summary is written to the "standard error" or "stderr" filehandle, which is typically printed to the console.)

    To see the first few lines of the SAM output, run:

    head eg1.sam

    You will see something like this:

    @HD     VN:1.0  SO:unsorted
@SQ     SN:22:20000001-21000000 LN:1000000
@PG     ID:hisat2       PN:hisat2       VN:2.0.0-beta
1       0       22:20000001-21000000    397984  255     100M    *       0       0       GCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACT    IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:100        YT:Z:UU NH:i:1
2       16      22:20000001-21000000    398131  255     100M    *       0       0       ATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCT    IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:80A19      YT:Z:UU NH:i:1  Zs:Z:80|S|rs576159895
3       16      22:20000001-21000000    398222  255     100M    *       0       0       TGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAA    IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:16A83      YT:Z:UU NH:i:1  Zs:Z:16|S|rs2629364
4       16      22:20000001-21000000    398247  255     90M200N10M      *       0       0       CAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAACCGCTGCACCAGGAAGTCGATCCAG    IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:100        YT:Z:UU XS:A:-  NH:i:1
5       16      22:20000001-21000000    398194  255     100M    *       0       0       GGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCACTTGGT    IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:17A26A55   YT:Z:UU NH:i:1  Zs:Z:17|S|rs576159895,26|S|rs2629364
6       0       22:20000001-21000000    398069  255     100M    *       0       0       CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA    IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:100        YT:Z:UU NH:i:1
7       0       22:20000001-21000000    397896  255     100M    *       0       0       GTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGA    IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:31G68      YT:Z:UU NH:i:1  Zs:Z:31|S|rs562662261
8       0       22:20000001-21000000    398150  255     100M    *       0       0       AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG    IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:61A26A11   YT:Z:UU NH:i:1  Zs:Z:61|S|rs576159895,26|S|rs2629364
9       16      22:20000001-21000000    398329  255     8M200N92M       *       0       0       ACCAGGAAGTCGATCCAGATGTAGTGGGGGGTCACTTCGGGGGGACAGGGTTTGGGTTGACTTGCTTCCGAGGCAGCCAGGGGGTCTGCTTCCTTTATCT    IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:100        YT:Z:UU XS:A:-  NH:i:1
10      16      22:20000001-21000000    398184  255     100M    *       0       0       CTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATC    IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII    AS:i:0  XN:i:0  XM:i:0  XO:i:0  XG:i:0  NM:i:0  MD:Z:27A26A45   YT:Z:UU NH:i:1  Zs:Z:27|S|rs576159895,26|S|rs2629364

    The first few lines (beginning with @) are SAM header lines, and the rest of the lines are SAM alignments, one line per read or mate. See the HISAT2 manual section on SAM output and the SAM specification for details about how to interpret the SAM file format.

    Paired-end example

    To align paired-end reads included with HISAT2, stay in the same directory and run:

    $HISAT2_HOME/hisat2 -f -x $HISAT2_HOME/example/index/22_20-21M_snp -1 $HISAT2_HOME/example/reads/reads_1.fa -2 $HISAT2_HOME/example/reads/reads_2.fa -S eg2.sam

    This aligns a set of paired-end reads to the reference genome, with results written to the file eg2.sam.

    Using SAMtools/BCFtools downstream

    SAMtools is a collection of tools for manipulating and analyzing SAM and BAM alignment files. BCFtools is a collection of tools for calling variants and manipulating VCF and BCF files, and it is typically distributed with SAMtools. Using these tools together allows you to get from alignments in SAM format to variant calls in VCF format. This example assumes that samtools and bcftools are installed and that the directories containing these binaries are in your PATH environment variable.

    Run the paired-end example:

    $HISAT2_HOME/hisat -f -x $HISAT2_HOME/example/index/22_20-21M_snp -1 $HISAT2_HOME/example/reads/reads_1.fa -2 $HISAT2_HOME/example/reads/reads_2.fa -S eg2.sam

    Use samtools view to convert the SAM file into a BAM file. BAM is a the binary format corresponding to the SAM text format. Run:

    samtools view -bS eg2.sam > eg2.bam

    Use samtools sort to convert the BAM file to a sorted BAM file. The following command requires samtools version 1.2 or higher.

    samtools sort eg2.bam -o eg2.sorted.bam

    We now have a sorted BAM file called eg2.sorted.bam. Sorted BAM is a useful format because the alignments are (a) compressed, which is convenient for long-term storage, and (b) sorted, which is convenient for variant discovery. To generate variant calls in VCF format, run:

    samtools mpileup -uf $HISAT2_HOME/example/reference/22_20-21M.fa eg2.sorted.bam | bcftools view -bvcg - > eg2.raw.bcf

    Then to view the variants, run:

    bcftools view eg2.raw.bcf

    See the official SAMtools guide to Calling SNPs/INDELs with SAMtools/BCFtools for more details and variations on this process.

    hisat2-2.2.1/docs_jhu/manual.shtml000066400000000000000000000021231370663763200170450ustar00rootroot00000000000000

    Table of Contents

    hisat2-2.2.1/docs_jhu/sidebar.inc.shtml000066400000000000000000000306171370663763200177620ustar00rootroot00000000000000

    Site Map

    News and Updates

      New releases and related tools will be announced through the Bowtie mailing list.

    Getting Help

      Please use hisat2.genomics@gmail.com for private communications only. Please do not email technical questions to HISAT2 contributors directly.

    Releases
    Please cite:

    Kim D, Langmead B and Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nature Methods 2015

    Indexes (see note)

    H. sapiens, GRCh38
    genome 3.9 GB
    genome_snp 4.6 GB
    genome_tran 4.1 GB
    genome_snp_tran 4.6 GB
    H. sapiens, UCSC hg38
    genome 4.1 GB
    H. sapiens, UCSC hg38 and Refseq gene annotations referred to in the Nature Protocol paper
    genome_tran 4.2 GB
    H. sapiens, GRCh37
    genome 3.9 GB
    genome_snp 4.5 GB
    genome_tran 4.0 GB
    genome_snp_tran 4.5 GB
    H. sapiens, UCSC hg19
    genome 3.9 GB
    M. musculus, GRCm38
    genome 3.7 GB
    genome_snp 4.0 GB
    genome_tran 3.7 GB
    genome_snp_tran 4.0 GB
    M. musculus, UCSC mm10
    genome 3.5 GB
    R. norvegicus, UCSC rn6
    genome 3.7 GB
    D. melanogaster, BDGP6
    genome 0.2 GB
    genome_tran 0.2 GB
    D. melanogaster, UCSC dm6
    genome 0.2 GB
    C. elegans, WBcel235
    genome 0.1 GB
    genome_tran 0.1 GB
    C. elegans, UCSC ce10
    genome 0.1 GB
    S. cerevisiae, R64-1-1
    genome 0.01 GB
    genome_tran 0.01 GB
    S. cerevisiae, UCSC sacCer3
    genome 0.01 GB

    * genome: HFM index for reference
    * genome_snp: HGFM index for reference plus SNPs
    * genome_tran: HGFM index for reference plus transcripts
    * genome_snp_tran: HGFM index for reference plus SNPs and transcripts
    more indexes

    Related Tools

    • HISAT-genotype: Next-generation analysis of human genomes
    • HISAT: Fast and sensitive spliced alignment
    • Bowtie2: Ultrafast read alignment
    • TopHat2: Spliced read mapper for RNA-Seq
    • Cufflinks: Isoform assembly and quantitation for RNA-Seq
    • StringTie: Transcript assembly and quantification for RNA-Seq

    Publications

    Contributors

    Links

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See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "dp_framer.h" using namespace std; /** * Set up variables that describe the shape of a dynamic programming matrix to * be filled in. The matrix is built around the diagonal containing the seed * hit: the "seed diagonal". The N diagonals to the right of the seed diagonal * are the "RHS gap" diagonals, where N is the maximum number of read or * reference gaps permitted (whichever is larger). The N diagonals to the left * of the seed diagonal are the "LHS gap" diagonals. * * The way the rectangle is currently formulated, there are another N diagonals * to the left of the "LHS gap" diagonals called the "LHS extra diagonals". It * might also be possible to split the "extra diagonals" into two subsets and * place them both to the left of the LHS gap diagonals and to the right of the * RHS gap diagonals. * * The purpose of arranging and these groupings of diagonals is that a subset * of them, the "core diagonals", can now be considered "covered." By * "covered" I mean that any alignment that overlaps a cell in any of the core * diagonals cannot possibly overlap another, higher-scoring alignment that * falls partially outside the rectangle. * * Say the read is 5 characters long, the maximum number of read or ref gaps is * 2, and the seed hit puts the main diagonal at offset 10 in the reference. * The larger rectangle explored looks like this: * * off=10, maxgap=2 * * Ref 1 * off: 67890123456 0: seed diagonal * **OO0oo++---- o: "RHS gap" diagonals * -**OO0oo++--- O: "LHS gap" diagonals * --**OO0oo++-- *: "LHS extra" diagonals * ---**OO0oo++- +: "RHS extra" diagonals * ----**OO0oo++ -: cells that can't possibly be involved in a valid * alignment that overlaps one of the core diagonals * * The "core diagonals" are marked with 0's, O's or o's. * * A caveat is that, for performance reasons, we place an upper limit on N - * the maximum number of read or reference gaps. It is constrained to be no * greater than 'maxgap'. This means that in some situations, we may report an * alignment that spuriously trumps a better alignment that falls partially * outside the rectangle. Also, we may fail to find a valid alignment with * more than 'maxgap' gaps. * * Another issue is trimming: if the seed hit is sufficiently close to one or * both ends of the reference sequence, and either (a) overhang is not * permitted, or (b) the number of Ns permitted is less than the number of * columns that overhang the reference, then we want to exclude the trimmed * columns from the rectangle. * * We need to return enough information so that downstream routines can fully * understand the shape of the rectangle, which diagonals are which (esp. which * are the "core" diagonals, since we needn't examine any more seed hits from * those columns in the future), and how the rectangle is trimmed. The * information returned should be compatible with the sort of information * returned by the routines that set up rectangles for mate finding. */ bool DynProgFramer::frameSeedExtensionRect( int64_t off, // ref offset implied by seed hit assuming no gaps size_t rdlen, // length of read sequence used in DP table (so len // of +1 nucleotide sequence for colorspace reads) int64_t reflen, // length of reference sequence aligned to size_t maxrdgap, // max # of read gaps permitted in opp mate alignment size_t maxrfgap, // max # of ref gaps permitted in opp mate alignment int64_t maxns, // # Ns permitted size_t maxhalf, // max width in either direction DPRect& rect) // out: DP rectangle { assert_gt(rdlen, 0); assert_gt(reflen, 0); // Set N, the maximum number of reference or read gaps permitted, whichever // is larger. Also, enforce ceiling: can't be larger than 'maxhalf'. size_t maxgap = max(maxrdgap, maxrfgap); maxgap = min(maxgap, maxhalf); // Leave room for "LHS gap" and "LHS extra" diagonals int64_t refl = off - 2 * maxgap; // inclusive // Leave room for "RHS gap" and "RHS extra" diagonals int64_t refr = off + (rdlen - 1) + 2 * maxgap; // inclusive size_t triml = 0, trimr = 0; // Check if we have to trim to fit the extents of the reference if(trimToRef_) { maxns = 0; // no leeway } else if(maxns == (int64_t)rdlen) { maxns--; } // Trim from RHS of rectangle if(refr >= reflen + maxns) { trimr = (size_t)(refr - (reflen + maxns - 1)); } // Trim from LHS of rectangle if(refl < -maxns) { triml = (size_t)(-refl) - (size_t)maxns; } rect.refl_pretrim = refl; rect.refr_pretrim = refr; rect.refl = refl + triml; rect.refr = refr - trimr; rect.triml = triml; rect.trimr = trimr; rect.maxgap = maxgap; // Remember which diagonals are "core" as offsets from the LHS of the // untrimmed rectangle rect.corel = maxgap; rect.corer = rect.corel + 2 * maxgap; // inclusive assert(rect.repOk()); return !rect.entirelyTrimmed(); } /** * Set up variables that describe the shape of a dynamic programming matrix to * be filled in. The matrix is built around the diagonals that terminate in * the range of columns where the RHS of the opposite mate must fall in order * to satisfy the fragment-length constraint. These are the "mate" diagonals * and they also happen to be the "core" diagonals in this case. * * The N diagonals to the right of the mate diagonals are the "RHS gap" * diagonals, where N is the maximum number of read or reference gaps permitted * (whichever is larger). The N diagonals to the left of the mate diagonals * are the "LHS gap" diagonals. * * The purpose of arranging and these groupings of diagonals is that a subset * of them, the "core diagonals", can now be considered "covered." By * "covered" I mean that any alignment that overlaps a cell in any of the core * diagonals cannot possibly overlap another, higher-scoring alignment that * falls partially outside the rectangle. * * |Anchor| * o---------OO0000000000000oo------ 0: mate diagonal (also core diags!) * -o---------OO0000000000000oo----- o: "RHS gap" diagonals * --o---------OO0000000000000oo---- O: "LHS gap" diagonals * ---oo--------OO0000000000000oo--- *: "LHS extra" diagonals * -----o--------OO0000000000000oo-- -: cells that can't possibly be * ------o--------OO0000000000000oo- involved in a valid alignment that * -------o--------OO0000000000000oo overlaps one of the core diagonals * XXXXXXXXXXXXX * | RHS Range | * ^ ^ * rl rr * * The "core diagonals" are marked with 0s. * * A caveat is that, for performance reasons, we place an upper limit on N - * the maximum number of read or reference gaps. It is constrained to be no * greater than 'maxgap'. This means that in some situations, we may report an * alignment that spuriously trumps a better alignment that falls partially * outside the rectangle. Also, we may fail to find a valid alignment with * more than 'maxgap' gaps. * * Another issue is trimming: if the seed hit is sufficiently close to one or * both ends of the reference sequence, and either (a) overhang is not * permitted, or (b) the number of Ns permitted is less than the number of * columns that overhang the reference, then we want to exclude the trimmed * columns from the rectangle. */ bool DynProgFramer::frameFindMateAnchorLeftRect( int64_t ll, // leftmost Watson off for LHS of opp alignment int64_t lr, // rightmost Watson off for LHS of opp alignment int64_t rl, // leftmost Watson off for RHS of opp alignment int64_t rr, // rightmost Watson off for RHS of opp alignment size_t rdlen, // length of opposite mate int64_t reflen, // length of reference sequence aligned to size_t maxrdgap, // max # of read gaps permitted in opp mate alignment size_t maxrfgap, // max # of ref gaps permitted in opp mate alignment int64_t maxns, // max # ns permitted in the alignment size_t maxhalf, // max width in either direction DPRect& rect) // out: DP rectangle const { assert_geq(lr, ll); // LHS rightmost must be >= LHS leftmost assert_geq(rr, rl); // RHS rightmost must be >= RHS leftmost assert_geq(rr, lr); // RHS rightmost must be >= LHS rightmost assert_geq(rl, ll); // RHS leftmost must be >= LHS leftmost assert_gt(rdlen, 0); assert_gt(reflen, 0); size_t triml = 0, trimr = 0; size_t maxgap = max(maxrdgap, maxrfgap); maxgap = max(maxgap, maxhalf); // Amount of padding we have to add to account for the fact that alignments // ending between en_left/en_right might start in various columns in the // first row int64_t pad_left = maxgap; int64_t pad_right = maxgap; int64_t en_left = rl; int64_t en_right = rr; int64_t st_left = en_left - (rdlen-1); ASSERT_ONLY(int64_t st_right = en_right - (rdlen-1)); int64_t en_right_pad = en_right + pad_right; ASSERT_ONLY(int64_t en_left_pad = en_left - pad_left); ASSERT_ONLY(int64_t st_right_pad = st_right + pad_right); int64_t st_left_pad = st_left - pad_left; assert_leq(st_left, en_left); assert_geq(en_right, st_right); assert_leq(st_left_pad, en_left_pad); assert_geq(en_right_pad, st_right_pad); int64_t refl = st_left_pad; int64_t refr = en_right_pad; if(trimToRef_) { maxns = 0; } else if(maxns == (int64_t)rdlen) { maxns--; } // Trim from the RHS of the rectangle? if(refr >= reflen + maxns) { trimr = (size_t)(refr - (reflen + maxns - 1)); } // Trim from the LHS of the rectangle? if(refl < -maxns) { triml = (size_t)(-refl) - (size_t)maxns; } size_t width = (size_t)(refr - refl + 1); rect.refl_pretrim = refl; rect.refr_pretrim = refr; rect.refl = refl + triml; rect.refr = refr - trimr; rect.triml = triml; rect.trimr = trimr; rect.maxgap = maxgap; rect.corel = maxgap; rect.corer = width - maxgap - 1; // inclusive assert(rect.repOk()); return !rect.entirelyTrimmed(); } /** * Set up variables that describe the shape of a dynamic programming matrix to * be filled in. The matrix is built around the diagonals that begin in the * range of columns where the LHS of the opposite mate must fall in order to * satisfy the fragment-length constraint. These are the "mate" diagonals and * they also happen to be the "core" diagonals in this case. * * The N diagonals to the right of the mate diagonals are the "RHS gap" * diagonals, where N is the maximum number of read or reference gaps permitted * (whichever is larger). The N diagonals to the left of the mate diagonals * are the "LHS gap" diagonals. * * The purpose of arranging and these groupings of diagonals is that a subset * of them, the "core diagonals", can now be considered "covered." By * "covered" I mean that any alignment that overlaps a cell in any of the core * diagonals cannot possibly overlap another, higher-scoring alignment that * falls partially outside the rectangle. * * ll lr * v v * | LHS Range | * XXXXXXXXXXXXX |Anchor| * OO0000000000000oo--------o-------- 0: mate diagonal (also core diags!) * -OO0000000000000oo--------o------- o: "RHS gap" diagonals * --OO0000000000000oo--------o------ O: "LHS gap" diagonals * ---OO0000000000000oo--------oo---- *: "LHS extra" diagonals * ----OO0000000000000oo---------o--- -: cells that can't possibly be * -----OO0000000000000oo---------o-- involved in a valid alignment that * ------OO0000000000000oo---------o- overlaps one of the core diagonals * * The "core diagonals" are marked with 0s. * * A caveat is that, for performance reasons, we place an upper limit on N - * the maximum number of read or reference gaps. It is constrained to be no * greater than 'maxgap'. This means that in some situations, we may report an * alignment that spuriously trumps a better alignment that falls partially * outside the rectangle. Also, we may fail to find a valid alignment with * more than 'maxgap' gaps. * * Another issue is trimming: if the seed hit is sufficiently close to one or * both ends of the reference sequence, and either (a) overhang is not * permitted, or (b) the number of Ns permitted is less than the number of * columns that overhang the reference, then we want to exclude the trimmed * columns from the rectangle. */ bool DynProgFramer::frameFindMateAnchorRightRect( int64_t ll, // leftmost Watson off for LHS of opp alignment int64_t lr, // rightmost Watson off for LHS of opp alignment int64_t rl, // leftmost Watson off for RHS of opp alignment int64_t rr, // rightmost Watson off for RHS of opp alignment size_t rdlen, // length of opposite mate int64_t reflen, // length of reference sequence aligned to size_t maxrdgap, // max # of read gaps permitted in opp mate alignment size_t maxrfgap, // max # of ref gaps permitted in opp mate alignment int64_t maxns, // max # ns permitted in the alignment size_t maxhalf, // max width in either direction DPRect& rect) // out: DP rectangle const { assert_geq(lr, ll); assert_geq(rr, rl); assert_geq(rr, lr); assert_geq(rl, ll); assert_gt(rdlen, 0); assert_gt(reflen, 0); size_t triml = 0, trimr = 0; size_t maxgap = max(maxrdgap, maxrfgap); maxgap = max(maxgap, maxhalf); int64_t pad_left = maxgap; int64_t pad_right = maxgap; int64_t st_left = ll; int64_t st_right = lr; ASSERT_ONLY(int64_t en_left = st_left + (rdlen-1)); int64_t en_right = st_right + (rdlen-1); int64_t en_right_pad = en_right + pad_right; ASSERT_ONLY(int64_t en_left_pad = en_left - pad_left); ASSERT_ONLY(int64_t st_right_pad = st_right + pad_right); int64_t st_left_pad = st_left - pad_left; assert_leq(st_left, en_left); assert_geq(en_right, st_right); assert_leq(st_left_pad, en_left_pad); assert_geq(en_right_pad, st_right_pad); // We have enough info to deduce where the boundaries of our rectangle // should be. Finalize the boundaries, ignoring reference trimming for now int64_t refl = st_left_pad; int64_t refr = en_right_pad; if(trimToRef_) { maxns = 0; } else if(maxns == (int64_t)rdlen) { maxns--; } // Trim from the RHS of the rectangle? if(refr >= reflen + maxns) { trimr = (size_t)(refr - (reflen + maxns - 1)); } // Trim from the LHS of the rectangle? if(refl < -maxns) { triml = (size_t)(-refl) - (size_t)maxns; } size_t width = (size_t)(refr - refl + 1); rect.refl_pretrim = refl; rect.refr_pretrim = refr; rect.refl = refl + triml; rect.refr = refr - trimr; rect.triml = triml; rect.trimr = trimr; rect.maxgap = maxgap; rect.corel = maxgap; rect.corer = width - maxgap - 1; // inclusive assert(rect.repOk()); return !rect.entirelyTrimmed(); } #ifdef MAIN_DP_FRAMER #include static void testCaseFindMateAnchorLeft( const char *testName, bool trimToRef, int64_t ll, int64_t lr, int64_t rl, int64_t rr, size_t rdlen, size_t reflen, size_t maxrdgap, size_t maxrfgap, size_t ex_width, size_t ex_solwidth, size_t ex_trimup, size_t ex_trimdn, int64_t ex_refl, int64_t ex_refr, const char *ex_st, // string of '0'/'1' chars const char *ex_en) // string of '0'/'1' chars { cerr << testName << "..."; DynProgFramer fr(trimToRef); size_t width, solwidth; int64_t refl, refr; EList st, en; size_t trimup, trimdn; size_t maxhalf = 500; size_t maxgaps = 0; fr.frameFindMateAnchorLeft( ll, // leftmost Watson off for LHS of opp alignment lr, // rightmost Watson off for LHS of opp alignment rl, // leftmost Watson off for RHS of opp alignment rr, // rightmost Watson off for RHS of opp alignment rdlen, // length of opposite mate reflen, // length of reference sequence aligned to maxrdgap, // max # of read gaps permitted in opp mate alignment maxrfgap, // max # of ref gaps permitted in opp mate alignment maxns, // max # Ns permitted maxhalf, // max width in either direction width, // out: calculated width stored here maxgaps, // out: max # gaps trimup, // out: number of bases trimmed from upstream end trimdn, // out: number of bases trimmed from downstream end refl, // out: ref pos of upper LHS of parallelogram refr, // out: ref pos of lower RHS of parallelogram st, // out: legal starting columns stored here en); // out: legal ending columns stored here assert_eq(ex_width, width); assert_eq(ex_solwidth, solwidth); assert_eq(ex_trimup, trimup); assert_eq(ex_trimdn, trimdn); assert_eq(ex_refl, refl); assert_eq(ex_refr, refr); for(size_t i = 0; i < width; i++) { assert_eq((ex_st[i] == '1'), st[i]); assert_eq((ex_en[i] == '1'), en[i]); } cerr << "PASSED" << endl; } static void testCaseFindMateAnchorRight( const char *testName, bool trimToRef, int64_t ll, int64_t lr, int64_t rl, int64_t rr, size_t rdlen, size_t reflen, size_t maxrdgap, size_t maxrfgap, size_t ex_width, size_t ex_solwidth, size_t ex_trimup, size_t ex_trimdn, int64_t ex_refl, int64_t ex_refr, const char *ex_st, // string of '0'/'1' chars const char *ex_en) // string of '0'/'1' chars { cerr << testName << "..."; DynProgFramer fr(trimToRef); size_t width, solwidth; size_t maxgaps; int64_t refl, refr; EList st, en; size_t trimup, trimdn; size_t maxhalf = 500; fr.frameFindMateAnchorRight( ll, // leftmost Watson off for LHS of opp alignment lr, // rightmost Watson off for LHS of opp alignment rl, // leftmost Watson off for RHS of opp alignment rr, // rightmost Watson off for RHS of opp alignment rdlen, // length of opposite mate reflen, // length of reference sequence aligned to maxrdgap, // max # of read gaps permitted in opp mate alignment maxrfgap, // max # of ref gaps permitted in opp mate alignment maxns, // max # Ns permitted maxhalf, // max width in either direction width, // out: calculated width stored here maxgaps, // out: calcualted max # gaps trimup, // out: number of bases trimmed from upstream end trimdn, // out: number of bases trimmed from downstream end refl, // out: ref pos of upper LHS of parallelogram refr, // out: ref pos of lower RHS of parallelogram st, // out: legal starting columns stored here en); // out: legal ending columns stored here assert_eq(ex_width, width); assert_eq(ex_trimup, trimup); assert_eq(ex_trimdn, trimdn); assert_eq(ex_refl, refl); assert_eq(ex_refr, refr); for(size_t i = 0; i < width; i++) { assert_eq((ex_st[i] == '1'), st[i]); assert_eq((ex_en[i] == '1'), en[i]); } cerr << "PASSED" << endl; } int main(void) { /////////////////////////// // // ANCHOR ON THE LEFT // /////////////////////////// // ------------- // o o // o o // o o // o o // <<<------->>> // 012345678901234567890 // 0 1 2 testCaseFindMateAnchorLeft( "FindMateAnchorLeft1", false, // trim to reference 3, // left offset of upper parallelogram extent 15, // right offset of upper parallelogram extent 10, // left offset of lower parallelogram extent 16, // right offset of lower parallelogram extent 5, // length of opposite mate 30, // length of reference sequence aligned to 3, // max # of read gaps permitted in opp mate alignment 3, // max # of ref gaps permitted in opp mate alignment 13, // expected width 0, // expected # bases trimmed from upstream end 0, // expected # bases trimmed from downstream end 3, // ref offset of upstream column 19, // ref offset of downstream column "1111111111111", // expected starting bools "0001111111000"); // expected ending bools // ******* // <<===----- // o o // o o // o o // o o // <<=----->> // ******* // 012345678901234567890 // 0 1 2 testCaseFindMateAnchorLeft( "FindMateAnchorLeft2", false, // trim to reference 9, // left offset of left upper parallelogram extent 14, // right offset of left upper parallelogram extent 10, // left offset of left lower parallelogram extent 15, // right offset of left lower parallelogram extent 5, // length of opposite mate 30, // length of reference sequence aligned to 2, // max # of read gaps permitted in opp mate alignment 2, // max # of ref gaps permitted in opp mate alignment 7, // expected width 3, // expected # bases trimmed from upstream end 0, // expected # bases trimmed from downstream end 7, // ref offset of upstream column 17, // ref offset of downstream column "0011111", // expected starting bools "1111100"); // expected ending bools // ******* // <<===--->> // o o // o o // o o // o o // o o // <<=----->> // ******* // 01234567890123456xxxx // 0 1 2 testCaseFindMateAnchorLeft( "FindMateAnchorLeft3", true, // trim to reference 9, // left offset of left upper parallelogram extent 14, // right offset of left upper parallelogram extent 10, // left offset of left lower parallelogram extent 15, // right offset of left lower parallelogram extent 5, // length of opposite mate 17, // length of reference sequence aligned to 2, // max # of read gaps permitted in opp mate alignment 2, // max # of ref gaps permitted in opp mate alignment 7, // expected width 3, // expected # bases trimmed from upstream end 0, // expected # bases trimmed from downstream end 7, // ref offset of upstream column 17, // ref offset of downstream column "0011111", // expected starting bools "1111100"); // expected ending bools // ****** // <<===----- // o o // o o // o o // o o // <<=----=>> // ****** // 012345678901234xxxxxx // 0 1 2 testCaseFindMateAnchorLeft( "FindMateAnchorLeft4", true, // trim to reference 9, // left offset of left upper parallelogram extent 14, // right offset of left upper parallelogram extent 10, // left offset of left lower parallelogram extent 15, // right offset of left lower parallelogram extent 5, // length of opposite mate 15, // length of reference sequence aligned to 2, // max # of read gaps permitted in opp mate alignment 2, // max # of ref gaps permitted in opp mate alignment 6, // expected width 3, // expected # bases trimmed from upstream end 1, // expected # bases trimmed from downstream end 7, // ref offset of upstream column 16, // ref offset of downstream column "001111", // expected starting bools "111100"); // expected ending bools // -1 0 2 // xxxxxxxxxx012345678xx // // ******* // <<===----- // o o // o o // o o // o o // o o // <<=----->> // ******* // // xxxxxxxxxx012345678xx // -1 0 2 testCaseFindMateAnchorLeft( "FindMateAnchorLeft5", true, // trim to reference 1, // left offset of left upper parallelogram extent 7, // right offset of left upper parallelogram extent 2, // left offset of left lower parallelogram extent 7, // right offset of left lower parallelogram extent 5, // length of opposite mate 9, // length of reference sequence aligned to 2, // max # of read gaps permitted in opp mate alignment 2, // max # of ref gaps permitted in opp mate alignment 7, // expected width 3, // expected # bases trimmed from upstream end 0, // expected # bases trimmed from downstream end -1, // ref offset of upstream column 9, // ref offset of downstream column "0011111", // expected starting bools "1111100"); // expected ending bools // <<<<==-===>> // o o // o o // o o // o o // <<<<------>> // ****** // 012345678901234567890 // 0 1 2 testCaseFindMateAnchorLeft( "FindMateAnchorLeft6", false, // trim to reference 8, // left offset of left upper parallelogram extent 8, // right offset of left upper parallelogram extent 10, // left offset of left lower parallelogram extent 15, // right offset of left lower parallelogram extent 5, // length of opposite mate 30, // length of reference sequence aligned to 4, // max # of read gaps permitted in opp mate alignment 2, // max # of ref gaps permitted in opp mate alignment 6, // expected width 4, // expected # bases trimmed from upstream end 2, // expected # bases trimmed from downstream end 6, // ref offset of upstream column 15, // ref offset of downstream column "001000", // expected starting bools "111111"); // expected ending bools /////////////////////////// // // ANCHOR ON THE RIGHT // /////////////////////////// // <<<------->>> // o o // o o // o o // o o // <<<------->>> // 012345678901234567890123456789 // 0 1 2 testCaseFindMateAnchorRight( "FindMateAnchorRight1", false, // trim to reference 10, // left offset of left upper parallelogram extent 16, // right offset of left upper parallelogram extent 11, // left offset of left lower parallelogram extent 23, // right offset of left lower parallelogram extent 5, // length of opposite mate 30, // length of reference sequence aligned to 3, // max # of read gaps permitted in opp mate alignment 3, // max # of ref gaps permitted in opp mate alignment 13, // expected width 0, // expected # bases trimmed from upstream end 0, // expected # bases trimmed from downstream end 7, // ref offset of upstream column 23, // ref offset of downstream column "0001111111000", // expected starting bools "1111111111111"); // expected ending bools // 0 1 2 // 012345678901234567890 // ******* // <<------>> // o o // o o // o o // o o // <<===--->> // ******* // 012345678901234567890 // 0 1 2 testCaseFindMateAnchorRight( "FindMateAnchorRight2", false, // trim to reference 6, // left offset of left upper parallelogram extent 11, // right offset of left upper parallelogram extent 13, // left offset of left lower parallelogram extent 18, // right offset of left lower parallelogram extent 5, // length of opposite mate 30, // length of reference sequence aligned to 2, // max # of read gaps permitted in opp mate alignment 2, // max # of ref gaps permitted in opp mate alignment 7, // expected width 3, // expected # bases trimmed from upstream end 0, // expected # bases trimmed from downstream end 7, // ref offset of upstream column 17, // ref offset of downstream column "1111100", // expected starting bools "0011111"); // expected ending bools // Reference trimming takes off the left_pad of the left mate // // ******* // <<------>> // o o // o o // o o // o o // o o // <<===--->> // ******* // 0123456789012345678901234567890 // -1 0 1 2 testCaseFindMateAnchorRight( "FindMateAnchorRight3", true, // trim to reference 0, // left offset of left upper parallelogram extent 5, // right offset of left upper parallelogram extent 7, // left offset of left lower parallelogram extent 11, // right offset of left lower parallelogram extent 5, // length of opposite mate 30, // length of reference sequence aligned to 2, // max # of read gaps permitted in opp mate alignment 2, // max # of ref gaps permitted in opp mate alignment 7, // expected width 3, // expected # bases trimmed from upstream end 0, // expected # bases trimmed from downstream end 1, // ref offset of upstream column 11, // ref offset of downstream column "1111100", // expected starting bools "0011111"); // expected ending bools // Reference trimming takes off the leftmost 5 positions of the left mate, // and takes 1 from the right mate // // ***** // <<------>> // o o // o o // o o // o o // o o // <<===--->> // ***** // 0987654321012345678901234567890 // -1 0 1 2 testCaseFindMateAnchorRight( "FindMateAnchorRight4", true, // trim to reference -3, // left offset of left upper parallelogram extent 2, // right offset of left upper parallelogram extent 4, // left offset of left lower parallelogram extent 10, // right offset of left lower parallelogram extent 5, // length of opposite mate 30, // length of reference sequence aligned to 2, // max # of read gaps permitted in opp mate alignment 2, // max # of ref gaps permitted in opp mate alignment 5, // expected width 5, // expected # bases trimmed from upstream end 0, // expected # bases trimmed from downstream end 0, // ref offset of upstream column 8, // ref offset of downstream column "11100", // expected starting bools "11111"); // expected ending bools // Reference trimming takes off the leftmost 5 positions of the left mate, // and takes 1 from the left of the right mate. Also, it takes 2 from the // right of the right mate. // // *** // <<------>> // o o // o o // o o // o o // o o // <<===--->> // *** // 0987654321012345678901234567890 // -1 0 1 2 testCaseFindMateAnchorRight( "FindMateAnchorRight5", true, // trim to reference -3, // left offset of left upper parallelogram extent 2, // right offset of left upper parallelogram extent 4, // left offset of left lower parallelogram extent 10, // right offset of left lower parallelogram extent 5, // length of opposite mate 7, // length of reference sequence aligned to 2, // max # of read gaps permitted in opp mate alignment 2, // max # of ref gaps permitted in opp mate alignment 3, // expected width 5, // expected # bases trimmed from upstream end 2, // expected # bases trimmed from downstream end 0, // ref offset of upstream column 6, // ref offset of downstream column "111", // expected starting bools "111"); // expected ending bools // ****** // <<------>>>> // o o // o o // o o // o o // <<====-=>>>> // ****** // 012345678901234567890 // 0 1 2 testCaseFindMateAnchorRight( "FindMateAnchorRight6", false, // trim to reference 6, // left offset of left upper parallelogram extent 11, // right offset of left upper parallelogram extent 14, // left offset of left lower parallelogram extent 14, // right offset of left lower parallelogram extent 5, // length of opposite mate 30, // length of reference sequence aligned to 4, // max # of read gaps permitted in opp mate alignment 2, // max # of ref gaps permitted in opp mate alignment 6, // expected width 2, // expected # bases trimmed from upstream end 4, // expected # bases trimmed from downstream end 6, // ref offset of upstream column 15, // ref offset of downstream column "111111", // expected starting bools "000010"); // expected ending bools // **** // <<<<==---->> // o o // o o // o o // o o // o o // <<<<====-=>> // **** // 012345678901234567890 // 0 1 2 testCaseFindMateAnchorRight( "FindMateAnchorRight7", false, // trim to reference 6, // left offset of left upper parallelogram extent 11, // right offset of left upper parallelogram extent 14, // left offset of left lower parallelogram extent 14, // right offset of left lower parallelogram extent 5, // length of opposite mate 30, // length of reference sequence aligned to 2, // max # of read gaps permitted in opp mate alignment 4, // max # of ref gaps permitted in opp mate alignment 4, // expected width 6, // expected # bases trimmed from upstream end 2, // expected # bases trimmed from downstream end 8, // ref offset of upstream column 15, // ref offset of downstream column "1111", // expected starting bools "0010"); // expected ending bools testCaseFindMateAnchorRight( "FindMateAnchorRight8", true, // trim to reference -37, // left offset of left upper parallelogram extent 13, // right offset of left upper parallelogram extent -37, // left offset of left lower parallelogram extent 52, // right offset of left lower parallelogram extent 10, // length of opposite mate 53, // length of reference sequence aligned to 0, // max # of read gaps permitted in opp mate alignment 0, // max # of ref gaps permitted in opp mate alignment 14, // expected width 37, // expected # bases trimmed from upstream end 0, // expected # bases trimmed from downstream end 0, // ref offset of upstream column 22, // ref offset of downstream column "11111111111111", // expected starting bools "11111111111111");// expected ending bools } #endif /*def MAIN_DP_FRAMER*/ hisat2-2.2.1/dp_framer.h000066400000000000000000000221431370663763200150350ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /* * dp_framer.h * * Classes and routines for framing dynamic programming problems. There are 2 * basic types of dynamic programming problems solved in Bowtie 2: * * 1. Seed extension: we found a seed hit using Burrows-Wheeler techniques and * now we would like to extend it into a full alignment by doing dynamic * programming in the vicinity of the seed hit. * * 2. Mate finding: we would a full alignment for one mate in a pair and now we * would like to extend it into a full alignment by doing dynamic * programming in the area prescribed by the maximum and minimum fragment * lengths. * * By "framing" the dynamic programming problem, we mean that all of the * following DP inputs are calculated: * * 1. The width of the parallelogram/rectangle to explore. * 2. The 0-based offset of the reference position associated with the leftmost * diagnomal/column in the parallelogram/rectangle to explore * 3. An EList of length=width encoding which columns the alignment may * start in * 4. An EList of length=width encoding which columns the alignment may * end in */ #ifndef DP_FRAMER_H_ #define DP_FRAMER_H_ #include #include "ds.h" #include "ref_coord.h" /** * Describes a dynamic programming rectangle. * * Only knows about reference offsets, not reference sequences. */ struct DPRect { DPRect(int cat = 0) /*: st(cat), en(cat)*/ { refl = refr = triml = trimr = corel = corer = 0; } int64_t refl; // leftmost ref offset involved post trimming (incl) int64_t refr; // rightmost ref offset involved post trimming (incl) int64_t refl_pretrim; // leftmost ref offset involved pre trimming (incl) int64_t refr_pretrim; // rightmost ref offset involved pre trimming (incl) size_t triml; // positions trimmed from LHS size_t trimr; // positions trimmed from RHS // If "core" diagonals are specified, then any alignment reported has to // overlap one of the core diagonals. This is to avoid the situation where // an alignment is reported that overlaps a better-scoring alignment that // falls partially outside the rectangle. This is used in both seed // extensions and in mate finding. Filtering based on the core diagonals // should happen in the backtrace routine. I.e. it should simply never // return an alignment that doesn't overlap a core diagonal, even if there // is such an alignment and it's valid. size_t corel; // offset of column where leftmost "core" diagonal starts size_t corer; // offset of column where rightmost "core" diagonal starts // [corel, corer] is an inclusive range and offsets are with respect to the // original, untrimmed rectangle. size_t maxgap; // max # gaps - width of the gap bands /** * Return true iff the combined effect of triml and trimr is to trim away * the entire rectangle. */ bool entirelyTrimmed() const { bool tr = refr < refl; ASSERT_ONLY(size_t width = (size_t)(refr_pretrim - refl_pretrim + 1)); assert(tr == (width <= triml + trimr)); return tr; } #ifndef NDEBUG bool repOk() const { assert_geq(corer, corel); return true; } #endif /** * Set the given interval to the range of diagonals that are "covered" by * this dynamic programming problem. */ void initIval(Interval& iv) { iv.setOff(refl_pretrim + (int64_t)corel); iv.setLen(corer - corel + 1); } }; /** * Encapsulates routines for calculating parameters for the various types of * dynamic programming problems solved in Bowtie2. */ class DynProgFramer { public: DynProgFramer(bool trimToRef) : trimToRef_(trimToRef) { } /** * Similar to frameSeedExtensionParallelogram but we're being somewhat more * inclusive in order to ensure all characters aling the "width" in the last * row are exhaustively scored. */ bool frameSeedExtensionRect( int64_t off, // ref offset implied by seed hit assuming no gaps size_t rdlen, // length of read sequence used in DP table (so len // of +1 nucleotide sequence for colorspace reads) int64_t reflen, // length of reference sequence aligned to size_t maxrdgap, // max # of read gaps permitted in opp mate alignment size_t maxrfgap, // max # of ref gaps permitted in opp mate alignment int64_t maxns, // # Ns permitted size_t maxhalf, // max width in either direction DPRect& rect); // out: DP rectangle /** * Given information about an anchor mate hit, and information deduced by * PairedEndPolicy about where the opposite mate can begin and start given * the fragment length range, return parameters for the dynamic programming * problem to solve. */ bool frameFindMateRect( bool anchorLeft, // true iff anchor alignment is to the left int64_t ll, // leftmost Watson off for LHS of opp alignment int64_t lr, // rightmost Watson off for LHS of opp alignment int64_t rl, // leftmost Watson off for RHS of opp alignment int64_t rr, // rightmost Watson off for RHS of opp alignment size_t rdlen, // length of opposite mate int64_t reflen, // length of reference sequence aligned to size_t maxrdgap, // max # of read gaps permitted in opp mate alignment size_t maxrfgap, // max # of ref gaps permitted in opp mate alignment int64_t maxns, // max # Ns permitted size_t maxhalf, // max width in either direction DPRect& rect) // out: DP rectangle const { if(anchorLeft) { return frameFindMateAnchorLeftRect( ll, lr, rl, rr, rdlen, reflen, maxrdgap, maxrfgap, maxns, maxhalf, rect); } else { return frameFindMateAnchorRightRect( ll, lr, rl, rr, rdlen, reflen, maxrdgap, maxrfgap, maxns, maxhalf, rect); } } /** * Given information about an anchor mate hit, and information deduced by * PairedEndPolicy about where the opposite mate can begin and start given * the fragment length range, return parameters for the dynamic programming * problem to solve. */ bool frameFindMateAnchorLeftRect( int64_t ll, // leftmost Watson off for LHS of opp alignment int64_t lr, // rightmost Watson off for LHS of opp alignment int64_t rl, // leftmost Watson off for RHS of opp alignment int64_t rr, // rightmost Watson off for RHS of opp alignment size_t rdlen, // length of opposite mate int64_t reflen, // length of reference sequence aligned to size_t maxrdgap, // max # of read gaps permitted in opp mate alignment size_t maxrfgap, // max # of ref gaps permitted in opp mate alignment int64_t maxns, // max # Ns permitted in alignment size_t maxhalf, // max width in either direction DPRect& rect) // out: DP rectangle const; /** * Given information about an anchor mate hit, and information deduced by * PairedEndPolicy about where the opposite mate can begin and start given * the fragment length range, return parameters for the dynamic programming * problem to solve. */ bool frameFindMateAnchorRightRect( int64_t ll, // leftmost Watson off for LHS of opp alignment int64_t lr, // rightmost Watson off for LHS of opp alignment int64_t rl, // leftmost Watson off for RHS of opp alignment int64_t rr, // rightmost Watson off for RHS of opp alignment size_t rdlen, // length of opposite mate int64_t reflen, // length of reference sequence aligned to size_t maxrdgap, // max # of read gaps permitted in opp mate alignment size_t maxrfgap, // max # of ref gaps permitted in opp mate alignment int64_t maxns, // max # Ns permitted in alignment size_t maxhalf, // max width in either direction DPRect& rect) // out: DP rectangle const; protected: /** * Trim the given parallelogram width and reference window so that neither * overhangs the beginning or end of the reference. Return true if width * is still > 0 after trimming, otherwise return false. */ void trimToRef( size_t reflen, // in: length of reference sequence aligned to int64_t& refl, // in/out: ref pos of upper LHS of parallelogram int64_t& refr, // in/out: ref pos of lower RHS of parallelogram size_t& trimup, // out: number of bases trimmed from upstream end size_t& trimdn) // out: number of bases trimmed from downstream end { if(refl < 0) { trimup = (size_t)(-refl); //refl = 0; } if(refr >= (int64_t)reflen) { trimdn = (size_t)(refr - reflen + 1); //refr = (int64_t)reflen-1; } } bool trimToRef_; }; #endif /*ndef DP_FRAMER_H_*/ hisat2-2.2.1/ds.cpp000066400000000000000000000062221370663763200140370ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "ds.h" extern MemoryTally gMemTally; /** * Tally a memory allocation of size amt bytes. */ void MemoryTally::add(int cat, uint64_t amt) { ThreadSafe ts(&mutex_m); tots_[cat] += amt; tot_ += amt; if(tots_[cat] > peaks_[cat]) { peaks_[cat] = tots_[cat]; } if(tot_ > peak_) { peak_ = tot_; } } /** * Tally a memory free of size amt bytes. */ void MemoryTally::del(int cat, uint64_t amt) { ThreadSafe ts(&mutex_m); assert_geq(tots_[cat], amt); assert_geq(tot_, amt); tots_[cat] -= amt; tot_ -= amt; } #ifdef MAIN_DS #include #include "random_source.h" using namespace std; int main(void) { cerr << "Test EHeap 1..."; { EHeap h; h.insert(0.5f); // 1 h.insert(0.6f); // 2 h.insert(0.25f); // 3 h.insert(0.75f); // 4 h.insert(0.1f); // 5 h.insert(0.9f); // 6 h.insert(0.4f); // 7 assert_eq(7, h.size()); if(h.pop() != 0.1f) { throw 1; } assert_eq(6, h.size()); if(h.pop() != 0.25f) { throw 1; } assert_eq(5, h.size()); if(h.pop() != 0.4f) { throw 1; } assert_eq(4, h.size()); if(h.pop() != 0.5f) { throw 1; } assert_eq(3, h.size()); if(h.pop() != 0.6f) { throw 1; } assert_eq(2, h.size()); if(h.pop() != 0.75f) { throw 1; } assert_eq(1, h.size()); if(h.pop() != 0.9f) { throw 1; } assert_eq(0, h.size()); assert(h.empty()); } cerr << "PASSED" << endl; cerr << "Test EHeap 2..."; { EHeap h; RandomSource rnd(12); size_t lim = 2000; while(h.size() < lim) { h.insert(rnd.nextU32()); } size_t last = std::numeric_limits::max(); bool first = true; while(!h.empty()) { size_t p = h.pop(); assert(first || p >= last); last = p; first = false; } } cerr << "PASSED" << endl; cerr << "Test EBitList 1..."; { EBitList<128> l; assert_eq(0, l.size()); assert_eq(std::numeric_limits::max(), l.max()); assert(!l.test(0)); assert(!l.test(1)); assert(!l.test(10)); for(int i = 0; i < 3; i++) { l.set(10); assert(!l.test(0)); assert(!l.test(1)); assert(!l.test(9)); assert(l.test(10)); assert(!l.test(11)); } assert_eq(10, l.max()); l.clear(); assert(!l.test(10)); assert_eq(std::numeric_limits::max(), l.max()); RandomSource rnd(12); size_t lim = 2000; for(size_t i = 0; i < lim; i++) { uint32_t ri = rnd.nextU32() % 10000; l.set(ri); assert(l.test(ri)); } } cerr << "PASSED" << endl; } #endif /*def MAIN_SSTRING*/ hisat2-2.2.1/ds.h000066400000000000000000002625271370663763200135200ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef DS_H_ #define DS_H_ #include #include #include #include #include #include #include "assert_helpers.h" #include "threading.h" #include "random_source.h" #include "btypes.h" //added by Joe Paggi to implement EList.push_back_array #include /** * Tally how much memory is allocated to certain */ class MemoryTally { public: MemoryTally() : tot_(0), peak_(0) { memset(tots_, 0, 256 * sizeof(uint64_t)); memset(peaks_, 0, 256 * sizeof(uint64_t)); } /** * Tally a memory allocation of size amt bytes. */ void add(int cat, uint64_t amt); /** * Tally a memory free of size amt bytes. */ void del(int cat, uint64_t amt); /** * Return the total amount of memory allocated. */ uint64_t total() { return tot_; } /** * Return the total amount of memory allocated in a particular * category. */ uint64_t total(int cat) { return tots_[cat]; } /** * Return the peak amount of memory allocated. */ uint64_t peak() { return peak_; } /** * Return the peak amount of memory allocated in a particular * category. */ uint64_t peak(int cat) { return peaks_[cat]; } #ifndef NDEBUG /** * Check that memory tallies are internally consistent; */ bool repOk() const { uint64_t tot = 0; for(int i = 0; i < 256; i++) { assert_leq(tots_[i], peaks_[i]); tot += tots_[i]; } assert_eq(tot, tot_); return true; } #endif protected: MUTEX_T mutex_m; uint64_t tots_[256]; uint64_t tot_; uint64_t peaks_[256]; uint64_t peak_; }; extern MemoryTally gMemTally; /** * A simple fixed-length array of type T, automatically freed in the * destructor. */ template class AutoArray { public: AutoArray(size_t sz, int cat = 0) : cat_(cat) { t_ = NULL; t_ = new T[sz]; gMemTally.add(cat_, sz); memset(t_, 0, sz * sizeof(T)); sz_ = sz; } ~AutoArray() { if(t_ != NULL) { delete[] t_; gMemTally.del(cat_, sz_); } } T& operator[](size_t sz) { return t_[sz]; } const T& operator[](size_t sz) const { return t_[sz]; } size_t size() const { return sz_; } private: int cat_; T *t_; size_t sz_; }; /** * A wrapper for a non-array pointer that associates it with a memory * category for tracking purposes and calls delete on it when the * PtrWrap is destroyed. */ template class PtrWrap { public: explicit PtrWrap( T* p, bool freeable = true, int cat = 0) : cat_(cat), p_(NULL) { init(p, freeable); } explicit PtrWrap(int cat = 0) : cat_(cat), p_(NULL) { reset(); } void reset() { free(); init(NULL); } ~PtrWrap() { free(); } void init(T* p, bool freeable = true) { assert(p_ == NULL); p_ = p; freeable_ = freeable; if(p != NULL && freeable_) { gMemTally.add(cat_, sizeof(T)); } } void free() { if(p_ != NULL) { if(freeable_) { delete p_; gMemTally.del(cat_, sizeof(T)); } p_ = NULL; } } inline T* get() { return p_; } inline const T* get() const { return p_; } private: int cat_; T *p_; bool freeable_; }; /** * A wrapper for an array pointer that associates it with a memory * category for tracking purposes and calls delete[] on it when the * PtrWrap is destroyed. */ template class APtrWrap { public: explicit APtrWrap( T* p, size_t sz, bool freeable = true, int cat = 0) : cat_(cat), p_(NULL) { init(p, sz, freeable); } explicit APtrWrap(int cat = 0) : cat_(cat), p_(NULL) { reset(); } void reset() { free(); init(NULL, 0); } ~APtrWrap() { free(); } void init(T* p, size_t sz, bool freeable = true) { assert(p_ == NULL); p_ = p; sz_ = sz; freeable_ = freeable; if(p != NULL && freeable_) { gMemTally.add(cat_, sizeof(T) * sz_); } } void free() { if(p_ != NULL) { if(freeable_) { delete[] p_; gMemTally.del(cat_, sizeof(T) * sz_); } p_ = NULL; } } inline T* get() { return p_; } inline const T* get() const { return p_; } private: int cat_; T *p_; bool freeable_; size_t sz_; }; /** * An EList is an expandable list with these features: * * - Payload type is a template parameter T. * - Initial size can be specified at construction time, otherwise * default of 128 is used. * - When allocated initially or when expanding, the new[] operator is * used, which in turn calls the default constructor for T. * - All copies (e.g. assignment of a const T& to an EList element, * or during expansion) use operator=. * - When the EList is resized to a smaller size (or cleared, which * is like resizing to size 0), the underlying containing is not * reshaped. Thus, EListss never release memory before * destruction. * * And these requirements: * * - Payload type T must have a default constructor. * * For efficiency reasons, ELists should not be declared on the stack * in often-called worker functions. Best practice is to declare * ELists at a relatively stable layer of the stack (such that it * rarely bounces in and out of scope) and let the worker function use * it and *expand* it only as needed. The effect is that only * relatively few allocations and copies will be incurred, and they'll * occur toward the beginning of the computation before stabilizing at * a "high water mark" for the remainder of the computation. * * A word about multidimensional lists. One way to achieve a * multidimensional lists is to nest ELists. This works, but it often * involves a lot more calls to the default constructor and to * operator=, especially when the outermost EList needs expanding, than * some of the alternatives. One alternative is use a most specialized * container that still uses ELists but knows to use xfer instead of * operator= when T=EList. * * The 'cat_' fiends encodes a category. This makes it possible to * distinguish between object subgroups in the global memory tally. * * Memory allocation is lazy. Allocation is only triggered when the * user calls push_back, expand, resize, or another function that * increases the size of the list. This saves memory and also makes it * easier to deal with nested ELists, since the default constructor * doesn't set anything in stone. */ template class EList { public: /** * Allocate initial default of S elements. */ explicit EList() : cat_(0), allocCat_(-1), list_(NULL), sz_(S), cur_(0) { } /** * Allocate initial default of S elements. */ explicit EList(int cat) : cat_(cat), allocCat_(-1), list_(NULL), sz_(S), cur_(0) { assert_geq(cat, 0); } /** * Initially allocate given number of elements; should be > 0. */ explicit EList(size_t isz, int cat = 0) : cat_(cat), allocCat_(-1), list_(NULL), sz_(isz), cur_(0) { assert_geq(cat, 0); } /** * Copy from another EList using operator=. */ EList(const EList& o) : cat_(0), allocCat_(-1), list_(NULL), sz_(0), cur_(0) { *this = o; } /** * Copy from another EList using operator=. */ explicit EList(const EList& o, int cat) : cat_(cat), allocCat_(-1), list_(NULL), sz_(0), cur_(0) { *this = o; assert_geq(cat, 0); } /** * Destructor. */ ~EList() { free(); } /** * Make this object into a copy of o by allocat */ EList& operator=(const EList& o) { assert_eq(cat_, o.cat()); if(o.cur_ == 0) { // Nothing to copy cur_ = 0; return *this; } if(list_ == NULL) { // cat_ should already be set lazyInit(); } if(sz_ < o.cur_) expandNoCopy(o.cur_ + 1); assert_geq(sz_, o.cur_); cur_ = o.cur_; for(size_t i = 0; i < cur_; i++) { list_[i] = o.list_[i]; } return *this; } /** * Transfer the guts of another EList into this one without using * operator=, etc. We have to set EList o's list_ field to NULL to * avoid o's destructor from deleting list_ out from under us. */ void xfer(EList& o) { // What does it mean to transfer to a different-category list? assert_eq(cat_, o.cat()); // Can only transfer into an empty object free(); allocCat_ = cat_; list_ = o.list_; sz_ = o.sz_; cur_ = o.cur_; o.list_ = NULL; o.sz_ = o.cur_ = 0; o.allocCat_ = -1; } //Added by Joe void swap(EList& o) { assert_eq(cat_, o.cat()); T* temp_l = list_; size_t temp_sz = sz_; size_t temp_cur = cur_; list_ = o.list_; sz_ = o.sz_; cur_ = o.cur_; o.list_ = temp_l; o.sz_ = temp_sz; o.cur_ = temp_cur; } /** * Return number of elements. */ inline size_t size() const { return cur_; } /** * Return number of elements allocated. */ inline size_t capacity() const { return sz_; } /** * Return the total size in bytes occupied by this list. */ size_t totalSizeBytes() const { return 2 * sizeof(int) + 2 * sizeof(size_t) + cur_ * sizeof(T); } /** * Return the total capacity in bytes occupied by this list. */ size_t totalCapacityBytes() const { return 2 * sizeof(int) + 2 * sizeof(size_t) + sz_ * sizeof(T); } /** * Ensure that there is sufficient capacity to expand to include * 'thresh' more elements without having to expand. */ inline void ensure(size_t thresh) { if(list_ == NULL) lazyInit(); expandCopy(cur_ + thresh); } /** * Ensure that there is sufficient capacity to include 'newsz' elements. * If there isn't enough capacity right now, expand capacity to exactly * equal 'newsz'. */ inline void reserveExact(size_t newsz) { if(list_ == NULL) lazyInitExact(newsz); expandCopyExact(newsz); } /** * Return true iff there are no elements. */ inline bool empty() const { return cur_ == 0; } /** * Return true iff list hasn't been initialized yet. */ inline bool null() const { return list_ == NULL; } /** * Add an element to the back and immediately initialize it via * operator=. */ void push_back(const T& el) { if(list_ == NULL) lazyInit(); if(cur_ == sz_) expandCopy(sz_+1); list_[cur_++] = el; } void nullify() { free(); } /** * Add an element to the back. No intialization is done. */ void expand() { if(list_ == NULL) lazyInit(); if(cur_ == sz_) expandCopy(sz_+1); cur_++; } /** * Add an element to the back. No intialization is done. */ void fill(size_t begin, size_t end, const T& v) { assert_leq(begin, end); assert_leq(end, cur_); for(size_t i = begin; i < end; i++) { list_[i] = v; } } /** * Add an element to the back. No intialization is done. */ void fill(const T& v) { for(size_t i = 0; i < cur_; i++) { list_[i] = v; } } /** * Set all bits in specified range of elements in list array to 0. */ void fillZero(size_t begin, size_t end) { assert_leq(begin, end); memset(&list_[begin], 0, sizeof(T) * (end-begin)); } /** * Set all bits in the list array to 0. */ void fillZero() { memset(list_, 0, sizeof(T) * cur_); } /** * If size is less than requested size, resize up to at least sz * and set cur_ to requested sz. */ void resizeNoCopy(size_t sz) { if(sz > 0 && list_ == NULL) lazyInit(); if(sz <= cur_) { cur_ = sz; return; } if(sz_ < sz) expandNoCopy(sz); cur_ = sz; } /** * If size is less than requested size, resize up to at least sz * and set cur_ to requested sz. */ void resizeNoCopyExact(size_t sz) { if(sz > 0 && list_ == NULL) lazyInit(); if(sz <= cur_) { cur_ = sz; return; } if(sz_ < sz) expandNoCopyExact(sz); cur_ = sz; } /** * If size is less than requested size, resize up to at least sz * and set cur_ to requested sz. */ void resize(size_t sz) { if(sz > 0 && list_ == NULL) lazyInit(); if(sz <= cur_) { cur_ = sz; return; } if(sz_ < sz) { expandCopy(sz); } cur_ = sz; } /** * If size is less than requested size, resize up to exactly sz and set * cur_ to requested sz. */ void resizeExact(size_t sz) { if(sz > 0 && list_ == NULL) lazyInitExact(sz); if(sz <= cur_) { cur_ = sz; return; } if(sz_ < sz) expandCopyExact(sz); cur_ = sz; } /** * Erase element at offset idx. */ void erase(size_t idx) { assert_lt(idx, cur_); for(size_t i = idx; i < cur_-1; i++) { list_[i] = list_[i+1]; } cur_--; } /** * Erase range of elements starting at offset idx and going for len. */ void erase(size_t idx, size_t len) { assert_geq(len, 0); if(len == 0) { return; } assert_lt(idx, cur_); for(size_t i = idx; i < cur_-len; i++) { list_[i] = list_[i+len]; } cur_ -= len; } /** * Insert value 'el' at offset 'idx' */ void insert(const T& el, size_t idx) { if(list_ == NULL) lazyInit(); assert_leq(idx, cur_); if(cur_ == sz_) expandCopy(sz_+1); for(size_t i = cur_; i > idx; i--) { list_[i] = list_[i-1]; } list_[idx] = el; cur_++; } /** * Insert contents of list 'l' at offset 'idx' */ void insert(const EList& l, size_t idx) { if(list_ == NULL) lazyInit(); assert_lt(idx, cur_); if(l.cur_ == 0) return; if(cur_ + l.cur_ > sz_) expandCopy(cur_ + l.cur_); for(size_t i = cur_ + l.cur_ - 1; i > idx + (l.cur_ - 1); i--) { list_[i] = list_[i - l.cur_]; } for(size_t i = 0; i < l.cur_; i++) { list_[i+idx] = l.list_[i]; } cur_ += l.cur_; } /** * push first l_size objects of T array l to top of stack * added by Joe Paggi */ void push_back_array(const T* l, size_t l_size) { if(list_ == NULL) lazyInit(); if(l_size == 0) return; if(cur_ + l_size > sz_) expandCopy(cur_ + l_size); size_t bytes = l_size * sizeof(T); std::memcpy(list_ + cur_, l, bytes); cur_ += l_size; } /** * Remove an element from the top of the stack. */ void pop_back() { assert_gt(cur_, 0); cur_--; } /** * Make the stack empty. */ void clear() { cur_ = 0; // re-use stack memory // Don't clear heap; re-use it } /** * Get the element on the top of the stack. */ inline T& back() { assert_gt(cur_, 0); return list_[cur_-1]; } /** * Reverse list elements. */ void reverse() { if(cur_ > 1) { size_t n = cur_ >> 1; for(size_t i = 0; i < n; i++) { T tmp = list_[i]; list_[i] = list_[cur_ - i - 1]; list_[cur_ - i - 1] = tmp; } } } /** * Get the element on the top of the stack, const version. */ inline const T& back() const { assert_gt(cur_, 0); return list_[cur_-1]; } /** * Get the frontmost element (bottom of stack). */ inline T& front() { assert_gt(cur_, 0); return list_[0]; } /** * Get the element on the bottom of the stack, const version. */ inline const T& front() const { return front(); } inline T* begin() { assert_gt(cur_, 0); return &list_[0]; } inline const T* begin() const { return begin(); } inline T* end() { return begin() + cur_; } inline const T* end() const { return begin() + cur_; } /** * Return true iff this list and list o contain the same elements in the * same order according to type T's operator==. */ bool operator==(const EList& o) const { if(size() != o.size()) { return false; } for(size_t i = 0; i < size(); i++) { if(!(get(i) == o.get(i))) { return false; } } return true; } /** * Return true iff this list contains all of the elements in o according to * type T's operator==. */ bool isSuperset(const EList& o) const { if(o.size() > size()) { // This can't be a superset if the other set contains more elts return false; } // For each element in o for(size_t i = 0; i < o.size(); i++) { bool inthis = false; // Check if it's in this for(size_t j = 0; j < size(); j++) { if(o[i] == (*this)[j]) { inthis = true; break; } } if(!inthis) { return false; } } return true; } /** * Return a reference to the ith element. */ inline T& operator[](size_t i) { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline const T& operator[](size_t i) const { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline T& get(size_t i) { return operator[](i); } /** * Return a reference to the ith element. */ inline const T& get(size_t i) const { return operator[](i); } /** * Return a reference to the ith element. This version is not * inlined, which guarantees we can use it from the debugger. */ T& getSlow(size_t i) { return operator[](i); } /** * Return a reference to the ith element. This version is not * inlined, which guarantees we can use it from the debugger. */ const T& getSlow(size_t i) const { return operator[](i); } /** * Sort some of the contents. */ void sortPortion(size_t begin, size_t num) { assert_leq(begin+num, cur_); if(num < 2) return; std::sort(list_ + begin, list_ + begin + num); } /** * Shuffle a portion of the list. */ void shufflePortion(size_t begin, size_t num, RandomSource& rnd) { assert_leq(begin+num, cur_); if(num < 2) return; size_t left = num; for(size_t i = begin; i < begin + num - 1; i++) { uint32_t rndi = rnd.nextU32() % left; if(rndi > 0) { std::swap(list_[i], list_[i + rndi]); } left--; } } /** * Sort contents */ void sort() { sortPortion(0, cur_); } /** * Return true iff every element is < its successor. Only operator< is * used. */ bool sorted() const { for(size_t i = 1; i < cur_; i++) { if(!(list_[i-1] < list_[i])) { return false; } } return true; } /** * Delete element at position 'idx'; slide subsequent chars up. */ void remove(size_t idx) { assert_lt(idx, cur_); assert_gt(cur_, 0); for(size_t i = idx; i < cur_-1; i++) { list_[i] = list_[i+1]; } cur_--; } /** * Return a pointer to the beginning of the buffer. */ T *ptr() { return list_; } /** * Return a const pointer to the beginning of the buffer. */ const T *ptr() const { return list_; } /** * Set the memory category for this object. */ void setCat(int cat) { // What does it mean to set the category after the list_ is // already allocated? assert(null()); assert_gt(cat, 0); cat_ = cat; } /** * Return memory category. */ int cat() const { return cat_; } /** * Perform a binary search for the first element that is not less * than 'el'. Return cur_ if all elements are less than el. */ size_t bsearchLoBound(const T& el) const { size_t hi = cur_; size_t lo = 0; while(true) { if(lo == hi) { return lo; } size_t mid = lo + ((hi-lo)>>1); assert_neq(mid, hi); if(list_[mid] < el) { if(lo == mid) { return hi; } lo = mid; } else { hi = mid; } } } private: /** * Initialize memory for EList. */ void lazyInit() { assert(list_ == NULL); list_ = alloc(sz_); } /** * Initialize exactly the prescribed number of elements for EList. */ void lazyInitExact(size_t sz) { assert_gt(sz, 0); assert(list_ == NULL); sz_ = sz; list_ = alloc(sz); } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ T *alloc(size_t sz) { T* tmp = new T[sz]; assert(tmp != NULL); gMemTally.add(cat_, sz); allocCat_ = cat_; return tmp; } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ void free() { if(list_ != NULL) { assert_neq(-1, allocCat_); assert_eq(allocCat_, cat_); delete[] list_; gMemTally.del(cat_, sz_); list_ = NULL; sz_ = cur_ = 0; } } /** * Expand the list_ buffer until it has at least 'thresh' elements. Size * increases quadratically with number of expansions. Copy old contents * into new buffer using operator=. */ void expandCopy(size_t thresh) { if(thresh <= sz_) return; size_t newsz = (sz_ * 2)+1; while(newsz < thresh) newsz *= 2; expandCopyExact(newsz); } /** * Expand the list_ buffer until it has exactly 'newsz' elements. Copy * old contents into new buffer using operator=. */ void expandCopyExact(size_t newsz) { if(newsz <= sz_) return; T* tmp = alloc(newsz); assert(tmp != NULL); size_t cur = cur_; if(list_ != NULL) { for(size_t i = 0; i < cur_; i++) { // Note: operator= is used tmp[i] = list_[i]; } free(); } list_ = tmp; sz_ = newsz; cur_ = cur; } /** * Expand the list_ buffer until it has at least 'thresh' elements. * Size increases quadratically with number of expansions. Don't copy old * contents into the new buffer. */ void expandNoCopy(size_t thresh) { assert(list_ != NULL); if(thresh <= sz_) return; size_t newsz = (sz_ * 2)+1; while(newsz < thresh) newsz *= 2; expandNoCopyExact(newsz); } /** * Expand the list_ buffer until it has exactly 'newsz' elements. Don't * copy old contents into the new buffer. */ void expandNoCopyExact(size_t newsz) { assert(list_ != NULL); assert_gt(newsz, 0); free(); T* tmp = alloc(newsz); assert(tmp != NULL); list_ = tmp; sz_ = newsz; assert_gt(sz_, 0); } int cat_; // memory category, for accounting purposes int allocCat_; // category at time of allocation T *list_; // list pointer, returned from new[] size_t sz_; // capacity size_t cur_; // occupancy (AKA size) }; /** * An ELList is an expandable list of lists with these features: * * - Payload type of the inner list is a template parameter T. * - Initial size can be specified at construction time, otherwise * default of 128 is used. * - When allocated initially or when expanding, the new[] operator is * used, which in turn calls the default constructor for EList. * - Upon expansion, instead of copies, xfer is used. * - When the ELList is resized to a smaller size (or cleared, * which is like resizing to size 0), the underlying containing is * not reshaped. Thus, ELListss never release memory before * destruction. * * And these requirements: * * - Payload type T must have a default constructor. * */ template class ELList { public: /** * Allocate initial default of 128 elements. */ explicit ELList(int cat = 0) : cat_(cat), list_(NULL), sz_(S2), cur_(0) { assert_geq(cat, 0); } /** * Initially allocate given number of elements; should be > 0. */ explicit ELList(size_t isz, int cat = 0) : cat_(cat), list_(NULL), sz_(isz), cur_(0) { assert_gt(isz, 0); assert_geq(cat, 0); } /** * Copy from another ELList using operator=. */ ELList(const ELList& o) : cat_(0), list_(NULL), sz_(0), cur_(0) { *this = o; } /** * Copy from another ELList using operator=. */ explicit ELList(const ELList& o, int cat) : cat_(cat), list_(NULL), sz_(0), cur_(0) { *this = o; assert_geq(cat, 0); } /** * Destructor. */ ~ELList() { free(); } /** * Make this object into a copy of o by allocating enough memory to * fit the number of elements in o (note: the number of elements * may be substantially less than the memory allocated in o) and * using operator= to copy them over. */ ELList& operator=(const ELList& o) { assert_eq(cat_, o.cat()); if(list_ == NULL) { lazyInit(); } if(o.cur_ == 0) { cur_ = 0; return *this; } if(sz_ < o.cur_) expandNoCopy(o.cur_ + 1); assert_geq(sz_, o.cur_); cur_ = o.cur_; for(size_t i = 0; i < cur_; i++) { // Note: using operator=, not xfer assert_eq(list_[i].cat(), o.list_[i].cat()); list_[i] = o.list_[i]; } return *this; } /** * Transfer the guts of another EList into this one without using * operator=, etc. We have to set EList o's list_ field to NULL to * avoid o's destructor from deleting list_ out from under us. */ void xfer(ELList& o) { assert_eq(cat_, o.cat()); list_ = o.list_; // list_ is an array of ELists sz_ = o.sz_; cur_ = o.cur_; o.list_ = NULL; o.sz_ = o.cur_ = 0; } /** * Return number of elements. */ inline size_t size() const { return cur_; } /** * Return true iff there are no elements. */ inline bool empty() const { return cur_ == 0; } /** * Return true iff list hasn't been initialized yet. */ inline bool null() const { return list_ == NULL; } /** * Add an element to the back. No intialization is done. */ void expand() { if(list_ == NULL) lazyInit(); if(cur_ == sz_) expandCopy(sz_+1); cur_++; } /** * If size is less than requested size, resize up to at least sz * and set cur_ to requested sz. */ void resize(size_t sz) { if(sz > 0 && list_ == NULL) lazyInit(); if(sz <= cur_) { cur_ = sz; return; } if(sz_ < sz) { expandCopy(sz); } cur_ = sz; } /** * Make the stack empty. */ void clear() { cur_ = 0; // re-use stack memory // Don't clear heap; re-use it } /** * Get the element on the top of the stack. */ inline EList& back() { assert_gt(cur_, 0); return list_[cur_-1]; } /** * Get the element on the top of the stack, const version. */ inline const EList& back() const { assert_gt(cur_, 0); return list_[cur_-1]; } /** * Get the frontmost element (bottom of stack). */ inline EList& front() { assert_gt(cur_, 0); return list_[0]; } /** * Get the element on the bottom of the stack, const version. */ inline const EList& front() const { return front(); } /** * Return a reference to the ith element. */ inline EList& operator[](size_t i) { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline const EList& operator[](size_t i) const { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline EList& get(size_t i) { return operator[](i); } /** * Return a reference to the ith element. */ inline const EList& get(size_t i) const { return operator[](i); } /** * Return a reference to the ith element. This version is not * inlined, which guarantees we can use it from the debugger. */ EList& getSlow(size_t i) { return operator[](i); } /** * Return a reference to the ith element. This version is not * inlined, which guarantees we can use it from the debugger. */ const EList& getSlow(size_t i) const { return operator[](i); } /** * Return a pointer to the beginning of the buffer. */ EList *ptr() { return list_; } /** * Set the memory category for this object and all children. */ void setCat(int cat) { assert_gt(cat, 0); cat_ = cat; if(cat_ != 0) { for(size_t i = 0; i < sz_; i++) { assert(list_[i].null()); list_[i].setCat(cat_); } } } /** * Return memory category. */ int cat() const { return cat_; } protected: /** * Initialize memory for EList. */ void lazyInit() { assert(list_ == NULL); list_ = alloc(sz_); } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ EList *alloc(size_t sz) { assert_gt(sz, 0); EList *tmp = new EList[sz]; gMemTally.add(cat_, sz); if(cat_ != 0) { for(size_t i = 0; i < sz; i++) { assert(tmp[i].ptr() == NULL); tmp[i].setCat(cat_); } } return tmp; } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ void free() { if(list_ != NULL) { delete[] list_; gMemTally.del(cat_, sz_); list_ = NULL; } } /** * Expand the list_ buffer until it has at least 'thresh' elements. * Expansions are quadratic. Copy old contents into new buffer * using operator=. */ void expandCopy(size_t thresh) { assert(list_ != NULL); if(thresh <= sz_) return; size_t newsz = (sz_ * 2)+1; while(newsz < thresh) newsz *= 2; EList* tmp = alloc(newsz); if(list_ != NULL) { for(size_t i = 0; i < cur_; i++) { assert_eq(cat_, tmp[i].cat()); tmp[i].xfer(list_[i]); assert_eq(cat_, tmp[i].cat()); } free(); } list_ = tmp; sz_ = newsz; } /** * Expand the list_ buffer until it has at least 'thresh' elements. * Expansions are quadratic. Don't copy old contents over. */ void expandNoCopy(size_t thresh) { assert(list_ != NULL); if(thresh <= sz_) return; free(); size_t newsz = (sz_ * 2)+1; while(newsz < thresh) newsz *= 2; EList* tmp = alloc(newsz); list_ = tmp; sz_ = newsz; assert_gt(sz_, 0); } int cat_; // memory category, for accounting purposes EList *list_; // list pointer, returned from new[] size_t sz_; // capacity size_t cur_; // occupancy (AKA size) }; /** * An ELLList is an expandable list of expandable lists with these * features: * * - Payload type of the innermost list is a template parameter T. * - Initial size can be specified at construction time, otherwise * default of 128 is used. * - When allocated initially or when expanding, the new[] operator is * used, which in turn calls the default constructor for ELList. * - Upon expansion, instead of copies, xfer is used. * - When the ELLList is resized to a smaller size (or cleared, * which is like resizing to size 0), the underlying containing is * not reshaped. Thus, ELLListss never release memory before * destruction. * * And these requirements: * * - Payload type T must have a default constructor. * */ template class ELLList { public: /** * Allocate initial default of 128 elements. */ explicit ELLList(int cat = 0) : cat_(cat), list_(NULL), sz_(S3), cur_(0) { assert_geq(cat, 0); } /** * Initially allocate given number of elements; should be > 0. */ explicit ELLList(size_t isz, int cat = 0) : cat_(cat), list_(NULL), sz_(isz), cur_(0) { assert_geq(cat, 0); assert_gt(isz, 0); } /** * Copy from another ELLList using operator=. */ ELLList(const ELLList& o) : cat_(0), list_(NULL), sz_(0), cur_(0) { *this = o; } /** * Copy from another ELLList using operator=. */ explicit ELLList(const ELLList& o, int cat) : cat_(cat), list_(NULL), sz_(0), cur_(0) { *this = o; assert_geq(cat, 0); } /** * Destructor. */ ~ELLList() { free(); } /** * Make this object into a copy of o by allocating enough memory to * fit the number of elements in o (note: the number of elements * may be substantially less than the memory allocated in o) and * using operator= to copy them over. */ ELLList& operator=(const ELLList& o) { assert_eq(cat_, o.cat()); if(list_ == NULL) lazyInit(); if(o.cur_ == 0) { cur_ = 0; return *this; } if(sz_ < o.cur_) expandNoCopy(o.cur_ + 1); assert_geq(sz_, o.cur_); cur_ = o.cur_; for(size_t i = 0; i < cur_; i++) { // Note: using operator=, not xfer assert_eq(list_[i].cat(), o.list_[i].cat()); list_[i] = o.list_[i]; } return *this; } /** * Transfer the guts of another EList into this one without using * operator=, etc. We have to set EList o's list_ field to NULL to * avoid o's destructor from deleting list_ out from under us. */ void xfer(ELLList& o) { assert_eq(cat_, o.cat()); list_ = o.list_; // list_ is an array of ELists sz_ = o.sz_; cur_ = o.cur_; o.list_ = NULL; o.sz_ = o.cur_ = 0; } /** * Return number of elements. */ inline size_t size() const { return cur_; } /** * Return true iff there are no elements. */ inline bool empty() const { return cur_ == 0; } /** * Return true iff list hasn't been initialized yet. */ inline bool null() const { return list_ == NULL; } /** * Add an element to the back. No intialization is done. */ void expand() { if(list_ == NULL) lazyInit(); if(cur_ == sz_) expandCopy(sz_+1); cur_++; } /** * If size is less than requested size, resize up to at least sz * and set cur_ to requested sz. */ void resize(size_t sz) { if(sz > 0 && list_ == NULL) lazyInit(); if(sz <= cur_) { cur_ = sz; return; } if(sz_ < sz) expandCopy(sz); cur_ = sz; } /** * Make the stack empty. */ void clear() { cur_ = 0; // re-use stack memory // Don't clear heap; re-use it } /** * Get the element on the top of the stack. */ inline ELList& back() { assert_gt(cur_, 0); return list_[cur_-1]; } /** * Get the element on the top of the stack, const version. */ inline const ELList& back() const { assert_gt(cur_, 0); return list_[cur_-1]; } /** * Get the frontmost element (bottom of stack). */ inline ELList& front() { assert_gt(cur_, 0); return list_[0]; } /** * Get the element on the bottom of the stack, const version. */ inline const ELList& front() const { return front(); } /** * Return a reference to the ith element. */ inline ELList& operator[](size_t i) { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline const ELList& operator[](size_t i) const { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline ELList& get(size_t i) { return operator[](i); } /** * Return a reference to the ith element. */ inline const ELList& get(size_t i) const { return operator[](i); } /** * Return a reference to the ith element. This version is not * inlined, which guarantees we can use it from the debugger. */ ELList& getSlow(size_t i) { return operator[](i); } /** * Return a reference to the ith element. This version is not * inlined, which guarantees we can use it from the debugger. */ const ELList& getSlow(size_t i) const { return operator[](i); } /** * Return a pointer to the beginning of the buffer. */ ELList *ptr() { return list_; } /** * Set the memory category for this object and all children. */ void setCat(int cat) { assert_gt(cat, 0); cat_ = cat; if(cat_ != 0) { for(size_t i = 0; i < sz_; i++) { assert(list_[i].null()); list_[i].setCat(cat_); } } } /** * Return memory category. */ int cat() const { return cat_; } protected: /** * Initialize memory for EList. */ void lazyInit() { assert(null()); list_ = alloc(sz_); } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ ELList *alloc(size_t sz) { assert_gt(sz, 0); ELList *tmp = new ELList[sz]; gMemTally.add(cat_, sz); if(cat_ != 0) { for(size_t i = 0; i < sz; i++) { assert(tmp[i].ptr() == NULL); tmp[i].setCat(cat_); } } return tmp; } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ void free() { if(list_ != NULL) { delete[] list_; gMemTally.del(cat_, sz_); list_ = NULL; } } /** * Expand the list_ buffer until it has at least 'thresh' elements. * Expansions are quadratic. Copy old contents into new buffer * using operator=. */ void expandCopy(size_t thresh) { assert(list_ != NULL); if(thresh <= sz_) return; size_t newsz = (sz_ * 2)+1; while(newsz < thresh) newsz *= 2; ELList* tmp = alloc(newsz); if(list_ != NULL) { for(size_t i = 0; i < cur_; i++) { assert_eq(cat_, tmp[i].cat()); tmp[i].xfer(list_[i]); assert_eq(cat_, tmp[i].cat()); } free(); } list_ = tmp; sz_ = newsz; } /** * Expand the list_ buffer until it has at least 'thresh' elements. * Expansions are quadratic. Don't copy old contents over. */ void expandNoCopy(size_t thresh) { assert(list_ != NULL); if(thresh <= sz_) return; free(); size_t newsz = (sz_ * 2)+1; while(newsz < thresh) newsz *= 2; ELList* tmp = alloc(newsz); list_ = tmp; sz_ = newsz; assert_gt(sz_, 0); } int cat_; // memory category, for accounting purposes ELList *list_; // list pointer, returned from new[] size_t sz_; // capacity size_t cur_; // occupancy (AKA size) }; /** * Expandable set using a heap-allocated sorted array. * * Note that the copy constructor and operator= routines perform * shallow copies (w/ memcpy). */ template class ESet { public: /** * Allocate initial default of 128 elements. */ ESet(int cat = 0) : cat_(cat), list_(NULL), sz_(0), cur_(0) { if(sz_ > 0) { list_ = alloc(sz_); } } /** * Initially allocate given number of elements; should be > 0. */ ESet(size_t isz, int cat = 0) : cat_(cat), list_(NULL), sz_(isz), cur_(0) { assert_gt(isz, 0); if(sz_ > 0) { list_ = alloc(sz_); } } /** * Copy from another ESet. */ ESet(const ESet& o, int cat = 0) : cat_(cat), list_(NULL) { assert_eq(cat_, o.cat()); *this = o; } /** * Destructor. */ ~ESet() { free(); } /** * Copy contents of given ESet into this ESet. */ ESet& operator=(const ESet& o) { assert_eq(cat_, o.cat()); sz_ = o.sz_; cur_ = o.cur_; free(); if(sz_ > 0) { list_ = alloc(sz_); memcpy(list_, o.list_, cur_ * sizeof(T)); } else { list_ = NULL; } return *this; } /** * Return number of elements. */ size_t size() const { return cur_; } /** * Return the total size in bytes occupied by this set. */ size_t totalSizeBytes() const { return sizeof(int) + cur_ * sizeof(T) + 2 * sizeof(size_t); } /** * Return the total capacity in bytes occupied by this set. */ size_t totalCapacityBytes() const { return sizeof(int) + sz_ * sizeof(T) + 2 * sizeof(size_t); } /** * Return true iff there are no elements. */ bool empty() const { return cur_ == 0; } /** * Return true iff list isn't initialized yet. */ bool null() const { return list_ == NULL; } /** * Insert a new element into the set in sorted order. */ bool insert(const T& el) { size_t i = 0; if(cur_ == 0) { insert(el, 0); return true; } if(cur_ < 16) { // Linear scan i = scanLoBound(el); } else { // Binary search i = bsearchLoBound(el); } if(i < cur_ && list_[i] == el) return false; insert(el, i); return true; } /** * Return true iff this set contains 'el'. */ bool contains(const T& el) const { if(cur_ == 0) { return false; } else if(cur_ == 1) { return el == list_[0]; } size_t i; if(cur_ < 16) { // Linear scan i = scanLoBound(el); } else { // Binary search i = bsearchLoBound(el); } return i != cur_ && list_[i] == el; } /** * Remove element from set. */ void remove(const T& el) { size_t i; if(cur_ < 16) { // Linear scan i = scanLoBound(el); } else { // Binary search i = bsearchLoBound(el); } assert(i != cur_ && list_[i] == el); erase(i); } /** * Get */ T& get(const T& el) { size_t i; if(cur_ < 16) { // Linear scan i = scanLoBound(el); } else { // Binary search i = bsearchLoBound(el); } assert(i != cur_ && list_[i] == el); return list_[i]; } const T& get(const T& el) const { size_t i; if(cur_ < 16) { // Linear scan i = scanLoBound(el); } else { // Binary search i = bsearchLoBound(el); } assert(i != cur_ && list_[i] == el); return list_[i]; } /** * Return a reference to the ith element. */ inline T& operator[](size_t i) { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline const T& operator[](size_t i) const { assert_lt(i, cur_); return list_[i]; } /** * If size is less than requested size, resize up to at least sz * and set cur_ to requested sz. */ void resize(size_t sz) { if(sz <= cur_) return; if(sz_ < sz) expandCopy(sz); } /** * Clear set without deallocating (or setting) anything. */ void clear() { cur_ = 0; } /** * Return memory category. */ int cat() const { return cat_; } /** * Set the memory category for this object. */ void setCat(int cat) { cat_ = cat; } /** * Transfer the guts of another EList into this one without using * operator=, etc. We have to set EList o's list_ field to NULL to * avoid o's destructor from deleting list_ out from under us. */ void xfer(ESet& o) { // What does it mean to transfer to a different-category list? assert_eq(cat_, o.cat()); // Can only transfer into an empty object free(); list_ = o.list_; sz_ = o.sz_; cur_ = o.cur_; o.list_ = NULL; o.sz_ = o.cur_ = 0; } /** * Return a pointer to the beginning of the buffer. */ T *ptr() { return list_; } /** * Return a const pointer to the beginning of the buffer. */ const T *ptr() const { return list_; } private: /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ T *alloc(size_t sz) { assert_gt(sz, 0); T *tmp = new T[sz]; gMemTally.add(cat_, sz); return tmp; } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ void free() { if(list_ != NULL) { delete[] list_; gMemTally.del(cat_, sz_); list_ = NULL; } } /** * Simple linear scan that returns the index of the first element * of list_ that is not less than el, or cur_ if all elements are * less than el. */ size_t scanLoBound(const T& el) const { for(size_t i = 0; i < cur_; i++) { if(!(list_[i] < el)) { // Shouldn't be equal return i; } } return cur_; } /** * Perform a binary search for the first element that is not less * than 'el'. Return cur_ if all elements are less than el. */ size_t bsearchLoBound(const T& el) const { size_t hi = cur_; size_t lo = 0; while(true) { if(lo == hi) { #ifndef NDEBUG if((rand() % 10) == 0) { assert_eq(lo, scanLoBound(el)); } #endif return lo; } size_t mid = lo + ((hi-lo)>>1); assert_neq(mid, hi); if(list_[mid] < el) { if(lo == mid) { #ifndef NDEBUG if((rand() % 10) == 0) { assert_eq(hi, scanLoBound(el)); } #endif return hi; } lo = mid; } else { hi = mid; } } } /** * Return true if sorted, assert otherwise. */ bool sorted() const { if(cur_ <= 1) return true; #ifndef NDEBUG if((rand() % 20) == 0) { for(size_t i = 0; i < cur_-1; i++) { assert(list_[i] < list_[i+1]); } } #endif return true; } /** * Insert value 'el' at offset 'idx'. It's OK to insert at cur_, * which is equivalent to appending. */ void insert(const T& el, size_t idx) { assert_leq(idx, cur_); if(cur_ == sz_) { expandCopy(sz_+1); assert(sorted()); } for(size_t i = cur_; i > idx; i--) { list_[i] = list_[i-1]; } list_[idx] = el; cur_++; assert(sorted()); } /** * Erase element at offset idx. */ void erase(size_t idx) { assert_lt(idx, cur_); for(size_t i = idx; i < cur_-1; i++) { list_[i] = list_[i+1]; } cur_--; assert(sorted()); } /** * Expand the list_ buffer until it has at least 'thresh' elements. * Expansions are quadratic. */ void expandCopy(size_t thresh) { if(thresh <= sz_) return; size_t newsz = (sz_ * 2)+1; while(newsz < thresh) { newsz *= 2; } T* tmp = alloc(newsz); for(size_t i = 0; i < cur_; i++) { tmp[i] = list_[i]; } free(); list_ = tmp; sz_ = newsz; } int cat_; // memory category, for accounting purposes T *list_; // list pointer, returned from new[] size_t sz_; // capacity size_t cur_; // occupancy (AKA size) }; template class ELSet { public: /** * Allocate initial default of 128 elements. */ explicit ELSet(int cat = 0) : cat_(cat), list_(NULL), sz_(S), cur_(0) { assert_geq(cat, 0); } /** * Initially allocate given number of elements; should be > 0. */ explicit ELSet(size_t isz, int cat = 0) : cat_(cat), list_(NULL), sz_(isz), cur_(0) { assert_gt(isz, 0); assert_geq(cat, 0); } /** * Copy from another ELList using operator=. */ ELSet(const ELSet& o) : cat_(0), list_(NULL), sz_(0), cur_(0) { *this = o; } /** * Copy from another ELList using operator=. */ explicit ELSet(const ELSet& o, int cat) : cat_(cat), list_(NULL), sz_(0), cur_(0) { *this = o; assert_geq(cat, 0); } /** * Destructor. */ ~ELSet() { free(); } /** * Make this object into a copy of o by allocating enough memory to * fit the number of elements in o (note: the number of elements * may be substantially less than the memory allocated in o) and * using operator= to copy them over. */ ELSet& operator=(const ELSet& o) { assert_eq(cat_, o.cat()); if(list_ == NULL) { lazyInit(); } if(o.cur_ == 0) { cur_ = 0; return *this; } if(sz_ < o.cur_) expandNoCopy(o.cur_ + 1); assert_geq(sz_, o.cur_); cur_ = o.cur_; for(size_t i = 0; i < cur_; i++) { // Note: using operator=, not xfer assert_eq(list_[i].cat(), o.list_[i].cat()); list_[i] = o.list_[i]; } return *this; } /** * Transfer the guts of another ESet into this one without using * operator=, etc. We have to set ESet o's list_ field to NULL to * avoid o's destructor from deleting list_ out from under us. */ void xfer(ELSet& o) { assert_eq(cat_, o.cat()); list_ = o.list_; // list_ is an array of ESets sz_ = o.sz_; cur_ = o.cur_; o.list_ = NULL; o.sz_ = o.cur_ = 0; } /** * Return number of elements. */ inline size_t size() const { return cur_; } /** * Return true iff there are no elements. */ inline bool empty() const { return cur_ == 0; } /** * Return true iff list hasn't been initialized yet. */ inline bool null() const { return list_ == NULL; } /** * Add an element to the back. No intialization is done. */ void expand() { if(list_ == NULL) lazyInit(); if(cur_ == sz_) expandCopy(sz_+1); cur_++; } /** * If size is less than requested size, resize up to at least sz * and set cur_ to requested sz. */ void resize(size_t sz) { if(sz > 0 && list_ == NULL) lazyInit(); if(sz <= cur_) { cur_ = sz; return; } if(sz_ < sz) { expandCopy(sz); } cur_ = sz; } /** * Make the stack empty. */ void clear() { cur_ = 0; // re-use stack memory // Don't clear heap; re-use it } /** * Get the element on the top of the stack. */ inline ESet& back() { assert_gt(cur_, 0); return list_[cur_-1]; } /** * Get the element on the top of the stack, const version. */ inline const ESet& back() const { assert_gt(cur_, 0); return list_[cur_-1]; } /** * Get the frontmost element (bottom of stack). */ inline ESet& front() { assert_gt(cur_, 0); return list_[0]; } /** * Get the element on the bottom of the stack, const version. */ inline const ESet& front() const { return front(); } /** * Return a reference to the ith element. */ inline ESet& operator[](size_t i) { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline const ESet& operator[](size_t i) const { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline ESet& get(size_t i) { return operator[](i); } /** * Return a reference to the ith element. */ inline const ESet& get(size_t i) const { return operator[](i); } /** * Return a reference to the ith element. This version is not * inlined, which guarantees we can use it from the debugger. */ ESet& getSlow(size_t i) { return operator[](i); } /** * Return a reference to the ith element. This version is not * inlined, which guarantees we can use it from the debugger. */ const ESet& getSlow(size_t i) const { return operator[](i); } /** * Return a pointer to the beginning of the buffer. */ ESet *ptr() { return list_; } /** * Return a const pointer to the beginning of the buffer. */ const ESet *ptr() const { return list_; } /** * Set the memory category for this object and all children. */ void setCat(int cat) { assert_gt(cat, 0); cat_ = cat; if(cat_ != 0) { for(size_t i = 0; i < sz_; i++) { assert(list_[i].null()); list_[i].setCat(cat_); } } } /** * Return memory category. */ int cat() const { return cat_; } protected: /** * Initialize memory for ELSet. */ void lazyInit() { assert(list_ == NULL); list_ = alloc(sz_); } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ ESet *alloc(size_t sz) { assert_gt(sz, 0); ESet *tmp = new ESet[sz]; gMemTally.add(cat_, sz); if(cat_ != 0) { for(size_t i = 0; i < sz; i++) { assert(tmp[i].ptr() == NULL); tmp[i].setCat(cat_); } } return tmp; } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ void free() { if(list_ != NULL) { delete[] list_; gMemTally.del(cat_, sz_); list_ = NULL; } } /** * Expand the list_ buffer until it has at least 'thresh' elements. * Expansions are quadratic. Copy old contents into new buffer * using operator=. */ void expandCopy(size_t thresh) { assert(list_ != NULL); if(thresh <= sz_) return; size_t newsz = (sz_ * 2)+1; while(newsz < thresh) newsz *= 2; ESet* tmp = alloc(newsz); if(list_ != NULL) { for(size_t i = 0; i < cur_; i++) { assert_eq(cat_, tmp[i].cat()); tmp[i].xfer(list_[i]); assert_eq(cat_, tmp[i].cat()); } free(); } list_ = tmp; sz_ = newsz; } /** * Expand the list_ buffer until it has at least 'thresh' elements. * Expansions are quadratic. Don't copy old contents over. */ void expandNoCopy(size_t thresh) { assert(list_ != NULL); if(thresh <= sz_) return; free(); size_t newsz = (sz_ * 2)+1; while(newsz < thresh) newsz *= 2; ESet* tmp = alloc(newsz); list_ = tmp; sz_ = newsz; assert_gt(sz_, 0); } int cat_; // memory category, for accounting purposes ESet *list_; // list pointer, returned from new[] size_t sz_; // capacity size_t cur_; // occupancy (AKA size) }; /** * Expandable map using a heap-allocated sorted array. * * Note that the copy constructor and operator= routines perform * shallow copies (w/ memcpy). */ template class EMap { public: /** * Allocate initial default of 128 elements. */ EMap(int cat = 0) : cat_(cat), list_(NULL), sz_(128), cur_(0) { list_ = alloc(sz_); } /** * Initially allocate given number of elements; should be > 0. */ EMap(size_t isz, int cat = 0) : cat_(cat), list_(NULL), sz_(isz), cur_(0) { assert_gt(isz, 0); list_ = alloc(sz_); } /** * Copy from another ESet. */ EMap(const EMap& o) : list_(NULL) { *this = o; } /** * Destructor. */ ~EMap() { free(); } /** * Copy contents of given ESet into this ESet. */ EMap& operator=(const EMap& o) { sz_ = o.sz_; cur_ = o.cur_; free(); list_ = alloc(sz_); memcpy(list_, o.list_, cur_ * sizeof(std::pair)); return *this; } /** * Return number of elements. */ size_t size() const { return cur_; } /** * Return the total size in bytes occupied by this map. */ size_t totalSizeBytes() const { return sizeof(int) + 2 * sizeof(size_t) + cur_ * sizeof(std::pair); } /** * Return the total capacity in bytes occupied by this map. */ size_t totalCapacityBytes() const { return sizeof(int) + 2 * sizeof(size_t) + sz_ * sizeof(std::pair); } /** * Return true iff there are no elements. */ bool empty() const { return cur_ == 0; } /** * Insert a new element into the set in sorted order. */ bool insert(const std::pair& el) { size_t i = 0; if(cur_ == 0) { insert(el, 0); return true; } if(cur_ < 16) { // Linear scan i = scanLoBound(el.first); } else { // Binary search i = bsearchLoBound(el.first); } if(list_[i] == el) return false; // already there insert(el, i); return true; // not already there } /** * Return true iff this set contains 'el'. */ bool contains(const K& el) const { if(cur_ == 0) return false; else if(cur_ == 1) return el == list_[0].first; size_t i; if(cur_ < 16) { // Linear scan i = scanLoBound(el); } else { // Binary search i = bsearchLoBound(el); } return i != cur_ && list_[i].first == el; } /** * Return true iff this set contains 'el'. */ bool containsEx(const K& el, size_t& i) const { if(cur_ == 0) return false; else if(cur_ == 1) { i = 0; return el == list_[0].first; } if(cur_ < 16) { // Linear scan i = scanLoBound(el); } else { // Binary search i = bsearchLoBound(el); } return i != cur_ && list_[i].first == el; } /** * Remove element from set. */ void remove(const K& el) { size_t i; if(cur_ < 16) { // Linear scan i = scanLoBound(el); } else { // Binary search i = bsearchLoBound(el); } assert(i != cur_ && list_[i].first == el); erase(i); } /** * If size is less than requested size, resize up to at least sz * and set cur_ to requested sz. */ void resize(size_t sz) { if(sz <= cur_) return; if(sz_ < sz) expandCopy(sz); } /** * Get the ith key, value pair in the map. */ const std::pair& get(size_t i) const { assert_lt(i, cur_); return list_[i]; } /** * Get the ith key, value pair in the map. */ const std::pair& operator[](size_t i) const { return get(i); } /** * Clear set without deallocating (or setting) anything. */ void clear() { cur_ = 0; } private: /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ std::pair *alloc(size_t sz) { assert_gt(sz, 0); std::pair *tmp = new std::pair[sz]; gMemTally.add(cat_, sz); return tmp; } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ void free() { if(list_ != NULL) { delete[] list_; gMemTally.del(cat_, sz_); list_ = NULL; } } /** * Simple linear scan that returns the index of the first element * of list_ that is not less than el, or cur_ if all elements are * less than el. */ size_t scanLoBound(const K& el) const { for(size_t i = 0; i < cur_; i++) { if(!(list_[i].first < el)) { // Shouldn't be equal return i; } } return cur_; } /** * Perform a binary search for the first element that is not less * than 'el'. Return cur_ if all elements are less than el. */ size_t bsearchLoBound(const K& el) const { size_t hi = cur_; size_t lo = 0; while(true) { if(lo == hi) { #ifndef NDEBUG if((rand() % 10) == 0) { assert_eq(lo, scanLoBound(el)); } #endif return lo; } size_t mid = lo + ((hi-lo)>>1); assert_neq(mid, hi); if(list_[mid].first < el) { if(lo == mid) { #ifndef NDEBUG if((rand() % 10) == 0) { assert_eq(hi, scanLoBound(el)); } #endif return hi; } lo = mid; } else { hi = mid; } } } /** * Return true if sorted, assert otherwise. */ bool sorted() const { if(cur_ <= 1) return true; #ifndef NDEBUG for(size_t i = 0; i < cur_-1; i++) { assert(!(list_[i] == list_[i+1])); assert(list_[i] < list_[i+1]); } #endif return true; } /** * Insert value 'el' at offset 'idx'. It's OK to insert at cur_, * which is equivalent to appending. */ void insert(const std::pair& el, size_t idx) { assert_leq(idx, cur_); if(cur_ == sz_) { expandCopy(sz_+1); } for(size_t i = cur_; i > idx; i--) { list_[i] = list_[i-1]; } list_[idx] = el; assert(idx == cur_ || list_[idx] < list_[idx+1]); cur_++; assert(sorted()); } /** * Erase element at offset idx. */ void erase(size_t idx) { assert_lt(idx, cur_); for(size_t i = idx; i < cur_-1; i++) { list_[i] = list_[i+1]; } cur_--; assert(sorted()); } /** * Expand the list_ buffer until it has at least 'thresh' elements. * Expansions are quadratic. */ void expandCopy(size_t thresh) { if(thresh <= sz_) return; size_t newsz = sz_ * 2; while(newsz < thresh) newsz *= 2; std::pair* tmp = alloc(newsz); for(size_t i = 0; i < cur_; i++) { tmp[i] = list_[i]; } free(); list_ = tmp; sz_ = newsz; } int cat_; // memory category, for accounting purposes std::pair *list_; // list pointer, returned from new[] size_t sz_; // capacity size_t cur_; // occupancy (AKA size) }; /** * A class that allows callers to create objects that are referred to by ID. * Objects should not be referred to via pointers or references, since they * are stored in an expandable buffer that might be resized and thereby moved * to another address. */ template class EFactory { public: explicit EFactory(size_t isz, int cat = 0) : l_(isz, cat) { } explicit EFactory(int cat = 0) : l_(cat) { } /** * Clear the list. */ void clear() { l_.clear(); } /** * Add one additional item to the list and return its ID. */ size_t alloc() { l_.expand(); return l_.size()-1; } /** * Return the number of items in the list. */ size_t size() const { return l_.size(); } /** * Return the number of items in the factory. */ size_t totalSizeBytes() const { return l_.totalSizeBytes(); } /** * Return the total capacity in bytes occupied by this factory. */ size_t totalCapacityBytes() const { return l_.totalCapacityBytes(); } /** * Resize the list. */ void resize(size_t sz) { l_.resize(sz); } /** * Return true iff the list is empty. */ bool empty() const { return size() == 0; } /** * Shrink the list such that the topmost (most recently allocated) element * is removed. */ void pop() { l_.resize(l_.size()-1); } /** * Return mutable list item at offset 'off' */ T& operator[](size_t off) { return l_[off]; } /** * Return immutable list item at offset 'off' */ const T& operator[](size_t off) const { return l_[off]; } protected: EList l_; }; /** * An expandable bit vector based on EList */ template class EBitList { public: explicit EBitList(size_t isz, int cat = 0) : l_(isz, cat) { reset(); } explicit EBitList(int cat = 0) : l_(cat) { reset(); } /** * Reset to empty state. */ void clear() { reset(); } /** * Reset to empty state. */ void reset() { l_.clear(); max_ = std::numeric_limits::max(); } /** * Set a bit. */ void set(size_t off) { resize(off); l_[off >> 3] |= (1 << (off & 7)); if(off > max_ || max_ == std::numeric_limits::max()) { max_ = off; } } /** * Return mutable list item at offset 'off' */ bool test(size_t off) const { if((size_t)(off >> 3) >= l_.size()) { return false; } return (l_[off >> 3] & (1 << (off & 7))) != 0; } /** * Return size of the underlying byte array. */ size_t size() const { return l_.size(); } /** * Resize to accomodate at least the given number of bits. */ void resize(size_t off) { if((size_t)(off >> 3) >= l_.size()) { size_t oldsz = l_.size(); l_.resize((off >> 3) + 1); for(size_t i = oldsz; i < l_.size(); i++) { l_[i] = 0; } } } /** * Return max set bit. */ size_t max() const { return max_; } protected: EList l_; size_t max_; }; /** * Implements a min-heap. */ template class EHeap { public: /** * Add the element to the next available leaf position and percolate up. */ void insert(T o) { size_t pos = l_.size(); l_.push_back(o); while(pos > 0) { size_t parent = (pos-1) >> 1; if(l_[pos] < l_[parent]) { T tmp(l_[pos]); l_[pos] = l_[parent]; l_[parent] = tmp; pos = parent; } else break; } assert(repOk()); } /** * Return the topmost element. */ T top() { assert_gt(l_.size(), 0); return l_[0]; } /** * Remove the topmost element. */ T pop() { assert_gt(l_.size(), 0); T ret = l_[0]; l_[0] = l_[l_.size()-1]; l_.resize(l_.size()-1); size_t cur = 0; while(true) { size_t c1 = ((cur+1) << 1) - 1; size_t c2 = c1 + 1; if(c2 < l_.size()) { if(l_[c1] < l_[cur] && l_[c1] <= l_[c2]) { T tmp(l_[c1]); l_[c1] = l_[cur]; l_[cur] = tmp; cur = c1; } else if(l_[c2] < l_[cur]) { T tmp(l_[c2]); l_[c2] = l_[cur]; l_[cur] = tmp; cur = c2; } else { break; } } else if(c1 < l_.size()) { if(l_[c1] < l_[cur]) { T tmp(l_[c1]); l_[c1] = l_[cur]; l_[cur] = tmp; cur = c1; } else { break; } } else { break; } } assert(repOk()); return ret; } /** * Return number of elements in the heap. */ size_t size() const { return l_.size(); } /** * Return the total size in bytes occupied by this heap. */ size_t totalSizeBytes() const { return l_.totalSizeBytes(); } /** * Return the total capacity in bytes occupied by this heap. */ size_t totalCapacityBytes() const { return l_.totalCapacityBytes(); } /** * Return true when heap is empty. */ bool empty() const { return l_.empty(); } /** * Return element at offset i. */ const T& operator[](size_t i) const { return l_[i]; } #ifndef NDEBUG /** * Check that heap property holds. */ bool repOk() const { if(empty()) return true; return repOkNode(0); } /** * Check that heap property holds at and below this node. */ bool repOkNode(size_t cur) const { size_t c1 = ((cur+1) << 1) - 1; size_t c2 = c1 + 1; if(c1 < l_.size()) { assert_leq(l_[cur], l_[c1]); } if(c2 < l_.size()) { assert_leq(l_[cur], l_[c2]); } if(c2 < l_.size()) { return repOkNode(c1) && repOkNode(c2); } else if(c1 < l_.size()) { return repOkNode(c1); } return true; } #endif /** * Clear the heap so that it's empty. */ void clear() { l_.clear(); } protected: EList l_; }; /** * Dispenses pages of memory for all the lists in the cache, including * the sequence-to-range map, the range list, the edits list, and the * offsets list. All lists contend for the same pool of memory. */ class Pool { public: Pool( uint64_t bytes, uint32_t pagesz, int cat = 0) : cat_(cat), cur_(0), bytes_(bytes), pagesz_(pagesz), pages_(cat) { for(size_t i = 0; i < ((bytes+pagesz-1)/pagesz); i++) { pages_.push_back(new uint8_t[pagesz]); gMemTally.add(cat, pagesz); assert(pages_.back() != NULL); } assert(repOk()); } /** * Free each page. */ ~Pool() { for(size_t i = 0; i < pages_.size(); i++) { assert(pages_[i] != NULL); delete[] pages_[i]; gMemTally.del(cat_, pagesz_); } } /** * Allocate one page, or return NULL if no pages are left. */ uint8_t * alloc() { assert(repOk()); if(cur_ == pages_.size()) return NULL; return pages_[cur_++]; } bool full() { return cur_ == pages_.size(); } /** * Clear the pool so that no pages are considered allocated. */ void clear() { cur_ = 0; assert(repOk()); } /** * Reset the Pool to be as though */ void free() { // Currently a no-op because the only freeing method supported // now is to clear the entire pool } #ifndef NDEBUG /** * Check that pool is internally consistent. */ bool repOk() const { assert_leq(cur_, pages_.size()); assert(!pages_.empty()); assert_gt(bytes_, 0); assert_gt(pagesz_, 0); return true; } #endif public: int cat_; // memory category, for accounting purposes uint32_t cur_; // next page to hand out const uint64_t bytes_; // total bytes in the pool const uint32_t pagesz_; // size of a single page EList pages_; // the pages themselves }; /** * An expandable list backed by a pool. */ template class PList { #define PLIST_PER_PAGE (S / sizeof(T)) public: /** * Initialize the current-edit pointer to 0 and set the number of * edits per memory page. */ PList(int cat = 0) : cur_(0), curPage_(0), pages_(cat) { } /** * Add 1 object to the list. */ bool add(Pool& p, const T& o) { assert(repOk()); if(!ensure(p, 1)) return false; if(cur_ == PLIST_PER_PAGE) { cur_ = 0; curPage_++; } assert_lt(curPage_, pages_.size()); assert(repOk()); assert_lt(cur_, PLIST_PER_PAGE); pages_[curPage_][cur_++] = o; return true; } /** * Add a list of objects to the list. */ bool add(Pool& p, const EList& os) { if(!ensure(p, os.size())) return false; for(size_t i = 0; i < os.size(); i++) { if(cur_ == PLIST_PER_PAGE) { cur_ = 0; curPage_++; } assert_lt(curPage_, pages_.size()); assert(repOk()); assert_lt(cur_, PLIST_PER_PAGE); pages_[curPage_][cur_++] = os[i]; } return true; } /** * Add a list of objects to the list. */ bool copy( Pool& p, const PList& src, size_t i, size_t len) { if(!ensure(p, src.size())) return false; for(size_t i = 0; i < src.size(); i++) { if(cur_ == PLIST_PER_PAGE) { cur_ = 0; curPage_++; } assert_lt(curPage_, pages_.size()); assert(repOk()); assert_lt(cur_, PLIST_PER_PAGE); pages_[curPage_][cur_++] = src[i]; } return true; } /** * Add 'num' objects, all equal to 'o' to the list. */ bool addFill(Pool& p, size_t num, const T& o) { if(!ensure(p, num)) return false; for(size_t i = 0; i < num; i++) { if(cur_ == PLIST_PER_PAGE) { cur_ = 0; curPage_++; } assert_lt(curPage_, pages_.size()); assert(repOk()); assert_lt(cur_, PLIST_PER_PAGE); pages_[curPage_][cur_++] = o; } return true; } /** * Free all pages associated with the list. */ void clear() { pages_.clear(); cur_ = curPage_ = 0; } #ifndef NDEBUG /** * Check that list is internally consistent. */ bool repOk() const { assert(pages_.size() == 0 || curPage_ < pages_.size()); assert_leq(cur_, PLIST_PER_PAGE); return true; } #endif /** * Return the number of elements in the list. */ size_t size() const { return curPage_ * PLIST_PER_PAGE + cur_; } /** * Return true iff the PList has no elements. */ bool empty() const { return size() == 0; } /** * Get the ith element added to the list. */ inline const T& getConst(size_t i) const { assert_lt(i, size()); size_t page = i / PLIST_PER_PAGE; size_t elt = i % PLIST_PER_PAGE; return pages_[page][elt]; } /** * Get the ith element added to the list. */ inline T& get(size_t i) { assert_lt(i, size()); size_t page = i / PLIST_PER_PAGE; size_t elt = i % PLIST_PER_PAGE; assert_lt(page, pages_.size()); assert(page < pages_.size()-1 || elt < cur_); return pages_[page][elt]; } /** * Get the most recently added element. */ inline T& back() { size_t page = (size()-1) / PLIST_PER_PAGE; size_t elt = (size()-1) % PLIST_PER_PAGE; assert_lt(page, pages_.size()); assert(page < pages_.size()-1 || elt < cur_); return pages_[page][elt]; } /** * Get const version of the most recently added element. */ inline const T& back() const { size_t page = (size()-1) / PLIST_PER_PAGE; size_t elt = (size()-1) % PLIST_PER_PAGE; assert_lt(page, pages_.size()); assert(page < pages_.size()-1 || elt < cur_); return pages_[page][elt]; } /** * Get the element most recently added to the list. */ T& last() { assert(!pages_.empty()); assert_gt(PLIST_PER_PAGE, 0); if(cur_ == 0) { assert_gt(pages_.size(), 1); return pages_[pages_.size()-2][PLIST_PER_PAGE-1]; } else { return pages_.back()[cur_-1]; } } /** * Return true iff 'num' additional objects will fit in the pages * allocated to the list. If more pages are needed, they are * added if possible. */ bool ensure(Pool& p, size_t num) { assert(repOk()); if(num == 0) return true; // Allocation of the first page if(pages_.size() == 0) { if(expand(p) == NULL) { return false; } assert_eq(1, pages_.size()); } size_t cur = cur_; size_t curPage = curPage_; while(cur + num > PLIST_PER_PAGE) { assert_lt(curPage, pages_.size()); if(curPage == pages_.size()-1 && expand(p) == NULL) { return false; } num -= (PLIST_PER_PAGE - cur); cur = 0; curPage++; } return true; } protected: /** * Expand our page supply by 1 */ T* expand(Pool& p) { T* newpage = (T*)p.alloc(); if(newpage == NULL) { return NULL; } pages_.push_back(newpage); return pages_.back(); } size_t cur_; // current elt within page size_t curPage_; // current page EList pages_; // the pages }; /** * A slice of an EList. */ template class EListSlice { public: EListSlice() : i_(0), len_(0), list_() { } EListSlice( EList& list, size_t i, size_t len) : i_(i), len_(len), list_(&list) { } /** * Initialize from a piece of another PListSlice. */ void init(const EListSlice& sl, size_t first, size_t last) { assert_gt(last, first); assert_leq(last - first, sl.len_); i_ = sl.i_ + first; len_ = last - first; list_ = sl.list_; } /** * Reset state to be empty. */ void reset() { i_ = len_ = 0; list_ = NULL; } /** * Get the ith element of the slice. */ inline const T& get(size_t i) const { assert(valid()); assert_lt(i, len_); return list_->get(i + i_); } /** * Get the ith element of the slice. */ inline T& get(size_t i) { assert(valid()); assert_lt(i, len_); return list_->get(i + i_); } /** * Return a reference to the ith element. */ inline T& operator[](size_t i) { assert(valid()); assert_lt(i, len_); return list_->get(i + i_); } /** * Return a reference to the ith element. */ inline const T& operator[](size_t i) const { assert(valid()); assert_lt(i, len_); return list_->get(i + i_); } /** * Return true iff this slice is initialized. */ bool valid() const { return len_ != 0; } /** * Return number of elements in the slice. */ size_t size() const { return len_; } #ifndef NDEBUG /** * Ensure that the PListSlice is internally consistent and * consistent with the backing PList. */ bool repOk() const { assert_leq(i_ + len_, list_->size()); return true; } #endif /** * Return true iff this slice refers to the same slice of the same * list as the given slice. */ bool operator==(const EListSlice& sl) const { return i_ == sl.i_ && len_ == sl.len_ && list_ == sl.list_; } /** * Return false iff this slice refers to the same slice of the same * list as the given slice. */ bool operator!=(const EListSlice& sl) const { return !(*this == sl); } /** * Set the length. This could leave things inconsistent (e.g. could * include elements that fall off the end of list_). */ void setLength(size_t nlen) { len_ = (uint32_t)nlen; } protected: size_t i_; size_t len_; EList* list_; }; /** * A slice of a PList. */ template class PListSlice { public: PListSlice() : i_(0), len_(0), list_() { } PListSlice( PList& list, TIndexOffU i, TIndexOffU len) : i_(i), len_(len), list_(&list) { } /** * Initialize from a piece of another PListSlice. */ void init(const PListSlice& sl, size_t first, size_t last) { assert_gt(last, first); assert_leq(last - first, sl.len_); i_ = (uint32_t)(sl.i_ + first); len_ = (uint32_t)(last - first); list_ = sl.list_; } /** * Reset state to be empty. */ void reset() { i_ = len_ = 0; list_ = NULL; } /** * Get the ith element of the slice. */ inline const T& get(size_t i) const { assert(valid()); assert_lt(i, len_); return list_->get(i+i_); } /** * Get the ith element of the slice. */ inline T& get(size_t i) { assert(valid()); assert_lt(i, len_); return list_->get(i+i_); } /** * Return a reference to the ith element. */ inline T& operator[](size_t i) { assert(valid()); assert_lt(i, len_); return list_->get(i+i_); } /** * Return a reference to the ith element. */ inline const T& operator[](size_t i) const { assert(valid()); assert_lt(i, len_); return list_->get(i+i_); } /** * Return true iff this slice is initialized. */ bool valid() const { return len_ != 0; } /** * Return number of elements in the slice. */ size_t size() const { return len_; } #ifndef NDEBUG /** * Ensure that the PListSlice is internally consistent and * consistent with the backing PList. */ bool repOk() const { assert_leq(i_ + len_, list_->size()); return true; } #endif /** * Return true iff this slice refers to the same slice of the same * list as the given slice. */ bool operator==(const PListSlice& sl) const { return i_ == sl.i_ && len_ == sl.len_ && list_ == sl.list_; } /** * Return false iff this slice refers to the same slice of the same * list as the given slice. */ bool operator!=(const PListSlice& sl) const { return !(*this == sl); } /** * Set the length. This could leave things inconsistent (e.g. could * include elements that fall off the end of list_). */ void setLength(size_t nlen) { len_ = (uint32_t)nlen; } protected: uint32_t i_; uint32_t len_; PList* list_; }; /** * A Red-Black tree node. Links to parent & left and right children. * Key and Payload are of types K and P. Node total ordering is based * on K's total ordering. K must implement <, == and > operators. */ template // K=key, P=payload class RedBlackNode { typedef RedBlackNode TNode; public: TNode *parent; // parent TNode *left; // left child TNode *right; // right child bool red; // true -> red, false -> black K key; // key, for ordering P payload; // payload (i.e. value) /** * Return the parent of this node's parent, or NULL if none exists. */ RedBlackNode *grandparent() { return parent != NULL ? parent->parent : NULL; } /** * Return the sibling of this node's parent, or NULL if none exists. */ RedBlackNode *uncle() { if(parent == NULL) return NULL; // no parent if(parent->parent == NULL) return NULL; // parent has no siblings return (parent->parent->left == parent) ? parent->parent->right : parent->parent->left; } /** * Return true iff this node is its parent's left child. */ bool isLeftChild() const { assert(parent != NULL); return parent->left == this; } /** * Return true iff this node is its parent's right child. */ bool isRightChild() const { assert(parent != NULL); return parent->right == this; } /** * Return true iff this node is its parent's right child. */ void replaceChild(RedBlackNode* ol, RedBlackNode* nw) { if(left == ol) { left = nw; } else { assert(right == ol); right = nw; } } /** * Return the number of non-null children this node has. */ int numChildren() const { return ((left != NULL) ? 1 : 0) + ((right != NULL) ? 1 : 0); } #ifndef NDEBUG /** * Check that node is internally consistent. */ bool repOk() const { if(parent != NULL) { assert(parent->left == this || parent->right == this); } return true; } #endif /** * True -> my key is less than than the given node's key. */ bool operator<(const TNode& o) const { return key < o.key; } /** * True -> my key is greater than the given node's key. */ bool operator>(const TNode& o) const { return key > o.key; } /** * True -> my key equals the given node's key. */ bool operator==(const TNode& o) const { return key == o.key; } /** * True -> my key is less than the given key. */ bool operator<(const K& okey) const { return key < okey; } /** * True -> my key is greater than the given key. */ bool operator>(const K& okey) const { return key > okey; } /** * True -> my key is equal to the given key. */ bool operator==(const K& okey) const { return key == okey; } }; /** * A Red-Black tree that associates keys (of type K) with payloads (of * type P). Red-Black trees are self-balancing and guarantee that the * tree as always "balanced" to a factor of 2, i.e., the longest * root-to-leaf path is never more than twice as long as the shortest * root-to-leaf path. */ template // K=key, P=payload class RedBlack { typedef RedBlackNode TNode; public: /** * Initialize the current-edit pointer to 0 and set the number of * edits per memory page. */ RedBlack(uint32_t pageSz, int cat = 0) : perPage_(pageSz/sizeof(TNode)), pages_(cat) { clear(); } /** * Given a DNA string, find the red-black node corresponding to it, * if one exists. */ inline TNode* lookup(const K& key) const { TNode* cur = root_; while(cur != NULL) { if((*cur) == key) return cur; if((*cur) < key) { cur = cur->right; } else { cur = cur->left; } } return NULL; } /** * Add a new key as a node in the red-black tree. */ TNode* add( Pool& p, // in: pool for memory pages const K& key, // in: key to insert bool* added) // if true, assert is thrown if key exists { // Look for key; if it's not there, get its parent TNode* cur = root_; assert(root_ == NULL || !root_->red); TNode* parent = NULL; bool leftChild = true; while(cur != NULL) { if((*cur) == key) { // Found it; break out of loop with cur != NULL break; } parent = cur; if((*cur) < key) { if((cur = cur->right) == NULL) { // Fell off the bottom of the tree as the right // child of parent 'lastCur' leftChild = false; } } else { if((cur = cur->left) == NULL) { // Fell off the bottom of the tree as the left // child of parent 'lastCur' leftChild = true; } } } if(cur != NULL) { // Found an entry; assert if we weren't supposed to if(added != NULL) *added = false; } else { assert(root_ == NULL || !root_->red); if(!addNode(p, cur)) { // Exhausted memory return NULL; } assert(cur != NULL); assert(cur != root_); assert(cur != parent); // Initialize new node cur->key = key; cur->left = cur->right = NULL; cur->red = true; // red until proven black keys_++; if(added != NULL) *added = true; // Put it where we know it should go addNode(cur, parent, leftChild); } return cur; // return the added or found node } #ifndef NDEBUG /** * Check that list is internally consistent. */ bool repOk() const { assert(curPage_ == 0 || curPage_ < pages_.size()); assert_leq(cur_, perPage_); assert(root_ == NULL || !root_->red); return true; } #endif /** * Clear all state. */ void clear() { cur_ = curPage_ = 0; root_ = NULL; keys_ = 0; intenseRepOkCnt_ = 0; pages_.clear(); } /** * Return number of keys added. */ size_t size() const { return keys_; } /** * Return true iff there are no keys in the map. */ bool empty() const { return keys_ == 0; } /** * Add another node and return a pointer to it in 'node'. A new * page is allocated if necessary. If the allocation fails, false * is returned. */ bool addNode(Pool& p, TNode*& node) { assert_leq(cur_, perPage_); assert(repOk()); assert(this != NULL); // Allocation of the first page if(pages_.size() == 0) { if(addPage(p) == NULL) { node = NULL; return false; } assert_eq(1, pages_.size()); } if(cur_ == perPage_) { assert_lt(curPage_, pages_.size()); if(curPage_ == pages_.size()-1 && addPage(p) == NULL) { return false; } cur_ = 0; curPage_++; } assert_lt(cur_, perPage_); assert_lt(curPage_, pages_.size()); node = &pages_[curPage_][cur_]; assert(node != NULL); cur_++; return true; } const TNode* root() const { return root_; } protected: #ifndef NDEBUG /** * Check specifically that the red-black invariants are satistfied. */ bool redBlackRepOk(TNode* n) { if(n == NULL) return true; if(++intenseRepOkCnt_ < 500) return true; intenseRepOkCnt_ = 0; int minNodes = -1; // min # nodes along any n->leaf path int maxNodes = -1; // max # nodes along any n->leaf path // The number of black nodes along paths from n to leaf // (must be same for all paths) int blackConst = -1; size_t nodesTot = 0; redBlackRepOk( n, 1, /* 1 node so far */ n->red ? 0 : 1, /* black nodes so far */ blackConst, minNodes, maxNodes, nodesTot); if(n == root_) { assert_eq(nodesTot, keys_); } assert_gt(minNodes, 0); assert_gt(maxNodes, 0); assert_leq(maxNodes, 2*minNodes); return true; } /** * Check specifically that the red-black invariants are satistfied. */ bool redBlackRepOk( TNode* n, int nodes, int black, int& blackConst, int& minNodes, int& maxNodes, size_t& nodesTot) const { assert_gt(black, 0); nodesTot++; // account for leaf node if(n->left == NULL) { if(blackConst == -1) blackConst = black; assert_eq(black, blackConst); if(nodes+1 > maxNodes) maxNodes = nodes+1; if(nodes+1 < minNodes || minNodes == -1) minNodes = nodes+1; } else { if(n->red) assert(!n->left->red); // Red can't be child of a red redBlackRepOk( n->left, // next node nodes + 1, // # nodes so far on path black + (n->left->red ? 0 : 1), // # black so far on path blackConst, // invariant # black nodes on root->leaf path minNodes, // min root->leaf len so far maxNodes, // max root->leaf len so far nodesTot); // tot nodes so far } if(n->right == NULL) { if(blackConst == -1) blackConst = black; assert_eq(black, blackConst); if(nodes+1 > maxNodes) maxNodes = nodes+1; if(nodes+1 < minNodes || minNodes == -1) minNodes = nodes+1; } else { if(n->red) assert(!n->right->red); // Red can't be child of a red redBlackRepOk( n->right, // next node nodes + 1, // # nodes so far on path black + (n->right->red ? 0 : 1), // # black so far on path blackConst, // invariant # black nodes on root->leaf path minNodes, // min root->leaf len so far maxNodes, // max root->leaf len so far nodesTot); // tot nodes so far } return true; } #endif /** * Rotate to the left such that n is replaced by its right child * w/r/t n's current parent. */ void leftRotate(TNode* n) { TNode* r = n->right; assert(n->repOk()); assert(r->repOk()); n->right = r->left; if(n->right != NULL) { n->right->parent = n; assert(n->right->repOk()); } r->parent = n->parent; n->parent = r; r->left = n; if(r->parent != NULL) { r->parent->replaceChild(n, r); } if(root_ == n) root_ = r; assert(!root_->red); assert(n->repOk()); assert(r->repOk()); } /** * Rotate to the right such that n is replaced by its left child * w/r/t n's current parent. n moves down to the right and loses * its left child, while its former left child moves up and gains a * right child. */ void rightRotate(TNode* n) { TNode* r = n->left; assert(n->repOk()); assert(r->repOk()); n->left = r->right; if(n->left != NULL) { n->left->parent = n; assert(n->left->repOk()); } r->parent = n->parent; n->parent = r; r->right = n; if(r->parent != NULL) { r->parent->replaceChild(n, r); } if(root_ == n) root_ = r; assert(!root_->red); assert(n->repOk()); assert(r->repOk()); } /** * Add a node to the red-black tree, maintaining the red-black * invariants. */ void addNode(TNode* n, TNode* parent, bool leftChild) { assert(n != NULL); if(parent == NULL) { // Case 1: inserted at root root_ = n; root_->red = false; // root must be black n->parent = NULL; assert(redBlackRepOk(root_)); assert(n->repOk()); } else { assert(!root_->red); // Add new node to tree if(leftChild) { assert(parent->left == NULL); parent->left = n; } else { assert(parent->right == NULL); parent->right = n; } n->parent = parent; int thru = 0; while(true) { thru++; parent = n->parent; if(parent != NULL) assert(parent->repOk()); if(parent == NULL && n->red) { n->red = false; } if(parent == NULL || !parent->red) { assert(redBlackRepOk(root_)); break; } TNode* uncle = n->uncle(); TNode* gparent = n->grandparent(); assert(gparent != NULL); // if parent is red, grandparent must exist bool uncleRed = (uncle != NULL ? uncle->red : false); if(uncleRed) { // Parent is red, uncle is red; recursive case assert(uncle != NULL); parent->red = uncle->red = false; gparent->red = true; n = gparent; continue; } else { if(parent->isLeftChild()) { // Parent is red, uncle is black, parent is // left child if(!n->isLeftChild()) { n = parent; leftRotate(n); } n = n->parent; n->red = false; n->parent->red = true; rightRotate(n->parent); assert(redBlackRepOk(n)); assert(redBlackRepOk(root_)); } else { // Parent is red, uncle is black, parent is // right child. if(!n->isRightChild()) { n = parent; rightRotate(n); } n = n->parent; n->red = false; n->parent->red = true; leftRotate(n->parent); assert(redBlackRepOk(n)); assert(redBlackRepOk(root_)); } } break; } } assert(redBlackRepOk(root_)); } /** * Expand our page supply by 1 */ TNode* addPage(Pool& p) { TNode *n = (TNode *)p.alloc(); if(n != NULL) { pages_.push_back(n); } return n; } size_t keys_; // number of keys so far size_t cur_; // current elt within page size_t curPage_; // current page const size_t perPage_; // # edits fitting in a page TNode* root_; // root node EList pages_; // the pages int intenseRepOkCnt_; // counter for the computationally intensive repOk function }; /** * For assembling doubly-linked lists of Edits. */ template struct DoublyLinkedList { DoublyLinkedList() : payload(), prev(NULL), next(NULL) { } /** * Add all elements in the doubly-linked list to the provided EList. */ void toList(EList& l) { // Add this and all subsequent elements DoublyLinkedList *cur = this; while(cur != NULL) { l.push_back(cur->payload); cur = cur->next; } // Add all previous elements cur = prev; while(cur != NULL) { l.push_back(cur->payload); cur = cur->prev; } } T payload; DoublyLinkedList *prev; DoublyLinkedList *next; }; template struct Pair { T1 a; T2 b; Pair() : a(), b() { } Pair( const T1& a_, const T2& b_) { a = a_; b = b_; } bool operator==(const Pair& o) const { return a == o.a && b == o.b; } bool operator<(const Pair& o) const { if(a < o.a) return true; if(a > o.a) return false; if(b < o.b) return true; return false; } }; template struct Triple { T1 a; T2 b; T3 c; Triple() : a(), b(), c() { } Triple( const T1& a_, const T2& b_, const T3& c_) { a = a_; b = b_; c = c_; } bool operator==(const Triple& o) const { return a == o.a && b == o.b && c == o.c; } bool operator<(const Triple& o) const { if(a < o.a) return true; if(a > o.a) return false; if(b < o.b) return true; if(b > o.b) return false; if(c < o.c) return true; return false; } }; template struct Quad { Quad() : a(), b(), c(), d() { } Quad( const T1& a_, const T2& b_, const T3& c_, const T4& d_) { a = a_; b = b_; c = c_; d = d_; } Quad( const T1& a_, const T1& b_, const T1& c_, const T1& d_) { init(a_, b_, c_, d_); } void init( const T1& a_, const T1& b_, const T1& c_, const T1& d_) { a = a_; b = b_; c = c_; d = d_; } bool operator==(const Quad& o) const { return a == o.a && b == o.b && c == o.c && d == o.d; } bool operator<(const Quad& o) const { if(a < o.a) return true; if(a > o.a) return false; if(b < o.b) return true; if(b > o.b) return false; if(c < o.c) return true; if(c > o.c) return false; if(d < o.d) return true; return false; } T1 a; T2 b; T3 c; T4 d; }; /** * For assembling doubly-linked lists of EList. */ template struct LinkedEListNode { LinkedEListNode() : payload(), next(NULL) { } T payload; LinkedEListNode *next; }; /** * For assembling doubly-linked lists of EList. */ template struct LinkedEList { LinkedEList() : head(NULL) { ASSERT_ONLY(num_allocated = 0); ASSERT_ONLY(num_new_node = 0); ASSERT_ONLY(num_delete_node = 0); } ~LinkedEList() { ASSERT_ONLY(size_t num_deallocated = 0); while(head != NULL) { LinkedEListNode* next = head->next; delete head; ASSERT_ONLY(num_deallocated++); head = next; } // daehwan - for debugging purposes // assert_eq(num_allocated, num_deallocated); } LinkedEListNode* new_node() { ASSERT_ONLY(num_new_node++); LinkedEListNode *result = NULL; if(head == NULL) { head = new LinkedEListNode(); head-> next = NULL; ASSERT_ONLY(num_allocated++); } assert(head != NULL); result = head; head = head->next; assert(result != NULL); return result; } void delete_node(LinkedEListNode *node) { ASSERT_ONLY(num_delete_node++); assert(node != NULL); // check if this is already deleted. #ifndef NDEBUG LinkedEListNode *temp = head; while(temp != NULL) { assert(temp != node); temp = temp->next; } #endif node->next = head; head = node; } LinkedEListNode *head; ASSERT_ONLY(size_t num_allocated); ASSERT_ONLY(size_t num_new_node); ASSERT_ONLY(size_t num_delete_node); }; #endif /* DS_H_ */ hisat2-2.2.1/edit.cpp000066400000000000000000000305461370663763200143640ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include "edit.h" using namespace std; /** * Print a single edit to a std::ostream. Format is * (pos):(ref chr)>(read chr). Where 'pos' is an offset from the 5' * end of the read, and the ref and read chrs are expressed w/r/t the * Watson strand. */ ostream& operator<< (ostream& os, const Edit& e) { if(e.type != EDIT_TYPE_SPL) { os << e.pos << ":" << (char)e.chr << ">" << (char)e.qchr; } else { os << e.pos << ":" << e.splLen; } return os; } /** * Print a list of edits to a std::ostream, separated by commas. */ void Edit::print(ostream& os, const EList& edits, char delim) { for(size_t i = 0; i < edits.size(); i++) { os << edits[i]; if(i < edits.size()-1) os << delim; } } void Edit::complement(EList &edits) { for(size_t i = 0; i < edits.size(); i++) { Edit& e = edits[i]; if(e.type != EDIT_TYPE_SPL) { assert_in(e.chr, "ACMGRSVTWYHKDBN-"); assert_in(e.qchr, "ACGTN-"); e.chr = asc2dnacomp[e.chr]; e.qchr = asc2dnacomp[e.qchr]; } } } /** * Flip all the edits.pos fields so that they're with respect to * the other end of the read (of length 'sz'). */ void Edit::invertPoss( EList& edits, size_t sz, size_t ei, size_t en, bool sort) { // Invert elements size_t ii = 0; for(size_t i = ei; i < ei + en/2; i++) { Edit tmp = edits[i]; edits[i] = edits[ei + en - ii - 1]; edits[ei + en - ii - 1] = tmp; ii++; } for(size_t i = ei; i < ei + en; i++) { assert(edits[i].pos < sz || (edits[i].isReadGap() && edits[i].pos == sz)); // Adjust pos if(edits[i].isReadGap() || edits[i].isSpliced()) { edits[i].pos = (uint32_t)(sz - edits[i].pos); } else { edits[i].pos = (uint32_t)(sz - edits[i].pos - 1); } // Adjust pos2 if(edits[i].isReadGap()) { int64_t pos2diff = (int64_t)(uint64_t)edits[i].pos2 - (int64_t)((uint64_t)std::numeric_limits::max() >> 1); int64_t pos2new = (int64_t)(uint64_t)edits[i].pos2 - 2*pos2diff; assert(pos2diff == 0 || (uint32_t)pos2new != (std::numeric_limits::max() >> 1)); edits[i].pos2 = (uint32_t)pos2new; } } if(sort) { // Edits might not necessarily be in same order after inversion edits.sortPortion(ei, en); #ifndef NDEBUG for(size_t i = ei + 1; i < ei + en; i++) { assert_geq(edits[i].pos, edits[i-1].pos); } #endif } } /** * For now, we pretend that the alignment is in the forward orientation * and that the Edits are listed from left- to right-hand side. */ void Edit::printQAlign( std::ostream& os, const BTDnaString& read, const EList& edits) { printQAlign(os, "", read, edits); } /** * For now, we pretend that the alignment is in the forward orientation * and that the Edits are listed from left- to right-hand side. */ void Edit::printQAlignNoCheck( std::ostream& os, const BTDnaString& read, const EList& edits) { printQAlignNoCheck(os, "", read, edits); } /** * For now, we pretend that the alignment is in the forward orientation * and that the Edits are listed from left- to right-hand side. */ void Edit::printQAlign( std::ostream& os, const char *prefix, const BTDnaString& read, const EList& edits) { size_t eidx = 0; os << prefix; // Print read for(size_t i = 0; i < read.length(); i++) { bool del = false, mm = false; while(eidx < edits.size() && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { os << '-'; } else if(edits[eidx].isRefGap()) { del = true; assert_eq((int)edits[eidx].qchr, read.toChar(i)); os << read.toChar(i); } else { mm = true; assert(edits[eidx].isMismatch()); assert_eq((int)edits[eidx].qchr, read.toChar(i)); os << (char)edits[eidx].qchr; } eidx++; } if(!del && !mm) os << read.toChar(i); } os << endl; os << prefix; eidx = 0; // Print match bars for(size_t i = 0; i < read.length(); i++) { bool del = false, mm = false; while(eidx < edits.size() && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { os << ' '; } else if(edits[eidx].isRefGap()) { del = true; os << ' '; } else { mm = true; assert(edits[eidx].isMismatch()); os << ' '; } eidx++; } if(!del && !mm) os << '|'; } os << endl; os << prefix; eidx = 0; // Print reference for(size_t i = 0; i < read.length(); i++) { bool del = false, mm = false; while(eidx < edits.size() && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { os << (char)edits[eidx].chr; } else if(edits[eidx].isRefGap()) { del = true; os << '-'; } else { mm = true; assert(edits[eidx].isMismatch()); os << (char)edits[eidx].chr; } eidx++; } if(!del && !mm) os << read.toChar(i); } os << endl; } /** * For now, we pretend that the alignment is in the forward orientation * and that the Edits are listed from left- to right-hand side. */ void Edit::printQAlignNoCheck( std::ostream& os, const char *prefix, const BTDnaString& read, const EList& edits) { size_t eidx = 0; os << prefix; // Print read for(size_t i = 0; i < read.length(); i++) { bool del = false, mm = false; while(eidx < edits.size() && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { os << '-'; } else if(edits[eidx].isRefGap()) { del = true; os << read.toChar(i); } else { mm = true; os << (char)edits[eidx].qchr; } eidx++; } if(!del && !mm) os << read.toChar(i); } os << endl; os << prefix; eidx = 0; // Print match bars for(size_t i = 0; i < read.length(); i++) { bool del = false, mm = false; while(eidx < edits.size() && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { os << ' '; } else if(edits[eidx].isRefGap()) { del = true; os << ' '; } else { mm = true; os << ' '; } eidx++; } if(!del && !mm) os << '|'; } os << endl; os << prefix; eidx = 0; // Print reference for(size_t i = 0; i < read.length(); i++) { bool del = false, mm = false; while(eidx < edits.size() && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { os << (char)edits[eidx].chr; } else if(edits[eidx].isRefGap()) { del = true; os << '-'; } else { mm = true; os << (char)edits[eidx].chr; } eidx++; } if(!del && !mm) os << read.toChar(i); } os << endl; } /** * Sort the edits in the provided list. */ void Edit::sort(EList& edits) { edits.sort(); // simple! } /** * Given a read string and some edits, generate and append the corresponding * reference string to 'ref'. If read aligned to the Watson strand, the caller * should pass the original read sequence and original edits. If a read * aligned to the Crick strand, the caller should pass the reverse complement * of the read and a version of the edits list that has had Edit:invertPoss * called on it to cause edits to be listed in 3'-to-5' order. */ void Edit::toRef( const BTDnaString& read, const EList& edits, BTDnaString& ref, bool fw, size_t trim5, size_t trim3) { // edits should be sorted size_t eidx = 0; // Print reference const size_t rdlen = read.length(); size_t trimBeg = fw ? trim5 : trim3; size_t trimEnd = fw ? trim3 : trim5; assert(Edit::repOk(edits, read, fw, trim5, trim3)); if(!fw) { invertPoss(const_cast&>(edits), read.length()-trimBeg-trimEnd, false); } for(size_t i = 0; i < rdlen; i++) { ASSERT_ONLY(int c = read[i]); assert_range(0, 4, c); bool del = false, mm = false; bool append = i >= trimBeg && rdlen - i - 1 >= trimEnd; bool appendIns = i >= trimBeg && rdlen - i >= trimEnd; while(eidx < edits.size() && edits[eidx].pos+trimBeg == i) { if(edits[eidx].isReadGap()) { // Inserted characters come before the position's // character if(appendIns) { ref.appendChar((char)edits[eidx].chr); } } else if(edits[eidx].isRefGap()) { assert_eq("ACGTN"[c], edits[eidx].qchr); del = true; } else if(edits[eidx].isMismatch()){ mm = true; assert(edits[eidx].qchr != edits[eidx].chr || edits[eidx].qchr == 'N'); assert_eq("ACGTN"[c], edits[eidx].qchr); if(append) { ref.appendChar((char)edits[eidx].chr); } } eidx++; } if(!del && !mm) { if(append) { ref.append(read[i]); } } } if(trimEnd == 0) { while(eidx < edits.size()) { assert_gt(rdlen, edits[eidx].pos); if(edits[eidx].isReadGap()) { ref.appendChar((char)edits[eidx].chr); } eidx++; } } if(!fw) { invertPoss(const_cast&>(edits), read.length()-trimBeg-trimEnd, false); } } #ifndef NDEBUG /** * Check that the edit is internally consistent. */ bool Edit::repOk() const { assert(inited()); // Ref and read characters cannot be the same unless they're both Ns if(type != EDIT_TYPE_SPL) { assert(qchr != chr || qchr == 'N'); // Type must match characters assert(isRefGap() || chr != '-'); assert(isReadGap() || qchr != '-'); assert(!isMismatch() || (qchr != '-' && chr != '-')); } else { assert_gt(splLen, 0); } return true; } /** * Given a list of edits and a DNA string representing the query * sequence, check that the edits are consistent with respect to the * query. */ bool Edit::repOk( const EList& edits, const BTDnaString& s, bool fw, size_t trimBeg, size_t trimEnd) { if(!fw) { invertPoss(const_cast&>(edits), s.length()-trimBeg-trimEnd, false); swap(trimBeg, trimEnd); } for(size_t i = 0; i < edits.size(); i++) { const Edit& e = edits[i]; size_t pos = e.pos; if(i > 0) { assert_geq(pos, edits[i-1].pos); } bool del = false, mm = false; while(i < edits.size() && edits[i].pos == pos) { const Edit& ee = edits[i]; assert_lt(ee.pos, s.length()); if(ee.type != EDIT_TYPE_SPL) { if(ee.qchr != '-') { assert(ee.isRefGap() || ee.isMismatch()); assert_eq((int)ee.qchr, s.toChar(ee.pos+trimBeg)); } } if(ee.isMismatch()) { assert(!mm); mm = true; assert(!del); } else if(ee.isReadGap()) { assert(!mm); } else if(ee.isRefGap()) { assert(!mm); assert(!del); del = true; } else if(ee.isSpliced()) { } i++; } } if(!fw) { invertPoss(const_cast&>(edits), s.length()-trimBeg-trimEnd, false); } return true; } #endif /** * Merge second argument into the first. Assume both are sorted to * begin with. */ void Edit::merge(EList& dst, const EList& src) { size_t di = 0, si = 0; while(di < dst.size()) { if(src[si].pos < dst[di].pos) { dst.insert(src[si], di); si++; di++; } else if(src[si].pos == dst[di].pos) { // There can be two inserts at a given position, but we // can't merge them because there's no way to know their // order assert(src[si].isReadGap() != dst[di].isReadGap()); if(src[si].isReadGap()) { dst.insert(src[si], di); si++; di++; } else if(dst[di].isReadGap()) { di++; } } } while(si < src.size()) dst.push_back(src[si++]); } /** * Clip off some of the low-numbered positions. */ void Edit::clipLo(EList& ed, size_t len, size_t amt) { size_t nrm = 0; for(size_t i = 0; i < ed.size(); i++) { assert_lt(ed[i].pos, len); if(ed[i].pos < amt) { nrm++; } else { // Shift everyone else up ed[i].pos -= (uint32_t)amt; } } ed.erase(0, nrm); } /** * Clip off some of the high-numbered positions. */ void Edit::clipHi(EList& ed, size_t len, size_t amt) { assert_leq(amt, len); size_t max = len - amt; size_t nrm = 0; for(size_t i = 0; i < ed.size(); i++) { size_t ii = ed.size() - i - 1; assert_lt(ed[ii].pos, len); if(ed[ii].pos > max) { nrm++; } else if(ed[ii].pos == max && !ed[ii].isReadGap()) { nrm++; } else { break; } } ed.resize(ed.size() - nrm); } hisat2-2.2.1/edit.h000066400000000000000000000235761370663763200140360ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef EDIT_H_ #define EDIT_H_ #include #include #include #include "assert_helpers.h" #include "filebuf.h" #include "sstring.h" #include "ds.h" #include "splice_site.h" /** * 3 types of edits; mismatch (substitution), insertion in the * reference, deletion in the reference. */ enum { EDIT_TYPE_READ_GAP = 1, EDIT_TYPE_REF_GAP, EDIT_TYPE_MM, EDIT_TYPE_SNP, EDIT_TYPE_SPL, // splicing of pre-messenger RNAs into messenger RNAs }; /** * Encapsulates an edit between the read sequence and the reference sequence. * We obey a few conventions when populating its fields. The fields are: * * uint8_t chr; // reference character involved (for subst and ins) * uint8_t qchr; // read character involved (for subst and del) * uint8_t type; // 1 -> mm, 2 -> SNP, 3 -> ins, 4 -> del * uint32_t pos; // position w/r/t search root * * One convention is that pos is always an offset w/r/t the 5' end of the read. * * Another is that chr and qchr are expressed in terms of the nucleotides on * the forward version of the read. So if we're aligning the reverse * complement of the read, and an A in the reverse complement mismatches a C in * the reference, chr should be G and qchr should be T. */ struct Edit { Edit() { reset(); } Edit( uint32_t po, int ch, int qc, int ty, bool chrs = true, uint32_t snp = std::numeric_limits::max()) { init(po, ch, qc, ty, chrs, snp); } Edit( uint32_t po, int ch, int qc, int ty, uint32_t sl, uint8_t sdir, bool knowns, bool chrs = true) { init(po, ch, qc, ty, sl, sdir, knowns, chrs); } /** * Reset Edit to uninitialized state. */ void reset() { pos = pos2 = std::numeric_limits::max(); chr = qchr = type = 0; splLen = 0; splDir = SPL_UNKNOWN; knownSpl = false; snpID = std::numeric_limits::max(); } /** * Return true iff the Edit is initialized. */ bool inited() const { return pos != std::numeric_limits::max(); } /** * Initialize a new Edit. */ void init( uint32_t po, int ch, int qc, int ty, bool chrs = true, uint32_t snp = std::numeric_limits::max()) { chr = ch; qchr = qc; type = ty; splLen = 0; splDir = SPL_UNKNOWN; pos = po; if(qc == '-') { // Read gap pos2 = std::numeric_limits::max() >> 1; } else { pos2 = std::numeric_limits::max(); } snpID = snp; if(!chrs) { assert_range(0, 4, (int)chr); assert_range(0, 4, (int)qchr); chr = "ACGTN"[chr]; qchr = "ACGTN"[qchr]; } #ifndef NDEBUG if(type != EDIT_TYPE_SPL) { assert_in(chr, "ACMGRSVTWYHKDBN-"); assert_in(qchr, "ACGTN-"); assert(chr != qchr || chr == 'N'); } #endif assert(inited()); } /** * Initialize a new Edit. */ void init( uint32_t po, int ch, int qc, int ty, uint32_t sl, uint32_t sdir, bool knowns, bool chrs = true) { assert_eq(ty, EDIT_TYPE_SPL); init(po, ch, qc, ty, chrs); splLen = sl; splDir = sdir; knownSpl = knowns; } /** * Return true iff one part of the edit or the other has an 'N'. */ bool hasN() const { assert(inited()); return chr == 'N' || qchr == 'N'; } /** * Edit less-than overload. */ int operator< (const Edit &rhs) const { assert(inited()); if(pos < rhs.pos) return 1; if(pos > rhs.pos) return 0; if(pos2 < rhs.pos2) return 1; if(pos2 > rhs.pos2) return 0; if(type < rhs.type) return 1; if(type > rhs.type) return 0; if(chr < rhs.chr) return 1; if(chr > rhs.chr) return 0; return (qchr < rhs.qchr)? 1 : 0; } /** * Edit equals overload. */ int operator== (const Edit &rhs) const { assert(inited()); if(type != rhs.type) return false; if(pos != rhs.pos) return false; if(type != EDIT_TYPE_SPL) { return chr == rhs.chr && qchr == rhs.qchr; } else { return pos2 == rhs.pos2 && splLen == rhs.splLen && splDir == rhs.splDir /* && knownSpl == rhs.knownSpl */; } } /** * Return true iff this Edit is an initialized insertion. */ bool isReadGap() const { assert(inited()); return type == EDIT_TYPE_READ_GAP; } /** * Return true iff this Edit is an initialized deletion. */ bool isRefGap() const { assert(inited()); return type == EDIT_TYPE_REF_GAP; } /** * Return true if this Edit is either an initialized deletion or an * initialized insertion. */ bool isGap() const { assert(inited()); return (type == EDIT_TYPE_REF_GAP || type == EDIT_TYPE_READ_GAP); } bool isSpliced() const { assert(inited()); return type == EDIT_TYPE_SPL; } /** * Return the number of gaps in the given edit list. */ static size_t numGaps(const EList& es) { size_t gaps = 0; for(size_t i = 0; i < es.size(); i++) { if(es[i].isGap()) gaps++; } return gaps; } /** * Return true iff this Edit is an initialized mismatch. */ bool isMismatch() const { assert(inited()); return type == EDIT_TYPE_MM; } /** * Sort the edits in the provided list. */ static void sort(EList& edits); /** * Flip all the edits.pos fields so that they're with respect to * the other end of the read (of length 'sz'). */ static void invertPoss( EList& edits, size_t sz, size_t ei, size_t en, bool sort = false); /** * Flip all the edits.pos fields so that they're with respect to * the other end of the read (of length 'sz'). */ static void invertPoss(EList& edits, size_t sz, bool sort = false) { invertPoss(edits, sz, 0, edits.size(), sort); } static void complement(EList& edits); /** * Clip off some of the low-numbered positions. */ static void clipLo(EList& edits, size_t len, size_t amt); /** * Clip off some of the high-numbered positions. */ static void clipHi(EList& edits, size_t len, size_t amt); /** * Given a read string and some edits, generate and append the * corresponding reference string to 'ref'. */ static void toRef( const BTDnaString& read, const EList& edits, BTDnaString& ref, bool fw = true, size_t trim5 = 0, size_t trim3 = 0); /** * Given a string and its edits with respect to some other string, * print the alignment between the strings with the strings stacked * vertically, with vertical bars denoting matches. */ static void printQAlign( std::ostream& os, const BTDnaString& read, const EList& edits); /** * Given a string and its edits with respect to some other string, * print the alignment between the strings with the strings stacked * vertically, with vertical bars denoting matches. Add 'prefix' * before each line of output. */ static void printQAlign( std::ostream& os, const char *prefix, const BTDnaString& read, const EList& edits); /** * Given a string and its edits with respect to some other string, * print the alignment between the strings with the strings stacked * vertically, with vertical bars denoting matches. */ static void printQAlignNoCheck( std::ostream& os, const BTDnaString& read, const EList& edits); /** * Given a string and its edits with respect to some other string, * print the alignment between the strings with the strings stacked * vertically, with vertical bars denoting matches. Add 'prefix' * before each line of output. */ static void printQAlignNoCheck( std::ostream& os, const char *prefix, const BTDnaString& read, const EList& edits); #ifndef NDEBUG bool repOk() const; /** * Given a list of edits and a DNA string representing the query * sequence, check that the edits are consistent with respect to the * query. */ static bool repOk( const EList& edits, const BTDnaString& s, bool fw = true, size_t trim5 = 0, size_t trim3 = 0); #endif uint8_t chr; // reference character involved (for subst and ins) uint8_t qchr; // read character involved (for subst and del) uint8_t type; // 1 -> mm, 2 -> SNP, 3 -> ins, 4 -> del uint32_t pos; // position w/r/t search root uint32_t pos2; // Second int to take into account when sorting. Useful for // sorting read gap edits that are all part of the same long // gap. uint32_t splLen; // skip over the genome due to an intron uint8_t splDir; bool knownSpl; int64_t donor_seq; int64_t acceptor_seq; uint32_t snpID; // snp ID friend std::ostream& operator<< (std::ostream& os, const Edit& e); /** * Print a comma-separated list of Edits to given output stream. */ static void print( std::ostream& os, const EList& edits, char delim = '\t'); /** * Merge second argument into the first. Assume both are sorted to * begin with. */ static void merge(EList& dst, const EList& src); }; #endif /* EDIT_H_ */ hisat2-2.2.1/endian_swap.h000066400000000000000000000101071370663763200153630ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ENDIAN_SWAP_H #define ENDIAN_SWAP_H #include #include /** * Return true iff the machine running this program is big-endian. */ static inline bool currentlyBigEndian() { static uint8_t endianCheck[] = {1, 0, 0, 0}; return *((uint32_t*)endianCheck) != 1; } /** * Return copy of uint32_t argument with byte order reversed. */ static inline uint16_t endianSwapU16(uint16_t u) { uint16_t tmp = 0; tmp |= ((u >> 8) & (0xff << 0)); tmp |= ((u << 8) & (0xff << 8)); return tmp; } /** * Return copy of uint32_t argument with byte order reversed. */ static inline uint32_t endianSwapU32(uint32_t u) { uint32_t tmp = 0; tmp |= ((u >> 24) & (0xff << 0)); tmp |= ((u >> 8) & (0xff << 8)); tmp |= ((u << 8) & (0xff << 16)); tmp |= ((u << 24) & (0xff << 24)); return tmp; } /** * Return copy of uint64_t argument with byte order reversed. */ static inline uint64_t endianSwapU64(uint64_t u) { uint64_t tmp = 0; tmp |= ((u >> 56) & (0xffull << 0)); tmp |= ((u >> 40) & (0xffull << 8)); tmp |= ((u >> 24) & (0xffull << 16)); tmp |= ((u >> 8) & (0xffull << 24)); tmp |= ((u << 8) & (0xffull << 32)); tmp |= ((u << 24) & (0xffull << 40)); tmp |= ((u << 40) & (0xffull << 48)); tmp |= ((u << 56) & (0xffull << 56)); return tmp; } /** * Return copy of uint_t argument with byte order reversed. */ template static inline index_t endianSwapIndex(index_t u) { if(sizeof(index_t) == 8) { return (index_t)endianSwapU64(u); } else if(sizeof(index_t) == 4) { return endianSwapU32((uint32_t)u); } else { return endianSwapU16(u); } } /** * Return copy of int16_t argument with byte order reversed. */ static inline int16_t endianSwapI16(int16_t i) { int16_t tmp = 0; tmp |= ((i >> 8) & (0xff << 0)); tmp |= ((i << 8) & (0xff << 8)); return tmp; } /** * Convert uint16_t argument to the specified endianness. It's assumed * that u currently has the endianness of the current machine. */ static inline uint16_t endianizeU16(uint16_t u, bool toBig) { if(toBig == currentlyBigEndian()) { return u; } return endianSwapU16(u); } /** * Convert int16_t argument to the specified endianness. It's assumed * that u currently has the endianness of the current machine. */ static inline int16_t endianizeI16(int16_t i, bool toBig) { if(toBig == currentlyBigEndian()) { return i; } return endianSwapI16(i); } /** * Return copy of int32_t argument with byte order reversed. */ static inline int32_t endianSwapI32(int32_t i) { int32_t tmp = 0; tmp |= ((i >> 24) & (0xff << 0)); tmp |= ((i >> 8) & (0xff << 8)); tmp |= ((i << 8) & (0xff << 16)); tmp |= ((i << 24) & (0xff << 24)); return tmp; } /** * Convert uint32_t argument to the specified endianness. It's assumed * that u currently has the endianness of the current machine. */ static inline uint32_t endianizeU32(uint32_t u, bool toBig) { if(toBig == currentlyBigEndian()) { return u; } return endianSwapU32(u); } /** * Convert int32_t argument to the specified endianness. It's assumed * that u currently has the endianness of the current machine. */ static inline int32_t endianizeI32(int32_t i, bool toBig) { if(toBig == currentlyBigEndian()) { return i; } return endianSwapI32(i); } template index_t endianizeIndex(index_t u, bool toBig) { if(toBig == currentlyBigEndian()) { return u; } return endianSwapIndex(u); } #endif hisat2-2.2.1/evaluation/000077500000000000000000000000001370663763200150725ustar00rootroot00000000000000hisat2-2.2.1/evaluation/build_indexes.py000077500000000000000000000060011370663763200202620ustar00rootroot00000000000000#!/usr/bin/env python import sys, os use_message = ''' ''' # GRCh38 release 84 def build_indexes(): # Build indexes if not os.path.exists("indexes"): os.mkdir("indexes") os.chdir("indexes") aligners = ["HISAT2", "HISAT", "Bowtie", "STAR", "GSNAP", "BWA", "minimap2"] genomes = ["22_20-21M", "22", "genome"] for genome in genomes: for aligner in aligners: if genome == "genome": dir = aligner else: dir = aligner + "_" + genome if os.path.exists(dir): continue os.mkdir(dir) os.chdir(dir) if aligner == "HISAT2": cmd = "../../aligners/bin/hisat2-build ../../data/%s.fa %s" % (genome, genome) cmd = cmd + "; ../../aligners/bin/hisat2-build -p 4 ../../data/%s.fa --snp ../../data/%s.snp --haplotype ../../data/%s.haplotype %s_snp" % (genome, genome, genome, genome) cmd = cmd + "; ../../aligners/bin/hisat2-build -p 4 ../../data/%s.fa --ss ../../data/%s.ss --exon ../../data/%s.exon %s_tran" % (genome, genome, genome, genome) cmd = cmd + "; ../../aligners/bin/hisat2-build -p 4 ../../data/%s.fa --snp ../../data/%s.snp --haplotype ../../data/%s.haplotype --ss ../../data/%s.ss --exon ../../data/%s.exon %s_snp_tran" % (genome, genome, genome, genome, genome, genome) elif aligner == "HISAT": cmd = "../../aligners/bin/hisat-build ../../data/%s.fa %s" % (genome, genome) cmd = cmd + "; ../../aligners/bin/tophat -G ../../data/%s.gtf --transcriptome-index=gtf %s; rm -rf tophat_out" % (genome, genome) elif aligner == "Bowtie": cmd = "../../aligners/bin/bowtie-build ../../data/%s.fa %s" % (genome, genome) elif aligner == "Bowtie2": cmd = "../../aligners/bin/bowtie2-build --threads 6 ../../data/%s.fa %s" % (genome, genome) elif aligner == "STAR": cmd = "../../aligners/bin/STAR --runMode genomeGenerate --genomeDir . --genomeFastaFiles ../../data/%s.fa" % (genome) cmd = cmd + "; mkdir gtf; ../../aligners/bin/STAR --runMode genomeGenerate --genomeDir gtf --genomeFastaFiles ../../data/%s.fa --sjdbGTFfile ../../data/%s.gtf --sjdbOverhang 99 --runThreadN 4" % (genome, genome) elif aligner == "GSNAP": cmd = "../../aligners/bin/gmap_build -B ../../aligners/bin -D . -d %s ../../data/%s.fa" % (genome, genome) elif aligner == "BWA": cmd = "../../aligners/bin/bwa index -p %s.fa ../../data/%s.fa" % (genome, genome) elif aligner == "minimap2": cmd = "../../aligners/bin/minimap2 -x sr -d %s.mmi ../../data/%s.fa" % (genome, genome) elif aligner == "VG": assert False else: assert False print >> sys.stderr, cmd os.system(cmd) os.chdir("..") os.chdir("..") if __name__ == "__main__": build_indexes() hisat2-2.2.1/evaluation/generate_reads.py000077500000000000000000000122631370663763200204230ustar00rootroot00000000000000#!/usr/bin/env python import sys, os, random from argparse import ArgumentParser, FileType from multiprocessing import Process def shuffle_reads(read_fname, random_list): reads = [] read_file = open(read_fname) for line in read_file: if line[0] == ">": reads.append([]) reads[-1].append(line[:-1]) read_file.close() read_fname_out = read_fname + ".shuffle" read_file_out = open(read_fname_out, "w") assert len(random_list) == len(reads) for i in random_list: read = reads[random_list[i]] print >> read_file_out, "\n".join(read) read_file_out.close() def shuffle_pairs(read1_fname, read2_fname): read1_file = open(read1_fname) num_reads = 0 for line in read1_file: if line[0] == ">": num_reads += 1 read1_file.close() random_list = [i for i in range(num_reads)] random.shuffle(random_list) shuffle_reads(read1_fname, random_list) shuffle_reads(read2_fname, random_list) def simulate_reads(): if not os.path.exists("reads"): os.mkdir("reads") os.chdir("reads") if not os.path.exists("simulation"): os.mkdir("simulation") os.chdir("simulation") _rna, _mismatch, _snp, _constant = True, True, True, True _dna = not _rna datasets = [ ["22", 1000000, _dna, not _snp, not _mismatch, _constant], ["22", 1000000, _dna, not _snp, _mismatch, _constant], ["22", 1000000, _dna, _snp, not _mismatch, _constant], ["22", 1000000, _dna, _snp, _mismatch, _constant], ["22", 1000000, _rna, not _snp, not _mismatch, not _constant], ["22", 1000000, _rna, not _snp, not _mismatch, _constant], ["22", 1000000, _rna, not _snp, _mismatch, not _constant], ["22", 1000000, _rna, not _snp, _mismatch, _constant], ["22", 1000000, _rna, _snp, not _mismatch, not _constant], ["22", 1000000, _rna, _snp, not _mismatch, _constant], ["22", 1000000, _rna, _snp, _mismatch, not _constant], ["22", 1000000, _rna, _snp, _mismatch, _constant], # ["22_20-21M", 1000000, _rna, not _snp, not _mismatch, not _constant], # ["22_20-21M", 1000000, _rna, _snp, not _mismatch, _constant], ["genome", 10000000, _dna, not _snp, not _mismatch, _constant], ["genome", 10000000, _dna, _snp, not _mismatch, _constant], ["genome", 10000000, _dna, _snp, _mismatch, _constant], ["genome", 10000000, _rna, not _snp, not _mismatch, not _constant], ["genome", 10000000, _rna, _snp, not _mismatch, not _constant], ["genome", 10000000, _rna, _snp, _mismatch, not _constant], ] data_dir_base = "../../../data" def generate_reads(cmd): print >> sys.stderr, cmd os.system(cmd) random.seed(0) print >> sys.stderr, "shuffle reads sim_1.fa and sim_2.fa" shuffle_pairs("sim_1.fa", "sim_2.fa") shuffle_reads_cmd = " mv sim_1.fa.shuffle sim_1.fa" shuffle_reads_cmd += "; mv sim_2.fa.shuffle sim_2.fa" os.system(shuffle_reads_cmd) pid_list = [] for genome, numreads, rna, snp, mismatch, constant in datasets: if rna: molecule = "RNA" else: molecule = "DNA" if numreads >= 1000000: dirname = "%dM_%s" % (numreads / 1000000, molecule) else: dirname = "%dk_%s" % (numreads / 1000, molecule) if mismatch: dirname += "_mismatch" if snp: dirname += "_snp" if rna and constant: dirname += "_constant" dirname += "_reads" dirname += ("_" + genome) if os.path.exists(dirname): continue os.mkdir(dirname) os.chdir(dirname) genome_fname = data_dir_base + "/%s.fa" % (genome) if rna: gtf_fname = data_dir_base + "/%s.gtf" % (genome) else: gtf_fname = "/dev/null" if snp: snp_fname = data_dir_base + "/%s.snp" % (genome) else: snp_fname = "/dev/null" cmd_add = "" if not rna: cmd_add += "--dna " if mismatch: cmd_add += "--error-rate 0.2 " if rna and constant: cmd_add += "--expr-profile constant " cmd = "../../../aligners/bin/hisat2_simulate_reads.py --sanity-check %s --num-fragment %d %s %s %s sim" % \ (cmd_add, numreads, genome_fname, gtf_fname, snp_fname) """ print >> sys.stderr, cmd os.system(cmd) random.seed(0) print >> sys.stderr, "shuffle reads sim_1.fa and sim_2.fa" shuffle_pairs("sim_1.fa", "sim_2.fa") shuffle_reads_cmd = " mv sim_1.fa.shuffle sim_1.fa" shuffle_reads_cmd += "; mv sim_2.fa.shuffle sim_2.fa" os.system(shuffle_reads_cmd) """ #generate_reads(cmd) p = Process(target=generate_reads, args=(cmd,)) p.start() pid_list.append(p) os.chdir("..") os.chdir("..") # wait for p in pid_list: p.join() if __name__ == "__main__": parser = ArgumentParser( description='Generate reads using simulate_reads.py in HISAT2') args = parser.parse_args() simulate_reads() hisat2-2.2.1/evaluation/get_data.py000077500000000000000000000073051370663763200172240ustar00rootroot00000000000000#!/usr/bin/env python import sys, os from argparse import ArgumentParser, FileType def get_data(small = False): data_root = "http://www.ccb.jhu.edu/software/hisat2/downloads/evaluation" # Download the reference human genome, SNPs, and gene annotations if not os.path.exists("data"): os.mkdir("data") os.chdir("data") genome_files = ["genome.fa", "genome.fa.fai", "genome.gtf", "snpCommon.txt", "genome.snp", "genome.ss", "genome.exon"] small_genome_files = ["22.fa", "22.fa.fai", "22.gtf", "22.snp", "22.ss", "22.exon", \ "22_20-21M.fa", "22_20-21M.fa.fai", "22_20-21M.gtf", "22_20-21M.snp", "22_20-21M.ss", "22_20-21M.exon"] files = [] if not small: files += genome_files files += small_genome_files for file in files: if os.path.exists(file): continue wget_cmd = "wget %s/data/%s" % (data_root, file) print >> sys.stderr, wget_cmd os.system(wget_cmd) os.chdir("..") # Download indexes if not os.path.exists("indexes"): os.mkdir("indexes") os.chdir("indexes") aligners = ["HISAT2", "HISAT", "Bowtie", "STAR", "GSNAP"] for genome in ["genome", "22", "22_20-21M"]: if small and genome == "genome": continue for aligner in aligners: if genome == "genome": aligner_dir = aligner else: aligner_dir = aligner + "_" + genome if os.path.exists(aligner_dir): continue cmd = "wget %s/indexes/%s.tar.gz; tar xvzf %s.tar.gz; rm %s.tar.gz" % \ (data_root, aligner_dir, aligner_dir, aligner_dir) print >> sys.stderr, cmd os.system(cmd) os.chdir("..") # Download simulated and real reads if not os.path.exists("reads"): os.mkdir("reads") os.chdir("reads") for type in ["simulation", "real"]: if small and type == "real": continue if not os.path.exists(type): os.mkdir(type) os.chdir(type) if type == "simulation": files = ["1M_DNA_reads_22", "1M_DNA_mismatch_reads_22", "1M_DNA_snp_reads_22", "1M_DNA_mismatch_snp_reads_22", "1M_RNA_reads_22", "1M_RNA_constant_reads_22", "1M_RNA_mismatch_reads_22", "1M_RNA_snp_reads_22", "1M_RNA_mismatch_snp_reads_22", "1M_RNA_reads_22_20-21M", "20M_DNA_reads_genome", "20M_DNA_snp_reads_genome", "20M_RNA_reads_genome", "20M_RNA_snp_reads_genome"] else: files = ["108M_RNA_wgEncodeCshlLongRnaSeq", "62M_RNA_SRR353653", "80M_DNA_SRR345300", "5M_DNA_NA12878D"] for file in files: if small and file.find("20M") != -1: continue if os.path.exists(file): continue cmd = "wget %s/reads/%s/%s.tar.gz; tar xvzf %s.tar.gz; rm %s.tar.gz" % \ (data_root, type, file, file, file) print >> sys.stderr, cmd os.system(cmd) os.chdir("..") os.chdir("..") if __name__ == "__main__": parser = ArgumentParser( description='Get reference genome, annotations, and indexes') parser.add_argument('-s', '--small', dest='small', action='store_true', default=False, help='small testset') args = parser.parse_args() get_data(args.small) hisat2-2.2.1/evaluation/get_programs.py000077500000000000000000000140641370663763200201450ustar00rootroot00000000000000#!/usr/bin/env python import sys, os use_message = ''' ''' def get_aligners(): mac = (sys.platform == "darwin") if not os.path.exists("aligners"): os.mkdir("aligners") os.chdir("aligners") if not os.path.exists("bin"): os.mkdir("bin") programs = ["HISAT", "Bowtie2", "Bowtie", "TopHat2", "STAR", "GSNAP", "BWA", "StringTie", "Cufflinks", "minimap2"] for program in programs: if program == "HISAT": dir = "hisat-0.1.6-beta" if os.path.exists(dir): continue fname = dir + "-source.zip" url = "http://www.ccb.jhu.edu/software/hisat/downloads" bins = "hisat-align-s hisat-build-s hisat-inspect-s" installs = bins + " hisat hisat-build hisat-inspect" cmd = "wget %s/%s; unzip %s; cd %s; make %s; cp %s ../bin; cd .." % \ (url, fname, fname, dir, bins, installs) elif program == "Bowtie2": dir = "bowtie2-2.3.4.3" if os.path.exists(dir): continue fname = dir + "-source.zip" url = "http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.3.4.3" bins = "bowtie2-align-s bowtie2-build-s bowtie2-inspect-s" installs = bins + " bowtie2 bowtie2-build bowtie2-inspect" cmd = "wget %s/%s; unzip %s; cd %s; make NO_TBB=1 %s; cp %s ../bin; cd .." % \ (url, fname, fname, dir, bins, installs) elif program == "Bowtie": dir = "bowtie-1.1.2" if os.path.exists(dir): continue fname = dir + "-src.zip" url = "http://sourceforge.net/projects/bowtie-bio/files/bowtie/1.1.2" bins = "bowtie-align-s bowtie-build-s bowtie-inspect-s" installs = bins + " bowtie bowtie-build bowtie-inspect" cmd = "wget %s/%s; unzip %s; cd %s; make %s; cp %s ../bin; cd .." % \ (url, fname, fname, dir, bins, installs) elif program == "minimap2": dir = "minimap2-2.12" if os.path.exists(dir): continue fname = dir + ".tar.bz2" #https://github.com/lh3/minimap2/releases/download/v2.12/minimap2-2.12.tar.bz2 url = "https://github.com/lh3/minimap2/releases/download/v2.12" bins = "minimap2" cmd = "wget %s/%s; tar jxvf %s; cd %s; make ; cp %s ../bin; cd .." % \ (url, fname, fname, dir,bins) elif program == "TopHat2": if mac: dir = "tophat-2.1.0.OSX_x86_64" else: dir = "tophat-2.1.0.Linux_x86_64" if os.path.exists(dir): continue fname = dir + ".tar.gz" url = "http://ccb.jhu.edu/software/tophat/downloads" installs = "gtf_juncs juncs_db prep_reads segment_juncs tophat tophat_reports sra_to_solid samtools_0.1.18 map2gtf fix_map_ordering bam_merge long_spanning_reads sam_juncs gtf_to_fasta bam2fastx" cmd = "wget %s/%s; tar xvzf %s; cd %s; cp %s ../bin; cd .." % \ (url, fname, fname, dir, installs) elif program == "STAR": dir = "2.5.2b" if os.path.exists("STAR-" + dir): continue fname = dir + ".tar.gz" url = "https://github.com/alexdobin/STAR/archive" if mac: add_cmd = "awk '{if($1 ~ /^CXX/) {print \"CXX =/opt/local/bin/g++-mp-4.8\";} else {print;}}' Makefile > Makefile.tmp; mv Makefile.tmp Makefile" make_arg = "STARforMac" cmd = "wget %s/%s; tar xvzf %s; cd STAR-%s/source; %s; make; make %s; cp STAR ../../bin; cd ../.." % \ (url, fname, fname, dir, add_cmd, make_arg) else: cmd = "wget %s/%s; tar xvzf %s; cd STAR-%s/source; make; cp STAR ../../bin; cd ../.." % \ (url, fname, fname, dir) elif program == "GSNAP": dir = "gmap-2015-07-23" dir2 = "gmap-gsnap-2015-07-23" if os.path.exists(dir): continue fname = dir2 + ".tar.gz" url = "http://research-pub.gene.com/gmap/src" installs = "gmap gmapl get-genome gmapindex iit_store iit_get iit_dump gsnap gsnapl uniqscan uniqscanl snpindex cmetindex atoiindex sam_sort ../util/*" cmd = "wget %s/%s; tar xvzf %s; cd %s; ./configure; make; cd src; cp %s ../../bin; cd ../.." % \ (url, fname, fname, dir, installs) elif program == "BWA": dir = "bwa-0.7.17" if os.path.exists(dir): continue url = "http://sourceforge.net/projects/bio-bwa/files/%s.tar.bz2" % (dir) installs = "bwa" cmd = "wget %s; tar xvjf %s.tar.bz2; cd %s; make; cp %s ../bin/; cd .." % (url, dir, dir, installs) elif program == "StringTie": dir = "stringtie-1.0.4" url = "http://ccb.jhu.edu/software/stringtie/dl" bins = "stringtie" cmd = "wget %s/%s.tar.gz; tar xvzf %s.tar.gz; cd %s; make release; cp %s ../bin; cd .." % \ (url, dir, dir, dir, bins) elif program == "Cufflinks": cmd = "" elif program == "vg": version = "v1.13.0" dir = program + "-" + version url = "https://github.com/vgteam/vg/releases/download/%s" % (version) cmd = "wget %s/%s.tar.gz; tar zxvf %s.tar.gz; cd %s" % (url, dir, dir, dir) cmd += "; source ./source_me.sh; make; cp bin/%s ../bin; cd .." % (program) else: assert False print >> sys.stderr, cmd os.system(cmd) files = ["hisat2", "hisat2-align-s", "hisat2-build", "hisat2-build-s", "hisat2-inspect", "hisat2-inspect-s", "extract_splice_sites.py", "hisat2_extract_snps_haplotypes_UCSC.py", "hisat2_simulate_reads.py"] os.chdir("bin") for file in files: if os.path.exists(file): continue os.system("ln -s ../../../%s %s" % (file, file)) os.chdir("..") os.chdir("..") if __name__ == "__main__": get_aligners() hisat2-2.2.1/evaluation/real/000077500000000000000000000000001370663763200160155ustar00rootroot00000000000000hisat2-2.2.1/evaluation/real/calculate_read_cost.py000077500000000000000000001744671370663763200223750ustar00rootroot00000000000000#!/usr/bin/env python import sys, os, subprocess, signal import multiprocessing import platform import string import re from datetime import datetime, date, time from collections import defaultdict from argparse import ArgumentParser, FileType osx_mode = False if sys.platform == 'darwin': osx_mode = True MAX_EDIT = 21 signal.signal(signal.SIGPIPE, signal.SIG_DFL) cigar_re = re.compile('\d+\w') """ """ def parse_mem_usage(resource): if osx_mode: resource = resource.strip().split('\n') for line in resource: if line.find('maximum resident set size') != -1: return int(line.split()[0]) / 1024 else: resource = resource.split(' ') for line in resource: idx = line.find('maxresident') if idx != -1: return line[:idx] return '0' """ """ def reverse_complement(seq): result = "" for nt in seq: base = nt if nt == 'A': base = 'T' elif nt == 'a': base = 't' elif nt == 'C': base = 'G' elif nt == 'c': base = 'g' elif nt == 'G': base = 'C' elif nt == 'g': base = 'c' elif nt == 'T': base = 'A' elif nt == 't': base = 'a' result = base + result return result """ """ def read_genome(genome_filename): chr_dic = {} genome_file = open(genome_filename, "r") chr_name, sequence = "", "" for line in genome_file: if line[0] == ">": if chr_name and sequence: chr_dic[chr_name] = sequence chr_name = line[1:-1].split()[0] sequence = "" else: sequence += line[:-1] if chr_name and sequence: chr_dic[chr_name] = sequence genome_file.close() print >> sys.stderr, "genome is loaded" return chr_dic """ """ def read_snp(snp_filename): snps = defaultdict(list) snp_file = open(snp_filename, 'r') for line in snp_file: line = line.strip() if not line or line.startswith('#'): continue try: snpID, type, chr, pos, data = line.split('\t') except ValueError: continue assert type in ["single", "deletion", "insertion"] if type == "deletion": data = int(data) snps[chr].append([snpID, type, int(pos), data]) print >> sys.stderr, "snp is loaded" return snps """ """ def extract_splice_sites(gtf_fname): trans = {} gtf_file = open(gtf_fname) # Parse valid exon lines from the GTF file into a dict by transcript_id for line in gtf_file: line = line.strip() if not line or line.startswith('#'): continue if '#' in line: line = line.split('#')[0].strip() try: chrom, source, feature, left, right, score, \ strand, frame, values = line.split('\t') except ValueError: continue left, right = int(left), int(right) if feature != 'exon' or left >= right: continue values_dict = {} for attr in values.split(';')[:-1]: attr, _, val = attr.strip().partition(' ') values_dict[attr] = val.strip('"') if 'gene_id' not in values_dict or \ 'transcript_id' not in values_dict: continue transcript_id = values_dict['transcript_id'] if transcript_id not in trans: trans[transcript_id] = [chrom, strand, [[left, right]]] else: trans[transcript_id][2].append([left, right]) gtf_file.close() # Sort exons and merge where separating introns are <=5 bps for tran, [chrom, strand, exons] in trans.items(): exons.sort() tmp_exons = [exons[0]] for i in range(1, len(exons)): if exons[i][0] - tmp_exons[-1][1] <= 5: tmp_exons[-1][1] = exons[i][1] else: tmp_exons.append(exons[i]) trans[tran] = [chrom, strand, tmp_exons] # Calculate and print the unique junctions junctions = set() for chrom, strand, exons in trans.values(): for i in range(1, len(exons)): junctions.add(to_junction_str([chrom, exons[i-1][1], exons[i][0]])) return junctions def to_junction_str(junction): return "%s-%d-%d" % (junction[0], junction[1], junction[2]) def to_junction(junction_str): chr, left, right = junction_str.split("-") return [chr, int(left), int(right)] def junction_cmp(a, b): if a[0] != b[0]: if a[0] < b[0]: return -1 else: return 1 if a[1] != b[1]: if a[1] < b[1]: return -1 else: return 1 if a[2] != b[2]: if a[2] < b[2]: return -1 else: return 1 return 0 # chr and pos are assumed to be integers def get_junctions(chr, pos, cigar_str, min_anchor_len = 0, read_len = 100): junctions = [] right_pos = pos cigars = cigar_re.findall(cigar_str) cigars = [[int(cigars[i][:-1]), cigars[i][-1]] for i in range(len(cigars))] left_anchor_lens = [] cur_left_anchor_len = 0 for i in range(len(cigars)): length, cigar_op = cigars[i] if cigar_op in "MI": cur_left_anchor_len += length elif cigar_op == "N": assert cur_left_anchor_len > 0 left_anchor_lens.append(cur_left_anchor_len) cur_left_anchor_len = 0 for i in range(len(cigars)): length, cigar_op = cigars[i] if cigar_op == "N": left, right = right_pos - 1, right_pos + length if i > 0 and cigars[i-1][1] in "ID": if cigars[i-1][1] == "I": left += cigars[i-1][0] else: left -= cigars[i-1][0] if i + 1 < len(cigars) and cigars[i+1][1] in "ID": if cigars[i+1][1] == "I": right -= cigars[i+1][0] else: right += cigars[i+1][0] junction_idx = len(junctions) assert junction_idx < len(left_anchor_lens) left_anchor_len = left_anchor_lens[junction_idx] assert left_anchor_len > 0 and left_anchor_len < read_len right_anchor_len = read_len - left_anchor_len if left_anchor_len >= min_anchor_len and right_anchor_len >= min_anchor_len: junctions.append([chr, left, right]) if cigar_op in "MND": right_pos += length return junctions def get_right(pos, cigars): right_pos = pos cigars = cigar_re.findall(cigars) for cigar in cigars: length = int(cigar[:-1]) cigar_op = cigar[-1] if cigar_op in "MDN": right_pos += length return right_pos def get_cigar_chars(cigars): cigars = cigar_re.findall(cigars) cigar_chars = "" for cigar in cigars: cigar_op = cigar[-1] cigar_chars += cigar_op return cigar_chars def get_cigar_chars_MN(cigars): cigars = cigar_re.findall(cigars) cigar_chars = "" for cigar in cigars: cigar_op = cigar[-1] if cigar_op in "MN": if cigar_chars == "" or cigar_chars[-1] != cigar_op: cigar_chars += cigar_op return cigar_chars def is_non_canonical_junction_read(chr_dic, chr, left, cigars, canonical_junctions = [["GT", "AG"], ["GC", "AG"], ["AT", "AC"]]): pos = left for cigar in cigar_re.findall(cigars): cigar_op = cigar[-1] cigar_len = int(cigar[:-1]) if cigar_op in 'MD': pos += cigar_len elif cigar_op == 'N': right = pos + cigar_len donor = chr_dic[chr][pos-1:pos+1] acceptor = chr_dic[chr][right-3:right-1] rev_donor = reverse_complement(acceptor) rev_acceptor = reverse_complement(donor) # print donor, acceptor # print rev_donor, rev_acceptor if [donor, acceptor] not in canonical_junctions and [rev_donor, rev_acceptor] not in canonical_junctions: return True pos = right return False def is_canonical_junction(chr_dic, junction): chr, left, right = junction donor = chr_dic[chr][left:left+2] acceptor = chr_dic[chr][right-3:right-1] rev_donor = reverse_complement(acceptor) rev_acceptor = reverse_complement(donor) if (donor == "GT" and acceptor == "AG") or \ (rev_donor == "GT" and rev_acceptor == "AG"): return True return False def is_junction_read(chr_dic, gtf_junctions, chr, pos, cigar_str): result_junctions = [] junctions = get_junctions(chr, pos, cigar_str, 0, 101) for junction in junctions: junction_str = to_junction_str(junction) is_gtf_junction = False def find_in_gtf_junctions(gtf_junctions, junction): l, u = 0, len(gtf_junctions) while l < u: m = (l + u) / 2 assert m >= 0 and m < len(gtf_junctions) cmp_result = junction_cmp(junction, gtf_junctions[m]) if cmp_result == 0: return m elif cmp_result < 0: u = m else: l = m + 1 return l # allow small (<= 5bp) discrepancy for non-canonical splice sites. relaxed_junction_dist = 5 chr, left, right = junction gtf_index = find_in_gtf_junctions(gtf_junctions, [chr, left - relaxed_junction_dist, right - relaxed_junction_dist]) if gtf_index >= 0: i = gtf_index while i < len(gtf_junctions): chr2, left2, right2 = gtf_junctions[i] if chr2 > chr or \ left2 - left > relaxed_junction_dist or \ right2 - right > relaxed_junction_dist: break if abs(left - left2) <= relaxed_junction_dist and left - left2 == right - right2: canonical = is_canonical_junction(chr_dic, gtf_junctions[i]) if left == left2 or not canonical: is_gtf_junction = True break i += 1 result_junctions.append([junction_str, is_gtf_junction]) is_gtf_junction_read = False if len(result_junctions) > 0: is_gtf_junction_read = True for junction_str, is_gtf_junction in result_junctions: if not is_gtf_junction: is_gtf_junction_read = False break return result_junctions, len(result_junctions) > 0, is_gtf_junction_read def is_junction_pair(chr_dic, gtf_junctions, chr, pos, cigar_str, mate_chr, mate_pos, mate_cigar_str): junctions, junction_read, gtf_junction_read = is_junction_read(chr_dic, gtf_junctions, chr, pos, cigar_str) mate_junctions, mate_junction_read, mate_gtf_junction_read = is_junction_read(chr_dic, gtf_junctions, mate_chr, mate_pos, mate_cigar_str) junctions += mate_junctions junction_pair = len(junctions) > 0 if junction_pair: gtf_junction_pair = True if junction_read and not gtf_junction_read: gtf_junction_pair = False if mate_junction_read and not mate_gtf_junction_read: gtf_junction_pair = False else: gtf_junction_pair = False return junctions, junction_pair, gtf_junction_pair """ """ def getSNPs(chr_snps, left, right): low, high = 0, len(chr_snps) while low < high: mid = (low + high) / 2 snpID, type, pos, data = chr_snps[mid] if pos < left: low = mid + 1 else: high = mid - 1 snps = [] for i in xrange(low, len(chr_snps)): snp = chr_snps[i] snpID, type, pos, data = snp pos2 = pos if type == "deletion": pos2 += data if pos2 >= right: break if pos >= left: if len(snps) > 0: _, prev_type, prev_pos, prev_data = snps[-1] assert prev_pos <= pos prev_pos2 = prev_pos if prev_type == "deletion": prev_pos2 += prev_data if pos <= prev_pos2: continue snps.append(snp) return snps """ """ def check_snps(snps, check_type, ref_pos, read_seq): found = False for snp in snps: snp_type, snp_pos, snp_data = snp[1:4] if snp_type == check_type: if snp_type == 'single': if snp_pos == ref_pos and snp_data[0] == read_seq[0]: found = True break elif snp_type == 'insertion': if snp_pos == ref_pos and snp_data == read_seq: found = True break elif snp_type == 'deletion': # snp_data and read_seq are length of sequence deleted if snp_pos == ref_pos and int(snp_data) == int(read_seq): found = True break return found def extract_reads_and_pairs(chr_dic, sam_filename, read_filename, pair_filename, unmapped_read_1_fq_name, unmapped_read_2_fq_name, snps_dict = None): temp_read_filename, temp_pair_filename = read_filename + ".temp", pair_filename + ".temp" temp_read_file, temp_pair_file = open(temp_read_filename, "w"), open(temp_pair_filename, "w") unmapped_read_1_fq, unmapped_read_2_fq = open(unmapped_read_1_fq_name, "w"), open(unmapped_read_2_fq_name, "w") hisat2 = read_filename.find("hisat2") != -1 or pair_filename.find("hisat2") != -1 vg = read_filename.find("vg") != -1 or pair_filename.find("vg") != -1 read_dic = {} prev_read_id, prev_read_seq = "", "" sam_file = open(sam_filename, "r") for line in sam_file: if line[0] == "@": continue fields = line[:-1].split() read_id, flag, chr, pos, mapQ, cigar_str, mate_chr, mate_pos, template_len, read_seq, read_qual = fields[:11] if 'H' in cigar_str: continue flag, pos, mate_pos = int(flag), int(pos), int(mate_pos) if read_seq == "*" and prev_read_id == read_id: read_seq = prev_read_seq read_seq = read_seq.upper() if read_seq == "" or read_seq == "*": continue if flag & 0x04 != 0 or \ chr == "*" or \ cigar_str == "*": """ if flag & 0x80 != 0: print >> unmapped_read_2_fq, "@%s\n%s\n+%s\n%s" % (read_id, read_seq, read_id, read_qual) else: print >> unmapped_read_1_fq, "@%s\n%s\n+%s\n%s" % (read_id, read_seq, read_id, read_qual) """ continue if mate_chr == '=': mate_chr = chr if len(read_id) >= 3 and read_id[-2] == "/": read_id = read_id[:-2] if read_id.find("seq.") == 0: read_id = read_id[4:] if read_id != prev_read_id: read_dic = {} HISAT2_XM, HISAT2_NM = 0, 0 if hisat2: for field in fields[11:]: if field[:5] == "XM:i:": HISAT2_XM = int(field[5:]) elif field[:5] == "NM:i:": HISAT2_NM = int(field[5:]) prev_read_id = read_id prev_read_seq = read_seq if snps_dict != None and chr in snps_dict: chr_snps = snps_dict[chr] else: chr_snps = [] snps = None XM, gap = 0, 0 read_pos, right_pos = 0, pos - 1, junction_read = False cigars = cigar_re.findall(cigar_str) for i in range(len(cigars)): cigar = cigars[i] length = int(cigar[:-1]) cigar_op = cigar[-1] if cigar_op == "S": if i != 0 and i != len(cigars) - 1: print >> sys.stderr, "S is located at %dth out of %d %s" % (i+1, len(cigars), cigar_str) if cigar_op in "MS": ref_pos = right_pos if cigar_op == "S" and i == 0: ref_seq = chr_dic[chr][right_pos-length:right_pos] ref_pos = right_pos - length else: ref_seq = chr_dic[chr][right_pos:right_pos+length] ref_seq = ref_seq.upper() if length == len(ref_seq): for j in range(length): if ref_seq[j] != "N" and read_seq[read_pos+j] != ref_seq[j]: if snps_dict == None: XM += 1 else: if snps == None: snps = getSNPs(chr_snps, pos - 1, pos + len(read_seq)) found_snp = check_snps(snps, 'single', ref_pos + j, read_seq[read_pos + j]) if not found_snp: XM += 1 if hisat2 and cigar_op == "S": HISAT2_XM += 1 HISAT2_NM += 1 else: XM += length if cigar_op in "I": if snps == None: snps = getSNPs(chr_snps, pos - 1, pos + len(read_seq)) found_snp = check_snps(snps, 'insertion', right_pos, read_seq[read_pos:read_pos + length]) if not found_snp: gap += length if cigar_op in "D": if snps == None: snps = getSNPs(chr_snps, pos - 1, pos + len(read_seq)) found_snp = check_snps(snps, 'deletion', right_pos, length) if not found_snp: gap += length if cigar_op in "MND": right_pos += length if cigar_op in "MIS": read_pos += length if cigar_op == "N": junction_read = True NM = XM + gap if hisat2: XM, NM = HISAT2_XM, HISAT2_NM if NM < MAX_EDIT: print >> temp_read_file, "%s\t%d\t%s\t%s\t%s\tXM:i:%d\tNM:i:%d" % \ (read_id, flag, chr, pos, cigar_str, XM, NM) found = False me = "%s\t%s\t%d" % (read_id, chr, pos) partner = "%s\t%s\t%d" % (read_id, mate_chr, mate_pos) if partner in read_dic: maps = read_dic[partner] for map in maps: if map[0] == me: mate_flag, mate_cigar_str, mate_XM, mate_NM = map[1:] if mate_pos > pos: flag, chr, pos, cigar_str, XM, NM, mate_flag, mate_chr_str, mate_pos, mate_cigar_str, mate_XM, mate_NM = \ mate_flag, mate_chr, mate_pos, mate_cigar_str, mate_XM, mate_NM, flag, chr, pos, cigar_str, XM, NM print >> temp_pair_file, "%s\t%d\t%s\t%d\t%s\tXM:i:%d\tNM:i:%d\t%d\t%s\t%d\t%s\tXM:i:%d\tNM:i:%d" % \ (read_id, mate_flag, mate_chr, mate_pos, mate_cigar_str, mate_XM, mate_NM, flag, chr, pos, cigar_str, XM, NM) found = True break if not found: if not me in read_dic: read_dic[me] = [] read_dic[me].append([partner, flag, cigar_str, XM, NM]) sam_file.close() temp_read_file.close() temp_pair_file.close() unmapped_read_1_fq.close() unmapped_read_2_fq.close() sort = False if vg: sort = True if sort: command = "sort %s | uniq > %s; rm %s" % (temp_read_filename, read_filename, temp_read_filename) os.system(command) command = "sort %s | uniq > %s; rm %s" % (temp_pair_filename, pair_filename, temp_pair_filename) os.system(command) else: command = "mv %s %s; mv %s %s" % (temp_read_filename, read_filename, temp_pair_filename, pair_filename) os.system(command) def remove_redundant_junctions(junctions): temp_junctions = [] for junction in junctions: temp_junctions.append(to_junction(junction)) junctions = sorted(list(temp_junctions), cmp=junction_cmp) temp_junctions = [] for can_junction in junctions: if len(temp_junctions) <= 0: temp_junctions.append(can_junction) else: chr, left, right = temp_junctions[-1] chr2, left2, right2 = can_junction if chr == chr2 and \ abs(left - left2) == abs(right - right2) and \ abs(left - left2) <= 10: continue temp_junctions.append(can_junction) junctions = set() for junction in temp_junctions: junctions.add(to_junction_str(junction)) return junctions def read_stat(read_filename, gtf_junctions, chr_dic = None, debug = False): read_stat = [[0, 0, 0] for i in range(MAX_EDIT)] temp_junctions = [set() for i in range(MAX_EDIT)] temp_gtf_junctions = [set() for i in range(MAX_EDIT)] alignment = [] prev_read_id = "" read_file = open(read_filename, "r") for line in read_file: read_id, flag, chr, pos, cigar_str, XM, NM = line[:-1].split() flag, pos = int(flag), int(pos) XM, NM = int(XM[5:]), int(NM[5:]) read_junctions, junction_read, gtf_junction_read = \ is_junction_read(chr_dic, gtf_junctions, chr, pos, cigar_str) if junction_read: for junction_str, is_gtf_junction in read_junctions: if NM < len(temp_junctions): temp_junctions[NM].add(junction_str) if is_gtf_junction: temp_gtf_junctions[NM].add(junction_str) if read_id != prev_read_id: if prev_read_id != "": NM2, junction_read2, gtf_junction_read2 = alignment if NM2 < len(read_stat): read_stat[NM2][0] += 1 if junction_read2: read_stat[NM2][1] += 1 if gtf_junction_read2: read_stat[NM2][2] += 1 alignment = [] prev_read_id = read_id if not alignment: alignment = [NM, junction_read, gtf_junction_read] elif alignment[0] > NM or \ (alignment[0] == NM and not alignment[2] and junction_read): alignment = [NM, junction_read, gtf_junction_read] read_file.close() for i in range(len(read_stat)): temp_junctions[i] = remove_redundant_junctions(temp_junctions[i]) temp_gtf_junctions[i] = remove_redundant_junctions(temp_gtf_junctions[i]) for i in range(len(read_stat)): read_stat[i].append(len(temp_junctions[i])) read_stat[i].append(len(temp_gtf_junctions[i])) if alignment: NM2, junction_read2, gtf_junction_read2 = alignment if NM2 < len(read_stat): read_stat[NM2][0] += 1 if junction_read2: read_stat[NM2][1] += 1 if gtf_junction_read2: read_stat[NM2][2] += 1 return read_stat def cal_read_len(cigar_str): length = 0 leftmost_softclip = 0 rightmost_softclip = 0 cigars = cigar_re.findall(cigar_str) for i in range(len(cigars)): cigar = cigars[i] cigar_length = int(cigar[:-1]) cigar_op = cigar[-1] if cigar_op in "MIS": length += cigar_length if (i == 0) and (cigar_op == "S"): leftmost_softclip = cigar_length if (i == (len(cigars) - 1)) and (cigar_op == "S"): rightmost_softclip = cigar_length return length, leftmost_softclip, rightmost_softclip def is_concordantly(read_id, flag, chr, pos, cigar_str, XM, NM, mate_flag, mate_chr, mate_pos, mate_cigar_str, mate_XM, mate_NM): concord_length = 1000 segment_length = sys.maxint pairs = {} pairs[0] = [flag, chr, pos, cigar_str, XM, NM] pairs[1] = [mate_flag, mate_chr, mate_pos, mate_cigar_str, mate_XM, mate_NM] if chr != mate_chr: return False, segment_length if (flag & 0x10 == 0x10) or (mate_flag & 0x10 == 0): return False, segment_length assert pos <= mate_pos left = pairs[0] right = pairs[1] left_start = left[2] left_len, _, _ = cal_read_len(left[3]) # cigar right_start = right[2] right_len, _, right_soft = cal_read_len(right[3]) segment_length = (right_start + right_len) - left_start - right_soft assert segment_length >= 0 if segment_length > concord_length: return False, segment_length return True, segment_length def pair_stat(pair_filename, gtf_junctions, chr_dic): # pair_stat = NM, junction_pair, gtf_junction, concordant_alignment] pair_stat = [[0, 0, 0, 0] for i in range(MAX_EDIT)] dis_pair_stat = [0 for i in range(MAX_EDIT)] temp_junctions = [set() for i in range(MAX_EDIT)] temp_gtf_junctions = [set() for i in range(MAX_EDIT)] alignment, dis_alignments = [], [] prev_read_id = "" con_file = open(pair_filename + ".con", "w") discon_file = open(pair_filename + ".discon", "w") pair_file = open(pair_filename, "r") for line in pair_file: read_id, flag, chr, pos, cigar_str, XM, NM, mate_flag, mate_chr, mate_pos, mate_cigar_str, mate_XM, mate_NM = line[:-1].split() flag, pos, XM, NM, mate_flag, mate_pos, mate_XM, mate_NM = \ int(flag), int(pos), int(XM[5:]), int(NM[5:]), int(mate_flag), int(mate_pos), int(mate_XM[5:]), int(mate_NM[5:]) pair_XM = XM + mate_XM pair_NM = NM + mate_NM pair_junctions, junction_pair, gtf_junction_pair = \ is_junction_pair(chr_dic, gtf_junctions, chr, pos, cigar_str, mate_chr, mate_pos, mate_cigar_str) # check concordantly concord_align, segment_len = is_concordantly(read_id, flag, chr, pos, cigar_str, XM, NM, mate_flag, mate_chr, mate_pos, mate_cigar_str, mate_XM, mate_NM) print >> (con_file if concord_align else discon_file), line.strip(), ('none', 'first')[(flag & 0x40 == 0x40)], ('none', 'last')[(mate_flag & 0x80 == 0x80)], segment_len if junction_pair: for junction_str, is_gtf_junction in pair_junctions: if pair_NM < len(temp_junctions): temp_junctions[pair_NM].add(junction_str) if is_gtf_junction: temp_gtf_junctions[pair_NM].add(junction_str) if read_id != prev_read_id: if prev_read_id != "": NM2, junction_read2, gtf_junction_read2, concord_align2 = alignment if NM2 < len(pair_stat): pair_stat[NM2][0] += 1 if junction_read2: pair_stat[NM2][1] += 1 if gtf_junction_read2: pair_stat[NM2][2] += 1 if concord_align2: pair_stat[NM2][3] += 1 for NM2 in dis_alignments: if NM2 < len(dis_pair_stat): dis_pair_stat[NM2] += 1 alignment = [] dis_alignment = [] prev_read_id = read_id if not alignment: alignment = [pair_NM, junction_pair, gtf_junction_pair, concord_align] elif alignment[0] > pair_NM or \ (alignment[0] == pair_NM and not alignment[2] and junction_pair): alignment = [pair_NM, junction_pair, gtf_junction_pair, concord_align] if mate_chr != chr or ((flag & 0x10) != 0 or (mate_flag & 0x10) == 0): if len(dis_alignments) == 0: dis_alignments = [pair_NM] elif dis_alignments[0] > pair_NM: dis_alignments = [pair_NM] pair_file.close() con_file.close() discon_file.close() # process last line if alignment: NM2, junction_read2, gtf_junction_read2, concord_align2 = alignment if NM2 < len(pair_stat): pair_stat[NM2][0] += 1 if junction_read2: pair_stat[NM2][1] += 1 if gtf_junction_read2: pair_stat[NM2][2] += 1 if concord_align2: pair_stat[NM2][3] += 1 assert len(dis_alignments) <= 1 for NM2 in dis_alignments: if NM2 < len(dis_pair_stat): dis_pair_stat[NM2] += 1 for i in range(len(pair_stat)): temp_junctions[i] = remove_redundant_junctions(temp_junctions[i]) temp_gtf_junctions[i] = remove_redundant_junctions(temp_gtf_junctions[i]) for i in range(len(pair_stat)): pair_stat[i].append(len(temp_junctions[i])) pair_stat[i].append(len(temp_gtf_junctions[i])) return pair_stat, dis_pair_stat def sql_execute(sql_db, sql_query): sql_cmd = [ "sqlite3", sql_db, "-separator", "\t", "%s;" % sql_query ] # print >> sys.stderr, sql_cmd sql_process = subprocess.Popen(sql_cmd, stdout=subprocess.PIPE) output = sql_process.communicate()[0][:-1] return output def create_sql_db(sql_db): if os.path.exists(sql_db): print >> sys.stderr, sql_db, "already exists!" return columns = [ ["id", "integer primary key autoincrement"], ["reads", "text"], ["genome", "text"], ["end_type", "text"], ["aligner", "text"], ["version", "test"], ["use_annotation", "text"], ["edit_distance", "integer"], ["mapped_reads", "integer"], ["junction_reads", "integer"], ["gtf_junction_reads", "integer"], ["junctions", "integer"], ["gtf_junctions", "integer"], ["runtime", "real"], ["host", "text"], ["created", "text"], ["cmd", "text"] ] sql_create_table = "CREATE TABLE Mappings (" for i in range(len(columns)): name, type = columns[i] if i != 0: sql_create_table += ", " sql_create_table += ("%s %s" % (name, type)) sql_create_table += ");" sql_execute(sql_db, sql_create_table) def write_analysis_data(sql_db, database_name, paired): if not os.path.exists(sql_db): return if paired: paired = "paired" else: paired = "single" aligners = [] sql_aligners = "SELECT aligner FROM Mappings WHERE end_type = '%s' GROUP BY aligner" % (paired) output = sql_execute(sql_db, sql_aligners) aligners = output.split() database_fname = database_name + "_" + paired + ".analysis" database_file = open(database_fname, "w") print >> database_file, "aligner\tuse_annotation\tend_type\tedit_distance\tmapped_reads\tjunction_reads\tgtf_junction_reads\tjunctions\tgtf_junctions\truntime" for aligner in aligners: for edit_distance in range(MAX_EDIT): sql_row = "SELECT aligner, use_annotation, end_type, edit_distance, mapped_reads, junction_reads, gtf_junction_reads, junctions, gtf_junctions, runtime FROM Mappings" sql_row += " WHERE reads = '%s' and aligner = '%s' and edit_distance = %d and end_type = '%s' ORDER BY created DESC LIMIT 1" % (database_name, aligner, edit_distance, paired) output = sql_execute(sql_db, sql_row) if output: print >> database_file, output database_file.close() def calculate_read_cost(single_end, paired_end, test_aligners, fresh, runtime_only, verbose): sql_db_name = "analysis.db" if not os.path.exists(sql_db_name): create_sql_db(sql_db_name) full_workdir = os.getcwd() workdir = full_workdir.split("/")[-1] num_cpus = multiprocessing.cpu_count() if num_cpus > 8: num_threads = min(8, num_cpus) desktop = False else: num_threads = min(3, num_cpus) desktop = True verbose = False sql_write = True is_large_file = False gz_file = False if os.path.exists("1.fq.gz"): gz_file = True if os.path.getsize("1.fq.gz") > (1024 * 1024 * 1024): is_large_file = True elif os.path.exists("1.fq"): gz_file = False if os.path.getsize("1.fq") > (2 * 1024 * 1024 * 1024): is_large_file = True else: assert(False) aligners = [ # ["hisat2", "", "", "", ""], # ["hisat2", "", "", "", "--sensitive"], # ["hisat2", "", "", "", "--very-sensitive"], # ["hisat2", "", "", "", "-k 50 --score-min C,-50,0"], # ["hisat2", "", "snp", "", ""], # ["hisat2", "", "snp", "", "--sensitive"], # ["hisat2", "", "snp", "", "-k 50"], # ["hisat2", "", "", "205", ""], # ["hisat2", "", "snp", "205", ""], # ["hisat2", "", "snp_tran", "205", ""], # ["hisat2", "", "tran", "", ""], # ["hisat2", "x1", "snp", "", ""], # ["hisat2", "x1", "", "", ""], # ["hisat2", "x2", "", "", ""], # ["hisat2", "", "tran", "", ""], # ["hisat2", "", "snp_tran", "204", ""], # ["hisat2", "", "snp_tran", "", ""], # ["hisat2", "", "", "210", ""], ["hisat2", "", "rep", "", ""], # ["hisat2", "", "rep", "", "--read-lengths 101"], # ["hisat2", "", "rep", "", "--sensitive"], # ["hisat2", "", "rep-100-300", "", ""], # ["hisat2", "", "rep-101-300", "", "--sensitive"], # ["hisat2", "", "rep-101-300", "", "-k 10 --score-min C,-50,0"], # ["hisat2", "", "rep-150-300", "", ""], # ["tophat2", "", "", "", ""], # ["bowtie", "", "", "", ""], ["bowtie2", "", "", "", ""], # ["bowtie2", "", "", "", "-k 10"], ["bwa", "mem", "", "", ""], # ["bwa", "mem", "", "", "-a"], # ["bwa", "sw", "", "", ""], # ["star", "", "", "", ""], # ["star", "x2", "", "", ""], # ["vg", "", "", "", ""], # ["vg", "", "", "", "-M 10"], # ["vg", "", "snp", "", ""], # ["vg", "", "snp", "", "-M 10"], # ["minimap2", "", "", "", ""], ] # sql_write = False verbose = True debug = False genome = "genome" cwd = os.getcwd() RNA = (cwd.find("RNA") != -1) chr_dic = read_genome("../../../data/" + genome + ".fa") snp_dic = read_snp("../../../data/" + genome + ".snp") gtf_junction_strs = extract_splice_sites("../../../data/" + genome + ".gtf") gene = "no" gtf_junctions = [] for junction_str in gtf_junction_strs: junction = to_junction(junction_str) gtf_junctions.append(junction) gtf_junctions = sorted(gtf_junctions, cmp=junction_cmp) print >> sys.stderr, "aligner\tuse_annotation\tend_type\tedit_distance\tmapped_reads\tjunction_reads\tgtf_junction_reads\tjunctions\tgtf_junctions\truntime" for paired in [False, True]: if not paired and not single_end: continue if paired and not paired_end: continue type_read1_fname = "1.fq" if gz_file: type_read1_fname += ".gz" if paired: type_read2_fname = "2.fq" if gz_file: type_read2_fname += ".gz" else: type_read2_fname = "" aligner_bin_base = "../../../../aligners/bin" def get_aligner_version(aligner): version = "" if aligner == "hisat2" or \ aligner == "hisat" or \ aligner == "bowtie" or \ aligner == "bowtie2": if version: cmd = ["%s/%s_%s/%s" % (aligner_bin_base, aligner, version, aligner)] else: cmd = ["%s/%s" % (aligner_bin_base, aligner)] cmd += ["--version"] cmd_process = subprocess.Popen(cmd, stdout=subprocess.PIPE) version = cmd_process.communicate()[0][:-1].split("\n")[0] version = version.split()[-1] elif aligner == "tophat2": cmd = ["%s/tophat" % (aligner_bin_base)] cmd += ["--version"] cmd_process = subprocess.Popen(cmd, stdout=subprocess.PIPE) version = cmd_process.communicate()[0][:-1].split()[-1] elif aligner in ["star", "starx2"]: version = "2.4.2a" elif aligner == "gsnap": cmd = ["%s/gsnap" % (aligner_bin_base)] cmd_process = subprocess.Popen(cmd, stderr=subprocess.PIPE) version = cmd_process.communicate()[1][:-1].split("\n")[0] version = version.split()[2] elif aligner == "bwa": if version: cmd = ["%s/bwa_%s/bwa" % (aligner_bin_base, version)] else: cmd = ["%s/bwa" % (aligner_bin_base)] cmd_process = subprocess.Popen(cmd, stderr=subprocess.PIPE) version = cmd_process.communicate()[1][:-1].split("\n")[2] version = version.split()[1] elif aligner == "vg": cmd = ["%s/vg" % (aligner_bin_base)] cmd_process = subprocess.Popen(cmd, stderr=subprocess.PIPE) version = cmd_process.communicate()[1][:-1].split("\n")[0] version = version.split()[5] elif aligner == "minimap2": cmd = ["%s/minimap2" % (aligner_bin_base)] cmd += ["--version"] cmd_process = subprocess.Popen(cmd, stdout=subprocess.PIPE) version = cmd_process.communicate()[0][:-1].split("\n")[0] return version index_base = "../../../../indexes" index_add = "" if genome != "genome": index_add = "_" + genome def get_aligner_cmd(RNA, aligner, type, index_type, version, options, read1_fname, read2_fname, out_fname, cmd_idx = 0): cmd = ["/usr/bin/time"] if osx_mode: cmd += ['-l'] if aligner == "hisat2": if version: cmd += ["%s/hisat2_%s/hisat2" % (aligner_bin_base, version)] else: cmd += ["%s/hisat2" % (aligner_bin_base)] if num_threads > 1: cmd += ["-p", str(num_threads)] # cmd += ["-k", "5"] # cmd += ["--score-min", "C,-18"] # daehwan - for debugging purposes # cmd += ["--score-min", "C,-50"] # cmd += ["--pen-cansplice", "0"] # cmd += ["--pen-noncansplice", "12"] # cmd += ["--pen-intronlen", "G,-8,1"] # cmd += ["--metrics", "1", # "--metrics-file", "metrics.out"] if version == "204": cmd += ["--sp", "2,1"] if not RNA: cmd += ["--no-spliced-alignment"] if type in ["x1", "x2"]: cmd += ["--no-temp-splicesite"] # DK - for debugging purposes # cmd += ["--dta"] """ if index_type == "tran": cmd += ["--no-anchorstop"] cmd += ["-k", "100"] """ if options != "": cmd += options.split(' ') if type == "x2": if cmd_idx == 0: cmd += ["--novel-splicesite-outfile"] else: cmd += ["--novel-splicesite-infile"] cmd += ["splicesites.txt"] # "--novel-splicesite-infile", # "../splicesites.txt", # "--rna-strandness", # "FR", if version: index_cmd = "%s/HISAT2_%s%s/" % (index_base, version, index_add) + genome else: index_cmd = "%s/HISAT2%s/" % (index_base, index_add) + genome if index_type: index_cmd += ("_" + index_type) cmd += [index_cmd] if paired: cmd += ["-1", read1_fname, "-2", read2_fname] else: cmd += ["-U", read1_fname] elif aligner == "hisat": cmd += ["%s/hisat" % (aligner_bin_base)] if num_threads > 1: cmd += ["-p", str(num_threads)] # cmd += ["-k", "5"] # cmd += ["--score-min", "C,-18"] if version != "": version = int(version) else: version = sys.maxint if not RNA: cmd += ["--no-spliced-alignment"] if type in ["x1", "x2"] or not RNA: cmd += ["--no-temp-splicesite"] """ cmd += ["--rdg", "100,100", "--rfg", "100,100"] """ if type == "x2": if cmd_idx == 0: cmd += ["--novel-splicesite-outfile"] else: cmd += ["--novel-splicesite-infile"] cmd += ["splicesites.txt"] # "--novel-splicesite-infile", # "../splicesites.txt", # "--rna-strandness", # "FR", cmd += ["%s/HISAT%s/" % (index_base, index_add) + genome] if paired: cmd += ["-1", read1_fname, "-2", read2_fname] else: cmd += [read1_fname] elif aligner == "tophat2": cmd += ["%s/tophat" % (aligner_bin_base)] if num_threads > 1: cmd += ["-p", str(num_threads)] cmd += ["--read-edit-dist", "3"] cmd += ["--no-sort-bam"] cmd += ["--read-realign-edit-dist", "0"] cmd += ["--keep-tmp", "%s/HISAT%s/" % (index_base, index_add) + genome, read1_fname] if paired: cmd += [read2_fname] elif aligner == "star": cmd += ["%s/STAR" % (aligner_bin_base)] if num_threads > 1: cmd += ["--runThreadN", str(num_threads)] if type == "x2" and cmd_idx == 1: cmd += ["--genomeDir", "."] else: cmd += ["--genomeDir", "%s/STAR%s" % (index_base, index_add)] if desktop: cmd += ["--genomeLoad", "NoSharedMemory"] else: cmd += ["--genomeLoad", "LoadAndKeep"] if type == "x2": if cmd_idx == 1: cmd += ["--alignSJDBoverhangMin", "1"] cmd += ["--readFilesIn", read1_fname] if paired: cmd += [read2_fname] if paired: cmd += ["--outFilterMismatchNmax", "6"] else: cmd += ["--outFilterMismatchNmax", "3"] elif aligner == "bowtie": cmd += ["%s/bowtie" % (aligner_bin_base)] if num_threads > 1: cmd += ["-p", str(num_threads)] cmd += ["--sam", "-k", "10"] cmd += ["-n", "3"] if paired: cmd += ["-X", "500"] cmd += ["%s/Bowtie%s/" % (index_base, index_add) + genome] if paired: cmd += ["-1", read1_fname, "-2", read2_fname] else: cmd += [read1_fname] elif aligner == "bowtie2": if version: cmd += ["%s/bowtie2_%s/bowtie2" % (aligner_bin_base, version)] else: cmd += ["%s/bowtie2" % (aligner_bin_base)] if num_threads > 1: cmd += ["-p", str(num_threads)] #cmd += ["-k", "10"] #cmd += ["--score-min", "C,-18"] cmd += ["-X", "1000"] if options: cmd += options.split(' ') if version: cmd += ["-x %s/Bowtie2_%s%s/" % (index_base, version, index_add) + genome] else: cmd += ["-x %s/Bowtie2%s/" % (index_base, index_add) + genome] if paired: cmd += ["-1", read1_fname, "-2", read2_fname] else: cmd += [read1_fname] elif aligner == "gsnap": cmd += ["%s/gsnap" % (aligner_bin_base), "-A", "sam"] if num_threads > 1: cmd += ["-t", str(num_threads)] cmd += ["--max-mismatches=3", "-D", "%s/GSNAP%s" % (index_base, index_add), "-N", "1", "-d", genome, read1_fname] if paired: cmd += [read2_fname] elif aligner == "bwa": if version: cmd += ["%s/bwa_%s/bwa" % (aligner_bin_base, version)] else: cmd += ["%s/bwa" % (aligner_bin_base)] if type in ["mem", "aln"]: cmd += [type] elif type == "sw": cmd += ["bwa" + type] if num_threads > 1: cmd += ["-t", str(num_threads)] if options: cmd += options.split(' ') if version: cmd += ["%s/BWA_%s%s/%s.fa" % (index_base, version, index_add, genome)] else: cmd += ["%s/BWA%s/%s.fa" % (index_base, index_add, genome)] cmd += [read1_fname] if paired: cmd += [read2_fname] elif aligner == "vg": # vg map -d 22 -t 6 -M 10 -f ../sim-1.fa -f ../sim-2.fa --surject-to sam > result.sam cmd += ["%s/vg" % (aligner_bin_base)] cmd += ["map"] cmd += ["-t", str(num_threads)] cmd += ["--surject-to", "sam"] index_cmd = "%s/VG%s/" % (index_base, index_add) + genome if index_type: index_cmd += ("_" + index_type) if options: cmd += options.split(' ') cmd += ["-d", index_cmd] cmd += ["-f", read1_fname] if paired: cmd += ["-f", read2_fname] elif aligner == "minimap2": # minimap2 -a -x sr 22.mmi sim_1.fa sim_2.fa > result.sam cmd += ["%s/minimap2" % (aligner_bin_base)] cmd += ["-a"] cmd += ["-x", "sr"] index_cmd = "%s/minimap2%s/" % (index_base, index_add) + genome if index_type: index_cmd += ("_" + index_type) index_cmd += ".mmi" cmd += [index_cmd] cmd += [read1_fname] if paired: cmd += [read2_fname] else: assert False return cmd for aligner, type, index_type, version, options in aligners: skip = False if len(test_aligners) > 0: skip = True for test_aligner in test_aligners: if aligner == test_aligner: skip = False if skip: continue aligner_name = aligner + type + version if (aligner == "hisat2" or aligner == "vg") and index_type != "": aligner_name += ("_" + index_type) if options != "": option_name = options.replace(' ', '').replace('-', '').replace(',', '') aligner_name = aligner_name + '_' + option_name if paired: aligner_dir = aligner_name + "_paired" else: aligner_dir = aligner_name + "_single" if fresh and os.path.exists(aligner_dir): os.system("rm -rf %s" % aligner_dir) if not os.path.exists(aligner_dir): os.mkdir(aligner_dir) os.chdir(aligner_dir) out_fname = "accepted.sam" aligner_cmd = get_aligner_cmd(RNA, aligner, type, index_type, version, options, "../" + type_read1_fname, "../" + type_read2_fname, out_fname) duration = 0.1 mem_usage = '' if not os.path.exists(out_fname): if not os.path.exists("../one.fq") or not os.path.exists("../two.fq"): if gz_file: os.system("gzip -cd ../1.fq.gz | head -400 > ../one.fq") os.system("gzip -cd ../2.fq.gz | head -400 > ../two.fq") else: os.system("head -400 ../1.fq > ../one.fq") os.system("head -400 ../2.fq > ../two.fq") # dummy commands for caching index loading_time = 0 if aligner not in ["tophat2"]: for i in range(3): dummy_cmd = get_aligner_cmd(RNA, aligner, type, index_type, version, options, "../one.fq", "../two.fq", "/dev/null") start_time = datetime.now() if verbose: print >> sys.stderr, start_time, "\t", " ".join(dummy_cmd) if aligner in ["hisat2", "hisat", "bowtie", "bowtie2", "gsnap", "bwa"]: proc = subprocess.Popen(dummy_cmd, stdout=open("/dev/null", "w"), stderr=subprocess.PIPE) else: proc = subprocess.Popen(dummy_cmd, stdout=subprocess.PIPE, stderr=subprocess.PIPE) proc.communicate() finish_time = datetime.now() duration = finish_time - start_time duration = duration.total_seconds() if verbose: print >> sys.stderr, finish_time, "duration:", duration loading_time = duration # align all reads if paired: sweep_read_cmd = "cat ../%s ../%s > /dev/null" % (type_read1_fname, type_read2_fname) else: sweep_read_cmd = "cat ../%s > /dev/null" % (type_read1_fname) print >> sys.stderr, datetime.now(), "\t", sweep_read_cmd os.system(sweep_read_cmd) skip_alignment = False if paired and aligner == "olego" and os.path.exists(out_fname + "1"): skip_alignment = True if not skip_alignment: aligner_cmd = get_aligner_cmd(RNA, aligner, type, index_type, version, options, "../" + type_read1_fname, "../" + type_read2_fname, out_fname) start_time = datetime.now() if verbose: print >> sys.stderr, start_time, "\t", " ".join(aligner_cmd) if aligner in ["hisat2", "hisat", "bowtie", "bowtie2", "gsnap", "bwa", "vg", "minimap2"]: proc = subprocess.Popen(aligner_cmd, stdout=open(out_fname, "w"), stderr=subprocess.PIPE) else: proc = subprocess.Popen(aligner_cmd, stdout=subprocess.PIPE, stderr=subprocess.PIPE) _, mem_usage = proc.communicate() mem_usage = parse_mem_usage(mem_usage) finish_time = datetime.now() duration = finish_time - start_time duration = duration.total_seconds() - loading_time if duration < 0.1: duration = 0.1 if verbose: print >> sys.stderr, finish_time, "duration:", duration if verbose: print >> sys.stderr, finish_time, "Memory Usage: %dMB" % (int(mem_usage) / 1024) if debug and aligner == "hisat" and type == "x1": os.system("cat metrics.out") print >> sys.stderr, "\ttime: %.4f" % (duration) # break if aligner == "star" and type in ["", "gtf"]: os.system("mv Aligned.out.sam %s" % out_fname) elif aligner in ["hisat2", "hisat"] and type == "x2": aligner_cmd = get_aligner_cmd(RNA, aligner, type, index_type, version, options, "../" + type_read1_fname, "../" + type_read2_fname, out_fname, 1) if verbose: print >> sys.stderr, start_time, "\t", " ".join(aligner_cmd) start_time = datetime.now() proc = subprocess.Popen(aligner_cmd, stdout=open(out_fname, "w"), stderr=subprocess.PIPE) proc.communicate() finish_time = datetime.now() duration += (finish_time - start_time).total_seconds() duration -= loading_time if duration < 0.1: duration = 0.1 if verbose: print >> sys.stderr, finish_time, "duration:", duration elif aligner == "star" and type == "x2": assert os.path.exists("SJ.out.tab") os.system("awk 'BEGIN {OFS=\"\t\"; strChar[0]=\".\"; strChar[1]=\"+\"; strChar[2]=\"-\";} {if($5>0){print $1,$2,$3,strChar[$4]}}' SJ.out.tab > SJ.out.tab.Pass1.sjdb") for file in os.listdir("."): if file in ["SJ.out.tab.Pass1.sjdb", "genome.fa"]: continue os.remove(file) star_index_cmd = "STAR --genomeDir ./ --runMode genomeGenerate --genomeFastaFiles ../../../../data/genome.fa --sjdbFileChrStartEnd SJ.out.tab.Pass1.sjdb --sjdbOverhang 100 --runThreadN %d" % (num_threads) print >> sys.stderr, "\t", datetime.now(), star_index_cmd os.system(star_index_cmd) if verbose: print >> sys.stderr, "\t", datetime.now(), " ".join(dummy_cmd) proc = subprocess.Popen(dummy_cmd, stdout=subprocess.PIPE, stderr=subprocess.PIPE) proc.communicate() if verbose: print >> sys.stderr, "\t", datetime.now(), "finished" aligner_cmd = get_aligner_cmd(RNA, aligner, type, index_type, version, options, "../" + type_read1_fname, "../" + type_read2_fname, out_fname, 1) start_time = datetime.now() if verbose: print >> sys.stderr, "\t", start_time, " ".join(aligner_cmd) proc = subprocess.Popen(aligner_cmd, stdout=subprocess.PIPE, stderr=subprocess.PIPE) proc.communicate() finish_time = datetime.now() duration += (finish_time - start_time).total_seconds() duration -= loading_time if duration < 0.1: duration = 0.1 if verbose: print >> sys.stderr, "\t", finish_time, "finished:", duration os.system("mv Aligned.out.sam %s" % out_fname) elif aligner == "tophat2": os.system("samtools sort -n tophat_out/accepted_hits.bam accepted_hits; samtools view -h accepted_hits.bam > %s" % out_fname) elif aligner == "vg": index_name = '%s/VG%s/' % (index_base, index_add) + genome if index_type: index_name += ('_' + index_type) os.system("echo %s %s %f >> runtime" % (str(datetime.now()), aligner, duration)) if aligner in ["star", "tophat2", "gsnap"]: os.system("tar cvzf %s.tar.gz %s &> /dev/null" % (out_fname, out_fname)) if runtime_only: os.chdir("..") continue suffix = aligner read_sam, pair_sam = suffix + ".read.sam", suffix + ".pair.sam" unmapped_read_1_fq, unmapped_read_2_fq = suffix + ".unmapped.1.fq", suffix + ".unmapped.2.fq" if not os.path.exists(read_sam) or not os.path.exists(pair_sam): if index_type == 'snp': extract_reads_and_pairs(chr_dic, out_fname, read_sam, pair_sam, unmapped_read_1_fq, unmapped_read_2_fq, snp_dic) else: extract_reads_and_pairs(chr_dic, out_fname, read_sam, pair_sam, unmapped_read_1_fq, unmapped_read_2_fq) out = '' if gz_file: out = subprocess.check_output("gzip -cd ../%s | wc -l" % type_read1_fname, shell=True) else: out = subprocess.check_output("wc -l ../%s" % type_read1_fname, shell=True) numreads = int(out.split()[0]) / 4 done_filename = suffix + ".done" if not os.path.exists(done_filename): done_file = open(done_filename, "w") if paired: sum = [0, 0, 0, 0, 0, 0] # mappep_read, junction_read, gtf_junction_reads, concord_mapped_read, num_junctions, num_gtf_junctions dis_sum = 0 stat, dis_stat = pair_stat(pair_sam, gtf_junctions, chr_dic) output = "" for i in range(len(stat)): for j in range(len(sum)): sum[j] += stat[i][j] dis_sum += dis_stat[i] mapped_reads, junction_reads, gtf_junction_reads, concord_mapped_read, num_junctions, num_gtf_junctions = sum output += "%s\t%s\tpaired\t%d\t%d\t%.2f%%\t%d\t%d\t%d\t%d\t%f\t%d\t%d\t%.2f%%\n" % \ (aligner_name, gene, i, mapped_reads, float(mapped_reads) * 100.0 / numreads, junction_reads, gtf_junction_reads, num_junctions, num_gtf_junctions, duration, (numreads / max(1.0, duration)), concord_mapped_read, float(concord_mapped_read) * 100.0 / numreads) if sql_write and os.path.exists("../" + sql_db_name): sql_insert = "INSERT INTO \"Mappings\" VALUES(NULL, '%s', '%s', '%s', '%s', '%s', '%s', %d, %d, %d, %d, %d, %d, %f, '%s', datetime('now', 'localtime'), '%s');" % \ (workdir, genome, "paired", aligner_name, get_aligner_version(aligner), "no", i, mapped_reads, junction_reads, gtf_junction_reads, num_junctions, num_gtf_junctions, duration, platform.node(), " ".join(aligner_cmd)) sql_execute("../" + sql_db_name, sql_insert) print >> sys.stderr, output, print >> done_file, output else: sum = [0, 0, 0, 0, 0] stat = read_stat(read_sam, gtf_junctions, chr_dic) output = "" for i in range(len(stat)): for j in range(len(sum)): sum[j] += stat[i][j] mapped_reads, junction_reads, gtf_junction_reads, num_junctions, num_gtf_junctions = sum output += "%s\t%s\tsingle\t%d\t%d\t%.2f%%\t%d\t%d\t%d\t%d\t%f\t%d\n" % \ (aligner_name, gene, i, mapped_reads, float(mapped_reads) * 100.0 / numreads, junction_reads, gtf_junction_reads, num_junctions, num_gtf_junctions, duration, (numreads / max(1.0, duration))) if sql_write and os.path.exists("../" + sql_db_name): sql_insert = "INSERT INTO \"Mappings\" VALUES(NULL, '%s', '%s', '%s', '%s', '%s', '%s', %d, %d, %d, %d, %d, %d, %f, '%s', datetime('now', 'localtime'), '%s');" % \ (workdir, genome, "single", aligner_name, get_aligner_version(aligner), "no", i, mapped_reads, junction_reads, gtf_junction_reads, num_junctions, num_gtf_junctions, duration, platform.node(), " ".join(aligner_cmd)) sql_execute("../" + sql_db_name, sql_insert) print >> sys.stderr, output, print >> done_file, output done_file.close() os.chdir("..") if os.path.exists(sql_db_name): write_analysis_data(sql_db_name, workdir, paired) if __name__ == "__main__": parser = ArgumentParser( description='test HISAT2, and compare HISAT2 with other popular aligners such as TopHat2, STAR, Bowtie1/2, GSNAP, BWA-mem, etc.') parser.add_argument('--single-end', dest='paired_end', action='store_false', help='run single-end only') parser.add_argument('--paired-end', dest='single_end', action='store_false', help='run paired_end only') parser.add_argument('--aligner-list', dest='aligner_list', type=str, default="", help='comma-separated list of aligners (e.g. hisat2,bowtie2,bwa') parser.add_argument('--fresh', dest='fresh', action='store_true', help='delete existing alignment related directories (e.g. hisat2_single)') parser.add_argument('--runtime-only', dest='runtime_only', action='store_true', help='run programs without evaluation') parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') args = parser.parse_args() aligners = [] for aligner in args.aligner_list.split(','): if aligner == "": continue aligners.append(aligner) calculate_read_cost(args.single_end, args.paired_end, aligners, args.fresh, args.runtime_only, args.verbose) hisat2-2.2.1/evaluation/real/init.py000077500000000000000000000052221370663763200173360ustar00rootroot00000000000000#!/usr/bin/env python import sys, os, signal import string, re signal.signal(signal.SIGPIPE, signal.SIG_DFL) use_message = ''' ''' osx_mode = False if sys.platform == 'darwin': osx_mode = True def make_cat_cmd(gzmode, read_dir_base, read_dir, fq_name, num_read): cmd = [] if gzmode: if osx_mode: cmd += ["gzcat"] else: cmd += ["zcat"] else: cmd += ["cat"] cmd += ["../../%s%s/%s" % (read_dir_base, read_dir, fq_name)] cmd += ["|", "head", "-n", "%d" % (num_read * 4)] if gzmode: cmd += ["|", "gzip"] cmd += [">", fq_name] return ' '.join(cmd) def init(): read_dir_base = "../reads/real/" read_dirs = os.listdir(read_dir_base) for read_dir in read_dirs: if os.path.exists(read_dir): continue gz_file = False fq_1_name = '1.fq' fq_2_name = '2.fq' if os.path.exists(read_dir_base + read_dir + "/1.fq.gz") and \ os.path.exists(read_dir_base + read_dir + "/2.fq.gz"): gz_file = True fq_1_name = '1.fq.gz' fq_2_name = '2.fq.gz' else: if not os.path.exists(read_dir_base + read_dir + "/1.fq") or \ not os.path.exists(read_dir_base + read_dir + "/1.fq"): continue print >> sys.stderr, "Processing", read_dir, "..." os.mkdir(read_dir) os.chdir(read_dir) RNA = (read_dir.find("RNA") != -1) tests = [ ["1M", 1000000], #["5M", 5000000], ["10M", 10000000], #["20M", 20000000], ["whole", 0], ] for dir_name, num_reads in tests: if os.path.exists(dir_name): continue os.mkdir(dir_name) os.chdir(dir_name) if dir_name == "whole": ln_cmd = "ln -s ../../%s%s/%s ." % (read_dir_base, read_dir, fq_1_name) print >> sys.stderr, ln_cmd os.system(ln_cmd) ln_cmd = "ln -s ../../%s%s/%s ." % (read_dir_base, read_dir, fq_2_name) print >> sys.stderr, ln_cmd os.system(ln_cmd) else: cmd = make_cat_cmd(gz_file, read_dir_base, read_dir, fq_1_name, num_reads) print >> sys.stderr, cmd os.system(cmd) cmd = make_cat_cmd(gz_file, read_dir_base, read_dir, fq_2_name, num_reads) print >> sys.stderr, cmd os.system(cmd) os.system("ln -s ../../calculate_read_cost.py .") os.chdir("..") os.chdir("..") if __name__ == "__main__": init() hisat2-2.2.1/evaluation/simulation/000077500000000000000000000000001370663763200172565ustar00rootroot00000000000000hisat2-2.2.1/evaluation/simulation/calculate_read_cost.py000077500000000000000000003144631370663763200236260ustar00rootroot00000000000000#!/usr/bin/env python import sys, os, subprocess import multiprocessing import string, re import platform from datetime import datetime, date, time import copy from argparse import ArgumentParser, FileType from multiprocessing import Process import bisect mp_mode = False mp_num = 1 cigar_re = re.compile('\d+\w') osx_mode = False if sys.platform == 'darwin': osx_mode = True """ """ def parse_mem_usage(resource): if osx_mode: resource = resource.strip().split('\n') for line in resource: if line.find('maximum resident set size') != -1: return int(line.split()[0]) / 1024 else: resource = resource.split(' ') for line in resource: idx = line.find('maxresident') if idx != -1: return line[:idx] return '0' """ """ def reverse_complement(seq): result = "" for nt in seq: base = nt if nt == 'A': base = 'T' elif nt == 'a': base = 't' elif nt == 'C': base = 'G' elif nt == 'c': base = 'g' elif nt == 'G': base = 'C' elif nt == 'g': base = 'c' elif nt == 'T': base = 'A' elif nt == 't': base = 'a' result = base + result return result """ RepeatDB """ class RepeatAllele: def __init__(self): self.repeat_name = '' self.allele_idx = 0 self.repeat_pos = 0 self.repeat_length = 0 self.positions = [] return def __repr__(self): return '[' + ','.join([str(self.repeat_name), str(self.allele_idx), str(self.repeat_pos), str(self.repeat_length), str(len(self.positions))]) + ']' def add_position(self, chr, pos, strand): self.positions.append([chr, pos, strand]) def __lt__(self, other): if self.repeat_pos < other.repeat_pos: return True elif self.repeat_pos == other.repeat_pos: return self.repeat_length < other.repeat_length else: return False class Repeat: def __init__(self): self.repeat_name = '' self.repeat_length = 0 self.repeat_pos = 0 self.allele = [] return def add_allele(self, allele_idx, repeatAllele): #self.allele[allele_idx] = repeatAllele self.allele.append(repeatAllele) def allele_sort(self): self.allele = sorted(self.allele) def cmp_repeatmap(a, b): if a[0] < b[0]: return -1 elif a[0] == b[0]: return 0 else: return 1 def read_len_cigar(cigar_str): cigars = cigar_re.findall(cigar_str) cigars = [[cigar[-1], int(cigar[:-1])] for cigar in cigars] read_len = 0 for cigar in cigars: cigar_op, length = cigar if cigar_op in "MISH": read_len += int(length) return read_len def read_repeatdb(repeat_filename): repeat_db = {} if os.path.exists(repeat_filename): for line in open(repeat_filename, 'r'): if line[0] == '>': line = line.strip()[1:] name, rptRefName, pos, rep_len, _, _ = line.split()[:6] pos = int(pos) rep_len = int(rep_len) rptName, allele_idx = name.split('*')[0:2] allele_idx = int(allele_idx) repeatAllele = RepeatAllele() repeatAllele.repeat_name = rptName repeatAllele.allele_idx = allele_idx repeatAllele.repeat_pos = pos repeatAllele.repeat_length = rep_len if rptRefName not in repeat_db: # new rptRefName repeat_db[rptRefName] = {} if rptName not in repeat_db[rptRefName]: # new rptName assert allele_idx == 0 repeat_db[rptRefName][rptName] = Repeat() repeat_db[rptRefName][rptName].repeat_name = rptName repeat_db[rptRefName][rptName].add_allele(allele_idx, repeatAllele) else: coords = line.split() for coord in coords: chr, pos, strand = coord.split(':') pos = int(pos) repeat_db[rptRefName][rptName].allele[allele_idx].add_position(chr, pos, strand) else: print >> sys.stderr, 'Cannot open file', repeat_filename print >> sys.stderr, 'Build repeatMap' repeat_map = {} for rptRefName, repeats in repeat_db.items(): #print 'Processing', rptRefName repeat_pos_list = [] for repeatName, repeat in repeats.items(): #print 'Common Allele:', repeatName, repeat.repeat_name repeat_left = sys.maxint repeat_right = 0 #for allele_id, repeatAllele in repeat.allele.items(): for repeatAllele in repeat.allele: left = repeatAllele.repeat_pos right = left + repeatAllele.repeat_length if left < repeat_left: repeat_left = left if right > repeat_right: repeat_right = right repeat.repeat_pos = repeat_left repeat.repeat_length = repeat_right - repeat_left #print repeat.allele #repeat.allele_sort() #print repeat.allele #print repeat_left, repeat_right repeat_pos_list.append((repeat_right, repeatName)) repeat_map[rptRefName] = sorted(repeat_pos_list, cmp=cmp_repeatmap) #print repeat_map[rptRefName] return repeat_db, repeat_map def find_leftmost_pos(rmap, left): pos = bisect.bisect_left(rmap, (left, None)) #print pos if pos == len(rmap): return pos if rmap[pos][0] == left: while pos < len(rmap): if rmap[pos][0] != left: break pos += 1 return pos def repeat_to_genome_pos(repeat_db, repeat_map, rptRefName, pos, cigar_str = ''): assert rptRefName in repeat_db readlen = read_len_cigar(cigar_str) #readlen = 101 # pos in sam-result. pos is 1-based left = pos - 1 right = left + readlen repeats = repeat_db[rptRefName] rmap = repeat_map[rptRefName] #print len(rmap) #print rmap i = find_leftmost_pos(rmap, left) if i >= len(rmap): print >> sys.stderr, 'Value Error' return if right > rmap[i][0]: print >> sys.stderr, 'Not repeat' return repeat = repeats[rmap[i][1]] #print 'Allele Size:', len(repeat.allele) #print repeat.allele for allele in repeat.allele: rpos = allele.repeat_pos rlen = allele.repeat_length if (left >= rpos) and (right <= rpos + rlen): offset = left - rpos for genome_pos in allele.positions: print genome_pos[0], genome_pos[1] + offset + 1, genome_pos[2], genome_pos[1] """ """ def read_genome(genome_filename): chr_dic = {} genome_file = open(genome_filename, "r") chr_name, sequence = "", "" for line in genome_file: if line[0] == ">": if chr_name and sequence: chr_dic[chr_name] = sequence chr_name = line[1:-1].split()[0] sequence = "" else: sequence += line[:-1] if chr_name and sequence: chr_dic[chr_name] = sequence genome_file.close() print >> sys.stderr, "genome is loaded" return chr_dic """ """ def extract_splice_sites(gtf_fname): trans = {} gtf_file = open(gtf_fname) # Parse valid exon lines from the GTF file into a dict by transcript_id for line in gtf_file: line = line.strip() if not line or line.startswith('#'): continue if '#' in line: line = line.split('#')[0].strip() try: chrom, source, feature, left, right, score, \ strand, frame, values = line.split('\t') except ValueError: continue left, right = int(left), int(right) if feature != 'exon' or left >= right: continue values_dict = {} for attr in values.split(';')[:-1]: attr, _, val = attr.strip().partition(' ') values_dict[attr] = val.strip('"') if 'gene_id' not in values_dict or \ 'transcript_id' not in values_dict: continue transcript_id = values_dict['transcript_id'] if transcript_id not in trans: trans[transcript_id] = [chrom, strand, [[left, right]]] else: trans[transcript_id][2].append([left, right]) gtf_file.close() # Sort exons and merge where separating introns are <=5 bps for tran, [chrom, strand, exons] in trans.items(): exons.sort() tmp_exons = [exons[0]] for i in range(1, len(exons)): if exons[i][0] - tmp_exons[-1][1] <= 5: tmp_exons[-1][1] = exons[i][1] else: tmp_exons.append(exons[i]) trans[tran] = [chrom, strand, tmp_exons] # Calculate and print the unique junctions junctions = set() for chrom, strand, exons in trans.values(): for i in range(1, len(exons)): junctions.add(to_junction_str([chrom, exons[i-1][1], exons[i][0]])) return junctions """ """ def read_repeat_info(repeat_filename): repeat_info, repeat_dic = {}, {} repeat_pos = {} if os.path.exists(repeat_filename): for line in open(repeat_filename): if line[0] == ">": line = line.strip()[1:] allele, rep, pos, rep_len, _, _ = line.split()[:6] pos, rep_len = int(pos), int(rep_len) common_allele = allele.split('*')[0] if rep not in repeat_info: repeat_info[rep] = [] repeat_dic[rep] = {} repeat_pos[rep] = {} repeat_info[rep].append([allele, pos, rep_len]) if allele not in repeat_dic[rep]: repeat_dic[rep][allele] = [] repeat_pos[rep][allele] = set() else: coords = line.split() for coord in coords: chr, pos, strand = coord.split(':') pos = int(pos) if pos in repeat_pos[rep][allele]: continue repeat_dic[rep][allele].append([chr, pos, strand]) repeat_pos[rep][allele].add(pos) for rep, repeats in repeat_info.items(): def my_cmp(a, b): if a[1] < b[1]: return -1 elif a[1] == b[1]: return a[2] - b[2] else: return 1 repeat_info[rep] = sorted(repeat_info[rep], cmp=my_cmp) return repeat_info, repeat_dic """ """ def find_repeat(repeat_info, pos): if len(repeat_info) <= 0: return -1 l, r = 0, len(repeat_info) while l < r: m = (l + r) / 2 _, rep_pos, rep_len, _ = repeat_info[m] if rep_pos <= pos and pos < rep_pos + rep_len: return m elif pos < rep_pos: r = m else: l = m + 1 return -1 def reverse_cigar(cigar_str): cigars = cigar_re.findall(cigar_str) cigars = [[cigar[-1], int(cigar[:-1])] for cigar in cigars] cigars[::-1] read_len = 0 cigar_str = "" for cigar in cigars: cigar_op, length = cigar cigar_str += ("%d%s" % (length, cigar_op)) if cigar_op in "MISH": read_len += int(length) return read_len, cigar_str def to_junction_str(junction): return "%s-%d-%d" % (junction[0], junction[1], junction[2]) def to_junction(junction_str): fields = junction_str.split("-") if len(fields) > 3: chr, left, right = "-".join(fields[:-2]), fields[-2], fields[-1] else: assert len(fields) == 3 chr, left, right = fields return [chr, int(left), int(right)] """ """ def junction_cmp(a, b): if a[0] != b[0]: if a[0] < b[0]: return -1 else: return 1 if a[1] != b[1]: if a[1] < b[1]: return -1 else: return 1 if a[2] != b[2]: if a[2] < b[2]: return -1 else: return 1 return 0 """ # chr and pos are assumed to be integers """ def get_junctions(chr, pos, cigar_str, min_anchor_len = 0, read_len = 100): junctions = [] right_pos = pos cigars = cigar_re.findall(cigar_str) cigars = [[int(cigars[i][:-1]), cigars[i][-1]] for i in range(len(cigars))] left_anchor_lens = [] cur_left_anchor_len = 0 for i in range(len(cigars)): length, cigar_op = cigars[i] if cigar_op in "MI": cur_left_anchor_len += length elif cigar_op == "N": assert cur_left_anchor_len > 0 left_anchor_lens.append(cur_left_anchor_len) cur_left_anchor_len = 0 for i in range(len(cigars)): length, cigar_op = cigars[i] if cigar_op == "N": left, right = right_pos - 1, right_pos + length if i > 0 and cigars[i-1][1] in "ID": if cigars[i-1][1] == "I": left += cigars[i-1][0] else: left -= cigars[i-1][0] if i + 1 < len(cigars) and cigars[i+1][1] in "ID": if cigars[i+1][1] == "I": right -= cigars[i+1][0] else: right += cigars[i+1][0] junction_idx = len(junctions) assert junction_idx < len(left_anchor_lens) left_anchor_len = left_anchor_lens[junction_idx] assert left_anchor_len > 0 and left_anchor_len < read_len right_anchor_len = read_len - left_anchor_len if left_anchor_len >= min_anchor_len and right_anchor_len >= min_anchor_len: junctions.append([chr, left, right]) if cigar_op in "MND": right_pos += length return junctions def get_right(pos, cigars): right_pos = pos cigars = cigar_re.findall(cigars) for cigar in cigars: length = int(cigar[:-1]) cigar_op = cigar[-1] if cigar_op in "MDN": right_pos += length return right_pos def get_cigar_chars(cigars): cigars = cigar_re.findall(cigars) cigar_chars = "" for cigar in cigars: cigar_op = cigar[-1] cigar_chars += cigar_op return cigar_chars """ """ def get_cigar_chars_MN(cigars): cigars = cigar_re.findall(cigars) cigar_chars = "" for cigar in cigars: cigar_op = cigar[-1] if cigar_op in "MN": if cigar_chars == "" or cigar_chars[-1] != cigar_op: cigar_chars += cigar_op return cigar_chars """ """ def is_small_anchor_junction_read(cigars): cigar_list = [] for cigar in cigar_re.findall(cigars): cigar_op = cigar[-1] cigar_len = int(cigar[:-1]) cigar_list.append([cigar_op, cigar_len]) if len(cigar_list) < 3: return False if cigar_list[0][0] != 'M' or cigar_list[-1][0] != 'M': return False if cigar_list[0][1] > 10 and cigar_list[-1][1] > 10: return False if cigar_list[1][0] != 'N' or cigar_list[-2][0] != 'N': return False return True """ """ def is_canonical_junction(chr_dic, junction): chr, left, right = junction donor = chr_dic[chr][left:left+2] acceptor = chr_dic[chr][right-3:right-1] rev_donor = reverse_complement(acceptor) rev_acceptor = reverse_complement(donor) if (donor == "GT" and acceptor == "AG") or \ (rev_donor == "GT" and rev_acceptor == "AG"): return True return False """ """ def is_small_exon_junction_read(cigars, min_exon_len = 23): cigars = cigar_re.findall(cigars) for i in range(1, len(cigars) - 1): cigar = cigars[i] cigar_op = cigar[-1] cigar_len = int(cigar[:-1]) prev_op = cigars[i-1][-1] next_op = cigars[i+1][-1] if prev_op == 'N' and cigar_op == 'M' and next_op == 'N': if cigar_len <= min_exon_len: return True return False """ """ """ def repeat_to_genome_alignment(repeat_info, repeat_dic, rep, pos, cigar_str = ""): assert rep in repeat_info left = pos - 1 # convert 1-based offset to zero-based repeats = repeat_info[rep] l, r = 0, len(repeats) while l < r: m = (l + r) / 2 rep_allele, rpos, rlen = repeats[m] if left >= rpos and left < rpos + rlen: while m > 0: rep_allele, rpos, rlen = repeats[m-1] if left < rpos: break m -= 1 break if left < rpos: r = m else: l = m + 1 alignments = [] while m < len(repeats): rep_allele, rpos, rlen = repeats[m] if left >= rpos + rlen: m += 1 continue if left < rpos: break assert rep_allele in repeat_dic[rep] coords = repeat_dic[rep][rep_allele] assert len(coords) > 0 for coord in coords: cchr, cpos, cstrand = coord adj_left = left - rpos if cstrand == '+': rep_left = cpos + adj_left rep_cigar_str = cigar_str else: if cigar_str: read_len, rep_cigar_str = reverse_cigar(cigar_str) else: read_len, rep_cigar_str = 0, "" rc_adj_left = rlen - adj_left - read_len; rep_left = cpos + rc_adj_left alignments.append([cchr, rep_left + 1, rep_cigar_str]) m += 1 return alignments """ def repeat_to_genome_alignment(repeat_db, repeat_map, rptRefName, pos, cigar_str = ''): assert rptRefName in repeat_db readlen = read_len_cigar(cigar_str) #readlen = 101 # pos in sam-result. pos is 1-based left = pos - 1 right = left + readlen repeats = repeat_db[rptRefName] rmap = repeat_map[rptRefName] #print len(rmap) #print rmap alignments = [] i = find_leftmost_pos(rmap, left) if i >= len(rmap): print >> sys.stderr, 'Value Error' return alignments if right > rmap[i][0]: print >> sys.stderr, 'Not repeat' return alignments repeat = repeats[rmap[i][1]] #print 'Allele Size:', len(repeat.allele) #print repeat.allele for allele in repeat.allele: rpos = allele.repeat_pos rlen = allele.repeat_length if (left >= rpos) and (right <= rpos + rlen): offset = left - rpos for coord in allele.positions: cchr, cpos, cstrand = coord if cstrand == '+': rep_left = cpos + offset rep_cigar_str = cigar_str else: if cigar_str: rep_read_len, rep_cigar_str = reverse_cigar(cigar_str) else: rep_read_len, rep_cigar_str = 0, "" rc_offset = rlen - offset - rep_read_len rep_left = cpos + rc_offset alignments.append([cchr, rep_left + 1, rep_cigar_str]) #print genome_pos[0], genome_pos[1] + offset + 1, genome_pos[2], genome_pos[1] return alignments """ """ def extract_single(infilename, outfilename, chr_dic, aligner, version, repeat_db, repeat_map, debug_dic, hash_idx): infile = open(infilename) if hash_idx == -1: outfile = open(outfilename, "w") else: outfile = open(outfilename + "." + str(hash_idx), "w") prev_read_id = "" num_reads, num_aligned_reads, num_ualigned_reads = 0, 0, 0 prev_NM, prev_NH, NH_real = 0, 0, 0 for line in infile: if line[0] == '@': continue cols = line[:-1].split() read_id, flag, chr, pos, mapQ, cigar_str = cols[:6] read_seq = cols[9] if len(read_id) >= 3 and read_id[-2] == "/": read_id = read_id[:-2] if read_id.find("seq.") == 0: read_id = read_id[4:] if aligner == "gsnap": chr = chr.replace("_", ":") if hash_idx != -1: hashval = hash(read_id) if hashval % mp_num != hash_idx: continue if read_id != prev_read_id: num_reads += 1 flag, pos, mapQ = int(flag), int(pos), int(mapQ) if flag & 0x4 != 0 or \ 'H' in cigar_str: prev_read_id = read_id continue NH, NM, XA = "", sys.maxint, [] for i in range(11, len(cols)): col = cols[i] # "nM" from STAR if col.startswith("NM") or col.startswith("nM"): NM = int(col[5:]) elif col.startswith("NH"): NH = col elif col.startswith("XA"): XA = col[5:].split(';')[:-1] if NH != "": NH = int(NH[5:]) if aligner == "hisat2": if prev_read_id == read_id: assert prev_NH == NH if NH == 1 or mapQ == 60: assert NH == 1 and mapQ == 60 if read_id != prev_read_id: num_aligned_reads += 1 if aligner == "hisat2" and \ NH == 1: num_ualigned_reads += 1 else: # In case of Bowtie2, only consier the best alignments if aligner in ["bowtie2", "bwa", "vg"]: if NM > prev_NM: continue def adjust_alignment(chr, pos, cigar_str): NM_real = 0 read_pos, right_pos = 0, pos - 1 cigars = cigar_re.findall(cigar_str) cigars = [[cigar[-1], int(cigar[:-1])] for cigar in cigars] for i in range(len(cigars)): cigar_op, length = cigars[i] if cigar_op == "S": assert i == 0 or i == len(cigars) - 1 if i == 0: assert cigars[i+1][0] == "M" ref_seq = chr_dic[chr][right_pos-length:right_pos] else: assert cigars[i-1][0] == "M" ref_seq = chr_dic[chr][right_pos:right_pos+length] ref_seq = ref_seq.upper() if length == len(ref_seq): for j in range(length): if ref_seq[j] != "N" and read_seq[read_pos+j] != ref_seq[j]: NM_real += 1 else: NM_real += length if cigar_op in "MND": right_pos += length if cigar_op in "MIS": read_pos += length if cigars[0][0] == "S": assert cigars[1][0] == "M" pos -= cigars[0][1] cigars[1][1] += cigars[0][1] cigars = cigars[1:] if cigars[-1][0] == "S": assert cigars[-2][0] == "M" cigars[-2][1] += cigars[-1][1] cigars = cigars[:-1] cigar_str = "" for cigar in cigars: cigar_op, length = cigar cigar_str += ("%d%s" % (length, cigar_op)) return pos, cigar_str, NM_real alignments = [[chr, pos, cigar_str]] if aligner == "bwa" and len(XA) > 0: for alignment in XA: alt_chr, alt_pos, alt_cigar_str, alt_NM = alignment.split(',') alt_pos, alt_NM = abs(int(alt_pos)), int(alt_NM) if alt_NM > NM: break alignments.append([alt_chr, alt_pos, alt_cigar_str]) # Convert repeat alignments to genome alignments if aligner == "hisat2" and chr.startswith("rep") and len(repeat_map) > 0: alignments = repeat_to_genome_alignment(repeat_db, repeat_map, chr, pos, cigar_str) for i, alignment in enumerate(alignments): chr, pos, cigar_str = alignment pos, cigar_str, NM_real = adjust_alignment(chr, pos, cigar_str) p_str = "%s\t%s\t%d\t%s\tNM:i:%d" % (read_id, chr, pos, cigar_str, NM_real) print >> outfile, p_str if aligner == "hisat2": if prev_read_id != read_id: if prev_read_id != "": assert prev_NH == NH_real NH_real = 1 else: NH_real += 1 prev_NH = NH prev_NM = NM prev_read_id = read_id if aligner == "hisat2": if prev_read_id != "": assert prev_NH == NH_real outfile.close() infile.close() # Sanity check for HISAT2's alignment summary if aligner == "hisat2" and os.path.exists(infilename + ".summary") and (not mp_mode): hisat2_reads, hisat2_0aligned_reads, hisat2_ualigned_reads, hisat2_maligned_reads = 0, 0, 0, 0 for line in open(infilename + ".summary"): line = line.strip() if line.startswith("HISAT2 summary") or \ line.startswith("Overall"): continue category, num = line.split(':') num = num.strip() num = int(num.split(' ')[0]) if category.startswith("Total reads"): hisat2_reads = num elif category.startswith("Aligned 0 time"): hisat2_0aligned_reads = num elif category.startswith("Aligned 1 time"): hisat2_ualigned_reads = num else: assert category.startswith("Aligned >1 time") assert hisat2_reads == hisat2_0aligned_reads + hisat2_ualigned_reads + num hisat2_aligned_reads = hisat2_reads - hisat2_0aligned_reads assert hisat2_reads == num_reads assert hisat2_aligned_reads == num_aligned_reads assert hisat2_ualigned_reads == num_ualigned_reads """ """ def extract_pair(infilename, outfilename, chr_dic, RNA, aligner, version, repeat_db, repeat_map, debug_dic, hash_idx): read_dic = {} pair_reported = set() infile = open(infilename) if hash_idx == -1: outfile = open(outfilename, "w") else: outfile = open(outfilename + "." + str(hash_idx), "w") num_pairs, num_conc_aligned_pairs, num_conc_ualigned_pairs, num_disc_aligned_pairs = 0, 0, 0, 0 num_aligned_reads, num_ualigned_reads = 0, 0 prev_read_id, pair_list = "", set() prev_NM = sys.maxint prev_NH1, prev_NH2 = 0, 0 NH1_real, NH2_real = 0, 0 for line in infile: if line[0] == '@': continue cols = line[:-1].split() read_id, flag, chr1, pos1, mapQ, cigar1_str, chr2, pos2 = cols[:8] read_seq = cols[9] if len(read_id) >= 3 and read_id[-2] == "/": read_id = read_id[:-2] if read_id.find("seq.") == 0: read_id = read_id[4:] if hash_idx != -1: hashval = hash(read_id) if hashval % mp_num != hash_idx: continue if aligner == "gsnap": chr1 = chr1.replace("_", ":") chr2 = chr2.replace("_", ":") if read_id != prev_read_id: num_pairs += 1 pair_list = set() prev_NM = sys.maxint flag = int(flag) canonical_pos1, canonical_pos2 = int(pos1), int(pos2) left_read = (flag & 0x40 != 0) pos1 = canonical_pos1 mapQ = int(mapQ) if flag & 0x4 != 0 or \ 'H' in cigar1_str: prev_read_id, is_prev_read_left = read_id, left_read continue concordant = (flag & 0x2 != 0) NH, NM1, YT, XA = sys.maxint, sys.maxint, "", [] for i in range(11, len(cols)): col = cols[i] # "nM" from STAR if col.startswith("NM") or col.startswith("nM"): NM1 = int(col[5:]) elif col.startswith("NH"): NH = int(col[5:]) elif col.startswith("YT"): YT = col[5:] elif col.startswith("XA"): XA = col[5:].split(';')[:-1] if aligner == "hisat2": if prev_read_id == read_id: if left_read: assert prev_NH1 == 0 or prev_NH1 == NH else: assert prev_NH2 == 0 or prev_NH2 == NH if NH == 1 or mapQ == 60: assert NH == 1 and mapQ == 60 unpaired = (flag & 0x8 != 0) or (YT in ["UU", "UP"]) if unpaired: if left_read not in pair_list: pair_list.add(left_read) num_aligned_reads += 1 if aligner == "hisat2" and NH == 1: num_ualigned_reads += 1 assert mapQ == 60 else: if read_id != prev_read_id: if concordant: num_conc_aligned_pairs += 1 if aligner == "hisat2" and NH == 1: num_conc_ualigned_pairs += 1 else: if aligner == "hisat2": assert YT == "DP" num_disc_aligned_pairs += 1 if chr2 == '*': continue def adjust_alignment(chr, pos, cigar_str): NM_real = 0 read_pos, right_pos = 0, pos - 1 cigars = cigar_re.findall(cigar_str) cigars = [[cigar[-1], int(cigar[:-1])] for cigar in cigars] for i in range(len(cigars)): cigar_op, length = cigars[i] if cigar_op == "S": assert i == 0 or i == len(cigars) - 1 if i == 0: assert cigars[i+1][0] == "M" ref_seq = chr_dic[chr1][right_pos-length:right_pos] else: assert cigars[i-1][0] == "M" ref_seq = chr_dic[chr1][right_pos:right_pos+length] ref_seq = ref_seq.upper() if length == len(ref_seq): for j in range(length): if ref_seq[j] != "N" and read_seq[read_pos+j] != ref_seq[j]: NM_real += 1 else: NM_real += length if cigar_op in "MND": right_pos += length if cigar_op in "MIS": read_pos += length if cigars[0][0] == "S": assert cigars[1][0] == "M" pos -= cigars[0][1] cigars[1][1] += cigars[0][1] cigars = cigars[1:] if cigars[-1][0] == "S": assert cigars[-2][0] == "M" cigars[-2][1] += cigars[-1][1] cigars = cigars[:-1] cigar_str = "" for cigar in cigars: cigar_op, length = cigar cigar_str += ("%d%s" % (length, cigar_op)) return pos, cigar_str, NM_real alignments = [[chr1, pos1, cigar1_str]] if aligner == "bwa" and len(XA) > 0: for alignment in XA: alt_chr, alt_pos, alt_cigar_str, alt_NM = alignment.split(',') alt_pos, alt_NM = abs(int(alt_pos)), int(alt_NM) if alt_NM > NM1: break alignments.append([alt_chr, alt_pos, alt_cigar_str]) # Convert repeat alignments to genome alignments if aligner == "hisat2" and (chr1.startswith("rep") or chr2.startswith("rep")) and len(repeat_map) > 0: if chr1.startswith("rep"): alignments = repeat_to_genome_alignment(repeat_db, repeat_map, chr1, pos1, cigar1_str) if chr2.startswith("rep") or (chr1.startswith("rep") and chr2 == "="): chr_tmp = chr1 if chr2 == "=" else chr2 alignments2 = repeat_to_genome_alignment(repeat_db, repeat_map, chr_tmp, int(pos2)) else: alignments2 = [[chr2, int(pos2)]] selected_alignments = [] for alignment in alignments: _chr1, _pos1 = alignment[:2] add = False for alignment2 in alignments2: _chr2, _pos2 = alignment2[:2] if _chr1 == _chr2 and abs(_pos1 - _pos2) <= 1000: add = True break if add: selected_alignments.append(alignment) alignments = selected_alignments for i, alignment in enumerate(alignments): chr1, pos1, cigar1_str = alignment pos1, cigar_str, NM_real = adjust_alignment(chr1, pos1, cigar1_str) chr2 = chr1 if aligner == "bwa": me = "%s\t%s" % (read_id, chr1) partner = "%s\t%s" % (read_id, chr2) else: me = "%s\t%s\t%d" % (read_id, chr1, canonical_pos1) partner = "%s\t%s\t%d" % (read_id, chr2, canonical_pos2) if partner in read_dic: maps = read_dic[partner] for map in maps: if map[0] == me: cigar2_str, NM2, pos2 = map[1:4] if aligner == "bwa": if abs(pos1 - pos2) >= 1000: continue if aligner in ["bowtie2", "bwa"]: if NM1 + NM2 > prev_NM: continue else: prev_NM = NM1 + NM2 if chr1 != chr2: continue # DK - debugging purposes if RNA: if aligner in ["bowtie2", "bwa"] and abs(pos1 - pos2) > 1000: continue else: if abs(pos1 - pos2) > 1000: continue if int(pos2) > int(pos1): p_str = "%s\t%s\t%d\t%s\t%s\t%d\t%s\tNM:i:%d\tNM:i:%d" % \ (read_id, chr1, pos1, cigar_str, chr2, pos2, cigar2_str, NM1, NM2) else: p_str = "%s\t%s\t%d\t%s\t%s\t%d\t%s\tNM:i:%d\tNM:i:%d" % \ (read_id, chr2, pos2, cigar2_str, chr1, pos1, cigar_str, NM2, NM1) if p_str not in pair_reported: pair_reported.add(p_str) print >> outfile, p_str if not me in read_dic: read_dic[me] = [] read_dic[me].append([partner, cigar_str, NM1, pos1]) if aligner == "hisat2": if prev_read_id != read_id: if prev_read_id != "": assert prev_NH1 == NH1_real assert prev_NH2 == NH2_real prev_NH1, prev_NH2 = 0, 0 if left_read: NH1_real, NH2_real = 1, 0 else: NH1_real, NH2_real = 0, 1 else: if left_read: NH1_real += 1 else: NH2_real += 1 if left_read: prev_NH1 = NH else: prev_NH2 = NH prev_read_id = read_id if aligner == "hisat2": if prev_read_id != "": assert prev_NH1 == NH1_real assert prev_NH2 == NH2_real outfile.close() infile.close() # Sanity check for HISAT2's alignment summary if aligner == "hisat2" and os.path.exists(infilename + ".summary") and (not mp_mode): hisat2_pairs, hisat2_0aligned_pairs, hisat2_conc_ualigned_pairs, hisat2_conc_maligned_pairs, hisat2_disc_aligned_pairs = 0, 0, 0, 0, 0 hisat2_reads, hisat2_0aligned_reads, hisat2_ualigned_reads, hisat2_maligned_reads = 0, 0, 0, 0 for line in open(infilename + ".summary"): line = line.strip() if line.startswith("HISAT2 summary") or \ line.startswith("Overall"): continue category, num = line.split(':') num = num.strip() num = int(num.split(' ')[0]) if category.startswith("Total pairs"): hisat2_pairs = num elif category.startswith("Aligned concordantly or discordantly 0 time"): hisat2_0aligned_pairs = num elif category.startswith("Aligned concordantly 1 time"): hisat2_conc_ualigned_pairs = num elif category.startswith("Aligned concordantly >1 time"): hisat2_conc_maligned_pairs = num elif category.startswith("Aligned discordantly"): hisat2_disc_aligned_pairs = num assert hisat2_pairs == hisat2_0aligned_pairs + hisat2_conc_ualigned_pairs + hisat2_conc_maligned_pairs + hisat2_disc_aligned_pairs elif category.startswith("Total unpaired reads"): hisat2_reads = num assert hisat2_reads == hisat2_0aligned_pairs * 2 elif category.startswith("Aligned 0 time"): hisat2_0aligned_reads = num elif category.startswith("Aligned 1 time"): hisat2_ualigned_reads = num else: assert category.startswith("Aligned >1 times") hisat2_maligned_reads = num assert hisat2_reads == hisat2_0aligned_reads + hisat2_ualigned_reads + hisat2_maligned_reads assert hisat2_pairs == num_pairs assert hisat2_conc_ualigned_pairs == num_conc_ualigned_pairs assert hisat2_conc_maligned_pairs == num_conc_aligned_pairs - num_conc_ualigned_pairs assert hisat2_disc_aligned_pairs == num_disc_aligned_pairs assert hisat2_ualigned_reads == num_ualigned_reads assert hisat2_maligned_reads == num_aligned_reads - num_ualigned_reads """ """ def is_junction_read(junctions_dic, chr, pos, cigar_str): result_junctions = [] junctions = get_junctions(chr, pos, cigar_str) for junction in junctions: junction_str = to_junction_str(junction) result_junctions.append([junction_str, junction_str in junctions_dic]) return result_junctions """ """ def is_junction_pair(junctions_dic, chr, pos, cigar_str, mate_chr, mate_pos, mate_cigar_str): junctions = is_junction_read(junctions_dic, chr, pos, cigar_str) mate_junctions = is_junction_read(junctions_dic, mate_chr, mate_pos, mate_cigar_str) junctions += mate_junctions return junctions """ """ def find_in_gtf_junctions(chr_dic, gtf_junctions, junction, relax_dist = 5): def find_in_gtf_junctions(gtf_junctions, junction): l, u = 0, len(gtf_junctions) while l < u: m = (l + u) / 2 assert m >= 0 and m < len(gtf_junctions) cmp_result = junction_cmp(junction, gtf_junctions[m]) if cmp_result == 0: return m elif cmp_result < 0: u = m else: l = m + 1 return l chr, left, right = junction gtf_index = find_in_gtf_junctions(gtf_junctions, [chr, left - relax_dist, right - relax_dist]) if gtf_index >= 0: i = gtf_index while i < len(gtf_junctions): chr2, left2, right2 = gtf_junctions[i] if chr2 > chr or \ left2 - left > relax_dist or \ right2 - right > relax_dist: break if abs(left - left2) <= relax_dist and left - left2 == right - right2: test_small = ":" in chr if is_canonical_junction(chr_dic, gtf_junctions[i]): if left == left2: return i else: return -1 else: return i i += 1 return -1 """ """ def compare_single_sam(RNA, reference_sam, query_sam, mapped_fname, chr_dic, gtf_junctions, gtf_junctions_set, ex_gtf_junctions): aligned, multi_aligned = 0, 0 db_dic, db_junction_dic = {}, {} mapped_file = open(mapped_fname, "w") first_mapped_file = open(mapped_fname + ".first", "w") file = open(reference_sam, "r") junction_read_dic = {} for line in file: if line.startswith('@'): continue read_name, chr, pos, cigar, NM = line[:-1].split() pos, NM = int(pos), int(NM[5:]) if read_name.find("seq.") == 0: read_name = read_name[4:] if len(read_name) > 2 and read_name[-2] == '/': read_name = read_name[:-2] multi_aligned += 1 if read_name not in db_dic: db_dic[read_name] = [] aligned += 1 pos2 = get_right(pos, cigar) db_dic[read_name].append([chr, pos, pos2, cigar, NM]) read_junctions = is_junction_read(gtf_junctions_set, chr, pos, cigar) if len(read_junctions) > 0: if read_name not in db_junction_dic: db_junction_dic[read_name] = [] for junction_str, is_gtf_junction in read_junctions: db_junction_dic[read_name].append([junction_str, is_gtf_junction]) if junction_str not in junction_read_dic: junction_read_dic[junction_str] = [] junction_read_dic[junction_str].append(line[:-1]) file.close() temp_junctions, temp_gtf_junctions = set(), set() for read_name, can_junctions in db_junction_dic.items(): if len(can_junctions) <= 0: continue # DK - for debugging purposes # 1. select the best candidate among spliced alignments if multiple def pickup_junction(can_junctions): junctions = [can_junctions[0]] for i in range(1, len(can_junctions)): def get_intron_len(can_junction): chr, left, right = to_junction(can_junction) return right - left - 1 intron, intron_cmp = get_intron_len(junctions[0][0]), get_intron_len(can_junctions[i][0]) if intron > intron_cmp: junctions = [can_junctions[i]] elif intron == intron_cmp: junctions.append(can_junctions[i]) return junctions # can_junctions = pickup_junction(can_junctions) for can_junction in can_junctions: found_junction_str = None junction_str, is_gtf_junction = can_junction if is_gtf_junction: found_junction_str = junction_str if not found_junction_str: junction = to_junction(junction_str) gtf_index = find_in_gtf_junctions(chr_dic, gtf_junctions, junction) if gtf_index >= 0: is_gtf_junction = True found_junction_str = to_junction_str(gtf_junctions[gtf_index]) if found_junction_str: temp_gtf_junctions.add(found_junction_str) temp_junctions.add(found_junction_str) else: if junction_str not in temp_junctions: None # assert junction_str in junction_read_dic # DK - for debugging purposes """ if len(junction_read_dic[junction_str]) <= 2: canonical = is_canonical_junction(chr_dic, to_junction(junction_str)) if not canonical: print >> sys.stdout, read_name, junction_str, len(junction_read_dic[junction_str]), can_junctions for line in junction_read_dic[junction_str]: print >> sys.stdout, "\t", line """ temp_junctions.add(junction_str) temp2_junctions = [] for junction in temp_junctions: temp2_junctions.append(to_junction(junction)) temp_junctions = sorted(list(temp2_junctions), cmp=junction_cmp) temp2_junctions = [] for can_junction in temp_junctions: if len(temp2_junctions) <= 0: temp2_junctions.append(can_junction) else: chr, left, right = temp2_junctions[-1] chr2, left2, right2 = can_junction if chr == chr2 and \ abs(left - left2) == abs(right - right2) and \ abs(left - left2) <= 10 and \ not to_junction_str(can_junction) in temp_gtf_junctions: continue temp2_junctions.append(can_junction) temp_junctions = set() for junction in temp2_junctions: temp_junctions.add(to_junction_str(junction)) file = open(query_sam) mapped, unmapped, unique_mapped, first_mapped, mapping_point = 0, 0, 0, 0, 0.0 snp_mapped, snp_unmapped, snp_unique_mapped, snp_first_mapped = 0, 0, 0, 0 for line in file: if line.startswith('@'): continue fields = line[:-1].split() read_name, chr, pos, cigar = fields[:4] trans_id, NM, Zs = None, None, None for field in fields[4:]: if field.startswith("TI"): trans_id = field[5:] elif field.startswith("NM"): NM = int(field[5:]) elif field.startswith("Zs"): Zs = field[5:] snp_included = (Zs != None) pos = int(pos) pos2 = get_right(pos, cigar) if read_name not in db_dic: unmapped += 1 if snp_included: snp_unmapped += 1 continue maps = db_dic[read_name] found = False found_at_first = False if [chr, pos, pos2, cigar, NM] in maps: found = True if maps.index([chr, pos, pos2, cigar, NM]) == 0: found_at_first = True # DK - debugging purposes if False and len(maps) > 0 and maps[0][-1] < NM: found = True if not found: for idx, map in enumerate(maps): if chr == map[0] and \ pos == map[1] and \ pos2 == map[2] and \ get_cigar_chars(cigar) == get_cigar_chars(map[3]): read_junctions = is_junction_read(gtf_junctions_set, map[0], map[1], map[3]) if True: found_list = [False for i in range(len(read_junctions))] for j in range(len(read_junctions)): junction_str, is_gtf_junction = read_junctions[j] junction = to_junction(junction_str) gtf_index = find_in_gtf_junctions(chr_dic, gtf_junctions, junction) if gtf_index >= 0: found_list[j] = True found = not (False in found_list) else: found = False if found: if idx == 0: found_at_first = True break if found: print >> mapped_file, read_name mapped += 1 if snp_included: snp_mapped += 1 if len(maps) == 1: unique_mapped += 1 if snp_included: snp_unique_mapped += 1 if found_at_first: print >> first_mapped_file, read_name first_mapped += 1 if snp_included: snp_first_mapped += 1 mapping_point += (1.0 / len(maps)) else: unmapped += 1 if snp_included: snp_unmapped += 1 file.close() mapped_file.close() first_mapped_file.close() # DK - for debugging purposes false_can_junctions, false_noncan_junctions = 0, 0 for junction_str in temp_junctions: if junction_str in temp_gtf_junctions: continue if junction_str in ex_gtf_junctions: continue if is_canonical_junction(chr_dic, to_junction(junction_str)): false_can_junctions += 1 else: false_noncan_junctions += 1 print >> sys.stderr, "\t\t\tfalse junctions: %d (canonical), %d (non-canonical)" % (false_can_junctions, false_noncan_junctions) return mapped, unique_mapped, first_mapped, unmapped, aligned, multi_aligned, \ snp_mapped, snp_unique_mapped, snp_first_mapped, snp_unmapped, \ len(temp_junctions), len(temp_gtf_junctions), mapping_point """ """ def compare_paired_sam(RNA, reference_sam, query_sam, mapped_fname, chr_dic, gtf_junctions, gtf_junctions_set, ex_gtf_junctions): aligned, multi_aligned = 0, 0 db_dic, db_junction_dic, junction_pair_dic = {}, {}, {} mapped_file = open(mapped_fname, "w") uniq_mapped_file = open(mapped_fname + '.uniq', "w") first_mapped_file = open(mapped_fname + '.first', "w") file = open(reference_sam, "r") for line in file: if line[0] == '@': continue read_name, chr, pos, cigar, chr2, pos2, cigar2, NM, NM2 = line[:-1].split() pos, pos2 = int(pos), int(pos2) NM, NM2 = int(NM[5:]), int(NM2[5:]) if read_name.find("seq.") == 0: read_name = read_name[4:] if len(read_name) > 2 and read_name[-2] == '/': read_name = read_name[:-2] multi_aligned += 1 if read_name not in db_dic: db_dic[read_name] = [] aligned += 1 pos_right, pos2_right = get_right(pos, cigar), get_right(pos2, cigar2) db_dic[read_name].append([chr, pos, pos_right, cigar, pos2, pos2_right, cigar2, NM, NM2]) pair_junctions = is_junction_pair(gtf_junctions_set, chr, pos, cigar, chr2, pos2, cigar2) if len(pair_junctions) > 0: if read_name not in db_junction_dic: db_junction_dic[read_name] = [] for junction_str, is_gtf_junction in pair_junctions: db_junction_dic[read_name].append([junction_str, is_gtf_junction]) # DK - for debugging purposes if junction_str not in junction_pair_dic: junction_pair_dic[junction_str] = [] junction_pair_dic[junction_str].append(line[:-1]) file.close() temp_junctions, temp_gtf_junctions = set(), set() for read_name, can_junctions in db_junction_dic.items(): if len(can_junctions) <= 0: continue # DK - for debugging purposes # 1. select the best candidate among spliced alignments if multiple def pickup_junction(can_junctions): junctions = [can_junctions[0]] for i in range(1, len(can_junctions)): def get_intron_len(can_junction): chr, left, right = to_junction(can_junction) return right - left - 1 intron, intron_cmp = get_intron_len(junctions[0][0]), get_intron_len(can_junctions[i][0]) if intron > intron_cmp: junctions = [can_junctions[i]] elif intron == intron_cmp: junctions.append(can_junctions[i]) return junctions # can_junctions = pickup_junction(can_junctions) for can_junction in can_junctions: found_junction_str = None junction_str, is_gtf_junction = can_junction # DK - for debugging purposes assert junction_str in junction_pair_dic if len(junction_pair_dic[junction_str]) <= 5: continue if is_gtf_junction: found_junction_str = junction_str if not found_junction_str: junction = to_junction(junction_str) gtf_index = find_in_gtf_junctions(chr_dic, gtf_junctions, junction) if gtf_index >= 0: is_gtf_junction = True found_junction_str = to_junction_str(gtf_junctions[gtf_index]) if found_junction_str: temp_gtf_junctions.add(found_junction_str) temp_junctions.add(found_junction_str) else: if junction_str not in temp_junctions: None # assert junction_str in junction_read_dic # print >> sys.stdout, read_name, junction_str, len(junction_read_dic[junction_str]) # for line in junction_read_dic[junction_str]: # print >> sys.stdout, "\t", line temp_junctions.add(junction_str) # DK - for debugging purposes filter_junction_db = {} temp2_junctions = [] for junction in temp_junctions: temp2_junctions.append(to_junction(junction)) temp_junctions = sorted(list(temp2_junctions), cmp=junction_cmp) temp2_junctions = [] for can_junction in temp_junctions: if len(temp2_junctions) <= 0: temp2_junctions.append(can_junction) # DK - for debugging purposes # assert to_junction_str(can_junction) in junction_pair_dic # filter_junction_db[to_junction_str(can_junction)] = len(junction_pair_dic[to_junction_str(can_junction)]) else: chr, left, right = temp2_junctions[-1] chr2, left2, right2 = can_junction if chr == chr2 and \ abs(left - left2) == abs(right - right2) and \ abs(left - left2) <= 10 and \ not to_junction_str(can_junction) in temp_gtf_junctions: # DK - for debugging purposes # assert to_junction_str(temp2_junctions[-1]) in junction_pair_dic # assert to_junction_str(temp2_junctions[-1]) in filter_junction_dic # filter_junction_db[to_junction_str(temp2_junctions[-1])] += len(junction_pair_dic[to_junction_str(temp2_junctions[-1])]) continue temp2_junctions.append(can_junction) # DK - for debugging purposes # assert to_junction_str(can_junction) in junction_pair_dic # filter_junction_db[to_junction_str(can_junction)] = len(junction_pair_dic[to_junction_str(can_junction)]) temp_junctions = set() for junction in temp2_junctions: # DK - for debugging purposes # assert to_junction_str(junction) in filter_junction_dic # if filter_junction_dic[to_junction_str(junction)] <= 5: # continue temp_junctions.add(to_junction_str(junction)) file = open(query_sam) mapped, unique_mapped, first_mapped, unmapped, mapping_point = 0, 0, 0, 0, 0.0 snp_mapped, snp_unique_mapped, snp_first_mapped, snp_unmapped = 0, 0, 0, 0 for line in file: if line.startswith('@'): continue fields = line[:-1].split() read_name, chr, pos, cigar, chr2, pos2, cigar2 = fields[:7] trains_id, NM, NM2, Zs, Zs2 = None, None, None, None, None for field in fields[7:]: if field.startswith("TI"): trans_id = field[5:] elif field.startswith("NM"): if NM == None: NM = int(field[5:]) else: NM2 = int(field[5:]) elif field.startswith("Zs"): if Zs == None: Zs = field[5:] else: Zs2 = field[5:] snp_included = (Zs != None or Zs2 != None) pos, pos2 = int(pos), int(pos2) pos_right, pos2_right = get_right(pos, cigar), get_right(pos2, cigar2) if read_name not in db_dic: unmapped += 1 if snp_included: snp_unmapped += 1 continue maps = db_dic[read_name] found = False found_at_first = False if [chr, pos, pos_right, cigar, pos2, pos2_right, cigar2, NM, NM2] in maps: found = True if maps.index([chr, pos, pos_right, cigar, pos2, pos2_right, cigar2, NM, NM2]) == 0: found_at_first = True # DK - debugging purposes if False and len(maps) > 0 and maps[0][-1] + maps[0][-2] < NM + NM2: found = True if not found: for idx, map in enumerate(maps): if chr == map[0] and \ pos == map[1] and \ pos_right == map[2] and \ get_cigar_chars(cigar) == get_cigar_chars(map[3]) and \ pos2 == map[4] and \ pos2_right == map[5] and \ get_cigar_chars(cigar2) == get_cigar_chars(map[6]): pair_junctions = is_junction_pair(gtf_junctions_set, map[0], map[1], map[3], map[0], map[4], map[6]) if True: found_list = [False for i in range(len(pair_junctions))] for j in range(len(pair_junctions)): junction_str, is_gtf_junction = pair_junctions[j] junction = to_junction(junction_str) gtf_index = find_in_gtf_junctions(chr_dic, gtf_junctions, junction) if gtf_index >= 0: found_list[j] = True found = not (False in found_list) else: found = False if found: if idx == 0: found_at_first = True break if found: print >> mapped_file, read_name mapped += 1 if snp_included: snp_mapped += 1 if len(maps) == 1: unique_mapped += 1 print >> uniq_mapped_file, read_name if snp_included: snp_unique_mapped += 1 if found_at_first: print >> first_mapped_file, read_name first_mapped += 1 if snp_included: snp_first_mapped += 1 mapping_point += (1.0 / len(maps)) else: unmapped += 1 if snp_included: snp_unmapped += 1 file.close() mapped_file.close() uniq_mapped_file.close() first_mapped_file.close() # DK - for debugging purposes false_can_junctions, false_noncan_junctions = 0, 0 for junction_str in temp_junctions: if junction_str in temp_gtf_junctions: continue if is_canonical_junction(chr_dic, to_junction(junction_str)): false_can_junctions += 1 else: false_noncan_junctions += 1 print >> sys.stderr, "\t\t\tfalse junctions: %d (canonical), %d (non-canonical)" % (false_can_junctions, false_noncan_junctions) return mapped, unique_mapped, first_mapped, unmapped, aligned, multi_aligned, \ snp_mapped, snp_unique_mapped, snp_first_mapped, snp_unmapped, \ len(temp_junctions), len(temp_gtf_junctions), mapping_point """ """ def extract_mapped_unmapped(read_fname, mapped_id_fname, mapped_fname, unmapped_fname, read2_fname = "", mapped2_fname = "", unmapped2_fname = ""): mapped_ids = set() mapped_id_file = open(mapped_id_fname) for line in mapped_id_file: read_id = int(line[:-1]) mapped_ids.add(read_id) mapped_id_file.close() def write_reads(read_fname, mapped_fname, unmapped_fname): mapped_file = open(mapped_fname, "w") unmapped_file = open(unmapped_fname, "w") read_file = open(read_fname) write = False for line in read_file: if line[0] == "@": read_id = int(line[1:-1]) write = read_id in mapped_ids if write: print >> mapped_file, line[:-1] else: print >> unmapped_file, line[:-1] read_file.close() mapped_file.close() unmapped_file.close() write_reads(read_fname, mapped_fname, unmapped_fname) if read2_fname != "": assert mapped2_fname != "" assert unmapped2_fname != "" write_reads(read2_fname, mapped2_fname, unmapped2_fname) """ """ def sql_execute(sql_db, sql_query): sql_cmd = [ "sqlite3", sql_db, "-separator", "\t", "%s;" % sql_query ] # print >> sys.stderr, sql_cmd sql_process = subprocess.Popen(sql_cmd, stdout=subprocess.PIPE) output = sql_process.communicate()[0][:-1] return output """ """ def create_sql_db(sql_db): if os.path.exists(sql_db): print >> sys.stderr, sql_db, "already exists!" return columns = [ ["id", "integer primary key autoincrement"], ["genome", "text"], ["head", "text"], ["end_type", "text"], ["type", "text"], ["aligner", "text"], ["version", "text"], ["num_reads", "integer"], ["mapped_reads", "integer"], ["unique_mapped_reads", "integer"], ["unmapped_reads", "integer"], ["mapping_point", "real"], ["snp_mapped_reads", "integer"], ["snp_unique_mapped_reads", "integer"], ["snp_unmapped_reads", "integer"], ["time", "real"], ["mem", "integer"], ["true_gtf_junctions", "integer"], ["temp_junctions", "integer"], ["temp_gtf_junctions", "integer"], ["host", "text"], ["created", "text"], ["cmd", "text"] ] sql_create_table = "CREATE TABLE ReadCosts (" for i in range(len(columns)): name, type = columns[i] if i != 0: sql_create_table += ", " sql_create_table += ("%s %s" % (name, type)) sql_create_table += ");" sql_execute(sql_db, sql_create_table) """ """ def write_analysis_data(sql_db, genome_name, database_name): if not os.path.exists(sql_db): return aligners = [] sql_aligners = "SELECT aligner FROM ReadCosts GROUP BY aligner" output = sql_execute(sql_db, sql_aligners) aligners = output.split() can_read_types = ["all", "M", "2M_gt_15", "2M_8_15", "2M_1_7", "gt_2M"] tmp_read_types = [] sql_types = "SELECT type FROM ReadCosts GROUP BY type" output = sql_execute(sql_db, sql_types) tmp_read_types = output.split() read_types = [] for read_type in can_read_types: if read_type in tmp_read_types: read_types.append(read_type) for paired in [False, True]: database_fname = genome_name + "_" + database_name if paired: end_type = "paired" database_fname += "_paired" else: end_type = "single" database_fname += "_single" database_fname += ".analysis" database_file = open(database_fname, "w") print >> database_file, "end_type\ttype\taligner\tnum_reads\ttime\tmem\tmapped_reads\tunique_mapped_reads\tunmapped_reads\tmapping_point\ttrue_gtf_junctions\ttemp_junctions\ttemp_gtf_junctions" for aligner in aligners: for read_type in read_types: sql_row = "SELECT end_type, type, aligner, num_reads, time, mem, mapped_reads, unique_mapped_reads, unmapped_reads, mapping_point, snp_mapped_reads, snp_unique_mapped_reads, snp_unmapped_reads, true_gtf_junctions, temp_junctions, temp_gtf_junctions FROM ReadCosts" sql_row += " WHERE genome = '%s' and head = '%s' and aligner = '%s' and type = '%s' and end_type = '%s' ORDER BY created DESC LIMIT 1" % (genome_name, database_name, aligner, read_type, end_type) output = sql_execute(sql_db, sql_row) if output: print >> database_file, output database_file.close() """ """ def calculate_read_cost(single_end, paired_end, test_aligners, fresh, runtime_only, verbose): sql_db_name = "analysis.db" if not os.path.exists(sql_db_name): create_sql_db(sql_db_name) num_cpus = multiprocessing.cpu_count() if num_cpus > 6: num_threads = min(6, num_cpus) desktop = False else: num_threads = min(3, num_cpus) desktop = True data_base = "sim" test_large_index = False verbose = False sql_write = True readtypes = ["all", "M", "2M_gt_15", "2M_8_15", "2M_1_7", "gt_2M"] aligners = [ # [aligner, two_step, index_type, aligner_version, addition_options] # ["hisat", "", "", "", ""], # ["hisat2", "", "", "204", ""], # ["hisat2", "", "", "210", ""], # ["hisat2", "", "snp", "203b", ""], # ["hisat2", "", "snp", "210", ""], # ["hisat2", "", "tran", "210", ""], # ["hisat2", "", "snp_tran", "210", ""], # ["hisat2", "", "", "210", ""], # ["hisat2", "", "", "", ""], ["hisat2", "", "rep", "", ""], # ["hisat2", "", "rep-100-300", "", ""], # ["hisat2", "", "rep_mm", "", ""], # ["hisat2", "", "", "", "--sensitive"], # ["hisat2", "", "rep", "", "--sensitive"], # ["hisat2", "", "", "", "--very-sensitive"], # ["hisat2", "", "snp", "", ""], # ["hisat2", "", "snp", "", "--sensitive"], # ["hisat2", "", "snp_noht", "", ""], # ["hisat2", "x2", "", "", ""], # ["hisat2", "x1", "tran", "", ""], # ["hisat2", "", "tran", "", ""], # ["hisat2", "", "snp_tran", "", ""], # ["hisat2", "x1", "snp_tran", "", ""], # ["hisat2", "x1", "snp_tran_ercc", "", ""], # ["tophat2", "gtfonly", "", "", ""], # ["tophat2", "gtf", "", "", ""], ["star", "", "", "", ""], # ["star", "x2", "", "", ""], # ["star", "gtf", "", "", ""], # ["bowtie", "", "", "", ""], ["bowtie2", "", "", "", ""], # ["bowtie2", "", "", "", "-k 10"], # ["bowtie2", "", "", "", "-k 1000 --extends 2000"], # ["gsnap", "", "", "", ""], # ["bwa", "mem", "", "", ""], ["bwa", "mem", "", "", "-a"], # ["hisat2", "", "snp", "", ""], # ["hisat2", "", "tran", "", ""], # ["hisat2", "", "snp_tran", "", ""], # ["vg", "", "", "", ""], # ["vg", "", "", "", "-M 10"], # ["vg", "", "snp", "", ""], # ["vg", "", "snp", "", "-M 10"], # ["minimap2", "", "", "", ""], ] readtypes = ["all"] verbose = True debug = False # sql_write = False cwd = os.getcwd() if len(cwd.split("reads_")) > 1: genome = cwd.split("reads_")[1].split("_")[0] else: genome = "genome" RNA = (cwd.find("RNA") != -1) test_small = (genome != "genome") if runtime_only: verbose = True chr_dic = read_genome("../../data/%s.fa" % genome) gtf_junctions = extract_splice_sites("../../data/%s.gtf" % genome) repeat_db, repeat_map = read_repeatdb("../../data/%s_rep.rep.info" % genome) align_stat = [] for paired in [False, True]: if not paired and not single_end: continue if paired and not paired_end: continue for readtype in readtypes: if paired: base_fname = data_base + "_paired" type_sam_fname = base_fname + "_" + readtype + ".sam" type_read1_fname = base_fname + "_1_" + readtype + ".fa" type_read2_fname = base_fname + "_2_" + readtype + ".fa" type_junction_fname = base_fname + "_" + readtype + ".junc" else: base_fname = data_base + "_single" type_sam_fname = base_fname + "_" + readtype + ".sam" type_read1_fname = base_fname + "_" + readtype + ".fa" type_read2_fname = "" type_junction_fname = base_fname + "_" + readtype + ".junc" assert os.path.exists(type_sam_fname) and os.path.exists(type_junction_fname) numreads = 0 type_sam_file = open(type_sam_fname) for line in type_sam_file: numreads += 1 type_sam_file.close() if numreads <= 0: continue print >> sys.stderr, "%s\t%d" % (readtype, numreads) junctions, junctions_set = [], set() type_junction_file = open(type_junction_fname) for line in type_junction_file: chr, left, right = line[:-1].split() left, right = int(left), int(right) junctions.append([chr, left, right]) junctions_set.add(to_junction_str([chr, left, right])) type_junction_file.close() aligner_bin_base = "../../../aligners/bin" def get_aligner_version(aligner, version): version = "" if aligner == "hisat2" or \ aligner == "hisat" or \ aligner == "bowtie" or \ aligner == "bowtie2": if version: cmd = ["%s/%s_%s/%s" % (aligner_bin_base, aligner, version, aligner)] else: cmd = ["%s/%s" % (aligner_bin_base, aligner)] cmd += ["--version"] cmd_process = subprocess.Popen(cmd, stdout=subprocess.PIPE) version = cmd_process.communicate()[0][:-1].split("\n")[0] version = version.split()[-1] elif aligner == "tophat2": cmd = ["%s/tophat" % (aligner_bin_base)] cmd += ["--version"] cmd_process = subprocess.Popen(cmd, stdout=subprocess.PIPE) version = cmd_process.communicate()[0][:-1].split()[-1] elif aligner in ["star", "starx2"]: version = "2.4.2a" elif aligner == "gsnap": cmd = ["%s/gsnap" % (aligner_bin_base)] cmd_process = subprocess.Popen(cmd, stderr=subprocess.PIPE) version = cmd_process.communicate()[1][:-1].split("\n")[0] version = version.split()[2] elif aligner == "bwa": if version: cmd = ["%s/bwa_%s/bwa" % (aligner_bin_base, version)] else: cmd = ["%s/bwa" % (aligner_bin_base)] cmd_process = subprocess.Popen(cmd, stderr=subprocess.PIPE) version = cmd_process.communicate()[1][:-1].split("\n")[2] version = version.split()[1] elif aligner == "vg": cmd = ["%s/vg" % (aligner_bin_base)] cmd_process = subprocess.Popen(cmd, stderr=subprocess.PIPE) version = cmd_process.communicate()[1][:-1].split("\n")[0] version = version.split()[5] elif aligner == "minimap2": cmd = ["%s/minimap2" % (aligner_bin_base)] cmd += ["--version"] cmd_process = subprocess.Popen(cmd, stdout=subprocess.PIPE) version = cmd_process.communicate()[0][:-1].split("\n")[0] return version index_base = "../../../indexes" index_add = "" if genome != "genome": index_add = "_" + genome def get_aligner_cmd(RNA, aligner, type, index_type, version, options, read1_fname, read2_fname, out_fname, cmd_idx = 0): cmd = ["/usr/bin/time"] if osx_mode: cmd += ['-l'] if aligner == "hisat2": if version: cmd += ["%s/hisat2_%s/hisat2" % (aligner_bin_base, version)] else: cmd += ["%s/hisat2" % (aligner_bin_base)] if num_threads > 1: cmd += ["-p", str(num_threads)] cmd += ["-f"] # cmd += ["-k", "100"] # cmd += ["--max-seeds", "100"] # cmd += ["--score-min", "C,-100"] # cmd += ["--pen-cansplice", "0"] # cmd += ["--pen-noncansplice", "12"] # cmd += ["--pen-intronlen", "G,-8,1"] # cmd += ["--metrics", "1", # "--metrics-file", "metrics.out"] if not RNA: cmd += ["--no-spliced-alignment"] if type in ["x1", "x2"]: cmd += ["--no-temp-splicesite"] # cmd += ["--no-anchorstop"] if version == "" or \ (version != "" and int(version) >= 210): cmd += ["--new-summary", "--summary-file", out_fname + ".summary"] if version == "" or int(version) >= 220: cmd += ["--repeat"] # cmd += ["--dta"] # cmd += ["--dta-cufflinks"] if options != "": cmd += options.split(' ') if type == "x2": if cmd_idx == 0: cmd += ["--novel-splicesite-outfile"] else: cmd += ["--novel-splicesite-infile"] cmd += ["splicesites.txt"] # "--novel-splicesite-infile", # "../splicesites.txt", # "--rna-strandness", # "FR", if version: index_cmd = "%s/HISAT2_%s%s/" % (index_base, version, index_add) + genome else: index_cmd = "%s/HISAT2%s/" % (index_base, index_add) + genome if index_type: index_cmd += ("_" + index_type) cmd += [index_cmd] if paired: cmd += ["-1", read1_fname, "-2", read2_fname] else: cmd += [read1_fname] elif aligner == "hisat": cmd += ["%s/hisat" % (aligner_bin_base)] if num_threads > 1: cmd += ["-p", str(num_threads)] cmd += ["-f"] # cmd += ["-k", "5"] # cmd += ["--score-min", "C,-18"] if version != "": version = int(version) else: version = sys.maxint if not RNA: cmd += ["--no-spliced-alignment"] if type in ["x1", "x2"]: cmd += ["--no-temp-splicesite"] """ cmd += ["--rdg", "100,100", "--rfg", "100,100"] """ if type == "x2": if cmd_idx == 0: cmd += ["--novel-splicesite-outfile"] else: cmd += ["--novel-splicesite-infile"] cmd += ["splicesites.txt"] # "--novel-splicesite-infile", # "../splicesites.txt", # "--rna-strandness", # "FR", cmd += ["%s/HISAT%s/" % (index_base, index_add) + genome] if paired: cmd += ["-1", read1_fname, "-2", read2_fname] else: cmd += [read1_fname] elif aligner == "tophat2": cmd += ["%s/tophat" % (aligner_bin_base)] if num_threads > 1: cmd += ["-p", str(num_threads)] if type.find("gtf") != -1: cmd += ["--transcriptome-index=%s/HISAT%s/gtf/%s" % (index_base, index_add, genome)] if type == "gtfonly": cmd += ["--transcriptome-only"] cmd += ["--read-edit-dist", "3"] cmd += ["--no-sort-bam"] cmd += ["--read-realign-edit-dist", "0"] cmd += ["--keep-tmp", "%s/HISAT%s/" % (index_base, index_add) + genome, read1_fname] if paired: cmd += [read2_fname] elif aligner == "star": cmd += ["%s/STAR" % (aligner_bin_base)] if num_threads > 1: cmd += ["--runThreadN", str(num_threads)] cmd += ["--genomeDir"] if cmd_idx == 0: if type == "gtf": cmd += ["%s/STAR%s/gtf" % (index_base, index_add)] else: cmd += ["%s/STAR%s" % (index_base, index_add)] else: assert cmd_idx == 1 cmd += ["."] if desktop: cmd += ["--genomeLoad", "NoSharedMemory"] else: cmd += ["--genomeLoad", "LoadAndKeep"] if type == "x2": if cmd_idx == 1: cmd += ["--alignSJDBoverhangMin", "1"] cmd += ["--readFilesIn", read1_fname] if paired: cmd += [read2_fname] if paired: cmd += ["--outFilterMismatchNmax", "6"] else: cmd += ["--outFilterMismatchNmax", "3"] elif aligner == "bowtie": cmd += ["%s/bowtie" % (aligner_bin_base)] if num_threads > 1: cmd += ["-p", str(num_threads)] cmd += ["-f", "--sam", "-k", "10"] cmd += ["-n", "3"] if paired: cmd += ["-X", "500"] cmd += ["%s/Bowtie%s/" % (index_base, index_add) + genome] if paired: cmd += ["-1", read1_fname, "-2", read2_fname] else: cmd += [read1_fname] elif aligner == "bowtie2": if version: cmd += ["%s/bowtie2_%s/bowtie2" % (aligner_bin_base, version)] else: cmd += ["%s/bowtie2" % (aligner_bin_base)] if num_threads > 1: cmd += ["-p", str(num_threads)] cmd += ["-f"] if options != "": cmd += options.split(' ') if version: cmd += ["-x %s/Bowtie2_%s%s/" % (index_base, version, index_add) + genome] else: cmd += ["-x %s/Bowtie2%s/" % (index_base, index_add) + genome] if paired: cmd += ["-1", read1_fname, "-2", read2_fname] else: cmd += [read1_fname] elif aligner == "gsnap": cmd += ["%s/gsnap" % (aligner_bin_base), "-A", "sam"] if num_threads > 1: cmd += ["-t", str(num_threads)] cmd += ["--max-mismatches=3", "-D", "%s/GSNAP%s" % (index_base, index_add), "-N", "1", "-d", genome, read1_fname] if paired: cmd += [read2_fname] elif aligner == "bwa": if version: cmd += ["%s/bwa_%s/bwa" % (aligner_bin_base, version)] else: cmd += ["%s/bwa" % (aligner_bin_base)] if type in ["mem", "aln"]: cmd += [type] elif type == "sw": cmd += ["bwa" + type] if num_threads > 1: cmd += ["-t", str(num_threads)] if options != "": cmd += options.split(' ') # if paired: # cmd += ["-T", "60"] if version: cmd += ["%s/BWA_%s%s/%s.fa" % (index_base, version, index_add, genome)] else: cmd += ["%s/BWA%s/%s.fa" % (index_base, index_add, genome)] cmd += [read1_fname] if paired: cmd += [read2_fname] elif aligner == "vg": # vg map -d 22 -t 6 -M 10 -f ../sim-1.fa -f ../sim-2.fa > result.sam.gam cmd += ["%s/vg" % (aligner_bin_base)] cmd += ["map"] cmd += ["-t", str(num_threads)] cmd += ["--surject-to", "sam"] index_cmd = "%s/VG%s/" % (index_base, index_add) + genome if index_type: index_cmd += ("_" + index_type) if options != "": cmd += options.split(' ') cmd += ["-d", index_cmd] cmd += ["-f", read1_fname] if paired: cmd += ["-f", read2_fname] elif aligner == "minimap2": # minimap2 -a -x sr 22.mmi sim_1.fa sim_2.fa > result.sam cmd += ["%s/minimap2" % (aligner_bin_base)] cmd += ["-a"] cmd += ["-x", "sr"] index_cmd = "%s/minimap2%s/" % (index_base, index_add) + genome if index_type: index_cmd += ("_" + index_type) index_cmd += ".mmi" cmd += [index_cmd] cmd += [read1_fname] if paired: cmd += [read2_fname] else: assert False return cmd for aligner, type, index_type, version, options in aligners: skip = False if len(test_aligners) > 0: skip = True for test_aligner in test_aligners: if aligner == test_aligner: skip = False if skip: continue aligner_name = aligner + type if version != "": aligner_name += ("_%s" % version) if aligner == "hisat2" and index_type != "": aligner_name += ("_" + index_type) if aligner == "vg" and index_type != "": aligner_name += ("_" + index_type) two_step = (aligner == "tophat2" or type == "x2" or (aligner in ["hisat2", "hisat"] and type == "")) if RNA and readtype != "M": if aligner in ["bowtie", "bowtie2", "bwa"]: continue if readtype != "all": if two_step: continue if not RNA and readtype != "all": continue print >> sys.stderr, "\t%s\t%s" % (aligner_name, str(datetime.now())) if options != "": option_name = options.replace(' ', '').replace('-', '').replace(',', '') aligner_name = aligner_name + '_' + option_name if paired: aligner_dir = aligner_name + "_paired" else: aligner_dir = aligner_name + "_single" if fresh and os.path.exists(aligner_dir): os.system("rm -rf %s" % aligner_dir) if not os.path.exists(aligner_dir): os.mkdir(aligner_dir) os.chdir(aligner_dir) out_fname = base_fname + "_" + readtype + ".sam" out_fname2 = out_fname + "2" duration = -1.0 mem_usage = '0' if not os.path.exists(out_fname): if not os.path.exists("../one.fa") or not os.path.exists("../two.fa"): os.system("head -400 ../%s_1.fa > ../one.fa" % (data_base)) os.system("head -400 ../%s_2.fa > ../two.fa" % (data_base)) if runtime_only: out_fname = "/dev/null" out_fname2 = "/dev/null" if not two_step: align_stat.append([readtype, aligner_name]) # dummy commands for caching index and simulated reads loading_time = 0 if aligner != "tophat2": for i in range(3): dummy_cmd = get_aligner_cmd(RNA, aligner, type, index_type, version, options, "../one.fa", "../two.fa", "/dev/null") start_time = datetime.now() if verbose: print >> sys.stderr, start_time, "\t", " ".join(dummy_cmd) if aligner in ["hisat2", "hisat", "bowtie", "bowtie2", "gsnap", "bwa"]: proc = subprocess.Popen(dummy_cmd, stdout=open("/dev/null", "w"), stderr=subprocess.PIPE) else: proc = subprocess.Popen(dummy_cmd, stdout=subprocess.PIPE, stderr=subprocess.PIPE) proc.communicate() finish_time = datetime.now() duration = finish_time - start_time duration = duration.total_seconds() if verbose: print >> sys.stderr, finish_time, "duration:", duration loading_time = duration # Align all reads aligner_cmd = get_aligner_cmd(RNA, aligner, type, index_type, version, options, "../" + type_read1_fname, "../" + type_read2_fname, out_fname) start_time = datetime.now() if verbose: print >> sys.stderr, "\t", start_time, " ".join(aligner_cmd) if aligner in ["hisat2", "hisat", "bowtie", "bowtie2", "gsnap", "bwa", "vg", "minimap2"]: proc = subprocess.Popen(aligner_cmd, stdout=open(out_fname, "w"), stderr=subprocess.PIPE) else: proc = subprocess.Popen(aligner_cmd, stdout=subprocess.PIPE, stderr=subprocess.PIPE) _, mem_usage = proc.communicate() mem_usage = parse_mem_usage(mem_usage) finish_time = datetime.now() duration = finish_time - start_time duration = duration.total_seconds() - loading_time if duration < 0.1: duration = 0.1 if verbose: print >> sys.stderr, "\t", finish_time, "finished:", duration if debug and aligner == "hisat2": os.system("cat metrics.out") print >> sys.stderr, "\ttime: %.4f" % (duration) if aligner == "star" and type in ["", "gtf"]: os.system("mv Aligned.out.sam %s" % out_fname) elif aligner in ["hisat2", "hisat"] and type == "x2": aligner_cmd = get_aligner_cmd(RNA, aligner, type, index_type, version, options, "../" + type_read1_fname, "../" + type_read2_fname, out_fname, 1) start_time = datetime.now() if verbose: print >> sys.stderr, "\t", start_time, " ".join(aligner_cmd) proc = subprocess.Popen(aligner_cmd, stdout=open(out_fname, "w"), stderr=subprocess.PIPE) proc.communicate() finish_time = datetime.now() duration += (finish_time - start_time).total_seconds() duration -= loading_time if duration < 0.1: duration = 0.1 if verbose: print >> sys.stderr, "\t", finish_time, "finished:", duration elif aligner == "star" and type == "x2": assert os.path.exists("SJ.out.tab") os.system("awk 'BEGIN {OFS=\"\t\"; strChar[0]=\".\"; strChar[1]=\"+\"; strChar[2]=\"-\";} {if($5>0){print $1,$2,$3,strChar[$4]}}' SJ.out.tab > SJ.out.tab.Pass1.sjdb") for file in os.listdir("."): if file in ["SJ.out.tab.Pass1.sjdb", "genome.fa"]: continue os.remove(file) star_index_cmd = "%s/STAR --genomeDir ./ --runMode genomeGenerate --genomeFastaFiles ../../../data/%s.fa --sjdbFileChrStartEnd SJ.out.tab.Pass1.sjdb --sjdbOverhang 99 --runThreadN %d" % (aligner_bin_base, genome, num_threads) if verbose: print >> sys.stderr, "\t", datetime.now(), star_index_cmd os.system(star_index_cmd) if verbose: print >> sys.stderr, "\t", datetime.now(), " ".join(dummy_cmd) proc = subprocess.Popen(dummy_cmd, stdout=subprocess.PIPE, stderr=subprocess.PIPE) proc.communicate() if verbose: print >> sys.stderr, "\t", datetime.now(), "finished" aligner_cmd = get_aligner_cmd(RNA, aligner, type, index_type, version, options, "../" + type_read1_fname, "../" + type_read2_fname, out_fname, 1) start_time = datetime.now() if verbose: print >> sys.stderr, "\t", start_time, " ".join(aligner_cmd) proc = subprocess.Popen(aligner_cmd, stdout=subprocess.PIPE, stderr=subprocess.PIPE) proc.communicate() finish_time = datetime.now() duration += (finish_time - start_time).total_seconds() duration -= loading_time if duration < 0.1: duration = 0.1 if verbose: print >> sys.stderr, "\t", finish_time, "finished:", duration os.system("mv Aligned.out.sam %s" % out_fname) elif aligner == "tophat2": os.system("samtools sort -n tophat_out/accepted_hits.bam accepted_hits; samtools view -h accepted_hits.bam > %s" % out_fname) elif aligner == "vg": index_name = "%s/VG%s/" % (index_base, index_add) + genome if index_type: index_name += ("_" + index_type) if aligner in ["gsnap", "tophat2"]: os.system("tar cvzf %s.tar.gz %s &> /dev/null" % (out_fname, out_fname)) if runtime_only: print >> sys.stderr, "\t\t\tMemory Usage: %dMB" % (int(mem_usage) / 1024) os.chdir("..") continue if not os.path.exists(out_fname2): debug_dic = {} pid_list = [] if paired: if mp_mode: for i in xrange(mp_num): p = Process(target=extract_pair, args=(out_fname, out_fname2, chr_dic, RNA, aligner, version, repeat_db, repeat_map, debug_dic, i)) pid_list.append(p) p.start() for p in pid_list: p.join() # merge os.system("mv %s %s" % (out_fname2 + ".0", out_fname2)) for i in xrange(1, mp_num): os.system("cat %s >> %s" % (out_fname2 + "." + str(i), out_fname2)) os.system("rm %s" % (out_fname2 + "." + str(i))) else: extract_pair(out_fname, out_fname2, chr_dic, RNA, aligner, version, repeat_db, repeat_map, debug_dic, -1) else: if mp_mode: # Prepare queues for i in xrange(mp_num): p = Process(target=extract_single, args=(out_fname, out_fname2, chr_dic, aligner, version, repeat_db, repeat_map, debug_dic, i)) pid_list.append(p) p.start() # wait for p in pid_list: p.join() # merge os.system("mv %s %s" % (out_fname2 + ".0", out_fname2)) for i in xrange(1, mp_num): os.system("cat %s >> %s" % (out_fname2 + "." + str(i), out_fname2)) os.system("rm %s" % (out_fname2 + "." + str(i))) else: extract_single(out_fname, out_fname2, chr_dic, aligner, version, repeat_db, repeat_map, debug_dic, -1) for readtype2 in readtypes: if not two_step and readtype != readtype2: continue type_sam_fname2 = base_fname + "_" + readtype2 + ".sam" if os.path.exists(type_sam_fname2 + ".done"): continue if paired: type_read_fname2 = base_fname + "_1_" + readtype2 + ".fa" else: type_read_fname2 = base_fname + "_" + readtype2 + ".fa" mapped_id_fname = base_fname + "_" + readtype2 + ".read_id" if paired: mapped, unique_mapped, first_mapped, unmapped, aligned, multi_aligned, \ snp_mapped, snp_unique_mapped, snp_first_mapped, snp_unmapped, \ temp_junctions, temp_gtf_junctions, mapping_point \ = compare_paired_sam(RNA, out_fname2, "../" + type_sam_fname2, mapped_id_fname, chr_dic, junctions, junctions_set, gtf_junctions) else: mapped, unique_mapped, first_mapped, unmapped, aligned, multi_aligned, \ snp_mapped, snp_unique_mapped, snp_first_mapped, snp_unmapped, \ temp_junctions, temp_gtf_junctions, mapping_point \ = compare_single_sam(RNA, out_fname2, "../" + type_sam_fname2, mapped_id_fname, chr_dic, junctions, junctions_set, gtf_junctions) proc = subprocess.Popen(["wc", "-l", "../" + type_read_fname2], stdout=subprocess.PIPE) out = proc.communicate()[0] numreads = int(out.split()[0]) / 2 assert mapped + unmapped == numreads if two_step: print >> sys.stderr, "\t\t%s" % readtype2 print >> sys.stderr, "\t\taligned: %d, multi aligned: %d" % (aligned, multi_aligned) print >> sys.stderr, "\t\tcorrectly mapped: %d (%.2f%%) mapping_point: %.2f" % (mapped, float(mapped) * 100.0 / numreads, mapping_point * 100.0 / numreads) print >> sys.stderr, "\t\tcorrectly mapped at first: %d (%.2f%%)" % (first_mapped, float(first_mapped) * 100.0 / numreads) print >> sys.stderr, "\t\tuniquely and correctly mapped: %d (%.2f%%)" % (unique_mapped, float(unique_mapped) * 100.0 / numreads) snp_numreads = snp_mapped + snp_unmapped if snp_numreads > 0: print >> sys.stderr, "\t\t\t\tSNP: reads: %d" % (snp_numreads) print >> sys.stderr, "\t\t\t\tSNP: correctly mapped: %d (%.2f%%)" % (snp_mapped, float(snp_mapped) * 100.0 / snp_numreads) print >> sys.stderr, "\t\t\t\tSNP: correctly mapped at first: %d (%.2f%%)" % (snp_first_mapped, float(snp_first_mapped) * 100.0 / snp_numreads) print >> sys.stderr, "\t\t\t\tSNP: uniquely and correctly mapped: %d (%.2f%%)" % (snp_unique_mapped, float(snp_unique_mapped) * 100.0 / snp_numreads) if readtype == readtype2: print >> sys.stderr, "\t\t\t%d reads per sec (all)" % (numreads / max(1.0, duration)) if RNA: print >> sys.stderr, "\t\tjunc. sensitivity %d / %d (%.2f%%), junc. accuracy: %d / %d (%.2f%%)" % \ (temp_gtf_junctions, len(junctions), float(temp_gtf_junctions) * 100.0 / max(1, len(junctions)), \ temp_gtf_junctions, temp_junctions, float(temp_gtf_junctions) * 100.0 / max(1, temp_junctions)) print >> sys.stderr, "\t\t\tMemory Usage: %dMB" % (int(mem_usage) / 1024) if duration > 0.0: if sql_write and os.path.exists("../" + sql_db_name): if paired: end_type = "paired" else: end_type = "single" mem_used = int(mem_usage) / 1024 sql_insert = "INSERT INTO \"ReadCosts\" VALUES(NULL, '%s', '%s', '%s', '%s', '%s', '%s', %d, %d, %d, %d, %f, %f, %d, %d, %d, %d, %d, %d, %d, '%s', datetime('now', 'localtime'), '%s');" % \ (genome, data_base, end_type, readtype2, aligner_name, get_aligner_version(aligner, version), numreads, mapped, unique_mapped, unmapped, mapping_point, snp_mapped, snp_unique_mapped, snp_unmapped, duration, mem_used, len(junctions), temp_junctions, temp_gtf_junctions, platform.node(), " ".join(aligner_cmd)) sql_execute("../" + sql_db_name, sql_insert) if two_step: align_stat.append([readtype2, aligner_name, numreads, duration, mem_used, mapped, unique_mapped, unmapped, mapping_point, snp_mapped, snp_unique_mapped, snp_unmapped, len(junctions), temp_junctions, temp_gtf_junctions]) else: align_stat[-1].extend([numreads, duration, mem_used, mapped, unique_mapped, unmapped, mapping_point, snp_mapped, snp_unique_mapped, snp_unmapped, len(junctions), temp_junctions, temp_gtf_junctions]) os.system("touch %s.done" % type_sam_fname2) os.chdir("..") print >> sys.stdout, "\t".join(["type", "aligner", "all", "all_time", "mem", "mapped", "unique_mapped", "unmapped", "mapping point", "snp_mapped", "snp_unique_mapped", "snp_unmapped", "true_gtf_junctions", "temp_junctions", "temp_gtf_junctions"]) for line in align_stat: outstr = "" for item in line: if outstr != "": outstr += "\t" outstr += str(item) print >> sys.stdout, outstr if os.path.exists(sql_db_name): write_analysis_data(sql_db_name, genome, data_base) """ """ if __name__ == "__main__": parser = ArgumentParser( description='test HISAT2, and compare HISAT2 with other popular aligners such as TopHat2, STAR, Bowtie1/2, GSNAP, BWA-mem, etc.') parser.add_argument('--single-end', dest='paired_end', action='store_false', help='run single-end only') parser.add_argument('--paired-end', dest='single_end', action='store_false', help='run paired_end only') parser.add_argument('--aligner-list', dest='aligner_list', type=str, default="", help='comma-separated list of aligners (e.g. hisat2,bowtie2,bwa') parser.add_argument('--fresh', dest='fresh', action='store_true', help='delete existing alignment related directories (e.g. hisat2_single)') parser.add_argument('--runtime-only', '--runtime', dest='runtime_only', action='store_true', help='run programs without evaluation') parser.add_argument('-p', '--multi-process', dest='mp_num', action='store', type=int, default=1, help='Use multiple process mode') parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') args = parser.parse_args() aligners = [] for aligner in args.aligner_list.split(','): if aligner == "": continue aligners.append(aligner) mp_num = args.mp_num mp_mode = (mp_num > 1) calculate_read_cost(args.single_end, args.paired_end, aligners, args.fresh, args.runtime_only, args.verbose) hisat2-2.2.1/evaluation/simulation/init.py000077500000000000000000000270251370663763200206040ustar00rootroot00000000000000#!/usr/bin/env python import sys, os import string, re use_message = ''' ''' def extract_pair(RNA): read_dic = {} pair_reported = set() out_file = open("sim_paired.sam", "w") hits_file = open("sim.sam") for line in hits_file: if line.startswith('@'): continue fields = line[:-1].split() read_id, flag, chr1, pos1, mapQ, cigar1, chr2, pos2 = fields[:8] flag = int(flag) assert flag & 0x4 == 0 pos1, pos2 = int(pos1), int(pos2) TI, NM1, Zs1 = "", "", "" for field in fields[11:]: if field.startswith("TI"): TI = "\t" + field elif field.startswith("NM"): NM1 = "\t" + field elif field.startswith("Zs"): Zs1 = "\t" + field assert NM1 != "" assert chr1 == chr2 me = "%s\t%s\t%d" % (read_id, chr1, pos1) partner = "%s\t%s\t%d" % (read_id, chr2, pos2) if partner in read_dic: maps = read_dic[partner] for map in maps: if map[0] == me: cigar2, NM2, Zs2 = map[1:4] if int(pos2) > int(pos1): p_str = "%s\t%s\t%d\t%s\t%s\t%d\t%s%s%s%s%s%s" % \ (read_id, chr1, pos1, cigar1, chr2, pos2, cigar2, TI, NM1, NM2, Zs1, Zs2) else: p_str = "%s\t%s\t%d\t%s\t%s\t%d\t%s%s%s%s%s%s" % \ (read_id, chr2, pos2, cigar2, chr1, pos1, cigar1, TI, NM2, NM1, Zs2, Zs1) if p_str not in pair_reported: pair_reported.add(p_str) print >> out_file, p_str if not me in read_dic: read_dic[me] = [] read_dic[me].append([partner, cigar1, NM1, Zs1]) hits_file.close() out_file.close() def init_reads(read_dir, RNA): sim1_file = open("sim_1.sam", 'w') sim2_file = open("sim_2.sam", 'w') for line in open("sim.sam"): if line.startswith('@'): continue fields = line[:-1].split('\t') read_id, flag, chr, pos, _, cigar = fields[:6] left_read = int(flag) < 128 NM, TI, Zs = "", "", "" for field in fields: if field.startswith("NM"): NM = "\t" + field elif field.startswith("TI"): TI = "\t" + field elif field.startswith("Zs"): Zs = "\t" + field print >> (sim1_file if left_read else sim2_file), \ "%s\t%s\t%s\t%s%s%s%s" % (read_id, chr, pos, cigar, TI, NM, Zs) sim1_file.close() sim2_file.close() extract_pair(RNA) def to_junction_str(junction): return "%s-%d-%d" % (junction[0], junction[1], junction[2]) def to_junction(junction_str): fields = junction_str.split("-") if len(fields) > 3: chr, left, right = "-".join(fields[:-2]), fields[-2], fields[-1] else: assert len(fields) == 3 chr, left, right = fields return [chr, int(left), int(right)] def junction_cmp(a, b): if a[0] != b[0]: if a[0] < b[0]: return -1 else: return 1 if a[1] != b[1]: if a[1] < b[1]: return -1 else: return 1 if a[2] != b[2]: if a[2] < b[2]: return -1 else: return 1 return 0 def classify_reads(RNA): if RNA: readtypes = ["all", "M", "2M_gt_15", "2M_8_15", "2M_1_7", "gt_2M"] else: readtypes = ["all" ,"M"] readtype_order = {} for i in range(len(readtypes)): readtype = readtypes[i] assert readtype not in readtype_order readtype_order[readtype] = i for paired in [False, True]: for readtype in readtypes: if paired: base_fname = "sim_paired" type_sam_fname = base_fname + "_" + readtype + ".sam" type_read1_fname = base_fname + "_1_" + readtype + ".fa" type_read2_fname = base_fname + "_2_" + readtype + ".fa" type_junction_fname = base_fname + "_" + readtype + ".junc" else: base_fname = "sim_single" type_sam_fname = base_fname + "_" + readtype + ".sam" type_read1_fname = base_fname + "_" + readtype + ".fa" type_read2_fname = "" type_junction_fname = base_fname + "_" + readtype + ".junc" if os.path.exists(type_sam_fname) and \ os.path.exists(type_junction_fname): continue read_ids = set() junctions = [] type_sam_file = open(type_sam_fname, "w") if paired: sam_file = open("sim_paired.sam") else: sam_file = open("sim_1.sam") cigar_re = re.compile('\d+\w') def get_read_type(cigar): cigars = cigar_re.findall(cigar) def get_cigar_chars_MN(cigars): cigars = cigar_re.findall(cigars) cigar_chars = "" for cigar in cigars: cigar_op = cigar[-1] if cigar_op in "MN": if cigar_chars == "" or cigar_chars[-1] != cigar_op: cigar_chars += cigar_op return cigar_chars cigar_str = get_cigar_chars_MN(cigar) if cigar_str == "M": return cigar_str elif cigar_str == "MNM": assert len(cigars) >= 3 left_anchor = 0 for ci in range(len(cigars)): c = cigars[ci][-1] c_len = int(cigars[ci][:-1]) if c in "MI": left_anchor += c_len else: break assert left_anchor > 0 right_anchor = 0 for ci in reversed(range(len(cigars))): c = cigars[ci][-1] c_len = int(cigars[ci][:-1]) if c in "MI": right_anchor += c_len else: break assert right_anchor > 0 min_anchor = min(left_anchor, right_anchor) if min_anchor > 15: return "2M_gt_15" elif min_anchor >= 8 and min_anchor <= 15: return "2M_8_15" elif min_anchor >= 1 and min_anchor <= 7: return "2M_1_7" else: assert False else: assert cigar_str not in ["M", "MNM"] return "gt_2M" # chr and pos are assumed to be integers def get_junctions(chr, pos, cigar_str): junctions = [] right_pos = pos cigars = cigar_re.findall(cigar_str) cigars = [[int(cigars[i][:-1]), cigars[i][-1]] for i in range(len(cigars))] for i in range(len(cigars)): length, cigar_op = cigars[i] if cigar_op == "N": left, right = right_pos - 1, right_pos + length if i > 0 and cigars[i-1][1] in "ID": if cigars[i-1][1] == "I": left += cigars[i-1][0] else: left -= cigars[i-1][0] if i + 1 < len(cigars) and cigars[i+1][1] in "ID": if cigars[i+1][1] == "I": right -= cigars[i+1][0] else: right += cigars[i+1][0] junctions.append([chr, left, right]) if cigar_op in "MND": right_pos += length return junctions for line in sam_file: fields = line[:-1].split() if paired: read_id, chr, pos, cigar, chr2, pos2, cigar2 = fields[:7] else: read_id, chr, pos, cigar = fields[:4] read_id = int(read_id) readtype2 = get_read_type(cigar) if paired: readtype3 = get_read_type(cigar2) assert readtype2 in readtype_order assert readtype3 in readtype_order if readtype_order[readtype2] < readtype_order[readtype3]: readtype2 = readtype3 if readtype == "all" or readtype == readtype2: read_ids.add(read_id) print >> type_sam_file, line[:-1] junctions += get_junctions(chr, int(pos), cigar) if paired: junctions += get_junctions(chr2, int(pos2), cigar2) sam_file.close() type_sam_file.close() # make this set non-redundant junction_set = set() for junction in junctions: junction_set.add(to_junction_str(junction)) junctions = [] for junction_str in junction_set: junctions.append(to_junction(junction_str)) # sort the list of junctions junctions = sorted(junctions, cmp=junction_cmp) # write the junctions into a file type_junction_file = open(type_junction_fname, "w") for junction in junctions: print >> type_junction_file, "%s\t%d\t%d" % (junction[0], junction[1], junction[2]) type_junction_file.close() def write_reads(read_fname, type_read_fname): type_read_file = open(type_read_fname, "w") read_file = open(read_fname) write = False for line in read_file: if line[0] == ">": read_id = int(line[1:-1]) write = read_id in read_ids if write: print >> type_read_file, line[:-1] read_file.close() type_read_file.close() if paired: write_reads("sim_1.fa", type_read1_fname) write_reads("sim_2.fa", type_read2_fname) else: write_reads("sim_1.fa", type_read1_fname) def init(): read_dir_base = "../reads/simulation/" read_dirs = os.listdir(read_dir_base) for read_dir in read_dirs: if os.path.exists(read_dir): continue if not os.path.exists(read_dir_base + read_dir + "/sim.sam") or \ not os.path.exists(read_dir_base + read_dir + "/sim_1.fa") or \ not os.path.exists(read_dir_base + read_dir + "/sim_2.fa"): continue print >> sys.stderr, "Processing", read_dir, "..." os.mkdir(read_dir) os.chdir(read_dir) os.system("ln -s ../%s%s/* ." % (read_dir_base, read_dir)) os.system("ln -s sim_1.fa 1.fa") os.system("ln -s sim_2.fa 2.fa") RNA = (read_dir.find("RNA") != -1) init_reads(read_dir, RNA) classify_reads(RNA) os.system("ln -s ../calculate_read_cost.py .") os.chdir("..") if __name__ == "__main__": init() hisat2-2.2.1/evaluation/tests/000077500000000000000000000000001370663763200162345ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CODIS/000077500000000000000000000000001370663763200170755ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CODIS/README000066400000000000000000000017161370663763200177620ustar00rootroot00000000000000To extract HLA alleles and variants ./hisatgenotype_extract_vars.py --base codis --inter-gap 30 --intra-gap 50 --min-var-freq 0.1 ./hisatgenotype_extract_vars.py --base hla --locus-list A --inter-gap 30 --intra-gap 50 --min-var-freq 0.1 To build genotype genome ./hisatgenotype_build_genome.py genome.fa genotype_genome -p 3 --verbose For DNA fingerprinting ./hisatgenotype_locus.py --verbose --base codis --locus-list D13S317 --fragment-len 200 --no-assembly --debug paired To run HISAT-genotype ./hisatgenotype_locus.py --base hla --locus-list A --simulate-interval 10 --perbase-errorrate 0.3 --perbase-snprate 0.1 --num-editdist 2 --debug paired for i in `ls -l ILMN/NA128*1.fq.gz | awk '{print $NF}' | cut -d'/' -f2 | cut -d'.' -f1`; do echo $i; ./hisatgenotype_locus.py --locus-list A --aligner-list hisat2.graph --num-editdist 2 --reads ILMN/$i.extracted.1.fq.gz,ILMN/$i.extracted.2.fq.gz --assembly-base assembly_graph_$i 2> $i.tmp; done hisat2-2.2.1/evaluation/tests/CODIS/genome.fa000077700000000000000000000000001370663763200244562../HLA_novel/genome.faustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CODIS/genome.fa.fai000077700000000000000000000000001370663763200257522../HLA_novel/genome.fa.faiustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CODIS/grch38000077700000000000000000000000001370663763200231542../HLA_novel/grch38ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CODIS/hisatgenotype_convert_codis.py000077700000000000000000000000001370663763200400372hisatgenotype_modules/hisatgenotype_convert_codis.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CODIS/hisatgenotype_extract_codis_data.py000077700000000000000000000000001370663763200420052hisatgenotype_modules/hisatgenotype_extract_codis_data.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CODIS/hisatgenotype_extract_vars.py000077700000000000000000000000001370663763200337752../../../hisatgenotype_extract_vars.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CODIS/hisatgenotype_locus.py000077700000000000000000000000001370663763200310352../../../hisatgenotype_locus.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CODIS/hisatgenotype_modules000077700000000000000000000000001370663763200306252../../../hisatgenotype_modulesustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CYP/000077500000000000000000000000001370663763200166675ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CYP/hisatgenotype_extract_vars.py000077700000000000000000000000001370663763200335672../../../hisatgenotype_extract_vars.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/CYP/hisatgenotype_locus.py000077700000000000000000000000001370663763200306272../../../hisatgenotype_locus.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/000077500000000000000000000000001370663763200200435ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/ILMN_StrandSeq/000077500000000000000000000000001370663763200225665ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/ILMN_StrandSeq/README000077700000000000000000000000001370663763200311432../ILMN_StrandSeq_original/READMEustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/ILMN_StrandSeq/SraRunInfo.txt000077700000000000000000000000001370663763200347612../ILMN_StrandSeq_original/SraRunInfo.txtustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/ILMN_StrandSeq_original/000077500000000000000000000000001370663763200244525ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/ILMN_StrandSeq_original/README000066400000000000000000000005611370663763200253340ustar00rootroot00000000000000Download SRA reads in the FASTQ format for i in `awk '{if(NR != 1) print $4}' SraRunInfo.txt`; do fastq-dump --gzip $i; done for i in `ls *.gz`; do echo $i; ../../../../hisat2 --no-hd -x ../hla.graph -U $i --max-altstried 64 2> /dev/null | grep "A\*B" >> temp; done scripts/get_haplotype_ILMN_StrandSeq.py ILMN_StrandSeq/SraRunInfo.txt ILMN_StrandSeq/temp hisat2-2.2.1/evaluation/tests/HLA_novel/ILMN_StrandSeq_original/SraRunInfo.txt000066400000000000000000000722261370663763200272520ustar00rootroot00000000000000Run spots bases download_path SampleName Sex ERR1429624 454580 23183580 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429624 NA12892 female ERR1429625 1164337 59381187 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429625 NA12892 female ERR1429626 446404 22766604 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429626 NA12892 female ERR1429627 1178769 60117219 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429627 NA12892 female ERR1429628 422539 21549489 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429628 NA12892 female ERR1429629 1158071 59061621 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429629 NA12892 female ERR1429630 1033423 52704573 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429630 NA12892 female ERR1429631 661547 33738897 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429631 NA12892 female ERR1429632 764557 38992407 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429632 NA12892 female ERR1429633 654774 33393474 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429633 NA12892 female ERR1429634 653700 33338700 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429634 NA12892 female ERR1429635 387115 19742865 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429635 NA12892 female ERR1429636 602942 30750042 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429636 NA12892 female ERR1429637 416442 21238542 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429637 NA12892 female ERR1429638 361789 18451239 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429638 NA12892 female ERR1429639 445019 22695969 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429639 NA12892 female ERR1429640 655382 33424482 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429640 NA12892 female ERR1429641 502098 25606998 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429641 NA12892 female ERR1429642 442140 22549140 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429642 NA12892 female ERR1429643 589718 30075618 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429643 NA12892 female ERR1429644 336049 17138499 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429644 NA12892 female ERR1429645 369398 18839298 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429645 NA12892 female ERR1429646 575850 29368350 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429646 NA12892 female ERR1429647 413988 21113388 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429647 NA12892 female ERR1429648 687172 35045772 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429648 NA12892 female ERR1429649 675486 34449786 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429649 NA12892 female ERR1429650 683774 34872474 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429650 NA12892 female ERR1429651 527860 26920860 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429651 NA12892 female ERR1429652 363994 18563694 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429652 NA12892 female ERR1429653 485829 24777279 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429653 NA12892 female ERR1429654 404689 20639139 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429654 NA12892 female ERR1429655 628956 32076756 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429655 NA12892 female ERR1429656 443843 22635993 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429656 NA12892 female ERR1429657 507127 25863477 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429657 NA12892 female ERR1429658 548507 27973857 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429658 NA12892 female ERR1429659 682499 34807449 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429659 NA12892 female ERR1429660 414572 21143172 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429660 NA12892 female ERR1429661 425025 21676275 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429661 NA12892 female ERR1429662 371171 18929721 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429662 NA12892 female ERR1429663 1092760 55730760 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429663 NA12892 female ERR1429664 651261 33214311 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429664 NA12892 female ERR1429665 643455 32816205 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429665 NA12892 female ERR1429666 657058 33509958 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429666 NA12892 female ERR1429667 527528 26903928 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429667 NA12892 female ERR1429668 941433 48013083 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429668 NA12892 female ERR1429669 635537 32412387 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429669 NA12892 female ERR1429670 493285 25157535 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429670 NA12892 female ERR1429671 1280258 65293158 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429671 NA12892 female ERR1429672 1765271 90028821 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429672 NA12892 female ERR1429673 936702 47771802 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429673 NA12892 female ERR1429674 876200 44686200 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429674 NA12892 female ERR1429675 1407029 71758479 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429675 NA12892 female ERR1429676 1703380 86872380 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429676 NA12892 female ERR1429677 1207785 61597035 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429677 NA12892 female ERR1429678 1061830 54153330 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429678 NA12892 female ERR1429679 1669149 85126599 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429679 NA12892 female ERR1429680 1476448 75298848 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429680 NA12892 female ERR1429681 1020002 52020102 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429681 NA12892 female ERR1429682 1356679 69190629 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429682 NA12892 female ERR1429683 915946 46713246 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429683 NA12892 female ERR1429684 1045332 53311932 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429684 NA12892 female ERR1429685 1525162 77783262 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429685 NA12892 female ERR1429686 471194 24030894 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429686 NA12892 female ERR1429687 1491856 76084656 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429687 NA12892 female ERR1429688 824709 42060159 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429688 NA12892 female ERR1429689 1112300 56727300 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429689 NA12892 female ERR1429690 874610 44605110 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429690 NA12892 female ERR1429691 697710 35583210 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429691 NA12892 female ERR1429692 582636 29714436 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429692 NA12892 female ERR1429693 761870 38855370 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429693 NA12892 female ERR1429694 616775 31455525 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429694 NA12892 female ERR1429695 680648 34713048 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429695 NA12892 female ERR1429696 557253 28419903 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429696 NA12892 female ERR1429697 456232 23267832 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429697 NA12892 female ERR1429698 1494318 76210218 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429698 NA12892 female ERR1429699 738330 37654830 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429699 NA12892 female ERR1429700 1328177 67737027 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429700 NA12892 female ERR1429701 741532 37818132 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429701 NA12892 female ERR1429702 1061965 54160215 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429702 NA12892 female ERR1429703 378981 19328031 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429703 NA12892 female ERR1429704 1117620 56998620 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429704 NA12892 female ERR1429705 1124119 57330069 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429705 NA12892 female ERR1429706 1339802 68329902 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429706 NA12892 female ERR1429707 2385699 121670649 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429707 NA12892 female ERR1429708 833880 42527880 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429708 NA12892 female ERR1429709 745854 38038554 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429709 NA12892 female ERR1429710 753263 38416413 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429710 NA12892 female ERR1429711 535518 27311418 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429711 NA12892 female ERR1429712 1299967 66298317 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429712 NA12892 female ERR1429713 899645 45881895 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429713 NA12892 female ERR1429714 739975 37738725 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429714 NA12892 female ERR1429715 1477561 75355611 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429715 NA12892 female ERR1429716 1411260 71974260 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429716 NA12892 female ERR1429717 1461893 74556543 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429717 NA12892 female ERR1429718 1167396 59537196 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429718 NA12892 female ERR1429719 455539 23232489 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429719 NA12892 female ERR1429720 691972 35290572 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429720 NA12892 female ERR1429721 1767814 90158514 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429721 NA12892 female ERR1429722 1089325 55555575 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429722 NA12892 female ERR1429723 1241483 63315633 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429723 NA12892 female ERR1429524 714521 36440571 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429524 NA12892 female ERR1429525 512294 26126994 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429525 NA12892 female ERR1429526 600138 30607038 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429526 NA12892 female ERR1429527 1026295 52341045 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429527 NA12892 female ERR1429528 485027 24736377 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429528 NA12892 female ERR1429529 610595 31140345 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429529 NA12892 female ERR1429530 634235 32345985 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429530 NA12892 female ERR1429531 588103 29993253 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429531 NA12892 female ERR1429532 2161798 110251698 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429532 NA12892 female ERR1429533 2008655 102441405 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429533 NA12892 female ERR1429534 2299802 117289902 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429534 NA12892 female ERR1429535 1667191 85026741 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429535 NA12892 female ERR1429536 1515614 77296314 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429536 NA12892 female ERR1429537 1800878 91844778 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429537 NA12892 female ERR1429538 1883585 96062835 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429538 NA12892 female ERR1429539 1390662 70923762 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429539 NA12892 female ERR1429540 1636297 83451147 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429540 NA12892 female ERR1429541 1547471 78921021 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429541 NA12892 female ERR1429542 1306043 66608193 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429542 NA12892 female ERR1429543 1545983 78845133 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429543 NA12892 female ERR1429544 1346888 68691288 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429544 NA12892 female ERR1429545 1672037 85273887 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429545 NA12892 female ERR1429546 1182992 60332592 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429546 NA12892 female ERR1429547 1110020 56611020 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429547 NA12892 female ERR1429548 673901 34368951 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429548 NA12892 female ERR1429549 418115 21323865 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429549 NA12892 female ERR1429550 700872 35744472 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429550 NA12892 female ERR1429551 839395 42809145 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429551 NA12892 female ERR1429552 574017 29274867 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429552 NA12892 female ERR1429553 1022930 52169430 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429553 NA12892 female ERR1429554 770504 39295704 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429554 NA12892 female ERR1429555 589137 30045987 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429555 NA12892 female ERR1429556 621838 31713738 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429556 NA12892 female ERR1429557 573305 29238555 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429557 NA12892 female ERR1429558 711033 36262683 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429558 NA12892 female ERR1429559 664808 33905208 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429559 NA12892 female ERR1429560 531349 27098799 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429560 NA12892 female ERR1429561 892253 45504903 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429561 NA12892 female ERR1429562 687951 35085501 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429562 NA12892 female ERR1429563 624558 31852458 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429563 NA12892 female ERR1429564 781236 39843036 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429564 NA12892 female ERR1429565 1372521 69998571 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429565 NA12892 female ERR1429566 1218765 62157015 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429566 NA12892 female ERR1429567 675274 34438974 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429567 NA12892 female ERR1429568 560720 28596720 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429568 NA12892 female ERR1429569 664298 33879198 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429569 NA12892 female ERR1429570 572222 29183322 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429570 NA12892 female ERR1429571 1350359 68868309 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429571 NA12892 female ERR1429572 1309402 66779502 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429572 NA12892 female ERR1429573 1088377 55507227 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429573 NA12892 female ERR1429574 1047059 53400009 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429574 NA12892 female ERR1429575 529587 27008937 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429575 NA12892 female ERR1429576 1169107 59624457 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429576 NA12892 female ERR1429577 699355 35667105 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429577 NA12892 female ERR1429578 698973 35647623 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429578 NA12892 female ERR1429579 567632 28949232 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429579 NA12892 female ERR1429580 880930 44927430 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429580 NA12892 female ERR1429581 682353 34800003 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429581 NA12892 female ERR1429582 800198 40810098 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429582 NA12892 female ERR1429583 564119 28770069 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429583 NA12892 female ERR1429584 765306 39030606 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429584 NA12892 female ERR1429585 690339 35207289 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429585 NA12892 female ERR1429586 559619 28540569 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429586 NA12892 female ERR1429587 922910 47068410 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429587 NA12892 female ERR1429588 467525 23843775 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429588 NA12892 female ERR1429589 638329 32554779 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429589 NA12892 female ERR1429590 690814 35231514 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429590 NA12892 female ERR1429591 477979 24376929 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429591 NA12892 female ERR1429592 560344 28577544 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429592 NA12892 female ERR1429593 784251 39996801 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429593 NA12892 female ERR1429594 747178 38106078 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429594 NA12892 female ERR1429595 827661 42210711 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429595 NA12892 female ERR1429596 950714 48486414 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429596 NA12892 female ERR1429597 5235515 267011265 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429597 NA12892 female ERR1429598 939966 47938266 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429598 NA12892 female ERR1429599 546226 27857526 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429599 NA12892 female ERR1429600 463247 23625597 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429600 NA12892 female ERR1429601 1024726 52261026 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429601 NA12892 female ERR1429602 1465503 74740653 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429602 NA12892 female ERR1429603 1174942 59922042 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429603 NA12892 female ERR1429604 921930 47018430 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429604 NA12892 female ERR1429605 1074963 54823113 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429605 NA12892 female ERR1429606 970622 49501722 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429606 NA12892 female ERR1429607 796770 40635270 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429607 NA12892 female ERR1429608 884429 45105879 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429608 NA12892 female ERR1429609 1034885 52779135 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429609 NA12892 female ERR1429610 579267 29542617 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429610 NA12892 female ERR1429611 497651 25380201 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429611 NA12892 female ERR1429612 538377 27457227 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429612 NA12892 female ERR1429613 1005381 51274431 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429613 NA12892 female ERR1429614 987462 50360562 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429614 NA12892 female ERR1429615 456782 23295882 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429615 NA12892 female ERR1429616 674193 34383843 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429616 NA12892 female ERR1429617 953023 48604173 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429617 NA12892 female ERR1429618 617758 31505658 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429618 NA12892 female ERR1429619 598055 30500805 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429619 NA12892 female ERR1429620 597972 30496572 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429620 NA12892 female ERR1429621 456116 23261916 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429621 NA12892 female ERR1429622 997159 50855109 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429622 NA12892 female ERR1429623 505516 25781316 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429623 NA12892 female ERR1429043 605410 30875910 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429043 NA12878 female ERR1429044 464531 23691081 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429044 NA12878 female ERR1429045 548098 27952998 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429045 NA12878 female ERR1429046 516069 26319519 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429046 NA12878 female ERR1429047 494917 25240767 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429047 NA12878 female ERR1429048 324250 16536750 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429048 NA12878 female ERR1429049 377454 19250154 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429049 NA12878 female ERR1429050 439003 22389153 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429050 NA12878 female ERR1429051 527178 26886078 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429051 NA12878 female ERR1429052 445298 22710198 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429052 NA12878 female ERR1429053 466364 23784564 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429053 NA12878 female ERR1429054 417270 21280770 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429054 NA12878 female ERR1429055 408573 20837223 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429055 NA12878 female ERR1429056 350813 17891463 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429056 NA12878 female ERR1429057 532542 27159642 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429057 NA12878 female ERR1429058 547294 27911994 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429058 NA12878 female ERR1429059 289030 14740530 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429059 NA12878 female ERR1429060 471157 24029007 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429060 NA12878 female ERR1429061 493958 25191858 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429061 NA12878 female ERR1429062 362477 18486327 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429062 NA12878 female ERR1429063 267605 13647855 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429063 NA12878 female ERR1429064 277873 14171523 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429064 NA12878 female ERR1429065 384164 19592364 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429065 NA12878 female ERR1429066 360900 18405900 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429066 NA12878 female ERR1429067 470448 23992848 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429067 NA12878 female ERR1429068 529681 27013731 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429068 NA12878 female ERR1429069 352233 17963883 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429069 NA12878 female ERR1429070 571067 29124417 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429070 NA12878 female ERR1429071 504820 25745820 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429071 NA12878 female ERR1429072 309267 15772617 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429072 NA12878 female ERR1429073 425635 21707385 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429073 NA12878 female ERR1429074 454845 23197095 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429074 NA12878 female ERR1429075 202007 10302357 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429075 NA12878 female ERR1429076 612703 31247853 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429076 NA12878 female ERR1429077 590290 30104790 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429077 NA12878 female ERR1429078 447433 22819083 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429078 NA12878 female ERR1429079 338973 17287623 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429079 NA12878 female ERR1429080 487364 24855564 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429080 NA12878 female ERR1429081 545881 27839931 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429081 NA12878 female ERR1429082 522177 26631027 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429082 NA12878 female ERR1429083 475914 24271614 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429083 NA12878 female ERR1429084 587003 29937153 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429084 NA12878 female ERR1429085 587009 29937459 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429085 NA12878 female ERR1429086 547645 27929895 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429086 NA12878 female ERR1429087 459050 23411550 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429087 NA12878 female ERR1429088 270489 13794939 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429088 NA12878 female ERR1429089 547181 27906231 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429089 NA12878 female ERR1429282 478706 24414006 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429282 NA12891 male ERR1429283 1604643 81836793 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429283 NA12891 male ERR1429284 1102261 56215311 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429284 NA12891 male ERR1429285 1337037 68188887 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429285 NA12891 male ERR1429286 845172 43103772 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429286 NA12891 male ERR1429287 1255934 64052634 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429287 NA12891 male ERR1429288 781311 39846861 https://sra-download.ncbi.nlm.nih.gov/srapub/ERR1429288 NA12891 male ERR1429289 784261 39997311 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femalehisat2-2.2.1/evaluation/tests/HLA_novel/README000066400000000000000000000024071370663763200207260ustar00rootroot00000000000000 hisatgenotype_locus.py --aligner-list hisat2.graph --base hla --locus-list A --num-editdist 2 --assembly -1 ILMN/NA12892.extracted.1.fq.gz -2 ILMN/NA12892.extracted.2.fq.gz for i in `ls -l CP_80/LP*1.fq.gz | awk '{print $NF}' | cut -d'/' -f2 | cut -d'.' -f1`; do echo $i; ./hisatgenotype_locus.py --locus-list A --num-editdist 2 --assembly --reads CP_80/$i.extracted.1.fq.gz,CP_80/$i.extracted.2.fq.gz --assembly-base assembly_graph_$i 2> $i.tmp; done hisatgenotype_locus_samples.py -p 3 -v --max-sample 400 --region-list hla.A --read-dir CP_400 --out-dir test_A > cp_400_A.txt hisatgenotype_scripts/compare_HLA_Omixon.py cp_400_A.txt ../CAAPA/omixon_caapa_hla.txt hisatgenotype_locus.py --aligner-list hisat2.graph --locus-list A --num-editdist 2 --assembly -1 CP_80/LP6005106-DNA_H02.extracted.fq.1.gz -2 CP_80/LP6005106-DNA_H02.extracted.fq.2.gz --display-alleles A*23:17,A*02:02:01:01 hisatgenotype_extract_reads.py --base-fname genotype_genome --reference-type genome --read-dir ILMN_12062016 --out-dir ILMN hisat2 --no-hd -x genotype_genome -c CAGCTGGGATGTGGAGTGGTGTGAGGAGTGGCCACAGGGGAGCAGAGGAGGTGGCAGAAGCCGGAGGTAAAGGTGTCTTAAA On MARCC - https://www.marcc.jhu.edu/getting-started/basic sbatch script-name squeue -u dkim136@jhu.edu scancel job-id sinfo hisat2-2.2.1/evaluation/tests/HLA_novel/hisatgenotype.py000077700000000000000000000000001370663763200273712../../../hisatgenotype.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/hisatgenotype_build_genome.py000077700000000000000000000000001370663763200346132../../../hisatgenotype_build_genome.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/hisatgenotype_extract_vars.py000077700000000000000000000000001370663763200347432../../../hisatgenotype_extract_vars.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/hisatgenotype_locus.py000077700000000000000000000000001370663763200320032../../../hisatgenotype_locus.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/hisatgenotype_locus_prev.py000077500000000000000000004251021370663763200255500ustar00rootroot00000000000000#!/usr/bin/env python # # Copyright 2015, Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # import sys, os, subprocess, re import inspect, random import math from datetime import datetime, date, time from argparse import ArgumentParser, FileType from copy import deepcopy from hisatgenotype_modules import typing_common, Gene_typing, assembly_graph """ Align reads, and sort the alignments into a BAM file """ def align_reads(ex_path, aligner, simulation, base_fname, index_type, read_fname, fastq, threads, out_fname, verbose): if aligner == "hisat2": hisat2 = os.path.join(ex_path, "hisat2") # DK - debugging purpose # aligner_cmd = [hisat2, "--mm"] aligner_cmd = ["/Users/infphilo/work/hisat2/hisat2", "--mm"] if not simulation: aligner_cmd += ["--no-unal"] DNA = True if DNA: aligner_cmd += ["--no-spliced-alignment"] # no spliced alignment aligner_cmd += ["-X", "1000"] # max fragment length if index_type == "linear": aligner_cmd += ["-k", "10"] else: aligner_cmd += ["--max-altstried", "64"] # DK - debugging purposes aligner_cmd += ["--haplotype"] if base_fname == "codis": aligner_cmd += ["--enable-codis"] aligner_cmd += ["-x", "%s.%s" % (base_fname, index_type)] elif aligner == "bowtie2": aligner_cmd = [aligner, "--no-unal", "-k", "10", "-x", base_fname] else: assert False assert len(read_fname) in [1,2] aligner_cmd += ["-p", str(threads)] if not fastq: aligner_cmd += ["-f"] if len(read_fname) == 1: aligner_cmd += ["-U", read_fname[0]] else: aligner_cmd += ["-1", "%s" % read_fname[0], "-2", "%s" % read_fname[1]] if verbose >= 1: print >> sys.stderr, ' '.join(aligner_cmd) align_proc = subprocess.Popen(aligner_cmd, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) sambam_cmd = ["samtools", "view", "-bS", "-"] sambam_proc = subprocess.Popen(sambam_cmd, stdin=align_proc.stdout, stdout=open(out_fname + ".unsorted", 'w'), stderr=open("/dev/null", 'w')) sambam_proc.communicate() if index_type == "graph": bamsort_cmd = ["samtools", "sort", out_fname + ".unsorted", "-o", out_fname] bamsort_proc = subprocess.Popen(bamsort_cmd, stderr=open("/dev/null", 'w')) bamsort_proc.communicate() bamindex_cmd = ["samtools", "index", out_fname] bamindex_proc = subprocess.Popen(bamindex_cmd, stderr=open("/dev/null", 'w')) bamindex_proc.communicate() os.system("rm %s" % (out_fname + ".unsorted")) """ """ def normalize(prob): total = sum(prob.values()) for allele, mass in prob.items(): prob[allele] = mass / total """ """ def prob_diff(prob1, prob2): diff = 0.0 for allele in prob1.keys(): if allele in prob2: diff += abs(prob1[allele] - prob2[allele]) else: diff += prob1[allele] return diff """ """ def Gene_prob_cmp(a, b): if a[1] != b[1]: if a[1] < b[1]: return 1 else: return -1 assert a[0] != b[0] if a[0] < b[0]: return -1 else: return 1 """ """ def single_abundance(Gene_cmpt, Gene_length): def normalize2(prob, length): total = 0 for allele, mass in prob.items(): assert allele in length total += (mass / length[allele]) for allele, mass in prob.items(): assert allele in length prob[allele] = mass / length[allele] / total Gene_prob, Gene_prob_next = {}, {} for cmpt, count in Gene_cmpt.items(): alleles = cmpt.split('-') for allele in alleles: if allele not in Gene_prob: Gene_prob[allele] = 0.0 Gene_prob[allele] += (float(count) / len(alleles)) normalize(Gene_prob) def next_prob(Gene_cmpt, Gene_prob, Gene_length): Gene_prob_next = {} for cmpt, count in Gene_cmpt.items(): alleles = cmpt.split('-') alleles_prob = 0.0 for allele in alleles: if allele not in Gene_prob: continue alleles_prob += Gene_prob[allele] if alleles_prob <= 0.0: continue for allele in alleles: if allele not in Gene_prob: continue if allele not in Gene_prob_next: Gene_prob_next[allele] = 0.0 Gene_prob_next[allele] += (float(count) * Gene_prob[allele] / alleles_prob) normalize(Gene_prob_next) return Gene_prob_next fast_EM = True diff, iter = 1.0, 0 while diff > 0.0001 and iter < 1000: Gene_prob_next = next_prob(Gene_cmpt, Gene_prob, Gene_length) if fast_EM: # Accelerated version of EM - SQUAREM iteration # Varadhan, R. & Roland, C. Scand. J. Stat. 35, 335-353 (2008) # Also, this algorithm is used in Sailfish - http://www.nature.com/nbt/journal/v32/n5/full/nbt.2862.html Gene_prob_next2 = next_prob(Gene_cmpt, Gene_prob_next, Gene_length) sum_squared_r, sum_squared_v = 0.0, 0.0 p_r, p_v = {}, {} for a in Gene_prob.keys(): p_r[a] = Gene_prob_next[a] - Gene_prob[a] sum_squared_r += (p_r[a] * p_r[a]) p_v[a] = Gene_prob_next2[a] - Gene_prob_next[a] - p_r[a] sum_squared_v += (p_v[a] * p_v[a]) if sum_squared_v > 0.0: gamma = -math.sqrt(sum_squared_r / sum_squared_v) for a in Gene_prob.keys(): Gene_prob_next2[a] = max(0.0, Gene_prob[a] - 2 * gamma * p_r[a] + gamma * gamma * p_v[a]); Gene_prob_next = next_prob(Gene_cmpt, Gene_prob_next2, Gene_length) diff = prob_diff(Gene_prob, Gene_prob_next) Gene_prob = Gene_prob_next # Accelerate convergence if iter >= 10: Gene_prob2 = {} avg_prob = sum(Gene_prob.values()) / len(Gene_prob) for allele, prob in Gene_prob.items(): if prob >= 0.005 or prob > avg_prob: Gene_prob2[allele] = prob Gene_prob = Gene_prob2 # DK - debugging purposes if iter % 10 == 0 and False: print "iter", iter for allele, prob in Gene_prob.items(): if prob >= 0.01: print >> sys.stderr, "\t", iter, allele, prob, str(datetime.now()) iter += 1 """ for allele, prob in Gene_prob.items(): allele_len = Gene_length[allele] Gene_prob[allele] /= float(allele_len) """ # normalize(Gene_prob) normalize2(Gene_prob, Gene_length) Gene_prob = [[allele, prob] for allele, prob in Gene_prob.items()] Gene_prob = sorted(Gene_prob, cmp=Gene_prob_cmp) return Gene_prob """ """ def lower_bound(Var_list, pos): low, high = 0, len(Var_list) while low < high: m = (low + high) / 2 m_pos = Var_list[m][0] if m_pos < pos: low = m + 1 elif m_pos > pos: high = m else: assert m_pos == pos while m > 0: if Var_list[m-1][0] < pos: break m -= 1 return m return low """ var: ['single', 3300, 'G'] exons: [[301, 373], [504, 822], [1084, 1417], [2019, 2301], [2404, 2520], [2965, 2997], [3140, 3187], [3357, 3361]] """ def var_in_exon(var, exons): exonic = False var_type, var_left, var_data = var var_right = var_left if var_type == "deletion": var_right = var_left + int(var_data) - 1 for exon_left, exon_right in exons: if var_left >= exon_left and var_right <= exon_right: return True return False """ Report variant IDs whose var is within exonic regions """ def get_exonic_vars(Vars, exons): vars = set() for var_id, var in Vars.items(): var_type, var_left, var_data = var var_right = var_left if var_type == "deletion": var_right = var_left + int(var_data) - 1 for exon_left, exon_right in exons: if var_left >= exon_left and var_right <= exon_right: vars.add(var_id) return vars """ Get representative alleles among those that share the same exonic sequences """ def get_rep_alleles(Links, exon_vars): allele_vars = {} for var, alleles in Links.items(): if var not in exon_vars: continue for allele in alleles: if allele not in allele_vars: allele_vars[allele] = set() allele_vars[allele].add(var) allele_groups = {} for allele, vars in allele_vars.items(): vars = '-'.join(vars) if vars not in allele_groups: allele_groups[vars] = [] allele_groups[vars].append(allele) allele_reps = {} # allele representatives allele_rep_groups = {} # allele groups by allele representatives for allele_members in allele_groups.values(): assert len(allele_members) > 0 allele_rep = allele_members[0] allele_rep_groups[allele_rep] = allele_members for allele_member in allele_members: assert allele_member not in allele_reps allele_reps[allele_member] = allele_rep return allele_reps, allele_rep_groups """ Identify alternative alignments """ def get_alternatives(ref_seq, Vars, Var_list, verbose): # Check deletions' alternatives def get_alternatives_recur(ref_seq, Vars, Var_list, Alts, var_id, del_len, other_del_len, left, alt_list, var_j, latest_pos, debug = False): def add_alt(Alts, alt_list, var_id, j_id): if j_id.isdigit(): if var_id not in Alts: Alts[var_id] = [["1"]] else: if Alts[var_id][-1][-1].isdigit(): Alts[var_id][-1][-1] = str(int(Alts[var_id][-1][-1]) + 1) else: Alts[var_id][-1].append("1") else: if var_id not in Alts: Alts[var_id] = [[j_id]] else: if Alts[var_id][-1][-1].isdigit(): Alts[var_id][-1][-1] = j_id else: Alts[var_id][-1].append(j_id) Alts[var_id][-1].append("0") if not j_id.isdigit(): alt_list.append(j_id) alts = '-'.join(alt_list) if alts not in Alts: Alts[alts] = [[var_id]] else: Alts[alts].append([var_id]) if del_len == other_del_len: return var_type, var_pos, var_data = Vars[var_id] if left: # Look in left direction if var_j < 0: return j_pos, j_id = Var_list[var_j] alt_del = [] if var_id != j_id and j_pos < var_pos + del_len: prev_latest_pos = latest_pos # Check bases between SNPs while latest_pos - max(0, del_len - other_del_len) > 0: if ref_seq[latest_pos - 1] != ref_seq[latest_pos - 1 - del_len + other_del_len]: break latest_pos -= 1 add_alt(Alts, alt_list, var_id, str(latest_pos)) if latest_pos - 1 > j_pos: return j_type, _, j_data = Vars[j_id] if j_type == "deletion": j_del_len = int(j_data) if j_type == "single" and j_pos == latest_pos - 1: j_cmp_pos = j_pos - del_len + other_del_len if debug: print Vars[j_id] print j_pos, ref_seq[j_pos] print j_cmp_pos, ref_seq[j_cmp_pos] if j_data == ref_seq[j_cmp_pos]: add_alt(Alts, alt_list, var_id, j_id) latest_pos = j_pos elif j_type == "deletion" and j_pos + j_del_len - 1 == prev_latest_pos - 1: alt_list2 = alt_list[:] + [j_id] latest_pos2 = j_pos alt_del = [alt_list2, latest_pos2] get_alternatives_recur(ref_seq, Vars, Var_list, Alts, var_id, del_len, other_del_len, left, alt_list, var_j - 1, latest_pos, debug) if alt_del: alt_list2, latest_pos2 = alt_del if var_id not in Alts: Alts[var_id] = [alt_list2[:]] else: Alts[var_id].append(alt_list2[:]) alt_idx = len(Alts[var_id]) - 1 get_alternatives_recur(ref_seq, Vars, Var_list, Alts, var_id, del_len, other_del_len + j_del_len, left, alt_list2, var_j - 1, latest_pos2, debug) # Remove this Deletion if not supported by additional bases? assert alt_idx < len(Alts[var_id]) if Alts[var_id][alt_idx][-1] == j_id: Alts[var_id] = Alts[var_id][:alt_idx] + Alts[var_id][alt_idx+1:] else: # Look in right direction if var_j >= len(Var_list): return j_pos, j_id = Var_list[var_j] alt_del = [] if var_id != j_id and j_pos >= var_pos: # Check bases between SNPs prev_latest_pos = latest_pos while latest_pos + 1 + max(0, del_len - other_del_len) < len(ref_seq): if ref_seq[latest_pos + 1] != ref_seq[latest_pos + 1 + del_len - other_del_len]: break # DK - debugging purposes if debug: pos2_ = latest_pos + 1 + del_len - other_del_len print "DK: latest_pos:", latest_pos + 1, pos2_ print "DK: var_pos:", var_pos, "del_len:", del_len, "other_del_len:", other_del_len print "DK:", ref_seq[latest_pos + 1], ref_seq[pos2_] latest_pos += 1 add_alt(Alts, alt_list, var_id, str(latest_pos)) if latest_pos + 1 < j_pos: return j_type, _, j_data = Vars[j_id] if j_type == "single" and j_pos == latest_pos + 1: j_cmp_pos = j_pos + del_len - other_del_len if debug: print Vars[j_id] print j_pos, ref_seq[j_pos] print j_cmp_pos, ref_seq[j_cmp_pos] if j_data == ref_seq[j_cmp_pos]: add_alt(Alts, alt_list, var_id, j_id) latest_pos = j_pos elif j_type == "deletion" and j_pos == prev_latest_pos + 1: j_del_len = int(j_data) alt_list2 = alt_list[:] + [j_id] latest_pos2 = j_pos + j_del_len - 1 alt_del = [alt_list2, latest_pos2] get_alternatives_recur(ref_seq, Vars, Var_list, Alts, var_id, del_len, other_del_len, left, alt_list, var_j + 1, latest_pos, debug) if alt_del: alt_list2, latest_pos2 = alt_del if var_id not in Alts: Alts[var_id] = [alt_list2[:]] else: Alts[var_id].append(alt_list2[:]) alt_idx = len(Alts[var_id]) - 1 get_alternatives_recur(ref_seq, Vars, Var_list, Alts, var_id, del_len, other_del_len + j_del_len, left, alt_list2, var_j + 1, latest_pos2, debug) # Remove this Deletion if not supported by additional bases? assert alt_idx < len(Alts[var_id]) if Alts[var_id][alt_idx][-1] == j_id: Alts[var_id] = Alts[var_id][:alt_idx] + Alts[var_id][alt_idx+1:] # Check deletions' alternatives Alts_left, Alts_right = {}, {} for var_i, var_id in Var_list: var_type, var_pos, var_data = var = Vars[var_id] if var_type != "deletion" or var_pos == 0: continue del_len = int(var_data) if var_pos + del_len >= len(ref_seq): assert var_pos + del_len == len(ref_seq) continue debug = (var_id == "hv454a") if debug: print Vars[var_id] alt_list = [] var_j = lower_bound(Var_list, var_pos + del_len - 1) latest_pos = var_pos + del_len if var_j < len(Var_list): get_alternatives_recur(ref_seq, Vars, Var_list, Alts_left, var_id, del_len, 0, True, # left alt_list, var_j, latest_pos, debug) alt_list = [] var_j = lower_bound(Var_list, var_pos) latest_pos = var_pos - 1 assert var_j >= 0 get_alternatives_recur(ref_seq, Vars, Var_list, Alts_right, var_id, del_len, 0, False, # right alt_list, var_j, latest_pos, debug) if debug: print "DK :-)" sys.exit(1) def assert_print_alts(Alts, dir): def get_seq_pos(alt_list): seq = "" seq_left, seq_right = -1, -1 for i in range(len(alt_list)): alt = alt_list[i] if alt.isdigit(): assert i + 1 == len(alt_list) if dir == "left": if i == 0: seq = alt break alt = int(alt) seq = ref_seq[seq_left-alt+1:seq_left+1] + seq seq_left -= alt else: alt = int(alt) seq += ref_seq[seq_right:seq_right+alt] seq_right += alt break var_type, var_pos, var_data = Vars[alt] if dir == "left" and var_type == "deletion": var_pos = var_pos + int(var_data) - 1 if i == 0: if dir == "left": seq_left, seq_right = var_pos, var_pos else: seq_left, seq_right = var_pos, var_pos if dir == "left": assert seq_left >= var_pos if i > 0: seq = ref_seq[var_pos+1:seq_left+1] + seq if var_type == "single": seq = var_data + seq seq_left = var_pos - 1 elif var_type == "deletion": seq_left = var_pos - int(var_data) else: assert var_type == "insertion" seq = var_data + seq else: assert seq_right <= var_pos if i > 0: seq += ref_seq[seq_right:var_pos] if var_type == "single": seq += var_data seq_right = var_pos + 1 elif var_type == "deletion": seq_right = var_pos + int(var_data) else: assert var_type == "insertion" seq += var_data return seq, seq_left, seq_right for alt_list1, alt_list2 in Alts.items(): if verbose >= 2: print >> sys.stderr, "\t", dir, ":", alt_list1, alt_list2 out_str = "\t\t" alt_list1 = alt_list1.split('-') for i in range(len(alt_list1)): alt = alt_list1[i] var_type, var_pos, var_data = Vars[alt] out_str += ("%s-%d-%s" % (var_type, var_pos, var_data)) if i + 1 < len(alt_list1): out_str += " " for i in range(len(alt_list2)): alt_list3 = alt_list2[i] out_str += "\t[" for j in range(len(alt_list3)): alt = alt_list3[j] if alt.isdigit(): out_str += alt else: var_type, var_pos, var_data = Vars[alt] out_str += ("%s-%d-%s" % (var_type, var_pos, var_data)) if j + 1 < len(alt_list3): out_str += ", " out_str += "]" if verbose >= 2: print >> sys.stderr, out_str for i in range(len(alt_list2)): alt_list3 = alt_list2[i] seq1, seq1_left, seq1_right = get_seq_pos(alt_list1) seq2, seq2_left, seq2_right = get_seq_pos(alt_list3) if seq1.isdigit(): assert not seq2.isdigit() seq1_left, seq1_right = seq2_right - int(seq1), seq2_right seq1 = ref_seq[seq1_left+1:seq1_right+1] elif seq2.isdigit(): seq2_left, seq2_right = seq1_right - int(seq2), seq1_right seq2 = ref_seq[seq2_left+1:seq2_right+1] if dir == "left": if seq1_right < seq2_right: seq1 += ref_seq[seq1_right+1:seq2_right+1] elif seq2_right < seq1_right: seq2 += ref_seq[seq2_right+1:seq1_right+1] else: if seq1_left < seq2_left: seq2 = ref_seq[seq1_left:seq2_left] + seq2 elif seq2_left < seq1_left: seq1 = ref_seq[seq2_left:seq1_left] + seq1 seq1_len, seq2_len = len(seq1), len(seq2) if seq1_len != seq2_len: len_diff = abs(seq1_len - seq2_len) if dir == "left": if seq1_len < seq2_len: seq1 = ref_seq[seq1_left-len_diff+1:seq1_left+1] + seq1 else: seq2 = ref_seq[seq2_left-len_diff+1:seq2_left+1] + seq2 else: if seq1_len < seq2_len: seq1 += ref_seq[seq1_right:seq1_right+len_diff] else: seq2 += ref_seq[seq2_right:seq2_right+len_diff] if verbose >= 3: print >> sys.stderr, "\t\t", alt_list1, alt_list3 print >> sys.stderr, "\t\t\t", seq1, seq1_left, seq1_right print >> sys.stderr, "\t\t\t", seq2, seq2_left, seq2_right assert seq1 == seq2 assert_print_alts(Alts_left, "left") assert_print_alts(Alts_right, "right") return Alts_left, Alts_right """ Identify ambigious differences that may account for other alleles, given a list of differences (cmp_list) between a read and a potential allele """ def identify_ambigious_diffs(Vars, Alts_left, Alts_right, cmp_list, verbose, debug = False): cmp_left, cmp_right = 0, len(cmp_list) - 1 i = 0 while i < len(cmp_list): cmp_i = cmp_list[i] type, pos, length = cmp_i[:3] # Check alternative alignments if type in ["mismatch", "deletion"]: var_id = cmp_i[3] if var_id == "unknown": i += 1 continue # Left direction id_str = var_id total_del_len = length if type == "deletion" else 0 for j in reversed(range(0, i)): cmp_j = cmp_list[j] j_type, j_pos, j_len = cmp_j[:3] if j_type != "match": if len(cmp_j) < 4: continue j_var_id = cmp_j[3] id_str += ("-%s" % j_var_id) if j_type == "deletion": total_del_len += j_len last_type, last_pos, last_len = cmp_list[0][:3] assert last_type in ["match", "mismatch"] left_pos = last_pos + total_del_len if id_str in Alts_left: orig_alts = id_str.split('-') alts_list = Alts_left[id_str] for alts in alts_list: if alts[-1].isdigit(): assert type == "deletion" assert len(orig_alts) == 1 alts_id_str = '-'.join(alts[:-1]) alt_left_pos = pos alt_total_del_len = 0 for alt in alts[:-1]: assert alt in Vars alt_type, alt_pos, alt_data = Vars[alt] alt_left_pos = alt_pos - 1 if alt_type == "deletion": alt_total_del_len += int(alt_data) alt_left_pos = alt_left_pos + alt_total_del_len - int(alts[-1]) + 1 else: alts_id_str = '-'.join(alts) assert alts_id_str in Alts_left for back_alts in Alts_left[alts_id_str]: back_id_str = '-'.join(back_alts) if back_id_str.find(id_str) != 0: continue assert len(orig_alts) < len(back_alts) assert back_alts[-1].isdigit() alt_left_pos = pos alt_total_del_len = 0 for alt in back_alts[:len(orig_alts) + 1]: if alt.isdigit(): alt_left_pos = alt_left_pos - int(alt) + 1 else: assert alt in Vars alt_type, alt_pos, alt_data = Vars[alt] alt_left_pos = alt_pos - 1 if alt_type == "deletion": alt_total_del_len += int(alt_data) alt_left_pos += alt_total_del_len if left_pos >= alt_left_pos: if verbose >= 2: print "LEFT:", cmp_list print "\t", type, "id_str:", id_str, "=>", alts_id_str, "=>", back_alts, "left_pos:", left_pos, "alt_left_pos:", alt_left_pos cmp_left = i + 1 break # DK - debugging purposes if debug: print "DK: var_id:", var_id print "DK: cmp_list:", cmp_list print "DK: cmp_right:", cmp_right # sys.exit(1) # Right direction if cmp_right + 1 == len(cmp_list): id_str = var_id total_del_len = length if type == "deletion" else 0 for j in range(i + 1, len(cmp_list)): cmp_j = cmp_list[j] j_type, j_pos, j_len = cmp_j[:3] if j_type != "match": if len(cmp_j) < 4: continue j_var_id = cmp_j[3] id_str += ("-%s" % j_var_id) if j_type == "deletion": total_del_len += j_len last_type, last_pos, last_len = cmp_list[-1][:3] assert last_type in ["match", "mismatch"] right_pos = last_pos + last_len - 1 - total_del_len # DK - debugging purposes if debug: print "DK: id_str:", id_str if id_str in Alts_right: orig_alts = id_str.split('-') alts_list = Alts_right[id_str] for alts in alts_list: if alts[-1].isdigit(): assert type == "deletion" assert len(orig_alts) == 1 alts_id_str = '-'.join(alts[:-1]) alt_right_pos = pos alt_total_del_len = 0 for alt in alts[:-1]: assert alt in Vars alt_type, alt_pos, alt_data = Vars[alt] alt_right_pos = alt_pos if alt_type == "single": alt_right_pos += 1 else: assert alt_type == "deletion" alt_del_len = int(alt_data) alt_right_pos += alt_del_len alt_total_del_len += alt_del_len alt_right_pos = alt_right_pos - alt_total_del_len + int(alts[-1]) - 1 else: alts_id_str = '-'.join(alts) assert alts_id_str in Alts_right for back_alts in Alts_right[alts_id_str]: back_id_str = '-'.join(back_alts) if back_id_str.find(id_str) != 0: continue assert len(orig_alts) < len(back_alts) assert back_alts[-1].isdigit() alt_right_pos = pos alt_total_del_len = 0 for alt in back_alts[:len(orig_alts) + 1]: if alt.isdigit(): alt_right_pos = alt_right_pos + int(alt) - 1 else: assert alt in Vars alt_type, alt_pos, alt_data = Vars[alt] alt_right_pos = alt_pos if alt_type == "single": alt_right_pos += 1 else: assert alt_type == "deletion" alt_del_len = int(alt_data) alt_right_pos += alt_del_len alt_total_del_len += alt_del_len alt_right_pos -= alt_total_del_len if right_pos <= alt_right_pos: if verbose >= 2: print "RIGHT:", cmp_list print "\t", type, "id_str:", id_str, "=>", alts_id_str, "right_pos:", right_pos, "alt_right_pos:", alt_right_pos cmp_right = i - 1 break i += 1 return cmp_left, cmp_right """ Example, gene_name, allele_name (input): A, A*32:01:01 allele (output): single-136-G-hv47,deletion-285-1-hv57, ... ,single-3473-T-hv1756,deletion-3495-1-hv1763,single-3613-C-hv1799 """ def get_allele(gene_name, allele_name, Vars, Var_list, Links): allele_haplotype = [] for _var_pos, _var_id in Var_list[gene_name]: if allele_name in Links[_var_id]: _var = Vars[gene_name][_var_id] allele_haplotype.append("%s-%d-%s-%s" % (_var[0], _var[1], _var[2], _var_id)) allele_haplotype = ','.join(allele_haplotype) return allele_haplotype """ HISAT-genotype's mpileup """ def get_mpileup(alignview_cmd, ref_seq, base_locus, vars, allow_discordant): ref_seq_len = len(ref_seq) mpileup = [] for i in range(ref_seq_len): mpileup.append([[], {}]) proc = subprocess.Popen(alignview_cmd, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) prev_pos = -1 cigar_re = re.compile('\d+\w') for line in proc.stdout: line = line.strip() cols = line.split() read_id, flag, _, pos, _, cigar_str = cols[:6] read_seq = cols[9] flag, pos = int(flag), int(pos) # Unalined? if flag & 0x4 != 0: continue pos -= (base_locus + 1) if pos < 0: continue # Concordantly mapped? if flag & 0x2 != 0: concordant = True else: concordant = False if not allow_discordant and not concordant: continue read_pos, left_pos = 0, pos right_pos = left_pos cigars = cigar_re.findall(cigar_str) cigars = [[cigar[-1], int(cigar[:-1])] for cigar in cigars] for i in range(len(cigars)): cigar_op, length = cigars[i] if cigar_op in "MD": for j in range(length): if cigar_op == 'M': read_nt = read_seq[read_pos + j] else: read_nt = 'D' if read_nt not in mpileup[right_pos + j][1]: mpileup[right_pos + j][1][read_nt] = 1 else: mpileup[right_pos + j][1][read_nt] += 1 if cigar_op in "MND": right_pos += length if cigar_op in "MIS": read_pos += length # Choose representative bases or 'D' for i in range(len(mpileup)): nt_dic = mpileup[i][1] num_nt = sum(nt_dic.values()) nt_set = [] if num_nt >= 20: for nt, count in nt_dic.items(): if nt not in "ACGT": continue if count >= num_nt * 0.2 or count >= 7: nt_set.append(nt) mpileup[i][0] = nt_set # Sort variants var_list = [[] for i in range(len(mpileup))] for var_id, value in vars.items(): var_type, var_pos, var_data = value assert var_pos < len(var_list) var_list[var_pos].append([var_id, var_type, var_data]) # Assign known or unknown variants skip_i, prev_del_var_id = -1, "" for i in range(len(mpileup)): nt_dic = mpileup[i][1] ref_nt = ref_seq[i] new_nt_dic = {} for nt, count in nt_dic.items(): var_id = "" if nt == 'D': if i <= skip_i: assert prev_del_var_id != "" var_id = prev_del_var_id else: for var_id_, var_type, var_data in var_list[i]: if var_type != "deletion": continue del_len = int(var_data) del_exist = True for j in range(i + 1, i + del_len): assert j < len(mpileup) nt_dic2 = mpileup[j][1] if 'D' not in nt_dic2: del_exist = False break if del_exist: var_id = var_id_ prev_del_var_id = var_id skip_i = i + del_len - 1 break elif nt != 'N' and nt != ref_nt: assert nt in "ACGT" id = "unknown" for var_id_, var_type, var_data in var_list[i]: if var_type != "single": continue if nt == var_data: var_id = var_id_ break new_nt_dic[nt] = [count, var_id] mpileup[i][1] = new_nt_dic return mpileup """ """ def error_correct(ref_seq, read_seq, read_pos, mpileup, Vars, Var_list, cmp_list, debug = False): if debug: print cmp_list print read_seq i = 0 while i < len(cmp_list): type, left, length = cmp_list[i][:3] assert length > 0 if type == "match": middle_cmp_list = [] last_j = 0 for j in range(length): read_bp, ref_bp = read_seq[read_pos + j], ref_seq[left + j] assert left + j < len(mpileup) nt_set = mpileup[left + j][0] if len(nt_set) > 0 and read_bp not in nt_set: read_bp = 'N' if len(nt_set) > 1 else nt_set[0] read_seq = read_seq[:read_pos + j] + read_bp + read_seq[read_pos + j + 1:] assert read_bp != ref_bp new_cmp = ["mismatch", left + j, 1, "unknown"] if read_bp != 'N': var_idx = lower_bound(Var_list, left + j) while var_idx < len(Var_list): var_pos, var_id = Var_list[var_idx] if var_pos > left + j: break if var_pos == left + j: var_type, _, var_data = Vars[var_id] if var_type == "single" and read_bp == var_data: new_cmp[3] = var_id break var_idx += 1 if j > last_j: middle_cmp_list.append(["match", left + last_j, j- last_j]) middle_cmp_list.append(new_cmp) last_j = j + 1 if last_j < length: middle_cmp_list.append(["match", left + last_j, length - last_j]) assert len(middle_cmp_list) > 0 cmp_list = cmp_list[:i] + middle_cmp_list + cmp_list[i+1:] i += (len(middle_cmp_list) - 1) else: assert type == "mismatch" read_bp, ref_bp = read_seq[read_pos], ref_seq[left] assert left < len(mpileup) nt_set = mpileup[left][0] if debug: print left, read_bp, ref_bp, mpileup[left] if len(nt_set) > 0 and read_bp not in nt_set: read_bp = 'N' if len(nt_set) > 1 else nt_set[0] read_seq = read_seq[:read_pos] + read_bp + read_seq[read_pos+1:] if read_bp == 'N': cmp_list[i][3] = "unknown" elif read_bp == ref_bp: cmp_list[i] = ["match", left, 1] else: cmp_list[i][3] = "unknown" var_idx = lower_bound(Var_list, left) while var_idx < len(Var_list): var_pos, var_id = Var_list[var_idx] if var_pos > left: break if var_pos == left: var_type, _, var_data = Vars[var_id] if var_type == "single" and read_bp == var_data: cmp_list[i][3] = var_id break var_idx += 1 if debug: print left, read_bp, ref_bp, mpileup[left] print cmp_list[i] read_pos += length i += 1 # Combine matches i = 0 while i < len(cmp_list): type, left, length = cmp_list[i][:3] if type == "match" and i + 1 < len(cmp_list): type2, left2, length2 = cmp_list[i+1][:3] if type2 == "match": cmp_list[i] = [type, left, length + length2] cmp_list = cmp_list[:i+1] + cmp_list[i+2:] continue i += 1 if debug: print cmp_list print read_seq return cmp_list, read_seq """ Use Stranded-seq reads to resolve assembly ambiguity """ def stranded_seq_alignment(genome_name, sra_run_info, ex_path, ref_allele): read_dir = sra_run_info.split('/')[:-1] read_dir = '/'.join(read_dir) runs = [] for line in open(sra_run_info): line = line.strip() fields = line.split('\t') genome_name_, run = fields[4], fields[0] if genome_name == genome_name_: runs.append(run) run_alignments = [] for run in runs: read_fname, out_fname = "%s/%s.extracted.fq.gz" % (read_dir, run), "%s/%s.bam" % (read_dir, run) align_reads(ex_path, "hisat2", False, # simulation? "hla", "graph", [read_fname], True, # fastq? 1, # number of threads out_fname, False) # verbose? # Read alignments alignview_cmd = ["samtools", "view", out_fname] base_locus = 0 alignview_cmd += [ref_allele] alignview_proc = subprocess.Popen(alignview_cmd, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) plus, minus = [], [] # Cigar regular expression cigar_re = re.compile('\d+\w') pos_added = set() for line in alignview_proc.stdout: line = line.strip() fields = line.split('\t') read_id, flag, ref, pos, _, cigar_str = fields[:6] flag = int(flag) assert run == read_id.split('.')[0] if flag & 0x4 != 0: continue Zs = "" for i in range(11, len(fields)): field = fields[i] if field.startswith("Zs"): Zs = field[5:] vars = [] if Zs != "": for var in Zs.split(','): _, _, var = var.split('|') vars.append(var) left_pos = int(pos) - 1 if left_pos in pos_added: continue pos_added.add(left_pos) right_pos = left_pos cigars = cigar_re.findall(cigar_str) cigars = [[cigar[-1], int(cigar[:-1])] for cigar in cigars] for i in range(len(cigars)): cigar_op, length = cigars[i] if cigar_op in "MND": right_pos += length entry = [left_pos, right_pos, vars] if flag & 0x10 == 0: plus.append(entry) else: minus.append(entry) if len(plus) > 0 and len(minus) > 0: if len(plus) > 1 or len(minus) > 1: run_alignments.append([run, plus, minus]) return run_alignments """ """ def typing(ex_path, simulation, base_fname, locus_list, partial, partial_alleles, refGenes, Genes, Gene_names, Gene_lengths, refGene_loci, Vars, Var_list, Links, aligners, num_editdist, assembly, output_base, error_correction, allow_discordant, display_alleles, stranded_seq, fastq, read_fname, alignment_fname, num_frag_list, threads, best_alleles, verbose): if simulation: test_passed = {} report_file = open(output_base + ".report", 'w') for aligner, index_type in aligners: for f_ in [sys.stderr, report_file]: if index_type == "graph": print >> f_, "\n\t\t%s %s" % (aligner, index_type) else: print >> f_, "\n\t\t%s %s" % (aligner, index_type) remove_alignment_file = False if alignment_fname == "": # Align reads, and sort the alignments into a BAM file remove_alignment_file = True if simulation: alignment_fname = "%s_output.bam" % base_fname else: alignment_fname = read_fname[0].split('/')[-1] alignment_fname = alignment_fname.split('.')[0] + ".bam" align_reads(ex_path, aligner, simulation, base_fname, index_type, read_fname, fastq, threads, alignment_fname, verbose) for test_Gene_names in locus_list: if simulation: gene = test_Gene_names[0].split('*')[0] else: gene = test_Gene_names ref_allele = refGenes[gene] ref_seq = Genes[gene][ref_allele] ref_exons = refGene_loci[gene][-1] novel_var_count = 0 gene_vars, gene_var_list = deepcopy(Vars[gene]), deepcopy(Var_list[gene]) var_count = {} def add_novel_var(gene_vars, gene_var_list, novel_var_count, var_type, var_pos, var_data): var_idx = lower_bound(gene_var_list, var_pos) while var_idx < len(gene_var_list): pos_, id_ = gene_var_list[var_idx] if pos_ > var_pos: break if pos_ == var_pos: type_, _, data_ = gene_vars[id_] assert type_ != var_type or data_ != var_data if type_ != var_type: if var_type == "insertion": break elif var_type == "single" and type_ == "deletion": break else: if var_data < data_: break var_idx += 1 var_id = "nv%d" % novel_var_count assert var_id not in gene_vars gene_vars[var_id] = [var_type, var_pos, var_data] gene_var_list.insert(var_idx, [var_pos, var_id]) return var_id, novel_var_count + 1 if not os.path.exists(alignment_fname + ".bai"): os.system("samtools index %s" % alignment_fname) # Read alignments alignview_cmd = ["samtools", "view", alignment_fname] base_locus = 0 if index_type == "graph": alignview_cmd += [ref_allele] mpileup = get_mpileup(alignview_cmd, ref_seq, base_locus, gene_vars, allow_discordant) bamview_proc = subprocess.Popen(alignview_cmd, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) sort_read_cmd = ["sort", "-k", "1,1", "-s"] # -s for stable sorting alignview_proc = subprocess.Popen(sort_read_cmd, stdin=bamview_proc.stdout, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) else: alignview_proc = subprocess.Popen(alignview_cmd, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) # List of nodes that represent alleles allele_vars = {} for var_id, allele_list in Links.items(): for allele_id in allele_list: if allele_id not in Genes[gene]: continue if allele_id not in allele_vars: allele_vars[allele_id] = [var_id] else: allele_vars[allele_id].append(var_id) # Extract variants that are within exons exon_vars = get_exonic_vars(gene_vars, ref_exons) # Store nodes that represent alleles allele_nodes = {} def create_allele_node(allele_name): if allele_name in allele_nodes: return allele_nodes[allele_name] if allele_name in allele_vars: var_ids = allele_vars[allele_name] else: var_ids = [] seq = list(ref_seq) # sequence that node represents var = ["" for i in range(len(ref_seq))] # how sequence is related to backbone for var_id in var_ids: assert var_id in gene_vars var_type, var_pos, var_data = gene_vars[var_id] assert var_pos >= 0 and var_pos < len(ref_seq) if var_type == "single": seq[var_pos] = var_data var[var_pos] = var_id elif var_type == "deletion": del_len = int(var_data) assert var_pos + del_len <= len(ref_seq) seq[var_pos:var_pos + del_len] = ['D'] * del_len var[var_pos:var_pos + del_len] = [var_id] * del_len else: # DK - to be implemented for insertions assert var_type == "insertion" None qual = ' ' * len(seq) allele_node = assembly_graph.Node(allele_name, 0, seq, qual, var, ref_seq, gene_vars, mpileup, simulation) allele_nodes[allele_name] = allele_node return allele_node true_allele_nodes = {} if simulation: for allele_name in test_Gene_names: true_allele_nodes[allele_name] = create_allele_node(allele_name) display_allele_nodes = {} for display_allele in display_alleles: display_allele_nodes[display_allele] = create_allele_node(display_allele) # Assembly graph asm_graph = assembly_graph.Graph(ref_seq, gene_vars, ref_exons, partial_alleles, true_allele_nodes, {}, # predicted_allele_nodes, which is empty for now display_allele_nodes, simulation) # Choose allele representives from those that share the same exonic sequences allele_reps, allele_rep_groups = get_rep_alleles(Links, exon_vars) allele_rep_set = set(allele_reps.values()) # For checking alternative alignments near the ends of alignments Alts_left, Alts_right = get_alternatives(ref_seq, gene_vars, gene_var_list, verbose) # Count alleles Gene_counts, Gene_cmpt = {}, {} Gene_gen_counts, Gene_gen_cmpt = {}, {} num_reads, total_read_len = 0, 0 # For debugging purposes debug_allele_names = set(test_Gene_names) if simulation and verbose >= 2 else set() # Read information prev_read_id = None prev_right_pos = 0 prev_lines = [] left_read_ids, right_read_ids = set(), set() if index_type == "graph": # nodes for reads read_nodes = [] read_vars_list = [] # Cigar regular expression cigar_re = re.compile('\d+\w') for line in alignview_proc.stdout: line = line.strip() cols = line.split() read_id, flag, chr, pos, mapQ, cigar_str = cols[:6] node_read_id = orig_read_id = read_id if simulation: read_id = read_id.split('|')[0] read_seq, read_qual = cols[9], cols[10] total_read_len += len(read_seq) flag, pos = int(flag), int(pos) pos -= (base_locus + 1) if pos < 0: continue # Unalined? if flag & 0x4 != 0: if simulation and verbose >= 2: print "Unaligned" print "\t", line continue # Concordantly mapped? if flag & 0x2 != 0: concordant = True else: concordant = False NM, Zs, MD, NH = "", "", "", "" for i in range(11, len(cols)): col = cols[i] if col.startswith("Zs"): Zs = col[5:] elif col.startswith("MD"): MD = col[5:] elif col.startswith("NM"): NM = int(col[5:]) elif col.startswith("NH"): NH = int(col[5:]) if NM > num_editdist: continue # Only consider unique alignment if NH > 1: continue # Concordantly aligned mate pairs if not allow_discordant and not concordant: continue # Left read? if flag & 0x40 != 0: if read_id in left_read_ids: continue left_read_ids.add(read_id) if not simulation: node_read_id += '|L' else: # Right read? assert flag & 0x80 != 0 if read_id in right_read_ids: continue right_read_ids.add(read_id) if not simulation: node_read_id += '|R' if Zs: Zs = Zs.split(',') assert MD != "" MD_str_pos, MD_len = 0, 0 Zs_pos, Zs_i = 0, 0 for _i in range(len(Zs)): Zs[_i] = Zs[_i].split('|') Zs[_i][0] = int(Zs[_i][0]) if Zs_i < len(Zs): Zs_pos += Zs[Zs_i][0] read_pos, left_pos = 0, pos right_pos = left_pos cigars = cigar_re.findall(cigar_str) cigars = [[cigar[-1], int(cigar[:-1])] for cigar in cigars] cmp_list = [] likely_misalignment = False # Extract variants w.r.t backbone from CIGAR string softclip = [0, 0] for i in range(len(cigars)): cigar_op, length = cigars[i] if cigar_op == 'M': first = True MD_len_used = 0 cmp_list_i = len(cmp_list) while True: if not first or MD_len == 0: if MD[MD_str_pos].isdigit(): num = int(MD[MD_str_pos]) MD_str_pos += 1 while MD_str_pos < len(MD): if MD[MD_str_pos].isdigit(): num = num * 10 + int(MD[MD_str_pos]) MD_str_pos += 1 else: break MD_len += num # Insertion or full match followed if MD_len >= length: MD_len -= length if length > MD_len_used: cmp_list.append(["match", right_pos + MD_len_used, length - MD_len_used]) break first = False read_base = read_seq[read_pos + MD_len] MD_ref_base = MD[MD_str_pos] MD_str_pos += 1 assert MD_ref_base in "ACGT" if MD_len > MD_len_used: cmp_list.append(["match", right_pos + MD_len_used, MD_len - MD_len_used]) _var_id = "unknown" if read_pos + MD_len == Zs_pos and Zs_i < len(Zs): assert Zs[Zs_i][1] == 'S' _var_id = Zs[Zs_i][2] Zs_i += 1 Zs_pos += 1 if Zs_i < len(Zs): Zs_pos += Zs[Zs_i][0] else: # Search for a known (yet not indexed) variant or a novel variant ref_pos = right_pos + MD_len var_idx = lower_bound(gene_var_list, ref_pos) while var_idx < len(gene_var_list): var_pos, var_id = gene_var_list[var_idx] if var_pos > ref_pos: break if var_pos == ref_pos: var_type, _, var_data = gene_vars[var_id] if var_type == "single" and var_data == read_base: _var_id = var_id break var_idx += 1 cmp_list.append(["mismatch", right_pos + MD_len, 1, _var_id]) MD_len_used = MD_len + 1 MD_len += 1 # Full match if MD_len == length: MD_len = 0 break # Correction for sequencing errors and update for cmp_list if error_correction: assert cmp_list_i < len(cmp_list) new_cmp_list, read_seq = error_correct(ref_seq, read_seq, read_pos, mpileup, gene_vars, gene_var_list, cmp_list[cmp_list_i:], node_read_id == "#HSQ1008:176:D0UYCACXX:4:1304:19006:96208|R") cmp_list = cmp_list[:cmp_list_i] + new_cmp_list elif cigar_op == 'I': _var_id = "unknown" if read_pos == Zs_pos and Zs_i < len(Zs): assert Zs[Zs_i][1] == 'I' _var_id = Zs[Zs_i][2] Zs_i += 1 if Zs_i < len(Zs): Zs_pos += Zs[Zs_i][0] else: # Search for a known (yet not indexed) variant or a novel variant var_idx = lower_bound(gene_var_list, right_pos) while var_idx < len(gene_var_list): var_pos, var_id = gene_var_list[var_idx] if var_pos > right_pos: break if var_pos == right_pos: var_type, _, var_data = gene_vars[var_id] if var_type == "insertion" and len(var_data) == length: _var_id = var_id break var_idx += 1 cmp_list.append(["insertion", right_pos, length, _var_id]) if 'N' in read_seq[read_pos:read_pos+length]: likely_misalignment = True elif cigar_op == 'D': if MD[MD_str_pos] == '0': MD_str_pos += 1 assert MD[MD_str_pos] == '^' MD_str_pos += 1 while MD_str_pos < len(MD): if not MD[MD_str_pos] in "ACGT": break MD_str_pos += 1 _var_id = "unknown" if read_pos == Zs_pos and \ Zs_i < len(Zs) and \ Zs[Zs_i][1] == 'D': _var_id = Zs[Zs_i][2] Zs_i += 1 if Zs_i < len(Zs): Zs_pos += Zs[Zs_i][0] else: # Search for a known (yet not indexed) variant or a novel variant var_idx = lower_bound(gene_var_list, right_pos) while var_idx < len(gene_var_list): var_pos, var_id = gene_var_list[var_idx] if var_pos > right_pos: break if var_pos == right_pos: var_type, _, var_data = gene_vars[var_id] if var_type == "deletion" and int(var_data) == length: _var_id = var_id break var_idx += 1 cmp_list.append(["deletion", right_pos, length, _var_id]) # Check if this deletion is artificial alignment assert right_pos < mpileup del_count, nt_count = 0, 0 for nt, value in mpileup[right_pos][1].items(): count = value[0] if nt == 'D': del_count += count else: nt_count += count # DK - debugging purposes if del_count * 6 < nt_count: # and nt_count >= 15: likely_misalignment = True elif cigar_op == 'S': if i == 0: softclip[0] = length Zs_pos += length else: assert i + 1 == len(cigars) softclip[1] = length else: assert cigar_op == 'N' assert False cmp_list.append(["intron", right_pos, length]) if cigar_op in "MND": right_pos += length if cigar_op in "MIS": read_pos += length # Remove softclip in cigar and modify read_seq and read_qual accordingly if sum(softclip) > 0: if softclip[0] > 0: cigars = cigars[1:] read_seq = read_seq[softclip[0]:] read_qual = read_qual[softclip[0]:] if softclip[1] > 0: cigars = cigars[:-1] read_seq = read_seq[:-softclip[1]] read_qual = read_qual[:-softclip[1]] cigar_str = "" for type, length in cigars: cigar_str += str(length) cigar_str += type if right_pos > len(ref_seq): continue if likely_misalignment: continue # Add novel variants read_pos = 0 for cmp_i in range(len(cmp_list)): type_, pos_, length_ = cmp_list[cmp_i][:3] if type_ != "match": var_id_ = cmp_list[cmp_i][3] if var_id_ == "unknown": add = True if type_ == "mismatch": data_ = read_seq[read_pos] if data_ == 'N': add = False elif type_ == "deletion": data_ = str(length_) else: assert type_ == "insertion" data_ = read_seq[read_pos:read_pos + length_] if add: var_id, novel_var_count = add_novel_var(gene_vars, gene_var_list, novel_var_count, type_ if type_ != "mismatch" else "single", pos_, data_) cmp_list[cmp_i][3] = var_id if var_id not in var_count: var_count[var_id] = 1 else: var_count[var_id] += 1 if type_ != "deletion": read_pos += length_ # Count the number of reads aligned uniquely with some constraints num_reads += 1 def add_stat(Gene_cmpt, Gene_counts, Gene_count_per_read, include_alleles = set()): max_count = max(Gene_count_per_read.values()) cur_cmpt = set() for allele, count in Gene_count_per_read.items(): if count < max_count: continue if len(include_alleles) > 0 and allele not in include_alleles: continue cur_cmpt.add(allele) if allele not in Gene_counts: Gene_counts[allele] = 1 else: Gene_counts[allele] += 1 if len(cur_cmpt) == 0: return "" # DK - for debugging purposes alleles = ["", ""] # alleles = ["A*24:36N", "A*24:359N"] allele1_found, allele2_found = False, False if alleles[0] != "": for allele, count in Gene_count_per_read.items(): if count < max_count: continue if allele == alleles[0]: allele1_found = True elif allele == alleles[1]: allele2_found = True if allele1_found != allele2_found: print alleles[0], Gene_count_per_read[alleles[0]] print alleles[1], Gene_count_per_read[alleles[1]] if allele1_found: print ("%s\tread_id %s - %d vs. %d]" % (alleles[0], prev_read_id, max_count, Gene_count_per_read[alleles[1]])) else: print ("%s\tread_id %s - %d vs. %d]" % (alleles[1], prev_read_id, max_count, Gene_count_per_read[alleles[0]])) print read_seq cur_cmpt = sorted(list(cur_cmpt)) cur_cmpt = '-'.join(cur_cmpt) if not cur_cmpt in Gene_cmpt: Gene_cmpt[cur_cmpt] = 1 else: Gene_cmpt[cur_cmpt] += 1 return cur_cmpt if read_id != prev_read_id: if prev_read_id != None: # DK - debugging purpose debug_allele_id = "A*24:355" assert debug_allele_id in Gene_count_per_read debug_max_read_count = max(Gene_count_per_read.values()) debug_read_count = Gene_count_per_read[debug_allele_id] if debug_read_count == debug_max_read_count and \ Gene_count_per_read["A*24:02:01:02L"] < debug_max_read_count and \ Gene_count_per_read["A*01:01:01:01"] < debug_max_read_count: print prev_read_id None if prev_read_id == "HSQ1008:175:C0JVFACXX:7:1208:5604:41201": None """ for line in prev_lines: print line print Gene_count_per_read[debug_allele_id], max(Gene_count_per_read.values()) print Gene_gen_count_per_read[debug_allele_id], max(Gene_gen_count_per_read.values()) for allele_id, count in Gene_count_per_read.items(): if count == debug_max_read_count: print "allele max:", allele_id, count """ if base_fname == "hla": cur_cmpt = add_stat(Gene_cmpt, Gene_counts, Gene_count_per_read, allele_rep_set) add_stat(Gene_gen_cmpt, Gene_gen_counts, Gene_gen_count_per_read) for read_id_, read_node in read_nodes: asm_graph.add_node(read_id_, read_node, simulation) read_nodes, read_var_list = [], [] if simulation and \ verbose >= 2 and \ base_fname == "hla": cur_cmpt = cur_cmpt.split('-') if not(set(cur_cmpt) & set(test_Gene_names)): print "%s are chosen instead of %s" % ('-'.join(cur_cmpt), '-'.join(test_Gene_names)) for prev_line in prev_lines: print "\t", prev_line prev_lines = [] Gene_count_per_read, Gene_gen_count_per_read = {}, {} for Gene_name in Gene_names[gene]: if Gene_name.find("BACKBONE") != -1: continue Gene_count_per_read[Gene_name] = 0 Gene_gen_count_per_read[Gene_name] = 0 prev_lines.append(line) def add_count(count_per_read, var_id, add): alleles = Links[var_id] if verbose >= 2: if add > 0 and not (set(alleles) & debug_allele_names): print "Add:", add, debug_allele_names, "-", var_id print "\t", line print "\t", alleles if add < 0 and set(alleles) & debug_allele_names: print "Add:", add, debug_allele_names, "-", var_id print "\t", line for allele in alleles: count_per_read[allele] += add # Decide which allele(s) a read most likely came from for var_id, data in gene_vars.items(): if var_id == "unknown" or var_id.startswith("nv"): continue var_type, var_pos, var_data = data if var_type != "deletion": continue if left_pos >= var_pos and right_pos <= var_pos + int(var_data): if var_id in exon_vars: add_count(Gene_count_per_read, var_id, -1) add_count(Gene_gen_count_per_read, var_id, -1) # Node read_node_pos, read_node_seq, read_node_qual, read_node_var = -1, [], [], [] read_vars = [] # Positive and negative evidence positive_vars, negative_vars = set(), set() # Sanity check - read length, cigar string, and MD string ref_pos, read_pos, cmp_cigar_str, cmp_MD = left_pos, 0, "", "" cigar_match_len, MD_match_len = 0, 0 cmp_list_left, cmp_list_right = identify_ambigious_diffs(gene_vars, Alts_left, Alts_right, cmp_list, verbose, orig_read_id == "a45|L_441_89M8D11M_89|D|hv1,7|S|hv15") # debug? # DK - debugging purposes DK_debug = False if orig_read_id == "a46|L_451_88M12D12M_88|D|hv2": DK_debug = True print line print cmp_list print "positive vars:", positive_vars print "negative vars:", negative_vars print "cmp_list[%d, %d]" % (cmp_list_left, cmp_list_right) # Deletions at 5' and 3' ends for var_id, data in gene_vars.items(): var_type, var_pos, var_data = data if var_type != "deletion": continue if left_pos >= var_pos and right_pos <= var_pos + int(var_data): negative_vars.add(var_id) cmp_i = 0 while cmp_i < len(cmp_list): cmp = cmp_list[cmp_i] type, length = cmp[0], cmp[2] # Disable the following sanity check due to error correction # if num_editdist == 0 and type in ["mismatch", "deletion", "insertion"]: # assert cmp[3] != "unknown" if type in ["match", "mismatch"]: if read_node_pos < 0: read_node_pos = ref_pos if type == "match": read_node_seq += list(read_seq[read_pos:read_pos+length]) read_node_qual += list(read_qual[read_pos:read_pos+length]) read_node_var += ([''] * length) var_idx = lower_bound(gene_var_list, ref_pos) while var_idx < len(gene_var_list): var_pos, var_id = gene_var_list[var_idx] if ref_pos + length <= var_pos: break if ref_pos <= var_pos: var_type, _, var_data = gene_vars[var_id] if var_type == "insertion": if ref_pos < var_pos and ref_pos + length > var_pos + len(var_data): negative_vars.add(var_id) elif var_type == "deletion": del_len = int(var_data) if ref_pos < var_pos and ref_pos + length > var_pos + del_len: if base_fname == "codis": cmp_left, cmp_right = left_pos, right_pos else: cmp_left, cmp_right = cmp[1], cmp[1] + cmp[2] # Check if this might be one of the two tandem repeats (the same left coordinate) test1_seq1 = ref_seq[cmp_left:cmp_right] test1_seq2 = ref_seq[cmp_left:var_pos] + ref_seq[var_pos + del_len:cmp_right + del_len] # Check if this happens due to small repeats (the same right coordinate - e.g. 19 times of TTTC in DQA1*05:05:01:02) cmp_left -= read_pos cmp_right += (len(read_seq) - read_pos - cmp[2]) test2_seq1 = ref_seq[cmp_left+int(var_data):cmp_right] test2_seq2 = ref_seq[cmp_left:var_pos] + ref_seq[var_pos+int(var_data):cmp_right] if test1_seq1 != test1_seq2 and test2_seq1 != test2_seq2: negative_vars.add(var_id) else: negative_vars.add(var_id) var_idx += 1 read_pos += length ref_pos += length cigar_match_len += length MD_match_len += length elif type == "mismatch": var_id = cmp[3] read_base, qual = read_seq[read_pos], read_qual[read_pos] read_node_seq += [read_base] read_node_qual += [qual] read_node_var.append(var_id) if var_id != "unknown": if cmp_i >= cmp_list_left and cmp_i <= cmp_list_right: positive_vars.add(var_id) if read_id == "HSQ1008:175:C0JVFACXX:6:2207:13481:60924" and False: print "add positive var:", var_id print "\tcmp_list:", cmp_list_left, cmp_list_right, cmp_list cmp_MD += ("%d%s" % (MD_match_len, ref_seq[ref_pos])) MD_match_len = 0 cigar_match_len += 1 read_pos += 1 ref_pos += 1 elif type == "insertion": var_id = cmp[3] ins_len = length ins_seq = read_seq[read_pos:read_pos+ins_len] if var_id != "unknown" or not var_id.startswith("nv"): if cmp_i >= cmp_list_left and cmp_i <= cmp_list_right: # Require at least 5bp match before and after a deletion if read_pos >= 5 and read_pos + 5 <= len(read_seq): positive_vars.add(var_id) read_node_seq += ["I%s" % nt for nt in ins_seq] read_node_qual += list(read_qual[read_pos:read_pos+ins_len]) read_node_var += ([var_id] * ins_len) if cigar_match_len > 0: cmp_cigar_str += ("%dM" % cigar_match_len) cigar_match_len = 0 read_pos += length cmp_cigar_str += ("%dI" % length) elif type == "deletion": var_id = cmp[3] alt_match = False del_len = length read_node_seq += (['D'] * del_len) read_node_qual += ([''] * del_len) if var_id != "unknown" or not var_id.statswith("nv"): if cmp_i >= cmp_list_left and cmp_i <= cmp_list_right: # Require at least 5bp match before and after a deletion if read_pos >= 5 and read_pos + 5 <= len(read_seq): positive_vars.add(var_id) if len(read_node_seq) > len(read_node_var): assert len(read_node_seq) == len(read_node_var) + del_len read_node_var += ([var_id] * del_len) if cigar_match_len > 0: cmp_cigar_str += ("%dM" % cigar_match_len) cigar_match_len = 0 cmp_MD += ("%d" % MD_match_len) MD_match_len = 0 cmp_cigar_str += ("%dD" % length) cmp_MD += ("^%s" % ref_seq[ref_pos:ref_pos+length]) ref_pos += length else: assert type == "intron" if cigar_match_len > 0: cmp_cigar_str += ("%dM" % cigar_match_len) cigar_match_len = 0 cmp_cigar_str += ("%dN" % length) ref_pos += length cmp_i += 1 if cigar_match_len > 0: cmp_cigar_str += ("%dM" % cigar_match_len) cmp_MD += ("%d" % MD_match_len) # Sanity check if read_pos != len(read_seq) or \ cmp_cigar_str != cigar_str: # cmp_MD != MD: # Disabled due to error correction print >> sys.stderr, "Error:", cigar_str, MD print >> sys.stderr, "\tcomputed:", cmp_cigar_str, cmp_MD print >> sys.stderr, "\tcmp list:", cmp_list assert False # DK - debugging purposes if DK_debug: print "positive:", positive_vars print "negative:", negative_vars # Node if assembly: read_nodes.append([node_read_id, assembly_graph.Node(node_read_id, read_node_pos, read_node_seq, read_node_qual, read_node_var, ref_seq, gene_vars, mpileup, simulation)]) for positive_var in positive_vars: if positive_var == "unknown" or positive_var.startswith("nv"): continue if positive_var in exon_vars: add_count(Gene_count_per_read, positive_var, 1) add_count(Gene_gen_count_per_read, positive_var, 1) if read_id == "HSQ1008:175:C0JVFACXX:6:2207:13481:60924" and False: print "positive_vars:", positive_vars print "negative_vars:", negative_vars for negative_var in negative_vars: if negative_var == "unknown" or negative_var.startswith("nv"): continue if negative_var in exon_vars: add_count(Gene_count_per_read, negative_var, -1) add_count(Gene_gen_count_per_read, negative_var, -1) prev_read_id = read_id prev_right_pos = right_pos if num_reads <= 0: continue for f_ in [sys.stderr, report_file]: print >> f_, "\t\t\tNumber of reads aligned: %d" % num_reads if prev_read_id != None: if base_fname == "hla": add_stat(Gene_cmpt, Gene_counts, Gene_count_per_read, allele_rep_set) add_stat(Gene_gen_cmpt, Gene_gen_counts, Gene_gen_count_per_read) for read_id_, read_node in read_nodes: asm_graph.add_node(read_id_, read_node, simulation) read_nodes, read_var_list = [], [] else: assert index_type == "linear" def add_alleles(alleles): if not allele in Gene_counts: Gene_counts[allele] = 1 else: Gene_counts[allele] += 1 cur_cmpt = sorted(list(alleles)) cur_cmpt = '-'.join(cur_cmpt) if not cur_cmpt in Gene_cmpt: Gene_cmpt[cur_cmpt] = 1 else: Gene_cmpt[cur_cmpt] += 1 prev_read_id, prev_AS = None, None alleles = set() for line in alignview_proc.stdout: cols = line[:-1].split() read_id, flag, allele = cols[:3] flag = int(flag) if flag & 0x4 != 0: continue if not allele.startswith(gene): continue if allele.find("BACKBONE") != -1: continue AS = None for i in range(11, len(cols)): col = cols[i] if col.startswith("AS"): AS = int(col[5:]) assert AS != None if read_id != prev_read_id: if alleles: if aligner == "hisat2" or \ (aligner == "bowtie2" and len(alleles) < 10): add_alleles(alleles) alleles = set() prev_AS = None if prev_AS != None and AS < prev_AS: continue prev_read_id = read_id prev_AS = AS alleles.add(allele) if alleles: add_alleles(alleles) if base_fname != "hla": Gene_counts = Gene_gen_counts Gene_counts = [[allele, count] for allele, count in Gene_counts.items()] def Gene_count_cmp(a, b): if a[1] != b[1]: return b[1] - a[1] assert a[0] != b[0] if a[0] < b[0]: return -1 else: return 1 Gene_counts = sorted(Gene_counts, cmp=Gene_count_cmp) for count_i in range(len(Gene_counts)): count = Gene_counts[count_i] if simulation: found = False for test_Gene_name in test_Gene_names: if count[0] == test_Gene_name: for f_ in [sys.stderr, report_file]: print >> f_, "\t\t\t*** %d ranked %s (count: %d)" % (count_i + 1, test_Gene_name, count[1]) found = True """ if count_i > 0 and Gene_counts[0][1] > count[1]: print >> sys.stderr, "Warning: %s ranked first (count: %d)" % (Gene_counts[0][0], Gene_counts[0][1]) assert False else: test_passed += 1 """ if count_i < 5 and not found: for f_ in [sys.stderr, report_file]: print >> f_, "\t\t\t\t%d %s (count: %d)" % (count_i + 1, count[0], count[1]) else: for f_ in [sys.stderr, report_file]: print >> f_, "\t\t\t\t%d %s (count: %d)" % (count_i + 1, count[0], count[1]) if count_i >= 9: break for f_ in [sys.stderr, report_file]: print >> f_ # Calculate the abundance of representative alleles on exonic sequences if base_fname == "hla": # Incorporate non representative alleles (full length alleles) Gene_prob = single_abundance(Gene_cmpt, Gene_lengths[gene]) gen_alleles = set() gen_prob_sum = 0.0 for prob_i in range(len(Gene_prob)): allele, prob = Gene_prob[prob_i][:2] if prob_i >= 10 and prob < 0.03: break if allele in partial_alleles: continue gen_prob_sum += prob for allele2 in allele_rep_groups[allele]: gen_alleles.add(allele2) if len(gen_alleles) > 0: Gene_gen_cmpt2 = {} for cmpt, value in Gene_gen_cmpt.items(): cmpt2 = [] for allele in cmpt.split('-'): if allele in gen_alleles: cmpt2.append(allele) if len(cmpt2) == 0: continue cmpt2 = '-'.join(cmpt2) if cmpt2 not in Gene_gen_cmpt2: Gene_gen_cmpt2[cmpt2] = value else: Gene_gen_cmpt2[cmpt2] += value Gene_gen_cmpt = Gene_gen_cmpt2 Gene_gen_prob = single_abundance(Gene_gen_cmpt, Gene_lengths[gene]) Gene_combined_prob = {} for allele, prob in Gene_prob: assert allele not in Gene_combined_prob if allele in gen_alleles: Gene_combined_prob[allele] = 0.0 else: Gene_combined_prob[allele] = prob for allele, prob in Gene_gen_prob: Gene_combined_prob[allele] = prob * gen_prob_sum Gene_prob = [[allele, prob] for allele, prob in Gene_combined_prob.items()] Gene_prob = sorted(Gene_prob, cmp=Gene_prob_cmp) else: Gene_prob = single_abundance(Gene_gen_cmpt, Gene_lengths[gene]) if index_type == "graph" and assembly: allele_node_order = [] predicted_allele_nodes = {} for allele_name, prob in Gene_prob: if prob < 0.1: # abundance of 10% break predicted_allele_nodes[allele_name] = create_allele_node(allele_name) allele_node_order.append([allele_name, prob]) if len(predicted_allele_nodes) >= 2: break asm_graph.predicted_allele_nodes = predicted_allele_nodes asm_graph.allele_node_order = allele_node_order # Start drawing assembly graph asm_graph.begin_draw(output_base) # Draw assembly graph begin_y = asm_graph.draw(0, "Initial graph") begin_y += 200 # Apply De Bruijn graph asm_graph.guided_DeBruijn() # Draw assembly graph begin_y = asm_graph.draw(begin_y, "Asssembly") begin_y += 200 # Stranded-seq read analysis if len(stranded_seq) == 2: run_alignments = stranded_seq_alignment(stranded_seq[0], stranded_seq[1], ex_path, ref_allele) def get_best_alleles(left, right, vars): max_alleles, max_common = [], -sys.maxint for allele_name, allele_node in predicted_allele_nodes.items(): tmp_vars = allele_node.get_var_ids(left, right) tmp_common = len(set(vars) & set(tmp_vars)) tmp_common -= len(set(vars) | set(tmp_vars)) if max_common < tmp_common: max_common = tmp_common max_alleles = [[allele_name, max_common]] elif max_common == tmp_common: max_alleles.append([allele_name, max_common]) return max_alleles for run, plus, minus in run_alignments: print run print "\tplus:" for left, right, vars in plus: print "\t\t", left, right, vars, get_best_alleles(left, right, vars) print "\tminus:" for left, right, vars in minus: print "\t\t", left, right, vars, get_best_alleles(left, right, vars) assert False # DK - debugging purposes # """ # Draw assembly graph asm_graph.nodes = asm_graph.nodes2 asm_graph.to_node, asm_graph.from_node = {}, {} begin_y = asm_graph.draw(begin_y, "Assembly with known alleles") # """ # End drawing assembly graph asm_graph.end_draw() # Compare two alleles if simulation and len(test_Gene_names) == 2: allele_name1, allele_name2 = test_Gene_names print >> sys.stderr, allele_name1, "vs.", allele_name2 asm_graph.print_node_comparison(asm_graph.true_allele_nodes) def compare_alleles(vars1, vars2, print_output = True): skip = True var_i, var_j = 0, 0 exon_i = 0 mismatches = 0 while var_i < len(vars1) and var_j < len(vars2): cmp_var_id, node_var_id = vars1[var_i], vars2[var_j] cmp_var, node_var = gene_vars[cmp_var_id], gene_vars[node_var_id] min_pos = min(cmp_var[1], node_var[1]) cmp_var_in_exon, node_var_in_exon = False, False while exon_i < len(ref_exons): exon_left, exon_right = ref_exons[exon_i] if min_pos <= exon_right: if cmp_var[1] >= exon_left and cmp_var[1] <= exon_right: cmp_var_in_exon = True else: cmp_var_in_exon = False if node_var[1] >= exon_left and node_var[1] <= exon_right: node_var_in_exon = True else: node_var_in_exon = False break exon_i += 1 if cmp_var_id == node_var_id: skip = False if print_output: if cmp_var_in_exon: print >> sys.stderr, "\033[94mexon%d\033[00m" % (exon_i + 1), print >> sys.stderr, cmp_var_id, cmp_var, "\t\t\t", mpileup[cmp_var[1]] var_i += 1; var_j += 1 continue if cmp_var[1] <= node_var[1]: if not skip: if (var_i > 0 and var_i + 1 < len(vars1)) or cmp_var[0] != "deletion": if print_output: if cmp_var_in_exon: for f_ in [sys.stderr, report_file]: print >> f_, "\033[94mexon%d\033[00m" % (exon_i + 1), for f_ in [sys.stderr, report_file]: print >> f_, "***", cmp_var_id, cmp_var, "==", "\t\t\t", mpileup[cmp_var[1]] mismatches += 1 var_i += 1 else: if print_output: if node_var_in_exon: for f_ in [sys.stderr, report_file]: print >> f_, "\033[94mexon%d\033[00m" % (exon_i + 1), for f_ in [sys.stderr, report_file]: print >> f_, "*** ==", node_var_id, node_var, "\t\t\t", mpileup[node_var[1]] mismatches += 1 var_j += 1 return mismatches tmp_nodes = asm_graph.nodes print >> sys.stderr, "Number of tmp nodes:", len(tmp_nodes) count = 0 for id, node in tmp_nodes.items(): count += 1 if count > 10: break node_vars = node.get_var_ids() node.print_info(); print >> sys.stderr if node.id in asm_graph.to_node: for id2, at in asm_graph.to_node[node.id]: print >> sys.stderr, "\tat %d ==> %s" % (at, id2) if simulation: cmp_Gene_names = test_Gene_names else: cmp_Gene_names = [allele_name for allele_name, _ in allele_node_order] alleles, cmp_vars, max_common = [], [], -sys.maxint for cmp_Gene_name in cmp_Gene_names: tmp_vars = allele_nodes[cmp_Gene_name].get_var_ids(node.left, node.right) tmp_common = len(set(node_vars) & set(tmp_vars)) tmp_common -= len(set(node_vars) | set(tmp_vars)) if max_common < tmp_common: max_common = tmp_common alleles = [[cmp_Gene_name, tmp_vars]] elif max_common == tmp_common: alleles.append([cmp_Gene_name, tmp_vars]) for allele_name, cmp_vars in alleles: for f_ in [sys.stderr, report_file]: print >> f_, "vs.", allele_name compare_alleles(cmp_vars, node_vars) print >> sys.stderr print >> sys.stderr # Identify alleles that perfectly or closesly match assembled alleles for node_name, node in asm_graph.nodes.items(): vars = set(node.get_var_ids()) max_allele_names, max_common = [], -sys.maxint for allele_name, vars2 in allele_vars.items(): vars2 = set(vars2) tmp_common = len(vars & vars2) - len(vars | vars2) if tmp_common > max_common: max_common = tmp_common max_allele_names = [allele_name] elif tmp_common == max_common: max_allele_names.append(allele_name) for f_ in [sys.stderr, report_file]: print >> f_, "Genomic:", node_name node_vars = node.get_var_ids() min_mismatches = sys.maxint for max_allele_name in max_allele_names: cmp_vars = allele_vars[max_allele_name] cmp_vars = sorted(cmp_vars, cmp=lambda a, b: int(a[2:]) - int(b[2:])) print_output = False tmp_mismatches = compare_alleles(cmp_vars, node_vars, print_output) print >> f_, "\t\t%s:" % max_allele_name, max_common, tmp_mismatches if tmp_mismatches < min_mismatches: min_mismatches = tmp_mismatches if min_mismatches > 0: print >> f_, "Novel allele" else: print >> f_, "Known allele" """ allele_exon_vars = {} for allele_name, vars in allele_vars.items(): allele_exon_vars[allele_name] = set(vars) & exon_vars for node_name, node in asm_graph.nodes.items(): vars = [] for left, right in ref_exons: vars += node.get_var_ids(left, right) vars = set(vars) & exon_vars max_allele_names, max_common = [], -sys.maxint for allele_name, vars2 in allele_exon_vars.items(): tmp_common = len(vars & vars2) - len(vars | vars2) if tmp_common > max_common: max_common = tmp_common max_allele_names = [allele_name] elif tmp_common == max_common: max_allele_names.append(allele_name) for f_ in [sys.stderr, report_file]: print >> f_, "Exonic:", node_name for max_allele_name in max_allele_names: print >> f_, "\t\t%s:" % max_allele_name, max_common """ success = [False for i in range(len(test_Gene_names))] found_list = [False for i in range(len(test_Gene_names))] for prob_i in range(len(Gene_prob)): prob = Gene_prob[prob_i] found = False _allele_rep = prob[0] """ if partial and exonic_only: _fields = _allele_rep.split(':') if len(_fields) == 4: _allele_rep = ':'.join(_fields[:-1]) """ if simulation: for name_i in range(len(test_Gene_names)): test_Gene_name = test_Gene_names[name_i] if prob[0] == test_Gene_name: rank_i = prob_i while rank_i > 0: if prob == Gene_prob[rank_i - 1][1]: rank_i -= 1 else: break for f_ in [sys.stderr, report_file]: print >> f_, "\t\t\t*** %d ranked %s (abundance: %.2f%%)" % (rank_i + 1, test_Gene_name, prob[1] * 100.0) if rank_i < len(success): success[rank_i] = True found_list[name_i] = True found = True # DK - for debugging purposes if not False in found_list and prob_i >= 10: break if not found: for f_ in [sys.stderr, report_file]: print >> f_, "\t\t\t\t%d ranked %s (abundance: %.2f%%)" % (prob_i + 1, _allele_rep, prob[1] * 100.0) if best_alleles and prob_i < 2: for f_ in [sys.stderr, report_file]: print >> f_, "SingleModel %s (abundance: %.2f%%)" % (_allele_rep, prob[1] * 100.0) if not simulation and prob_i >= 9: break if prob_i >= 19: break print >> sys.stderr if simulation and not False in success: aligner_type = "%s %s" % (aligner, index_type) if not aligner_type in test_passed: test_passed[aligner_type] = 1 else: test_passed[aligner_type] += 1 if remove_alignment_file and not simulation: os.system("rm %s*" % (alignment_fname)) report_file.close() if simulation: return test_passed """ """ def read_Gene_alleles(fname, Genes): for line in open(fname): if line.startswith(">"): Gene_name = line.strip().split()[0][1:] Gene_gene = Gene_name.split('*')[0] if not Gene_gene in Genes: Genes[Gene_gene] = {} if not Gene_name in Genes[Gene_gene]: Genes[Gene_gene][Gene_name] = "" else: Genes[Gene_gene][Gene_name] += line.strip() return Genes """ """ def read_Gene_vars(fname): Vars, Var_list = {}, {} for line in open(fname): var_id, var_type, allele, pos, data = line.strip().split('\t') pos = int(pos) gene = allele.split('*')[0] if not gene in Vars: Vars[gene] = {} assert not gene in Var_list Var_list[gene] = [] assert not var_id in Vars[gene] left = 0 Vars[gene][var_id] = [var_type, pos - left, data] Var_list[gene].append([pos - left, var_id]) for gene, in_var_list in Var_list.items(): Var_list[gene] = sorted(in_var_list) return Vars, Var_list """ """ def read_Gene_links(fname): Links = {} for line in open(fname): var_id, alleles = line.strip().split('\t') alleles = alleles.split() assert not var_id in Links Links[var_id] = alleles return Links """ """ def construct_allele_seq(backbone_seq, var_ids, Vars): allele_seq = list(backbone_seq) for id in var_ids: assert id in Vars type, pos, data = Vars[id] assert pos < len(allele_seq) if type == "single": assert allele_seq[pos] != data allele_seq[pos] = data else: assert type == "deletion" del_len = int(data) assert pos + del_len <= len(allele_seq) for i in range(pos, pos + del_len): allele_seq[i] = '.' allele_seq = ''.join(allele_seq) allele_seq = allele_seq.replace('.', '') return allele_seq """ """ def test_Gene_genotyping(base_fname, locus_list, partial, aligners, read_fname, alignment_fname, threads, simulate_interval, read_len, fragment_len, best_alleles, num_editdist, perbase_errorrate, perbase_snprate, skip_fragment_regions, assembly, output_base, error_correction, discordant, display_alleles, stranded_seq, verbose, debug_instr): # Current script directory curr_script = os.path.realpath(inspect.getsourcefile(test_Gene_genotyping)) ex_path = os.path.dirname(curr_script) # Clone a git repository, IMGTHLA if not os.path.exists("IMGTHLA"): Gene_typing.clone_IMGTHLA_database() if not os.path.exists("hisatgenotype_db"): typing_common.clone_hisatgenotype_database() simulation = (read_fname == [] and alignment_fname == "") # Download human genome and HISAT2 index HISAT2_fnames = ["grch38", "genome.fa", "genome.fa.fai"] if not typing_common.check_files(HISAT2_fnames): typing_common.download_genome_and_index() # Check if the pre-existing files (hla*) are compatible with the current parameter setting if os.path.exists("%s.ref" % base_fname): left = 0 Gene_genes = [] BACKBONE = False for line in open("%s.ref" % base_fname): Gene_name = line.strip().split()[0] if Gene_name.find("BACKBONE") != -1: BACKBONE = True Gene_gene = Gene_name.split('*')[0] Gene_genes.append(Gene_gene) delete_hla_files = False if not BACKBONE: delete_hla_files = True if len(locus_list) == 0: locus_list = Gene_genes if not set(locus_list).issubset(set(Gene_genes)): delete_hla_files = True if delete_hla_files: os.system("rm %s*" % base_fname) # Extract HLA variants, backbone sequence, and other sequeces Gene_fnames = [base_fname + "_backbone.fa", base_fname + "_sequences.fa", base_fname + ".ref", base_fname + ".snp", base_fname + ".index.snp", base_fname + ".haplotype", base_fname + ".link"] if verbose >= 1: print >> sys.stderr, Gene_fnames if not typing_common.check_files(Gene_fnames): extract_hla_script = os.path.join(ex_path, "hisatgenotype_extract_vars.py") extract_cmd = [extract_hla_script] if len(locus_list) > 0: extract_cmd += ["--locus-list", ','.join(locus_list)] extract_cmd += ["--base", base_fname] if not partial: extract_cmd += ["--no-partial"] extract_cmd += ["--inter-gap", "30", "--intra-gap", "50"] # DK - debugging purposes extract_cmd += ["--min-var-freq", "0.1"] if base_fname == "codis": extract_cmd += ["--leftshift"] # DK - debugging purposes # extract_cmd += ["--ext-seq", "300"] if verbose >= 1: print >> sys.stderr, "\tRunning:", ' '.join(extract_cmd) proc = subprocess.Popen(extract_cmd, stdout=open("/dev/null", 'w'), stderr=open("/dev/null", 'w')) proc.communicate() if not typing_common.check_files(Gene_fnames): print >> sys.stderr, "Error: hisatgenotype_extract_vars failed!" sys.exit(1) for aligner, index_type in aligners: if aligner == "hisat2": # Build HISAT2 graph indexes based on the above information if index_type == "graph": Gene_hisat2_graph_index_fnames = ["%s.graph.%d.ht2" % (base_fname, i+1) for i in range(8)] if not typing_common.check_files(Gene_hisat2_graph_index_fnames): hisat2_build = os.path.join(ex_path, "hisat2-build") build_cmd = [hisat2_build, "-p", str(threads), "--snp", "%s.index.snp" % base_fname, "--haplotype", "%s.haplotype" % base_fname, "%s_backbone.fa" % base_fname, "%s.graph" % base_fname] if verbose >= 1: print >> sys.stderr, "\tRunning:", ' '.join(build_cmd) proc = subprocess.Popen(build_cmd, stdout=open("/dev/null", 'w'), stderr=open("/dev/null", 'w')) proc.communicate() if not typing_common.check_files(Gene_hisat2_graph_index_fnames): print >> sys.stderr, "Error: indexing HLA failed! Perhaps, you may have forgotten to build hisat2 executables?" sys.exit(1) # Build HISAT2 linear indexes based on the above information else: assert index_type == "linear" Gene_hisat2_linear_index_fnames = ["%s.linear.%d.ht2" % (base_fname, i+1) for i in range(8)] if not typing_common.check_files(Gene_hisat2_linear_index_fnames): hisat2_build = os.path.join(ex_path, "hisat2-build") build_cmd = [hisat2_build, "%s_backbone.fa,%s_sequences.fa" % (base_fname, base_fname), "%s.linear" % base_fname] proc = subprocess.Popen(build_cmd, stdout=open("/dev/null", 'w'), stderr=open("/dev/null", 'w')) proc.communicate() if not typing_common.check_files(Gene_hisat2_linear_index_fnames): print >> sys.stderr, "Error: indexing HLA failed!" sys.exit(1) else: assert aligner == "bowtie2" and index_type == "linear" # Build Bowtie2 indexes based on the above information Gene_bowtie2_index_fnames = ["%s.%d.bt2" % (base_fname, i+1) for i in range(4)] Gene_bowtie2_index_fnames += ["%s.rev.%d.bt2" % (base_fname, i+1) for i in range(2)] if not typing_common.check_files(Gene_bowtie2_index_fnames): build_cmd = ["bowtie2-build", "%s_backbone.fa,%s_sequences.fa" % (base_fname, base_fname), base_fname] proc = subprocess.Popen(build_cmd, stdout=open("/dev/null", 'w')) proc.communicate() if not typing_common.check_files(Gene_bowtie2_index_fnames): print >> sys.stderr, "Error: indexing HLA failed!" sys.exit(1) # Read partial alleles from hla.data (temporary) partial_alleles = set() for line in open("IMGTHLA/hla.dat"): if not line.startswith("DE"): continue allele_name = line.split()[1][:-1] if allele_name.startswith("HLA-"): allele_name = allele_name[4:] gene = allele_name.split('*')[0] if line.find("partial") != -1: partial_alleles.add(allele_name) # Read HLA alleles (names and sequences) refGenes, refGene_loci = {}, {} for line in open("%s.ref" % base_fname): Gene_name, chr, left, right, length, exon_str, strand = line.strip().split() Gene_gene = Gene_name.split('*')[0] assert not Gene_gene in refGenes refGenes[Gene_gene] = Gene_name left, right = int(left), int(right) exons = [] for exon in exon_str.split(','): exon_left, exon_right = exon.split('-') exons.append([int(exon_left), int(exon_right)]) refGene_loci[Gene_gene] = [Gene_name, chr, left, right, exons] Genes = {} if len(locus_list) == 0: locus_list = refGene_loci.keys() read_Gene_alleles(base_fname + "_backbone.fa", Genes) read_Gene_alleles(base_fname + "_sequences.fa", Genes) # HLA gene alleles Gene_names = {} for Gene_gene, data in Genes.items(): Gene_names[Gene_gene] = list(data.keys()) # HLA gene allele lengths Gene_lengths = {} for Gene_gene, Gene_alleles in Genes.items(): Gene_lengths[Gene_gene] = {} for allele_name, seq in Gene_alleles.items(): Gene_lengths[Gene_gene][allele_name] = len(seq) # Read HLA variants, and link information Vars, Var_list = read_Gene_vars("%s.snp" % base_fname) Links = read_Gene_links("%s.link" % base_fname) # Test HLA typing test_list = [] if simulation: basic_test, pair_test = True, False if debug_instr: if "basic_test" in debug_instr: basic_test, pair_test = True, False else: basic_test, pair_test = False, True test_passed = {} test_list = [] genes = list(set(locus_list) & set(Gene_names.keys())) if basic_test: for gene in genes: Gene_gene_alleles = Gene_names[gene] for Gene_name in Gene_gene_alleles: if Gene_name.find("BACKBONE") != -1: continue test_list.append([[Gene_name]]) if pair_test: test_size = 500 allele_count = 2 for test_i in range(test_size): test_pairs = [] for gene in genes: Gene_gene_alleles = [] for allele in Gene_names[gene]: if allele.find("BACKBONE") != -1: continue if "full" in debug: if allele in partial_alleles: continue Gene_gene_alleles.append(allele) nums = [i for i in range(len(Gene_gene_alleles))] random.shuffle(nums) test_pairs.append(sorted([Gene_gene_alleles[nums[i]] for i in range(allele_count)])) test_list.append(test_pairs) if "test_list" in debug_instr: test_list = [[debug_instr["test_list"].split('-')]] for test_i in range(len(test_list)): if "test_id" in debug_instr: test_ids = debug_instr["test_id"].split('-') if str(test_i + 1) not in test_ids: continue print >> sys.stderr, "Test %d" % (test_i + 1), str(datetime.now()) test_locus_list = test_list[test_i] num_frag_list = typing_common.simulate_reads(Genes, base_fname, test_locus_list, Vars, Links, simulate_interval, read_len, fragment_len, perbase_errorrate, perbase_snprate, skip_fragment_regions) assert len(num_frag_list) == len(test_locus_list) for i_ in range(len(test_locus_list)): test_Gene_names = test_locus_list[i_] num_frag_list_i = num_frag_list[i_] assert len(num_frag_list_i) == len(test_Gene_names) for j_ in range(len(test_Gene_names)): test_Gene_name = test_Gene_names[j_] gene = test_Gene_name.split('*')[0] test_Gene_seq = Genes[gene][test_Gene_name] seq_type = "partial" if test_Gene_name in partial_alleles else "full" print >> sys.stderr, "\t%s - %d bp (%s sequence, %d pairs)" % (test_Gene_name, len(test_Gene_seq), seq_type, num_frag_list_i[j_]) if "single-end" in debug_instr: read_fname = ["%s_input_1.fa" % base_fname] else: read_fname = ["%s_input_1.fa" % base_fname, "%s_input_2.fa" % base_fname] fastq = False tmp_test_passed = typing(ex_path, simulation, base_fname, test_locus_list, partial, partial_alleles, refGenes, Genes, Gene_names, Gene_lengths, refGene_loci, Vars, Var_list, Links, aligners, num_editdist, assembly, output_base, error_correction, discordant, display_alleles, stranded_seq, fastq, read_fname, alignment_fname, num_frag_list, threads, best_alleles, verbose) for aligner_type, passed in tmp_test_passed.items(): if aligner_type in test_passed: test_passed[aligner_type] += passed else: test_passed[aligner_type] = passed print >> sys.stderr, "\t\tPassed so far: %d/%d (%.2f%%)" % (test_passed[aligner_type], test_i + 1, (test_passed[aligner_type] * 100.0 / (test_i + 1))) for aligner_type, passed in test_passed.items(): print >> sys.stderr, "%s:\t%d/%d passed (%.2f%%)" % (aligner_type, passed, len(test_list), passed * 100.0 / len(test_list)) else: # With real reads or BAMs print >> sys.stderr, "\t", ' '.join(locus_list) fastq = True typing(ex_path, simulation, base_fname, locus_list, partial, partial_alleles, refGenes, Genes, Gene_names, Gene_lengths, refGene_loci, Vars, Var_list, Links, aligners, num_editdist, assembly, output_base, error_correction, discordant, display_alleles, stranded_seq, fastq, read_fname, alignment_fname, [], threads, best_alleles, verbose) """ """ if __name__ == '__main__': parser = ArgumentParser( description='test HLA genotyping') parser.add_argument("--base", "--base-fname", dest="base_fname", type=str, default="hla", help="base filename for backbone HLA sequence, HLA variants, and HLA linking info (default: hla)") parser.add_argument("--locus-list", dest="locus_list", type=str, default="", help="A comma-separated list of HLA genes (default: empty, all HLA genes in IMGT/HLA database)") parser.add_argument('--no-partial', dest='partial', action='store_false', help='Include partial alleles (e.g. A_nuc.fasta)') parser.add_argument("--aligner-list", dest="aligners", type=str, default="hisat2.graph", help="A comma-separated list of aligners such as hisat2.graph,hisat2.linear,bowtie2.linear (default: hisat2.graph)") parser.add_argument("--reads", dest="read_fname", type=str, default="", help="Fastq read file name") parser.add_argument("--alignment", dest="alignment_fname", type=str, default="", help="BAM file name") parser.add_argument("-p", "--threads", dest="threads", type=int, default=1, help="Number of threads") parser.add_argument("--simulate-interval", dest="simulate_interval", type=int, default=10, help="Reads simulated at every these base pairs (default: 10)") parser.add_argument("--read-len", dest="read_len", type=int, default=100, help="Length of simulated reads (default: 100)") parser.add_argument("--fragment-len", dest="fragment_len", type=int, default=350, help="Length of fragments (default: 350)") parser.add_argument("--best-alleles", dest="best_alleles", action='store_true', help="") parser.add_argument("--random-seed", dest="random_seed", type=int, default=1, help="A seeding number for randomness (default: 1)") parser.add_argument("--num-editdist", dest="num_editdist", type=int, default=0, help="Maximum number of mismatches per read alignment to be considered (default: 0)") parser.add_argument("--perbase-errorrate", dest="perbase_errorrate", type=float, default=0.0, help="Per basepair error rate in percentage when simulating reads (default: 0.0)") parser.add_argument("--perbase-snprate", dest="perbase_snprate", type=float, default=0.0, help="Per basepair SNP rate in percentage when simulating reads (default: 0.0)") parser.add_argument("--skip-fragment-regions", dest="skip_fragment_regions", type=str, default="", help="A comma-separated list of regions from which no reads originate, e.g., 500-600,1200-1400 (default: None).") parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') parser.add_argument('--verbose-level', dest='verbose_level', type=int, default=0, help='also print some statistics to stderr (default: 0)') parser.add_argument("--debug", dest="debug", type=str, default="", help="e.g., test_id:10,read_id:10000,basic_test") parser.add_argument("--output-base", "--assembly-base", dest="output_base", type=str, default="assembly_graph", help="base file name (default: assembly_graph)") parser.add_argument("--no-assembly", dest="assembly", action="store_false", help="Perform assembly") parser.add_argument("--no-error-correction", dest="error_correction", action="store_false", help="Correct sequencing errors") parser.add_argument("--discordant", dest="discordant", action="store_true", help="Allow discordantly mapped pairs or singletons") parser.add_argument("--display-alleles", dest="display_alleles", type=str, default="", help="A comma-separated list of alleles to display in HTML (default: empty)") parser.add_argument("--stranded-seq", dest="stranded_seq", type=str, default="", help="Stranded-seq data (e.g.,: NA12892,ILMN_StrandSeq/SraRunInfo.txt") args = parser.parse_args() if args.locus_list == "": locus_list = [] else: locus_list = args.locus_list.split(',') if args.aligners == "": print >> sys.stderr, "Error: --aligners must be non-empty." sys.exit(1) args.aligners = args.aligners.split(',') for i in range(len(args.aligners)): args.aligners[i] = args.aligners[i].split('.') if args.read_fname: args.read_fname = args.read_fname.split(',') else: args.read_fname = [] if args.alignment_fname != "" and \ not os.path.exists(args.alignment_fname): print >> sys.stderr, "Error: %s doesn't exist." % args.alignment_fname sys.exit(1) if args.verbose and args.verbose_level == 0: args.verbose_level = 1 debug = {} if args.debug != "": for item in args.debug.split(','): if ':' in item: fields = item.split(':') assert len(fields) >= 2 key, value = fields[0], ':'.join(fields[1:]) debug[key] = value else: debug[item] = 1 if not args.partial: print >> sys.stderr, "Warning: --no-partial will be no longer supported!" if args.read_len * 2 > args.fragment_len: print >> sys.stderr, "Warning: fragment might be too short (%d)" % (args.fragment_len) skip_fragment_regions = [] if args.skip_fragment_regions != "": prev_left, prev_right = -1, -1 for region in args.skip_fragment_regions.split(','): left, right = region.split('-') left, right = int(left), int(right) assert left < right assert prev_right < left prev_left, prev_right = left, right skip_fragment_regions.append([left, right]) if args.display_alleles == "": display_alleles = [] else: display_alleles = args.display_alleles.split(',') if args.stranded_seq != "": stranded_seq = args.stranded_seq.split(',') if len(stranded_seq) != 2: print >> sys.stderr, "Error: --stranded-seq is incorrectly specified" sys.exit(1) else: stranded_seq = [] random.seed(args.random_seed) test_Gene_genotyping(args.base_fname, locus_list, args.partial, args.aligners, args.read_fname, args.alignment_fname, args.threads, args.simulate_interval, args.read_len, args.fragment_len, args.best_alleles, args.num_editdist, args.perbase_errorrate, args.perbase_snprate, skip_fragment_regions, args.assembly, args.output_base, args.error_correction, args.discordant, display_alleles, stranded_seq, args.verbose_level, debug) hisat2-2.2.1/evaluation/tests/HLA_novel/hisatgenotype_modules000077700000000000000000000000001370663763200315732../../../hisatgenotype_modulesustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/scripts/000077500000000000000000000000001370663763200215325ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/HLA_novel/scripts/run_extract_ILMN_HiSeqX.sh000077500000000000000000000007751370663763200265000ustar00rootroot00000000000000#!/bin/bash -l #SBATCH --job-name=infphio.HLA.ILMN.extract.genome #SBATCH --nodes=1 #SBATCH --cpus-per-task=8 #SBATCH --mem=80G #SBATCH --partition=shared #SBATCH --time=166:0:0 #SBATCH --workdir=/home-1/dkim136@jhu.edu/infphilo/hisat2/evaluation/tests/HLA_novel /home-1/dkim136@jhu.edu/infphilo/hisat2/evaluation/tests/HLA_novel/scripts/extract_reads.py --base-fname genotype_genome --reference-type genome --read-dir /home-1/dkim136@jhu.edu/ssalzbe1/users/infphilo/ILMN_HiSeqX --out-dir ILMN_HiSeqX -p 8 hisat2-2.2.1/evaluation/tests/genotype_genome/000077500000000000000000000000001370663763200214205ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/genotype_genome/hisatgenotype.py000077700000000000000000000000001370663763200307462../../../hisatgenotype.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/genotype_genome/hisatgenotype_build_genome.py000077700000000000000000000000001370663763200361702../../../hisatgenotype_build_genome.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/genotype_genome/hisatgenotype_extract_vars.py000077700000000000000000000000001370663763200363202../../../hisatgenotype_extract_vars.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/genotype_genome/hisatgenotype_locus.py000077700000000000000000000000001370663763200333602../../../hisatgenotype_locus.pyustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/genotype_genome/hisatgenotype_modules000077700000000000000000000000001370663763200331502../../../hisatgenotype_modulesustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/genotype_genome/hisatgenotype_prev.py000077500000000000000000001346551370663763200257320ustar00rootroot00000000000000#!/usr/bin/env python # # Copyright 2016, Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # import sys, os, subprocess, re import inspect, random import math from argparse import ArgumentParser, FileType import hisatgenotype_typing_common as typing_common """ Align reads, and sort the alignments into a BAM file """ def align_reads(base_fname, read_fnames, fastq, threads, verbose): aligner_cmd = ["hisat2", "--no-unal", "-p", str(threads)] # aligner_cmd += ["--mm"] aligner_cmd += ["-x", "%s" % base_fname] assert len(read_fnames) > 0 if not fastq: aligner_cmd += ["-f"] single = len(read_fnames) == 1 if single: aligner_cmd += ["-U", read_fnames[0]] else: aligner_cmd += ["-1", read_fnames[0], "-2", read_fnames[1]] print >> sys.stderr, "Aligning %s to %s ..." % (' '.join(read_fnames), base_fname) if verbose: print >> sys.stderr, "\t%s" % (' '.join(aligner_cmd)) align_proc = subprocess.Popen(aligner_cmd, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) sambam_cmd = ["samtools", "view", "-bS", "-"] sambam_proc = subprocess.Popen(sambam_cmd, stdin=align_proc.stdout, stdout=open("hla_output_unsorted.bam", 'w'), stderr=open("/dev/null", 'w')) sambam_proc.communicate() print >> sys.stderr, "Sorting %s ..." % "TBD" bamsort_cmd = ["samtools", "sort", "--threads", str(threads), "hla_output_unsorted.bam"] bamsort_proc = subprocess.Popen(bamsort_cmd, stdout=open("hla_output.bam", 'w'), stderr=open("/dev/null", 'w')) bamsort_proc.communicate() print >> sys.stderr, "Indexing %s ..." % "TBD" bamindex_cmd = ["samtools", "index", "hla_output.bam"] bamindex_proc = subprocess.Popen(bamindex_cmd, stderr=open("/dev/null", 'w')) bamindex_proc.communicate() os.remove("hla_output_unsorted.bam") """ """ def genotype(base_fname, fastq, read_fnames, threads, num_mismatch, verbose, daehwan_debug): # Load genomic sequences chr_dic, chr_names, chr_full_names = typing_common.read_genome(open("%s.fa" % base_fname)) # variants, backbone sequence, and other sequeces genotype_fnames = ["%s.fa" % base_fname, "%s.gene" % base_fname, "%s.snp" % base_fname, "%s.index.snp" % base_fname, "%s.haplotype" % base_fname, "%s.link" % base_fname, "%s.coord" % base_fname, "%s.clnsig" % base_fname] # hisat2 graph index files genotype_fnames += ["%s.%d.ht2" % (base_fname, i+1) for i in range(8)] if not typing_common.check_files(genotype_fnames): print >> sys.stderr, "Error: some of the following files are missing!" for fname in genotype_fnames: print >> sys.stderr, "\t%s" % fname sys.exit(1) # Align reads, and sort the alignments into a BAM file align_reads(base_fname, read_fnames, fastq, threads, verbose) # Read HLA alleles (names and sequences) genes, gene_loci, gene_seqs = {}, {}, {} for line in open("%s.gene" % base_fname): family, allele_name, chr, left, right = line.strip().split() gene_name = "%s-%s" % (family, allele_name.split('*')[0]) assert gene_name not in genes genes[gene_name] = allele_name left, right = int(left), int(right) """ exons = [] for exon in exon_str.split(','): exon_left, exon_right = exon.split('-') exons.append([int(exon_left), int(exon_right)]) """ gene_loci[gene_name] = [allele_name, chr, left, right] assert chr in chr_dic chr_seq = chr_dic[chr] assert left < right assert right < len(chr_seq) gene_seqs[gene_name] = chr_dic[chr][left:right+1] # Read link information Links, var_genes, allele_vars = {}, {}, {} for line in open("%s.link" % base_fname): var_id, alleles = line.strip().split('\t') alleles = alleles.split() assert not var_id in Links Links[var_id] = alleles for allele in alleles: if allele not in allele_vars: allele_vars[allele] = set() allele_vars[allele].add(var_id) gene_name = "HLA-%s" % (allele.split('*')[0]) var_genes[var_id] = gene_name # gene alleles allele_names = {} for gene_name in genes.keys(): if gene_name not in allele_names: allele_names[gene_name] = [] gene_name2 = gene_name.split('-')[1] for allele_name in allele_vars.keys(): allele_name1 = allele_name.split('*')[0] if gene_name2 == allele_name1: allele_names[gene_name].append(allele_name) # Read HLA variants, and link information Vars, Var_list = {}, {} for line in open("%s.snp" % base_fname): var_id, var_type, chr, pos, data = line.strip().split('\t') pos = int(pos) # daehwan - for debugging purposes if var_id not in var_genes: continue assert var_id in var_genes gene_name = var_genes[var_id] if not gene_name in Vars: Vars[gene_name] = {} assert not gene_name in Var_list Var_list[gene_name] = [] assert not var_id in Vars[gene_name] Vars[gene_name][var_id] = [var_type, pos, data] Var_list[gene_name].append([pos, var_id]) for gene_name, in_var_list in Var_list.items(): Var_list[gene_name] = sorted(in_var_list) def lower_bound(Var_list, pos): low, high = 0, len(Var_list) while low < high: m = (low + high) / 2 m_pos = Var_list[m][0] if m_pos < pos: low = m + 1 elif m_pos > pos: high = m else: assert m_pos == pos while m > 0: if Var_list[m-1][0] < pos: break m -= 1 return m return low # HLA gene allele lengths """ HLA_lengths = {} for HLA_gene, HLA_alleles in HLAs.items(): HLA_lengths[HLA_gene] = {} for allele_name, seq in HLA_alleles.items(): HLA_lengths[HLA_gene][allele_name] = len(seq) """ # Cigar regular expression cigar_re = re.compile('\d+\w') test_list = [[sorted(genes.keys())]] for test_i in range(len(test_list)): test_HLA_list = test_list[test_i] for test_HLA_names in test_HLA_list: print >> sys.stderr, "\t%s" % (test_HLA_names) for gene in test_HLA_names: ref_allele = genes[gene] ref_seq = gene_seqs[gene] # ref_exons = refHLA_loci[gene][-1] # Read alignments alignview_cmd = ["samtools", "view"] alignview_cmd += ["hla_output.bam"] base_locus = 0 _, chr, left, right = gene_loci[gene] base_locus = left alignview_cmd += ["%s:%d-%d" % (chr, left + 1, right + 1)] bamview_proc = subprocess.Popen(alignview_cmd, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) sort_read_cmd = ["sort", "-k", "1", "-n"] alignview_proc = subprocess.Popen(sort_read_cmd, stdin=bamview_proc.stdout, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) # Count alleles HLA_counts, HLA_cmpt = {}, {} coverage = [0 for i in range(len(ref_seq) + 1)] num_reads, total_read_len = 0, 0 prev_read_id = None prev_exon = False for line in alignview_proc.stdout: cols = line.strip().split() read_id, flag, chr, pos, mapQ, cigar_str = cols[:6] origin_read_id = read_id if read_id.find('|') != -1: tmp_read_id = read_id.split('|')[0] try: read_id = int(tmp_read_id) except ValueError: None read_seq, qual = cols[9], cols[10] num_reads += 1 total_read_len += len(read_seq) flag, pos = int(flag), int(pos) pos -= 1 if pos < 0: continue if flag & 0x4 != 0: continue NM, Zs, MD = "", "", "" for i in range(11, len(cols)): col = cols[i] if col.startswith("Zs"): Zs = col[5:] elif col.startswith("MD"): MD = col[5:] elif col.startswith("NM"): NM = int(col[5:]) if NM > num_mismatch: continue # daehwan - for debugging purposes debug = False if read_id in ["2339"] and False: debug = True print "read_id: %s)" % read_id, pos, cigar_str, "NM:", NM, MD, Zs print " ", read_seq vars = [] if Zs: vars = Zs.split(',') assert MD != "" MD_str_pos, MD_len = 0, 0 read_pos, left_pos = 0, pos right_pos = left_pos cigars = cigar_re.findall(cigar_str) cigars = [[cigar[-1], int(cigar[:-1])] for cigar in cigars] cmp_list = [] for i in range(len(cigars)): cigar_op, length = cigars[i] if cigar_op == 'M': first = True MD_len_used = 0 while True: if not first or MD_len == 0: if MD[MD_str_pos].isdigit(): num = int(MD[MD_str_pos]) MD_str_pos += 1 while MD_str_pos < len(MD): if MD[MD_str_pos].isdigit(): num = num * 10 + int(MD[MD_str_pos]) MD_str_pos += 1 else: break MD_len += num # Insertion or full match followed if MD_len >= length: MD_len -= length cmp_list.append(["match", right_pos + MD_len_used, length - MD_len_used]) break first = False read_base = read_seq[read_pos + MD_len] MD_ref_base = MD[MD_str_pos] MD_str_pos += 1 assert MD_ref_base in "ACGT" cmp_list.append(["match", right_pos + MD_len_used, MD_len - MD_len_used]) cmp_list.append(["mismatch", right_pos + MD_len, 1]) MD_len_used = MD_len + 1 MD_len += 1 # Full match if MD_len == length: MD_len = 0 break elif cigar_op == 'I': cmp_list.append(["insertion", right_pos, length]) elif cigar_op == 'D': if MD[MD_str_pos] == '0': MD_str_pos += 1 assert MD[MD_str_pos] == '^' MD_str_pos += 1 while MD_str_pos < len(MD): if not MD[MD_str_pos] in "ACGT": break MD_str_pos += 1 cmp_list.append(["deletion", right_pos, length]) elif cigar_op == 'S': cmp_list.append(["soft", right_pos, length]) else: assert cigar_op == 'N' cmp_list.append(["intron", right_pos, length]) if cigar_op in "MND": right_pos += length if cigar_op in "MIS": read_pos += length """ exon = False for exon in ref_exons: exon_left, exon_right = exon if right_pos <= exon_left or pos > exon_right: continue else: exon = True break """ if left_pos < base_locus or \ right_pos - base_locus > len(ref_seq): continue def add_stat(HLA_cmpt, HLA_counts, HLA_count_per_read, exon = True): max_count = max(HLA_count_per_read.values()) cur_cmpt = set() for allele, count in HLA_count_per_read.items(): if count < max_count: continue """ if allele in exclude_allele_list: continue """ cur_cmpt.add(allele) if not allele in HLA_counts: HLA_counts[allele] = 1 else: HLA_counts[allele] += 1 if len(cur_cmpt) == 0: return # daehwan - for debugging purposes alleles = ["", ""] # alleles = ["B*40:304", "B*40:02:01"] allele1_found, allele2_found = False, False for allele, count in HLA_count_per_read.items(): if count < max_count: continue if allele == alleles[0]: allele1_found = True elif allele == alleles[1]: allele2_found = True if allele1_found != allele2_found: print alleles[0], HLA_count_per_read[alleles[0]] print alleles[1], HLA_count_per_read[alleles[1]] if allele1_found: print ("%s\tread_id %s - %d vs. %d]" % (alleles[0], prev_read_id, max_count, HLA_count_per_read[alleles[1]])) else: print ("%s\tread_id %s - %d vs. %d]" % (alleles[1], prev_read_id, max_count, HLA_count_per_read[alleles[0]])) print read_seq cur_cmpt = sorted(list(cur_cmpt)) cur_cmpt = '-'.join(cur_cmpt) add = 1 """ if partial and not exon: add *= 0.2 """ if not cur_cmpt in HLA_cmpt: HLA_cmpt[cur_cmpt] = add else: HLA_cmpt[cur_cmpt] += add if read_id != prev_read_id: if prev_read_id != None: add_stat(HLA_cmpt, HLA_counts, HLA_count_per_read, prev_exon) HLA_count_per_read = {} for HLA_name in allele_names[gene]: if HLA_name.find("BACKBONE") != -1: continue HLA_count_per_read[HLA_name] = 0 def add_count(var_id, add): assert var_id in Links alleles = Links[var_id] for allele in alleles: if allele.find("BACKBONE") != -1: continue HLA_count_per_read[allele] += add # daehwan - for debugging purposes if debug: if allele in ["DQA1*05:05:01:01", "DQA1*05:05:01:02"]: print allele, add, var_id # Decide which allele(s) a read most likely came from # also sanity check - read length, cigar string, and MD string for var_id, data in Vars[gene].items(): var_type, var_pos, var_data = data if var_type != "deletion": continue if left_pos >= var_pos and right_pos <= var_pos + int(var_data): add_count(var_id, -1) ref_pos, read_pos, cmp_cigar_str, cmp_MD = left_pos, 0, "", "" cigar_match_len, MD_match_len = 0, 0 for cmp in cmp_list: type = cmp[0] length = cmp[2] if type == "match": var_idx = lower_bound(Var_list[gene], ref_pos) while var_idx < len(Var_list[gene]): var_pos, var_id = Var_list[gene][var_idx] if ref_pos + length <= var_pos: break if ref_pos <= var_pos: var_type, _, var_data = Vars[gene][var_id] if var_type == "insertion": if ref_pos < var_pos and ref_pos + length > var_pos + len(var_data): add_count(var_id, -1) # daehwan - for debugging purposes if debug: print cmp, var_id, Links[var_id] elif var_type == "deletion": del_len = int(var_data) if ref_pos < var_pos and ref_pos + length > var_pos + del_len: # daehwan - for debugging purposes if debug: print cmp, var_id, Links[var_id], -1, Vars[gene][var_id] # Check if this might be one of the two tandem repeats (the same left coordinate) cmp_left, cmp_right = cmp[1], cmp[1] + cmp[2] test1_seq1 = ref_seq[cmp_left-base_locus:cmp_right-base_locus] test1_seq2 = ref_seq[cmp_left-base_locus:var_pos-base_locus] + ref_seq[var_pos + del_len - base_locus:cmp_right + del_len - base_locus] # Check if this happens due to small repeats (the same right coordinate - e.g. 19 times of TTTC in DQA1*05:05:01:02) cmp_left -= read_pos cmp_right += (len(read_seq) - read_pos - cmp[2]) test2_seq1 = ref_seq[cmp_left+int(var_data)-base_locus:cmp_right-base_locus] test2_seq2 = ref_seq[cmp_left-base_locus:var_pos-base_locus] + ref_seq[var_pos+int(var_data)-base_locus:cmp_right-base_locus] if test1_seq1 != test1_seq2 and test2_seq1 != test2_seq2: add_count(var_id, -1) else: if debug: print cmp, var_id, Links[var_id], -1 add_count(var_id, -1) var_idx += 1 read_pos += length ref_pos += length cigar_match_len += length MD_match_len += length elif type == "mismatch": read_base = read_seq[read_pos] var_idx = lower_bound(Var_list[gene], ref_pos) while var_idx < len(Var_list[gene]): var_pos, var_id = Var_list[gene][var_idx] if ref_pos < var_pos: break if ref_pos == var_pos: var_type, _, var_data = Vars[gene][var_id] if var_type == "single": if var_data == read_base: # daehwan - for debugging purposes if debug: print cmp, var_id, 1, var_data, read_base, Links[var_id] # daehwan - for debugging purposes if False: read_qual = ord(qual[read_pos]) add_count(var_id, (read_qual - 60) / 60.0) else: add_count(var_id, 1) # daehwan - check out if this routine is appropriate # else: # add_count(var_id, -1) var_idx += 1 cmp_MD += ("%d%s" % (MD_match_len, ref_seq[ref_pos-base_locus])) MD_match_len = 0 cigar_match_len += 1 read_pos += 1 ref_pos += 1 elif type == "insertion": ins_seq = read_seq[read_pos:read_pos+length] var_idx = lower_bound(Var_list[gene], ref_pos) # daehwan - for debugging purposes if debug: print left_pos, cigar_str, MD, vars print ref_pos, ins_seq, Var_list[gene][var_idx], Vars[gene][Var_list[gene][var_idx][1]] # sys.exit(1) while var_idx < len(Var_list[gene]): var_pos, var_id = Var_list[gene][var_idx] if ref_pos < var_pos: break if ref_pos == var_pos: var_type, _, var_data = Vars[gene][var_id] if var_type == "insertion": if var_data == ins_seq: # daehwan - for debugging purposes if debug: print cmp, var_id, 1, Links[var_id] add_count(var_id, 1) var_idx += 1 if cigar_match_len > 0: cmp_cigar_str += ("%dM" % cigar_match_len) cigar_match_len = 0 read_pos += length cmp_cigar_str += ("%dI" % length) elif type == "deletion": del_len = length # Deletions can be shifted bidirectionally temp_ref_pos = ref_pos while temp_ref_pos > 0: last_bp = ref_seq[temp_ref_pos + del_len - 1 - base_locus] prev_bp = ref_seq[temp_ref_pos - 1 - base_locus] if last_bp != prev_bp: break temp_ref_pos -= 1 var_idx = lower_bound(Var_list[gene], temp_ref_pos) while var_idx < len(Var_list[gene]): var_pos, var_id = Var_list[gene][var_idx] if temp_ref_pos < var_pos: first_bp = ref_seq[temp_ref_pos - base_locus] next_bp = ref_seq[temp_ref_pos + del_len - base_locus] if first_bp == next_bp: temp_ref_pos += 1 continue else: break if temp_ref_pos == var_pos: var_type, _, var_data = Vars[gene][var_id] if var_type == "deletion": var_len = int(var_data) if var_len == length: if debug: print cmp, var_id, 1, Links[var_id] print ref_seq[var_pos - 10-base_locus:var_pos-base_locus], ref_seq[var_pos-base_locus:var_pos+int(var_data)-base_locus], ref_seq[var_pos+int(var_data)-base_locus:var_pos+int(var_data)+10-base_locus] add_count(var_id, 1) var_idx += 1 if cigar_match_len > 0: cmp_cigar_str += ("%dM" % cigar_match_len) cigar_match_len = 0 cmp_MD += ("%d" % MD_match_len) MD_match_len = 0 cmp_cigar_str += ("%dD" % length) cmp_MD += ("^%s" % ref_seq[ref_pos-base_locus:ref_pos+length-base_locus]) ref_pos += length elif type == "soft": if cigar_match_len > 0: cmp_cigar_str += ("%dM" % cigar_match_len) cigar_match_len = 0 read_pos += length cmp_cigar_str += ("%dS" % length) else: assert type == "intron" if cigar_match_len > 0: cmp_cigar_str += ("%dM" % cigar_match_len) cigar_match_len = 0 cmp_cigar_str += ("%dN" % length) ref_pos += length if cigar_match_len > 0: cmp_cigar_str += ("%dM" % cigar_match_len) cmp_MD += ("%d" % MD_match_len) if read_pos != len(read_seq) or \ cmp_cigar_str != cigar_str or \ cmp_MD != MD: print >> sys.stderr, "Error:", cigar_str, MD print >> sys.stderr, "\tcomputed:", cmp_cigar_str, cmp_MD print >> sys.stderr, "\tcmp list:", cmp_list assert False prev_read_id = read_id # prev_exon = exon if num_reads <= 0: continue if prev_read_id != None: add_stat(HLA_cmpt, HLA_counts, HLA_count_per_read) HLA_counts = [[allele, count] for allele, count in HLA_counts.items()] def HLA_count_cmp(a, b): if a[1] != b[1]: return b[1] - a[1] assert a[0] != b[0] if a[0] < b[0]: return -1 else: return 1 HLA_counts = sorted(HLA_counts, cmp=HLA_count_cmp) for count_i in range(len(HLA_counts)): count = HLA_counts[count_i] print >> sys.stderr, "\t\t\t\t%d %s (count: %d)" % (count_i + 1, count[0], count[1]) if count_i >= 9: break print >> sys.stderr def normalize(prob): total = sum(prob.values()) for allele, mass in prob.items(): prob[allele] = mass / total def normalize2(prob, length): total = 0 for allele, mass in prob.items(): assert allele in length total += (mass / length[allele]) for allele, mass in prob.items(): assert allele in length prob[allele] = mass / length[allele] / total def prob_diff(prob1, prob2): diff = 0.0 for allele in prob1.keys(): if allele in prob2: diff += abs(prob1[allele] - prob2[allele]) else: diff += prob1[allele] return diff def HLA_prob_cmp(a, b): if a[1] != b[1]: if a[1] < b[1]: return 1 else: return -1 assert a[0] != b[0] if a[0] < b[0]: return -1 else: return 1 HLA_prob, HLA_prob_next = {}, {} for cmpt, count in HLA_cmpt.items(): alleles = cmpt.split('-') for allele in alleles: if allele not in HLA_prob: HLA_prob[allele] = 0.0 HLA_prob[allele] += (float(count) / len(alleles)) """ assert gene in HLA_lengths HLA_length = HLA_lengths[gene] """ HLA_length = {} # normalize2(HLA_prob, HLA_length) normalize(HLA_prob) def next_prob(HLA_cmpt, HLA_prob, HLA_length): HLA_prob_next = {} for cmpt, count in HLA_cmpt.items(): alleles = cmpt.split('-') alleles_prob = 0.0 for allele in alleles: assert allele in HLA_prob alleles_prob += HLA_prob[allele] for allele in alleles: if allele not in HLA_prob_next: HLA_prob_next[allele] = 0.0 HLA_prob_next[allele] += (float(count) * HLA_prob[allele] / alleles_prob) # normalize2(HLA_prob_next, HLA_length) normalize(HLA_prob_next) return HLA_prob_next diff, iter = 1.0, 0 while diff > 0.0001 and iter < 1000: HLA_prob_next = next_prob(HLA_cmpt, HLA_prob, HLA_length) diff = prob_diff(HLA_prob, HLA_prob_next) HLA_prob = HLA_prob_next iter += 1 """ for allele, prob in HLA_prob.items(): allele_len = len(HLAs[gene][allele]) HLA_prob[allele] /= float(allele_len) normalize(HLA_prob) """ HLA_prob = [[allele, prob] for allele, prob in HLA_prob.items()] HLA_prob = sorted(HLA_prob, cmp=HLA_prob_cmp) success = [False for i in range(len(test_HLA_names))] found_list = [False for i in range(len(test_HLA_names))] for prob_i in range(len(HLA_prob)): prob = HLA_prob[prob_i] print >> sys.stderr, "\t\t\t\t%d ranked %s (abundance: %.2f%%)" % (prob_i + 1, prob[0], prob[1] * 100.0) if prob_i >= 9: break print >> sys.stderr """ if len(test_HLA_names) == 2: HLA_prob, HLA_prob_next = {}, {} for cmpt, count in HLA_cmpt.items(): alleles = cmpt.split('-') for allele1 in alleles: for allele2 in HLA_names[gene]: if allele1 < allele2: allele_pair = "%s-%s" % (allele1, allele2) else: allele_pair = "%s-%s" % (allele2, allele1) if not allele_pair in HLA_prob: HLA_prob[allele_pair] = 0.0 HLA_prob[allele_pair] += (float(count) / len(alleles)) if len(HLA_prob) <= 0: continue # Choose top allele pairs def choose_top_alleles(HLA_prob): HLA_prob_list = [[allele_pair, prob] for allele_pair, prob in HLA_prob.items()] HLA_prob_list = sorted(HLA_prob_list, cmp=HLA_prob_cmp) HLA_prob = {} best_prob = HLA_prob_list[0][1] for i in range(len(HLA_prob_list)): allele_pair, prob = HLA_prob_list[i] if prob * 2 <= best_prob: break HLA_prob[allele_pair] = prob normalize(HLA_prob) return HLA_prob HLA_prob = choose_top_alleles(HLA_prob) def next_prob(HLA_cmpt, HLA_prob): HLA_prob_next = {} for cmpt, count in HLA_cmpt.items(): alleles = cmpt.split('-') prob = 0.0 for allele in alleles: for allele_pair in HLA_prob.keys(): if allele in allele_pair: prob += HLA_prob[allele_pair] for allele in alleles: for allele_pair in HLA_prob.keys(): if not allele in allele_pair: continue if allele_pair not in HLA_prob_next: HLA_prob_next[allele_pair] = 0.0 HLA_prob_next[allele_pair] += (float(count) * HLA_prob[allele_pair] / prob) normalize(HLA_prob_next) return HLA_prob_next diff, iter = 1.0, 0 while diff > 0.0001 and iter < 1000: HLA_prob_next = next_prob(HLA_cmpt, HLA_prob) diff = prob_diff(HLA_prob, HLA_prob_next) HLA_prob = HLA_prob_next HLA_prob = choose_top_alleles(HLA_prob) iter += 1 HLA_prob = [[allele_pair, prob] for allele_pair, prob in HLA_prob.items()] HLA_prob = sorted(HLA_prob, cmp=HLA_prob_cmp) success = [False] for prob_i in range(len(HLA_prob)): allele_pair, prob = HLA_prob[prob_i] allele1, allele2 = allele_pair.split('-') if best_alleles and prob_i < 1: print >> sys.stdout, "PairModel %s (abundance: %.2f%%)" % (allele_pair, prob * 100.0) if simulation: if allele1 in test_HLA_names and allele2 in test_HLA_names: rank_i = prob_i while rank_i > 0: if HLA_prob[rank_i-1][1] == prob: rank_i -= 1 else: break print >> sys.stderr, "\t\t\t*** %d ranked %s (abundance: %.2f%%)" % (rank_i + 1, allele_pair, prob * 100.0) if rank_i == 0: success[0] = True break print >> sys.stderr, "\t\t\t\t%d ranked %s (abundance: %.2f%%)" % (prob_i + 1, allele_pair, prob * 100.0) if not simulation and prob_i >= 9: break print >> sys.stderr """ # Read variants with clinical significance clnsigs = {} for line in open("%s.clnsig" % base_fname): var_id, var_gene, var_clnsig = line.strip().split('\t') clnsigs[var_id] = [var_gene, var_clnsig] vars, Var_list = {}, {} for line in open("%s.snp" % base_fname): var_id, type, chr, left, data = line.strip().split() if var_id not in clnsigs: continue left = int(left) if type == "deletion": data = int(data) vars[var_id] = [chr, left, type, data] if chr not in Var_list: Var_list[chr] = [] Var_list[chr].append([left, var_id]) var_counts = {} # Read alignments alignview_cmd = ["samtools", "view", "hla_output.bam"] bamview_proc = subprocess.Popen(alignview_cmd, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) for line in bamview_proc.stdout: cols = line.strip().split() read_id, flag, chr, pos, mapQ, cigar_str = cols[:6] read_seq, qual = cols[9], cols[10] flag, pos = int(flag), int(pos) pos -= 1 if pos < 0: continue if flag & 0x4 != 0: continue if chr not in Var_list: continue assert chr in chr_dic chr_seq = chr_dic[chr] NM, Zs, MD, NH = "", "", "", "" for i in range(11, len(cols)): col = cols[i] if col.startswith("Zs"): Zs = col[5:] elif col.startswith("MD"): MD = col[5:] elif col.startswith("NM"): NM = int(col[5:]) elif col.startswith("NH"): NH = int(col[5:]) assert NH != "" NH = int(NH) if NH > 1: continue if NM > num_mismatch: continue read_vars = [] if Zs: read_vars = Zs.split(',') for read_var in read_vars: _, _, var_id = read_var.split('|') if var_id not in clnsigs: continue if var_id not in var_counts: var_counts[var_id] = [1, 0] else: var_counts[var_id][0] += 1 assert MD != "" MD_str_pos, MD_len = 0, 0 read_pos, left_pos = 0, pos right_pos = left_pos cigars = cigar_re.findall(cigar_str) cigars = [[cigar[-1], int(cigar[:-1])] for cigar in cigars] cmp_list = [] for i in range(len(cigars)): cigar_op, length = cigars[i] if cigar_op == 'M': chr_var_list = Var_list[chr] var_idx = lower_bound(chr_var_list, right_pos) while var_idx < len(chr_var_list): var_pos, var_id = chr_var_list[var_idx] if var_pos >= right_pos + length: break if var_pos >= right_pos: assert var_id in vars _, _, var_type, var_data = vars[var_id] contradict = False if var_type == "single": contradict = (read_seq[read_pos + var_pos - right_pos] == chr_seq[var_pos]) elif var_type == "insertion": contradict = (right_pos < var_pos) else: contradict = True if contradict: if var_id not in var_counts: var_counts[var_id] = [0, 1] else: var_counts[var_id][1] += 1 var_idx += 1 if cigar_op in "MND": right_pos += length if cigar_op in "MIS": read_pos += length for var_id, counts in var_counts.items(): if counts[0] < 2: # or counts[0] * 3 < counts[1]: continue assert var_id in vars var_chr, var_left, var_type, var_data = vars[var_id] assert var_id in clnsigs var_gene, var_clnsig = clnsigs[var_id] print >> sys.stderr, "\t\t\t%s %s: %s:%d %s %s (%s): %d-%d" % \ (var_gene, var_id, var_chr, var_left, var_type, var_data, var_clnsig, counts[0], counts[1]) """ """ if __name__ == '__main__': parser = ArgumentParser( description='HISAT2 genotyping') parser.add_argument("--base", "--base-name", dest="base_fname", type=str, default="genotype_genome", help="base filename for genotype genome") parser.add_argument('-f', dest='fastq', action='store_false', help='FASTA file') parser.add_argument("-U", dest="read_fname_U", type=str, default="", help="filename for single-end reads") parser.add_argument("-1", dest="read_fname_1", type=str, default="", help="filename for paired-end reads") parser.add_argument("-2", dest="read_fname_2", type=str, default="", help="filename for paired-end reads") parser.add_argument("-p", "--threads", dest="threads", type=int, default=1, help="Number of threads") parser.add_argument("--num-editdist", dest="num_editdist", type=int, default=2, help="Maximum number of mismatches per read alignment to be considered (default: 2)") parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') parser.add_argument("--daehwan-debug", dest="daehwan_debug", type=str, default="", help="e.g., test_id:10,read_id:10000,basic_test") args = parser.parse_args() daehwan_debug = {} if args.daehwan_debug != "": for item in args.daehwan_debug.split(','): if ':' in item: key, value = item.split(':') daehwan_debug[key] = value else: daehwan_debug[item] = 1 if args.read_fname_U != "": read_fnames = [args.read_fname_U] else: if args.read_fname_1 == "" or args.read_fname_2 == "": print >> sys.stderr, "Error: please specify read file names correctly: -U or -1 and -2" sys.exit(1) read_fnames = [args.read_fname_1, args.read_fname_2] random.seed(1) genotype(args.base_fname, args.fastq, read_fnames, args.threads, args.num_editdist, args.verbose, daehwan_debug) hisat2-2.2.1/evaluation/tests/genotype_genome/paper_sensitivity/000077500000000000000000000000001370663763200252015ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/genotype_genome/paper_sensitivity/sensitivity.py000077500000000000000000000137121370663763200301540ustar00rootroot00000000000000#!/usr/bin/env python # # Copyright 2017, Daehwan Kim # # This file is part of HISAT-genotype. # # HISAT-genotype is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT-genotype is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT-genotype. If not, see . # import sys, os, subprocess, re, resource import inspect import random import glob from argparse import ArgumentParser, FileType """ """ if __name__ == '__main__': aligners = [["hisat2", "graph"], ["hisat2", "linear"], ["bowtie2", "linear"]] genes = ["A", "B", "C", "DQA1", "DQB1", "DRB1"] samples = ["NA12878", "LP6005041-DNA_A01", "LP6005045-DNA_D07"] for sample in samples: for aligner, type in aligners: sample_dir = "%s_%s" % (sample, aligner) if aligner == "hisat2": sample_dir += (".%s" % type) if not os.path.exists(sample_dir): continue fq_fnames = glob.glob("%s/*.fq.gz" % sample_dir) assert len(fq_fnames) == 2 regions, region_loci, region_count, region_read1_count, region_read2_count = {}, {}, {}, {}, {} for line in open("%s.locus" % ("genotype_genome" if type == "graph" else "genotype_genome.linear")): family, allele_name, chr, left, right = line.strip().split()[:5] # DK - debugging purposes if family != "HLA": continue region_name = "%s-%s" % (family, allele_name.split('*')[0]) assert region_name not in regions regions[region_name] = allele_name left, right = int(left), int(right) if chr not in region_loci: region_loci[chr] = {} region_loci[chr][region_name] = [allele_name, chr, left, right] aligner_cmd = [aligner] aligner_cmd += ["-x", "genotype_genome" if type == "graph" else "genotype_genome.linear"] if aligner == "hisat2": aligner_cmd += ["--no-spliced-alignment"] aligner_cmd += ["-X", "1000"] aligner_cmd += ["-1", fq_fnames[0], "-2", fq_fnames[1]] # print >> sys.stderr, "Running:", ' '.join(aligner_cmd) print sample, aligner, type align_proc = subprocess.Popen(aligner_cmd, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) prev_read_name, extract, read1_extract, read2_extract, read1_first, read2_first = "", set(), set(), set(), True, True for line in align_proc.stdout: if line.startswith('@'): continue line = line.strip() cols = line.split() read_name, flag, chr, pos, mapQ, cigar, _, _, _, read, qual = cols[:11] flag, pos = int(flag), int(pos) - 1 strand = '-' if flag & 0x10 else '+' AS, XS, NH = "", "", "" for i in range(11, len(cols)): col = cols[i] if col.startswith("AS"): AS = int(col[5:]) elif col.startswith("XS"): XS = int(col[5:]) elif col.startswith("NH"): NH = int(col[5:]) if read_name != prev_read_name: for region in extract: if region not in region_count: region_count[region] = 0 region_count[region] += 1 for region in read1_extract: if region not in region_read1_count: region_read1_count[region] = 0 region_read1_count[region] += 1 for region in read2_extract: if region not in region_read2_count: region_read2_count[region] = 0 region_read2_count[region] += 1 prev_read_name, extract, read1_extract, read2_extract, read1_first, read2_first = "", set(), set(), set(), True, True if ((aligner == "hisat2" and NH == 1) or (aligner == "bowtie2" and AS > XS and read1_first if flag & 0x40 else read2_first)): if chr in region_loci: for region, loci in region_loci[chr].items(): _, _, loci_left, loci_right = loci # there might be a different candidate region for each of left and right reads if pos >= loci_left and pos < loci_right: extract.add(region) if flag & 0x40: read1_extract.add(region) else: read2_extract.add(region) break if flag & 0x40: # left read read1_first = False else: assert flag & 0x80 # right read read2_first = False prev_read_name = read_name for gene in genes: gene = "HLA-" + gene if gene not in region_count: continue print "\t%s pair: %d, left+right: %d" % (gene, region_count[gene], region_read1_count[gene] + region_read2_count[gene]) hisat2-2.2.1/evaluation/tests/one_snp_test/000077500000000000000000000000001370663763200207345ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/one_snp_test/evaluate_one_snp_reads.py000077500000000000000000000216321370663763200260220ustar00rootroot00000000000000#!/usr/bin/env python import sys, os, subprocess import multiprocessing import string, re import platform from datetime import datetime, date, time import copy from argparse import ArgumentParser, FileType """ """ def evaluate(read_fname, verbose): aligners = [ ["hisat2", "", "", ""], ["hisat2", "", "snp", ""], ["bowtie2", "", "", ""], ] num_threads = 3 cwd = os.getcwd() genome = "genome" align_stat = [] # for paired in [False, True]: for paired in [False]: base_fname = "common_snp_reads" type_sam_fname = base_fname + ".sam" type_read1_fname = base_fname + "_1.fa" type_read2_fname = base_fname + "_2.fa" type_read1_fname = read_fname def get_aligner_version(aligner, version): version = "" if aligner == "hisat2" or \ aligner == "bowtie2": if version: cmd = ["%s_%s/%s" % (aligner, version, aligner)] else: cmd = ["%s/%s" % (aligner_bin_base, aligner)] cmd += ["--version"] cmd_process = subprocess.Popen(cmd, stdout=subprocess.PIPE) version = cmd_process.communicate()[0][:-1].split("\n")[0] version = version.split()[-1] elif aligner == "star": version = "2.4.2a" elif aligner == "gsnap": cmd = ["%s/gsnap" % (aligner_bin_base)] cmd_process = subprocess.Popen(cmd, stderr=subprocess.PIPE) version = cmd_process.communicate()[1][:-1].split("\n")[0] version = version.split()[2] elif aligner == "bwa": cmd = ["%s/bwa" % (aligner_bin_base)] cmd_process = subprocess.Popen(cmd, stderr=subprocess.PIPE) version = cmd_process.communicate()[1][:-1].split("\n")[2] version = version.split()[1] return version def get_aligner_cmd(aligner, type, index_type, version, read1_fname, read2_fname, out_fname, cmd_idx = 0): cmd = [] if aligner == "hisat2": cmd = ["hisat2"] if num_threads > 1: cmd += ["-p", str(num_threads)] cmd += ["-f"] cmd += ["--no-spliced-alignment"] if index_type: index_cmd = "../grch38_snp_hisat2/genome_snp" else: index_cmd = "../grch38_hisat2/genome" cmd += [index_cmd] if paired: cmd += ["-1", read1_fname, "-2", read2_fname] else: cmd += [read1_fname] elif aligner == "star": cmd = ["%s/STAR" % (aligner_bin_base)] if num_threads > 1: cmd += ["--runThreadN", str(num_threads)] cmd += ["--genomeDir"] if cmd_idx == 0: if type == "gtf": cmd += ["%s/STAR%s/gtf" % (index_base, index_add)] else: cmd += ["%s/STAR%s" % (index_base, index_add)] else: assert cmd_idx == 1 cmd += ["."] if desktop: cmd += ["--genomeLoad", "NoSharedMemory"] else: cmd += ["--genomeLoad", "LoadAndKeep"] if type == "x2": if cmd_idx == 1: cmd += ["--alignSJDBoverhangMin", "1"] cmd += ["--readFilesIn", read1_fname] if paired: cmd += [read2_fname] if paired: cmd += ["--outFilterMismatchNmax", "6"] else: cmd += ["--outFilterMismatchNmax", "3"] elif aligner == "bowtie2": cmd = ["bowtie2"] if num_threads > 1: cmd += ["-p", str(num_threads)] cmd += ["-f"] cmd += ["-x ../grch38_bowtie2/genome"] if paired: cmd += ["-1", read1_fname, "-2", read2_fname] else: cmd += [read1_fname] elif aligner == "gsnap": cmd = ["%s/gsnap" % (aligner_bin_base), "-A", "sam"] if num_threads > 1: cmd += ["-t", str(num_threads)] cmd += ["--max-mismatches=3", "-D", "%s/GSNAP%s" % (index_base, index_add), "-N", "1", "-d", genome, read1_fname] if paired: cmd += [read2_fname] elif aligner == "bwa": cmd = ["%s/bwa" % (aligner_bin_base)] if type in ["mem", "aln"]: cmd += [type] elif type == "sw": cmd += ["bwa" + type] if num_threads > 1: cmd += ["-t", str(num_threads)] cmd += ["%s/BWA%s/%s.fa" % (index_base, index_add, genome)] cmd += [read1_fname] if paired: cmd += [read2_fname] else: assert False return cmd for aligner, type, index_type, version in aligners: aligner_name = aligner + type if version != "": aligner_name += ("_%s" % version) if aligner == "hisat2" and index_type != "": aligner_name += ("_" + index_type) two_step = (aligner == "tophat2" or type == "x2" or (aligner in ["hisat2", "hisat"] and type == "")) print >> sys.stderr, "\t%s\t%s" % (aligner_name, str(datetime.now())) if paired: aligner_dir = aligner_name + "_paired" else: aligner_dir = aligner_name + "_single" if not os.path.exists(aligner_dir): os.mkdir(aligner_dir) os.chdir(aligner_dir) out_fname = base_fname + ".sam" duration = -1.0 # Align all reads aligner_cmd = get_aligner_cmd(aligner, type, index_type, version, "../" + type_read1_fname, "../" + type_read2_fname, out_fname) start_time = datetime.now() if verbose: print >> sys.stderr, "\t", start_time, " ".join(aligner_cmd) if aligner in ["hisat2", "hisat", "bowtie", "bowtie2", "gsnap", "bwa"]: proc = subprocess.Popen(aligner_cmd, stdout=open(out_fname, "w"), stderr=subprocess.PIPE) else: proc = subprocess.Popen(aligner_cmd, stdout=subprocess.PIPE, stderr=subprocess.PIPE) proc.communicate() finish_time = datetime.now() duration = finish_time - start_time duration = duration.total_seconds() if verbose: print >> sys.stderr, "\t", finish_time, "finished:", duration assert os.path.exists(out_fname) correct_reads, correct_multi_reads, num_reads = 0, 0, 0 prev_read_id = None for line in open(out_fname): if line.startswith('@'): continue read_id, flag, chr, pos, mapQ, cigar = line.split()[:6] if chr.startswith("chr"): chr = chr[3:] pos = int(pos) - 1 true_chr, true_pos, true_cigar = read_id.split('_')[1:4] true_pos = int(true_pos) if read_id != prev_read_id: num_reads += 1 if true_chr == chr and pos == true_pos and cigar == true_cigar: correct_multi_reads += 1 if prev_read_id != read_id: correct_reads += 1 prev_read_id = read_id print >> sys.stderr, "\tfirst: %d / %d (%.2f%%)" % (correct_reads, num_reads, float(correct_reads)/num_reads*100) print >> sys.stderr, "\tall: %d / %d (%.2f%%)" % (correct_multi_reads, num_reads, float(correct_multi_reads)/num_reads*100) os.chdir("..") """ """ if __name__ == "__main__": parser = ArgumentParser( description='test HISAT2, and compare HISAT2 with other popular aligners such as TopHat2, STAR, Bowtie1/2, GSNAP, BWA-mem, etc.') parser.add_argument('read_fname', nargs='?', type=str, help='input read file') parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') args = parser.parse_args() evaluate(args.read_fname, args.verbose) hisat2-2.2.1/evaluation/tests/one_snp_test/simulate_one_snp_reads.py000077500000000000000000000331661370663763200260440ustar00rootroot00000000000000#!/usr/bin/env python # # Copyright 2015, Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # import sys, math, random, re from collections import defaultdict, Counter from argparse import ArgumentParser, FileType """ """ def reverse_complement(seq): result = "" for nt in seq: base = nt if nt == 'A': base = 'T' elif nt == 'a': base = 't' elif nt == 'C': base = 'G' elif nt == 'c': base = 'g' elif nt == 'G': base = 'C' elif nt == 'g': base = 'c' elif nt == 'T': base = 'A' elif nt == 't': base = 'a' result = base + result return result """ """ def read_genome(genome_file): chr_dic = {} chr_name, sequence = "", "" for line in genome_file: if line[0] == ">": if chr_name and sequence: chr_dic[chr_name] = sequence chr_name = line.strip().split()[0][1:] sequence = "" else: sequence += line[:-1] if chr_name and sequence: chr_dic[chr_name] = sequence return chr_dic """ """ def read_snp(snp_file): snps = defaultdict(list) for line in snp_file: line = line.strip() if not line or line.startswith('#'): continue try: snpID, type, chr, pos, data = line.split('\t') chr = chr.split()[0] except ValueError: continue assert type in ["single", "deletion", "insertion"] if type == "deletion": data = int(data) snps[chr].append([snpID, type, int(pos), data]) return snps """ """ def getSamAlignment(chr_seq, read_len, snp): snp_id, snp_type, snp_pos, snp_data = snp # Define MD, XM, NM, Zs, read_seq read_seq, MD, Zs = "", "", "" left_read_len = read_len / 2 pos = snp_pos - left_read_len assert pos >= 0 if snp_type == "single": cigar_str = "100M" read_seq = chr_seq[pos:pos+left_read_len] + snp_data + chr_seq[pos+left_read_len+1:pos+read_len] MD = "%d%s%d" % (left_read_len, chr_seq[pos+left_read_len], read_len - left_read_len - 1) Zs = "%d|S|%s" % (left_read_len, snp_id) elif snp_type == "deletion": del_len = int(snp_data) cigar_str = "%dM%dD%dM" % (left_read_len, del_len, read_len - left_read_len) read_seq = chr_seq[pos:pos+left_read_len] + chr_seq[pos+left_read_len+del_len:pos+read_len+del_len] MD = "%d^%s%d" % (left_read_len, chr_seq[pos+left_read_len:pos+left_read_len+del_len], read_len - left_read_len) Zs = "%d|D|%s" % (left_read_len, snp_id) else: assert snp_type == "insertion" ins_len = len(snp_data) assert ins_len < read_len cigar_str = "%dM%dI%dM" % (left_read_len, ins_len, read_len - left_read_len - ins_len) read_seq = chr_seq[pos:pos+left_read_len] + snp_data + chr_seq[pos+left_read_len:pos+read_len-ins_len] MD = "%d" % (read_len - ins_len) Zs = "%d|I|%s" % (left_read_len, snp_id) if len(read_seq) != read_len: print >> sys.stderr, "read length differs:", len(read_seq), "vs.", read_len print >> sys.stderr, pos, cigar_str, MD, Zs assert False ref_read_seq = chr_seq[pos:pos+read_len] return pos, cigar_str, MD, Zs, read_seq, ref_read_seq """ """ cigar_re = re.compile('\d+\w') def samRepOk(genome_seq, read_seq, chr, pos, cigar, MD, Zs): assert chr in genome_seq chr_seq = genome_seq[chr] assert pos < len(chr_seq) # Calculate XM and NM based on Cigar and Zs cigars = cigar_re.findall(cigar) cigars = [[int(cigars[i][:-1]), cigars[i][-1]] for i in range(len(cigars))] ref_pos, read_pos = pos, 0 ann_ref_seq, ann_ref_rel, ann_read_seq, ann_read_rel = [], [], [], [] for i in range(len(cigars)): cigar_len, cigar_op = cigars[i] if cigar_op == "M": partial_ref_seq = chr_seq[ref_pos:ref_pos+cigar_len] partial_read_seq = read_seq[read_pos:read_pos+cigar_len] assert len(partial_ref_seq) == len(partial_read_seq) ann_ref_seq += list(partial_ref_seq) ann_read_seq += list(partial_read_seq) for j in range(len(partial_ref_seq)): if partial_ref_seq[j] == partial_read_seq[j]: ann_ref_rel.append("=") ann_read_rel.append("=") else: ann_ref_rel.append("X") ann_read_rel.append("X") ref_pos += cigar_len read_pos += cigar_len elif cigar_op == "D": partial_ref_seq = chr_seq[ref_pos:ref_pos+cigar_len] ann_ref_rel += list(partial_ref_seq) ann_ref_seq += list(partial_ref_seq) ann_read_rel += (["-"] * cigar_len) ann_read_seq += (["-"] * cigar_len) ref_pos += cigar_len elif cigar_op == "I": partial_read_seq = read_seq[read_pos:read_pos+cigar_len] ann_ref_rel += (["-"] * cigar_len) ann_ref_seq += (["-"] * cigar_len) ann_read_rel += list(partial_read_seq) ann_read_seq += list(partial_read_seq) read_pos += cigar_len elif cigar_op == "N": ref_pos += cigar_len else: assert False assert len(ann_ref_seq) == len(ann_read_seq) assert len(ann_ref_seq) == len(ann_ref_rel) assert len(ann_ref_seq) == len(ann_read_rel) ann_Zs_seq = ["0" for i in range(len(ann_ref_seq))] Zss, Zs_i, snp_pos_add = [], 0, 0 if Zs != "": Zss = Zs.split(',') Zss = [zs.split('|') for zs in Zss] ann_read_pos = 0 for zs in Zss: zs_pos, zs_type, zs_id = zs zs_pos = int(zs_pos) for i in range(zs_pos): while ann_read_rel[ann_read_pos] == '-': ann_read_pos += 1 ann_read_pos += 1 if zs_type == "S": ann_Zs_seq[ann_read_pos] = "1" ann_read_pos += 1 elif zs_type == "D": while ann_read_rel[ann_read_pos] == '-': ann_Zs_seq[ann_read_pos] = "1" ann_read_pos += 1 elif zs_type == "I": while ann_ref_rel[ann_read_pos] == '-': ann_Zs_seq[ann_read_pos] = "1" ann_read_pos += 1 else: assert False tMD, tXM, tNM = "", 0, 0 match_len = 0 i = 0 while i < len(ann_ref_seq): if ann_ref_rel[i] == "=": assert ann_read_rel[i] == "=" match_len += 1 i += 1 continue assert ann_read_rel[i] != "=" if ann_ref_rel[i] == "X" and ann_read_rel[i] == "X": if match_len > 0: tMD += ("{}".format(match_len)) match_len = 0 tMD += ann_ref_seq[i] if ann_Zs_seq[i] == "0": tXM += 1 tNM += 1 i += 1 else: assert ann_ref_rel[i] == "-" or ann_read_rel[i] == "-" if ann_ref_rel[i] == '-': while ann_ref_rel[i] == '-': if ann_Zs_seq[i] == "0": tNM += 1 i += 1 else: assert ann_read_rel[i] == '-' del_seq = "" while ann_read_rel[i] == '-': del_seq += ann_ref_seq[i] if ann_Zs_seq[i] == "0": tNM += 1 i += 1 if match_len > 0: tMD += ("{}".format(match_len)) match_len = 0 tMD += ("^{}".format(del_seq)) if match_len > 0: tMD += ("{}".format(match_len)) if tMD != MD: print >> sys.stderr, chr, pos, cigar, MD, Zs print >> sys.stderr, tMD assert False """ """ def simulate_reads(genome_file, snp_file, base_fname, \ paired_end, read_len, frag_len, \ num_frag, sanity_check, verbose): if read_len > frag_len: frag_len = read_len genome_seq = read_genome(genome_file) snps = read_snp(snp_file) chr_ids = genome_seq.keys() sam_file = open(base_fname + ".sam", "w") # Write SAM header print >> sam_file, "@HD\tVN:1.0\tSO:unsorted" for chr in genome_seq.keys(): print >> sam_file, "@SQ\tSN:%s\tLN:%d" % (chr, len(genome_seq[chr])) read_file = open(base_fname + "_snp_1.fa", "w") ref_read_file = open(base_fname + "_ref_1.fa", "w") if paired_end: read2_file = open(base_fname + "_snp_2.fa", "w") ref_read2_file = open(base_fname + "_ref_2.fa", "w") cur_read_id = 1 for chr in snps: assert chr in genome_seq chr_seq = genome_seq[chr] chr_len = len(chr_seq) if chr in snps: chr_snps = snps[chr] else: chr_snps = [] for snp in chr_snps: # SAM specification (v1.4) # http://samtools.sourceforge.net/ flag, flag2 = 99, 163 # 83, 147 pos, cigar_str, MD, Zs, read_seq, ref_read_seq = getSamAlignment(chr_seq, read_len, snp) # pos2, cigar2_str, MD2, Zs2, read2_seq = getSamAlignment(chr_seq, frag_pos+frag_len-read_len, read_len, snp) if sanity_check: samRepOk(genome_seq, read_seq, chr, pos, cigar_str, MD, Zs) # samRepOk(genome_seq, read2_seq, chr, pos2, cigar2_str, MD2, Zs2) if Zs != "": Zs = ("\tZs:Z:{}".format(Zs)) # if Zs2 != "": # Zs2 = ("\tZs:Z:{}".format(Zs2)) read_id_str = "{}_{}_{}_{}".format(cur_read_id, chr, pos, cigar_str) print >> read_file, ">{}".format(read_id_str) print >> read_file, read_seq print >> sam_file, "{}\t{}\t{}\t{}\t255\t{}\t{}\t{}\t0\t{}\t*\tXM:i:0\tNM:i:0\tMD:Z:{}{}".format(read_id_str, flag, chr, pos + 1, cigar_str, chr, pos + 1, read_seq, MD, Zs) print >> ref_read_file, ">{}_{}_{}_100M".format(cur_read_id, chr, pos) print >> ref_read_file, ref_read_seq """ if paired_end: print >> read2_file, ">{}".format(cur_read_id) print >> read2_file, reverse_complement(read2_seq) print >> sam_file, "{}\t{}\t{}\t{}\t255\t{}\t{}\t{}\t0\t{}\t*\tXM:i:0\tNM:i:0\tMD:Z:{}{}".format(cur_read_id, flag2, chr, pos2 + 1, cigar2_str, chr, pos + 1, read2_seq, MD2, Zs2) """ cur_read_id += 1 sam_file.close() read_file.close() ref_read_file.close() if paired_end: read2_file.close() ref_read2_file.close() if __name__ == '__main__': parser = ArgumentParser( description='Simulate reads from GENOME (fasta) and GTF files') parser.add_argument('genome_file', nargs='?', type=FileType('r'), help='input GENOME file') parser.add_argument('snp_file', nargs='?', type=FileType('r'), help='input SNP file') parser.add_argument('base_fname', nargs='?', type=str, help='output base filename') parser.add_argument('--paired-end', dest='paired_end', action='store_true', help='single-end reads (default: paired-end reads)') parser.add_argument('-r', '--read-length', dest='read_len', action='store', type=int, default=100, help='read length (default: 100)') parser.add_argument('-f', '--fragment-length', dest='frag_len', action='store', type=int, default=250, help='fragment length (default: 250)') parser.add_argument('-n', '--num-fragment', dest='num_frag', action='store', type=int, default=1000000, help='number of fragments (default: 1000000)') parser.add_argument('--sanity-check', dest='sanity_check', action='store_true', help='sanity check') parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') parser.add_argument('--version', action='version', version='%(prog)s 2.1.0') args = parser.parse_args() if not args.genome_file or not args.snp_file: parser.print_help() exit(1) simulate_reads(args.genome_file, args.snp_file, args.base_fname, \ args.paired_end, args.read_len, args.frag_len, \ args.num_frag, args.sanity_check, args.verbose) hisat2-2.2.1/evaluation/tests/repeat/000077500000000000000000000000001370663763200175145ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/repeat/commands000066400000000000000000000047131370663763200212450ustar00rootroot00000000000000date; time bowtie2 -X 800 --extends 1000 --score-min C,0 -p 3 -x 20 -f -1 1.fa -2 2.fa > bowtie2.sam date; time hisat2 --no-spliced-alignment --score-min C,0 -X 800 -p 3 -x 20 -f -1 1.fa -2 2.fa > hisat2.sam # Chromosome 22 /usr/bin/time -l hisat2-repeat -p 3 --save-sa --load-sa --repeat-length 51-300,76-300,100-300,101-300,151-300 --repeat-count 5 22.fa 22_rep /usr/bin/time -l hisat2-build -p 3 22.fa --repeat-ref 22_rep.rep.fa --repeat-info 22_rep.rep.info 22_rep hisat2_simulate_reads.py --dna --num-fragment 1000000 --repeat-info 22_rep.rep.info 22.fa /dev/null /dev/null sim hisat2_simulate_reads.py --dna --num-fragment 1000000 --error-rate 0.2 --repeat-info 22_rep.rep.info 22.fa /dev/null /dev/null sim cp 22_rep.rep.info ../../data/ cp 22_rep*ht2 ../../indexes/HISAT2_22 # Human genome /usr/bin/time -l hisat2-repeat -p 3 --save-sa --load-sa --repeat-length 100-300,101-300 --repeat-count 5 genome.fa genome_rep /usr/bin/time -l hisat2-build -p 3 genome.fa --repeat-ref genome_rep.rep.fa --repeat-info genome_rep.rep.info genome_rep /usr/bin/time -l hisat2-repeat -p 3 --save-sa --load-sa --repeat-edit 0 --repeat-length 101--300 --repeat-count 5 genome.fa genome_rep-101-300 /usr/bin/time -l hisat2-build -p 3 genome.fa --repeat-ref genome_rep-101-300.rep.fa --repeat-info genome_rep-101-300.rep.info genome_rep-101-300 hisat2_simulate_reads.py --dna --num-fragment 1000 --repeat-info genome_rep.rep.info genome.fa /dev/null /dev/null sim hisat2_simulate_reads.py --dna --num-fragment 1000 --error-rate 0.2 --repeat-info genome_rep.rep.info genome.fa /dev/null /dev/null sim hisat2_simulate_reads.py --dna --num-fragment 10000 --repeat-info genome_rep.rep.info genome.fa /dev/null /dev/null sim hisat2_simulate_reads.py --dna --num-fragment 10000 --error-rate 0.2 --repeat-info genome_rep.rep.info genome.fa /dev/null /dev/null sim hisat2_simulate_reads.py --dna --num-fragment 10000000 --repeat-info genome_rep.rep.info genome.fa /dev/null /dev/null sim hisat2_simulate_reads.py --dna --num-fragment 10000000 --error-rate 0.2 --repeat-info genome_rep.rep.info genome.fa /dev/null /dev/null sim cp genome_rep.rep.info ../../data/ cp genome_rep.*ht2 ../../indexes/HISAT2 # Bisulfite-treated sequencing /usr/bin/time -l hisat2-repeat -p 3 --repeat-length 100 --repeat-count 2 --repeat-edit 0 --forward-only --CGtoTG 22.fa 22_rep /usr/bin/time -l hisat2-repeat -p 3 --repeat-length 200 --repeat-count 2 --repeat-edit 0 --forward-only --CGtoTG genome.fa genome_rep hisat2-2.2.1/evaluation/tests/repeat/generate_repeats.py000077500000000000000000000131721370663763200234120ustar00rootroot00000000000000#!/usr/bin/env python import sys import struct chr_name = "20" chr_seq = "" for line in open("%s.fa" % chr_name): if line.startswith('>'): continue line = line.strip() line = line.replace('N', '') chr_seq += line chr_seq += '$' chr_sa = [] f = open("%s.sa" % chr_name, "rb") while True: fourbytes = f.read(4) if fourbytes == "": break num = struct.unpack('I', fourbytes)[0] chr_sa.append(num) if len(chr_sa) % 5000000 == 0: print len(chr_sa) f.close() assert chr_sa[0] + 1 == len(chr_sa) chr_sa = chr_sa[1:] assert len(chr_seq) == len(chr_sa) # for i, num in enumerate(chr_sa): # print "%10d\t%s\t%d" % (i, chr_seq[num:num+100], num) seq_len = 100 i = 0 repeats = [] while i < len(chr_sa) - 1: pos = chr_sa[i] base_seq = chr_seq[pos:pos+seq_len] for j in xrange(i+1, len(chr_sa)): pos2 = chr_sa[j] cmp_seq = chr_seq[pos2:pos2+seq_len] if base_seq != cmp_seq: break if j - i >= 200: repeats.append([base_seq, sorted(chr_sa[i:j])]) i = j if i % 5000000 == 0: print i found = False print len(repeats), "repeats" deleted = set() for i in xrange(len(repeats) - 1): for j in xrange(i + 1, len(repeats)): if j in deleted: continue num_close, num_close2 = 0, 0 pos_seq, pos_set = repeats[i] pos_seq2, pos_set2 = repeats[j] k1, k2 = 0, 0 while k1 < len(pos_set) and k2 < len(pos_set2): _pos, _pos2 = pos_set[k1], pos_set2[k2] if abs(_pos - _pos2) < 300: num_close += 1 if abs(_pos - _pos2) < 1100: num_close2 += 1 if _pos <= _pos2: k1 += 1 else: k2 += 1 if num_close > min(len(pos_set), len(pos_set2)) * 0.95: deleted.add(j) if num_close == 1 and num_close2 < 5: found = True print pos_set print pos_set2 print pos_seq print pos_seq2 file1 = open("1.fa", "w") file2 = open("2.fa", "w") pos_seq2_rc = list(pos_seq2) pos_seq2_rc = pos_seq2_rc[::-1] for k in xrange(seq_len): nt = pos_seq2_rc[k] if nt == 'A': nt = 'T' elif nt == 'C': nt = 'G' elif nt == 'G': nt = 'C' else: assert nt == 'T' nt = 'A' pos_seq2_rc[k] = nt pos_seq2_rc = ''.join(pos_seq2_rc) for k in xrange(1000000): print >> file1, ">%d" % k print >> file2, ">%d" % k print >> file1, pos_seq print >> file2, pos_seq2_rc file1.close() file2.close() break if found: break print i chr_seq = "" for line in open("%s.fa" % chr_name): if line.startswith('>'): continue line = line.strip() chr_seq += line N_ranges = [] prev_nt = None for i in xrange(len(chr_seq)): nt = chr_seq[i] if nt == 'N': if prev_nt != 'N': N_ranges.append([i, i]) # inclusive else: assert len(N_ranges) > 0 N_ranges[-1][1] = i prev_nt = nt to_joined_list = [] for N_start, N_end in N_ranges: if len(to_joined_list) == 0: if N_start > 0: to_joined_list.append([0, 0]) else: to_joined_list.append([N_end + 1, 0]) else: N_size = N_end - N_start + 1 to = N_end + 1 - to_joined_list[-1][0] + to_joined_list[-1][1] - N_size assert to > to_joined_list[-1][1] to_joined_list.append([N_end + 1, to]) to_genome_list = [[y, x] for x, y in to_joined_list] N_ranges_tmp = [] for i in xrange(len(to_genome_list)): to_genome = to_genome_list[i] if i == 0: if to_genome[1] > 0: N_ranges_tmp.append([0, to_genome[1] - 1]) else: to_genome_before = to_genome_list[i-1] N_ranges_tmp.append([to_genome_before[1] + to_genome[0] - to_genome_before[0], to_genome[1] - 1]) assert N_ranges == N_ranges_tmp file = open("%s_rep.info" % chr_name, "w") def print_rep_info(rep_name, rep_pos, rep_len, pos_set, pos_seq): print >> file, ">%s*0\trep\t%d\t%d\t%d\t0" % (rep_name, rep_pos, rep_len, len(pos_set)) for i in xrange(0, len(pos_set), 10): output = "" for j in range(i, i + 10): if j >= len(pos_set): break if j > i: output += " " def convert(pos): for i in xrange(len(to_genome_list)): if i + 1 == len(to_genome_list) or (pos >= to_genome_list[i][0] and pos < to_genome_list[i+1][0]): return pos - to_genome_list[i][0] + to_genome_list[i][1] assert False pos = convert(pos_set[j]) assert chr_seq[pos:pos+seq_len] == pos_seq output += ("%s:%d:+" % (chr_name, pos)) print >> file, output print_rep_info("rep1", 0, seq_len, pos_set, pos_seq) print_rep_info("rep2", seq_len, seq_len, pos_set2, pos_seq2) file.close() chr_seq = chr_seq.replace(pos_seq, 'N' * seq_len) chr_seq = chr_seq.replace(pos_seq2, 'N' * seq_len) file = open("%s_mask.fa" % chr_name, "w") print >> file, ">%s_mask" % chr_name for i in xrange(0, len(chr_seq), 60): print >> file, chr_seq[i:i+60] file.close() file = open("%s_rep.fa" % chr_name, "w") rep_seq = pos_seq + pos_seq2 print >> file, ">rep" for i in xrange(0, len(rep_seq), 60): print >> file, rep_seq[i:i+60] file.close() hisat2-2.2.1/evaluation/tests/repeat/test_repeat.py000077500000000000000000000061601370663763200224130ustar00rootroot00000000000000#!/usr/bin/env python import sys, os, subprocess, random from argparse import ArgumentParser, FileType """ """ def reverse_complement(seq): result = "" for nt in seq: base = nt if nt == 'A': base = 'T' elif nt == 'a': base = 't' elif nt == 'C': base = 'G' elif nt == 'c': base = 'g' elif nt == 'G': base = 'C' elif nt == 'g': base = 'c' elif nt == 'T': base = 'A' elif nt == 't': base = 'a' result = base + result return result """ """ def read_genome(genome_filename): chr_dic = {} genome_file = open(genome_filename, "r") chr_name, sequence = "", "" for line in genome_file: if line[0] == ">": if chr_name and sequence: chr_dic[chr_name] = sequence chr_name = line[1:-1].split()[0] sequence = "" else: sequence += line[:-1] if chr_name and sequence: chr_dic[chr_name] = sequence genome_file.close() print >> sys.stderr, "genome is loaded" return chr_dic """ """ def generate_random_seq(seq_len): assert seq_len > 0 random_seq = "" for i in xrange(seq_len): random_seq += "ACGT"[random.randint(0, 3)] return random_seq """ """ def test_repeat(verbose): random.seed(1) backbone_seq = generate_random_seq(500) mm_seq = backbone_seq[:] mm_seq = mm_seq[:50] + ("A" if mm_seq[50] != "A" else "C") + mm_seq[51:] mm_seq2 = backbone_seq[:] mm_seq2 = mm_seq2[:450] + ("A" if mm_seq2[450] != "A" else "C") + mm_seq2[451:] del_seq = backbone_seq[:] del_seq = del_seq[:50] + del_seq[52:150] + del_seq[152:] del_seq2 = backbone_seq[:] del_seq2 = del_seq2[:350] + del_seq2[352:450] + del_seq2[452:] indel_seq = backbone_seq[:] indel_seq = indel_seq[:30] + indel_seq[32:130] + "AAA" + indel_seq[130:] indel_seq2 = backbone_seq[:] indel_seq2 = indel_seq2[:30] + "AAA" + indel_seq2[30:130] + indel_seq2[132:] seqs = [ # dummy_seq, ["bb01", backbone_seq], ["bb02", backbone_seq], ["bb03", backbone_seq], ["bb04", backbone_seq], ["bb05", backbone_seq], ["mm01", mm_seq], ["mm02", mm_seq], ["dd01", del_seq], ["dd02", del_seq], ["dd03", del_seq2], ["dd04", del_seq2], ["id01", indel_seq], ["id02", indel_seq], ["id03", indel_seq], ["id04", indel_seq], ["id05", indel_seq], ["id06", indel_seq], ["id07", indel_seq2], ] for id, seq in seqs: print ">%s" % id print generate_random_seq(20) print seq print generate_random_seq(20) """ """ if __name__ == "__main__": parser = ArgumentParser( description='') parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') args = parser.parse_args() test_repeat(args.verbose) hisat2-2.2.1/evaluation/tests/the_small_example/000077500000000000000000000000001370663763200217175ustar00rootroot00000000000000hisat2-2.2.1/evaluation/tests/the_small_example/COMMAND000066400000000000000000000001611370663763200227160ustar00rootroot00000000000000../../../hisat2-build --offrate 0 --ftabchars 1 --snp small.snp small.fa small ../../../hisat2 -x small -c GCTAG hisat2-2.2.1/evaluation/tests/the_small_example/small.fa000066400000000000000000000000161370663763200233340ustar00rootroot00000000000000>small GAGCTG hisat2-2.2.1/evaluation/tests/the_small_example/small.snp000066400000000000000000000001071370663763200235470ustar00rootroot00000000000000snp1 single small 1 T snp2 deletion small 4 1 snp3 insertion small 5 A hisat2-2.2.1/example/000077500000000000000000000000001370663763200143565ustar00rootroot00000000000000hisat2-2.2.1/example/index/000077500000000000000000000000001370663763200154655ustar00rootroot00000000000000hisat2-2.2.1/example/index/22_20-21M_snp.1.ht2000066400000000000000000222121761370663763200202210ustar00rootroot00000000000000  @BL D@A@D@ @!@0U@@ @BA D0@# \  A0@@0Q=@ @@ 0@@4@@@|oQ1(@@  !r  P?f $ #!0O 0 o'Ux.L L@0 1P@A   @  9@@@ Š5 N0` @1 @pGQP0])J\dP@D "  DB@~.9aA@0&TD !O0 2<2EPPSEUPTAH@AU KqL sG1P2D@qD"ӼpW ar Z ~ Ќt3 :$PNs"TU*`_ovY * b1 0/H x >8  '2~5 ) cETP| $4,H$T` #tE. -$@ $ |@?52 ?$:Jߣ0<3`t1? 30Lu0. 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" $ $ & ( ) * * * + - . / / / / 2 2 4 4 : ; ; < > > @ @ @ A A A B B C C C D D E G G I I I J J J M P Q Q S S T T U U U V W Z [ [ [ [ \ \ \ b b b g i i k k l n o r r r r r r r u x {  ̃ ΃ Ճ ك            ! % ' - 9 < < = A C E H Q W \ ` b f f i k l m u ~ Ä Ƅ Ȅ Ȅ ˄ ˄ ΄ τ Є Є ф Մ Մ Մ ք ք ք ք ۄ ܄ ܄ ܄ ܄ ܄ ބ ߄ ߄        " # % & ( ) 0 3 3 6 8 ; @ B D G R U U U U V W W W [ \ ^ b g i j j m q u v x y | | } }  … Dž Ʌ ˅ Ѕ օ օ ؅ م ݅ ߅     # % * 2 6 7 < ? @ L U V c g j k m o s ~ † Æ ņ ʆ ͆ ҆ ׆ ۆ ( + 0 ? H R a u x      , 1 > J ] ` f g s w y | } ~  ˆ ƈ Lj ʈ ͈ ψ ψ ш Ո ֈ ؈ ؈ ؈ ؈ و و و ݈ ߈               ! ! % % & ' ( ( ( * * * + + + - - . . . . . 0 0 1 1 1 1 2 2 2 2 2 2 2 2 2 3 5 6 6 9 : < = = ? ? A A A A A B B C C C C E E E E E F F F F H H J K K K K N Q Q S T V Y [ [ ^ ^ ^ _ _ b c e g i k k m m m m p t t t t u u u u u x x x x y y { { { { { { | |   ‰ ȉ ʉ ˉ ˉ ω щ ҉ Չ Չ ډ ݉ ߉                                       ! ! 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C D E E F G G G G G G I I J J L M M P P S X Y Z Z Z Z [ [ [ [ [ [ \ \ ] ] ^ ^ ^ ^ ^ ^ ^ _ a a a a b b b d d d e f f f i i j j j l n n o s u v v w y z z z | | | } } } } ~ ~        “ “ “ Ó Ó ē ē ē ē ē ē œ Ɠ Ɠ Ɠ Ɠ Ɠ Ɠ Ǔ ȓ ȓ ȓ ɓ ɓ ɓ ɓ ̓ Γ ӓ ӓ ӓ ӓ ӓ ӓ ӓ ԓ ֓ ד ٓ ۓ ۓ ݓ ݓ ݓ                % ) . 1 9 = D D S U ` ` c h i k n o o r v ~ ” ” ” ” ” Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ô Ĕ Ĕ Ĕ Ĕ Ŕ Ŕ Ɣ Ɣ Ɣ Ɣ Ɣ Ɣ ǔ ǔ ǔ ǔ ǔ ǔ ǔ ǔ ǔ ǔ ǔ ǔ ǔ ǔ ǔ ǔ Ȕ Ȕ Ȕ Ȕ Ȕ Ȕ Ȕ Ȕ Ȕ Ȕ Ȕ Ȕ ɔ ɔ ɔ ʔ ˔ ̔ ̔ ̔ ̔ ̔ ̔ ͔ ͔ Δ Δ Δ Δ Д Д Д Д є є є Ԕ Ԕ Ԕ Ԕ Ԕ Ԕ Ԕ Ք Ք Ք Ք ֔ ֔ ה ה ה ה ה ה ؔ ؔ ؔ ؔ ؔ ؔ ؔ ؔ ڔ ۔ ܔ ܔ ܔ ܔ ܔ ܔ ݔ ݔ ݔ ޔ                                                           ! ! ! ! ! ! ! ! ! " " " " " " $ % & & ' ' ( * + . . . . . . . . / 4 6 6 7 7 7 7 7 7 7 9 9 9 9 9 9 9 9 9 9 : : ; < < = ? ? ? ? A A A B B B C D D D D D E E E F F H H I I I N N O O O O P P P P Q R R R R S S T T T U X Y Z [ [ [ [ [ [ [ ] ] ] ] ] ] ] ^ ` ` ` ` a b b b b b b b b b b b b b b f i j j j j l l l q q q q q q r r s s s s u u u v x x z } } Õ ƕ Ǖ ȕ ʕ ˕ ˕ ͕ ѕ ؕ           ! % - . 8 @ F L S T Y c f g g h i j k k l m n s v v z | ~ F I K O Y \ _ g p p p p u | ė ͗ ؗ ޗ           " % & & & ) ) ) * * * * - - . . . 1 2 3 3 4 4 4 4 5 5 5 5 5 5 6 6 7 8 8 9 : ; A E G G I N N S S T U V X ] b d i i p r { Ę ǘ Ș ɘ ɘ ɘ Ϙ ј Ԙ Ԙ Ԙ ՘ ؘ ۘ ߘ ߘ       ]        " & + . / 4 8 : : : : ; ; < < < < = @ B C F F I L N N O P Y Z ^ e k s x x y | } Ù ř ř ƙ ș ʙ ʙ ̙ ̙ ͙ ͙ ҙ ҙ ԙ ؙ ݙ      # - = C D G P S U [ e i p  ˚ Κ њ ۚ    " ) 8 @ C K U Y Z Z [ [ ] ` a d h r }  › Û ě ƛ ɛ ˛ ϛ ӛ ՛ ݛ                       " " " " # # $ % % & ' ' ' ' ' ( ( ( ( ( ) ) * * * + , , , . . 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 4 5 7 7 ; < < = B B D H K N P P R S T [ [ [ ] a e f m p p p r v | œ Ü Ü Ĝ Ĝ Ĝ Ŝ Ȝ ќ Ԝ ՜ ל ܜ ܜ                ! ! # # & & & & ' ' ' ' ( ) ) , - - / / / / / 0 2 3 7 9 : : : < > > B E I P Q Q Q U X Z [ [ [ ] _ _ d e g j k k l n t t t t t t t t t t t t t t t t t t t t t t | | | | | | | | } } } } } } } } } ~ ~ ~ ~ ~   ڝ ۝ ۝ ܝ ޝ ߝ \                                                                                                  # $ $ $ $ % % % & & & * * * * * * * * * * * * * * + , , - . . . . . . . . . . . . / / 0 0 2 2 2 2 4 4 4 4 4 4 5 5 6 6 6 7 8 8 8 8 : ; ; ; < < < < < < < ? ? ? ? ? ? ? ? ? ? ? @ @ @ A B C C D D D D E F F F F G G G G G I J J J K L L L O P Q S S S S S S T T T U Y Y Y [ [ \ ` b e e e f k k k l l l m m m m n n n n n o r r r r r r r r r r r r r r r r r r r r r s s t u u u u u w w w w w w w w w w w w w x { ~   ž ž ž ž ž ž ž Ğ Ğ Ş Ş Ş Ş Ş Ş Ş Ş ƞ ƞ ƞ ƞ ƞ ƞ ƞ ƞ ƞ ƞ ƞ ƞ ƞ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ǟ Ȟ ɞ ɞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ʞ ˞ ˞ ˞ ˞ ˞ ˞ ˞ ˞ ̞ ̞ Ξ Ϟ Ҟ Ҟ Ҟ Ҟ Ҟ Ӟ Ԟ Ԟ ՞ ՞ ՞ מ מ מ                                           ! ! " ' * + , - - - - - - - - . . . . 1 : : : : ? @ @ @ @ @ @ A A B B B B B B B B B B B C C F F F G G G G G G G H H H I I K L ^ ^ _ ` a c d d d d d d d d d d f f g h h h h h h h i i i i i i i i i j l m o o o o o o o r r t v v x x y { { { { | | | } } } ~  ˟ ̟ ϟ ϟ џ ԟ ՟ ՟ ֟ ֟ ֟ ֟ ֟ ֟ ֟ ֟ ؟ ؟ ڟ ڟ ڟ ڟ ۟ ۟ ۟ ۟ ۟ ۟ ۟ ۟ ۟ ۟ ܟ ܟ ܟ ޟ                       ! & ( J K K K K K K K K K K M M M N N N P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P Q Q R S T U U U U U U U U U U W W X k k k l l l l l m m m m m n q q q s t v v v v v v v w y y y { { } }          à Ơ Ƞ Ƞ Ƞ ɠ ʠ ʠ ʠ ʠ ʠ ʠ ˠ ˠ ˠ ˠ ̠ ͠                                                                               ! ! ! ! ! " " " " # # $ $ $ % % % % & ' ' ( ( ( ) * + , , . / / / / 2 2 3 5 6 7 8 ; ; ; ; ; < ? ? A A A C F G H I J L L L M S T T T T T T U U W W X [ ] ] ] ^ ^ _ _ _ _ _ _ _ _ _ _ _ _ _ _ b c d d d d e f f f f f f f g i i i i i i i i i i i i k m m o p p p p p p s s u u w w x y y y y y { { | } } } } } } } } } ~           ҡ ҡ ӡ ԡ ա ١ ۡ ܡ ޡ ߡ ߡ                                   ? C I I I J J J J K K K L N N O P P P P Q R R S T U V V V V V V X X X X Y Z Z Z Z [ [ [ [ [ [ [ [ [ [ [ ] ] ] ] ] ] ^ ^ ^ ^ _ _ b b c c c c d d e f f f f h h i k n n p q s s v x | ~  ¢ ¢ â â â â â â â â â Ģ Ģ Ģ Ģ Ģ Ģ Ţ ɢ ɢ ʢ ˢ ̢ ͢ ΢ ΢ Т Ѣ Ң Ԣ ܢ ߢ                          " ' ( ( ( ) ) ) * + + , , , , , - / / / / / / / / 2 2 3 4 5 7 7 7 8 8 8 9 9 : = = > ? A A A B K R T T U U Z [ [ [ [ [ [ \ ] a a a b b b b b b d d f f i q u v ǣ ͣ ԣ ԣ ԣ ԣ ף أ أ أ ٣ ٣ ٣ ڣ ڣ ܣ ޣ      ( ( ( , 1 2 4 7 ; > A E H M P Q R U U Z c d d d d d i k k o o o u y y y y { { { { { { ~ ~  ¤ ä ä ä ä ä Ĥ Ĥ Ĥ Ĥ Ĥ Ĥ Ĥ Ť Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ƥ Ȥ ɤ ɤ ɤ ɤ ɤ ɤ ʤ ʤ ˤ ˤ ˤ ͤ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Τ Ϥ Ф Ф Ф Ф Ѥ Ѥ Ѥ Ѥ Ѥ Ѥ Ҥ Ҥ Ԥ Ԥ ֤ ֤ פ פ ڤ ڤ ڤ ڤ ڤ ۤ ܤ ݤ ޤ ޤ ߤ           ! ! # ) ) ) ) * * * * * + , , - - - . . . . / / / / / / / 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 2 3 3 3 3 3 3 3 4 4 4 4 4 5 5 5 6 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 < < < < = = = = = = = = = = = > > > > > > ? ? ? ? ? ? @ @ @ @ @ C C C C C C C C D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D D E E E E F F F F F F F F F F F F F F F F F F F F F F F F F F G G H H H H H H H H H H H I I I I I I I I I I J K K K K K K K K K K K K K K L O O O O O O O O O O O O O O O O O O O O O O O P P P P P Q Q Q Q Q Q R R R R R R R R R S S S S S S S S S S S S S S S S S S S S S S S S S S S S T T T T T T T T T T T U U U U U V V V W W X X X X X X Z Z Z [ [ [ [ [ ] ] ] _ _ _ ` ` ` ` a a a a a b c c c c c d d d d d d e e e e e f g g g h i i i i j j k k k k k l l l m m n o o o o q q q q q q r u u u u u x x x x y z { | } } } > > > > > > > > > @ @ A A A A A A B B B B B B B B B F F F F I I P P P P P P P P P P P P P P Q Q Q Q R R R S S T V V V V V V V V V V X Y Y Y Y Y Y ^ ^ _ _ ` ` ` a b c d d e e e f f f f f g g g g g g g h i i i k ܦ ܦ ܦ ܦ ݦ ߦ                                          ! " " " " % % % % % % % & & & & & ) ) ) ) * * * * * * * * * * + - - . . . . . / / 0 0 2 3 6 7 8 8 8 8 = = C D D D D D H R U U X [ a b c c g g g g g g h h i j k l ç ç ç ç ħ ǧ ǧ ɧ Χ ӧ է ֧ ֧ ק ڧ ڧ ڧ ڧ ڧ ڧ ڧ ڧ ڧ ܧ ݧ ݧ ާ ߧ ߧ ߧ ߧ [                                                                                       ! ! ! ! ! ! ! " " " " " " " " " " # # # # # $ & ' ' ' ' ' 0 3 G I J J K L L L [ [ \ ] ^ ^ _ ` ` a a a a a a b b b c c f f f f g g g g g g h h i i i i i j j j j j j l l l l l m q q r s u v v v v w x x x x x z z z z z z { { { } } } ~ ~   è ƨ Ǩ ʨ 2 3 3 3 4 4 4 4 4 4 6 7 7 7 7 7 7 7 9 ; < = > > @ @ @ @ A D D E E F G N N O R R T Y [ ^ _ ` ` f f h h h j j j j j k k l m n n o o p r s s t u v w x z | | | | } }   é ĩ ĩ ǩ ǩ ȩ ȩ ʩ ˩ ˩ ˩ ˩ ˩ ˩ ˩ ̩ Ω ϩ Щ Щ Щ ѩ ѩ ө ө ԩ թ ש ة ة ة ة ة ة ة ة ܩ ݩ ߩ                                                         ( * * * * , , . 0 0 1 1 1 2 2 3 4 4 4 5 7 7 8 8 : : ; < < > ? C D E E F G I I I I I I J J J L L O R S T U U U U U V V V V V V V V W Z Z Z Z Z Z Z Z [ \ \ ] ] ] ] ] ] a b e f f h i i m m n n n n n n o Zr r r r r r t t t t w x x x y y z z z { } } } } } } } Y ê ê ê ê ê ê ê Ī Ī Ī Ī Ī Ī Ū Ū Ū Ǫ Ǫ ɪ ɪ ɪ ɪ ɪ ʪ ʪ ʪ ʪ ʪ ʪ ʪ ʪ ʪ ʪ ̪ ̪ ̪ ͪ ͪ ͪ ͪ ͪ ͪ ͪ ͪ ͪ ͪ Ϫ Ѫ Ѫ Ѫ Ѫ Ҫ Ԫ Ԫ Ԫ Ԫ Ԫ ժ ժ ժ ժ ժ ժ ժ ժ ժ ժ ժ ֪ ת ت ت ت ڪ ڪ ڪ ڪ ڪ ڪ ܪ ݪ ߪ       % % ' ( > ? @ @ @ A C C C E E F F F F F F F F F G G M | } « « ī ɫ ͫ ͫ Ϋ Ϋ Ϋ ϫ * * + + - - - - 0 0 0 1 5 5 6 7 ? ? B F ¬ Ƭ ɬ ɬ ˬ ά ά Ϭ Ь Ь Ҭ ׬ ٬ ެ ߬ ߬ ߬        $ & - 9 ; ? D K M Q R T Z [ [ [ [ [ \ ] ] ] _ e j m n o p q q r r r r r r r r s s t t u u u u v { } ~        ˭ ˭ Э ѭ խ ڭ ݭ ݭ                   ! " % . 8 > B D H J R \ c e g l l l l m m n r r w y |  î ʮ ʮ Ϯ Ю Ӯ Ԯ ծ ۮ ޮ ޮ ޮ ޮ      ! $ ( * * + + + , - . . . . . . . / / / / / 1 1 1 2 2 3 3 4 5 5 5 5 7 7 8 : ; ; ; ? ? A C D J M N N O P P P R S T U U U V W Z ^ ^ ^ _ _ _ ` ` ` a c d e g h i k k l l m n r w {  ȯ ͯ ѯ ݯ                                                                                               ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! " # $ ( ( ) ) + , , , , , , , , , , . 0 2 2 2 2 2 2 2 2 2 2 2 5 8 9 9 : : ; < > ? ? @ A B B D H H I I K L M M M O Q Q Q R R R R R T U W X X X Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z [ [ [ \ \ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ _ ` ` a b b b c c c d d f h h h h h h h i i i i i m n n o o o p r r s s t t u u v x x x y z z { { { | } ~ ~ ~ ~ ~ ~ ~ ~ İ İ Ű Ű Ű Ű Ű Ȱ Ȱ Ȱ Ȱ ʰ Ͱ ΰ ΰ ΰ ΰ ϰ ϰ ϰ Ѱ Ѱ Ӱ հ հ ٰ ڰ ۰ ۰ ܰ ܰ ް ް ް        ! & / 4 : ; < ? ? ? A G L O P U U V ^ _ c k s | ± ± ñ ñ ñ ı ű DZ ɱ ˱ ̱ α Ա ձ ر ر ر ٱ ٱ ٱ ܱ ݱ ޱ ޱ ߱ ߱ ߱ ߱              " $ $ ( ( ( * , - . . . . . / 3 4 4 7 : > > J O T U U Y _ ` a b i l l l p q q r s s s t x x x x x | | | } } } ~   ò IJ Բ ܲ ޲      & & ' * , , 1 : = C I R V   & 0 2 6 ? @ k q q r s s t y z ~ ´ ô Ŵ ɴ ˴ ˴ ˴ ˴ ˴ ̴ ̴ ̴ ϴ д Ѵ ״ ״ ״ ٴ ٴ ܴ ݴ ݴ        ! - 0 4 = @ H O X i q u y ŵ ȵ ̵ ε ϵ ҵ ڵ     # # $ ( ) , 2 @ D F F I I J K L O P R X [ [ ] b b b f h h m s s w w ¶ Ķ ƶ ȶ ɶ ϶ ׶ ׶ ׶ ׶ ض ض ض ܶ ܶ ܶ ݶ ޶         ) + 0 7 9 > C O \ _ b h t v w w x y z | | ŷ ȷ ȷ η η Ϸ Ϸ з ѷ շ շ ٷ     ( * 8 9             + 5 = = ? C E I N Z \ b f i m o p q q s y Ĺ ι ҹ Թ ׹ ݹ ޹ ޹       * , ? S Z d v | ú ɺ ˺ ֺ  * 9 G Q \ _ j x z  ɻ ϻ ϻ л ӻ ׻ ػ ػ ٻ ڻ ڻ ڻ ۻ ۻ ۻ ۻ ۻ ۻ ۻ ݻ ݻ ޻ ޻ E L ¼ ü ż Ƽ ȼ ɼ μ ϼ Ѽ Ѽ Ҽ Ҽ Ӽ Ӽ Ӽ Ӽ Ӽ Լ ռ ռ ּ ּ ټ ټ ټ ݼ ޼ ޼ ޼                   ! $ % ( 0 4 5 7 9 9 : ; ; = A E E F F F F G G H I K L L L L N N N O P P T W W [ [ \ ^ ^ _ a c d d e g h k l m m p x  Ž Ƚ ɽ ̽ ν Ͻ н Խ ޽ ߽         " & & ) . 4 < E F H K M N N N P Q R S T U V W W W W W W X Z \ \ ] c c f f f g h j j k k k l o p p q q t x x { { { } ~   ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ ¾ þ þ þ ž ž ž ž ʾ ʾ ˾ ̾ ̾ ̾ ̾ ; ξ ξ ξ ξ ξ ξ ξ ξ Ͼ Ͼ о о о о о о о о о о Ѿ Ѿ Ѿ Ѿ Ѿ Ѿ Ѿ Ѿ Ҿ Ӿ Ӿ Ӿ վ ׾ ׾ ؾ ھ ۾ ۾ ݾ ߾                              ! $ % % & ' ' ( ( ) ) ) , - / / 2 4 5 < > @ D G H J P Q Q Q Q R R S S U U U V W Z Z [ [ _ ` d e f f g h i n r t u x y y ¿ ο ѿ ӿ ؿ ۿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿ ߿               ! % & & + / 8 < < > @ B D D D E I J L O P R U V W X [ ^ ^ ^ ^ ^ _ e g g g g g h i i o p s s s s s t t t t t t t t t u v v w w x x       # % % ' . 2 7 : : : = = = > ? ? C D E H J K T V X [ [ \ ] _ ` ` a b b c f f i j j j o p p p r t x { | ~ ~ ~ ~ ~ ~                          % % & * + . . . 0 0 4 6 ; < = = @ A A D D E H I N N N P V V V V W X X [ [ \ \ ` d g g i j k p q q r t t t v v v w w w w w y y | }  X                 ! $ & & * + , , . . / / / 4 5 8 : : ; ; ; = = @ A A C C C C C C C C C C C C C C E E E E E F F F L L O Q Q S T T X Y Y [ \ ` ` c d e g h h i j j j l o o p s t v v v v w w x { { } ~                     ! # # & ) * - / 3 4 7 8 9 ; < < ? @ @ B B B C C D E I I I K M P P P Q R S S T U X ] ] ^ ^ ` ` c c e f i m m q r r s s s t x y y y z z { { { { { { | } } } } }    W                     $ & ( ( - / 2 6 9 @ F H K N O P Q Q R R S S T T V Y _ b b c e f g g i j l n n q y }            " $ % % + - 9 ; @ A F L M Q Z c l p q s u u w w y y y { { | ~                                                                " # $ % ( ) ) + + + - - 4 7 7 7 ? ? ? @ B D D E E E E E E H H I J K M N R R U V V X Y [ _ a a g i j k m n n n n n o p p r t t u v v { | } ~ ~ ~ ~ ~ ~ ~                                   " & ' ' ' * + + , / 0 1 3 4 5 : = C G I J P P P P W Y \ a a d e g j n n p x z }                             - - 1 2 4 7 8 ; < < = = = > B C E F J N O U V Z _ a c c d d d d f f g i i j k m p q q q q q r s s t y z | }                                        ! # # # ' ) ) - / 2 4 6 6 ; ; ? H J N P U \ ] ] ^ _ ` a a a a a e e e e e g j j j k k k k k k k k k k m m m m m m m m m m o o o o o o o p p p p p p p q r r r r r r r u y y { { { { { { { { { | | | | | | | | | } } } ~ ~   < < < < < < < < = > > > > C D E F G H I I I I K K K K K K L L L ] ^ ^ ^ _ ` a a a a a a a c e e e e g i k m m p r t t t w x y z                   ! ! ! ! ! ! ! ! " " # # % & ( ( ) ) ) * * * , , - . 2 5 6 8 ? ? @ @ A D D F J M N O Q R R R S S T T T U X X Z Z Z [ \ \ d e i j j k l n p t v x y }                       ! " # # $ $ ' ' ) ) + - - - / 0 1 1 6 6 7 ; K K M M M M M P P P W Y _ a b d i m t x { { }                      ! ! & ( , - . / / 0 4 4 5 6 7 7 7 8 : = ? ? ? ? @ C F H J M M N N N P Q R R R R R R S U V [ ^ ` c c e f f h h h i k l n s v v {                 " " # # # $ $ ' ' ) - / 4 = @ H M M N R S S S S T T T V V V V V V V V W W W X Z Z [ [ [ ] ] ^ ` ` ` b e e e e f f j m n p r s u v v v w w w w w w w x x x x x x z z z z z { { } ~ ~      ! $ $ % ( ) . 0 4 9 < @ @ B B D E F G I O O U ^ g h p y }  2 : P V W Z [ \          ! " % ' * S X _ b b b d f t t w z K U e h j n s u w y       + 2 5 > F U Z ` b p    # % % + , - - - / / 1 1 2 8 8 9 : = @ A C D D D E E E E E G H I I I I I J J J L M N N N O O O O P Q R R R U V W X X X X Y Z [ \ ] ] ] ^ ^ _ _ ` ` a a a a a b b c d g g g h h i i j j l l o p u u u u v v x x | }                 " ' ) ) ) , - - . / / / / / / 0 0 0 0 0 0 1 1 7 7 8 8 ? @ @ @ F F G G G G H I K L N N Q R R U V W ] ` ` ` a b b b d f f g g h j j l m m p q q q q s s v v v w w x x { | }  V                                " # # # % & ' ) * , 1 1 1 2 2 3 3 3 4 4 7 7 7 7 9 9 9 9 9 9 9 9 ; ; ; ; ; ; ; ; = = = D D D J M P R R S U U U U V V V V V V V V V V V V V V V V W W X Y Y Y Y Z [ [ [ [ [ \ \ _ _ ` a a a b b b b b b b b b c c c c c c c c c e e e e e e e f g g g g g g g g h j k l l l l l o r s t u u x x x { { { |                                  " ( + + / 0 0 3 4 5 5 5 6 6 7 8 9 ? ? @ B D D D D I I I L O S T U W X Y \ _ _ f i o s z ~ ~                             ! ! ! ! ! ! , 0 ) - . . 0 1 1 2 9 9 9 9 9 9 : : = = = = = > ? @ E E F F F F I I b b b b b b b c h h h i j j m o p p p r r r s s t } } ~                        # $ % & & & & & ) ) ) ) ) - . . 1 3 6 9 ; ; ; < B F F G L N N O P Q Q T U V V Z \ _ _ _ _ _ _ l l { }                           # & ) / 1 7 ; = > ? @ A D F F F F F H L M N P R Y \ \ \ \ \ \ \ ^ ^ ^ _ _ _ ` ` ` ` b d e f f f g g g g g g g g g g g h h i i i i i j k k n n n o o o q q q q r r r r r r r s s s s s s t t t t y y |                      ! ! # + + . 0 2 2 2 7 = ? @ C D G G H K M O S W X Y Y [ [ \ _ ` ` a d h i i j m m m p q q r s x }           ) * 1 8 9 9 : @ A B B B B D D E F F H M O P Q R S W W X Y Y ^ ^ ^ b f k n o q t u v v ~ ~          ) , 1 3 : H U W W ] e g g l t y |       ! % & ( 1 9 I L L P T V Y a m q t |    ! ( 7 < < < ? A F H I P T [ k r r s t x z { ~ ~                                                     " $ $ & & ' ( ) - / / 0 2 4 4 4 4 4 6 6 7 : : : ; ; < < @ A A A A A U ] }                ! " % & ' + . / 0 1 4 8 < A H I N P                     " % ( * / 4 5 : ; < = = = = = = = = > ? ? ? @ A B B E E F F J N Q [ d h r u u w y } }        $ / 2 7 7 7 8 ; ; @ E F G S ^ e p         " ' ) * - 2 2 2 2 2 2 2 2 2 5 6 6 7 7 8 8 : ; < < > > ? A B C D H O Q Q S U U W Z \ _ _ d d f h h h h h j r t v {      % + , / 4 5 6 > E H K P P R T X Z \ \ _ j m o r u v w { { } ~                                  " & ) 1 2 5 5 6 9 9 9 ; @ C G I N O T ^ b e i r U                       ! ! ! ! # # $ $ $ $ $ & ' ' + . / 0 1 1 1 1 1 1 1 1 4 6 7 7 9 ; < < < < > > ? ? E E F F I K M O O O O Q T T V W Y Y ` h o r u          # ( / / 6 6 6 7 ; ; A I K K L M N N O P T T U Y [ [ [ \ \ \ \ ^ ^ ` c d h n |   $ 3 ; G \ d e h v w x x z {                                                     ; < ? @ A B B B B B C D D D E E E F G G G G G G G G G G G G H H I I J K K L L L L L L L L M M N N N O O O O P P P P S S S T T X X X [ [ [ \ ] ] ] ^ ^ ^ ^ ^ _ _ ` ` b b b c c c d e e e f h h j m o q q r r r r r r r r s t t t t w w w x z z z { { { { { { { { } } } } } } } } } ~                            ! ! " % ) - . 0 3 4 4 5 L O O O O O O O P P P P Q Q Q Q Q Q Q Q R R R R R R R R R R R R R R R R R R R U V V V V V V V W W X Y Y Z Z Z [ \ \ ] ` a e e e f f g g g g h j j j j j k m p p q q q q r r r t u v v v x y y y y y y y z z z { } } } } } } } } } } ~ ~                        " " # & - . . / 0 0 0 3 7 9 = M X ^ d n q x x | 6 6 7 7 7 7 7 8 ? ? ? ? ? ? ? ? ? ? B B E E E F F F F F F F G G G G H K M M M R S T T V W X Y Y Z Z Z [ ] ] ] a a a a b b c e e q q w                                  $ & ' ' ( ( ) ) * , , , , , , , - - - - - - . . 4 5 7 7 8 8 8 : < = = > A C C C D D D F F F H I I L L L P P Q Q Q Q Q Q Q Q Q Q U U U U U U U U U U U V V W W W X X X Y Y Y Y Y Z Z [ \ \ \ ] ` c c c d e f f f f f f f f f f g g g h h h h j l m n o o o r u                  ! " , 4 > B C F K M S [ b b j m q r r s w w y |                      # # # * * 0 0 1 2 2 2 8 : : ; ? F H P V W ] a d s y |     " # ( - . . / / 0 2 5 5 5 8 8 : < < = ? ? A B C D I R V [ _ d s x x x { }          " $ & . 6 : F M O Q U Z Z ] m p z |    / F Q T _ w | } } } }     ! " % 0 3 5 5 6 6 : = = ? B E H L L M N P P Q Q S S S S S U V W Z Z Z Z Z Z Z \ ] ^ ^ ^ _ _ _ _ _ _ _ ` ` a a a a a a a a b e g i i j k k k k k k k l l m m m o o p p p s s s u u x } ~                # $ + 2 5 = > ? B J K L L N O P T T V W Y ^ _ _ _ _ ` ` ` a a a a a a a b c c c d g h i m o o r u u x x y y y |                          ! ' ( ) ) ) * , , / / 0 2 3 6 6 7 8 8 ; ; F F F F G G H I I I L L M M O O O O O O O O P R R S T V V V W Y [ \ ] ^ a c c c c h m q w x x x                     # # $ $ $ $ $ % ( * * * * * , - 1 4 4 5 6 6 6 = B F H I J J J J K M M O U V X X b e j l q s v |                             ! $ $ % % % & & ' ( ( ( + , - - . / / / / 0 2 3 4 5 5 5 6 8 8 8 8 8 8 9 ; ; ; < @ E E E E F F G G H H J J J J K L N N N N N N O P R T W W W X X X Z Z Z [ ] ^ ` b g g h i j o r r u v v w {                    " % ( + - - / 0 1 1 1 1 2 3 5 7 ; A R T U W X Y \ ^ d e k q r x x x y y z }       ! ' ( ) . ; B G I Y ` n p s y                   ! # ) * + + . / 0 0 0 0 0 1 1 1 3 4 8 8 9 9 9 : < @ C D D D E F F G H H I J J J K K L M M O O O O O O O P P Q Q R R S T T U U U V V V W W Y Y Z Z Z Z Z Z Z Z Z Z \ \ \ \ ] _ ` ` ` ` ` a a a e g i l m m n q r r u z { }                       " # $ % % % % & & ' ' ' ' ( ) * + + , - / 2 2 2 2 2 2 3 3 4 4 5 5 5 5 6 6 8 9 : ; = = = > > > ? @ @ A E G K K L M O O Q S V W X [ [ \ \ ] ^ ^ ^ ^ _ ` b e g h j k k k p s u w w w | | | | | } ~ ~ ~      ! ! ' ( , . = @ H P S \ \ _ c d f j p w { |            ! " % % ' ( ( ( ( ( ) ) ) ) * * * * - - . 0 0 1 1 3 3 4 5 5 6 : ? A C E K M N T U U U Y ^ c c e k o o p p q r s t u v x y z z | } ~    ( , - / 4 5 5 5 7 7 9 ; ; > ? A F I I J K L M N O O T V V Y a f y { { |                                                                     " $ ( - . 0 1 2 4 4 4 4 8 9 9 ; = ? B B B E F O Y Y Y Y [ [ [ [ [ [ [ [ [ [ ] ` b b c c e f h h i j j l l m n o p q r r s s u y                                              # # # $ $ $ $ $ % % % & & & & & & & ) ) ) * * + + + + + + + + + + + + + , , - - - . / 3 3 3 4 4 5 5 5 5 5 5 5 5 8 9 9 ; ; < < < < < < < < = > > ? @ A E F G G G G G G M M N O Q Q R R U U U U V V V V W X [ [ ] ^ a d f i i j k k l l l l l m m n o o r r s u v v v w w w w x x x y | ~                                                                                                                                        ! ! # $ $ & ' ) ) ) ) ) * * * - 0 0 1 1 1 1 3 4 5 8 8 8 : = > > ? ? ? @ @ @ @ A B D D D D D F F H H K Q Q S U _ c c c e h i l l v z                                                                  ( , 6 7 7 9 ; > C N Q R W ^ d m u y                     " # ( , , , , , , - - - . / / 2 2 2 3 4 4 5 < A A D J K Q Q R S U V Y ] a a a d e f h i k o s u w w { ~                                                                                                           # # # # $ ' ' ' ( ) ) ) ) ) ) ) ) + . . 1 2 3 3 3 4 4 4 4 5 6 : < > ? ? @ B C E E E G I J L N R T [ ^ ^ _ b d d d j l m q r u u u u u v z } }                                                                              ! 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" " " " " " " # # # # # # # # # # # $ $ $ ( ( ( ( ( ( ) , , , , , , , , , , , . . . . / 0 0 0 0 1 1 1 1 1 1 1 1 2 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 7 7 7 7 7 8 9 9 9 9 9 : : : : : < = = ? @ @ @ A A B B B B C E E E H H H H H H H H I K M M M M M O O O R U V V W W Z Z Z Z Z Z Z \ \ ] ] _ _ _ ` ` ` a c f i i i j k k m m n n o o o o o p s s s s s s s s s s t t t t u v v v w w w w w x x y y { ~ ~                                                                                                                                                                                                                                                                                                                                          # # $ ( ) * - 0 4 6 8 8 > > > ? ? @ @ A B B D F I I I I K K K K K K K K K K K K K K K L L L L L L L L L L L L L L L L L L L L M M M M M M M O O P P P Q R R R R S S S T T T T T T T T U V V V V V V W W W W W W W X X Y Y Y Y Y QZ Z Z Z [ [ [ [ [ [ [ [ [ [ [ \ \ \ ] ] ] ^ _ a a d d f f h i i k k m m m n n n o o q q s t u u u u u u u u u v v v v w w w x x z z { { | | | |                                                                                                                                                                                                                                                                             ! ! # $ $ $ % & & & & & ' + , , , - . 2 3 6 7 8 8 : < = > ? @ @ @ E I Q U V V X X Y ^ d f g i i p p r r t t u | ~              P                                                                                                                            % ' ' ' + - - . . . . / / 0 1 1 2 3 3 3 3 4 4 5 7 7 9 > > > G O V Z [ \ ^ ` d i n s w w w z | ~                                                                                                                                                 & - - - 4 6 7 8 9 9 9 9 9 < < ? B E E F G I I I I I I I I I I I J J J J J J J K K K K K K L L L L L L L L L M M M M M M M N N N P Q Q Q Q Q Q Q R R R R R R R R R R R R R R R R R R R R S S S T T U U U V V W X X X Y Y Y Y Y Y Z [ \ \ ] ] ] ] ] ] ] ] ] ] ] ^ _ _ _ _ _ ` ` ` ` ` ` a a a b b c c c c c d d d d d e e e e e f g g g h h h h h h h i i j l l m m m m n o o o o p p q r s s y {                                                                                                                                                                                      ! ! ! ! " " $ % - / 0 1 2 2 2 3 3 3 4 4 5 9 9 ; ? A B L N O P S T U W Y Y Y Z Z ] ` f j l m m n o q w z |                                                                                    & ' + 1 3 3 3 4 5 6 7 7 8 8 : : ; ; > ? @ @ B B C C C E E H H H H J L O P Q R T W X X Y Z Z Z \ ^ ^ ^ _ _ _ a b b e j j k n o o p r u { } }                                                                                    " % * + - . . . / 4 8 9 9 : = = ? 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" " # # # # # # # # # # # # # # # # % % % ' ' ( ( ( ( ( ( ) ) ) ) ) ) ) * * + + + + , . / 0 1 3 6 6 6 6 88 8 8 8 8 8 8 9 9 9 9 : : : : ; ; ; ; ; ; ; ; ; ; ; ; = = = > > ? @ @ A B B B B B B B B B B D D D D D D D D D E F F F G H I I I I I I I I I I L M M N N N O P S T U W W W X X Y Y Z Z Z Z [ [ \ \ \ \ \ \ \ \ \ \ ^ _ _ a a a a b b b b b b b b b b b b b c c d d d d d e e e e f f g g h i i k k l m m m m m m m n n n n n n r t x x x z z z { { | } } } ~  7 € ǀ ǀ Ȁ Ȁ ɀ ɀ ʀ ʀ ʀ ΀ Ѐ Ԁ ׀ ڀ ۀ ܀ ݀ ހ ߀                       ! ! " " " # # # $ & ( ) ) ) * + , , - - - 0 0 0 1 1 1 2 3 3 3 3 4 4 4 4 4 6 < < < < < > > ? ? @ @ @ A B B C D D D D E E E H H H H H H H H H H H J N O O P P P S S S S S V W Y Y Y Y b b b d d f f f g j l n o q q q r r r u v x x x y z { | | | | | | |  Á Á Ł Ł ǁ ȁ ́ Ё с ҁ ԁ ׁ ځ ځ ہ ہ ہ ݁                              ! " & ( - - . 5 6 7 < @ E F H H I K K L L L Q Q R V W X \ ] ] ] ] ^ ^ ^ a a b j k o r s w x y z { { | | } } ~    à à à à ł ł ł ł Ƃ Ƃ Ƃ Ƃ ǂ Ȃ ɂ ɂ ʂ ̂ ̂ ͂ ͂ ͂ ͂ ΂ ΂ ς т ւ ւ ؂ ۂ ܂ ނ ߂ ߂ ߂ ߂ ߂ ߂                         ! ! " & ( ( ) ) - 0 0 0 0 1 2 2 2 3 4 7 : < ? i l r r r r s { ڃ ܃ ܃ ܃ ܃ ܃ ܃ ݃ ރ ރ ߃ 6                                ! # % A A C C C C D G J K L P Q S V V X [ [ ] ` a a d e g j m n p p p q r s w  Ą DŽ Ʉ ̈́ ΄ ΄ ΄ Є ӄ ӄ ӄ ӄ ӄ ӄ Ԅ Ԅ Մ Մ Մ ք ؄ ؄ ڄ ۄ ݄ ߄ ߄ ߄                                                    ! # # # $ & ' * * + , 0 1 3 4 5 6 ; < < < < < < < = > A B B B D D H I K N Q R T U U V V V V X Y [ [ [ ] ^ ^ ^ ^ ` a c c c d d d e e e e e f f f f f g h h h h i i k k l l l l l l m o s t t u u u u u v x y y { { { { } } } } } ~   ą ƅ ƅ ƅ ƅ ͅ ΅ Ӆ ԅ ԅ ԅ ԅ ׅ م څ ۅ ݅ ݅ ݅ ݅ ݅ ݅ ޅ ޅ                                        " " " $ & ' ( ( 1 3 3 3 6 6 6 9 ? @ B B 5D D D E E E F I I I I I I I I I I I J J J J J J J K L L L L L L N P R T V W Z Z [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ ] ] ] ] ] ] ] ] ] ] ] ] ] ^ ^ ^ _ _ _ _ _ _ ` a b b b b b b b c c c c c c d e e e e g g g h l p t t t u u u v v v v x y y { | |  † † Æ Ć Ć dž Ȇ Ɇ Ɇ ʆ ʆ ˆ ˆ ͆ Ά Ά Ά Ά φ φ φ І І ц ц ӆ Ԇ ؆ ؆ چ ܆ ܆ ܆ ݆ ކ ކ ߆ ߆ ߆                            " " " & & & & ' ' ) ) * * + + + . . / / / / / / / / 1 1 1 1 1 1 1 1 2 2 2 3 4 5 5 6 7 8 8 8 8 : = > ? ? 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" # # # # $ $ $ % % % ' ( * + / / / / / / 0 0 1 3 3 3 4 4 4 4 5 5 5 6 6 7 8 9 9 < = @ @ @ @ @ A A G G I J K L P S T T U V Y Z Z [ ] ] ] ] ] ] _ a b e e g k k k l m m m n p p q s s t t v w w x x y z z { | } ‰ É É ĉ Ɖ ȉ ̉ ͉ ω Ӊ Ӊ Ӊ ԉ Չ ؉ ډ ߉              " & & ( , - - / 6 6 < ? @ E E G H H K Q ] _ c d h j            ! ! $ & & & ( ) ) + + + + + , , , - - - . . / / / / / 0 0 0 0 0 0 0 0 2 2 5 6 6 7 : : : ; = @ A D E H L L M M M M O O R R S S S S T U W W X X Y Y Y Y Y Y Y Z Z \ ] ] ] ] ] ] ] ] ] ] _ _ _ _ _ _ ` ` ` ` a c c c c c g k k k k k l l l l m n n p p p p q q r s s s s s s s s t t v v w x x y z z z z { { } } ~ ~   ‹ ċ ċ ċ ŋ ŋ ȋ ˋ ΋ ΋ ϋ ы ҋ Ӌ ԋ ԋ Ջ Ջ Ջ ֋ ֋ ֋ ؋ ܋ ߋ                                    ! ! ! ! ! " " " " " " " " " # % % % & ' ' ' ( - - 1 2 3 : : < > B C C D F G J N N S S T T U W [ ^ _ _ _ ` ` ` ` b b b c c d d e e e e e e f g h h k k l p s s s t u w y { |   $ % * . 2 6 9 ? A G J R U V V X Z Z Z Z Z [ \ _ _ a b e h n r r r s s t t t u u v v v v x x x z ~  Í Ǎ ͍ ΍ ΍ Ѝ Ӎ ԍ ׍ ڍ ߍ         # - 4 6 = ? ? A F F H H H H I Q R U U X \ ` ` c d e i j j j l s u w { Î Î Ď Ď ǎ Ȏ Ȏ Ɏ Ɏ Ɏ Ɏ ʎ ̎ ̎ ̎ ̎ ͎ ͎ ͎ Ύ ю ю ӎ ؎ َ ڎ ڎ ڎ ڎ ڎ ێ ܎ ގ ގ ގ ގ ގ ގ                  " " " & & & ) - / 1 4 8 8 9 > ? @ B E J J J M N P P Q R R R S T X Z \ ] _ _ _ a c c c c c c c c c c d e e e e e e e f f f f h h h h h h h h h i i i i l l l m o o q s t t t t t t t t t t t t t t t t t t t t t t t u u u u u v w w w x x x y y y y | ~  Ï ď ď ď ď ď ď ď ď ď ď ď ď ď ď ď ď ď ď ď ď ď ď ď ŏ ŏ ŏ ŏ ŏ ŏ Ə Ə Ǐ Ǐ Ǐ ȏ ɏ ʏ ˏ ˏ ˏ ˏ ̏ ̏ ̏ ̏ ̏ Ϗ Ϗ Ϗ Џ я я я ҏ ҏ ҏ ҏ ҏ ҏ ӏ ӏ ԏ ֏ ֏ ֏ ֏ ׏ ؏ ؏ ؏ ُ ڏ ڏ ڏ ڏ ܏ ܏ ݏ ݏ ݏ ߏ ߏ                                ! " % & , . / / 1 1 2 2 3 5 9 : : : : ; A B J J J K L P S \ ] _ j k m m m o p s w | ~  Đ Đ Ő ʐ ː ͐ ͐ ѐ א ܐ ސ ߐ ߐ                     ! ! % & ' ( ) ) + - 2 2 2 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 : : : : : : : ; ; ; ; ; ; ; ; ; ; ; < < < < = = > > > > ? ? ? @ A A B B C C C C C C C D D D E E E E G G G H H J J J J J K K K K K K K K K K K K L L L L L L L L L L L L L L L L L M M M M N N N N N N N N N N N N N N N N N N P P P P P P P Q R R R R R R R R R R R R R R R R R R R R R R R R R R R T T T T T T T T T T T T T T T T T T T T T T U U U V V V V V V V X Y Y Y Z \ \ \ \ \ \ \ \ \ \ \ \ ] ] ] ^ ^ ^ ^ ^ ^ ^ _ _ _ _ _ _ d g h h i i i i j j j j j k l l l l m n n o o o o p p p p p p p p p p p p p p p p p p p p p p p s s s s u u u w w x x x x x x x x x y y y y y y y y y y y y y y y z z z z z z z z z z z z z z z z { { { { { { { { | | | | | | | } ~ ~ ~                             ‘ Ñ Ñ ő Ǒ ȑ ȑ ɑ ʑ ʑ ʑ ʑ ʑ ʑ ʑ ʑ ʑ ʑ ʑ ʑ ˑ ˑ ˑ ˑ ˑ ˑ ˑ ˑ ˑ ̑ ͑ ͑ ͑ ͑ ͑ ͑ ͑ ͑ ͑ ͑ Α Α Α Α Α ϑ ϑ ϑ ϑ ϑ ϑ Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б Б ё ё ё ё ё ё ё ё ё ё ё ё ё ё ё ё ё ё ё ё ё ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ґ ӑ ӑ ӑ ӑ ӑ ӑ ӑ ӑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ ԑ Ց ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ֑ ב ב ב ב ב ב ב ّ ڑ ڑ ڑ ܑ ݑ ݑ ݑ ݑ ݑ ݑ ݑ ݑ ݑ ݑ ݑ ޑ ޑ ޑ ޑ ޑ ޑ ߑ ߑ ߑ ߑ ߑ                                                                   ! # # # $ $ $ $ $ $ $ $ $ $ $ % % % & & & ' ( ( ( ( ( ( ( , , , , , , . 0 0 3 3 3 3 3 4 6 6 9 9 : : : ; ; < = = > @ @ @ @ @ A B B D D D D D D D D D D D D D D F F F G G H J K K L L L L L L L L N N O P P Q Q Q Q Q Q Q R R S S S S S S T T T T T T T T T T V V Y Y Y [ [ [ \ \ ] _ b d f f g g g g h k l t x z | | | } ~ ~ ʒ ʒ ˒ ̒ ̒ ͒ ϒ ϒ ђ Ԓ Ւ ג ؒ ے ݒ ޒ ߒ                           ! ! # ( , / 2 4 5 8 9 9 9 : : : : : : : : < < < = > > > ? ? ? @ A C C C C E E E E I J J J K K K L L M M M O O O O O P P P P P Q Q Q Q Q R U V g h l l l m m m m m m m n n n o o r r r s t u u { | } ~   Ó ē œ œ œ Ɠ Ǔ Ǔ ȓ ȓ ɓ ʓ ͓ ϓ ϓ ϓ Г Г Г ғ ӓ Փ ד ٓ ܓ ܓ ܓ ܓ ܓ ܓ ܓ ܓ ݓ ݓ ݓ ޓ ޓ                    ! & ) - 1 3 ; ; ; < < = F K L L L L L L N N O P R T T T U U U U W W X Z \ \ _ a b h i i i j l r t u v w { { { } Ɣ ˔ Д Ӕ ה ۔                             ! # # # $ $ % % % % % % % ( . 0 2 2 2 4 7 7 < > @ H O P S V V V W Y Y Y Z Z Z [ [ ^ b b f g i j j k k k k k k k k k l l n o o o p q q r r r t t t t t t u v v v v w w w w w w w w x y z { ~ ~   • ĕ Ǖ ɕ ɕ ̕ ϕ Е ҕ ҕ ӕ ԕ ԕ ԕ ԕ ԕ Օ ֕ ֕ ו ו ؕ ڕ ڕ ە ޕ ޕ ޕ ߕ                 + , - - - - - - - - - - - - . 0 0 1 1 1 1 2 3 3 3 4 4 6 6 6 6 6 8 9 ; < < = > ? @ @ @ B C D D E E G H I J J K L M N N Q Q Q R V W X Y \ \ ] ] ] ] ] ] ] ] ] ] ] ] ] ] ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ _ _ _ ` ` a a b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b b c c c c c c c c c c c c c c c c c c c c c c c c c c c d d d d d d d d e e e e e e e e e e e e e e e e f f f f f g g g g g g g g g g g h h h h h h h h h h h h h h h h h h h h h h i i i j j j k k k k n n n n n n n n n n n n n n n n n n n n o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o p p p p p p q r r s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t u u u u v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v w w w w w x x x x x x x x x x x x x x x x x x x x x x y y y y y y y y y y y y y y y y y y y z z z z z z z } } } } } ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~       – – – – – – – – Ö Ö Ö Ö Ö Ö Ö Ö Ö Ö Ö Ė Ė 4Ė Ė Ė Ė Ė Ė Ė Ė Ė Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ŗ Ɩ Ɩ Ɩ Ɩ Ɩ Ɩ Ɩ Ɩ Ɩ Ɩ Ɩ Ɩ ǖ Ȗ Ȗ Ȗ Ȗ ʖ ʖ ʖ ˖ ˖ ̖ ̖ ؖ ؖ ؖ ٖ ږ ۖ ۖ ۖ ܖ ܖ ݖ ޖ ޖ ޖ ޖ ޖ ޖ ޖ ޖ ޖ ޖ ޖ ޖ 3                                                                        D D D D D D D E E E F F F F G G G H H H J J J J J J J J J M M N O O O O O O P P P P P P P P Q Q Q R R R R R R R R R R R a a a a a a a b c c c c c c d 2 × × × × × × × × × × ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ė ŗ ŗ ŗ Ɨ Ɨ Ɨ Ɨ Ɨ Ɨ Ɨ Ɨ Ɨ Ɨ Ɨ Ɨ Ǘ Ǘ Ǘ Ǘ Ǘ Ǘ Ǘ Ǘ Ǘ Ǘ Ǘ Ǘ Ǘ ȗ ȗ ȗ ȗ ȗ ȗ ȗ ȗ ȗ ȗ ȗ ȗ ɗ ɗ ɗ ɗ ɗ ɗ ɗ ɗ ɗ ɗ ɗ ʗ ʗ ʗ ʗ ʗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ˗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ̗ ͗ Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η Η ϗ ϗ ϗ З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З З ї ї ї ї ї ї ї ї ї ї ї ї ї ї ї ї ї ї ї ї җ җ җ җ җ җ җ җ җ җ җ җ ӗ ӗ ӗ ӗ ԗ ԗ ԗ ԗ ԗ ԗ ԗ ԗ ԗ | | | | ~ ~ ~ ~               ˜ ˜ ˜ ˜ Ø Ø Ø Ø Ø Ø Ø Ę Ę Ř Ř Ř Ř Ř Ř Ř Ř Ř Ř Ř Ř Ř Ř Ř Ř Ř ǘ ǘ ǘ ǘ ǘ ǘ ǘ ǘ ǘ ǘ ǘ ǘ ǘ Ș Ș Ș Ș Ș Ș Ș Ș Ș Ș Ș Ș Ș Ș Ș Ș Ș Ș ɘ ɘ ɘ ɘ ɘ ɘ ɘ ɘ ɘ ɘ ɘ ɘ ɘ ɘ ɘ ɘ ʘ ʘ ˘ ˘ ˘ ̘ ̘ ̘ ͘ Θ Θ Θ Θ Ϙ Ϙ Ϙ Ϙ Ϙ Ϙ ј Ә Ԙ Ԙ ՘ ՘ ՘ ֘ ט ט ט ט ט ט ט ט ט ט ט ט ט ט ט ؘ ؘ ؘ ؘ ؘ ؘ ؘ ؘ ؘ ؘ ؘ ؘ ٘ ٘ ٘ ٘ ٘ ٘ ٘ ژ ژ ژ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ۘ ܘ ܘ ܘ ܘ ܘ ݘ ݘ ݘ ݘ ݘ ݘ ޘ ޘ ޘ ޘ ޘ ޘ ޘ ޘ ޘ ޘ ޘ ߘ                                                                                                              ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! " " " " " " " # # $ % & & ' ' + + + + + + + 0 0 1 1 1 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 4 6 6 6 7 7 8 8 8 8 8 8 9 9 : : : : : ; = = > > > > > ? @ @ @ @ @ @ @ @ @ @ A B B B C E E E F F F F G G H H H I I I I K K K N P Q Q Q Q R R S W W W W X X X X Z [ \ ] ] ] ] ^ _ _ ` ` a b b b f f f f g i j k m n n n n p p p p p p p p p r r s s t t t t t t t t w w w w w w x x x x x x x x x x y y y { { { { | | | } } } } ~ ~ ~ ~ ~   ę ę ę ę ę ę ę ę ř ř ř ř ř ř ř ř ř Ǚ Ǚ Ǚ Ǚ Ǚ Ǚ Ǚ Ǚ Ǚ ș ș ə ə ə ə ə ə ə ə ə ə ə ə ə ə ə ə ə ə ə Й Й Й Й ٙ ٙ ݙ ݙ ޙ ޙ ޙ ޙ ޙ ޙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ ߙ 1                                                                                                                 ! ! ! " " " " " " " " # # # # # $ % % % & & & ' ' ( ( ( ( ( ( ) ) ) ) * * + + + + + + + + + + + + + + + + + + + + + + + , , , , , , , , , , , , , , , , , , , , , , , , , , , - - / 0 1 2 4 4 4 4 4 5 5 5 6 7 7 7 8 8 8 9 9 9 : : ; ; ; ; < < < = = = > ? ? ? ? B B B C C C C C C C N N N N N N N N N N N N N N N N N O O P P P P P Q R R R R R R S U U U U U V W X X X Y Y Y Y Y Y Y Y Y Y Y Y Z Z Z Z Z Z Z Z Z Z Z [ ` ` ` ` ` ` ` a b b b b b b b b b b b b b b b b b b b b b b b b b c c c d d d d e e e e e e e f f f f f f f f f f f f f f f f f f f f f f f f f f f f f g g h h h h h h i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i j j j j j j j j j j j j j j j j j j j j j j j j j k k k l l m m m n n n n n n n n n n n n n n n n o o o p p p p p q q q q q q q q q q q q q q q q q q r r r s s s t t t t u v v v w w w w w x x x x x x x x x y y y z z z z z | | | | | | | | š š š š š š š š š š š š š š Ú Ú Ú Ú Ě Ě Ś Ś ƚ ƚ ƚ ƚ ƚ ƚ ƚ ƚ ƚ ƚ ƚ ƚ ƚ ǚ Ț ʚ ʚ ʚ ̚ ̚ ̚ Κ њ њ њ Ӛ Ӛ Ԛ Ԛ Ԛ ך ך ך ך ך ך ؚ ښ ښ ښ ښ ښ ܚ ܚ ܚ ܚ ܚ ܚ ܚ ܚ ޚ ޚ ߚ ߚ                                                                                                                                               ! ! ! " $ $ % & & & ( ( ( ) ) ) * * * + + + , , - - - . . 0 1 1 5 5 5 6 6 6 6 6 6 6 7 8 8 8 8 8 8 8 8 9 : ; ; ; < < = > ? ? ? @ @ A A A A A B B C C C C C C C C C C C D D D E E F F F F F F F F F F F F F F F F F F F F F G G G H H H H H I I J J J J J J J J J J J J K L L M M M M M M N N R S V W W Y Y Y Y Y Y [ \ \ \ ^ a a a b c e e e e e e e f g g h h h h h h h h h h j k m n o p q q q q q q r t t t u u v w w w w x x y z ~                       Û Û Û Û ț ͛ ͛ ͛ ͛ ͛ ͛ ͛ ͛ ͛ ͛ ͛ ͛ ͛ ͛ Λ Λ Λ Λ ћ ћ ћ ћ ћ қ қ қ қ қ қ қ қ ӛ ӛ ӛ ԛ ՛ ՛ ֛ ֛ ֛ כ כ כ כ כ ؛ ؛ ؛ ؛ ؛ ؛ ؛ ؛ ٛ ٛ ڛ ڛ ڛ ڛ ڛ ݛ ݛ ݛ ޛ J O S Y ] ^ ^ Ü Ü Ü Ĝ Ĝ ʜ ˜ ͜ ͜           " " " " # $ $ $ & & & ' ' ) + , 0 3 3 A A A B B F J M N P P Y Y ] 0` c d e f g g g h i i i i l m m m n p q r t v w x x z { { { { { { | | | | | } ~ ~    Ý ĝ ĝ ĝ ȝ ʝ ˝ ̝ Н ԝ ԝ ם ם ؝ ٝ ٝ ޝ ߝ                  " ) ) + - - / 0 2 ? A B D H I I L N Q R S S T U W X [ [ _ b c c c h h h i i j k l m n n o o o p p q w y } } ž Þ ƞ Ǟ Ȟ Ȟ Ξ Ϟ О Ӟ Ӟ ٞ ܞ ߞ         / / / / / / / 0 1 1 1 1 1 1 1 1 4 4 4 4 4 4 4 4 4 5 6 6 6 7 8 8 ; < < < = = = = = > > > > > > > ` ` ` a a a a a b b b d d e f f h i i j j j j j k k l l m n q q v v v v v v v v v v v v v v v v y y y y y y y y y z z { Ÿ Ÿ ş ş ş ş ş ş ȟ ȟ ȟ ȟ ȟ ȟ ȟ ɟ ʟ ˟ ͟ ͟ ͟ ͟ ͟ Ο Ο ϟ ϟ ϟ ϟ ϟ ϟ ϟ ϟ ϟ ϟ ϟ ϟ П џ џ џ ҟ ҟ ӟ ӟ ӟ ӟ ӟ ӟ ӟ ӟ ӟ ӟ ӟ ԟ ԟ ԟ ԟ ԟ ՟ ՟ ՟ ՟ ֟ ֟ ֟ ؟ ؟ ؟ ؟ ؟ ؟ ؟ ؟ ؟ ؟ ؟ ٟ ٟ ٟ ٟ ٟ ٟ ٟ ٟ ڟ ڟ ڟ ڟ ۟ ۟ ۟ ۟ ۟ ۟ ۟ ۟ ۟ ޟ ޟ ޟ ޟ ޟ ޟ ޟ ޟ ޟ         l n q s s s s t w w w x x x y y | | | ~   à à à Ġ Ġ Ġ Ġ Ġ Ġ Š Ơ Ǡ Ǡ ɠ ʠ ʠ Ѡ Ӡ Ԡ ٠ ܠ ݠ ޠ                  " % ' ( ( * - 1 2 2 3 3 4 8 9 A G K L R R S U X \ \ \ ] _ _ ` b c d e e g g g h i j m n q s u v v v w w x y z z z z z z z ġ ǡ ȡ ʡ ˡ ̡ ̡ ̡ ̡ ̡ ̡ ͡ ͡ ͡ Ρ Ρ ϡ ѡ ѡ ӡ ԡ ԡ ԡ ա ֡ ֡ ס ١ ١ ١ ١ ١ ۡ ۡ ۡ ۡ ۡ ۡ ۡ ߡ ߡ ߡ ߡ ߡ ߡ ߡ                ! " $ * * * - 1 1 2 3 3 3 4 6 6 7 7 7 7 7 : ; ; ; ; < < < = = = > A A B B B C C C C C D D D F F F F F F F F H H H I K L L L L L M N N O P P P R R R R S S S S S T T U U U W W Y \ ] _ b b b c c h j k k l l l n r s w z z ~ Ƣ ͢ ΢ Ϣ Т Т Ԣ ֢ ע ٢ ٢ ڢ                              ! & - 5 9 = > @ @ @ @ @ @ @ @ @ @ A A A A A A A A A A A B B B B B C C C C C C C C C D D D F F G I J K K K K L M M M M M M N N O O O P Q Q R R T U U U U U V V V V V W W W X Y Y Y \ ` ` a d e g g h h h j k s u x { ~ ~ ~ £ ̣ ̣ ͣ ͣ ͣ ϣ ϣ ң գ գ ֣ ף ڣ ݣ ݣ ݣ ߣ                      ! " # # # $ $ % & & ( ) + + / 1 7 = ? C C F G M O R R U V W W W X \ ] ] ^ _ a i m n p t y z |  ¤ ä ä Ť Ƥ Ƥ Ƥ Ȥ ʤ ʤ ˤ Ҥ դ ֤ ٤ ٤ ۤ ܤ ߤ ߤ ߤ ߤ ߤ ߤ ߤ ߤ ߤ ߤ                      ! ! " # * + + , - . 0 0 0 1 3 3 5 5 5 9 : ; ; ; ; ; ; < > ? ? @ @ B E E F G H H H H H I I I I J K K M M M M M M M M N N N N N N O O P Q R S U V Y Y Z \ a b f f g g g h k l m m m m n n n n n n n n o o p q q q q q r r v v x { ¥ ¥ å ĥ ʥ Х ԥ ץ ٥ ݥ ޥ ޥ ޥ ޥ ߥ                                           ! $ % % % & & & & ) ) ) ) * + 0 2 3 5 6 7 7 7 8 8 ; ; < < > ? @ @ A A A A A B B B B B B B B B C C C C C C E E E F H H H I J J J J J J J J J J J J J J K K L M N O P Q Q Q Q Q Q Q Q Q Q Q R R R R S S S S S S S S W W W W W W Y Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z [ \ \ \ _ _ _ _ ` ` ` ` b c c c d d d d e e e f g h i i j j j j j k o o q q q q q q q q q q q r u u u u x y y y y y y y y y y y y y y y y y y y z z | | } } } ~ ~ ~ æ ɦ ʦ ʦ ʦ ͦ Ϧ Ц Ц Ц Ѧ Ӧ զ ئ ٦ ܦ ݦ ߦ ߦ                                   ! % % ' ) * * * * * + + + + + + + + + + + + + + + + + + + + + + + + , - - - - - / / 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 4 4 5 5 5 6 6 6 6 7 8 8 9 9 9 9 9 9 9 9 9 9 9 ; ; = > ? B B F F F F F G I P S S V X X Y Y Y \ \ ] _ ` ` e g h h h h h h l l m m n o p q q s s w y y z { { { § § § § § § § ç ç ç ŧ ǧ ȧ ȧ ʧ ʧ ʧ ˧ ˧ ˧ ̧ ̧ ̧ ̧ Χ Χ                     : : = A I I J M W X Y Z Z Z Z Z Z \ ^ _ _ _ a a a a b e g i i i n n n o o o s s t u z z z { } } ~  Ĩ Ĩ Ĩ Ĩ Ũ ƨ Ǩ ɨ ɨ ̨ ̨ ̨ ̨ ̨ ̨ Ψ Ψ Ψ Ш Ш Ѩ Ҩ Ө ը ը ֨ ר ٨ ٨ ڨ ڨ ۨ ۨ ި                                 ! ! ! " " " " & ' ( , , , , 0 0 0 3 7 9 9 : : : : : < < < = > > ? ? ? @ @ C C C F F J J J J J N S S T V W Z [ [ [ \ ^ ^ ` b c d d d f h h h j l l l l l m m m p t t t w x x x x x x x x { { ~   ɩ ɩ ɩ ɩ ͩ ϩ ѩ ѩ ѩ ѩ ѩ ѩ ө ө ө ө ԩ թ ة ة ک ܩ ީ ީ                     " " " " " $ $ % & -   Ī Ū ˪ ͪ Ϊ Ϫ Ѫ Ѫ Ѫ Ѫ Ӫ ت ٪ ٪ ٪ ڪ ۪ ۪ ۪ ۪ ު ު ު ߪ /                                       ! " " $ $ $ & & ) - . . 0 2 4 5 7 7 7 9 9 : .< = = @ B B B E E F H I I I J K L M N N O O O O O R S T U U U U V V W W W Z Z ^ _ b c h k l n p q s t t t v w y z { { | | | ~   « « « ū ū ū ū ū ƫ ƫ ƫ ǫ ǫ ǫ ȫ ɫ ˫ ˫ Ϋ Ϋ Ϋ Ϋ Ы Ы ԫ ի ի ׫ ׫ ث ث ث ث ث ٫ ٫ ٫ ٫ ٫ ٫ ګ ګ ګ ګ ۫ ۫ ۫ ۫ ܫ ܫ ܫ ܫ ܫ ܫ ܫ ݫ ݫ ݫ ݫ ݫ ݫ ݫ ݫ ޫ ߫ ߫                       -   " # # ( * * + + , , , , - - . . / 1 3 6 9 : < = = @ A A E G H H H I J J J J L M N Q Q Q Q Q R R S S U U U U U V W X Z Z Z \ a b d e f g h h j j k l l l n p q q q q q q q q q q q q r r r r r s v x x y y y | }  ì Ƭ Ǭ Ǭ Ȭ ̬ ̬ ̬ ͬ ͬ ͬ ά Ϭ Ϭ Ϭ Ҭ Ҭ Ӭ Ӭ Ԭ Ԭ լ լ ֬ ֬ ֬ ֬ ֬ ֬ ֬ ֬ ׬ ׬ ׬ ٬ ٬ ٬ ڬ ڬ ڬ ڬ ݬ ެ ߬ ߬                                              ! ! # % & & & & ( ( ) ) * * * + + , , - - . . . . / 0 3 4 4 4 6 6 6 6 6 6 6 7 7 9 9 9 9 9 : : : : : : : : : : : : : ; ; ; ; ; ; ; ; ; < = ? ? ? ? E F J K N P Q Q U X \ \ ] ] ^ _ _ _ a a d d e f g j j j j k m o p p q t u u v v v w w y y y y z { { { { | | } } ~  ĭ ĭ ǭ ǭ ǭ ɭ ɭ ɭ ʭ ʭ ʭ ˭ ˭ ˭ ˭ ̭ ̭ ͭ ͭ ͭ ͭ ϭ ϭ Э ѭ ֭ ֭ ֭ ֭ ֭ ׭ ׭ ح ٭ ۭ ܭ ܭ ݭ ݭ ޭ ޭ ޭ ޭ ޭ ޭ ޭ ޭ ޭ ޭ ߭                                       C H f f i j n n p s u y } }  ® Į Ʈ Ȯ ɮ ʮ ʮ ʮ ʮ ʮ ʮ ʮ ˮ ͮ ͮ ή ,Ϯ Ӯ ծ ծ ծ ׮ خ ٮ ڮ ޮ ޮ ޮ                    ! # ' ' ) , 0 1 6 9 : > > ? A B B B B B C C E E E E E I J J K M M M O Q Q R V W Y \ _ ` b b e g g g j l m r s s t u u u u u v y y y y z z { | | | | | } } } } ~ ~   ï ï ů ͯ ͯ ѯ ү ү ׯ ٯ گ ۯ ܯ ݯ ݯ ݯ ߯ ߯                                                 ! ! ! ! ! ! ! ! ! " " " " " " # $ $ $ $ $ $ $ $ % % % % % % % % % % % & & ' ' ' ' ' ' ' ' ' ' ( ( ( ) ) ) ) ) * + + + - - - - / 2 4 5 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 8 8 8 8 8 9 9 9 ; ; = > > ? @ @ @ @ @ @ @ B B B B B C D D E E E E E E E E F F F F F F F F F F F I I K K K L L L L L L L L L L L L T U V V \ ^ c d d e g h i i m m n o o o o o o o o r u v x x x x x x x { { { { { { { { { { { { { | | | |   Ű Ȱ ɰ ˰ ̰ ̰ ΰ ΰ ϰ ϰ а Ұ Ӱ ԰ ԰ ְ ڰ ۰ ݰ ݰ ߰ ߰                             # $ $ $ $ $ $ $ ' ) * + - / 0 6 9 ; < > > > > > ? @ D D E E I I L O P P Q R T W Y [ ] ] ] ] ^ ` a a a b b e f g g g l l n n p q q w w y |  Ʊ ȱ ױ ױ ر ر ݱ ޱ           ! " & ' ( , , . 1 6 8 8 8 ; ; ; ; ; < > ? @ @ @ @ @ @ @ @ A A A C C D D D E E E E E E G I J K K L L N N O O O P Q Q R S T V X Y Y Y [ ` c g i i i m n n o o r s w w x x z z } } }  Dz ɲ в Ӳ Ӳ Բ ײ ݲ      4 7 < ó dz dz ˳ γ γ س     E G G G G I I J O O O O U U U U Z Z [ a a c e f l n o p p q q q q s t t t t w x x x y y z } } } } ~  ´ ´ ´ ô Ŵ Ǵ ȴ ʴ ̴          # % % % % % & ( ) + + + + . / / 1 1 3 4 6 6 8 8 8 8 = ? C C C D E E G L R S U X X [ [ [ [ [ \ ^ c c e f i p r w x z { } ĵ ȵ е е ӵ ֵ ׵ ٵ ڵ ڵ ڵ ܵ ݵ ޵              $ ( ( ( , , - 3 : < = A B D D F F F H I P R T U W [ _ d f j j n p t v z  Ŷ Ŷ ʶ ж ض ض ڶ ۶ ܶ ܶ ܶ ޶ ޶ ޶ ߶ ߶ ߶ ߶                          " # % % ' ' ' ' ( ) ) ) + + + - - - - - - - . . . . . . . / 0 0 2 2 2 2 4 5 5 8 9 9 : : ; ; = = B C G H I M N P R R S U V W W X Z [ \ ] ] ] _ _ _ _ ` d e e f k n q s s w y | · ÷ ÷ ÷ ÷ ÷ ÷ ķ ŷ ŷ ŷ Ƿ Ƿ Ƿ ȷ ȷ ɷ ʷ ̷ ̷ ̷ Ϸ Ϸ з з з з ѷ ҷ ӷ Է Է Է Է շ շ շ շ շ շ ׷ ط ۷ ۷ ۷ ۷ ۷ ۷ ߷ ߷ ߷ ߷ ߷                                                                             ! ! ! ! ! ! ! ! $ % ' ' ' ( ( ( ) ) ) * * * * + + + , - - - - - - . . 0 0 0 0 0 0 0 0 1 2 2 2 4 4 7 7 7 7 7 7 7 8 8 9 < < < < < < < < < < < = = > > > > ? ? C D D E F F F F F F G G I I J L M O O P Q Q T T V Z Z Z \ \ \ ^ d d e g h j k k l l l m n n n n n n s Ƹ Ƹ Ƹ ʸ ʸ ʸ ˸ ˸ ̸ ̸ ̸ θ Ը Ը Ը Ը Ը Ը Ը    - - 4 4 7 8 8 8 9 9 9 9 9 9 9 9 : : : : ; ; ; ; ; ; ; ; ; = = = = = = > > ? ? ? ? ? ? ? ? ? ? ? @ @ @ @ @ @ @ @ @ @ @ @ @ A A A A A A A C C E E E E E E E E E E F F G G G H H H H H H H H W X X X Y Y Y Y a a b c d f f i i i j ¹ ¹ Ź Ź Ź ǹ ǹ ǹ ǹ ǹ ǹ ȹ ʹ ʹ ʹ ʹ ̹ ͹ Ϲ Ϲ Ϲ й й ѹ ӹ չ չ ֹ ع ع ۹ ۹ ۹ ܹ ܹ ݹ ݹ ݹ ߹ ߹                        ! " & ( ) * , . . . 0 5 7 8 8 : : = ? A F I J L O O P P Q U U U W Y Y Z _ a g h j l m o s v w w w y y y z { } } º º º ú ú ú ĺ ź Ǻ ˺ ̺ Ϻ к к к к к к Ѻ Ѻ Ѻ Ѻ Ѻ Ӻ Ӻ Ӻ պ պ պ պ պ ֺ غ ٺ ٺ ٺ ٺ ٺ ں ں ۺ ۺ ۺ ۺ ۺ ܺ ܺ ܺ ܺ ܺ ܺ ܺ ܺ ܺ ܺ ܺ ݺ ݺ ݺ ݺ ݺ ߺ          ! # % ( ) * , , - / 0 1 3 3 5 5 6 6 8 8 9 : : : : : ; ; ; ; ; ; ; < < < = = > > @ A A A C D D D D D E E E F I J J J M N R R R R T V V V Y [ [ [ \ \ \ ] ^ ^ ^ ^ ` ` ` b b c d d e e e e e e f f f g g g h i i j j j l l l m m n n n q r r t u u u u w w y { { } » » » » » û û û Ļ ǻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ˻ ˻ ˻ ˻ ˻ ̻ ̻ ̻ ͻ ͻ λ λ λ λ λ λ ϻ ϻ ϻ ϻ ϻ ϻ ϻ ϻ ϻ ϻ л ѻ ջ ֻ ֻ ֻ ׻ ׻ ׻ ׻ ۻ ܻ ݻ ݻ ޻ ߻                                            ( ( + / / 0 1 1 3 3 3 7 J J Y Y [ [ [ [ [ ] ] ] ] ] ] ] ] ] _ _ _ _ _ a b c c c c c c d f g h h h h h n u { ¼ ¼ ļ ļ Ǽ ˼ м м м Ѽ Ӽ Ӽ Ӽ ռ ռ ּ                            " # & & ( ( ( ( ( * + + + + + + , , , , , , , , , , , - - . 4 4 4 4 4 4 4 5 8 8 8 8 8 : : : ; ; ; ; ; ; < = > > ? @ @ @ A A B B E F I I I J J J J J J J K L L L L L L M M M M M N P P S T V X X X Y Z Z Z ^ ^ ` a a b b c d e e i k l m n q r r r r s t t t t u w x x x z | } } ~ ǽ ˽ ҽ ׽ ׽ ׽ ڽ ۽ ۽ ۽ ۽ ۽ ܽ ܽ ܽ ݽ ޽          $ ' ( , - - . 2 4 4 5 7 7 9 ; < < < ? ? B B B D D D E E F J P Q R T V Y ] ] _ ` ` c d h j o r r s w y ¾ ƾ ̾ ξ Ҿ Ծ ޾          ! ! & ' - - - . . . 4 8 9 : ; < < < = > ? ? A B D F O V Z \ ` b f j $ ' ( * * , , , 0 0 0 2 2 3 3 4 5 5 6 6 6 6 6 7 7 7 8 9 9 : : : : : : = = > > ? ? @ C D D D D D D E E E F F H H H H I J J K M M M M M M N N N N O O P Q Q R R R R R T T T T U V X X X X Z [ [ \ ] _ ` ` c e f f h i j j j j j j k n n n n n o p p q q q q q q q r r r r r s t u v v v v x y z { } ~                                    ! ! # # # $ $ $ % % % & ' ' ( ( ( ( ( ( ) * + + + , - - - - - / 1 7 9 : : A A A A B B B B B B D D D D D D F F G H H H L O O Q U [ ] _ a b c c e w { +           ! ! + 4 5 < ? D F H K N Q R R R S S S U U U U X Y Z Z Z [ ^ ^ ^ _ _ _ _ _ _ _ _ ` ` ` ` ` ` ` a b f g i i n n n n n n p s z z { } }    # , 9 < > ? B B B B C E I L L O P P S T T V V X Z [ ^ ^ a h h i l q v v w w x x z }                " # $ % % & * , / / 0 4 7 7 ; ; @ E K O Z [ ] b f g g h h i l l n o q r s s u w w w w w w w w w w x y y y y y y y y y { { { { { | | | | | | }                               " # # + , . / / / 0 1 1 2 3 8 ; < A A A A A A G R U U U V V W W W X X Y Z [ ] ] ^ ^ _ ` a b d f g h j o q s t u v v x y { { { { { | ~ *      ( * - 3 4 6 7 : < < < < = = = = = > @ A B C F F G L L L L L L N N N N N O O Q S S S U V W Z Z ] ` a d f l r t t t t v v v x x { { { { } } ~                                                                                                                           ! ! ! ! ! ! ! ! # $ $ $ % % & & ' ' ' ' ' ( ) ) , , , , - - - - - - - . . . . 0 1 1 1 1 3 4 4 4 6 6 6 7 8 8 9 9 9 9 9 : : : : ; ; ; ; > > > ? B C C C C C C C C G G G G G H H H L M N N N N N N N N N O P Q Q Q S S S S V V V V X X \ ] ` b c e e f f h h i i i i i i j )j j j l l l l l l l l l l l l l m m m m m m n n n o o q q q s s s s s s s s s u u u v x y y y y y y                                                       ! ! ! # # $ $ $ $ $ $ $ $ $ % % & ' ) * + + + 9 : : ; ; > ? @ B B B B B B B B B B B B B C C F F F F G H H K K K K K K L M M M M N N N Q Q Q Q R R R R S S S S T U V V W W W W W X X X Y Z Z Z Z [ ^ ^ ^ ` a a a a b b b c d e e e e e e g g h j k l l m m o o s s s s s s u y z }                                            ! ! ! ! ! " " " " " # # # # # # # # # # $ $ % % & & & & & & & & ' ' ' ( ( ( ) ) ) ) * * * + + , , , . . / / / 0 1 3 5 6 7 < = @ C C F G M P V W Z b b b b b b b e e f f g i i i l l l l n q q q t t u u y { | ~           % & & & & ( ) ) ) , - 1 2 2 3 3 3 4 5 5 5 5 8 : ; = @ F J M M P T U W \ ] g i m m p r w ~ {      $ - . . / / 0 2 3 3 5 5 7 9 : : < @ @ @ @ @ @ @ @ @ A A A A B C C C C C C D D E F F F F F F F F F G G H H H H H H H H I I I I K K O P Q Q R R R R R R S S S S S T U U V W W X X Y Y Z Z ^ ^ b b b c d d e e g h k l o o o p q r t w x x x x x z { { { { | } ~ ~ ~ ~ ~ ~ ~ ~ ~                       # $ % & ' ' ' ( ( + , - - - - - - - - - - - . / 0 1 2 4 6 8 8 9 = > A B D D D D E E F I I I I I I I J L L N N P P P P P P P P Q Q Q S S S S S S T T W W X Y Z \ \ \ ^ ^ ^ ^ ^ ^ ^ ^ _ _ _ _ _ _ ` ` ` ` ` ` ` ` a a b b c d d f i i j j j k m m o s u u u w w w y | ~               ! # $ ' ) ) ) + - - - - - / 0 0 0 0 1 2 5 6 6 9 ; ; ; < < > ? ? ? ? ? B C C D E E F H K L M N O P P P Q Q Q Q S W W W W W W W W W X Y Z Z Z Z Z Z Z Z [ [ [ \ ] ] ` i l m m m m m n o o o o o o o p p q q q q q q q r r r s s t u u u u u u u v v v v y y y y z z z } } } } } } } } ~ ~                 ! ! ! ! ! ! ! ! ! $ ' . / / 0 1 1 1 2 3 5 9 ; < = @ F L L L N Q Q R U Y \ \ a a f f f g g g j n n n n n n o o o o o o r r r r s s s s t u v v w w y }      " # % ( ( ( ) ) ) . . / 2 4 = A C D ^ ` ` ` b b b b b b c c f g g g h h k n p p p p q r r r s s s v x y z z { |                              ! ! ! ! ! " # # # $ $ $ $ $ $ % % & ' ( + , - / / / / 0 3 3 4 4 7 7 8 9 : : : ; < = ? ? ? ? ? C E E G J K Q Q T V X Z \ h l l m p p q q r r s t t t u x y { { | | } } ~ ~ ~ ~ (      # $ & ) 8 : : = B H K K L L L L N O O R X Z \ \ \ m r r s s t u x x { { ~       ! $ + 0 6 8 : L M M N P P Q V W X Y Y ` b b c f g g g g g h h j j j j l l m m o p r r r s s v v { { | ~              # & . 3 : A E F K N S S U X Y Z Z [ ] a g j k k l r s u u u u |                          # # # ' ' ) ) + - - . / 1 1 1 1 1 2 3 5 5 5 9 ? A B B B C C C C C C C D D E F H J K K L M M O O O O O Q Q Q Q Q R R R R S S T V Z ^ c h h j j k o p p u y y y         # $ ' ' ( ( ) ) * + - - - - - . / / / / 0 5 8 : ; ; ; < = = > @ B B B B B C D D D D E E F H K L O O P Q R R U U U W Y Y Y Z \ ^ e h |  + -                             ! ) . 3 5 7 8 = ? @ @ @ B C D E H M V V V Y [ \ _ a m s v { |                        ! " $ ' + - 2 2 2 4 4 4 8 9 : ; ; ; ; = = = = @ A D E F G I J K K K K N P P P R S U W W Z \ \ ^ ^ a c c d d j l p u v w x z {          '  $ % ( ( ) * - . . 0 2 2 7 9 : = = > > > > > I K N U d ~                     # $ % & & & & ( * + + + , , . 0 7 7 9 ; > H H H I K K N Z Z a f m         & ) - / 1 4 5 6 ; ; = ? F I I J M P Q R R R R R T V V W Z [ \ \ ^ ^ a c c h k m      $ % * / 0 4 : G Z ^ ^ a g g g g g h l p p q r w      ' + / 4 8 9 : : ; ; < = > ? B C K N T T T U X X Z \ b f h i m t          ! $ % ) 0 2 9 < C E K N N P \ \ _ c f h h l o o q s                  ! # % ( + , 5 6 9 = > @ E L i m m o x y z z z z { } ~            ! % & ( ( ( + , , . 3 8 : ; ; = ? B B H J N T W a a d f g h i r t u v y                            ! ! " % % % & & ' ' ) , . . . / 0 3 4 5 6 ; ; ; > @ @ C E E F G G H H H I I I I I J K M N N O P Q Q R R R S S S T T T T T T T U U U V X X X Y Y Y Z [ ^ ^ ^ _ _ _ _ _ _ _ _ _ _ _ ` c d d d d d e e e g i m p p p r s u u u u z | | | } ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ &                                      $ & & ' ) * + , 5 5 9 : : ; < < > > > @ C E E E E E G H H H H H H H H H H H J J K K L O O U V X _ _ _ d j j k k k k k k k k k k k k k k l l l m m m m m m n n n n n n n o o o o p q s s t t t w y y y y y y y y y y y y y y | | ~ ~ ~ ~ ~                " " # # $ $ & ' ' * - 1 4 4 4 4 4 4 4 5 6 ? D D J K N Q Y ] ] ] m o o r t   * 1 5 6 8 : J f i j         & 8 > > ? @ B B B C C F F F H H I M M N P P Q R S U Y [ \ ] ` c e e f j k ~                                  ! " " % & ' ' * , , , - / 1 2 2 2 4 5 6 6 6 6 6 6 7 7 8 9 ; < = > > ? ? ? @ A D F G H J J K K K K M M R W W W Z Z Z Z Z Z \ \ \ \ ] ] ^ ` ` ` a a a a a a a a a b b b d e e f f f g h k q q q r s s t t t u v x x y y y { }                           ! ! ! + + , , , - - - - - - . . . 0 0 1 2 3 3 4 5 5 6 6 7 7 7 8 8 8 9 9 9 : : : : = = = = = > > > > @ @ @ @ @ @ @ @ A A A A A A A B B B B B C C C C C C E F F G G G G G G G G G G G G G G G G G G G G H H I I I I I I I I I I I I I I I I I I I I I I I I I I J L L L P P P P P P P R R S S S S T U U U U U U U U W W W W W W W W X Y Y Y Y Y Z Z Z [ [ [ [ [ [ [ [ \ \ ] ] ^ ^ ^ _ _ _ _ _ ` a b b c c c c c c d e e e e e f g g i i i j j j j j k k k o o o o o o o o r s u u w x x x y y y y y y y y y y y y y z z z | | | | ~ ~ ~                                                                 ! " " " " " " " " " " " " " $ % % % % % & ' ' ' ' ' ' ' ' ' ' ' ' ' ( ( ) , , , , , , . 1 3 3 4 4 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 9 9 9 9 9 9 : ; > > ? ? @ @ A A B B B B B B C C C C E E E E E E F H H I L M Q X X X X X Y Y Y Y Y Y Y Z Z Z ] ^ ` ` ` ` ` ` a a a a a c c c c c d g g i i k k l l l m m m m n n n p q r s w x y z { { { | | } }                                    # & & * * * . 3 4 6 9 < > @ @ @ C D G H H J K N P P P R T T U U U V V V V V V X X [ ^ ^ ^ e g h j k m n n n n n o p p p r r s t w y z | } }              " $ $ ' * + , . 1 6 7 9 9 ; < < ? @ @ A D G L L L L L N O O O O O O O O O R R R S S T W ^ ^ _ e l p u v x y z z z z z { { { { { | ~ ~ ~ ~ ~ ~ ~ ~             " # + + / 0 1 1 1 1 1 1 1 1 2 3 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 7 7 7 7 7 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 : : : < = = = > > ? ? ? ? C C C D D E F G G H I I I I I I K L L L L L M M M M M M M M M M M M M N N N N N N N N N P P P P P Q Q Q R R R R S T T T T T V V V V V V V V W W W W W W W X X X X X X Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Z \ ] _ ` a a b b b b b b b b b b c d d e e e e e f f f f f f f f f f f g g g g g i i i i i i i i i i i i i i i j j j k k k k k k k k l l l l l n n o o o o o o o o o o o o o p q q r r r r r r r r t t v w w w w x x x { | } } } ~              $ ( 8 ; ; ; F K K M Q Q R V V Y [ \ c k k o r t v v x x y y y z z z z { { { | | | |                       ! % & ) ) + - - . . . . 2 5 6 6 6 6 6 7 7 7 7 8 8 ; ; ; ; = = = = > > ? ? ? ? @ @ C D E F G G I J K M P P P P S S U U X Y ] _ ` f j m            ! " ' ) ) + , - . . / 1 7 : > @ A D E G G G G G G G G I K K N P P T T T W Y Y Y Z Z Z Z \ ^ b b c f g i l n r |                   ! ! " # # # $ $ $ $ % ' ' ' + , . 0 4 7 @ @ A C C C C C C D D D E E F F F G H I I I I J J K K K K K M M M M M N N N N N P P P Q S V V W W W Y Z Z Z Z Z Z Z Z [ \ ] ^ ^ ` ` ` ` ` ` ` ` ` c d d d e f g g h h k k l o r v w x y ~ ~ ~               ! & ' ' ( ) ) . 0 1 5 6 < A D D H J J J M T W ] _ a e e f k l l n t u v v v w w w w x y }        # * * 1 3 3 4 = ? @ A B J L O O R V Z ] f f h l l m n n p q q q r u w x x y z { { }   ' + + + + + - - . / / 0 5 5 6 : ; < < < < > > > > > > ? ? @ @ @ A B B C D E F I J K L L L L M N N N N N N N N P P Q S T W W X X \ ] ] _ ` a a d e f f g g n q t t v y z z { ~       ' . / 5 5 6 6 6 8 ; @ A D M O S V X [ a d h m z | - 0 1 8 > > > ? ? @ @ A A A A B B B C C D D F J K N P P Q Q Q Q Q S U W Z Z Z \ \ _ ` ` c d f i j j l m m n r t u w | |                                                                       ! ! # # # # $ $ $ & ) + , , , , , , , , - / 0 0 0 0 0 1 1 1 2 3 4 6 7 8 : : = = > > A C D E G J J J K K K L M O O Q R U V V V V V W X X X X Z ] ] ] ^ ^ b b b b b c c d f h h j k k k l l p s u u y { |                                     %! ! ! " $ % & ' ' . . . . . / / 0 2 3 3 3 3 3 3 3 3 3 3 3 5 5 5 5 5 5 5 5 5 6 6 6 8 8 8 9 < C G G I J K K L M O P P P P Q R T T U U U V W W X [ \ b d d g g g j j j k m n r r s                     " # # # # # $ ) * * * * * + + + , , , , , , , , , - - . 0 4 6 6 ; > @ D L M M N P P P R R S S S V Y Y Y Y Y Y [ [ \ \ ] ^ ` b b b b b b b b b d e g g i j k m m o o o s t t u w { | | | | } } } ~            " % & ' ( * , , 0 3 3 3 3 4 4 4 4 5 6 6 7 7 8 8 8 8 8 8 8 8 9 : ; ; ; ; ? ? @ @ @ @ @ @ A C C C C C C D F F F F F F G G G G G G H I J L L O O O O P P P P S V V V V V V W W W W X X X Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Z Z Z Z Z \ \ ] ] ] ] ^ ^ ^ ^ _ c c d d f h h h i j j j j j j j j j l p p p p p p p p p p p p p r r r r r s s t t t t t t t u u u u u u v v v z z { { { { | | }                                                                           ! ! ! ! ! ! ! ! " " " " " # # $ % ( ) ) * * * * * * * * * * * * * . / / / / / / / 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 4 5 6 6 7 7 7 7 7 7 7 7 7 7 7 8 9 9 9 9 9 9 9 9 9 9 9 : : : ; ; ; ; ; ; ; ; < < < < > > ? ? ? ? ? ? ? ? ? ? ? A A A A A A A A A D D D D D D D D D D D D D E E E E E E F F F H H H H H H I K K K L L L L L L L L L L M M P P P P Q R S S S V V V V W W W W W X X Y ^ ^ a a c d d d e e e f f g g g g g g i j j j j j j j k k k l m n n n n n n n o o o o o o o r r r r s s s s s s s s s s s s s t t t w x x x x x y y y y y y y y z z z | } } } }                                            " # $ % & ' ' ' ' ' ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ) ) * * * * * * * * + , , , . . . . / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 6 7 7 8 8 9 9 9 9 9 9 9 9 : : : : : ; < = = = = = > @ @ @ @ @ @ @ @ @ @ @ A A A A A B B B C C C C C C C C C C C C E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E F F F F G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G G H H H H H H H H H H I J J J J J J J J K K K K K K K K K K L L L L L L L L L L L L M M M M N N N N N N N N N N N N O O O O O O O O O O O O P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P Q Q Q Q R S T T T T T T T T T T T T T T T T T T U U U U U U V W W W W W W W W X X X Y Y Y Y Z Z Z [ [ [ [ [ [ [ [ [ [ [ \ ] ] ] ] ] ] ] ^ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ` ` ` ` ` ` ` ` a a c c d d d d d d g g h h h h h i i j k l l l l l l l l l l l m m m m m m m m m m n n n n n n n o o p q r r r r t t t t t t v w w z z } } } ~                                                                                  # # # # # $ $ $ $ $ $ $ $ & & & ' ' + + - . / / / 0 1 1 1 4 5 6 7 9 9 9 9 9 9 : : < < = = = = ? A B B C D D D D D D F I z z z | | | | } ~ ~                                                                                     ! ! ! ! ! $ $ $ $ $ % % % % % % % % % % % % % % % % % % % % % & & & & & & & & & ( ( ) ) * * * + + + + + + + + + , , - - - - - . . . . . . . / / / / / / / / / / / / 0 0 0 0 1 1 1 1 1 1 2 3 4 4 4 6 6 6 8 8 9 ; ; ; < < A A A A A A A A A A A A A B B C C C E E F F G G G G H H H I J J J J J J J J K K K L M P R R R R R R R R R R T T T T T T T T T T U U U U U U V V V V V V V V V W W X X X X X Y Y Y Y Y Z Z [ [ [ [ [ [ \ \ \ ] ] ] ] ] ^ ^ a a a a a c d d f f f f f f f g g o o q q t u u u u u u u u u u u u u v v w w x x z z z z z z { { { { { { { { | ~         $ #                                                                             ! ! " " " $ % % & & & ' ( ( ) ) , - . . . . . . . . . . . . . . . . 0 0 0 0 0 4 4 4 5 7 7 7 7 7 8 8 8 : : : : : = ? ? ? ? ? ? ? ? ? ? ? @ @ @ @ @ @ @ A B C D E F G G G G G G H H H H H H H J J L L L L L L M N O O P Q R R S T T T T T T T V V V V V V V V V V V V V V V V V V W W W W Y Y Y Y Z Z Z [ [ [ [ [ [ \ \ \ \ \ ] ^ ^ ^ _ _ ` ` a b b b b c c c c d d d d d d e e e e e f f h h i i i i i k k k l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l m m o o o o o o o o o p p q s t u u u u u u u v v v w x x x y y z z z } } } } }    - - 0 3 3 3 3 3 8 8 < c c h i l m m m                             $ & & ' ' ' ( ( - / 0 0 2 3 5 5 5 5 6 7 7 8 8 9 ; < < = = = > > > B B E G G G G G G G I I I I I I I K K K K L L L M N O O Q T T U U U U U U U U V V V W W W W W W W W W W W X Y y { { ~ ~ ~ ~                                                        ! " " " " " # $ $ $ ' ( ) * - . . . / / 0 0 1 4 4 4 7 9 9 > > A A C C D D D D H H I J K K K L L N N N N N N N O O P P P P Q S S T T U U U V V W W W W W W W W W W W X X Y [ ] ] ^ ^ _ ` ` ` a c g i i j j j j j j j j k k k k k l l m o o p p p p p p p p p p p p p q q q r s t t t t u u u u u u u u u { { { { { { | | | | | | | } } ~ ~                                                                                                                                                                                                                                                                             "              ! " " # $ % ( ) ) + , . . / / / / / / 1 2 3 3 4 4 4 4 4 5 5 5 6 6 6 6 6 6 6 7 7 8 8 9 : : : ; = @ @ @ @ B B D E G G G K K L O Q R T V V W W Z \ ` c h k l n p p p q x z z { | | | | |                                                                                                                                                                     $ ( ) , . 0 1 1 3 6 6 9 9 9 9 : ; ; ; < > > ? ? ? ? ? ? A A A A C C D D E E F H H I I I L M O P R S S T U U U U U U U V V V W W X Y Z Z Z [ [ [ [ \ \ \ ] ] ^ ^ _ _ _ _ _ ` a a a b b d g h i i i i i i i i j j j j j j j j k k l m m m m m m m o o q q q q r t t v w w w w w x z { | | ~ ~                                                                                                                                                                                                                                                                                                                                                                                                                               ! " $ % ) * , , , , , . . 1 3 3 3 3 5 9 9 < > ? ? @ A C D I I I K N P P P P Q Q R T U V X ] ` a e f h p v x                                                               !                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         ! ! ! ! ! ! " " " " " " " " # # $ % % % & & ' ( ( ( ( ( ) ) ) ) ) * + + , , , , . . . . . . . / / / 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 : : : : : : : : : : : : : : : : : ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; < < < < < < < < < < < = = = = = = = = = = = = = = > > > > > ? ? ? ? ? ? ? ? ? ? ? @ @ @ @ @ @ @ @ @ @ A B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B C C C C C C D D D D D E E E E E E E E F F F H H H H H H H H J J J J K K L L M M N N N N N N N N N N N O O O O O O O O O P P P P P P P P P P P P P P P R R R R R S S S S S S S S S S S S S S S S S S S S T T T T V X X Y [ [ [ [ [ [ [ \ ^ _ _ _ _ _ ` ` ` ` ` ` ` ` ` ` ` a a a a a a a b c d d d d e e e e f f g g i i j j j j j j k k k k k l l l l m m o o p p q q r u u u u u v v w w w w w x x x x x x x x x x x x x x x x x x x x x x x x x x x x x y y y z z z z z z z z |                                                                                                                                                                                                                                        " " " " " " " " " " " # # % ' ) + , 2 5 6 8 < < = > > > > > > > > > > ? ? ? A A A A A A A A A B C C C C C C C D D D D E E E E E E E E F F F G G G G G G G G G G G G G G G G G G G G G H I I I I J K L M M N O O Q Q T T U X Y Y Z Z Z Z Z [ ] a d d d f h i j k k l l o p s u v x x x x x x y { ~                                                                                                                                                                             " " " & ( - 1 6 6 6 9 9 9 9 9 9 9 < < < = @ @ B C C G I I I I I J J J J K K N O O O O O O P R T W Z Z [ [ \ ] ] ] b d e e f h j k m m n n p r s s s v x y y y z z } } } ~                                                                                                                                                                                                                    ! $ % & ' ) * * + , - - . . . . / 1 2 6 6 6 7 7 7 7 : : : ; < = @ A A C C C D D E E E E F G J L O Q W X X [ ] ^ ^ ^ ^ ` ` ` a a e f f f f g j j k k k k k k k k l n n o o p p q s s s s s u v y z z { | } } } ~ ~ ~                                                                                                                                                                                                                                                                                                                                                                                                    " " " " " " " " " # # # # % % % % % % & ( ) ) ) * * + + + + , - 1 1 1 1 3 6 6 6 7 7 7 7 7 7 7 8 9 9 9 9 : : ; ; ; < = = = = = ? ? B C C C C C C E F G G G H I I I I I I I I I J J J J J J K P P R S S T V V W W X X Y Y Z [ \ \ ] ] a b b c d e e f g h h i k l o p q s s s t v v w z { { | } } } } ~                                                                                               " " # # $ & ' ' ' ' ( ( ( + + + + + + + + . 1 1 1 1 1 3 3 3 3 3 3 3 3 5 5 5 5 5 8 9 9 9 9 ; ; ; < < = = = > > > > > > > > A B B B C E G J M M M P P Q Q Q Q W W X X Y Y Y Y Z Z [ [ \ \ \ \ \ \ \ \ \ \ ^ ^ _ a a a b b b b c d e e f f f f f f f f f f f f l l l l l l l l l m n n o r r r t u u v v v v x x | } } } ~ ~   = > @ B E E E G G I I I I I I I J J J J J J J J J J J J J J J K L L L L L M M M P P V W W W W W Y Y Z Z [ [ [ \ ] ] ^ ` ` ` ` d d d e e e h i j j j k k k k k l m n p p p p p p q q r r r s t v v v v w x y } } } }                        ! ! ! ! ! ! ! " " " " " " " " # # # # # # # $ & * , - . . . . . . . . . . / / / / / / / / / / 0 0 0 0 6 6 6 6 6 6 6 6 7 8 8 8 8 9 9 9 ; ; ; ; ; < = = = = = = = = = = = = = = ? @ @ @ A B B C C C D D E E E F F N N N N N N N O O P Q T T V V V V V W X X Y Z \ _ ` ` b b b b b f i j k n o o o o o p q r r r t t w w y z { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { { | | | | | | | | | | } } } } ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~                    " ' , / / 0 1 1 2 2 3 6 8 : : : ; < ? A A A B F H H I I I I J J J J K K K M O O O Q Q R R T T U U U U U V V W W X X Y Y Z [ [ \ ] ^ ` a a a a a e f f f i i i i i i j j j j l n n n o o q r s s s t t v v v y z z z z { } } } } } } } } } ~                                                      " " " # $ ' ' ' ' ' ' ' ( ( * , . . 0 1 3 4 6 9 ; ; ; < < < = = = = > > > ? ? @ A B B B C E E I J K M M M M O                                                                                                                                                                                                           # # # # # # # # & & & ( * + - / 0 1 2 3 7 8 = = > > > C C C D D E G G H I I I J \ \ ^ ^ ^ ^ ^ ^ _ _ ` b c d g g g h j k k l m n p p p q s t t t t u x y y y y y y y z z { { ~ ~ ~ ~                                                                                                                       ! # # ' ) + / 0 2 3 9 9 : : : : : < < < < = = ? ? @ @ D F F G H H H H H H I I I I I I I J J J J J J J J J J J J K K K L L L L L M M N N O O P P P P P P P R R S S S S S T T T T T U U V X X Y Y Z \ \ \ ] ^ u v v v w w w w w x x x x x x x x x x x y y y y y y y y y y y y z z z z z z z { | | | ~                                                                                                                                                                                                                                                                                                        ! ! ! ! ! ! ! " " " " " " # # $ $ $ $ $ $ % % & & ' ) ) ) ) * + + , / 0 1 2 3 4 4 4 4 4 4 5 5 5 7 7 7 7 7 8 8 9 9 : : : : : : ; ; < < < < < < > ? ? ? ? ? ? @ A B C C C C G G H H H H H H H I J J M M N O P P P P P P P P P P Q Q Q Q Q T U U U U U U U U U V W X X Y [ \ \ \ ] ] ] ^ ^ ^ ` ` ` ` ` ` ` ` a c c c d f f g h i i l m m m n n q q q q q t                                                                                                                                                                                                                                                                                                                                              " " " " " " " " " # # % % ' ) ) + + - / 2 2 3 7 8 8 8 8 8 9 : : > J K P P P P T U V X X X a a b b b b c d d d d d d d d e e i i j j j j j k k o p r s u v w x x y { ~                                                                                                                                                                                                                                                                          ! ! ! ! % ( ( ( + - . . 7 ; < < > > ? ? A A A C C K K K K K L L L L L L L L L L L M M N S V X Y Z Z ] ] ] ] ] ] ] ] ] ^ ^ ^ ^ ^ _ a a a a a a b b c c d f h j j j k k k k p q q u v w w w w w x y y z z z z z z z z z | | | | | | | | | | | | ~ ~ ~                                                                                                                                                                                                                                                                                                                                                                                                 ! " " " " # % & & & & & & ( ( * + + . . . / 0 0 0 0 0 0 0 0 0 0 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 5 5 5 5 7 7 7 9 : ; ; ; = = = ? B B B B B B B B B C C C C C C C C C C C C C C C H H H I I K K K K K L M N N N N N N N N O O O O O O O P P P P P Q Q Q Q R R R R R R R S S S T X X X X X X Y Z Z [ \ ^ ^ ^ ^ _ _ ` ` ` ` ` ` a a b b b b c c c c c c c d d d d d f f f f f f g i i i j j k k k k k k l l l l l l m m m m o o o o p p p p p p r r r r r r r s s s s s t t t t t t t t t t t u u w w w x x x x x x y y z z { | | } } ~                                                                                                                                                                                                                                                                                    " " " " # $ $ % ' * * * + + , , , , - - . . . . . / 0 0 0 0 0 0 3 4 5 5 6 6 6 8 8 9 9 9 = = = = = = = = B C C C E E F G H J K K L O Q U V W W W X Y ] `                                                                # $ ' , , - . . . . . . . 1 2 3 3 9 9 9 9 9 : = > > > @ B B C D D E E E E G M M O Q S V W Y Y Z Z \ \ ] ] ^ ^ ^ ^ _ _ _ _ _ ` ` ` f h j j l l l l m m m o o p p q t u v v w w w w { { | | | } } } ~                                                                                                                                                                                                                ! ! ! ! ! ! ! ! ! ! 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C C F G H J O R R T T V Z Z \ \ \ ] _ a b b d e g j k l q u u u u u u u u v v w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w w x y y y y y y y y y y y y y y z { { { { { { { { { { | ~                                                                                                                                                                                                                                                                                                                           " & & ' * / / 2 3 3 4 4 4 4 4 6 7 ? ? @ @ D D F K K L T T V Y Z Z ^ _ ` e h m o p r v x y y |                                                                                                                                                                                                          ; ; > > > > > > @ B C D D E F I M M N O O O O P P P P Q R S S S U U \ \ ] ] _ ` a c d d d d d d d d e f h h j j j j k l m n n n p p q q q q q q q q q u v x x x x x y y z z z z z { } } }                                                                                                                                                                                                                                                                                                          ! ! ! ! ! " # # $ & & & & & & & & & & ( ( ) * + - - . . / / 2 2 6 6 7 9 9 : : : : : : : ; ; ; ; < < = = > > > > ? @ @ @ @ A A A D D D F F I J J J K K K L L L L O O O P P P Q Q Q Q Q Q R R R R S S S S S S S S S U U U X Y [ _ a a a b b d d f f g g g h h h p s t u v v v v w w w w w z z z z z z { | | ~                                                                                                                                                                                                                                   & & ' ' ' ' ( ( ( ( ( * + + + + , - - - . . . . / 1 3 5 5 5 5 5 6 7 8 8 8 8 9 ; ; ; ; ? ? @ @ @ A A B B D D G M R R R S Y Y Y [ [ ^ _ _ _ ` a a a b b b e e e e g g g h i i i i i i i k k n o o o o q q r r s v v x x y | | |                                                                                                                                 ! ! ! ! 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" # # % ' ' ' ' ( ( ( ( ( ( - . / 0 1 1 1 1 1 3 3 4 6 6 7 9 : : : ; < < < < < < > ? ? ? @ @ @ @ @ @ B C C C C C C D D D D D D D D D E E F F G G G J J J J J K N O R R U U V V V V V V V Z Z [ [ [ \ \ _ _ _ ` ` ` ` ` ` ` ` ` ` a a a a a b b b b c c c c c c c c d d d f f f f h h h i i i i k k k n o o o o o o q q q q r s s s t t t t u u u v w w w w z z z z { { { { { { { | | | | ~ ~ Ń Ń ȃ ʃ ʃ ʃ σ σ σ Ѓ у у ҃ ҃ ҃ ҃ ҃ ҃ ҃ ҃ ҃ ҃ ҃ ҃ ҃ փ ׃ ׃ ׃ ׃ ׃ ׃ ك ك ك ۃ ۃ ܃ ݃ ރ ߃ ߃                                            ! # # $ $ % % ) ) ) ) ) ) ) ) ) ) ) * - - - - - - - . . . . / / / / 0 0 0 0 0 1 2 2 3 3 5 6 6 6 6 7 8 8 9 9 ; < < < < < < = = = = > > > > ? B B C C C D D D D D D D D D D F F G G G H H H H H H H H K K L L L L L L L M M N N O O O O P P P R R R R R R S S S T T T T T W W X X [ [ [ [ \ \ \ ^ ^ ^ ^ ^ a b c d d e e h h h h h h h h h h i j j j j j j j j j k k k k k k k k l l l l l l l l m n p q r s s t u w w w w w x x x y { { { | | | | | } } } } } ~ Ä Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą Ą ń ń ń ń ń ń ń ń ń ń ń ń ń ń ń ń Ƅ Ȅ Ʉ ʄ ʄ ʄ ʄ ʄ ˄ Є ф ҄ ӄ ӄ ӄ Մ Մ Մ ք ք ׄ ׄ ׄ ׄ ׄ ل ڄ ڄ ܄ ݄ ݄ ބ ބ ߄ ߄ ߄ ߄ ߄ ߄ ߄ ߄ ߄ ߄ ߄                                                                            ! ! " " " % & ' ' ' ' ) ) * * + + + + - / 0 1 1 1 1 2 2 2 2 3 3 3 3 3 3 3 3 3 6 6 8 8 9 9 9 : : : ; < < < < = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = > > > > ? B B B B B C D D D D E E F F F F F F F F F H H H I L N N P P P Q Q Q Q Q Q Q Q Q Q R S S U U X X X X X X X X X X Y Z Z [ [ [ ^ ^ ` ` ` ` ` ` ` a a a a a b b c d d d d d d d e e e e h h h h j k k k k l m m n n n n n n o o o o o o o o p p p q q q q q q q t t t t t t t t t t t t u v w w x x x y y y y y y y y y y z z z z z z z z z { { { { { { { { { { { { { | | } } ~ ~ ~      Å Å ą ą ƅ Dž Dž Dž Dž Ʌ Ʌ Ʌ Ʌ Ʌ Ʌ ʅ ͅ ΅ ΅ ΅ Ѕ Ѕ Ѕ Ӆ Ӆ Յ ׅ ׅ ؅ م م م م م ۅ ۅ ܅ ܅ ܅ ܅ ܅ ܅ ܅ ܅ ܅ ܅ ܅ ܅ ݅ ݅ ݅ ݅ ݅ ݅ ޅ                                                % & ( ( ( ( ( ( ( ( + - 0 5 6 6 6 6 6 : : ; ; < < < = = ? B G G G H I J J N O O O R R T U U ^ ^ ^ e f g g i k k l q v v w  Æ Æ Æ Ć Ć ņ ņ ņ Ȇ Ȇ Ȇ Ɇ ʆ ˆ ˆ ̆ Ά Ά Ά φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ ц ҆ ҆ ҆ ҆ ӆ ӆ ӆ ӆ Ն ֆ ֆ ކ                    # & ' ( ( ) ) , , , , , , , / 0 0 0 4 5 5 6 6 6 6 6 7 7 9 < = = A B D E E E K K K K N N P P P P R T U U W W X X X Y Y Y Z Z ] ] ] ] b c c e e e e e f f f g g g g i k m m m m m n n n n n n n n n n s t t v w w x x y y y y z z z z z z z { { { { { { { { { { | | | | } } } } } } ~ ~ ~ ~ ~ ~ ~  ‡ ‡ ‡ ‡ ‡ ‡ Ç Ç Ň Ň Ň Ň Ň Ň Ƈ Ƈ Ƈ LJ LJ LJ ȇ ȇ ȇ ȇ ȇ ȇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ɇ ˇ ͇ ͇ · · · · · · · · · · Ӈ ԇ ԇ ԇ և և և և և և և և և և ؇ ؇ ۇ ۇ ۇ ۇ ۇ ܇ ܇ ܇ ܇ ܇ ݇ އ                                   ! ! " " " " " " " # # $ $ $ $ $ $ % % % ' * * * * * * + + T ] _            ' ' ' ( ( ( ( ( - - - / / / / / 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 3 9 9 9 9 < < < < = = @ A A A A A B B B C C C C C C C C D F G G G G H H H H H H H I I I I J K K M M M M N N N N O P P P P T U V Z Z Z Z Z Z [ [ [ ^ ^ ^ _ c d d f g g g h j m n n É ĉ lj lj ̉ ҉ Չ ؉ ى ܉ މ                 # # & - / / / / / / / 0 3 3 5 5 5 6 6 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 : : : : : : : : : : : : : : : : : : : : : : ; < < < = = > > > > > > > > ? ? ? @ @ @ @ B C D D H H H H I I I I T T T T U U U U U U U V V V W W X Y Z Z Z Z Z [ [ ] ] ] ^ ^ ^ _ ` ` a b b e h j t w w w y | | | | Ċ Ċ Ŋ Ɗ Ɗ Ȋ Ɋ ʊ ̊ ̊ ͊ ͊ ϊ ϊ ϊ ϊ ϊ ϊ Њ ъ Ҋ Ҋ Ҋ Ҋ Ҋ Ҋ Ҋ Ҋ ӊ ӊ ӊ ӊ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Ԋ Պ ֊ ֊ ֊ ֊ ؊ ؊ ي ي ي ي ي ي ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ڊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ۊ ܊ ܊ ܊ ފ ފ ފ ފ ފ ފ ފ ߊ ߊ ߊ                                                                                                                                                                       % % % & & & & & & & & & & & & & & & & & & & & & ( ( ( ( ( ( ) ) * * * * * * * * * * * * * * * * * * * * + + + + + + + + + + , , , - . . . . 1 2 2 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 6 6 6 6 6 6 6 6 6 6 A A A A B B B B B C D D E E E E F H H H H K L L L L L L M M M N N O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O P P P P P P P P Q S T U W W W W W X X X X Y Y Y Y Y [ [ [ [ ] ] ] ] ] ] ] ] ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ _ _ _ _ ` ` ` ` ` b c c f f f f f f f f f g g g g g h h h i k k k k k k k k k k m m m n n o p p p p p p p q q q r r s s t u u v v v v v v w w x x } } ~ ŋ Ƌ Ƌ Nj Nj ̋ ͋ ͋ ϋ Ӌ Ӌ Ӌ ԋ ֋ ֋ ׋ ׋ ׋ ׋ ׋ ׋ ׋ ׋ ׋ ׋ ׋ ׋ ׋ ؋ ً ً ڋ ڋ ڋ ڋ ڋ ڋ ڋ ڋ ڋ ڋ ۋ ܋ ދ ދ ދ ދ ދ ދ ߋ ߋ ߋ ߋ ߋ ߋ ߋ ߋ ߋ ߋ ߋ ߋ ߋ                                             ! ! ! " # # % % % % % % % % % % % % % % % % ) ) ) ) ) ) ) ) ) * * * * + - - - - - - / / 0 1 2 3 3 3 3 4 4 4 4 6 8 8 8 8 9 9 ; ; < < < < < < < < < < < < < < < < < < < < < < < < < < = = = = = = > > > > > > ? ? @ @ @ @ @ A A A A A A A A A A A A A A A A A A A C C C C C C C D D D D D D D D D D D D D D D D D E E E E E F F F F F F F G G G G G G G G G G G G G G G G G G G G G H H K L L N P R R R R U U U U U U U U W Y Y Z Z Z Z [ [ [ [ [ [ \ _ ` ` ` ` a a a b b b b b c c d d d d d f g g g h h h h h i i i i i i j j j j j j k k k l l l l l l l l l l l m m m m m m n n n n o p t t t t t t t t t t v w w x x y { | | } } ~ Œ Ì Č Č Č Č Ō Ō Ō Ō Ȍ Ɍ Ɍ Ɍ Ɍ Ɍ Ɍ ʌ ʌ ʌ ʌ ʌ ʌ ʌ ʌ ʌ ˌ ˌ ˌ ˌ ˌ ̌ ̌ ̌ ̌ ͌ ͌ ͌ Ό ό ь Ҍ Ҍ Ҍ Ҍ Ҍ Ҍ ӌ ֌ ܌ ߌ ߌ ߌ                                                                ! " " " " " " # # # # $ $ $ $ $ $ $ $ $ % & & ( ( ( ( ) ) ) ) ) ) + + + + + + + + + , , , , - - - - - - - - - . . . / / / / / / / / / / 0 0 0 1 1 1 1 1 4 4 5 5 5 5 5 5 5 5 6 6 6 6 7 7 7 7 7 9 9 : : : : : : : : : : < < < ? A B B B C F G K O Q S Z [ [ [ \ _ _ ` c j j n r t w w            / / / / / / / 1 : f i k { | ~ Ž Ď Ď Ŏ ʎ ʎ ʎ ʎ ʎ ˎ ͎ ͎ ͎ Ύ Ύ Ύ Ύ Ύ Ύ Ύ Ύ Ύ Ύ Ύ Ύ ώ ώ ώ ώ ώ ώ Ў Ў ю ю ю ю ю ю ю Ҏ ӎ ӎ ӎ ӎ ӎ ӎ Ԏ ֎ ֎ ֎ ֎ ֎ ֎ ׎ ؎ ݎ ߎ ߎ ߎ s } } % % & & 6 8 9 9 9 9 9 9 : < < @ B I K K c c d d f i i j j q v x { {            ' C C C C C D D D E E E E E F F G H I I M O O O O O O O O O O O P P Q Q R R R R R R R R R R R R S S W _ _ x x x x | ~ ~   ‘ ‘ ‘ Ñ Ñ Ñ đ đ ő ő ɑ ʑ ʑ ʑ ˑ Α Α Α Α ґ ґ ӑ ב ב ב ב  1 1 2 2 2 4 4 5 5 5 7 8 8 : : : : = = = B D D D D D D E E E F G H H H H H H H H H H H I I I I I I I I I I I I I I I I I I J J J L L L L L M M M O P P P Q V V V W W W W W X Y Z [ ] ^ _ _ ` a a c c c c c c c c c c c c c e e e e e f f f f f f g h h i i i i j l l l l l l l l n n n o o o p p y z z z { { | } ~ Ē Ē Ē ג ؒ ؒ ؒ ؒ ؒ ؒ ؒ ؒ ؒ ؒ ؒ ؒ ْ ْ ْ ݒ ޒ ߒ ߒ ߒ ߒ ߒ ߒ ߒ ߒ ߒ ߒ ͓ Γ Γ Γ Г Փ ֓ ד                                                             ! ! ! # $ % & & & & & & ( ( ( * + + + + . . . . . / / / 0 0 0 1 1 6 8 : < < < = = = = ? @ A A B D E F F H L P P Q Q Q R R S S V Z [ ^ ^ e e e e e g g j j n o r t v w x Ô Ô Ĕ Ĕ Ĕ Ŕ Ŕ Ŕ Ŕ Ŕ Ŕ Ɣ Ɣ ǔ Ȕ Ȕ Ȕ Ȕ ɔ ɔ ɔ ˔ ˔ ̔ Δ Δ Δ Δ Δ Δ Δ Δ Δ Δ ϔ ϔ Ԕ Ԕ Ք ה ה ה ٔ ٔ ٔ ڔ ڔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ۔ ܔ ݔ ݔ ݔ ޔ ޔ ޔ ޔ ޔ ߔ ߔ                                                       " " # % % % % % & ' * + + , , / / / / / 2 9 9 9 9 9 = > > B C C F F J K M N O O O O O O O O P Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q R T T T T T U U U U U U U U U U U U W W W W W W W W W W X X X X X X X X X X X X X X X Y Y Y Y Y Y Y Y Y Y Y Y Y Y Z Z Z Z Z Z Z Z Z Z Z Z Z Z [ [ \ ] ] _ _ _ ` ` ` ` ` ` a d f g g h h i i i i i i i i i i i i i i k l l n n n n n p p r t t t t t u u u u u v v w y y y z z { } ~ ~ • • • • • • • Õ ŕ ŕ ƕ ʕ ʕ ʕ ʕ ʕ ʕ ʕ ˕ ˕ ˕ ̕ ̕ ̕ ̕ ̕ ̕ ̕ ̕ ̕ ̕ ̕ Ε Ε ϕ ѕ ҕ ӕ ӕ ӕ ӕ ӕ ԕ Օ Օ Օ Օ ٕ ە ە ە ە ە ݕ ݕ                           * * * * , , , , / / / / / / 0 2 2 3 5 5 5 5 5 5 5 5 8 8 : : : = = > > > ? ? ? ? ? @ B C C E E E E E G G G G G G G G G G G G G G L M M M Q Q R S U U V W Y Y [ [ \ \ ] ] ^ ^ ^ ^ ^ _ ` ` c e e f f g g g h h h h h h h h j k l m n o o o p p q r r r s s s s s s t t t t t u u x x x x x y { { } ~      – Ö Ė Ɩ ǖ ǖ ʖ ˖ ˖ ˖ ̖ ̖ ̖ ̖ ̖ ͖ ͖ ϖ ϖ ϖ ϖ ϖ ϖ ϖ ϖ ϖ ϖ ϖ ϖ ϖ ϖ ϖ і і і Җ Җ Җ Җ Җ Җ Җ Җ Җ Ӗ Ӗ Ԗ Ԗ Ֆ ֖ ٖ ږ ܖ ߖ                                                        ! ! ! ! " $ $ & & & & ' ' ( ( ( ( ) ) ) * * * , - - . / / / / / / / 0 0 0 0 0 3 3 3 4 5 6 8 8 ; ; ; < = ? @ @ @ @ @ B B B B B C C C D D D E F I I I L L M N U U V V W X X \ ] ^ a a a b c c c c c c c c d e e e e e e e e e e e e e e f g g h h h h i k k l l l l l l l n n n n n n n n n n n n n o o o o o o o o o o o o p p p p p p p p p p q q q q q q q q q q q q q r r r r r r r r r r s s s s s s s s s s s v w x x x x y y y y z z { | | | } } — — — × × ŗ ŗ ŗ ŗ Ǘ ȗ ɗ ʗ ͗ ͗ ͗ ͗ ͗ Η Η З З З З З З ї ї ї ї җ җ җ җ җ җ җ җ ӗ ӗ ӗ ӗ ӗ ӗ ӗ ӗ ӗ ՗ ֗ ٗ ٗ ۗ ۗ ۗ ۗ ۗ ޗ ޗ ߗ ߗ ߗ ߗ ߗ                                              # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # $ $ $ $ $ $ $ $ % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % * * * * * * * * * * * * + , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , - - - - - - - - - - - - - - - - - . . . . . . . . . . / / / / / / / / / / / / / / / / / 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : : ; ; ; ; ; ; ; ; ; ; ; ; ; ; < < < < < < < < < < < < < < < < < = = = = = = = = = = = = = = = = = = = = = = = = = = ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @ @ A A A A B B B B B B B B B B C C C C C C C C C C C C C C C C D E E E E E E F F F F F F F F F F F F H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I J J J J J J J J J J J J J J J J K K K K K K K K K K K K K K K K K K K K K K K K K K K L L L L L L L L L L L L L L L L L L L L L L L L L L L M M M M M M M M M M M M M M M N O P P Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q R R R R R R R R R R S S T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T T U U U U U U U U U U U U U U U U W W W W W W W W W W W Y Y Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z ] ] ] ] ] ] ] ] ] ] ] ] ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ _ _ ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` b b b b b b b b b b b b b c c c c c c d d d d d d d d d d d d d d d d f f f g g g g g g g g g g g g g g g g g g g g g g g g g g g g g i j j j j j j j j j j j j j k k k k k l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l l m n n n n n n n n n n n o o o o o p p p p s s s s s s s s s s s t u v v w y | | | | | } }  ǘ ǘ Ș Ș ɘ ʘ ʘ ˘ ˘ ˘ ̘ ̘ ̘ ̘ ̘ ̘ ͘ ͘ ͘ ͘ ͘ ͘ ͘ Θ Θ Θ Θ Θ Θ Ϙ И И Ҙ Ҙ Ҙ Ҙ Ԙ ט ט ט ژ ܘ ݘ ݘ ݘ ݘ                                                                                                             ! " " " " " " " " & & & & & & & & & & & & & ( ) + + + / / 0 2 2 3 5 7 7 7 7 9 9 9 9 9 9 < < ? ? ? B C F H H K K K K K K K K K O O P Q Q Q S T T U W W X X X X X X X Y Y Z Z Z Z [ \ \ ^ a a a a a a b d d d d j j k k k m m m m n n n n o o o q q q q q q r t t t u v v w x x y { | | | | | | | | } ~ ~ ~ ~ ~ ~ ~ ~ ~ ~      ™ ę ę ę ƙ ƙ ̙ ͙ Й љ ҙ ԙ ՙ י ۙ ۙ ۙ ۙ ݙ ݙ ݙ ݙ ޙ ߙ ߙ ߙ                                                                                 $ ' ) ) * + 1 1 2 2 2 2 2 2 5 6 6 6 7 8 9 ; < < = = ? ? ? ? @ @ @ @ @ B B E F F F F G G H H K K K K L M M M N O O O P Q R R R R R T T U U V V X X Y Z Z [ [ [ [ [ [ \ ` a b e f i i k l l n q q q q q q t w x { {    Ú Ě Ě Ś Ś ǚ ǚ ǚ Ț ɚ ɚ ɚ ɚ ̚ ̚ ̚ ͚ Κ Ϛ Ϛ К њ њ њ њ њ њ њ њ Қ Қ Қ Қ Қ Қ Қ Қ Қ Қ Қ Қ Қ Қ Ӛ Ӛ Ӛ Ӛ Ӛ Ӛ Ӛ ՚ ՚ ՚ ՚ ՚ ՚ ՚ ՚ ך ؚ ؚ ؚ ؚ ؚ ٚ ݚ ޚ                                          ! ! $ $ ' ' ' ' ' ) * * , . . / / / 0 0 0 0 0 0 0 0 0 0 1 1 1 2 3 4 6 6 6 6 6 7 7 7 8 9 9 9 : : ; ; < > > @ A A A A A A C D D D E F J V W X Y Z Z Z [ _ _ ` ` ` d d e e f g g g h h h h h j j j k m m m m m m m m m p p q q q q q q q q q q q q q q q q q q q r r r r r s s s s s s s t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t t u v v v v v v v v v y y y z z z z z z z z z z | | | | } ~ ~       Û ƛ Ǜ ț ɛ ɛ ɛ ɛ ɛ ɛ ɛ ɛ ɛ ʛ ʛ ʛ ʛ ʛ ʛ ˛ ˛ ̛ ̛ ̛ ̛ ̛ ̛ ̛ ̛ ̛ ̛ ͛ ͛ ͛ ͛ ͛ Λ Λ ϛ ϛ Л Л Л Л Л қ қ ӛ ԛ ԛ ՛ כ ؛ ؛ ؛ ؛ ٛ ٛ ٛ ٛ ٛ ٛ ٛ ٛ ٛ ڛ ۛ ޛ ޛ ޛ                                                      " $ ' ) ) ) ) ) ) ) ) ) ) ) 0 4 E s u u u u u u u u y y y z z z z z ~ œ œ œ œ Ü Ü Ü Ü Ĝ Ĝ Ĝ Ĝ Ŝ Ɯ Ɯ ɜ ʜ ̜ ̜ ̜ ̜ ̜ ̜ ̜ ̜ ̜ ̜ Μ Μ Μ Μ Μ Μ Μ Ϝ Ϝ Ϝ Ϝ Ϝ Ϝ Ϝ Ϝ М М М М ќ ќ ќ ќ ќ ќ Ҝ Ҝ Ҝ Ҝ Ҝ Ҝ Ҝ Ҝ Ҝ Ҝ Ҝ Ӝ Ӝ Ӝ Ӝ Ӝ Ӝ Ӝ Ӝ Ԝ Ԝ ֜ ֜ ל ל ל ؜ ؜ ؜ ܜ ݜ ݜ ݜ ݜ ݜ ݜ ޜ                                            & ( ) * + + + + - - - . 1 1 1 1 1 1 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 5 6 6 6 6 7 9 : : ; ; ; ; < ? @ @ A A B B C D D D D D E E E H J K L L L L L M M M M O Q Q R S S S S S T T U [ [ [ [ [ [ [ [ [ [ ] ] ] ] ] ^ _ b b g j l l n o p r r r r r r r r r s s s t u v x z z | | | | } ~ ~   Ý ĝ ŝ ǝ ǝ ǝ ǝ ǝ ǝ ǝ ǝ ǝ ǝ ʝ ʝ ʝ ʝ ͝ ͝ ͝ ͝ ͝ Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν Ν ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ ϝ Н Н Н Н Н Н Н ѝ ѝ ѝ ѝ ѝ ѝ ѝ ѝ ѝ ѝ ѝ ѝ ѝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ҝ ӝ ӝ ӝ ӝ ӝ ӝ ӝ ӝ ӝ ӝ ӝ ӝ ԝ ԝ ԝ ԝ ԝ ԝ ԝ ԝ ԝ ԝ ՝ ՝ ՝ ՝ ՝ ՝ ՝ ֝ ֝ ֝ ֝ ֝ ֝ ֝ ֝ ֝ ם ם ם ؝ ؝ ؝ ؝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ڝ ۝ ۝ ۝ ۝ ۝ ۝ ۝ ܝ ܝ ޝ ޝ ߝ ߝ ߝ ߝ                                        " # # % & & & ( * . / / 1 1 3 5 5 5 5 6 6 6 6 6 6 6 6 7 7 8 9 ; = > > > ? @ @ C C C C C D E F G G G G H H I I J L N O O O O O O Q Q Q R S S S S S S U X Y [ \ ^ a b d h h j j k l l n n p p q s s t t t t t t t t u u u u u u u v v v v w w w x y y | | } ~ ~ ~ ~ ~      Þ Ğ ƞ Ǟ Ǟ Ȟ ʞ ˞ ˞ ˞ ˞ ˞ ̞ ͞ ͞ Ξ Ξ Ξ Ξ Ξ Ξ Ξ Ξ Ξ Ϟ Ϟ Ϟ О ў ў ў ў ў ў Ӟ Ӟ Ӟ Ӟ ՞ ֞ ٞ ٞ ٞ ٞ ٞ ٞ ڞ ܞ ޞ ߞ ߞ ߞ                                                           # # # # % % % % % % & ' ) ) * * * * + , , , , , - - . . . . . . . . . . . . . . / / / / / 0 0 0 0 0 0 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 7 7 7 7 7 8 : ; ; = = = = = = > > > ? ? @ @ A B B C C D H H K K L L L L M N N N N N N N N N N N N O V V W W W X X Y Z Z [ [ [ \ ] ] ] _ _ _ ` ` ` b b b b c c c e e e e f f f g g g h j l l l l l m m m m m m m m n n p p p p p p r s s s s s s s s t t w w w y | | | | Ÿ ß ğ ğ ğ ş ǟ ǟ ȟ ɟ ʟ ʟ ʟ ʟ ʟ ʟ ʟ ̟ ̟ ͟ ͟ ͟ Ο ϟ ϟ П џ џ ҟ ֟ ֟ ֟ ן ן ؟ ؟ ؟ ؟ ؟ ٟ ٟ ٟ ڟ ۟ ܟ ܟ ߟ ߟ ߟ                               # # $ $ % % % % % & & & & ' ( ( , / 1 3 6 8 < > > > C D D D E E H W W X Y Y Y [ [ [ [ [ [ [ \ \ ] ] ] ] ] ^ ^ ^ ^ _ _ _ _ _ _ _ ` ` ` ` ` ` ` ` ` ` ` ` ` ` a a b b b c e e f f f f g g g h h i i j j j j j k l m n n n q q r u u u u v v v { }             à à Ġ Š Š Ơ Ǡ Ǡ Ǡ Ǡ Ǡ Ǡ Ƞ Ƞ Ƞ Ƞ ɠ ɠ ɠ ɠ ɠ ɠ ʠ ˠ ˠ ̠ ̠ ̠ ͠ Π Π Π Р Р Ѡ Ѡ Ҡ Ҡ Ҡ Ӡ Ӡ Ӡ Ӡ Ӡ Ӡ Ӡ Ӡ Ӡ ՠ ؠ ٠ ݠ ݠ              # $ $ % ' ' ( ) ) * * * * * * + , , - - - - 1 3 3 3 3 3 3 4 5 7 8 9 : ; ; < < < < > ? ? ? ? ? ? ? ? A E E E H H I I N O Q Q S S S S U U U U U U V V V W Y \ ^ _ _ ` a b c c d e f f f f f g g g h h h h h h h h h h h h i i i i i i j j j j l l m m p p p q q q q s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s u v v v v v w w w w w w w w w w w w w w x x x x x x x x x x x y y y y y z z { { { { { } } } } ~ ~ ~ ~  ¡ á á š ơ ȡ ȡ ɡ ɡ ɡ ɡ ʡ ʡ ʡ ̡ Ρ Ρ ϡ ϡ ϡ ѡ ҡ ԡ ա ա ֡ ء ء ء ١ ڡ ڡ ܡ ܡ ݡ ݡ ߡ                                         ! " # $ $ & & & ' ) * * * * * , - - - 0 2 3 4 4 6 7 8 8 8 8 8 8 8 9 : = > B B G G H H H H J J L N R R X \ \ ] ] ] ^ ^ ^ ^ ^ _ ` ` c d d i j l o o o r r r r r r s s s u v v x { } ~ ~ ~ ~ ~ ~ ~ ~ ~ ¢ ¢ Ƣ آ آ ܢ ܢ ܢ ܢ ܢ ܢ ݢ ߢ ߢ                       ! ! ! ! ! ! ! ! " # # $ % & * * / 7 7 9 9 < < < < @ G H I N W X Y ` ` a a f g g i i i i j k l m o o p p p p p q q q q r t w x x x y y y y y y z z z { | } }  £ ã ģ ƣ ɣ ɣ ʣ ͣ ͣ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ Σ ϣ ϣ ϣ ϣ ϣ У ԣ ԣ ԣ գ ף ף ף ٣ ܣ ܣ ݣ ޣ ߣ ߣ          ! " % % & ' ' * , , , , 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 4 4 4 5 5 5 5 6 6 6 7 7 7 : ; ; ; ; ; ; ; ; ; ; ; ; < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < < = = = = = = = = = = = = = > ? ? @ A A A D D D D D E E E I I I J J J J J J J J J J J J J K L L L N N N N N N N N O R R S T T T U U V V V X X Y Y Z [ [ \ a b c e i j j j j j j j j j j j j j j k k k k k l l l l l l l l l l l l m o o o o o p p q q r r r s s s v v v v v w w w w { { { { ~ ~ ~ ~      Ƥ Ǥ Ǥ ʤ ͤ Ԥ Ԥ ڤ ۤ ۤ ۤ ܤ ܤ ܤ ܤ ܤ ݤ ݤ ݤ                                        " " " " " $ $ $ $ $ $ $ $ $ % ' ) ) - . 0 8 9 9 ; ; ; ; < = > ? @ A B C D F F F F H I J J L O T T p p q t t u u u v v v v v v v y z ~  ¦ ¦ ¦ Ʀ Ǧ Ǧ Ǧ Ǧ Ǧ Ǧ Ǧ Ǧ Ǧ Ȧ Ȧ Ȧ ɦ ɦ ʦ ʦ ʦ ʦ ʦ ̦ ͦ ͦ ͦ ͦ Φ Ц Ѧ Ӧ Ӧ Ӧ Ԧ Ԧ ֦ ֦ ֦ ֦ ئ ٦ ٦ ٦ ٦ ٦ ٦ ٦ ٦ ٦ ڦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ܦ ݦ ݦ ݦ ݦ ݦ ݦ ݦ ݦ ݦ ݦ ݦ ަ ަ ަ ߦ ߦ                                            ! ! ! ! ! ! ! # % % & & & & & & & & & & & & ( ) * , - . . . / 1 2 3 6 ; ; ; ; ; ; = > > > > > @ @ A A A A A A B B C E E E E F F F F G G G H I I J J J L L L L N N N P P P Q Q Q Q R R S S S V V V Y Z Z Z Z ] c d d d d d d d g g i i j k k k k k m m m m m n n n n n n n p q q q q q q q q q q q q q q q q q q q q s t t t t t v v v w w w x x x x x x x x x x x x x x x x x x x x x z z z z z z z z z z z z z z z z z z z z z z z z z z z z z z z z z z z z z | | } } ~ ~ ~ ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ç ħ ħ ħ ħ ħ ħ ħ ħ ħ ħ ħ ħ ŧ ŧ ŧ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ Ƨ ȧ ȧ ȧ ʧ ʧ ˧ ˧ ˧ ̧ ̧ ̧ ̧ ̧ ̧ ̧ ͧ ͧ ͧ ͧ Χ ϧ ҧ ҧ ҧ ҧ ҧ ҧ ҧ ӧ ӧ ԧ ֧ ק ק ק ק ا ا ا ٧ ٧ ۧ ܧ ܧ ݧ ݧ ߧ                                                            % & ( ( ( * * * * * * * + + + + , . . 0 0 0 0 3 3 3 6 6 6 6 6 8 8 8 8 8 9 9 9 ; < < < < = = = @ A B F H H H H J J J J J K L M N O O Q Q T T T T W W X X X X Y Z ] ^ ` b c d d f g g i i k l n p q r r t t t t u v w w w | ~   è è è è è Ũ ƨ ƨ ɨ ɨ ɨ ɨ ɨ ɨ ɨ ɨ ɨ ɨ ɨ ̨ Ψ Ϩ Ш Ш Ш Ш Ѩ Ѩ Ҩ Ҩ Ҩ Ө Ԩ Ԩ Ԩ ֨ ֨ ֨ ֨ ֨ ֨ ר ר ب ب ۨ ۨ ި ߨ                                                   ' ' ( ) * * + + + , - - - - - / 0 0 1 5 7 8 8 8 8 8 8 8 8 8 8 : : : : : : : : : : : : : : : : : : : : < < < < < < < ? ? @ A A B C E E F F F F F F F H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H J J J J J J J J J J J J K K K K K K K K K K K K N O O O O R R R T U U U V W X Y Y [ ] ^ ^ _ _ b b b b b c c c c c c d f g h h h h h h j k k l p p p q t t t t u v x x x x x x y y y y y z z z z z z z z z z z | | | } } } } } } } } } } } } } ~ ~ © © ĩ ĩ Ʃ ǩ ǩ ȩ ȩ ȩ ȩ ȩ ȩ ȩ ȩ ȩ ȩ ʩ ʩ ʩ ˩ ˩ ˩ ˩ ˩ ̩ ̩ Ω Ω Ω Ω Ω ϩ Щ ѩ ҩ ԩ թ թ ֩ ֩ ة ٩ ٩ ک ک ܩ ܩ ܩ ݩ ީ                                                                    ! " " # $ % ' ' ( ( * + - - . . . . . . / / / / 0 1 1 4 4 4 4 4 4 4 5 6 7 7 7 7 7 8 8 8 8 8 : < > ? ? ? ? A A D D D D F F F F H H H I J J J J K K L L L M M M M N N N N N O Q Q Q Q Q Q Q Q R R R S S S S U U U V V V Y Y Y Y Z Z Z \ ] ] ^ ` l p t w x x x y z | } }  ª ª ê ê ê ê Ī Ī Ī Ī ƪ ƪ ƪ Ǫ Ǫ Ǫ Ǫ ˪ ̪ ̪ ͪ ͪ ͪ ͪ ͪ ͪ Ϫ Ϫ Ϫ Ъ Ъ Ъ Ъ Ѫ Ѫ Ѫ Ѫ Ѫ Ѫ Ѫ Ѫ Ӫ Ӫ Ӫ Ӫ Ӫ Ӫ Ӫ Ӫ Ӫ ժ ժ ժ ٪ ڪ ڪ ڪ ڪ ܪ ު ު ު ު ߪ ߪ                          ! $ % % & , - . / h i o « ū ƫ ǫ ǫ ɫ ɫ ɫ ̫ Ϋ Ы Ы ԫ ׫ ۫ ۫ ޫ                                                                                                 ! " $ & ) ) , - - - . / / / 1 1 4 5 6 7 7 7 < < < < < > > A A C C F F G J J J J K M O R V V W X X X Z [ [ \ ^ ^ ` c c m o r w w w y z | | | | } }   ¬ ¬ ì ì ì Ĭ Ĭ Ŭ Ǭ Ǭ Ǭ ˬ ˬ ˬ ˬ ˬ ˬ ˬ ͬ ͬ ͬ ά ά ά ά ά Ϭ Ϭ Ь Ь Ь Ь Ѭ Ѭ Ѭ Ѭ Ѭ Ѭ Ҭ Ӭ լ լ ݬ ݬ ݬ ެ ެ ެ                              ! " # % & & ' ' ' ( ( ) * , - 1 5 A C H N N O O O T U U W Z [ ] b c c d g m o p p p p p p q r r v x x { | ~ ~ ~ ~ ѭ ҭ ֭ ح ٭ ٭ ٭ ٭ ۭ ݭ ޭ ߭                  $ + 1 1 3 7 7 7 7 8 @ @ F H J J K N N P Q S U V W X X X X X Z [ [ ] _ _ _ a a b b b c c d e i i i i k k q x y z z { } ~ ~ ® R Z ^ ` n r t w y z z z ~ ~ ~ ~ Ư Ư ʯ կ ֯ ׯ ׯ ޯ    4 ư Ӱ ԰ ԰ հ ݰ ݰ ݰ ݰ                  ! ! " $ % ( ( ( * 6 7 7 7 8 9 : ; = > @ A A B B B D E G G I J J J J J K K K L L L M M M M M N N N N N O O O O O P P P P Q Q T T T W X X X Y Y Y \ a b c c e e e e f f h i j k m n q t x z z } } ~ ~ ~ ~ ~   ñ ı ȱ ʱ ˱ ˱ ˱ ͱ ͱ ͱ ͱ ͱ ϱ ϱ ұ ұ ֱ ֱ ֱ ر ڱ ڱ ۱ ܱ ܱ ܱ ޱ ޱ ޱ ޱ                                ! " " " # # $ $ % ' ( ( ) ) * + + , , , , - . . / / / 1 1 1 1 1 1 2 2 2 2 2 2 3 4 6 6 7 7 8 ; < = = = = ? B B B B B B B B B C C C C C C C C C C D D D F G G H I L L L L L L M M M M M M M M M M M N N N O O Q R R R R R S S S S S S S S S S T T T T T T T T T T T U U U U U U U U U U V V V V W W W W W W W W W W W X X X X Y Z Z Z Z ] ] ] _ _ _ d g j n n ² ² IJ IJ Ȳ ɲ ɲ ɲ ɲ ɲ ʲ Ͳ Ͳ β β β β β ϲ ϲ ϲ ϲ ϲ ϲ ϲ ϲ ϲ ϲ ϲ ϲ ϲ в Ѳ Բ Բ Բ Բ Բ ղ ײ ز ز ز ز ز ز ٲ ٲ ٲ ڲ ܲ ܲ ܲ ݲ ޲ ޲                                                  " $ % % & ) ) , , , . / 0 0 1 3 3 3 3 3 3 3 3 9 9 9 9 ; ; ; ; ; ; < < < < < = = = = = = = = = = = = = > ? B B C F F G G I I I K K K K M N O P P P P T T W Y Z Z Z \ ^ ^ _ _ _ ` ` ` b b c d d e e e e f f h j j j j j k k l l m m n n o o o o o p p p p p p p q q r s s s u u u v v w x x x x x x | | |  dz ˳ ˳ г г ҳ ҳ ҳ ӳ ٳ ٳ ٳ ٳ ٳ ٳ ڳ ڳ ޳ ޳ ߳ ߳ ߳ ߳ ߳                                  ! ! # $ % & ( ) ) ) ) * * , . 0 0 3 3 3 4 6 < < > > ? @ B B C C C C E E F I J K K K K K K K K K N N N N O P P P P P R R R S S T U U V V V V V V V V W W W Y Y Y Y [ \ \ ] ] ^ ^ ^ ^ _ _ b d e g i j j j k p r t t t t t u v v y { { { { { { { { { { { { { { { { { { { { { | } ~ ~  Ĵ Ŵ ƴ Ǵ Ǵ ɴ ʴ ʴ ʴ ˴ ˴ ˴ ʹ δ δ δ δ δ ϴ д Ѵ Ѵ Ѵ Ѵ Ѵ Ҵ Ӵ Ӵ Ӵ մ մ մ ܴ ߴ                                              " $ $ $ ' ' ) * * * + + , , , , , , - - - - - - - - - - - . / / 0 0 0 0 0 1 1 3 4 5 5 6 6 7 7 8 ; < > > > > > > > > > > > > > > > ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ A A B B B B B C C C C C C C D D D F G H L X Y c c c c d e h j k k k k k l m m m n o r t t t u u u u u v v x x x y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y y z z z z z z z z z z { { { { { { { { { { { { { { { { { { { { { { { { { { { { | | | | | | | | | } } } } } } } } } } } } } } } } } } } } } } } } ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~     µ ĵ ĵ ĵ ĵ Ƶ Ƶ Ƶ ȵ ȵ ʵ ʵ ̵ ͵ ѵ ҵ ӵ ӵ յ ֵ ׵ ٵ ٵ ۵ ܵ ܵ ߵ ߵ ߵ ߵ                                              ! " " " # # $ & & & & ( ) ) , , , . / / / 1 2 2 3 5 5 5 7 7 7 8 8 : : : : : ; ; ; ; ; < = > @ A C D E F F F F F F G K K K M M M N N N O R R R U U U U U U Y Z ] ] ] _ ` b b b b c e e e e g g l n ¶ Ķ Ķ Ķ Ƕ Ƕ Ƕ ȶ ɶ ɶ ɶ ʶ ʶ Ͷ Ͷ Ͷ ζ ζ ϶ Ӷ Զ ض ض ٶ ڶ ڶ ڶ ۶ ۶ ۶ ۶ ۶ ۶ ۶ ܶ ޶                                                        " " 5 6 [ [ [ _ ` ` a a a a a a a a a b c d d d d e g h h k k k n n v v x y z z z z z z { } } ~ ~ ~ ~ ~ ҷ ҷ                                                            ! " # % & & & ( ( * * * * + + + + , / 1 3 3 3 4 4 4 6 6 6 7 7 9 9 > ? ? A B D F F G G J L L L M M N N N N N S V V Y Y \ \ \ \ ^ ^ _ _ _ _ _ a a a c c c d f f g h i m m o o q q q q r t w w } } ~ ~  ¸ ĸ Ƹ ɸ ˸ ˸ ̸ ̸ ̸ ͸ θ θ ϸ ϸ ϸ Ѹ Ҹ Ӹ ָ ׸ ׸ ظ ٸ ۸ ۸ ݸ ޸ ޸ ޸ ޸ ߸ ߸ ߸ ߸ ߸                                               ! ! ! ! ! ! " " " " " " " " " " " " " " " # $ $ ' + + + + + + + , , 0 0 0 0 0 0 0 0 0 1 1 1 1 2 2 4 4 4 5 6 7 7 7 9 : ; < < = = ? @ A D D G J J K L L L L L L L M M M M M M M M M M M M M M M M M N O P P P P P P R W W W W Z [ [ \ ^ ^ _ _ a a b b d e e f ¹ ¹ ¹ ¹ ¹ ¹ ù Ź ƹ ƹ ƹ ƹ ǹ ɹ ˹ ̹ ι Ϲ ѹ ѹ ѹ ҹ Թ ׹ ׹ ع ع ܹ ܹ ݹ ߹ ߹ ߹                            " # $ ( - - 1 4 5 5 5 8 9 9 9 < < ? A B B J J J K K K L L L L L L L L M M M M M M M M M M M M M M M M M M M M M M M M N O O O O O O R U V W X X X X X X X X X X X X X X X X X X X X X X X Y Y Y Z [ [ [ [ [ [ \ \ \ \ \ \ \ \ \ \ ] ] ] ] ^ _ _ _ _ _ _ _ _ _ _ _ _ _ b b b e f f f f f f f f f f g g h i j j j j j k k k k l l l l m m m m m n n n n o t t t t t t u u u u w w w w x x x x x x x x x x x x x x x x y y y z } }  º º ú ú ú ú ĺ ĺ ĺ ĺ ź ź Ǻ Ⱥ ʺ ʺ ʺ ʺ ʺ ʺ ʺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ˺ ̺ ̺ ̺ ͺ ͺ ͺ ͺ ͺ κ Ѻ Ѻ Ӻ Ժ պ պ պ պ պ պ պ պ պ պ պ պ պ պ պ պ պ ׺ ׺ ׺ ׺ ׺ ׺ ׺ ׺ ׺ ׺ ׺ ׺ ׺ غ غ غ غ ٺ ٺ ٺ ٺ ٺ ٺ ٺ ٺ ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ں ۺ ۺ ۺ ݺ ޺ ߺ                                                  " % & & & & ) ) * + + + , , , , , , / / / 0 0 0 0 0 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 3 3 4 5 5 5 7 7 : = = = = = > > > > ? @ @ B B B B B B B B B C E G G H I I I I I I I J K L L N P P P Q R R R R R S T T T T T T W W W X X X Z \ \ ] ] ] ] ] ^ _ _ _ _ ` ` ` ` ` ` ` ` a c d d e e f g j n n o p q q q q q q r u v v x x y y y z z { { { { | ~      » » Ļ Ļ Ļ Ż Ż Ż ƻ ƻ ƻ ƻ ǻ ǻ ǻ ǻ Ȼ Ȼ Ȼ Ȼ Ȼ Ȼ Ȼ Ȼ Ȼ ɻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ʻ ˻ ˻ ̻ ̻ λ λ λ λ λ λ ϻ ϻ л ѻ ѻ ѻ һ һ һ һ ӻ ӻ Ի Ի ջ ۻ ۻ ۻ ܻ ߻ ߻                    $ $ % % % % % % & & ( 1 2 4 4 4 4 4 6 7 : ; < = = @ A A A A B B B B B E E E H Q S S T U U U U U U V V V V W X Y Z Z Z Z Z Z [ [ [ [ [ [ [ [ [ [ \ \ \ \ \ \ \ \ \ \ \ \ \ \ ] ] ] ] ] ] ] ] ] _ b c d e g g g g g g g g i i i j k k m o p q q q r r r t u w w x z { } } } } } } } } ~  ¼ ¼ ¼ ¼ ¼ ü ü ü ü ü ü ü ż ż ż ż ż ż Ƽ Ǽ Ǽ Ǽ ȼ ʼ ̼ μ Ҽ Ҽ Ҽ ռ ׼ ؼ ؼ ݼ ݼ ݼ ݼ               # & ' * * . 2 4 7 7 9 9 < ? @ D E F F H J L N O P Q R T U V X Y ^ _ _ q q s u w x z |                                 " $ $ % & * 0 1 1 4 : = = = = B B D E F F K K M M M N N S W Y Z [ \ \ ^ a b c e e f g h h j k l n p p q q q r r r u v v w z { | } ¾ ž ɾ ˾ ; Ͼ о о о Ӿ Ӿ ׾ ھ ھ ܾ ܾ ݾ ߾ ߾               # $ % % % ' ' + . 6 : = = @ @ A A A A D H J J L Q Q U W W [ ^ b b b b e e g h i i j l m n o s v v y y | | ~ ~ ~ ~ ~    Ŀ ǿ ǿ ɿ ɿ ɿ ʿ ˿ ˿ ˿ ˿ Ϳ ο ѿ ӿ Կ տ ֿ ׿ ؿ ڿ ۿ ܿ ܿ ܿ ܿ ܿ ܿ ܿ ݿ                                     ! " & ' ' ( ) ) , - 0 1 3 6 6 : < = = > ? B B B B B D D E E E I O P T U V Y Z \ ^ ^ ^ _ b e f g i k k k k m o q s v w x x y y z { { { | | }                B D L M O P R T U U U U W \ ^ ^ ^ ^ _ _ a a b b b b b c e f i j l l n n q q q r w y | | } ~ ~ ~                   H K T U W W c c c d g g i j u v v y y }                 $ % % ( , . 1 6 |                                           " " $ $ $ $ % % & & & & ( ( ( ( ( ( ( ( ( ( ( ( ( + , , . . . . . 1 1 3 4 4 4 4 ; @ @ F L L V V W X X X Y ` a b f f h j q q s x {                                        = = = = = = = = = = > > > > > ? ? ? ? @ @ @ @ @ A A A B B C D D D E E G G H I I I I I I I J L M M M N P Q Q Q Q T U Y [ \ \ ] ] ] ] ] ^ ^ _ _ _ b c e g g j j j k o q r r r s t w z  $ $ $ & & ' + 0 1 5 = = E E F F G I I K P T V Z Z [ ` a e i k p s u y ~   ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! " # # # # # # # # # # # $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % & & & & ( ( ( ( ( ( ( ( * * * * + + + + + + + + + + , , , , , , . . 0 0 0 0 0 0 0 1 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 4 4 5 5 > > > > > > > ? d d e h i j j l n n o o o o o r s s s s t t t t t u u u u u u u u u u v ~                                     ! ! ! ! ! ! ! ! ! ! ! ! ! ! " # $ $ $ $ $ $ $ $ $ % & & & * * * - . / / / / 0 0 1 1 1 1 1 1 2 2 2 2 2 3 3 3 3 4 4 5 5 5 5 5 5 6 7 7 7 8 9 9 9 9 9 9 9 9 9 9 9 9 : : : : : : < < < < < = = = = = = = = = = = = > ? ? ? ? ? ? ? ? ? ? @ A A A A A A A A A A C E E F F F F G G G G G G G G H H J J J J L L M M M O O O O O O P R R S S T T T T T T T T T T T T T T T T T T T T T T T T T U U U V V V V V V V V V V V W W W X X X X X X X X Y Y Y Y Y Y Y Y Y Y [ [ [ [ [ [ \ \ \ \ \ \ \ \ \ ] ^ ^ ^ ^ _ _ _ ` ` ` ` ` ` ` ` ` a a b b b c c c d e e e e e e e f g g g g g g g g g g g g g g g g g g g g g j j j j j j j j j l l l m n n n o o o p q q q t t u v v x x x x x x z z z z z { }  " " " # $ $ $ $ $ $ $ & & ' ' ' ( ( ( ( ) ) ) ) ) ) ) ) ) ) ) * * * * * * * * . / / 1 1 1 1 2 2 2 2 2 4 4 4 4 4 4 5 6 6 6 6 6 6 6 6 7 8 8 8 8 : ; < < = = = = > > > ? A B B B B B C C C C D D F F F F G G G H J L L L L L L L L L L L M N N O O O O O O P Q S S \ \ b c e e f f f f f f g k k k k k k l p p r r u u v w z |                                    $ % % ( * * * * * * * * * * * + + + + + + + + + + + + + , , , , , , , , , , , , , , , , - - - 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 2 2 3 4 6 6 6 7 9 9 : : > A B D F F F F F F F G K R S S T U W [ b c c h h j k n                      ! )                                 ! ! & * - / 1 2 3 5 7 8 9 ; < = = = A E H I K M N O Q R T X [ [ ^ ^ ^ ^ _ _ _ b d e h m o p r r t u y }                                " " " $ $ $ & ' ' ( ( ( + + + + / / / 0 0 1 1 2 4 4 5 5 5 5 5 5 6 : = > > @ B B B H J L L O O R R R T T W _ g h h h i i i i i i i i j j j j j j j j j j m m m n o q r u v z { ~                                     " $ & & ' ' ( ( ( ( * - . / / 2 2 3 3 4 4 6 9 : : < ? C E H I J L M N N P P Q Q R R S T W _ _ ` a f g g j k m n n o s u w x z z { { | | } ~ ~ ~ ~ ~                     # ' ( * , - / 0 0 0 0 2 2 2 2 2 2 2 2 3 3 7 7 7 8 8 : : : : : : ; = = ? @ @ A A B B C D E H I J K O P R R R R S S S S T U U U V V W [ \ \ \ \ \ \ \ ^ ^ ^ a b b c d g h j j l m m m n n n n n n o p s t u u v x y y y z }                        ! ! # $ % ( + . 0 3 4 5 5 6 8 ? C E E E E E E F F F G H L M N N O O O P Q Q Q R S T V V V \ ] ` b e e g g i l q s {                " " " " # & ' ' ' ' ' ) ) ) ) ) + + + , , , , - / 3 3 5 8 ; < = = = = > > D E F G J J K M M M O Q Q Q S U V Y Z Z Z [ [ ] ] ] ] ^ ^ ` ` a b b d d e e e f f f g h h j k k k l l l n o p q q q q s s s s v v v v w x x x x x x x y y z ~                                   & & ) ) ) ) + + , / / 0 0 1 1 1 1 1 1 2 5 6 6 6 7 : < > > @ A B B B C E F F G G G G G H H I I L M M N N N N N N N O P P R U X [ ] ] _ ` b b b d g k o p s x x x z z z }      " " # % % & ( ( ( * + - - 0 2 4 6 6 6 7 7 9 : ; ; = = > > > ? ? @ C E I K K M M N O P Q Q R T V Y Z [ \ \ _ a a e h m o o q r u w x x y y | ~ ~                 " " % * + + 0 5 9 B F G P Q Q R U X a d f f f f f h j j j j j l m m m n n o o p p q u u w z    $ ' ' * . 2 5 5 6 7 9 C C K L M U Y Y Z \ _ _ a a a a b b b c c d d d d d d d d d d d d d d d d d d d d e e f f f g g g h i j k l l l l m m m m n n n n n o o o o o o o o o o o o o o o o o o o o o o o o p p r s s s t u u u u u u u w y y y y y y y y y y y y y z z z z z z z z z { { { { | } ~              & & ) . 1 4 4 5 6 6 7 9 A B B C C C C F F G G G G H H H I I I I I I J K K L N R U Z \ ] ^ ^ ^ ^ _ a a a a a f g g i i i k l l n q s y                          " " $ $ & * + , / 1 1 6 9 < ? @ B B D F G I J J N R T V X X X Z [ \ ] ] ] ^ _ ` e g g h i m n p s u    5 7 D G H J K P U X ^ _ _ ` ` b f g g h j n n o w z + > > > > E G H I I L L L M M N O O Q R S T W W W Y Z Z Z Z Z Z \ ] ^ _ ` ` ` a a c d d h j j k m m m m m m m n n q q s s s s s s s v w x x y { { { { | ~ ~ ~               " " " # # $ % & & ( ) ) ) ) ) * * * * * - 0 4 6 ; ; ; B M M O P R R R S S S S S S T T T T T W W Z Z Z [ [ \ ] ^ _ _ _ ` ` ` ` a c c c c c c c c f g g i i i j j j j j j j j k k l l l l m m m n n n o p p p q r s s s s u u v y z z           ! ! " " % * - - 1 3 5 7 ; > > @ C E H J K M S T T T T T T V V W W X [ ^ ^ _ _ a b b e e i l m o t x                      " " # $ $ $ ' ' ' ( - / / 1 2 4 6 6 9 : ; ; ; ; ; = > > > @ D E E E F G H K L Q Q Q T T T T T V V V W Z ^ ^ ^ _ a d h i i l p r t u u z | | ~ ~ ~ ~ 8 8 9 > @ A B B D D E E E E E F G K L N O S U W X Y [ ] b g h i k m n p p p p q s s t w w }                                                         ! " " " " " " " " " " % & & & & & ' ' ( ) ) ) ) * * , - / / 0 0 1 2 2 2 2 3 3 3 3 4 5 6 ; ; ; ; = ? @ @ @ @ @ @ @ A B C C C C E H H J K P P Q Q Q R T V X Y Y [ [ ` a a e e i k k k k k l l m m n n n n q q q s t t t t w w w w x y z | | ~                              $ ( ) + , / 0 0 3 5 7 7 7 7 7 7 7 9 9 9 : > B C D E G H I J L L M R R R S T U V V W X Y Y [ \ a g h h l m o q q r t x y y y y z z { { { { { { { { } } } } } ~ ~              ! ! ! ! ! $ & * + , , / / / 0 2 2 3 3 4 6 6 8 < = ? ? A A D E E E F G J K N O O V V V V V V W W W X Y Y Z Z Z ] _ ` b b e f g g g g g h h h h i j j j k k l m m n n n p p p p p p q q q r r r r s s t t t t u v y z z z { }                           ! " # $ + 1 2 2 3 4 5 6 6 7 9 9 ; < < = @ B C E E G G G G G G G G G G G G G J J K K M M M M N O O O O O R R S T V V V W W W X X X X X X [ \ \ \ \ \ \ \ \ ] ^ ^ ^ ^ ` ` ` ` c c c c d d d d d d d d d d d d d d d d d d d d d d d d d d d d e f f f g i i j j j j k q q q r r r r r r r r r r r v v v v v v w w w w w w w w w w w w w w w w y z { { { { { | | | | | | } ~ ~                                                                                ! ! ! " " $ $ $ $ $ $ $ $ % % % & ( ) ) * * * * * , . . . / / / / / / / / / / / / / / / 2 2 3 3 B B C G G H K K L L L L L L M O P P P P P P R S S S S T T T U U U U U V V V V V V W X Y Y Y Y Y Y Y Y Z Z Z Z Z Z Z Z [ \ \ ] ] _ ` ` ` ` ` b b b c c d d d d f f f f f f f g g g g g h h h j j k l l n n n n n n n p r s s s s s s s s s s s t t t t t u u u u v w w w w w w x x y z } ~                                         " " % ( ( ( , - . . 0 3 5 9 9 : : > > > ? B C C E E E E E E E E F F F F F F G H K L L L L L L L L L L M M M M N N O Q S U W W W W X X X X Y Z \ ^ ^ ^ a e i i i i i i k s t                              ' ' ) * * * , , , , , - . 2 4 4 5 7 7 7 7 8 8 8 8 8 < < < < > > > ? @ A D D E E J K M N N O S U V W Y \ \ ] a a b c n n q q s u v v v v x x { { | | ~                     ! " " # $ % & ) ) , - - - - - / 4 4 4 4 4 4 5 5 5 6 6 6 6 6 6 6 6 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 9 9 : : : ; ; < < < < < < < < < < < < < < < < < < = = ? @ @ @ @ @ A A A A B B B B B B B B B B B B B B B B B B B B B B B D D D E E G I I I J J J J L L L L L L M M M M M M M M M M N N N N N N N N N N N N N O P P P P P P P P Q R R R S S S S S S S S S S S S S S T T T T T T T T T T T T T U U U V V V V V V V W W W W W W W W W W W W W W Y Y Y Y Z \ \ \ \ ] ] ^ ^ ^ ^ ^ ` ` ` b b b c c d d d d d d d d d e f f f f f f f j j j j j j j k l m n n o o o o o o o o r t t u u u u u w x x y y y y { { { } }                 ! # $ ' ( , / 2 2 2 2 2 2 3 3 3 4 5 7 8 9 9 9 9 ; = = > A D G G L N N N N O P P R T T U U U X Y Y Y \ ^ ` ` ` a b c c d d d e e g g g h k m r w x z z { ~                       # # $ $ $ & & & ( * * + , . . . . . 5 6 9 : : ; ; ; @ B I L M M M M M N N P R R R S S V W X X \ \ ` a a a b b b b c c c c c d d d d g g g h i i k k k k k k l l l l l n n o o o o o o o p q u v w x { } } ~ ) , - . 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 7 7 7 8 9 9 9 9 9 9 : ; ; < < = = > @ A A D D E E E E F F F G G G I I J J J J J L L N O O S T U U U U V V Z Z [ [ \ \ a a d d i i j j l n p p r s s u u u v w y z z { }                      " & & ' ( ( ( ( ) + 2 2 2 2 2 2 2 3 3 3 5 6 8 9 : : ; = > > > > > > ? ? ? @ B B B B B B B B E H H J J J J J K L L M M M O P Q R R S S T U V W Z Z Z Z Z [ \ _ ` b e e h h i j j j l l l l m n p p q q r r s s s s s v y y y z z z | | ~ ~ ~ ~ ~ ~ ~                      " % % ( ( ) ) * / 0 1 4 4 4 6 6 8 9 < = = ? @ C C C C C C D E F I L O O O O O O O O O O O O O P P P P P Q Q Q R R R S U U U U U V V V W W W Y Y Z Z \ ] ^ ^ a d d d g j j j k l l l l l m n o o o p q t u v y y z z z ~ ~ ~ ~ ~                                        " % & & ' ( ) ) * + + , , , , , , , - - . 0 0 1 3 4 = ? D D D D E G I I I I I L M N O R R S T T X \ \ \ \ \ ] ^ ^ ^ a a e e e e e e e e e e e e e e e e e e f f f h h i k n n q q q t u w w w w w w w w w w w x x x x x x x x y y y y y y y y y y z z z z z z z z z z z z z z z z z z z { { { | | } } } ~ ~ ~ ~ ~                                                           ! " " " " $ $ $ $ $ $ $ $ % % % % % % ( ( ( ) + + + + + , - - - - - - - . / 8 8 : ; ; ; ; ; < = = @ A A A A A C C C C D E H H I I K K K K K K K L L L M M M M N O O O Q Q S T T T V V X X X Z \ \ \ _ _ _ b b c c c d d h h o o r s t t u u u v v v x z }                 ! $ & ' ' ( ( ( ( ( * - . / / / 2 2 3 5 5 5 5 6 7 8 : : : : : ; ; ; < = = ? @ A A E F J K N Q T T V W W W W W Y Y [ \ \ ` b c c c f l l n n n n n p q s x y y z |                        ! ! ! $ ( ( ) ) + . 0 0 2 2 3 3 3 3 3 3 3 4 4 5 6 8 ; < = = > A B B B B D D D F G H I I I J U W X Y Y Y Z \ \ ^ ^ ` ` ` a a a a a b b c c c e e f f h i k k l l m m m o p t t u u u w w x x x y y y y y y { | | }                                 ! " " " % % ( ( ) . / 0 0 2 2 2 2 2 2 2 3 3 4 4 5 5 5 7 7 8 = ? @ @ @ @ A A A A A A A A A C D D E G G G G H J J J K L M M P Q R R T U V W W Y Y ] ] _ e e f f f f f f f g i i i j j j l l n n n n n n n n n n p p p p q q t v x { { { | } ~                                                                " " # $ $ $ $ % % % ( ( ( ) * + , - . / / 1 1 2 2 2 4 6 7 7 8 9 9 : : : : : ; < < < < < < ? ? ? ? ? ? ? ? ? ? ? @ A A A C D D D D D D E E E F G H H H H H I I J M N O P Q Q Q Q Q Q Q R S S S S S S S S U U X X Y Y Y Y Z Z ] ^ _ ` ` ` ` ` ` ` ` ` ` b c c c d d d d d d d e f f f h i i m n n o o o o o p p p p p t t t u u x x x x x x y z { { | | } ~                          ! $ $ $ % & ' ( ( ) + 1 2 5 6 7 ; > ? @ B B C G K Q R U V ` ` a c h i k r t u x z z }                           ' ' ) ) + + - 0 1 2 4 7 : ; ; < = > > > > > > C C F H L M O R R T T U U V V [ t u | ~                                                     " ' , . 5 ] _ l l l o p p s t x x y { | ~                                                 ! ! # # $ $ 9 9 : < < = = = = > > > ? @ @ A B B C C C C C C C C F F F F G G H H H I I I I J J J J J J J J K K K K K K K K K K L N O O O O O P P P P P R S S S T T T W W Y Y Z Z Z [ [ [ [ _ _ ` ` ` ` ` ` b b b b b b h h h h h h i i i j k k k k k k k k l n q r r s s s s s s s s s s s s u u u u u u u u v v v v v w w w x x x x x x x y y y { { { | } ~                        ! ! " $ % & ) * , 0 2 2 3 4 8 8 9 9 : < = = = = > ? ? ? ? ? ? ? A C C G J J O P P P P S S U V V V Y Y Z ] ] ^ _ a a d d d d h h i l l l p q r s s t t t t t t v z | ~                                 # $ $ + , / 0 0 0 3 3 5 : ? @ B G H M M O O Q T X ` c d j q q t { } ~          " % ( + , . 2 9 9 : > @ B D D D I O P U U V X Y Z \ \ ^ ` b d d f k m o p s { | | ~           " " # % % ' ( ( , / 1 9 : ; > @ @ @ @ A E L N N Q S W a b b d f g g o q t x y z | }              $ ( / 2 5 8 ; B C C E F I R \ a a a a b c d e f f i m m m m p p p q s u {  ! & & / 0 1 4 z z             ! # # ) + 1 6 8 9 : A B C C D F I J K M T U X X X X X Z Z [ \ \ ] ] ] ] _ _ a b b b b c c c d e e f f g g h h h i i j k k k l l l m m m m p r t t t v v v v v w w w x y z | | | | } } } }             ! ! # % % % & ' ' ' ' ' ' ' ( ) * - - . 0 0 1 3 3 3 3 3 4 8 9 : : : ; ; < < > > ? A A A A B B C C C C C E E G H H I K M P P P Q Q Q R S V V W W W X Y Z Z \ ` a b c c c c c c c c c c c e e e e e e g g h h h h h i i j j j j j j k n n o o o p r r r r t t t t t t u v v v w x x x x x x x x x z z z { } } } } } } } } }                                        * + - 0 3 4 5 9 ? @ B B B B B B F O R S S S S S S S S S S S S S S T T T T T T Y ^ ^ ` b d j j j m m p p p p p p p p p p p r r r r r r s s s s t w w w w w x x y y y y y z z z z z z z { { { { { { { | | | | | | | | | | | | | | | | | ~ ~                         ! ! ! " & ) ) * , , - / / 1 1 1 1 1 1 2 4 7 9 9 : : = > > > A D G H H H J K M M M M M Q Q T T T U V V W X Y [ [ [ \ ^ _ _ ` ` a c d d g i i i k n q t v w y y y y y z { ~                           $ $ $ $ $ & & & ( ( ( 0 3 3 3 4 6 8 : : ; ; ; ; < < < < < < < = ? ? @ A A C C C C C C C C C C C C C C C C D D D G H H I J J J J J K K K L L M M N N P P P P P Q R S U U W W Z Z [ \ ] ] ^ _ e g h i k k l m q q r r r s t t t w x x x x x x x x x x x x x x x x x z { { { } ~ ~                                                                                                                                                        ' ( ) * * * * - - . . 0 0 0 1 4 7 9 9 ; < < B E H I K L S U [ ]                $ + / / 0 0 1 1 1 1 G G I L L O O Q T U V V W \ ` ` d d e e e e i k l l l l l l l l l l l l l l l l m m t t t t t t u u v w w w x x y y y y z z                                                       " # ( ( ) + + + + + - - 0 1 2 2 2 3 4 4 5 5 5 5 6 8 9 : : : : : : ; ; < < < ? ? ? A E G G H H I I I I I I I L L L P P P P P P P P P P P R S S T U W X X Z \ _ b b b b d d d g h i i m n t t u v v v |                                                                 ) / / / / / 1 2 3 3 3 4 4 4 4 6 6 6 7 ; = > g q w |                                                                                                                             & ( ) + - . 0 2 3 5 9 < = ? @ C E G G J J N R T U V Y Y [ a c f i k k n n n n n o q r u y y z |                                    j n x |                                                                      ( + + 2 9 < A B B B C D M ^ f i m r z                   # % ( ( ( ( ( ( , , . 1 2 5 5 6 ; ; B B H N O O P V W W ^ ^ ^ ` b b c c c c d d f g g g g h h i j j j k l l l l l l l n n n n n n n o o p p r r t t t t t t t t v v w y { { { } ~                " $ $ % & ' ( , . . / 4 4 7 8 8 : ; ; = ? ? ? ? A A A A A E H I K K K L L N P P Q Q R S U V V V V W Y ^ _ ` ` a b c f f g i i j k m n o q r r t u v z z { } ~            % * / 5 C D I I J J J M R X ] ] ] ] ` b d d f g i j l l l l l m m s t x       ! ' / 4 7 = C E J Q Z d g k n w z z { ~                  ! ! " " # # & & & ' ( ) . / 1 5 G H N O Q T V W [ [ ] ^ a e g n                                                                                                                            ! ! " # $ $ $ % % % % % % % % % % % % % & ' ' ( ( ( ) ) ) * * * * * * * * 0 0 2 4 4 4 4 4 4 4 6 9 < ? @ F K M U U U U W W [ [ ^ a c e e h o p q t v y { } ~                                                                                                                                                                                           # & ' ' + + + + + + + , - - - - - . 1 1 3 4 4 4 4 4 4 4 4 4 5 5 6 7 7 7 7 7 7 7 8 9 : = ? ? @ @ @ C C D H H H H J K L L O O O Q Q T T T U W Y Y Y Y Y Y Y Y Y Y Z Z Z Z \ \ ] ] ^ ` ` a b b c c d d d e e f h i i j l m o o p r r r r r r r s t t w x x x y { ~                                                                                             # # % ( * - . . . / / 2 3 : < ? F G P P R R R S X Z \ ^ d g i j t t z }                                                                                                                                                                                                                                           " " # # $ $ ' ( ( ( ) ) ) + , 0 0 0 2 2 3 4 5 7 8 9 9 9 9 : : p q r s s s s s x y z | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | ~ ~                                                                                                                                                                                                                                                                                                                                                                           # & & ) + , . . . 0 5 5 5 5 5 5 5 5 5 6 7 8 9 9 : : : : ; < < < > C C F H K L O O R T V W X Y Y Z Z Z Z \ ^ b b b b b b b f g g h h j k k l l l l n r t t u u u x                                                                                                                                                                                                                                                                                                                                                ! ! ! ! ! " " " " " " " $ % ' ( ) + + + , - - / / / 1 1 1 1 1 2 2 3 3 3 3 3 3 3 3 3 6 6 6 6 6 6 7 9 9 9 9 : : : : ; ? ? ? ? ? ? ? B B C F H H I J J L M P Q Q Q Q R R U U X Y Y Y Y Z Z Z [ [ \ ] ^ ^ ` ` ` ` ` ` c c e e e e e e e e e e e e e e e e e g g h i i j j k k l l m m m o o o o t u v w w x y y z { { | | | | | | ~ ~                                                                                                                                                                 $ % ' ( ( ) ) + - / 0 1 3 4 4 ; @ @ C E I K K K Q T U Y ^ b c p v y | }                                                                                                                 ! " " $ $ & ) + 0 0 7 ; ; ? ? A A A B G K O P P U Y \ j l m m p s v w | ~                                                                                 & ' . 2 4 8 = = B I N X [ \ ^ b b d i o q w                                                              ( + / : < B C D H H L R Y _ d i o v |                                                                   $ ( / 6 9 ; ? 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" " # % & & & & & ' ' ' ' ' ' ' ) ) ) ) ) ) * * * * * * * * * * * * * * * * * * * + + + + + + + + + + + + + + + + + + + + + + + + + + + + + , , , - - - - - - - - - - - - - - - - - - / / / / / / / / / / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 7 7 8 8 8 8 8 8 8 8 8 8 9 9 : ; ; ; ; ; < = = = = = = > ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ @ A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A B B B B B B C C C C C C C C C D D D D D D D D D E E F F F F F F F F F F F F F F F F F F G G G G G H H H H H H H H H H H H H J J J J J J J J J J J K K K K L L L M M M M M M M M M M M N N N O O O P P P Q Q Q Q Q Q Q Q Q Q Q Q R R R R R R R R R R R R S S T T U U U U U U U U U U U U V V V V W W W W W W W X Y Y Y Z ^ ^ a a b b b b b b b b b c c c g h i i j k k k k k k k k l l l m m m m m m m m m n n n n n n n n n n n n n n n o o o o p p q q q r r r r r r r r r s s s s s s s s s t t t t t t u u v v v v v v v v v v v v w y y z z z z z z z z z z z z z z z z z z z z                                                                                                                                                                                                    ) ) ) + + , - - . . . . . / A A B D F H H H H I J J J L L L N N N P P P R R S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S S U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U U V V V V V V V W X X X X Y Y Y Y Z [ \ \ \ ] ] ] ] ] ] ] ] ] ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` a b b b b c c c d d d d d d d d d d d d d e e e e f f f f f f f f g g g h h k k k k k k k k k k k k k k k k k k k k k k k k k k k l m m m m n n n n n n n n n n p p r r t t t t t t t t t t t t t t u u u u u u u u u u u u u u u u u u u u u u u u v v v v v v v v v v v v v w x x y y y y z z { { { { { { { { { | | | | | | | | | | | | | | | | | | | ~                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     " " $ $ % % & & & & & & & & & & & & ( ( ( ( ( ( ( ( ( ( ) + , - - - - - - . / / / / / / / / / / / / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 3 3 3 3 3 3 3 3 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 6 6 6 6 8 ; ; ; = = = = = = = = > > ? @ @ @ @ A B B C D E F F G G G G G H I I I I I I I I I J J J J K K K L L M M M M N N N N N O O O P Q R R R R R R R R R S S T T T U U U U Y Y Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z \ \ ] ] ] ^ ^ _ _ _ _ _ a b b d d d e f f f g i i j n n n o o p q q q t u u u u u u u u u u u w w w w w w w w w w w z z { { | ~ ~ ~ ~ ~ ~                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                ! ! ! ! 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} } ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ $~ &~ &~ '~ +~ ,~ ,~ -~ -~ -~ -~ -~ .~ 2~ 3~ 5~ 8~ :~ ;~ ;~ >~ @~ @~ A~ C~ D~ F~ F~ G~ G~ H~ H~ I~ I~ I~ J~ J~ K~ K~ N~ P~ Q~ S~ U~ V~ V~ X~ Z~ ^~ ^~ b~ c~ e~ m~ n~ s~ v~ |~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~                $ / 1 4 9 9 B F F I L R S S S T W [ [ ^ ` a d e f i j k k k k k k k k k k k k k k k k k k m m m m m m m m m m m m n n n n n n n n n n n p p q q q r r r r r r r r r r s s s s t t t t u u u u u u u u u u u u u u u v v v v x x z z z { { { { { { { { | | | | | | | | | | | | | | | | | } } } } } } } ~ ~                                                                                                                                                                                                                                                               & ( , / 0 1 1 1 3 6 7 8 9 ; ? ? @ @ A E G J M Q R R ] ] ] ^ ^ _ ` b e i j k k k n n r r s v v x z z } } } } }  À À À ǀ ǀ ǀ ǀ ɀ Ѐ Ѐ р Ҁ Ӏ Ԁ ؀ ڀ ۀ ݀ ހ                     W X Z [ \ ] ^ _ _ _ _ a c c e j k l l l l m m o p p q r t t t u z { | | | ~  Á ā ā ā ā ā ā Ł Ł Ł Ł Ɓ ǁ ȁ ȁ ȁ Ɂ Ɂ Ɂ Ɂ ʁ ʁ ́ ́ с ҁ Ӂ Ձ Ձ ׁ ځ ځ ܁ ܁ ܁ ݁ ށ             ! " " # $ $ $ & ' ' ' ) * * , - - - - . / 0 1 1 1 2 2 2 2 2 2 2 2 6 6 6 : < = ? ? @ @ @ @ A B B B D F H K K K K K K K K K L L L L N N N O O R R V V W W W X Y \ \ \ \ \ \ \ ] ] ] ^ ^ ^ _ _ a a b d e f j k l m o r r r t y z { | ~  ‚ ‚ ‚ ‚ ‚ ł ł ł ł Ƃ Ƃ ǂ ǂ ǂ Ȃ Ȃ Ȃ ɂ ʂ ʂ ʂ ʂ ʂ ʂ ˂ ˂ ˂ ˂ ˂ ̂ ̂ ̂ ͂ ͂ ͂ ΂ ΂ ΂ ΂ ΂ ς ς ς Ђ т т т ҂ ҂ ӂ ӂ ӂ ؂ ؂ ؂ ق ق ق ق ۂ ܂ ݂ ނ ߂ ߂ ߂ ! % ) * + . / 3 4 8 9 ; B C C C K P S T T U V W W X X \ _ ` ` d n p s t u y y y y z ~ ƒ ǃ Ƀ ʃ ʃ ʃ ̃ σ Ѓ Ӄ ԃ                      ; > ? A B D I K Q R U U W Y Z Z \ ] ] _ a a g j j j k m m n o r s t t u v v v v w z { | | | ~  Ą Ą Ą ń ń ń ń Ƅ Ȅ Ȅ Ȅ Ȅ Ȅ Ʉ ̈́ τ Є ф ҄ ӄ Մ ք ؄ ؄ ۄ ۄ ܄                              " $ & & & ' ' ' ' ' ' + + , , , . . 0 2 3 4 5 6 7 8 9 9 9 = = > > > > ? @ @ @ @ B B C F I K K M M M M M M M Q R R R R R R S S S S S U U U U V V V W Y Y ^ ^ ^ i i k k k l o q w x x z z | | | | | | ~ ~ … … … Å ą ą ą ą ą Ņ Ņ ƅ Dž Dž Dž ȅ ˅ ̅ ̅ ̅ ̅ ̅ ̅ ͅ υ х ԅ ԅ ԅ ԅ ԅ Յ                        " " " # # & & ( ( ) ) ) ) ) + + - . 1 2 3 3 4 5 6 6 6 7 8 9 9 = = > @ @ @ A A B C H H J K K K N Q R S S S S S S T U U Y Y Z Z Z Z Z [ [ [ [ \ \ \ \ \ \ ] ] ^ b b b b b b b b b b b b b b d d d m m m m m m m n q r s u u u x y { { { | } } } } † † ņ dž Ȇ Ȇ Ȇ Ɇ Ɇ ʆ ͆ Ά Ά Ά ҆ Ԇ Ԇ Ԇ Ԇ ׆ ؆ چ چ ۆ                          ! " " " # $ $ $ $ % % & ' ( ( ) * + , , , . / 0 3 8 < @ A A A B C G G I J M O Q S T W Y Y Y Y Y Y Y Z \ _ a a b b d e e e e e e f f f g g g h h i i k n n t w z | ‡ ć Ň Ƈ ɇ ɇ ʇ · · ч ҇ Ӈ ԇ ׇ ؇ ݇  ) + + + . . . . . 3 4 4 5 7 8 : : : ; ; ; ; < < > > > > > ? ? ? B F H I J J J J L L O P P P P S S V V V X Y [ [ \ a a a b b d d e g i j j k m m m q r v y z z z | | } ~ ~ È ƈ Lj Ɉ ̈ ͈ ψ ψ Ј Ј Ј ш ҈ Ԉ Ԉ ֈ ֈ ֈ ֈ ׈ و و ۈ ވ                      ! ! # $ & ' ) * * + , , / 0 1 2 3 3 8 9 ; > A A C D G O R R R R S S T T T T W W W [ \ ] ] ` ` ` ` ` ` ` ` ` ` c c c d e f g g h i i k k k k l n p s s u u u u v w w x y y | É ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ʉ ʉ ˉ ˉ ˉ ˉ ˉ ̉ ω ԉ ։ ։ ډ ܉ މ                            " % & & ( * * + + , , - 2 2 2 2 2 2 3 3 6 8 : < = A C C E E G I L R T W Y ^ _ _ _ _ _ ` a b b g j r v x y Ê Ê NJ NJ Ԋ ֊ ֊ ؊ ߊ d k l m n q q q r r t u v z |  ‹ ŋ Ƌ ɋ ̋ ΋ ҋ ԋ ֋ ֋ ֋ ֋ ֋ ֋ ؋ ݋ ݋ ߋ ߋ                  ! ! " " " # % % & ) ) * * , , , . . 0 0 0 1 3 4 6 6 9 : : ; ; ; ; ? ? @ A C D D F J J K L S S S S S S T T V Y Y Y ] ^ ^ _ a b b c c d e e e e i i q s v y { } } ~ ~  Œ Č Č Č Č nj Ȍ ͌ Ќ ь Ҍ Ռ ֌ ֌ ֌ ׌ ڌ ڌ ی ی ی ݌ ݌ ތ ތ ތ ތ ߌ    0 1 [ ^ ^ ^ ^ ^ b c  Í ȍ ʍ ʍ ˍ ̍ ̍ ͍ ΍ ؍ ؍ ۍ ܍ ݍ ލ                                 " ( * + + / / 4 5 6 7 8 9 < < < = = = = = = ? ? ? A C C K N N N P T X Y Y [ [ _ b b b b c c c d e e e e e f g i i i i i i j j j j k k k k l l l l l l m m n n n n o o q q r s t u w y y { { | | | | |    Ž Ŏ Ǝ Ǝ Ɏ Ɏ ˎ ˎ ˎ ˎ ͎ ͎ Ў Ў Ў Ў Ў Ҏ ӎ ӎ ӎ ӎ ӎ Վ Վ ڎ ݎ ݎ ގ                                   " % & & & & & & ' ) * + , - 0 2 2 5 6 6 8 8 9 9 < = = > ? ? B F F G G I I L M O O O O O S Z [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ ] ] ] ` a a a b b b b b c c d f g h i j j j k m p q r s u w y y z { | }  ď Ǐ ɏ ̏ Ώ Ϗ я ԏ ԏ ԏ Տ ֏ ׏ ؏ ڏ ڏ ڏ ڏ ۏ ۏ ݏ ݏ                                   ! ! " " " " " # $ $ $ $ % % & ( * , / 3 4 5 5 7 7 8 9 9 ; ; ; ; ; ; ; < = = > > > > > > ? ? @ @ @ @ C D D D D D E E E E E F G L N O P P S T T W Y Z ] ` ` a a g h j j j k n o p s u v v w w z { | } ~    Đ Đ Đ Đ Ɛ ɐ ɐ ɐ ɐ ː ː ̐ ͐ ϐ ϐ ϐ ѐ ѐ ѐ ѐ Ґ Ԑ ֐ ֐ ؐ ؐ ؐ ؐ ؐ ؐ ې ې ې ܐ ܐ ܐ ݐ ݐ ݐ ߐ                     " " " $ & Y Z ] ^ a a d i p p q q q r t t x x x y { { { { { { | | } } ~ ~ đ ő Ƒ Ƒ Ƒ Ƒ Ƒ Ƒ Ƒ Ƒ Ƒ Ƒ Ǒ Ǒ Ǒ ɑ ɑ ɑ ɑ ɑ ɑ ɑ ɑ ɑ ʑ ʑ ʑ ʑ ʑ ʑ ʑ ʑ ʑ ̑ ̑ ̑ ̑ ̑ ̑ ̑ ̑ ̑ ̑ ̑ ̑ ̑ ̑ Α Α Α Α Α Α Α Α Α ϑ ϑ Б Б Б Б Б ӑ ԑ ԑ ֑ ֑ ֑ ֑ ֑ ב ב ّ ܑ ߑ  ! ! ! " # # # & & ( ( * * * * * * + . 0 0 1 1 1 1 1 1 1 2 2 3 4 4 4 5 5 5 6 6 6 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 9 9 9 : : : ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; < < < < < < < < < < < < < = = > A A A A A A A A A A C C C C C C C C C C C C C C C C C D D D D D D D D D D D D D D D D D D D D D D D D D E E E E E E F F F F H H I I I I I I J J K K K K K K K K K K K K L L L M M M M M M N N N N N N N O O O O O O O O O O O O O O O O O O O O O O O O P P P P P Q Q Q Q Q Q Q Q Q Q Q Q R R R R R R R R R R R S S S S S S T T V W W W W W X X X X X X X X X X X X X X X X X X X X X X Y Z Z Z Z Z Z [ \ ] a a b c c d d d d e e e f f i i i i j l l m m n n n o o o p q q q r r r s s s s u u v v v v v w x y z z { } } } ֒ ג ג ג ג ג ג ג ג ג ؒ ؒ ڒ ڒ ڒ ے ے ے ܒ ܒ ܒ ܒ ܒ ܒ ܒ ݒ ߒ                           ! " $ & ' ' ( * + + + + , , - - . 1 1 2 5 6 6 6 6 6 7 7 : : : : : : ; ; < < < = ? ? @ A C E F F G G G G G G I L L L M M M N N O O R S S S S U V V V V W X Y Z Z Z Z Z ] ^ a a a f j m m p r s s s v w x x } ~ Ó œ Ɠ Ɠ ɓ ɓ ɓ ɓ ˓ ̓ ͓ Γ Г ѓ ֓ ؓ ؓ ؓ ړ ۓ ޓ ޓ                   ! ! ! " " " $ & & * + , , , 1 4 5 7 ; < = = > ? A C C C C E F G I J K N P R T U V [ [ \ ] ] ] ] ] ] ] ^ ^ _ a a a a a a b b b b b b c c c c d f h h j k l n o o q r u y y z { | | | } ~     ” ” ” Ô Ô Ô Ĕ Ĕ ǔ ǔ ǔ ǔ ˔ Д Д Ҕ Ҕ Ӕ Ӕ ٔ ڔ ۔ ܔ ݔ ߔ                                ! ! # $ $ $ $ $ $ $ $ $ $ $ $ $ $ % % % % % % & & & ' ' ' ( ( ( ( ( ( ( ( ) ) ) ) ) ) ) ) ) * , , , , . . . . . . . . . . 1 1 1 1 1 1 1 1 2 2 3 4 7 : : : : < > > > > > > A A B C E F G G G G G I I J M O P P R R X [ [ \ _ b e i j j k m m m m o o p p q r s t u u w x y | | | } ~  • • • Õ ĕ ƕ ƕ ƕ ƕ Ǖ ȕ ȕ ˕ ̕ ̕ ̕ Е ѕ ѕ ѕ ҕ ԕ ԕ Օ Օ ֕ ֕ ֕ ڕ ܕ ߕ                     " # # # $ & ( + - - . . . 1 1 1 4 8 8 9 = > > A B B C E I J J J J L O O O P R R S S T V Y ] _ _ ` a b e e f f g g g h h k k k k l m n n o p q q r r s s s s s t u v w y y y { { | } }  – Ŗ Ŗ Ŗ Ŗ ǖ ǖ ʖ ږ ږ ݖ ޖ ޖ ߖ ߖ                     " " % ' ' ' + - 2 3 4 4 6 8 8 9 9 9 9 = > > @ A D G G G G I L L M Q Q Q Q Q R R S S S U U W W X Y Y Z \ \ \ \ \ \ \ ] ^ ^ ^ ^ ` b c c d d f g i i i j j k l n n n n n p p q q q q r r r s s w | } — Ɨ Ɨ ȗ ȗ ʗ ʗ ˗ ˗ З З З З З З З З З З ӗ ֗ ח ח ؗ ؗ ٗ ٗ ٗ ٗ ڗ ڗ ڗ ڗ ڗ ڗ ڗ ڗ ڗ ݗ ݗ ޗ ߗ ߗ ߗ ߗ                                                   ! " " " " % & & & ' ' ' ' ' ' ' ' ( ( ) * * * , , - . . 1 2 5 8 9 < = = = = = @ A A B B F J J K M O Q Q Q Q Q R R T V W W W W W W W W W W W X [ [ [ [ [ \ \ ] ^ ^ ` a a b d d g g g g h h h h h h h h h h h h h h h i i i i i i i i j j j j j k k k k k k k k k k k k k k k l l m m n n n n n o o o o o o o p p p p p p p r r r r r s s s t w y } } } ~         ǘ Ș ɘ ɘ Θ Θ Θ Ϙ И И И И И И ј ј ՘ ՘ ֘ ؘ ژ ژ ޘ ޘ ޘ ߘ                             ! ! " # $ & & ' ' ( ( , . 1 1 6 6 6 6 7 8 ; < < < < < < = = B D D E E F F F G G G K M M M M P P Q Q Q R R R R R S T V W W X Y [ _ _ _ ` ` ` ` ` b b b c c c c d e g i i i j k n n n o o o r u u v x { } } ~  ™ ę ř ƙ ƙ Ǚ Ǚ ə ə ˙ ˙ ˙ ̙ Ι ϙ ϙ ϙ ϙ љ ԙ ԙ ԙ ԙ ՙ ՙ ՙ ؙ ٙ                                        ! $ & ' ( ( ( ) ) ) ) ) ) ) , , , 1 3 3 5 5 5 7 7 : : : ; ; = = ? ? A C D E F F F F F F F H I L L L N P R T U U U U W W W Y ] ^ ` c c d d g g g g j k l n n n p p q q r r r r t t u u u u u v v v v ~ š š Ú Ě ǚ ǚ ǚ ɚ ʚ ˚ ˚ ̚ ̚ ̚ ̚ ̚ К К њ Ӛ Ӛ Ӛ Ԛ Ԛ Ԛ ֚ ך ܚ ޚ ޚ ޚ                                      # # # # ' ) + 0 0 2 2 5 6 6 6 7 ; ; > @ @ B C D D E G K K M Q S S V W W Y Z [ ` ` ` ` ` c d f f f h h m p s t v w x x x y z z z z { { { { { | | | | } } } } › ě ƛ Ǜ ɛ ʛ ʛ Λ Λ ћ қ қ қ қ ՛ ՛ ֛ ֛ ֛ ֛ כ כ ٛ ڛ ۛ ۛ ۛ ۛ ۛ ܛ ݛ ݛ              # 1 2 5 7 9 ; > @ [ a c c c c d d e g j j k l n r t t u v w y { } } ~ ~ ~ œ œ Ü Ü Ĝ Ĝ Ɯ ǜ ǜ Ȝ ɜ ˜ Ϝ М ќ ќ ֜ ֜ ֜ ؜ ڜ ڜ ޜ ޜ ޜ                                         # ' * * + , - . 0 1 2 3 3 4 4 4 7 7 8 9 9 : : ; > A A B E F F F G H H H I I I I I I K K K K P P P P P P P P S S S S S T U U U U U U Y Y Y Y Y Z [ \ \ ] _ _ _ _ _ _ ` a c e e f g g h k m o o o q s w x z z { }   Ý Ý Ý ĝ ŝ ȝ ɝ ʝ ˝ ˝ ͝ Ν Н ѝ ҝ ӝ ם ؝ ٝ ٝ ٝ ڝ ڝ ڝ ۝ ܝ ߝ                          $ * + + + , , , - 0 1 1 3 4 5 7 9 N O P P Q Q Q Q Q S S S S S T V X Y Y Y Y [ \ ] ` ` ` c c f g h m m o o p p p q r r r r s v { ~ ~ Ş Ǟ ˞ ̞ Ξ О О ў ў Ӟ Ӟ ՞ מ מ ؞ ڞ ڞ ܞ ݞ ݞ ߞ                     ! " # # # $ % & & & & & & & & & & ' ' ' ' ( ( ( ( ) - - . 0 0 0 0 0 0 0 0 1 3 6 7 8 8 8 8 9 9 9 9 9 ? @ @ B C E H H H I I I I J K K L M N P Q R V V V V V [ [ [ ^ ^ _ _ ` ` ` ` ` a b f l s s u u w x x x |  ß ß ß ȟ ˟ ˟ ˟ ͟ ϟ џ ӟ ԟ ؟ ۟ ܟ ݟ ޟ                                                                     ! # $ ' * - - - 0 1 1 2 3 8 9 < @ B E I I N Q T ] ` ` a c e e e e f h i k m n o o p p s s u u u u u u v v v v v v v v v w w w w w w w w w w w w w w w w w w w w w x x x x x x x x z z z z z z z z z z z z z z z z { { { { { { | | | | ~ ~   Ơ Ơ ۠ ۠     ) * + 0 3 3 ; = = > > > > @ A B D F F F F F F F G G H I M M O O   ġ š š ȡ ȡ Ρ ҡ ԡ ١ ۡ ܡ ߡ                        & * 3 3 5 8 G H J J J K M M M O S T U W W X Y Y Y Y Y Y Y Z Z Z Z Z Z Z Z [ [ [ [ [ [ [ [ [ [ [ [ [ \ \ \ ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ^ _ _ ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` a a a a a a a a a a b b b b b b b b b b b b b b b b b b c c c c c c c c c c c c c c c c c c c c c c c c d d d e e e e e e e e e e e f f f f f g g g g g g g i i i i i i i i i i j k k k k k k m n o o o o o p p p p p p p q q q q q q q q q q q r r r r r r r r r r r r r r r r r r r t t t t t t t t t t t t t t t t t t t u u u u u u u u v v v v v v v v v v v v v v v v w w w w w w w w w w w w w w w w w w w w w w x x y y y y y y y y y y y y y y y y y y z { { { | | | | | | | | | | } } } ~ ~ ~ ~         Ģ Ģ Ģ Ţ Ƣ Ƣ Ȣ Ȣ Ȣ ɢ ͢ ͢ ͢ ͢ ΢ ΢ ΢ ΢ ΢ ΢ Т Ѣ Ӣ Ԣ ע ע ע ע آ ٢ ۢ ܢ ܢ ݢ ݢ ݢ ݢ ޢ ߢ ߢ ߢ                                                                              # $ % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % & & & & & & & & ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ( ( ( ( ( ( ( ( ) ) ) * + + - - / / / / / / / / / / / 2 2 3 3 4 5 5 7 7 8 8 ; ; ; ; < ? ? ? ? ? ? ? A A B E E E G G G G I I I L M M M M P Q Q Q S S S S S S T T T U U U U U U U U U U U U V V W W W W W W W W W W W W W W W W W W W X X Y Y Z Z Z Z Z Z Z Z Z [ [ ] ^ ^ ^ a a b b b b b b b b b c d d d d d d e e e f f f h h i i j j j l l l m n o o p p p p p p q q q q q r r r r r r r u u u u u u u v v v v w z z z z z z z z z { | } } } } } } } } } } } }  ã ã ţ ƣ ǣ ȣ ȣ ȣ ˣ ˣ ̣ ̣ ̣ Σ Σ У У У ѣ ң ӣ ڣ ݣ ݣ ݣ ݣ ߣ                                                      # % % & ' ' ' ' ' * * - 0 3 4 5 7 9 9 < < = @ @ B E G I J K K L L L L M O O O P P Q R R S T T T U V V V X X Y Y Y Y Y Z \ ] ^ a b c c d e f g h k n o p p q q q s t t u u v v x x z { ¤ Ĥ Ĥ Ť Ȥ ʤ ˤ ˤ ͤ Ф Ф Ҥ Ӥ դ ֤ ֤ ٤ ۤ ܤ ߤ                                           ! " % ' + / / 0 1 2 4 5 8 < = = = @ @ @ C C D E E E E I I I I J J K L M N N N O O O P P P R R R R R S S S S S T T V V V W W X Y Y Z Z Z Z Z _ ` ` ` ` a a a a a a b b c c d d e e f h i i i j j j k l n s s s t u v w x { { { | ~ ~ ~ ¥ å å ĥ ĥ ȥ ȥ ȥ ɥ ˥ ˥ ͥ ͥ ͥ ϥ ҥ ҥ ҥ ӥ ӥ ӥ ӥ ӥ ӥ ե ե ե ֥ ֥ ֥ ֥ ֥ ֥ ץ إ ڥ ۥ ݥ ݥ ݥ ݥ ݥ ݥ ޥ ߥ                         ! $ & ( * * , - / 1 3 6 6 8 8 9 9 9 9 9 9 : : ; = = = = @ @ @ @ @ @ A A B C E E F G I J N P P T V W Y [ [ [ \ ] ^ _ a b c d e g h h j k k l t v x y } } }   Ħ Ħ Ħ Ħ Ħ Ŧ Ʀ Ʀ Ȧ ɦ ɦ ̦ ̦ Ϧ Ѧ Ӧ ֦ ئ ئ ަ                ! % ' ' ' + , 1 3 4 4 6 7 9 ; ; < ? A B B C D D D D D E E E F F H I N P P Q T V V V V [ \ ] ] ] ] e g k l m n n n n o r r x x y | | } ~ ~ ~ ~  ͧ ͧ ߧ         3 6 9 ; D E E F H P R e h l o o o o p s x ܨ ܨ ܨ ܨ ܨ ݨ ި ߨ   " # # # + + . . / / 1 8 : = ? A A B B B C F F F I J J K L L N P U V Z Z Z ] ^ _ _ b c e f h j l l m m o p t w x } } } Ʃ ȩ ȩ Ω ѩ ҩ ҩ ҩ ө ө ԩ թ ک ک ک ک ک ک ݩ ݩ ݩ ީ ީ ߩ ߩ ߩ ߩ ߩ ߩ ߩ ߩ            $ $ , / 5 6 6 7 ; ; < = G H J M O O Q S S T Z ^ ^ ^ _ ` e f i i k k m m m o r t x | | }     ª ª ª ª ª ª ª ª ê Ū Ȫ ɪ ɪ ʪ ˪ ˪ ˪ ̪ ̪ ̪ ̪ ͪ ͪ Ϊ Ϊ Ϊ Ϊ Ϊ Ϊ Ϊ Ϫ Ϫ Ҫ Ӫ Ԫ Ԫ Ԫ Ԫ ժ ժ ת ת ت ت ت ت ت ڪ ۪ ۪ ۪ ۪ ۪ ۪ ۪ ۪ ܪ ܪ ܪ ܪ ܪ ܪ ܪ ݪ ު ު ߪ                           # # # $ & & ' ' ' ( ) ) + + , - . . / / 0 2 6 9 9 ; = ? B B B B C D D D J J K L M N N N N N N N R R W X ҫ ҫ ӫ ӫ ֫ ֫ ׫ ׫ ث ګ ޫ                             ! ! " " # # % ' ' ) ) ) + , . / 4 4 6 6 6 6 6 7 7 8 8 8 9 9 < = > > > ? ? ? @ A E F G H J K L L M P Q Q Q Q Q Q T U V V V V Y Z [ [ _ ` b c d e h l l l m n o r x y z | | } }   Ȭ ɬ ˬ ̬ ͬ ͬ ͬ Ѭ ڬ         ! # # # # $ % % % % & + 1 ; < = ? A B B B B B B B B C C C D G G I K K K M Q R U V V V V V V V X [ ^ a a a d d e f f g g k p s t t y { { { { | ~ í í ǭ ǭ ˭ ˭ ϭ ѭ ѭ խ ۭ ߭   \ b d d h h n n Ů ʮ ˮ ͮ Ϯ Ѯ Ѯ Ѯ Ѯ Ү ֮ ݮ           ( \ b b e f g i m u x  ¯ ï ï ï ï ï į į į                                        " $ % % % % & & & & ' ) ) ) ) ) 0 0 7 7 7 7 7 7 : < D E I L M S T T X X Z [ b d i k l l p s u w ° ð İ ư ư ǰ Ȱ Ȱ Ȱ Ȱ ɰ ɰ ʰ ʰ ʰ ʰ ʰ ˰ ˰ ˰ ˰ ˰ ˰ ̰ ̰ Ͱ Ͱ Ͱ а а а а а Ѱ Ѱ Ѱ Ѱ Ѱ Ѱ Ѱ Ѱ Ұ Ӱ Ӱ Ӱ Ӱ ԰ ԰ ԰ հ ְ װ װ װ ذ ذ ٰ ٰ ٰ ٰ ۰ ް ߰ ߰ ߰                                                                         ! ! # # # $ $ % % & & & ' ( ) + + + / / 0 0 0 0 0 2 2 2 2 3 4 4 4 5 6 7 7 8 8 8 : C C C D E G H H J J T W X Y Z Z Z _ o s s v w y { | ± ñ ñ ű ű DZ DZ DZ ȱ                  % & & ' 1 2 5 5 6 8 < A L Q R S T V V V W W X X X \ g i n q r t t t u u u u u u u u u u w w x x x y y y | } } ~ ~ IJ IJ IJ Ų Ʋ Ʋ Ȳ Ȳ ̲ β β ϲ ϲ в в в в в в Ҳ Բ ղ ܲ ޲                   , - 4 5 ; A D D E H J J L Q S W ^ ` b c c c c d g l l l m m m m y z ȳ ȳ γ ֳ ֳ ׳ س س ٳ ٳ ٳ ٳ ݳ ޳                % % & ' ' ) ) ) + , . . . . 1 1 2 2 3 3 4 6 7 9 ; ; > ? ? @ @ @ @ @ A B B B C C E F J N N U Y [ ` a d f h k n n n n n n n n n n s s t v w y z | Ŵ ȴ ˴ ̴ ̴ Z c k y z | ŵ                  ! ! ! $ ' - - - - / / 0 0 0 0 3 6 7 8 < ? H M M N R S S S S S T T T T T T U U U W W W X Y [ [ \ \ \ \ \ \ ] ] ] ^ _ _ ` a b c d f g g n y { Ķ Ķ ˶ ζ ߶          " " " & & ' + + - 0 2 5 6 7 7 8 9 9 : < < = = @ @ A A B B F I K M O O Q R R S T T U Z \ _ c f h h l l o v z } Ƿ ɷ ͷ Ϸ ӷ Է ַ ٷ ۷ ݷ ݷ ޷            ! % ( * - / / 0 0 3 5 6 7 7 9 ; < A F H Q S S Y Y Z [ \ ] b j l l n p p s v x | } } ~ Ǹ ˸ ͸ ϸ Ӹ ڸ ۸                                     ! ! ! # , , / 1 1 2 2 4 5 5 6 6 6 7 7 7 8 8 < < > > ? @ A A C D E F G H M Q ¹ ù ù ù ù ù ƹ ɹ ʹ ˹ ͹ Ϲ ҹ ӹ Թ չ ׹ ع ع ع ٹ ٹ ڹ ۹ ܹ ܹ           > H P [ \ a f g h h h i j l l n q s x { { { } } } ~ ~ ú ú ú ú ú ĺ Ǻ ɺ ɺ ˺ ̺ ͺ к к Һ Ӻ պ ٺ ں ں ۺ ۺ ܺ ܺ ܺ ܺ ܺ ܺ ܺ ܺ ܺ ݺ ݺ ޺ ޺                                            ! # % & & & & ' ' ' ' ' ) , q r s s s s s s s s t v v v v w y { { { | } } } } } } }  » Ļ Ȼ ɻ ̻ ͻ ѻ ѻ ӻ ӻ Ի ջ ջ ֻ ֻ ֻ ֻ ֻ ׻ ׻ ׻ ۻ ݻ ޻ ޻ ߻ ߻ ߻                                     ! ! # $ & & & & & ' ' ' ( * * + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - . / / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 2 2 3 3 4 4 5 5 6 6 7 8 8 9 9 : ; > ? @ @ B C E E E E F F F K K K K L M Q R R T U W ] ^ ` b b b b d e e e g g h i j j k l m o r s s s s u v v w w z { | |  ļ ż ż Ƽ ɼ ɼ ɼ ɼ ͼ ͼ μ μ μ м Ӽ ּ ټ ۼ ۼ ߼               ! $ ' ) + + . . . 2 3 4 4 4 4 4 9 : A A B D D H H J M O R Y ] a a a e e f f g g g g h i i k n q r r t t t t t v v v v w w w w x x x x y z ~ ~ ý Ľ Ž Ž ƽ ƽ ƽ Ƚ ɽ ɽ ɽ ɽ ɽ ˽ ˽ ͽ ν ν Ͻ Ͻ н н н н н ѽ ѽ ѽ ѽ ѽ ӽ ӽ ս ս ֽ ֽ ֽ ׽ ׽ ׽ ٽ ڽ ڽ ۽ ۽ ݽ ݽ ޽ ޽                                                   # # # $ $ $ % ' ' ) ) + + + + + + , . / / 0 1 1 1 1 2 2 3 5 6 9 9 ; < > > > > @ C C C E F F J L M N N P P P P P Q Q R R S S S S S T T W Z [ [ [ [ \ \ ^ ^ _ _ _ _ _ b e f h j k l l l l l n p p q q r r s s s w x x x x z z }   ž ž ž Ⱦ ɾ ɾ ɾ ɾ ʾ ˾ ; ξ ξ ξ ξ ξ ξ ξ ξ ξ Ͼ Ͼ о о о о Ѿ Ѿ Ҿ Ҿ Ҿ Ҿ Ӿ ־ ־ ׾ پ ھ ھ ھ ھ ھ ھ ھ ھ ھ ۾ ۾ ۾ ۾ ۾ ܾ ܾ ܾ ܾ ޾ ޾ ޾ ޾ ޾ ޾ ޾ ߾                                                   ! ! ! ! ! " " # $ $ % & & ' ' ' . 0 2 2 2 2 3 3 4 5 6 : = ? ? ? ? ? ? ? ? ? A A A D D D D E F F G H I I J J J J J J K N P P S S S S S S S S T T T T T T T T U V V V V V W W X Y Z Z Z ] ^ ` ` ` ` ` ` ` ` ` ` ` ` ` ` a a a b b b b b b b b b b b b b b b c c c c c c c c c c c c c c c c c c c c c e e e e e e e e e e e f f h h h h h h h h i i i i i i i i i j j l l l l l l l m m m m m n o p p q q q r s s s u w w w w w w w x x x x z { { | | | | } } ~ ~ ~ ~ ~ ~ ~ ~ ~ ~    ÿ Ŀ ſ ſ ɿ ɿ ɿ ɿ ɿ ɿ ɿ ɿ ɿ ɿ ɿ ɿ ɿ ɿ ʿ ʿ ʿ ˿ ˿ ˿ ˿ ˿ ˿ ̿ ̿ ο ο п п ѿ ѿ ҿ ҿ ҿ ҿ ҿ ҿ ҿ ҿ ҿ ҿ ҿ ҿ ҿ ҿ ӿ ӿ ӿ ӿ ӿ ӿ ӿ ӿ Կ տ ׿ ؿ ڿ ۿ ۿ ۿ ܿ ܿ ܿ ݿ ݿ ݿ ݿ ޿ ߿ ߿ ߿ ߿                              % , , \ i i j j j k l n q q q r r s s s s t t u u w y y { { { { { { | } } } ~ ~ ~ ~ ~ ~ ~                                                  ! ! # # % & & ' ( * + + , , . . / / 0 0 3 3 3 3 3 3 3 4 4 5 5 5 8 9 : < < = = = = ? @ B C D F F G G G H I I I I I I K M O O O Q Q Q Q R R R R T U ^ _ a a a a a b c d f f h h h h h i i i l o o q r s s t w w w x { { { | | | } } } } ~                                  " # % % % % & & & & & & ' ' ( ) ) ) ) ) ) + + , , / / / 0 3 6 8 9 9 9 9 9 9 9 9 9 9 9 9 9 9 : : : : : : : : ; ; ; < S T d d d d d d e f f f f f f g h h h h h h h h h h h h h h h h i i i i i i i i i i i i i i i j l m n q q r r r t t v v x { |                                                      " # # $ $ & * 7 9 9 ; ; < = > > > > ? ? @ A A C C C E H T T U V V W W X X X X h h j j j j j j k k k m p p p q r u v x {                 & ( ( ( ( ( * * + + + + - - / / / 1 1 1 3 4 5 7 7 7 ; ; < = > @ @ B D E E F G G G G H H H H H H H H H H J J L L L O P Q S S T T V W X Y [ ] ] ^ ^ _ _ _ _ ` ` ` a c c c e e f f f f h k n n o q s u u u u u v x { { | } ~ ~                                                      ! # % ' ' ) * + , - 0 0 0 0 0 4 4 5 7 7 8 8 8 9 : < > ? ? A D F H K K L N R R W W \ d d e i j j k l s s t w w x y y z { |                          ! # $ $ % & ' ( ( ( ( ) + , . 0 0 2 3 4 5 6 9 = > ? C F J J J L L L N P P P U U W W W X X X Y ^ ` ` ` ` ` a a a b c c f f f f f f f g h i l l l m o p u w w z | ~ ~ ~                                           ! ! ! ! ! ! # # # # # # # $ $ $ % ' ' ' ( ( ( ( ( * * * * + + - - . 1 1 1 1 1 1 2 2 3 5 7 7 7 8 8 9 A A C D F G G I I J J K M N N P S S S S S S S S S S U U U U U V V V V V W X X Z Z Z Z Z Z Z Z Z [ [ [ \ ^ a c e f q u u v v v z { { { { { } ~ ~                                                                         ! $ % % & & & & & & & & & & & ' ' ' ' ' ( ( ) ) * * * + + + + + + + + - - - - - - - - - . / / / / / / 0 0 0 0 0 0 0 0 0 2 2 4 4 4 4 6 6 6 7 7 7 8 8 8 8 8 8 9 : : = = = > ? ? @ @ @ @ @ B B B B B B C C D F F H H H H H H K M N N O O P S S S S T T T T T U W X ] ] _ b f f g g g g g g h k k k l l l l m m m s s t v v v v w x x x y y y z z { { { { { { { { | | | |                            ! # # # # # # # # # $ % ' ( ( ( ( ( ( ) ) * + + + , - / / / 0 0 1 3 3 4 4 5 5 6 6 7 7 8 8 8 8 8 9 = > ? @ A A A A A B D E F H H H I I I I I I I I I I I I I I I J K K K K K L L L L L L M O O O P Q Q R R R R S T T U U [ [ \ ] ] ] ] ] ] ^ _ _ _ _ ` e e e k k k m n o o o p p p p q r u u v v v v v v v w x y y z z | | } } } ~ ~ ~ ~ ~                                    ! ! ! " " $ $ % % % % % & & ' ( ) * * * , , , , . 1 2 2 2 3 3 5 7 7 7 8 ; = = > A A A A C C C C C C D G H I J K R U W W W X X Y Z [ ] ^ ^ b b d i j j j s t u x }                            $ ( ( * + . . . . 0 1 2 2 2 5 5 5 6 6 8 9 : ; = = = ? ? @ A B B C D D D E E E E F F G L M N N O P R R S S S S S T T U U V V W W Y Y Y \ ] ] _ ` ` ` b c d d e f g h h i i i i i j j j k l m m o p p p r r r r s s s s s s t v w w x x x x x x x x x { { | } ~          # # # # # # # # # # # $ % % % ' ' ' ' ' ' ' ' ( ( ( ( ( ( ( ( * + + + + + + + + + + + + , , , , , , , , , , - - . . . . . . . . . . . . . / 0 0 0 1 1 1 1 2 2 2 2 2 3 5 5 5 5 5 9 9 9 9 9 : ; ; ; < < < ? C D D G H H K M M N N P P Q Q Q Q S T W W W Y Y [ ] ] ] ^ ^ ` b c d d i j j j j k k l l l m n n q q q q q r r r s u v v v w w w x x y z { { ~                          ! " # # $ $ $ $ $ $ % % % & & & & ' ( * + , , , , , , , , , , / / / 0 4 4 4 6 6 7 8 8 8 8 9 : = ? ? ? ? ? A A A B B B B B B B C C F I I M M M M M M N O O O Q Q Q Q R T T T U U V V V V V V V V V V W W Z [ [ \ ] ^ ^ ^ ^ ^ ^ ^ ^ ^ _ _ _ _ _ _ _ _ _ _ ` c c c c d d e e g g g g g g g g h h h i i i j j n o p q q q q q q r r r r r r r r r r r r r r r r r r r r r s t u w                            " # # $ % ' ( * * * * * . / 2 6 ? B B E H Q U W Y Y m p t y ~ ~                  ! " # % & ' ' ' ) ) ) , , , - . . . / / / 5 6 7 9 9 ; > > A A B B B B B D D D E F F F K K N Q R U W \ ^ f g h h h h i i i i k l m n n q r s s s s s t t t u v x | } } } ~         $ % % ' 3 < ? D G H K K L L L N S Z Z Z Z Z Z Z Z Z [ ] c e e e e f f g h i i k n q v x                  ! ! ! " " " " " " # % % % % % & ( ( ( ( ( ( ) ) ) ) ) ) ) ) ) + + , , , , , - - - - - / 0 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 3 4 4 4 4 4 5 5 5 5 5 6 6 6 6 6 8 8 8 < = > ? ? @ @ @ B C C D E L L L L L L L L M M M M M O O O O O O O O O O O O O P P Q Q R R S T T T T T U U U U V V V V V V W W W W X Z Z [ [ \ \ \ \ \ \ ] ^ ^ ^ _ _ _ _ _ _ _ a b d d d e e e e e f f f f f g g g h i i i j l l l l l m m m n n q q q q q q q q r s s s t t u u u u v v v x x x y y z } ~ ~ ~ ~ o s u u w y y y | ~ ~                          " $ $ & & & ' ( ( ( ) ) * * - - / 2 2 3 3 6 7 9 9 9 : : : ; < < = > > @ A D I I J K M N S S S U U W X Y Z [ \ a b b b d e h k l m o o o o o o q r s w x | }                 ! $ ' , . / 2 4 4 5 9 ; = @ K L N O O P P Q S \ ^ d h o p w z ~                     ! " $ $ % % ( , . . / 1 4 > B D H O T Y [ ] _ c f f f g g j j j m m o r t t t v v x | } }          ( , 3 7 @ O T V Y c f g g g h h h h i k p p s s s s t u w x x x { { | | } ~           ! # % ) + - / / 1 7 9 ? ? @ B B B C F I K L L M R R R R R R T X Y \ \ a b f h j n s y              $ ' ' ( , , . . / / 2 5 6 = A G O S T W Y [ ^ ^ c e l r t y ~      ! " & & ' ' ' ' ) * - - 0 6 ; C F F G G K K K K K L N O R X [ [ ] ^ ^ _ _ ` c d e g k k l l m m n n n s t u v | ~                         " # # & ' ( ( , . / 4 6 6 ; < < = > @ C H H H H I I I J J J J J L L L L M M M N N O S U X Y Z Z ^ _ ` ` ` a b m q q y ~ ? B H K M P R U V [ [ _ a b d d g i i j l o q s v y z    $ + , Y ^ _ _ _ ` b e f h i j q s t u u x x y { } } } ~ ~                                                           ! ! ! " " # # # # # # # $ $ $ $ $ ( ) ) ) * * * * * , , , - - - - - . . . . / / / 0 0 1 1 1 1 2 2 2 4 4 4 5 5 6 6 7 7 7 7 7 7 9 : ; < = @ @ B C C C G G G G G G H H J J J M N N N P P P P P Q S S T T T U U U U U U U U V V V W Y Y Y \ \ ] _ a a b b b b b b b d e e e e e g g g h h j j l l l l l m m n n p q r t u u v w y y y z z z { }                              ! ! ! # % ' ' ' + , . 1 4 7 8 8 9 = = > > > > ? ? ? ? ? ? ? ? @ @ @ D E E F H L M N P R S T U V V d d e h k l n r r s t v w w z |                    ! ! ! " $ % + + + - . . . 0 0 2 2 2 2 3 4 7 : : = A A C H I J J L P S S S T ] b c c e g l m m n o o p q q q s t w w x y y { { { { { } } } ~ ~ ~ ~ ~                                    # # $ % & ( ) + 1 2 2 2 2 2 3 5 8 ; > @ A D D E G G I M O P Q R R R R U X Y Z e f f h i j k n p q q t v v w y | | | | ~                  $ & & ( ) + , , / 1 6 7 ; ; < < = @ @ @ @ @ @ @ @ @ @ @ A A A B B C C C C E E E F F G G G H H I I L L N N O O O P P P P S T T V V V V V V V W W X X X Z Z ] ^ ^ _ _ ` a a b c c c c d e g i j l l m p u v {    3 4 5 9 ? @ F M N R R S T U W Z ` ` a b c c g g i j j k l l l l n n n n p q r { | ) - - 2 3 6 : ? A B G G G G G H K K K M M R V V V V V V Y [ [ [ [ \ \ _ ` ` d f g i i j k m m m n q q t t t t t t t t u u x x z { ~ ~           # # ) + 1 3 6 7 : < @ B D F G L O Q S W ] c d e e e f g p }                          " " # % ) ) ) ) ) ) * * + , . . . 0 0 2 9 : ; ; > @ @ @ @ E G G G H H H J J K K L M P Q T U X X X X X Y Y Z Z Z [ [ \ \ \ ] ] ^ ` ` ` a a a b b b b b b d e g g g g g g i i i i k k k k k k l l m m n o o o o o o o o o p s t t t u u v z z } ~            ! " # # $ % % ' ) , . 1 5 7 A H I I K K K M M M N O P P Q R R R T U V V W Y Y Z Z Z Z [ [ d d d d d d f z } }         " $ % - 6 7 @ @ @ B B B J P Z [ [ \ \ _ b b c e g i j j l l o q r r s x { { | ~       8 @ K Q T Z _ d l ~       ! ! " $ ) ) . 4 8 ; < = > @ B D D D D E G H I M O O O Q T W W W Y Z ` c g g m m n n n n r v z z ~     # ) 0 0 0 0 3 4 4 4 6 6 6 : ; ? ? D G J J K K K M Q S S V d e k m x | } ~                           " " " % % & ' ' ' ' ' ) ) + , - / / 2 4 7 7 8 8 : : ; ; @ H M N \ ^ b b c d f g g p r t w } }                     ! ! " " " " $ ' ( ) ) * + + + / / 3 6 ; C C D F F R R S W X Y ] ] ^ _ _ ` ` a b b b d g h i k l l l l l m m n o o o o o o o o o o o o o o o o q q q q q q q t w w y { { { { } ~ ~    " " % % ' ( ) + 0 5 = = = > > > > ? @ @ A C D D D D D E E G H J O O X Z e h k m p r t t u u u w | |   ! ' ) , 6 = ? ? B C E M O W X ` h o r v x z | | | | | | | } }                                                                            ! " # # # # # # # # # # # # # # # # # # # # # # # # # # # $ $ $ $ $ $ $ $ $ $ $ $ $ & & & & & & & & & & & & & & & & & & & ' ( ( ( ( ( ) ) ) ) ) ) ) ) ) ) * * * + + + + - - - . . . . / / 2 4 4 4 4 4 4 4 4 4 5 5 6 8 9 9 9 : ; = ? @ A B B D D E E E E E F H H J J J J J J K K K K L L L N O O R R T V W W W W W ^ q                                               ! " # $ $ & & & & ' ' ( ) * + , , , - . 0 2 2 3 3 6 6 8 9 9 9 9 9 ; < = = > > @ @ B B C D D F F F F F F F F F F F F F G G G G G H I K L L L L L L L L L O O O Q Q Q U V V W W W X X X X X X X X X X Z Z Z Z Z [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ \ \ \ ^ ^ ^ ^ _ _ _ ` ` ` ` ` ` ` d d d d d d d d d d d d d d d d d d g g g h i i i i i i n n p t t u w w w w w w w w x { { { { | | } } } } } } } } }                                       ( ( ) ) ) ) ) + + , . 0 0 4 6 7 9 9 C D D D E E F I L L L N N N O O P Q R R R S T T U Y Z Z g h h h h i i j m m m m m m m n n p p p q q r r r r r r s t v v v x { { { | | | | | ~ n o z { ~    % % & / 5 : : : ; ; ; @ @ @ A A A A A B B B C D F G I M O W Z b z        $ ( ) 0 0 5 : ; < > @ E E G G G I L M N P Q S T V V W W X Z [ [ [ [ [ _ ` ` a b b b b b d d d g g n n p q q q q r s w y y {       ( - 8 8 8 8 8 9 ; ; ; < > > ? @ A E E G J K L N U V Y [ a g l l o q s v w z       $ % - . / 2 4 > @ B B C C H M N U Z ` e f i k o u u y y       # # # # # & ) . 2 3 7 = B G K M S W ] e g h l s w w w w y z ~ ~                      $ % % ' ' ' ) ) * - / 2 2 2 3 4 5 5 6 7 8 : ; ; A A A A A C E I K M N P R R n n s v y ~                                                                                                                                                                      ! ! " " " # # & & & & & ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ' ( ( ( ( ( ( ( ) * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + , , , , , , , , , , , - - - . . . . . . / / / / / / / / / / 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 3 4 4 4 4 4 4 4 5 5 5 6 6 8 8 8 8 : : : : : ; ; ; ; ; < < < < < < < < < < = = = = = = = = = = = = = = = = = = = = > > ? ? ? @ @ @ @ A A A A A A B B B B B B B B B B B B B C C C C C D D D D D D D D D D D D D D D D D D D D D D D D D E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E F F F F G G G G G G H H H I I I I I I I I I I I I I I I I I I I I I I I I I I I I I K K K L L L M M M N O O O O O O O O O S S S S T T T T T T U U U U U V V V V V V V V V V V V V W W W W W X X X X Y Y Y [ [ [ \ ] ] ] ] ] ] ] ] _ _ _ _ ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` a b b c c c c c c d e e e e e e e e e e e e e e e e e e e e e e e g h j j j k k k k m m m n n o o p p p p r s s s s s s s s s t t t t u v w x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x x y y y z z z z z z z z { { { } } } } } } } } ~ ~ ~ ~ ~ ~ ~ ~ ~ ~                  ! ! ! ! ! ! ! " " # $ $ $ $ $ $ $ $ $ $ $ $ % % % % % % % ) ) ) * + + . . 0 0 3 7 8 8 8 8 8 8 8 9 : : ; < < < < < < < = = > > > > > > ? @ @ @ @ J J J J J J K K K K K L M M M M P Q T T T T T T T U U U V V W W X X X X X X Y Y Y Y Z Z [ [ \ \ ] _ ` a a b b b b b b c d d d d e e e e e e e e e f f f f f f f f f f f f f f f f f f f f f h h h h h h h h i i m n n n o o p r r r r r s u w w w x x z z z z {                                                                                                                                   " $ ) * * * + + + , - - . / / 0 0 0 0 0 0 1 1 1 1 1 1 2 2 2 2 3 3 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 6 7 8 8 8 8 8 8 8 8 8 8 8 8 8 9 : ; < = > > ? ? ? ? @ @ @ @ C D D E F F F F F G G H I J J J K K L M N N N N P R R R S S S S S S S S S S S S T W W W W W W W W W X X Y Y Y Y Y Y Y Y Y Y \ \ \ ] ] ] ] ] ^ ^ _ _ _ _ _ _ _ _ _ _ _ ` a b c c c c c c c c c c c c c c d d d d d d d d e e e e e e e e j j j j j j k k k k m m n n o o o o o o p p q q u u u w w x y                                              ! $ $ $ & & ' * , , , - - - - / 2 4 4 4 6 7 7 8 8 8 8 ; < = > ? @ A A B C C C C C C C C E F F F F F F G G H H H H H H H H H H H I I I I I I I I J J J K K L L M M M M O Q Q S S S S U U V W X Z \ \ \ \ _ _ _ _ _ _ _ _ _ _ _ ` ` ` ` ` ` ` ` ` a b b b b b b b b b b b b b b b b b b b b b b b b b b c c c c c d d d d d d e e e f f f f f f f f f f f g g g g g g g h h i i j j j j k k k l m n p q q q q q q q q s s s t t t u u u u u u u u u u u u u u u x x x x x x x x x x x y y y y y z z { { } } } } } ~ ~                                                            # # # # % % % % % % & & & & & & ' ' ' ' ' ' ' ' ' ' ( ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) * * * + , , - - - - . . . 0 2 2 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 6 6 7 8 8 8 9 ; ; ; = > ? A B B B B B C C C D J J J J J J J J J K K K K L L L M M M M M M N N N N N Q R S S S S S T U U U U U U U W Y \ \ ] ^ ^ ^ ^ _ a b b d d e e f f f f h h l l l l l n o o q r s s t u v w x x y y y y z | | | |                                                   ! ! " # # $ % % % ' ' ' ' ( ( ( ) ) ) ) * * - . . . . . 1 3 4 5 6 7 7 7 8 8 : : : : : ; = > > @ @ @ @ A A D D D D D D D G G G G G G G H H H K K K M M M M M M M M M M M P R U X X X X X Y Y Y Y Y Y Z ] ^ _ _ ` a b b b b c d e e e g p q t t t u v v v v v w x x x x x x x x | | | | | | | } } } } } ~                                                                 # # # $ $ $ % % % & ) * * + - - . / 1 1 1 1 4 5 5 5 5 6 7 8 F F F I J J N N N O O O O O O P P P Q T W X Y Y Y Y Z [ [ ] ^ b d d d d d d d d e e e f f f f g g g g g g g h h h h h h h i i j j k k k k l m m m m m m m m n o o o o q r r s s t t w x x y z { | | } } } } ~ ~ ~                              " " # $ % & ( ( ( ) ) ) ) ) * * + + + + + + + + . . / / 0 1 1 1 3 6 6 6 6 6 6 7 7 7 8 < < < < < < < = @ @ @ K N P R Y \ ` b b d j j j m p q t v w y } }             " % % & ) + . / 0 2 4 5 5 5 6 : ; < < = > B E E E E H I I J J J K K K L L L M M O P P Q R R S T T T W W Y \ \ \ \ \ \ _ _ d i j n o p p s s s t t t u v v v x { } } } ~                                                                                                 # ' ( ( * + + , - - / 2 3 3 3 3 3 3 3 5 5 5 5 8 8 : ? ? E G G G I N N N N N O O O O O O P R R R R R R R T T U U U U U U U U U U V V V V W W W X X Y Y Z Z Z Z [ ] ] ^ ` ` ` a a a a a a a a a a a a b b d d d d d d d f g h h h j j l p x | ~                                                                                                                                                                                                   " " " # # $ $ $ $ % % ( ( ) ) * , , . / 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 5 5 8 9 9 9 9 9 : : : : : : : : : : : : : < < < < < < < < < < = = = = = = = = = > > > ? ? ? ? @ @ A A C D D D D D E E F G G G G H H H L L L M N O O O O O O O Q R S S U V V W W W W Z Z Z Z [ [ ] ] ^ ^ _ _ a a a c c d d e e g g g g h h i m m m o q w w w w y y z }                                                                                             # $ % % ' ( A F O Q T \ h p p r }                                                                                                                          " # # & ' ) ) * + + - - - . / 1 2 4 4 6 6 8 8 < > A C E G K L S S S S T T W [ \ \ \ \ \ \ ] ] a a a b c c c d d f g h h h i i k k n t u u v w w z { }                                                             ' ' ' ) * + + + + + + , , . . . / / / 0 0 3 4 4 5 5 5 5 5 5 5 7 7 7 : = = ? C D E H K O Q Q T X X Y Z Z [ ^ ` ` e e f i j j l n o o o p q s t t t t v w w y y z z | ~                                                                  ! ' 0 6 > ? ? B E E J V Z [ [ ] ] ] _ ` b b d e f f h j j m m o r x                                                                                                                    ! ' ( , - 2 6 ; ; ; = = @ F F J K L O U V [ ^ c e f h k m p r v x |                                                                 ( ) 0 6 : > D G J K K O O O T Y Z [ [ [ \ \ ] ^ _ _ _ _ ` ` ` b d g h k k p u w { ~        # % ' ( + + 1 6 7 < > B J S U V W W X X Z Z [ \ \ ] _ ` b b c d e h i m s u w z z }                    " # $ $ & ' * , , 4 4 5 7 8 8 : : : > D G G G G H H H H H H H I J J N N N N P P Q T X X Y [ ] ` c d f f i l n s t x { | }           " # % ' 5 : < = @ J N O O O P P Q R R R R R S U U U V V Z Z Z ] a a e e e f g g i k n p r u u v y y |                                                              ! " # # # % % % & ' ' ( + , - . 4 5 5 6 6 9 : : ; < < = > ? ? @ @ @ A B B B D D E E E G G G H L N N O P R T _ _ ` ` ` c d f h k m o o o o o o p p p r t v w x x z { { } } } } } ~                                                                                                                                                      # & & , . / 0 5 ? ? D E H L O Q S T Y Y Y Z Z ] ^ _ ` a b d f h n r u x y y y z                                                                                                                     ! $ % ( + . 0 2 4 7 7 8 : : ; < = A B D F G K N O P Q Q S U X X X X X Y ] b d f g l m n o o q r v { ~ ~ ~                                                                                                                                                   # $ ( * , - - 0 0 1 2 8 8 9 < ? ? A C E I K L M M M M M P S U V V V V V V V V V V V V V V V V V W Y \ \ ^ ` ` d e j j n n r r v x x x x x | | | ~ ~ ~ ~ ~ ~                                                                                                          " & & ( + - / 1 ; B B C C D D D D F G H I J J L P S T V Y \ ] ] ] ^ ^ ^ _ ` b d e h l p s t u u w x { { | | |                                                                                                                                              ! ! " # & ' ( ) + + + + - - . 0 2 4 4 5 : : ; ; = > A A E E F F F G G I I L L N N O Q Q S U U X [ \ ` b c e e e f f h h k m m m n o q v x z { } ~                                                                                                                                                                                                                                     ! " $ $ $ % & & & & ( ( ( ( ( + + , 0 0 1 1 2 2 4 4 6 7 7 9 : = ? 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A A B B B C D D H H I I I I I I I I I J J L N N N N N N N N N Q Q Q S T T T V V V W Y Z Z ] ^ b e e l l l m m m p t v y z Ň Ň LJ ɇ χ χ Ӈ և ڇ ڇ ڇ ڇ ڇ ڇ ڇ ڇ ڇ ڇ ڇ ڇ ۇ ۇ ۇ ۇ ۇ ۇ ۇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ ܇ އ ߇ ߇ ߇ ߇                                                                                                                 $ % % % & & & & ' ( ( ( ( ( ( ( ( ( ) , - . 0 0 0 2 2 2 4 5 6 6 8 9 9 9 9 : : : ; = = @ @ C K L O R V Z Z Z _ c r t w z ~ ň Ȉ Ȉ ʈ Ј Ј Ј ш ш ҈ Ԉ Ԉ Ԉ ؈ ڈ ܈ ݈ ݈ ވ ߈            " ( . 1 1 4 7 7 8 8 8 : > > > D E M P R U U Z [ \ \ \ \ ] ^ ^ _ _ _ ` b d f f h n o p r v y z ~ ~ ~ ~ ~ ~ ~ ‰ Ɖ Ɖ Ɖ Ɖ Ɖ Ɖ Ɖ lj ȉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ɉ ʉ ʉ ˉ ˉ ˉ ˉ ˉ ̉ ̉ ̉ ͉ ͉ ͉ ͉ ͉ ͉ Ή ω ω ω ω ω ω Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ Љ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ щ ҉ ҉ ҉ ҉ ҉ ҉ ԉ Չ Չ Չ Չ Չ Չ Չ Չ Չ Չ Չ Չ Չ ׉ ׉ ׉ ׉ ׉ ׉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ؉ ډ ډ ډ ډ ډ ډ ډ ډ ډ ډ ډ ۉ ۉ ܉ ܉ ܉ ܉ ݉ ݉ ݉ ݉ ݉ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ މ ߉ ߉ ߉ ߉ ߉ ߉ ߉ ߉         0 0 1 2 3 3 3 3 3 3 3 3 4 4 4 ? ? ? ? ? ? @ @ A B B B B B B B C D D D F F G H H H H I I I I I I I I I I I I I I I I I I I J J J J J J J J J J J J J K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K K L L L L N N N N N N N N N N N N N P P P P Q Q Q Q Q R R R R R R S U U V W W W X Y Y Z Z [ ] ] ] ] ] ] ] ^ _ _ ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` b b b b b c c c c c c c c c c c c c c c c c c c c c c d d e f f h h h h h h h h h h h h h h h h h h h h h i j j j j j k k k k k l l l l l l l l m m n n n n n n n n n o o p p p p p p q q q q r r r r r r r r r r r r r r r s s t t t u u u u u u u u u u u u v y y y y y y y z { { { | | | | | | } ~  Ê Ê Ê Ê Ê Ê Ê Ê Ê Ċ Ċ Ċ Ċ Ċ Ċ Ċ Ċ Ċ Ċ Ċ Ċ Ċ Ċ Ċ Ŋ Ɗ Ɗ Ɗ Ɗ Ɗ Ɗ Ɗ Ɗ Ɗ Ɗ Ɗ Ɗ Ɗ Ɗ Ɗ NJ NJ NJ ʊ ʊ ʊ ʊ ʊ ʊ ʊ ʊ ʊ ˊ ̊ ̊ ̊ ̊ ̊ ͊ ͊ ͊ ͊ Ί ϊ Њ Њ Њ ъ ъ ъ ъ Ҋ Ҋ ӊ ӊ ӊ ӊ ӊ ӊ ӊ ӊ ӊ ӊ ӊ ӊ Ԋ Ԋ Պ Պ Պ ׊ ׊ ׊ ׊ ׊ ׊ ׊ ׊ ׊ ׊ ׊ ׊ ׊ ׊ ي ي ي ڊ ڊ ڊ ڊ ڊ ڊ ڊ ۊ ۊ                                            " " % * * + + + + , , , , , , , 0 0 0 0 0 1 1 1 1 1 1 1 4 4 4 4 4 6 6 7 7 8 8 8 8 8 8 8 9 ; < < < < > ? C D D E E E F H I I J J J K K K K K K K L L M M M N O P P R R S U W W X Y Y Y Y Y [ [ \ ] _ _ _ _ _ _ _ a a a a a b b b c c c c c c e e e e e e e f g g g g g g h h i i j j j j j j k k k m n q q q q u u u u u u v x y y | Ћ Ջ Ջ Ջ Ջ Ջ Ջ Ջ Ջ Ջ ֋ ֋ ֋ ֋ ֋ ֋ ֋ ֋ ֋ ֋ ֋ ֋ ֋ ڋ ڋ ڋ ڋ ۋ ۋ ދ                 ! " $ % ( - . 3 4 8 : < = A B C E F M S T V W Z \ \ ] ^ ` b f g g g g g h h h h h h h j j l l l l m n n r v v { } ~             ! " $ & ( + , / 2 4 8 9 ; > G J N N P Q R W X X X \ _ b c f g m o o o o q q q r r s x x x z | } ~ Ǎ ʍ ʍ ̍ ύ э ԍ ׍ ؍ ܍ ܍ ܍ ݍ ݍ ލ             ! ! ! ! ! ) + , / 1 1 3 4 4 8 9 = @ C D F F F H I I K N S W Y [ ] _ c c e h p ~  Î Ď Ȏ Ɏ Ɏ ʎ ˎ ̎ ͎ ݎ            # & ' ( + + + + + - - . . . . . / / / 0 1 1 3 3 3 3 3 3 3 4 5 5 6 7 7 7 9 9 < < < ? A B B B B C E E H H I J K K K K K L O O O O P S U V V X Z [ \ ] ` b c e f h m m r r t x y { | | | | }   Ï ď ŏ Ǐ ̏ ͏ ͏ ͏ Ϗ Џ Џ ҏ ҏ ԏ ԏ ׏ ۏ ܏ ܏ ޏ                 ! ! ! $ % % % % % ) + 0 1 3 4 4 5 : : = A B B C E F F K L L N Q S V V [ \ \ _ a e e f t w x x x y { { |  Ð Đ Ɛ Ȑ ː ̐ ͐ ϐ Ґ Ԑ ֐ א ܐ ܐ ܐ                    ! ! ! ! ! $ % ' ' * + , / / 2 2 3 3 4 6 9 9 9 9 9 9 9 < < < = > > > ? A C E F G G G I J K L L N N N O O Q S S T V V X Y Z _ ` ` c c c c c d d e h j o p q u w w z z z z z z | Ñ Ñ Ƒ ȑ ɑ ʑ ˑ ̑ ͑ ͑ ͑ ͑ Α Б Б Б Б ґ ֑ ֑ ֑ ֑ ڑ ڑ ܑ ݑ ޑ                                  " " # $ ' ( ( ( ( ( ( ( ( ( ( ( ( ( ) ) ) ) ) ) ) * * * * , , , , , , , , , , , - . / / / 1 3 5 5 5 5 5 6 6 6 6 6 6 6 6 7 7 7 9 ; = = = = = > > > > > > > ? @ @ A A D D D D D F I J K K K L L L L L L L M O Q R U V Z [ [ \ ] b e f h h k n n o p p p q q r t t v v v v w y z { { }  ƒ ˒ ˒ ϒ ђ ג ڒ ݒ ݒ ޒ   # ( ( ) ) + + - - 0 6 8 9 : : = = > ? ? ? @ A B B C D D D F F G N R R T V X ] a a c e e e e h i m p p t x { | | | Ó Ó Ɠ ͓ ϓ ϓ ϓ ѓ ѓ ѓ ֓ ד ד ٓ ܓ ޓ               ! " " ( ( ) ) ) ) * * - 0 1 2 2 2 3 3 5 5 5 6 7 8 9 9 9 : : ; @ @ A B C C C C C E F G H H H H K K L L M P P P Q Q Q R U U U U X X Y Y Z Z \ \ \ _ b b f f f f f j l l o p r v w |     Ĕ Ɣ Ɣ Ɣ ʔ Д Д Ԕ Ԕ ֔ ڔ ܔ ޔ ޔ ޔ              " " $ & ' * . / 0 4 6 6 7 9 ; ; ; ; ; ; ; < < > > ? A B B B C C C C C C C C C C D D D G H J L N O O O O R S T U U Z [ ] ^ _ a a b b b d f h j l l n q q q s t v v y  ƕ Ε ϕ ѕ ԕ ו ڕ ܕ ܕ ݕ                            ! $ $ % & & ( + , - / 0 0 4 9 : = ? ? A C J P R R S V W W X ] ` f l q r r r r r t x ~ ~ – Ė Ė Ŗ Ɩ Ɩ ǖ Ȗ Ȗ Ȗ ʖ ʖ ˖ Ζ Ζ Ж Ж і і Җ Ԗ Ԗ Ԗ ֖ ٖ ۖ ݖ ݖ ݖ ݖ ݖ ݖ ݖ ݖ ޖ ߖ                      ! " " " " " % ( * F J K P Q R R S U X p q r s t t v x x y ~ Ɨ ˗ ԗ                  # ( - . 1 7 9 H L L N P T T T T T T T T U V V V W W X X Y Y Y Z Z \ ] ] _ _ _ _ _ ` a c c c c c c c c d e e f h h h h i i i i i i i j j k k k m m m m o o o o o q r r r u v w { |   Ř ɘ Θ И ؘ ژ ۘ ܘ ޘ                                                                               ! ! " " & & & ' ' ' ' ' ( ( ( ( ( ( ( ) ) ) + + + + , , , - - - - - . . . . . . . . . / / / / / / / 0 0 0 0 0 0 0 1 1 1 2 2 2 2 2 3 3 4 5 5 5 6 7 7 8 9 9 = = ? A C C C C C C C C E E E E E F F G G I I I J J J J K L L L L M M M N P Q S T W W W W W W W W Y Y Z [ [ \ \ _ _ _ _ ` a b d d d d d e e e f g g h h i i j k o o p q q s u x x | Ù Ù Ù ę ř ƙ ƙ ƙ ƙ Ǚ Ǚ Ǚ Ǚ ș ə ʙ ˙ ˙ ̙ Й љ ֙ ڙ ڙ ۙ ܙ ܙ ݙ ݙ ޙ             " $ & * * / 0 4 7 8 B I O Y ` a c e g g i i k k m m m n q w ~ ~ ~  Ě ƚ ƚ Ț ˚ Ϛ Қ Ӛ Ԛ ՚ ՚ ՚ ՚ ֚ ؚ ۚ ܚ                      ! # # # & ) ) + , - / 0 0 1 1 1 2 2 2 4 = = = = ? ? @ A A A A A B B D D E E E E F G G I K L O Q T V W Y ] ^ b b g h i o p r v w { { { { ~ ~  Û ě ě ś ś ɛ ˛ ˛ ˛ ˛ ̛ Λ Λ Λ Λ Λ ϛ қ қ қ ӛ ԛ ԛ ֛ ֛ ٛ ٛ ٛ ۛ ݛ ޛ ߛ                   ! # % & ' ) + + + - - . / 0 2 8 : = > @ C D D F F H H N P U V Z \ _ c g i m n t z z  Ɯ Ϝ Ϝ ќ Ҝ ؜ ؜ ߜ                  ! ! " # # $ ) , - 0 4 9 E G G I M P Q Q R R R R R R R S S S S S T T T T U W W X X Y Y Y Y Z Z Z ^ ^ _ ` a a f h h j k p r r r t u u u u u u u v v v v v w x x x x y z z z { } ~ ~   Ý Ɲ ͝ ϝ ϝ ҝ ۝              ! " & ' ) + + . 0 1 3 6 7 9 = = = > ? ? ? D D G G G G H H H I J J K O R R W X \ e n s v v x } ~ ~ ~ ~ ~ ž Ȟ ў Ҟ ڞ ܞ ߞ             ( 6 : : ? G J L N Q S Y _ a c n s x { | } ß ş ɟ ˟ Ο ҟ ӟ ԟ ܟ ݟ ݟ ߟ               ! $ % % % * , - 4 4 7 7 : : : ; @ H J N N P R V Y Z _ b f o r t w       à à Ġ Ġ Ġ Ġ Ġ Š Ƞ Ƞ ɠ ɠ ʠ ʠ ʠ ʠ ͠ ͠ ͠ Π Р Р Ѡ Ҡ Ҡ Ԡ נ ؠ ڠ ܠ ޠ                            # $ ( , , 0 3 8 = @ A C E E E H I Q T T V Z ^ ^ _ _ d e j l n p p r s t v y ~ ܡ ݡ      ( 0 0 4 6 7 7 = ? ? ? @ @ A A A A A D D D G H I L L L O O P Q Q S S S T U V X \ ^ ` b d d e e f f g g o q q r r r r s t u } ~  Ƣ Ȣ Ȣ ɢ ˢ ˢ ̢ ͢ ΢ Ϣ Т Ѣ Ӣ Ӣ Ԣ Ԣ բ բ ע آ آ آ آ آ ٢ ٢ ٢ ٢ ٢ ٢ ٢ ٢ ڢ ڢ ۢ ܢ ݢ ݢ ޢ ޢ ޢ ޢ ߢ ߢ ߢ ߢ ߢ                                      ! " " " " $ $ ( ( ) ) ) ) ) * + , - - . / / 1 ; ; ; ; > > > @ @ C C D D F J J K N R S S S T T T W W W W W X X Y Y Z Z ] _ ` j k n n n o q r r u v v v w w x z { } £ ţ ǣ ͣ Σ ԣ ף ڣ            # & ) - 3 6 6 8 : : : ; ; ; ; ; < < = = = = = = = = = > > > > > > > ? ? ? ? ? ? @ @ @ @ @ @ @ @ @ @ A C D D D F G G G G G G G G G G G H I I I I I J J K K L M M M M M M N N P U U V V X Y \ _ _ a a a c c d d g l l l n n q v w y } ¤ ʤ ͤ Τ Τ Ӥ Ԥ ؤ ٤ ڤ        # % ' + + - . 0 1 3 8 : : > @ C D J L R Y ^ _ b e m o q w   ¥ å ʥ ͥ Υ Х ӥ ֥ ܥ          # & * + , 1 1 1 6 B D G H J L O U W ` g k p q x } æ Ŧ Ǧ Ȧ ˦ Ѧ Ҧ զ ٦ ܦ     ( . / 6 ? D N S \ f ʧ ˧ ͧ ͧ Χ ϧ ӧ ӧ ֧ ֧ ٧ ާ ߧ                                 " " " " " $ % & ' ' ( ) * * - 0 4 8 8 : > > @ D D D E H H J Q Q U U U V V V Z _ ` ` ` ` ` ` ` ` ` ` b b b b b b b c d d d g i i k m o v y z ~ è Ĩ Ĩ Ĩ ƨ ƨ ƨ Ǩ Ǩ Ǩ Ȩ ɨ ̨ ͨ Ϩ Ҩ ը ר ۨ ܨ ި ި                   $ ' ' ) + + 0 7 7 D E H J N [ a d n r x z { | | | | } ~ ~ ~ é é é é é é é é ĩ Ʃ Ʃ Ʃ Ʃ Ʃ Ʃ Ʃ Ʃ Ʃ Ʃ ǩ ǩ ɩ ɩ ɩ ʩ ʩ ˩ ˩ ˩ ͩ ͩ ͩ ͩ ͩ Ω ϩ ϩ Щ Щ Щ Щ Щ Щ ѩ ѩ ѩ ѩ ҩ թ թ թ թ ٩ ٩ ٩ ٩ ٩ ٩ ٩ ٩ ۩ ݩ ݩ ݩ ީ ީ ީ ߩ ߩ ߩ                                      ! ! & ( ( ) ) ) ) ) ) * * + , - - - - - - - - / / / / / / 0 0 0 0 0 0 0 1 1 1 2 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 6 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 : ; ; ; ; ; ; ; ; ; < < < < < < < < < < < = = ? A A A B B B B B B B B B C C C E G G H J K L M M O T V X Y ] ^ ^ ^ _ _ _ _ _ _ _ ` ` a b d f g k l n o o o o p q r r s s s s s s u u u u u u u u u u u u u v w w w x z { { }  Ϫ Ъ Ъ Ѫ Ѫ Ѫ Ѫ Ѫ Ҫ Ҫ Ҫ Ҫ Ӫ Ԫ Ԫ ٪ ڪ ۪ ۪ ܪ ݪ ݪ ݪ ݪ ݪ ު ު ު ު ު ު ު ު ު ު ߪ ߪ                                                   ! ! " # $ $ $ % % & ' ' ) ) ) * + + + - - - - - - - - - - - - . 0 1 1 3 4 4 4 4 4 4 4 6 7 7 7 7 7 7 7 > > > > > > > > > ? ? ? ? ? ? ? ? @ @ @ A A A A A A B B B D D H H I I I I I I K K K N N N N N N N N N N N N N N N N N N N N N N O O O O Q Q Q R R T V W W X X Y [ \ \ \ \ \ \ \ ] ] ] ] ] ] ] ^ ^ _ _ _ _ _ _ _ _ _ _ _ a b b d e e f g g h h j j j j j j m n o q u u u u u v x x x x y y z { | ~ ~ ~ ~ ~ ~ ~ ~     « « « ū ū ƫ ǫ ǫ ǫ ǫ ɫ ɫ ʫ ʫ ʫ ʫ ʫ ˫ ̫ ̫ ̫ ̫ ̫ ̫ ̫ ̫ ͫ Ϋ Ϋ Ϋ ϫ ѫ ҫ ҫ ӫ ӫ ӫ ԫ ֫ ګ ګ ۫ ݫ ޫ ޫ ߫ ߫            ! " % & & ' ( ( , , , - . 1 3 3 3 4 6 6 6 6 6 6 6 6 7 7 7 7 7 8 9 9 ; = ? A A B D E E G N N N N P P P Q R R R R R R S T T W W W W X Z Z Z Z Z Z [ [ ` ` a a c d e e e e g h i j k k m m m m m o q q r r r s v w w w y z | | | } Ĭ Ĭ Ĭ Ĭ Ŭ Ŭ Ƭ Ƭ Ƭ Ǭ Ǭ Ǭ Ǭ ɬ ɬ ɬ ʬ ͬ ͬ ͬ ά Ь Ҭ Ӭ լ ׬ ۬ ܬ ߬                         ! ! $ $ ( ( ( ( ( ( ( ( ( ) ) ) ) ) ) ) * * * * * + + + , , , , , , , , , , , , , , , - - - - / / / / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 2 2 2 2 4 4 4 7 7 7 8 8 8 9 < ? @ H I J J J J J J L P P T T X X Z [ [ _ b h u w w y { | | | | | | ~ ~ ­ ­ ­ í ĭ ĭ ŭ ŭ ŭ ŭ ŭ ƭ ƭ ƭ ƭ ƭ ƭ ȭ ȭ ȭ ȭ ȭ ȭ ȭ ɭ ɭ ɭ ̭ ̭ ̭ ͭ Э ѭ ѭ ѭ ѭ խ ׭ ۭ ۭ ޭ ޭ                               " " # % ( ) * * * + , - / 1 1 5 5 5 7 7 7 8 9 9 : = @ D D F G H J J N W Y ] ] ] ` a a a b e e h k k o q t u w x z { ® ® Ů Ů Ů Ǯ ͮ ͮ ή ή ή Ю Ю Ю Ѯ Ү Ӯ Ӯ Ӯ Ӯ ծ ծ خ ٮ ڮ ڮ ڮ ڮ ڮ ڮ ڮ ۮ ۮ ۮ ܮ ܮ ޮ ޮ ޮ                                                     ! ! ! " % ' ' ( ) . . / 2 3 4 4 5 7 7 7 7 7 7 9 9 9 ; < ? ? @ A A E F G H J L L L L L L M O O O Q Q R U U V V V W X Y Y Y Y Y Y Y Z Z [ [ \ \ \ \ \ \ \ ] ] ^ ^ ^ a a a a a a b b c c c c d d e e e e e f f f f h k k k k k k m m m n p p q q s u y | } ǯ ȯ ȯ ʯ ʯ ˯ ѯ ԯ 6 8 8 8 8 8 : : : : : : ? A A A A A B B B C C C C C C C C E E F O O O Q Q S V W Z \ \ \ \ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ _ _ _ _ _ ` ` ` ` ` ` ` ` ` ` ` ` ` ` a b d d d d d d d d d d d d e e e e e e e e e e e e e e e e e e e e e g h h h i j k k k k m m m m n u v x y y | | | | | } ~ ° ° İ ư ǰ ǰ ɰ ɰ ɰ ˰ ˰ ˰ ˰ ˰ ˰ ̰ Ͱ Ͱ Ͱ Ͱ ϰ а Ѱ Ұ Ӱ հ հ հ װ ٰ ڰ ڰ ڰ ڰ ۰ ܰ ܰ ܰ ݰ ݰ ݰ ݰ ݰ ݰ ް ް ް M P R T U V V V V W W Z ] ^ ^ ^ _ _ _ _ _ ` ` a b c c c d e g j j j m n n o q r v w w y z z | |  ± ± ñ ɱ ̱ ̱ ͱ ͱ ͱ ͱ ϱ б б б ұ ر ۱ ܱ ޱ                       " # % & ' ' ' ' ( ) ) . . . 1 2 4 5 5 6 8 8 8 8 8 8 : : : : : : : : : < < < < < < < = = = = > > > > > @ A C D D D D D D E F G G G H J K K L N N N N O O R T T T X Y Y Y Y Z Z Z [ ] ^ a a a c d f f h t u x z z } } } } } } ~  IJ Ų ϲ в в Ѳ Ӳ Ӳ Ӳ ղ ղ ײ ٲ ٲ ٲ ٲ ߲ ߲              ! $ $ $ + + + + . 2 4 4 5 5 7 8 : = = @ A C D D F F F H H H H H K L L O O O O O O O O O P Q S T U U U V W W W W X [ \ \ ^ _ _ _ a d d d e f f f f f i i j j k k o o o q q t t t u u u u u v x x x x x x y y y y y z z z z z z z { { | } } ³ ³ ³ ³ ij ij ų ų ų ų ų ų ų Ƴ Ƴ Ƴ ȳ ȳ ȳ ȳ γ γ ѳ ӳ Գ ճ ֳ ׳ ׳ ٳ ڳ ڳ ܳ                        ! ! ! $ % & ' ' ( + + - . 1 3 4 5 5 5 7 7 8 < < @ A B C D G H H I J K O O Q R U V X X Z Z \ ^ _ e h l m m t t t t v v z |   ô Ĵ ʴ δ д Ѵ Ҵ Ҵ Ҵ Ӵ Ӵ մ ڴ ޴             " ' ' , / 0 2 2 3 4 4 4 5 8 8 : = ? ? ? B B C C C C E E E F F F G J J J J J O O Q R S T T U W W Y Y Y \ \ ] ] ] ] a a a b c e f f g i i k k k k l o p r t u u u v w w x x y z { { | ~ ~ ~ ~ ~ ~ ~ ĵ ĵ ĵ ǵ ǵ ǵ ǵ ɵ ɵ ͵ ε ҵ ӵ Ե Ե ֵ ׵ ׵ ׵ ׵ ۵ ݵ ݵ ݵ ߵ ߵ                     ? X ] _ b d d d d f h h i i k k o s s s t u u v w x x x y y y y { } ~ ~ ¶ ȶ ȶ ɶ Ӷ ׶ ׶ ض ض      % ( + 0 = = > > > > > > I I M M O Q V W X X [ \ \ ^ ^ a a a a a b c e h j j l m m m m n o p v w w x y { | | · Ʒ ȷ ɷ η ѷ ӷ ӷ ۷       ! $ % & & ( * , , , - / / : A A B D D D D H J L P ^ b g q Ǹ ͸ ׸    # # & & ( * * * * * * * - / 0 2 3 8 ; < ? @ @ A E F H H Q S T \ h k m r v v v w x y | ~ ù ù ù ù Ĺ Ĺ Ĺ ɹ ʹ ʹ ʹ ʹ ̹ ̹ ̹ ̹ ̹ ͹ ͹ ͹ ͹ ͹ ͹ ͹ ͹ ι ι й ѹ ҹ ֹ ׹ ݹ ޹                             " & & & ( ( * , , , , - . 5 6 8 > B D D G K L P R T Y Z a c d d f f f f f f f f f g g i i i i k m m m m o p p p p s s t v } º ú ĺ ĺ ĺ ĺ ĺ ĺ ĺ ź ź ź ƺ ƺ ƺ ɺ ɺ ɺ ɺ ˺ ˺ ̺ к Ѻ Ѻ Ѻ Ժ պ ֺ ֺ ں ں ۺ ۺ ܺ ܺ ܺ ܺ ܺ ݺ ݺ ݺ ޺                                      ! ! " " $ & ' ( ( ( ) * , , - . 0 2 5 8 : < < > > > A A B B B B B B I K L L N P P P P P P P S S T T W X Z Z [ \ \ \ ] ] ] ] ] ] ] _ a a b d d d d e f f i j k k k k k l l l m n n n n n o q t w w z z { { { | ~ ~ ~ ~ ~ ~ ~ ~ ~ ʻ ͻ ͻ ͻ λ λ λ λ ӻ ջ ջ ֻ ٻ ܻ ܻ ݻ ݻ ߻                                             $ % % % ( * , , 0 4 5 7 < < < = @ A F F G G G H I J O O O O P P P P P P Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q Q R R R R S X Y Y Y Z Z Z Z [ [ ] ^ _ ` i i l n p p p p p p q q s s s s t t v w w w w w w w w w x y y y { | } ļ ż Ǽ ȼ ȼ ȼ ȼ ȼ ɼ ˼ ˼ ˼ ̼ ̼ ̼ ̼ ͼ μ ϼ м м м Ѽ Ѽ Ҽ ռ ռ ؼ ؼ ؼ                                                                         ! ! " # $ $ % % % % % % % ( ( ) ) ) * - - - - / / 0 1 1 2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 5 5 6 8 8 9 9 9 9 9 : : : ; ; ; ; ; ; < < < @ A A B D E F H H H I I I I J J J J J J K K L L M M N O O O O O O Q S S S T V V V V V V V W W W W W W Z Z Z \ \ ] ] ] ] _ _ _ _ ` a b e e e g h h h h h i i i k l l l m o p p q q q q r r r r r r r r r r r r t v v v v w { } } ~ ~ ~ ~ ~ ~ ~ ~ ý Ž ƽ ǽ ɽ ɽ ɽ ɽ ɽ ɽ ɽ ɽ ɽ ɽ ɽ ɽ ɽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ʽ ͽ ͽ ͽ ͽ ν ν ν ν ν ν Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ Ͻ н н н н н н н н н н н н н н н н н ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ѽ ҽ ҽ ҽ ҽ ҽ ҽ ҽ ҽ ҽ ҽ ҽ ҽ ҽ ҽ ӽ ӽ ӽ ӽ ӽ ӽ Խ Խ Խ ս ս ս ս ս ս ս ս ս ս ս ս ֽ ֽ ֽ ֽ ֽ ؽ ؽ ؽ ؽ ؽ ؽ ؽ ؽ ؽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ڽ ۽ ۽ ۽ ۽ ۽ ۽ ܽ ݽ ݽ ݽ ݽ ݽ ݽ ݽ ݽ ݽ ޽ ޽ ޽ ߽ ߽ ߽ ߽ ߽ ߽ ߽ ߽ ߽                                                        % % % & & & ' ' ' ' ' ' ' ' ' ( - - . . . . . / / / / / / / 0 0 1 1 1 1 1 3 3 5 5 5 6 7 7 7 7 7 7 8 9 : : < < < < < < < < = > > > > @ B C F G H H J J J J J K K K K K K M M M M M M M M O P P Q Q R S S S T U U U Y Y Y Z Z ^ ^ _ b h h k m n n n r u v v v v v w z { | | | | | | ~ ~ ~ ~ ~ ~ ¾ þ ľ ľ ž ž ž ž ž ž ž Ǿ Ⱦ Ⱦ ɾ ɾ ʾ ʾ ; ; Ͼ Ͼ Ѿ Ѿ Ѿ Ҿ Ҿ Ҿ Ҿ Ӿ Ӿ Ӿ Ӿ Ծ վ վ վ վ վ ھ ھ ھ ھ ܾ ܾ ܾ ݾ ݾ ݾ ޾ ޾ ޾ ޾ ޾ ޾ ޾ ޾                                                               ! % % % % % % % % % % % % % % % % & & & & ' ' ' ) ) ) ) ) + + + + + + + + + + + + + + + + + + + - - . . . . . . . . . . . . . . . . / 0 0 0 0 0 2 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 6 6 6 6 6 6 6 7 9 9 : : < < < > > > > > > > > ? ? ? A A B B B B B C C C C F G H H H I J L N N N N N N N N N O O P P P P S S S U V V W W X X X Z Z \ \ \ \ \ \ ^ ^ ^ ^ ^ _ _ ` ` a b b b b c c d d e g g g h h h h h h h i i i i i i i j k m m o o o o p p p p p r s t t u u v v v x { { | } } ~ ~     ¿ ¿ ¿ ÿ ÿ ÿ ÿ ÿ ÿ ÿ Ŀ Ŀ Ŀ ſ ſ ſ ſ ſ ſ ſ ƿ ƿ ƿ ƿ ȿ ɿ ɿ ˿ ̿ ο Ͽ Ͽ п п п п п ѿ ѿ ѿ ѿ ѿ ѿ ҿ ҿ ӿ ӿ ӿ ֿ ֿ ֿ ֿ ֿ ׿ ׿ ٿ ڿ ڿ ڿ ڿ ڿ ڿ ڿ ڿ ڿ ڿ ڿ ڿ ڿ ۿ ۿ ۿ ۿ ۿ ۿ ܿ ܿ ܿ ܿ ܿ ܿ ܿ ޿ ޿ ޿ ߿                                         " " $ $ $ $ $ $ % % % ) * * , / 0 0 1 4 4 4 4 6 6 7 ; ; ? ? B D D D E E G G G K K M M N N N N N N N O O O O O O O Q R S S S S S U U U U V V V V V V V Z Z Z Z Z Z [ \ \ ] ] ] ` ` ` ` ` ` ` ` ` ` ` ` d e e g h h h i i i i j j j j j j j k k k k l l l l l l l l l l l l l l l l l l l m o p q q q q q s s s t u u x | | | }                                                                                                                                               " " " " # # $ & & & & ( ( , . . . / / / 0 0 2 3 3 4 4 5 6 8 8 8 8 8 8 9 : : : < < < < < < < < = A A B B B B C C D D F F F F I J J L L L L L L L L L L L L M N O P P Q Q Q Q Q S S T U X X X Y Z Z ^ ^ ^ ^ ^ ^ _ a a b c c c c c d d e f f f h h i i j k k p r s s t u w x x y y y y y { { } } } } } } } } } } } } } } } } } } } } } ~ ~                                    ! # ' ' ' ( ( ) ) ) ) ) ) ) * , - - / / / / 0 0 1 1 2 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 6 : ; ; < ? ? @ A E G G M N N P P S T V V W X Z Z [ [ [ [ \ \ \ ^ ^ ^ ^ ` ` ` ` a c c c c c c d d d e e f f f f h h h h j j k l o o p p p p p p q r v v y z | } } } ~ ~ ~                                       $ $ $ % % % % % & & ' ( ( ) * * * + . . . / 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 5 5 5 5 5 5 5 6 7 7 9 : = = > > ? ? ? @ B B B B E E E E F G G I I K K M M M M N O O O P Q R S U U V V V X Y Y Y Y Y Y Y Y [ [ \ ] _ _ _ ` a a b b b d d e e e f f f f f f f f f f f f f g g g h h j j j j j j j k k k l m n n n o o o o o o o o p p p p p q q s t t t t u u u u u w w w w w x y z z z z z z { | | | | ~                                      ! " " " # $ $ % % % % % % % % % % & & & & & & & & & & ( ) ) , , - . . . . . 0 1 1 1 2 4 4 4 4 5 5 5 : : : ; < = R T ! " ' ) ) * * + , , . / / / 2 2 3 3 4 4 6 7 ; ; ; ; < ? E F | | } }                                         ! ! ! ! ! # $ $ $ $ $ $ % % % % ' ' ' ( ( ( * * - . 0 1 1 2 3 3 4 4 5 5 5 7 8 8 : < > @ C D E E F I I I I J J J J J J K M M M M M O O O O O O Q R R R S S T X Y Y ] ^ a a a e e e e i m p p q q s s s s s t t t t t u u u v v v v v v w w w w w w w w w z { | | | |                                  ! # # % & & ) * + + + + + , , - - / / / / / 0 0 1 3 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 7 8 8 9 9 : : : ; ; ; ; ; ; ; ; ; ; ; ; < < < < < < < < < < < < < = = = = = = = = = = = = = = = = = > > > > > > > > ? ? ? ? ? ? ? ? @ @ @ @ @ @ @ @ @ @ @ A A B B B B B B B B B B C C C D E F G G H I I I I I I J L L M M O O R R R S X X Y Y Z [ [ [ [ [ [ [ [ [ [ \ \ ] ] ^ ^ ^ ^ ^ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ` ` ` ` ` ` ` ` ` ` ` a a a a a a a a a a a a a a b b b b b b b b b f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f f g g g g g g g g g g g h h h i i i i i i i i i i i i i i i i i i i j j k k k k k k k l m m m m m m n n n n n n n n n n n n n o o o o o o o o o o o o o o o o o o o o p p p p p p p p q q q q q q q q q q q q q r r r r r r r r s s s s s s s s s s s s s s s s t t t t t t t t t t t t t t t t u u v v v v v v v v w x x x x y y y y { { { { { | } } } } } ~ ~                                                                                                                                                                                                                                                                                       ! ! ! ! ! ! ! ! ! ! ! ! ! ! " " " " " " " " " # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # $ $ % % ( ( ( ( ( ( ( ( ( ( ( ( ( ) ) ) ) ) ) ) ) ) ) ) * * * * * * * * . . . . . . . / / 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 8 8 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 : : : : ; ; ; ; ; ; < < < < < < < < < < = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = = ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? @ @ @ @ @ @ @ @ @ A A A A A A A A A A A A A B B B C C C C C C C C C C C C C C C C C D D D E E G G G G G G G G H H H H H H I I I I I J J J J J L L L L L L L L L L L L L L L L L L L L L L M M M M M M N O O O O O O O O O O O O O O O O O O O O O O P P P P Q Q Q Q R R R R R R R R R R R S S Y Z Z Z [ [ [ [ [ [ \ \ ] ^ ^ ^ _ _ _ _ a a a a b b b b b b c f g g g g h i i i i i i i i i i i j j k k k l l l l l l l l l l m n n p p r r r r r r s s s t v w x x x x x x x x x x x x x x x x x x x x x x x y y y y y y y y y y z z z z z z z z z { ~ ~                                     ! ! ! # # $ $ $ ' ) ) ) * * * , , , - . . 0 1 1 2 5 6 6 7 7 7 8 8 9 : : : < < < < < < < = = = > ? ? ? A C D H I J K K K L M O O O P Q Q T U U U U V W W W W W W W W X \ \ \ \ \ \ ^ ^ ^ _ _ _ ` ` ` ` a a c c c c c c c d e e e f f f g g g h i i i i i j l l l l m n n n n p p p s s s t t t t t u u v w x y | | ~ ~ ~ ~                                             ! ! ! % % & * * * * , , - - . / 0 0 3 4 4 4 5 7 7 7 : : : : ; ; ; ; > > > > > > @ @ @ @ A A A C E F I K M Q Q Q R R R R T T V Y [ \ ] _ _ ` a c f f g g h m m n n o p s u u u w x x x x x y y y z { { } } ~                                                        " " # # # # # # $ $ % % & & & ' ' ' ' ' ' * + + + + + + + + + + + + , , , , , , , , , , , , , , , . . . . / / 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 5 5 5 5 5 5 5 5 5 5 6 6 7 7 7 7 7 8 8 8 8 8 9 ; ; ; < = = > ? ? ? @ @ @ @ A A A A A A B C E E F G I I I I I I K K L M M M S S ` ` d d d e e e f f f f f g g g k k l m m n p q s t t t u x y y z z z z z z | |                                                                                                    ! ! ! " " $ & ' ( ( ( ) ) ) ) ) ) * , , - - - - - - - 1 1 1 1 1 1 1 2 4 5 6 7 8 9 : : < < > @ @ @ @ B B D E F F F H H I I J L L L M N N O O O P P P P P P P P P P P S S S T T T U U U U Z Z [ \ \ \ ] ^ ^ _ _ _ _ _ _ _ _ c c n n n n o o o o q s t u u w w x x x { | | | | } } } } } } } } } ~                    D n p                                                          $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ % & & & & & & & & & & & & & & * + W W W W W W W W W W W W W W W W W W W W W W X X X X Y Y Z Z Z Z Z Z Z Z Z Z [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ \ \ \ \ \ \ \ \ \ ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ] ^ ^ ` ` b c c c c d e e e e e e e g g g g g g g g h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h h i i j t t t t u v v w x y y y y y y y y y y y y y y y y y y y y y z z z z z z } } } } } } } } } } } } } }        / / 1 1 1 1 1 1 1 1 1 1 1 1 1 2 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 8 8 8 8 : : : < > ? @ @ @ @ @ @ @ @ A B B B C C C C C D D D F F H I J J J J J J K K L L M M N O O Q Q Q R R S T U U W Y [ [ \ ] ] ] ] ^ _ ` ` ` a a a a a a a a b e g g g g g g g g h h j j j j k l l l l n n n n n n n n n n p p r s t t t t t u u u w x x { { | | | } ~                                                                                                                      ! ! " " " # # # # # $ $ % % & ' ( ( ( - . 2 3 3 3 4 4 5 5 5 5 6 6 6 7 7 7 9 : : : : : ; ; = A C E E F G H I J K K L M M M N N N N N O O O O O O O O O P P P Q T T T U V [ ] ] ] ] ^ _ ` ` ` a c c c j k k l m m n n o o o p p p p p p r r t u u u u v v w z z | | | | | } } } } } ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~                                                              # # # $ % % & ( ( ( ( ) - - - - 0 0 1 1 1 1 2 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 7 8 8 8 : : ; < < < < = = = ? ? ? @ @ B B D D E E E E F F F F F F I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I J J L L L Q Q Q V X X X X                                                                        " $ $ $ & ' ' ' ' ' ' ' ' ' ' ' ( ( ) ) ) ) + + , , , , , - . 0 1 2 2 2 2 3 4 4 4 6 7 7 9 9 9 9 : : : ; ; < > > > ? ? A B C C C C D D E E F F F F F F G H H H H H H H I J K K K K K K K K K K L M M M M M M N N N N N O O O O O O P P Q Q Q Q Q Q Q Q Q R R R R S S S S S S T T T T T T T T T T T T T T T U U U U U U U U U U U U V V V V V V V W W Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Z Z Z [ [ [ [ [ [ ] _ ` a a a a a a a a a a d d d g g g g g j j j j j j j j j j j j k k k k k m m n n n n n p q q r s s s s s t t t v x y | | } }                                                                                   ! ! ! ! ! ! " ' ' ' ' ( ( ( ( ) ) + + + + + + + , , , , , , , , , , , - - - - . . . . . . . / / 0 0 1 1 1 1 1 2 2 5 6 8 8 8 9 9 : : : : : ; ; ; ; ; ; ; < < = = = > > > A B B B B B B B C C E G G G H I J M M N N N N N N P S T T T T U U U U V V V V V V W W Z [ [ [ ] ^ ^ ^ c c d d f f h h h j k l p r s s s v w x x } ~                                              ! ! " # # $ % + + + + 5 6 6 6 8 8 8 8 8 9 9 9 9 9 : ; < < < < < < < = = = = = = = = = C F G K K K K K L L M M O R R V V W Y [ \ \ \ \ \ \ \ \ \ \ ^ a b b c c c c d d d d d d d e e e e e e e e e e e e e e e e e e e f f f f f f f f f g h h j j l l l l l o o o p p p p p p p p p p p p p p p p p p p p p p p q q q q r r r r r r r r r r r r t u u u u u u u u u u v v v v v v v v v v w y z z z z z z z { { { | | |                                                            $ % ' * - - - / 0 0 1 2 I I L P h h i l l m m m m n n n q r r s s s s s s s s t u v v v x x x z       K L L b b c c d g g g g g g g g k k m o o o p q r r r r r r r r r r r u u u u u u u u v v v v v v v v v v w w w x x x y } ~ ~ ~                                           $ $ % % ' ( ( ( ) ) ) ) * * * * + - - - 1 1 1 1 1 1 1 1 1 1 2 2 2 2 3 3 3 4 4 4 5 5 5 5 5 6 6 9 < = = > > > > > > > > > > > > > @ @ @ @ @ A A A B C D D D F F H H H J J L O R R R R R R R R R R S S S S S S S S S T T T T U U U U U W W Z [ [ [ [ [ [ [ [ \ \ ^ _ c c c f f g g i i i i j j l l m o o o p s s s x x x y y | } } } } } } } ~ ~ ~ ~ ~ ~ ~ ~ ~                                                                                                                                                                          " " " " # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # # $ $ $ $ % ' ' ' ' ( ( ) ) * * * * + + + , - - - . / / / / / / / / / / / / / / / 0 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 4 5 5 5 5 5 5 5 5 5 5 8 9 < < = = > ? ? @ A A B B D H K K K N N N N P P P P P Q Q Q Q Q Q S U V V V V V V V V W W W X Y Y Y Y Y Y Z [ [ [ \ \ \ ] _ a b b b c g i i k k l m m m n p p q s s s s s s s u u v v v v v v v v v v v v v w w z { { } } } ~                                                               ! ! " # # # ) , , , - - . . . . . . . . 6 8 8 ; ; ; ; ; ; ; ; < < < < < < < = = = = = > > ? @ @ @ @ @ B B B B C D D D G G G G H H H H H H H I I I J J K K L L L L L L L L L L L L L L M M N N N N N N N N N N N O O P P P P P P Q S T T T T T T T U U U U U U U U V V W X X X Y Y Z [ \ \ \ \ ] ^ ` b c c c d g g g g g g g g g g h h h n o o o o o o o o o o o o o o o o o o o p q q q q r r s s t t v x x z | } } } } } } } } } } } } } } } ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~                                ! ! ! ! ! ! " $ & & ( + + , , , , , , , , , , , , - - - - - - . . . . . . . . . . . 1 3 5 6 6 7 7 7 7 7 7 7 9 < = = ? @ @ @ @ @ @ @ @ @ @ @ A B B C C D E I I K M M M M N O O O P T U U U V V V Y Y Z Z Z Z Z Z Z Z Z Z [ [ [ [ [ \ \ \ ] ] ^ ^ ^ ^ ^ ^ a f f f j j j j j j j k l p q q u v v v w x y z } ~                            ! " # $ $ $ $ $ $ $ $ $ $ $ $ $ $ $ % & ' ' ' ' ' ' ' ' ( * + , . . 1 1 1 1 1 1 1 1 1 1 8 8 8 ; ; > > > > > > > > > > > > > > @ A A A B B B B B B B B B B B B B B C C E E E E E E E E E F H H M Q Q Q Q R R R R S S T U V V V V X X X Y Y Y Y [ \ \ \ \ \ \ \ \ ] ] ` ` a a a a a a a a a a a a a c e f f f f f f h h h h i i k l n n p p q q q r s s s x x x                          ! ! ! ! ! " # # # # # # # # # # # # # # # # # # # # # $ ( . 0 1 3 3 4 7 : : ; ; A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A B B B B B B B B B B B B B B B B B B B B B B C C C C C C C C C C C C C C D D E E F F F F F H H I I I I I I I I I I I I J J L L L N N O Q R R R W Y Z \ \ \ \ ^ ^ _ _ b b c e f h k k l m m m t v v w z z { { }                        ! # # % ' ( * * * * * * + + , , . 1 @ @ B B C D D D E E E F J J J N N N P P Q R R S S S S S T T V W W W Y Z Z \ \ \ ` ` a b c c c e f j l p r t t t u u v {                      ! & & & & / 0 0 0 3 4 4 4 4 7 8 8 8 ; ; ; < < > ? ? B B B B B B B B B B B C F F F F G H I J L L M N O P Q R S U W [ \ ] ] ] ^ ^ _ c c c d j n n u y z { {       # * + , 0 < @ I c f g h l m n n p s u u v x y y z | | } } ~                                        ! % ) ) ) * 0 2 2 3 5 5 5 6 7 8 : = > > ? @ D D E J J J K K K K L L L N P P P R T V V V V V V V V W W W W W W X X Y Z Z Z Z \ \ ^ ^ ^ _ _ _ _ _ ` ` a a a a a b d g g h i j k k m n p p q r s t t t t t u v v v x x x x y y y { { { | | | | | | | | | | | | | | | | | | | | } } } ~    ! ! ! ! " & & ' ' ' ( ( + + , , . . / 0 1 1 1 4 4 4 4 4 4 9 9 9 : > ? ? ? ? @ C C C D F j j j j l l l n t v w w { | | }            * . . / 0 2 2 3 4 4 6 6 7 9 9 < < ? ? ? @ C C D F F G H H M M M P Q R R R S S S S S S T T V V V V V V W W W W W X Z Z Z Z \ \ \ \ \ \ ] ] ] ] ] ] ] ] ] ] ] ] ] c d e e f f f f f h j j j j j j k k l m n n n n o o o o q r r u v v y y z z z z z { { | } } } } ~ ~ ~ ~ ~ ~ ~                                        " $ ) ) + 0 1 1 4 5 7 9 @ C D D D D D D D D H I J J J J K L L M N O P P P Q Q R S T V V V V X X X X Z ] ] _ a d d g g h i k l m m o o q s s s t y { |                 ! " " " " ) * * + 5 5 : :                       " " $ ' ( + + , . . / 0 2 3 5 6 6 6 7 > ? ? B C F F F F G I J K K L M N N P Q Q Q R R S S V V V V V V W W W W W W W W W W W W W W Y Y Y Y Y Y Y Y Y Y Z Z Z [ \ \ \ \ \ \ _ _ _ _ _ _ _ _ _ _ _ ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` a a a a a a a c c d d e e f f f f                                    & ) * * * + + - - . 1 6 9 < = ? A A D D P Q R V V Z Z Z Z Z [ [ \ \ \ \ \ ^ _ ` ` b b d g h h h i i j k l o o p p q r r s | | |                                               " " $ ' ' * * , , - . 0 2 3 3 3 5 6 8 8 9 : : > A C G G G G G I I I I K K N N P R R S T W Z [ \ ^ _ a a b c d g h h l l m n q q q s s t t v w w w w w w w x y y | } } ~ ~                                   " 2 2 3 3 6 @ C E H J L L M M M M M M M M N O O O O O O O O O P P Q Q S S S S S S S U U U U U U W W W W W X X X X X X X X X X X X X X X X X X X X X Y Z [ \ ] ] ^ _ _ a b b d d e e f f h i k l o s s w w y z z z { }                           " " " " " # $ $ % % ) ) ) * - - - - / / / 0 0 0 0 0 0 0 2 2 3 6 7 7 7 8 9 ; < < > > ? ? B F G K L L N P U V V V V X Y Y Z ^ ^ a c c g g g g g g i p t v w x x z { { ~ ~                                    # ' ) ) , , , , , , 1 2 3 3 4 8 9 9 : : : < ? B D D D D D D E F G H H J J N O O Q S X ] a a c e e f g h i j p p q q r s u v v w y y y { | } } } ~ ~                                                    ! ! ! " " " " " # # # # $ % & ' ) , - / 1 2 3 3 4 4 4 4 4 4 4 5 5 5 7 ; ; > @ @ A A B B B D D E H I I J K K L L L N O P P Q Q T V V W W Y Z [ [ [ \ \ \ a a b c f g g h i m o p u v y z z { ~                             " " " $ ' ' ( ( * , - . . / 0 0 2 2 2 2 7 8 8 : ; ? ? ? ? @ @ @ @ @ @ @ @ A B B D D D F H H H J Q R S S U U V V W W Y \ ] ] ] ] ^ ^ ^ _ _ _ _ _ _ _ _ _ _ _ _ _ ` ` a a a b b c c c d g i i i i k l m m n p p p r r s s t t t u v y y y { | | | | } ~                                      $ $ ) * + - - - . 0 ; ; < > @ @ A A A A A A A A B B E E E E E E E E E E E E F F F F G H J K K L L L L L N P P R S Y _ _ _ ` ` ` a a a d d f f i m n o q q q q r s s s s s s s s s s u v v v v w w w x x x y z z z { { ~ ~ ~                                 # # % ( - / 2 3 4 4 4 6 8 8 9 : F G G H H I K L L L L L M M N O P P Q R R R S U W W W X X Y Y [ [ \ \ \ \ ^ ` a a a a a a a b c d d d d d d d d d d d d d d d e f f g h h i i j k l l m o q q q q q s t u u w x x x x y z z z z { | } ~                    " " # # % ' ( ( + . . 0 3 5 : : = > ? C E E I L O O P T T V Y Y Y Y Z Z [ [ \ ] ^ ^ ^ ^ ^ ^ ^ ^ _ _ _ _ _ _ _ _ _ ` ` a a d i j m n p q r r u w { { { { ~ ~                           ! ! " " $ % + - 0 5 5 6 < < = ? A C E L N N O O O Q Q S U i k k k k m n n n n n o o o o o p p q } }                         ! $ $ % % % ( ( ( ) * + 0 2 2 6 8 ; ; ; < = = = ? ? @ B B B D F G G G I K L L L M N P P P P P R S S S S S S T T T T T T T T V V W W W X X Y Y Y Z [ \ \ \ \ ] ^ ^ _ _ _ _ _ ` ` a b j j j j j j k l l l n p r s t u v w w y y y y z | | | ~                     ! ' ) 1 4 5 7 < = > > A C G J O O P R [ [ ] ^ c d g g h h h h h h h h h h j k m p u u v w x z |              # & * * , , - / 6 9 ; A C P P P R S S T W [ ^ a e g k q q y }               " ' ' , - - / / / 1 3 6 7 7 8 8 9 C C M Q T \ a b e i k n o o p q s s w w y {                        ! " ' / / 1 4 7 : ; ; ; > > > @ A B E J J M M M N O O S S T T T T T T T T T T U U U U U U U U U U V V W W X X X X X X X X Y Y Y Y Y Z [ [ [ [ [ ] ] _ _ ` ` ` ` ` ` ` ` ` ` ` a b c c c c c c d f f f f f f h i j j k k k k k k m m n p q r r s s s t t v v v w w w w x x y y y z { | | | | | | | | | | | } ~             # $ $ $ $ $ % & & & & ' ' ' ' ' ( ( + , , - 1 3 3 5 9 = A A C F J M O Q S T T Y Y Y [ b f h i i k s s u x y z                  " $ ( ( * + , - . 5 : = C D D D D D D D D D D D D D D D D D E E E E E E E E E E E E E E E E E E E F G G G G H H H H H J J J J J J K K K K K K K K L L L L L L L L L L L L L L L L L L L L L L L L M M M M M M M M M M M M M M M M M M M M M M N N N N N N N N N N O O O O O O O O P P P P Q Q Q Q R R R S T T U U U U U U U U U U W W W W X X X X X X X X X Y Z [ \ \ \ \ ] _ ` ` ` ` ` ` ` ` ` ` ` ` ` ` ` b b b b d d d d d d d d d d d d d d d d d d e e e f f f f f f f f f f f f f f f f f g h i i i i i j k k k k l l l l l l m m m m m m m m m n o o o o o o o o o o o p p p r r r r r r r r s s s s s s s s s s s s s s s s s s s s s s s t u u u u u u v v v v v v v v v w w w x y y y y y z z | ~ ~ ~ ~ ~ ~ ~ ~ ~ ~                                                                    " " # # # # # # # # # & & & & & ' ' ' ( ( * + / 1 2 2 2 2 3 3 3 6 7 9 9 9 < = = ? ? A A A C C C C D F F F F H H H H H J L L M M M O Q Q Q Q Q Q Q Q Q S S T T T T T T T T T T T T T T T T T T T T T T T T V V V V X X Z Z [ [ [ ] ] _ ` ` ` a a b c e f f g g g i l l m m n n n n n o o p r u u x z } } }                   " % ' ( + - 0 1 5 = ? C H I O O O P P Q W ^ ` ` ` b b b d d d d e e e e e f f f f j j o v w y {             ! $ $ % % % % ' ) - . 0 0 1 5 5 9 > > > > C C I I J M N N R R X ] _ c e f h i j m u { ~               # & , . 0 2 6 9 : : = ? F J M M P R T T V X Y ` f g o q r r s t x }                  ! " % * + 3 3 6 = > ? C G I J K K K N R S W W X Y \ ] a b e f h h j j j k l l m m m n n o p p p q r r s s u u u u u u w x x x x z z | | | | } ~ ~ ~ ~ ~ ~                      ! 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rs7511235 rs187454438 rs685627 rs115803514 rs144972331 rs853125 rs138298773 rs114688043 rs56075775 rs12166518 rs142358110 rs117381628 rs34814924 rs545280536 rs80296680 rs138098180 rs684057 rs34443648 rs12166099 rs12166147 rs165785 rs527831543 rs165919 rs147181459 rs74466416 rs12165536 rs143814858 rs626568 rs546419 rs165703 rs566704625 rs5759527 rs144048639 rs552823 rs111348451 rs9612414 rs117727383 rs60128632 rs58090831 rs5759533 rs74756466 rs111822094 rs9612416 rs35725919 rs9612417 rs12170292 rs79337706 rs74476273 rs5751562 rs5751562 rs9620323 rs9624321 rs165897 rs78264877 rs5751564 rs8141468 rs13340094 rs75679744 rs526942 rs34877965 rs528634 rs731272 rs3747078 rs73877712 rs731273 rs76481083 rs34944089 rs879953 rs115786135 rs561595 rs879955 rs13340098 rs13433634 rs73386169 rs73386169 rs17819593 rs73386171 rs67888103 rs67695329 rs510270 rs73159154 rs143565951 rs5759551 rs17819599 rs514648 rs556083381 rs6003435 rs6003437 rs1557798 rs602808 rs544887 rs79101340 rs58396621 rs59007955 rs680548 rs112940211 rs493316 rs5759559 rs5759560 rs5759562 rs115257028 rs650538 rs1108099 rs1108101 rs738040 rs6003459 rs6003460 rs7285254 rs7287256 rs16988585 rs472409 rs473304 rs12484196 rs5759567 rs145036582 rs853127 rs619770 rs58136388 rs76937208 rs114671525 rs7285851 rs78344658 rs112669588 rs617867 rs148735102 rs142285441 rs141897641 rs80189286 rs147731752 rs602738 rs11090296 rs9624340 rs562536 rs113244786 rs853131 rs853131 rs853131 rs569817 rs570795 rs482165 rs483273 rs112133338 rs543463705 rs75156636 rs5759575 rs143269077 rs143269077 rs143269077 rs143269077 rs118139208 rs112372337 rs490749 rs5759576 rs512705 rs79438615 rs79882895 rs11912543 rs654526 rs67589781 rs8138867 rs653181 rs114547898 rs2027369 rs8141122 rs199593637 rs201122671 rs116432563 rs202212126 rs11090300 rs623679 rs111319236 rs9620335 rs9620335 rs73386179 rs73386180 rs78003513 rs111455684 rs557958 rs143901038 rs148714004 rs77793416 rs564493 rs34404739 rs687164 rs687106 rs531775638 rs578134684 rs115769480 rs144037599 rs480471 rs116434024 rs685380 rs142051118 rs74607071 rs79591662 rs73159169 rs16988610 rs28690416 rs17818796 rs670380 rs8135803 rs8135829 rs8142362 rs72050218 rs115556005 rs115556005 rs554397435 rs10427922 rs112030875 rs1861078 rs4822364 rs73159173 rs4822365 rs150412261 rs111445826 rs2539916 rs149404181 rs623999 rs117536765 rs77465539 rs9620348 rs112467558 rs376886796 rs5759632 rs7285862 rs597996 rs597996 rs597996 rs597617 rs596235 rs7286749 rs115287577 rs150886737 rs117082775 rs12483894 rs555454 rs115952739 rs578356 rs375348388 rs78263078 rs2039822 rs61417886 rs5759640 rs16988629 rs673440 rs114160819 rs741192 rs602269 rs73159179 rs6519496 rs12627833 rs565048 rs7364324 rs11090304 rs41277303 rs7364305 rs10854776 rs585467 rs9624384 rs583184 rs1974310 rs1974309 rs853108 rs4822376 rs664193 rs5759659 rs113275556 rs149210767 rs662758 rs1003718 rs139226225 rs661440 rs79261470 rs57517020 rs75042821 rs648936 rs145875329 rs17757910 rs12166248 rs580204 rs5751615 rs5751616 rs633773 rs9612546 rs2080195 rs78488852 rs5996510 rs741193 rs741194 rs73877727 rs5996512 rs741195 rs2016016 rs9612548 rs117692845 rs113977853 rs2539918 rs78464105 rs9624394 rs7292155 rs56772365 rs78817229 rs9754375 rs117437821 rs7288217 rs12484849 rs113982879 rs10522714 rs505603 rs759226 rs510954 rs201135342 rs141761092 rs658793 rs61351538 rs9624401 rs673017 rs35333818 rs491264 rs674393 rs759228 rs115900295 rs5759729 rs520609 rs11090308 rs2003574 rs113572801 rs526119 hisat2-2.2.1/example/reads/000077500000000000000000000000001370663763200154545ustar00rootroot00000000000000hisat2-2.2.1/example/reads/reads_1.fa000066400000000000000000003166451370663763200173210ustar00rootroot00000000000000>1 GCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACT >2 AGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCAT >3 TTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCA >4 CTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTG >5 ACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCC >6 CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA >7 GTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGA >8 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG >9 AGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGT >10 GATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAG >11 CAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGG >12 TGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTT >13 CTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCT >14 CCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCT >15 ACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCA >16 TACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCC >17 CCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTC >18 AACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCAC >19 GCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCCCA >20 AGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAG >21 TACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGT >22 CCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCT >23 TCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCA >24 CGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACT >25 CAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATG >26 TGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGA >27 CAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAG >28 CAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCC >29 TCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTG >30 TGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTG >31 TGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTT >32 CAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGA >33 CAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAG >34 CCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTG >35 AGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAG >36 CACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGT >37 GGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGG >38 CAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCA >39 CCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGC >40 GCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACA >41 CAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCT >42 AGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGG >43 TCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGG >44 AGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCT >45 CCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCC >46 GGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGG >47 ACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGAT >48 GGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGAC >49 AACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCAC >50 GCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGG >51 TTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCG >52 ATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGT >53 AGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGT >54 CCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCG >55 CCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACAT >56 GGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGG >57 CTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCA >58 TGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTG >59 AGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCT >60 CCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGG >61 CGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGA >62 CGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAG >63 CTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAAC >64 TCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCC >65 CGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGA >66 CATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTG >67 AGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGT >68 GTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTG >69 CCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCG >70 ACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTA >71 GCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGG >72 AGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGG >73 CCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCT >74 AGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTA >75 CCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCC >76 ATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCT >77 CATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTG >78 CCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTG >79 TCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTA >80 TGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAA >81 TGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTC >82 GAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTC >83 CCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTG >84 GCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACT >85 AAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGT >86 CTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAA >87 CGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGA >88 GCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCG >89 TCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACC >90 CATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCC >91 GAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAG >92 TGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGA >93 ACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGAT >94 CCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGT >95 ATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGC >96 GCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAA >97 CTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGC >98 TAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCA >99 TCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTG >100 GCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACT >101 GTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGG >102 CTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGG >103 TGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAG >104 CCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGG >105 CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA >106 CCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCAT >107 CAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCA >108 AGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGG >109 AACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCAC >110 TCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTC >111 AGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGA >112 TTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTA >113 GGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACA >114 AGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTC >115 GCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGA >116 TAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCG >117 TCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGG >118 TGAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGT >119 GGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGG >120 GTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGC >121 ACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTG >122 TGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAA >123 GAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATG >124 CCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGT >125 CCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCC >126 CAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAG >127 CCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGG >128 TGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTC >129 TGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTT >130 CACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGC >131 CCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCAC >132 ATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGG >133 CACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGT >134 CAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCA >135 CTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGG >136 CCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTT >137 GGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTC >138 TGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGC >139 CCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCAT >140 TCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTA >141 AGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTG >142 ATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACA >143 CTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATC >144 AGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTC >145 TGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTG >146 AGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCG >147 GGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAG >148 CAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGC >149 ACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCA >150 TCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTG >151 GGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAG >152 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG >153 ATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCT >154 AAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAG >155 CAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGT >156 TCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCAC >157 AAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACG >158 AAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTT >159 AAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGA >160 TCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGG >161 CAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGC >162 GAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCA >163 ACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCAC >164 ATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTT >165 AAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGAC >166 TGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAA >167 AAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGA >168 GGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGG >169 TGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAA >170 TCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCA >171 GCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGA >172 CAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAG >173 AAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTG >174 TGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGC >175 GCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATAC >176 TGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGG >177 GAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGC >178 GCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGG >179 CTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACG >180 GGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTC >181 GATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGA >182 TCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCAC >183 GGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGG >184 CACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGC >185 TCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCC >186 TTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCG >187 AGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTG >188 CCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCA >189 CCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTG >190 GTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCA >191 ATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCT >192 AGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGG >193 TGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAAC >194 ATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCT >195 TGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACA >196 AAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGC >197 AGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGA >198 ATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCC >199 AAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCC >200 CAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACA >201 CACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGT >202 TTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGG >203 CCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAG >204 GTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGC >205 TGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTC >206 CAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCC >207 AAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGC >208 GGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGG >209 CCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGA >210 GTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCA >211 CCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCT >212 GAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGA >213 AGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAG >214 GGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGC >215 CCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTG >216 CATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGT >217 GCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTC >218 ATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCT >219 CTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCA >220 GCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTC >221 AGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAG >222 AGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAA >223 TGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTT >224 CCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGC >225 GGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGG >226 CACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGT >227 TTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTA >228 GAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCA >229 CGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAG >230 TCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGG >231 TGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAG >232 GCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACG >233 GGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGG >234 CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA >235 CCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCC >236 ATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGG >237 CCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTC >238 CCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCC >239 CACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCC >240 GTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTG >241 GCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCG >242 GCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCG >243 GACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGA >244 CACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCC >245 GAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCC >246 TCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGG >247 GGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGA >248 CTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGC >249 CAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAG >250 GGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGT >251 TACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCC >252 GAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCC >253 CTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGG >254 AGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCC >255 TTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCT >256 CAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGA >257 TGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGC >258 CATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTG >259 CAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCC >260 ACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGA >261 ACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCC >262 CCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTC >263 GGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGC >264 ACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGAT >265 CCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCC >266 CCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCC >267 TGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGT >268 GAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGC >269 TTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTA >270 GCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAG >271 ACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCC >272 CCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAG >273 GCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCC >274 CCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCC >275 ATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTT >276 GAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCC >277 GTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGC >278 AAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGC >279 CTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCC >280 TTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTA >281 TCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAG >282 CAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACA >283 CAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCA >284 AGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAA >285 AGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCT >286 GTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGG >287 ATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGC >288 GTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTG >289 CAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTT >290 CACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCA >291 GGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGG >292 ACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGG >293 TCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCAC >294 TGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTG >295 CCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGA >296 AGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGC >297 CAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAG >298 GGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGG >299 GTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAA >300 ATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACA >301 ATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCC >302 GCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGA >303 GCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAG >304 GTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGC >305 AGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTG >306 GCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAG >307 GGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTT >308 GGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGT >309 TCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCC >310 TTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGC >311 CATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGT >312 GATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGA >313 GATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAG >314 CCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCT >315 TTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGC >316 CCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCC >317 TCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCA >318 CAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCC >319 GAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTG >320 GAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGG >321 CACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGT >322 CACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCA >323 CTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTT >324 GAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGAT >325 GTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGA >326 CCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGC >327 AGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGAC >328 CCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCC >329 GCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGAC >330 GGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGC >331 TGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGG >332 CTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTT >333 GGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTC >334 TCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGC >335 TCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTA >336 CAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGA >337 CCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCT >338 GTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGG >339 AGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCAC >340 GCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAG >341 CCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAG >342 CAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTC >343 CCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAG >344 CTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCC >345 CCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGC >346 AGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGAC >347 GCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATC >348 GCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCC >349 GTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAA >350 GGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTG >351 GCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGC >352 ACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACT >353 CCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCA >354 TCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCC >355 CCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAG >356 CCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTC >357 CTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCG >358 AGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGG >359 CGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACT >360 CGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGT >361 CCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCT >362 TCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACAC >363 CATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTG >364 GAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCA >365 AGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAA >366 AGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACC >367 ACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACC >368 GGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAG >369 GTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATA >370 GGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGT >371 GTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGC >372 TGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCT >373 GCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGA >374 GAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCT >375 CGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAG >376 CTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCT >377 GTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTT >378 AACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGT >379 CTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGG >380 ACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGG >381 GAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCC >382 GGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGG >383 CTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGG >384 CACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGAT >385 CCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGG >386 TTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGT >387 TGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGA >388 CACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTT >389 CCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCC >390 ACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCA >391 ACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGC >392 GACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCG >393 AAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACG >394 ACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGAT >395 CAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCAC >396 CCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCG >397 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG >398 CGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACG >399 CACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGC >400 TCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCC >401 CGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACG >402 CAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGG >403 GCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGAC >404 GCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCT >405 CATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTAT >406 GGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCC >407 TTTCTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGA >408 AATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACT >409 ATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATG >410 TACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGT >411 CATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCC >412 GGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTG >413 AGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGT >414 GCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACT >415 GCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAA >416 TGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAA >417 ACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCA >418 CTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTA >419 AAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACG >420 TGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTG >421 GGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGG >422 AGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCT >423 CTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTA >424 GCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTC >425 CACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGC >426 TTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGC >427 CATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTG >428 GGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTG >429 CTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTC >430 CACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCA >431 AAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGC >432 TCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGG >433 GCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGG >434 CGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGT >435 GGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAG >436 GTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTT >437 AGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGAC >438 GAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCC >439 GAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGC >440 GGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGAC >441 TCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTC >442 GCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGG >443 CCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCG >444 CAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGT >445 GTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGC >446 GACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCT >447 ACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCC >448 CAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTC >449 AACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGAT >450 TGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCA >451 CCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTC >452 GTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGG >453 CCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATC >454 ATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCT >455 ATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGG >456 CTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTT >457 GCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCT >458 CCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGG >459 GTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTG >460 GAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGG >461 ATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCT >462 GGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCCC >463 CGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACG >464 TTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCT >465 ACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTG >466 TCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGC >467 ATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACA >468 ACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGG >469 ATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTG >470 ACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATC >471 CCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGC >472 GGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCC >473 GCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTC >474 GCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTG >475 CACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGT >476 AAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCAC >477 CTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCA >478 CAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGA >479 AAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAG >480 GGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGT >481 AGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCT >482 CCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCC >483 TCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCG >484 CAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCC >485 CGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAG >486 GCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGG >487 GGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGT >488 CAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCC >489 TGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAA >490 AGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGC >491 CTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATC >492 TGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTAC >493 AGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACC >494 TGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTC >495 AGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGG >496 ACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTA >497 ATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGC >498 CTGAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGG >499 GATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGC >500 GTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGT >501 ATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACA >502 CATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGT >503 CAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGG >504 CGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGG >505 CAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCC >506 GGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACA >507 GCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCG >508 AGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGC >509 CCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCT >510 GCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACT >511 GGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTAT >512 CTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGT >513 GGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGC >514 AGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAG >515 CCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCT >516 ATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGT >517 GATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAAC >518 TCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACA >519 AGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAG >520 TACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCG >521 TGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGC >522 TTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCC >523 AAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTT >524 ACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTT >525 GAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCA >526 CCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGG >527 CGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGA >528 TGAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGT >529 GGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGC >530 CCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCT >531 GCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCC >532 AAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAG >533 CAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGG >534 GGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGC >535 ACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATG >536 GTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACA >537 TACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCG >538 AGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCT >539 GTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCC >540 CGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGC >541 ACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATC >542 CCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCA >543 TGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTT >544 ATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACA >545 TGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCT >546 CCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCA >547 GGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGC >548 TCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTT >549 TCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTA >550 CAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTC >551 CCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGG >552 ATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCC >553 CATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGT >554 CCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAG >555 CCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATC >556 CCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTT >557 GGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGAC >558 ATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAA >559 AGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGC >560 AGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATA >561 TCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCG >562 TGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTT >563 GCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCC >564 CATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTG >565 CCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTC >566 TCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCC >567 GGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTAT >568 TGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGG >569 AAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGA >570 CGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAG >571 GGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGT >572 GCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACA >573 CCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACA >574 TGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTC >575 ACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATG >576 GGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGA >577 ATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGT >578 GAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCA >579 TGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGC >580 ACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTA >581 CTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCG >582 ACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGC >583 ATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGT >584 ATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGG >585 ACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGAT >586 CCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTC >587 TTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCT >588 CCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCC >589 CTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGG >590 GGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTAC >591 CCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCC >592 TGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGC >593 TACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGT >594 CCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGG >595 CCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAG >596 AGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAG >597 TGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAA >598 CAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCA >599 GGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCC >600 TTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAG >601 GACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTG >602 CTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAA >603 AACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGT >604 GCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCG >605 TTCTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGAC >606 CCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCA >607 TTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCT >608 CAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACA >609 GACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCG >610 CCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCT >611 AGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGC >612 AAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGC >613 CTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAAC >614 CCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACA >615 CATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCA >616 CAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAG >617 CCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGG >618 AGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGG >619 CAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCA >620 ACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTT >621 GACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTG >622 CAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAG >623 CAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGA >624 CCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACAT >625 TCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCC >626 TGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCC >627 GCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACG >628 ATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTG >629 CATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTAT >630 GACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCT >631 GGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTC >632 GGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGA >633 AGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCG >634 CTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTT >635 TGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTG >636 GGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGA >637 CAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGG >638 TGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAAC >639 CCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTAC >640 TCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGG >641 TGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGA >642 AGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTT >643 GACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTG >644 CCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGA >645 TTCTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGAC >646 ATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCT >647 TCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGG >648 TCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACAC >649 GGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGA >650 CTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGG >651 TGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCT >652 GAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGA >653 CGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGC >654 AGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGG >655 TCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCA >656 GCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCG >657 GTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGT >658 CCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCC >659 GTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAA >660 TGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAAT >661 CCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCAT >662 CATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCC >663 TCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACAT >664 ATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGC >665 GGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTC >666 GAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCA >667 CAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCA >668 CACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCA >669 CTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCT >670 GGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGG >671 TGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTG >672 TTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACAC >673 AACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGT >674 CAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAG >675 CCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTC >676 TTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCA >677 CACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACAC >678 GTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACT >679 ACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTC >680 GATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGC >681 GGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGG >682 GGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTG >683 CACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCC >684 GAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCA >685 AACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCAC >686 GGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGA >687 TCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACA >688 GCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTG >689 AAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGAC >690 CCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCC >691 CGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGA >692 AGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATT >693 AGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGT >694 TGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCT >695 CCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCT >696 TGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCT >697 AGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAG >698 AGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCT >699 AAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGC >700 AGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGG >701 CTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTG >702 TTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGC >703 TCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTC >704 AGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCT >705 CCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGC >706 CAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGG >707 TGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCT >708 CACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGG >709 CCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTC >710 TGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAA >711 GAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCT >712 GGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCCC >713 CTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGC >714 GAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTG >715 GGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGG >716 CAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGC >717 CCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCC >718 TTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCG >719 CACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGT >720 GATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTG >721 ACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTG >722 GGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCC >723 AGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAG >724 ACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTG >725 ACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGG >726 CTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGA >727 TGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTG >728 CCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCC >729 CCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCC >730 GAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCT >731 GAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCA >732 CCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTAC >733 TCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGG >734 CCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCC >735 CCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCT >736 ACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCC >737 CATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAG >738 GATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAG >739 GTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAG >740 TCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGC >741 GCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTC >742 CTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCT >743 TGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGC >744 TGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTG >745 CATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCC >746 TGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCAT >747 TCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGG >748 GGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTA >749 TTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCT >750 GACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGA >751 AAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTG >752 CTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGA >753 GACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGA >754 CCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCC >755 CATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCC >756 TTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCAT >757 GAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATG >758 TGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAAC >759 GTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCA >760 ACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCC >761 CGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAG >762 GTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAG >763 GCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGC >764 GCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACT >765 CACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCA >766 CCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCT >767 AGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCT >768 ACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATG >769 CCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTAC >770 ACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGG >771 GGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGC >772 TTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCAT >773 GTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGA >774 CCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGC >775 GCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAA >776 CTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCA >777 GGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGT >778 TTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAG >779 AGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCA >780 CTGAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGG >781 GAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATG >782 CTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAAC >783 GATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAAC >784 TTTCTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGA >785 TCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCC >786 CGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAG >787 GCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCC >788 GGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGG >789 CTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTT >790 GCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAA >791 CGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAG >792 TCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACAC >793 ATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGT >794 CGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGA >795 GAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGC >796 GCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCC >797 CGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCA >798 CCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAG >799 CATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCC >800 GCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTG >801 TACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGT >802 GTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGC >803 GGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGG >804 CTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGC >805 TCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGC >806 AAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGC >807 CTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCT >808 GAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGG >809 GAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCT >810 GACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCT >811 ACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTC >812 AGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGC >813 GTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCC >814 GAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTC >815 TGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGC >816 TGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTG >817 CCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCT >818 AGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGG >819 GCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAG >820 TCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTA >821 TTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCT >822 GGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAG >823 GTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGC >824 CCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCG >825 AGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAG >826 CAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAG >827 GTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCC >828 TGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAA >829 CTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTT >830 CCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTT >831 TGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAA >832 CCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGA >833 ACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATC >834 AGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGG >835 TCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGG >836 GGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGG >837 ATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTT >838 GTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGC >839 GGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTAC >840 CGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGA >841 ATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCT >842 ACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCC >843 CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA >844 CCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATC >845 GTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTG >846 CCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGC >847 TTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCAT >848 CCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGG >849 TGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCAT >850 GGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCC >851 TGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTG >852 TAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCA >853 TGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAAC >854 GGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGC >855 GCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAA >856 AGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCT >857 TGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAA >858 TGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTG >859 ACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCC >860 CTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGA >861 GCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCC >862 TGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTC >863 ATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCT >864 GGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGG >865 CAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCAC >866 ACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGA >867 CCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACA >868 GCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTAC >869 CACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGC >870 TTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACAC >871 TTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCC >872 TCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACA >873 GACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAG >874 GGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGC >875 CCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAG >876 CCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCC >877 CATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCA >878 AAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACG >879 GAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGA >880 GCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTG >881 CCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCG >882 GAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCA >883 CCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGG >884 GCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTG >885 GTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGC >886 TGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGA >887 CGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAG >888 GACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCT >889 GCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTG >890 AGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAA >891 GTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCC >892 GGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGAC >893 GCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTG >894 CTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGA >895 CCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGC >896 GGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGG >897 GAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGT >898 TGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTT >899 CCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCC >900 TTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCG >901 GGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGC >902 CAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCC >903 CGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGT >904 CTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCA >905 CCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCC >906 GATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTG >907 AGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGT >908 AGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGT >909 ATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTC >910 CCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGG >911 TTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCG >912 GTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTC >913 GTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTT >914 AAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCA >915 TCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCC >916 GGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGT >917 TACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCG >918 CAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAG >919 CTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGG >920 AAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAG >921 GCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATAC >922 GGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGA >923 GTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGC >924 CAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCA >925 GCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTC >926 AAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGA >927 TGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGC >928 AGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGG >929 GAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGG >930 AGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCT >931 CAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTC >932 TGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTC >933 CACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCC >934 TACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCG >935 CCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGG >936 TGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAAC >937 GCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTAC >938 TGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAG >939 GAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTT >940 GGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAA >941 AGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGC >942 GGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCCC >943 TCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGG >944 ATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTG >945 CCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCC >946 AGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCA >947 AGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTG >948 TACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAA >949 TGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCT >950 ACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGAT >951 AACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGT >952 AGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGG >953 CTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGG >954 GAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGA >955 GCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTC >956 TGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGG >957 CAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCA >958 CAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCT >959 CTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGT >960 TCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAA >961 TGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAA >962 TTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTA >963 TTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCA >964 GGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATG >965 TGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCAT >966 AAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCAC >967 GACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCC >968 AAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTG >969 GTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCC >970 CCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAG >971 CTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGG >972 CCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGC >973 CCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCC >974 GGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGG >975 CAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCA >976 GCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCG >977 AAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCC >978 GTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCC >979 ACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCA >980 CGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCA >981 GCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTG >982 GGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTC >983 GCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTC >984 GGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGT >985 AGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGC >986 GATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAG >987 AGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTA >988 GGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTG >989 GGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTC >990 GCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGG >991 CCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCC >992 CACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACAC >993 TCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAG >994 GAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTC >995 CTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATC >996 GGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGG >997 TTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAG >998 CTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAAC >999 GCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCA >1000 TCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACAT hisat2-2.2.1/example/reads/reads_2.fa000066400000000000000000003166451370663763200173220ustar00rootroot00000000000000>1 CCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGT >2 GCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTC >3 GAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGA >4 CGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGC >5 TTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTA >6 GAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGA >7 TGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCAT >8 TCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCA >9 CCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGA >10 CAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATG >11 TACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAA >12 TTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACAC >13 GAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGAT >14 CTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACAC >15 TCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTC >16 CCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGC >17 CTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTC >18 CGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGA >19 CTGAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGG >20 GCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGG >21 ACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGAT >22 CATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTC >23 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG >24 GCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCT >25 CTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATG >26 CCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGG >27 CTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGT >28 CCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTG >29 GCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCA >30 TCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAG >31 TTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACAC >32 CAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCA >33 GACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTG >34 GCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTG >35 TCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTT >36 CCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCT >37 CCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTG >38 CAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGA >39 AGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAG >40 GAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATG >41 CGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTA >42 ACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCAT >43 GGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCC >44 GGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATG >45 CTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGT >46 CTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGC >47 CCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCT >48 ATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGT >49 CGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGA >50 AGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAG >51 ATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTT >52 GGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGAC >53 AGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTG >54 ACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCA >55 GCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGT >56 GGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGT >57 AGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCA >58 GGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGA >59 GGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAG >60 AAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCAC >61 TCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTA >62 ATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGG >63 GGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTC >64 ACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTA >65 TCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTA >66 GCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACT >67 AGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTG >68 CCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCC >69 TGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTG >70 TCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCC >71 AGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTG >72 CCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAG >73 AGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGT >74 GCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGC >75 AACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGT >76 AACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGAT >77 TAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCA >78 GACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGA >79 CGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGA >80 GGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTG >81 CTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATC >82 GGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCC >83 CAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCC >84 CCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGT >85 GTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTG >86 GAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGG >87 CAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGA >88 GGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAG >89 GCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAA >90 CTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCG >91 GAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAG >92 CCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGG >93 TACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGT >94 AGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTT >95 GCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGA >96 TCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACC >97 GGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGG >98 CATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTG >99 GCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCA >100 CCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGT >101 AGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAG >102 CTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCG >103 GTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCA >104 CACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTT >105 GAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGA >106 ATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGT >107 AAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCA >108 TCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGG >109 CGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGA >110 ACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCA >111 CATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGT >112 ACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCC >113 TCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCG >114 GGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTA >115 ATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCT >116 CTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGA >117 AGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGG >118 GGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCCC >119 TTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCAT >120 CCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTAC >121 GCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTC >122 TGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGG >123 GCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACA >124 GCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACT >125 GGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTC >126 CTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGT >127 ACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACC >128 TCTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACT >129 TTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACA >130 CCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGG >131 ACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGC >132 GTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGC >133 AAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTC >134 CAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGA >135 CAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGG >136 CCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCC >137 GTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACA >138 TGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTC >139 ATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGT >140 CGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGA >141 AGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGT >142 TCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCG >143 TGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTC >144 GGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTA >145 GGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTG >146 CCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTC >147 AGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCT >148 GGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGG >149 TCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTC >150 GCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCA >151 GCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCG >152 TCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCA >153 GCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTC >154 GGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTG >155 CAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGG >156 TCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAG >157 CTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTG >158 GAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCA >159 AGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAA >160 AGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGG >161 GGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGG >162 AGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGAC >163 GGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTG >164 TTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCG >165 TACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCG >166 CACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCC >167 TGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTT >168 AGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGG >169 GGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTG >170 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG >171 AGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACC >172 GACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTG >173 TGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGA >174 GTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCA >175 TGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTC >176 TGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAA >177 CACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCA >178 GCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAG >179 TCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGC >180 GTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACA >181 CCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGA >182 TCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAG >183 TTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCAT >184 CGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCA >185 GATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAAC >186 ATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTT >187 GATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGC >188 GAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTG >189 GACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGA >190 TGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAG >191 GCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGA >192 CGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGA >193 TCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCC >194 GCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTC >195 GCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTC >196 AGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCT >197 CATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGT >198 TCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCC >199 CCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCC >200 TTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAG >201 AAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTC >202 GAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCA >203 AGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCA >204 TGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTG >205 TGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGC >206 GCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGA >207 AGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCT >208 TTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCAT >209 ACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGG >210 GAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGG >211 CACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGT >212 TTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCA >213 GCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTG >214 AGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGC >215 CAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCC >216 AGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGA >217 AGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCAT >218 GCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTC >219 GTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGA >220 ATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCT >221 TCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTT >222 GTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGC >223 TTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACA >224 GCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGC >225 GCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAG >226 AAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTC >227 ACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCC >228 GCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAA >229 TGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAAT >230 TGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGG >231 CCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCC >232 ACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTA >233 TCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAG >234 GAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGA >235 AGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAA >236 CGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAG >237 CTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTC >238 CCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCT >239 TGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAA >240 CCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCC >241 GGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAG >242 AGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGG >243 CCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTG >244 TGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAA >245 TTCTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGAC >246 AATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACT >247 CTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCC >248 GGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGG >249 GACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTG >250 AGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCC >251 CCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGC >252 GTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTC >253 CAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGG >254 GGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGT >255 TTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCC >256 CAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCA >257 TGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTC >258 TAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCA >259 GCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGA >260 CTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCT >261 CAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAG >262 CTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTC >263 AGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTT >264 GCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCC >265 CCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTT >266 CATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAG >267 GGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCC >268 TACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCG >269 ACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCC >270 GGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAG >271 CAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAG >272 TGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCT >273 ACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTG >274 CCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGAC >275 TTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCG >276 GTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTC >277 GTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACT >278 GGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAA >279 GGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGA >280 ACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCC >281 CAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCC >282 TTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAG >283 CGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAG >284 GTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGC >285 AAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGC >286 GAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCC >287 TCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTC >288 AAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGT >289 GCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTAC >290 GGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGC >291 CCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTG >292 CGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTG >293 TCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAG >294 CACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGG >295 AGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGT >296 GGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGC >297 CTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGT >298 TCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAG >299 CAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCT >300 TGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGG >301 AAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCT >302 AGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACC >303 GCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGG >304 CCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTAC >305 AGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGT >306 GCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGG >307 TGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTC >308 GTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAA >309 ACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATG >310 ACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGC >311 AGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTC >312 CCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGA >313 CAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATG >314 CGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGG >315 ACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGC >316 GAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGG >317 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG >318 TCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAG >319 CCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCA >320 CTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAA >321 CCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCT >322 GGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGC >323 TTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCT >324 CTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCT >325 CTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCA >326 GCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGC >327 GAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCA >328 CCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTT >329 TGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTAC >330 AGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTT >331 TGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAA >332 CCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACA >333 GTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACA >334 CTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACG >335 CGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGA >336 CAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCA >337 CGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGG >338 GAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCC >339 CAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTC >340 GCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGG >341 TGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCT >342 CCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGC >343 TGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCT >344 GGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGA >345 CAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTC >346 GAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCA >347 GACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCC >348 GTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTT >349 CAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCT >350 TGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAA >351 CTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGG >352 AAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCG >353 GAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTG >354 TGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAAC >355 ATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGAC >356 GGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGC >357 TCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAA >358 ACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCAT >359 GCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCT >360 CTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCT >361 CACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGT >362 TTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGT >363 GCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACT >364 TTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGG >365 CCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCC >366 GCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGA >367 GCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCAT >368 GCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAG >369 AGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGG >370 CTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGA >371 GTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACT >372 CCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAG >373 AGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACC >374 ACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTT >375 CAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCA >376 GAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGAT >377 GCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCC >378 CCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCC >379 GCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGC >380 GTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGA >381 GTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTC >382 TTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCG >383 TGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACAC >384 GCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTG >385 TGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGA >386 TCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACAC >387 CCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGG >388 CCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGG >389 GTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTG >390 TCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTC >391 TTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGC >392 TGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAA >393 CTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTG >394 GCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCC >395 TGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGT >396 ACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCA >397 TCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCA >398 GACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAG >399 CGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCA >400 ACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTA >401 AGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCT >402 CTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCT >403 TGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTAC >404 TGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCT >405 GGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGAC >406 TGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGT >407 AAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCA >408 TCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGG >409 GGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGA >410 ACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGAT >411 ACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATC >412 AAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAG >413 CCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGA >414 CATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTG >415 TCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACC >416 GACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCG >417 TCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTC >418 CCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACA >419 CTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTG >420 TCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAG >421 TCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAG >422 GGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATG >423 CCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACA >424 ACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTG >425 CGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCA >426 TCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACAT >427 GCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACT >428 ACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCAC >429 CCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTC >430 GGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGC >431 GGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAA >432 GGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCC >433 GCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAG >434 CTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCT >435 CTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATG >436 GCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCC >437 GAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCA >438 GTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTC >439 AGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCG >440 ATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGT >441 ATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGC >442 AGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTG >443 ACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCA >444 CTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCA >445 AGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAA >446 CCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCC >447 GTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCA >448 CCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGC >449 ATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCT >450 GTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGT >451 CTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTC >452 AGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAG >453 TCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGC >454 GCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTC >455 GTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGC >456 TTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCT >457 CGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACT >458 ACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACC >459 AAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGT >460 CTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAA >461 GCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTC >462 TGAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGT >463 AGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCT >464 TTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCC >465 GCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCC >466 GCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAG >467 TGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGG >468 CGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTG >469 GAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTT >470 CATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCC >471 AGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGC >472 TCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGG >473 AGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCAT >474 TCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTG >475 AAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTC >476 CCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGG >477 CAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGT >478 ACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGA >479 GAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGT >480 AGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCC >481 CGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACG >482 CTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGT >483 CTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTC >484 TCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAG >485 GAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTAC >486 AGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTG >487 CTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGA >488 GCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGA >489 GGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTG >490 GGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGC >491 GACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACAC >492 GCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGAC >493 GTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCC >494 CTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATC >495 CGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGA >496 GCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACG >497 GCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGA >498 GCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCCCA >499 AGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTG >500 TGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCA >501 TGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGG >502 AGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGA >503 CTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCT >504 CCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCT >505 CCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTG >506 TCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCG >507 GGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAG >508 GGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGC >509 CGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGG >510 CATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTG >511 ACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCC >512 CAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAG >513 CCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTC >514 GCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTG >515 CACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGT >516 GGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGAC >517 CCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGG >518 GTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCT >519 TCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTT >520 AAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGAC >521 TGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTC >522 TTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCT >523 GAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCA >524 GAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCT >525 AGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGAC >526 ACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACC >527 AGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGG >528 GGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCCC >529 CCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTC >530 GTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGC >531 GTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTT >532 GGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTG >533 GGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGA >534 CCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTC >535 TCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCC >536 GGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTC >537 AAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGAC >538 ACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATG >539 AGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACC >540 CTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTG >541 CATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCC >542 GAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTG >543 TTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACA >544 TCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCG >545 CCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAG >546 GACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCAT >547 CCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTC >548 AGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAG >549 CGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGA >550 CCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGC >551 GATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAAC >552 AAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCT >553 AGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTC >554 ATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGAC >555 TCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGC >556 CCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCC >557 CATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTAT >558 AAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACAT >559 CACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACAC >560 GCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCC >561 GGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACA >562 TTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACA >563 GTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTT >564 GCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACT >565 CTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTC >566 GATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAAC >567 ACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCC >568 ATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACA >569 TGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTT >570 CAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCA >571 CTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGA >572 GAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATG >573 GTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAA >574 TGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGC >575 AGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCT >576 CAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGG >577 CCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCAT >578 TTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGG >579 CTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGG >580 GCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACG >581 TCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAA >582 CCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCAC >583 GGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGAC >584 GTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGC >585 TACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGT >586 CTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTC >587 TTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCC >588 GAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGG >589 TGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACAC >590 AGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGT >591 GTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTG >592 TGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTC >593 ACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGAT >594 CACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGC >595 AGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGG >596 GCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTG >597 TGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGG >598 CGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAG >599 CTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCT >600 CAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACA >601 CAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAG >602 GAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGG >603 CCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCC >604 GGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAG >605 GAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCC >606 GAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTG >607 CTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTT >608 TTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAG >609 TGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAA >610 CTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACAC >611 GGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGC >612 AGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCT >613 GGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTC >614 GTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAA >615 AGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATT >616 CTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGT >617 CACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTT >618 CGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGA >619 CGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAG >620 GAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCT >621 CAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAG >622 CTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGT >623 CAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCA >624 GCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGT >625 TGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAAC >626 GGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGC >627 ACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTA >628 GAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTT >629 GGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGAC >630 CCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCC >631 GTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACA >632 CTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCC >633 GAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGC >634 CCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACA >635 CTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGC >636 CAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGG >637 CTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCT >638 TCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCC >639 GTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGC >640 AGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGG >641 AAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTG >642 CCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGT >643 CAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAG >644 AGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGT >645 GAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCC >646 GCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTC >647 AATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACT >648 TTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGT >649 CTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCC >650 GAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCA >651 GCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCT >652 CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA >653 GTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTC >654 GTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATA >655 AGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAG >656 AGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGG >657 TGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCA >658 AACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGT >659 GGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGT >660 CGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAG >661 ATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGT >662 CTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCG >663 TTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGC >664 GCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGA >665 CCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCC >666 AAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTT >667 CAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGA >668 GGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGC >669 ACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGA >670 TTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCG >671 GGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTG >672 TGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTT >673 CACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCC >674 ACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCC >675 AGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCG >676 GAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGA >677 AGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGC >678 GTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGC >679 TACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGA >680 AGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTG >681 TCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAG >682 AAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAG >683 AACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGT >684 GCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAA >685 CGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGA >686 ATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATG >687 GTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCT >688 CCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCA >689 TACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCG >690 ACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTT >691 AACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCAC >692 CATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCA >693 CCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGA >694 CTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGC >695 CCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCC >696 CCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAG >697 TGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGC >698 GGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAG >699 GGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAA >700 CGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGA >701 CGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGC >702 ACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGC >703 ACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCA >704 GGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAG >705 GCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGC >706 ATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTC >707 CCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAG >708 TGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTG >709 AGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCG >710 GACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCG >711 ACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTT >712 TGAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGT >713 GGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGG >714 CCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCA >715 CTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGC >716 GGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGG >717 CCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTT >718 GGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGG >719 CCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCT >720 CCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAG >721 ACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCC >722 TTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAG >723 TGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATG >724 ACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCC >725 CCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGA >726 GGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCC >727 GGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTG >728 CCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGAC >729 AAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCC >730 ACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTT >731 AGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGAC >732 GTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGC >733 AGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGG >734 GACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCT >735 CTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACAC >736 CAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAG >737 GGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGA >738 CAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATG >739 CGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATC >740 GCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAG >741 ACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTG >742 GGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCC >743 TGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTC >744 CCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCG >745 CTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCG >746 GTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGA >747 GGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCC >748 AGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTC >749 CTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTT >750 CCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTG >751 TGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGA >752 TAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCG >753 CCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTG >754 TGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAG >755 CTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCG >756 GGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGG >757 GCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACA >758 TCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCC >759 TGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAG >760 ACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTG >761 GAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTAC >762 CGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATC >763 CCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGC >764 CCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGT >765 GAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGC >766 CCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCC >767 CGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACG >768 TCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCC >769 GTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGC >770 CCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGA >771 AGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGC >772 GGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGG >773 CTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCA >774 GCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGC >775 GAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCA >776 CAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGT >777 GTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAA >778 CAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACA >779 GGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGA >780 GCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCCCA >781 GCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACA >782 GGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTC >783 TCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCC >784 AAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCTCCA >785 ATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCC >786 ATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGG >787 GTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTT >788 AGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGG >789 TTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCT >790 TCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACC >791 GAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTAC >792 TTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGT >793 CCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCAT >794 TCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTA >795 AGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCG >796 AGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATA >797 CACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGC >798 ATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGAC >799 CTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCG >800 CCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCA >801 ACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGAT >802 CCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTAC >803 TCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAG >804 GGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGG >805 CCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATC >806 AGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCT >807 CAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGG >808 GTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCA >809 ACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTT >810 CCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCC >811 TACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGA >812 CCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGC >813 AGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACC >814 GGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCC >815 GTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCA >816 CTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGC >817 CCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGG >818 GCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCG >819 GGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAG >820 CGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGA >821 TTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCC >822 GCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAG >823 CCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTAC >824 TGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTG >825 CCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGC >826 GACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTG >827 TCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCT >828 GACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCG >829 TTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAGATCT >830 CCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCC >831 TGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGG >832 AGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGT >833 CATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCC >834 GGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGG >835 TGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGG >836 CTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGC >837 TTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCG >838 CCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCT >839 AGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGT >840 CAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGA >841 GCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGA >842 TTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTA >843 GAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGA >844 TCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGC >845 CCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCC >846 CAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTC >847 GGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGG >848 CACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGC >849 GTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGA >850 CTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCT >851 CCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCG >852 GGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGG >853 TCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCC >854 CCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTC >855 GAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCA >856 GGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAG >857 TGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGG >858 GTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGC >859 GGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTAT >860 TAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCG >861 AGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATA >862 GCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATAC >863 GCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTC >864 TAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCA >865 TGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGT >866 CAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGA >867 CTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTA >868 CAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTT >869 CCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGG >870 TGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTT >871 ATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACC >872 GTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCT >873 CGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACG >874 CACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCA >875 TGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCT >876 AACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGT >877 AGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATT >878 CTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTG >879 TTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCA >880 CACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGAT >881 TGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTG >882 TTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGG >883 CACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGC >884 AGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAG >885 AGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAA >886 CCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGG >887 GAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTAC >888 CCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCC >889 CACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGAT >890 AAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGA >891 TGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAG >892 CATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTAT >893 AGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAG >894 GGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGT >895 AGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGC >896 GGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGT >897 AAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAG >898 TTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACAC >899 AGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAA >900 GGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGG >901 CCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTC >902 TCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAG >903 CTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCT >904 GTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGA >905 AACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGT >906 CCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAG >907 GGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTAC >908 CCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGA >909 CAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGG >910 CACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTT >911 GGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGG >912 CGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGC >913 GCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCC >914 TTTCTGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGA >915 ATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCC >916 TCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGT >917 AAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGAC >918 CTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGT >919 GCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGC >920 GGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTG >921 TGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTC >922 GCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACT >923 CCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCT >924 CAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGA >925 TGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACA >926 AGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAA >927 GTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCA >928 GGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGG >929 GTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCA >930 GGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAG >931 AGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCAC >932 GCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATAC >933 AACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGT >934 AAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGAC >935 CACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGC >936 TCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCC >937 CAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTT >938 GTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCA >939 ATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTG >940 CCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGG >941 GGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGC >942 TGAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGT >943 GGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCC >944 GAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTT >945 GACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCT >946 CCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAG >947 GATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGC >948 CAGTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGG >949 CCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAG >950 CCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCGCCGATGTTCAGGGACATGGAGCGCT >951 CCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCC >952 CGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGA >953 CTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCG >954 CAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCA >955 ATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCT >956 TCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGG >957 CGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAG >958 GTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAA >959 CAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAG >960 CTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACGTGCCGGTTCATGCTCCCCTTGGCCCCG >961 CACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCC >962 ACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCC >963 GAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGA >964 AGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCT >965 GTGGAGTAGATCTTCTCGCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGA >966 CCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGG >967 GCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATC >968 TGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGA >969 TCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCT >970 TGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCT >971 TGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACAC >972 GCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGC >973 AACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGT >974 CCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTG >975 CGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAG >976 AGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGG >977 CCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCC >978 TCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCT >979 CCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTGCAGCTCTGACCAAGTGGAGATCTTCTCCAGCCTGCTGCAGCG >980 CACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGC >981 TCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTG >982 CTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAAC >983 TGGCCTGGGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACA >984 GGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGG >985 CACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACAC >986 CAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATG >987 GCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGC >988 AAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAG >989 GTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACA >990 GCGAAGCACATTGCAGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAG >991 AACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGT >992 AGTGGAGATCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGC >993 GGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTGAGGGCCCTCTTCCTGGGGAGCACAGGGCCCCTGGTGTGTACAGTGTGTCATGGG >994 GGGCGTCTGTGTATCACGGTACTTCTGTAGACGGGGAACACATTTCAGTGAGCTGCTCCTGGAAGGGCAGGGAAGAGAGTCCATAAGGTGAACTTGGCCC >995 TGCTGAGATAAAGGAAGCAGACCCCCTGGCTGCCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTC >996 CCTCGGAAGCAAGTCAACCCAAACCCTGTCCCCCCGAAGTGACCCCCCACTACATCTGGATCGACTTCCTGGTGCAGCGGTTTGAGATCGCCAAGTACTG >997 GGGCCAAGTTCACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCC >998 GGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCAGCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTC >999 TCTTCTCCAGCCTGCTGCAGCGCTCCATGTCCCTGAACATCGGCGGGGCCAAGGGGAGCATGAACCGGCACGTGGCGGCCATCGGGCCCCGCTTCAAGTG >1000 TTCCCCGTCTACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGTGCTCCCCAGGAAGAGGGCCCTCACTTGAAGC hisat2-2.2.1/example/reference/000077500000000000000000000000001370663763200163145ustar00rootroot00000000000000hisat2-2.2.1/example/reference/22_20-21M.fa000066400000000000000000037015611370663763200177220ustar00rootroot00000000000000>22:20000001-21000000 TGGGAAGGTGGGGAGGTGCTCACCTTGGGGAGCCCCCCTGAGTGCTCTGCAAGCTCTGGG GTCACCTGCCTGGCCAAGGACCCAGTACCAGCCGCCTCAAAGCGGGGGAATGACCACGCC CTTTATTGGGCCCTTTTCCCAGTGAGGATGGCCTGGGCCTACCCATGCCATGGGACCCCC ACACTGGGTCTCCCCACCAAACTGTCCAGACCCGATGGCTGGCCATGGGGCTCTGTCAGC CCCTGGCTACACCCCACTGATGCCCTAGTAAGCCCTGTGGTTCTGGCACGGTATCCATGG TCCAACCAGAGGGCTGAGAGGTCTCACACTGGGGCATAAGCCTGGCCCAGGCCACACAGC CAGATTGGCAAGCTACAGTCCTTTGGATAGCTGACGGCACAGGGCAGCTCCCATGGGTAC ACACTGGGCCCAGAACTGGGATGGAGGATGCAGCCTGGGCTGGGTAGGCAATGAGGGGCC CAGGTGAGCTCGCTCTGCACTCCCTGCAGTGTGGCCTCAGCAGGCCACCCCCTCCCCAAG TCTCCTCGCTTCCTGCTCTATGAGCGCGAGGTGACACTGACTGTAGGTAGCTGAGAACAT GCCCTGGCAACAGCCAATGCAGGCAGGTGAATGGAGTGCCCCGTGGCCAGTGGACAGGGA AGGTCCATGCTGCTCAATGACAATGCTGTCCACTACAGCAAAACCGAGTGTTCTCCTAGG CCTGCTGCCACCCTGGGCACATAGTGAGAACACGCCCACTTCTGCTGTGGACATTGAGGC ACAGACCACACCTGGGAAGGCTTGGGAGGCCCCTGGGCACACACTTGAAGGACACTGCAC TTTTGGGGCTACGCAGGCACAGGGCTAGTCCTTCACTGTCCCTGCTGAGCCTGTGTGTGG TCACCCACCAGCTGGCCTTCAGGCTTCCTTCTGCCTGCACCAAGCATGTTGGGGGTACCG AGGGGCCTTGCTTGGCTCCTTTCCAACTCCACGCCCTCAATAGGCGGCTTGGAGGCCGAG CTCCGGTCATCCAAGCCAGGTGTCTGGGGAAGGGTCCTGGTCTGCCGCAGGAAGCTGGGT GGAGCCGGTCTTGCCTGCTGTGGCGGTCCCAGCTGCCCTCTGGTGGCCATGCTCAGCTCC CCGTTGCTCTGCCAGAGGCGGCAGCCCCTCAATCAGCAGGGGCAGTGGGAAGGGCCTGTG CATAACTGGTTATGGAGTGGGGGGAACTCTGTGGCCCCAGGCACCCACCAGCTCTGGGTC ATCTTCCCGACCTGAAACAGGCCAAGATATGAAGGCCCTGAGCCAGGAAAACCTACTAAG GGATCCCTGATCCCAAGTCCCCTCAGAGTGACCCAAGACTTCATCTTGGGTTTTTCTGTT TGTTTTTTTGTGGGTATTTTTTTTTCTTTTTTTGAGACGGAGTCTCACCCTGTCACCCAG GCTGGAGTGCGATGGCACAATCTTGGCTCACTGCAACCTCTGCCTCCCAGGTTCAGGCAA CCCTCCTGCCTCAGTCTCCCCAGCAGCTGGGATTACAGGCATATGCCACCAGGTCCAGCT AATTTGTTTTGTATTTTTAGTAGAAACAGGGTTTCACCATGTTGGCCAGGCTGGTCTCAA ACTCCTGACCTCAGGTGATCTGTCTGCCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAG GTACGAGCCACCGTGACCAGCCCATCCTGGTTGTTTTGTTTGTTTTTGTTTTTTGGAGAT AGAGTCTCGCTCTATCACCCAGGCTGGAGTGCAGTGGTGTGATCTTGGCTCACTGCAACC CTCACCTCCCCGGTTCAAGCGATTCTCCTGCCTCAGCCTCTTGAGTAGCTGGGACTACAG GCGCATGCCACCATGCCCGACTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATG TTGGCCAGGCTAGTGTCAAACTCCTGACCTCAGGTGATCCACCTGCCTTGGCCTCCCAAA GTGTTGGGATTACAGGCATGAGCCACTGCGCCCGGCCTCATTCTGGTCTTAATTGAAGCT GCTGACAAAAGAACAGACACCTCCCCATGACTAAGCCACCATCAGGGAAGTCCCCAGGAC CCCCAAGGCCCAGCTGGTGCTCTGCAGTGCTGTCCCTGGACCCCCACCCTCCATTCCTGG CCCTGTCTACAGAATGGGCCCTGACTCCCTATCCCAGCTCTACCCTGTCACTCCCTCTGC CAGGGCCAGGCGGGGAGCACGACAGATGGGCTGGACAGAGGGTGGTGCCTGGCATCCGTG TGCAGAGCTCCCTGCTCCACCCAGAGACCGGGGATCCTGCTGCTCCTGCACCTTCTGTCC TTACTCAGGACAGCCTCATGCTCCCCCAGGCCAGACTCCTGGGGTGAAGGAGCACAGGGA ACTCACCCTGAATCCCTGTGTAGCACAAACAGAAGGAGGGGTGCTACCCTACCCTCACAG AGCCACAGAGATGCAGAGGGCCCCGGGACTGGGGGGAGACTACAGGGCCACCTCTGGAGG GAGTGCCAGAGCAAACATGGGAGCCAAGCAGCCCAGATGTGGTGGGTGGGGAGACTCAAA TTTAGCAGGATTGAGGGTCAGAACTTGTTCTCCCAGGAGGAACCTTGGTCAGAAGGTCCT TGTGTTTATGCTGAAAAGCCTGACAGGGCAGCTCAGCCAAGAGCTGGGCAGGGGCTGGCT TGGCTGTGCTCAACTACCCTCTCAGCTGGGACTTGAGTCCCTTAGCGAATGTTCTGTGCC CAGTCCTCTGCTGGGCGCTGGTGGACCCCAGAATGGGGGTAGGGTGGGGACAGGGGACAG CTGGGAGCAGACATTGCGGATAGGATGGGGCAAGACTGGGGGAAATGCCTTCCCAGACAC GCTGCGGCTCACACTGTCGCCTGATCTAAGGCCTGGATCACCTGCTTAGGAGCTGACCGG CCACTGGCATGGCACACCGGGCAAGAGAGCGCTGACTGCCCCCCTGCATTCAGCACCATC CCAGCTCCAATAGAGTGAGTACAAGATAGGTAACACCTGCCTTGTTTGGACCAGACCTCT AAACACCGCCCAGATCCCAGAGTAAAGGCAGATAAGGCAGTAGTTAAGAAGTAGGAAGAA GTAAAGGCAGCTACCCCAGAGAAGCTAAGGTCGGGAGAGGTGGAGGCTCCCACTGCACCC CCCTGGCCCATACCATAGGGGAGGTTTCCCCCACAGCAGCCTGGGTACTCACCTGAGGTT ATTAGACAGCAGCATTAAGAGCCTACTCTAAAAAATACCCACGGGGCACCTTTCTTTTCC TTATTTCTAAATATGCCTGGATGTAGGGAAAGGGAGCTGAAGTCCTTTAGTTCCATTTTA AGTATGTTACATTACACATAGCTAAGAAACGGTGAAATATTGTAAGCAGCCAATATTTAA TTATTTAAAAATTAAAATAGGCTGGAGCTCTCAGGCCACCTGCCCAGGGCGACTCCCCAG CAAACCGAGCAGTCTCCCTCAGAGTGGAGATGACATGTCTCCGCACCGGCACCGAGGGGG CGCAGGGCGGCGTGGGGAGGGGGACCCAGGCCGGGGGTCCGGGTGGCGCGCCCTCTCTCA CCCCCGAGGCGGCAGGAGCAGCGCGGCGCGAGGACACCTCCGGGCCGTCGGTGCCCCTGA GCTTCCCGGCGGCGCGCTGCCCCAGGAAAGTTCCCGGCCTGCCGCCCCTCGGGCTGTTGG AAAGTTTCGGCGGCTGCAGGGCCCTGGCAGCTAGGCGCCAAGCGTCGCGCGGGGCGGGCA CGGCTGCGGGGTCGGGCCGCGCTCGGCTGGAGTGCTCAGTGCAAGACGCCCGGACGGGTA GGGTAGGGCCCCAAGGCCACGCACAAAGAGACCCGCCCCCGCCGGCCCGGGGTCTCGGAC GCCGCAGAGCGCGCGGACAAGACGCGGGTGGGGCGGAGATTGGCTCCGACGTCCGGCCCA GACCCTCGCCCGGATCTCGACTCCAGGGATAGGACCCTGTCCGGACCGCTGCCCGGAATG CGTCCCATATCTGCAGCCCCGCCCCCAGTCCCGGCTCCGACCCGAGGCCCCAGCCCCGGG TCCCACACCCCGCCCACTGGCTCAGGCTTACCTGGAGCGCAGCGTGGGCGGCCCCGCAGC GCGGCCTCGGACCCCAGAAGGGCTTCCCCGGGTCCGTTGGCGCGCGGGGAGCGGCGTTCC CAGGGCGCGGCGCGGTGCGGCGCGGCGCGGGTCGCAGTCCACGCGGCCGCAACTCGGACC GGTGCGGGGGCCGCCCCCTCCCTCCAGGCCCAGCGCGCAACCCGAGACCCCGGGCCAGCC CTCCCGCCCTCACGCGGCCCGTGCGCAAAGCGGACCGGGCCGGCGACGCGAATCTCCGCC CCGCCCCATCTGACAGCAAATTTTCCCATTGGTCGAGAGACCCGGCGCGGCACGGCAACC GGCGCCGATTGGGCGGATGCGCGGAGATGACGGCTTGCCATTGGGCCTCCGGGATGTCAG TCCCGGCGGATGACCGGCCCCCTCGCGGAGGGGCCGCGGCCCCGCACCTGCCCGAACCTC TGCGGCGGCGGTGGCAGGGTACGCGGGACCGCTCCCTCCCAGCCGACTTACGAGAACATC CCCCGACCATCCAGCCCCGAGCAGGGACTGGCAGAGCCGGCGCCCCTTCCCCGGACTCTG GCCGCGTAGCCTCCGCCACCACTCCCAGTTCACAGGTATCCTTCTTCAGCGCCTGCTACT CGCTGCGCGCTGCGCGCCGCTCCCTCCTGTCGACATCGAATTGTATTCTTCACGCAAGCC GCGAGAGAGAGTTCTGCTGCTATCCCCATTTTACAGTTGGGAAACAGCCTCAGAGAGGTT CTCACTTGCTTGAACACAGTCGTCTGACCCTGGGGCCTGGGGGCTCTCTCATCCTCACGC CCCTGGGTCCCCGCAGCGGGGAGACGATGGAGGTAGGAGCTGCAGTATTTCCGAGGGCAG GAAGGGACCCCCTCGGTAAACCAGGGGCTCACCTCCCCCACCAGGCCTGGATGTAGAGAC CTCTTGGGCTTGTCCCACCCTGCAGGCCCCCCATGGGGCCGCCCTTTCTTACCTCCTCCT GGCATTCTCACAGCCCCACAGCTCTGCGTCTGGGGAGGCAGAGTTGTGCGTTTATGCCCG CACCCCGTACTACTGTCCCATTTTCTTCACCCTACCTGGAGTCAGGAAGAAACGGTGCTT CACTGCCTGTTGAGATTCACGTAGGTTACATTTCCTTGCCAGATTAAGAGCACCTGACAT GAGTGGGACCATGACCCTCCGCCAGCTCTGCGTAACCTTGTGGTGTAGGGGGGAACCAGC AGGGATGCTGCCCAGTGGGCTTTTCTAGGGGGAGGTCCTGAGATGACTTCTTAGCCAGTG ACCCTGGCTCACGCATGCTGTCATCCTTTCCTGCCCTGGATGCACCTGGAGATGACCAGT TCCTCGGCTCATTGCATTCTCCAAGGCCGCCTGGGGAACCAAACTGCCCACAACCATGCT TATGAGTGGTGGAGGGGTGTGACCAGACACTCTTGTCTTATCCACCCATATTGGGAGTCC AGTGTGTTCATCCAATCATTCCTTGGCCAGGACAAAGAAAATGAAATGGCCCAAGGCAAG AACTCACTGCTGGGACCACCGCTGGGTCCATCTGTGTGCTGGCAGCATGGTGAATCGATC CCAGCTGCCTTTCAGAGCCAGGCAGCTACTATCAATCGATTGGAGCTGGCACTTGACGTG CCAATTGGCCTCCTGGCATAGGGTTGTTGCTTGCTGCTCTGGGAGGGCAGAATGAAGTAG CTGTCCCTAGAATAGGGGCCCTCACAAACCAGGTAGCTGTCATCCTGTGGAGCAGCACCA TGGTGACATTCTGGCTCAGGTGCTGTGTAGCTGAGGGGTCGTTTCCTCCATTTAGGAGTA GACGTAATGAGTCCAGCTGTCCCCAGCCATCTCTTCAAGGCCATTGGACCCTGGTTCTGC TGGAGCCCCTCTTCTCACAGTTACCTCATAGTCCTGCACCACCCCCTCCCCACACTTGCA AGAGGGACTGGACAGACCTCTGTTGAAAGAGTGCAAAAGACAGAACCATACACCAAAATC TGTGCAAGTGAGACTACCTCTCCTCTCATCACCACCCCTACCACCCATAGGGTTGCAGAG ACCTGTCTGCCCTCGGCAGGCAGGATCATTAGCTGAGGTCAGAGGATGTCGGGGCAGCAC CAATTCAAACAGGACTCAGGACCGCTGTCCAGCAGTCGGCCCATTAGGCTGCAGCCAGGG GTCAGAAACACAACAAGGGAGGCTGGGCGCAGTGGCTCATGCCTGTAATCCCAGCACTTT GGGAGGCCAAGGCAGGCAAATCACCAGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACAT AGCAAAACCCCATCTCTACTAAAAGAATACAAAAAAAAGTAGCGGGGCATGGTGGTAGAC GCCCATAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGATCGCTTGAACCTGGGAGGCGG AGGTTGCAGTGAGCTGAAATTGCACCACCGCACGGCAGCCTAGGAGACAGAGCGAGACTC CATCTCAAAAAAAAGGAAAAAAGAAAAAGATAAGAAAAACACAGCAAGGGAGACCACAGG GACCAGTAGCCCAGTGTTCTCCCTCTCTCATCCTCTCACCTAGCTCCATGCGGACAGCTG TGAGGCCCCCGGAGAAAGCCACAGAGAGAAAATGCTTTCAGCTGGGTGTCAAGAACTTTT CAAGAGTGGCAGCTGTTGAAATCAAAAGAGTGGCCACAGAAGGTAGTGAGCTTCCTATCT GAGTTTGTGTGCAATTAGAGGTTGGATGCCAGTGAGCCTGTTGCAGCCCCAAGATCCCAG GGCTTAGGTGCCCATCCTAGACCCATTTCATCTGCCGTCACCTGCTTCCTGGCATCGCAT GTCATCCAGACTGGAGTGTCAGTGGTGACAAAGAAGATCAGAGTGTCTGCTCTGGCTCTG TCTAGCTGGGAAAAGTATGGCTGGGGGTGGGGATGGCCCAAGGCCACAAGGTATGACAGA ACAGGATGCAAACCACCATCTGACATGTTACTGCATCCCTCTTTGTGGGCAAAGGGAAAC CTTGGTCAACTCATGTCCAGCTTTGAGGTGCATGGCCTGGTGCTTTGGGGAAGCCCATTC TCCAGGTGCTGCCATGAACCCCAGGGCACAGTCCATCACCCCCAAGCCCCAGATCCCACT TGTCCACCACCACCACCTCTTGGCCCTATAGATTGCATAGGTTTGGCTGCTCAGAGGGAA ATCACTGGTACCAGCCCCACATGATCCCACTGCTGGATGGTGGGAGAGACAACCATGTCT GGCTGGGTCTGCGGCCCGCTGGCCATGAGTCCCTGGGCATCTGGAGTCCTTGTAAAAGGT TCTGTTGGGTTCCAGGGGAAACACAGATAAGATAGCAAAAGTGTGGAGCACAACGGGCCC GCATGTAGGTCCAGGTTAGACTCTTGCCGTTGCTGATTACCAGCCAGGCTCTTCCCATCC CTGAGCCCCAGTTTTCTGCAGGGCATCAGGGCACCTAGGTCTTCAACACAGGTTGAAGCT AATGTGGCCTCTCTTGCTTGCCACCTTTTTCAACTTCAACTGAATGACATAACTGCTCCA AGTCTCAGTGTTTACATCTATAAAGTGGGATGAAAGAACTGCTGACCCAGACATTGGGTT TAGAGTGCTTTACACATTATCATTATCCCTAATGTCTTTGTCATCCATGTCATAGTTTTT TGAATGCTCACTTTGAGGCAGGGCCTGAGCCTCTGGGCTTTGACTCTCCACCTTTCTGCC ACATAGGCCCAGGCACAGCCCAGAGGCTACAGGAGCAGCGGTCCCAGCTAATGCCTCTGT CCTGTTGGGAGGGCCCCAGTCAGTGCCCACCTGCTCACCCTGCACAAGTCAAGCTGGGCC AGCCCCTCGTCGCTGGGGCCTGGACCTGGATGTGAGCTCAAAGGCTACACTGGGGGGTGG AAGTGGGGGAGGTGGTGGGTGTGCAGGGTATAGTGCTCAAATGCCTAGTCCAGGGTCTGG GGGTACATACTGGGAATGGCAGGGACATCAGGGAGGAAAGAGCCGACCTGGAGGCCATAA AGTGAAGGAAGGGATGACCAAGAGGAAGATGGCCAGGTCCAGAGGTGGGGTTGAGTGAAC AGGATGGGGAGACCAGGACGCAGTCTGAGGGGGCCAGTCCGGAAGTGAGCATTCCTGTAC CTCTTCCGAGAGAGGCCGCAGGGTGAGCAGGTGACAGATGGTGTCCATGAGGGCATGGAC TCAGGGGCGGCTGCGACCTCCCATGGCGTCCCTGCAGGCAGGGAGCCGCGGAGTCCCCGA AGGCCGCTGGATCAGGAAGCGCTCAGACGCCCGCGCAGCCCGAAGCTTTGGCGCAGCCCC GGGGGCGGGGCGCCAGGAGGTGGCAGCCTCCGAGCGACAGCGCGCCCAGCCAACGGGACG CCGGCATGCGGCGCGCCGCCCCGCCCCTCGCTGCGCGGCCCAATGGTGAACGCGCCGGCT TCGGGCTGGGCGGTACTGGGCTGGCTGCGGTGGCGCGCGGGCGCGGCACCCGGAAGTCGG CGGCGGTGGCGGAGGCGGTGAGTGCGCGGCTCCGGGGCTGGCCGACTCCGCTAGTGGCCC GGCCGGCCTGGGCTCGGGGGCTCCGGGCTCTGGGCTCTGGGTGCGCGGACCGGGCCAGGC TGCTTGAAGGTGGGTGGGCTGGCCGCACTTCCTCACCCTGCCTCAGTTTCCCCTTTCTGA GGTATGGTATAGAATCAGGCGGCTGGGTCAGGGGCAACCACGTCGATCCCGGCCGACGAA AATGCCCCGCGCGTTACTGGAAGGTGTTTTTGATAATCCCCGCTGGCACGGGATGCGAGG GACTCTGGCAGGTGGGACCAGACTCGCTGCGGGGAGGCTGAGATCGGCAGGGTTGGGAGC TGCGTGGTCCCTGCAGGGTGTGTGGGCTGCTCGGCCTTGGCCAGCATCAGGGACAGCTCT GGCGCCCGGTCACTCTGCCCCCTACCCGCGGCCTGCTGCGGGCCAGCAGGGTGACAGCTA ATGTGGGTGTGAGTAGATAGTAAGTGCTGGGTCATCCCTGCGCCTGGATGCGGCCTTCCT GGGGAGGTTTGGGTAGTTAGATCCCCCACCCCCAACTCCAGAGCGTCAGAGGTAGGGGCT GGCAGGGCTGCACTCAGGGCAGTCCTTGCCCCACCGCTGGGCTTTGTAGCAGCCTGGGCT TTGCCCGTTTTCCTCTAGGAAGTGGGCACAGAGGGCTGTTTGCTGAGACCCCCACCTTCC CTGCCCCTAGCACCTCTCCTGTGCTCCCTTGGCTTCCTGCTGGACCACAAGCTTTAGAGT GAACCGTCTGCCCTTGGACAAGTCCCTGGGCCTCAGGCCCCCTTCCTCTTCAGGAATGTC ACATGCCCTGCCCTGGCCAGGCCTGTTTGCACTGTCGTGTGGCTCTAGACCTGCTGACCA CTGTTTTGCTGCCACTCTGGGCACAGTGCCCTCTTCCATGAGGCAGATGTGACGGCAGCT ATAAGGGGGTAGCTGAGTAGCTCTTTGGAAGGCTTTCTCACGCTAGCTCATTTGCCTCTG GGCAGCCTTTTGGGGCAAGCATTCATTGATCCAGTCCTGCTCATACAAGGAAAACTCAGG CTAAGGTGGCAGATCGTGTTCCCAAGGTCATTCAGAGCCAGGGCCTGCTCCCAGTGCCTC TACTACTCCTGGGCCACTCTCTTAGGAGTGTCCCTGTGTCTGTGTCCTGCGTCTGTGTTG GGTGGGCTGGGAGCAGCTGGAGAGGGTGACCATGTTGTTAGATGGACACAGCAGAGCCTC TGTTCCCACAGGATCCTAGTGCTCTGGTGGTCCAGGGTAGGGTGCCCTGGTGCCCCCACC CCCTTTGACCCCTACACAGCTTTGTGTTACTTGCATGTCCTCAGACTGCAGGCCCCTCAG ACCAAGCCCCTGCCTGCTGTGATGTGTGGTGGCCATAGCCCTTCCCTGAGCTAAGGTGAC CAGCTGTCAGCTCTTCCTGGGCTGCCCAGCCTCTTGTGCTTCCTTGGTCTTCACTTGTGG GCTGTCATTCTGATTACGTTTTGCAGTGTGTGGAATCACGTGCAGTGTCCTGGGAACTTG ATCCTGTGCCCGGCTTCTGCGCCAGACGGCGAACTTGGAGGGCACCCCGGTGCCCACCCA AGGGTGCTGCTTCTGAAAACACCCACAGGGATATGAGGGGCTTCTCAGGGACCCCAGAAG GGGTCTGGAGAGAAATGGCCCGGGATAGGGAGGCTGGGAAGACTTCTCGAAGTAGGGCCA TGCTTGTCATTGACACTCCAGATGCATGAGAAGCAGGGCCCATCCTTTGGTGACGTGTCA GTCAGCACATGGCAGCAGCATGGCAGAGCCTGGCCTTTGTTGGCCCCTACCATAGGGCTT GGGCTGTCTTGAAAACAAGCCTATCCCTACCATGAGGATGGGCTCCGTGAAAACAAGGTC ACCCCAGAAAGACTGAGCTGCTTTTTGAGGTTATCACAGCTTTTTCCCCTTTTTCTCTCA ACTTCCCTTTTAGACTGCAAAAATAGTTTATGCATGCTTAAAGATAAAAAAGGCTGGCCT CCTTCTCCCAGCGGCTGCTCCCCTGGTGCACTTGCTGACATTTGGGGGTACATCCATTGA GATCTTCAGTGCCATGCTAACTTGAAACTTGAAGTGCTTGCCTTCATCCTCTTTGCGGGC ATGGTAGGAGTGCCCCCCTTTTTTTTTGTTTTTACTAACCACTCAGGCCGCAGGGGCCGC TGCGAGGCTGGCCTGTCCCTGGCTTGTGAGGAAGCTGAGTGTTCCTCCCAGGAGGGCCTG AGGATGGGGCCACCCTGTGTCCAGAGCTTGCTGTCTGCTCACGATGCCTCTTGTTTGTGC TATTCCTTGGAACTGGGAAGAGACAGTGATCCTCTGGTGTGAGGCTGAAGAAATGCCCCG GAAGCATTGTGAAGATCTGAGGAGAGAGTACCACTGTAGAAAATGTGTCATGATAGGCCA AGCACGGTGGCTCACGCCTGTAATCCCAGCAGTTTGGGAGGCCGAGGCGGGTGGATCACG AGGCCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATA CAAAAAAATTAGCCGGACGTGGTGGTGGCAGGCACCTGTAGTCCCAGCTACTCGGGAGGC TGAGGCAGGAGAATGGCGTGAACCCAGGAGGCAGAGCTTGCAGTGAGCCGGGATCGTGCC ACTGCACTCCAGCCTGGGCAACAGAGCGAAACTCCATCTCAAAAAAAAAAAAAGAAAATG TGTCATAATAAAATGCCATTTAGGCCCGGCACGGTGGCTCACACCTATAATCCCAGCACT TTGGGAGGCCGAGACAGGTGGATCACCTGAGGTCAGGAGTTCGAGATCAGCCTGGCCAAC ATAGTGAAACCCCGTCCCTACTAAAAACAAAATACAAAAATTTGCTGGGCATGGTGGCAG GTGCCTGTAATCCCAACTACTCGAAAGGCTGAGGCAGGAGAATTGCTTGAACCCAGGAGG CGAACGTTGCAGTGAGAGAGGTCACCCTCTCCAGCTGCCCCCAGCCCACCCAACAGGACC ATTGCACTCCAGCCTGGGCGACAAGAGCAAAACTCCATCTAAAAATAAAAATAAAAAATG CCATTTAATAGCCAGAGAGTGTAAACTGGTGAGCTCTTGCTGCGGAGATAGGAGGGAGCA GGGCATTGCAGTGGACTGGAGACTCAGAGGGTTTCCTGACGTCAGGACTTTCTTCGCCCA GGCCCCGTGAACAGCGACACTGTGTTCCCACGTGTGTTTACCTCCTCTGCTGACAGCCAC CGGGAGCCCGCGTCACATGTCTGTGTCTGCCTTTGAAAAGAGCAACTCGTGCTTTCAAGC TCTGCCCCTGGGGGCTGCCACCGCTGATGGAGTGGGGATTACAAGGGATGGGGATTGGGC GCCCGGCTGCTCAGCCCTGCAGCACCAGCCCTTGTGGCCATTGAGGCCATTAAATGGGGG CAGCAGACTGTAGGGCTTAGTCACCCTCTGTTCAGGGACTCACCATGTGCAGCCCCAATA GGAGCCCGACTTGATGGCTACCCTCCCAAGTGTCTAGGTCCCCTTGACCCTGTGGTACTA ATAGCTTGCCATTCCTCTGTCACCACTGCTTTCTGCTTAGCACACATCCCCTCTGCCCAT GTGCTATGCTTGCCTTTTCGCTTGCCAAAGGGAAGTGGTTCCGCCAAAAGCGTGTGTCCC ATCCCCTCTGGCCCCCAGGGTCTCCAGGTGTGGTGTTGCTGCGACAGCCCAGCTACACCC CGTCCTATGCAGAGCTGTCTTGCACCAGGGCTGATTGGGTCTTCTCTTGCTTTCTGTGAA CAAGTGACTCTTGGGTTCTTGCCCTTCCTTCCCTCTCTTCCCTGCCCATAGAAATGTCCC AGCCCTGGCAGGGAAGGAGGGCAGGATCTGTACATTTTCTTTTTTTTTGCGTTTTGAGAC AGAGTCTTGCTTTGTCACCTCAAAAGGCTGAGGCAGGAGAATCGCTTGAATACCGGAGGC AGAGGTTGCAGTGACCTGAGATCGTGCCACTGCAACCTCTGCCTCTCAGGTTCAATGCGA TTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCGTGCATCACCACGCCCTGCT AATTTTTGTGTTTTTAGTAGAGACGGGGTTTCACCATTTTAGCCAGGCTGGTCTTGAACT CCTGACCTCAGGTGATCCACCCACCTCGGCCTCCCAAAGTGTTGGGATTACAGGTGTGAG CCACTGTGCCTGGCCAGTGCACTTTCTTTTGAGGGTAGACATTACTGTTCCCCGCCGGGA AGCATGCTTACTGGGTGTGCACGCTTTCAGTGTCTGGCGTGACAGATTGTACAAAGTGAA CCCAGCCACACAGCTACCGTCCATATCACACCACCACCAGCACCCAGATATCTCCCTCGT GTCCCCCAGTGGCTGCCTAGACCAGGTGGCCCTGCCCTGCCTCCTGTCCCCATGGGCAGA TGTTGCTTATCTCTGGCCTCTGCATGAAAGACCAAGCAGAGTGCTATCTTCTGGCTGAGA CCTCCCTGCTGCCTCATCACCATGGAAAGGCACAGGCTGGCTTCTTTTCCAGGTTATGGA CTTTCTTACTGTGTCACTTTGTGGACTTGGGCACCTGCAGAGTGCCTGGCATGGTGAGCA GGAGTTGGGGCATGGCGTGATCTAGGCAGGGTTTGGAAGGTTCTTTGAATAGCCAAGGGG GACAGTCTGGAGGGTGCCCTCCAGACACCTGAGAATGCATTGAAGCTAGGGTGACTACTG CAGCATCAGAGGCCTGGTGACACATTTAAGGCCTCAAGGGGCCCATGTGACTAGAAGTCC AGGATGTGGCTGAGGTGGGCCAGACTGGATGGCCAGCGGCCACCTAAGGACAATTGGCTG TGCGTTTGGGAAGCATGGGTGGTCCCCACCTCACAGCAGTCGCACAGGCAGACAGTGGGC AGATCCAAGAGCTTCACCAAGGCTGGGCGGGGTGGCTCACGCCTATAATCCCAGCATTTT GGGAGGCTGAGGCGGGGGGATCATGAGGTCAGGAGTTCAAGACCAGCCTGACCAACATGG TGAAAACCTGTCTTTACTAAAAATACAAAAATTAGCCAGGCATGGTGGCACGCACCTGTA ATGACAGTTACTTGGGAGGCTGAGGCAGGAGAATTGCTTGAACCCAGCAGGTGGAGGTTG TAGTGAGCTGAGATCGCGCCGTTGAACTCCAGCCTGGGCGACATAGCGAGACTTCGTCTC AAAAAAAAAAAAAAAGAGCTTCACCACTGTGCAACCATACAAGGTAGATAAGAAAAAGGA GTTCTCATTTTGGGACAGGGAGGGCCTTTAGCAGGACGGGACATGACTGCTGTACAGTAA TGATGGGAAAATCCCATGTAATATAAGGCTATTTCTACTAGACAGCATCACAGGCAGAGC TGAAAGGCAGGGGATGGCTAGGGAGAAAACAGTTGCAGGCCTGGGACAGGCACATGGTTG CCCACATCACACCAGCTGCACTACGGGTCAGTGAGACAAAGGCCTAGCAGTCAGTACCCA TGTTGGTGAATTGTTGGCAAGAACATACAGGAGGTCACAAGTTGCCCAGCCAGTGGGGGA TGCCCATAAAACCCTGGCGGGAACATTTCCCACACTGCATACACCCTGGAGGCAGGTGAG TGGCAACAGGTTCTGGTGCTAGACGTTAGCGGGGACTTGGCCAAGTCTGTTTATCTGAAA GCGTCACCCATGGATGGAGCAATTCCACCTCTTGCGTCTATCCTAGGAATGTCCTCAGCT GCTCTCCCACTGTTGTAAAGAAATACCTGGGACTGGGTCATTTATTAAAAAAAGAGGTTT AATTGGCTCTCGGTTCTGCAAGGTGCACAGGAAGCATGATCCTGGCATTTGCTCAGCTTC TGGGGAGGCCTCAGGAAACTTACAATCATGGCAGAAGGAAAAGCAGGAGCCAGCACTTCA CACGGCTGGAGCAGGAGGAAGAGAGAGATGGGGGAGGTGCCACACACTTAAAACCAGATC TCGTGAGAACTCTATCACTGTACAGCACCGGGGGGGATGGTGTTAAACTTTCATGAGAAT TCTGCCCCCATGATCCAATCGCCTCCCACCAGGTCCTGCCTCCAACACTGGGAATTAAAT TCAACGTGAGATTTGGTGGGGACACAGATCCAAGCCAAATCAGCCCATGTGCAGAGCATG TGCTAGGGATGGTCGCTTGATGGGACGAAGACACCAGCCACAGCTGAGAGGCAGAGCAGC GGGAAAGCTGGGCAGTGGTGAACCAGTGGGGACATGGCTCTGGTGGAGACAGACCTCATG GCCAATCAGGGAGAACGGAGCAATGGGACCCTAGCGTGTTAACCCCACAGCATACATGTG TCTGCTCACATGTGTGTGCAAGCAGGTTAAGAACACTCTGGCGCCACAGGCAGCCCTTGG CTGAGGCTTTTTTTCCTTTTTTTTTTGAGACTCAGAGTCTTGCCTTGTCACCCAGGCTGG AGTGCAGTGGTGCTATCATAGCTCACTGCAGCCTCAAACTCCTGGGCTCGAGCAATCCTT CTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGATGCATGTCACCATGCTCGGCTAATTT TATTTTATTATTATTTATTTTATTTTACTTTACTTTTTTGAAACGGAGTCTCGCGCTGTC ACCCAGGCTGGAGTGAAGTGGCACAATCTCGGTTCACGGCGCCCTCCGGCTCCTGGGTTC AAGTGATCCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCCTGCACTACCATGC CTGGCTAATTTTTGTATTTTTAGTAGAGACCGGGTTTCACCGTGTTGGCCAGGCTGGTCT TGAATTCCTGACCTCAAATGATCTGCCCGCCTCAGCCTCCCAAAGTGCAGGGATTACAGC CATGAGCCACTGCACCCTACCTTATTTTATTATATTATTAGTATTATATTGAGACAGAGT CTCCCTCTGTTGCTGTGTTGCGGAAGTGCAGTGACATGATCATAATTCACTGCAGCCTTG ATCTTCTAGGCTCAAGCCATCCTCCTACCTCAGCCTCCCAAGTAGTTGGGACCACAGGCA TATGCCACTGCACCTGGCTAATTGTTTTTATTTGCTTTTTGTAGACAGGGACTTGCTGTG TTTCACAGGCTGGACTTAAACTCCTGGCCTCAAACAATCCTCCTGCTTTAGCCTCCCAAA GTGCTGGGATGACAGACATGAGCCCTGAAGGGATGGGGCTTTGGCCCGTTAAATGTCACT GGAAATGTTTCCCTCCAGAATGTGCTCCTGGGTGACCTGAGCGATGGTATTAACAACTAT GGGTCCCACCAGCCTCCAGAGCTGCCAGTCGTGGCTACAGGAGACTCAGCCTTAGGCTGG GGCACAGAGAGGCTGAATAGAGGGTGAATGAACAGGCAGCGTATGGGAGGGACCAGCGTT TCTTGGTATTATACAAAAGCTAGTGGAGGTGGTGCTGGTTGGTGTGGGGTGGGCGACTGG CACGGTCAGATGCCTGATGCCTGTTGGCCTCCTCAGAAGGGGAGGCTGGCAGTAGTGGGT TCTGGGACATGCAGGAGGGTGGCCTGGATTTGCCCTGCAGCACTGAGGTCTGCCTGTTCC CCACTGGGTCCTCCATTTGAGGCCCTGTCAGCATGGCCCTCGTCCCCCTTGCTTCTTTTC CCCATGGGCCTCTGCAAGGTTACTGCCCACCCTCCACCCCACCTACCTGCCTGTGGTGGG CACTGAGAGCCAGGGAAGCAGATCCTCACTGCCAGGCAGCAGGCCATGTGCGCATCCATC AGGGTGCCAGACCTTGTCCCCACGCAGCCCTGGCTGGGGTCACTGCTGCTGCTGCTCTCT GCCTGTGGCTCTGGCCCATCTTTGCCCAGGCGTTCTGTCTGAATGGTTTTCTTGTCGTGC CTTTTTTAGACACCGCATGGAGTGAGGTTTGGGGCTTGCCTCAACTGGAGTCTCATTCTT CTTAGAGAGGGAGTTTAGCCCGTTTCCATTTGGCCTTAGGACAGAAGGCCCTGTTGGTTT CTCTGAGAAGCCAGGGGAGGAGCCCTAGGCTGGAGAGGGGATTCTAGAGCGGGGTGGCTC TCAGAAGCAGAAGGTGGGCAGAGAGGACGGTGCACGCTGGGAGAGTTCTCGAAAGCATCA GCAGATGGGCCCAGAGTGCCTGGAAAGCTTCCGTCCTTCACAGACAGCGCCATGCTCTAA GAATAACCCATGCCAAGTCCAGGCTTTCTTCCTGTTTTGCAGAATTACCAGGTTGTTAGG CTAGGGGAACGTATGCTGGGATTCTGGGAAGAATTCCACCCGCACTGGTATTCTTAGAGG ACGGGAAGCCCTTGGCTGCGGTGTGAAGAGGGCTCTCTCTTTCCATTCATATCTCATAGA TCCTCTGGCCAGGCTTGGGGGTGTTGAGGGACTCCAGGAGTGTGGGGGGAACTTCCTCCC TGGAAGAGGCCTGTGCTCTGTCCCTGCTGAGGCTGGTGGCATTTGCAGTCACATGGTGAG CTCACAGTGACTGTCCCCCTCTTACCTCCCTGGCAGGGTTGGCAAAGACCACACCAAAGT CCCAAGTGATTAGTTCCCCGGCCTCCCTGCACAGAGGCTGTGCATGTCCCCTGGGCATTC ACTCTAGCTGTCTGGGCTGCAGGGAGGCAGGGTGCCTGGCCCAACTGGAGATGGCACTCT ACTGGGGGTGGTTAAGTGTTGACCATCACAAAGGCGACCTCTTTTGTCACATCTGTGTGA GAGGAGCTGGACTTTCTCAGATATGGATGAGCCTCACGACGCACATCCTTTCGTCCCTTG CTTGTCTACCAAGTCCCTATGTGCTCCAGGAGGCAGTGGTACAGGCCAGCCGCTCCGTAT GACCAGAGAACCAGTGCCTCACACTCCAGCGCATTTCCTTCCCAGGGTGCACCAGGACTC TTGTAAAGAGGGCATTCTCGTGTAAGCAGTTGCTTTTTTTTTTTTTTTTTTTAATTTTTA TTTTTTTGAGATGGAGTTTCCCTCTTGTCTCTTAGGCTGGAGTGCAATGGCACGATCTCG GTTCACTGCAACCTCCACCTCGTGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCTCAGTA GCTAGGATTATAGGCACGCACCACCACACCTGGCTAATTTGTTTGTTTGTTTGTTTGTTT GTTTTTTGAGATGGAGTCTTGCTCTGTTGCCCAGGCTGGAGGACAGTGGTACGATCTTGG CTCACTACAACCTCTGCCTCCTGGGTTCAAGCAAGTCTTCTGTCTCAGCCTCCCAAGTAG CTGGGACTACAGGCGCCCGCCACCACGCCCGGCTAATTTTTGTATTTTTAGTAGAGACAG GGTTTCACCATATTGGCCAGGCTGGTCTCGAACTCTTGACCTCGTGATCCACCCACCTTG GCCTCCCAAAGTGCTGGAATTACAGGCGTGATAATTTTTGTATTTTTAGTAAAGATGGGG TTTCACCATGTTGGCCAGGCTGGCCTCAAACTCCTGACCTCAGGTGATCCACCCGCCTTG GCTTCCCAAAGTGTTGGGATTACAGGCGTGAGCCACCTCGCCCAACCAGCAGTTGCTGTA AAATTTTTTAAAAGGATGTCACTTATACTACCCAGTCATTAAGACTTCCTGAGGTGGAAA GATCACTTCAGCCCATGAGTTTGAGAATAGCCTGGGCAACATAATGAGACCCCACCTCTC CAAAAAAATTTAAAAATTAGGGCCAGGCGTGGTGGCTCATGTCTGTAATACCAGCACTTT GGGAGGCCAAGGTGGGTGGGTCACAAGGTCAGGAGATCGAGACCATCCTGGCCAACATGG TGAAACCCCATCTCTACTAAAAATACAAAAATTAGCTGGGTGTGATGGCACGCACCTCTA GTCCCAGCTATTTGAGAGGCTGAGGCAGGAGAATCGCTTGAACCAGGGAGGAGGAGGCTG CAGTGAGCTGAGATCGCGCCGCTGCACTCCAGCCTAGGTGACAGATCGAGACTCTGTCTC AAAAAAAAAAAATTAGCCGGGTGTGGTGGTATGCAGCTGTAATCTTAGCTACTTGGGAGG CTGAGGTGGGAGGATGGCTTGAGCCCAGGAGTTCGAGGCTGCAGTGAGCTATGATCACAC GACTGCACCTGGGCGACAGAGTGGAGGTGATGTGCTTCCTCAAATAGCCATGAGAACAAC GCTTCTTTTCTTGTCCTGGCTCAGGAACCTCACCACCATGCCACGTTGCCCTGCTGCGCT GTGCTGGTGTCGTAGGTCGGTGGCAGGACATTTGCCCACCTCACAGGCTCTGCTTTCAGT CTTGAAGTATGGCTCCTCTCGACTGCTGCCCACACGGCCTCCTGTTGTCAGCCTGGGCTA CATATCTGGGCTCTTCCATCCCTGTGGGCCTGGTGAGGCCTGGCCATGTGCCTGTGTCAT GCCAGTTGCTTTCAAGATGATGTTTCTAGTAGGACCACATGTGAACACAGTGACCACCAT GGCCAGGAGGAAGCTGGCTGCCCATCACCAGCTTGCCCAGGGACCTCTTGCACCCTGGCG GGGATCAGCAGATCCTGCTATGAGAGAGCAGAAGGCAGCATGCTGACTTCCAGGAGTGTC CACAGCGTGGCTGGGCAGAGCAGAGCTACAAATGAACAGATCCAGTGCCGAGGCCATCCT GACTCTCTCAGGGGCTGCCAGGCCGTGGGTGCAGGGTCCAGCAGGCCCACTTGATCCCAG CATCCCACGTGTGCCTGCTCGGGGTGGCCAGCTCACTGCTTCTTCACCTCTAGCCCAACC AGCAGGTGCCCCACCATGAGCCCCTCCAGGCTCACCTTGCCCATCCCTGCCATGTGACCT GGGACTCTTGAGATGAGATCACAGCTGCAGAGCACACAGCACAGGGAGGAGCTCATCAGC ATCTTCCCACCTGTGGTGCCCTGGCCCAGCCTCCTTCCGGTTGGGGTTGGACCCGCCCTG GCAGCCAGGTTTTGCGTCACCTGCCCCACCCGCCCTGTAACCTACTTGCCCAACTGCTAT CCCGGTGGCCGTGAGGCCAGTTGACAGAATGGAGAGGGGTAAGCATTTTCCGCCTGGAGA GGATGGGCGTGTGGCCATCTGACTGCTTCTGCAAGCCTGGTGGTTGTGTGCAGGCTGCCC TGGTGGTCTTAGCCCCTTGGGGCAGGCGGGGTCCTGTGCAGGATGCAGAGACTGGGCGTG GCTTCCTGCAGATGTTCAGATGCTTCTGGGCTGTGGCCTCTGAGACCCAGGGCAAGGCTG CTGACTGGACCCCAGACCCTCCTGCCTGCCTCTTCCTCCAGCTGCTCTTTGTGGTCTGGT TCTGGCCGCCTGGGCCTCAGGGCCAACCCTGCATGGCTTTGACCACTCTGGCCTTCTTGG ACTGCCAAGGACATGCTGAGAGTTGGTTGGGCTTGGCCCAAGTGCTGAGCCCAGGGCAGG GGTGGGGACAGGCAGGGCGGAGCCTCACCTGGAAAGCCAGGCACGATTCTCCCTGGGACA CCAACCCTCCTGTAGAGTTGGCCAATTCGGGCAACCTTGAGGGCGAGCTCTGTGTGGCAC AAGAGATGCCCTGAGGAGCCCAGCCTCACCTGGTGTGATGGGACCTGTGGAAGTTGTGGG AAGTGGTCCTGGCCACCCGTGTGCCCACATGCCCTGCGCCCAGATCAAGATATGGTGACC AAAAGGACAAGGACTATATCCATGGCTCCCTCCCAGAGCCTGGCAAGTCAGCAGGCATGA GAGGGTGTTGGAATGCTGTGGTGGGCCTGTGTGGGGACCTTGTAGGACCTAGGGAACCTG CAGGGCTTGGCTTAGGGAGCACACAGGGGCCCAGGCAAAGGCAAGGTCACAGGTCGGGGG AGGTGAAGCTGGCAGGGGGAGGGGGAGACCTGCTGGCTAGAGCTGGGTTGGGGGCCGGTG GGCAGTGGGCCTGGCTCGAGCAGGGGGCGAGGGATTGGAGAGAAAGGCAGTTCCTGATGG TCCCCTCCCCAGGGGCTGGCTTTCCTCTGGTCCTTCCCTCCCAATGACCGCGTCTTCGTC GAGGCCACAGCCCTTGGCTCTGCGCCCACACCTCCAGTGCCAGGCTGTCCGGAGATCTGT TTATGGCCTTCCCTTGGACCATGGAGCCCTCCCTGCCACTGGTGCCTGGAGGGCTGGTCT GCTGCCCCTGCACCCTGGCCAGCTAGGATGGTGGGGTCCTGCAGCTGAACTGGGGGTGCC TTTCTGCCTGTCTGTCCTACGTGTATCAGTGGCCAGAACTGCCCTCATCCATGGCCTTGC CAGCAGCTGGGCGTGCAGCCCAGGGAGGGCTGAGGTCGGCTGAAGTTGATTGAGACCAAC GGCCTTCTCTAACACCCCAGAGGAGCATGTCCCTGGTGAGCAGGCCTTGGGCAGCCCTGG GCTCCAGGCCAGTGGGATTCCAGTAGAGACCAGCTGGCAGGGCTTCTCCAGTGACTGCAG CCAGGGGTTCTCATGTGCAGGAAAGACCAGCTCCTCCAGAGTAAAGGGACTTGTGTCTTC TGGCCACATGGTGAGTGCTGGGCCCACCCCATCAGTGGCACCTTGGGTGCACTCCCAAAG CCCAGCGTGTTCCCCATCTTCCTGCACTGCATTCGGGATGGTTCTGCATCATCAGCTGCA CCTGATTGCTCTTAAACCCACAGCTGAGCTTTAATGTGCAGTCACTGAAGCTTTCATTTC TAAAATAGCTTCCTAAGAGTTCTTTTGGCCAGGCATGGTGGCTCATGCCTGTAATCCTAA CACTTTGGGAGGCCGAGGTGGGTGGATCACCTGAGGTCAGGAGTTGGAGACCAGCCTGAC CAACATGGAGAAACCCCGTCTCCACTAAAAATACAAAATTAGCTGGGCGTGGTGGCGCAT GCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATTGCTTGAACTCGGGGGGCG GAGGTTGCGGTGAGCCGAGATTATGCCAGCCAGCCTGGGCAACAGGAGCAAAAACAAAAC AACAACAACAAAAAAAAACCGAGTTCCTTTTGCTGCTTTGTAAACCCAGTTTGCTCTTCA TCACTTCCTTTGCCCTGCAGATGTTTCTGAGCTGTCTGTTTTCTTTCTGCCATCAGAGCA TGGTTGGTTGATGATGCATGTACGGTTATCTTTCTGCTGGTTCTTGCTCATGGTGCTTCT TTCATTATGTGGTTCTTTCTGACTATGTACTGCTTCTGGTCTTTGAAAAATGATTTTGAG ACTTCTTTGAAGTATAGGATGAAGGTATCTTTCTCCAGAGAGAACTTCAGGCACAGGGGC ACTACGAGTGCTGGAACCCCTTAAAACTGTGCTTGGCTTGTGGGTCCTTGTACCCCCAGT GTGGACCTGGACGGCTGGCTGTGCCACAGCTTCTCAGAAACTTCCCTTCACTTATGTGCA ATGTCCCCATAACCCCTGGGGGTGGCTGCGGCTGTAGTTCATCTCTCCCTAAGCATCGCT CTTTGGAGTCCCAGCCTAACTTAGAGGAGAGTCTTCTGTTAGGTTCTTCATCCTGTGGGG GTGACTGGACCTTGTCTTCTGTCTCCTTCGCCCCCGAGGCCACCAGAGTATGAGCCCCAG TGTGTTCAGTGGGCAAACGCTCTCAGGCAGGATTGGCCACTGTGTTCTGCTGTCCTCTGT GGGCCACGCTCTCCGAACTGTTGGCCAAATGCTGCATGAGCTCATCAGCTCCTGGCAATT TTTAAGATGTTTTCATATTTCATCCAGCATCTTGAGTTGTTTTTACCAAAAGGATTGCCC CTAGTAGCCTGCCCACCCTCGGATGATGGCCCCCTGTGGGCGCTTCCAGGGCACCCGGCA AGGGGACCCAACCCACTTCCAGCCCCACAGTCTGCTTCCCACAGGGCCCCCTTGCAACAA CACACCCTTTAGCTTCCCATCCTATGCAGAGTGGTGGCCAGCAGACCATCTCAAATGGCT GCCCATCCCCCTGACCCAGTGCGTCTGCACGTGGCGCTTATTGCTATCCCAATACTTCAA CTGCACAGCCACCAGCTCCTCACTGGAGCAGATGGTCCCCAATGGCCATCCCTAATGCCC GTGCCTATGGAGGCACATACACTGAGCCCTCTTCACCTCCGTCCCCACTCATTAGACTTT TCTCAGAGGAATCTTTTTGGGTCTCTTCAGTATTTGAACAGCTTCTTGGCTGTCCCATGT CCCAGCTGTGAGCACTGGCTCTGCCCTGGAGCCGTGTCACTAAATCATTGCACATGTGCA CATTTCAGGGAACTTGGGTGTCACCCTTGGGTGGCCTCGGAATCTAATCTCCGTTCTGCA CTCGTTTGCATTGTGGAAAACTGCCTGCTCTGGGCCCCAGAGGTCCCTGGGTGGGGACGA GGTGTATGCTAAGTGTGCAGTCACCCAGCGTGTCCCCGGCACGTGTCCATGCCAGCTGCT GTGGGTGTGTGGGGCCTTCGGGTCCTGTGGGATGGCGAGTGTGTGTGCTGAGCAGGACCT GTAGGGCCTGCATCCCAGGTGAGTGGGCCATGCTGTTGGAGGCGGAGGCAGGGCTGCCCC CACTGGTGGCGTGTGGGGTGGGTTTGGTGCTGTTGCAGGGGAGGAGATGTGCATGTGGGG GACAGTCTTGAAGGAAGAATGTATACAGTGGTCTATTTCCAAGACAAAGTGTCTTAAATT GGCTTAGCTCAGCAAACTACAGAAGAAACAGGATATACTAGGCCACCCTGCTTGGATAGC TGATGTGTGCTTGTCGGCCTCCACTTTCCCCGCACTTAGTTGCCTTCACCCGAACCAAAG AAGTTCAGTCTAAGCTGAAAGTTTACTAGCCTGCAAAATAGCTCATTTTGTCTGTTCTTA TCAGCCTGCCTGCTACTTAGGTCGTAAGTTAAATACTTAAAAAGTCCCTGAGCTAACTAG GACTGCAATGCATTGTGGGCTGCAACAAAATACAGCAAGACAAGCCCCCAAAAAAACACC TGAAGCCCCTACCCAACAATTAATAGGCGACGTCCGGGAAAATTGTGACCCCACAGTACT CGGCCTATGAGGAACTGGGGGAGGGACCTGCGTACTAGGGGATAAATTGCTTGTCGAAAC TGTGCTGGGTGTGCCTGTCTGACACCTGATCTTGCAAGACTGTCATTAAAAGTCTCACTT TCGCTGTTCTCTGGGTCTGAGTTCATTCTTTAGATTTGGATGGGTAAGTTTGTTTCTCAC AGTGTGGCTCCTGCCCTCACTTCTGCTGCCCCCCGGGGACCCCCTCGGCACCCCAGCTCG GTGTGTCCAGGCCTTAAGACTACCATGGTGCTGATGGGATGCTTCTGTTCAGGGCTGGTG TTGCAGGTGGTGGAGAGACAGTCTGGCCAGTGCCAATGAGTGCATGGGTTTGGGAGTTGT TTGTGTGGCACCGGCCAAGAGTGTGGTGTCCAGTTCCCCCACACCCAGCAAGTAGTACAG ACACCACAGAGGTGGTTCTCTCTGTTCTGGCCTGTTGCAGGTTCGGAGGGCAGCCCTGAG TGTCTGCCATCCGCTCAACTCAGTGTTTTCCTTTTCCCGCAGACCTCGCGACCTGTGTCA GCAGAGCCGCCCTGCACCACCATGTGCATCATCTTCTTTAAGTTTGATCCTCGCCCTGTT TCCAAAAACGCGTACAGGTAACCCCCTCGCTCTGCATCTGCTGCGCCCTGCAGGGTCCTG GGTGCCCAGCCAGTTCTCATGCCACCCAAGCTGCTGTGTGCAGGAAGGTGTGTGGGCCAG GACGGGGCTGCACAGGCCTGGCACTGCCCTCCAGGACAGGGTCACTCAGTGTGGGATGCT GTCAGAATGCCTCTCGGGGCGGGGACTCCAGTCAATGTACAAAGACGTGAAGACTCAGCC ACAGAAGGCAGCCACAGGTAGGACAGAGGAGTGACATGGGTCCAGGTGGGCTGCAGTTCT ATGGGCTTTGAGGAGGGTCGGGCGGCAGAACTGGGACAACGGCGTCAGTGAGTGAGAGCC AGCTTGGGCTGGCGGGTCACAGCTGCTGGTGAGAAGTCAAAGTGGCGGCCAGGCTAGGGG TCTGTGGGTGGGTCCTGAGCTGAGGAAAAAGAACCTGGGGAAGAGGGGACGCTGAGGACC AGCCAGAGGAGTCAGGAAATCGAGGCCAGAAGCCACCCCTGACCCTGGACTCAGGACGGC CACACCTCCATTCCCCAGGACACCCCAGCATTTATCCACACAGAGAACGAGGCAGAGATA TAACCCACATCTAAAATGAAACGTGTAACCACTCTCTAATGCAACTGCTCGGGAAGTTTT CATCATTTTAAAATCTGTACCAGAAATGATTTGAACCATTTCAAGAGGGTCTCAGATTTG AGTCACTGCAAAGTTGCATAGACACCATGCTGCTTGACTTCAAGGCTGAGTGAAGTCATT TCAGGCCAAGGAATCAGGAAGGAAGGGTGTCTGGTGTTTTCCCTTTTTGCAGTTGGGTGA GTGGCAGGTCCCAGCAGCAGACAGCATGCTGCAGGGAAGGTGGAAGAAATGTTAGGAAAG AACTATTGACTGCTACAGGGACTGGAGTAGTGTTGGGCTGGCTAGTGAGTGAACAGAACT CTAAAGAATGTAGGGGCTGGCCGAGCACGGTGGCTCACGCCTGTAATCCCAGCACTTTGG GAGGCCAAGGTGGGCGGATCACAAGGTCAGGAGATCCAGACCATCCTGGATAACACATCT CTACTAAAAACACACAAAAAAATTAGCTGGGCATGGGGGTGGGCGCCTGTAGTCCCAGCC ACTTGGGAGGCTGAGACAAGAGAATGGCGTGAACCTGGGAGGCAGAGCTTGCAGTGAGCC GAGATTGCGCCACTGCACTCCAGCATGGGCAACAGAGCGAGACTGCATCTCAAAAAAAAG ATGAAGGTAAAATAAGGTCATGTGGGTAGGCCCTAATCAATGTGATGGGTGACTCAGATA CGCACAGGGGAAGCTGGTGTGTATACAGAGATAGAAGCAGTCGTCTGCATACCGAGAAGA CCCTCAAAAGGAGCCAGCCCTGCTGACGCCTTGATCTTGGATCTCCAGCATCCAATGCTG CGAGACAATAGTGTCTGTTGTTGCAGCCGCCTGGTCTGTGGTGCTTAGTTACGGCAGCCC CAGGAAACTAGCACGCTGAGGAGAGCCAGCCAGCTCTGTATGCGTTTCAGGGCTGCCTCC TGCAGGCTGCTGAAGAGACTCAACCCACCCCGAGTGCTCCCATCTCTGGAAAGGAAGCTT GATGGCTGGCCTGCGGGTGCTGGTCAGTGCCACCCAGCCCCTTGGGTGGAGAGGAGGAGG GTTGACATGGGCCCCTCTTGCTTTCACTCAGAACCCGTGTGTTGCTGCCAGGACCTGTCC TGCGGGTCCTGCCATTCCAGTTGGCCTCGGGATTTCTCTGGACTATTTTCCGAAGCCCTA AGCCAAAGACGAAATGTGCCACCTCTGGGTGACTGAGCAAATGTGCGCTGTGTTTTCCTG ACTTCATGGGAGGGTGTGAGACACTTCTGGTGGCTGCTTCTGCTCTGCCTGTGGGGTGTC CAGCCAGGGGAAGGGCACGCTGCAGGCTGGGAGCACAGACACTTCTATGGAGCCCCTGAC AGCACCTCTGCAGCACTTTGGGAGGCCTCGTACAGGTACCCAGCTAGGCTGGGCTGGACT CCTCACCTGTGGACTGGAGTAAAAAGTAGGGTGCTTTAGGCCACTATGTTTTTTTTTTTT ATTATTATTATTATTATTTTAGAGACAGGATCTTGCTCTGTTGCCCAGGCTGGAGTACAG TGGTATGATCATAGCTTACTGCAGCCTCAAACTCGCCGGCTCAAGCGATCCTCCCACTTC AGCATGCCTAAGTAACTGGGACTACAACACCATGCCCGGCTAATTTTTTTTTTTTTGGTA GAGACAAGTTCTCACTATGTTGTCCAGGGTGGTCTCGAACTCCTGGGCCCAAGTGATCCT CCTGCCTCAGCCTCCCAAAATGCTGGGATTACATGCGTGAGCCACCATGCCCGGCCTCGG CCACTGTATTTGGTAATTTGATACCCATCACGGAAAACCAAGACCCTGCAGCAAGATAAC ACAATCACTTCATGATGTCACCCTATCTCTTTCAAAACTGCCTGTTTACAGCCGGGTGCG GTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGTGGGTGGATCACTTGAGGT CAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATACAAA AATTAGCCTGGCATGGTGGCGCACACCTGTAATCCCAGCTATTAGGGAGGCTAAGGCAGG AGAATTGCTTGAGCCTGGGAGGCAGAGGTTACAGGGAACTGAGATAGCGCCACTGCACTC CAGCCTGGATGACAGAGTGAGACTCCATCTTAAAAAAAAGAAAAAACGAAAAAAAACCTG CCTGTTTACAGCCCCACGTGATGCCACTTGCTGGCTTCAGCCTAGAGTGGCTGCAGGACA AGCTGTTACTCCATGGGACAGCTTCCATGGGGTCACCACTGAGGGTTTGATCACTGGATC TCAGGCCAGAGTACCTCCTATCTGTCACCAAACTGTCCCCCAGACAGTGCACACACTCAC CCCCCGGCAGAACACACTCACCCCCCGAGATGATGCACATACCCCCCGGACAGTACACGC ACTCACCCTGCCTAGCAGCGTGGGTGTCAGGTGGTGATGGATAATTAGTGGTTTCAGTTA TTGGAACAAAATAAAGCCGAGGTGACTTGCCATCCTCAGGGACCTCACCGTGGTGGGTGC ATCCCTGTCTCTCTGGACGTGGGGCGCAGATAGGCTTCTACCCATGTGCAGCCCCACCAG TGATGACTGAGAGAGCTACTTCCTCCTCCCTTTGGGGCTTGATCTGGGTGCTCAGCATGC TGGCTTGTCCCTGAATTATGTATTCAATGAAATCCCTGGCAAACCACCAGCAATTCAGCT AAACATGACTGCTGGAACAAAAATAACCTGGCAATTAATGTTCCTGAGTTATTAGGCAAA CATATATAATTGCTCCTTGTTTAAAAACAGACAGCTGAACATCTTAGGACAAAAGTCCAT GCTCAGTCAGCTTTTCTTCCTACTTTTCTCCCCTTTATTTTCCCTTTGTAAGAGCATTAT GTCAGAATCAGCACCCGAAGCCAACAGCCCCAGCCTGGTTTGGGGCTGGACTGGGATGAG GTGTCCCTGGCTCCCCCGACCCCTGTTCACCTGCAGGGCTGTGGGACGTGGAAGCAGAGG AGGAAGGTATTTAAGAGAGAATGCTGAGCAGAACCACATGGAGCTTGACCCCACCCATAG GGTGGAGGAGCTGTCATCCCAGACCCTGGCACATCCCCACCATAGCACTCAGGGACTGCG GTGTTCCCCCTTCTCCTGCCCGCTGGCCAGGACACAACCAATGGGCACCTACAGGACTGG CCCATCCAGGTCTGGGGCAAGTGCACTGCCTTCTACTAGCACTGTCCAGCCCAGGGAAGG TGGGCTCTCCTGCCATCCCTGCTGCAGAGGTTGGGGGGCACTTCTGCCCAGAAGGGACAT GGAGACTTGGGCTGGGTCTGGGTTGAAACATGGCAGTTAGAGGTACAAAGGCTGAGGCTC ATCAGGGACAGAGACCACCTCCCCCTACCCCAGGCAGCACATCCCTGAGACCTGGTGCAG CCCTGGGGCCAGCTGGGTGTCAGACCAGGCTGAATGGGGCACCCTGGAGGTGGTAAGGCT TCCATAGCAGACCCCAAGGGCTCTAGGGCATGGCACAGTCCCTGGCAATGACCAAAAGCA TTTCCCCAGTTCTGCTGGCAAGAAGCTTCAGGGGCAGGCACTGAGTGGGGGGCACACATG TGCACACAGGTGTGAATGCCTCCAAGGGCATGAATGGTGGGTGCTAGCACCATGTACTTT TTTGTTTGAAATGGAGTTTCGTTCTGTCGCCCAGGCTAGAGTGCAGTGGCACGATCTCAG CTCCCTGCAACCTCCGCCTCCCGGGTTCAAGCAATTCTCTGCCTCAACCTCTCGAGCAGC TGGGATTACAGGCGCCCGCCACCACACCCGGCTAATTTTTTTTTTTTTTTTTTGAGACGG AGTCTTGCTCTGTCACTCAGGCTGGAGTACAGTAGCATGATCTCGGCTCACTGCAAACTC TGCCTCCCGGGTTCACACCATTGTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGC GCCCGCCACCACACCTGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTTACCGTGT TAGCCAGGATGGTCTCGATCTCTTGACCTCGTGATCCTCCCACCTTGGCCTCTCAAAGTG CTGGGATTACAGGCGTGAGCCACCGGGCCCGGCCTAATTTTTGTATTTTTAATAGAGATG GGGTTTCACCATCTTGGCCAGGCTGGTCTTGAACTCCTGACCTCATGATCCACCCACCTC GGCCTCGCAAAATGCTGGGATTACAGGCATGAGCCACCGTACCCAGCTGTGAGCACCACT TTCACTCCCAATTTTATGAATGTCTGTTATTTCACATTTAATCAGGAGCAAATGTTGTTT TATGAAATTGATGTGCAATGATCAATATGGAAATTATTTTTAAACACTTGTCGGGCTCTC ACCACTGCAACTTGGTTTCAGACTCAGATTGTGATGGCAGGACAGCTGAGCTCTGCTCCT TGGAGCTGCCTCTCCGGCACAAGTGCTCAGGGGGTCTTCAGCCATCACTTTTTTTTTTTT TTTAGAGACAGGGCTTCTCTGTTCCCCAGGTTGGAGTGCAATGGTGCAGTCACAGTTCAC TGCAGCCTCAAACTCCTGGGCTCAAATGATCCTGCTGCCTCAGCCTCCCCAGTAGTTGGG AATACAGGCATGCACCACCACACCTGACTAATTTTTGTATTTTTTTTTTGTAGATATGGG GTCTTACTTTCTTGCCCAAGCTGGTCTCAAAACTCCTAGCCTCAAGTGATCCTCCCACTT TGGCCTCCCAAACTGCTGGGATTACAGGTGTCATCCACTGTGCCTGGCCCAGCCATCGCT TCTTAGACCCTCAGGTCTAATGGACATTGCAGGAACTGACCCCTTCCACTCAGTCACTGC TGTGTCCTTATTTGTTTTCTTCTAATGTCCAAGCTGGGACTTGAAAGTCAGTTTTCTTTC CTCAAGACCAGGGCTCTTAAAGTTTAGATGTCTCTCCTAATTCCAAAAGAAATATTTGTT TGATTAGGCTGGATGTGGTGGCTCACACCTGTAATCCTCCGCCTCGGAGGCCGAGGCGGG TAGATCATTTGAGGTCAGGAGTTCAAGACCAGCTTGGCCAACATGGTGAAACCCCATCTC TACTAAAAATACAAAAAATTAGCTGGGTGTGGTGGCGGGTGCCTGTAATCCCAGCTACTC AGGAGGCTGAGGCGGGAGAATAGCTTGAACCCGGGAGGCGGGAGTTGCAGTGAGCCGATG TCACGCCACTGCACTCTAGCCTGGGTGACACAGAGAGACTCCATCTCAAAAAATAAAAAT AAATAAAAATGGAAATATTTGTTTGATTCATAATGCAAAAAAACTAGTAAAACAGTGAAG TTCCCATCCACTCCCAACCAACCCGAGAAATTAACTCCAAAACACACAGAATGGAGCAGC CCTCAGAGGCCCACATGCTGTGCCATTCAATCTGTATCCTTGAGCTCACGCTTCCAAGAG CCCACGGGTTTGTTGTGGGTGTTCCCAGGGCTGGGGACAGATGGACAGAAGGAGGAACAG CCCCCCACCCTCCAGCCTGGGCTCCCGGCATGGAGCCCAGGCTCCATGGTGCCCCTAAGG GCCTGGCAGACCCGTCTCCCGCCATTGAATGGTGTGGATGTGTGTCCCTGCCTAGCATCC TCCTGTTTCCCGTGCGGTCCCAGCTGCAGGCTCCCGAGAGCTCACCTGGAGCCACGGTGC TCCTTCCTCCAGTGTGGGTGCAGCAGGACTGCCGGCTTCCTGCCCTCTCTGAGACTGGCC CTTGTTGCCACTGAGGCCAGTTTTGTGTGTGAAAAGAGAAAAAGACTTGGCTCGCTCGTT TCCATCTGAAGCCATCAGTGATGCTTTCCTCTTGCAGGCTCATCTTGGCAGCCAACAGGG ATGAATTCTACAGCCGACCCTCCAAGTTAGCTGACTTCTGGGGGAACAACAACGAGATCC TCAGTGGTGAGTCTTCCTGCGTGCTCAGCGGTGGCTGCGCCTTGTTGCTTCCCTAGCCCC TGCGTGGCCCGAGTGACCCTAACTGAGTGGTGCTGCTTCCAAGTGTGATGGCGGGGTGTA GAGGTGGAACTGATGGGGCTCGTGGTCGGGGCCAGCCCCAGCACCGGGCATTGCCAGAGC TGCCCACCCAGCACTGTCTGCGTGGCTTCCTGTGCCATGCGTCTGCATGGGGGCCCCATC ATGTGAAAGCATCAGTTGGTTTTATATGGATCCCTCCTTTTTGCTGAGGGCATGTGGGTG GATTTGTGTGATGTACAGATCCACACTCAGTCACTTTGTGAAGCAACCCAGTCATATGAG TGTGTGCATGCGTGTGTATGCGTGCGTGTGTGTGCCTGTGTTTGTGTTTCTGTTTGTATA TGAGCACCCTTGCTTCAGGGATGGTGGGGCTGAGTTTGGGAAGCTGGTGGTGCTGAAGCC CCGTCACCTGCACCATGCCTGTGTTGCATTAGGTGTACTTATAGGGCAGAAGTAGGGGTG AGAACCAGGTCCTCCCCACCAAAGATACCAGCCTCATTGAAGTCCTGTTGGACAGGAACC AGTTCCTGCCCATGGGGTGGTGGGAGTCTACCTGGAAGGTGCGCATAGATGGCTGGTGAG GCCACGGGTTACTCTGCCTGCGTACCAGGCGCCAAGTGGGCCCGAGGCATCCTGGCATGC TCCAGGGTGCCCACTCAGGCCAGATGTGTGACCTCGTGGGCCCATGGAGGCTGTGTGTGC CCTGTGCTGCCTCCCAGGCCGGGAGGGCAGGTGTTGGGATTATAGGTGTGAACCCAACAC TTAAGGAGGCTGAGGCAGGAGGATTACTTGAGCCCAGGAGTTCAAGACCAGTCTGGGCAA CATGGTAAAACCCTGTCTCTACAAAAAAAAATACAAAAATTAGCCAGGCGTGGTGGCATG TGCCTGTGGCCCCAGCTACTCAGGAGGCTGAGGCTAGAGAATTGCCTGAGCCCGGAAAGC AGAGGTTTCAGTGAGCTGAGATTGTGCCACTGCACTCCAGCATGGGTGACAGAGCGAGAC CCTGTCTCATTCAATCAGTCAATCAATCAATCAAATAAAAGCTCAGCTGTGGGGTTTTGT GAGGACTCCTGTGTAGGCCTGTGGCCTGGCAGTAGTGTGAGAATGCTGCTGAGGCTGGGT GGTCCCGGCACCGTGTGGGAGGAGGACGGGTGCATGGAGACTGAGGCAGGGTGGTCCTAG GCCTGCTGGTATAGGAGGATGCAGTGGGTGCAGGGCTGGTCAGGACGGGTGGGTGAAGGC ACAAGACTGATGTCCTTGGAAGGTGGAAGGTTTTCATAGATGAAGATGTGGCGGCTCAGA GGGTTACCCTCTTCTGGTTCCTTGACTATTGTGCCCTCCTGGGCTCCATCCCACCCTGGC ACCCAGGTCTGTCAGCTGCCTCTGAACTAGTGCCAACCCCAGCAGCTCCAGTCTTGTGGA ATTGTTCACCCCTGTGGTGTTTTGATGGTGGCAGCTGGTGCATGGATGTACACGGGCTTC TGCCAAGTGTCGGGGTGACCATATCACTAAGGCCCACATCCTCTCACTCTCTGGCGTCAC CGGATTGTATGCAAAAGTTTAGGACAGTGGTTCTCCAGGTGTGGTCCTTGGGCCTGTAGC ACCAGCTTTACTTGAGAACTTGTTAGAAATGAAATCACCCTGGGCCGGTGGCTCACACCT GTAATCTCAGCACTTTGGGAGTCCAAGGCAGGAGGATCCCTTGAGGCCAGGAGCTTGAGA CCGGCCTGGGCAACGTAGTGAGTCCCTGTCTTTACAAAAAAAAAAAAAAAAATTAGCCAG CTGTGGTGGCATATGCCTGTTGTCCCAGTTACTCAGGAGGCTGAGGTGGGAGGATCGCCT GAGCCTGGAAGGTAGAAGCTGCAGTGAACCGAGACTGAGCCACTGCAGTTAGCCTAGGCA ACAGAGTGAGACCCTGTCTGAAAAAAAAGAAAAGAAATGCAAATCAAAATCATTAAATTC ATAAAAATTTGGCCATCACTTCTTTTTTTTTTTTTTTTTTTTTGAGATGGAATTTTGCTC TTGTTGCCCAGGCTGGAGTGCAATGGTGCCATCTCAGCTCACCGCAACCTCCATCTCCCG GGTTCAAGCTGTTCTCCTGCTTCAGCCTTCCTAATAGCTGGGATTACAGGCATGCACCAC CACACCAGGCTAATTTTGTATTTTTAGTAGAGATGGGGTTTCTCCATGTTGGTCAGGCTG GCCTTGAACTCCCGACCTCAGGTGATCCGCCCGCCTTGTCCTCCCAAAGTGCTGGGATTA GAAGCATGAGCCATTGCACCCAGCGGCCATTACTTTTTCAAGTAGTTTTTCTGTCTTCTC TCTCACTCTCTCCTTCTCTCCAGTTACACACATATATTAGTTCACTTGCCCTTGTCCCAC ACCTCTGCGATGCTCTCTGCGTACTTTTCTCGGTGCCCCTAGATCTTGAGGTTTTCCAGC CTGGTGTCTGGGAGCCAGCCCTACTCCCTGCCCTGTGTGAGCACTGGTGGTTCTTGCCCC AGTGTCATATAGTCTCCTCCCTGAGATGCTGATCAGACATTCTCTGTGTAGCTCCTTCCT CTCTGGACTTTTGTGCCACTGTCTTAGTTCGTTTGTGTTGCTCTAAAGGAAAAAGCTGAG TCTGGGTAATTTTTTTCTTTTTTTTTGAGATAGGGTCTCACTCTGTTGCCCAGGCTGGAG TGCAGTGGCACAATCCTGTCTCACTGCAGCCTCTAACTCCTGGCCTCAAGTGATCCTCCC ACCTCAGCCTCCCTAGTAGCTGGGACTACAAGTGCACATCACCATGCCCAGCTAATTTTT AAAAATAATTTGTAGCGACAAAGCCTTGCTGTGTTGCACAAGCTGGTTTCAAAGTCCTGG CCTGAAGTGACCCTCCTTTCTTGGCCTCCGAAAGTGCTGGGATTACAGATAATAAGCCAT GGTGCCCAGCCAAGGCTGGGTAATTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTTGC TCTGTCGCTTAGGCTGGAGTGCAATGGTGCGACCTTGGCTCACTGCAACCTCCACCTCCC AGGTTCAAGTGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCACCTGCCA CCACACCCTGCTAATTTTTGTATTTTTGTATTTTTTTATTATTATTCTTTAAGTTCTAGG GTACATGTGCAGAACGTGCAGGTTTGTTACATAGGTATACATACATGTGCCATGTTGGTT TGCTGCACCCATCAACTCATCATTTACATTGGGTATTTCTCCTAACGCTATCCTTCCCCC AGCCCAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCAGGTTGGCTGGGCTGGTCTC AAACTCCTGACCTCAAGTGATCCACAAGGCTGGGTAATTTATAAAGAAAAGAGGTTTATT TGGCTTAGAGTTCTACAGGGGACCTGAAGCACAGTGTCAGCATCTGCTTCTGATGCAGGC CTCAGGAAGCTTCTACTGATGGTGGAAGGGGAAGGGGAGGTGGTATGTGCAGATCACACA CGGAGGGAGGAAGCCAGAGAGAGGGAGGAGGCGCCACGCTCTTTTAAACAACCAGTTCTC AAAGGAACTAATAGAGCTAGAACTCATCACTGTGAGGACAACACCAAGCCGTTCATGAAG GATCTGCCATTAGGTAGATCTGTCTCCCATTAGACCCCACCTCCAACACTGGGGATCAGT TTTTTGGTTTGTTTGTTTTTTTTTTTTTTTTTTGAGACAGAGTTTCACTCTTGTTGCCCA GGCTGGAGTGCAATGGCACGATCTCGGCTCACCACAAGCTCCGCCTCCAAGGTTCAAGCA ATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCGCCTGCCACCATGCCCAGC TAATTTTTTGTGTTTTTAGTAGAGACGGGGTTTCACCATGTTGGTCAGGCTGGTCTCGAA CTCTTGACCTCAGGCAATCCACCCGCCTCAGCCTCCCAAAATGCTGAGATTAGAGGCGTG AGCCACCTCACCCAGCCCGGGGATCAGATTTTAACATGAGGTCTGGGGGACAAACATCCA AACTATGGCAGCCATGAACTCTGGTTGTCTTAGTTTCCCCAAACACTAAGCCCCTCTTCT CAGCTCAGGGAGTCTGCTGAATTCGGCCTGGTCCCCCCACTCCCTGGGCTGGGTCCTGGA GCCTCTCTAATCATGGGCTCACTTTGTTTCCTGTCTGTCATATCCTTTTTTGTAAAACCC ATTTGATATGGTTTGGCTGTGTCTCCACCCAAATCTCATCTTGAATTATAGTTCCCCAAA TCCCCACATGTCCTGGGAGAGACCCAGTGGGAGGTAATTGAATCATGGGGCATTACCCCC ATGCTGCTATTCTCATGATAGTGAGTTCTCATGAGATCTGATAGTGTTTTGTTTTTTTTT TTTTGAGACTGAGTTTCGCTCTTGTTACCCAGGCTGGAGTGCAATGGTGCGATCTCGGCT CACTGCAACCTCCACCTCCCAGGTTCAAGCAATTCTCCTGCCTCAGCCTCCTGAGTAGCT GGAATTACAGCCGCTTGCCACCACGCCCAGCTAATTTTTGTATTTTTAGTAGAGATGGGG TTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCCGCCTGCCTCG GCCTTCCGAAGTGCTGACATTACAGGCATGAGCTGCCGCTCCCAGCCAAGATCTGATGGT TTTATAAGGGGCTTTTCCTGCTTTGCTTGGAACTTCTCCTTCCTGCCACCATGTGAAGAA GGACATGTTTGCTTCCCCTTCTGCCATGATTGTAAGTTTCCTGAGGCCTCCCCAGCCATG CTGAACTATGAGTCACATATCTTTCCTTTATAAATTACCCAGTCTCGGGTATGTCCTTGT AGCAGCATGAGAATGGACTAATAAATACACCATTGTTTCTTGAGTTTTATTTATTTATTT ATTTTTTGGTTGTTTTATGCAGGAGGGTAAATCTGGTCCTTGTTGCTTGGGCAATTTGGA AGTTTTGTTCTTGTTTTAATTATTGATTTGTAAGAGTTATTTACCCATTCTGGATATGAG TCCTTTGTGCCTACTCATTTCCTTAATGATAGCTTATGGTGAGCAGAATTTTTTTTTTTT CTTTTGAGTGGGTGGAGTCTTACTCTGTTGCCAGGCTGGAGTACAGTGGTGCGATCTCAG CTCACTGCAACCTCCGCCTCCCAGGTTCAAGTGATTCTTCTGCCTCAGCCTCCTGAGTAG CTGGGACTACAAGCATGCACCACCATGCCCAGCTAATTTTTGTATTTTTAGTAGAGATGG GGTTTCACCATGTTGGCCAGGATGGGCTCAATCTCTTGACCTCTTGATCTGCCTGCCTTG GCCTCCCAAAGTGGTAGGATTACAGGCGTGAGCCACTGCGCCCGGCCAGAATATTTCATT TTAACAAAGTACAGTTTATCTTATCTTTTATGAGTTGTTCTTTTTGTGCCTTATCCAAAA AGTCTTTGCCTACCCAGAGGAAAATATATAACAATATTTTCCTCCATTTGCTTCTAGAAG CCTAATTTTTGTTTTTATGTTTGGTCTATGATTTATCTCAAGGTTTTGTACATGGTGTGA GGTAGGGGTTGAGATTTACTGCCTCCAACCCCCAAATCTCTCTAGTTGTTCCAGCATCAT TTATTAAATGGATTATCTTTTGCCTAGTTCATTACTATGGCAACTTGGTTGTACATCAGT TGAACATATATGTGTGGGTCTATTCCTGACTCTGTTGTATCCCATTGATCTATGTATCTG TCTTGATGCCAACACCATGCAGTCTATGAGTGCTGCTGCTTTTTAGTAAGTCTTGAAATC AGGCTGAGCACTGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGACCGAGGTGGGCAG ATCACCTGAGGTTGGGAGTTCAAGACCAGCCTGACCAACATGGAGAAACCCCGTCTCTAC TAAAAATACAAAATTAGCTGGGTGTGGTGGTCCATGCCTGTAATCCCAGCTACTCGGGAG GCTGAGGCAGGAGAATCACTTGAACCCGGGAGGCGGAGGTTGCGGTGAGCTGAGATCACG CCATTGCACTCCAGCCTGGGTAACAAGAGCGAAACTCTGTCTCAAAAAAAAAAAAAAAAA GAAAAAAGAAATCAGGCAATGTGGGTCTGCCTTCTTTTTCGGGACTATTTTGGCCATCCC AGTTCCTTTGCATATGCATAGATATTTCAGCCAGTCTGTTAATTGCTATAAAACGAAAAG CCCCCTGGGATTTTGACTGGGCAGGTTTTGAATCTATGGGTCATGTGGGGAGAAGTGTCA CCTTAACAATACTGAGTTGTCTAAGAAGTACCCAGTGAAACAAATAATAACAAGAAGAAA CACTGTTGAGGCTGGGCACGGTGGCTCACGCCTGTAATTCCAGCACTTTGGAAGGCCGAG GCGGGCCGATCATGAGATCAGGAGACCGAGACCATCTTGGCTAACACGGTGAAACCCTGT CTCTACTAAAAAAATATGAAAAAATTAGCTAGGTGTGGTGGTGGGCACCTGTAGTCCCAG CTACTTGGGAGGCTGAGGCAGGAGAATGGCCTGAACCCGGGAGGCGGAGCTTGCAGTGAA CTGAGATCACACCACTGCACTCCAGCCTAGGCGACAGAGCAAGACTCTAAAAAACAAAAA CAAACAAAAAAACACTATTGAATCTTTCAGTGAATATGCATGTCTCTCCTTAAATCAGGT CAGCTCAACTTGAGACCCTCAACAGTGCCCTGGAGTTTTCAGTCTTTGTCACTCATTTTT CATTAAATATTTTCCTGGTGGTTTTTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTTGGT CTGTTGCCCAGGCTAGAGTGCAATGGCGCCATCTCTGCTCATTGCAACCTCCACCTCCCG GGTTCCAGCAATTCTCCTGTCTCAGCCTCCCGAGTAGCTGGAACTACAGGTGCCTGCCAC CATGCCCAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCATCATATTGGCCAGGCT GGTCTCGAACTCCTGACCTCGTGATCCGCCCGCCTTGGCCTCCCAAAGTGCTGGGATTAC AGGCGTGAGCCACTGCACCCGGCTTTTTTTTTTTTTTTTAATCACACTAAGGATTTTAAA AATGCTGTTATAAATGGTATATTTAAATGCTCACTTTCCAGTTCTTGCTAGCATTTACTT ATTAGTTCTAATACATCGGTTTTGGTATTCCTTTTGGGTTTTCTTCATATATGATCCTGC TGGTTGCAAATACAGTTTTGTTGGGTTTTTTTTTTTTTTTTGAGACAGAGTTTCGCTGAG AGTGCAATGGTGTGATCTCGGCTCACTGCAACCTCCACCTCCCGGGTTCAAGCAATTCCC CTGCCTCAGCCCCCCGAGTAGCTGGGATTATAGGCTGCCTCAGCCTCCTAAGTAGTTGGG ATTACAGGCATGCACCACCACGCCTGGCTAATTTTGTATCTTTAATAGAAATGGTGTTTC TCCATGTTGGTCAGGCTGGTCTTGAACTCCCAACCTCAGGTGATCTGCCCGCCTCGGCCT CCCAAAGGGCTGGGATTACAGGTGTGAGTCACCGCACGGGGCCACAAATACAGTTTTATT TCTTTATTTTTGATCTTTACATCTTTTCTTTTTCTGTTATAATTAATTATTGTTATAAAA ATAATATCTTAGGGCCAGGCACGGTGGCTCACACCTGTAATCCCAGCACTTTGGGACGCC AAGGCGGACGGATCACCTGAGGTTGGGAATTCGAGACCAGCCTGGCCAACATGGAGAAAC TCCGTTTCTACTAAAAATACAAAATTAGCCAGGTGTGATGGCACATGCCTGTAATTCCAG CTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAGGGAGAGGTTGCAGTGAG CCGAGAGCACACCATTGCACTCCAGCCTGGGCAACAAGAGCGAAACTCTGTCTCAAAAAG TAATAATAACAATAATAAATAAAATAACATCCTAGCCAGTGCAGTGGCTCACTCCTGTAA TCCCAGCACTTTGGGAGGCTGAGTCGGGAGGATCGCTTGAGCCCAGGAGTTCGAGACAAG CCTGGGCAACACAGCAAAACTTTGTCTGTACAAAAAAATGCAAAAATCAGCCGAGTGTGA TGGTACCTGCCTGTGGTCCCAGCTACTTGGGCTGAGGTGGGAGGATCGCTTGAGCCTGGG AGGTGGAGGTTGCAGTGAACTGTGTGATTGCCACTGAACTCCAGCTTGGGTGACAGAGTG AGACTCTGTCTCAAAAAAATAAAAAATGAAATAAAATAATAAAAATAACATCTTAATGAC AGCCTTTTTTGCCCCAATCTTTGGGTAAAATGACCCCCTGGTCTAAGAAGGAGCCCTCCT GTGGCTTTCAGAGCCCTCCAACCTTGTGGCATTAGCAGTGGCCCATGCCCTGCAGATTTG GCGGGGGTGAAGGTCCTGGTTGTGTGCTGGGCTCCTAGTGAAGGGCCTGCCCTTTGGGCC TGGTGCATCCTGCTGGGTGAGGACAGCCAGCGTCCCTGCACCCCATTCATGCGATTGGAG CCTAGTGCTGCCCCTCAGCCTTGACGCTGGCCCAAGTGGAAGGATGTGTTCTGCAGGGCA TCTGTTGGGATAGGTGTGGTTGGTGTCATAGTTGTCCTGCAGGACAGGGATCTCACTTGC ACGACCCATGAACCCCAGGGGCTGCCTGGCCTTGAAGAGTTCCAGCCACGATTCCATAGC TGGGGCTGCTGGGCCAAGCCGCATGTCGAACGTCCTCGAGGAGCTTGAGGCAGGAGCTGG GCCGAGTATGCGTCTCCCAGGGCTGGGAACCAGGTGGGGGACTGGAATGGGTGGCATCAA TGGTGGTAACACTGTCATCTGCCACAGGGCTGGACATGGAGGAAGGCAAGGAAGGAGGCA CATGGCTGGGCATCAGCACACGTGGCAAGCTGGCAGCACTCACCAACTACCTGCAGCCGC AGCTGGACTGGCAGGCCCGAGGGCGAGGTAAGGCGAGTGGGGTGGGGCCAAGGTGAGACA GGGTGGGGTGGGGCAGGCCTAGGTGAGACAAGGTGGGGCCAAGGGACTACAGGACTGGCC AATGTATGGTGGAGTGGGGCGGGCCAAGGTAGGAAGTGCCAAGGTGCTTGTGCTTGGGAG TGGGATGGGGCAGGGCTGAGGTACCGTGGGGCAGGGCCAGGTTACAGAGTGGGCGGTGAC AAAGCGTGGCAGGGCAGGGCCCAGGTAAGGGTGGGGACAAGAAGGGGGAAGCTGGGCCCC GTGGCAGGTGGGTGGCTTCCGTGGGCATATCACTGCCGCCACTGAGGTGTGCTGCAGGGG TATGCGGCAGAGACAGGAAGAGTGAGGAGGGGAGCATCCCATTTCCCACACCCTGGCCCT CTGGCGTCGCAGCCAGGAGCCCACTCCCACCTGACCCACGCAGCACTGTTTACTCCTCTC AGGGCGCACTCTCCCCTTGTCCTCATGCATGGCACTCCTGACTGCTGTGTCACTGTCCCC TGCCTGGAATGTTTGTAGAGTAGATGCCTTCCCCGAGACGACAGCTCTGTGGACACACAT TGTCCCCACTGAAAGGTGTTGTTTACTTCATGCTGGATTTCCATAGGAAGACATAGTGGG GAAATTTTTTTTTTAATAAAATGAGCAGTTTTCAGTGCCGACCTGAGTGGCAGCCAGGCT AATGAGGTGTGGCTGACTCTTGCGGCAGTCATGACAGGCCAGGGCTCCAGTGGGACCCCT CATCTTCCTGCATCCTCCCCAGGGGGCCCCATATCAGTGTTCCTGGGAGAAGAGCACATG CCTGCAGCTGTGGACACAGCATCTGTCCCCTGCCTACAGGTGAACTTGTCACCCACTTTC TGACCACTGACGTGGACAGCTTGTCCTACCTGAAGAAGGTCTCTATGGAGGGCCATCTGT ACAATGGCTTCAACCTCATAGCAGCCGACCTGAGGTGGGTCTGCCAGAGGGGGATGGCGG CTCTGCCCAGCACTGCCTCGGGGGCAGGCCTCAGGCTCATCAGGAAGTGGGGTTCCACTG GGGGCTGTGGCAGCCTCTCCAGGGACGTTGCTCCTCGTGACTTGGCAAACATTCTGAGGA CTGAGCCTGTCACAGATGTACCAACTCGGGTATTTGGGGGTGAAGCCCAAGGCTTCCTGT GATGGCATGAGGGCTGCCGGATCCCTGGAGAAAGCTGAGGCTGTGAAGAACAGTCACATA GAGTGGCTCTGGCCGGGTGTCCTCCCTCCCACGGGCAGGCTGTGCAGAGCAGCCCTCCGC AGCCCTGGCTTGGAGTGACTGGGATGCTGTGCTCCCAGGGAAGCTCCTGTTGGAGATGAG GCCGCAGGAACTGCGGAGGTTTTGGTGTTTTGCCCTTCCGTGAAGTGAGCATTCCTCCCT CCTGCCCTCCTCCCCCTCCAAAGCCCCAGAACTTGGCCAATGCTGTTAGTCCGGGGCCTT CTGCCAGGACAGGCAAACGGGAAGTGGTCGGCACATTGTGGCGGGCCCTGAAGCTGCCTC CTTCCACAAGGGCAGGTCCATGGGCCTCTCTCCTGGCTCCCGAGCTCCAGGCCTGCTGTT CGTTGAGCATCCCTTGTGTGTGGCATGCCCAGCCCTGCCCCACATGGGTCTGCACACTTG CTATGGAGGCTGGGAGAGTTGCAGTGTCCCCTAGGTGGAAGGCCGAGGGCTGGTGCGGGG CACTTGCTGTCCGGAGCCGTTCTTCCTGCCCATGCCTTCGTGACTCCAGGTGGCAGAGCC CCTGTGCTCTCTGCATGCCCCCATGCTCAGGAACCACTTTTCCCGGTCAGTAGTTCCATG TGGGTCTGATTAGTCTCAGGAGAGTTCCTCTGCGGCCGTGGCACCAGCAGGACTTGGCAC TCAGCAGCTGTGTGGAGGTGGGGTGCCAAGGGTGCCATGCCTTGAGCAGATACACGGGCT CCTGTGGTTGGGGGTTGGGGGTTTGGGCTCCTCAGAGCCTGTGCAACTCTCCCGGCTCCA AAGGCTGCAGAGTGAGGGTCAGCCTCCTGGACCTAGCCTGAGGATACAGGCAGTGGCCAG GAGAGCATACCACAGGTCAGGCAAAAGCTGGTGCCAGGGCCGAGGCTGGAGGGCGGGGGC ATCGGAGGTCAGTGAGCAGCCCTGCTGGTAGGTGCAGATGAGGAAGCCAAGGCCTGGAAG GGAGAGAGGCTTGTCATGGCCACAGCTATCCCTGGGTGAGGCCTGCCCTCCGCCTGGCCT GTGGTATAGTGCTGGCAGCATGGGTGTCCAGTTCTGTAGGTCCTTACACAGAAAGCCCAG AGGAGATTTTTGCCTGCAAACAGTCTCTGAAATTATGAACCAAGTATCTTAGGAAAGTTG CCAAAGACCATGGTGAGGAAGAACGGGAGTGGAATGTGAACATAGACGGGGGGGCCCTTG AAGCCTACCAGGGGTGGGGAAGGTGGGTCCCACCTGGGGGCAGCACTTCGGGGAGGAAGG CACCATGCCTGGCTCTTTCTCTGCATCTGTTCCATCTTGAAGGCCCAGGGCTCCATGCCC AGCATCTGGCCTGGGAGGGCATCTCACAGGCCACAAAGAGATCCTCTTCACTTTGCAAAA AAGGCACTCCAAGTTCTAGAAGACTTACTGTGGTATTGAGGATTGGAATATTTTCAGCTG GGGAGCATTTTGAACTATTTTCTTTTTAAAAAATGATTTATTTATTTTGTAGAGATGGGG TCTTGCTATGTTGCCCAGTCTGGTTTCAAACTCCTGGCCTCAAGCAGTCCTCCCTCCTTA GTCTCCAGATTAGCTGGGACTATAGGCATGAGTCACTATGCCCAGCTCTACTGTCCCTTA ACTATTTTAAAGTGTACACTTAACACTTGAGAGTAGGAAAATGTGGTCTTTGGCGCTTTC TTTGGAAGCCTCGATCACCCTGGGGCCACACTGGGGCCACCTGTCCAGAAACCCTCATAG ATGAGCTGTGGTAAGGGGCCTGATGGAGAGGTTGGTGATGTGGGGATGGGGCTCAGCCCT GCAGTTGTCCCCATGGGCAGCCTCTGTGTGTCCATGCCATGGGGCATTCGGCATGGCAGG AGGGCATTTTAGATCCTGTGCCTCCTGGGCTGGTGGGCCCCGAGGTGAGCTGGGAACATA CCCGTGATGCTGGACAAGAGCTGGGGCCTCCATGACCCCGCCTGCCAGGCACCTGAGGGG CTGGGTCCTGCAAGCTCCTGACCTGGCCGCAGCGGGCTACTGCGCACATCGCTAGCCTCC AGAAACAGGGCTGTGAGATCACCGGGCAGTGTCGGGGAGGACGCCCTATGGCTGTAGTCT GACATTCTCTCCCCTTGGCCTGCAGCACAGCAAAGGGAGACGTCATTTGCTACTATGGGA ACCGAGGGGAGCCTGATCCTATCGTTTTGACGCCAGGTGAGCCTGCCCTGGCAGCCTGAT GGGGTGGGGGACTGTTTCTATGCAGAGGTCACCCTTGTGCTTTTTAGAGACCAGGCACTT GTCATATTACTCTTCTGAGATGACTTTGTGGCCATTTAAGTGCCTTATGGTCTGTGGGCA CCTTGCCCTGCACCTGGACACTTCAGAGAGCCCTGGCTCCCTGCTCACCCCCTCCCCATG GGCTTTTCCTGCTGAGCAAGGTGCGCCCCTGTTCTGGGCTTCCCACCGCAGCCCTGCAGC CCAGCCGGCCACCCCCAGCCTCACTTCCTGTGCCGTCCCAGTGTGCCCTGTGCCTGTTCC TTCTCAGAACCTCTGGAGCCATCACCACCTGGGGTTCTTGCCTGGTCTGCTCCCTGACGC ACAAGTACACACCCCACTCAAGCCCCACACAGATGCACGCCTCCTGCACCCAGTCCCCAG TCTGATCCCCGGTCCCCATCCTCAGGGCCTCGAGCCCCTCTGCGCCAGGTCACACTCCAC GGCTGGGCCCTTCCTGCCCTGGCTCTGCACCACTGCCCAGGCTGTGCCTCCTGCTACATG CCCTTCCTTTCCCCCTCCTCGGGTGCTGCGATTTCCTGGAAGCTTCCTCTGCCAGTGCCC CAACCTGGAGAGAATGTGTCACATTGCCCCCCTCTGCAGCCATGTTCTGGGAGGGAGGTC TGCATCTTGTCCCAGGTCTGGTGCCAGGACAAAGCAGCACTCCTGGGAAATGGCATTTCC CCTGGGTGCACAAAACTGTAGGGACCATAGCCTAGGTTCCGGGGACTGCTGGCTCACTCA CTCCCACACTCGCTGCCCACAGACACTCCTCGCTGGTGGCATCGTGGAACTCTGTGCCTT AAACGCCCGCTCTGGTCCCTCTACTCAAGCTGCTGGCTCACACAGGGTGTTCCGGCGCCT GGGGAGTGTGTCAGTGCCCTTAGCGCCACCCACACCCCTCTGTGAGGGGGCTCCCTCACA CCCCTGGCTCCTCTGAAGCTCCATGCCCACGTCACACAGACTTTCTTTTGCATTTGGCAC AAATCACCAAGTGAGGTTGCAGGTCACAGGTGCACACCTTCCCACTGGGTGTACACGGTG GAACTGAAGCCCCAGGCGTCCCTGGAGGGGCCCGAGGGAGATGATAAGCAGTCCCCAGGA CAGCTGAGTGGCCCCATCCCACCTGCATCTTAGGGGGCTGGTGGTGCTGACGGGCTCATC ATGAGTCCTGTGGTCCTTGCCTGGCCCACACTGGAGCTGGCTTCCAGCTTCCCACGTCTG CCCAGTGATGTTTCATCCACAGATAGGCCCAGCCCCCATTTGCTAGGGTGGTGCCCAGAC CCGAACCCCTGGGCTAGTTGGGATCTCCAGAGGAGGCATCTGCCTGGAGGGTGGCTTCCA GTGTGTCCCACCCACTGCCCCCACGCCTACCAGCTGGTGCCCTCTCCTCCACCTGGGGCT TATCCTTCCGGCCACCCTCTGTGACCCTGGTCTTCCCCTCCCTACCTTGGATACAACTGT GGGCAGCCCACCTGAGCTCCCAAGAGCTGGGCAGTGGCACAGACACAGAGACTACCAGCG AGGCAGGGATGTGGCCCCAGAAGGTCCTGAGCTTGCAAAGGATTGTTCTGGCTGTCTCAG CCAGCTCTTGCTGTTCCACCTGCCCCAGAACCTCCGCATCAGTTGAGGGTCGTGGGGTTG GAATGTGTGGCGAAGTAGGTCTGTGGCAGTGGAGTTGGGCCATGCTGAGCCTGTGACTGC CCAGGAGAGAATTGGAAACTAAAAGGTGCAAAAGCAGTCAGAGACTCCCTACCACCCCCT CACTGTGAACATTGGCCCAGCGGCCTCTGGGGCAGTCGCTGTAACCATCCACGTGGCATC CTCGGTTCTCAGAGGCTGAGACCTCCGTGGCTGGAGATGCTTATTTTCTGCTCAGTTGTT GTTCTGTAGAACTTATTTCTGTCTACATCCTGCCTCATCCCTGAGCCTGCCCCACCTGGT CCACATTGCCTTTTTTTTTTTGATAAAGTTTTATTGAGATATGACTGACTAGCGGCACAG TTCACCCATTTAAAGTGTATCATTCAGTAGGTTTCGTTTCATTCACAGAGTTGAGCAACT GTCATCACTACCTAATTCCAGGACATTTCTTGATCCCCAAAGAAAGCCATACCTGTCAGC AGCCACTCCCCATTCCCCGTCTCTTCAGCGCCTGCAACCTTTAATCTGCTTTCTGTCTCT GGATTTGCCTATTCTGGACACTTTGTATAAAGGGAATCCTATAATATGTAGTTTAGGGGA CTGACTTTTTTCACTTAGTGTTGTCGTCTAAAAACTCACCAGTGGCGAGGCACGGTGGCT GACGCCTGTAATCCTAGCACTTTAGGAGGCCAAGGCGGGCAGATCACGAGGTCAGGAGAT CGAGACCATCCTGGCTAACACGGTGAGACCCCATCTCTACTAAAAATACAAAAAATTAGC CGGGCGTGTTGGCAGGTGCCTATAGTCCCAGCTACTTGGGAGGCTGAGGCGGGAGAATGG CGTGAACACAGGAGGCGGAGCTTGCAGTGAGCTGAGATCGCGCCACTGTACTCCAGCCTG GGCGACAGAGTGAGATTGCATCTCAAAAAAAAAAAAAACAAAGAAAAACTCACCAGCATG GCTAAATGATAGAAAGGAGAGTTTTATTTGCCATATCAGTTTGCAGACCCGGAAGAGAGA GTCTCTAGCATGGACTGAGGTGCTCTCTTTGAAGAGGGGAAGGGCAGTTTGAGTTTTATG CCTCCCAAGGTCCATATCACACAATAGAGTCATACATATTCAGCAGGTTTGGGGAAAAGC CATACATATTTATGAGGGGACCCGAGAACATATGCAATAGATAAACATACATGTAACATA CATCCCATGTTCACTTTGGGGCAGGGTTTTAGCATTAAAATGAGGTAGAATCTGGATCTT TCCATCAGAAGGTGAGCTCTAGGACACAGTTGGAGTGCAGCCTCTATGAACTGCTGAAAC TGGCTTATGGTCTGTAGTTGCTTATCAGAAAAGAGTGTTTGAAGGCTGGTTCTCTGTCCA GTCAGGGTTGTGGTGGTCTGGGCTGTCAAGAGTTAGGAGGGTCTGATAACGTGTGTGATC TCTACTGTTGTGAGGGAGTTTATCAAGACTGTGGTTTTTCTTGTAGCTGTAGGAATTTAG GGAGTTGCTGAGACAGCTGCCCTGAACCCTCGAGCCAGTTGGTACTTTTTGTTTCTTCCC TTTATCCATTAATGGACACTTGAGTTTTTTCCACCTTTTGGCTGTTGGAAACAGTGCTGC TCACAGTGTGACCTCACAGCATTTGTGTGGTCATATGTTTTTATGGCTCTCATGTGAATA CGTAGGAATGGAGTTGTTAAGTCGTATGGTAACCCTGTTTAACCTTTTGAGGAACATCCA AACTGTTTGCCAAAGCAGCCGCAGCATTATACATTCCCACCAGCAGTGTATGCGGGTTGC AGCTTCTCTGAATCCTCACCAAAACTTGCTATGTTCTGTGTTTTTGATGATAGCCACGAT AGTGGGTGTGAAGTTGTGTGGACTCGTGGTTTGGTTTGCGTTTCTCTGATGGCTATGATG TTGAGCAGCTATTCTCATGCTCGTTGGCCATTTGTATGTCTTCTTTGGAGAAATGGTTAT TCAGACTGGGTTGTCTTTTTGAGTTATAGGAGTGACTTATACATTCATTCTAGCTACAAG TCTCTTATTTAAAGCACAAAGCTTAAAATTTTTAAGTCCTATGTCTCTCTCTTTTTTTTT CTTTTGTTGCTCATGCTTTTGGTTTCTTATCTAAGAAACCATTGCCTAATCTAAGGTCAT GAAGCTTTCCCCAATACTTTCTTCTTAGAGTTTTATAGCTTTTGCTCTTTCACTTAGGTC TTTGGTTTGACTTAAGTTAACTATTTTTTTTTTTTTGAGACAGGGTCTGGCTCTGTCACC CAGGCTGGAGTGCAGTGGTGCAGTCATGGCCCACTGCACTGTCAACCTCCTGGGCTTAAG AGATCCTCCCAGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAA GGCGGGTGGATCACAAGGTCAGGAGATCGAGACCATCCTGGCTAAACCGGTGAAACCCCG TCTCTACTAAAAATACAAAAAATTATCCAGGCGTGGTGGCGGGTGCCTGTAGTCCCAGCT ACGTGGGAGGCTGAGACAGGAGAATGGCGTGAACCCGGGAGGTGGAGCTTGCAGTGAGCC GAGATTGCGCCACCGCACTCCAGCCTGGGCGACAGAGTAAGACTCCGTCTCAAAAAAAAA AAAAAAAAAAAGAGATCCTCCCTCCCAAGTAGCTGGGACTGCAGATGTGTGCCACCATGT TCAGCTAACTTTTATGTTTTGTAGAGATAAGGCCTCACTATTTTGCTATTTTGCCGAGGC TGGTCTTGAACTCCTGGGCTCCAGTGATCCTCCTGCCCCAGCCTCCCGATGTGCTGGGAT TATAGGAGTGAGCCACCTCTCTTGACCTGAGTTAACTTTTGTGTATGGTGTCAGGTAGAG TCCAACTCCCTTCTTTGCCCATGACTATGCAGTTCAATGGTCTTCCATGTTGTTCATGAT GGAAAAAGCACTGTGACACCTGGGGCTGGCGGCTTGGCTCTGGGGGCCCCAGCAGAGCCA GGCTGTCTCCCTGGGAAATGCCTCGGCCAAGATATTCTCCTGGAAGTCTGACAGGGAGAG GGTGGTGTCTTGGTCTCCCTGGTGCATCATTTGGGGTGCTTGGCTCCCATCAAGCCCACC CCTGCTGCCCTGGCCTCTAGAGAAGAGCCTCTGAGGTGAGGTGCTTCTCCACCTCAAGTC CCTGCCCTTCACTTTCTGTGACATGAGAAACCCTTTTCCTGTTGGCCATGCCTTCTGCCA ACACTCTGGGAGCTTGTTCAGAGTCTGTGCAGAGCCCCCCGGCTCTCCTAACCTGGGGCC TGGCTGGTACGCAGAGGTGGGCCATGCTCTGCTGAGGCCCAGCCTACACCTGTGCCCACT GTGTGGCGCTGTTGGGGGTTCCCCAAACAGGGACATGATTGTGGTGGGTGCCTCCTGGCC TCTGGTGGCTAATTCTCCCACTCTTGAACTTCTCAGGTGGCCCCCCAACCTGGCGTGAGG TGAAGAGGTTCCCTCTTGGCTGTCCCCTTTGCCTGTGAGATCTCAGGTTGCTCATTTCTT ATTCATCCATCCATCGAGGCTGCTGGGCATCTGTGCCTTGTGGTCTCCTGTGGGTGCCCG CTTCCGCCTACTGCTGAGTTCTCCTCTCCTTGCCATGCCATCAGGTCAGGAATGAGCATG GGGAAGGAGCTGGAGCATGGAGCGGCTTGGCCTCAGTCTGGCCTGATTTCATGGTCCCCA GCTGTACCCCGTGTTAGGTGGGTGGCAGGTGGCAATTTGCCCTGACATGGCACAGCAGGG CCTCTGCATGGCCCGCTGATTGCTCCTCACAGGCACCTACGGGCTGAGCAACGCGCTGCT GGAGACTCCCTGGAGGAAGCTGTGCTTTGGGAAGCAGCTCTTCCTGGAGGCTGTGGAACG GAGCCAGGCGCTGCCCAAGGATGTGCTCATCGCCAGCCTCCTGGATGTGCTCAACAATGA AGAGGCGTGAGTGGGCGGGTCCTGCTGGGGTGAGCCCCAGTGTCCCGCCACCAGGGCAGA GGGAAAGGCAGGCCCTGCTGCCCCGGGAGGCCCATGGAAGTGGCCAGGCTGGGTCCCCAG CTGCAGGGAAGCTGATGGATATCCTGTCCTCCCTGCCTGTCCCCTTGAGCCAGCTGAGGT TTCCAACACCAGGGACTTTTGATGACAGAAGATGGGCCCAGGCCCAGGCCCAGGCCCAGG GGAGGCTCAGTGCACCCAGAGGCTTGATGCAGCCACGTGGGGCCCAGAGCAGATGGATGC TCCCCTCTGCATGCCGAGAGCTTGTGGGAAGTTCCAGAAGATTCTAGACACTCAGCAAAC CCAGGCGGAAGAGACACATCATGGAAATGAGTGGCTAGGGTCAGCACGCTCAGGGTTGAT GGGCTTCCCGCAAGCGGTCTGTCCCCAGCGACCCTGGGCTGAGTGCTGGGCAGGGACTTG CAGTTCCGGGAACTGCCTGGGTGTAGGAGCTCCCACCCACTTCCTTGCCCGTAGGCCTCA AGGCCCCTCAGGGGCCAGGCACTGGGGTCTTCGTTGCCCCAGCCGGGGGTCCTGGTGCTT GCCATGCCAGCCTTTCTCCCACTGGGCGAGATGCCCCACCCATCCCTCGCTCCCCACCCA GGTCAGACCACTGCCCTGCCCAGGTGATCCAGCGTCCACTCCCACCCTGTCCAGCTTGCC CCCTTCAGAGAAGCACCCATGGACCATCTCCCACTCCTCCCCCTGGGGCCCCTTTCTGCC CCCTGCCTACCCTCCTTCTCCTCTGGGCCTCTGATAGGCGGCCTCGTCCTTCAGCCTCAG CATCGCTGCCTCACTGGAAGAGCCCCACCTGGGCATGCCCAGCAGCCCCTCCCAGCTGTA AGTCCACACAGCCCCCATCAATGTGCCATCTGGGGCAGGCCGAGGACCGGCCCTGGCTGC CCTCACTGATGGCTGGAGCCCCGATGCTAGCTACAATGGCTGGCTTGAGTGGACTTCTGT CACAGAGGCAAATGCCCACAGGGCACCTCTTGGGGCAGGCAGGGAGCTTGTATGTGCCAT CTGGGGCCAAAGCTCCACGATTGAGTCATAAGTTAAGGGTAGATGGAGATTTTCCGCCTT GAGCAGAAACGTGGAGGAAACACGCCTGGTGTGTGACTCACGCCTGTAATCCCAGCACTT TGGGAAGCCGAGGTGGGCGGATCACCTGAGGTCAGGAGGTCAAAACCAACTTGGTCAACA TGGTGAAACTCTGTCTCTACTAAAAACACAAAATTAGATGGCTGGGTGGTGCACGCCTGT AATCCCAGCTGACGGCTGGAGCCCCGATGCTAGCTACAATGGCTGGCTTGAGTGGACTTC TGTCAGGAGAATTGCTTGAACTCGGGAGGTGGAAGTTGCAGTGAGCCAAGATCGTACCAT TGCGCTCTAGTCTGGGCGACAAGAGTGAAACTCCTCCTCAAAAAAAGAAAATAAGAGAAA AGAGAAGAGAAGAGGAAAAAGAAAAGAAGAAAAGAAACAACCCTTGCAGTTCCCCGGCCA CTCCCCAGAGCCAGTTAGGCCACCAGGTGGGCTGGGGCTAGACCCGGCTGCGGGCGGGCC ACAGAGGGACTAATGGCCACCACTTCTCTCCATCCTGCAGGCAGCTGCCAGACCCGGCCA TCGAGGACCAGGGTGGGGAGTACGTGCAGCCCATGCTGAGCAAGTACGCGGCTGTGTGCG TGCGCTGCCCTGGCTACGGCACCAGGTATTGCAGCACCGTGGGTGCGCCACCTCCTATCC CATGTCCCACCTCCCACGCTAGAGGGCCGGCAAAGAAGCCAAGTGCCCATAGCCAGAGCC AGGCTTCTTCCTCGCCTGAGTGGATTCCAGAGCTTCTGCCCTGTCCAGACGCAGCTGCAG GGTGAGCAGATACCACAGGCTCCCGGGGCCCCACAGGCTGGGCTTAAGGAGCCAGTACAC AGCCACACAGGGCCAGGATGGCTTCAGGGCTGCCTGCACATTGGTGGATCCCTGCGGGCA AACGCCCCGGCACCATGGCACTAGGCCTGCAGGCTGTACCCTTGGGTGGTGGCTGCTGGC CAGCCTTTCCTGCCTGGGGCTGGCTGGCCCATTGGCAGGTGCAGGGTGTCATTTCCTTCA GCCCCTCCTCTGTCATAGGCTCATCTTCCTGTGTGTGAGGGGAAGGCAAGTGCCAACTCC ACTCCCTGGCAGGCCGCATAGCCGCTGGGTCCCACCCCACGGGGTGCACTGCGTATGTGT CTGCCCTGGGAACCAGCCTGTGCTGGGGCTGTTGGGGGATCCAGGGCCACCCTCCGTCTG CAAAGCCCACGGCAGAGAGGGTTGGAGGAGAGGAGAGTGTGCTGAATGTTTGCTTCAGGT GGGCTGGCATGGCCTGGGCAGTGGAAGAGAGGGCACAGGAGGGAAGCCTTCCAGACAGAG GAGGCCTGGGCCCCAAGTGGTCAACAGCTTGACCTCTTGGAAGAACCAAGCATGGCTGTG TCTGGGAACCTGGGCCTCCAAAGGGGAAGTGTGTGGTCAGCCCGCAGGCTGCCCTATGCC TTGGCTCATGCTACACGGGGAGAGCAGGGCAGGGTGGGGTGGTTCCCAGTGAAGGTGGCA GGACTCAGCAAGTGTGGGCCTGTGGAGCCAGCCAAGAGCTCACACGCCACTGATGGCCCC TCATCATCCCCAAGACTGAGGCTGCTGCTCTCTGTGACTTGGCACAGCCTCCAGGCATGG GGTTCCTAACTCTACCTGCCTGCATTCTTGCCCTATTTGTGGCTGTGACAGGCAGGGCAG GGCTGAGGGACACCAGGTGAACGAGGGCCCCTGCTCTCTTTCAGAACCAACACTATCATC CTGGTAGATGCGGACGGCCACGTGACCTTCACTGAGCGTAGCATGATGGACAAGGACCTC TCCCACTGGGAGACCAGAACCTATGAGTTCACACTGCAGAGCTAACCCCACCTCTGGGCC TGGCCAGTGGGCTCCTGGGGGGCCCTGCCTTGAGGGGCACTGTGGACAGGAAACCTTCCT TTGCCATACTGCATTGCACTGCCCGTGGCTTGGCCAGCATCCCCCGGATCAGGGCCCTGT GGTTTGCGTGTTACCCATCTGTGTCCCCATGCCCAGTTCAGGGTCTGCCTTTATGCCAGT GAGGAGCAGCAGAGTCTGATACTAGGTCTAGGACCGGCCGAGGTATACCATGAACATGTG GATACACCTGAGCCCACTCTTGCACATGTACACAGGCACTCACATGGCACACACATACAC TCCTGCGTGTGCACAAGCACACACATGCAAGCCATATACATGGACACCGACACAGGCACA TGTACGTGCACAGGTGTGCTACACATGTGCACACATGCACAGTTGCACAGACACACACAC ACAGGTGCACACACACGATGCCGAACAAGGCAGAAGGGCGACTCTCACCTCTCATGTGCT TCTGGCCAGTAGGTCTTTGTTCTGGTCCAACGACAGGAGTAGGCTTGTATTTAAAAGCGG CCCCTCCTCTCCTGTGGCCACAGAACACAGGCGTGCTTGGACTCTTGACAAGCAGACCTG CTCCTGCAGAGGAGACAGCCACATTTGGAATTGGGCACCGAGAAGACCTGAGAAAAACCC ACTCTCTCTTTTTTTTTTTTTTGAGACGGAGTCTTGCTCTGTCACCCAGGCTGGAGTGCA GTGGCACGATCTCGGCTCACTGCAACCTCCGCCTCCCAAGTTCAAGCAATTCTCCTGCCC CAGCCTCCTGATTAGCTGGGATTACAGGCGTGTGCCACCATGCCCAGCTAATTTTTGTGT TTTTAGTAGAGACGGGGTTTCACCATGTTGGTCAGGCTGGTCTTGAACTCCTGACGTGGT GATCCACCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCACGCCTGG CCGAAAAACCCACTCTCATAACAGAAGTGCAGACTCATTGCTAGATTCAGTGCCCTTGAG TGTGCCAGAGGTCCTCTGTGTTTGAGACAATCCTGTGTGTGCCAGGAGGCTCCGTGTGCA CCAGGGGCTCTCAGAATCCCGCTTACCCAGCTGGAGACCATGCCTCTGGCAGCCCCATCT CAGCCAGCCCTGCTCTCTCCCTCTTCCCTCCAGGTGAGGCAAACTTCATAGGAATCTGTA CCTGAATGTGAGCTCCTGATAATAAAACTCTGAGGCTTTGGTGAGCGCATTTCGAGGCCT TTCCCTTGTATGCAGGGTGCCAGTGGGAGCTGCTATGTCTCCTGTGCAGCCAGACACCCT GGACGAGGCCCTTCCCACCTCCCCCTTTCCTCACGGCTTCTTCAGCACCACATGTGGATG CCTGTGGGTCAGTCCATCTGTCCATGGGCAGGGGCCGCTGTGAGAGACCCCATGCGCGTG GGATGTGGTAGATGCTGATTATGGGGCCAGCCACCCATACAGGTCTATCAGGTCGCAATG GAGGCCCCACTGGAGCGGGAGGCAGGCTGGCCTGGACTTCTGTTGGATGATGGGGGCCAG GGGATGGTAGGAGGGGCAGGAGAGCAGGTCTGCATCTGTCTCAGCCCCAGGAGCTCCTGG TTTGGGGCCAGCTCTAAGGAGCTTCATCCCTGAGCCTGGCCGAGTATAAGGGGGTCCCGG ATATCCAATGCTGAGACCTAGACTTTTCTAAGCCTCTGCACCCAACCCCTAACTTGGCCT TTTCAGACCCATGAGAGTGCAGGGTGGGTGTGGACCTGCGCCAGGGCTTCCCAGCCTGAT TGTGACATGATGTGAGTCCACCTAGGGAGGAGCAGGGGCCTCCCCGTCTCTTCCCCTTCT TGCCCAGACCACTTTGCTGGGCTCAGAGCCTTCCATGGCTCACTTTGAACTCAGAAGAGT TTCCCAGCTGCGACCCAGGCTGCACCCCCAGGCCTGAGGAGAAACTGAGGCCCCACATCC CATACGTGTCCTCTGAGTCAGGACAACAGGCTCCCGGCTGGGGGCACTGCCCTTGCTGCC AACACATTCACCTGCCCCAGTGGCTACCATGTCCTGTGCCGGTGACCAGTGCCAGTCCCC AACCTGGATGGTGCCCTGCCCTCATGAAGAGCCCCCCGGATGTTCACCTCTGCCCAGAGA CCCGGCCTGCCCAGGAAGAGCAGTCTCTGCTCTTGTGTGGGTCCCTCTGCCCCCACCCTG CCCAGGGCTCCCCAGCAGCCTGAGGCCCCTAAGGAAGAACAGCCCTCAATGGTGATTCTA TTGGTGTTATGAGTTGTGTCCCCAAAGAGATATATCAGTGTCCTGACCCACAGTACCTCA GAATGTGACTGCATTTGGAGATAGGGTTCTTCCAGGGGTAACCAAGGTAGAATGAGGTCG TTAGTGTGGGCCCTAATCCAATATGACGGCTATCCTTATAAAAAGAGGAACATTTAGGCT GGATACAGTGGCTCATGCCTATAATCCCAGCACCTTGGGAGGCTGAGGCAGGAGGACTGC TTGAGCTCAGGAGTTCAAGACCAGCCTGGGGCAACATGTCGAAACGCTGTGTCTACCAAA AAAAAAAAAAAAAAATACAAAAATTAGCTGGCCATTGTGACTCACACCTGTAGTGCGAGG CTGAGGTGGGAGAATCGCTTGAACCCAGGAGGCAGAGGTTGCAGTGAGCCAAGATTGCAC TACTGCACTCTGCCTGGGCAACAGAGGGAGACTCTGTCTCAAAAAAACAAAAAAGGAAGA TTTGGGCTGGGCGCAGCGGCTCACATTGTAATCCCAGCCCTTTGGGAGGCCAAAGCTGGA GGACTGCTTGAGCCCAGATTAGCCTGGGCAACATAGCAAGACCCCATCTCTAAAGAAAAA TGAAAATTAAAAAATTAGCAGGGCATGGTGGTACACACCTGTAGTCCCAGCTACTTGGGA GGCTGAGGTAGGAGGATCACTTGAGCTCAGGAGGTGGAGGCTGCAGTGAGCCGTGATTGC ACCACTGCACTTCAGCCTGGATGATGGAGAAAAACCCTGTCTCTTAAAAAAAAAAAAAAA GGAAAAAGAAAGATTTGGACACGGACGGATACGCGCACAGGAAGAACACCGTGTAAAGAC TGGAATTCTGCTCCACGAGCCAAGGCATCACCAGAAGCTGGGAGAGGGGCCTGCTTCAGA GCCTCTCTTAGAGCCTTCAGAGGGAGCAGACCTGCTGTCACTCTGATCATGGACTTCCAG CATCCAGAGCTGTGTGACAATAAATGTCTGTTATCTTAAGTGGTTCAATTTGTGATGCTT TTTTTTTTTTTTTTTTGGAGACAGGGTCTCACTCCATTGCCCAGACTGGAGTGCAGTGGC AAGATCACAGCTCACTGCAGCCTCCACCTCCTGAGGTCAGGCAATCTTCCCACCTAGTCC CCTGAGTAGATAGGACTACAGGTGCATACGACCACGTCCAGCTAATTTTTTGTATTTTTT GTAGAGACGGGGTTTTACCATGTTGCCTGAACTGGTCCCAAACTCCTGGGCTCACATGAT CCTCCCACCTTGGCCTCCCATAGTGCTGGGATTACAGGTGTGAGCTACTGAGCCCAGACT GTGGTGCTTTTTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTCACTCTGTCACCCAGGCT GGAGTATGGTGGCACAGTCTTGGCTCACTGCAACCTCCACCTCCCGGGTTCAAGCAATTC TCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGGCACCTGCCACCACGTCCAGCTAAT TTTTTGTATTTTTAGTAGAGACGGGGTTTCACTGTGTTAGCCAGGATGGTCTTGATCTCC TGACCTTGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCAC CGCTCCCGGCCGACTGTGGTGCTTTTTTAAGGCGGCCCTAACAGACTAACACAGTAGTGA TCTTCACTTTACAGATGAAGGAACTGCGGCTTGGGGAGATACCATAACGGCCCCCCGGTT CCGAGACAGGTGGAGAGGCAGAACCATGGGCCAGGACAGAATAGGATGAGTACAAGGTGG CATCTCTGGGGGTGCCTGCCCCACAGATCCATGGTCTTCCCATGCTGGTGTCCCCACAAT GTCTGGCACTGCTCCCCGCTTCACCTTACTTGCCCAGGCCTACCCCCATACCCTCAGCTC ACAGCTACACACTCTGCACAGCTCCCACCCCAGGGTCTTTGTGCTCACAGTTCCCCTGCA GATACCCAAACCACACTCTTCCCCCAGTTCCTGCAGGTCTTTACTGAAGTCTCGGGCAGG CAACTCATTTAAAGTTTCAGTTTAGCCCAGCACGGTGGCACACACCTGTAATCCCAGCTA CTTAGGAGGCTGAGGTGTGAGCGTCGCTTGAGCCCAGGAGTTTGAGGCTGTAGTAAGCCA TGATTGCACGACTGCACTCCAGCGTGTGCGACACAGCGAGCCCCCATCTTGAAAGAAAGA AAAAAGAAATTAAAAAGGAGGCTGGGCACTTTGGCTCACACCTGTAATCCCAGCACTTTG AGAGGCCAAGGTGGGCAGATCACTTGAGTTCGAGACCAGCCTGACCAGCATGGTGAAACC CCATCTCTACTAAATATACAAAAATTAGCTGGACATGGTGGCGGATGCCTGTAATCCCAG CTATTCGGGAGGCTAAGGCAGGAGAATTGCTTGAACCTGGGATGCGGAGGTTGCAGTGAG CTGAGATCGTACCACTGCACCCCAGCCTGGGTAACAGAGCTAGATTGTATCTCAAAAAAA AAAAAAAAAAAAAAAAAAGGAAGGAAGGAGGAAGGAAAGAAGGAGGGAGGAAGAAGGAAG GAAGGAACTCTCTTTTTCTCTATGGCAATGAGCACGTACCATCTGGCATGCTGTGTGCTA GGATTGGTTCTTGCCTGTCTCCATCAGAATGGGAGCTTCAGGAAGGCAGGGACCTTCCTT CTCTTGCCCACTACTGTACTCTGATGCCCAGGCCAGGCAGGCATTTGAGCCAAGAATGAC AGTGCTGGTGCTTCCCAAGCCCCCATCTATTTGGGCCAACCCCTTGATGCAAGGCCCTTG TGCTAATAGAGGGTATAGGGGTCCAAAGCCAGTGCCTTCTAGAAGAGCTTTGGACGTGAC ACCAGGAATGGCTGGGCCTGGACAATGGCCTTCCTGGCACTGCCCAGCCAGTCCTCTGTG CTTGTTGCCAGATGCACCTGATCCCTGAGTCCCCAGCATGGGTCTCTTTCTCCCAGGCAG ATGGGGAACTCACCTTCTTCTCAGATGCCCCTTCCCAACATAAGCTGCTTCTGCCCTCTG GCCCCGAGATGGCCAAGGTGAGCTGTGTAGACATAGAGTCTGGAGGCATGGGGCTGGCTA ACGTGGAGGCAATTCTATGAACAGACATGGCCAGGCCAGGAATGCGGCATGGCCTCAGGT CTCATCCTTGAAGGCCTGGGCTCCTGGTCTGCCTGCAGACTTCCTGAGACCACGTCTTGT TCTCATCTGCCAGCAGATGCCTGGCTCATGGGTGAGCCGGGCTTAGGTGGAAACACAGGA GCCCAGAGGGGAAAGGATCCTCCCCCAGCTTGGGCTGGAGTCCCAACTTCCAATCATCAA TCTTTGCAACCCTGGGGCTGGACCTGGCTAGTTCCATGGTAAGGATCCTTCTCCGTCACC CAGTCAGTCCCAGGCTGTGTGGGAAACTGGCTGTGGTGGGCAGGTCTGAGGTCTCAAGTC TGACAGGGGCCACTGGAGCCTCCAACTCACCAATCAACCAAGTGTGAGAGGTTGCTTTGG TTGAATGGCCATGTGCTGGGGTCTGACTGGCCCAGCCACAGGGAGGCTGGCATCCCCTAG CTGAGTCCTGGACCCAGACCCTCCAGGGCATGGAGCCCATTGTGAGGTGTCTGGTGCTGA AGTGGTGGGGGAGGCCCGTGCAGGCCTACAGCTTTGTCATCTGCAACATTCCTCTCCCCA CTTTCTTTAAACTTTTTCATTTAGAAATGATTTCAATGGCCAGGCGTGGTGGCTCATGCC TGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGTGGATAACCTGAGGTCAGGAGTTCGCG ACCAGCCTGACCAAAATGGAGAAACCTTGTCTCTACGAAAAATACAAAATTAGCTGGGCA TGGTGGCACATGCATGTAATCCCAGCACTTTGGGAGGCTGAGGCAGGTAGATAACCTGAG GTCAGGAGTTCGCGACCAGCCTGACCAAAATGGAGAAACCTTGTCTCTACAAAAAATACA AAATTAGCTGAGCATGGTGGCACCTGCCTGTAATCCCAGCGACTTGGGAGGCCGAGGCAG GAGAATCACTTGAACCCGGGAGGCAGAGGTTGCAGTGAGCCGAGATCATGCCATTGTCCT CCAGCCTGGGTGACGAGAGTGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAATGAAACCT CGTCTCTACAAAATATACAAAAATTAGCTGGGCATGGTGGCAGGTGCCTGTAATCCCAGC TACTTGGGAGGCTAAGGCAGGAGACTCGCTTGAACCCAGGAGGCAGAGGTTGCGGTGAGC CAAGATGGCACCACTGCACTCTAGCCTGGTGACACGGCAAGACTCCATCTCAAAAAAAAA AATTTTTTTTTCAATTTACTAAAGGATTTAAAAAGTAGAACAAAAAATTCTAGTGTCTTT CACCCAGTTACCCCAAATGTGAATGCCGCACCCAATCACAGAAAAATGATTACAACCAGG AAATTAACACTGATAATGTTAAAGAAAGAATTACTGAATGACATTTGCTAAAGCACTGTA GGGAAGACTTTATTCAGGACCGTCTTGCTAGGTCTAGGGACCACAGGAATGAGATTCTGC AGTGGGGGAGAGAGATTAAGCTCAAGTCCGAATGGGCAAGTGGGAACTGATGATAGCTGA GGAGCAGGGCGGTGGGAGCTGGGTCAGCACATGGAAAATGACTAAGAAGAAACACCCCGG GTGAGGGAGATTCTGGCTACACCAATTAACAGGATTTTTGCTGAAGACAGGCCACAGTGA CCGGACATCTTCACCTGGGGGACAGTGTAGGAACGGATTAGATAAGGAGGGTGATCAGAT GTCGAGGGTCAGGGGATGCTTCTTGCTAAAGTGACTTTGCAGGGTTCTTGCTCTAAGTGA AATTTACAAGGAAGTGCACAGATGGGCCTAGAAGAAGGTTCAGAAGCCTGGTTTGGCCAA GCAAAGAATCTTTGTCGGTCCCTCCTCTTGTTCAAGGAAAGAAGAGGTTATCTTTCCTTG AATAGTACGAGTCCATTTCCTTGCTGGGCTGCCTTTTGTTCATTAAAGGCCAGCTGAATC ATCTGCTGGGACCGGCAGTAGAGGAGATATCCTCCATGGTGTGAGCCATCAGGCGTTGCA TGTTGAAGATAAAAGAAAAAGATATTAGTTGGAACCAACTATAAACTCAGTTTCTGAGTT CAGAGGGCAGCTACTCAAGATTTCCAGGTGCTGTGCTTGAAGCATCTTCAGATGGAGTAG GGGCTAACAGACTATCATGGACGGCAGTTTGCGTGTCATGAAGTCTGCCCACAAATTATC TGTTGTGGTGCCATCTTAAAGTTTCTTTCTTTTTTTTTTTAGACAGAGTCTTGCTGTTGT TGCCCAGGCTGGAGTGCAGTGGCGCAATCTCAGCTCACTGCAACCTCTGCCTCCCAGGTT CAAGCGATTCTCTTGCCTCAGCCTCCAGAGTAGCTGGGACTACAGGCACACACCACCATG CCCAGCTAATTTTTGTAATTTTTGTAGAGACGGAGTTTCACCATGTTGGCCAGGATGATC TCAATCTCTTGACCTCGTGACCTGCCCACGTTGGCCTCCCAAAGTGTTGGGATTACAGGC GTGAGCCACCGTGCCTGGCCTAAAGTTTCTATCAAGTTGTCCGGGTTCAGCTTAAAGGGC TTCAGGAAATGGGAAGTTTTAATTTTTAGCGGTTCCAGGCCAGAAGGGTGGGAAAAAAAC TGGAAACATCAGTTTAGAGAAAATCAAATATTGGAGGAAACTAGAAGAATTCAGGATCCA ATCCAGTTTGTAGGTAAATATCAAAACCTCAAAAACAATGAACAGGGCTAGAATCTGATA ATGGGTGCACTATAGTTTTCTTCTGAAACTAATTTTTCTCTATATAGTCACCCCACTTCT ATCAAAGATGTTGAAGTAAGACTAATTTGTTTGTAGCTAAGTTTAGGTTGATTAAACTTG GCCTAATTACTTACATAAGTGCCGCAACAATAGTGATTGAACACATAGGCTTTCTTTAAG TTTCCTTTGCTAGGACTTTTTTTTTTTTTTTTTTTTTTTTGTCACGGAGTCTCGCTCTGT CGCCCAGGCTGGAGTGCAGTGGCGTGATCTCCGCTCACTGCAAGCTCCGCCTCCCAGGTT CACTCCATTCCCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACCCGTCACTATG CCCGGCTAATTTTTTTTTGTATTTTTTTTAATAGAGATGGGGTTTCACCGTGTTAGCCAG GATGGTCTTGATCTCCTGACTTCGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGCAT TACAGGCGTGAGCCACCGTGCCCAGCCTGCTAGGACTTTTAATAAGGCATCTCAGATTGG GCTTTTAAAAGCCTTGCCAGGCTAGGAAGCCAAGCCGAAGCTTCACCATCATACTTCATT TGCAATCATTATAGGTTTGGGTGAATTCCTCTCTTTTTGAGGTCCCCCAAAATATCCTGA CATCCTTGGGCCTGTCAGGAAGTGACTTTCCTTACTCACCTACAAGGCAACCACATGACC CATGTATTCAAGGTTTAAAACCCATTTTTCCCCAAGGGCTTTATTGGTTCCATAAAGTCA TCTTAGTTACTTAAAGTTGTCTGGCCATAGCTAAAAATATGGCATTCTAGTCAAAGCTTT GGTAATATAAACAGCATTTCCAATTGTGTCCTGTTACAAAGAGAACAGGTTCTTACTGAA CTTATGTAAATAACCATATTGCCATTAAAATAAAAATACTCATGAATAGTTTCCGAATTA TGGAAGGATAAGGTACAGAGAAAAAGTAATTGTTTCACTTTTGTTTACAAAGGCATACTT TACCAAACTGCTGTAAGCTATGGATAACTTAAAAGAGAAAAAAAAGTTTCCTTAAATCTG GAAAACAAAACATTAAAGAACCAGCAATGTTTCAAACAAAACATAAAAAAATTATCCTGG CTGGGTACAGTGGCTCACACCTGTAATGCTTAGGGAGGCTGAGATGGGAGGATCATTTGA GGCCAAGAGTTTGAAACCAGCCTGGTCAACATAGTGAGACCCTATCTCTACAATAAAATT AAATAAATAAATAATTGTAATTGGTTTATTCATTGCCATGTAATTTATTCTTGTTCTGTT TGATCTTGATCAGCAGTTTCACGAACCCCTCAGTGTCTTCATTCAAGTTCTGGAAATTCT GGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGCAGA TCAGTTGAGGCCAGGAATTTGAGACCAGCCTGGCCAATATGGTGAAACCCTGTCTCTACT AAAAATATAAAAATTAACAGGGCGTGGTGGTGGGCGCCTGTAATCCCAGTTACTTGGGTG GCTGAGGCAGGAGAATCGCTGGAACCCGGCAGGCAGAGGTTGCAGGGAGCCAAGATTGCA CCACTGCATGCTGGCCTGGGTGACAGAGTAAGACTATGTCTCAAAAAAAAAAAAAAAAAA AGAGTTCTGGAAATTTTTTTTTTTTTTTTTTGAGACGGAGTGTCAGTCACCCAGGCTGGA GTGCAATGGCACGATCTTGGCTTACTGCAAGCTTCGCCTCCTGAGTTCAAGTAATTCTCC TGTCTCAGCCTCCAGAGTAGCTGGGACTAAAGGCACACACCACCACACCCAGCTAATTTT TGTGTTTTTAGTAGAGATGGGGTTTCACCATATTGGCCAGGATGGTCTCAATCTCTTGAC CTCGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTATAGGTGTGAGCCACTGCG CCTGGCCCCAGCCACATTTCTTAAGTAATCAAAAACCTAATAAAAGACAATATGAAGAAC AAGGAACTATCTTGATATAACACAAAAATCTTTGTTTCCAAGGTCAATTATTTAAAAGGT AAACAGAGGCCAGGCGAGGTGGCTCACGCCTGTAATGCCAGCACTTTGGGAGGCCGAGGC GGGCGGATTACGAGGTCAGGAGATCGAGACCATCCTGGCTAACACAGTGAAATCCCGTCT CTACTAAAAAAATACAAAAAATTAGCCGGGCATGGTGGTGGGCGCCTGTAGTCCCAGCTA CTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCAGGAGGCGGAGCTTGCAGTGAGCCG AGATCGTGCCATTGCACTCCATCCTGGGCGACAGAGTGAGACTCCGTCTCAAAAATAAAA AGTAAAATAATAATAATAATAATAAAAGGTAAACAGAAATCTTCATAATCTCAAATACTG TAAGAAAACTTTGTCATTTCAACAGAGAAGATCAAGTTAAAGTTCTGCATCATAGCACTA CTAATAAAGCTAATTTTAACAAAACCTTATAAATGAATCCATCCAATCTCATGCAAGATA ATATTTCTTTTCCAAGATTTCTTTTCTATAGATCTTTTACAACTTAAAAAAAATATCTGG CCGGGCACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGGATC ATGAGGTGAGGAGATCGAGACCAGCCTGGCTAACACGGTGAAACCCTGTCTCTACTAAAA ATATAAAAAATTAGCCAGGTATGGTGGCGGGCAGCTGTAGTTCCAGCTACTCGGGAGGCT GAGGCAGGAGCATGGCGTGAACCCGGGAGGCAGAGGTTTCAGTGAGTCGAGATCTTGCCA CTGCACTCCAGCCTGGGTGACACAGAGAGATGCCATCTCAAAAAAAAAAAATCTATCAGT ACTTTTTTTCCCCTACATTTTTTTTCTTTCTCATTCTGGAACAACCAGTTGCTATATATA TTAGCACAGAGTCAGTTATTCCTTTAAGGGATTTTATAAATTAATTTGGTGCTACTATCT GGAGGTAGAAAAATATCATGTATATATAACATACAAACATACATATGTAGTTGCTCTGCA ACTGAAGTTGGCAGAGGCCAGACCCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAG GCCAAGGCAGGCAGATCACTTAAGGCCAGGAGTTTGAGACCAGTCTTGCCAACATGGTGA AACCTGGTCTCTACAAAAATACAAAAAAATCAGCTGGGCGTAGTGGCGTGCGCTTATAGT CCCAGCTACCTTGGAGACTGAGGCAGGAGAATAGCTTGAACCCAGGAGGTGGAGGCTGCA GTGAGCCGAGACTGCGCCACTGCACTCCAGCCTGGGTGACCCAGTGAGTCTCTGTCTTAA ATAAATACATAAATAAATAAACAAACAAATAAATAAAGTTGGCAGAGTATATAGTCTGAA GGGGTTTGAGGAAAGGGGATTCAGGTGACTGAGAAGTTCCGATGGGAGAAGTAGGATCCA ATAGAGAGAACAGAGAGGCCTCACAGTGGGAAGAAAAGACTGCCAGCCCAGGAGTCAGGG AGTGAATCCCCATTCAGAATGAAGAGCCACAAGGAAGACCTTCCAACCCAGGAAGTATTA TACCTTTCAAAAGAAGGCTGGGACCTTAAAAATCAAAATCTGTCCTTACCAGCTAAAATA AAAGTTTTTCTACAGTCATGGCTCAGGAGTTTGACTCACCAGTAGATCCCCAATCAGCCA GTCAGAAGACAAAGGCTGCAAAGTTGCACATTGAGAGTCCTGGGTCAGAGGCCTGTGGCA CCACAACCATTAAAGAAAAAAATTATTCAGCCCTTGTCAAAGCACGGTCGGGAAGGCCTT ATTCAGGAGCATTGCCACTGGCATATGGACCACAGGCAATGGGGTTTTGCTGCAGGGGAG AGATTGGGCTGAACTCTGAATACAGCATGGACAAGTGTGAACTGATAGCCAAGGAGCAGG GCGGGGGTCAGTGGATGAAAAATTACTAAGAAGAAACATAAGTGATAAGGGGGATACTGG CTAAGCTGACCTAAGAGGATTCTTGCTGAAGACAGGCTGGGGTGATCAGACATCACCTAG AGGATGGTGGAGGGATGAGGAATTTCATCAGTATTGAGGGCGATCAGATATCAAAAGCAG GGTGGTTCTTGCTAAACTGGAATTCTCAGACGGGCCTAGAAGATTTAGGAGCCTGACTAA AGTTTGGCCAAGCAAATAATCTTTTGTCAATACAACAACACGATACTTTTTACTGTTTGT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGAGTCTCCTGCTGTCTCCCAGGC TGGAGTGCAGTGGCGCGATCTCAGCTCACTGCAACCTTTGCATCCCGGGTTCAAGCAATT CTCCTGCCTCAGCCTCCCAAGTAGCTGCAACTACAGGTGCGTGCCACCATGCCCGGCTAA TGTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCCTGTTAGCCAGGATGGTCTCGATCT CCTGACCTCGTGATCTGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCC ACCGCGCCAGGCCAATACAATAGTATTAACTAATCTACAGACCTTGTTTGAATTATATCA ATTGTCCCATTAATGTCCCTTTTCTGAGCCAGGATCCCATCTATCGTCTGGATTCCACAC TGCATTTTGTTATCTCTTTTAATTGAAGATGGCTCCTCAGTCCATCTTTGACTTGAATGA TCTCGATACTTGAAAAGCACTGGCTGGTAATTTTGCAGAATGCCCCTAATTTGGGGTTTG TCTGCTCTTCATTCAGATTCTGCCTTTTGGGCAAAAATACCCACAAGTGAACTTGTGTTC TTCTCAGGAGGTCCACAGTATGTCCTCTTACTGGAACTAACTTTTTTCATTTCTATTGAG GCAGAATTTATATACAATGCAATACACATGTTTAAATGGTACAATTTGCTAAGTTTGCTG GTGACCTTGATGGCTTGGTTAAAGGTGGTGTCGCCCAAGTTTTCTTCCACTCTAAAACTA CTATTTTCCCTAGGCAATTAGTATCCTGGGGGAGAAACTTTGCAACACTGAAAACAAAAC AAAACCTGTTTGTCATGTTTTTGCCCACTATTTTTTTTTTTTTTTGAGATGGAGTCTCAC TCTGTCACCCAGGCTGGAGTGCAGTGGCGTGATCCCGGCTCACCACAACCTCTGCCTCCC AGGTTCAAGTGATTCTCCTGCCTTGGCCTCCCGAGTAGCTGGGACTACAGGCACGTGCCA CCATGCCCAGCTAATTTTTTTTGTAGTTTTAGTAGAGACGAGGTTTCACCATCTTGGCCA GGATGGTCTTGATCTCTTGACCTCGTGACCCACCCGCCTCGGCCTCCCAAAGTGCTGGGA TTACAGGCGTAAGCCACTGTGCCTGGCCTAATTTTTTTGTAGGGGGTTGGGGGCCGGGGA CCGAGTCTCGTTCTGTTGCCCATGCTGGAGTGCAGTGGCTGGATCTTGGCTCACTGCAAC TCCTGCCTCCCAGGTTCAAGCAATTCTCCAGCTTCAGCCTCCGTAGCTGGGACAACAGGC ACCTGCCACCAAGCCCTGCTATTTTTTTTTGTATTTATAGTAGAGACAGGGTTTCACCAT GTTGGTCAGGCTGGTCTCGAACTCCTAACCTCAAGTGATCCGCCCGCCTCGGCCTCCCAA AGTGCTGGGATTACACGCATGAGCCACTGTGCCTGGCCTTGCTCACTATTTTTTCTGGAG ACGGTCTCGTTCTGTCACCCAGGTTGGAGTGCAGTGGCCCGATCTCAGATCATTGCAACC TCCACCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAG GTGCCTGCTACCACGCCTGGCTACTTTTTGTATTTTCAGTAGAGATAAGGTTTCGCCACG TTGCCCAAGCTGGTGTGGAACTCTTGAGCTCAGGTGATCCGCCTGCTTCGGCCTCCCAAA GTACTGGGGTTACAGGCGTGAGCCACAGGGCCCGGCCCACTATTTTTATCACTCAAGATT TTCGTCTACAGCAGCTATTATTGGGGTGGCTACCCAGTGGAAAATTTTTCTATTTCCACT ATTCCTTCCACATTTATTGACAGGAAAGCTAGTCTAAGGAAGAGCTGTACCCTCACTTAC TCAGACCCTCGTTTTCCTTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTCACTCTGCCGC CCAGGCTAGAGTGAAAGTGGTGCTATCTCGGATCACTGCAGCCTGGAACTCCTGGACTCA GGTGATCCTTCCACCTCAGCCTCCCGAGTAGCTCGGACCACAGGCGCCCGCCACCACGCG GCTAATTAAAAATAATTTTGGGCCCGGCGCGGTGGCTCATGCCTGTAATCCCAGCACTTT GGGAGGCCGAGGCGGGCAGATCAATTGAGGCCAGGAGTTTGATACCAGCCTGGCCAACAT GGTGAAACCCCGTCTCTACCAAAAATATAAAAATTAGCCGGGCGTGGTGGTGGGCGCCTG TAATCCCAGTTACTCGGGTGGGTGAGCCAGGAGAATCGCTTGAATCCGGGAGGCAAAGGT TGCAGTGAGCTGAGATCGCGCCACTGCACCCCAGCCTGGGAGACAAAGCGAGACACCGCC TCAAAAAAAAAAAAAAAAAGAAAGAAAATTTGTAGATACTGGGCTCTCACTATGTTAGCC AGGCTGGTCTCGAACTCCTGGGCTCAAGTAATCCTCCTGACTCGACCTCCCAAAACTCTG GGATTACAGGCGCAAGCCACCGTGCCCGGCCGAACCACCGTGCCCGGCCGAACCCTCCCT GTTTTGGAAATCCTGTATTAGCAAAGGGGACAGCGCCAGACAGTTGAGCACACGCGTTCT TTATAAATGATGATGTCTGATACAGTGATGCTTTTAACAGGGTTGTGTAACGCCTGTGTG TGCACGGTCTCTTCTGCAGTGTCCACCGAAGGTGGCCCGTCTGCGTGTCCGCTCCCGGCG TACTCCGCGCTCCCTGTTCCCGGCATTCCCCGCGCTCCCCGAGCTTCCCGCGCTCCCCTG CTCCCGGCATTCCCGGTGCTTCCCGTTCCCGGCGCTCCTCGCGGCCGCCGCCCTCAGTCC AGCGAGCCCCGCCTGGGCAGCGACTGTTAGCGCGACCGCGGCCTGGGCGCCTGGTCCCTC CCGCGCGGCCGCGCTATTGGGCCGCGCGCGGCGGACACACGCGCTACGCGCCGCCGATTG GCGGCCTCCGCAGCGCGCTCTGCGGCTCCCCCAATGCGGGGCGGCCGCGCGCGGGGATTG GCCGCACGCCGCGAAGGCCCGCCCTCCGCTCGCCCGGCGCGGCAGGCGGGTGCCGGCGAC CGGAGAGCCTGGACAGGCTTTCCAGATGGCTGCGGCGGTCGGTCGGTGAGGCTTTCCCGG CTGTGGTTTGGCTGCGGGCGGCTTGGGCAGCCCGCGGGCGCCTCAGGTAGGTGCGGGGCG CGAGGGGCGCCCGCGGGCGGTTGGGCGGGCGCCGCGGCCTGCGCGAGGGCGCGCCCTTCC CTCCCGCCTCCCTCCGGGACCGAGGCCGCGGGCGGGGCGGGGGCGCGGAGCCCGGCCTTT GTGAGGCAACATGGCCGCGGGCGGTGGAGAGCGCGGGGTGGGGACGCCTCCGGGGCTGGG GGGCGGGCCCCGGGCCCAGGCGTTGCCGACTCTCGTCGCTGTCCGCCCGGGTAAAGAGGC TCCTCTCCCGGAGGGCCCGAAGCCTAGCATCGCCTTCAGGCGGGAAAGGTGTGGACAGGG CCAGTGCTCGCCTGGGACCCTGCCGGCTTTAACACAGTTTGGGAAATCCAAGAAGTCGCC TAAGTCTTTTTGTCCACAGAATGCTCACGGGTTGAGTGTCACGTAGATAATTGGGATACG GTTTTATTGTAACAATGAGCATGGCGGAAATAAATGGAAGGTGGCAACCTGTTGACAGTG ATGCCTGTCTGGTGTCAACCGATGGACTTGGGTTTCTTGTGGCAGGAGTCTGATTTGCCT TTTTGCATGCTTGCATGTACCTTGTTCCTGCACGGCAGTCCTGTCTTTGGCTGTCGACTG TTCGAGGCCAACCACCAGGCGAGGATCCACTGTTTCCTGGCCAGACTAGACTCCCAGCTG TGGGAGTGGCCCAAACCCTGCTGCAGGCCTTCACTCACAAAAGGGAGTGAATGGCCAGCC CGGGGTACCCAGTGTTGCCTCTAAGTGCGGGATATGAGGCCTGTTGGAGGATGACAAAAG GAACGGATGAGGTCATACTGCCGCTGGGTTGGGCGGGAGGCTGCAGCGGGAGAGACTTGA CCAAAAGGCAGCCTCCATGGCGCTGGCACTGGTGCTGGCCCGTCACAGCCAGTGCCTCTG TTAGCCAACACAGCAAGTGGGCTAGGAGGAAGGGTGCTGTCCAAGTGTACGGGTTTTCTC CCCGTGCCAAGGGTCCAGGGGAGCTTGCCGTGAGCCCGTTCCTGTCCGATAGTCCTGTTG GCAAGGCAGGGTTTGCATGCGGTGTTTGGTCAGCTGGAGCAGTGGGGAGGCCTACGGGCA GAGAGCTCCCCTTTGAGCCCTTTGCGGCCAGGCGCTGTGTGGAGCACTTCCCAGGGGTTG TTTTGTAGTCTCACCGTGACCTTGGGGTAGGAAATGTGCTTCTGGCTGGCTTGCCCAGTC TTAGACTCTCTTGTTGTAGCCCTCTCCTTTGTGATGTCACCCAGCCTCCTGACTTTAATG CCAGCTTTCCTTGACATCTTCACAGCTCCAGCCCTGGCCTCTCTTCTGATTTCCAGCCTG GTGTTTCTGGCCACCTCTTCCATATCTCCATGTGTTACTTTGGCGCCAAGTAGCCATATC CAAACCAAACTCAAATCTTCACCCATGCCAAAACATTCCTTGGCCTATCTAAAGTCCCTT TGATTCCTCTCTTTCTTACACCTTCATGCAGTCCTAAGGAGGCCCCGTCAGCTCACCTAT TTCTAAGCACAGACCATTTCTCACCACCTTCAAGTTAACATCCTGGCCCAGCCTACCTGG TGAGCCTCTAATTCTCTTTACTGTCACCTTAGGTCCTCACTCCCCATCCACCATCTATAG TTTTTCTGGACACAGAGTGATTCTTCTTTCTTTCTTTTTTTTTTTTTTTGTTTTTTTGAG ACAGAATCTCGCTCTGTCGCCCAGACTGGAGTGCAGTGGCGTGATCTCAGCTCACTGCAA CTTCTGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTGC AGGCGTCCACCACCACCACGCCCGGCTGATTTTTGTATTTTTAGTAGAGACGGGGTTTCA CCATGTTGGCCAGGCTGGTCTCGAACTCCTGGCCTCAAATGATCTGCCTGCCTCAGCCTT CCAGAGCGCTGGGATTACAGGCGTGAGCCACTGTGCCTGGCCACAGAGTGATTCTTTTTA TTTTTTTGAGACAGAGTCTCGCTTTGTTGCTCAGGCTGGAGTGCAGTGGTGCGATCTCCC GCTCACTGTAAGCTTCGCCTCCTGGGTTCAAGCCATTCTCCTGCCTCAGCCTCCCGAGTA GCTGGGACTATAGGCGCCCACCACCACACTCGGCTAATTTTTTGTATTTTTAATAGAGAC GGGGTTTCAATGTGTTAGCCAGGGTGGTGTCGATCTCCTGACCTTGTGGTCCGCCTGCCT CAGCCTCCCAAAGTGCTGGGATTACAGTCGTGCACCACCGCGCCCGGCCCAGAGTGGTTC TTTTAAACCATAAGATGGTATAAAACAGAGGCGGTGTAGATGCAAGCCAGCTTGCCTGCT TGTAAAGCAGCTCCATCACTTCCTGGCTGTATGACCTTGAGCATGTGGGTGAACCTTTCT TCCATAATGCTATGTGAGACCTGGGTGAGTTAAAATGCTTAAAGTGCTTAGAACAGGGCC TGGCTGAGGTTTGGTTGCCACTGCTATCCTGGAATCAGCTGATGCCACTGTGTGGGCCTT CCTATCCGGTGGCCCATCTCTCTCACTAAGAGCCAAAGTCCTGGCAGCAGCCTGGGAGGA CCAGCCCTGATCCATCTGCCCTCATGGACTTGGCCCACTGTCACGTCCCCTCAGCACACT GCCCCAGCTGGCCACACCACATTCCTGACCCCAGCCCTCTGCTTTTCCTCTCAGTGTCAC CTGGAACATCCCCTCCTCCCAGCCCTGTAACCCACCTGGCTCACTCCTGCCTTCACGTGG ATGCAGGCCATGAGGCCTTCTTGGATCCCCCTTCCTAAAACTGCCTCCCCTTCTTCAGGC GCTTTCCTTGTTTTGTTTTTGCTTGAATACTTCTTGGCATGTGACAGGGTGCATGCTTTT CTTAGTTGTTAGCGGTCCATCTCCCCCACTAAAATGTGAGCTCTGTAGGGCAGGGTTTTG TCCACCTTGTGTCTGTAGCATCTAGGATGGTGTGTGTGTGTGTGATGAATGGAGAAAGCT TATAGAGGGGAGGAAGGAACTTGCCCAGGACTGCCCACCACTGAGGTCTGGGGCTGGGCA GGTTTCCTGCTTTCCTAAGGAGCTCACTCACTTGTAAGGCACCAGGCTTTCCTGCTGTGC ATTTCTTCAGTGCCGCCGCTTGGCCCCATTAGCACCTTTGCTTGGAACGTCTACTCTAGC TGCTTCTTCGCCTTCTGTGGTAGCAGGCTTCCCCATTCCGTCCCACTGTTGGTCTCTAAT CCCCTGCTCCTTGTGGCTTCTTCATGTAGCTTTTTGTGTCCCAGAATCATGGATTGACTC ACCAGGGTCTCTATGGGACTGCCAGGTTCTGAGGTGTGTGGCCCTGGTCTTCCTTGTAAT CTGTGGCACTGGGCGCATTGACCCCTCGGTCTGTTTTCTCTAATGGTCTTTCTTGGTTGC CAGGAGACTGACCTCACCCCGCTTAGTGTTCCTCATGCCTGCCTGGTTGCTGCCCACAGC CTCTTGAGCGTCTTCAGCACTTTATAGTCAGTTGTTAGCTCTGTGCTGTGTTTCCTGGAT CCTCAGCACCTTTGATGGACACATCTTTGGGAACATACGGTGTGAAATTCCCATTAGATT CAGAAATGTTAGGACATCTTAGAATCCAGGAAATGGGGTGACAGTGCAGTGGTCATTGAC CTGCCAGTGTGTCCAGGCCCTGTGACCTTGTCTCTTGTCCATATAGTGCGTTCCTCGGAG TAGATGGCCAGTGGATAGTTGTTAAATGTATTGATTAAACCTAAACTAGTCTCTGGTTTT CATGTTGTGTCCCCTTCGAAGTACATCCACCATGCCTTGTCCCTTGTTAAATGTGTGATG TCTGCATCTCCTGTTCTTTCCGGGAATCTTGGGAGGATGTTAGGTCATGCAGTGCGGTTT GCATGTGTGCATCTTTACTGGTATGCACGCATGTGCATGCATGCGTGTTCCTGTGCACGT TGTGGGAGTGTGCATGTGCGTGTGTCTGCATGTGTTCCTTCTGCTCTGATGCAGGGTTTT CTGGCTGCCCCCTCAGACCCATCTTCCCTGCTTCATGTTGTCCCTTTCTTCATGCTTCCT CTGTAGTAAGGCTTCCTAGAGTTGCATCCTTAACCCTCATCTCTGGGTGGCCTTCCTCCT GTGGGTTTGGCGACTACCTCCTCAGTGCGTCCTGGCCTTCCGTATCTGGACCAGTCATGG CTCCAGAGTTGTGGCCTCGGCACCTTCAGAGCCTGCCTGGAGGTCCCCTCTGTTCTCCTC GCCAGGCTCCTGCCTGCATGCTGGAATTTGGGTGTCCTTCCTTGTCTTTAGTTCCCTCTT TTCTCTGCTATCCACCTCCGTTGGGTCAACAAGTGCGGTCAGTTCTGTGACACTGTTCTT GGCCCATTGCCAGGGCCTCTTTCACACCTTTGTCCTCTGGCCTGGAGGACTGTCCCCGTG CCTTGGATGCAGTTGCTTCTGCCTGGCACAGCTGTGGGTCTCCCCTCTCAAGTAGGAACC TACTTGTGGCATGAGAGTCGCTTCACCTTCAGTGGCTCCTTATTGTCCCCAGGGCGGAAC GGGCTGCAGGTGGCACCCAGGCCTCTCTGTGCCTCGGCTCCAGCTGTCTCTTGTGGCTCC CCACCCCAAATCCTGGCAGGTCCCTTCCCCACAGCCACCCACCCCTCTCCTCCATCGGGA ACAGAACTATGCTGCCACCTCTGGTGACCTCAGCACGCTGCATCACTGTCCCCGTCCACG TGCTACCCTGTGGGCCCAGGAGAGCCCTGGGGTCCCTGGGTAGCAGAGCGCCTGGCCATG CCTCTGAGGCCCCTAGTGCCGCAGAGTTGAGCTGAGGGTCTCGCGCTCGCCCTCTGACTG ACCCAGCCCTTGCAGGTGAGTGGATTGCTGTGCTCTGGTGGCCTGAGGGAGGCCACGCGC CTTCTGTGTGTTCCAGAAAGGGTGCCTCCCACTGCATGCTTGCTTATCTGAGTTAGAAGA ATGCTGTGGTGGAGTTTAGTGTAAATTTTTAAAATATTTTTTGAGCCTTATGATTATATA GTTTTTGTGTTTCTGAAGTAGGAATTAAAGTGGGCATTAACAAAATATTTAACTTTGGAC TTAAGTTATAATTCAGGTTCTGAAGAATAAAAGTAAGGTTAGTTTGTTTTGATGCCTAAA AAGTCCTCTTAGGGAATATTATTTTGAAGCCCTTTACTATGCTGTTAATAGTGCTTGGCT TTTAACTTGGTACCAGGGAATTGGAAGGTTTCTGTCATTTTGTGACGATATTTTTAAATT TCTTTGCAGGTAGAAGAAGAAAGGTGCCACTCCGGCATGAAGACAGACTCGCTTAGTCGC CAGTCACTTAAGCTGAGTGCATTGTGATTTCCAATAATTGAGGCAGTGGTTCTAAAAGCT GTCTACATTAATGAAAAGAGCAATGTGGCCAGCTTGACTAAGCCGCCAGCGCACAGCGCG GCAGGACGCGCCCGGGTCTCAGCGGACTTGTGCATGTTAGCTGTGTAGATTTATGTGAGG GCTTGTAAAACTCTGGTCTTGTAAACTAGTCTTAAGCGCTTTTAATATGGAGACAGATGA GAGCCCCTCTCCGCTCCCGTGTGGGCCCGCAGGAGAAGCGGTGATGGAGAGCCGAGCTCG CCCCTTCCAAGCGCTGCCCCGTGAGCAGTCTCCACCACCTCCCCTGCAAACGTCCAGTGG TGCAGAGGTAATGGACGTTGGCTCTGGTGGTGATGGACAGTCCGAACTCCCTGCTGAGGA CCCCTTCAACTTCTACGGAGCTTCTCTTCTCTCCAAAGGATCCTTCTCTAAGGGCCGCCT CCTCATAGACCCGAACTGTAGTGGCCACAGCCCGCGCACCGCCCGGCACGCACCTGCGGT CCGGAAGTTCTCCCCTGACCTTAAGTTGCTTAAGGATGTAAAGATTAGCGTGAGCTTTAC CGAGAGCTGCAGGAGTAAGGACAGGAAGGTGCTGTACACAGGAGCAGAGCGCGACGTGCG GGCGGAGTGCGGTCTGCTCCTTAGCCCTGTCAGTGGGGACGTGCATGCTTGTCCCTTTGG CGGGAGTGTTGGTGACGGGGTAGGCATAGGGGGTGAGAGTGCTGATAAGAAGGATGAGGA GAATGAGCTGGATCAGGAAAAGAGAGTGGAGTATGCAGTGCTCGATGAGTTAGAAGATTT TACTGACAATTTGGAGCTAGATGAAGAAGGAGCAGGCGGGTTCACGGCTAAAGCAATCGT TCAGAGAGACAGAGTGGATGAAGAGGCCTTGAATTTCCCCTACGAGGTATGTTGGCAGCC CCTCCTCTAGAGGGCTCTTAGCAAAACCCAAAGAGAGATTTGGGAATTGCAGCATCTTTT GAAAGCAGGGAAATTAAAAAAAAAAAAAACAAAAACCAAAATCCCCTCTGAGGTGGAATA ATGTTAATGTGGAGAAGAGAAAGATGTAAGGAGTCCAGATTTTTAAAGTTTCCTAATGAA AAGTTTGGCCCATGGGTAGGCCCTGCATCCCTGATCTAGCGCGTGGGGCAGCAGGTGCTG CTGAGTTACGCTCCTTGGCAGTGTGTGCCCCTGGACCAGGTGTGTTGGTGTCAGCTGGTA GCTTCATCCTGTTTGTTTTTCAGATGATCATGCACCCTAAGGGCACATCTAGGCCCCCTG AGAGCACCTCCTTTCTGTGTCTGTTCTCAGGAATGCTGTTGAGCTCTCCTGTTGCAGGAG CATGAGCGCCAGGGGCTCTGGTGTCTGAACAGCGTGTTTTGCAGGATGACTTTGACAACG ATGTGGATGCTCTGCTGGAAGAAGGCCTTTGTGCCCCCAAAAAGAGGCGAACAGAGGAAA AATATGGCGGAGACAGCGACCATCCGTCCGATGGAGAGACAAGTGTGCAGCCGATGATGA CCAAGATTAAAACAGTGCTCAAAAGTACGTGTGTGGGTCAGAAGCAGTGGGTGTTCCAGG GCAGTGGAGGGGTGGTTGCTTCCTTAGCAGAAATGCTTTGAGAGAGCTCTGGTGCCAGGT AGTGGGCTGGGTGGAGGCATGTTTGAGGACAGGACAGAAATATCTGAGGAGGGAAACACA GGATGGGAGGACGTCCTGATGCATGACCCAGATGCGGATCCTGGTGTGTGCTATGGAAAC CACAGAGCAGCAGGCACTGTGCAGAGGAATGGGAGGGGACTTCTGAGGGGGTCAGGAGGG GATGTTGGTATATGCCAGGAGCCAGCATGACCATCAGCTGGAGGGTGTGGAGACCAAGGG CAAGGTGGGTACACAGTGAGATGGCAGGTGATAGTTGTGATCCAGGTTCTAGTCCTGGAC CAAGTGTCACAGGAAGCCACCAGAAGCAAGTGGTGTTACCTGATTTTTGTTTAACAACAT GGCTTCTGGGCCACAGCATGGTGTGTGCACTTCAGCTGGGCAGGGTGGAAGTGCCCTGGT CGACGTGGCACTTCCTCAACTTTGAGTTGAGGGTAGTGGGGAGAGCACTTGAGAGGCGCC CAGAGTTGCACTGTGAGGCTCTGTCATCGATGTGGTGCAGGTGTTGGGGGAGTCGGAGTT GGTGGCTTGGAGGCACCTATGAGGGTCCTGGTGGAGATGTGGGGCTCGGTAGAGGGACTG AAGAGATGCACGGGGACTCAGGCTCACAGGTGAGACCCCAGGCTGAGGGCTGGGGTGCAC AAGGTGGCAGGGTGCGACTGGGAGCAAAGCAAGGGGCGGGTGGCACATGGCCATCCTCCT TGGGCCATTGAGGGGACAAGTTAGCTATGCCAGGTGCCACATGTTGCTGCCTATGTCGTG TTGGTAGCAGTTAAGCAGGAGGGCTTTTGGTCTAGACGGTTGCAGGCAGGGTAAACCTGG GGCTTCAGCTTCTGGGGCTGGGGGCAGGAGTCTTGACCTCGGGGCCTCTAGGAGGCTGGT TTGGTGCTGCATTTTCAACAGGCTCATTCAGAAGGTAAACCCACATTCGTCCTGCACAGC TCACTGGTCAGCAGCCATTCTGGCCTGGATGCCTGGATGTTCACAGACGGACAGCTGCAT GTGACCCCCACCTCAGGTGGCTGGGCCAACTTTGTGACCACTCACTGTCACAGAGGAGCA GCCCCAGCGGACCCGTGGCCCGTCTCTTGCTTGGCTCCGTATTCCCTTTGCTTCTTTTCC TGACTTAACCTAAAGCTGGCAGGAGGCAGCTCCATCTTGGCCTGACTCTGTGGGGAGAGA GCCCACGCAGTGTGCATGCACCCCTGACCTTTAACACTCCCTCCCTGAATCATCACCTGA TGGTGGCCTGGGTACAGGTGTCAGGTCCAGGGTCAGGGCCACTTGGTGGGTACCTAGCCC TGAGAGTCACTGGCCCTTTTTCCCCTTATGTATATTTTTTAAGGTTTCATTTGCTTTCTT CTGTTTTTTAATTTTTTTTTTGAGGCGGGGTCTTGCTGTTACCCAGGTTGGAGTAGCTCA CTACAGCCTCAGCCTCCCAGGCTCAAGTGATCCTCCCACCTCAGCTTCCCAAGTAGCCAG GACTATAGGTGTATGCCACCATGCCTGGATAATTTTAAAAAGTTTTTTGTAGAGTCAGGG TCTTGCTGTGTTGCCTAGGCTGGTCTTTCTAAGTTTTTAACTTGTTCCCAATACCTGTGA GAAAATTAAGTTTTAGTTACATCTTTGCCTTACGCTCTTCCCAGCAGTCAGGCCTGGTGG CAGGTGTCAAGTTGATAAGTCGTAAACCTCTTCCTCACAACTGCCTATAAAGCAAAGAAG GGGTTGGGAGTAGTGTATTAAACCAACTTAGAAACGGAATCATTTGAAGCAGTGTTTTGT ATGCTGCTGAGAGTGCAGCTCAGTTACAATAAAGACTTGTCACCTGGTCACTGGGAGCAG TGGCTGACAGCCATGCAGTTTATTAGCAAAGCTCATCCAGCCCCAAGGCGCCTCTGGCCA CAACCAGACAGGCAGCAAAGGAGTCCAGTGTCCACAGTGCTTACCAGATGTGGAGGCAGG AGCTGAGGCTGGGACTTATATCTTGCACAGACCTGTTCTGTGCTCCTTCATGTGCTGGTT TGACACGTGTAAATTCCCACCTCAGGCCACATGCTATCTGTCCTAGGCCTGGAGGCATAG CAGTGAGCAAGGCAGAGAGGAATTTCGATTATCTGTGAAGACCCAGTTAAACACATGCTA GTACCCAACAATTTCTTGTGCAGGAGGTGCTGTGGCAACAATTCCAAGTGTTTTTACAGT GATTATAGGATGTTCTTGTCTTCCTGTGCAGGTCGTGGCCGCCCACCTACAGAGCCGCTG CCCGACGGGTGGATCATGACATTCCATAACTCTGGAGTCCCGGTGTACCTACACAGAGAG TCTCGGGTGGTCACCTGGTCCAGGCCATACTTCTTGGGAACGGGAAGCATACGGGTAGGG GAGGCATCAGTCGTGACTTTAGGCTTGTAAGTTCTCAGACCAAAACGTGCACATCCACAC ACATGGCACCGCAGCCAAGCAGAGGCCGGTGAAAGGCATCAGAGTTTCAGACTCTCGGGT TGTCCTTGTAATCCATATTGCAATTTCACTATCCACAGAAACACGACCCTCCTCTGAGTA GCATCCCTTGTCTGCATTATAAGAAAATGAAGGACAACGAGGAACGGGAGCAAAGCAGTG ACCTCACCCCTAGTGGGGATGTGTCCCCCGTCAAGCCCCTGAGCCGATCTGCAGAGCTGG AGTTTCCCCTGGATGAGCCTGACTCTATGGGTGCTGACCCGGGGCCCCCGGACGAGAAAG ACCCACTAGGGGCTGAGGCAGCCCCTGGGGCCCTGGGGCAGGTGAAGGCCAAAGTCGAGG TGTGCAAAGATGAATCCGTTGGTGAGTTTTTGAAGGACTCTTCCCTTCTTGCCTCCTGGG ACCCATGAGCTTTGTTTTTCTAATGAAGGCCATAATTGTTCATAGTTCCTAGTTGTACTT GTGAGCAAGGGGCTGTGCACCTCCACTGTTGGTGTGGCTGAAAGGCTTGTCTTCCTGTCC TTGCAAGAACCTGCCTGGGTGCAGCACCCTAGGCTCCTGTCTGTGGGCTCTGTGGTGTCT GCATGGCTGAGGCTGCTGGGGAGATGGCTGCTGGCACTGGCAGTGGGCCGTTTTCTCTCT GGTTGTTTGTATGATCTTTCCCTAAGTCTTGCTTTTTCAGTAGGATGTGCTCATGTGGCA TTGTTTTGATTTGTCTTGATGAGCTCAGTGAGCCTCTTTAATCCATGACTCAGTGTTGGG AAACTGTCACCATTCTGTGGATAGTTCCCTCTTCAATCCTGCAATTGCTTGCTGGATGTG TACTGGTGTTTCCCATTCTCTCTTCCATGTCTTTTAGCCTAGTCCCGCCATTTAAGTCTT AGTTCTCTCTTCAGGTGTGGCAGGTCTCTGTGAACCCACCTGCTGGATTACTTTCAAAGA GCACTTTCCATTTTCTAGGTTTCTATTTGTCTGTACATCACTACCCAGCCTGTCTCATAA CTCCGTGGAGTGTGGGGTGCCCTAGAAGAGCCTGTGATGGGGGCTGTTGCTGAGGATGGG GAAGTGGCCCGGGAGGAAATGTGACAAATGCGAGGCCCTGTGCAGGGAGCTGCCTGGGTG TGTTGCTGACTACAGAGGTGGCCAGGATGGCCAGAAGGAAAAGAGTGAGGTGGTCAGAGA GGTGGCGGGCGCATGGAGCCCGGGTGCTGCTGGCATTCGGGCACCTGTTTTGATACAGGG AGGAAGTCCCGAAGGCCATCTGACTTTTTTAAGCTGTCTTTTTTCCTTTTCATTTTTGTA TTCTTCCTGCCCCAAATAAAGTAAAAGAACATCTCAGAAATTCATCCAGAAGGGACCTGC CAGAATAATCTGAGCTGGTGTAGGGAGGCCTTCATTCTTTGCTTCCCTCCCCTTTGAAAG GGACGGGGAAAGGAAATAGTGTCATGTGGCCTGGGCCTGCCCCATGCACTGGGCTGTGAG AACCTGACTCCTATGTTGGAAGTTAAGTAATTTGTTTCTCTGGTAAATCTGGGACAGATC TCGAGGAATTTCGAAGCTACCTGGAGAAGCGTTTTGACTTTGAGCAAGTTACTGTGAAAA AATTCAGGACTTGGGCTGAGCGGCGGCAATTCAATCGGGAAATGAAGCGGAAGCAGGCGG AGTCCGAGAGGCCCATCTTGCCAGCCAATCAGAAGCTCATTACTTTATCAGTGCAAGATG CACCCACAAAGAAAGGTATAAGCCTCTGCATTTTAACATCAGCAGACTGGTTATGCTGTT ATTTCTAATGTGATGTGTTGAGGGCATGTTTTATGAGTTGCATTTCGTTCAAAGTTTATG TTTATCCCATGAATGCAGGGGTCTGCCACATTCACGGTCGTGAGCCCCTAGTTACTGACA TGGTAACAGGAAGCTGTGTGCTAGCTTGTGGCACTGCTTCACACTTGCTGAGATGGTTCT TTTTGTCACAGAGTTTGTTATTAACCCCAACGGGAAATCCGAGGTCTGCATCCTGCACGA GTACATGCAGCGTGTCCTCAAGGTCCGCCCTGTCTATAATTTCTTTGAATGTGGTAAGTC TAACCTTCCCCATTTCAGTCCTAAAGAATCACAAGGTGTAACTTTGGTCAGGGCTGGGGA GGCAGGCGCTGACCGCTAGCCCTACTCTACTGGAACGGGAGGAAAGGGAAGGGTAGAGAG CAGCGTGCTGCAGATGGGTGGCTTGTTTCCTGGAGCCGCGCCCCATCTGAGTGGGTGGCT CTGCTTTTCCCTTCCAGTCTGTCCCCTCTCCCCACCTTCCTCTTGTCCTGCCCCATCTGC CTTGCTGGTTTCTGATCCTGCGTTGTCATGGACAGCCTTATTGCGAGCAAGGCCTCCCCA CTGTGCACTGGGTCCTTTCCCTGGTCTCCTGGTGCCCCTGGGTGTCTGAAGGGTTCTTGG GCATGCATTTCCCACCAGCTCTGTCTTCCTTGGGTGCCCTCATCACACCTTTGTGAAGAC CAGTCTGTCTTCCCCTTGGAAGCTGGAGCCTGGGGGTCACTCATCCTTTGAGGTGGTGTT TCTTTGGCAAGCCACATCTTGTGCTTTCCCATTCCGGCAGTCCTGTGTCCGTCTTTCGTA GACCTTGGTCCCTGTCGTAGGAGGCTCTGCCCCCAGCCTCCTGCTGCCCCTCTGGCTCAC CATCTCCCACGTGCCTCATTTCCCTCCACTTTCCCAGCTGTGCCCTTTGCTGCTGCTTGT CTGCTGCCCGTCTCCAGTCCCAGGGAGCCCCAGGTCTCTGCAGTCAAGCACAGGCCCACT CTCTGCAGGGACAGCCCCTGACTGCGCCTTGTCTGTCGGTGTGGGCAGACTGTGCACACG CTTTTGATGTCTTGACTCGTATGTTTTAAAACAAGTAATTTTAATTTTAAGAGAACCCAA GTGAGCCTTTTGGTGCCTCGGTGACCATTGATGGTGTGACTTACGGATCTGGAACTGCAA GCAGCAAAAAACTTGCGAAGAATAAAGCTGGTAACGTGCTTGCTTGGGTGTCAAAGATAC GTGCTGCCTGCTGTGTCTGCCTCGCTGCTTGGTTAGGGAGGGGCTGAGCAGTGGTGACAA GTGGGGGATGTGGGTGGGGCATGTTTATTTTATTTATGTAATCATCTACTTTGATTTTTA AAATTACATGCTCATTGGAAAAGACTCAAATAGAGCAAAATGTGAGACTTAAAAGTAGAA GGCCTCGGCCGGGCGTGGTGTCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAAGTG GGCAGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCCTCTCTC TACTAAAAATACAAAAAATTAGCCGGGTGTGGTGGCGGGCACCTGTAGTCTCAGCTACTC GGGAGGCTGAGGCAGGAGAATGGCGTGAACAAGCGAGGCAGAGCTTGCAGTGAGCTGAGA TCGTGCCACTGCACTCCAGCCTGGGCGACAGTGCAAGACTCTGTCTAAAAAAAAAAAAGT AGAAGGCCTCTCTACTGAGTGACAGCTGGGCCAGTATGTCCCTCCCTAGTCCTGGCCCTG CACATATTCAGGGGTGTGCCTAGAGCCAACTGGGCTGTGCTGGGCACATGCTGTAGTGCT TGTTTGTTTTTAAACTGAGCAGCATGTGGGCATGCCAGGAGGGAGGAGAAGCGGGCCAGC ATGCTGTTGGCATCCTCCCCCAGTGGTGCTGGCTCTTACCGGCACTATTTCCTGAGTCCT CCCACTCCATGTTTGGGCCTAGCCACACAGAACACCTGGCACAGCGCAGCTCTGGGAAGG CTGTGTGTCCTGGCGAAGACCCACTGCCCTTTTGGACCATGCCCTTGCCGTTTGCATTCA TGCCCTGCATTCGTGGTAGTTGACATTCTTTCACAAAAAGCCATTTGCCTCAGCGTCCAG GGTTAGTTGGCTTCTTGTAGGGTGGTGTTGGGTGTACCCCAGGCCCTCGCACCCACAGGT TGATTCTCGAGGTATCTGTCATTGCCGTGTGTTTTCCAGAAGGCCTGCTGCCCATCTTAG ATTCTGCCTAAGAGACTTTTGCAGCTCAACAAAGGGGGTGTGGTGGAGGTTTTGACAGAT AACTTGCAGTGGGGCCTTTTGTGGGGGTGGAGATGCCTTGGGCGTGGCTGTGTGCTCTTC CCTCTGCGGGATCACCCGCTTTCCTCCCTATGTGCTGGAACCCACTTCCCTTGCCCAGAC TCTTGGCTGCCCTATAGACAGCTTGGCTCCATGTCTTGCCAGCACTGATAGCTCTGTGGG GTCTATGGAGCCATCAGCCCTTGGCTATGGCAACTCCGACCCCAGTGGGCATATCCGTGC CCTGCCCTCTAGCAGCTCCCTCTAGCGTCTCTCAGACCTGGCTGCATCTCTCCACCCACG CCAGCCACTGCTGCTGCCACCTGGTCTAGGTACCTGCAGTAGCCTGCTCTCAGGCCTCCC TGCATGGTCCAGGGCCCGCAAAGCTGTTTTCTCACGTGCCACCGAAGTGGTTCCCTAACA TACAGCTCCGAGCTCCTCCCTGCCTGCTCCCAGGACCCCAGGGTGAGAGCGCACACACCC AGTGTGGCCCGTGAGGCTTTCTGGTAGCCTGCCACACGTCTGGGCTTTTCCTTCTGTCCT CTCCTGACCCTGTCACTGAAGTGCTGTCTCTCCTCAGGGCCCTCGCTCAGCCTCTGAGCT GTGGGGATGGGAGGCAGCAGTGCACAGTTCACTCTGCAGGGTGGTGAGAAGAGGGCAGTG CCCAAGCCTCACCCTCGGGCTCTTTTTTTTCATAGCCCGAGCTACACTGGAAATCCTCAT CCCTGACTTTGTTAAACAGACCTCTGAAGAGAAGCCCAAAGACAGTGAAGAACTCGAGGT GAGTGTTGTGGTCCTGCCCTGCTGGGAGCTGTGTGTGCAGTGCGGCTACCCTGCCCTGTT AGTGTGAGCTGGGGGTGTCCGTGGGCTGTGCTCATGCCTTCCATGCCCTGTAGGTTAGTC TCATCCAGCCTCCAGGTTCTTCTGTTTGTATTTCATAAAGAGGAAACTTGCCTGTCTTTG TATAGGCAGGGAAAAAATGTCCTTTTTGTTAGAGATTTTTGTCCTCTTTTGCAGCATTGA GTGTGCACCGTAAATGTCGGGTACACATAGGTTTGTGATGGGAATGAATCTTTGAGGCAG TCTACAAGATTTAGATTGTTCTTTTTTTTGAGACAAAGTCTTGCTCTGTCACCCAGGCAG TGACAGAGTTTGATCTTGACCCACTGCAACTTTCACCTCCCGGATTCAAGTGATTCTCCC ACCTCAGCTTCCTGAGTAGCTGGGATTACAGGTGTGTGCCACCATTTCTGGCTAATTTTT GTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACC TCGGGTGATCCACTCGCTTCCGCCTCCTAAAGTGCTGGGATTACAGGCATTAGCCACTGC GCCCGGCCCTAGATTGTTCTTTATATACTTGACCCTTGAGTAACGTGGAAGTTAGGGGCA CCAGCCTCTCACGTGGTCGAAAATTCACGCATCACTTTCAGTCCTCCCAAACCTTAACTA CTTACTGGTAACCTACTCTTGACCAGAAGCCGTACCAGTAACAAACAATTGATTAATACA TATTTTGTTATGTACATATTACTACATCTGTCCCCAACCTAGTTTTGGCACCAGAGATCA GTTTCAGGGAAGACAATTTTTCCACAGACGGTGTTGGGGGGCGGGGATGGTTTCAGGATG ACTCAAGCACATCACATTTATTGTGCACTTTTTATTTCTATTATCACTACACTGTAATAT GTAATGTAATCACTATACAACTCACCATAATATAAAACCAGTGGGAGCCCTGAGCTTGTT TTCTTGCAACTAGATGGTTCCCTCTGATGACGATGGGAGACAGTGACAGATCATCAGGCA TTAGATTTGCATAAGGAGTACACAACCTAGATCCCTTGAATGCACTGTTCACAATAGGGT TCACACTCCTATGAGAATCTAATGCTACTGCTGATCTGACAGGAGACAGAGCTCAGGTGT AAAATGAGTGATGGGGAGCAGCTGTAAATACAGATTAAGCTTCACTTGCTCACTTACTCC CCCTGCCCCCACCACTCATGTCCTGCTATGCAGCCCGGTTCCTAACAGGCCATGGATTGG TACCAGTCCATGGCCTGGGGGTTGGGGACCCCTGATATATACTGTATTCTTACATTAAAG TAAGTTACAAAAAGAATGTTATTAAGAAAATCATAAGGAAGAGAAAATTTATTTACTGTT CTTGAAATGGAAACATCATCATGAAGGTCTTCATCCTCCACATCTTCATGTCGAATAGCT GAGGAGGAGGGGGAAGAAGAAGAGTTGGCCTTGGTATCTCAGGGGTGGCAGAGGCAGAAG AAAATTTTTGTATAAGTGGGCCCATGGAGTTCAAATCCATGTTGTTCAAGGGTCAGCCAT GTGAAGAACCATTTAAAACTAGCTGTAAACCTTTTTGGGTAAATGGTTATTTGTAGCAAG GGCTTGTGGAATTTTGAAGGGTGCATTCTTATTTATATAATAGCTTTATTGAGATAGTTT ACATACTATAAATTTGGCCTTTTAAACTATATAAGTGGATTTTTATATATTCAGAGTTGT ATAACCATCATAACTACCTAATTTTAGAGTATCTCAACACCCCCAAAAGAAACTCCATAC CCATTAGCATTCCCTCTTAGTTCTCTCCAACCACCAAAAGCCACTAATCTGCTTTCTGTC TCTATATATCTGCCAACTCTGGACATTTCATATTAGCGGAATCATACAATATGTGGTCTT TTGTGACTGGCTTCCTTCACTTAGTGTGCTATGTCAGCTGTAGGTTTTTATCAGAATAAG GAAATTTCCTTTTGTTCCTGGTTTGCTGAGAGTTTTTGTAGGAATGATGTTAGATTTTGT CAAATGTTTTTCTGCTTCTATTCAGATGATCATGTTTTTTTTTGTCTTTTATTTTAGTAA TATAGTGTATTATATTGGTTTTTGAATGGTGAGCCAACCTTGCATTCCTGGGATATATCC CACTTGGTCATGACATATAATGATTTTTTTAATATGTTGCTGGATTCATTTTGCTGGTAT TTTGTTAAGGATTTTTGCATTTGTAGTTATAAGCAATATTGATCTGTAGTTTTCTTGTGA TATCTTTGTCCAGTGTAACAGCAGGTACAGGTATCAGGATAATACTGGCCTCATAAAATG GTTGGAACATGTTCCATTTTCTTCTGTTGTGTGGAATAGTTTGTGAAGGATTGGTGTTCA TTTTTTAAATGTTTTGTAGACTGCGTTCAGTGAAACCTTCTGGTCCCAGGCTTTTCTTTG TCCAGAGCTACTTAATTGCTAATTCAATCTTACATGTTATAGGTCTATTGAGATTATCTA TTTCTTCTTGAGCCAGTGTTGATAGTTTGTGTCTTTTTTGGAATTTTTTTCCCATTTGAT GTAGATTATCTAATTGATTAGCATATATTTGTTCCTAGTATCCCCATATGACCCTTTTTT ATTTCTATATGGTTGTGATGGTCCCTTGCTGTCATTCCTGATTTCAGTAATTTGAGGCTT TCTTCTTTTTCTTGATCAGTAAATTGTTACACTTGATGATACATCACAGGTCTCTGAGGC TCTGTTGTTTCTTCTCTGTACTTTTTTCTTTCTGTTCCTCAGTGTGGATTATTTCAATTG CCCTGTCTTCAAGTTTGCTCCTTTATCTTCTGTTGGCTCACATTCTGCTGGTAAACCCCT CTAGTGACTGTTTCATAATTTCTGTCTTTTTTAAAAAAATTCTTTATTGGGTAATGCATT GTTTTTATACTTTAATTCTTTTTTTTTTTTTTTTTAATTATATATATAGGGTCAGGCATG GTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCAGATCACAAGGTCA AGAGATCGAGACCATCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAA TTAGCTGGGTGTGGTGGTGGGCACCTGTAATTGCAGCTACCCGGGAGGCTGAGGCAGAAG AACCACTTGAACCCAGGAGGTGGAGGTTGCAGTGAGCTGATACTGCGCCACTGTGCTCCA GCCTGGTGACAGAGCGAGACTCCTGTCTCAAAAAAAAAAAAAGGAAAAAGAAAAAAAATA TATATATATATGGGGTAGAGATGGGGTTTCACCATATTGCTCAGGCTGGTCTCGAACTCC TAGGCTCAAGCAATCTGCCCACCTCAGCCTCCCAAATTGTTGAGATGACAGACGTGCCAC TGTGCTGGCATTATACTTCAGTTCTTTAGACATGGTTTCTTAGTCCTTTGAGTATGTTTT CTGTAGGTGATGTAAAATCTGTTTCTGGTAAGTCCAGTTCTCAGAGAGCGTGTATATTAA ACTGCCTTTCCCTTCATGTATGGGCTGTCTCTACTTTCCTATTTCTTTGCATATCTCAGA ATTTTTTGTTATTGTCAAGAATTGGACATTTAAAATATATGGCAACTCTGGAAATCACAT TACCTCACCTCCCTTAAGTTTATAGTTTTTGTCATATGTCCTTGCTCTCTATTTGTGACT GTATCAGTTTGCTAGGGATACTGTAACAAAGTGTCACAGACTGGGAGGCTTAAATGACAG AGATGTATAGTCTCAAAGTTCTGGAAGCCAGAAGTCCAAAATGAAGGTGTCAGAAGGCTA AGAGAAGGCCTTTCTCCGTGGGTGGAGGATAGCGATCTCCATGTCCATGCCTTTCTCTCT GTGTGCATGCTGGTCTCCACATTGCCCATTTGATAGAGACACCTGTCTCACTGGATTAGG CCCTACCCTAATGACCTCATTGTAATGTGATGACCTCTGTAAAGACCTTGTCTCCAAATA CTGTTGCATTCTGAGGGACTTCAACATGTAAATTTGGGGGTGACAGTTTACCTCATAATA GTGACTTTCCTAGATTGATTCTGTGAAGTGTGTATTCTTTATGATTTGTGGCCACTGAAT ACACGGGCCCTTTAGCTGAGTGGTCTCCTGATGCTTGGACAGGGATTTTCGTAAATTCCC TGAGCCAGTAAGTCTCCCGGCCTGTGATGAGAGCTGTGGGTCTGTGGGGCTCACCTGGTG TGCTCCGGTGGCTGCCAACTGTCTTAGCCTTTGTGTGCCACCTGCACAGAGTCTCCAGGT CAGTCAGCTGGGAGGTTTTCCTGGGCCTGTGCGCAGTCATCTCCAGACATACGCTAGAGC CTTTCAGAGCCCCATGGACATCTCCAGCTTTTCCTTCTTGGGTTCTTGGCCAGCCTCTTG TTTGCCCAACTAGTGCCCCAGCCTCTGGCAGCTGCAGGGTGAAACAGTTGCCACTGGTTC CTTTTGACAAAAGCCCTCAGGAACTGGGCTGTTTTCACTGAGTGAGATCAAATAAAGACA AATCCTGCCATGAGGCTTTTCCAGAGGGTGGCCAGGTAAACCCAACAGTGACAGTTCTCT GGGCCTGGGGCCTTGAGGACCCGTTCTCCATTGAGTGGCTACCACTGAGCTGGGGAGAAG GGCTGGGAATGGGGCGAGTTAAGATGCCAGCAACTCCTTGTTCTTAGCAAGACTCGTTGT CTTTCTTGAATCAACACTCCTCAGATTGTTGAAAGCCTTTCTTTAGTTTCCAGGGTTCTG AAAGAATTAATTCTGACCATTTTTGCTAGTGTTCTTGTTGCTTTAATGGAGGAGAGCAGA TTTTGGAGGTCCTTATTCGGCCATTCCAGAACTACTTTAGGATGGGTTTTGTAAAGTTTT CTCATCTTTGTGGACTGGTGCTGTTATAAAATATGGTTAAGTGTCTATGGTAGTACTAAC TTGCTCTTAAGTTTCTTGAACTCCTGGCTTCAAGCAGTCCTCCTGCCTCAGCCTCCCAGG ATGATGGGATTACAGGCGTGAGCCACCACACCTAGTCTCTTGCTCTCAAGTTTCTACATG TTTTCTGTGCTTTATGTAACTGCTTCATCGTGGACAGGCTACAGACAGATTGCAGACCAC ATGTGGATCAAGCACAGTCTCCAAGTACTAGCTGTTCCAGGAAGAGTCACATTTGTCCCT TTTTTTTTGAGATGGAGTCTCGCTCTGTTGCCAGGCTGGAGTGCAGTGGTTCGATCTCTG CTTACTGCAACCTCCGCCTCCTGGGTTCAAGCGATTCTTCTGCCTCAGCCTCCTGAGTAG TTGGGACTACAGGTGCACGCCACCACGCCCGGCTAATTTTTGTATTTTTAGTAGAGATGG GGTTTCACCGTGTTGGCCAGGATGGTCTCTATCTCTTGACCTCGTGATCCGTCCGTCTTG GCCTCCCAAAGTGCTGGGATTATAGGCGTGAGCCACTGCACCTGGCCACATTTGATTTTA TTTCTGAGTGACCCAACCGTGGGAAATATGGCTCCCAGGGAGGCTTCCAAGGCCCTCTGG GGAAGTGCTTGCGTAGACCCTGGCCCACCAAAACTCTTAGTATGGGTTGCTGCAGACGAT GGTGCCTGAGTTGTGTAGGTCTCACGCTGCCTTCCTGTCTGAGTAAAAGCCCTGCCTGTT CTTTGCTATGGGGTGGTGGAAATGTCCATGAACCACTGTGGTTTCTGGTGCCAGGAGAGG GCCCAGCGGGGAGTCAGGTTCTCAGCCTCCACTCTGGGGTTCTTCTGTGAAGTGGAGAGG CTTTGCAGAAACCACATTGCAAAGTAGTCTGAGAAAGAAGAGGTTTCCCCAGGTGGAGAA GGACATGGGGGTGGGGCATGTGGTGGCTTCCGCTGTAGACTCCCCTGCCTGGGGCTTTTT CCACACCCCTACTCCGATCCTCCCACACTGCAGCCTGTCTGGAGCTAGCGTCATATCTCC CCAGGTAAGGAGCTCAGTTTCGTGGGACTGCCCCCACTTCCTGTGCCAGCCGCAAATGGG GGCCCCTGCCACCCGGCACAGGACCCCTCCTCGTGCTTGGAAATTTGCTAGGACTCAGGA ACTCAGGAAAAATTTTACTTATGAAACTTATTTTTCATGAAGGGCTGAAAGTTCCTCCCC TCTACTGACACATTTGATTTCTCCGGGGAGCCCACCCTGGCCACCTCCTCAGCATAAGCT CTGTGGTGGGGTCGTTTTGAATCACAAAAGACAAACCTAGCACTCAGGAAATCCTCAGGG TTTTAAGAACTCTGTTCTGGGAACCTGAGAGAAGACCGTGTGTTTTTATGATGCCATGCC TTGGTTTAGGGTGCCTTTTGTATGTGCCCCTCCTGAGAAGCTCTTTGACCCTCTTATTTG TATATATGTCATCTGTGGGCTGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGG AGGCCGAGGCGGGCAGATTACGAGGTAAGGACATCGAGACCATCCTGGCTAACACGGTGA AACCCCGTCTCTACTAAAAACATACAAAAAATTAGCCGGGCATGGTGGTGGGCGCCTGCA GTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCAGGAGGCGGAGCTTG CAGCGAGCTGAGATTGCGCCACTGCACTCCAGCCTGGGCGACGGAGCGAGAATATGTCTC AAAAAAAAAAAAATGTATATATGTCATCTGTGATGTCCCCATTTAAAAATCTGTCAGAGG GGAAAGTAGCTTTTCCCTCCGTTGTAGGGTGGTGGGGGTGTTTAGACAGGGGTGGCCATC TCCTGTTCCTGGGAAGTTTCTATTTGCTGGTCCTCCCTAGCGAGGCTCTCAGGTTGTGTC CCCGCCCCCTGCTCCTCCCAGCTGCCTGTGCAGAGGTGCCAACACCTGTCCTTGGACTGT CCCCTGGACAGCCTGGGGCGTGGGGTAGTATCCCCTCCTTGTGTGGACTCCGGAGGTGGG GTTGACATGTCAGGAGGGGCGGGTTGATGCCTGTGAGGTGTGGTGGCCACGCGTGTATGC TACTTGATTAAAAAGGGAAAAAAGGAACAACTGGCATCATTTCCTTTTCACAAAGCTTGT GGTGATCTCTTGGTATTTTGTTGCTAGCTGTGGGTTGGTGGTTCATGGCTATTAAATTTA GGTATTAGTGACCAAGTGTTTTCTTTTTTTTTTTTCATCTTATTTTAAAATTTACATAGA GTTAAATTCACTTTTTGGTACAGTACTGTGAGTTTTGACAAATACACAGTCATGTAACCA ACACTGCAATCAAGATGCAGAACATTTCTGTACATTCCAACATTCTTCTTTGTTGGTCAA ACTCTCCCACCTGTATCCTCTGGTGGGCACTGAACTGCCCCTGTAGTTTTGCCAGATCCT GAATGTCCTCTTAGTGGAGTCACCTGGTACGTAGCCTTTGAACCTGGCCTCTGGCTTGGT GTGGTGTGACGCATCCGTCGTGTGGTCATTTGTACTCCTGAGTGCTGGCCACCAGGTGGC TGTGCCCCTCAGTGCGTCTCTCCATTCTTCCTTGAGGGACACTGACAGGTTTCTGGGTTT TGGTGATGGGCAGTAAGGCTGCTGTAAGGTCGTCCAGGTCTGTGTGTGATGGGCGGATTC GTTTCCTTCAGGTAAACCCTTGGGATGGGGTTGCTGGGCTGTGTGGCAGGTGCACGCTTA TGTTAGCATTGCCCTCACACCTTTCTCAGTCACTAGGTGACCGTGGAAGTGTGTCTGTTT TGGGGCTGTCTACTGTCTTTCGAGCTGTGTGTCCTTTTCCTGATGCCACACTTTCCTGAT GACTTGAAGTCTTGATACTGGGAGTCCTCCAACTTCGTTATTCTTTCTCAAAAAATTGTT TTGGCTATTCTAGCTCCTTTGCTGTTCCATGTAGACTTTAGAAACAATATGTCAGTTTCT ATAAAAAATTCTGGGCTGGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGC CGAGGTGGGTGGATTACCTGAGGTTAGGAGTTCGAGACCTGCCTGGCCAACATGGTGAAA CCCCATCTCTACTTAAATACAAAAATTAGGTGGGCGTGGTGGCGGGCACATGTAATCCCA GCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAGGCGGAAGGTGAGGTGG CAGTGAGCCGAGATTGTGCCATTGCACTCCAGCCTGGGCAACAAGAGCGAAACCCCATCT TAAAAAAAAAAAAAAAATCTGCCTGCATTTTGATTGGAATTGATCCTGTAGATCAATCTG GGGAGAATGGACATCCTAACAATATTGGATTTTCCAAATCATAAACATAGTTCATCTGTT CATTTATTTAGGTCTTCTTTGATATCTTTCATGGGTGGTCGGCAGTTTTCATCACACAGG CCCTGTGTATATTCTACTAGATTTATAGTTAAATATTTTATGTATGGCACTGTGATGAAT AGTACCTTAGTTCCAATTGATTGTTTATTGCTAGCTTACAGAGCTAGAATTTGTTTTATT TGCACTGATTTATTCTAGTAGTTTTTCTGTAGATTCCTTATCCAGTTTTCTTCTCTAATT TGTTAATGTAGTTAATTACGTTGTTTTTTTAAAAATGTGTTCCTGGGATAAGTCCTACTT GGGCATGGTATATGTTCCTTTTCATGTGTTAATGGCTTTAATTTGCTAGTTCCCTGCTGC ATCTGTGTTCTTGGTGTTAAGGTCATGGTACGGGTAGTGTCATCAAGCGAAGTGGGAAGT AGCCCTCCTCCTGAGTTTTTCAGAAGGGCAGGACAGATATAGGATTGGTATCATTTCTTC CTTAAAATTTGGGCCTGGAGTTTTCCTAATGGGAAAGTTTTAAACTACTACTGTATGAAC TACTAACTATAACTATGACTACTCTGAACTACTGTTGAGTTTCTTGAACAGGTGTAGGGC TGTTGAGGCTACTGAGATAGTTTGTGTCTTGCCAGGCATTGGTCCATCTCATATAGATAG TGGAATGTAGAGGTGTCGGGCGTCTGTGGTGCTCCCACACGGTCCTTTGGCATCTGCCAG GCACTGATTGCTTAGGTTTCTCCTGTGCTGTTTGTCAGAAGCAACAGGGATTTGTCTGCA GGTCACTTCTGCCATCTGTCTGCTCTCATGAGCTGGAGCCTCCACCTGTCCCTGGCGGGT TAACCCTCAGTAGCACATGTGGCCTCAGGCAGCTTGGTGCAGGGCATCCTGGTGTCGGTC TTTTTTTTTTTTTTTTTTGAGACGGAGTCTCGCTCTGTCGCCCAGGCCGGACTGCGGACT GCAGTGGCGCAATCTTGGCTCACTGCAAGCTCTGCTTCCCGGGTTCACGCCATTCTCCTG CCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGCCTGCCACCGCGCCCGGCTAATTTTTT TTTGTATTTTTAGTAGAGACGGGGTTTCACCTTGTTAGCCAGGATGGTCTCGATCTCCTG ACCTCGTGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCG CGCCCGGCTGGCGTCGGTCTTTTGCCAGAAACTTCTGAGCGTGCTCTCTAGCTGTGTGTG TGTCAGCCACTGCCTCAGCTTCTCCAGGGCCAGTCCCGAAGCCGTGGGGTATGTGGGTTC TTGTGGGCCGACAGCACTGGACGTTGCTAATTGGTTGGCTGGTTGACAAACCCTTGAGGT TCCAGGGCCTGATGTCTTGGTGAGTGACCTTCCCTGGAAAAAGACCCATGTTGATTTGTG CACTTGAGAGGAGCAGTGTCTTCTGGGGACCTATTTGAAGACAGTAGCCAAGCCATACCT CGAGGATTTTTCGCTTGCCAACCTCAGGGCTAGTGCCTTCCTCTTCTATGCAGTTGCAGC GAGTTGGCCTTGCTGCTTGCTGGAGCGGCGAGGGCAGAGGAGCTGCCCACTGCCCTGTCT GTAGGGCCAGTGTCCCTGTGGAGCGGGCTTGGCACACTGTGGAGCCTGCACAGCGAGGCG TGTGAATCATTTGATTCTAGGTTTCCTCGCTGCCCATGGGCCAGGCAGGCTCCAGGTCCC TGCAGACCCCTCCAGAACAGGGCCAGTGGGGGAAGCACTGTGAGGGTGTGGCTGATGGGT AATCTCCAGTGCTTCAAAGTGTGGTCTTTTTTGGAAAGAGGATGATTGCAGATAGAATTA GGTTAACATGAGATCACACTGAAGCAGAGCAGTGGTGTGATCCAGTGTGTCTGGTGTTTC TCTAAGAAGAGGGAGGAGGGGCAGGGCTAGTAGTCCATATGAAAATCGGAGCAATGCTGC CATAGCCAAGGGATGCCGGGGCCACCAGGAGCTGGCAGAGGCTCCCTAGAGGCTTCTGAA AGAGGATGGCCCTGCTGAGGCCTTGATTTCAGACTTCAGAATGGTGAGAGAATAAACCTC TTGTTCTAAGCCACTGACTTTGTGGTATGTTGTCACAGGAGCTCCACCAGGGAGGTTGTC CATGCTCCAGAGTGGGCTGGATAGCATCTTCCTTTCACCCAGGAGGCACTCTCAGCTTCA GCCAAAGTCCTAGTCTGGGAAGGCACAGTGGGCAATGTCTCTGCACCTCAGCCTGCTGCA GCTTCCCTATGCCTCACTGTGTGGAATCTCCCCATCACCTCTTGTTTCCCTGTCTGCCTC TCTTGTCCTTCCTGTGTGTTCTTGGGTTGGCTGCAGAGCCAGGGTGATGACACTGTTGGA AGCCTTGCTGGGCTGGGCCTCTGCTGGCCACCTGGGGCTGGGCTCTGTGTAGCTGTGTGG GGCTTTGGCCTTTGGGGAGCTGGGCCTTTTAAGGCCAGCTTTTGGGTCTGGCTGACTTTC CCTCTCCTGTAGTCTCAGTTGTGGCAATAGTAAAGCCTCTCTGCAAAGGTGCTTTATCTT TATAGAGTTCTGAGAAGGGTGCCTTATGGATCAGAATCAGATCCAAATAAGGTGGGCCTG GCCAGCCAGCCATGTACCTGGGGCCCTCTTAGGATACCAGACTCCATCCCACAGTGCTCA GGGTGGAAGGGTGCTGTGGGCTCTGGAGATGGGCAACTGGACGGTGAGGTACGGAGCATG TCTTGTACCCGGTGGGTTCCTTGCAGAGTTGAGGTATGAAGTGCTAGGCGAGCTCACAAG AACAGACATTAAGCATCTGGGGAAAGCCAACCGCAGGCCCAGCCATGAAGAGGCCGGTGG GCCTGGTGGGGACTCACAAGCCTCTGCTTTGTGTTGTAGTATTTTAACCACATCAGCATC GAGGACTCGCGGGTCTACGAGCTGACCAGCAAGGCTGGGCTGTTGTCTCCATATCAGATC CTCCACGAGTGCCTTAAAAGGTAGGGTAGGGGGGTGCCTCCCCCCATGAGTCAGGTCGGG GGAGCTCCTCTCTGGCGTCTCATATTCTTGTGGCTGTTTGTCCCAAGGCAGAGGCATGGC CAGGTTTCCCTGGGTACACAGCAGCCCCTTGGCCCTGGCCACCAGTCAGTCCCACAGGCC TGAATCGGGCGTGTGGAGAATTCCCTCCTCTGGCTGAGATGCAGTCTGGGGAGAGGCTTG GTCATTTCCTAAGGGCTTCCCAGAGCAGGCCTCCTCAGAGGCAGCTGGCTGCTCTGCTCA GGGGACGCCATCTGTTGTCTGTTTTCTCTTAAAGAAACCATGGGATGGGTGACACGTCTA TCAAGTTTGAAGTGGTTCCTGGGAAAAACCAGAAGAGTGAATACGTCATGGCGTGTGGCA AGCACACAGTGCGCGGGTGGTGTAAGGACTCCTTCTCTGCTGCCTGGTGGCCGCTGGGCG GGCGGCCCCTGGTGTGGCCCTGCGCCTCTGTGGCTTGGTCTCAGCTGTTGCCCTGGCATT GTCCCTGAGCCCAGCTGTGTGTGCTGGCCACCACACGTTCAGCCTAAGGCGGACTGGCAG CCGTCCTGCCTGCACTTTCCCTGTCTTTTCCCTTTCCCCCGCTGTCACCCCTGTCTCCGT CCAGCCCTTGTCCCTGTGGCAGGAGGAAAGGCCCTCCTCCTCGAACAGCCAGCAGAATCC TGCTGCTCCCTGTTTCTGGAGGTGTGCGTCTTGGCGGGAGGGAGGCTCTCGGCTGCGTGG CTTTGAGTGGTAGTGTCCTCAGCTCCTAGACTCTGAGGGCCACTCTCTCAAGGTGGCAGC TGGGTACTGTGAGACTCAGGTGCCCAGAGCAGTGGCAGAGTGCTGCCTATTCCTGTAGAA TGTGCCCTGGCTGGCCCTCGGGGTGTGGGTCAGGAGGGCTGGGAGCGGCAGGGGGCCCCA TGAGCACTGGGTGTTTGTGACCCCCTCTCCATGCTTGCTCTTTCTCTGTGTAGGTAAGAA CAAGAGAGTTGGAAAGCAGTTAGCCTCACAGAAGATCCTTCAGCTGCTGCACCCACATGT CAAGAACTGGGGGTCTTTACTGCGCATGTATGGCCGTGAGAGCAGCAAGATGGTCAAGCA GGTAACTGGCCATCAGCAGGTCCCAGGGCAGCCTGTGCTGCCACCCTGGAGCGTATTCCT GAGGCTCTGTGCTCAGAGGGGGCAGAGCTGGGCAGCTCTGCTGTTGCTCACAGCTGCCTC TCTCCTGGGCCACTTGTGTGGCTTTGTGGGCAGGGGTGGGGTCGTCCCAGCTACATCTCC TATCCCAGCCTGCTGGGCATGGTCAGTGAAGTGTGGGTGATTGGGAACGCAGGCTGGGCC CAGCTATGTGGCTTGTAGGGAGATGGCCAAAGTGCAAGGCAGGGCCAGGCAGGAGATGCT GAAGGTGCGCCCGGCTCGGAGGTGTGCAAAGGCTCAGGAGCCAGGGCCAGCTCTCCCTGC TTGAAGGAGAGCGAGTGTGTCAGGGTAGCTCTCCCTGGTACCAGTGCCGGTCCATCTCTA ACAGAAGAGCATATGTGTACCATCTGTCTTGTTTCTGCTGTTGTGGCCAACATCCTGCAG TGTAAGCCAGTGCCCACTCCTGAGGGGTGGAGGGTAACATGGGGAGGTGAGTGTAATCAT TTTCCTCAAATGATTACAGAAACTCTCTGGGTTCTTGCCCTCCTGGTCATTGGAAGGGGA AGTAGTTGTGGAGGGGTTAGGCCTGCAGGTGGGGCAGCCCTAGGACTGTGTGGTGGGTGG GCCCTCTGCCCAAGCCAGCTGCCAAGAGCAGGGCCTGCCTTTAGGTCCCTGAGACCGAGG CCTCGGCACTGTCCCCGAAGAGGCCTTTACTCTGGACATGATAAGGGAGGGTGAGGGGGC TCACACGGGCCACCAGGGCATGTTAAGGAACTTGAGAGAGATTTTTGGGAAGCAAGTTGA AATGTAGGTAGCACTTGCTTCTGCCCTTGCTGTGCATGGTCCTTGGCTGTGACGGAGGGA GAGAAGGCAAGCCCCACTCTGGCCCGGGGGTGGGAAACTTCCCTGAGCAGGAGGGGGAAA GGTCCCAGCCAGGCACAGCCACAGCTTGGGGGAGCTCTGGCATCCCGAGGAACCACAGCA CCCTTGGGGCTGGCCTAAGACGGGAAGGGGCTGAGGCCAGCCTGATGCTGCTCTGCATTC ATGGAGTGTGCAGAGAGCTCAGTGCCTGAGGCTGATGGGCTGGGCTGCAGCGTGCTGTTC TTACTGGCTATGGCCACACTCTGGTTTCACTCCTTGATGAGGACTCTGGGCACCACGAGC ACCTCACCTCCCTACTCTGCCCATGGCAGGAGGCCATATAAGTGGCGTGGTGTGGGCAGA AAGGCCTGGCAGCTATGGACTGCCCCTCGCTCTCTGCTGCTGTCAGTGGGCCTGGCATCA CTGAGGCGGGCCAGTCAGCATGCAGTTATGTTGCCACAGCTCCTGGCTGTTTCGTGTCTG CCAGACCCTGGGCGCGCCTGCCTTCAGGGTCCCAGGCACACAGCTGCTGGGCAGCGGGCA GGCCGTAGAGCTACAGCCTGCAGTCCTGAGCGTGAGGTGCTATACTTCCCAGGAGACATC GGACAAGAGTGTGATTGAGCTGCAGCAGTATGCCAAGAAGAACAAGCCCAACCTGCACAT CCTCAGCAAGCTCCAAGAGGAGATGAAGAGGCTAGCTGAGGAAAGGGTGAGTGCCACCCT AGCGGGAGGGCTGCCGGGCCACGGCCATTCCTGTGGCACCTGTGGAGGGACAGCAGACCC CAGGACCCGTGCCTGAGCTGACACCTTGGAAATGCTTGGATCCTCCTTCCAAAATCAGAT ACAGGCTTTTCTTTGAGCTTCGACATGTGTGATAGTTTTATAAATCACTTCAGTTTTGGT TCTGTTGTTAGAGAGCTGGCAGGCCAGCCTGTTGGTGGACCATGAAGGTCACAGTCTTGG TGGGCCACCTGAGTCCCATGTCCTCCCTGGGTGTTTGGAGGTAGGTCAGTGCACCTGCCA GGGTTGAATCTGTAGGGCTTGGTCCTGATGAAGCCTCCTAAAGCCACCTTGGGAACCCCA CTCAAAGAGGGGCCTGTCAGGCTGGCAGTGGTGTCTTCTGTCTTCAGGCATCGTCTGCCT GGCCACGGCGTGACCTGCCCACCTGGGCCTTCTGGGTGGGAGCTTTCTTAAGTGTGGCAG TCCCCTGGCCTGGGTGAGAGCCCCAGGAGGTATCTGCTGCCGCCCAGGCCAGCAGGTCCC TTGCTGCTCCCTGGGGTGGGGCTTGGCCTCCTGGAGCCAGTGGGACTGCCTGGTTTGCCC CTGGTCGGAAGAGGGGCTGTGGGGCTCAAGGGCAGCAGGTGCAGGACATAGTGCTCCTGG CTGGGCCTGGGGCAAGCAAAAGCTGGGCAAGGGAGGAACACCCAATGGGGAGGCCTGGCC TGTCCCTGTAACTGCACCCAAAATTCTTCACTTCTTGGTACATGTCCTGATTGCCAGGGA GGCTGCACATGTTGTACTCTGATGGCAATGCAGGGGGCCTCTGCAATCTCAGGCTGGCCT GCCCCAGGCCTTCCCTGCTCCTCCTAGCCTGGCATCACAAGCACTGGTGCCGTTGGGGCT CCAGGGCCTCCTGGCATGATCTGCTGGGCTCTCCCTCCACCTTGTGTCTTCCCGAGCCTC TGCCAAGCCCACCTCACTGGTACCCCTGACCTTTGTTGTTCTTTCAGGAGGAGACTCGAA AGAAGCCCAAGATGTCCATTGTGGCGTCCGCCCAGCCTGGCGGTGAGCCCCTGTGCACCG TGGACGTGTGAGGGAGGTGGCACGGGCCAGGGCGCGGGGGCCGCCAGCCGCACTTCTGAG GAGACCAGCAGTCATGCATCGTGCACCACAGTGTCAGGCCTCCAACCCACGCTCCTTCCC TGTGGCCAACCTGTGGGCCCGGCCTTAGGGTGGAGGCTTTAGTGTACAGGGACAGCCATG GCCACACAGCACACATGTGGAGCAGCGGCTCTCCCTGGAAAGCTCCAGGCCTGAATGGAT GGACTCAGCGACTGCACCAGTGGCAGCTGGTGACTGTGGACAGTGGTGGACCCTGCTTCT GTGCACCTGCTGCAGGCTCTTTTTATGAAGGCTTTCATGAATTTTAGTATGTAATACGCA CTGACGACACATGATGCTTGGATGACAGATGAGAGGGGATGGCTGAGTCCTGTGGCTGGC CCGTGATGCCAGGTGGCCCATGTGCCCAGGGCGCCTGCAGGGCTGCTACAGGGACCTGGT CAGGAGGTGCACATGGTGCCCTGCCCTCACCCACCCTCTGTGTTTCCCCTTCTTTGAAAA GGTAGAAGAGAAAGGAATATTTTAAACCTTTTTGGCTTAAACAGAATTTTAGCATCAGAA CTAGCTTTCTGGGATTGGAGGCAAACCATCAAGGTGGTCCCTCTCCAGTCTGGACACGAT GCCAGCAAGGATGACGTCCTGCCACCTCCTGGAGTTACCCTGGCCTCCTAGGGTCCCTTT TTCTGATGAAGTCTTAATTCCCTAAAAGCGCCTCTTTGGACACTGAGGCCCTCTCTGCCT TTCCTGGCCTCCGGCAACAGTTTTTTACAAAGATTTTTTGCAGTCGAGTCCATATGTCCA CCCATTGATTTTTAAAGCTTTTGTGATATTTTAGCATTTTGAAAGACTTTCACAGTGAGA GTAGAAGGTAGATTTGGAATCATGCATTTTAGCAAGTGGACTTGTTGAAACAGGAAGCAA GGGCCTTCAGTGTAGCCCATTCTTGATCCAGAGCTGTTGCCTGTGACAGCGGTTTCTCTG GATGTCAAAGGCAGCTGCCTGGTGCCCAGCTTGCTTCTCGACTGGTGGCCCCTATGGGTG GGTGTGCGATGGAAATGTGTTCCTGCCGGAGTCTGAGGCACCAGGGTGTGCTCAAAGGCT GGCCCTGGTGGTGGACTGGCACCTGTGCAGAGTGCCGTGTGCTTGTGGTGCGCCATCTGA AGCAAGAGTCCAGCGTTCTGCCGTGTCTGTCCCCCACCATGCCCCCTACAGGCGGTACTG ATGGCGCTTTTTTTTTTTTTTCTGTCAGGAAAACAATGTTGGCCTGTGGGCCGCCCACAA CATATCCTTCCCTCACTACCTGTGTGACCAAGGTTGGCTTCTGTTGACCTTTAAAAAAGA AACCCTCAACTCAAATTGCTATAATTAGACACTTGCTTCTGTCTTGCCTCCTGTCTGCAG CTGTGAATAGTCATTTGACTGTGACTGTTGCCCTTAGCCAGCCAGATGCGCCTGTGAACC AAAGCTTCGTGCACATGTGTTCCCCTAAAGGTTGGGGAGCCTCGCTGTGTCTTGCTGTTC CCAGGCACCACCACAGCAGGTGCTGCCATACTCTTGTGGTCTCTGTGCGCCCCCCCCCCC CCCCCACCCGTCTGCCAAGCATGGGTATGAATCGTGCACACAGCCATGCTTCAAGGCCGG GGCAGGGGAGCCTGTGCTGATGCCATCCAGGGCACTGGGCTGTGCCTGGAAGGCGAGCCT TGATTGTCTGAACACATAAAGCAAACTGTCCAGAAGGGAATGGCTGATGTCTTTATTCTG AGGGTGAGAGGCTGGCACCCTGTGGACCCTTGACTGGCCAGATCCCCTCCTGGGGCCTCC CTGCAGGCTGGACCCTGCCTTTGTAACAGCTGAGCAGCACCCCAGCATGGCCCCTTCCTT GGTTGATGCCCTCTCTGCTGGGCTGAGGGCCAGGCTGGCCACAGAGGGGCCAACTCTGCC CAGTGTTCTCCACCCCATGCCCCCAGCTGGTGCAGTTCCCAGACCTCTCTCTGCCTCTCC TCAAGTTTAAGTATCAAATCGAACATTCTTTTTTAAAGTAAGCTCTGCCCTGACTCCCCC AGCCATGCAGAGAGGCTTGCAGAGCTGCCTAGGCCTAGTTTGGCCCTTTCCCTGGCACCC AGGCCCTGTGTTCAGACCCCTGGCTCTCATCACAGAAACACCCTTTGTTGAGCAGTTGTT TATTCTGGCCCTCACAGCCTTGGCTAGTCCACAAAGGCCCTGGGGATGGGCAACAGGCTA CAGGAACCCACCTGTCTTCCTGGTCAGGGCCCCTGGCCCCTAGCAGCAGGCCAATCCTGG TGGGGCACAGGGTTCTGTGCCCTTTGGCTGCCTACCTCTGAATATCCTGGCCAGCAAGCC ATGCCTTCCCCGCCCCTGGGGCCCTGGGAGCCCTTCAGCTCCTGTCCCCATAATGGGTCC TGGGCCTAGGATGAGGGGAAGGTCCCAGTTTCTTGTAGGGTGTTATCTGGGGGTCCTGGT GTGGGTTGAGCTGGAGGGCTGTGGGGCCCCAAGACCCCTGTGCCATTGGGGTGCTCCACC CTCTCAAACAGGATGAGCATCTCACAGTGCGGGGTCTGCGGGAACAGGTCCACTGCCACA GCCTTGACCGGCCGGAAGGGAATGCCCTTCACCCGGTTAGATGGGGCTCTGCAGAGGCTG TGGGGGGAAAAGGGGGGCGCTAAGGTCAGCCGATAGGCTAATCAGGGGCTCCTGTGGGAG CTGGGGGCTTGGTAGCAGGCCTAGCCCCCACCCAGGCCCCTGCAGACACTCACTCCACAA AGTTGCCCATGGCTGCCCGGGGGTTGCATGAGACGTACAGCAGCCGCCTGAGGTTCTTAG CTCTCCGGATGGCCAGGATCACCTTGGAATCTGAGGAGGCAAACACCAGCTGGGTCCCCA CAGCCAGAGAGTGCATAACATGGTGAGCCACCCCATGTGTCCTCGTTCCCGTGCCACCTC CTTAAGCAAAAGCCACTCACGCAAGCCAGCACGGGGTGGGTCCAGGATGGCCACGAGGTG CTGGGAGGCCAGTCTGCTCACCAGGGTGGGCACCAGGTCCTCGGCCCTCCCGCAGTGGAA CTCCACATTACTCAACTCTGAAGAGATGGCACCGTGGCCTGTCAGAGGGCCACATGCCCT CCCAGCAGGGCCAGCCCTGGGGAACCCCTAGCCAAAGAGCTTGCTCACTTGCTCTACCTG TCGGGCAGCCCCCAAGCCCTGGCTGTGGCTGAGGCTTGCAGCAGGGGTGGCGGCTGCCCC CATCCCCACCCCCACCCACGGCCTTGCCACTCACCATTGTCCTGGGCGTTCACCCGGGCG TCCTCCACAGCCTCTGGGCATAGCTCGACCCCAATGACCCTCTTTACCTTCTGAAACAGG AAAGCGTGGTGTCGCCCCACCCAGGGAGGGGAGTACATGGGGCCCTGTGGGCAGCAAAGA GGAGCTGGGCCTGGGGCATGATGGAGTCAGCTGTGGTGACTTTGAGCTGGGGTCAATGGG GGTCTTGGGGTGGGGAGAGGGTGCCCTCAGCAGGGCAGGAGGGCTCACCCGGGCCAGGGC CAGGCCAATGGTGCCGGTGCCACAGCACACGTCCAGCACCATGCTCCCCGCATCCAATTG GGCCCAGTCCTGGATGACTGTGTAGAGCACCTCGGCTGCGGGTGTGTTCACCTGGGGGCA GGCGGTGCCCAGGATGTCAGTGGCTCCTCTCCTGTGGTGCCACTGGTGCCCCGACGCCCA CATTCCTGGTCCCTCCATCCTGCCGGGACCTCCGGCGCCCACTCCCCACCTTGGTGCCCA ACGTCTTCCCTGACCCCACCTTGGACCAGCCTTGCTGATGCCCTCATCCAGCCCCTGGCT TTAAAAGCCAAGCCCGTGCTAAGCACAGCCTCCTCCTTGCCCCACTTGGTTGAGGGCTCA TGAGGCCCCGCAGGCCAATAGGCTGTACCTTCCCTGCCCCAGCAAGGGGGCTTTGCCTCC CACAACTCACACCGGGGCCCCTGGAATCACCCAACTGCAGCTCCCACCCCCACACCTAGC CCTCCAGGAGGTCCTGCCAGGCCTGTCTCCAAAAACGGCCGCTCCAGGTCCCCACAGCCT TCCTGCAGCAGTGAGCAAACAGTGAAACAGTATTTCCCAGCCCAGAGGCCACCATGGCCT CCCTCCTGCCGAGCCCAGAGCAGTGCCCTGTGGTAATGCACTGAGGGCCACTCCTCATCC TCCCTGTGACAAGGGCCACTGCCGTGTGTACCTCTAGGTTATCCCGAGTCAAATCAGTGA TGACCAACCTCTTTCTTACTTCACCCTCCCCTGTGGCTCACCTGCCTGGATACTCCCCCA AATCTCACCGCCTGAGCCCACTGTTCCCATCACGTCCTGATGGTGCGTCAGGCCTCCCTC AACATGTCCCCATGCCCACCAGGGACTCATGGCTACCTGGAAGAAGGCGTGTGGAGAGAT CCGGAAGGTCAGCCCTAGCAGGTCCTCGTGGATGCACCGGTCCCCAGCCACATGCTCCAG GGGCAGGCCCTCCTGGCTAGGAGTCTTTCTGTGGGCGAAGGTGCAGGTCCTTCAGTGTCA CAGTCCCTGCAGGGACCTGCCCCGCCCCACTCGGGCTCCTTACCGCTGTCCCTCCTCCAC GAAGTAGAGGCAGGTCACTCCACTGGCCCTGCCTGGCCCTGCTGTGAAGTGCTGCGCTAG GGAGGTCTTCAGCTCTGCCAGCTCCTCAGGGCTCAGCTTCTGGAGTAAGTGGTGAAAAGT TCCCATCAGGCTGTGCTGAGGCCCACCTAGGCTAGGCACCCTCCCCCAGCAGGGCCCCCG TTGAGACCTGGGGGTGGAAGTAGGCAATGGCCATGGCCTGGTGGCGGCGGCTGGTGCGCA CAGTCAGCTGCTTCCAGTGGCCTGTGTACGTCTCTGGGTCGTATGCCGAGTATGGAGTGG ACCTGTGGGAATCACGAGCTGGCCCAAGTGCCCACAACTGGGCAAGCAGTCCCCCTGCTC TCTCCTCTGGCCACACCTTCCTTCATCTGGCTGAAATGGCAGGCACTCCAGAATTCCCGG GGCAGGATCACCACCCTCTGCTCCCACACCGCATAGGACTTCCCGGGAGCCCCGTCACAG GTCCTCACCGGATGAACTCCTGGAAGGCCTTCACCACCTGCTTGGTGGCTTCGGGGATGT GCACGGTGTCAAACGGGGCTGCCACAGCACACGTCCCGCCCTTGTACTTGCCGAGCCGAC AGCCCACGGTGTTATCCTCCCCATCCACCCCGACGCCAACCAGAAACTCACACTTATTAC GATACTCAGTCTGCAGGGAGAGAGAGCTGCACCTTACCCTGGCTGCCCAGCGCTTGGGCC TGGAAACCCTGATTTCCCCACAGGGGCTGGCAGTCAAACAAGAGGAACAGCTGACAACTA TGTGATGTTACCAGAAGAAAACACAAAGCAGGGAGATCAACAGGAAGGGTGAGCAAAAGC AAAGTTTTTCAAAACCCAGGATAGGGGTTTGTGGCCACCGAAGTCTAGTCTGACCTGCTG GGGTGATGGCCTGACCCCCTCCAGCGGGCAGCAGGCCTTGTTGTGCTTGTGCCTCTGCTC GAGCAGCCAGGGCAGCAAGGCACGGTTGGTGCTCCCGATTTCCCTGTAAGAGGAGCAGAT CGGTGGTTGGACTCCACCTACTGCCCCCGACCCATGCCAGGCCACTCCCCAAATGTCCAG GCTCCTGACCCCTCCTTTGGGATGTCATTGTGCCCACTGTGACGTGGGTGTCTTGCTGGC TAGCTCCCAAAGGGGCCGCCAGATCCTGTGCTGGGCCTCCCAACTGGTGCATGGACAGGC TGAGGAAGCACAGGCCGGGCAGAGCCCTGGCCAAGAGGGGCGTCTTGCGCCCACACTCAC TTGGCAAGTTTCTGCAGCACCTGCTCGCACTCCAGCTGCTTCCGCTCAAGCTGCTCAGCA TAGGGCACTGTCCATAGAGGGGTCACCACGTCGGCCACTCGTGTTACTGGTGGCTCACTC TCACCCTCCTGTCGCCTCCTCCTGGCCATGGGGTCGGCCTTGGGCCGGGCCAGGCGCACA CTGAGTGGGCGGCCTTTCCAGAGGGCACCATGCAAAACGCGCAGGGCCTTGTCCCTCTCT GCAGCGCTGCGGAATGTCACAAAGGCGCAGGGTGGTTGCCCAAAGAGTTTGGTTTTGTGG GGCTGCAGACCAAAGCGGCCCAGGAAGCGCCGGACGTCGCTGAAGCTGGCGTGGCGAGGC ACGTTCTGCAGCTCCAGTTTAAAGATCTCAGAGGTAAACAAGTCATCCCTGATGTAGCTG TAGAGCCCGGGCTGAGGCCCCGGCCCTGTAGCCGCCCCAGCGCCCTCTTTCTCCACCTCC TCCAGGGCTGCCGGGGCTGCAGGGGGCACCGAGACGGTAGGGCAGCTCAGGGCACTGCTG CTCTCCTGGCCACAGCTCTCCATGGGCTTCGGGCCCTGTGTGGGGACAGATGGGGTGCTA GGGTGAGGACTAGGCCCTGGGGGCCTGGCCCCCCAAGCTTCCTTATGGGACACCCAGACC TCCGTCGGTGCACTCTGCACTCAGCCCTGCCCCTCCCCCAGTCTACCCTCCCGACCCCAT TCCCGTCTCCTTTCCTCCCCTATCGCACCTGCCTCGTCCCCACCTATTCTCTGGCAGGTT TCCTGACCCACCCCGCCTCCTCCCACCCCATCCCCGTCTCTACCCAGCGTCTCCCCGCAC TCTACCTCGTTGTCGAGGTTCTCACTCATCGCCCAGGCGGTTCTCCGCCTAGACCAGGGA CGCCATGGGGGCCGCCTGGCCACCTCGTCCTGGGCCTGTCACAAGGGAAGTGGCCTGTCA CAAGGGAAGTGCTCAGAGGGGAGGTGCTCACAGAGCCGGTGCAACGCCGCGAGGTCGCCG CCACCCCCGGCTTCGCCCCAGGCGGGGCGGGACTCGAACCTGCGATGCTCAGGTCCGGGT CTCAGGCTTGGGGCTGTACCGCCCGCCCGCCAGGGGCCCGCGCCGGCCGCTCGCTTCGCC AGCCACTCTTAGTCCGCCAGCGCGTGCGGCGGAGGCCGAGCGTCTCTATGATCCTGGCTT CTGGCAACGTCATCGTCACGCGCCGGATCCAACCCCCAACCACTTTAGCCAGCTCTAGAG GCGCGCGTGGCCGGGACGGAAGTGCGCGCGGGTGTCGCCGGGAGTGCGCGCTCCTCTGGC TGACGGGCGGGCCGGGCATGCGCCGCGGGCGTTTTGGCGGGAAGCGCGGGGCGGGCCGGA CAATGAGAGTGTCCGCCTCCTGAGCCAATAAAGCTGTACTGGTTTTGAATCGCGGCGCGT TTCCCGCCGCTGGGGTCAGGGGTCGAGGTTCGGGTCGTGGGGCGGAGGGAAGAGCGGGCG GGCGGGAGGCGCCGGCGCCAGACGCGGAGGGAAGGAGCTACGAGTAGCCGCCGAGAGGCC GCGGAGCCAGCGACGACCGACCCAGCCGAGCCGCCGCCGCCGCCGCGCCCCCATGGCGGC CGCCAAGGTGCCGCCGGGCCCAAGCGGGGACAGGGTGGGCGGGCGGGGCCGGGGCCGTTA TCAGCGGCCACGTGGGCGGCCCGCGCCGCCGCCACCAACCTCCGCCGGCCTGCAGGCTCG GCCGTCTGCTGGGCCTCGGTGGCGCGGAGTCGGGGCGCGTGGGGTTTGGGGGCTCGAGGC CTGCGGAGTTGGGGCGTTTGGGGGTGCAGGCTCTGCAGAGGCCTGGGGGCTGCAGGGCCC CCGCGTGACCTTGGGGTGGGTTGGGGAGCGGCCGTCGCCACGCGGGACGGCCCCAGCCTC CAGGAACTGGTGCGGGCGGCGGGTTTCCTCCGGCTCGGCCCACTTGGGGCTCCCCACTAG TACGGTGCATTTATTCTTTCCGTCGCCCACGCAGCACTTCATTCATTCGGTTCTTACGTC TGGAACCGCCACTGGCCTCTGTGGCAGAAGCTCGCGTTCCAGCGTGGGGCACAGGTCCTA GATGCGCCGACCCAGGCGGCGGCTTTGTTGGCTGCGGTGTCTGGTTGAGTGCGGGCCGCT GCAGAGCCGCAGTGCTGTGGGGAACAGTGAAGAGCAGCGAAAAGCTTATGTTTTCTTTTC CAGTTTGAGTCTAGTGGTGAAGTCTTGGGTCTCTTACGGACTTCTGGGTGACCAAGGCAT GTGTTCTGGAAGACTCGAACCTAAAATATGATTATTGGTGCTCTCTTATCCTCTAAATTG GGCCAGGATTTCATGACTTTCACCGGTACAATTTATCTAGTTATTAACTTAGTTTTCTGA ACTGTGAAAAAATTGCTGGCAGAGCTGTGAATCCAGTGTAAATACTAGCAGTCCTGTAGC GACTTTTTTCGTTTTACTAAGCAAGAGCACAACTATTTCCTAATTAGTCAAAACTGAAAG AGTTACATTTTGCTTTAAAGTAATATTTCAAAATCTATTTCGACCCATCATTGTGTCCTT TGATCTTCACACAGCAGGTATCATCTGCATTTTATGGATGAGGAAGAAACTGATGCTCAA AGAGATGAGGCAGTTTCTGCCAGGCCACTCTCTGCAAGTGTTGGCGGCTTGTAGGGTCCC CCATCAGTCTGTGCACAGATCTGATTGATGTCTGTGAATCTGGGGGAGGGGCCCCAAGTT CTGGTGTCTCACTCTGTGCTCACATGGAGCTGGGCATAAACGCAGAGCTTTCCTAGTGTG GAAGAGTGGGCCTAATTTCTTGCTTTGGGGTTGGGCCCATCCTTGTATCTGAAGTCCCTT CATTGTCGGAGGGCTGACCACTGCAGGCACTGGTTGGGCTCTGGTTGTGAGCCAGATCCA TAGGCCAGCAGTGTAACCTCTTTGTATCTCCACAGGACACTCATGAGGACCATGATACTT CCACTGAGAATACAGACGAGTCCAACCATGACCCTCAGTTTGAGCCAATAGTTTCTCTTC CTGAGCAAGAAATTAAAACACTGGAAGAAGATGAAGAGGAACTTTTTAAAATGTAAGTAG GCTGATGTCCCAGAAATAGGACTCTTTAAGGTTGAGGTTACAACTTTTATGGGTGTCCAG GTTTTGGCCTGACTTTTAATCAGACTGAGGCTGCTTAAAGAAAGGGCCTAAAGTTGGTGA CTAATCCCATGTGGAAATTTAACCCGTTCCTTTCATGAATCTTCACCAGATGCCCTTGCT GCCCTGCAGCCAGTTGCATGAAGGGCATCCCATCTTGCTGCTCTGTCCACTGGCATGGCG GCCCCACTGCCCTTTTTTGCCACTGGCTTCTGAGGATCCAGGGAGTCCCTTGGTTGAGCA GGGGGTTTTAAGTTTTTGCAGCATAGACTTCTTTGGCATCGTGAAGCTTATAAATCCCCC ACCCGGTGTCTGTTGTTGTTGTTGTTTTGAGATGGAGTCTCGCTCTGTCGCCCAGGCTGG AGTGCAGTGGTGCGATCTTGGCTCATTGCAAGCTCCGCCTCCCAGGTTCACGCCATTCTC CTGCCTCAGCTGGAACTACAGGCACCCGCCACCACGCCCAGCTAATTTTTTGTATTTTTA GTAGAGACGGAGTTTCACCGTATTAGCCAGGATGGTCTCGATCTCCTGACCTCATGATCT GCCCACCCCAGCCTCCCAAAGTGCTGGGATTACAGGCGTCAGCTACCGCGCCCGGCCCAC CCTTAATATACAACATGATGCTTGCGATAACACATGAAAATAGGTGATGGTTTAGTTATT AAAACCACTTAAAAAAATTCTGTACATGTGCTTCTTGACGCTTTCAGTTAAGAGCTGGCT ACAGTGGTGGGTACCAGCCTTGTGTCTTGTGACAGTGTCACAGCACCACAGAGACTGTGG CCTGTCGCCTACACTCAGGATTGCAACAAACATCATGTTTAGTTACACACCTCAGTGAAA ATGAGGTTGTAGTTTTTCCCTGTTCAAGTTCATGGAATCCCCCCATCCTTGAAGTTGCTT TAAGAGTCCAGGTTAAGGTTTTGTTGCTCTGGGGTGGGAGAGGGAAGCAGAGAGAGTCAT TCCTACTGTGAGTTTCCTATCCAAGGAAGGGGGCCAGGAGGGATACCTGGGAAAGGAGAG CTGTGTGTCACCCTTTGGTCACAGAGGGTGATGGACATCTCCCTGAAACCACTGGGTGGC CATGGCTCCACTCTGCTGATGCATTGCCGTAGCCCCTGTTTGGGAGTAGCTCTTTGCTGT GGTGAAGTGCTTGCCTCTGTTCCCTCACTGCCCATGTCGGGTGGGAGGTGCTCCTGGTGG CCTGGGCCCAGGCCCTTCCTGAGGGAGGTGAGGCCAGCAGGGTGGGCCCGGGTCTTTGCA GTAAAGCTCCAGGGGCATCCTGTTGGGGAACCCTGTGAGACAGTCTCTTGCCAGAATCAT GTTGGGGCAGAGAGCAGAGTCGTCTGAGAGAGGCAGGCAGAGCTGTGCACAGAGGGAGGG CGCCAGCCTGAGCCCTGAGTGCACTGCTGGATTCTGGGTGCCCTGGTCCCCAGCGGGAGG GGCTGCTCCTGGGCAGGAGGGCCAGGTGTTGGCCAGGGCCTGTTGCCTGATTAGAGGCCT GGGCCTCTTCTCTGGGTCATCTGACCTCAGATGACCCGCCCGCCTCAGCCTCCCAAAATG CTGGGATTACAGGTGTGAGCCACCGCGTCCGGTCATCTAATTCTCTTGGTTGTGGAAGTG GAATTCAAGATGACCTGGAGTTTTTTGGGCTGCTGTGCTTCCTGGTGGTGTCGTGACAGA GGTCCTTGGCCTGCACCTGATCCATGTTGTCCCTGCAGATCTCTGGGGCTGGGGTGCTCT TGCACAGTGACCATGGGACGGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAGTGGCGCG ATCTTGGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCAATTTTCCTGCCTCAGCTTCT TGAATAGCTGGGGCTATAGGCGCATGCCTCCATGCCTGGCTGATTTTTTGTATTTTTAGT AGAGATGGGGTTTCACCGAGTTAGTCAGGATGGTCTGGATCTCCTGACCTCATGATCCAC CTGCCTTGGCCTCCCAAAGTGCTGGAATTACAGGCGTGAGCCCCCGCGCCTGCTCTCTCT TTTTTTTTTGAGATGGAATTTCGCTCTTGCTGCCCAGGCTGGTGCGCAATGGTGTGATCT CAGCTCGCCGCAACCTCTGCCTCCCGGCTTCAAGGGATTCTCCTGCCTCAGCCTCCCGAG TAGCTGGGATTACAGGCATGTGCCACCACACCCGGCTAATTTTGTATTTTTAGTAGAGAG GGGGTTTCTCCACGTTGGTCAGGCTGGTCTCGAACTCCCGACGTCAGGTGATCCACCCAC CTCAGCCTCCCAAAGTGTGGGATTACAGGCGTGGGCCACTGTGCCCTGTTTTGTTTTGTT TTTTTTTAAAATAGGGTCTCACTCTCACCCAGGCTGGAGTGCAGCAGTGGTATCATGGCT CACTGCAGCCTTGAACTCCTGCGCTCAAGGGATCCTCCTCCCTCTGCCTCTTGAGTAGCT GAGACTACGGGTGTTAGCCATCATGTCCAGCTAATTTTTATCTTTTTGTAGAGATAGTGT CTTGCTATGTTGCCCAGGCTGGTCCCCACTGAGGGAAATATTGTGACTGTAGGAGCAGTG GGGCAATCTCCACTCACTGTATCCTCTGCCTCCTGGGCTCAAGTGATTGTCGTGCCTCAG CCTCCTGAGTAGCTGGGATTACAGGCGTGCATCACCACACTCAGATGATTTTTGTATTTG TAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTTTTGAACTCCTAACCTCAGATG ACCTGCCCGCCTCGGCCTCCCCGAATGCTGGGATTACAAATGAGCCACCGCGCCCGGTCG TCTAATTCTCTTGGTTGTGGAACCGGAATTCAAGATGACCTGGAGTTTGTTGGGCTGCTG CGCTTTCTGGTGGTGTTGTGACGGGCAGTTCTTGGAGCCTGAAGAAGGGCTGGGGCGTTC GTGGAGGCATGGGGTCCTGCAGTGCAGAGTTGTCCTGTGTCCTCCTGCGCAGGCCCTGCA TGTGGGCTGCAGGTCTGCACTCTTAACCTCACGGCTTTTCTTGCAGGCGGGCAAAACTGT TCCGATTTGCCTCTGAGAACGATCTCCCAGAATGGAAGGAGCGAGGCACTGGTGACGTCA AGCTCCTGAAGCACAAGGAGAAAGGGGCCATCCGCCTCCTCATGCGGAGGGACAAGACCC TGAAGATCTGTGCCAACCACTACAGTAGGTGGCATGAACGACCACCTCGACAGTCCCCAG CAGCTTGGCCTGGGACCTTTGGGAAGATTCAGGGCTGATTGGGAACTGGGGAGCGTGTTA TTTCATGGAGTGCAAGTTTGTGGTGTGTTTGTTGAATTTAAAACCAGAAATACGTTTTTC AGACGTGCCTCTGAACTCAGAGCAGGCCCGCAGCGAAGCTTAGAGCACACGGGGGTGTGG AGTCAGCCAGACGGGCCCTGATGGGAGGACGCAGCGCCCAGGCTGGGCTCGGGACGAGGA GTGAGTGCTGCAGGGCGAGGGGGTGCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTG TGTGTGGTGTCTGGGGGGTGGGGGTTGGTGTCTGGGGGGGGTTAATGGGGGGTATGTGTA GTATCTAGGGGGCTGTGGTGTCTACTGGTGGTGGTGTATAGGGGTGTGTGGTTCGGTGTG TGTGGTGTCTGGGGCAGGGGGCTGTGTGGTGTCTGAAGGTGTGTGGTGTCTGGGGCGGGG GTTGTGTGGTTGGGTGTGGTGTTTGGGGGTGTGTAGTTGGTCTGGTGTCTGGGGGGTGTG GTGGGGTATGTGGTTGGGTGTGGTGTCTGGCGGGGGTGGTTGGGGGTGTGTGGTTGGGGG TCTGGTGTCTGGGTGTTTGGTGTTTGGGGTGTGTATGTGGTGTCTGGGGGTGTGTGTAGT GTCTGAAGGTGTGTGGTCTGGGGGGCTGTGTGGTGTCTGAAGGTGTGTCGGGGCATGTGT GGTGTCTGAAGGTGTGTGGTGTCTGGGGAGGTGTACAGTGTGTGGTGTCTGGGGGGCGGG TGTGGTATCCGGGGTGTGTGTAGGGGTCATCTGGTGTTTGGTGTCTAGGGGGGTTTGTGG TGTCTAAGGGGTGTGGTTGGGTGTGTGTGGTGTCTGGGGGTGTTGGGGTGTGTGGTGTCT GGGGGTGTTGGGGTGTGTGGTGTCTGGGGGTGTTGGGGTGTGTGTTGTCTGGGGGTGTTG GGGTGTGTGGTGTTGGGGGGGGTGTGTGGTGTCTGGGGGGGTTTGGTGTCTGAGGGGGTG CGGTATCGCGGTGGGGGTGTGTGTGAGAGAGATTGGGGGTGTTCGGGCAGTGTAAGAGCA GATGTGTGAGGTGCCCAGGCACCAGCCTCCCCAGTTATGGTGTGTTCTACCCTCACCAGG ATCTTCCTGGGGAGGAAACGGGAGGGGAACGGGGCTGAGAAGGTGGCCAGGTCCTGGGCG CCTCTGAAGCTACATAAGTAGAGGCCCTTGGGCAGGTTCAGCCAAGAGCTCCAGCCACCC TGTGAGGGGTCCAGCCTGGCCTGCAAAGAAGTGGCAGTAGGTTGTCCTGAGTATGGCTGG CTGCCAGCTGTCTTTAGATTGAACCTGCACTGCCCAGCTTGTTGGCATGTTTGGAAGTAC ACTTGTCCTGGGGTGTCCCTTGGTAAGCTTCCTAGTGTCGTTTGCTGAGTGTCATGCGTG TCCGGCGGGGCAGGGTCCCTCATCCCAGGTGCAGGATGACTGCTGGCAGGAGCCTGAGTG GCTCAGCCCTTGAGGGCGATGCAGAGGGGTGGGTGGTGGTCTGCCTATGCCATGGGCACC TCACTGCACGGCCTGGCCTGCCAGACTTCCTTGGGAACTGGCTCCTGGGTTCCCCTCCCT CCCTGCTCAGCATGTCTAGTCCTGGGGAAGCTTGCACTCTGGCTGGTCCTCCTCCTCGAG CCGTTTGAATCATTTCCTTGGGGGTCTTCCCTGATGCCCTGTGCTGCTCCCAGAGCCTGG GTCAGGTTGTGGGTTGTTGTGTGTGCATCATAAGTTGGTTCTGGGCTCCTGGGCATCTGG GCAGAGCCATCTGTGTAGCCAGCAGTGTCTTGTGCCCATCATGGGCCCTTGGTAGACAGG GAAAGTGGCAGTGGGTAGAGGCTGGCTTCTGGGTAAGGTGTCGAAGAGGCTGTGGGCTGG GTCGGGGCGGGGTGTGAAGGGGGCATGCAGGGGGCTTGGCATCCGCTGTGGCAGGCTGCA GTAGACGAGACCCCCCCCCAGCTCCCAGTTCAGATAGTGACGTGTAGGGGTATCCTGGGG GTGCATGTGGCCTGGACTGGGCATGGGCACCTGGGATGGTCTGGGTTCTCAGGGAGCTGT TCTGAAGAGTGGTCACCTGCAGGGCCATCTCCCCTTGGGGCTGCCTGTGCCTGGGGAGTT GGGGTGGAGGCGTCCATTGCAGGCCGTGGCTCTGTCTCCTGTGCCCTCCAGGCTTCAGTC ATGGGGATGCAGGTGGGCACTCCTGCATCAATCATGGGGATGCATGGCCACGGCCCCCAG TGGGCTCCCTTCTGGCAACTGCCCCACTTTCTAGCCTTCTGCTGCCCCCCACTTTCAGAT AGGCTGGCTCTATGTGTTGGGTTCCCTTAGGAAAGACTGGGCCCCCTGGGCCTTGTGCCC TGATATCTGACCCTGGGCTTCTGACTTCACGGCCAGGGTCAGAATCAAGGGGATCAGGGT TGGGCTTGAGTGCAGCCCTGGGATACAGCTGGGGCCTGCTTTGCCAGCTCTGCTTCTCTG CATTTCTGGAGGTGGGGGTCTGTGTGCCAAGTGATCTTGAACACAGCTCAGGAGCTCCTC GTGGAGATGGCCTTACGGTCAGGGAACAGGTGCCCATTCCTATTTAGGATGGTCCTGGTT TTAAGTGAATATGGAGGATTTTCAAAGAGGTTTGAATAAAACTCAGGCGCCGTGGTACTT TGTCTAAGCCTGGTTCTCCAACCCCAGACTGTCCCCGTGTTGTCTGAAGCAGACCCCTCA GGTTGGTGAGCAGGGTGCTCTGTGGCCGAGGGCTGGGTGGGCTGGCCTTTGCAGTCATCT CACCATCTTCACGAGCTTCTCTCTCTTCAGTCACGCCGATGATGGAGCTGAAGCCCAACG CAGGTAGCGACCGTGCCTGGGTCTGGAACACCCACGCTGACTTCGCCGACGAGTGCCCCA AGCCAGAGCTGCTGGCCATCCGCTTCCTGAATGCTGAGAGTGAGCCAAGGGCCCTGGGGA CCTGCCTGACTTGGGGCTCACCTGCGTGGCAGCGTGGCGGGTTATGTGCAACAAATGCTG TTGCCTTTTGGGGAGAGGGGGCAGTAACTGGGAAGTGTGTCGTGTGGGCTGCCCTGCCCT GCTGTTTGGGGGCCATGCCCAGACTGAAGCCATGAGCAGCGCCTTCCCCCTTAAACTCAA AGCTGTGCCAAGTAGCTGGTGAACAGCAGTGCCCGCTCAGCCGCCTTGTGGCTGGCACTT TGGTTTGCTCTCCAAGCTCCGGTTCCCATTAGTTGTTGCGGAGCCTGAGGCCCAACCCAG GGCTTCCTCCCAGTGCTGATCCTCGTGCCAGGCTTCTGGTTCACTGGGGCCAGGGGCCCC TCATGGTTGCCTGGGTGCCAGCAGGGGCTGGGCAGAGAGATCCACAGGCCCAGGCTGATC TCTTCCCTGCTCAGAGGTCTTTCCAGTCCAGATCGTCCAAGCTGCGTCCAGTGGCTCCTG GAGGAGTCAGTGGGTACTGTGGCCTGGCGCCTGGTAGCTGGCCGTTTGGTGCCAAGTGAC CTTGTCCTTGCTGTGGGCGTGGCTGCCCCAGCAGCCTTTCCAACACGGTTGGGAGCCGAG CCTGTGGCAAAGAGGCCCATGCCCCTTGGTTGCCTGAGGGTCCGTGCTCTACCGCTCTGC CCAAATGGGTGCAGTCAGATGAAGGAGACAGAAGGCCAGGGCCGATGTCAGGTGGACCAG TCCTTTCTTAGGTCAGCAATTACCATGAAATGGGTCTTCTGTGAATCCTGACTCTTCAGA CCCGGCAGGGCATGTCTCTTCCACTGCTCACAGCTCTTAGGAAGTCTTCGCTCTCCCTTC CACAGATGCACAGAAATTCAAAACAAAGTTTGAAGAATGCAGGAAAGAGATCGAAGAGAG AGAAAAGAAAGGTGACGTGGTGCCATGGGTTGGGGGGCTTCTTTGCAGACTCACTCTGCA TCTGACTATACTTCCAGGCGGGTGCTTTTTCTGTCTGCCAGATAAACATTCCAGGGTGCT GTGGCCGCCTCACGTATCCAGAGTGATGCAGCTCCCTGGGGACACAGGTGCTTCCTGGGG AGCCCTGAGCACTTGTCAGTGTTGCTGGCCTCAGTCCAATTGGGCAGGCATTGGAGTCCG GGCACTGCCTGGAGCTCACCAGAAGGTGCTTTATGATGGTCCTCGGTTTGTCTCTCAGAG GGCTTGGTCACCTCTGCCATGAGGCTGGACTGCAGCTGTGGTGGCAAGTGACCAGATGTC ACTGAGCAGCCTGACCATGGTCTAGGCAGTGCTTGAGTGCATCCACCTGGCTTCCTTTCG TCACTGAATTTCAAGAAGACAGACTCCAGGAGGCGGCTTGGCCAGGCAGGAGTCTGTCCC GAGGGCGGGCAAGCCGCTGTGGGTGGGTGACGGCACTTGGGACCAGCCTGGCGAGGGCCA TGTGCTTGAATGCACCGTGCTTCTGTTTTCTCACTGTGCTGCTTGTGTTTTTAGCAGGAT CAGGCAAAAATGATCATGCCGAAAAAGTGGCGGAAAAGCTAGAAGCTCTCTCGGTGAAGG AGGAGACCAAGGAGGATGCTGAGGAGAAGCAATAAATCGTCTTATTTTATTTTCTTTTCC TCTCTTTCCTTTCCTTTTTTTAAAAAATTTTACCCTGCCCCTCTTTTTCGGTTTGTTTTT ATTCTTTCATTTTTACAAGGGACGTTATATAAAGAACTGAACTCAACATTCAGGTTGTTT TTTTTTTTTGTTTCTAAGTTTTTGCCCTATTGAAGATGACTTCAGAAAATCCATTCCCCA GTCATGAAAATGTACTGTGCTAACTTTCTTTTCCATAGTGGAAACACTTATTTATAGTCA TCAAAAATAGTGAATAAAAAACACATTTGGAACCTGGGCCAGATGGCAGGACTGTGTGTG TACAGGCCCCTGCGCTGTGAGCGCCGCTCGCCTAAGCCTCCCGGGTGTATGTCCAGGCCT GTCCGCAGCACAGGTGGGTGCTGCACCTCACGGTAGCCTGGGTTCCTGGGAGAGACGCCC TGGTGTCTGAGGTCGTGCATGTATCTTGCTGTGGGCCTTGAAGCTGAGGTGTCTGAGCTG GCGCTGGTGAGGGAGCCTGGCCAGGGGTGTCTGGTGCCGGGTGTGGTACTGATGCACGAC TGTGGGCTGGGCAGCGTGGGCTCTGCCGAGGCAGGCTCCACTGTGCCTTATGGCCTGAAC AGCTACTGCTTTTTTTCTATGGTGAGGAGGGGTGGGCTTTCTGTGTTTTGAGGAGAGAAA GGAAGACCACTGGAGTACCTTGGCTTTGGAGCAGGGAGGCAGTAGCCAACTGAGCAGAGT CTCTAGCATCGGCCAAGGCTGACTTTGGCCCCTGTGGCAGAGTGGCCCCTGTGGAGGGAA AGGCCAGTCTGGTGTGCTGGGGGTGGGGGCTCGGCCCTGTAGCTCTTCTTTTGGAAAGCC TATTGTACCCTGGCCTTGGAGAAAACTGGGGTCTTGGCCTCCTGGACTTGAAGTCCACAC TTGGCAGTGTTGGAGTTATGGGCATCTGCAGTAACTTTAAATAAGGAGATATTTTCCAGT ACGCCATTGGGGGTTGTCATGTGTAATGGATTGTCTCTGCAAATACCATCTTAAGTGTGT GGTCCCAACTGCCATAACGTGCCCTGTCACCGTTTGTCATGAACACCTACCAGACGTGCT TACTGTGAGGCTGGCTGCCTGCTGCCATCACTTGCTCTTTCGTGCCCTCCACCTGGGCCT GGGCCCTGTTCTCTCTGACCTCCGTGTGCTGTGTCCTGTCTGGTGACTGCCTGCTGGTTT AAACACCAGTCCTTTGTCTCTCTGGGGCAGGGGTCCCCAATCCCTGGCCTGCCGACCGGT ACAAGTCTGGCCTGTTAAGAACTGGGCAACACAGCAGGAGGTGAGTGGTGGGCGAGCGAG CTTTACCACCTGAGCGCCTCCCCTCAGATAAGCAGGGCATTAGATTCCTATAGGAGTGCG AACGCTAACGTGAACTACGCATGTGAGGAATCCGTGTTGTGCACTCCTTATGAGAACCTA GTGCCTGATGATCTGGGGTGGACAGTTTCATCCTGAAACCACTCCCCCACCCCTGGTCCA TGGAAAAATTGTTTTCCACCAAACTGATCCTTGGTGCCAAAAGGGTTGGGGACTGCTGCT CTGGGGGATGTACTTCCAAAGTCAACTACTCTCAGGTTTGTTTATTTTTACATAAATGGG AACCACTTTATGGTCTCTAGCATCAGCCAAGGCTGACTTTGGTGGGTCCCTGTGGCAGAG TGGCCCCTGCAGAGGAAAAGGCCAGCTCAGGAGAGAAGGCTGTGCCGACTCCTCATGGTT TCCAGTGCCCTACATGTTCAGAGAAACGTCTTTGGTAATGAACTATGGAAATGGTCCCTG AAGGTATGGTCTTTGCTCTAGAAAAAGCCCAACTGATGACTCCTCACTGGCATCTTGACT GTGTTCTGTGAGCTGTGGCCCTCATGCTCATGGACTGCTTGCCTCCAGCAGGTTGGCAGG GGGACAGTGGTGCACTTTGCACCCAAGAAGAGGTGAAGCCTGTGCCCTATGCCCTGTGCC GAGTGAGGTCCTCGTGTTGGGAGGGTGACGCCGTCCACTGCAGATGGTGCCTTGCTGTGC TGCACTCTTGGTGACACCCGGGGCCCCAGGTCGGGTACAGGTGTCAGTTGGAGGCTTTGA TGTCTGTGAAAGGGAGGGCCCCTTGCACTGGCTGGAGCTTCCAGGAGAGAAGCCCTTGGC TAGGACAGGCACCCCCCAGCACAGAAAGGATGTTTAGCATTCTGTATTTGATTTGGGGTT CCAATAAAATTCCAGGCTGACTTCCTTCATCTGGGGATGGGAGAAATGTCTCCAAGGGAG AGTCCCCAGCAGTGAGAAGGCCCCTCGGAATGGCTGGGAGACAGTGCCATCCTGTGGTGG GGCTGTGTTGCAGGGTGCATTTGAGGATGCAGCTTGTTGACACTGCAGGATGCGATGGTG CTGCGGGAAGGGAGCTGTGCTGGGAGACAGAATGCGTCCGCGTGCTGGCCCCACGGGACC ACAGGGTAGGCTATACAAGGACGCCCATGTGGGCACTCGGGCCAGCCGTCCAGCCCAGTG TGTTTCCATTGGCTTCCCTTTGGCAGACAATGGTGAGCAGAATGGGGTCCTGCCGGTGCT GGGGACTGTGTGAGAATGCAGTGTGCTTCAGCCTTCCATCCCATGTCTGTCTGCCGGGCT GCAGGCTGGGGAGCCTCCTGCTTTCTGCTTCATTTTCCTTTTCCCTGGGATCTTAGCTCT TCTTGAGGAGGAAACTAGGCCATGGGCTTGGCAGCCATGTTGGGTGGGGAGGCCGGTGCC AACCTCGCTTGGTTCTCTGTCTCCAGTAGTGGGTCTGCACTACTTGGGACCCCTCTGCCC AGGCCTCTGTGTTAAGGAAAAGGGATCCCAAGGCCAGATGCAATGGTTGACACCTGTCAC CCCAGCACTTTGGGAGGCAAACGTGGGAGGATCACTTCAGGCCAGGAGTTCAAGGCCAGC TTGGGCAACATAGTGAGAGACCCTATCTCTACAAAAAAATATAAAAAAAATAGCCTGGCA TGGTGGTGCACGTCTGTGGTCCCAGCTACTTGGGAAGCTGAGGCAGGAGGATCACTTGAA CCCTGGAGGTTGAGGCTTCAGTGAGCCATGATTGCACCACTGCACTCCAGCCTGGGTGGC AGAGTGAAACCCTGTGTTAAAAACTCAAGGGAAAAGGGATCTCACCAAGTAGGCCTGATT CCGCTTCTTCCCACTGTCAGCATTTGGCAGTTTACTCCAGGGTTCAGCTGGTGGAGGAAG GTGACAGTCCTGGCACCCTGCACAGAGCATGGGCATGGAGTCAGACCTGGATCCTGAGGG GCTCTGTGGCATGTGCCACTAGAGGAGGCACTTCATGGGGGAAGGTAGGAGCAAGGGCAA GGCTGGAGATGGGCACCAGGTGGCAGGTGTGTTGCCAACTGAGAGGCTCACTTGTGGCCA CTGCAGAGCCCAGGTAACGAGCCATCTGCTCCAGTGGTGTCTGCTCTAGGCTCACCATTG TGCCCTGAGACCCCTCCACTGCCTCAGAGCTACCATCAGTGCTTGGGAGCGCTCCAGGCT GCACCTCCTGGAGCAGGGCACCCTGAGAACAGAGCTTACCACTACGGCTGCCTGGGAGAA AGCCCATCACCATTTCTGCTGTGCAGTAGTTAGTTGCATTAAGGGCTGGGTCCCGGCCCC AGTCCTGGGTTCCTCCTTCCAATAGCCACAACCCTGGCCTGCCCACCCTGAGGAGCTGCG GCCGGGTGCTTCCCCAGGGGAGCAGGGGCCTGCAGGCCAAGGAGCCTCATGCCTGTGCCG CAGGGTAGGGCCGCTCGGGGTTCTGGTTGCAGAGGAGCAGACACTCAGGGCTGGGGGCGA TGGGTGAGGTCCCCCCACAGATCTCCACCTAGGGAGGGGTCTAATGGGTCTGGGCTTCAG AACAGGGGACGCTCGGGGCATGTTGGCGTGGCCGAGGACAATGACCCTCACTGGGGACGC TCCGGAGCTTGACTGAGGGTCTCCCGCTGGCACCGCCTCCTCTGCCGGCGTGTGTGTGTC TGGGGGGGGGACCCACACGAGGCCCCGCCCCACCCCGCCCACAGCGCCGTCGGCCCCGGC CCGGTCCACCTTAAGCGGCGGCGGGGCGGGTGGGATTTCCTGCGGGAGGAGACGCGCCGG GCCTATCGGGCAGGGCCGGGGCGCCGGGCTTCCCAAAGGGCCCCGCCTCGGGGAGGATGG GGGCTCCAGCCGACCGTCTCTCAAGCCTGCAGCTCGGGCCAGGACGTGGAGGGCCCGGCG CCCGCTGGCCGGGCTCCTCCAGCCCAGTGGCCCGAGGGAGGGCTGCGAGGCGGTCAGAAG GCAAACAGCCACGACGCCGCTGCGGTGTCACACCCGCGCCCCCTCGTGAATTCCAAGGGG CTTCAGACCAGAGGGGTCGTTAGGGAAAGGGCTGCGCCCACAGACGCCCCCGAGGCCACC TGCACGGCTCCGCGGCCCGGGACCAGGGGGGCCTGGCCTTCGCCGTGGTGCCCACCACCC GGGCCGCAGGGGTGCGTGGGTGCCCGGCCCCCGAGAGCCGCGGGCGCCCGCCGGGCTGGG GTCCCCGAGGCCGCGGGGTCCTGCCCCCTCCGAAGGTCCCGCGAACCGGTGGCGGTCCCC GGGCCCCATCAACTCGCCCCGCCCGGTCCCGCCCCGCCGCGGCCCCCAGCGCCCGGTCCA CCTTAAGGGGCGCGCTGACATAAGAGCGCAGCAGCCGCCGCCGCCCGTGGGGTGGGATTT CCTCCGCCGCCGCCGCCGCCGCCGCCGCGGGTCCTGCGCCGCGTCCAGCCCGCCCGCCCG ACCCCGGCCCGACCCCGGCCGGCCCTGCCCGCCCGGCCCCGGGGAGGGATGCGGCGGCGC GGCGCCCAGGATGCCCCGCAGCCCCGGGACGCGCCTCAAACCCGCCAAGTACATCCCGGT GGCCACGGCCGCCGCGCTGCTGGTCGGCTCCAGCACCCTCTTCTTCGTGTTCACGTGAGT CGGCGCCGCGTCTGGGGGCACGCGGGCAGCGATGGGCAGGGCCCGCACAGCCCGGGCACG GGGGCAGCGGTCGCGGGGGCTGGCGGGGCCCCGGGCAGTGGGCGGGCGGGGCGCCGGCTG CAGGCGATGGCAGTCCCTCTCCACCCGCTGGGGCTGGGGGAGTTGAAGCGGTGTGCCGGC GTCCCGGGCACCGGGGTGAGTCCCTTGTGTGGGGACACCAGTGCGGGCTGGGCACCTCGG GAGTGTCCAGGCAGGCCCTGGCGGGGAGCGCTGGGCCCTGGCAGGGGAGGAGGCCGGGCT GCCCTGGGTGTGGCTGCAGCCCTTCCCTCCCCATGGGACCCCCGAGGGGACGTGGGCCAC CCACAACCATGAGTCTTCTCTGCAGGTGGCCGTGGGTGCCTCTCGGGTCTCCTCCTGGCT GCCCGCCCCGGGCCAGAGTCAAGTTGTTCCCACGTAGGTGTGGCCTGGCCTCGTTGCCAC GTGCCTGCCCCACAGCCACTGGTGACCCGGGCCTGGCCCTTGGCCTCCTTCTCAGGAAGA CCCAGTGGTCATCACAGCTGGAGGGCCCAGGCCAGGCCACACCCAGGGCCCGATGTTAGC ACATCACCTCCGTGGGCTGGCCATGGTGCACACCAGGACCCTTGAGGTCCTGTTTCTGGG AGTGAGAGCCCGCGCCTGCAGGACACTGGGCCCCTCCAGCCTCAACTTCTTCACCCAAGA TACAGGCTGGCTTAGGACCAGAGAGGCATGCGGACAGCACAGCCTCCCTCTGTGGCTGTG GGGTCACCCCAGGGCTCCTTCAGGCACAAGGAGGGTTGGGGGCTCCAGGCTTCGGTTGCA CAGATCTTCAACCACCCACCCCCCTTCCACCAATCTGGGGTTGAGACATAGCCCAGGTGG CACGAACGATGGCCACCTGTTCCAAGGTGGGGCCACCCCTGAGCATGTGAGCCTGGAGAG GGGCAGGTAGGGGCTGCCTGGCGCTCGTCACCTTTGCAAGAGTTTGGGGGGCCCCCACGT GCATCGCAAGGCTGAATCGGGTGCCCGTGGAGGCGAGAGAGTTACCTGCAGGCTGGGGCT GGGTGGGCACCGCAGGCTGCCAAGAGCATGTTTAAAAAAGGCCTGTGTGGACGGGCTTAT TTCTGGCTCGGGCAAGCCAGTGTGTTCTTGTCACTTAGTTGCTGCTGGTGTGGGGCAGAG AGAGGTGGGTGGGGGGCAGCCTGGGGAGCCTGGCCTGGGGGGACACATCCCACCAAGGCT TCTGGAGGGGTGGGCCCAGCCCAGTGCTGGTCCCCAGGGCCTCTGTCCTTGGGCGGGGGG TCTGGCTTGGTTCCTTACTCTGTCCATGCAATTCAGTGTCCTTTCTCATGACTTTAAAAG TAATGTGTGGGCCGGGCGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAG GTGGGCAGATCACCTGAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGCGAAACCCC GTCTCTACTAAAAGTACAAAAACTAGCCAGGCGTGGTGGTGGGCACCTGTAATCCCAGCT ACACAGGAGACTGAAGCAGGAAAATCACTTGAACCCGTAAGGCGGAGTGAGCCGAAATCG CACCACTGCACTCCAGCCTGGGTGACAGAGCAAGACTCTGTCTCAAAAAAAAAAAAAAAA AAAGTATGTTCTTTATGGGGAACTTGTGAAATACAGAAAAAAAAAATGCTAAGAAATTAA AATCAACCCCACACACCCCCCATCTCCTGTCCTCCTTGGAGGGCTCCTCTGTTCCTGGCG CCGCGTCTGCAGCCTGGCTGTGTGGTGGCTGCAGCTGCTGGTTCCCTCACGCGTGATGTC AGAGGGCCCTTTACGTGACTCTGACTGTGAACCTGACCCAGCCCTAGGCATGGAGTGCTT GGGTGGCTCCCACTCTGCTGAGCACCAGTTTCCCCTCCTCTGTTCTGGGGTTCTGGTTTC GGCGACACCAGCAGTCTTGAGGTATGGGGTTCCCTCCTGGGGCAGTGCAGGCCATGCCTG CACCCAACCCTACACCTGGCCTATGCTGACCTCTGCCGGCCCTGCTCCTGGGAGAAGATG CCATGTGTCCCTGTAGCAGGATGGTGGCCTGGGTGCTCACCTGGGTTTGATGTGCAGTGG CCACCTAGCAAGGCTAAGAAGCCCTGCTTGCCCCCTCAGGGCAGGTGAGGCCACACCCCT GGGCTGAATGGCTGTCAGTGATCCTGGCCTCTTCCCCGACCCGCCCCGCCTCTGCTGAAC TCCTCCGAGACTGCTCAGTTCCTGAGAAGGGGTTCTGTTGGGTCGGCTGGCCCATTGATG CCACCCATAGCAGCTGTGCTGATTCTGGGTAGGCAGGGGTCCAGGCGGCCCCCTTCTTGG GGCAGGCCACTTGTCTACCCGTTGCCCCACCTCCTGCCCTGTATCTGGTCCAGGCACACA GGAAGGGGAGCAGGTTGCTCTTGTGCCCCGAATCCCTGCCCTTAGTGCCTATACAGGGTT CCCGACTTCACTGTTTGCTCCTCGGACGTTACCAGACAGTGAGTGGCCCCAGCCCCCTCT CTGCACTCAGTAAACATGAGTTGCTGCCGGTGGGGTGGCTTCACCCATGCACCAGGTGGG CAGGAGTTCTGGGTGTCATGTGGTGTGGGGAGCTGGAGGCCTGAGAACCAGGGACCCCTC CCTCAGCCAGATCGCCAAGGGTCTTGGGACCAGCAGGACATTCACACATCTCTCTAGGAG GGTCTGGGCATGGAAGGATGCCCAGTTGTGAAGAGCACCCTCTCAGGATGGGGCACATAC AGCAGGGCAGTTTCTCAGAGGCCCTCTTCCTTCAGGAAGCCTTCCTAGGATTTTGTTGTT GTTTGTTTGTTTGAGACAGGGTCTCACTCTGTCACCCAGGCCAGAGTGCAGTGGTGCTAC CTCGGCTCACTGCAGTCTCGACCTCCTGGGCGCAAGTGATCATCCCACCTCAGCCTCCTG AGTACCTGGGATGACAGGTGGGCACCCTCACACCTGGCTAATTTATTTATTTTTTGTTTT TTTGTTTTTTTTTAGTAGAGATGGGGTCTCGCTATGTTGCCCAGGCTATTCTCCTGAGCT CAGGTGATCTGCCAGCCTTAGCCTCCCAAAGTGCTGGGATTACATGTGTGATCACTGCTC CCGGCCCCTTCCTATGTTTCTGGGGGTTTTGTATCCCCCTGGGCTTCAGAAGCCCTTTAT ACCATGGTATAATGCTAGGCTGGGTCAGCTGCTGGCAGAGGGTTCGAAGACCCCTAGGCC AGGCTCCCGACCCTGCCCTGACTTCACTCCAGAGCCACATTGCAGAATGACAGGGACCCT GTGAATGTCCGAGGGCTGGGGCTGCCTGGCAGGCTGCCAGTGCCTGGGGCTGAGCCAGGC TGGCTTATCTCTGTCTGGGGCATCTGGGATGGTGTGGCTGGTCCCTAGATCGTCCTGAGC CCTCTCCTTGCCTGGAGGCCCAGCTCAGGACAGCAGTCAGAACAGGCCCTGGGCCAGACT AGCCTTGGTGGGCCAGGAGGGACTGAACTGCTGACTGGGACCTCATGAGTCTGGGGAACC TGGGAGTGAAAACTCAGTGTGAGCCCCCCTGCTGGGACCCACCGCATGTGGCCTGGGGTA TTCCTGGCCCCCGAGCCTCAGTTTCCCCATCTGTCCAGGTGTCTATAGGGATTGGACAGG TAGGTGCAGGTACTTCGAGGCCCAAAGGCCTGGCCTGACAAGGGCGCAGCAGCTTCTGCC CCGCCGGTCCCGAGCGCCCCACGGGCGAGTGCCACTCATACACAAACAGTGCTAATGAAG AACCTGATTTAAATGAAAACGTCTGAGTTAAAATTTTCCATTCCACATTTAGCTGTGGCG GACATTCTGCCCCTAAGCCACCCCTCTTGCTCGGCGGTATCTCCCTGCCAGCCCAGGCGG GACTTGGGGCCTCAGCCCGGGTGGGGCCTATGGTGGTGGTGCATGTCCCTGCCTGTGGCC CTGGAGGAGGGATTGGAACCTCGGGGCTGGGCCCCCTGGACCTGCCCTCCACCCGTAGCT TGGCTGGAGCCAGCCAGGAGGGGCTCAGAGGGACATGGCGGGCCAGACACTCCACCTCCA GCCCAGGCCCAGAGCCTGTCTTTGCCTGGCCCTCCGGCAGGCGAGCCTGGTGTGCACCAT CAGGTCTGGGTGGGAGTGGAGCCTCCCCGAGCCTGGACCCAGACTACTGCCTGCTAGGCC CTGCCTGTGGGAATGCGTGTCTTTGAGTGTCCCTGAGCATCCACACATGGCCTCCCCTGT CCCCATGTGTCCTCATGCATCCTTGCATGTCCTTGTGTGTCCTCCCGTGTTCTTGCGCAC CCTGAGAGTGCTTGTGTGTCCTCACACTTGCCCTATCCACAGAGGGGCAAAGGGAAGCCC AGAGAGTGGGAGGGCCGTGTCTGAGGCCCAGAGTGAGGGGCAGCCGAGCCGAGGCTGGAT AGGGCTGGGTGCAGTGTGAGGAGGCATGTGTCTATGCATGCCTGTGTTCACGCGTGTTCA CACATAGGTCTTGGTGGGTGAGTGATGTGCAGGCGTGTCTGCGTTCATGCATGTGCACCA GTGTGTGCACGTGTGCATGTCTGCATGCAAGTGTTCATGCATGTATATGTGTTTTGTATG CAGACATGATTGCATGTGTGCATGTGTGCCTTCTGTGCACCAGAACAGGTTGTCTGTGTG TTTATGTGTGTACATGCCTGTATATTTGTGTGTGTGCACACATACCCATGCACATGTGCC CTGGAGCATGTTTGTGTGTGTGTCCATGTCTTCACACGTGTCTGTAGGTGCCTGCGTGTG CTCCTCATCTTCTCTCCCACTGAGCCCTGAGAGCTCTGGGCCCTGGTGGGTTGGCCTGTG AGCTGGTCCCTGTTAGGCAGGATCTGACCTTGGGGACTACAGAGAGGACAGTTGCCTCAC TCTGGAGGACTGGAGGAGTGGGCAGCAGCTCAGGTCCCAGTCAGAGAACCCACAACAAGC CTCCCCAACCCCTGGATTAGGGCCCATATGGGCCCTCACAGGCATGTGGCAGGTACCCGC ACTGGGCAGGAGGGACCCGGTCACACTGCAGATTTCCCAAAGTAGCCCCACGGCTGCAAG GCTGCTGTCCAGCCCGCCTGGTGTGGGGGAGTCCAGGCTCAGGGAGGACACAGCAGTACC GCCTGCTTGGGCTGGCAGGGAGCTCAAGTCAGCATGGGGTTCAGGCCCCGCCTGCCTGAT GGGCGAACACACCCCCAAGGCCCCCGTTAGGGCCAGGAGGCAGGCTGGCAGCCAGCGCAG GTGCAGAGGGGCCCCGAGGCTTCCATCCTGAGGTCAGCACTGGGAGGACAGAGGCTGCCT CCTTGCAGCCCGAGGGTTGACCAATGGCACTCGCCGCATCCCTTCCCAGAGCAGGCCAGC CCCACGGGCATCATGAGGGCCTGCGTTTGGTTTAATGCTCCGCTGTCAACATCTTGAAAT TCTCTGTAATTTTTAAATAAGGGGCCCGCAGTTTCATTTTGCACAGACTATGTAGCCAGC CTTCCCCAGAAGGCTCGTGGAGAGCTCCACAGGCCCTGCCTCTCCCTGGGGGAGCTGCTC ACCTGCCTGTGCCCATCTTGTTGGGCCGACCCTGCCTTAGGGGCCTGTGGTCCTGGCTGA ATGGCGTTGCATTGGAGGCCAGGCACATGTTTGAGTGCTGCCTGTGTGCCAGGCAGGGCT GGGGAGGGTGGAAGGAAGCCTGGGGGCTGAGCCACTGGGCAGGGACAGGGAGAGCCCTCC CAGCAGAGGGAGGTGTGGCCGCAGCTGTGTCCTCACCCTGGTGTCTACCTCCACGGGCTG GGCCCTGGACAAGAGGAAGGGTAACTTGGGCTCCCAGCCCCGCCGAGGATGGCTGTCGTG GGAAGCGGAGTCTTTAAATAGCCTCTTGCTTTCCACTAAAAATAGTGAAGCTCTGCTGCC GTGGAGGTGGTTGCCGGGGCGGCGGGGCTGGCTCTTGGCCAGGCGGGCGTGGGTGCAGCA GCGTGGCCACGTGCGCTGGAGTGGTAGGCAGCGCGTGGGCCCCATCCACTGCTAGTGGCC ATAGAGGTCAGGGCTGCTGTGGGGGATCCCCACTAGCCCCCCGACTATGCAGAGGGCCTG GGTGCAGGGAGGGACGCAAACCCCTGTGTTCCTGTCCCTGCTGCTTGTCACAGCAGCTGT CTCAGGGTGCGCAGGGGAGAGACAAGGAGCCTGAGGCCAGAGGGGCTTCTTGGAAGCTGA GTGGGTGGATTTAGGAACCCGCTGCAGGCCGTGTGGCCCGAGGACCGGCAAGTCTCTTCA GAGGGGACTCGGGCCAGGCCCGTTAAGGGCTGAGGAAGGCGCCTTTCAGGAAGGTGTGTG AGGGCACACCTTATAGGGAAGCTGCAGAGGGCAGCCTGGAGTCCTGGAAAGCTTCCCGGA AGAGGGGGTGGCCTTGGAGCCAAGTGCTGATGGCTGGGAAGATGGGAAGTGGTGGGGGGT GTTGTGCATTCTAGAGTCCCTAAGTGTTCTACTTTGTCATCTGGGGAGGGAGGTGACAAA CTAAAAAGAAGGGGCTGTCTGGATGGGGATGTGCCTGACTGGGAAGAGTAGGGGAGACAT GCCCCATGCACTTACTGACCAAGGACTGGGGCTCCCCACACCCCAGAATCTGCCTGGTGA GAGGCCCCACTGGGCCAGGGACAGGCCTGGATGAGGCATCGTGGCGCATTTTCCATCTGG GGCTCCCACCTCCAGCCTGTGTACCGCCCCCGCCCCCTACCCCGCCCCAGTCATGGAGCC TAGGTCGCTGGAATGGGGTGACCTTAGGTCTGTGGGCCTCTCTGAGACGGCTGCCCTGAG AGGTTGCCGGGTCAGGGCACGGGTAGCCCACATGCTGTACTGATACGCACAGCCTCTGGC CGTTACTCGAAGGAGCGATGTGGCTGAGGTTTCTTGGCAGGGCTGTGGCAGCCTCCCCAC TGCCTCTGAGCTCTGAGGGTGACCTTGCAGGCCCAGCCTGCCGCACCACATAGCTCTGCG CCTGCATCCGCTCTGCACCCCCAGACTCCACTCTCTGCCCCCACTGTCTACTGCAGGTGC CCGTGGTTGACACGAGCTGTGTCCCCAGCTGTTCCCGTCTACAATGGCATCATCTTCCTC TTTGTCCTGGCCAACTTCAGCATGGCCACTTTCATGGACCCTGGTGTTTTCCCCCGAGGT AGGGCCCTGTGCTGCGGCAGCTCCTCTCACTTTCACTAGAGTGTGAGTGGCTAACTTGAG ACAGCCTTAGGGATTCCTGGTGGGTGGGCTGGACTTGGGGGAGGGATTCACCTGCCAGGA AAGTCAACTTCCTGCTCACTGCCTGGCTCCTGGTCTGTAGCGGATGAGGATGAGGACAAG GAGGACGACTTCCGGGCTCCGCTGTACAAGAACGTGGATGTGCGAGGTATCCAGGTCCGC ATGAAGTGGTGTGCCACGTGCCACTTCTACCGCCCGCCGCGCTGCTCCCACTGCAGCGTC TGTGACAACTGTGTAGAGGTGACCGCCCTGCTGCCCCGCGCTCCCCCAGCCCTGTGCCAC ATCCCTGCCTGCCATGTGCCCTGATGCACTGCTCCCGCCCAGGACTTTGACCACCACTGC CCCTGGGTCAACAACTGCATCGGGCGTCGAAACTATCGCTACTTCTTCCTGTTCCTGCTG TCACTCAGTGCACACATGGTGGGCGTCGTGGCCTTCGGCCTGGTCTACGTGCTGAACCAC GCTGAGGGGCTGGGAGCCGCGCACACCACCATCACGTATCCTTGGTTCCTGTGGGCCTCA TTGCAGAGGCGATGTGGACCGGGGACACGTTCACCAGGGAGATTGAGCCTCAGAAGGCTT GGTTCCTGCCCAGGGATCCCCAGTGGGCAGGTGGCCGTCCCTAGGTTGGGAGGAGGTTTG TCCACAGGCACCTGCTCTGTGCTGCTGCGATGGGGTCTGCGGTGTCCTGGCCTGCACCGT TGGCCTTAACGGGCTAGCATGGCTGTCATGTGTGTGGCCGGCCTCTTCTTCATCCCTGTC ATTGGCCTCACTGGCTTCCATGTGGTGCTGGTCACTCGGGGGCGCACCACCAACGAGCAG GTGCAGACCCCATGGGGGATGGGTGGCCCCAAAGGGCCGAGAGAGTTGGGGCTGCATGGG GACAGGGTGGGGGCTGGGGCACCCTTGCCTGAGGTAGCTTGTGCAGCTGTGTTGAGACGA GGCTCCAACTCTGAGACCCCCACGTGGGGCTACGCCTGCCCCGTCCCCTGCGCACATCCT GCCTGTGGACACTGTGGCCACCAGTGACCCAGAAATCCCACAGCTCCCTGCAAGTTCAGG CTGGGTAACCTATGTGAGGGGCAGCCCTGGGCTGATCCCACCTAAGCCACTTCCCGCAAG TATCAGCTTCTAAGGAACCCCAGCTCCCTTGCCCAGCCCCCTGCCCACCCTGGCCTGGAC CTTGGGAGGCCAGGCTGGCTGAGGGTCCTGCATCCACAGGTGACTGGGAAGTTCCGCGGG GGTGTGAACCCTTTCACCCGAGGCTGCTGTGGGAATGTGGAGCACGTGCTGTGTAGCCCC CTGGCGCCCCGGTGAGGCCCGGCCTGGGCAGGGTGGAGGGGGGCCTCTGCTGGGTGTGGG GCGGGCAGCCTTAGACCCTCTTGGTCCGTTTGGCTCTTGCCAGGTATGGCCAGCCCTGCC CTTGTGTGTTTGTGGCAGGTGGGCTGGCTACTGACCCCTGTGCCCTGGTGCAGGTACGTG GTGGAGCCACCCCGGCTGCCGCTCGCGGTGAGTTTGAAGCCGCCTTTCCTTAGGCCTGAA CTCCTGGACCGAGCTGCACCGCTCAAGGTCAAGCTTAGTGACAACGGGCTGAAGGCTGGC CTGGGCCGTAGCAAGGTGGGCGCCTGGGCTTAGGGATGGGCTGGCAGTCAGGCCCTTGGA TGGGGAAACAGGCAGGTTGGTGGGGGCCTAGAAGAGGTGGATGGGGCAGACAGAGCCGTA GACAGATTCTTGGGAGGGGGCTAGGTCCCAGGCTGAATCCCTAGTGAAGCCCTGCCCCTC ATCCAAGCCCCACACACCACTGACCCCGCTCCCTCCCAGTCCAAGGGCAGCCTGGACCGG CTGGATGAGAAGCCACTGGACTTGGGGCCACCACTGCCCCCCAAGATAGAGGCTGGCACG TTCAGCAGTGACCTGCAGACCCCGCGCCCAGGCAGTGCTGGTGAGGTTGGGGCAGCCACG ACTAGGGAGGGATATGGGTTGACCCTCCTCTGACCTCAGTCTGACAGTGGTCTGCATCCC CAGAGAGTGCCCTGTCGGTGCAGAGGACCAGCCCCCCGACACCTGCCATGTACAAGTTTA GGCCGGCTTTCCCCACGGGTCCCAAGGTGCCCTTCTGTGGACCAGGCGAGCAGGTAAGGA GGCCTAGCCTGCCCCCTGGCAGGCCTCGTCCCCCAGGCCCGCCTCTAGGAGCTCGCCCCA CAGCCTTCACCCCGTGAAGGCCCCACCTCCAGAGCCCCATCTCTCCAGCAGAGGCCACAT CCCCTGAGGCCATGCCCCTCAGGGCTCTGCCTGGGTCACCAAGGGCCTTGTGTGACTGGT AGGACCCTGTCTGTGTGCACCCAGAGCTGGGACCCACAGAGGGGAGCAGGGTGTCCTGGG AGGGGCTGGCGTGGGCCATCCAGGCTTGGCGTCTGTCCCTTGGCCAGCATTGCCCTAGGC CAGGTGGGCGGCTGCCCCCAGGAGCACTGTGTACCAGGATGCCTGGAATTCCACCATTAG TCACTCTGTAAGGCCCACGGGAAGACTTTGGAAGAATTTGAGACGTGCTTGAGAAAGCAC ATTTCTCTTCCCTGAGTTGCATGAGTGCTCTGTGGTCACTGGAATGGTGGGCAGTGCGGG GCGGTCTGTGCAGTGATCAGGAACACCAAACAGTCCCAGGGTGCCATCCTTCCTTCTGGG CCACCCCGAGAGGCAGGCCTGTGACTGGGCCCTGTGCACCTAGACCAGCCCCATCCCCAG GGACCTCTCCCCTCAGGACTGATGTCTTTCCTGGAGAGAGTCACTGGGTGAAGCAGTGAG TCTGAGCCTCCTTGGCCAAGGGTCTGAGTCTGCAGGCATGTAGGGGCCGTGCCTCGCAGG AGGAGGCATCCCTGTTTGGAGATGAGGGCTCTGGGTATGTCATCTTCCTGGGACTCCTGG GTCCAGTGGGGGATTGGGTCTGGTTGGCCAAGTGCATTGAGCCCACATTTGCCCTGATCC ACTTCTGGCTTTGTGGTTCCTGAGGCTCTTGTCCCAAGCCCAAGCCCAAGCCCATGCCTA TGTGCCCGTCACAGGGGCTAGACGAGGCTCCTCCTCCCTCTGGCTGCCCTGAGTGGTGGG CCCCTGGGGCTGCATGGCTCCAAGGGCAGAACCCCAGGTGTCTGCAGTAGCCAGCTGTGG CCTCCCTCCTGTTGCAGAGCCCCTGCCCCTCAGCCGTGGATGCTCAGGGACAGGGCCATG GGTCACTATGTGGCTCTTATGGCTCCTTGAGGCCCAGTTCCTGAGGCCACGGTGCCATGT GTGGCCCCCGTGGGTGCTGACTGGCGGCTGCAGGCGGGGTGGCAGGTGGGCAGTGGTGAG AATGCCTTCTCCCTACAGGTTCCAGGCCCTGATTCCCTGACCCTGGGGGACGACAGCATC CGTAGCCTGGACTTTGTGTCCGAGCCGAGCCTGGACCTCCCTGACTATGGGCCAGGGGGC CTGCATGCAGCCTACCCGCCATCCCCACCGCTCAGCGCCTCTGATGCCTTCTCGGGCGCT TTGCGCTCCCTGAGCCTCAAGGCCTCGAGCCGGCGGGGCGGGGATCATGTGGCCCTGCAG CCCCTGCGCTCTGAGGGGGGGCCCCCCACGCCCCACCGTAGCATTTTTGCCCCCCATGCA CTGCCCAACCGCAACGGCAGCCTGTCCTATGACAGCCTGCTCAATCCTGGCTCGCCTGGT GGCCACGCCTGCCCTGCCCACCCAGCAGTTGGCGTGGCCGGATACCACTCACCCTACCTG CATCCTGGGGCAACGGGCGACCCGCCACGGCCCCTACCCCGCAGCTTCAGCCCCGTGCTG GGCCCCCGCCCCCGGGAGCCCTCGCCTGTGCGCTACGACAACCTGTCCAGGACCATCATG GCATCCATCCAGGAGCGCAAGGACAGGGAGGAGCGTGAGCGCCTGCTGCGCTCCCAGGCC GACTCACTCTTCGGCGACTCAGGCGTCTATGACGCTCCCAGCTCCTACAGCCTGCAGCAG GCCAGTGTGCTGTCCGAGGGCCCCCGAGGTCCCGCGCTGCGCTATGGCTCCAGAGACGAC CTTGTGGCTGGGCCCGGCTTCGGTGGCGCCCGCAACCCTGCCCTGCAGACGTCACTGTCC TCGCTGTCCAGCTCCGTGAGCCGTGCACCGCGGACGTCGTCCTCCTCCCTGCAGGCTGAT CAGGCCAGCAGCAACGCCCCGGGGCCCCGGCCCAGCAGTGGCTCACACAGGTCACCTGCA CGCCAGGGCCTGCCCTCCCCGCCCGGCACTCCCCACTCACCATCCTACGCGGGCCCCAAA GCTGTCGCCTTCATCCACACGGACCTCCCAGAGCCACCGCCCTCGCTGACCGTGCAGAGG TGGGTGCCGGGAGGTGCGGGTGGGCTTCCTGGCACAGGGCAGCTGTCCAGCCGTCTCCAG GGCCCCTCTTGGCTGGGCACTCATAGGTCATCCCAGGGGCCTGAGTTTTTCTCTGGGGAC GGGACAGGGAGGATGCGCCTCACCCTGCCTTGAAGGCCTTGGCTAGCCCGTGTGGCCCAG CTCTGAGGATGTCCTGGGCACGCTCTGGGGACAGGGTATGAGAATTTGTGTCCTGAGCCC GACGACTCGTTCTTCTAGAGCTGGCACAGACCCAGCATGTGGGAGGCCATGGGGCTTTCT CTTTGGGGCCAGTATCATGTGGCAGCCTGGGCCTCCTTTAGGCTGTGGCTCTGGCTTCCT GGGTCACAGTGGCCCTGTCCACCCAGGGCTCTCCCTGGGCCTGCTTGGCTGCATCTCTAG GAGGCTGGGGCTGAGGATATTACCTAGCAGCAGAATGTGCCACCCTGAGTGGCCGGGCCC TGGCCCTCCCTGTAGGACATAGTTCTAGGAAGCAGGGGCTTCCCTGCTCGTAGGTGGGGA AGCTGAGGACTGGAGAAAGCAGGCTGGCTCCAGGCACCCGGCAGGTTGGTGACTGAGAAG GCGGGGTCAGCTGGCTCACAGGCCCAGGTCCAGGTAGGAGAGCTGCACACCTGCTGGGGC CAGGACCCACGCCACAGACGCATGGTGTCCTCACGGCATCTGTCGCCCTGTCCGGGCCTG GAAACCTCAAGCACAGGCGCAGTTGTTTCCCGTCGGCAGATGCAGCCCCAGAGGGAGAGC CCAGCCCAGCCCAGCCCAGGCCACTTGGCCACAAAGGGGCTGAGGGCACAGCAGTCCAGC CCTGAGCGGGTCTCCTCTACCCCACAGGGTGGGACCTGTTCAGCTTTTGTGGAGTTGGGG AGGAGGCTGCCACTGGGAGTGATGAGTGGGGAGCCCGAGACCTAGAAGTGGGCAGCATTG CAGCCAGGCAGGGATCGCAGCCCGGGGACAAAGGGCTCTCGCCTCAAGAAGGTTTAAGTA GCAAAGATACAGCTGCTGTTGGTCAGCGCTGGTGGGCAGGAGGGCAGCCCCTCGTTTTCC TGAGAGAGAGCAGGAATGAGGCCAGGCAGAGTGGCTGGGCTCCCAGTGCAGGGCACGGCA GGGGCTCCATTCAGTGCCCACCTGGCTGCCCCATGCTCTCTGTGGCCAGTGCTGGCTAGG AGGGACCCAGGGACTCGGGAGGGTGGTGGCAGTTCTGACAACATCTGTCTGTCCTGTGAC AGCACAGCTGCTCCCCAGGCAGGCTCTGCATCCCTCACCTGCTTTCCAGGTGAACCCTCC AAGCCCAGAGGGCAGGGACTGCACGGGGCCCCACAACTCGGCTGCACTAGTCCACGTCCG CGTCGTCTCTGTCTTGCTGCCTCCTCCGTCCTCTGTCCCCGTGTTCGTGTGTTCACCGTG TGCATGTGTGTATGTGCTTGTTTTGATGCAGGGACCACCCTCAGCTGAAGACTCCCCCAA GTAAGCTTAATGGGCAGTCCCCGGGCCTGGCCCGGCTGGGACCTGCCACCGGCCCCCCAG GGCCCTCTGCCAGCCCTACACGGCACACGCTGGTTAAGAAGGTGTCCGGCGTGGGTGGGA CCACCTACGAGATCTCGGTGTGAGGACTGACTGCCACACATCCGCCATGGTGCCACGGGG ACCAGGACCCCACAGCGCACCCCCCCTCCCCACCAACTTCTCTGCCCCAGGGACCCGAGG CCACCCCAGCCTGGTGTGGACCCATCGGCGGGAGAGAGTGCCACGCCTCCACAGCTTGCC CCAAGCGCTCTGCCTGCCCGTCCACTCATCTGCCCATGGGGAAGTCGGCTCACTGGGACA AGGGCCACTGGGCTGGTCTGTGTCTGGGCCTGTCCCATGGCTGGGGCAGTGAGGGGGCCC AGTCAGCCTCTTTGGGGCACCCTCTCTCAGCCAGGCTTGGCCCACTGCCATCACCCAGCA CCCCAGATCACCGCCAGGCCAGCCCCCAATGGTCCCCTTACGGACAGGTCCCAGAGATGG ACAGAGGCACCCAGGGCCCCCACCGTCCTTCTGACACAGCCTGTGGGCTCCCGGACCGAG TGTCCCCCGCCAGGCTACTCCTAACTAACGCGTTGCCTTTCACGGACCCCGCTGGAAGCT TGTAGCTTGGCAAGGCTGATGCTTCTGCCCTGGCCTGCTCTGGGTGGTGGTGGATAGGTG GACAGACGGCCAGCCAGCCAGCTGTGGCCGGGGGCCCGGCTCCATGTGTCCCGTGTCTGT GTGCTGTGCTGCCGCGCCGTGTCTGATGTGTCAGTGCTCCGGCCGCCGCTGTCCCTTTCA TCAAAGCCTTAACCTTTGCTTTATGCTCTTGTGGGAGGCGACGGGGGGGCAGGCGGGAGC AGGCACGGGGGTGATGCTGCCACAGGGGGCTGGTGACACCCAGAGCCCCCTCCCCAGCCC TCAGGCCCTCCCTGCCAAACTGGAGAACCCCACCCCAAGGCATGCCACGTCCGCAGCCCC GGCCTGGCTGCGGTGCTCGCGCCGTGGGAAAGCACACTGGGGAGGGGTCAGTGCTTCCCT TGGTGTCAGGGACCTGAGAGTAAGCACATGACAGCGTCTGCTTGCGTTGTGTCTGTTTTA TGTTTTTATATCTACATCTATATATCTATAATTTTATTAAAAAAAAGAAAAAGAGTTATT TTGATTCTTTCCTTGGGCAAGGCCAGGGCTATTGCTGCAGGATCCCGAGTTAGGGGAGGG CAGGGGCCGGGCCCCAGAAGAGGGAGTGGCTGCTGTCTGGTGTGCAGGGGTCGGTGGGTT CCTCAGGGGCATTTCTGCCGACTTGGGCTGAGTCAGAGGCTTGGGAAGAGGGGGCGGCCG ATGGTCTGTGGCTGGGAAGTGTGAGGGTCTCTCGCCTTGGGTCTGTGTCAGTTCTGGCAG TGACAGGGTGTTTGGGGGAAAGACTTGGGTCTGCCGCTACCCACAGGGGACTCTCAGGAA CCCGAGAGCTTGGGGAGATGAAATGGGGGTGCATAGGGGCCACCTGTTGGCTCAGGGCCC TGTGGGGGCCGCTGAACCTGCTGGAACTCTCCTGCCTGCCACATGCCAGGGGCAGGGCTG AGGGAGTGTGAGGGATGCACAGCTGTTCAGGGCTGAGACATTCTGGGCTGGGGCTGTCCC AGCGTGGGAGGCGTGCGGGCTGTAGGGCCCCGAAGCTGACCTCCACCTTTCTGCTTCTCT CTCACGGACGCCGCGGCCCGCAGGGGGCGGATTGGCACCTGCACCCGTGGATGGGGGCGG CGTGGCCAGCCTTGGGTGCCTCCTGGGCTGCACCTGTGCCACCTTGGCCGCCCGGAGGAC CGCCCACCACTGCGGGCCCCCTGGAGCCAGGCCGCCGGGGCACCCCCACGCGGGGCCATG TGCCGCCTGCACTTGGCTGCCTCCAGTCTTTTCCCCAGCCTCTCGGGGCCCTAGCAGGAT GACAAGTAGGCGGCTCTGGGGCCCAGGACAGCCCAGCTGGGGACCCAGGAGGTCAGACTG CAGTGGACCCTGGGGCAGGGCTGGGGGTGGGCTGGGCTCTCTGCTCCACCAGCCACAGCT TGACAGATTCCCAGCCTGCCAGGGCCTGAGACCCTGTGTCCACATGACCTCAGGGAGTCC CCCACCTGCTGCAGGGGGTCCAGCACCCCACAGGGGGGCAGTCCCAGAGCTGTGGGGACC GGCACGACCTTTGCCCAGCCTCCCTACCCAACCAAGCACTTTAGACTAAGCCACTTCCTC CTCGGGGAGCCCAGGCCTCCGTGGGTTGGGCTGGGTGGGGGGGGGGTCTCAGGTTGCCCC TGAAGGTCTCTGCACTCCTCCTGCCCTTCCCCTGACACATGAACAGATGCCTTAACTTCC TGGAGCCACCAGCCTGGTGAGCCATTGGCCTCTGCCTGCCACCAAGGTCCTGTGGTCTTG GCCAGCTCCGCCTGGGCCCCACTGGGGCTGCCTGCACCCAGAGACGATGCCGGCGGGATC TCAGAGGGCCTGAGGCCCAAGCCCTGTGTCCTCCAGCAGTGGTACGGCCTGCGGCAGGGT GGCACTCCGGCCAGCCCTTCTCCGTCACAGGGTCCCTGTCCCTGGGTCCACCCTGGGCTG TGGCTCTACATCTCCCATTTGGGGACGAGAAAGCCACAAAACCATTCTCTATTGTTCTTA AGGGTACCCCTGCTAATTAATTCCCCAAATAAAATTTTTGGTGTTGATCAGCTCTGTCTA CCAATGTGGTCCCCTGGACCGGACAGGGCCCTGGGTTGGGGAGGTGACAGGACCTGGGCA GGGAGGGTGGTGGCCAGGAAGGGAGGGGAGTTGCATGAGGGGCATGAGGTCAGTGGATCA GCAGGTTCAGCGCTGAAAACCTGACCTCTGAGGTCCCTGGGAGGGCCTGGCCCAGGGGGC AAGACCTGAGGACCCCCCAGTCCTTACTGCTGTTGCCTTTCAGTGGAGACCCTCCTAAGA CGCCTTGGGGGGTCAGAGCCCCGTGCAGAGGGGCTGCCTTCTGGAGGGCAGGTAGGATGT GGGGTGGGAAGCCGGCCCTGAGCTGGGGTCTCCACCCTGAGGGACCCTCGCTGCAGGATC GCCTGGGCATCCCTCCCCCAATTTCCACAGAGACGTGCTCACCGGCCACTTATGTCATGA TTCTATGTCCAGCCACAGGCCCAGCCCTCAGGACAGACCCCACCTCCACCCTTCTCTGCA GCTTCTGGCAAGGATGGTGCACAGTGGTGCCATGTGCAGGGGGCTGGCACGGCCACTGGG CATCCGGGGCAGTGCTGGTCGCCTGGCCTCCTTGCTGGGGCCGCTGGGCCTCTGGCCTAG AAGGGCAGGAAGCCGTCCACTTTGAGGCGCAGGAAGGGGTCCAGCAGGGCCCGGAGCTTC CAGACGGTGGCACGGCTCAGCAGGGGTGGCACCAGCCCCTCGAAAGGTGTGGGGTTCACC ACGTACACGTAGGCAGCGGTCCAGACCAGCAGGGCGCCCAGCAGCAGCCTCAGGGTCAGC AGCCTGCGGGCGGCGGTGGTCAGGGGCAGGGGCGCGCGGGGGGCCTGGGGGCTGGGCTCT GCCGTGCAGTCACCTACCAGAGCTGGCCTGCCAGGCCCTTGGAGCTTCCCAGCTTTGGCC TCCCTTTCTGTCGGGGAGGGGCTGGATCAGAGCCCACCCCGAGTGTCCAGGCTGAGGGCA GGGATCCCTGGCTTTGGGAAACCCTCCTCAGACAGGGCTCCCCGCACCGCAGCCCTGGCC CCTTCCTCCTGGACAGGCTCCTGCAGCCACTTTTCCGATGCCCCCTGCCCTGGACCTGCC CTCCTGGAGCCCATTTCTCCCTCTCGCCCAAGCAGGGCCCTGGGTGGGGGGCTGGTCTCC AGACCCAGAGTGGGGCGTGGGGGGCTCACCGACATGCTTGCCATCTGCTTCTCCTTCCTG GCTCGAGTGCGGCACTGGGCCCGCTCCCTGCGGGGGCCTCACTGTCAGGACCCCACACCC TCCACCCCCAGCCTGGCACCCACCCCCTGGCTCGGTGCTGTGGAGGTGGGGGGTCAAGGA TGGGTCTGCCCTGTCCTGACCAGGACACCTTCTGAGACCCTGCTCAGCTGGCCCGGCCCC TGCCCCCGTGTTACCTGAGAATCTCCAGCTGGATGTCCTCCATCTGGTCCAGCACGCCCC GGATGTCCTTCTTGAGGTTCTGGGGGCCAGAGGAGGTAAGCACAGCAGGCCCCTCGCCCC GCCCTCCGTGGCCTCTCGCCCGCCGGGCCCCGTCCTTCTGGGTGCTGCCCTGTGGGGACC TACCAGGAGCCCGGTGGCCTGGTTGTTCAGGGCCATGTGCATCTCAGCCACGTTGTCCCA CACCTAGGGGGAGGTGGGGTCACGCCTGAGGAGCAGGACCCACTGGGTGGGCCCCCAGCA CCCCTCCCCCTCAGCCAGGCGGGCACCTCTGTGATGACCATCTTCTTCCTCTCAAAGCAC AGGCGGATCTGCTGCAGCTCGTTCTGAGGGTGGGGCCCAGGATGGGCTTGGGCAGCATCA GCAGGGTGGCCAGTACCTCCCCGCTGGGCCCACTGGCCTACTGCACGAAGACCTCCCTCC CTAATGAATCCCTCAGGCCCACCTGGAGTGCTGTTACGAAGCTTCCTCCCCCCCCACAGC CCCTCCCCCAGGCCCACCTGGGACTGTTGCACGAAAGCCTCTTGCCCCTGGCACAGCTCC CGCCGCAGCAGCTGCAGAGGTGCCCTGTTCCCCAAGGGCTGCGTGCCAGCCGGGTCGGGG GGCTGTGGGAGAGCCCCCAGATCAGCCCTAAGACTGCGGGCTAGAGGGGCGTTGGCTGCA TGGGGTCCCTGGGCCCTGTATTTACCCAGGCCAGAGCAGTGCAGCTTGAGTGTTCGGGAC ACAGGGGCAGGAACTGCTGCCCCGGCCCCAGGAGGGCCCCCAGCTGCTCCCGAAGTTCCT GATTCTGCCTTTTCTGGGGTGGGGGGCAGAGAGAGGGGGCTGCCTGTCATGGCCGCTCCT GCGGCTGGGGCTGGGGCAGGGGCAGGGGCGCCAGGTGGTGGGTGGGGCTGGGGCTGGGGC AGGGGCAGGGGCAGGGGCGCCAGGTGGTGGGTGGGGCTGGGGCTGGGGCAGGGGTACCAG GCGGTGGGTGGGGCTGGGGCTGGGGCTGGGGCGGTGGGTGGGGTGCTCACCAGGCTGTGG TTCTCCTGCTCCAGCTGCAGGAAGGACTGTTCTGCAGAGCCCAGCAGCCCGCACTGAAGC TGGGACAGGGCTACCCTGGGCGAGGCCAGGGCCCCTCCCTCCCGTGACAGGGGTGGGCAT GGGCAGGGTCTGGTCCCTGAGCCAATGGATCCCCCCTGCCTGGGAGCCCCCTGGTTCGAC CCTGGGTGCAGGGGCCAGCCCCACCCAAGCCCTGCCTCCAGGTCTCCTTGTCTCGGCCCC TCAGTGTCTCTCGCTCTCTGCTCCTGGCTGGACAGAAAGAGCCCGGGAGCCTCGCCCTCC ACTGTGGGCCCACTGCCCCCTGCCCCTGGGACTGGGAAAGGTCTCTGGGACTGCACTGAG TGAGCAGAGGAGATGGGGCCCAGGCAGGGCCACCCTACAAATCCCTGGCAGGGCTGGTCC AGGCCTCCTCCATGGCTGCTGGAGGCTGGGGTTGGGGGACACTGGGGGTGGGGGTGGCCG CAGGGGCCCAGGGTTTTCAGCCCCTCCATCCCTCCCTGCAACCCCTTCCTGATCCCAGGT CCAGGCTCTGGCCTGGCCCAACCTGCCACCCTCTTACTGAGGGGGCTGCCCCAGGCAACA GTCACAAAGGCCTGGGTGCAGCCAGGGCGGTCACAATGGGCCCCTCCCACCTGGTCCTGA AGCTGCCCCGCCTGATGACCTCACTGCCTCTGACCCACTGACCCACCATGCGAGTTTTCT TTCTTTCTTTCTTTTATGGGAAGGGGACTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGC ACAAGGTGTCTCATTGCAGCCTCCAACTCCTGGGCTCAGGCAATCCTCCCACCTCAGCCT CCCAAGTAGCTAGGACTACAGATGTGTGCAACCATGCCCGGCTAATTTTTAAGTTTTTTG TAGAGATAAGGGTCTCGCTATGTTGCCCAGGCTGGTCACAAACTCCTGGCCTCAAGCGAT CCTCTTACCTCAATATCTGAAAGTGTTGGGATTACGGATGTGAGCCACTGCACCCAGCCT AGTTATAAAACAGATAATACACACACTCACAGTGTGGATTCAAAAAGTATCAAACAATAA AAATGGTGTCCAAGCTTTTCCCACCTTTATCTCCTGTCCCTGCAGACAGGAGCTTCCTCT TTAAGCTCCTAGAGGTGGGCTGCCGTCCCCTGCCCCTGGTTCACACAGAAGAGGTCATCC TGTAGCCCAGCTTTGCTTTTCACCTAATATATCTCTGAGGTTTTAAAACATTCTTTTCTA GCTATGTATTGAATATTTACATTATGCCAGATCCAGTACATGCCATTGTTATCCTCTGTT TTTTTTTTTTTTTTTGAGACGGAGTCTTGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGC AATCTCGGCTCACTGCAAGCTCCGCCTCCCAGGTTCACGCCATTCTCCTGCCTCAGCCTC CCGAGTAGCTGGGACTACAGGCGCCTGTCACCGCGCCCGGCTAATTTTTTTGTATTTTTA GTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCATGATCC ACCCATCTCCACCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCTGGCCTGT TATCCTCTGTTTATGGGTGGGAAAGCTGAGGCAGGAAGCCCTTTAGTCACTTGCCGAAGG CCACGCTGTTAACGGTCGGACCAGATTTGGGTGGCGGAAGAGAACTCATCCGACAGTAGG GCCAGATTCACGCCCCAGGCCCGTTCCCTCCTGCTCCCTGGTGCTCCTCACACCATCGGC CCTGCTGCCCCTCACTGCCCCTGAGTCCCTGTGCCCGTGTCCTCCTTCTTGAACCCATCA GCCGTCAGTTAACCCTCAGAAAGCTGGCTCGGAGAAGTCCTCATATGGTAGGAAGAGCCC CGTTCCCTCCTCTCCCGGTGTCCTGGCCCCCCTGCAAGGTGACTTCACCTCCTACATCAA GAGGCGGGTACTGGTCTCCACCTCTGGGCTCTGGGCCGGCCTCGGATGCACTTGGCCAAT GGGCTGCTCCTAAGCTGCTGTGGGCAGAGGTTGGCACGACACTTGTGCGCTGGGCCTGGA CCTCTCTTGCTTGGCTCTGGGGACTGTCCTCCCTGCAGGAGGCCTGGGCTGGGCTGCAGG ATGAGAGGCCATGGGTCAGGAGAGGCTTCTGTCGTCCCAGCCGGTCCCCAGCTGCCTAAA GATGGGTGGACAAGCCCAGCGAGGCCCCCAGAGAACCGGCCCATACGTTAGCCAAGTTGC CGGCCATGTACCATCGAGCCCCGGGCTGTCATGGTGGTTTTGCCACTGAGTTTTGGAGTG TGCTGTGACGTGGGAGGAGACATTGAGAGGCCTCACAAGCCTAGAAGCGTCTTACCCTGA CCTCACACCTGCTGGACTTCGGGCTGGGAAGAGAACTGGGGCCGAGGGCGTTTTCCCACA GGACTGGAAGGCCGGTCGTGGTCCTTGGTGGTGAGAGCTGGCCAGGCTGCCAGCCTTCTC CCCGGAAGCCTGCAGGGTCCCCTCTGATCCCTGCGCCTCTGGGGGTCACACCAACTGTGG GTCTTTCCTTCCTCACCACGCTGGGCACTTGGTGAGCACATTCTGCCTTGAGACGGGGCA TTTCTTTGATAACATTTCTTCTCATTTCCTGCTTGTTCTGGAACTCCTGTCAGTCAAATG TTGGGCCTTTTGAATTGATCCTGTGAGTCCTTCTTTCTACCTTTGCTTGTAGATAGACTT CCCTGAACCCAAACACAAACACTTTCTCTCTGCTTTTTAAAAAATTGGTTTTGGGGCCGG ACGTAGTGGCTCACGCCTGTAGTCGCAGCACTTTGGGAGGCCAAGGCAGGCAGATCACTT GAGGTCAGGAGTTCTAGACCAGCCTAGCCAACATGGTGAAACCCCGTCTCTACTAAAAGT ACAAAAATTTGCTGGACGTGGTGGTGTGCGGATGTAATCCCAGCTACTCGGGAGGCTGAG GCAGGAGACTGGCTTGAACCTGGAAGGCGGAGGTTGCAGTGAGCCGAGATCAAGCCACTG CCCTCCAGCCTGGGTGACAGAGCCAGACTCTGTTTTAAAATAAATAAATAAATAAAAATA AAAAATTGGTTTTGGATTTTAGCAGTCCTGTTTTTCATTTCCAAGACATTTCCATTGTTC CCTGAGCTCCCCTCTTCTTGTTCCAGAGAATTCTGTTCTGCTCTTAGGGATAAAGTGGCT TTGGGGGGAGGCGAATCTTTTGCTTTCTGCATTTTCTCTCTTGCGGTTCTTTTTTGTTTG TTTAGTCTTGGAGCTTCACATGAGACTCAGTGCTCAAATGTCTGGCTTTTCTTTCTTTTC AATCCCTGTTTATAGGGGAGACAGGCAGAGCTGCCGGGCACTCCTCTTTCCTTGTTAACT TGAGGAAGCCGGACAGCCAGGTCTTGGAAGGGAGGCCTCCTGCCTCCAGGCGGCATCTCG GGCTGCAGGGCTGTGGTCTGGAGTGCGGACAGTACCAGCTCCCACTGCTGTCCTGCCATG CTTGTTCGCAGTACTCGTCTCCTCACACCAGTGACGAGGCCACTGCTTGCCAGCCGCTTG CCTTCTTCCCGCCAGGCTGTCCTGCTACGTCAGCCTCCGCTCTCCACCTCAAGAAGTGTC TCGAAATATCTTCTCTGCAGATGACCTCTCTCCTGTTCTCAGTGCCACGTGGGGCCTCCC CAGCAGAGGCGGCTGGACACCTGGCCCTGGCTGCTGGAGGTTGTTTAGCTGCCAGAGGGT GGCGGTAGCCTCCAGGACCCTCCCCTGGCCTCTGCCCTGTTCATCCTTTGCCGGAATGTC ATGGGTTTCAGGATGTGCTGAGCTCCGAAACCCCATGGGGCAACTTGGGATGGGGGCTAA GGAAGGCCTCTTGGAGGAGCCCTGAGGCGAGGTGGCCCTGGGGTGGGGTGGAGGAGGCCC TGAGCAGCAGGGGAGGGGGTTGCACCCTGCTCTCTGTGTCCCTGAGTGAATGGGGCAGGG GCTGCACCCTGCTCTCTGCGTCTCTGAGTGAGTGGGGCGGGGGCTGCACCCTGCTCTGTG TGTCCCTGAGTGAGTGGGGCAGGGGCTGCCCCCTGCTCTCTGCATCTCTGAGTGGGATGG GGGCTGCCCCCTGCTCTCTGCGTCCCTGAGTGAGTGGGGCGGGGGCTGCCCCCTGCTCTC TGTGTCCCTGAGTGGGGTGGGGGCTGCCCTCTGCTCTCTGTGTCCTTGAGTGAGTGGGGT GGGAGCTGCACCCTGCTCTCTGTGTCCCTGAATGAGTGGGGTGGGGGCTGCACCTGCTCT GTGTGTCCCCAGCCTGCTAAGTGCCCCACAGGGGCCAGGATGCTGAGGAGTGAGAGGTCT TCACAGAGACAGGTGGCTCCTGTAATAAGGAAGACAACACTGGCTTCCTGGGCGAACAGC AGGGCGGGGTCCTTAGGTCCCACTGTAGGTGCCCAGGCCCGGATGAGATTCCCCAGTGGC CCTGAGGGGGCGCCCGGGACACCCTGGATGGCCCAGTGTCCTCCCGTCACCCTCGCCCCT GGGCACCATCCTCCTTGGCCTGGAAACCCCAAGCTCCCTGACGCCCCCGCCCCTCTCCCC GGGCCTGTGCCGGGGAAGGATACTGCTGCTCCGTCAGTCAGCCATAGAGCCCCTCCTCCC CCAGCCCCTCTCACCCCTCCCTGTGGTCCTTGCCAATCGCCCTGAGCCCGAAAAGGTGGG AGATTCTTGGAGAGATGGGGCCCTGGATCCCCGGCCCTGGATCCCCATCCCTGGGGTCCC CCACTCACGGGGTCAGATACGCTAACAAAGGCCAAGCGCCCTGCCAAGCGGTGTCCTGGA TTGGGGTGGGCGTGGGGGTTCGCCTCAGGTCCAGGCTCCGGGATGTCGTGCAGGACAGGG CGCGGTTGCGGCGGGGGCACTGGCTGGAAGCCGGAACTCCCGGCCTCGAGGGCGGAAAGT GTGGCCAGCGCAGCGTGCTGGGGGGGCCATGGCCCCGTTGCCAGGCAGCGCCTCCCTGGA CGCCAGTTGCCATGGAAGCCGCTTCCTGCCCATGTTGCCAGGGCGTCCAGATGGCCTCGA GGAGCCTGGTTCAGAGGGGCCGCCCTATGCCCTTGCCCCTCCTCCGCTGCCCAGGGATGG GCTGGGAGGACAAAGAGGAGGAGAGGCTGGGACTCCCAGCATGGAGATCCGAGTCCTTCC TGCTGGAGCCAGGCTGCGGCCCAGGGCAGGGGCCAGATGGGGCTGTTCTGCTGTCTGCAC CGCGGACTTGATGTTCATACCCCAAGCTCATTTTATTTATTTTCTATTTTTATTAAGAAA GAGTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGCGCGATCACAGCTCACTGAAGCCT CCAGTTGGGCTCAAGGGATCCTCCCACCTCAGCCTCCCGTGTATTGGGGACCACAGGTAT GTGCCACCATGCCCAGATAATTCTTTATTGTTTTATTTTTGTAGAGACTGGGTCTCGCTA TGTTGCCCAGGCTGGTCTTGAACTCCTGGCTCAAGTGATCTGCCCGCCTCAGCCTCTCAA AATGCTAGGATTACAGGTGTGAGACACCTCGCCTGTGCTTGGCTGCTCCCGAGCATATCT CAGAAGCCAGGAAACAGGCCCTGAATGGATGGAGCTCGGGGTCACTGGGCCAGCCACAGC CCTCAGAGGACCCCATCTCTGTGCCCTCTAACCTGGGCTCCTGGCCCACACCCACCCAGG CTGCCTGTCAGGATCTGCTCCCTCTCCAGCCCTCAGAGCTGCCCTATGTGGAGGGTTGGG GGTCCCAGAAAACCTGGGAGAAACGGGTCCTGGAAACAGGGTCGGGGGGAAGGGCAGAGT GCTGGGGATGCTGGGCCCACGACCTCCCTGTCCCTGGGGCTTCCCCATGTCAGAGGTGAG GCTCAGAGAGGTAAGGGCGCACCCTTTGTCTGCCCCCTCCCCAGCATGGCCAAGTCGCTC CCAGGGTGCAGGCGATGGCAGGCCATTTGTCTCCCTCCTGGGTGAGTCTCTGGACATGGA TTCTCACATTTTTTATTTAAGAATCAGAGAGATATAAGAATGTCAAGGAAAAATCCTCTC ATGGACAATGCAGCCCCAGTAAATGACTGTCAGCCGGCGTGTCCAGGGCTTCAAGGCCCC AGGAAGTGGCCATGCTGGGGCTGCCAGGCCTCTGGCTCCAGGGTCACTGGGGCTGAACTG TCTGCCCAGGCCCGGAGACACCCTGCCCCTGAGGAGCCCACCGGCTTGGCCAGTCCATCT TCTTGGCACTCCCTGACCACCACTTACCTTCTAGTCGGACAGTGGAGCCTGGGGGGACAG CGTGGCAGAGTGCCTGATGGTCGGTGACAAAGTCATCTCCAAAGTCCTTGCTGGGGCCAA GAGCCAGGACTCCTTGCCCGTCCCCGTCACTGCCCTGTGCCCGCCCAGCACCTGCTGGGG ACTAGGCTGCCCATTGGAGAAGGAAACACAGTGCTGGGCTGTGAGCTCCTGAAGCCTGTC TCTGTGCCCCAGGACCAGGCTCCTGGGTGGAGGGAGAGACCAGGGGCAGGTGAGGAAAGG CAGGGCCCCCAGAATCCCTCCATGCCTGCCCCTCAGTCTCCAGGACTTATGTGCAGGTAC CGTTTGGAGCTGTGGTGCAGTTCCCAGTCTCACCACCAGATGGCACCATGCCCCTGCAGA AGCAGTGCCCAGAGCAGGCCAGGTGGTTCTCGGGGGCTGCGGTGGAGGAATCCACCCAGC CGAAGCTCTGGCAGGGAAGGGGCAGTGCTAGGGGGAGCCCCCTCCCCACTTGGATCCGAA GTTCCCATGGGGCCTAGGGCAGGGGCTTGCCAGGTAGCCAGAAGTTCCCTTGCTGAGTCT AGGGTGTCTATCCCACATTTCACATGGAGACGAATCATCCCAGACACCCCGGCACTGTCC CGGGGCCGTTTGCCACCCCTCCCAAGGGGCAGGGCTCCTGTCTTGGGGCCTTCCCAAGCA TAGTAGCCTTCGGGCAGCCCCGGCCTGGCCCCAGGTTGTCCCACTTGTCAGTGGGCAGCT GGGGCTGGGGGAGGGGTCCCAGCTCCTGTCCAAGGTCACCATTGCCCCTGACCAGGGTGT GGCAGCTGCTGTGGCCTGGGTCAGTGAGGCCGTGGACGGAGGGTGGCCTGGGTGTAGGCA CTGCTCTCAGCTAAGAGAGAGCCCATCTGGAGGAAGAACCAGGAACCAGAGGGATGGCCG GGTTCCCAGCGTGCAGGAGGCCAGCTTAAGTTCAGTAGGGTCCCCCTGCAGGGACTCCAA GAGGCAAACCACATGGAGAGTCACCTGAGAGGGAATGATGGAGGCTGGGGAGGTGGAGCC CCCACCTCCAACTGCTGCCTCGGCCCCCACAGCAGGGGAGGGCGGGAGGCTGGCACTGGC ACTGCCCTGGGAGATGCCACCTGGGCCTTGGTCTGGGGAGGAGGGCTGGCCAGGAGGCTG GGTACGGCTACTGCTCTGGAGTGACCGAACCATTTCCACCCTGCTTGCCTTATTCTTCTC ATGAGGGCCTCAAGGGGACACACCAGAGGTTTACTGGGGACATGGCAGGGGAGGGATCTC AGTGCTGGCCTTCTGGGGCCCTGCGTCCACTCCGCCCCTTTCTGACCTGGGCTGGGGCCT CTGCCCTCCCCGGGGCCTGGAGACAGAGCCCTTTTGGAGGCCCTCAGGCTTGACAAGCCT ACTTCTTAGAGCCAGGCTGTGGGCTGGGCTGTATCCTGGTGGGATGAGGGATCTGGGGCT TGGGTGGGAGTGGGGCTGGGAGCAGGTGGAAGTTGGTGCCTCAGGCTCTGAGGAATGGGC CTGGGATGGCTGCAAAGTGGAGGAGAGGCTCTCTCAACCCTGGGCTTTGGGGTCTGAGCC CAGCCCTGTCCTCAGGCAGTCTGCCCCTGCAGCCCCGCATTGCCATGTTGGTTGGAGTTG CGATGAGAGGTGCTGGGGCCTCCCTTGCCTCTGCCCAGCACAGCTGAGAAGTTACGTGCT GCTGCCCAACTGTGGGGGTTTCAAACCCCGCTGGAGCCTCCAGGTGGGCCCTTGGGCAGC AGGGCCTTGTGGCGGGAGGAGGGGACCCTGGGAAGTCAGGGACCAGGCGGGGGAGGGAGA GAAGGTGGGAGCACAGTTGTGGGTGTGGGTACCCGCCCCTTCCTGAACTGTGTTCCTCCC GGCCCACCTGACTCTAGTCCTCTGTGCCGGCTGCCAGGGGATACCCGAGGCCACCTGCGT CCCCAAAAGAAGGTCTCACCTTCAGAGCCAGCCTTTGAGGCTCCTGGGTGACCTCAGCCT GTTACCCTTGACCCTGAGTTCACCTAGATCAAGTGGGAGGCCGCTCTTTGGGCCTCAGTT CTCCCATCCTCAGGGAGTAGGTCCTCTGTCCCCTGACAGCACCCCTCCTCGCTGTGCCAC CACCACTGCCAGGGTTCTGATCTGTGGATGTTCCCCAGGGGGTGGCTCTGGGCTCTCAGG CAACCGGCTACTGTGGATGGTGGTTTGCACGAGAATAGTGTCACTCTTGGGTCCTTTGCT TGAAGTTGGCTCTGCTGAGTGCAGCCTGCAGACATTTTCAGGAAACTGAATCCACAGGCT GGAGCCTTCTGCTTCCCCACAACCCAAACGGCCCCCAGCCAGCCAAACAGCCACGAGAAC CCAGCTGCTAGCAACTGCCAGCAGGAAACCCACTACCAGCCGCTCACAGATGGACAGCCC ATCCCAGGAGCTGGCCCCACCTCCCCGCCCCTCTGCTGACCAACCCAGCAGCTCCTTGGG TGGGTGTGGCCACCCTGCTGCCCCAACCTGCTCCCGGACCCTGTGCCAGGAGGTGCTTCG CCTCAGCGGGGCCTTGCCTCCCCCAGAGCCGCTCTGCTGCCTGACCTTGCAATGTCATGG GTCCTCTGGGCACTGGCGCAGTTGGGTGGGATCAGCTTTGGCCAGCAGGTGGGGGGCAGG TGGGTAAATCCTCCCTCTCTGCCATCTCCGCTTCCTTCCTCCTGCCCGGCTTCCCTGCCT TTTTCCTACTCTGGCTCCAGGGTTGCACTGCTCAGAAAGCTGTTGCTGCCTGGCCAGCAC GCAGGCCTGATTTTCCTGGGAGCCTGGACTAAGACCCTGACCAGTCCTCCCACCCACCAC CCACACCACCACCCAGCTAGCCAGCAGCTAGGCCAGGAGTGATCAGGAGGGGGCCCAACC GACCACCCACCCGGCGTCTCCAGCCGGCCAACTCTGCCCACTCAGGGCCGTGAGCTGGAG GTGCCAGGAGCCTCCCGGGAGGCAGTGTGAGGCATTCTTCTTAGTAAGTCCCGCATCGAT GATAAAGGGCAGTATCAGCCCCTCTCTGATGCGCAGTGAGCGGCTCACCATACAGGGGAG GCGTTTAATTTATCTCGGGAACTGGGCGCAAGTTATAAAAATGGTAATTTCTTGGAGATT CAATTACATGTTTTCATTGTTACCCTCGATAATCAAGGAAACGATTTTTTATAAAAGGCT GTGGGATGATACTGATCTCAGGAGAGGATCTGGGCCGGGTGGGGGCTTTGGGGACGAAGA GGAGGGCAGTGTCTGCTGGGGGCAGGCAGGGCAAGTGGAGCTGGGCCGGCCGGCCCACCA GCTCCTGCAGCCCACCAGTTCCTCCAGCTCCTCCAGCCCTCCAGTTCCTCTAGCTCTCCA GCTCCTCCAGCCCACCAGTTCCTCCAGCCCTCCAGCTCCTCCGGCCCTCCAGCTCCTCCA GCCCACCAGCTCCTCTAGCCCTTCAGCTTCTCTGGCCCACCAGTTCCTCCAGCCCTCCAG CTCCTTCAGCCCTCTCTGGCCTCAGTCTTCCCATCTGCCGACGGGCCCATCTCTGGTAGG GAGGGTCTGCGTTTTGGCATGAGGGCTGATGAAGTGAGGCGTGACTCCTGGGCCGTGAGG GTGGCTGCGTCCCTGCCCCTCCCTTCCTGGTGCCCGAGCAGTCTCGTCCATCTCCTGAGG GCTCTGCAGGGAATAGAGAGGGGTTCCTGGTGGAGTGTGGACCTGATTTTTTCCCCAAGT GATGTCTTGGCTCTAAATAACATCTCCAGGATTAATGATGCCGGCCCCCAACAGCACCAG GTGATTGGAAAAAGGGTTTTATGGCCCCGTGGGTGAAATTGAACTTGCATCTGAGGTGTA TTTCCAAGATGGGATTTTCCTCCCTGGCAGAGGCGGCACATTTCACGCTAATGATAATGG CAGCTCCTCGACATTCATCACCTATAGTTGGGGGGACACCCGTCTGGGGCACAGGGGCAG GGTGTCCCTGGGGTAACCTCCAGGTCTCCTAATCTCAGCAGACTCTGGGGGGCTGGTTCT AGGAGATGCGCCCTCACCCAGTGCAGGGCCAGGGAAGGGGGAGGCAGGAGCCCCCAGGGG ATGAACGAGCTCTAGCTGGGCTGCACCAGGGAGGAGGCTGGAAGGAGGCCCCAGGCTGAG GGTAGGACCCGGCTAGTGCTTGCCCAGGGACCCACAGGCCCCTTGCTGAGCAGACTCCAG CGAGGGGCCTGGCCCATCTTGTTGCAAAGGGCTAGAGTCCTGGGCTGAGCTGGGCAGTTG ACCTTGGATCTGACTCTGTCTCCCCTGAAAGCTGTTCCACCCCAATGCAGGCTCCTGCAG CCTAGCACAGCGCCCAGCCAGGATGGGCAGGAGGAAGCCCTTCTTCCGACGGGCTGTCAC CGCGCCCTGCCTGAGGGGCCTCAAGCCCCAGGGCTCTTGTCTCAGAACTGGGCCGTGGAG ATTCAGGAACTATGGGAGTTTGGGGGATCTTGGTGGCTGAGGGCTGTGCTCATGGAGGAC TCCTTGAGTTCAGAAGATGAGGCCCACCTTCTGCATAGCCAATAGGTCATTGTCACAGGG ACACTGACCACCAGCACCAACCTCCATCCCCCGGCACCTGCCTTTATCACCCATTTCCCA GGTGAGGACAGGGGCTCAAAGAGGGGCAGGGTTCCTCTGAGGTAACACAGCAGGTGAGCA ACAGAGCCAGTGAGGGCCTGGGGGTGTCCTGCCACGGGCTGAGGTGTGCCCACATGCAGG AATCCAGGGGTCCGTGGGCCAGGACACTGCCAAGAGTTGGGGGCTGGGGGAGGGAAGGGG GCATTGCCAATGCTGCAGCCCCTGCCCTGGGGGGTAGGAGGGAGATGCCCCTCCGGGGGC CCAAGGGCAGGGTGGGCCCATGCTGTTCCCTCCCACCTGCCACGGGTGAACCTGACAGGA GTGGGGATGGAGCCTGGCATCCCGAGGGAACCTCCTGGAGTAATCACTCCACATGCTCGC GGTAAATACTGGGCCCTTCTTCCCCTCCCAGTAATTGAACTTATTTACTTTTAACTAAGA TTAAATGTTCTCTGCGAGCTCAAGATACTGACTGCCCTGTCAGGCTCAGTCCACGCTCTG CTCTTCCTGCCTCTGGGGACCAACCCCAGGCAGGGAGGGACCAGGCAGCCCTGGAGAAGC ACAGAAATTGTTTTTCCAGGAAACTCTGGGTGGATAATCCACTTACATCATCACCACTGT CACCATGAGCATGGCCATAACCACCATCATGGTCAGCTCCACCAGCATCACCACCACCAC CACCACCATCACCATCACTGCCACCACCACCGCCACCATGACTACCACCACCATCACCAC CACCACCACCATCACTACCACCACCACCATCACTACCACCACCACCACCACGATCACTAC CACCACCACCACCATCACCATCACTACCACCACCACCACCATCACCATCACCACCACCAT CACTACCACCACCACCACCACCACCATCACCATCACTACCACCACCATCACTACCACCAC CACCACCACCACCACCATCACTACAACCACTACCACCACCACCACCATCACTACGACCAC CACCATAACCATCACTACCACCATCACCACCACCACCATCACCATCACCACCACCATCAC CATCACTACCACCACCACCACCACCACCATCACTACCACCACCACCATCACTACCACCAT CACCATCACTACCACCACCACCACCACCACCATCACTACCACCACCACCACCACCATCAT CACCACCACCACCACCACCACCACCATCACTACCACCACCACCACCATCACTACCACTAC CACCACCATCACTGCCACCACCACCACCACCACCATCACTACCACCACCACCACCATCAC TGCCACCACCACCACCACCACCATCACTACCACCACCACCATCACCACCACCACCACCAT CACTACCACCACCACCACCACCATCACTACCACCACCACCACCACCATCACTACCACCAC CACCATCACCACCACCACCACCATCACTACCACCACCACCACCACCACCATCACTACCAC CACCACCACCACCATCACTACCACCACCACCACCATCACTACCACCACCACCACCACCAC CATCACTACCACCACCACCACCATCACCATCACTACCACCATCACCACCACCACCACCAT CACCACCACCACCATCACCATCACTACCACCACCACCATCACCACCACCACCACCATCAC TACCACCACCACCACCACCACCACCATCACTACCACCACCACCACCACCATCACTACCAC CACCACCACCATCACTACCACCACCACCACCACCATCACTACCACCACCACCACCATCAC CATCACTACCACCATCACCACCACCACCACCATCACCACCACCACCATCACCATCACTAC CACCACCACCACCACCATCACTACCACCATCACCACCATCACCACCACCATCACTACAAC CACCATCACTACCACCATCACCACTACCACCATCACTACCACCACCACCACCACCATCAC CATCACTTCCACCACCACCACCACCACCATCACTACCACCACCACCACCACCATCACCAT CACTTCCACCACCACCACCACCACCATCACTACCACCACCACCACCACCATCACCATCAC TTCCACCACCACCACCACCACCATCACTACCACCACTACCACCAGGACCACCATCACTAC CACCACCACCACCACCATCACTACCACCATCACCACCACCACCACCACCATCACTACCAC CACCACCACCACCATCACTACCACCACCACCATCACCATCACCACCACCACCACCACCAC CATCACTACCACCACCACCACCATCACCATCACCACCACCACCACCATCACTACCACCAC CACCACCACCATCACTACCACCACCACCATCACCATCACTTCCACCACCACCACTGCCAT CACTACCACCACCACCACCATCACCATCACTACCACCACCCCACCACTACCACCATCACC ACCACCACCACCATCACTACCACCACCACCACCACCATCACTACCACCACCACCATCACC ATCACTTCCACCACCACCACTGCCATCACTACCACCACCACCACCATCACCATCACTACC ACCACCACCACCACCATCACCATCACTACCACCACCACCACCACCACCATCACTACCACC ACCACCACCATCACTACCACCACCACCATCACTACCACCATCACCACCATCACTATCCCC ACCATCACTACCACTGCCACCACCACCACCATCACTACCACCACCACCACCATCACTACC ACCACTACCACCACCACCACCATCACCACCACCACCACCATCACTACCACCACCACCACC AGCACCACCACCATCACTACCACCATCACCACCACAACCACCATCACCACCACCATCATC AGAACCACCACCATCACCATCACTACCACCACCACCACCATCACCACCACCACCATCACC ATCACTATCACCACCACCATCACCATCGTTACCATCACCATCACCATCACCACCACCACC ACCATCACCATCACTATCACCACCACCATCACCATCGTTACCATCACCATCACCATCACC ATCACCATCACCATCACCACACCTCCATCACTATGGTCGCTGTGACTGAGCCCATCCTCA CTACTGTCATCAGCACTGTCACAGCACCATGCTGACAAGCCCACAGTGGGTTTGAGGGCT GCCATTGCTTGGGACTAGTTCCTCAGGCACAGGAGGCTCCACGCTGGTCTGATTGCATTG GAGGGGATGCCTGACAAAGGTCTTTATGGGGTGTGCATTGTGGAGTGGGGAGGGGGAGGC TCAGGGAGGTCTCGAGGACTTCATGCCGTGGGCATTCTCCCCGGCCCCCTTCTGTCTCAG CAGCTCCCCACGCTTCTTGACTGAGCTGGAGCAGAGGAAGCAGGCAGGGGTGGGAGGAGG GGCAGGGGCAGGAGAAGGGGCAGGGGCAGCCACAACCACCTCCACTTAGGTTGGAGCACA GCCTCCTGGCCTCCCTAAAGTGAAGGGCCACCCAGAGGAGGCACCTAACCATTCTCCCCT CCGGACTCCACCCAGTGCTTGGATAGCCAGCGGGGCAGCTTGGGGTCGGGGCTGGGCACT CAGCCAAAGGGGCTGGAGAGCATGCCGTGTGCAGAGCCTGCTCCCATGAGGTGGTGAGAG CCTGCTGCACTGGTCTCCTGCCCCGAGCCTTGGCTTGCTCATCTATGAACGGGTGACGTG GTGCCTGCTGCACAGCAGGCCACAAGAGCAGAGCGACAGGACACAGTGAGGGGCGGTAAG GTGCTGCCTGGTAAGAGAGCTGTGGGCTGGCCTCAAGGGGTAGCCTGTGTCCTCTTGCTG AGAATAGTGGGAAGCGAGCTGCGAGCTTGGGCCCACAGTGGTGGTCTTAGGCTGCCGTGG TCACATAACGCCACCTCCCAGTGCCTGCGGCACCACCCCTGGGCTCCGCAGCCCCAGCCA GCCGTCCATGTGGCCCATCCTGTCCTCTCCAGGCCTGTCTGTGTGCACCCAGTGGAGGGT CTCCTGTCTCCTGTGGCTCCCTGAGGCTGCTGGTCACAGAGCTGATGAGGACACTAGAGA CCGCTGCGGGGCTGGGCGGGGGCCCTGTGGGAGCGCAGCAGCCTGGGATCTGCAGCTGGG GCTGACCTAGCACAGCAGGCCACTGATGCCACCTCTCAAGTGGCAGGGGCTTCACAGCCT CCCACTGCAATCCCGGCAGGCCTCGGTGCCAGGGCGGCCCCATCCTGGGGGCCACTGAGG ACCTGCATCGGCCACAGGGCTCGTGAGCATGGGCACCCCAGGGCTCAGTGGGGGCAGCTA GAGGGAGGGTTCAGTCACCTAGGCCTGCCCAGAGCCCTGCTCAGGGCCCAGGATGAACTG ATGGGGGCAACCTGCAGGCGGTGCCAGCGAGAGTGGGCGCTCCCCACAACAGGGGAGCCA CAGCCAGCCTCACCGAGAGCAGGGAGGGAATGTGTGGAGATGCAGGAAACCTCTGCCACG TCCAGGAAATATTATTAACAATTAAAAATTTTAAATCTTGGTAACATTCCCGATGGCCAG ATAATTAATATAAGAGCAGATAATAATTACTCAATGATTAATAACTGCACTGAATAGTTT TCAAAGTAATTGTGATAAAGAGAATGTTAAATGAAAACCCATCAAAGTTGCTGATGCCCT GCACAGCGAGGGCACCTTAGTGCTGGGGGTGGCCATGTCGACTGGGCTGAGACTGGCCCC CCGGGCCCACTGTGTCCTGGTGGCACGTATGGAGTGTGAGGACCACCTCCCCAACACACA CGGGGTGAGGACCCTGCACGCACATGCAAAGGCAGGGGTCTCTCTTAGTATTGGGGCAGG TGGGCAGAACCTGTCTCTCTTCCTGCCTCACTGGGATTGTGGATGACCATGGGGTTGGCT CAGGAGTCTGCCCTGTGCCTGCAGATGTTCTGGATATCTGGGCTAGTGTTGGGGGGTATT ATCTGACCCATGGACCAGTCTCTGGTTCTTCTATTGCCCCAGCCACTCCAGGCCTTTGGT GAACCCCCTCTACCATTCAACTTGACCTGCCATGTTCCTGAGATCTAGCCCAGGGCTACT CCATCCACCAATTCTCACTAAAGTGTGAGTGGTTGCTTGGCACTACCCTAATCTTTGCCG CCTCATGTGTTTATTGGTGTGAAGCCTTCAGCTGCAGGGGGATGAAGAAGGTGATTCCAG GCAGAGCAGGCCTGCTCCCTCCACCCTTAGGCTACTGACTCTGCAGTCCTGGACAGTGGC TGGGAGCTACCAGGAGCCCAGGTTGGGGCAGGACCTGGGGACCTACTGCCCCTCACATAA GCCAGTTTCTCCAATCCTGGTTCCTTGCCCTGTCTGCCATGCCTCCCAGCTGACACCACC GTGCGGCTCCCTCACCTCATCCTCTGAACAGGACCATCCTGAGTTGTCACAGCTTCCAGA CCCCCCGAAGCCACCTGGTCCCCTATCAGGCTGAGGCTGTCCAGACATGGCCTGCCACGA GGCCCTGAGTGGATATGCTGGCCTGGACACCAAGGATGAGGGACCCTGGCACTTCGCATC TCAGGGATGCCGGGTGCTCACCCGCAAAGACCCAGAGGAGAGAAGGGGAGAAAAAGTCTG AAGGGCTCGTGACCACCCTGGGGTCCTGATGGGGCCAGGCACGGTGGCCATACTTGTAAC CCTAGCAGTTTGGGAGGCTGAGATGGGAGGACTGCTGAAAGCCTGCCTGTGCAACAGGCA TAGAAGGCCCTGTCTGTATGAAAAATAATTAGCTGGGTGTGGCAGCAAGTGCCTGTAATC CCAGCTACTCAGGAGGTGAGCCCAGGAATTGGAGGCTGCAGTAAAAGTCATGATTGTGCC ACTGCACTCCAGTCTAGGTGACAGAGTGAGAGCCAGTCTCAAAATAAAAAAATAAAAAAT AAAAAAAAAGAAGGACTCCTGGATCACCCTGGGGCCGCGCAGGGATGGGTGGGCTCTGGA GGCCACAACTCTATTCTAACCACAGTGGTAATGATGGCTTCGGGCAGGACCACTGATGGC ACAGAAATCACCGGGTGAAAGGCTGATGCAGATGGGCCTGTGGCTGAGCTCAGCTTCCCC ACAAGATCCTGCTTCCAGAAAGAAGAAGGCGTCCTCCTGGTGGAGAGCCCTCGGAGCTCA GCCCATGAAGGAGGGACATGTGGTGCCCCTGCTGCGGCTGCCGCTCCCCAAGTCACCAGG AAATGCAGACCACTGTCTGCGGCCACTGCATTGCTTCCCTTGTTCAGAGGTCGTCATCCC ACAGTGATGGCAAAGATGGCAGTGACCACGGGGCAGGGGAGAGCCCAGTAGGCGATGGGC CAGACAGGTGCGGGCCCGGGCAGGGCAGATGGGGCGCTTAAACTGCTCTTGCTGCTTCTT TGAGAGTTAAATGTTTCAAGGCACAAGACTTTAAAAAGGAGGCACCTGGAGACATGAAGG CCAAGGGTGACAGCCTCGTGGGGAGGGCCGTTGGGGGCCTTCCCGATGCTCGGCACAGAT GGGCACTGTGGGCTATGGGCCGGGCAGGTGTGGTCCTGGCCTCAGGTTCCTGTCTCCCAC TTCTGGGAGCTCTGCCTCCCTTGTGGCCTCTGTTTCAAGGTCATTTCTGGATGGGCAAAC TGAGGCCCCAGTGGCTGGGGTTGTCCAAGGTCCTGAAGGCCAGTGTTGGAGCCTCCGGGC ACTTGGCCCCTGCGGCCCCCACGCCGCTACCCCCAGCCCAGGCCCTCAGGTCCAGAACCT CTTGCCCTCCAGCCTGGGGCCACTGTTCTGAGAGTGTGCCCCGAACGACACTCCCTCATT AGCCGAGTCCCAGCTCTGTGTTCTGGGGTTGGAGCTCTGGGGCTGTTGTCCTCTGGGCCT GGCTGCTCACTGAGGCTCCTCCAGGGGCCCTGCCACCCACCTTGTGTGCCTGCAGATGTC CTTCCCATGCGAGCACCAGTGCCACAGCCCTGACCCCCCGCCCTGCCTACCCCATCATGG CTCGCCACCAGCCCAGCTGAGCTGCTACCCCGAGCCCACCACTTCCCTAAGCCCCAGCTT GCTCCTGGCACCTGGCATCAGCCTGGGAGGGGCTGATGATCTTGGGGGAGGATCCTGCGA CAACTGCCGGGCCTGGGACAGTGATGGAAGTCCAGGCCTCCAGGGAGGCTAGGAGGCTGG TCCCAGTCCCTGCTCAGCCATTAGGGCTGCCCCTCCCCATGTTGGCCTCAGTTTCCCTGT GCGTAGCATGGGTTTGCGGTAAAGGTGATGCACTGCCATGGCAAGGTTTGTTATTGAGGG TCCTTGGAGCGGGGCCACTGCAGCAGGAAGTCCCCGTGGGGCCCCCTGCGTGGGGTTGGC CTGGCTCAGGAGATGGTCCCGGCTGCTATTTCATGTATCGTGAGGACACCGAATCCTACC CCAAGGCCATGAGGGCAGAAGGCCAAGGCTGTGAGCCTTCCCTACTTGGAGACATTTGCC CTGGGGAGCGGCTGTTCCCAGACCCTACATGCCTCCCCCACGAGTGATCTGCGGACTGGG GAGGGCACTGCCTACATCTGCCCCAAAGGCAGTGGCAGAGGCTTTGTGGGATGAGGGTGG TTTGTGCTCTCAGAGGCACTGGGCTCAGCCAGCGGCTGAGGTAGAGCTGGGGCCCTCCGG GGCAAAACATCAAAAGTCTCTGAGCTCCTGTCCAGGGCTTGGATGAGTTGGGATTGGTGC TGCCCCAGCACCCAGGATTCCCCAATGCACCTGCCACCTGGCAGTCCCTCCCCAGGGCTC AGCACCCACCATGAGTCCTCAAGGATTTGGGTGATTCAGTGGCCTGCCTCGCTGGGCCTC CCCCTTAATAATGCATTCTTGCTAATAACCTTCTTTCCTGTGTGTCATTAAACACATCCT CCTGCTGGTGGGTAGTGGAGCAGCCGGTGGTGGCTGGGGAGGCCCGGGCCCCCAGGAGAG AGGGAACCCTGGGAGGCTGGGGGGTAGGAGGGGACAGGGCTGGTGCACCACCTGAGAATA GGTACAGGGGCTCCAGCTCTGGGCAGCATCATGAGACGCTTGTGGGGTCGGGGGCGGCAG GGCAGAAAACAGCCTACAGGGGCCAGGCTGGGGGCTGCCCTCCTCGTCCTGTGGTCACTG GGCTATGGGAATGGGAATGAGGGCAGGGCAGTGCTAGTGGCCCACAGCCCTGGGGGGCAG CAGCCCAGGGATGGTTACAGGACAAGGTTTGCGTAAAACCAGGCTTGGAGGCCGGTGTGG GGTGATCCCTCTTGGAGGTGAAGGAAGGGAGCAGTGTGGCAACGTCCTGGAAATCATGGT CAGAGGGCCCAGCTGGGGCGTGTGGCCTGGAAAAGGCACTGCCCCCACCCCCACCAGCCA GGGTCTTTCCCTTGCCCCTGCCCCCTCCTCAGGGCCCCGCCCTGTCAGCCCTGCCTGTGG GTTTCAGCCCCATGGGCAGAGCATCAGACTTAGCCCGGGTGGAGGACATGGTCGGTCCTG CGGGCAGGTCGGGGGAGTCCCAGGCCAGGGCCCAAGGCCACCCGGGGGCAGCCCTGCCCA CGCCTCATCTGGTGGACCAGGGAGAAGTCCTCCATCAGGCATGGTTACAAAACTTCTCAG ACTTAATTAAAGTCAATCTCCAGTTACTCAAATGCCATTGATTTGGATCTCGACTGATGG GGAAAATATGATCATCATCATGGGAAACATCGATAGGAAGGCGCTGAGTACCTGGGCACA GGGCCGATCTGGAGCCAACACAGTCACGGAGCCCCTGCTCTGGGCCCGACCACGGGGCCC TCATGCAGAACCCTTGTCCAGGCCCTTCCGGAAGGGTCTGCCACTTGCTGCCTGGGTGTC CCTGAATTCTCACCCCCTGGCTGAACCCAGGGGCCTCACAGAACCGGGATCCTGCAGGGC TGATGGGAGGCTCAAGGGTGCGTGTCTTGGGGCCGGCAGCAGCCAGCGCTCTGTGGAGAC CGTCTACCCTCGTCACGTGGCTGGGGTCCTCTTGATGTTGCACCGGGGACCCTGGGCTGG CAGGACTAAGTCTAACTGGGGATCCAGGGCTCCTGCCTATCAGCCCCCAGTGAGTGAGTG CAAGAGGGAAGGCCCTTTGGCCAGTGCTGCACCTTTTACTTGCGTGGGCTTTGAGGCTTG GACCCAGCCCGAGGCCCATTGGGCAGCAGATGTCAGCACCTCTGTAGAGGGCAGCTGAGG CCAGACTTCTCCATCCAGCCTCAGCAGAGAAGCAAGGGCGGAGAGCAGGTAGAACATCAA GGCGGAAGGAGGTGATGCTGTTCGGGGGCTGGGGAGAAGTGCCTTCAACCAGAGCACCTG AGGTTCTCTTCTGGCCTTGCAGCCTGGAGCCTGGTCTGGGGCTAGGAGGTGTCGCAGCTG TGAGGCCCCAAGTGCAGGCCCAGGCAGAGGAGACCTGGAGGCAGCTAATGTGGGGGAGAG TTGACTGGGGATCGTAATCTAGTTTTCTGTTTTTCCTTGGAAAAAGAAAGACAAAGTCCC CAACTCACCTGCCCTTAAAATAAAAAGTCACAGGCTGGGTGCATTGCCTCATGCCTGGAA TCCCAACACTTTGGGAGGCTGAGGCAAGTGGATTGCTTGAGCCCAGGAGTCGAAGACCAG CCTGGGAAACATAGAGAGGGAAACCCCCTCCCTACAAAAAAATAGAAAAATTAGCCAGGC ATGGTGTCGCATGCCTGTAGTCCCAGCTACCTGGGGGCCTGAAGTGGGAGGATCCTTTGA GCTCTGGAGGTGGAGGCTTCAGCGAGCTGGGATTGCACCATTGCACTCTAGCCCGGGTGA CAGAGTGAGACCCTGTCTCAAAAAACAAAACAAAACAAAAAAAACCACCAAAAACCAAAG TCACATAAAATGTCTTCATAAATACAATAGAGGCAAACCTCATGTGATTTGTGTTCATGG ACTTGGAGGGGCCAAAGGCCCAACAGAAGTGAGAGGAGGAGGGAGGGGAGGGGTGCAAGG GCCAAGGGTAGAGGCCGGGAGGGGCCAGCATGGTGGCCTGAGTCCACTGCAGGTGGCACT GCTGGCTTTGGTGTCCTCATTTCATCTCCTGTGACCTCTGCCCTTGGCATGTCCCCAGGT ACCTCCTGTGGGCTCCTGGCTGGGGTGGGGTAGGAGGAGGAAGCTGAGGGTGGCCAGGGT CTGTGTCCATCGAGGGGTGTCCAAATTCTAGTGGAGGGAGGGCTGGAGGGGAGTGGGGAG GGGCCTTTGGCCAAAGCCAGGTGCTGGGGTGTGACGGATCTCCACGGTTGCTGGGACCTT GTGGCCATTCCTGGGACATGGCTTTCACCTTAACAAGGGCCCTGGGTGTTCCAGGGGTCC CCATCCCCTGCCCCTGGGGGCTCAGCACACAAAGCCCAGCCTGGGGCCTGGGCCATGGTA GCCAGGGAGGTCAGGGTGTGAGGCCTTGGTGGCCTCCAGGTGGGCGGTGAGCCAGGGGGT GTGGACATGTGGACAGTGCCGGGCCTGGGTCATGCACTTCGTCAACCCTTCACGCCTAGA CCACAGCCTCTCTGAGCAGGGCCCAGCTTGCGTTGTGCTGAGTCTTTTGTTACTTTAATT AATGCCCAGCTTCCTGACGCTTAATTAAACTGGAATTGCTCCATGTCCAATAGCCAAGCA TCCCATTAGATACAAGTTAATGCAACTGTCGCAGTGGCCATGAAATGAGGCTGGTGCGTC CCCCAGGGAGGGATGCAGAATGCATCTTGGAGGACAGCATGGCCTGACCGGGCAGCTGCA CACAGGGCCAAACTGAGCCCGTACTGGACAGCCAGCCCTCAGCCCACCCAGTACTTCCAC AAGACCCTTCCCAGGCCCCTCGAGGAGGACTCCCCATGGGGGCCCTGGCCTCCAGCTCCT CTGCACGCCCGAGGGCAGCCTGCTTGCTTCCTGCTGAGGTTTGCTGTTCCAGCTCCATGA GAAACTCATGCTAGTGTATCAACATTTGGAAAATGCAGAAAAATTAGAGAAAGGAAAGTA GGGACTCCCCACCCCAGAAAGGTGCCATCCTCCCATGGGCCTGCGTTTTGGGCTCCGTAG GGGCTACAAGGTGCTCTCTGGGGCCTGCAGTGGGGGCCGGGGCAGGAGCTCTGGGAAGGC GTGGGGGCAGAGGCCACATTGTGAGGAGCCATTGGTTCTTCAGGAGCCGGAAGTTCTAGG ATGGGGGGCAGGGCTGGGAGCCAGCGGGGGTGAGGCAGGCTGGGGATGCTGGGGTGATAC CTTGCACCCACCAATCTCCAGTGCCAGCCTCTCACCCTGATGGCCCCGTGTCCAAGGCAG CCTCACGGTGCCCTCTGCCAGTGGCCAAAGGCATTGTGCCTGGTTGGTCCATCACCCCAG AGTCTTTTAAAATTAAATTAAATTAAATTAAATTATAATTAATTTTAGAGATAGGGTCTC GATCTGTCACCTAGGCTGAAGAGCAGTGGCACCATCACAGTTCACTGCAGGCTTGAACGC CCACGCTCAAGTGATCTTCCCACCTCAGCCTCCCAAGTAACTGGGACTGCAGGTGTGCAC CACCACTCCCGGCTAATATTTAAACACTTATTTATTTATTTATTTATTTATTTGTTTTTA GATGGGGTCTTGCTCTGTCACCCAGACTGGAGTGCAATTGTGTCATCACAGCTCACTGCA ACCTTGAACTCTTGGGCTCAAGTGATCCTCCAGCATCAACCTCCCAAGTAGCTGGGACTA TAGGTGCAGGCCACCACACCTGGCTAGTTTTTAAAGTTTTTTGTAGCGAGGGGCTTTCAC TATGTTGCCTAGACTGGTCTCGAATTCCTGGCCTCAAGCGATCCTCCCACCTTGGCTTCT CAAAGTGCTGGGATCACAGGCCTGGGCCACCACACCCGGCCAGCCCAGACTCTTGGTTCA ATCCCCTAGAGGGCTTGAGTGGGGCAAGGAGGTGGCAAAGTGGGCAGCAAGGGGGCCGCC CTGGACAGGTCTCCCTGTCCCCCTACCCGAGGGTACTGAGCGGCCTCTGCATTGGGCTCC CAGGCTGGGCTGGGTGGGTCTGAGGCAGGAGCAGCCTCCCCCTGGAGGCCAGCTCAGGTG GCAGAGGCCCGGTCTGCCCCGGGTCTGCGGAGCCGAGGGCAGGTCTCTGGTGCGTAGGTT GCTGGGCCTGGCTCTCTCTCAAGCTTGTTTGCTGTTTGGGTCCTCCTCTTTGGGGCCAGG GCCACACGCACCTCTGCGACATTAGCTTGGTGCCTGTGAGGGGCTGGGTATCTCAGGAGA TCGCAGCAGGTCCTAGCACTACCCTGTTGCATTCTTCAAGGACTCCCCGCAGCCCAGTGT CTGTGGAGTGGGGCGTGAGGTGCTGCCTCCCATGTTGCTGCTTAGAAGGACGCAGCCCTG GAAACCCTCACTGTGGAGTCTCTGAGCCCCTCATCCGCAGAGCAGCAGTTGCTGCTGTTT GCACAGACAGCAAATCCGGGGCATGTTTGTTTAGTAAGATTACCTCTGCTGGGGCCTGGC CCAAGGGACAGGCCAGGCACAGAGGGGTGGGGGCTCTGTGCATACTGAACACCCCCCCGA GAGTGGCTCTGTCTACAGCCGGACCACTCTGGAAGTGACGGCACTGCGGGCCTGGAGGGC TGGGGCAGTCCAATTGGGCACCATGCCCTCACAAACAACACACACACCAGCGGTGGGGTT GCGAGGGGGCGCTGAGGGAGGATCCTGGCTGCCCTGCTTAAGCCCTGCAGCCTGTGGGGC TGTGTCCACCCGGGTCGCATGTTGCTTCTACCTGCACGGTGGCCCCACCACACAAGGTGC CGTCCTGCCCTCCAGTCAACTTCCTGGGGCTCTGAGCAGCCTGTGGTGGCACCTCACCTG GGCAGAGGGTGGGAGGAGCAGCGCCCTTCCTTCTGTGCAGGGAGTCTGATCAGGGTTCTG TCTCACTGTGGGACAGTGGGGGCCGGGGAAGAGCGGCAGGTACCAAGTGCAGACAGCAGA TGTAGATGCATGCGGGGGTCCGGGCTGGGGCCAAGAACACAGCCCTGGAGTCAGGGACAC CGGCTTCATGTCCTCACTCTGCCATTCAAGAGCTGGGGGACCTCGGGCAGGTGGTGTAGG AACAGGGAGGAGTCTGCCTGCAGCACGCAGAGGAGGGCTGTGCTGGTCAGGGGGTGTTTT GGGGAGAGGACCTCAAAGGGAGGTTGACTTTGGGAAAGGGGTCAGAGTACAGAAAAAGTG GGTCCAAATCCTGCTTCAGCACTGAGACCCTGCCTCGGCAGGGGTCTCCAAGCTTGGCTC CACCCCCTGAGGGGCCAGTTACTGCATACCTCGCCCACCCAGGAGCTGGGGGCCCTGGTC CTGTTTCCCAGGGAGCTGTTGGCACTTGTGGGCTCCTGGCTGGGGTCCAGGTGGGCTCTG CTCCTCTGCCCTGCCCAGCGCTTTGTACTGAGCCCATCCTGCAGAGACTCAACGGGGCCA AACGCACATTCCTTGACCTTCTGTGAGTCTGTCAGTCTTCATCCCTCCCTGGACCTAGGA AGGCCTGGACAGCTTTGACCATTGGTGTTGGGTGGACATGGCGCTGCGCTGCACCCCAGG CCCTCAGAGCCCAGCTGTTCTGCTTTCTGCTTTTGGTCCTGAGCTGCCTTGAAGGAATCT GACCAGCAGGGAGAGCCCACGGAGCGGGAGGGCCTGTGGGGCTCAGCCCTGCAGCCGGTC TGCCGAGGCCAGGCCTGGGGGCACAGCTGGGGCTGAGTACACTGAATGCCCCCTCGATGC CATGCACAGCAGAAGAGCTGCCCACACCGCCCTGCTGGGCCCTGCCCGGGCTCCAGACCC ACAGACCAAGCACACAAGAGACTGGGCTGCTTTGTCATGCAGCAGCAGGTAGCCGGACAC ATGTTTCCCCATTCCCCTGCCATCCTCTGAAATCCTGTGGGGAGAAAGCATGTGAGGTCC AGTGGGGGTTGGGGTGAGGGTCTGACTGGGGGCTTTCTAACCCACGTCTGAGTCCCATCC GGCGCCAGTGCCACAGTGGCGGGTGACTGGTGCTGCTCACACTGGCAAGCTCCTACCTAG ACTTTGGAGCCCAACTCCACAAGGCCCCTGAACTACCCCTGTAGGCACCGCAGCCCCCTT GCCTTCCTCTGCTACGACATCAACCACTCTGATGGTATTTGTGGTTTGATGTGTTTAAGT AAAAGGCTTGTTGCAGAGTTTAATTGACCCCATATGTCTGCCTGAGGAGGCCGTGAGCTG CAGAGGGCAGGTGTGAGCTGGGCTCTTTCCTCAGTCCCCATGTCTTGCTGGGTACTCACA GGGCACACACGAGCACGTGCGGCAGGGGCAGGGGCCAGCGGGGGATGGGGAGGCTTGGCG CCCACGGCCCCGCTGCCCCATGTTTCCACTGCAGCCGAGGAGAAAACGAGGACTTTACGG CTGTCACAAACATTTCATCTTGTGCATTTTACAGTCCACGACTGCTGTGAACACTGGGGC TCCTGTCGGAGCTAGAAGATTCATAAAATGCATGTGAATTATTTTATTTTATTTTATTGT TTTGAGATGGAGTCTCACTCTGTCACCCAGGCTGGAGTGCAGTGGCGCGATCTTGGCTCA CTGTAACCTCCACCTCCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCAAGTAGCTGG GACTATAGGCGTGCGCCACCACGCCTGGCTAATTTTTTTGTATTTTTAGTAGAGACGGGG TTTCGCCATGTTAGCCAGACTGGTCTCAAACTCCTGACCTCAAGTGATCCTCCCACCTCG GCCTCCCAAAATGCTGGGATCACAGGTGTGAGCCACTGCGCCTGGCCAAATGCATGTGAA TTTTTATGTTCAATTATTCCAGGTTCCCTGCTCGGGGCCCATGGGCTTCCCCATGTACAA CCCACCGGTAGGTCCCCGCCCAGTCCATGCTCCTCAAAGGCCAGCCTCACACCCATCCCA CGTCCTCTTGGCTGGTAAACACTCTCCCCCTTCTTCCTTCTCCCTCCACATCTCAACTGC TGGAAGGGAAGAGCTCCTCAGTCTACTTGGAGTCCCTCCTGCACAAGAGGATCCAGGGTG GTCCAGGAGGGCCAAACCCACCTCTTTGGGTGCACCCCTGGGCAGTGGATTGGGGATCCA GGGCTTGTGGGGGTATGCACCCACCAGGGCAGGTCCCTCAGGCTCACTCTCCCTGCGCCT CAGTTTCCTCTCCTGAAAAATGGGCTTGTGACTAAATGTTGCAGGGAGCTGCTGAGAAAA CGCCTGCTGAAAGCTTGGCAGGAGGTCATTGTGACCAGGGATGGGGCTGGGTGCTTCGGT CAGGCCAGGGCTGGAGGCAGGGGCTCACACTGGGGTCAGCAGTGCAGGGGCGTGGCTGGC GTGGCAGGCGCATGGGGATTGGCTCATCAGTTGCGGCCTCACGTCCCTTCGTTATGGCGC CTGCAGAAGATGTTCTCTGATTAGTCTGCTGAATTATGAATTAGGTTCCGGGTCCCTGTG CAGCTGTTCCCGGCTTGTCATCCCCACCACTTTATTTCCCCATCTGTTTATAAAACCAGG AGCGCCGTCCTGCAATAAACACTTCAAACGCTTTTTTTAGGGGCTAATTAATTGTGTTGC ATTTCTTCGACACAGCATGCAGGCCGGACCTGCCCCGAATTCCCAATGCGGGTGTGGCTC CTGCTGAGCGCCTGGGGAGGGAGCCAGGCCTGGAGCCTGGGGGGCTGCATGTGGCCGCCC CAAGTTTGTTTTCTCCAAGGCAGCCCTCCACCTTTATGTGGCGGGCTGGGCTGTCTCAGG AATGCGCCGGCCATGTCAGGTTCCCCCTAGATTCCCCCTCCGCACCCTTTGGCTGAAGTC CTCCTGCTCGGACCATAGGACACTCTTGAAATTATCTGTGGCACATCCAAGGTCAAGGTC ACGCCCTGCTGCCAGCTACAGTGCTGACCCTGAGGGCTCCACCTCCCAGGCCCACTGAGC ATCACGCTGTCTTCCCTGCCTTGCCTGCCCAGAGCACCTCTGCCCTCCAGGACCCCCTCA GCGAGTGCGGTGGGCGAGTGCAGTGGGTGGGTGGCCCATCTAGGCAAGCCAATCTCTCCT CGGATCCGTGCACCTCTTTGGAGAAGGCCTCTGGGAGCCCCAGCCCTGCTGTGCACCCTG AGGCCTCACTCCGTCTGAGGAACCCAGAGAGCATCGCGGAGGAGGCTTAAAACAATGACA ATAAGAAAACAAAATCACAATCACCAAGTCACGAAACAGCTCAGAGGCAATTGAGTTAGG CAGCCCCCTGCCAGTGCCCAATGCCGGTAAAGTGGCTCTTTAGGAACTGAGTCCTAATGT GGGCTGCGCAGCGGGTCAGAGGAGGTGTTCAGCCTGTGGCCAGGCATTGGGGCCCCAAGC CAGTCCTCCACTGCGGACAGAACCTTGCCCAGCTGCAGCTGGAGCCTGAAGGCATCCACT GCGTGCATCTGGAGGGGTCGGGGGTTCTTTGTCCCCAAGGCCACTGTGGCTCCTGTGGGC CACCGTCATGTCCACTGGGACTGGTGGGAAACTGGGTCTGGGGACTTGTACCCTCCGCTT CCTCATGTCCTCATCCTCAGGTGCCCTCCCAGCCCTTCAGGGGTCCTGTTTTCCAGCTGT GGGCAGGATGTTGCTCCTCTGCCCTGTCTGTCCACGACTCAGGGTGCCAAGGCTGGTGGA GCCTCCCTGCAGCCCCAAGGGAGGAGGCTGGGGCCAGAGACGCCCTGGGGTGGGGCACGT CTCTTCCCAGTGAACCTCAGATCCCTCGGGCGCCCACGCTCCCCACTGAGGGCCTGGGGC CACAGCACTCTGCCTGTGAGGGGTGGGCCCTTGGAGGCTTGGGGACATGCGCGCCCACAG GCACGTGGGGAGAGCCTGCTGAGCGCTGTCTCAGTGATGGTTCCCCACACTGGCCCCTGA CATGGGGCTTGGTGTGGCAGCTTGTTGGGGAGGTGGTGCGTGGAGCAGGATGGGGACCGG GAGGTGGTTGCTGTGGGCTCTGCGGGGCGTGAGGGCCGGTGTTTGCCCCAAGCTCAGTGC CTGCCTGTGACCACACCAGCTCATAAGTGACCTCTGGGTGGCCCAGGGGTGAGCGGAGAG GGGACCTGAGCAGGCAACACGGGCCTGGAGAGGAGCTTTGTCTCTCTAGGAGAAGGCAGC CTGTCCTGCCAGACCTGGTATCACGCCACCCCAAGGCAGGCCCCGCAGAGCCCTCGGGAG CTCCACTCGGAGATGGAGACGCGCTTCCCTGTGTCGGGGCTTCGCTCAGAACATTTTTAT GGGCTGTTAATTGTTATGCTTTCATTTAATTCTGGGTGTCATTCCATGTCAGCCAAGCCC GACAGGCTGGGATTTATGCTTCCAAATTATGGGGCTCGGGCTGTTCAGAGGTAAAACAAT TACCCATCAATCAGCCACCAACCGGCTTGTGCTGGGGCTGGCAGCCCCATTAGCGGGCGT GGGCAGCCATGGCCCAGCAAGACCCCTTGAGGGACAGGACTGCCTTCGAGGGCAAGGCAG TCTGGGTGAAGTCTCGCCCCAGGTTGGCCCTGTGCTGTGGCAGGGCGCAGCTCTCTGGGG AGGCGGCCGCTCAGGGAAGGACCCCAGGGTCCACATTCACACCCAACTGGCTCCAGTGCT GCAGCCCTCGGCCTCCCATATGATGGTGTGACCCTAGGTGGGCTGAGGGTGACCCGCCGG AAGACGCTGCAGCATCAAGGTGGGGGGACCATGGGTCCCATATGCTGGGGCCTGGGGAGC CTTGGCCAGCCATGGTCACTCAGTGGGAGGGGCAGGGTCACCTGCCTCCTGAGCACCCCA TCCCCAGGAGGGAGGGACCAGGTGTGACCCACTTCACAGGGAGGGAAGCCTTGGCTGTCA GAGGGGACACTGCGTCTCTCACTGGGAGGACCTCCCGGCCTGGGCTTGAGGCCCACCTGG ATCTGTGCAAACGTAGATGTGGGTAGGGTGGGGCAGGGGAGGGGAGGTGGGCAGGGGAGG TGGAGTGGGCTGGCTGTCAGCTCAGGAGTGGGGGTCGGGGAACCTGGGCCACAGAGCGGA TCCTGTCCCACCTTGACCCTGGTATGGTTGAGTCGGCGGCTAAGCCAGGCCCCGTGGTCC TTCACCCAGGGAGAGCCTGTGCCAGGCCAGGCACTGCCAGGCCAGCACACAGCCTTGACT GAGCGCCTCCCCGTCTCAAGTTGTCTCCTGGATGCCCGGCTGGCTGCTCATGGGGACAGG GGCAGGATGGGACAGCGAGAAGAGGTGGGCTCCCCCAGCCCCAGTGACCCTCCAGGCCAG GGATTGGCTTCTCTCCCTGCAGAGATCGCCCTTCCCTGCCCCTCTGCCCAGCTGCCCTGG GACTTGTGTCCCAGCCCTGCCCAGCATTCATCCTGGGTGATCTGCACGTGGGCATGGCGC CTCACCGGGTCAGGGTGGTGTCCAGGTGAGCAGGCGGAGGACTCTCCTCACTGCCCAGCA GTGTCCTGCTCCCCCAGCTGCTCTGACAGCCAAACTGGTCCCCAGCTGGGGCATCTCACG CCCAGACCTGCTGCCCGTTGGTGCAGTGCAGCCCCCGCCAATGCAGACACTGTACCTCGG TCTCCGGGACCCCCTCTGCCCCTGCAATTCGACGCTCTGCTCCTGCCCTGCTGGGATCCC ACGTCCCTGGACCCTCCCTATGGCAGTCTCGCCCCGCGACCCTGCTACAGGCCTGCCCTG CCACCCCCGGGCACACTCCCTCATCCAGAGTCTGTGCAGTCACATGTGGACCCCTGAGGC CGGCCTGCGGCTCCCTGCTCACGGGCGGCTAAAAGGGGGCTTTCCAGTCACGCCCTGCCC TGGCCTTCTGCCTAAGGCCCTGGCCCTCATGGGCCAGGCATGGTCCTCAGGCCCTTGGCT CTGCCAAGGCTCTGAGACCCCACCCTAGAGAGACCCTGGCCCACCCAGCCTGGCCCTGCA GCAGTGCCCCAAGTGCCCCCGAGCCCGGCCTGGCTGCTGCCAGGGTGACCACCAGGGGCA GTAGTCAGATAGAGGGCGGTGGCCGGGCCCAGTGTGTCTGGGTGACCGCAGGGCCTGAAG GTGTGGTGGCCCACCCGGGTCAGAGTGAAACTGAACTCCTAGGTCAGGGTCGGGTCCTCC CAGGGTCGTGTCCTCTGGAACTTGGGGCGAGTCACTACGCCTCAGTGTCATCGTGGGCAG AACCTTCCACATGTGGCATTGAAATCTTTCAAATTGAATAGGAAAAGCCCCACGTTTTCT GAGCCCTATATATAGACCAGTACTGTCCTAAGTGTATTCTAGGGAAAGACAGTGGCCAGC CCTGTAGCTCAGTTATTTCTGTTTAAAGTGTGAACGGGCTGGGTGCAGTGCTTGAAAATC AAGCATGAAGTGAAACCCAGTGGGCCGTGCATGGCTCTGACACCCATGAGTCATTCACAC GATGCCACTTGCTCATTGCTGTCCCACCCCAAATGCAGCACTGGTGGGCGAGACCCTCTG GGCATATTACCTGCGGGATGTGGCTCCAGCGCCTCACTTCCAGCTCACTGCTGCCTCAAA CTCCTGGACTCAAGGAATCCTCCTGCCTCAGCCTTCTGAATAGCTGGGACTACAGGCATG TGCCGCCACACCCGGTGAATTTTTTTTTTGTAGAGAGGGGTTCTCTCTATGTTGCCCAGG CTGGTCTCAAATTCCTGTGCTCAAGCGATCCTCCCACCTTGGCCTCCCAAAGTGCTGGGA TTACAGGTGTGAGCCACTGCACCCAGCCTCTTCACCCTTTAAACAGAAACAACAACTGAA CATACAGGGCAGACCAGTGTCTGTCCCCAGTGCCTGTGCTGGGGGTGACTTTCCAGCTGG GGCTCAGCATTGCGAGTGGGAAGACCTCCTGGGGCCAACATGGCTTGGATTGTGAGGGCA CGTGGGTCGCCGTGGGGCCAGAGTCCAGCAGTGGCAGAGCTGCACAGCAGTGGGGAGGGG CAGGGCAGGGCAGGGAGAGGGCCCCGGCTGGCCCGGCTGGCCCAGCCTGCCCAGGGCTGC ACGGTTTCCATCCCTGTCCCCAGCTGCAGTGGGACACCTGCCATGGGGGAGGCTGGGGGA GGCCTGGGGTGGCGCCTTGGGCTCTCAGCCAGGAGTTCGCTCTCTCCAGGTCAGGCTGGC AGAGCAGCCCCAGGGCCCCTGCACCCCCACTGCCCATGCCCACCTTGGGCTCTCTGATGC CCGGCAGGGCCTCTGGGCTTCTCCCTCCCAAGCTTGGGTGGCAACGCTGGCATTTGGAGC CTGGGGAGCTGCTGACAGTGCTGGTCCTGCCCGCGAAGGAGGCGGTGCCAGGCTGTTTGG AAATGTCAGGCTGATGGGTGGTGGCACTGCCCAGCACCCCACCCCCGCTCCCACCCCAAG AGGCTGGACACGGGCTGGGGCCCCGGTGATGGAGCCAGGCCGGGAGCCAGGAGATGCTGC TGAGCAGGGATAGCTGGCTGAGGGCTCCTGGGCGGCAGGGGATCCGAGGCTGATGGACAA GGGGCCTCTCCTTGCCACCGCAGCTGCGGCCTTGGTGGGCCGGTCGATGCGGAAGAGTGA TGGTGCACGTGGGGCCGTGGGGTGTCGGAGGGAGGTTTATTGATGTCGGCTGATTTCCCT GGGGGCCCCTTGCTGGCAGCAGCCACCATGCTCCCCGAGAGCTAACCGTATCAGGCCGGA AGGGTGGTGGGCAGTGTGATGGATGGACTCCGGCTGTCGGCTGGCTGGGCCTGGATGGAG CCCCGAGATGGGCACACTGGGCCGTGACAAGAGTGCCGTGGGGTCCCTGGGGGAGCCCAA AGAGGCCTCTGAGAGGGTCTGTGCTGGGAGGTAGACCTGAGAGAGGGGAGACACCATCCC AGGGTGGGCACAGGCATGGGGCAGAGGGCACGTGACGCCAACCTAGGGCCCCAGGGGGTG GGACGGAGGGGCCTGGTTCTGGGGCATTGGGAGAGGCCTGGGACTCTGGACCTGGTCTGT CTTTCTTGTCCCCAGTGTGAGCACCAGCCCTTGCAGCTGAGCCCTGGGAGGCGCCTCCTG CCCACGCAGCAAAGCTCAGAGCTCTGTGGCCTCCTCTGGGCTCCTGCACAGCGGGTTTGC TCCCAGCTGCTCCCCTGACACGGACACCTTCAAGGGCCACGTGACTGCCATCCCCAGGCC TTGGCCCTCGATGAGGAAGGGCCCTTCTCAACCTGTGGCTCTGAAATAGGTCTCATGATG GTGCCAGTTTAATGACAGGAGCAGTGGCTAATCCAGTGGTCATTATTGCATGGGCTGTCA TGCCCTTCTCTATCCGTGCTCCCTGAAGAGGCGAGGGTCCACTGCCGGCTGCCACACAGA GAAGCATGGCCACAGAAGGAGGCTCCTCTGCTCTGAGTGGGATGTTGGCTCCTCGGGCCA CACTGGGCTGGAGGACAAGGGGAGGCAGGGGTGGGAGGGCCCTGGGCTGGGGTGGATGGG GTGGGCCTCACAGATGGCATGGCCCTGTGGACAGCGAGTGGCTTTTCCTCCCCAGGTCTC GTCCTGAGCCCTGCTCCTCCTCCTGGCCTCCCCTGGCCCCACACCTCTCCCACTGGCTTC CTTTCCTGGCCTCTTTCTCTCTGCTCCTTGTCTCCATCTCTCATTCCTCTGGGCCTTGCT TCACGGAGTCACCAGCCCGTGTGACAATGTCCACTTTCCCTGGCCTCATGTCTCCAACTT AGTGTGGGACCTGCCTTTCCAGGCTCTTTGGCCACATGAGATCATGGAAACACATGGCTG CTGTACCCACGCTGACCTGGCTCAGCCCTGGCTCATGATGCTTCCTGTGCACCCTGGTCC CATCTGTACCTCAGTTTACCCATCTGCCACACAGGGCTGAGGGCATCTGCCTTGTGTGTG GCGGTCAGGCTGGCCCAGTGACCGCCGAGCCATTTTGGCTTCCTCAGGTCTGGCCACTGT GAAGGACATGGCTGTGCCCCCCGTCTGCCCTGTGTGGCCCTGGGCACATTAACCCCTTCC ATAAGCCTGGTCTGCAGACGAGCGCCCACCGGGCTGGCAGTGCCTGAAGAGTCTCGGGCG ACGTCCACTCACCTCCCAATCTGGTCCCAGCTCTGCTCAAACTCAGACTGGAGGATCCCC CAGGGCAGTGACTCCAGTTCAGAGTCTAGCTGACTGCGTCCCTGGCAGCCAGGCACGGCC CAAGGTCCAGAGCTGGAGACTGACCCCCCTGTGTAGGACCTCTGGGAAGTCCCCAGGACC CAAGACGGCTCAGGGCTAGTGAGGTAGGGACAGGTGGCTATTTCAAGCCCCCAGCGGGCT TCCTGCCTGGGGCACCTGGGCACAGTGGAGAAAGATGGCTCCCGGCAGAACACATGCCCC CATCCTTCGGGGCCAGATGCCTTGGGTTGGGCACGGAGCAGGGCCCCCAGCAGACACAGA GGCCAGGGTGGGAAGGGCCAGGGCACCGCGCAGTCTCTTGCCCTGTGTCAGGGCTCCAGG CCTATGGTGGGGCTGGGCCTACCCACCCTGTCACTTTCCCCATACGGCTTTGGGGTCCCA GGGGTTCAGGGGCCCCTCCTGCCTGACCCAGCCTTAACTTTTTGTTGTTTCTTCTCATTA CAAAAGAAGCATGTTCAAAAGAACCCCACAGTCCTCCATGAACCCCACAGTACCCCGTGA ACTCCAGAGTCCTCCATGAACCCTACAGACTCTCATGCACCCCACAGTCCCCCATGAACC ACATAGTCCCCTATGTACCCCACAAGCTCCCATGTACCCTATAGTCCCCGATGCACCCCA CAGTCCCCTATGTACCCCACAAGCCCCCATGCATCCCACAGGCCCTTATGTACCCCACAG GCCCCCATGCACCCCACAGTCCCACATGCACCCCACAGGCTCCCATGAACCCCACAGGCC CCTGTGCACCCCACAGTCCCACATGCACCCCACAGGCCCCCATGAACCCCACAGGCCCCC GTGCACCCCACAGTCCCCTATGTACCCCACAGGCCCCCATGTACCCCACAGTCCCCTATG TACCTACCCCACAGGCCCCCATGCACCGCACAGGCCCCCATGAACCCCACAGTCCCTCAC AGCCCCCATGCGGGGAAAGGGCCTCACTTCTGGCCGCTGACTGACTTGCCAGTTTCCTGG AGTTCTTTATCCAACTTTCAAGGTTGCTGAGTCCCGGGAACACCTCAGAAACCTCACGAA ACAGTGACAGGTTGTCAATCAAGCAGCAAGGTTGACATGTTTGAGCAACGTTCAGCCTGG GAGGCGCTGGGGCCAATTAGCAGAGACTCCCAGGTAGCAGAGCCCCCGGGCTCATGGCTG GGGGGTGGATGGTCAGCCCTGACGCTGCTGCCCCCACCCGAGGATTTCTCCCCAGCCATG GACTTGGGGCTGAGGGATTCCTGTACCCTCCCGGCCACCTGGACAGGGTCCCCGCCGTGG CCCGGCCTAGGGCGTTGGCTCAGACCACACCTGCCCAAGGATGGAGCCTGACACCCAGGA AGCAGCTCCTTGCAGCGCCCTGGGCCTCCGTGGGGCCCCTCTTGGCAGAGTGACCTGGGG ACCAACCCCCTTCTGTGAGGCAGGTGAGGATGCTGCCTTGTGGGCTGAAGGCCAAGGGGA AGAGGCATACTGGGGCCACTCAGGCTGAGCAGTGGTCACAGCGGCCACCCCTCCCTCTCC AAGGCTCTGGCGACCACTGTTCTGTTTAGCCCCTACCCCTCCCCACCTTTTAAAAAGACA GGGTCTCGCTCTGTCACCCAGGCTGGAGTGCAGTGACACAATCAGAGCTCACTGCAGCCT CCAACTCCTGGGCTCAAGCGATCCTCTTGCTTAAGCCTCCGAGTAGCTGGGACAACAGGT GTGTGCCACCATGCTCAGATAATTTTTTTAAAATTATTTTTTGTAGAGATGGCGGTCTCA CTTTGTTCCTCAGGCTGGTCTCAAACTCCTGGCCTCAGGCAATCCTCCCTTCTTGGCCTC CCAAAGCCCTGGGATTATGGGAGCCAGGTTCCCAAACACTCTCTGCTCTTCCCTCCACCT GGCATCTCCCTCCCCAAAGCTCTGCCCCATCCTGAAGTCCCCACTGCAGATGCTGCCCCT CCCAGAAGGTAGCTGGGCATGGGCTCTGCAGCTGCAGAGTTATGGACTGGGCCTCCCTGG GCAGCTTCCTGGGAGCTGCACCATGCACGGGTCACGGGACCCTCCTGGGTAGGTTGAACT GGGAGGCGACAGAGACCCTTCTAAAGTCCCTATGCCGGGTGCTGGGCTGTAGTCACATGG TGCCCTCCCCAGGAATCCCTGAGGGGTGGGTGCGTCTCTCTCAGCAGCACCAGCAAGGAC CAGCGGGGAGGGGTGAGCATCTTTGTTCTGTCCAAGAGCCTCTTTGGAGCACGAGCACTT GCCACATGCAGGAGAGAGCCATGTTCACTTTCCTTTTATGGCTGAGTTTGAGATGTCTGG ATAGAGAGTTATCAGCCAAGGCACTGATAAGGTCTGCGGGGGGAATATACTGTAAAGGGC TGAGCGTGGAGTGGGGGCTACCTCTCCTGCTCGGGCTGGTGTTGGGGGTTGGGGTGGGCT GGGTGAAGCTGCCACTCCTGGCCTGGATATATGGAGCCCTGGGCAGTCCTGCCTGTCCCA TGGCTTTGCTGGACACAACAAGAACTGCCAGATGTCATTTGTGTGGGAATCATTTTGCTG TTTGAGCGAAGTTGGATTTGTTAGTGTGCTCACCAGTGTGGATGGATGAGGTCTGACCAG GAGGCTGCCTGTTGTCTGTCTTTCACAGAGGGCCCTCCACACCCAGGGTAGAGCTGGGGA GAAGGAGTCCTGGGGAAATACTGAGTTAAACAAGGCTGATGTATCCTCTGCAGGACTTCT CAGTGCCTTTAGCATGGCGGGTGTAGGTGAGTCTAAGAGATGGCCAGGTAGCCTAGGTGT GGATATCACCAGGCTAGGGCTGCTGTCTGTCTCCTCTGCTCACGCTCCTGTGCAGGGAGG GACTTGAGGGTGGGCCTGGGGAAACTGTGGAAAGTAGGAGTCTCTGTCCCCAGACACTGT CTCTGAACTGGGGCCAAGATTCAGTCATTTCATGTCCCCACTAGGCAGTGCTTACAGATG TTGCCCAGTATCAGCTGCATTGATTAAAGGAAGAGCTTTCTGACCATCAGGTGGGGGCGC TCTCCTGGTGAGTGGGGCTGGGAGGCAGAGCCCTGTCCTCCGCGGTGTGACTGGGACGTA ATCCCCTCCTGGGGCTGAGTGGAGCAGGGGCTTGCGCCTCCCGCAGGCCTAGCTGTGTGG GATCTCATGGCACTCTGCTTTTATTCTGTTTGCTCCAATACCTTCTGCTTGCCACCTGCG ATGCTTATTTTCCATTTCCAGTGGTGCTGTAAGACTTCCTTTCAAAATATAATCACTTAA GTTAAAAAAGCCCATTCGAAGAAAGCCCTCGAGTAAACAATAGTGGAGCAGGTGGGCAGG TGGAGCTGGCCCTTGGCCGGTGTGTGGGCTGGGGGCAAGTGGGAAGGGACAACGTGGGAA GGAAAGGGGAGCAGGGGGTGTGGGGACCCTCCCTGGGAAACTCCCAGCCCGCCCTGGCCT CTGTTACTGCCTGGCCCTGCTGAGCTCCCTGGGCTGCTGTCTGGTGGCCAACACTGGCGT TGGCTGCCCACATGCCATGGGGTCTGCACACAGCAGGGGCTGGGGGACAGCGTCACAGGA GGAGGAAGGCCACCTGTCACAGGAGAAGGAAGGGCACACAGAGGCTTGTCTGAGCCCCAC GTGAGGATTAACAGAGGGGATGCATTGCAGTAAAGAGCACGAAGCTGCTGTGACCTTTGC CTCTGACCGCCCCCCTTGCAGGGCCACGCCTGCACGTAGCCTCTGGGCAGGTGCCATGGG GTCTGCCAAGGCTGGCATTGGCTGCCCAGATGCTGTCCTGCCCTGAGGTTCCATAGTCCT CCGGCCACTGTGGCCTCACACTGTGTCCAGCTATGCTTTTTATTTACCTGTTAACCTCCC AGCATACTTATTCATGATAACAGTGACAGGGAATGGGGCGAAAGTCTGAAGTGTCCTTCA CCAGCAAGGGGCTGGTGGAAAAGTGGGACTCCTGTCTGAGGCCACTCTGGGTGGTGTCTG TGTCCCAGGACTCTACAGCCAAACCCAATGCAGCTGGGGGCCTTCCATCCACAGACCCAC ATGAGCAGTGGTAAGGACCTGCTATATTGAGCCAGCTCCTCACACACGCCCTCTTCCCAG AGGGTAAACTGAGGCCCAGGATGTTGCCTCTTGCCTGAGGCTGCATGGCTGGGTGGAGCC AGGCCCTGACCTCTTTGCTTCTTCACCTGCCCAGGGCAGGTGTTTGGATGTTGAGTCGAC TGATGCCCCAGGGCGCCAGGCCCTCCTTCCATCTCACGGTCCCTGCCCCTGAGGCCCGTG TGTTGGGGCTGGGTGGAGGTGGCCCTGTCTCCTTTCCAGGTGAGTGGTGCGCACCCTGGT GAGCAAGGACCCCAGGCCCTGGCTGCAGGGCCTCCCTGTGGCTGGAGGTGTCTTCCACAC GGCCAGTGTCCGGCACGAGCACTCTGGGGTCGGACACTTGAGATGGGCTGGAAGGAGGGG GCATAGAGCCTTGCACAGACAGGGGCAGCTCAGGCCTGGGTTTGAATCCTGACCTAGGCA CCCTGTGGTGAGGACAGCCACAGGCTGAGGACACTGGTGTCCAGCATGTGCCCAGCTGTC ACCTCCACCCTGGGTTTGGCATGGGGCTGGCAGCAGGCCCAGGAGGGATGGGAGTCAGAG TGTCAGGGAAGCAGTGGGAACTCGGGACACTCTGCTGCTCCATGGGAACAGCATTGGCCC TGTGGTGTGGGCAGCCTCAGCAACTGGGCAGGGGCTGTCAGAGCCCAGGGGATGCTCCCA GAAAGTGGGATGGGAGCCCAGAGCCCACAGCCCCTGATATCACTCAGCCTGGGAGCAGGC CCACTGCCTCCACCGTGCAGACGAGGAAACTGAGGAGGGCCCTTGGGTCCCCTGCCCAGG CCTCACAGAGAGCAGGTGACAGCTGGAGCCACCCCAGGCTTGTCCACCCCCCTGAAGGGA TCCCCAGGTGCCCACCCTCTTCCTGCCATCTGGGGGCATGACTCCTACTGCTGACTTGAG AGTGAAGCTACCGCCACCGCCTCCCTCCACACCTGGGCCAGCCTGGGCCATGCCAGGGGC ATGCCTGCTTCTGGCCCCATGGGCCTACTGGTCTTGACCATTAAAAAGCATGTGTTGGGG CTGGGCGCGGTGGCTCACACCTGTAATCCCAGCATTTTGGGAGGCCGAGGCAGGCAGATC ATTTGAGTTCAGGAATTTGAGACCAGCCTGGCCAATAGGAAGAAACCCCGTCTTTACTAA AAATACAAAAATTAGCTGGGCCTGGTGCCTCCCACCTGTAGTCCAGCTACTTGGGAGGTT GAGGCAGGAGAATCGCTTGAACCTGGGAGGTGGAGGTTGCAGTGAGCCGAGACTGCGCCA TTGCACTCCAGCCTGGGCGACAAAGTGAGACTCTGTCTCAAACAAACAAACAAACAAACA AACAAAAAAGCACGTATTGGGACTACAGTCAATGATAATTTATTGTGCATTTAAAAATAA CTAAAAGAGGCTGGGCACAGTGGCTCACGCCTGTAATCCCAGCGCTTTGGGAGGCCGAGG TGGGCGGATCATGAGGTCATGAGAACGAGACCAACCTGGCTAACACAGTGAAACCCTGTC TCTACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTAGCTAGGCTTGGTG GCAGGCACCTGTAGTCCCAGCTACTCCAGAGGCTGAGGCAGGAGAATGGTGTGAACCTGG GAGATAGAGCTTGCAGTGAGCCAAGATGGAGCCACTGCACTCCAGTCTGGGCGACAGAGC AAGACTCCATCTCAAAAAAACAACAAAAAACAAACAGACAGAAAACTAAAAGAGTATAAT TGGGGTGTTTGTAACACAAAGAAATGATGAATGCTTGAGATAATGGACACCCCATTTCCC CTGATGTGACACTAACCATTGTATGCCTGTATCAACATATCTCATGTACCCCGTATCTCA TGTACCCCATAGATATACCCACCTACTATGGTCCCACAGAAAAGGCATGTATTGGGGCCA GGCCCAATGGCTCACACCTATAATCCCAGCACTGGTTTGGGAGGCCGAGGCAGGAGGATC ACTTGAGGCCAGGAGTTCAAAACCAGCCTGGACAACATAGCGAGACCCCCATCTCTACCA AAAACAAACAAAACAAAAAAACTTAGCCAGGTATTGTGGTCTGCACCTATAGTCCCAGCT ACTTGGGAGGCTGAGGCAGGATTGTTTAAGTCCAGGAGTTTGAGGCCACAGTGAGGTATG GTTACACCATGGGTGACAGAGCAAGACTATTTCTCTAAAAAACAAACAAAAAACAAAAAC AAAACACAAAAAAGCATGTGTTTTCCTGTTCTCCCACCACCCCACGGCAAAGACAGTACT GGCTATTGACACAAAAGGTGTGAAACGCCCACATGCTAAGGAAGACAGAAGCCCCCAGAA TCCAACCTGGGGGTGTTTCAGAAGTGCTGAAAGGGAACAGTGTGGTCCCTTTAAATGATA CGAAAGAGAGGAAGGGGGCTGGGCGCGGTGGCTCACGCCTGCAATCCCAGCACTTTGGGA GCCTGAGGCGGGCGGATCACCTGACGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTG AAACCCTGTCTCTAATAAAAATATGAAAATTAGCCGGGCGTGGTGGTGGACTCCTGTAAT CCCAGCTACTTGGGAGGCTGAGGCAGGAGAATTGCTTGAACCTGGGAGGCAGAAGTTGCA GTGAGCCGAGATCAGGCCATTGCACTCCAGCCTGGGCAACAGGGCAAGTCTCCATCTCAA AAGAGGGGAAGGAAAGTGCTGGGTAGAGGAAGGCATGGTCCCTGGCTAGGGTTCCACCCC CATGGACCTAGGTGAGGACAGGCATTTCCTGCCCACATGTTGTGGCATTTCCCAAGACTA CCCGGGCCTGCCATGCTCCATCTTGTGCCTATAAAAACCCCTGAGACCCTAGTAGGCAGA TACTCTAGCGGCTGGACATTGAGAGGAGTGGATCAGTGGAAGAACGCACAAGTGGCTGGA CGTCAAGCGGAATCCACAGACAGGCACTGGCAGGCCTGGCATTTGTGAAGCCGGCTGCGG CAGAATGATGTGGAGTTTGGCTGGGGCAGTCGTAGGAGAGCTCAGGCCGCCCAGGGGAAA ATCATCTCCCTTCTGGCTCCCCCATCTGCTGAGAGCTACTTCCATTCAATAAAACCTTGC ACTCATTCTCCAAGCCCACGTGTGGTCAGATTTACACAAGGCAAGAACCCCAGGATCCAG AAAGCCCTCTGTCCTTGTGATAAGGCAGGGGTCTAGTTGAGCTGGTTAACACAAGCTGCC TATAGATGGCTAAACTAAAAGCACCCTGTAACACGCCCAGTGGGGCTTCACGAGCTGTCA ACATCCACCCCTAGACACTGCCGTGGGGTCAACACCCCACAACTTGCCCGTCTGTATGCT CCCCCAGAGGTTTGAGCAGCAGGGCACCGAAGAAGTGAGCCACACCCCCATTGCATGCCC TGCGAAGGGGACAAAGGAACTTTTCCTGTTTCAGGGGAATGGTCCTCACTGCCTCCTCCA GATTTTCTGCCCTGAGTTGTGAGGACAGCACAGCTTGGGCTCTGTCCACAGCTTTTACTG GGAGCCTGAGGCTCCGTCACTCACTCGGCTGCTTTTTCCTGTGCTGGATGAAGCCCAGGA GCTGGCTGTGCAGGTGTGTCTGGGATTTCATGGGCATAGATGACTCCAGGACAGCTCCTT AAACACCAAGCTCAAGCAGGTATTGCCAGCTAAATGAGGTCAATTCCTGGCAGTGGATCT CAGGGCGCCGGCCTTGGACCCCACCTGCTTGCCCACCCCCAGCAGGCAGAGACCTTTCTA GAAGGTCACAGATGCTGCTGCGGGATGGATCGGGTCCCAACCCTGGCTGGGAGTGATGCC CGACAAGCTGCTGTAATTCTCAATTCTGCTCCGGAGGCCTTGGAGATGCTGCAGGCGGGC AGGTGGGGTTGGGGGTCAGCGTCACAGGAGGGGAAGGCCACCCATGGGGGCACATAGGGG CTAGTGTGAGCCCTGCATGAGGATTAACAGGGCATGCATTGCAATACAGAGCACGAAGCT GCTGTGACCTTCCCCTCTGATCGCTGTCACAGGGCCTAGCCTGCACGTAGCCTCCTGGGC AGGCACACCCCTGGACCCCTCCCTGGTGCTCTGCTGGAGGCCACGCTGGGCTCTGGAATC TTACTGCACAGAGCTCCTGGAGACCCACCCTCAACCCTTGGGCCCCCTCTTCCTTCCTGG GTCCTGGAGGGGGCTGTTAGGGGGCATGAGGCCACGCAGCCCTTGAGCACCTACTGTGTG CAAGGGCCTGGCGTTTGTGAAGCAGGCTGCGGCCATCCTGGGTGGGAGTCAGGGAATGGG GGTCCCGGAAGTGCTGGCTTTTCTGGTGGCTGTGGGGGATCAGGGGGCCTTGCTGTGTAT CTGTGAGCTGAGCCATCCTCTGAGGGGCTGAGCGCTGGCAGGAACCTATCACGTTTCCCA GGGGCAGGGTTGGGCATGAGAAGCCCTATCCTGCCTCTCAGCTAGGCCATGTTTCATACC CCACAGATGCTTTGGAGGTGACAGATCTGGGTCAAGACTGGCCTTCCTGGTGACCAGCCT TCAGATGGACACTGGGCGGCAGGTGTTCCTGGGCTGCCATGGCTGGTCTCCTGTGCTGGT CCCCAGAGCAGCCTTCCTCTCCCCTCCCCAGACACCCCATCCTCTGCCGAGGCCTGTGAG CACCTGCTGTGTGCCCACCTTTTGCTTAAACCCCAGGACGGCCTTACCCAGATGGGGAGC CTTGGTTGGATTGAAGGGGCTTGCTGGAGGTGCAGGGGGAGGGGAGGTCAGAGCCCACAT GCATCCTGAGCTCCCTCATCTTCCACACCCCATGCTCATCCCCCCAACCTGGCCCCGATG CTATCAGCCCTTAGCTGGGGGAGGGCGGACCCCGGGGACCTCAGAGGCAGGGCTCCTGCA AGCACTTTGCTGCACATGTCTCTGTTCCTGAAAATGCCCCAACCCCACCAGGGCAGCAGG CAGCAGGGTCTGTGGGAAAGTCAGGAGCCCCGCTGATGGTCCTGAGGGGCCACCCCAGCC TCAGGAGGAGGACCTGCCCTTCTCAGGGCCCTGCTGTTGCTCCTTCAGCTCGCCTCCTCC ATGCTGGCCTCCGGGCTGCTCACACGAGGATTAGAGGCAGGAATGAAGAAAACCAAAGTC AGACCACTACCAGGACCAGGGTACAAGAGACCAGGCCTGGACACCACTTATGGCTGGGGG CCTTGGGCTGGTCAGCGCCTCCCCCTCAAGGCATATACACAGTGGATCGGTAAAGGCTGT CCCATCTGGGGCTGTGTCAGGCCTCTGTGGGAGGAGGCATGTGGCGGGCCCAGCTCAGTT GATGGATTCAGACCCATGCATTTGGCCAGCCCTAGTCAGGGAACATTGCAGCCACTGCCT GCCCAGCACTGGGCAGGAGGAAGTGTCCTTTCTAAGAGTGGAGGGAGCTCCTGCTCTCAG GCCTCAGAAACGAGGTGCTAAGGGGGCTTTCAGGCAGCAGCCTCAGGCAGGAGGAGCTCC CGTGCCATGCGAATGGCTTTGCCCAGACACCGGGTGGCTCAGGCAGGTCACTCTGCCTTC TGTGTCCTCCTCCTTGGTGGGCTGGTGGGCTGAGCCAGGAGTCGGGCTTGGGGCCTGTTC CAGGCTTGCAGGCAGGAACAGGCCCGGGACACCCCAGAGACAATATTGATGCTGTTTCTG CCCAAACAAATGCCAGCTACTAATTCCGGTCTCTGTTGGAAACACAGAAATCAATGCTCA CAGAACAGGGAGGCTGGCCCTCGAGGAGCAGTGTGGTGTTGGGTCCCCAGGCAGTGATTA CGACAGGACAGGCAGGCAGGAGAACAGAGCTCTCGACCTTAGACACGTCCCATGCGGCCC TCACCTGGCGACCTGGACTCGGCAGCACGTTGGGGGTCAGAACAGTGGGTTCTCAGGGAC CTGGCCTCGGGGGGCACCCGTCATCCCTCAGCACCCTCCCCTGCAAAAGGGAAGCAGGGC CTGGACATGACCCCCAACCCTGGCTCCTGCCACATTCCCCTGACTTTATTTACCACCCGA GAGACTCATAAATAATAGTTGAGGGGTCAGTGAGCGAATGAACTAGCCAAAAAACCAGCT GGGGAATGCGTGTGCAGGTGGAGAGAATGGGCGCTGGGCCTGCCTGGGCACAGGTTCACG GGCCTCTGGATGCAGCTCACCCCGTGGGAGCCCACAGCCCTGCAGGCCGGGCAGCCTGAG GGCCAGGACAGCCCCACCCTTCTCTCAAGGTGCTGGTCCAGGGCCTGTTTCCCCATCCAT GTAGCATTCTTGATCTCTGGGTCCTACCACGTCCCTTGGATCTCAGGCTCCCAGCCAGGA GGGGCTCCCACCACCATCTCATGACCTTCTGAAACCCCAGCGCTGGGTGTGCACCTTCTT GCCAGGCTCCATGGGCAGCAAGCACCCGCCCCTGGCCTGCTGGGCACTGCACCCAGGAGG TGCCCAGTACTTGCTGGGACAGCTGTCTTCTGCTATGTCCTGGGCTCGTGTCCTGCCTGC AGCTTCCGGGCTGGGGAGCTTGGGCAAGGCTCTTGGCCTCCCCCACCCCCACCTTGGTAT CCCTCAACCCATCCCCCTGGACATGGGGTTGGTGCCTCTATTGGAGGAGGTGGTAGGTCA GGCAGCCTCTGGCCCGCAAAGTCGGCCCTGGGTCCACCAGCCGCCCCTCTGCCCTGCCTT TCCCGGGTCCCGCCTTCCCTTTCTAGGGCTATGGCTGCTTGTGGACAAGCGTCTCACGGG TAGGAAACGGGACCTCTGAGAGCTCCTGGCCTGGCTGGGTCCTCAGCATGTGGGTCTGTG AGCCAGGTCCACGCCAGGCAAGGGTTTTTACCTCTCTGAGCCTCGGTTTCCTCGTCTGTA AAAATCAGTGAGGGGATGTCTCCTTGTTGCACACACAGCAGGACTGAGATTAGAGCCACA GCCTGCCCCACCCCAGCCCCTGGTCAGGTGGGGGTGCTGCCATCTCCCAGCAGCCTGCCC AGGCCCCTCCCTCACCTGTTCCGAGACTTCCTGGCCCCACATGGGCACCCGGCATCCTTG TGGGCTCACCACAGCCTGATGCTGCCCACAGCCCATCTGTGTGGCCTGGCAGGGGGCCCC ACAGCTTGGAAATGCCGGTATCTCCTTGCAGACCCCAGGTCAACACTGTAGATTCCTCCC TCTTCTGTCATGGACCCCAGGAAGGAGGGTGGGTGGGGCCCTGGGCTGGTGCACCGCCCC AGAAGCCCTCAGCTCCTGGCAGGACCCCCCTCCCCCTGCAGGCCCTCCCCTGGGAGCAGC TGCTTCCCTGTTCATTCCAGGCTGGGTGGGGACCCCCAGGATGGCCCTTTCTCTGCTGTC TGTGGGCCTAGGGTGGGGTGGCCCTGGCCAGGCCCTGTATGGCCTCCCAGGCTTCCCCTT CACCCACTCGTTGTGGGCATGTGACACATTCATTCTAACTACAAACTGACACCCACTCAC TGAGGGCATTGAAAAACCGGAGAGCCCCAGGAGCCAGGTCACGTGGTGACCCCGACAGAG ACACCCCACAAGGGTGTCCCAGCCAGGTAGAGTGGCCCTGGGTTGGGGAGGCTTTTAGGT TGGATGCTTGGTTAGGCACCTGCCAGGTGAACCTCAGCTGGGGGCAGAGAACCTGGGGCC AAAATCTGGGGGCCGTGGTCCTGGGGCGCTTCCCTGGGCCCCACTGCAGGCACATGCCCT CCCACCACGCGGGGGAGCTCCCCCTGCCCGTGAGCCCTGCTTGCACTTCCTCACCCGTCC TCACCCTGCCCCAGCCTCCAGCCCGCCCTGGAGCAGGCTCTGTCCCTCAGGCCCTCTTGG CCATGGGGTCAGCCTGGTGCCTGCTATTTTGCCCTCAGGACCCCTTTCTCCTGACCCCTG CCAGGAGCCCTCAGCATTCACTGAGTTTTGGAGGAGCTCCCTAAACACAGCTGTGTTCAC TCTGGAAGGCTGAGATGTGGGGGCAGGTGCGGGTGGGGTAATGATGTTGGGTGGGCTGGC AGTGGAGCCAGAGAAGGCCCTCCCAGGACGAGGGCGCAGGGGGCTGGAGGGGAGGGCACT CGGGCCCTGCTTGAGATTGAACTCAGTTACTGAGCAGCCAGTCTGCCAAAGTCTGATCTC CCGGCTTTCATTCTACATTAAAAATTGATCTGAGATGGGAGAAGTGAAGGCAGTGGCCAT GCCACCGAGAAACACTAGCTGTTCTCTGAGGCTGCTGGAGACACAGCCTCCTCCAGGAAG GCTCCCAGAGGCAGCTCCAGACCACCAGGCCCGCTTCCTCCATGCTCATCTGCCCCACGC TTGCCTCAGACTCCTGGCAGATCACTAACCCCTCTGAGCCCCCAGCCAGGCCTGGATGGG TTTGTGTTTGCATGAGGAGGCCCTGGTCCTCACAGGCTCCCCCTGTAGCCAGGGCAGTCC ATCTCATCCTCACCCCCGTCCACTCTGCCGGGGCTCCTCTGTCCCTGAGTTCTGCCTGAG GGGCTGAACCACCAGCCTCGGGGCGGGGGAAGCCCACTGCACACCACCTCCCTGACATCC CGGGACATCTTGCAGAGAAGGAAACTGAGGCTTAAGAGAGGAAACAGTGTCTGCACAGGG CACAAAGCCACAGAGCCGCCTTCTGCACAGCTCCCACCTGCACCTGCGTCTCCAGGGGAG CTGGGATTTTCCAGGAGTAGGTCCTCAACTCTTGGGCATGTGGCTGGGCCCTTCCCGACC CTCAGAGGAGAGCAGGAGCCACGGGACAGGAGTAGACATGAAGAAGCCTCCACAGGGTTG GGCTTGCCCAGGGCAGCCTGTGGCCAGCGCTGGGGGTCACGGCCCCAGTGGCCTTCCGAG GGAATGGACAGGTCTGCCCCATCTCTCGCCTTGGCTCAGGCTCTGTGTGTGCTCACTCAA TGCAGCCGCCAGCTGCTGCTCCCCCACGGGGTCTCGCTCACTGCATGCGGGCTCTGCTGC TGCTGTCGGTCCTTGGCCTTGAGAATTCCTCTCTGGGAATGCGTGTTTCTCTCTGTGAAC ATGTTTTGCCTTCTACAGGCCTCCTTAGCCTCATCAGAGACTCCTGAAATGAGTTTCCCG GTCCTGTGTGGGCCAGGGGCCACCCGCCTCTCCAGACCCCCAGGGCCCATAGTGGCCTGT CCTTTTGTCCTGCGGCCTCGGGCCCCTGGGGTGTGTGGGGGGGTTTCCTGTCCTTCCCCT GAGCCCTGGGGCACGGGGACCCCTGGGTGGATGCCCCTCATCCTCATCCAGTGCGCTTGC TGCCGGCCCCAGGCCCCCCGCGAGGGGCCCTATGGAGTCGAGACAGGAGCCCACCTATAG GCCCTGTGTGGCCTGGAAGGTGGGGATGCCAGGGGAGCTCTCCTGCAACACCCCTGACTT CCAGACTCCCCTGAGGGAGGGACTTCCACAGGGAAATTAACCCCGGACGAGCTTGGTTCC CAGGAACTCACTTCGAGCCTCCAGGAAACAAACAGCCCCAGAGGGCAGGACACTCGGTGT CCGAGGCACAGGAGTGAGCAGGGTCTCCTGGGCACCGGTGACCTCCACCAGCTCATCCTT TGTCCACACCTGGGTGGGCTGTCCCTGGCGGGCTCTCAGGGTAGAGGCGGGGGCTGGGTG GGGGCAGAGCAGGTGCAGAGGCTGGTGAGGCCTGCTGGGTGGGAGGTGGGGGTGACCGCT CTCTGGCTGCCTTGACTCCATTCATCAGCCTGCAGACCCCCCTCTCCAACCCCCAGTCTT TGAAGCCACAATAAATATTGATTTGGGCAAATCGATGCATTTCCGAGGGCCTGGGTCGAT AGCAAAGTTACAAGGATCCGGGCAAAGTCATTCACGTGCCCAACTCCAGCTCTGAGAAGA GCCTGGCTAGAGCTAAAAGGTGACTGGCCATGTCCCGGGTGCCAGTGCCACCAGCTGGGC TACCCCAAGGGATGGGCAGTGTGGCCCCTCACTCTCACCTGGCCACAGCCAAGCTGGGGT TGACCGTCAGAGTCCGGCTGCCTGTCTCTGCGTCTCCCCGTGTCTGCCCCCTGCCCAGCT CCTGGCTGCATGGCTGCCTCTCCCCGTCCTGTCTGGGTGTCTTCCCGCCTGGATCCCAGA CACTCTTATGGGGCGCTTGGCACTGGGATCTGAGCAGTTCTGCACATGGTAACCACGGGT CCAGGGTAGAGCTGAGGGCCCTCATGCGACGCCTCCCTCGGGCTCTCACAGTGGCCCCTG CCCCTAGATGGGAACTCATTGCTTCCTAAGCTGCCTCTTCCATCTCCACCCGCAGCAGTG CCTCACCCTGACCCTGGGCCACAGAGGGGACATCTCCCCAAATGCACACACCAGCCTTGG CCTGCTTAAGGTCACCTGGCCATCTCCCCGCTCTGGCCTCAGCCTCTGTGTTTCTGCTGT CTTCTCTGACTCCCCGTGGGGTCTGTGGAGACCTGACCACTGCGGCCCCACCTGGGTGCT GTTTCGCACGTGGCTCCCTCTGCTCTGTCCACCGTCCACAGTTCAGCCCTTCATCCAAGC CCCCCAAGTGCCTGAACTGCCCTCTCTAGAGGGCAAGGCTGGGGCCCAGGACCCTGGGAG GAGGTGCCTGTCCTCTGAGTCCTCCTAAGCCAGAGGCTGGGCAGGGATGCGCTCAAGGGC GGTGGGCATCTGGGCTGAGCCCTGCACCCTCTTTTCTCCTGACATCCCTTGCCTCCCCCC ACCCCGCCTGGTCTCCCTCCCTCTCTAATCATTTCTCATGGCTCCAACCCACTCTGCCTT TCTTCCTCTCCTCGAACTACAAGGCTGGGTCTGGTGCACTCTGGTGGGGCCACCTTATTT ATGAGAATTTGGGAAATGCAGATGTGATGTTTCCAGCTAATAGCCCCAAATCACTTGCAC CGCCCCATTAGCGGGGAAACCAGCCTGTGTACATTACATACCACCTAAGTGGGAGCCGAG ACAGCGGTTTTATGACCCCATAAAACTTTGGGAGTTTATTGCTATTGAACAAACATGTAA TCCGATGCTTCAAGTTCAATATATATAGCCAGCAGCATCTAGAGCCAACCATGGCCGTGG CTCCTACAGCTGGGCCAAGCTGCTGAGACATCTAGGGGATTTGGAGCCGATTGGCAATTG ATCTGCTGTAGGACAGGGGCAGGATTTATGGTGGCCTTGCAGGGCTGGTCCTTCTGCATA CTAAACAGTCCAGAGGACAGCAAGGGGAGGGCAGCCGCCTGGAGCCCAGGGCAAGTGGCT GCTTGTGGGTTGCTGGGGCAGAGCCTTTAGCAAAGGGGTATCCACCTTGAGGACCTGCCC CTGTAAAAACCCAGCTTCCTTGGAGACTCAGCTGCGGTGGGAGCCATGCAGAAAGTGGCC CTGTGGCTGTGTGCCCTGCCCTTCAAGAGGCACCATTGCCTCCTGAAGCCTGTTTCCTTG TCTGCAAGATGTCCCAGGATGGTTTGTTGCCCAAGAGGTGGCCCAGCAGAGCTCCTGGAA GGAAGGGGCAAACACACAGGCCAGAGCAGCGGTCAAATGGCTAAGCCTTATCCACCAGAG CCCCTCACCCAGCTCCCATTCATCCACTGTCCACCATCCATTCATCTAAACACCCATCTA TCTAACCATCCATCCTTCTACCCATTGATCCTTTCACCCATCCACCTGCCACTGATTTAT CCATCCATCCCTCCACCCATATACCTATCCATCCATCCATCCATCCATCCATCCTTTCAT TCATCCATCCACCTGCCCATCCACCTGCTCACCCATCCATTCATGTGTCCATCCATCTAT CCATCCATTTACTCACCTGTCCATCTCTCTATTCATCCACTCACTCATCCATGCATCTAT CCACCCATCTGCTAGTCCATCTGCTCATCCGTCCATCCACTCATCTATCCATTCATCCAT CCCTCCCACCATTCATTCCTCTATTTGTCCACCTGTCCACCTACCATTCATCCACATAGC CATCTCTCTATGCACCCATCTGTTCATCCCCCATCTACTCACCCATCCATTTTCACCTAC TCACTGTCCATCCACCCACCTGTCCATTCACCCACCTATCTGCCCATATACTTGTGTGTC TGTTCACTCATTCATTCACCCTCCATTCATCTGCCCATATACTTGTATGTCTGTTCACTC ATTCATTCACCCTCCATCCATCTGCCCATCTGGCCTTCTATCCTTTCCCTCAAAGCTGCC CCTTGTCCAGGGACAGGGCTGTCCTTATAGTCGCAGAGATGATTGGAGCCTGAGAGAGCC AAGGTGACTTATCTCACCTTGCAGAAGGTGCTGGCATTATTGCTTGCTCAGGACACCCGC ATCCTCTTGCCCTGGGCTTCACACTCCAGGAATGCCTTTCTCATGTCTGTGCTGCATATA CTTATTGTAAGTAAAGGAGAACCTCATGGCCGATGATAGTTTGTTCATTTCCTCCCAGTC TTATTGTGTGCACCTATTCTTTCTTCCAAGTTCACAGTCTCTCTGGGCTTGTATGGCGTG CATTTCACTCACCACAGATGTCTCTTCGGGGCTGGTCCATGGTGTTACTCTCCCAGCATT GACTTAAACATTTCCCCACTGTTGGATAGTGTAGCTGTTTCCACCTCTTTGTGGTTATAA ATATGCTGTGGTCATTATCTGAGCTGGCAGAGTGAAGTCCGCCCCTCTACGTGTTTAATT AGCATAGACTCCAAGAGGGGGCATTCATGGGTTAAGCATCTCATGACTTGGGACACCCCT TGTCCGATGACAAAAACCTGGCCCTGTGATCCCTGCCCTCAGTGGAGGAGGGAGGGCCTT CCGTGGTGCCCGGCCGGAGGGGCTGGGATGTGTTCACTGTGAGTCTGGTGGGTCTCACAT GTAGGCAGAGTGGGGACAAGGCAGCCTGCTCTGTGGCGTGTCCCATGGCGTCCGGGTTGC CTTGTCTGCAGTTCACTCACATTGCCCTTGGTGTTGCTGCTTCAGATAAGTTCCTGCCGT GTTAATGCTATTAATTTTTTGTCATGTTAGTGTCAAAAATTTACCCGCCTCATTTCTTGT CTTTTAGCTTGATGTTTTCCTTTGAGCCACCTGAGAATTTAGCTTCCACACAGTCAAAGT GAACTCATCCTTCCCTTTATTCTTGGAAAGTTCCTTCTCACCAGGGATTCGGTAAATATT TGCTTCTGTTTACTTCCTGTTCCTTTCAATGGTTTAAGTTATTTTCTGTCAGCATCAACT TATTCCCCAGCCCATATCCGCAGGCCTCACACTCTAATTAAGAGGATGCCCTATGGCCGG GCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTAGGAGCCCGAGGCAGGCTGATCACTT GAGGTCAGGAGTTGGAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAAT ACAAAAATTAGCCGGGCATGGTGGTGTGTGCCTGTGATCCCAGCTACTCAGGAGGCTGAG GTAGGAGAATTGCTTGAACCTGGGAGGTGGAGATTGCAGGGAGCCGAGATCATGCCATCG CACTCCAGCCTGGGCAACAGAGTGAGACTCTGTCTCAAAAAAAAAAAAAAAGTAAAAAAT AAGAGGACACCCTGTGTCCCTCCCTCTCTCCCTCCCTACCCTTCCTTCCTTCCTTCCTTC CTTCCTTCCTTCCTTCCTTCCCTCTTCCCTCCCTCCCTCCCTCCCTTCCTTCCTCCCTTT CTGCCTAAACTTTTCTCAATAGCCTCAAGGCTGCAAATAAGACCTGCCCCTGGCCTCAGT GACTTGGTGGCACAGACGTGCAAAGCACAGAACGCAGAAATTCAAGTCAGGGCTGAGGAC TGGGGGTGGATGGGGGCCTGCTGGGGGGCTGGGGGAGAGAGTGGCAGGGTGAGCTGGGGA CATTGGAAAACTCTTGCAGCTGTGACTGATGATGGTGGGCAGGTGTAAGGGGTGGCTTTT CGGGGGAGGCACATTTTGGCACTGGGTTATGTGCTTCAGCACCAGGAGCAGGGGTGAGGT AGCAGACGGAGGGGAGGAGGCCATACACCTGGGCAGGGGCTGGATGGGACTGGCTGACAG CTGACCTCCCTCCTGTGCCCAGAGAGCCACAGCCTGCTCGGTGGCCCGCATGGGATGAAA CCTTTGTGGTCCCAGCTTCTGATCACTGGAGGTGAGAACTAGTTTTTGAGAGACGGGAGC AACTGCTGAAGACCCAGGGGCTGGGGCACGGCCCAGCGTTTGCTTTTGAGAGCTTCGCCA TGGCCCGTGTGGAGAGGACAGCAACCACCAGGGAGGGGGCTGGGTGCCCCAGCCTGTCTG CCAGCTTCTGAGCCCCACCTGCGTCTGCACCTACTGGAGACAGCTGGGTGGGCTCTAACC TGGATGGTCCTGGGTGGGAATCCCAGCTCTGCCTCATGTGAGATGAGGGCTGAGCCTCTG CTCATCTTAGAACCTCAGTTTTGTCACCCATACAATGGGATGGTCAGTTAGCAAGGAAAC GCGTGTAGGTGCCCCGTGTAGTGCCGGCTGCCTGGGAGGCGCTCCCAGAATGTTTCAGAG GTGATTCTTCTCAAGGACTTCCTGGGCCCCTTGGTCATAAGGCTCTGAATTCCCAGCCTC TGGCCTGAGTGCCACAGACCCTAGAGGGTTCCTGGCCACTCCTGGGGTCTGTGTGGACCT CGCTGCTTAAGGCCCCCCGTGTGCACTGAAGCGCGACACCCCGCACGCAGGGGCCATCAG GTCTTTGGGGATTGATAGAATTAAGCCCATGGAACGTGCATTTGCTTGCTGTGAAGCCGC AACAATCTCGCTGCCACAGGGCAGAGGACGTTCTGAACTGAGTGTCCCCTCGGCTCCCTC ACCGGCCTCCTGCACCAGCAGATGGTGCCTGATGTGGTGGCTGGCACTGGTGCCCCTTGC CCTCTGAGCAGCGGTACCATCAAAACCCATCTTCCCAGCAGCTCAGGATCACCGCGGTGG AGTTTCCAGTCGCAGATGCCCTTGGCAGGGGCTGTGGGACTGCAGGGGAGACGGAGGTCT GGGCCCTACCATGTGGGGGCAGCATGCAGGAGGGGCCCATTCCTGCCCCCCGGCAATACA AGAAGCCTACGGGGGTGCCCAGGGGTCAGGGACCTGAGAGATGTGCTCACTGAGACCTAG CATGCTCACCCCCAGCTGCCAGAGGGGGTAGTTCCCAAAGAGGGGCAGGCCCTCAGAGTG AGCTCCTGTCCTGAGATGGGCAAGGGAATATTCCTGGGACACTGCAGAACTGTTACCAAG CCAGGCACAGATGGGGCCAGGCCCCTCGGCCTCTAAGACAAGGCCGGCCCATGGATGGAG CACGATGGCCCTGACCTTGGGCACAGACCCGACGATTGAGTGAGATGGAGACACAGGCTT GGCTTGTCCCCAAGAAGAAAGAGGCACAGAAGCAGCTACAGAGTTTCTTGCTTCCCTGTT TGGGTTGCTGCTGGCTCAGGTACCACAGCTGGCAACAGAAACAGAGGTCAAGGCCCAGAG GGAGGGAGGGGTCGGGTCTGCTTTGTCCAGGGCTGGCTGGCCCCAGGAGGGCCCCGAAGC TGTGGCAGAAGCAGGTACCAGATGGTCAGTGTAGCACTGTGGCTAGCACCCAAAACTGGA CATGGGCTGGGAGCAGCCTGAGATGCAGGGCAGAGGACAGCAAGCCACGAGTCTGAATGG CCCCTGTTCTGGAGGCAGTGCTGGGAGGTGGAGGCTGGAGGGGGTGTCTCGCAGAGCCTG GTCTGCTGAGACATAGGCTGGGACAGGCTGCTGGGTGTACTGTCCCTGGGAAGTGCAAGG TGGCGAGTCAGGAAGGCCCCAGGAGCAGGGTTGGGGCAGGAGCCAGTGAGCAGGGCAGGA CCCTCATCTGGAGGGGCTGGGGGAGGTCTCCAGTCTGGGATGGGGGTGTTCCCGGCACCA GCCCAGTGTTTCTGGTGCAGCAGCCACGTCATTATGGTTGGAATGTGACCCCATTAGCCC TGGGGCGGGGCTGTTCAACATCCTGAGGATAATTGATCAGAGGGAGGAGGGGCTGCACCG CTTCCTAGCAGGTTGAGTTAATTATCAGCCTGCATTAGCACTTGACCTGTGGCATGTGTC AACTCATTACTGCTAATTGCAGGGACTCAGTGACCTCTCCGAACTTCTGTCACCCCTGTC ACGGCCCCTGCCCTGTGGAGGCCTGTGGAATGGGAAGTGGGGAGGGGAAGGAGAAGGGGG CGGAGTCAGCCGGAGCCTCCTGGCCAGGCCTGGAGTCACCCTGTCCTGCCACAGCTCTCA TGGGTTTAGAGTGGGCCCTGGGGCAGAGGGTTTGCAAGGATAGGGGTGGGTGGCTCTGCC GAGGGCGGTGTTCTGAGTAGGTCTCATTCCCCAGTCTGCCCAGCCTGGCCCCACCCCAGC TCTGCTGCCCTGTTACTTTGTCCCCCTGTGTTCACCCTCCACTTCCTGCCGGGGACACTC CAAGGCCTGCCAAGGTTGGAGCAGGGGTGGGACTCAGCCATTCAACAAACGCTTCCTTGG TTATGCAGGGCTGGGCATTGGGGTGGGGGGCTGACCTGGACCCTCCAAGACATCAGATTG TCCAACTGCTCAGTTTTCATTGGGGCAAGTGCCTCGCTGAGGGTGAAGGGGGCTGTGAGT GCCCATCAGGGAGCCTGGACAGGTCCCGCCTCTCACAGCTGTCCCACTCTCTGGAGCTGC CTTGGTGGCAGATGGCACTGTCCTAGAAAGGTGAGGCTGGAGCTGTCTCTTCCACCAGGG GTGGCCAAGGACGCTTCCAGGGTCTTCCATGCAGGCCTCCAGGGTCACCCTGGGCGTGGC TGAGGGCATGCAGACCACGCTGTGCTCCCCCCCACCCCCCATGCCCGTCGCTTGCCTGAC ATGCTCCTCAGCTGTCTTGCCCCGCAGAAGCTGTGTGGTCTCCCCTGTGAGTAGAGTCGG GCTCCATGATCCTCCGGAGCAGCGCCAGGGACTCTCCCCAGGCACAATTTTAAAGGGGGC CCTAGGACCTGCCAGGTCCACGGAAGTGGTCCTTGTAACTCTCTCCCTGGTGGCAGCCTC TGGTGCATTCCGGCAGGGGCAGCCCCTCCTCATCACAGGGACCCTGTGTGCCAGGCCCCA GCTGCGTTGCCGCTGAATTATGGATTTAATAGCAGAGAAGCCTTGCAGGCCGCCTGTGCT GGCTCGGGAAACCGGGGCCCAGCCGGGGCCTGTCACGGTCCTGGTGCCGCCCTGCTTCCT GTCGGCGCCACGGAGCAAGCGGAGAGATTTATCTTCCCGGCTTTAAGAGGTACTAACGGA GGAGGCTGAGAGGCAGGAGCTGAATAATTGATGGCCTTCTCTGGCTCAGGATCTGCTGAA AATGACACGGTGCAAGGCAGAGGGGCTGAGAGTGCTGACCACAAAATGCTGAGAAGCCAC TCCAGGGCGGTCACGGCCAGAAGGAGGACCCCCACTCAGGACGGGGTCACAGGCATTTGG GGCGGGTGTCAGAGGCTCCTGCCCTGGGTCATTCCAGGGGGCCAGGATACCCAGAAGCTG GGCCTAGAGGCCCACAGGGCTGGCTCACCCCCACTTCCCTCTCACACGCCTGCCATCACC GGTGCTTCAGCCCAGCAACCCCATGGCTCGTTGGCCCCGTCCACAGCCCCCACTGCCTCC TGTGTGCTGGGCGTGGGCCTCTGAGCTCTACTCTCAGCCTCCTTAGCCCCAGGAGAGGAG TGGAGGACAGGGGTTTAACGTGGGGTGTGGTGCGGAGCGGGGAGGGCTGTGGCGCCCCCT CAGCCCCTCCTTTCCTAACAGCCTCAGCCTCCCCCCTGGAGAATCCCCTACCTGCTGGGC ACATCTGGCCTCTCCCCACCATTCTGTCTCCAGCCGGGAGATCTCTACCCAGTGAAACAG TGAAACAGGAAGGGTTCCCTGGTTACCCCGCGCAGGGCGCAGGGCATGCAGCGGGGGAGT GGCTCGCTTCTTCAGTGCCCCACCGCTCACACCTCTAGGGGAGCATACAGACGGGCAGGC TGTGGGGCTCTGACCCCATGGCAGTGTCTGGGGGTGAATGTTTACAGCTCTTGAAACCCC AGTGGGAGTGCGTTACCGGGTGGTCTTTTAGTCTAGCGGTCTGTAGGTGGCTGGTGAGAG TGAGCTCAATTAGACCCCTGCCTTATAACAAGGACAGAGGAATTTCTGTATCCCAGGGTT TCTTGCCTTGGTGTACCAAAAAATTGGATCACACGTGGGCTTGGAGAATGAGTGCAAGGT TTTATTGAGTGGAAGTAGCTCTCAGCAGATGGGGGAGCCAGAGGGGAGATGGTTTTCCCC TGGAGTCAGGCAACATTCTCGGGATGTCCAGCAGCTTGTCTTCTTCCACCAGTGTGTTCC TCTCCCCACACAGAGGCTTCTGTCTCTGCCTTGCTAGGGTCTTGGGTTTTTATAGGCATA GGATGGAGGCATGGCAGGCCAGGGTGGTCTTGGGAAATGCAACATTTGGGCAGGAAATGC CTGTCCTCACCTAGGTTCGTGGGGGTGGAGCCCTAGGCAGGGACCACGCCCTTCTCTACG GAGCACTTCCCGTCCCTCCTCTGTATCATTTAAAGGGACCACGCTTTTCCCTTCTCAGCA CTTCCTTTCCGTATCAACAGGAGGCCAGGGACTGGCTGGACCCCTAGGAGCCAGGTCCTA CGGATAGGTGAGCCGGCCCCCCAGGGCAAACACCTCCACTGGGCAGACCTGACTGCCCAG AGCTGGATTCTTTACTTGTAAAAGGGGCCATGAGTGACACAGGCTCGGCCTGCTGTGCCT TGAGGAAAGGAGAGGCTAAAAATTCTGCCTTCTCTCCTAAAGCCAGCCGGGCTCTTTATG GTGGGGGAAGGAAGGATTTGTTTATTTGCACTCTTCTGAAATAAAAGTTCTCTATCCTCC CTCCTGGCTGGAGTTGGGAGGGCCCCATGGGTGGGCTCTGAGATGTGGCACACGGTACCC AGGGCAGGGCTGGGAAGGGTGGGGTGCAGGGGCCTCCCTGGCTGCAGGGCCCACCCACCA GGTGCCTGGGCTTGCTGCACTGAGCAGGCATGGGCTCTGGCCCCCCTCACTGTCCCTTCC AGGCCCAGCCTGTGTTCAGGGCCCAGGACAGGGTCTGGCCAGCAGGCCCCAGGCCTCCAA GATGTTCTCAGCCTCTGGAGTGTGTGTGATGATGAGCACTCATGGAGCTTCCTGTATGCT GGGCCCTGGGCTTCTGAGAGGGCCACCGGGTGGCGACTGAGTCACGAGGTGGAGGGCCAG TCCCAGGGACTCCAGGTGACCTCGTGTCGGCACCACGCCCTTCTCCCATGGGCAGTCCTG GGCGCCCCTTCCTGGCCTTGGGCCCAGTGGCCTGTGGCCTGACCCCGTCCACTCCTGCTT GGTTGTCCCAGCAGCAGCAGGAGCCCATGCTGGAGAAGGTGTTATGGGGCAGGTGGAGGG CCCGGCTGACTCAGATGATCCCCCAAGTTTCTTTTCAATGAACACTTGTTTAGTGCAGTT TTTCCTCCTCAAATCCCCCTCACACAGGCAGACACCCTCTTGGCCCAGGGTAATCTGCTG AGCCTTTGCTCAAATATAGCAGCTTTGCTGCTTTCTAAACACAACTAATCCAATTTGGGA TTTGGGGATTCGAGGTGGGCAGCACTGGGCCTCAGATGAGCCCCCATCACCCCAGACCCA TGCAGGAAGAGCCAGGCAGGGTGGGGCCCTGGGGCCACCTGGTGGGCTCAGGGCCAGGGC ATCGCGATGTCCCCATCCTTCACACTACGGGGCTTCCTGGCTGCAGAGTTGGGGACTCAG CTGGGAATGCTGGCCATAGAGGCAGAGAGCCCCCATCCTGCTGGGCCAGCCCTGACCTGG CTTCTCCAGGGTGGCCCAGTGTCCCCTTCTGCCCTTCCCCTGGCTCCCACCGTCCACATC CCTCTATCTGCCCACCCTGGACAACAACCAGCACAGTGCAGTGTGGCCCTGCAGTGACAC CACCTGCAGAGAGGTCAGGCCCAGCCTCCCCCGTCTCCTAAAAGGCCAGCAGGTCCCAGG AGCTGGGGGTGGGGCCTGGCTGTGGGGCCTCCATGCCCCAGGGGCCAGGGCAGACCCTCC TTAGCCGGCAACCACTGCAGATTCCTCCCCTGGCCAGCACTCGGGGACCTCCCTGCCCCT GCCCCGTCCCCCTACCTTGCCTGCTGAGACATAGGGCTCCACGGGTCTCTCTCCTGGGGC CGGGCTGACTGTGGCCTGCGAGGGGCAGTCATCGTGTTGGGTTTTCCTGCCAGAGGCAGA AACCACAAAATTACCTGGAACATACACGCCCCAAGTGACAGATTCAATTCAATTCCACAA ATATTGACCTCGCGTCTAATCCACTCGTCCTCCAGTGCCAGCTCATTCCTGAGAAGCCCA GCCTGGCAGCCGCAGATCCCATTTAATCCCAGCCCCACAATGTCACCAGCTCGCAGCTTG GGAGCTGGGCTCATCCAGGCCCTGGCAGGTGGGGCCAGAGCTGGGCTCTGACCTCTGTAC CCACACTTACCACCATGTAAAGTGCTGGGAAGAGGGGTGGGGAGTCACGGCAAGTTGAGG GTCCAGGCTTGCCCCAGCTCGCCAGATGCTCGCCCTGGCTGCCCTGACCTAGCAGCTAAT CTCTGCGGGGCACTGCGGCTACATGGTGACTCCAGCACGGGGACATTTATCAGAAATGGA ACTGATGGATTAAAGTGGAACCAAGCGAATTGATTTTTCTCCCTGGGCACTGAAATGCTG CGCTTAATCAAGCGATCAACTCCGTGGGCAGGGTGGCTGTGGGGACGGAGGTGGGGGACA AGGGGATGGTGGCCATGGTGGGGACTGAAAGGTGGCTATGGGGACAGATGAGGTGAGCCA GGTGGCCCTGGGGTCGAGCTCCAGCCCAGCCTCTGGCAAAGAGGCCCTGTGTGCAGAAGT CCGGGTGTGATGCCCTCCCCCCACCGATTGTGTGATACCGGCCAGTCCTGTCCCGGCTGA GCCTCGGTGGCCTTGATGTAACCCTGTGGGGTCTGGTGGCAGGGACTGATTCACTCATTT GCTCATTCATTCATGTGTTTGACACACACTGCAGCAAAGGAAAGGTGAGAGCAGCCACAG GCTGGCCAGGGACGATTCAGACTTCAGAGTGTAGCGTGCCCGCCCCTCCTCTTGTGAGGT CCAGCCTTGGTGTGTGTGGATTCCCAGGCACCCTCCAGGTCCACCCTCCACTCTGCAGTG CCCCGCCCACCTGTCCACATACTCTGTTCTGTGGTTTGATCGCCATCCTTCTGCTCTGCG GCCAACTGGATGGCCCAAGCGGACTGAGTTGGGCAGGGTGGTCATACCTGTGGAGCCTGT GGCTGTGGTTGGAGGCCCCGTGGGCAGCTGAGGCAGCCTGGGTGGGCATGTCCCACTGGG ACTTAAGCCCCTAGGTGGGCTGTCCGAAGTGACTCCTGAGTGGCTGGGGTGTGCTGTCCT TCCAGCCTGGCCGTGGTGGCTGGGTGGTGTTGGAAGGCACTGAAGGAAGGGCAGGTGAAT TCGTGCTGGACCTGGTCTCAGTGCCATGGGCCCCAGGAGCCCATGACATCACTGGCCTCA TGGACTGTGGGGATTAACCCTGTCACTGGACACCCCCAACACACCCCCTCCAAAAGAAAG GTGTTGGCACAAGTGAAACCCCTTGAGTGATGGCTGGGATTTCAAGCTCCAGACTGGTCC CCACTAGGAAGCTCTGGGTCTGGCTGGTGGAGCAATGTGTACCCGCGCCTCTGCGTCTCC CTGCATCTGGCAGCCCTCCCAACAGGGCTCATGTGGGGTGCAGTGAAGCAGCAAAGCCCC CCACCCCACCCCACCCCAGACTAAACACAGGCTGTAGACCTGGCCCAGGGTGAGAAGGGC CCCTGTGGACCACCTACCCAGGTGGTCTGTGGCAGCCTCTCCCAGCTTTCGGTCCAGGTG GCCCAGGGAGGTGGCAGCCCTGACCCTTCCTGGCGATACCCTGATTGATCATAGTGAAAC ACTGAGCTGGGAGAGCCAGGGGCACCCAGGGCTGAGCTCCCGGGGCAGCTCCCAGGAGCT GCAAGTGGACCTGGCACTAGGAAGGAGCAGTCCACCGAGCACCTAGGCCAGGGCCCGGGA CGGTGCCAGGCACCAGCTGAGGCCCTGCCAGGCCAGCCGCATCTTCACATTCAAGACCCC AGGCCCCAGCCACGGCTCTGAGCTCTGGGTGTCTGCAGCCACGGTTGGCAGGGTGGGCCC CACCTTGCCCTGACTGGGCCACGGACTGGTGTGAAGAGCTCTAAATTTCACTTACTCCCC CAAAGTAGAAGTGGCCTAGGACTGTGTGGTGGCTCCTGGTCTCCAGAACCCTGCCAGGCT CCAGGGCAAGCAGGGAAGTCAGGCTTAGCTGCAGGGCAGCACCTGTCTGCACACCCCGTC CTGCCACGCTCCCTGCAGGGACCTCCGCCTGTTGTCCATGGTCCAGCTTGCTGGATGGGG CAGCAGCTCCTACACTCAGAGCTCTGCCATCCCCAGCCCCAGGCCAGGCTCTCCCCACAG CCCGTTAGGCCCTGACGTCCCCTGACAACCAGGAGTTGGACAGGCCTGGCCCCCAGGCTG CCCCACACTGCCATGAGGACCCCAGCTGGCTGAGTCCCATGTCCAGGCTCACTTCCTCTG TCCCTGCAGCAGTGAGCACCTGGGACCATCATGGTGAGCACCACCCACCTTGCCCCTTGG CCCATACTCGGGGACCTGGGTTCCCCAGGTGATCAGGGTGGCCTGCAGGGCCTGAGGTGG CCATGCCAGCCAAACTCCCCCTGCCCCACAACTGGGTCACCTCTTCACCCTCCCGCAGCC CTTGGGAACCCAGTGAGCTCCTCCCCCTTGCTGCTTTGTGAGTGCTGGCAGTCCTGGGTT GCTGTCCTGGAGCTCCAAGACCTCCCTTCTGGAAGGCTGGGGCTTCCTGGTGGAAGATCC CGCTCACCTCAGCTGTGGCGTGGGCCTGGGCTTCAGCCTGGGGCAGCCCTCATGGGCAAC TGCTTCAGACTGGACCCCATGTGCTGGGGTGGCCAGGGCTGTGCATGGCAGGTTCAGGGC ACAGGGTGCAGGCAAGGGTGTGAGGGTGTGGGAGGGCTTCAGTGGGAGGCCCTGATGCTG AGCAGACACAGTACAGCCTGGTAAACAAGGCTCGTGTGCTGGGCACTCATCGATCTTGGC CATTCGGGCCTGGCCAACAGCCGGTGGGCTCAGGCCTGGGCGGGGGCTGGGGTGGGAGTG AGCTGCCTCAGGCTGGGCCGTGGGCACTGCAGGTGGCTTGTCCGGACAGCCCTTTCCCTG GACCCCTCATGCCCATGGCTCTAGCTGAAAACACAGCTGCTCTTCTCTACAGGCTCGTCC TGGGGTGAGGAGGGTGTGGGAGCTGCCTAGCCCTTCCTTTCCCAAGGGGTTGGGTGCCCC TGGCTGAGGAGGAGACCCTCATGGCTGGAACTTGGTGGTGGTCAGAGCCTCCTGGAAGTT GACCAGCTGAGCTCCAGCAAGTCCATGCCTCCTGGGACACAGCCTGGCCCCGATGAACCC TCAGCTGCCATCATCCTCAAAGTCGCAGCAGGCTGGATGCTGAAGGCACAGGGGGCCGCA TGGACTGTGGCTGCAGCTCTCTCATTGTCATGGGGAGGGAATGGCCTGCAGCCCCATCTT GTTCCCTTCTCTTAACAGCCTCTTTCCAAAGATGGCCCGAGCTCCCATCCCCGCCCCCAC AGGGCTCTTCCTGCACTTCCCCAGGCATCCTGGGAGGAGAGGTGGGCTGTGGCTGGAATG TGCCGCCCTCTCACCCTCCTGCCAGGACCACTGTGCTGGCTGCCTTGGTGGCCTGGAGGC CTGCAGAGCTGCCCAAGCGGGGGCTCCATGCGCAGCTCCCACTCCATGTTCTGTCCCTGG GGAGGCCCACACAGCACTGCCCCTCTGTCTCATTCCAGGTGCCTGGTCCCCAGCGATCCG CCTTGACCCCAGATGCTCAGACTTGCTAGCCCAGGGCCAGAGGGGCACAGGACAGGGCTT TGGAGAGGGGGCTTCCTAGGCTGGTGCTGGCCCAGCCTGCCCAGCCCTGCCCTGCTCGGC CCCTGCCTGTCTGGGTGCTGGACCTGGCCTGATCCATGAAGCTTCTCCTAGCGAGGTGTT TTCCAGGCCTTTGGAATCTTAGGGATGATTTCGTATGGGGAGGGGCCTTCTCCCCAGCCT GCTGTCTTATTTTTGTGCCGATTCCCCGCAGCCCGAGCTCCTGCCCCCAGCACCTGCCTT TCCAGCCTGTGTCCTGTCTTCCACCAGCGGACTCCAGCCTGGCTCCCACTGCCACGTGGC TGCCTGGCGCATGCTCCACCCCGTGTCCTCCCAGGGTCCCAGTGAGGGCCCAGCCCCGCA GTGCGCAGTCTCCGCAGCCATCGAGTAGGCGGCCAGCTGAGCAGAGGTGCTGGCCGGCTG CCAGCCCAGCACCCGTCCTGCTGAAGTTGCTCCTGGCACGCTTCCGGCCCCAGGCTTCCC TGGGTGGTGGGGCAGACTTTGTGTTGCTGGTGTGCAGGAAGGGCCCACGACCCCCACCCA ACATGTGAGAGGGGCATTTGGCAGCCATGGGCAGGACACCACCTTCGACCATCAGCTAGG GGTATGTGCAAAGGGCCTGAGTGAGAAACAAAAGACAAAATGGACCAAATGTGCTTGTGT GGCCGGGTGTGTGAATTGTGTGCAGGTGAGTGTGGAAAGATGTGTGAGCACAAGCCTGTC AGCATACACGTGTGGGCGTGGGCATGTGGGTGAGCATGCAAGTGTGTATACATAAGTGAG TGAGCACGTGTGTGTGGACGTGCATGTTATGTGGATGTGCATGTGTGCATATGAGTGTGT GAGTGCATGTTTGATGAGCATGTATGTGTGGGCACACGTGTGTGTGTCAGCGTGTGCTGA CTGCCATGGACGTGCACCTGGCTGAGTGTGGAAATGTGTGTGTGAGTGTACTGGCAGTCC AGGTTTTACACCCACACGGCTTTGCCTCCCCAGGAATCCGTTTTAATAGCAGAGTGGGCT GGAAGGGCTGGCCAGGCCCTGAGGCAGTTTTCAGGCCAGCAGTCCCTGGCTCCTCATATG CGGTGACAGCCCAGGAGGCCGGGGCCCTCAGAGTGCAGTCTTTGGACATGCCTGGTTGAT GGATCCGTCATTACCAGCCTGGGAGCTGGGCATGGGCTCCGGGGCTGGAGGACACCCCTT TCCATCCCTGGCCCTGGCGAGCCCGGCCCTCCTCTCGCCCCGTGATAACCTGCAGCCTCA TGGGGAAAGGCACGCGGAGGTGGTGCCGCACCCCCAGCCCTCACCCCTGCTTTGCCAATC CTGGATCCTGGCCTCCGCCCGCCAAAGTCCACCATTGGCCACAGGATGTGGGTGCTGTGC TGTGTCCTTGAAAATCTGAAGATGAGGACAGCTCAGTGGGCGGGGGCGTTTCACACATTC GACACATGCCCTCACGCCTGTGGGTGTGTGGCGGGCTGGTTTACAGTTACGTGAGGATTT CAGACCCAAAGTGCAGAGCCTGCAGCTGAGCAGTGCCCAGGGAGGGCTGGGGAGGAAGGT CCCAGCCAGGAAAGTAGGGTGACTCTCATCAGTGGCTGCGGTGTCCAGCTGGGATGATCA GGGCCGTGCCAGCCTGGTGCGCCTGGGGGACCCACAGCAGAGGTCCTGCATGCACTTGGC TGGCACGTGTAGTTCTGAAGGCCCCTGGGGAGGCCTGGCACATGCAGTGTTTGGGGACCC TCTCTGCTGCCTAGACACTGCCACAGACCCCACACCCAACCTTGCTGTTTTTCCCCCAAG TGAGGGGAGCTGCTTTTTCCAACTCGGATAGCCCCCCTGCACTGTGCAGCTCGCCCACCT CCCTCACAGCTATTCCAGCATCTCTGGGCGGGTGCGGGAAGCCGGATGCAGCCAGTACCG TTGCTGTTTTTGGAAAAGAGAGGTTCAAGGCAGGGAGGGCCCAGCCTATGTTCCAGCAGG GTTGTCTGGCTGGGGGGGTCTGCACCTCGCCCCTGCACCCCCAGGTTCCTGGCTCCCCAC CCCCGTGCACGAAGCCCTTCTTGCCTGGCAGGCCTGGGGGCCCCCGGCCGCCATGCGCCC CACCGCGGGTGTGATTTGGCAGCAGCGTTCTCCTCGCCAGCTTGTTTCTCACATAGGGGA TGTTTCCATTTTTACAGCAGAAAGAAACCGATTCCAAGCGCCTCAGCCACAGCCGGAGCG CGACGGCCCCCAGGGAGCTGCTGCCACGTCAGGTGCTTTGGGGGTGTGGGCTGCTCTGCC AGCCGTGGTCCTAATTGCTGTCACTCAGGGAGCCAGCTCTAGCCTGGGGCAGGCAGGAAC CATGAGGGGGGTCTTCGGGGCAGGCACTGGTCCCTGGCGGAATCCGAACCTCTGCCTGGT TCTCCTGGCACCCCCTCCCCAGAGACCTGGAGCACCTCCCGACTTTGGGTGGGATGGGCC TGTGGTTCTGGAACAGGAGCTTTGCCTGTGGGACCCTCCCATGGGGGCGGGGTGTCCTCT CTTAGCCCAGGTGGGTTTGAGCCCCACACTGCTCCTTCCACCGCTCAGATTCCCGCCTGC AGGTCCCTGTCACGGGGCTGCTGAGGACCGACCAAGGTGACTGCCCATAGCCCCACGCCA GCATGGGGCACAGCTGCCTGGCGGCTCTGCCGTCACCCCCCGTGCTCTGACTCTGTGCTG TGGATTTGTGTCACCTGCAGACTTGGGTGGCATTGGGCACAGGGGCACGGTGTCCTCTGT CACTTCCCCACCTTGCTTGGGGCTGGTCTCCCAGGATGCTGAAGCATTGAAGTGCCTCGA GGACTGTTAGAAAGATTTTCTTAGAAAGAAACATTTGCTACCCAGGGCACAGAACAGCTC CTCGAGGTCTCGTAAACCAGTGTGGACTCGGCCCTGCTCCCGAGACTTGGAGGAAAGGCA GAAAGGGCGGCAAGTCACACGCAAGGCAATTTTCAAAGAAAAGGGCACAACTCATAGGAG GCAGGGGAAGGATTCCATTTCACCTACAGTCATGGCCCCGCGTCTTCAGGGAATAGGTGC TGTCTGGTGCATCACATTATTAGTGCTGGAGGGCATCGCAGTCCTGGGGGAGGTCACTGC CAGCATGGCAGCCCATAGGAAGTGGGAGGGTGGGATTCAAGGGCAAGGCGGGTGGTGGGT CCTGGCCCCTGATGAGGCAGGTGGATGGTGGACCTGCCTCCACCCTACCCAGCAGGCTGG CCACCAGGGCGTGTCCACAGACTCCAGACAAGAAGGAGAGAGTAAATTGAGGCATGTGGG GCCCAGGCCCTGGGCGCAAGGCTGGAGGCCTGAAGTGGCATCCCATCAGCTGGAATCAGG ATTGCCTCGATCTTTGATTCTTGAGACAGGAGGAGTGTGGCGGTGGCCCCAGCAGGATCC CTGAGATCTTACACAGGTGAGGTGGTTGCAGAAGCACTACACGGGGCTGGGGCCTGAGGG TGCAGTGCTGGGTCTAGGTCTCCCTGAGAGTCCACTGACAGCTGTGGAACCAGGCCTCAG GGGAGCTGGGTGCAAATCCCACCCCTCCACAGAGCAGCCGTGTGACCCGAGGCTGTTTTG CCCTCTCTGCCTCAGTTTCTCTCTGGCAGCCGCTCCTCCCTGGGCCCCGACCTGACCCTT CAGGCTCAGGAGGCTGCTCCAGGTCCAGAACCTGGTGCTGCACTGGGTCCAGCCTCCAGG CCTTTGCTAATGTTGTGGTCTCTGCCTGTGATAACTCCTGCGAGAGCTCCTGATCTCTTA TGACCCTCCCCTGCCTGGGCAGATGCTCCTCCTGTCACCGAGGGTCCCATTAGCTGGCAG CTACCCTGCCACTTGGTGGACCCAGGTGATGTGGGTGGGCCTCACTCTTCATCCACAAGT GGGTGAGAAAAGGCAGGAAAGGAGGTGAAGCCAAAGATGGCATGCTCCCCCTGCACAGGG TGACTGTATGGGGGGGTCACGGCGGGGGAGGGAGGGGAGGAGAGTGGTCCATCTAAGGGC CCTGCCAGGGCCCAATGCAATGTCCCATCCTTAGATCCATCTTCAGTCACATCCCCACGC TAGGGGGAGCAGCATTTCCAAAACGCCTGCCCTGGGGGCTCTCAGAATTCAGGGGTTCCC TTTGAGGTCACACAGGCCCCACTGAGGCCAGACACAGCAAGTCCTTGCTCCTTGGCCTAA GCCTGCAACCCCTGAGCCTCAGTTTCCTGCAGTAAGTGGGACATGATGCCTTTCCACTCC TCTAGGCTTCTGCCCATCCTGTGTCACCGTCCCAGCAGATGAGTAGCTGGTCTGGTGGCC TGAGACCTTGCTTCACTGGGGGACAGGGCTGCCATCCTCGAAGGCCAGGGTCTGGGCCTC TGGGCCGGGAGCTCCTCCATCAGCTTGCAGCCCTGGGGGAGGCTTGCAGCCCTGGAGGCC ACTCCCTCTAGTGACAGAACCCATACCTCTGGGAACTGCTTCCTCTGCCTGCATGCCCCA AACTTGGACTTGGAGGTTGTAGATGGCCGTGGCAGGGCCCTTGCCTGGCCCCTATCCTCC TGCAGCCTGGCCTAGGCATGGGTGACTTACCCAGTGCCAGCAGGCTGCCTCAGCCCGTGC CCACTAAAAAATTTGTTCCTGGCCGGGCGCAGTGGCTCACGCCTGTGATCCCAGCACTTT GGGAGGCTGAGGCAGGTGGATCGTCTGAGGTCAGGTGTTTGAGACCAGCCTAACCAACAT GGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGATGGCAGGCGCCTG TAATCCCAGCTACTCAGGAAGCTTAGGCAAGAGAATCACTTGAACCTGGGAGGCGGAGGT TGCAGTGAGCGGAGATTGCGCCGCTGCACTCCATCCTGGACAATAGAGCAAGACTCCGTC TCAAAAAAAAAAAAATTGTTCCCATGATATTCCAAATACAGTCATATACCATAATGACAT TTCAGTCAACCATGGGCCGTGTATATAGCGGTGGTTCCATAAGATGAGAATACTGTATTT TTACTATACCTTTTCTATGTTTAGATACACAATACCACTGTGTTACGATCCCCTACAGTG TCCAATAGAGTAACACGCTGCACAGGTGTGTAGCCTAGGAGCAAAAGGCCAAACCAAATA GCCCAGGTGTGTAGTGGGCTCTGCCATCTATGCTTGCTTAAGTTACTCTATGATGTTTGC ACAATGACGAAATCACCAAACAATGCATTTCTCAGAACATAGCCCCATCATGTAATGGCA TGAGAGGACACGGGCAGAATTCTTGGATGCCCATTTTGGGGACAGCCACCATAGTTTGGA CATTAATACATTTGGGCACATGCACATGAGCACACACATGCTCACATGCACATGGATGCA CACGTATGCGCGTACCACTCACTCCCGGGCACATGCTCATTCCCGGGCACATACACAGTG GGGCGGGGCAGGAAGGCTGGATGGGGCCCAGGGGAGTCAGGTGCTGTGGTGCGCTGGGGC TCAGGCCCTTCCAGCATTCAAACAGAGAGTCTCCTAAGAAAGCAGGGAGCCCACAGGGCA TGGGATTTGGAATCTGAGTCAGGGATTTGAATCCCATCTTGTGCCCTGGGCATGCTAAGT CTCCCTCCTAGTGCAGTGGGTGCCTGCTGCCCAGGCTGCAGGAGCGTTTGAGGAGGCTTG TCAGCCTATAGGGAACTGCCTGGCGTCCTGCTCAGTGCTGCAGGCTGCATGAGGGGCCAA TTGAGGACCCCCTAAGCCCAGGAGGGGCAGAGGCCAGGGACCAGATGCCACAAGTATCTG TGGGTGGGCTTGGCTGTGATGAGCAGGACAGGGCTCAGCCAATGCCCAGGGGCAGAGCCC CCGAGGCACTGATGACCACAGTGAGGCCAGCGTCAGGCTCTGGGTCTTGGGTCTTGGTGC TGCTCCAGGAGTGGACCCACATGGCCCCTCAGATGCTGCTCTGTGACTCCAGGCCTGGGT CAAGCCGACCTCTCTGCTTGTCCCCCCACCTCCCTCTAGGTAACTAGGCCCCAAGATGTC ACAGCATCAGAAATACAGAGAAAAAAAAGCATAGTTAATACTGCTCCTGCTGGGAAGGCC CACAGCAGGGCGGATGGTAGTGCACCCAGCAGGTGTGACAGCCCAGTCTCCCCATCTGCA GGGAAACCCCATGCCCCCTCAACCCTTACTGCCTGTCACCAGCTGGACCCCGGTCTCTCC TGGCTGCTGTCTCACCTGCCTTCACATGGTTTCTGGTTCTTTCATCTTTTCATTATAATT TTTGTTTGGAGAAACTTTAAAAGTAAAAGAATGTGGGCCAAAAATATGTTAACCCATTCC CACCAGACATGCAACACTGGGGGGGCTGTTCCTCCCAGGCTCCTTGGGCCCCCCAGCTGC CCATGACCCCTGGGTGTGCCCAGACAGCAGGGGGGGTGTAGTCCACCCCCACCACGTCCA CAGCCCCTGACTCTCGGGTGGGTCTGGGGGCTGCTCGCCCTGGGAGAGAAGCCATCCCCT CCCGGCGGGGCCATCTATCAGGACCTCTCTGACATCAGCATTTTCCTCTGTGGCTCTAGC TTCCTGCCTCCTATCTCTGTCCTCCTGAGAATGTCCCATCTGTCCCATCTTTCCCATTAG CAGCCCTGTCCTTGAGCCCAGTGCAAGCTCCACAGTGGAGAGGCCACTCAGCATTGTAAG TGAATGCTTGTCCTCGTGCCTCCCTCCCCGGGAGCTCTGCCTGGAGCTCCTTCCCGGCTC CGGTGAAGCTGCAGGCAGCATTTCATTGCTTTGACCTGTCCCAGGGCACCTTCCTTCTAG AAGGGCGACTTGAAACCTTTTCATCACAGCACGACATATGCCCGGTCTTGCTTTGGTCAT CCAGTTTTATGTGTCCCATTTTATACTCATTTTCTTCCCCTCATGTTTTGTTTTTTTCTT TTAACTAAAACGATCCATTTATTGAGTTAAAAGATTGTATATTCAAGACGTTCACTCAGT ATGTGATCCTGCATAGTAAACCTCGGGTGAAACAAAGACAACTATTTTTATATATTTTTA AAAATTTAGTGGCATATAGACTTCTAATATTTTGGGGGGGGGGATATTCTTTTTTTCTTT AATTATGGTAAAATATCCATAATATAAAACTGACCACTCGAATCATTTCTAAGTGAGCAG TTCAGTGGCATTTGGCATACTTACATTGTTGTGCAGCCATCACCCCCATCCATCCACAGA ACTTTTTCATCTTCCCAAACTGAAACTCTGTATCCATTAAATAACAAATCCCCACCCTTC CCCGCCCCTGGCAACCACTGTCTACTTTCTGTCTCCATGGATTTGACTACAGTGTGTCTA AGTGGGATCATTCAGCATTTGTCCTTTTGTGACTGGCCTATTTCACCCCTCTGTTTTATA CCTTTCTGCTGCGTGGCGCTTTGGCTTTCTATGTTCTGCGGCGGTGGGTGGTAAAGTGGA TGTCCTGCTTTTAATTCTGAGGGCTCGCTTCTCAGTTTTCAAAAACACACTTTAATCTCT GATTCTCCAATTATCAAGCATTTGCCATGAAATGATACTTGCTGCTCCCCAGCACTGCAG ACTGTGTAAGAGACATCTCAGCCAGCATTTGGATTAAGTCAAACCCCAGGAATCCGCACC TCCTCCACCTCATCACCCAATAAAAAAGGTAACGCTGAGGTATTAGAACATTCACATTAG TTCCATGAGCATGCCCTCTTATGGGCCTGAGAGCACCCAGGCTCTGGTGCAAGCCCCCCA CAGCCCAGGGAAGTGTTTCAGGACTGGAGCCCTGCTCAGTGCTGTGGGCTGCACAAGGGG TCAACTGAGGACCCCTAAGCCCAGGAGTGGCAGAGGCTGGGGACCATCCCATGGGCTCCC AGGATGGCCTTGGGGCCACAGGGATTATGGCCAGGCTTGTACTTCCAAAGCCCTTCCAGG GAGGAAGGCGCTCCACTTCCTGCTTTGGCTTAAAAAAGTCCAGGGAAGCACCTGATTGGC CTGGCTCAGGTCACATGACACTCCCTCTGTCCAATCACTGTGGCCAAGAGAATGCGCTAT TGTGATTGGCCCAGCCTGGGTCAGAGCCTTCCCCTGCTGCCTGGAGCAGGGTCTGTCTCC AGGAGAGAGGAGTAGGGTTCAGCCATTCCAATGGCGCTGCCGCCTGAAGAGCTGTCAGTC CTACTTTGCAGTTGAGCTGGCTCTGAGAAGAAAGTGCTCTGCCTAGCCCTGTGATTGATC CACAACTTCATGGGGACTCAAACATGGGTTCTTTTATTCCAGGTGGAGAAATTGAGGCCC CAAGCAGGGGCAGACTCACTGGGGTCACATGGTGAGTTGGTGGCATGAATGGGTGCTCCG GTCTACACTGTACCACCTCCTCCAGGAAGCCTTCCTGGCTTGGTCAAGCCAGGCCTGGGG TGGTCACCACCTGATGTCACATCCCCTTGAGACAAAGCCCCCCCCCTCCCCCGCCACCAG GCACCTATCCCCAGTGTCCAGGACATAGCTACTGGATTCAGGCAGCTTCTGCAGATGAGC TCCCAGCCCTATGTCCAGGAGGTTTCTGGAATCGACAGCTCTCCATGCCACAATACCCTC CATGGCTATTTAGGGAACACCCAGTGTGTGCCAGGTTCGGCTGGCCTGGGAGAGTGCACA GTGGACACTGGTCACCAGGAGACACAGGGGGCCAGTCCACACCCTTCCGCAGTCGGGCCT GGGGAATGGGTTGGGTACTGACCTGGAGGAGGGAGGGACAGCAGGAAGGGCTGGCTGGGC CTCAGCTCTCTTGCTTACCTTCCATTCTCTCGATGGCCCTGGCTAGACACATGCTGGCTC GCCCAGGCCCTACTCATGTTTCGGCACAACGACCCTGCTCTGCCTCCGTCCCCAAGACCC TGGGCTGTGCAACCAGGGCCAGACCTGTTCTTCAGCCGCACCAGGGCCTGGCTGTGCCTC TGCCTGTCAGTGGCCAAGCCCTGAGCCTGGAGCCATCCAGAGCCGCACGGAACCCACAGC CTGCAGGGTGGGGCTAGACATGGCTCCTCCTGCCCGCCCGTGGGTCGTACACCAGCTCAT TAGAGCCAAAGCAGCATCGATTTTGTGAATGATGAGCATGTGTACGTGTTTTTGGAGCCC TGGTGTGATTTGGCGTGTGCAGCTGATGATTCAGGGGGGTCATGTGCAAACGACACGCCT TATAAATGTGAACCCACCAGAACGCCCCGTTACGGAGATTTACACTGATCCTTAAACATC GTAGATTAATTTTTGGAAAACATCTATAAAATAAGAGATTTATACCTAAAACAATACCCT ATGCTGTGACAGGAAGGGATTAGAGTCAATTTATTCCTGTATTAGTCAAAAACTCAATTT CATATATTGTGTGGAAAAATTAAAACCCTTTCTACATTTTATTACAAATTATGCATACTC CATATATGGAGATAAATAATTGATAGCATTACAAATACTTAATCTAGAAAATATTTATTA GGAACAGACAACTCATTTTTCATGTGTTTTGTTGAGAACAATATTGCACACTTTAAAAAA TTAATGATGACTCTTTGCAAAAATAAATGTCTACATCCTAATTCTTTTGTTTAGCATTGT GTCCAAGCCAGGCAGACGCAGCCGGGCAGTGGGCAGGTGGCCATGGAGCTGGGATGTCCA CTCCCTGGCCCCTGGCTGTGGATGGGTCAGAGGGCTCATGTGCTGTGGCAGTTGGCCAGG GAAGGTGCATTTGGAGAGTGTCAACATACTGTTTGTCTTCTGCTGTGCAAACTCTCCTCG ACTTGTAATTTAAAACTAAAAGTGTTTCCGACTTAATTTCTGAGAAAGCACCAAGTAATG GGATTTGTGGATGGACTTGAAGAGCCAAGAAATCAACACTGACTGCCTCTCGACCTGGAG AATTACGGAGCCCATGAATTCTCTGGGCCTCAGCTCTGGTCCCAGGCTCTGTGCACAAAG AGAGGTGGGCCTGGTGGGTGGTGCGGAGCTTGGCCTTGTACTTCGTGGCTGGCTCAGCCT GCCCTCCTGGGACAAGTGGCTCTGCCGGGTTCTGTGTGCCTCCTGGCATCTCTCTGAGGG GAGCTCCCAGCCTCTGCCTAGAATACCTGGGCAGCGTGTTCAGACCTGGGGCCCCCTGCA CCATCACCCCATTCCAGGCTGGGAAGCTTGAGGACCCAGGCAGGGAAGCTTGAGGACCCA GGCAGGGCCCCAGTGTGCCACAGGGCTTGGCCACTGTGCAACTGCACTGCAGCTCTGGCC TCTGGCCGGGCCTTGCTTACCGACCTCTGCCCTCTGCCGTGTGTCCTCCACCTTGGCCAT GCACAGCGGGGGCTAGGCGGGCACATGGTGGAGGCTGCCAATGGTGCCCTGGCAAGTTCT CTTCGCCCTCGTGCCAGCCACTCCAGGGAGCACCAGGGATTGAATAAATAACTGCCACGT TTATTAAAAAATAAATTTGGTGCCTAACGGAAGTGCTGTTTCTATACATAGAAAAAAATG TTTAAAATAAAAACCAAAACAGTTCTCAAAGGCGCTGTGGCATTAATTATTCATCGCTTG CTAATGAAAACATGGCTTTGCAGGCGTGGCCCCCACCTCCCTCCAAATAAGACTCGGGCT CAGGGAAAGCCTGGCCTTAAGGGCAGGGAGCTGGTGACCCCTGAATAAAATGGGGGTGGC TGTGATATCCTCCATGGGTATTTAGGGAGCACCCAGTGTGTGCCAGGCTCGGCTGGCCTG GGGGAGTGCACAGTGGGCACTGGTCACCAGGCAGGCACAGGGGGCAGTCTGCACCCCTCC TTAGTCAGCCTGGGGAATGGCTTGGGGTCTGACCTGGAGGAGGGAGGGAGCAGCAGGAGG GGCTGGCTGGGCAGCAGAGCCACCTCTTGGAGGAAAGAAATGGGGACAGAGGCCACTCTG GCCAGCACCAGAGGACAGTGGCTACATGTCTGGGCACCCAGGCCAGGGGCCTGGCAAACC CCCACAGGGTGGGTGGGTGAGGCCCTGTTCTAAGTCATTTGATGACCCAGCCACCCTCTC AGATGCTGCTGGATCCCAGACAGAGGGAGAGAGGGAGGCTGCAGGCCTTGCCGGAACCTA GGCGTGCCATGCATGCCCCAGCAGCCAGAACCAGCCATGTGGTGGTGCCACACCAGAAAG GGCCGGCCTGCCAGGCAGTCAGGGGCAGATGGGAGCAACTTGGGAAACAGCAGCAGGAGC CTCCACTCCCTCTGCCTCCACCTCTCCAGCAGACTATATGGCACACCCTGCCCAGCTGTG TGCAGCAGGGAAATTTGTGCCAGGCCTCGGGCACCTGAGCCCACACTGGGCATCGCTGTC CTGTCACTGATCCTGGGCACTGATAGGAACTGTGAACAAAGCTCGGTCCAGGGCCCTGTG GACTGAGCTCCCCATCCTCAGGGCCCGGGAGCCCCTCCTCCTGCAGTCACCTGGGGCAGC CCTATCTCTGTGGGGAGGCCTGGAGCATTTGTTCTTTTGTTCATTCATTCGTTTATTTAT CCATAAATGCCCCCGAGCTGGCTGCCTGGTCTCCTGGGGAGAAGGGATGCTGATCCCATC AGTCACCTGCCTGAGCAGGGGGTCTGGGAGGCTGAGAGGAAGCCTCTCTGAGCTGCTGTG GGTGCAGGTGGTATCCAGGGCGACAGCTCCTGGGCAGGAGATGAGCTGTGCCCTGAGGTG GTCAAGTGGAACCCCAGCAGGGGCCTTTGAAGCTCCGCCAGCCCCTGGGGCAGGTCAGGG ATGGGGAAGAGGCTGCAGGGGGCGGCAGGAGAGTGAGGCTCCATCTCTGCTGGGTGGCTC CGTCCTGGTTAGCAAGGTCTCTCGCGTCCTGGCCCCTGTGCTGGCTCCTGGGACGTGGAG TGGAACCCGCATCCCTGCTGCCCATGTGTGAGGACCCGGGCTGCCAGGGCTCCAGCTGGG CCCCAGGCTCAGGGTTTGTGTGCTGGAGACCCAGCAAAGCTGGACAGAGCCTGGCGGGAC CTGGCTCTGCGATGGCCCAGCTTGGTGTACTCAGAAGGCCTGATGGGAGCTAGCCTGGAC ACCTTGGCCTGATTTAAGCCTGGGAGCCGCGTAAGGGGAGGCAGTTGGGCCTGCCGCCGA ACCGACGCCCCGCTGCCCCCACTTGCCTCTCCCGCCGGTGGGCCTGACCTTGCTGGAAGC ATCCCTCAGCCTGGTATGGATTTCCTGTCTTCACCGAGGCCGCAGGGCCAGCCGGGCGCA GGAGCCGAGCGGTGGTCAGGCCCGGGGATGGTTTTTTGTTTGTGGGGGCAGGGGCTGAGG GGACCCAAACCTTCCAGAGCTGGGGGGTTTTCTTCTCACCTGTCACTCCGTGGCTCCCTT TGGTGGCTCTTTGTCTGGCATGGGTGCTGGGCCCACCCTGCGGTGTGGGCAGCTGTCCAG CCTAGAGCCTGCCTCTGACCCTGGTCCTATCCTCAGGGCCCCTTCCAGGGCTGGAGCAAC AGGGACACAATTGCTGAGCTCAGCCTGTGTCAGGTGCACAAGTGACCTCACTTGGGAGAC AGTGCAACTGCAGTGGGCCTAGAGAGGGCTCTTCAGCTCCCAGTCACACAGCCAGGCCTG GAGAGGTACACATCCTGGTCCCTGATTCCCCCATCGGCACTGGGTGGACTGAGGAGTGGA GTGGGGACCCTGGGTGGGGGAGAGGAAGGAACTGGGCACAGGGGGAGAGCCAGGGACCAG AGGGCATCATGGAGATGAAAGGACAGGCGAGCCCAGGAGGGCACACACCATGGCACGGCA GGCAGACAGACATAGACATAATGCCCATATGGCAGCATGATCTCTCACACACACACATGC TCACATGGGCAGCACACTCACACACACGTGCATATGGACGGCACGCTCACATACACACGC ACACATGGGCAGCACGCTCACACACACACGCACACATAGGCAGCACACACACACGCACAC ATGGACAGCAGGCTCTCACACACGCACACATGGGCAGCATGCTCACACACACACATGCAC ACATGGGCAGCATGCTCTCAGATACATGCACACATGAACATGGGCAGCACACACGTACAC ATAAGCAGCATGCTCACACATGCACACATGGGCAGCACGCTCACACACTTGCACACATGG GCAGCACACACACACGCGAGCAACACGCTCACACACACAACATGGGCAGCACACACATGT ACACATAAGCAGCACACACAATGCACACATGGGCAGCACACACATGCACACATGGGCAGC ACACACATGCACACATGGACAGCACACTCACACGTGTACACATGGGCAGCACGCTCACAC ACATGCACACATGGGCAGCATGCTCACACATGCACAGATGGGCAGCACACACACACATGC ACACATGGACATCACACACGGACATCACACACATGCACACGTGGACAGCATGCTCATACA TGCACAGATGAGCGGCACGCTCACACACTATGAGCAACAAGCTCACACACATGCACACAT GAGCAACAGACACGCACACAGGCACAACATGGGCAGCACACACACATGCATATGAGCAGC ATGCGCACACATATGCACACATGAGCAGCACGCTCACACATGTAAACAGGCAGCACACAC ACAAGCACACAAGAGCAACACACACACATGCACAACATGGGCAGCACACACATGCACACA AGCAGCATGCTCACACATATGAACACATGGGCAGCACGCTGACACACACATGCACACGTG AGCAGCATGCTCACACACGCACACGTGAGCAGCATGCTCACACATATGCACACATGGGCA GCATGCTCACACACACGCACATAGGAGCAGCACGTTCATACACACATGCATACATGAGCA GCACACTCACATGCACATGTGTGCACATGGACAGCACACAGATACACATGCACATGTGGG CCACACATACACACACATGCACACATTTATGCACATGGGAAGTATACTCACACACATGCA TGCACACATGGGCAGCACATTCACATGTGTACATATGTGCATACCTAGGCAGTATGCTCA CACACACACACACTCATGTGGGCAGCACACACACGCACACCTGGACAGCATATTTACACA TGCATGTGCAGCACACATACACGAGGCAGCATGCTCACAGACACACGTGCACACACACAA GCACACTCAGAACAAGCTGTGGGGCCTGCGGTGAGGACACCAGGCAGGCAAGGCGGCTGC TGCAGAAATAATTAGTCACTGGTAGACAGGCCTGGCTCACCTTCTCCAAAGCCAAAGGGG GGCTACCAGCCCCCAGCTCTGGGAAAGGGCTCTGAGGGCCCGCCCCCACCTTGCAGTGGT TCCTGGGACCTGCAGGGAGCATCCCAAGCACGGCCCGTGGGGTGCGGGACTCTGCCATGT GATCCCTGAGGCTGGGCCTGGCCGGACGTGCCTTGGCCCCTGCAAACCGTGCACATCCCC AGGTCCCCCAGCACAGCAGCAGTGCAGGGAGTGGAGACGTGAAGATGGGGAGTGGAGACG TGAAGATGGGGAAGAAGGAGGTTGGAGGTGCAGGGCCGCCCTGTGTTCTTGTTGGAGTGG GGGTAGGAGCACTCCAGGTCTGGTTGGGCCCTGTGAGGGTGTTGGGGACCCCAGGCCAGG CCTGGGTCGGATCCATCACTCCCTGGAGGAGGCATCTGAGTCCAGGGATGCCGGACAGGC TGGTCCCAGGTCCCCAGGCTGACTGGGCCTCAGGCATATGGGCATCTGCAGCTGTCTGGG GCCAGGCCCAGGCTGAGGGGCAGCCAGCAGGGTGCAGGAACTCCCAGGCCCTCAGCTTGC AGCGCAGAGAAGGGTGACTGCTCTGCTGTTAAAACAATTTATTTTGCTCTTCAAAAAGCT AACAGCTGTTTACAGTCAGCATGTGGGAGCTGCCCCCTGGGTGATGGATTCTGCCAATCC AGCCCTCCCTTGCCTTCCGGCGAGGAGGGGTCATCCCAGGCTTCGGGTCAGGATGGGCAG AGCAGACCACTGGGGGCCAGGCCATGATGTTCCTGGCCACTCCACGTCCCCTCGGCTGCC GTCCTGTGTGCTGCCCTGGGAGAGTGCAAGGAAGGGTGGCCCCCTGAGGCTGATACGAAA TGGTACGGTGATGTGAGGGGGTCAGGAGGGAGGACCACTGTGGGAGGCGTGAGGTGCAGT GGAAAGGTCAGGGAGACCTTCTGAGAAGGCCAGCAGGGAGGGCTGCCCAGGGCCTGGAGA GCGTGCATAGCTGGGGGCAGGGTGGGGGCAGCCGGCAAGGGCAAAGCCCAGTGGGTGTGG CGATGCCCTGGGGGATGGGCCCCGATGCCCCTAAACTGGTTCGGCTCTCTAGACCAGACT AGTGTGCTATGTCGGAGCCGAGCAACAGTCCCCCTCAACTGTATGCATGTACAGTAGGCG CTCAACAGACACTTGCTGGATTTGACAATAGCCCTGGACGGAGGACTGAGGCCTGGGCTG CACACATTCTGGGCTCCTGCAAGCCCCTGGCAGCCCTGAGCTGGTTTCTGAGTGCAGAGG GAGGGGCTGTCAAGGGTGGAACCCACACATGCCAAAGCCTGGAGGCCAGTGCAGGGGCAG GGCACCTGCTGCCTCGGGACAGCGCTGAGGTTCAAAGCCACCTGCTGGTTCCCACCTGCC TGGCACAGGACCCTGTGTCGGTGGGGGTGGCCAGATAGGGCAGCTCCTTGTCTCCCTGGA ACCTGTACCCTCAGAGGTCCAGGATCCCTGGGGGAGGTTCCGGGGCTGGAGAGACGGCCC CTGTGGCAGGGCAGGGACAGACGGGCTGATTCTATCTAGGATGGGGGCACACAAAGACTT CCTGAGGGGCGCTTCAAGGCCACAGCAATCTAGGTGGGACAGGGACATCAGTCAGAGGTG CCCTGTGGAGACCCCCAGGTCCAGCTCCTGGTACTGTGAGAAGCCAGTTGTTCCCTCGCA GGCCGCCAAGGCACCGCACAGTCCTGGCCTGAGTCGTTGTGGGGTCTTGGGGAGGAGGGT TCTCTGGAAGGACACTGAGCCTGGACTTTGGGGCAAGAGACCCAGAGCCCCTGGATGCTT GCGGCCTCAAGGCCCAACATGCAGCCAAGCAAAAGCTGGGGGCACAGGCAGGGGATCCCC TCTGGGGAGGATGCAGAAGCCGAGCCAGGCTGGGCCGAAGCTCCTTACCTGATCTGGTGT CCTCCTGTGCGATGGTACAAGAGCCTCAGGGCACATCTGGTCTCTGTGACACCCCCCGAG ACACCCGGGGAATGTTCCCAATGACCACTGTTGGGCCACTGCCCGACCACGTCACGGTTG CCCCTGCCAGGACTCAGGCAAAACCCAGCTGTCCCTGGCGCTGTCTCCCAACAGCCCTGC AGGGCCCAGTGCTGGACCAGGGCCTTGTCTCCAGCCTCTCCCCCATGGGACTCTGGGGCC TTTGGGGGCCCTTGGGGGCCTTCCTGCACCCTCACACCTACTCATCTCTGGACTCAGCCT CAGTGTGAATGCTCACCTCGGGCTGAGCCAACCCCTCCAGACACGTTCTGCAGCTCGAGG AAGGAACTGCCCTTTGCAGCTGGGATCTGCCCGAGTTTCAGCAGCAGCCTTGAATTGTAA TCCCGAGCGGCTGGCGAATAACACAGCACACAGCACTCGAGAGCCCAGGTGAGTCCTAAG TGGGGCTGGGCTATGGGATCTCTGGCTTCTGGGCCTTTCTCAGAACAGACCCTCCAGGCC ACTCTCCCCTACATCCCCGGTTGCTGCACTGGGTGCAGGGGTCCAGAGCCAGGTCAGCTG CTGCCCAGGGAGAGGGCCAAGACTGCAGACCCCGCCAAGCCTGGGGGCAGCCTTCCTGAC AACAGCACCATTTGCTGCTTGGGATCAGCCAGGCCGGCTCCCCGCAGATGGGAGGGCTGT GAGGTGAGCACCAGCGAGCCCCTGGAGGTGCCAGAAAATGCCAGTGTGGGCACCAGGGGG CTTCCTTGGGCTGGCATGTCAGGGGAAGCCCACTGGGGCGGCTCAGTGCCCATGATGGAA GCTCAAGTCCCACGGTGCTGCCCCAGGAGCTATACCCCAGTGAGGGTCGTTGGCTGGCGA TGTGTCTGGCTGGCATGGGCTGCCTGGTAACGAGGGGGGCCCATGTGTGAGGAGCCTTAG GTAAGGATCCAGCTGGCACACCGAAGACCCCAGGCCAGGGGACACAGGCTGGGGGAGGGG GCTACAGAGACATGAACCCAAAAGAGAATGTCTGCTGACATCCCAACCCTGACAGATAAC CAGCTGGGTGACAGGTGGGGGCTGTGCTCAGATCACCCTCCTCTGTCCTGTGCACATGTG TGGGTGCATGGGAGTCCATGCGTTGCTCCTGGCTTGCCCCCTCCTCTCCTCGGCCCAGCT TGTGGTGCTGCGCTGTGCATGGAGCTCGGTTTCTTAGCAAGTTCATGCCATGAGCCGCCA GCTAGGCTGAGGAGGAGTGGGTCTGGGTGCCACTGGGGCGGTCTGGGCTTCCCTCAGCTC CAGGGCTCTTGCCCTTGCTAGCATTGCTGTGATTCTCACCATTGCGCCCTGTTACAGAAG AGACAACTGAGGCATGGGGAGGTGATGGGACCCCCAGGGCCACCCGCAGGAGGTGGCCAG CCCAGCAGCCCTGCCCAGTCCCCCGCCGGCCTTGTGCTCCCATCTGGGCCCCTATCTGTA GTTTCCCCAGTTAACAAAATCACAACAGTCATTCCTGGTTGTTCTTGCTGAATTCTCTCC ATTTGAGGAGCAGCATTTTGGGGTCTGTGTCCTAAATGGACCCAATCACAGGAGTCACCC AGGGCACTGGCTCAGCCCAAGACAACACCTTCCCCCATGTGGACACACACACATGGGGGA AAGAAAACCTTGCCAACCCGCCCTCTAGCAAAGGGGCCTGGACTCGCATTTTTAACAAGA ACCAAGAGATCCCGTGGAGGAGGTAGTCCACAGGGGGAAAAACACCCTTGAGTCTCAAGT CCCCCGTGGCTCCCCAGCTCTGCCAGTGGCTGCATGGCGGGTGAGATGATGGTGGCTCTT GGGTCCCTCTGAGTCTTGCTGCCCAGGCTGGCACCGGCACATTTTGTCTTGGCTCCATGC TGTCGGGCCCTGAGCTGGGCACTGGGCTCCCTGAGTCTCGTGGGAGCTGTCTGGGTCCTG AAGCAAGTGGTGGCCCGAGCAGGGGAGGCGGTCCCCACCCTCGGGGACACTGTCTGCTCC GGCTGTGGGCTCCGGGGGCTGAGAGCTATTTCGGCTGCACTGGCCTCTCCACTCCTGCCA GGCTTCAGTGAGCTAAGATTGTGCCAAAGTCAACAGAACAGAAAAGAGAGGCTATGGCGC TCTGTCAGGGAGAAAGCCCAATTAGGAGGGGCTTGTGGGGGCAGGGCGGGAGTGCAGAGG GAGCCCCGGAACCTAGCCAGCCCACTGTGATGACAGGGCCTTCGACAGCAGCCCAGCACT TGGACTGTGCAGAACTGCGGAGATGGTGCTGTGAGCCTGGGGAGGGTGCCTTTCTCCCCA GGGGCCCGCATGGGGCCAAGTCTACCAGACTTCTGAGCCGGCATGTCTGGGTACAGGACC TGGCCCTGCAGAACTAGCCTGGGCATCAGAGCCCACCAGAGGTGACTGCCAGCGGCCAGG ACCCACCACGGAGTCTCAGGGAGCAGCACACGGGTGGGGAGCGGGTGGAGGGCACACAAC GCTGGTGGGGCATGGGCCTGGGCACCTCTCCAGCTGGACACTGCACATGACACCCCAGGC CAGGCCAGGGTCCCCTGACTCACAGACCCCAGGCACAGGGACATGCTGTTTTCCTTTTAG TCCAAGTGTACGGGGTGAGGAGCTGCAGCCCCCACCCTGAGGGCCTGCTCTGTTTAGAGG GGGTGCCAGGGTCCTGGGTCCCTCCCTCTGTTAGAGCTGTCTGGAAATCCTGAGTCGGGT AGGTGAGGTAGGAAAGGGAGACCTCCCCACCCACTGCCCTGCCTGAGCGGCTCCCACGGT CTAAACCCTGTCTTGGGGGTGGTTCCCAGGAGGAGGCAGCAGAGCCTGGTGAAAAGAGAC CAGACAGTGTGGACCTCCCTGGCCCTGGGATCAACAGCCTCTCCTGGGCCCAGCTTCCCC AGGCCTGAGCTTGGGGTTGGAGGATCGGCAGGAGAGTGAGTCCATGAAGGCACCATCCCC ACCCCTCAGCACCCTTTCCCTCCATGTCATCTCGGCCACCCATCTCAGAGCCCAGCTCAG CACCCGGGCCCTACTGGGCCACCCCGTCAGGCCCTGAGTCCCCATGGAGGGTTTGGAGGC TGGAGGCCGGTCAGCTGGGGAGGGGAAGGGCAGAGGAGCAGGGGTGAGCCACTTGGCTCA GGGCTGAGGACCTCTCGCACTCTGGCCACTGCAGAGGGTGTCCGGCTGGGGCAGGAGGGC TGTGGGAGAGAGATCACATCGTGTGTGGGAGGGGACAGTCCCCTCCTGCCCAGTCCCCTC CTGCCCAGTCCCCTCCCCACAAGGCCTTAGGGAAGGGTGGGGGTGCCACACACCATGGGC TGGTGCCCCCATCCTGGCTTTGCCTACCTAAGGCTCTGGGATTGGCAGGCATGGAGTTCC CCAGCCCTGAGCGTGGCAGGGGCAGGATGACAGACCCACGGTCCCCTAAGCCCTCCCTCC TCCACTTACACCCTCGCCCGCCAGACCCCAGTCATTCTCCTGCCCTCAGGGCCTGGATTC AGCCCCACCCCCAGCCTCCTTGTAAGCCCGGCCTTATTAGTGCTGCTTCACCCCGACTTT GCAAGTCAAATTCCGCATTACTGCGCTCCTGCTCTGTTTCCACCTTGATGCATGGAGAGC TGGAGAGCAGCTGGAGCAGAGAAGGCACTGCAATGAGGCGGCAACCCGTGCTGGCCAGGG GCCCCAGGGCCCAGAGCCAGGTGGGGTGGACCCCGAGGCCCTGTCTTTCCACAGGTGTCC AGGGTCACTCCCACCCCACCCTTGGCCTCCTGCTGGCTCAGCCCCACACCCCCACCAAAG GGCCAGGCTCCGGGTGAGTGATCCTCAGGGCCCCCGCACAGGCTGTTCCCTCTGCACACA GGCTATCCCTGCCTTCCTGTCCAGCCAGTGGGTGGGGAGAAGCGAGTCCCTCCTTTCAGG GATTCTCAGGAACCGAGTGGCTATCAGGGTATGCAGCTGTGATGCACGTGGCAGGAGAAG ACCTCAGACGCCACAGCCTAGCTCTCTGTGGGGCAAGTGTGTCTCAGCCTCTCTCTGAGG AGAGGAGAGCCCCTGGACTGGAGGGTGCCCTGCTCAGCTCTGCCTGGGTATTCTGTGGCT TGCAGGGTGGAAGGTGAAGGGCTGCAGGCGCTGGCCTTCCCAGGCCCCCCACTGTCTGCC CAGCTTTTCCTTCTTTGGCAGCCCCAGTTCCTTCCTTATACCCAGTGGGACAGCAGGGCC CAGCCCTAGGGGCCGCAGGAGACAGACATGCCCCAACTGAGACAGACAATGGCCAGCATG GCAGGTGGCCCAGTCTCGGCCACCAGGACGTCCTAGGAGGTACGGCCCCTCAGCTAAGCT GGCTGTCCTTAGTGGCCCAGCTCTGGCTCTGGCTGGTCCTGTGAACTCAAGATGGCAGAC ACTGTTCCTGGTCTCAGGCCAGGTCTTGGGTGAAGAACGTTGGGTGTAGGACCCTGTACC TCTCCTCCTGGAACACCCCCAGTGGATGGCGAGGTCACTGAGGCTCCATCCAACGGGCAG CCTCCAGGGCAGCCCTCGCTCCTCCCTTGCCCTGGAGGTGGGCACGGGTCAGTGGAAGGA TGGGACTGAGGGGCTGGGCCCTTCACCGACCCTGGAAGGTGGCTCCCTGTGGCCTTCCCT GCAGTTTCCTGGTCCAGTGTGGTTCAGCCTAAGACACAGGTGAAGGCCTGGTTATCACTT GGCTGCTTTTGCCAAAAGAGTGGCTTCCAGTGAAATCCTTTGGCTGCCGAAAGGCAAAGT CACCCTCCCTCCCTGTGGCCCCAACACCTGCAGGGTTTGTCAGCAGAAGGTTTTCACGGT CCACTGGGCATTCGCGGGCCTTTGGCATCCTCGAGGGCCTGCCTTTGATTAGCACACACC CTTTGCTTGACCCAGAGGGTCCAGATGGCACTGGCTCCACGGCATGGCCTTGAGAATTGA GGCCGGCTTCGGCGAGGATGGTCCCACCTACTCTGGCCTTCAGGCCTTCACCAGGAGAGA TTCAGGCTGCTGGCCCAGCCAGTCTCTGCAGGGACACCCCGGTGGGCCCAGGGAGGGACT GGGGTCAGCTGGGGCCCTGGACTTACAGTGGATCCCACTTCCCCCACGTCTTGCTTGGTT CTGGGCATAAGGGTCCTAGTTCTTAGGGCCTGGCCTGGCCCTCTCACTGCTCCCGGGAAA CCTCGGGAAACATGGCAGGCTGTGTTCCCCATCCTCAGGCTCCCAAGTCGCTTCTGTCCA GTCCTCTGCTGGTATCTGTGACTCAGCACCATGACGGGGCCCTGTCCAGGGCGTCCTGGG CAGAAGGCTGCTCCGAGTGCACAGCGGGCACCCCCATGGACAGGCTGCGTCCATGTGCCA CTCCTTGGCCGCCCTCTCTTCATCTGTAGTGATGACACAGTGACCCCCACCTGCAGGCCT GGTGGCACTGCTGTCACCAGGCACAGGGAGCCTGCCCCCAACCTGGGTGGTGTTGTGGGA GTGGCTGGGCAAGGAACGCTGCTATCCCTGCTGAGCTGGGTGGGCACTGATGGGCATGGG GACCTGGAGTGCTGACCCCTAGCCCTACTGTCGGCAGGCCACGCCTGCCCCTCTGGGGAA CTGCTGTGTCCCCCTTGGCTGGCCTAGAAGTGCCTAGAAATGTCTTTTATGTGGGGAGTC GGTGGGTGAGGGCCCCACTGTCCCATCCCTTGAGGAAGGGAGGGCTCTGGGCAGGGTCTG CACCAATTCCCCAGTGCCTGCTGAGGCCTGTGGCTGCCTCAGGGACAGTGTCAGTCACCC CTTCACCCTGGCATAGGTGTCCTATTTCCAGAGTGTAGTGCTGGATGCTTCTGTTTGGCT GTGTTAATTGAGACCAGCTTTTACAGAATGAGCCTCTTGGGGTGGGAAAATCTGCACAGC CTTGGTGTTTAGGGTAACCTGAAAGTGGAGAGTGAGCCTGGGGTGCAGTCTCACTCCCCC ATGCCCCTGGGCACAGCTACCCACAGGCCCACAGCAGGCAGGCAGGACTGATGCCATCCT CCAGATCCCCCGACCAGGCCCAGAGGTGGGGCCAGGAGAGATCTGGGGAGTTGGTGCCAC CAGATTTCCCAACCATGACAGGATGTCGGGGGGTGGGGGGCAACAGCAGGACTGCCCCAG GCTGGGTCCCCACCCAAGTGAGGCTTGGTCGTGGCCTCATGGCCAGTTTGTCCCTCTGCG GATCTGGAGGGGTCCTCGGCTGCAGCAGGGGGCCCAGATGATCCTGACCTGGGGATGGGA AGGCCTCTGTCCGTGGTTCCCCAGCAGAGCGGGGCCTGTTGAGAGGAGCAGGGGATGGTC TGTTGAAGGAGTCGGTGAACGGAGAGGCATGCAGGGATGGATATGGACAGGGCGGGTGCT CAGACCCTACAGTCTGAGGTGGAGGGGTGGGGTTGGAGGCTGGAATGGCCGCGGGGGAGC CTAGAGGGGTAGGGCTGTGGCTCCACCACTGTTGGCTAAGGGACCTCAGGGAGGGCTTCA GAGGCTGGGGCCCTGGCTCCCAAGGCCTCAAGACCCTCCTGCTCCCCCTGAAGGTAGCAG TGACCGCCTCTCAGAAACCCCTGGCATCCCTGGAGGCTGCAGTCCCCCGCCCCGCCTCCA CTCTTTCTCTCTGCTCTGTCATTTTTCAAAAATAATTGTGCAGCAGCACCAGGCTGTCTC TGTGCCCCTCCTGTAGGTTGCCCTACCCCACCAGGTCCCTGGCTCGGCGTCCCCAGACTC TGCCAGGCTGGCCTCACCGTGTGTATCGAGGTGTTCTCCCTGCAAGGTCTTCATCATTCC CCAATTGCTCGCATTTATTTTTCTCCTTCCTTGGTGAGTGGCCCTGCCAGCCTCAGCCTT GTCTCTCCTTGGTGAGCGTCATTAATGCAGCTTCCGCGCCACGATCAGAGGGGGCTGCCC CCACCCTGCCGGCCCTGCCGCTTCTCCTCTGCCCGCCTCGGGATGGATGTTCATTTATTG CCCATAAATCTCTGCACAGCCCTTTGTAAAGACAAATTGGAGGCCTCCTGTTCCCCGGTG TTGGTTGGGCCGATCGGTGGTGGGTTTAATTGGCCCTTCCCGTGGAGGCTGCCCCTGCAG GAGAGGCTCGTTGTGGGCTGGGCCAGATGCACTGCGTCTGCAGAAGGGGCCATTCTCTGC CCCTCATTTTGTCTAGCGTCTGAGGCTGGGCCATGTGCAGGGACAAAGTGTGGATCCTCC CTCGGGCTTCCAAAACATGGCCTCCCCGCATTCACGTGGCCTGGCAGCAGCCCAGGCCCA GGCAGGGCCCTCTCTCACCCAGGGGGTGATGCCTGCAGCTTCAACAGCAACCTCCAGGAA GGAGGCGCATGGGTTCGTTGAGGTGATACAGGAGTGGAAGAGCTTGGTCCCTTGAAATGA ATGATAGGGAAAAGGGGAAGTGCTGCGTAGAGGAGGGTGTGATCCCTGGCTGGGGCTCCA TCCCACAGACCTAGGTGAGGACAGGCATTTTTGCATTTCCCAAGACCACCCTGGCCTGCC ACACCCCCATCCTATGACTATAAAAACTCGCGAGACCCTGGCAGGCACGCACAAGCTGCT GGACGTGGAGAGGAGCGGATTGGCAGAGGAGCACTCAGGCGGCTGGACGTCGAGAGGAAC GCACTGACGGGCGCCGGCACCCCGGCAGGCTACCAACCGGCAGCAGAATGACCTGGAGTT TGGCTGGGGCAGCCAGAGGAGAGCCCGGGCCCGACTCCAGGGGAAAACCGTCCCACTCCA TCTTCTTCTGCCTTCCCCCATCTGCTACCTCCACTCAATAAAACCTTGCACTCATTCTCC GAGCCCACGTGTGGTCCAATTCTTACACCAAGGCAAGAACCTTGGGATGCAGAAAGCCAT CTGTCCTTGCGATAAGGCAGGGCTCTAACTGGGGTTTAATTGAGCTGAGTAACAGAAGCC ACCGATAGAGTTAAACTAAAACAGCAAAACTAAAAGAGCACCCTGTAACACGCTCACTGG GGCTTCAGCTATAAACATTCACCCCTAGACACTGCCGTGGGGTTGGAGCTCCACAGCCTG CCCGTCTGTATGTTCCCCTAGAGATTTGAGCAGCAGGGCACTGAAGCAAGCCACAACCCC ATTGCATGTACTGTGAGGGGAACAAGGGGACTTTTCCTGTTTCACCAGCACACAGCAGGT GCCTAATAAACGCACAGCGAACTGGTCAGAGAGAGAGGGACCCTCCACCGAGCCCAGCCA CTGCACAGGCTGGGAGGGCAGGACTCCTGCCGAGCTTGGCACCCTTTCCTGCACCCAGGT GGCTGTGAGCCTGGCCCTGCCCCGGTCAGCAGGCCTGTGCTGACCCCACATGCAATCTGC TGTTGGTGCCAAGGAAGTGCTACCTGTGCAGCTCTCAGAATGTCAGGCTGGGTGCTCCTC CACTGAGGTCAGGGTCAAGAGACCCTAGGCCAGCTCTGGGGGGCCCACAGGGCGTGAGGG GGGTCCAGGCAGCCAGCCCGGCCCAGCCTGGTTCCACTGCTTACTGTGTGGCCTTGGGGG GCTCCCTTGGCTTTCTGGGGCCTGGGGAACCAGGCGCGAGCTTGGTCCTGTGGTCTGAAT CCAAGAAGACAGGTTGTGGGTGGGGACATTTGGAGGGAGGGACAGCCTCGGCCTCAGCTT TGGGACCGTGTGGCAGAAGCACCCTGTCTGGCCCTCTATGAGCTCCTCCAGGGCTGGGCC CAAGCAGGGACCTCTTCTTCCATGAGGGTTTACTCATTCCTCAAAACAGGCACAAAAGCA GAGGGCTGTTGAGGGGTAGTGACCTGCCCCTGGCTCTGGCCATGCGGGTCTGGGCACTGT GGATCCACTCCATGGCCACCTGGCTGGGTGAGGACCAGCCTGGAGGATCAGGAAGAGCTT CTTGGAGAAGGGAGTGGTGTCAGAACTGAATACTGAGCAAAGAGGAAGGACCAAGAAGAA GATCCTAAGGAGTTCTGGGGCTGGGGGCTGGGGATTGGGAGCAAAAGGAGAGATGAGGCT GGACAGGTGCCTGTTCCTCCACCCACTGGCTCAGGACTCCAGGCATGGCCCAGCAAGGGT CCCCTGCCCCTGGGAGCAGGACTCCCCTGCAGCAGACAGAGGCAGGTGGGGAGTGCGACG CACCTGGAAGGGGCTTCCGGAGAGGTGTGCCCTGGCAGAGCCCCACCTCCCCCAGGAAAA GACAGTAGCACCAGGCCCCAGCCCTGGAGAAGCCTGACCAACCAGAGAAGTCCCACGGCC TCCTCCATGGAGTTTGGGGCTCCTCCTCTAGACCTGGGATTGGCACTTTGGGGTCAGGCC AGCTGCTGGGGAGCCCTGGGCCCCGACACATGCACCCTCCCAACAGGAGTGGGGCTCCGG GGGCCACTGGGCTCATGCCCATCGCAGCCACTCTCCAAGGCCTTGGCATAATGGGATCCT TGAGCTGCTGTGCCCGGCGGGGTGGGGGGGGATGGGCTGAGATACACCCTTTGCTGACAG TGGCCATTGCACTTTTCCCACCCAGGCCTCATGTGGGGAGCCCTGCCAAGCCCAGGCTGT GGTGTACACAGGGGCTGGGCTGCAGGGCAGGGCTCCGGAGCGGCCTAGCCCAGAGCAGGG TGGGAAAGGAGCCAGCTGGGGTCCAGGCAGAACAGAGGCCAAGGCCACCAGTGAGAAATC TGGGCATGAGGGTGGCCACCAGGCCGGCAACGTGGGGGAAATTGAGTTGAGGGCTGGGCC TCCTCACTGTGGCTGGAGGCGGCCCAAAGGCCAGCTGACCAGACACAGAGCTGCCTCAGT CACAGAGGTGCCACAGCTCAGGGTGCCACCCCGAGTGACATCCCTAGGCTGACTGACACA GTGGAGGGAATTGACACCCAGGGTCTTGTGATGTCCCCGGGCACTGGGATGGAGAAGCAG AGCAGCACTCTGGAGCACAAACACAGTTGGCAGCGAAGGGTCTGCTCCCGCGTGGCCTCG GGCCCAATCCTGCCTGACATGTTGCTTCATGTCTCTGGGCCTCCGTTTCCTCATCTGTGC CCTGGGGACATTAGCAGTGTCTTCCTGCGGGGATGTGAGTGTGAGGTGCCCTACCACCCC AGGGAGGCTGTGAGCTGCAGGCAGCCCGGCAGTGAGGAGCCCCCACCCCTGCCGCCGCCG CAGTGGCCCGCCCCGGGCCCCAGCTCACACCACAAACACTTGCCTCCCTCTCAACTTGTT TCCGGCATGTTCTCAGTCTCGCTGGATGTTAGTTTACCATCGGCTGCTGAACATTCGGCA GCTTCTATTATTTCAGAACTAATTTAAGGATTAAAAAGTAATTCTCCCAATTACTAGGCA AAGCCCTGGGCAGCCAGATAAACAGGGGGCAGGCGCTCCCATCCAAGGACAGGGCTGCTC CTGCAGCCCTGGGCAGACAGGTGTGCAGGGCAGCTCTGGTGCAGCTCAGCCCCCAGGGCC GCAGGGGTGGAGTCGGGGTGGTTGAGAGGAATCCCCAGGAGCAGCCCAGCCTGGGCCCGA CATCTGAGGGTTGCTTGCCCCTATCCCCCACCCAGGTCGGCTACAGCCACCTAGTGGTGA GCCCAGCCCCAGCTGTCACCTCCACCAATTGTGCTGGGTGGTGCTGCCCTGGCTAGGCCT GCAACACAGGGACAGGCCCATGAGCTGTGAAGGATTCGGTCCACAGGGCCCCCTGGGGCT GGCCAGGCATCTTTGGACGGGGCCAGGAGAGTCTGTGGTAGACCAGCAGCCCGCATGGGG TGGGGGCCAGGATTTGATACCCTCAAGGCTGATTCACGCAGCTTTAATTAGCTTTCTGCA CCTGCTCTGGGGTCTGGGTGATCAAGGCTGGACACCAGCTGCCACCCCCAACCCCTGCCT GGTCCAGCACCTGACCTCAGGGGCCCAGCCGGCCGGGGCTGGTCAGGCAGAGCTGGGGAG TGCCGGGGTCGGCGCAGGAAGGAGCTGAGTCCCTGGGCCCTGGCCCAACGCCCCGCCCAC TCCGGGCCCCTCGTCAGCAGCGGCCGGCCGGTGAGCTCATCTCTTCCTGGGCTGTAAATA TCACCCAAGTGCTCACGACTCCCAAATGTGTCCTCCAGCCGGGCCCCTCTGCTGAGCTCA GCCTGCACAGCCCACGCGACACCCCCGCCCAGATGTCTCCTGGGCCTCGACAAAGCGGCC GCTCCTCTCGGCGCCCCCACCTCAGGGACGGGCGCCCCGTGCCGGCTGCTCAGGCCTGGG AGCCCTGCCTGCCGCCTCCACCCCATGGGAGCGTCCATGCATCCTGCTGAGTCCAACTTA AAACTCGTCCACTTCCTTCCGGATCCTCTCACCCCGTCCTCAGGTCTGGTTGGACTGTGC CCAACTCCCCACCCCTGCCACTGCACAGGCCCCCACTGTGGCCTGGAGGGGAGGGTGCAG AGCCCTGAGGGCAGCTGGTGGTGACAGTCAGCCCCGAAGCTGGCCTGAGGGCGACAGGTG CCTGCTTCTTAGTTAGGAGTGGGCCCAGCAGAGGTAATGAACTCCTTGTCCTGGGAGGTG TGCAAGCTGAGGCAGGATCTGCTGTCCTTCCACAAGCCATTGAGGGCGCCAGCATCCACT GCAGTTGGGTCACTCAGGTGGGGCTCAAGGCTCTTTGGAAATGGGTGGCTTCGGTTTCCT GGCTCTGACTGCAAGGAGCCCCCAGCCTCAGCTGAGAGCACTAGCCCAGATCCAGCAGTT TAGCTGCCTCTTCTCAGGCATGACAGGGGCCACTTGGGAGAAAAACTGAATAAAACCCTG CCTATGCCAGTGCCCAGGCACAGCCTGGCAGAATCCTGGGCCTGGCTGTGGGGTCACAGA GACTGGGGGTGCCAGGGGTGCTGATGGTAGAGCTCCAGGCACAGGACAGAGACTGCACAA CATTTTGACATCAGGCCATCCATGGAGAGGCCTCTGCTGGGGCTGCCAAGGGTCTCCTGG GCCCAGAGGGGCTGCAGAGGGAGCCCTGGGCAAACAGCACCCCACCCCGGCAGGGCTGCC CTGCCTGAGCTGCCCCCAGCCTTGGGGCAAGGCTGCCGGCCCTGGATCTCAGGTGAGCAG AGGGGCAAACTGCAGGTGGAGGTGTGCGCAGCCACACGGGGGGCCTCAGCACCTGTGGGC ACAGGCACAGGGTCTCTCAGGAGAGACAGGACATGTGGGAGGACCCTGAGGAGGGTACAG GCCAGACAGGACCACTGCATCCTGGCTCTGCCCTGTGCTTGATGGGAGGGGTCTGAGCCT TGGGCCGCAGCACACGCACAGAGAAGCGGGCGGAGGAGCTGGGGCCTTGGCTGCTAAGCG CGTCCTCCCCGCTTCTTCTCCCTCCTTCTCCTCCCCCCAGGCTTCTGGAGCCCCGGCCGC CCCCACGACCATCTGGCTTCACCAGCAACGCTCCCCCACACCCTCCCCCACGTCACTGTT TTTCTCCCTCTCCCCCTCCCCAGCCACTGCCCTGGCGGGGAGGGGGCCCTTTCCTGGGAA GGCTTCCTGGCCACGTGGCCCTTCCCTTTTGTTTCTGAGTCCTCTTCGGCCCAGGCCGTG GGAAAGCGTGCCTGTGCATATTTCTGCCTGTGCATATTTTTGCCTGGATGCGTGTGCTGC TCTCGAGCCCCCGGTGCCTGCATGTGTGTGGACATGTGTTCCCTGCCCTGCTGTGTGTGC ATGCGTGTATGCATACATGGGTGCAGGGACATGCAGACCCACCACGGCCTATGTTTGGGG TGTGGGCATGCTGTAAGTGGGGGTGCCCCAGACACGCATGTGGGGTCTAGTGTGACCACC AGCAGCGGTGTGAGGGTGCATACAGCCCCCACTGGGCCTTGAGGCCAGGCCTGACCCGCC CAACTCTCCTTCAGGGCCTTCCTCTCCTGCCACCCACCAGGGGTAGGAACAGGGTCCCTC ATCCTCAGCCTCCTCCTGGGGGCTGCCCGGGCAGCCTGATTGGGGTGGGGGGCCCGAGAG CAACTGTCCCCACAGGGGCTGCTATGTCCAGACCCATGGACCTGGCATGTGCTCCATCAG CCAGGCTCAACGACGTCAGCCACCAAGCATGCTCCATCTGTGCGGCTGGGGGGCCAGGGG CACCTGACATTGGAGCTGCCCTTGGCCAAGGCTGTCCCACCTGTGGTGCCAGGCAACTCC CTTCCTCTGGGGGCCTTGTGATGGGGGAGAGGCTGCCCGTCCAGCACCCACCCCTGCTTC TCACCGCCCACAGCACCCTAGCCTGAGAGGCCACAGCTTAAGAAAAGAGCTCTTTATTCC ACGTCGTCCGATATTTTTACACAAGTAAAATAAAATGCATATCTCTATATACCGCGATCT GGGTGGGAGGCGGCGTTCTGGAACAAACGCTGCCGCCGAACCCTGTAAACATGATGGGGT GGAGATGGGGGTGGCGGGCGGCAGGCGTCCATCAGGGAGGACCTGGCCTGGCCTGCCCCA CGGGTCGGCCGCCCGGCTGGCTTGGCGGCGTGGAGAGAGACCCCGTATGTACACACACCT GGCTGCTGAGCACGCTCTTGTGTCCGCTGGGGGTCAGCAGGGCCCAAGCACTGTCCACAG CACCAGCGCCAGGCCCAGGGGGGTGAGGCTGCAGGTGAGGCTGGGTAGGGCACCTGAGCC TTCTGAGTCACCAGTCCCGCCACCCCCGCTGCCTGCCTGGCCCAGACGGCAGTGGCTGCG GGTGCGGTTCTTGCGTGAACAGCCTGGCCTCCGGCGAGGGCCCGAGGTGGGGAACCCTGG TGGCTCGGAGCCCTCGGGCCGCACTGCAGTGAGCGGGGGCTCAGCAGAGCCAGGCAGAGT CCCAAAGGGTGAGTCATTGATGTGCCGTGGGCCAGAGCCGTTGCCCGGCGGGCTGTCACC GGGCGGCACGCGTCCCTTCAGCGCATTGCCTGCCGAAGCTGGTCTTCCAGGCTCCAGTAC TGAGGCCTTGTCAGCGGCATCTGGCTGGCAGCACTTGGGAAGCCCCAGCGGCTCCTCATC GGTGGCCCTGCCGGTCCAGATGGGATGGTAAGGGCCGGTGGCCACAGCGCAGCCCTGCAG GTCATTGGCAGCTAGGCGTTTGAGGTCACGGCCAGCCAGGCGTTGCGGGAGGCTGCAGGG CACCTCGGAGGAGGAGCCGCGGAACTTCTGCAGCCAGGCCCAGAGTGGGCGTGCCCGGCA GTCACACACCCAGGGGTTGTCGTTGAGCCTCAGGTACTGCAGGGCACGCAGGGGGGCCAG GGCCTCAGTGGGCAGCGCTGATAGATTGTTGGCAAACAGATAGAGTGTCATGAGGCGGCC AAGGTCACGGAAGGCATGCGGGTGCACATGGGCCACGCGGTTCTGGTGCAGTAGGAGACG GTCGAGGCTGTGCAGCCCACGGAAGGCGCGCTCGGGCACGCTGGAGATGCGGTTGCCGTG CAGGAAGAGGTGTGTGAGGTTGCCCAGGTCGCGGAAGGTGTCATCAGGCAGTGCCTGCAG CGCGTTGTCCTGCAGGTAGAGGTACTGCAGGGCAGCCAGGCCGCGGAACAGCCCCGGGCC CAGCTCCTGCAGGCCGCAGCGGTCCAGGTGCAGCGTGTGTAGGCGGCCCAGGCCGTGGAA TGTGGCAGGGTCCACAGACCGGAGCTGTGCATTATCGCTGAGGTCCAGCTGCTCCAGGAG GGCCAGGCCAGTGAAGGCAGCCGCATCAATTCGGGCCAGCACATTCGAGTGCAGCCACAG GATGGTGAGGTTGCGGCAGGCACGGAAGCTGGCAGCTGGCACATGCGAGATGCGGTTGCC GTGCAGGAAGATGCGCTGGCTGGCAGCAGGGATGCCCACGGGCACAGCCTGCAGGCCCTG CTGGGGGCAGCTTGTCGTCACCTTGGGCTCATTGTAGCATACGCAGGCACCTGGGCATGG GGCTGCCACCTGCCAGGCCTGCAGCCACAGCACCCATGCCAGCAGCCGGCTCCCTGTGGG CAGAAGACAGAGTGGTTAGCAGGAAGGGCAGGGGTACTGGAGAAGCTGGAGGTTTTGCTA GAGCCAGGTGGGCCCCGGAGATTGGGTCTCAGGAGGACCTGGGGCTGGGTCCAAGATGTG GCCACCACCCCCGGGAATCGCAGTGTTGGGATCCCTGCTTCACAGGCCTCATGGACGATG CTGGGCTAGAACATGCCTTGGAGCCATGCACTCCACCCTGGAAGCCACATCTGCAGACTC CCCATACCACACCCAGGAGCAGCCCACTGGGTCACCAGGGGCACTCACTTGAAGGCCTCC TGGAATGGACACTGGTGACTGCAGGTACCTCCTCAGGACTCCAGTGAACACAGAGCCCGC ATTGCAGGAGTCCCCGGGGGGTCCCAGTGGGGGTTCCACCCACAGGGCCTAGGAAACACT GCAATGGGGCTGGGGTCCCTCTGGGCACGTCTATCTCAAAAACGGGCGCCTGGATGCCCG AGGGCGGGGGTGGAGGGCAAGTGTGTCCAGGGCTTAGGAGGAGTCGCGTCCCACAACCAC CCAGGCAGCCTGCTCCCTCCTCCACACATCCTCCTTTCAAGCACTTCCTGACTTGGCAGG AACCCTCCTGAAGCCTCCGCCCCCTGGCCTCATTCTCTGCTCCATCTCCTCCCTGGGAGG AGTTCACTCCTCCCCAGCCAACCCCTGCAGTGCCAGCTGCCTGCACCCCTCCCACCCAGA CGCCATCAGCAGGGATAGCCTCCCAGTGGGTTCTCCCCCAGCCCCAGGTGCCTGCAACCC CTCCTCCATTACAGGGCCAGATACACCTGGGGCTTCACCAAGGCCCTACATGGGCAGTAG TCCCTCCCGGGGTGCCGCATCCAGAGCTGGTCTCACCAGCGGGGTCACCCACTCCGGAGT AAGCTTTCCTGCCTCTCATTCCACCATTGAGCCCCCACCCCTTTGGAATGCTGAAGATTC TTTCTGCCTTCAGCAGTGAGTCTATTAAAATGTGAATCCCCCTGGGAAGGTTATGGCTTA TCAGCTAACACTTGAGTTATCAGCTAACACCTGCGTTGACAGCTAACACCTTCCACGGCA GCTGGCTAAGAGCAGGATTCTGCAGACATGAGGCCGGCTAAGTGCTGGGGTTGGAGCCGG GCAGGAGGAAGGAGTGGACCACAGGCCAGCTTCCCCAACCTAGAACTGTGACCCTGGGGC CCTCCTACCCACATGGGGCCCCTGCCCCACTGGGTCCTCTCTACTTGGGCAGACAGTGCA GTCGCCCTAAACGCAGAGCCCAGCAAGCCCAGCACGCGCAGTGACCGCTGGGCATGTGCG TCCTCCCAGACTCAGGTTCATAGGTCCTCAGACCCCCTCCCCACAGCCTGGGGGCCTCCT CTGCCAAACCCCCTGCCCCAGGGCCATTTGTTGGCTACCAGGGAGATGGCAGCCCAGGGA TGGGGGTCCGGAGGCCCCTCCCTGGGTGGGCTGGGTGAGGAAAGCACCTGGCCTCGTGTT CTGTTTAAACTGCGGACACCCTCTCTGCTCTCTTTCTACACACTCTTCCTAGGAAGGTGG GGCCTGACCATGTCAGCTCCCCACTGCTGTCTCACCTGCTGTCACCAGAGGGAAAACTGT GCTATGTAAATGGCACTTCCTGTCCTGGGGCCAGGTGGGGTGGAGGCCCCACATGCTCAT GGGGCAGGGTCAGGACTCACTGCCACATTATCGACCACACCACACCACTGACTGTGGGGG TCACCAACTGAGCCTTGTCACCAACCATACCATGCGGCTGACTGCACCCACCAAGACTAG CCACCCATGGTGGCCACGCCCGCCACATTACCACTGAGCCATGCCACCATCCCACCACTG TCCTGTGGCTCCAGGCCCGAAGCTGTGGGCTGTGAGTCTGGCTGTGGTCAGGGCTCAGCT CACTCGCCTTGAACCGAGCCCTGTGCTCATGGATCTCCCTCCCGAAGCAGCTGTGTGTCC ACTCTAAACATGCCCCCAGTGAGGTGCCAGAACTGAGCCACACGGTGGTGCGGGGTCGGG GGAGACAGGGGAGGACATGGGGGAGTGGCCACGTGGCGCTCAGTTCTGCCAGGAAGTGGG TATTGGGCGCAAAGACACGGCTCGGTTCCTGCCGAGACAACTGCTTTTCGTAAACCAACT TCTGAGAGTCGGGTGAGGTGGGCTCTCTCCTCGCTGCAGCCATGGTAATTGGCTCCAGCC TGGCCCCCCTTGGGGCCTGCCCGCTGGTCTCCATGGCAACCCCTCTGCTGAGCGAGCAGG AGGGGGCATCTTTTTATGAGATAATCAAACCCAACTTTGCAGCATGGGTTTGTCTTCACA GCCGGGGCCCTGGGATGAGGGAGACGCAGCCAGGTGGGGGGAGGGTGTCCTCTGACCCCA TCCCAGGGGCCAGACTGGTGACTCACCAGCCTCCTCCCTGGCCCCATGGTGGACTGCAGC CCCTCCCCACCCCAAAAGACCTCCCTGGCCCGTGGCCCCTGACCTGTCCCCTGGGCTCTG CAGCCTCAAGGCCACAAGGCCTCTCTGGCCCTCTCGCCCACCTTTCTCTCTCCTCTATCT GCCTGGCCAACTCTAATAGGATGCTCTACTGTCCTTCTGGAGATAGCATGGCCTCCCTGC CTGCCATCACCCCACTTTCCACTCTCACCCAGTCTCACCCTGGCCCCAGTTCTCACGGAG CCTCCAGTATGAGCTCTGTCCTTCCTGCCCCTTGCACACGGGGTGAGCAACCAGCCCTGC CATGGCCCCAGCTCTCACAGAGAAACCATGGATAGCGTGCCTGTCCACCGTCCCCACCCA GGTTGGCCCTGCGGCCACCTGAGCCTGCGAGGGCCCCAGAGGGCTCTCCTCAGCCTCCAA GCTGCACACAAACTGGTACATCTGTGCCTCTCCCACCCTCCCAGGCCAATTAACATCTCT TCTTTCCTCCAGGGTCTCCTCCTGGGACCTCCCCGGAGGATGGCCCTGCTGGCTGTCAGG GCTGGGTCATAGCTAGGAGCTCCTGTCTCAGCCCAGGCCTGGCTCAGGAGCCACAGTCAG GGGAGGGCTGGCTCTCCTGCCTGGAAGGCTCTGAGCTTTCCCAGCGGGGACAGTGACCGG ATACTGACCCAGAGCCTCTCCCTGCGGCAGCCAGACTGGGCAGGGTAGGACCCAGGATCA CCAGGCTGAAGGCCCAGGAGGCTGGGCAGCAAACAGTCCAGGACGCATCCCCCTGACGGG AGCAACAGGCAGCCCAGGTGGGCACCTGCACCATCAGTTCTGGTGAGTGGAGGGCTGTTC ACGTTCACTGCAGGAGGGAGTGTGGCGGGGGGAGGAGGAGGTGGGAGGAGAGCGAGGAGG GCAGGAGCAGAGGGGAGGGGTGGAGAAGGGGGATGGGGAGGTGGCGGTGGGAGGCTCTGG GGATATGCCCAGCCATTGGGCAGGCAGGCGGCTGCTGAGGTCAGCAGCTCACCCCAGCAG CCCAGGGAGCTGAGCCGAGCCGAGCAGGGATGGCCTTCTTCATGTGGTCCCTGGCCCCCA CTGTTCCTGGGGCCCCGCCCACCTCCGTGCCCTCCCAAGCTTGGGCTGAAGTTCCACAGG GATGCCTTTGCCTCTGCCCAGCGATGCCAGGAGGCAGCACAGGGCAGGCAGCTGTGGAGG CGCCGAGCACCGGCTTTCATCCACGCACAGCTTGGTGCCCTCGGGCCCCAATGCCGCCCC TGGGCTGGGATCACCGGAGGGTCCCATCACCTGCTATAAAAAGTGAGACAGCAATACATT TACAGATTAGGAAACTGAGGCAGTGAGTGGTTAAGCCCTGCCCTGCTCCCACTCCAGACT ACCCAGGTGGAGAGGCGGCCAAGGGAGACACTCACAGTGCTGCCCGGAGGGGCCACGAGG CATGTCCTGCCCAAGCAGGAAGGCTGGCGGCCTGGCTAGTAATGGGGGAGGGTGTGGAGG GGCTGGGCTGCAGGCCACGTGCAGAGGGGCAGGCAGCGCCCTCCTGTGTCATGGGTGGCC GGGAGGGGAATCTCCAGGGAGGCTCTGAGCTGCTGCTTCCCAGGGCTGGGGTAAGAGGCT GCAGAGGGCTGGGGCTGGGGGTGGGACCAGCTGTAGCACAGCCGGGACATCAGTCAGCTC AGGATCCTCTCGGACACCAGCCATGTCCCCGTCAGGCGGTCACGTCACAGACCACAGTTA TGAGGCAGAACAGGAGGCCACAGCTCAGTGCACCAAGGCCAGAAATAGCTCCAGGCGGGG GTGGCGGGCCTATGTCATCCCCACACCACAGAGGGTCAGGGCACCCCTCCCACGCCTGCT CCCCACTCCTGGCCCTCCACCACTGACTCACTCCCCCCGCCCCCCACCCATCTGTCCCCC GATCCTCGCTTGCTTCTCACATGTACACCCACCCGCCATCCATCCTCAGGCCCACAGACA CGTGCAGGGGCAGCCAGAATTCCCATGGTGTTTCCTGTGCCCTTCCAGGGCTCCAGGTGC TGCGGCAGGAGATTGGCAGCAGCTTCTGGTGGGGGCAGTCTGCCCTTGAGGGGTCCTGAG TCTCCTAGGGAAGTGAGTACCAGCCCCTGATGCCCTGGCCCTGGCCCCTGCTCCTTGTTG ATCTTGGCTGCAGGGGCAGGGTAGGCAGACCAGGTCAATAACAGGCAAGGACACCTGCCA TGGTGAGAGGTCACTGGACCCAGACTGAAGCAGCTCCAGGCCTAGCTAGCTACATGCTGT GGGGGAAACTGAGGCCCAGTGTGGGCAGAGCCTTCAGGACTGGCCTCCTGAGTGTGCCTA GTGCTTCCTCAGCCCCCTCGGCTCCACTCTTTGCCCTGGCCGGCAGGGCTCGTCCCTGCC CCGTGATGCTGGGCTCACCAGAGACAACGAGATGTAGGTAGGTCCAGGGCCAGCAGGCCA GGCCAGAGGCTGGGCCCTTCAATCCAGCAAATTCTCTTGGAGGGGGTGTCAAGTTTATTG ACCCATTTTACAAATGAGGAACATAAGAACTGGTGTGGGTATAATCAATAGCAAACACAA TCCAAGGTCAAATCCAACCATATGCTACTTACAAGAAACATGCCTAAAAGTACACAAAAT CTTGCAATTAAAGATATTATCAGAAAAATAATAACAAGTAAAGCAGGAGAAGCCACAGCC CCAGGGCTAGCGGGCTTCCCAGTCTCATGGCCCCAGGGGTGCGAAAAGGTCTTGCTGGGC AGTAAAGGCTCAGCCACCCCCGCTCCTAACCTGAGCTCCACAGTCCTGTTTCCAGCCAGC AGGGCACCTGAGAGGGTACACCTTTCAAGTCACAGACACTGAAGCTAGCAGAGGATGGCC TGGCCCAGGTGAGAGCTGGCAGCCCTGGATGGCACCCTCACCCCAAGGACACATCAACGT GGGCCACACCGTGCAGGATCATCCACTCGCTTGGCCCTCCCTCCGTACCTTGGCAAGGTG TGAAGTGGCCCCTGTCCCAGGTGCTGTGCTGAGTGCCGCCCATACAAGACCAGGCCGAGG AGCCGGGTCCCAGGCACTCACGGTCCAGGGCCAGCTGACCACGGGCAGCCACCCTGCTGC ATGGCAGGTGCCCTATGGGAGAGTTAGGAGCCACAGAGGCCTGAGCCAGCCCCAGGCAGC ATGAGGATGTTGGCAGGAGCCCAGGAGCCCACTACCAGCCAAAGGGTCTTGGCAGAGCCC CAAGTGGGCAGGATCCTTGGCCTAGCACCTAGGCCTCGAGGATTCATCTTTGGTCAATGC TGGGCTGCCTGCGTGGAGGGCTCCAGAACCTTGCCCCATCCAGCCCATACACCCCTCACT GGAGGTGCACCCTCCTCGGCTCCCACCCCAGTGAGCCCCACACTGGGGGCCCCTGGAGGC TCTGCCCAGTGGAGGCCTCGCCCAGCCCAGGGCACAGCCAGTGCCCCCTTGCCCCACAGG AGGCTTGAGCCCACCTGCTCCTCCATGCTCCACCAGGAGCCAACAAACACCTGCTGCCTA ATTATCTGGAGAATCCTGTCTCCACCAAGGGCTCATCTTGGTCCTGCAGCTAATTCAATC CAGCAGCCAGAGCAAGCTGAGTCCCCAGAGGACACTAATGAGCAAGTGACCGGTGGCCGG CCTGCTCCCACCCTGGGGACGCACTCTGAGCCAGGTCTGGGCCCAGCACTGCCATACACC TGCCTCCCATGCCCATCTGCCTGCCTGACCCTGCCCGCTACCTGGTGCCTAGCCCTGCAG GGCCCACATCCACTCCCATGGGCTAGCATCCAGACCTAAGCCCCAGCCCTCCCACACTCC CCCAGGACCCTGGATCTGCTGCATGCACAAGGCTGACCACTGCCTGTGAGCCCAGAGTGT CGGCCTTGACAGGGCAGGAGGAAGTACAAGAGCACATGCTCAGGGTGCACGAGCCAGGAG CACAGACTCAGTGCATGCAATGGGACCCCTGGGGGTGCACACGATAGGCGTGCATACCGG AAACACACAATAAGAGCCCCCAGGGCTGGTCAGGGCTCAGGCAGAGTCCACTGAGCAGTA GAGGGTTGCTGGGATGCGAGCCCCTCCCAGGAGCCAGGGCCCGGCTGGAGGGTCTGCATG CCGCTGCGGCAGAGGAAGATGTGTAGGCACATCCCCATGTGTTCTCTTGCCCTCAGCATT TGTAAAACATGATCAACCAGGCAGGCGAGCGGCCTCTGTCCGAGCAGAATTTATTCTGCT GTGAAAAGACACGTGTGGGTCCTGAGGCTCTTGCCGGCTTGTTCTCAGCAGCCTTGGATC AGCCTCAGTCAGCAGAACAGCAGCTGGACAGGCTCCCATTGGAGCCATCCCAGGGACTGC CCAGCCCTTCACATTAGAAACTGAGCTGGGGGCAGCAGCTGGCCCACGGCACATAAGCAG TGCCTGCTAAGACCACAGAGAAGCTCAGCCAGGCAGGGCCAGTGGGATCCAGCACTCCCC AGGGGATGAGGAATCTAAGAGGCAGGAGAGGATCCTCCTCAGCCTGGGATGTTCCCATTG GAATTCCCCCAGCCAAGGCCAGAGCTCCGTGTCCGGGGGCCAGAGTGGAACCCTTGTCAG AGCCGACAGGCCTTGGCCTCCTATTCTACTGGCACCTGGCAAACTGGCCCAAGGGCTGGC TGCTGCTTCTGTAAAGGATGTGCTATTGGCAGCCAGCCACACCTATGTGTTCACGCATTG TCCATGGCTGCTTTCCCGCAACAACAGCAGAGCTGAGTTGTTATGACACACTGTGTGGCC CACAAAGCTGGAATTATTTAGTATCACACCATTCACACAAACAGTTTGCCGACCTGGTTT GTGAGCAAGCCGAGTGTGGGTGCTGCCACCTTCACCCTCACAATGGCCCCAGCAGTTCAC TCAGACCCCCACTAATGAAAAGGAAACCAAGGGTCAGAGAAGACCACGGTCCTGCTGAAG TCACATAGCCAGGTGAACAGAGCCCAGCAACCTCAGTGCAGGCTCAGCTCTGTGGAGACC TGGGCAAGGTCAGCATCTCACGGAGGCCTAGGGTGAGGGGTTCTAGGCAAGAAAGAGACT CAGAAATACTGAAATACTCTGCAGGTCAAAACAGAAATGATCATTTTTCTAAAGAGGCTG TCAGGAGTCTCACAGTCAAATATGCATAAAATAGGACAACCTAACACAAATAAGTTGGGA GGGGTGGCTCACACCTCAGCCTCCTGAGTAGCTGGGATTACACACACACACATGCATGCA CACACACACACACACATACACACAAAGAGAACAAAATGGTTGTAAGGAGAGGGGGACAGC AGAGGAAGTGCTGAATGACACAGTCCGGCTTCGGAGGAAAGGACTTGGGTGGCACTTCAG AGGAGACAAATCCAGTGCAGGTAGTTTTGAAGAGGGCAGCCGTTTAACTGAGGTAGGGGG TCTCTGGGGACAGGGCCCCTCCATGCCAGTCCACCCACCTTGGAACCCTGCAGAGGCTGG GCCGAGGCCTCTCCCTGCACCTTGTATCAGGGACTCTGGCCAGGTCACACCAAGGTGGCT TATGACCCATTAACATCAAGCTGCCTCCCTGGGCTGGACAGGGCCTGGAAAGAGGTGACC TGGGCAGATCCGTGTCGATGTACTTCCCAGGACATCAGGATTCCAGCCTCTGCTAAAGGG ATCTTCACAGTTAATAACAAAGAAATATTAAAGATGAGAACACTTGGTGCAGGAAACAAG AGAGGAAAGGGAGTGCGGTCCCTCCTTGCAAACAGGATGCCCTGGACCACCCTCCACGGC CTCAGGCCTGGATGCTCCTGCAGACCCATAGCCAAGATTGTGAAGGAAAAGTAGGCTGTG GAGGCATTTTCTGTCCTTGTTATCATCATTGTAAACATACTCATCATCACCATCACCATC CTCATCACCATCACCACCACTATCACCACCATAATCATCAGCAGCAGCATCATCACCATC ATCACCACCACTATCATCATTACCATCCTCATTACCATCACTATCACCACCATCATCTTC ATCACCATCCTCATCACCATCACCATCCTCAACACCATCCTCAACACCATCATCACCATC ACTATCATCATCACCATCACCATCCTCAACACCATCATCACCATCACTATCACCACCACT ATCATCCTCATCACCATCCTCATCACCATCACCAGCCTCAACACCATCATCACCAACACT ATCACCATCATTATCATCATCACCATCCTCATCACCCTCACCATCCTCATCACCATCATC ACTATCACTATCACCACCATCATCATCACCACTATCCTTATCACCATCATCATCCCCAGG CGACCAGAGCAGCACCATCCTTATCCTCATCACTATCACCATCACCATCACCATCATCAC CATCCTCATCCTCATCACCATCATCCCATCACTATCATCACCATCCTCATCACAATCACT ATCATCACCATCACCATCCTCATCATCACCATCGTCCTCATCACCATCACCGCCATCAGC ATCCTCATCACTATCATCATCACCATCCTCATCCCTGTCACCATCATCGTCTTCATCATC ATCACCGTCATCATCACTATCATCACACCAGCTCTGTGCAAGGCCCTTAGATAAAGGGCA GCCTGGGGCTGACCTCACACAAGGATAGGAAGCCCCAGGTGAGCAATGGGTCAGTACTGG GTCGACACCAGATATCCCTGGGCAGAGGCCCTGGCCCAAGGATGGGGAAGGGAAGAGGAC AAGGGATGGGGAGTAAGAAATGAGGGCCCCTCGCCTCCCTGGGACAGCCCAAGGTGCAGC CTTTCCTCATTCCTGGAGGTTTCAGGATATCCCTGTGCTGCATCCTCCCATTGTACGGTG AGGGGGTGACTGCCAACCCACTCCTCAGATGAGGCACTGACTGAGGCAAGTGCCTTGCTG GCCACCTTGCAAGGCCAGGAGACAGTGCACACAATCTGGACTCCAAGATCTCTGCCCACC CCCATCTTCATCACCACTATCCTTATCACCATCATCATCCCCAGGCGACCAGAGCAGACC CTGAGCCCCAGGGATGACACAGCAGGGAGGGGCAGCCACTCTGGGCTCCCCAGTCCCCCA TGGCACCTCAACCTCAGCCCAGGAGGCTGCCCTCCCAGGGATGGCTGAAAGTGGGCCCTG GGGTCTTGCAGCCCTGGGCTCAGATGTAGCCTCTGCACCCCCTCACAGAGTAGGCAGAGA ATCTGCCAGCCCCCTCCAAATCTGTCCCCACACCAGTGATGTGAGGACAATGAGAGGCCA GCACCTACCTCCCCCCAGGACCCCAAAGAAACCCCACCCAGGCCCCCACCCAGCATCCTG CCTCCCCAGAGGCCTGCCAATGTGACCTGGGTCAGCCTCGTCTCAGGTGAGGAGTCCAGG GAGGCAAGGTACAGCTCACAGCGTCCCAGAGCCACACGGACCCCTCTGCTCCAGCGGTCT TGCAAGGCCACTCTGGCCTGGCCTGGCAGTAGCCCTGCTTTATTGAGGAGATATGGCAGG CAGGATCCTGGCTGCATCTGGGCTGGAAGAGGGGGCAGCAGCCAGGCTGTGCTACAAACC CACCTGGAGCCACTTCCTCGCTGCTTCTGCCCATCTCAGCCTCAGCAGAGAAAGAAGGCT TGAGGACAGGCCTGGTGCAGCCTGACTCATCTCATGAGAGACCCGGCCTGTGCTCAGAGT CGAGTTGCTGTGGCTTCTCATGCAACACTGATGGTCCCCCTCCCCTGCTCACCCACCATG TGTTAGACACGGGGATCTGCATGAAAGACACGTGTTCCTCCCCTTCCAGAGACCCACCGA AATGAGGGTGATGCAGAGTGAGGAGTGGGGAGCACAGTGGCAGGCTGCTCCCCTGGAGAA ACACCCAGAGAAAGGGGTTCCACCACTGGTATCCAGGAGCACCCAGGGCCGATGGGCAGA AGAAAAGCTCCATCTCAGCAGAGCAGCCAACCAGCTTCCCTCATAAAAGTGACCAGAAAA CCAGTGACCACCAGGCATCCGGAAAACATATCAATGCAGAAAAAGAGGGAGAATGATGCT AGAGGAAGCCGACAGTGTGGGAAAAAGACAAAAATTAGGAGAAACATCTAATTTGTGTCT TCCTAGGCATAGATGGGGATTTAAAACAAGAACAGGCTGCTATGAACAAGGAGGCATCAG ACAAAAAGAAAGAGTGCCTGGAAATTAAAAATAGAATCACTAAAATTAAAAAAAAAATCA ATACAAGTGTTAGAAAACAAGGTCAAAGAAATCTTCCAGAAAGAAAAACAAAGGAGAAAG ACAAATGAATGAGATTTGGGAGACAAATATAGGTTGAACATCCAAGGGGAGTTCCAGAAG AGAAAACAAAAAAACTGAGAAAAGCTAGCAAAGAAACAATACCAGAAAACTTCCCAAAGC TGAGTACCCAGTCCTCCACTGGGCATTCACAGTGCACTTTCAGGCCCCCAGGGGTCAAGG GAAGTCTGAAGCTCCAGGAGGAGAAGCTGGTCACAAAGAGGCAACACGAAGCCATGAGCA GCAGTGCACGCCGGTAATCCCAGCACTTTGGGAGACTGAGGTGGGAGGACTGCTTGAGGC CAGGAGTTTGAGACCAGCCTGGGTAACACAGTAAGACCCTGTCTCTACAAAAAATTAAAA AGTTAGCCGGGTGTGGTGATGCATGCCTGTAATCCCAGCTACTTGGGAGGTTGAGGTGGA AGGATTGCTTGAGCCCAGAAGTTTGAGGCTGCAGTGAGCCATGATCATGCCACTGCACTC CAGCCTGGGTGACAGAGTGAGACTTTCTCCAAAAAAACACACAACAAAGAAGATGCACTA TGGTATTGGCCTCCTTCATAGCACACTAGATGCCAGGAAACAACAACACTGCAACAGCTT CATAGCTCTGAGGGGAAACTAATTTCAACATGGAATTCTAGACCGAAACAATCCAGCTTG AACATACTTGTAAATACAGGAAGACTTCCGGAATGATTCTTCACCATATCTTCTTTAGAG AGTGAGTCAGGCTTATGTTTCAGCCAAAATGAGGTGCAAACCCAGAAAGAGGAAAACGTG GGGCCCAGGAAGCATTAACTGAAATTTCAACAGGTGGTAAAGGGAAGTTATGGGGCAGCA GGACAAGTGCTTGGGGTGAATCTTTATCCAGGGAAAAAAATGAGACCCCAATAGAAGCCC TGATGTGGGGAAGAGTAAGCCCATGAGGGTGGTGGATGGGTAAAAAACATCAACTGTAAG CCCCAAGGAAAACAAAAGGCCATGAAAGAGAAGGAAGGTCACCCATCCCACGGCAGGATT GAGAAGAACATTTATGGGACCTGGGTTGGGAAACACTGGCTGTGAATTTAACTGATGGCA GCGAGACAGCCATGCTGGGGAGTGTGCTGGAGGAGACAGAGCTGGAGTAGAAGCTGAGGA AATGTGGCGGCAACCACTGGCAAAGCCAGGGAACAGAGGCGACAGTGGCTGCCTCCCAGG AGGACGCGGGGCAGGGGCAGATTGAGCCCATTCTCCAGACCAGCCTCCGAGGCTGGGTGC AGGTGTTAACTCTGCAATATGTTGTTGACCGAATGTTATTATTTTTTAAAACAACTGCAT ATGTGGCACTAAAATCTCAGAAAACAGATAAATAAAAAGAAAAAAAAGAAAGTGTATCTT CCAGAAGGTGGAGACAAATGTAGGTTAGAAGCAGGTGTCTCCAAGGTGCAGGAGGAGAGC GAGGGACAGTGAGAGCTCAAGAGCACCTGGACATACAGCCCCAGGGCAGCTGGCATCTGG CCAGGGAGGATCCACATGCTGCTTTTTGGTCCCCTGCTCGGCCAAGGGTGGCTGAGGATA CATCTGGGTCTCCCAGGACACTATGGGCTCTTGGCCCCAGAGACTCTGCAATCCAGACAC CCGAAGGGGCAGCTTCTGGCCTAAAAGCCCAACTCTGTGCTTGGGCCTTCCCTGCAGGCC AGGCAGCTGCTTCCCTGAACCAGCCTCAGAGTTCCTGCCTGCCCACCCAGCCCAGCCCTC ACACTGTGATGAAGCCCCACCCCTGGATTCCCCTTACCCCTGCCCGCTCCTGTCCCCTCC TTGTCACAGATCTGAGCCCCCCACTCCAGGCTGGGGCCCCTCAGGAGCCGGGGGTAGGGG TGTGCGGTCAACACCCAGCAGGGGTCGCCTAGCACTGGGGTGGGTGTGCAGCCCCCAGCC TCCTTTCTGACCGTGCACACGGGCTTCTCTCAGCTGCTATGAGAGCCCCCAATGCGTCTC CACAACCAAACCGAGGCACGCACGCAGCGGCGACGTGAATAAGTAATTGCTCTTTTATTA ACAAGTGATAAATTATTCCTATATGATTGTGTGTGATAACTCTTCCTTATTTGAAATCAA TTTGGGATGGAAACTGTTGTCGGCTTCTCATCCACGAGGGTGGGAGGCCCAGGTGCCGCT GCCTCTTGGAAAACGAGGTCAGTGTATTTCAGCTACTTCCTGAGGCTGATGCGCCGCCCC ACTGCGCAGGGCCAGTGGAAGGGGGTCAGCTGCCCTCCGGGAGGCGGGGCAGTCCGGCCT TATTTCCTCAGCATCTCTGTCTGGGACCCACGCCACTGAGCCCAGGGCACAGAGGGGTCC CTCAAGTCCAAGGCCACACTGGGCGTCACCACAGGGTCATCAGGGGTCAGGGGGCCATGC ACAGGCCATACTGCATTCATCCCCCCATGTAAGCCCCAAAGACTGGGCAGCCTGACCTTT CATCCCACAGAGGACACTCCAGCACCCAGCTGGTCTCCTGGCCTGCCCGGGCCACCCCAC GGTGGCTTCTCAAACCTGCAAAGGCATAAGCCATTCTCCTAAAATTGTCCTGATGATTTT ACAACAGCCCTGGCCCCATCCCCTTTTCTTCCTCCCCCAGCCTCACAGAGGAGGGTTCAG GGACTCAGCCTGTTCAGGGCAGCCCCTGGGCCAGGCCTCAGCACCTCTTCTGTTAATGCA TATTTAAAACCTTGCAACCCACCGGGCAGCCAGCCAGCCGGTGCCTCCCTCCCAGGCCAG GCCTGCAGGAAGCCATGCCCCGTCCCGCTCACTCCTCCACTGGCTGCTTCTCCGACCCCG ATCATGGCTGCAGCAGGCCTGGAGGCCCGGTCACCCCCAGGCCCAGTCACATGGCCCTAC TCAGGCACCACCTGTGCTTCTTGGCTGGGTTTCTCATCTGAGAAATGGGAAGGCTGGGTC TGGGTGAGGCACTTCTGTGGGCCCACTCCCACGGTCTCCTTGACACCCCTCCACCTTCTG CCCACTGCCAGGGTGCCCATTCCACTCCACACCTCGCTGGCCCACTTAGCAGGCTTCTGT GTAGCTTGTTTCTGTCGGTTGAGGCACCCTGGGCTTTGGGCTCCACAGGGGCTGGTGACC CGCATACAGGAGGTGCTCCATAAATGTTAGTCATGTGAATGCACTAAATGCTCACCACTA AGGGCAAGTGGCAGGCTTTGTGCAGCTATAGAGACAGCATTCCCCAGGATGTCCAGCCCT GGGAGAGAGCCTTGGCCCACAGCCCATGAGGCAACGGATGTTCCTGGGTTGGGGGCATGG CTCCAGCACCCCAGTGCCAAGGGCACGGTCTCTAAGTAGCAACATGGATAGCTTTACTTC TGTTACAGCTCCATGTCTGCGTCTCCCCCAGATTCACGTGTCGAAGCCCTAACCCCCAGT GTGATGGTATTTGGAGGCGGGGCCTTTGGGAGGTGATTACGGTTAGATAAGGTCACAAGG CTGGGGCCCTCACCATGGCATTAGTGGCCTGGAAGAGAAAGAGACCTCAGAGAGTACATG CCCTCCTCTCTCTCCACCCTTCCATCCCCTACCACGTGAGGGCACGGTAAGGAGGTGGCT ACCTGCAAGCCAGGAAGGGAACTTCCACCAGAACCAGCCCATGCTGGCACCCTGATCTCA GACTTCCAGCCTCCAGAACTGTCAGGAAATGTGCTTCCGTTGTTTAGGACACCTTGTCTA TCGTATTTTGTCATGGCAGCTTGATCAGACTAATACAACTTCTTTCTTAAAACTTTGGTG TATTTTTCCAGATTTTCTACAATGAACATGACCTCATAAAATCAAGAGTGTTTTTCGTTT GTTCTGTTTGGTCTTATGCTGACACTGGGCCAGGTGGAAGGGTGGGTGAGTCCGTCTTGC AGGTGGCAGGAGCCCCCTCCATGAGCCCCCACACCCCAGGGTCCCTGCCCAACGTGATCG GCCCTGCAGGGCAGTCCTGGAACCAGTTCAGGGCTTGGGGCCCTGGCTCTGCCTAGGGAG GTCTCAGGCAGGCCCCCCAACGTCTCAGAACTGCTTCCCTCAGTTGGAAATGGAGATAAT AAGGCCCACCTCATGGCACCCACAAAGAATGGGGCAGCCTGCACCTGGCCTCTCCCTGGA GGACCTTCTCACATCACCCCCGGAGGGACCACCATCCCCACCCCACAGAGGAGAAAGCCC AGCTCCAAGGCCAAGTTGCAGCCAGGAAGTGCTCTGCTGGCATCCTACCTGGCCCTGACG CCAGGCCTGAAGCCAGCACCACCCGGGGCTCTGTTGCTCCAAGGCCATTGCCCCTGCTGG CGTTTGCTCAAGGAGCTCCCCCTCCAGAATGCCCTGCCCTATTCCCAGACACACTTCCTC TGGGGGGCTCTTCTCCCCCAGCCCCACCCCTGCCACCCCTAAGGAGGCTGGCCCCTTCCC AATCCCTTCCTCGTCTGCACCACTGCCTTGCCCAAGGTGTCCATCCTCAGACCCAATGGG GCCACCATGGGAGCCCGGCTGGGTAATGGCCCATAAGCTCCTGCCCATGGAGTACTCAGG CAGGGAGGGGCACAAGCTTGGCTGAGCATGAGTGACAGGCTGGGTGGGTCTCAACCTCAC CCTGGGCCTGGGGTAGCCCCTTGCAAGTGAGGATGTTGAACACGGTGGACCCCCAGGCAG AAAGTGAGCCACGGGGGGCTATGCAGGACTGCAGCCCTCCCCTCCCCTCAATTCACTGGT GAGTAGCCTGCTACTCCCCTGGCTCCCAGGGCAGGGCCCTCATCACCTCGAGACCCAGAA GCAGTGATGGAGACCGTCCTGCAACATCAAGCTCCCTGTGATCTGGGGGCCAGGGGATCC TGAGTACCCTGCCTCCTGGAGTGCTCTGCCTGCAGGGGGCAGTAGCAGCACCTTCAGGTG GGGTGGTTGGGGTGGCTTCCCGGAGGAAGTGATAACTCAGCTAGGAGTGGGGCGTGGGGG TGGTCCTGACATGTACCAAGGCTGGGGGTCAAGTGTGCACTGTCTCTCAGCCAGGAGGGT GGAGGCGCTGGACCAAGGCTGGGGGTCAAGTGTGCACCGTCTCTCAGCCAGGAGGGTAGA GGCACTGGACCACTGGGGCCTCAGGTGTCCAGGTCACTTGTCCCTGGAGTCCCTGGCTAT GACAGAGGCCCTGGTGGGCAGCTCGGGGTTGGGGACAGGCCAAGGCATAGGGGAGGGCAC AAAGGGAGGTTGGTGCCCCACCCATCACCCATCATCCGCCTCAGGCCACCCCAGGGGACC GGCCTCCGCTCCGGCAGCGGGTGAAGAATGGACTCCATTAGGGCCGCCATCTGTCTCAGC CATGACGGGTCCTCGCGGTGACATCCGGGAGGTATGTAGATGGAATATCTTTATGGGGAG AGTTTTTCTTTTGCCAGTTTTCAAATAGGAAGCCAGACACTTGTCACACCTGTCCCTGTG TCAGCCTCTGCCTGACATAAATTGGGGCCGAGGCAGGGAGATGAAAGACAGCCTGGAGGC AGCCGGGTGCGCAGGGCCCATGTGTCAGGCCGAGGGAGCACCAGAGCTGCAGGCAGCCTC CTGCTGGGGCCAAGGAGAGCTCGCCCCGCAGTCCACCTCCGCAGCCTCTGCCCCTCCCTC CACTCCCCTCTGGACCCACAAGTTCTTCACCTGCCCTCCCCCAACCCAGAGCCCTCCAAG CATCTCACCCGGGCAGTGGCCTGTCCTGCAGCCACTGGGCTTCTGTTAGGGGACACTGGA GCCTCCTCAGCCCCCAGGCCAGCAGCTCCAGCTCCTCTTCCAGACCTGGGGCCAGTGTGG CTTTCATGAGCGGTCAATGTGGGAGGCTCAGAGTCCTCAACCTCACCCCCGCTGCCACTC CTAGGGACCTTGTGGGCTTCAGCATGTTTCCCTGGAATCTGGGGGACTTGGGGGTAGAGC ATGTCAAGAACCTCAAGGTCCCTGCCGCCCAGCATGGGACTATCTTAGAACTGATGCCAG GAAGAATTTGCTAAGAGAATGAAGGGAGAAGCAAGTGACCCCTGTGGGCCTCCCCTGCAT CGGGGACTGGACCAGGGCCCACTGGGAGTGCATCTTGGTGCCTGAAACTGCACCAAGGAT GAGGAGGGGAGGACCCTCCCCAGGGGACACTTGGGGTTCACCTGGCAGGCCACACTCAAG TCCACAGTCCCCCATGGCCCCAGCGGGTGGGGCAGGGGACGCACCCAGCTGAGGGAACTG TGGCAGTCCTGCTGTTGTCTCACGTGGCCCTAGGGGGCTCATCTGTAGCCCCCACGGGAG CCCCAAGAAGCCAATCCCTGCTGGGCTCATGGGCTACCACCTCATACAGCCCCATCCCTC GGGAGGCCCTGAGGAGACCCTGGGGCTGGGCAGGGCGTGACCGGCCAGCTCCATGCTTCC TGACAAGGGGGCCTGGATGTCCAGGGAGAGCCAGGAGGGAAGCGGAGACTCAAGTGGAGA GTGTGTGGGAGGGAACGTTGCTGGGCTCCACAAAGGAGGGGGCACCAGGGCTGACTCCCA GGTTCCTGGTGGCATGCGTGGAGGCAGGTGGGGCCAGGAGCACCAGCGCAGCACATCCTG CGTGACCTTGGGCCAGCTCCTCAGCCCTCCCTCGACAGCCGTGCTGGTAGCTGGCCAGGC CCGGGTGAGTGAGTTCCCCAGGTAGGTGGAAGGGGCTTCTGGGGTAGGGGCCACGCCCGG GGGCAGGTGGCCCACCCTCCCAATACCCAGTCCCACCCCGCCCTACCACAACTGAGGACT CTGCCCAAGGACCAGTCAGGGACGAGGCATCCTCTCCAACTGCACTGCAAGGCCAGCACA AGTGAAGGGCGGGACAGAAGCCTCCACACAACAAAGGCAGAGGACCCCATACTTCTCTCT GAGTCAGTGCAGAGGTCCTGCGGCAGAGCTCCTGCTGGAACCCAAACTGCGCCGTAGGCT CCTCACTGAGAACTAGGGACTTCTCCACATGCACCCAGCCTCCCCTCCGGACTCCACAGC TGCTAGCCCTGCCCGGACCCTGTGCACCCCACTCCCACCCCGTCATAGCCTCTCAGCCCC ACTGTCATTTGGGCTCCCCTGGCATGAACTAACGTGTCTCTCAGGATGTCCTACGAGGAC CCTCACGGCAGAAGGACCGATGGGGAGGAGTGGCCCCAGAGAGCAGTTCCTTCTCTGTAC CCACCAGGGTTCAGCAGAGGCTCCTGCTCCAGGCAGTACAGTGGGGCCCACATGCCCATC CTCACTGGCCACATGGGCTGGTCAGAGTCCAGCCTGGATCCCCCCAGGGCAGGACAGGAC AGGTAAGAAGGGCTGAAAACCTGGGAGGCCTCCAGGAGGTGAGGGACCTGCCACCCCTTC CCACAAGGCCCTAAGGCCCCAATCACTAGTCTCGATTTCCACAGCTAGTTTAGTCCAGGC CAGGAAAGGGCGTGCAAGGGGCCTGGCCTGGAAGAAGGGGTCCCTGGAGCCAGGTGCACC AGGAGCCTCCAGCTTGCCCAAATCAAGCTGCCACTATGGCCGGGTCCTGTGGGCAGTGCT CACTCACCACCGCTCCAGCTTCACGATAGCCACGTGCGGACAGGGAGGCTCAGAGGGTAG GTGGCTCCCCGGGGGAAACAGAGCAACCCTTGTGCAGCCGGCTGGGCCAACTGATCTGCA CCTAACAGCCAGTCTTGAAGGCCACCAACCCTGGGGTCTCCCGGAGCTGACCCAGGGACA GAAGGGCACCAGGCACTGCCCACAAAGGTGCAACTGATCCTCCTCACCGGCGCCTGGGCC CTCCCTCCCTGCCAGTGTGGCTGTCCCACCCTTCATGGCTTTCTGCATCCTCAAGCTAGG CCTGGGCTCTTCAAGTGGAGAGGGGTAGGCAGACACAGGTGCCAGGCACTGGGAACTAGG GACGGGGGCTTCCCTGGTGAGGATGGAAAAGAAGGTTCCTACATGAGACCCTCTAGCCCA GGCCCTGAGAACGAAGGGGCCAAAGCTTCACCTAGGGGCAGCCATGGGGGAGTTTAGGCA GGCAGTGCCTGGCTGGAAACATGTCTTAGGGAAGCACTTGAATCCCGGGGTGGGGAACAA GATGGTAGGCAGGGAGCCGGGGCCGGCTGTGGCCACAGGCCGGGGACAGAGGCCAGGTCT GAGGATGGCCCTCAGTGAGGAGTAAAGGGGATGGACCGGGAGACAGGGTGCAGGTTTGAC ACTGATTTTGCCTTTGGGGATGGGTCGAGGGAAGAGGCTGAGACTCTGGGCTTGGACGTT GGGTACGGAGGATGTGACACTAGCAGACAGGCTGGATCTGGAGGCCCAAGGTGTGTAGTG TGGGGAGCTGTGTGTGCAGGAGAGATGCAGGGCACTAGGGAGCCTGAACCCCTGCTCCAG TGCACAGGCCGGGCACACGGCAGGGAGGAGGCAGTCACCATTCCCTCACTTGCCGGGACC TGGGAGGCAGGGCAGAGTGGGTGATGGGCCAGGCTGGGGCCTTGGAATGACATCTGGCAT GTGGCAGCGGAGGGGGGACATGTAACTGGGGACTGTGGACTCCCAGCTGGAATCCACCTC CACCTTCCACATCCACGCACTGCATTTTGGTCTTTACTGAGCACCTGCTGTGTGCAGGGG CTGCTCTAGCCCCTGGGACCACCAAGGGCCAGCCCTCCACAACCCCAGACCTGCACTCAG CCCCCACCCACTTCCTCCAGAGCCCCACACCCCACACACTGAGGACTCTAGGCATTTTCC CACTCTTGAAGACACTGAGCTGGGAACACCACCTCTGGCTACCAGCCGTGGACTGCAGAG AGGCCTACGGTGACCAGGAAGGGTGGAGCTGCCCAAGGTCACACAGCAAGCCAGCACTGC ACAAGCTTACCCATGGCTGCCCAGCCTTGGGGAATGGGGGACTTCTGGCATGTCCCCGAG CTGGGCCCTGGCAGAAGGCAAATAAAGGCAACATCTCAACAAGGTTGTGATAGCCGGGGA TCCAGGCAAGCAAGGGACAATCCAGGAAGGCTCCCAGGAGGAGGTGAGGGGCCTGGAGCC CACCCTGGCCTGAGCCCCTCCTTGGCACTCCAGCCTGCCTAGCCCTGCCCCCTTCCTTGA CGGGCCCCTCACCCAGCCTCAGCCTTGGCCTGACATTTCCCGTGACATTTGCTGCAAAGA AATAGAGTCATAAATCCAAAGGTTAAATTTGCACATCGCCTCAAATTGCTGGGAGAAGCA GTGGTCCCTCCACCCACTGGGCGCCCCGTGGGTGCGGGGTTTGTGTGGCAGGGTCTACCC ATCCCAGGGCCCTTGCTGGAGGCCCACAGGTCTCCTCCTCTGGTCCTCCGGGGCACTTCA ACCACTGTTTCCCAAAACAGGTTCCTGGGCACACCAGGCCCTGAGGTACATCGCATAGGG GTGCCTCTCAGAGAAGGTGCTGCCCACCCCTCTCCAAAACAGGGCCCAAGACGCCCTGCG GGAAAGGAGCACCATCAGCACCATTTATTCCAGCAGGTCCCACATTTGACCACAAAGCCC TTATGTGATACCCCCAACATTCCAAAGAATTTCAACTCCATGGACCGTGACCCGTGACAG CCAGCTAAGAATGCCAGGGTGCTGACTGGAGAGCCAAGAGGCACCCGGGGGCCCAGGGCA GGCTGCTCTGAGTGAGGCTGGGTCAGCACCCGACACCTCACTCCCAGATCAGCCCAGCTC CATGTGCCAGGGCCCTGAATGGGGCTTTCCACCTGCCAGGCCTGTGTAGACCTACATGGG GACCCTGGCCTGTGCCCACTCAGCCCCTCCTAAGGCCACTCTCGAGGTGTGCCAACTGCC CTGCCTTTCTTCTCCACCTCCTGGTCTGGCTGCTGTGGGTTGCCTCCATCGGACAGGACA CAGCTGCGGAACAGTGTCAGCATCACCCCATGCCCCGCTGCCCTGCTGCCCCGCAGAAGA GGCCCCGCAGGCCATAGCAGGCAAGACCAGAGGCTGTATGAAAGAGGCAGGGGAGAGGTG GCCCCAAGGCACCCACTGCACAAATGGCTGCCCAGACTCTACTCACTCACCATCACCTAC CCGGCCAGCCCACGGTTGTCAAAGCCTCAGTGGGCTAGGAGGGGCAGGATCTGGCCCTGC TGCCGAGTCCCCTGAAATCAGCCCAAGCATCGGGGGAGAGGCACGTGACCAGACCCCAGA CCTGGTGGGTGCCAGCTGAGGACATGGTGGGGTGGGTGTGCAGAGCTGCATCTCCCAGTG TCCAGCTCCACGGCTGCAGGCAGTGCAGGAGGCCGGTGCAGGTTTGGATAAATAAATGAT GGTGCATTCTCAGGAAGGGATTCCACACACACCAGAAAACCCATGATGCACAAGAATAGT TCCCGATGTGCAAAATGTTTAAAATACAGCGATAAAAGAAGAAACGGCAGCACAGAACAC CTGGTGTGGGGGAGCCCAGCTGGGCCCCAGGGACAGTGCAGGGAGATTTGTGGGGTGGGG CAGCATGCCCGCCGCTCCGATCGGGCTTCCCTGGGCCACTCTGTGACCTCCTATCCTTTA AAGCATTTAATCCTAGAGATGCCCGATGCCAAGGATCACACCCCTTGATTTGCCTGGCTC AGAAGAGAATCTGACAGGCTAGGAACGAACGAGTGAATGAATGAACAAATGAGAAAGTGA ATGGATGGTTGGGGCAAATGCAGCATCTGGGATCTGGCTTTGAGGAGGGTGCCTGTGACC TTGGCCTGGGTGCAGCCAGACCGCAGAGCAGGGACACTGAGGGGTGCAGAGAGGGACCCG AGAGGGCATCTGCACAGCTGCAGAGGCAGCAGGCGGCCAAGCCAGCTCCCGCAGACCCTG AGGCCCAGGCCCTGGGTGGCTGCAGAGGGTCAGGCAGACCAGCCTGGGGGTCAGAGGTCA GGCTCACCAGAATGGGGGCGGGACGGCATGAACCTCATTGATCAGATAGTAGCCAAGTGT CAGGAGCCCTCACTAACCCTGTGCCAGGGCCGGGCGGGGCGCCTGGCTCACACACGCATT TGTTCATTTCCAGGTCATCCTGAAACTCCCCGTGTGCTTCCTGCTCTTGACCCCTTTCCC AGGTGCACAGACCAAGGCTCCCGGTGAGAGGTTCCGGGCCACAGGCCCAGGACAGCAGGA GGGCTCCTCTGCCTCCCGCCCGTCCTGTCCTCACCAGCTGGACCCTCCTGGTGTTGCACC GGCTGCACAGGGCCGTCCGTGCCACAAAGCATGAATTCAAGGCTGGGGCAGGGACGAGGA GAGGGCAATGCTACAAGGCTGCACTGTTCCTGGGCAGAGGGAGGGGGAGAGCACGGTCCC ACACACCCTGGCCCTCACAGCCTGCCTGTCACTCACAGCAACAGCAAACAAACCCTGGCC CCTCCACAGGCTGTGGGCCAGTTCCCAGCCTCCCAGGACAGCAGGACCTTCCCCCACCTT CCAGGGCCCCAGCCCCTGTAGGGGTGTCCTCCATGGACCCTCCACAAGCCCTCTGGCTTT CAGAGACGTGCTGGGCCAGGCCCTCCCTCAGCCTCTCCCCTGCTTCTCACTCCATGCAAA CCTACGTTTCTGCCAGTCCCAGCAGAAGGACCCTGGCACGGGGAAACTGAGGCTCAGGGG TTCCACAGACCCCCAGGTGAGGAACAGGGGGAGGGCAGGGAGGGAGTGAAGGTACTATGG GAGGGTCTCCCAAGCTGGGCAGCATCCCTGCCCTGTCCCCTCTCCACTCACCTGTGGCCC GAGTTGCCTGACTAGGGGCTTTTATTGTCCCTTCTGCACATGGATGGGGTGAGGGGCCAG CTGGCAGTGCAAAGGTCCCAGGAGGACTCTGCTGAGTGGGGGTGTGGGGAGGTGTTGTGG GCATTCTGAGGCAGAAGCACCAGAGTCAGAACTCCACAGGGAAAGCCGGGCCAATTCCGC CCAGCCTCAGCCTGGAAGTGCCCCGATAATCCCTGACCACTGGCCCATGGTGCAGGTGAG GAAGACTGAGGCAGAGAGGCGACCTGTCACACCCAAGGTCACACCGCCAGCAGGTGGCTG AGCCAGGACCCAAATCCAGGTCACGGTTCTGCCACGGCAGGCCCTGCACGTGCCCCTCTG CCAGGTGGCGTCTAGGTGCTGATGGCCACCCATTCCCACCCCCGGCATGGGAGCTCACTT CCTCCTCACCTCAGTAGAGAAGAACAGGGAACAGTACCCCCTTTATAGATGGGAAGACTG AGGCTTAGAAAAAGAGACTGGCCAGCCCCACTGCTTCAGCTGCCCCTCTGCTAGTATGTT TCACCCTCACACAACCTACAAGGATAACCTGCTGGTCCCCACTCTATAGATGAGCCTCTG GTGGTGGCCAGGGGAGCAGGAGGTGTGGCCTGCCAATCCAAGCCCCAGCCCAGGGCCCTT GCCACAGTGCTAGTGGGCCACCCTTGCCCATCATGCAGCATGGCATCCATCTGCTCAGCA CCCTACACAACACCGGAGGTAGGGGAGCCCACCACAGAACCCCTCACCAAGAAGGTCCTC ACCATGGAGAAACTGAGGCACTGGAGGCGGGGCCTCTGCAGTCACGCTGGGTCACGTGCT CCAGTCAGCCAAGCTGCAGCGGCCACCAAGGGTGCCCAGGGTCTGGCCTGTTGGTCACAG TGTTGACCTCATGGCCTGAAATGGGTCTTCCCTGCCCAGACAGGTTCCAGCCCACCATCA TCTATCTGCCTCAGCCCTGGTGGGTAGGGAGTGGCTGGGAGACCCTGCCCCTCACACCCT GGGCTTTGCGGTATTCAACGAGCCCCCAACACACATTCCAGGGCCTCTGCTCCAGATTAG GCCACAGCCTGTCTAGGCACACAAGGGGCACCCACACCCTGGAGGCAAAGAGACATGCCT ATGTGCCCGCAGGCACACATGATCACGCGCACAGACACACGTGCAGATGCATACCAAGTT ACACACACGCACACAAGCCCGAGGCGGGGACCCCCAGGACACGGAGGCAGGCAGGCAGAG GTGGGGCCCAGGATGTCTTGCCCCTGTCTGTGGGCCTGGCCTCCCCACTCAACAGACGAG CATGTGGGTGCGGGAGTGGTTATTGCGAAAGGGGCCCCAGCATGTGCCACCCGTGGAGAC CACAGCAGGCATGCCGCGTGCATGCATGTGTGGCCGTGGGTGTGCAGGCGGGGGCGAGGA CACAGATATGCACCCTCAGAGTCCGTGTGTGCACCCAGTGTTTGTGCATGTGCACACTGG GGCGGCGTTGGCTTGCATGTGCACCAGAGGTGTGCAAGTGTGCAGTCCGTGTAGACAAGT GCACGTGCAAGGAAACGTGTGTGCAGCCTGCGGCACCATGGACATCAGCAGGGCAGCCTG GGTGGGCACCTGGGGAGCACCCTCCAGGCGGGGCATGCCCAACTCACATGGAGCCCACCC TCCCTTGCCCGCACCGCGGGACCTCACAGCCGGTGGGGCGGGGGAGTCCCACGAGGGCTG CTAGGGAGGGGTTGCTGATTGCCCCACCGCCCCTGATTCCCCCAGCCAAGCCTCTCAATG TCCCTCGGGCTGCAGATGGTGGGCGTTGCTGCCTCCTCTCACCGCCCGCCCTGGAGGCAG CGCTGGGACAACCCTGTCCCCCACGCCCTCTACTGCCGCCAACGCCCCCATTCATCCCAC TGTGGACTGACCTCGGAGAAACGGGAGGTTCTGAGCCCAGACCGTGAGGCTGGGGTGGAG GCGGCCCGACACCCCCACCCACCCTCGGTACCCACGTTTCTGCAAAAACCCGCTCGTTAA CCCCTTGGTGGCCGGGGCGCTCCGAGGCCGGGCCCCGCCCTTCCAGCCCACGGCCCCCGC CCTCGACTTGGCCCAGCATCGGGGACCCTCGGACCGCCACCCTCGGCCCTGCCGAAGCCG TCAACTTTCCCGGGAAGCGGCAACTTTTCCTGCCGCCTCCGGCTCCGCCCTCCCGGGGCC GGGGCGCCAGCGGCCCCAGCCTGGCGCGGCACTGCGGTGGCTTTCCCTGCCGCCCGGCTA CGCTTCCCTCGCCTCGGCAGCCCGGGACGGGCCCTGCGGCTCCGGGGAGGGGGCGAGCCG CCCCCCTCCGCGCCCCGCCGCCGGCCGGGCTCGGGTGCAGCGCGGACACTCACCTCCAGC GGACGCCCTCTTCATCGTAGGGGTTGGGCGGGGCGCGTCGGGGACTGAAAGTCGTTTCGG GGCGGGCGCCCGTCTCCCCGCGGCCGGGTCCGCATCCAGGCGCCGCCGCTACGGCCCGGC CCCGGCCCGGCCGCGGGACGAGGCTCGGCGCGCTCCGCTCCGCCCGGCTCTGGCTGGGCT CGGGCCGCGGGTGCGCAGGGCGCGCAGGGCGCACAGGGCGAGGGCGGCGGCGGCGCGGGG GTTGGGGCGTGGGCGGCGCGGCGGGCCGGGGCGCGCAGGCCGGGCCGCAGGAGGCGCTCA GCCCGCCCGGTGCACCGGCGCCGCGCGGTCCCAGACAAAGGCGGCGCATTCCTGAAGAGG CGGCGGCCCCTGCAGGCGGCGGAGGGGATGGCGGCCCGGGCCGGAGCCGGCCGGGGACGC GGGCCCGAGGGTGTGGCGGCCTAGGCTGGCCCGGTCCCCAAGCGGGCAAGGGCTTTGTCC CGAGGCCTAGGAATGGCCTCTGCCTGACCCACTCCCTTCCTGGCCTCACGAAACCCATGA ATCCCCCGCCCCCTGCACAGAAGCCCCTTGCTGCAGCACGATCGCCCCCATTTTTCAGTA AGAAACAGGCTCAGGGCTTAAGAGAGTTCTGGGGGCCCTCCTGGGCCTTTGTGGCCCTCT CTGCACCATCCTAGAGAAGGGACCTAGGCCTGATGTTACCCGTAACCCTGCAGGGGTCCC CCACCTCTTCCCTTCTGCGGAATGGGGGCTCCAAACCCAGAAGACGCTCTCCGAGGTCAC CTGCTTAGTATCCCCCCACTCTACCAAGCCAGCCCCCCACGGGCGGTGTGAGGCATGGGG CCAGAGAATCCAGTGACGCCCCTGCACACACCACACAGGGGTGCAGGTGTCCCTTCTCAA TCTTCTGCTCGGGGCACCTACCGGGCCTCACCGATGAGCTCCTAAGTGCATCCCGGGGTC TTCAGACAACTGCCAGGCTGCCCCTGCACCTGGAGAAGTGCTGGGAGTCAGCTCAGCCGG GCTGGGTCCTCCCCAGGCCCACCCTGCATGAGAGGACCATGCAGATGAGGGTCTTGCAGG CCCACCTGGTGTCAGTCTGTAGGACCCTGACTCAAGGACGGAGTGGGCCAGCCTGATGCC TTGATGGCCCCCCTCAGAGCCCAGGTCCACTGATATGAAGCAGAGAGACCTGGTGACTGG GCACCCCTGTGCCCTCTGCCTGCCCCTGGCCATGGAGGTGGCCCTGAGGGAGATGAGCAG GGCGCAGGGCCGGTGACCTGCCTGCCTTGTGCAGAGGAAGTGAGGACCTCCCAGAGCCCT GGTGCTCCCATCTGATCCCAGTGGCAGGCAGCTTCGCAGGGTGGAGCCATGCTGGGTGGG CCCTCTGCCTGGTGTGGAGGCTGCACTGCCCACATGCCTCTGGCTGCCGCTCTGTGGCCC TCATCACTTTGGCCTGCTTTATCCCTATCACCCTTGTCCCAGCCTAGGCTTATGAGGGCA TTGTGGAGTGCATGCCATTCCCAGCCACACAGAAGGTGAGGTGGGGCCCAGACACCCCCA GCCACTCTGTCCCCTGGCCTCCTGAAGGGGTGGGCTGTGCACACACTGCCACCTCCCCAG AGGGTGCTGGCTGCCGAGTGCCACCAGCTCAGCCCCTGAGGTGGCTCAACAGGTCCTCCC AGAGCCACACCAAGACGTCATCACCCATTGCACAGATAGGGAACCTCAGGCGAAGCTCAC AGAGCCAGTGGGAGGCTGAGGCCTGACCCCCAGACAAGCATTCTTCTCCAGGGTCCCTGC ACACAGCAGCCCCCAGGCTAGGAACTGAGCTCTCTCCACAGGGATGTATCAGACCCTAGA TGAGTCACGTGGCACTGACCATCAGTCTGAGGACAGCGCTGGGGTGGACCAGGCTCCAGT CTCGGGCTGCTCTGTGCTTGCAGAAAGGCCTTGAGAGGACACTTGCTTGCTTACTCCGCA GGCGTCTCTTCAATTACCAGTGACCCAGAGCAGCCCACTATAGCTGAAGATTGTGACCCC ACACCCTATCTCATAGGTTTAAAGCCCCTCCCACCCCTCCTGCCGCAGCCGAAGTGACCA TTCTGGGGCATGGGATGGGCATGGGGTGAAATGGGGAGCAGGGGTGGCCCTGGAGGCTTC TGGCACCAGCACCCTCTCCAGCCACCAACCTGGCTGTTCCCGGTTGCTCTGCGCTGGGAC CTGGGCCTCCACCTCCCCACAGGTCTCTGCATGGGAGCAGGGCTGAGGCCTGCAGAGGGA GCGCTGGGGGATGGGCTGGTTTCCTGTGTCCGCCACCTCGGTCAGCACGTTGGCCAGCAC CCTCCCCACTGGAAGCTGGGCCCACTCCTGGGTGCTAGCTGGGATGGTTAGGCGTCGGCC AGCGCTGATAGGGAGTTATGGGGGTGCAGAGGCAGAGTTGAGCAGGGCTAGGCAGCCGGT GGGTCTGCTACCAGCAAGGCACAGCCCACTTCTCAGGCTGCGCATGTGGGAGGGTTTGGT AAGCCACATTTTTCAGGGTGTAGCCGGGTGAGGGGTAGCATGGGGAGTGGTGTACTCTTT GGGCAGGCGACCACGGAGCCATCACCCCCCAGGGCCCAACGGGCAAGAAGGGGAGCAGCT GACGGCTGGTGAGGGGCTAGCAGGGAGGGCTTGACAAGGGCTGGAGCCCCTGGTCAAGGG GCACAACCAGTTCTATCGGGTCACCTTGTGGGAGGAGCCAGGGACCCTGTGGGCAGAGCC TGGCCGTCTGCTGCCCGGAGTCTACACCAGTCAGTTCCTAGGAAGCCCCATCAGGGGTGC TGGGAGGACAGGCCATCTGTGCCCAGCTGATGCAAGCTCCCAGGCTTTCTGGACACGTGG CCCTGGGGATCAGGAGTGAGCTGGAGACTGCAGACAGCTGTGCCCTCAGGGCCACACAGA CCTGTCACACCCTGCCTGGACTGGGTCAGCAGGGCCACACGGGCATCTGCCCGAATCGTT CCCCACCTCACACCCACCATGTGGGGCACATTCTTTGCCAGCTTCCCCAGCCCACCACCC CTCTACAGTGAGGCCAGGGGCTGTCTCACCTCCCACCCACACCCACACCCAAAATGTGGG GCTCAGCCTCCACGACCAGCATGCCGGTCATGAGGGCCCTGCCCAGCTGTGTGACCTCAG CCCAGTCCCTTGGCCTCGCTGAGCTTGCACAGCATGTGGACGAGGCAGACGCTAATGCCT CAGGTCCTGGTCAGCTGGTCACCATCCCTCCTTTGTTCACCTCGCCTGTCCCCTGAGAGG ATCATGCAGGGTTTTCACTCAAAACTCCAGGACTGCCCATAGAAGCCAGCTTGAGATGAG GGGAACTTTCGCGGTGGCCTGTCTTGGGCTGACAGGGACACGGTTCAGCCTCCTTGGAAG GTCACTGCTTCCCAGGGCTTGGGAAGACTCATCTCCTGCCTGGAGAGATAGGCGTGCCAT GGCTTTCCTTGGCATGCCTGTGCGGTACTCACGGACATCCAGGCCCTCAGTGTCTCCTGG GGAGGGATGGGGCAGCAAGGAAACTGGGTCTTTGCTCCACACATTCTGGGCCACTCCAGG CCAATGCAAGGAGAGACCAAGAAGGAAGCTAGAAGGTCATGTGGCAAGCACATGCCTGCT GCCCAAGTGTGTCAATGTGACTGACCTTCCCTGCACTCTGAGTGGGCGGCTGGGGATGTG GTCCCAGTGCCAGGGAGCTGGGGGGTGTTGGCCTGCCATCTGCTCTCACCATGGAGGGCG GCTGCATGCCCAAGGCCTAAGAGGACTCTGTGCCAGGCCTGCTGAGTGAGTCATCCCATT CAATCCTACAGTGTAGACACTACTACCACTACCCATTTTCCAGACCAGAGAAGGAGGTCT GGGGAGCTTAGGTGGACCCACATCTTGGCAGCCCCTTTCTGCCCTCTCTTTGTCCACAGC CTCCGCCTCCTCCAGAAGCCTCTCACCATCCCCCATCCCCGGGGCTGCAGAGGGTGTCCT GGCCTTCCCCTATCAGCTCTGCACCCCTGAGGACTCCCATCCCTAGGAGTCCCTCTGCCC AACTTTGGTGCATGTTAAGCCATAATTAATCTGCTTCCTCATCTCTTATTCATCAAAGCC ACAGCAGCAGCAAAGGGGGCTGGCACAAGGAAGGGACCGACAACCAAACCCTGGCAGTGG CCCCAAGAATCCCACTGGGCCTGCCCATCTCCAAATGCCTCTGACCCTTCACTAGCGAGG GCCTCCTCCCACAGAATGGCATCCTGGATTGTGCCACACTAGGCAGGCCCCCTGACGGAC CATGCGCACCTCCCACCACCTGGAGGCTGCTGGGGACCGTCAATGGGACGTATGCATTGC ACAGTGATGGCCCTTACCTCTGCGTCCCCCAGCCCTGGACTGGATCCCTTGTGGCCCATC TCCAGGATGGGAGCCTGAACGGAGATGAAAGGACACTCGGCCAAGTGGGACATGCCAGGC TGGCACCTGGACCCTGGTCCGCCACTCTTCCTGGTGCCTAGAGGAAGGGGTGAGTCCCCA GGGCTCCTGGACCTTGAGATGAGCCTCGGGCCACTTGTGTTTGCCAGCAGATGGGCTCAG GGTCTTCTCTCAGTGGGCACAACCGAGTTGGTGGCCTCTCTCTGGGGAGGCCGGCTGGTG GGGGCTGGGGAGTAGGCGGCTTCTGGTCCCTGAAGGCCCAAATCTGGTGCAGGGGCAGAG CTCCGGAAGGCGGGGGTCCCTGGGCTGTGCCCAAGGCATCCGCTGTGGCTTCACGCCCCT CCCCCGCCCCTGCAGGGAGTGTGGATCCTTCTAGAGCCACCCACCTGGCAGTGACAGCTA AATTGGGAGCCGCTGTTCACACCAGGAGCCCAGCGGGCAGTCAAAACCGGGTCGGGGGGG AGGGGCACCCGCTGAAAACACCCACAAGCTCCCAGTGGGGCAGGGAAGGCAGGGAAGGCA TGGTGATAGCTGTCAGCAGGAGTGGGTGGGGAGTGGAAGGCAGGGTAGACCGCCAAACCC ATCTTGGGGCCATAGTGAGATGGCCCCTGGGATGCTTCGGCTGGCCAGTGCCCAGCCTTG CTCTTGTAGGAGCTCCCTGGGCCCCTGGGTTCCCCACTCTGGGCCACCACAATCCATGCA GCATGCAGGTTTGTTCCGTTTCCCAGATGGAGAGGCTGAGGGCTTACCACAGGGTTCCGT CAAGGACGCAAAAGGGCCAGCCTGACTCTGAGCTCAGTAGCCCCACCCTCCACAGCCTCC CTTGCCGCCTTCATCTGCTCACTTCCTCAGTCACTCAACAAACACTCTGGGAGGCAGACT GTAACACCCTGCATACCTCTGCTGGACCCCCATTCCACATATCCCCACTGGTTACAGGTG GGGAAACTGAGTGCCTGGGAGGCGGGCCTTGGCCTGAGTCCCCCAGTCAGTGATTCCAGG ATAAAGGGCATAGCCATGCCATTTGGTCACTTGAAGCCCACAGGGGTGACTGGGAAGAGC CCACCTAGCACCCCACATTCTGCTAGGGTGACTCAGACCAAGTCCTTGGGGCTCTGCCCC CCAGCCAGTGGCTCTGCCTCTGAGAGCCATGGGACAGGGAGCTGTGCAGGGTGGGGCCAG CAGAGCCTGTGCCCCTGCCTGAGCTAGTCGTGATGGGGCCTGCCCTGACCCCTTCCCAGG CTCATGGGTGCGGAGCAGGGCGGGGCCTTACAGTGAGATGGGGGCCTCTGTGGGCAGTCC TGGAGGCCAGGCCCTGGGGCTGGGACTCACTGGCACAGGGCCTAAGTTTCCTCCTGTGTC CGCAGGCCTTATTGCCTGCAAGGGAGGTGGAGGGGCACAGGGGCGGTGCCCAGCGTGAGG AGGGGGCTCTGAGGAGGCTGCAGCAGCCGGCAGGCCTCTGAGACTGGGATATTTTTAGCC AGGATTCCTGCTGACGGTCCAGGCTGGCCGGGGTTTGCTGCCAGCCTTAGAGGCGCTTCT CCTAGTCTGTGTCCGGACATCTGGGGTCTGCTGGCCCCACCCACCCTCTGCTCAATGCCC CCGCCCCACTGGCCCATCCAGCCAGGCGAGGGGCTCCCAGGAGGCCGGCCATGGTGGGAG CAGAGCCTGGGGCCCTCGGGGTGTGGCTGAGAAGAGGCCACATGGGCTGCTGGGGGCTGC CTGTGTCCTGGGTCTCCAGCTCCCAGCTGCCACTCTTAGCCGACCCGCTTAAGGGGCAGG TTTCTGGAGGCTCCGGTTCATGCCCAGGCTCGAGTTCTGAACTGAGCATTCTCAGTCCGC TGCCTGCTGCCTGCTGGAACGGATGGCTCAGCAGAGCGGCCACAGCTTGGCTCCCTCCTG GGCACTCCTGCCTGCCCTTGCCGCTTAGGTGCTCTGGTCATCAGGCCTCTCTGGCTGGCT CTACCAAGGTGCATGTGTGCAGGGCCTGGGCTTGCAGGGCCTTGTGGCTCAGCCAGGACC ACCTCCCTTCCCATGGGCAAGTGAGGACATCCAGAAAGAGGGTCCTCTGCCTCGGCCACT CCCTTGGGACACCCCTCCACCCCAAGGCCTCTCGTGGAAACAGAGTCCAGCACTCAGCGT CATGCCTATGCACTCCACCGGCAGGACTGGCTGAGTCCCGAGGAGCCAGGCCCTGGGGCA CAGGCCAATCGGATGGAACCCAGATGAAAGCCCAGTGCTGGGACCCACGGCCCATTCTCT GGCCTGGCTGAAGCCTGATGGCTTGTTCCTTGAATGAATCAGGAGGCCCATCCAGAGCAA TTGTGAAGTTCACATGAAAGAATGAACGTACGGAGAGAGATGGGCCATGTCGCATCCTGA AACGCCTTATATTCTAAGACCATGATAATTAACACAGACCCTGGACAGTCACACGATGGG AAAGAGTCCAGAAAGAGGCTCAAATGCAAATATGAGAAGAGGCAGGTCAAATCCAAGAGG AGAGATGGAATATTCAACAAATAATCCCACATATACAATCAACACTTGGCCTTAAGACAA CGGAATTGACATATGGAGGAAACATTTGGATTCCTACCTCACTCCTTGACGCTAAAACAA GTTCCAAGTGGAACTAAGATTTAAAAAGTAAAAATGAAACCATAAAACTCCTAGAAGAAA GCATGGATGGCAAAAAACCAATCTTGAGTCACTCTAAGAATTATACAAAACCCAGAAGCC ATGAAGTAAAAGATGGGTATATTCAACCTCGTGAAAATAAAAGGCAGATGCATGGAGAGA AAAAAACCACCATAAAATGATAACCAACAAAGGGTAAAAACATCGGCAACTCAGTTCACA AAGGGCCGTTTTTGGTTCTCAATACAAATCAGTAAGAAAAAGAGCAACAGCCCAAGAGAA AAATGGGCAAAGGCATGAAAGGAACCTCCCATCACCCCCTGCTGACCTTCACTCAGAGTG AGAAACATGCGGATCTAAGCTGCAGTGAGATGCTGCCCCTCACCCACAGGGATGGCAGCC GCATCAGGCTGGGGGTTCCCTAGGGGGGCTGCTTAGGGGCTCCCACAGGTCTGGCTGTTG GAGGGCAGGGAGAGGCCTGCGGGTCTGGTGGGGCCGGGGTGAGGCACTCACAGATATGTC TAGTGGGGGTGAGGAGGGGGACCGCCTGCAGGTACAGCTGGGGGTGTCAGGTCGCTGGGG ATGGGGTGGAAGGAGAGGATACCCATTCAAAACAGGAAATATATATATTTTAAAGTAAAA CAGGCTAAGTGCAGTGACTCACACCTGTGATCCCAGCAGAGCGGGGAGAATCCCTTGAGG CTAGAAGTTCGGGACTAGCCTGGGGAACATAGTGAGTCCCAGTCTCTATAAAAATTAAAA AATAAAATTATCTGGCTGTTACGATGCACGCCTGTAGTCTCCGCTACTTGGGAGCTGAGG CGGGAGGATCACGTTAGCCCAGGAGTTCGAGGCTGCAGTGAGCCATGATTGTGCCACTGC ACTTCAGCCTGGGCAACAGAGCAAGACCCTCTAGAAAAATAATTAAAAAAAAATAAAAAT AAATATAAAATAAAATAAAAACCAGCTGACATCCATCCTCTCCTTGCCACCACTGACATG GCCGTGGGCCGAGTTCTCATGGCCATCCCACCGCGAGTCCCCAAGCGCTCCTGATGCCTG CTCACCCTTCTTGTCTTTGTTAGCGCCCCGTTAGCAGAGGCCTGCTTAACTTGTCTCTGG TCTTGCTTCTCAGGAGCCGTGTCACATTCAACAGTGGCAGCACAGAGGCCCTGCTCAGCC CCAGTCCCTCACCCTAGGCCTCCACTGCCAGATGTGGTCCTGGTGGTCCTGCGGACTCAA GCTGTCCCCCCCTGCCCAACCTTCACAGGCCACCAGCAAGTCCCAGGCCTCCCACAGGCA TGGCTCTGGGCAGTATCTTTACCTTCCCAGCCTCAGTTTCCCTGATGGTGAAACAGGGTG AGATCTACTTCCTAGCAGAGAATCCGGGGGAATGGAGGCCGTGGGAGGAAGAGGACTTGA GGAAAGGAGCATTCCTGAGGGGTCCCAGAGCGGGCTGGGGCTGACAGGACTGAGTTTGCA GGGCCTGGGGACATTCCCTGGGGAGGTGCTAAGATGCCGGTCAGCTAGAGCAGCGGGGAG TGATCCAGGATGGCTGCCCGGATGAGGGGCTGGGTCGCTGGTCCCTGAGTGCCAAGGTGG TCTGTCCATGTCTGCAGGAACACACCCACCCTGCTGCCTCCAGGAGGGGTGCTGGACAGG CCTGAGCTCCAGGATCATAGGAGGCAAGAGCCACTCTCACCAAAGAACAGCAAGTGTGCC TCCCCTGCCAGCAATCTGTCCGGTCCAGCCCCTGCCCAGCTATCTGGGGAAAAGGCCACC TTTTCAGTGGAGGCTGAGCTCAGGGCAGGAAGAGGTTGAGAGGGGAGAAATGGAGTGAGA ACTGGAAGGTGAGAGGTGTGAGTGAGAGGTAGGGGTGTGAATTGGGGGTGAGATGTGGTA GGTGAGAGCGGCAGGTGAGAGGTAGGGATGAGAGGTGGGGGTGAGAAGTGGAGGTGGGCA GTCATGGTGATAGCTTGTGGGGTGAGAAGTGGAGGGTGAGAATTGGGGTGGGAGTTGGGG TGTCACCTCCCAAACCCTCTCACCCTCAGATTAAGAAGCCCCTCCTATGGGCCCTGGAGG AACAGGCCTTTGACCCACCCCAACCCCACTGGTGTCTTGGGCAGGGAGATCTCGAGGTGG GGGCCTCAGCTCCCCGTAGGACCGTCTAGCCCTGGACCACCTGACCACAGCTACAGTGGG ACAAGGCCCCTGTTGCCTGTCTGCTGGGGGCCGAGCGTGGTATGGAGGAGCCAGCAGCCC CCGCCTTCATCACAGAGGAGGAATCCGAGGCCCAGGAGGGTGTCTCTGGCACCCAACACG TGAGACCCTTTGAAAATCCCCCGAGTGTGTGGGGGGCTGGGATGAACATCTGGGGGCGGC AGCAGCAGGTGGCCCACCTGGCCAAACACACTCCTGTGTCCCCTTGTTTTTGGCAATTTC AATGACCAAAATCAGATCCTGGTATGGAGAAAAAGACATCGGTAAGGACGAAGGAAATGT GAACACTGCATGGACGATGGCTGATAATAGTGTGCCGCTATTGGCTCAGCAACTCTAACC TACCAATGCCAGATGCTCACAACGGTGAAACTGGTGTGTGGTAGATGCGATCTCTCTGTA CTGTCTTCTCAATTTTTCAGCAAAGATAAAACTGTTCTAAAGTTGAAAAGTTTATTAAAA AAAAAAAAAGAAAAAAGACGGGCTTATTAGCAAAAATCTGAGATAGAACCAAGCAGTAGG GCCCAGCCCACCCCCCGCTCAGGGCGCACCAGCAGTACCCCTCTCTCAGGGACATGGCCA GGCCTGGGCCACTCCGAAGAGCTCTTCCTGGTGCTGGGGGGCCTGGTGGGTTTATTCCAC CTGTCAGTCTCACTGCAAAGTGTGGCAGTGGAGTCTGAGTTAGGCTGACTCTTACCCTGC CAAGGACACCCCTGCACTCAGGACCCAGCCCCGGCCCCCTTGCCCTGTGGGCTGGAGGCG ACCCTGTGTGCAGATGGCAGATGGAGGGGCATGGTGCTGTTGCTCTCAGGTCAGGGGCAG CAGCCTCAGCCCTGACCAGCCCTCTCCTGCGTGGTTGTGGTTGGGTCTCCTGGTCTCTGG CCTCCTTTCCATTCCAACCTGTAGAAAACAGTGTGGTGGGGGATGCTCGGCCTGCAGATC AGACAGGCCGGGGTCAAAGCCCCGAACTACCACCTGAACTAGTCCTGGCCGAGTGGCGCT GATGCTCTAGGCCGAGGGCCCCCTTGACAGAACAGCCATCATCTAGCTCCGCCGCAGGGT AGGGGAGGTGGCAGTGTGGGCCGCAGCCCTCGGCTGAAGCGGGGGCCAAGCGGGGCTCTG CCTGGTGCAGGGGGTTTCCTCTATGGGGCAGGGGCTCCCAAATTAGCCCTGGTGACATCT GGAGCTCACCTCTGGGGGCTCTGGGGGCTGAGAGAGCAGGCGGGGCCCAGTTCTATCAAT CATCTGCCTTGTTCCTGGGGGATCGCGGTCAGTCCCAGCCTCCCCACAAGTCCTGGGGCG GGGCAGGCCTGCTGGAAACCCAAGATGCAGAGAAGAGCGCCAAGCACCTGAGACAGGGCT CTGAGCACGGAAGGCAAGGACGCCTCCCTCAGCCTCAGCTGGGGAGCCAGTGGCCCTGCA TGCACTTTCTCCCGTCTGGGGAGCCTGAGCCCTGACCCAGTCCCGAGGCCCATGTGTGCA CAAAAGCCCCCTGCTTTCTCACCCAGGACACTGGCGATCAGTGGGTAGGCGAGCCGGAGC CCAGCTGCTGCGCTCCCTTCCCTGCCAGGACCGGGCCAGCTGCATCCTCATGCCTGGGTT TCAGGGCTTCTCGGGGGATGTGATGGCACTGCCTGAGGATGTGGGATCCTTCCACTGAAA AGGGCCACACGGCTGTCCCCAGCACCCTTCCAGAAGGGACAACCCACTCCTGGGAGGTCT TAATGTAGACATCGAGGTCCCCCGAGCTTCCTCCATGGCCTGGCCACTTCACAACCATAT TTCTTTATGTTCCACGCGCCTTGCCTGCTCCACGGCCAGGACCAGGGCCACCATCTTTGC AGCCGGTGAAGGGACAGCTGCCGTGGGCTGGGCTGCTGAGGCAGGACAGGGGTCCGGGCC AGGGAGGGGTGGATGGAGGCCAAGGATGGGCCCCTTGGAGACAGCCTTGGGGTGCAGCCG GCTCTGCCACCTCTCCCTGGCTTCCTTCCCTCCAACCTGTGACTCCTGGGAAGTCCCTGA TGGGCCAGCCACTTGGGGCCGGGAAGTAACTGTGGTGTGTGGTGGTCACCCTCACCAGAG TTCATGGGGTGGAGCAAGGGGGAAGAATCTCCCCCAAAATCAGGTGTCTTGTTCCCAGGC CCAGCACAGAGACCTGGCCCCCAGACATCTCTGGACATTTGTTAAGAGTGGACAACTGCT TCCATTACACAGGAGAGGAAACCGAGGCTCTAAGGGGTCCCAGACTTGCCTGGCTGCCAC AGTGAGCAGGCAACAAATACCAAGTGGCTGCCGCAATGGGGTAGGTGGGAGGCAGTGGCT GAACCAGAAAGTGCTGCTGTCGATTCTGCTGGTTGCATGAGTCTTAGGAGATCATCAAAG GGCAGATTGTGGGGAGATAAAAAGTATGAGTGGGGAACAGGATTGGGGGGAGCAGAAGAG CCTCAAAGACCCTGCGACATTCTCAACGTCTCGATGGTGACTCAGAGATAGCTCCAACCC GCATGGCGGGCAGAGTGCGGAGGGGACGGCTTTGTGGCTGGATTAGTCATCCCGGTGCAG CTCACCCCAAATCCCGGCTTCCAGCCCTGGGCCGTCCATGCTGGGATGGAGGAAGGGCAG CCTCTCCGCAGGGCCACGTCCTGGAGCCTGATAACCTGGCCAGCACACTGTGCAAATGGT GCATGAGACAGGGAGTTGGCTCTGTGTTCCCCACGGTGGCCAAGGGACCAGCTGTGCCTC AGTTTCCCTTATTCTATCAAACTTTGAAGAAGGTCCCGTAAAAGCGCATCTGTTTCCAGG GTAGGGGCCAGAGTCAGCCACCCACACCAGAGTGAGAATTGGTGGGTGCCCTAGCCCTGG CAGGATCTGGCATTTTGCTGGACGGAGAGAGGAGGCAGCACCCACTGGGGCTCGGGCAAC CATCCCGGCTACCCCCGCCCCGGCCCGCCAGGAGAGGAGGGAAGCCTTGAAGTGCCAGGC CTTTGAATCGCCCATCTCCATGGCAACGCGTGGGCACAAAGGGCCGGGCCGGCGAGCAGG CGGCGGCTGCGCAAGCTGGAAGGAGGAAGGGAATCTTTTATTTATGGGGAGGGAGAGTCG GGGAGCCAGGCTGCGCCCACGCTCAGGGCCAGGCCGCAGTCTTGAGGCTGGGCCCCCTGC CCCATAGCCAGGGCCTCTGAGCAGCACCTGCCCACACACCCCTGACCCTCCGTGCCTGGC TGGGTGGACGGAGCATGGGCAAATGGAGGGCAGAATGGAGTGAGTGTCCAGCATGCACCC AACCTGGAGATCGGCCCAGAGGGTCCCTGGGAGCCTGCCATCCTCCATACCCCAGTCTCT GTGATGCGGGCACCCGGGCAGATACCATGCTGAGAGCCAGCTGGGCCTGCAGTGAGGAAA CGTAGCCCAAAGCAGACTGCAGAGCTGGAACTCAGGTGGGGCTGCTGCCATAGGCTCTGA AGACAGCGCAGGAGACCGGGAGGGGTCACCAGTGCCAAGGCCCTGAGGCAAGCCAGAGCT GGGAGCATCTGAGGATGGGCATTAGCGTGGGGCTCCAGGGTACAGCCGGTCCACTGCAGG CAGCCTGGCCTCCCCAGAGACCCAGCCACGGCTCCTCACCTTGGACAGGGGGCCCTGGGC CTTGGGCATCAAATGACCTGCTTGCCGCGGTGGAGGCTGCCATACCAGGACGCCGGCCCA CGGCCTCCAGCGGCCCGTCCCCACCTTCCCAAGCCCCTCCCCTCATGGCTCACACCCTGT TCCCTGAGCCCCCAAACATGGGAAATGGCACCTGTTGTGCCCCAGCGGGGGAATTCTGAC CCCAGACCCTGGGGCCAGTGCAGAAACCACCACTGTATCAGATTTTATTTCATCTAAAAA ATATCTGAGCCAGATTGAAAGCAGCGGGGAAAGGGTCCCATGGCTGCTTCCCCCAGCCCC ACCCTCCACAGGCCCTGGGCCCTCCCAGCCCCCATTTCCTTTCCTTGAGAAAGTCTAAAG TGGGGCAATATTTTGTGAAATTGGAGGCACCACGTTGGAAATTCCAAACCCGTGTGTACG ATGAGCTGCACGCGCGTTTTGAACCTGCCCTGGTTTTGAGCTGAGGGAAGCGGAGGGTGC AGGAGTTGCCCACTCCAGAGGCCCTGGGTGTGCATGAGGAGCCCTGAGGCTGGGCCGAGA GCCGCTTGTGGGAGGCACTGTGCCCCCTGCCCCCGAGATCAGGGCCTGGGTGCCCAGGAC ACTACAGACCTTTTGGGGATCCTGGGGGTTGGGAAGCCTAGAGCTGGAACCCACAGGCCA GAGTCTGAGCTTTCATCCCCCACCCCCGAAGACGCTCCCCCAGCCACTGCCTGCGCATCG GGGGAGACAAGCTCCCCCCACGCCCACCTTGCCTCCAGCAGGGCTGTTCCTCCCCTCATC TGCTGGCATCAGTTTTTTCTTTAACTGAGCCCCATGCTGGGGAAGGGAGAGCTCCAGGGG ACCAGGCAGCAGAGAGGGGAGGAGAGAAGCCATCAAACAGGCCGGGGGCTGGGAGGCCAT GCACTCAGCATAGAGCCCCACATGGCAGCAGGGGGCTGGATGGCCGGCTTAGAGCCCATT CTCTCCTGGACACTGTTCTGGAATGGCCCCTGGCCCCTCCGTTCCAAAGATGGGACCCTC CGGTCCCCGGGGACCCGGGATGATGTCCCCCAAACCACCTCTCTTCCCAGACTTCATCAA TGGCAATGAATGGTGCATCCTGGCTGGGGCCGCCGCCCCTCACTGTGAGACTTCAGCCCA GACGAGCGCCCAGCCCCTCCTGGGGTCTCAGATAGTCAGAAAGAAAGGCTCACGGCTGCC ATGCACTTCCACAGGACTTTGTGGTTCCTCCTGCACCCCCGGTGATGCTTTCATCCCTCT CCAGCACCATTCCAGAGGCCAGAAAACCATGATGTTTGGGGTCAAACAGCTGGGAGAGGC AGTTGGAACTCAGGCCAGGCCTGTCTGACTGGACAGTGCAGAGACTGGGACCATGCTCCC CTACAAGTGCTGTCAGTGCACCCTCGATGCCCCCCAGAGCTTGCCATGCCCCGACAGTCC CTTCATGGCCAAAGTGTTCCCTGGCACAGGATACCCTGGGGTGCCATGTCTGCAAGAGCT CACACCCTTCTCTCCCCAGCTGCTGCTAACTGCTGCCAGAGGTGGGAGTGTGGAGGGCTC GGGGCCTCTTCACCCTCCCTGCACCAACTCAGCTCCAGAACATTCCCAGCAGTGAGAAGA GGGTGGCCACTGTGCTGCAGCCCCAAGGGATGGGCAAGGCCATGCCAGCTGGAGTCATCC ATGTCCCCTTGGACATGGTGGCCTCATTCAGAGGGCAGCATGGAGGAAGGTGCAATGAGG GTGGCTACCAGAGACAGTGGAGATGAGGCAGTCAGGAAGCCCAGGGACAGGACTGGAGGG GACACACGGCCAGCCTGGGACAGGGCATGAGGCCTTTGTTCTGCGTGCTGTGGCATACGG TGTTGGCTCTGGCCCTGGCCCATCTTTTGTTAGTGGTAGGAGGGTGCTCCTTTAATCAGC CAACTCTTTGTTTCAAATTGCCCCTGTCCTTGAAAACACTGCTTCAGGGAAGCGTGAGAC AAAGCCAGTTCAGTGGGGTGCCTACCTGGCCAGCAGCCCTCATCCGCAGGGGCTGCTCCT GGGGCACGTGCCAGAATCAGACAGTCCCACCGTCATGCAGGTGCTGGGGCCTTCAGTCAG CAGGACCAGGCAGCCCATGTCCTGGGGACCCTTGGGGAGGTGTGGGCATTTGCTGAGTTG GGCTGTCATTTGCTAGACAGGCGCTTGGCGAAGGGGCCCTTTTGTGCATGGATGCCCTGG GCAGGTGTCAGTCCTGAGTCCTGGAGAGGAAGGCAAGCTTTGGAGTTGATGCTGGGTCCT AGGAGGGAGCGTGTGGTTTCGCCAGCTTCTCAAAAGTCACCTGAGACCCTGCCAAGGGAC CTCCAGACATTCGGTGCAGGCTCCTGGAGGCTGGACAGAGGGCACCAAAGGGCGACGGGC CCCGAGGACAGAGGTGTGCAGCCAGGTGGGGCCATGGCCAGGCTGGGACCTGCTGGAGCT CTTGTGTGTGAGCCTGGAGGAGCTGCCAGGCTGGGGGAGATCGGGGCTGGGGCTGGGGGC GGGGCTGGGCGTCAGCAGGGTCTGCAGCTCCCATCTTCCCAGCTTATAGACCTGAATATC CAGGTTGAGAATAAACCATTCTCCCAGGGCACCATAGCCTCCAGGAAGCACCTCCTCCTT CCACCATCCGCCTTTCAGGAGGGGCCTCCCTGGGGGGCTGTCTCTGGGACTGCAGAGGCT GCTTGGTGTATCTGGGGCTCTGACATGAGTGAGATTGTTCCGGGGCCTGTGCTGCCCAGC CTCTTCCCCAACAGCTTCCTCTGGAGGCAAGGGGCTTGGCAGGAGCTGCAGGGAGCCAAT TATCCCTGAGGCAAGCTGGCTGCAGATAATTGGGAAGACAGGCTTTTCACCAGCAGCCAA GCTCTGGGGCGCACTCGCCACACCAGGACTTGGGACTTGAGGATCCAGAGGTCTGCCAAA CCCCACGCCTGATTGGGAGCCCCATGTGCTGCCATCTACCAGGGGAGGTGGCATTTTCTT GGCAACTGAGGGGGGAAGCCAGGTGCTGAGGTGTGGCCCACGGGACGCCTCGCCTGGGGA CCTCCCAGCCCTTCTCCTCTGCCTCCTAGGGGCCTCCTCTCCTTACACATGAAAACCTCC AATGTCTAGCCCAGGGCTCCAGATTAGGGGTCAGTGCCGCCCCCAGCCCAACTTCCTCTG GGAAGTGTGGGGTTCTCCCCGGGGCACTCAGGCCTCTTTCTTACAGGGCTTCCCAAGGGG AGGCATCCAGAGGAAGGCAAAGGCTGTGATCAGGTAGAACCCGCTGGACGCCCCCACCAT CTCCCTCCATCAACATCAAAAGACAAGCTGACCACCCCCAATGCAAGGTGGACATTAGGG TTAAACCCTGAGTGACCAGCACTCAGCTCCCAGCCTGGCCGGGATGCAGTGGGGTGGGGT GTGGTGGGCTCATGGGGCTGGTGAGAGCCCGGTGGGGTGGGTTTCAGGTGCTGCTTGTCC AGGGCTCAGCTCTGTTCCTTGGGGCTCCCTCAGCCCAAGGGCCCACCTCCCCTCTGGGCA GCCCCGATCGGCGTGTTCTGCCTCGGCCAGCTGTCAGCCACCTGCAGCTCCTCCCTCATT GCACCACCCTGGAGCATCACGGTGCCCAGGGCAGGCCGTGGAGACCTGGCTAGCCCATGG TCTGCTCAGTCCTCACCAGCTGTTGTGACAGAAACAAGTCTGTGCTGATGAGCTGAGCCG ACCAGGCTGCAGCCCTCACTTCAGGAGAGGGTCAGGCCCAGCCAGGCCTGGTGAGCAGGT GTGGGGAAGGATGGGTGCTCATGTGCGGGAAGTTTCTCAAGAGTGGTGGAAGGAGGATAG ACAACGAGGGCCCAGTGGATAGGGCGGGGAGGCAGGAAGAGGGGACAGAGAGGGGACCAG TGAAGGAGAGTGGAGGGTACTGCCCCCCTGGGGGAAGGATGGGGTGGGGCTGGTGTTAGC TGGGTTGGGTGTGTGGGTCTGGCAGGGAGCCGGGCATGGGAGTGGGTCCCGAGGAATGCT AGGTTGGAGCCAGGACCATGGGCCTCACCAGTCATAGAAGCCTCACCTCAGGGCAGAGGG GGCTGACAGACTGGGCTGACGGGAAGGGGGGCTTCCTGTCACCAGAGAACATAAAGGGAC CACTAGTGGCCTGGGCCATGAGGCTCTGTGGCCCTCAAGGTGCTGAGCTCAAGGTGGGCC CAGCCCATGGCTGCCTTTGTGGGGACCCCACAAGGAGATGCTGGCCTCTGAGGATGCTCA GACCTGGTTCAGCATGGCCAGGCCCTGGCCTAGCCCAAGGTCAGCACTGGCCACTACCCT GGGGGCTGAGTCTCCGTCTCGCCCTGTGACATGGTGCCCACACTATGAGCCAGTGTCAAC CCTTTGCCCATACACCGGGAGCCTTGTGTGTCCATACTCATGGCTGAGGGCCTGTTTGGG GGTCTCTGTGTTGGGGGAGGTGGGTCCAGGGTGCGTGGCCCCTGGGATTGTTCATCATGA GTGCCACCTGGAAGGTGCTGGAGCCTCACCCGTCCTCCAGAGTGTTTTGGGGAGATCCTG TGCTGGCATCTTGGCTGGGCCAGGATGGTGTCTAGGAGGGACCCTTGGGAGGGAAGCAGG GGCATCAGGGGGCCTGGTGCTGGGGTGGGTGGCAGGAGAGGCCTGGAGGGCGGCGGCCCA AGCCAGTGGCTGGGCAGGTGTCAGGAGGGAGAGGGGTGAGGCCTGGCCTCCCTGCCACAG CTTAGCCAGGGCCTGGGGTGTCTTGGGGTGGGACAAGGCCCAGGGCAGAGGCAGGGCTGC TTGCATGCAGGGTGGCCACATCCCGGCCTGGTGAGTGCCGGCCACTCAGTTCTGTGGCCC TCTGCTGCCCTCTGCCGGCCGCCTCTGTCCCTGCAGGCCCAGCCATCTACCTGCAGGTCT GGACAGACCCTCAGAGCCTAGGCTGGGAGGTCTCAGGAGTCAAGACAGGGTTGAATCCCA TTTGAGGGCATCTGAAGGAGTGAGAGCCTATGTCAATCCCCTGCCCAGCCTTGCCTCGGC CAGTGGCTCCCAGGGGACTCTGGTGGGCAGTGGCTAAAGATGACAGAGGGGCTCAGAGAG GCTGAGCTGCTCTGTAAGGTTGCACAGTGAGCAGGGGCCGAGTGGCCTTGGGAGAGGCAA CAGCAGGAGGGTCCTCGTCATGGCCACAGCCCAGGGACAGGAAACCCCTGCTGGGTTCCC CCACCAAGTAGCTCCCATTCCCAGCCCTTAGGGTGTCTCAGGAGAGGTTCCGGGGGAGGT CCCACCTGCCTCCTGCCTTCTCCCCCAACCACACCCTAAATGGGTCCCAGCTCCCCAGCC CCTCCCACAAGGGCTCACCCGCCAGAGCTCGGGGAGAGGCCCTGCAGGGCGGCTGTTCCT CCGTGTGCTGCAGTCCTCTGGACACTGAAAGCCAATCCCATCCATCTTAATGGCATTTAA TATGCACAGTGAAAGTCAGCAGCCACAGCCACGGGCGTGTGCTCAGGGCAAGGTTTACAC GCGGATGCCACCAGGCTTAGCCCATGAGTGGCCTCCTGGGTCCCAGGAGACCTGTGGGGT GGGGGTGCAGCTGGGTGGGCCATGGTAGCAGAGACCTGGGGAGGGGATGTGGGAGGTAGG AACTGGGCTAGGCAGGGACTCAGGGGAGGCTGAGGCCTGGAACAGGGAGACTGAGACCTC AAGGAGGCTGGGACATTGGGAGTCAAGACTTGGGGAAGCTGGAACATGGGCAGGCCGAGA CGCAGGGGTTTAAGAATGAGGGTCTGGTGCAGACCTGAGTTGGAGCCCAGCCGCTGCTGG CCCCACGTAGCTGGAGAGTGTGCTGACCCCATGGTGGCTAGAGAATGTGCTGTTGGTAGG GAAGGCAAGTGCTGGGAAGGGAAAAGTGTGGTCCATTTAAATGATACGGAAGCAGGGGAG GGAAGTGCTGGGTAGAGGAGGGCATGGTCCCAGACTAGGGCTCCAACCCCACGGACCTAG GTGAGAACAGGCACTTCCTACCCAAATGTTGCATTTCTCACGACCACCCTAGCCTGCCAC GCCCCCATCCTGTGCTTATAAAATCCCCCGAGACCTTAGCAGGCAGACACAGGCAGCTGG ACGTGGAGAGGAACACATCAGCGGAGACACACACGGGCGGCTGGATGTCGAGATGAACGC ACTGATAGGCACCGGCATGCTGGCAGGCCACTGACTGGCAGAACCACACGGAGTTTGGCT GGGGCAGTTGGAGGACAGCCCGGGTGCACAGCGGCCCCACTCCAGGGGAAAACCTTCCCA CTCCACCCCCTTCTAGCTTCCCCTATCTGCTGGGAGCCACCTCCCCTCAATAAAACCTTG CACTCATTCTCCAAGTCCACGTGTGATCTGACTCTTCTGGTACACAAAGGCAAGAACCCC AGGATCCAGAAAGCCCTCTGACTAACACAAGCCACCCTACAGACGACAAACTAAAAAAGC CCCCGTGACACACGCCCACTGGGGCTTCAGCTGTAAACCTTCACCCCTAGACACTGCTGT GGGGTCGGAGCTCCACAGCCTGCCCGTCTGTAAGCTCCCCTAGAGGTTTGGGCAGCAGAG CACTGAAGAAGCGAGCCACTCCCCTTCTCACACACCCTGCTAGGGCAACAAGGGAACTTT TTCCGTTTCACTGGCCCAAGGTGGCTGGACAGTGTGCTGGCCCCACGGTAGCTGGAGAGT GTGTCGCGCTCCCTGGCTTTCCTCCTGATGTATAAAATGAGTTAGCGGGTCCTGGGGGCT GTTCCCACAGTGACGTGGTGGAGGCTGACCCTGACACCCACCCCTGCTGAATGCTCAGTG AATGGTGGTTGCTGTCAGGAAGCATCATTGCTGTCCTCCATCAGTGTCTGTGGGGCTAGG TGACTCTTGGAGGATGCTTCCAGCAACGTTGCACTGCCAAGACAGAGGCCGTCCACCTGC CTCTGGGCTTCCTGGTGTGAACAGATGGATGGATGCGTGGGCCTGCTGCGTGCAGGGAGC TGAGGCTGGGTCTTCACCTATGGGCAGGAGGCTACACAGGGCTCTGGGAGAGACAGCACT GTGGCTGGGACAGAGCCCATGTGATGAGATGCCCTCGGTGCCCCTGGCCTCCCTCCTCGC TCCTGCCCCTCTGGAACATCACCCAGAGCCCCCCTTGCTCTTTTGGGGGATGTCTGGGTG TCTGCGGTTGCTGTCACTCTTGATCCCACCCACCCACCCCTGCAGGTTGCTGGCAGGACA AGTGCAGGAGTCATCTCATGGCTTTCCCTGGGATCTCTGTACCCGCTGAGAGCCGGGAGC TGCACACAGCCTGTCCACCATCCTGGGAACTCACCCTGTTGCTGCCGCTTGTCCTGGGTC ATCCTGGGGCTCACTCCTTGCCATTCAGACCCCTGTGAGGAGAGCACTGGCTGTGAGTTG CTCTGTGGCTAGTGATGGCAAGGCAGGGCCTGGAGTAGATCCATCACGAATAGTTCTTTT TTTTTTTTTTTTTTTTGAGATGGAGTCTCGCTCTGTCACCAGGCTGGAGTGTGCGATCTC GGCTCACTGCAACCTCCGACTCCCTGGTTCAAGCAATTCTCCTGCCTCAGCCTCCTGAGT AGCTGGGATTAAAGGCACATGTCCCTATGCCCAGCTAATTTTTGTATTTTTAGTAGAGAT GGGGTTTCACCATGTTGGCCAGGACGGTTTCGATCTCCTGACCTCGTGATCCACCTGCCT CAGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACGGTGCCCAGCTGAGAATTCTTA ATGAAATGGGAACATGCCTCCCACGAAATGCACAGGGGCCTGCAGTGATAGAGTGGCCTC AGGTATGTGCACAGGTGTGAACACGCCTTCCACAGAGGGCAGTGGACGTGCACATGCCTC ACACACACTGTGGCGCAGGGCAAGTGTCCCACATGTGTTCATGCTTGCGCACATGTCAGT GGGCTGGATGTGTGTGTGCACACCTGTGTCATGCCTGCAGAGAAGACAGTGGAGAGAAAT ATGCCCAAATCTCCCCTCTGGGTCCCTGCCCTGGGTGCTGGGATGTCAGAACAGGTCGAT GTCCTTTTATCCTTCCCCATATCCACCACGCTGTCTACCAAAGCACGCTTTAGTTTTATA ATCAGAAATGAAATGCACCGTTTCAAGAAAGCCATGGGCCAGCCCCTCTGTGCCCTGGTG TTGGCGGGGGCAGAGGGGTGGGGTGTGTAGGGGGAGATTAGCCTCATGCAGCCGTGGGGC CAGGGAGGCCAAGGTCAGCTTGCTCAGCACCTGGAGGTACAGCGAGTACCAACAACACAG CCTAGCTTGACCGCGGAAGGAAAATCAATGCTGGGCGCAAGGACTGACGGCAGACTCACT GGGAACACTGTCCACCGACGTCAGAGCTGCTCACAGGGCACCGGGAGCTTGCCAGCGAGC TTGGCTGCCTGTGTTTGGAACAACCCTGGTGGGCGAGCACGGGCCCCTCACCTGACTCCA CTCAGTGCCCTTCCCAGAGGGGAGACAAAGGCTGTGAACAGCTCTGGAGGCCCTCCCTGC CAGTGCCCCCCAGGCCTGGCTAAAGAAATCTCAAATACCCCGAAGGGCCTGGCTCCAGCG GCCCCTGCTATGGGTCAGCCTCAGGGGGATGCTCATCTTCAATTGACCCTGACTGTTCCA CATTACAAGATAAACTGTGGGGTTAACTCAAGGCGGTATTACTTTGTAAAGACTGCTCTT TATACAAGAGCGGTGGATGCAAAAACAAAAACATTGCCATGTAAGACGGGAGTCCTCCGA GTCCTGTCTTCCTCCATTCCGTGTCCTTGCACCCCATCCATGTGTGGCCTGCATGGACAG ACAGCTTCACACACTCAGACTCTTCCCTCCCCACGCATCAGAGATGTGTGTCCCTGTTAC TGACATGCAGGGAGGGGCTCATCATCTTGGGCACACCCCATGGTGCTCTGAGCCCATCAT CACCTATGGTGGACGTTGAAGGAGTTCCCAGGTTTTCCTTTTCAGGAGCCATGGCTCGGG CCAGTGCCTAGGAGGTCAGAGGTTGGGGGCCCAGGGAGAGGTCACTGCCCAACCGTGCCA CCTTGGACTCAGAGTGCCGGCCCAGGGAACCAGGCAGGCCCGGCTCTGACCGTGCCGCTG GGCGACCGCTGGCTGAGACCTGGGGCTGAAACACATCCCACATCAGCCAAGGAAACAGGC TCTGCATGAGGCCTGAAGCCCCTGAAGGGCTCTGGGACAGGAGGAGAGCCCAAGCCTTCA GCTGTGATGTGCACAGCACCGTCTGTCCTCCACCCAAGGGAACTCACCTTGGTGACCCTG CTGGAAGGCACAGGTTGCTGACATTCTGACCCCTGACATTCGGACCTCCAGCAGAGCCTG CAGTTTGAGACCTCTGGTTCAGGAGCTGGGACTGATGGGGCCCTTGGGGATTCCCGTGCT GCTGGAGGAAGATGGGGGCATGGGGGTGTCCACAGCAAAGAAGTGAGAAGTAGAGGCCGC CAGCCCCCCGCCCCGACCCTTCCCTGGCTAGAGTGGGGCAGGCTTGGGGGTGAGTCTGGG GTGCTGATTTCACCCTCCTGGGCCAAAGTCTCCCCGTGACTTTATGGACTGTCTCCCTGT GCTCCCTGAGACCCTGGTTCTCCCTCACCAGCCTGGGTCCTTCTTGTCTCAGCTCAGCTG TCACCTCCTCCGGGAAGCCCCCAAGCCCTTGAGTGGGTGCCCAGCAGGCATGGTCAGTGC TGAGGCTACAAGCCCTGCCGGCACCCGGCAGGGAACACAAGGCCAGGTCAGGCTCTGGGG ACAGTAGACAAGCTTATCCCTGCACACTGGGGACTGCCCACCTGGGGGAGAGAGCTAGGT CAGGCTGGGGAGACGTCAGGGGATGGCCGGGCACCCCTCCCTGAGGAGAGCTCCACCTGA GCAAGAGTGTCACCAGGCCCACTCCAGACACATGCACTGCCTCAGACGGGCTGGAGAGAG GGTGTGGCCGGGCCATGGGATGAACCATTCTGGGCGGTTGTCCTGGCATCACCAGACATG TGGCTCTATTAAGCATCTCGACTCCACAGGAGCCCATGTGGCATGTCCTCTGACCTCCTG TTCACTTCTGAAAGGGGCGGCCCTCTGAGATTCTGCCTGGAACCTGTCAGCCCTCCTGAC AAGGTCTCTGGTTCCCGGCCATCACAGGCACCCAGCCACCCTACAGAGAGGCTCTGGGAG GCTCAGCCTTGCCTGGGGCCCCAAGACTGATGCCAGCTGGGGCATCTCACAGGGAGCACC CCTACCTGTAAACCGCCCAGCTCAAGCCCTGCCTCGACCTCTCCAAGTCAGCAGCCGACC TTCCCCAGGGCCCCTCCCAGCCTGCAGGGGGTCTGAGGCTCTGAGGCTTTTCTGCAGCCT CTCAGTCCCCCACCCTGCTACCTGCTCAGGCAGAGAGATCATGGCTCTGGGTTATTGTGT GCTTAGTGGGTGCTGGAGCTCGTGCTAAACAACTTGGGTGCCCCCTGAATCTTGACAATA ATCTGAGGGGGCATTATTATTACTATTCTCACTTCCCCGAGGAAGAAGTCCCAAACCCCG TCTGGCACAAAGCCAGCGCCCCGAGTCTCCCTCTGCAGTCCATGTGCTCCCACGTGGGGC ATGCCTGGGACATCACAACATCTGGGGTCAATGCGGCTGCTGTTGTCACTGTGGCATTCA GCTTTCCCAAGGCTCACATCTGCTCCATCCAGCAAAGGGGTCTTGCACCCCAAGCCTGCC TCCCTCTTCACACACACAATCCCACTTCGTCAACCACAAAGATGCATGGGTTTACCCAGC CTCAACCCCATCGGCCCCTCCCCTTCCAGTTGCCGGAGGGCTCGGGCCCCTTCAGCTGGG CTCCCACATTCCTGACCCCTGGCCTGCAGGCCCTCAAGACTATGCCAGCCCGGGCTCACC TGGTGCCAGACACCTGCATCTCCCTGGGGTGGGGGTTGGGGGCAGTAGGCTCCGTGGACC TAGCACACGTATGATTCTTGGAAAATGTAAATGTCTCTGAGGACAGCGATCCCAGCTTCA AATATTGCTGTGGGCTCTCGGGCAAGTCCCTCCTCCTCTCTGAGCTTCAGTTTCCCCCGC ATAAAATGGAGCAGTGATCGTAGCTCCTGCATGGGAAGGTTGTGAACACCATGCTTGTCA GCAGAATATACCCTAATTTTGCAAGGAAACAATATGGCAGGGGTGGGGGGGATCTAAATA CCTTTCCTTATACACGCATTGAAAATGATTATATAAAGTGGTGAAGTGTGATTGCATTCA CATAAACAGTGGCATGTTCTGGGATCTAGCAGACAATCTTAACTTCAGGAAATGCAATCA TATTTTACTAGCTTTGAGAAAAGTGAATAAAATGTCATAGCTAAATAGAACTCAAGTAGG ATCTGTGAACGGCAGCATGGAGTGGATAAGGGGATAACGTACCGATGAGGGAATTTATAT ACCCAGCAGCCCAGGTTTGGTGCAGGCTGATAACGGAAAGACAAACTCCAAATTAAATGA ATGTGAATGAATTCCTTTGCCTGCGGACAGACCAAAAATATCGGTAAAAGTGGCTACAGG GCAAGCAGAAAAATGCAACAAATTAGAATGAACGCAGGGAACCATGTTTCTGGCAGAGAA ACAAATATTTCAGAGTACAGGTAAGAGGCACATGGCAAAAAAAACAACAACCTGACTTTG CCAGAGCTCCAATGTCATGCTCACGGCTGAGGAGAAACAATTATCTCGCTGGGAATGAGT GCAAGTGAGAGAACGGAAGGAGTCCTGAGCACGGTTGACTGCATGTGCCCAGGGGCCCGG ACAGGCAGCACAGCGGCCTCTCTACCCTCGAAGGTCTGTTTCTTGTCTGCTTCCTCCTTG AGAGTGGCAGGCCCTGGTAACTGGCCCAACCTCGGGGGCGCCCAGCAGCTGGGTCCCCAC CACATGCCCAAGTCTGGCCCTCTCAGATCTGCCTGTCTCCTGAGCTGGAGAACGGAGCCC ACCCCTCCATCCCCGACACTCATCTCCCATCCAGTTGATCTCCCTGAGGACCCCCCCACC GACAACTCCCACCTGGATCCCCTGGCCCTGGTGGGGCATCATTCTTTCAAAATAATCCAT GACTGTGTTCAGTGGCTCACGCCTGAATCCCAGCACTTTGAAAGGCCAAGGTGGGAGGAT TGCTTGAGCCCAGGAGTTTCAGATCAGCCTGGGGTAACACAGCCAGACCCTGTCTCCACA AAAAACTATTTTTTAAAAATTAGCTGGGTGCAGTCCAGCCTATAGTCCCAGCTACTTGGG AGGCTGAGGCAGGGGGATCACAAGCCCAGGAGTTCAAGGCTACAGTGAGCTGTGATCACA TCACTGCACTCCAGCCTGGGCAACAGAGACCTTGTCTCTTAAAAAAAAAAAAAAAAAAAA AGCCATCAGCTGGCTCAGGGTAGCCACCTGCCAAGGGTCCCCAGCTGAGCGTAAGACCTG CTTCCTAGGATTTACAGAGCTCCTGTGAAACTCGGTTGTTTCATTGATTGTCACATGGAG CCAGTGACTTTCAGGCCAGCCTTGTTCCCACAGGTGGGCTCTCCCGTGGGCCTGGGCACC CAGCGCTAGGCCTGCCCTGCTATGGTCTGGATGTGAGCAGAGATCTCTGTGATCCCTGTA CCCCTCGCTTCTCCCCCTGCCCTCTCTGCAGCCCTGCCCCTGGCAGCCCCTGCCCACCTT CGCCTCTGTGCAGGGCCACAGGCTTCCTTGAAGCAAATGCTAGAACTTTGAGCTCTTTGC TTTTTAAAAAAAGATAACAGCTTTATTGAGATATAATTCATTATACTATTCATTTATTAC CTTTTGCATTATACCATGCAATTTTTTCCATTTCAATGGCACAATCGAATGGTTTTTTAG TCTATACACAAGGTTGAGCAGACATCAACACTCTGTAGCCCCAGAGTATTTCCAGACCCC AAAGAAACTCCGTGGGCCTTGGCAGCCCCTCCCACTCCCCCTGTCCTCCACCTCCAGGCC CTTACCAGTGCCACAGTCACCAGGCTACATTATGGGCTTGTCTTCGTGTGACTTCTGTCT CTATGGGTTTGCCTATTCTGAACTCTTCATATAAACTGAACCATAAAACTTGTGGCCGTC TGTGTCTGGCATCTTTCTTTCTTTTCTTAAAAAAATTTCACTTTTATTTTAGAGACAGGG TCTCCTTCCATTGCCCAGGCTGGAGTGCAGTGGCACGATCATAGCTCACTGCAGCCTCAA CCTCCTGGGCTCAAGCAATCCCCTCACCTCAGCCTCCTGAGTAGCTGGGATAACAGACAT AGACCACCATGCCTGGTTAATTTTGTATTTTTTTATAGAGATGAGGTCTTATTATGTTGC CCAGACTGGTCTAGAACTCTTGGGCTCAAGTGATCCTCCTGCCTTGGCCTCCCAAATTGC TGGGATTACAGGTGTGAGCCACCACACCTGGCTATTTTTATTTTTTAATTGACACATAAT AATTGTACATATTCGTGGGGTACGTAGTGATGTTTCTATACATGCAATGTATGGTGATCA GATGAGGGTAATTAGCACACCTATCATCTCAAACATTTATCATTTCTTTGCATTACGAAC ACTCAATATTCTCTTTCTAGCTAGCTGAAAATACATAATTTTTGTTTTGGTTTGGATTTT GGTTTTTGTTTTTTGTTTTTGAGACAGGGCCTCACTCTGTTGCCCAGGCTGGAGTGCAGT AGCACCATCACGACTCACTGTAGCCTCTACCTCCCAAGGCTCAGGTGATCCTCCTGCCTC AGCAACCCGAGTAGCTGGGACTACAGGCATGTACCACCACACCTGGCTAACTTTTGTATG TTCTGTAGTGATGGGATTTGACCATGTTGCCTAGGCTGGTTGTGAACTGCTGGGCTCAAG CAATTCTACCCCTCTAGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCGTGACACCT GACCCATAATATGTTGTTGTTAACTAGAGTCATCCTACATTGGTGTAGAACACTAGAACT TATTCCTGCCATGTAGCTATAATTTTGTATCCTTCAACACATCTCTCCCTCCCTCTCCCT CCCTTCTTTCCACCCTTCCCAGCCTCTACTATCCTCTGTTCTACTTTTGACTTCTATGAG GCCAGCTTTTTTTAGCTTCCGCATATGAGCAAGAACATGCGGTGTTTAACTTTCTGTTCT GTCTCATTTCACTTAACATAAGTTTCTCCAGTTCCATGCATGTGACTGGCTTCTTCGATT TTGCATACTTTCAAAGTTCATCCATGTCATGGCATGCATTAGTACTTCATTCCTTTTTAT AGCTTAATAGTATTCCACTGTATGGATGGATCACGTTTTGTTTGCACCGTTTTGCTGTTG TGAATAGTGCTACTGTGCACCAGGGTTGTATTCCTCTGTGTGCCACTGGGTTGAGGGTTG GGGGATGGTAGGTGCTCCCATAAAGGGGGAAATTGACTGATACTGGCTGAAATGGACCAG GCTCTTCATTAAGCTGGCCTCTGAATGTTGCAAAAGCATTCTGTTGGTTTCCAGGATCAC TGCTTAAGACGGTTCCTTTCAGTACCACTGTTGTCCAGGGGAGGGATGGGTTCCCGGAGC TTCCTGATCTGCCACCTTCTCTGATGTCACCCTCAGACCCTCTGCTTTTGAGCCCCAGGC TACAGAGTAAAAGTCTGATGGTAGGATGTGAGGTGAGGGACCTTCCTTGACCCGGCACCC ACATGAGGCAGGACTGAGAACACCCTGAGGACTGCCCGGGTGGCTGGGGTGCTGAAGCCA GGCTTGGCCCCTCCACAGGCCATGCATGACAAGGACCACCCTGTCTCTGAGCTTTGCCTT GGCCTTGCCGTGACATTTTCAGGTGTGTTTCAGCTTAGATTCTGCTCTGGGGTTTCCCGT GTAAGAACAGGGATGACGTCTAAGCCCTTGTCTGGCATTTGTCAGGAATATTGGGAGAGG CAATGTGGCAGTGTGAGAAACAAGCTCACCCATCCAAACCTGAAGAATGGACTCAGAGGC ACGAGCAGTGAAAGTGAGACTTTTTTTTTTTTTTTTTTGAGACGGAGTCTCGCTCTGTCG CCCAGGCTGGAGTGCAGAGGCGCCATCTCGGCTCACTGCAAGCTCCGCCTCCCGGGTTCA CGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGCCCACCACCACACC TGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGGTCT GGATCTCCTGACCTGGTGATCCGCCCGCCTCCGCCTCCCAAAGTGCTGGGATTACAGGCG TGAGCCACCATGCCCGGGAAAGTGAGACTTTTAATAGTGGTCTTGCGAGATTGGTGTCTG GTGGATAGGCGCACTCAGGGCAGTCACAGCAAGTAACTTATGCCCTAGCACACAAGTCCC TCCTCCTCATTGGTCGAGTACTATGGGGTTACAATCTTCCAGGACATTGCCTAAGTTTCA TTATCCACCTTATAAGGTTATACCCCGTCGCCTTCCCCGCTTAAGTTTCAATTTTCCAAT AACGAAACTTTCTTCCCTTTTATGGGCTGACCCTTTTTCTACATTCTGTCTGCTTATTGT GATCTTCTACGTGCATGAGCCGTGCGGTTTGTTACATTCTCAGGCTGGCTGCCAGTACTT AGATTTATCATGCCTTGAAAATGGGCCATTTAAAATGTTTTCTCATGGCGGTAAATCGGT GAGCTGACTGTCATACACTCAGTCATTTTGCCCAAAGTGTGTGTGATGTGGAGGGCTGAC AGCCTTCTCCATTACCAGTGCAGTGAGAGGTGGCACAGCTGGTGGGACCCCCTCTCTCTC ATGGATATGAGTGCGTTAGAGGGGAGGTCTTGGGCCAGGAGCAGATCAGATCAACAGGAT TATCCAGAAAATGGCAGTCATGGCGAGTATTGGCTCCAGATGGGGAGGGTTAGGGTCAGG GTCGGCTCTTCTCACCTTCCTGAGCGAGTCACCAGCGTCGACAAGTTACTCCCAACATAC AGGGCCTGGGTGGAGGCTGAGTGCTAGGAACCCAGGCTCCAATTCTGGTAACCAGGTGCA GATGCTCAGGAGAAGAATGCAGCCCAGACAAGCGTGGTGTGCCCTCCAGAGGAGAGGACG TGGCATCAAAGGGCTGTCTTCATGGAGAGCAGGAACAGAGACTTTGGGAAGCCAGGGGGC CTTGGCCTCAGCCCTGCTGGCAGAGGGTCCCCACCATGTAGCTGAAGTGCCAGGGTGCTT GTGAGGACTGGGTGTGGTCCAGGGTGACTCAGGGCAAGGCCTGGGGGATGCATCTCAAAT CCACAGTCACCAGCCTAGAAACTGGGGTCACACTTCCCACATGGGTTCCCACATGTGGAA ATTTCATTCACTAAATAACGACACATCAAGGCAGCAGTATGGATGGGGCACAGCTGTCAA AGTCACCAGCAACAAGTGCAAGAAGAGAAGAGGGAGTTGCAGCCACATAGGTGCCCAGGT GGGCCCTGGGGGTCAGAGAGGGCCTGCCAGGGGTAGCACCCACCATGCTGACCCTGATGA GGAGAGGAAGGAGGGTGTGGGATGCAGGGGAGGGAAAGGTGCCTGAGAGCAGCATGCAAA CAGCCTGGGCTCAGGAAGAGCCGATGGCATGACAGGTGCTGGGGAGAAAGGCGTGATAGG TGTGGACAGGGGCCACAGGAGGAGGGATGGTCCTCAGGAAGGGGAGACTGGTGGCTATGG CCGGCTGCCGAGGCTAGCCTCTCTCCAGCAAAATGAGCCTGGATTCATCCACTCCTTTTG TCTCTCCCCTTTGCTCTGTGGTGCCGGCAGGAGGAAGAGGAGCAGAAAAACTGTTGCTCC ATTCATCAGGAAGCCTCTCTCTCTCTCGAGTGGTGGTGATGGTGTCCCCCAGGGGCACCT GGCAATGTCTGGAGACATTTACGGCTGTCCCGACTGTGTATGTCTGTGTGTGTGTGTACT ACAGGCCTCTGGTTCCTGGAGGCCAGGGATGCTGATACACAACCTGCAATGCACAGGGCA GCTCCCACAAGGAAATTATTCAGCCTCAAATTTCAACATTGCAGAACAGGTCAGGGAGTG CAAGGTACCTCCTGCTCTTTCCAGCAGGTGCAAGGTGCACAGGGAGAAGCAGGTGGGTGA GGTGGGAGGAGGCTGGAGCCAGACCCAGCGTCTGTGGGGTGCAGGAATATCGTGAGGTGA CGAGGGGCCCAGCGGTGAGTGATGGGGAGGGGTGCACTACGGCAGGCCGCCAGGGGCGGT TCCACCAAGAAAGTGACATCTGGGAGGGGCATTGAGGCAAAGGGGCCGCGAGAGGGTAGG TGCTCTAGAGGTGGCCCACGGGCGGGCGGCACTGAGGCCGGGCCTGCAGGGGAGGCGGTG TGGCCAGCGGACTCTTGTTGGGTGGCAAGGGAAGGGAAGAACCACAGGGCTGCACAGGTT CCGAGGGGGCCTAGTGGTCCGCATTTGCCCTTGGGTGGGCGGTGGGACTGGGAGGACAGG AGAGGAGCGCGGGGCAAGCCCCTAAGAGGCCTGAGGGGTGGGAGGGAGAGGACGGCTTCC CATACGCCCATTTAGACCAGAGGAGAAGCGGACCCACTTTGAGGGCTGGATTCTCAGCCG ATCCGGCCTCAGCTGTGGGTCCACGCGGCTCAAGGACCAGGGAGGAACCCAGGTGTGCGG CTGCGCTGATCGGCGGGCTCCGGACCCACCTGGCTGGCCTGGGGACCTGGGAAACCCGGT GTGGTGGGGAGACCGCAAGCCTGGTCAGGGGTCCCGCTCCCAATGGGGCGGGTCTCCTGT GAGGAAGGCGGAGCTCAGGGCGGGTAGCCCCGGGGACCGGGTCTCTCGCGGGGGTGTGGC CACGGCCCGGTGGGCGGGGCCTGCTGTGGACGCACAGAGGTCTGCAGCGCGCGGGGCGGA ACCTCCCGAGGAGAGGGCGCGGCTGGGAGCGCGGGGCGGGGCCTTCCGAGGAGAGGGCGA GACTGGGAGTGTGAGGCAGGGCTTCCCACGGAATGGGGGCGCGGAGCCGGTCCTCCCGAG GAGCGGGCGCGGCTGGGAAGGCGGGTCGGGTCCTCTCGAGGAGCGGGCGCGGCTGGGAGC GCGGGGCGCGGCCTCAAGAGGAGAAGGGGCGGTGGGTCCTGGGGCGTGCCAGAACCCTTG AGGAAGAAGCGCTGGTGTTCTGGAGTATGACCACCGGAGCCGGCCGGGCCTCGGGTTGGG GCGGGGCCTCAGGGGTGGGGTTGGGACTTCGGGATGGGTTCTCCAGAGGTGAGAGCTGGG GTCGTAGGGTGGACCGGCGTCTGCTGAGGAAAGGGAGCTGCTTGGAGCGCGGGGCGAGGG ACTCACGTGGAGAAGGCGTGGGCCCTACGGCGGGCGGGGCGGGGCCTCAAGAGGAGCGGG TGCAGATGCTAGGGAGGGGCGGGGCCCCTGGGATGGGGCGGTGTCTCCCGAGGAGTGGGC GCGGCTCGGAGCGTGGGACTTCTAAGGAGAGAGCGCGGCTAGGGGGCATGCGGGGCGGGG AGGGGCGGGGCGGGACCAATAGCGCATACTTAAGCGGCCCGGGCGGGTACCGGCGTCCCG CCATGGCTCTGCGGCGCGTCTTGCCCGCGCTGCGCCCCTACATTCCCCGCTTCGCGCCGC TGTCCACGGCGCCGGCCGCCAGCGAGCAGCCCGCCGCGGGCCCAGGGGCCGTGCCAGGAC GTGGGTCGGCCAGGGCAGTGCGGCCGCTGGTGCCCGCCGTGGACTTCGGCAACGCGTAGG AGGCGTACTGCAGCTGGCGAACCTGGGAGCTGGCGCGCAGCCTGCTGGTGCTGCGCTTCT GCGCCTGGCCCGCGCTGCTGGCGCGCCACGAGCAGGTGCGCGGGGTGCAGCCGGGGCGCG GGGCTTCTGCCCGTCCCGGGAGCCTTTACGGAGTTTCCTGGCGTGAAACGGGTGCCTCCC AAGCATCTCCTGGAGCAAGGGAAGCCAGGAAGGGAGGCGTTTCCTGCAGTCCCTTTCTGA TATCGCGAATTTTCCTACTTCTCGATGTCTACCAACTAAAAACAATGGTCGTTTTAATAT TTACGATATATACCACTTGTGGGAATACAGAGTTATCACTTGGAATTTAAAATGGAGTAT TCTTCATGTTGACACAAGAGAAGCCTGGTGTAGGGCTCATAGCCTGGAACCCCAGAGTTC AATCCCCGGGACCCAGCCGCTTCCCAGACACGGCCACTCTGAGAACACTGTTGGGGAAAA ATGGGAAGCCCCTCAAACGCCCAGCTTTGAACTCACCCATGGTAACTTTAAAAAGTGTCA GGGCTTTTGGTGGCGGTGGTGGGGGGCAACAAGCAGAACACTGTTTTAGCCGCATCAGCC TAGGGGTACTAGAGATGACGGTTATCTCCAGGTGACCCTGGGAAGAGTTTGCAAGGTTCC GCTTTTTGGCGCCAGGGTATCTGGTGGTGTTGATTTCTCAGGTGAGACTGTACAGAGGTC GGGGGCTGCACCCCTGGCCTCAACAGGAGGCGGGGGAAGGCGGGGGACGGTGTGAGATGA GAAGCACCGAGCCACACTTGAGCTTAGGTGAGGCCTCGGCGGGTGCGAGGTGAGGGCAGG GATCCACGAACTCAGAGTCAGATGCTGTTGGCCCGAATCCCATGGGGAGCGCCAAGGTCA GCCTAGGACCCGTGGTGGCGGGAGTGCGGAGGATGGTGGAGGAAGAAGGAGCTGAACTGG ACAGATGGAGGGCTCTGGGAGGGCACTGCTGTGCTTGTGCACACAAGGATGTCTTAAGGT CAAGTTTTGGCCATAGAGAGCCCCGGCCAAGGAGACAGGAGGGCGAGAAGGGACATCCCA GGTAGAGGAACTGGCAAGGTGAGGGACGGCGATGTGGAGGCTTTGGGGAGTGCCCACATC TAAGGGGCATCCCAAAAGAGGAGCCAGTGCTGGTGGTGCTGCAATAGGAGGAGGAGGCAG CAGGGCAGGAATGCCAGGGGAGGGAGTGGCCAGCCACAGGGCAAGGACGGGCATTCTAGG TGGCAGCTGAGTGGGACAGAGACTAGCCATGCAGAAGCCATGCCCACCTTGTGGCCAGGA TGTAGTGGGGGCTAGGAAGGCACCAGGTCAGGTTTCTTTTTTCTTTTTTTTCTTTTTCTT TTCTTTTCTTTTCCTTCCTTCCTTTTCTTTTCTTTTTTTTTTTTTTTATGGAATTCGCTC TGTCACCAGGCTGGAGTGCAGTGGCGCGATCTCTGCTCACTGTGACCTCCACCTCCCTGG TTCAAGCGATTCACCTGCCTCAGTCTCCCGAGTGGCTGGGATTACAGGCACGCGCCACCA CACCCAGCTAATTTTTGTATTTTCAGTAGAGACAGGGTTTCATCATGTTGGCCAGGATGG TCTCAATCTCCTGACCTCATGATCTGCTCACCTTGGCCTCCCAAAGTGCTGGGATTACAG GCATGAGCCACTGTGCCCAGGGAGCTGGTGCCGCATCTTCTGCCTGGGAATCCCTTCCTG ACACAGCCTCTGGGGGCCAGAGATGGGGGGAGCAGGGCTCCAGGGGTAGAGAAGCCAGAG GTCACTAAGGCCCAGTGTCCTTCCGTGTTAGAAACACCCGACCCTGCACAGCCCCATGCT GGCCCCAGCAGCCCTCCGTCAGGGGCAGCGGGTCCCAGGCCTGTTTATGTTTTTGTTTGT TTGTTTCAAATGAGACTGGGTCTTGCTCTGTTGCCCATGCTGGAGTGCAGTGGTGTGATC ATAGCTCACTGCAGCCTAGACCTCCTGAGCTCAAGCCATCCTCCCACCTCAGCCACTTGA GTAGCTGGGTCTACAGGTGCATGCCGCCACACATGGCCAAGTTTTTTTTTTTTTAATTTT TGTAAAGAGGAGGTCTCACAGTTGGGCGTGGTGGCTCATGCCTGTAATTCCAGCACTTTG GGAGGTGAGGCTGGTGGATTGTCTGAACTCAGGAGTTCAAGACCAGCCCGGGCAACAGGG TGAAATCCTGTCTCTAATAAAATACAAAACATTAGCCAGGCATGGCGGCAGGTGCCTGTA ATCCCAGCCACTCGGGAGGCTGAGACAGAATTGCTTGAACCCGGGAGGCAGAGGTTGCAG TGAGCCGAGATCACTCCACTGCACTCCAGCCTGGGTGACAGAGTGAGACTCCGTCTCAAA AAAAAAAAAAAAAAAGAAAAAAGATGAGGTGTCACTATGTTACCCTGGTTGGCCTGGAAC TCCTGGGCTCAAGTGATCCCCCCACCTTGGCCTCCCAAAGTGCTGGAATTACAGGCGCAC ACTACCACACCCAGCTAATTTTCATATTTTTTGTAGAGATGGTATTTCACCATGTTGCCC AGGCTGGTATCAAACTCGTGGCCTCAAGTGAACCTTCCACTTTGGCCTCCCAAAGTGCTG GGATGACAGGGCATACCACTGCATCTGGCCCTTCTGAGCAGTGCTTGGAAGCCACCTCCT GACGGGGGCTGGGGGTCAGAGTGGCCAGCTGAATGCTGGGCCATCAGGGTGAGGGGCGTT TTCTCCTCATCCATTGCCTGTTCTCCTGGGGCAGATGTAAGGGGAAGGCCCTGCACTGGG GTGGTGTTAAACAAGATGAGATGGTAAGATCTGCTTTCACTTTAAAGGAACATTTCTGGT TGTTGCTTGGAAAGAAGTGTCAGGGGCAAGAATGCTGCAGGAGGGCTGGCCTGGAGGGCA GGGTGGGACTGCAGGCTGGCTGCACGGGGGCAGGAGCCTGCGCTTGCGTCAGAGGTTCCT GAGAGACTTGGGCTCTGGGGCAGAGGCCTGAGCAAGGGGGCCTCGCTGGGGTGTCTCTGT CTTAGCCACACTCGGTGTTTCTGGGTCCTGTGACCCATGTGTGAGCAGCCTGTGGTGCTG GTGGCACAGGGGAGGAAACCGAGGCAGGGAGCCTGTGGGGGCTGCTCGGCAGGGGACAGG CAGCTCCTGTGCCCACTGCTGTCCCACACCTGCATGGAAACACAAACCCCACCACGGAGC CCCCTCTCATCTCTACAGGGCCTCCAGAGCGCCATGTGCAAGAACTTCATGGGCGGGGTG GGGTGGGGTCAGGAGCCTGCTGTGGGCTGGCCAGGCATGAACACCAAGCTGGAGGTGGCG GTGCTGCAGGTGGGGCAGCCTTCCCCTTCCCCACTCCTGTGGCTTGTGAGGGGGTGGGCA CCGTGGGGGCGCACAGGGGGCATGGTGGGCAGTGGTCGGAGGTGGTTTTGGGAACTCACC CAGGGCACGTGGCTCACTCTGCAGCTGCTGGGGTTGGCCTGGTGGGGAGGAGGAGGTCGG AGGGACTCTGAACAGAGAGGGGAGATGCTGGCAGGAGGGCTGGAGACCCAGCCCTGCAGG GCGTGGGCGTGCATCAGCAGCGGGGTGGGAATAGACCTGTCTATGCAGTAGAAACAAACC AGACATGGATCCTGTTCTTACTAACGATGTAATAAAGCCAGAAACAGCACCAGAGGCCAA TGGGTGGGGGGAAGAATAAAATGTGAGCGAATGGGGCTGATCCAGGCCTTCTCACTTCTC AAGGAAAGTGTCGCAAAGATGGGCATCGCATCCAGGGCTGAGATTGAGGACTGGTTCACG GCAGAGACCCTGGGAGTGTCTGGGTGAGAGCAGCCCCCAGGCTGAGAGGACACGAGCATC AGCCATGGTTCCAGCCAAGCCCCAGTGACGTCTCCCAGTCCTGGCTGCCCTGCCTGAGAG GGTGGGCCTTGGGATGACAGTGCTGAAGGAACTGCTTTTCACACAGCAGGACTTGATGGC CTCAGGAGTCCCTGAGGCTGGGGCTGGGCTGGGCAGGGGAGACACAGGGCCAGCACAGAC CCCATAGAGGGCTCTTTATAGAATTATCTGGGTCCAGAGCAAAGGTGGCACTCTCAGCCC TCCCTGGAGTCCACGCCGGCAGGTGAAGGATCAGAGCCCCATGCCCCATCCTTGGGGCCA GGGGCTGGGACCCAGGTCACCAGGAGCCTCCAGTTGAAGTGGGGAGGTGACTGGGGTGGT GCAGTAGTGAGCCATCCCCTCACACACTGCCAGCCTTGCCCACTGTGTCCTCCCTGGCCG CCCGGAGCTGTGTCCGCCTCCTGCCTGGCTGCCTGTGCTCGGCCCCTCCACAAGCCCAAA ACAGCCCTAGGGTACTCAGTGTTTTCAGAGCCGCCTAGATGCAGGACTGTTTGTGTTTTG GTTTTAAATTTTTTTAATTAATTTATTTTTTATAGAGACCAGGTCTCACCATGTTGCCCA GGCTGGTTTCAAACCCCTGGGCTCAAGTGACCTGCCCGCCGCAGCCTCCCAAAGTGCTGG GATTACAGGTTTGAGCTACTGCACGCCACTGGTTTTAAATTTTTAAACAGAAAACAATAT TTACATTTCCTCTGTCTTTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTCGCTCTGTCAC CCAGGCTGGAGTACAGTGGCGCGATCTCTGCTCACTGCAAGCTCCGCCTCCTGGGTTCAT GCCATTCTCCTGCCTCAGCCTTCTCGTCTTTTAAAACTGAACACTGAGGTGGGTTTTGTG GTGGTTGGTGAACGGACAGGTTTGGGCCTGGATCCCCCACCCGACCCTAGCACCCACAGG TGGGGTCGCCAACTCTGCTGATGTGCCTCCTCCCTCAGCACTGTGGACCTGCTGGACTGG AGCAGCCTCATCGACAGCAGGACCAAGCTGTCCAAGCACCTGGTAGTCCCCAATGGACAG GTAACACCTCCGCTCCTCAGTGAGGCCCAGCTCAGCAGGGCGCTGCGCTAAGAAGGGAAT TCAGCCTGCCACATGTTTCTCTTGTTGCCTACCCTGGGAACTTAACATGACCAAGATCAC TGCACACTATGGCCCCACAGACCCCCTATGGTCCAGGGGGAAAAGAAGACAAACCCACTA GTGTCCACAGAGTGCTTGGTGGGACAGAGACCCACGCGGAGCCTTAAAACACCACAAGGA GAGGGGGTCTGGGGGCCTTCCCCAAGTCATCTGTCTTGTGCATCAGGTCACCCCAAACTT CAGAGGCCTAAAACGGCAGCAGCCACTCTGCTTCTGCCTCATGGTTCTGGGGTTGACAGG GCTCAGCTGGGCAGTCCTCGCTTGGGGTCTCCTGGGTAGCTGTACTCACAGAGTGGTGGC GCTGGGACCTGGGGGAGGCGCCCACTCACATGTCTGGGAGTTGGTGCTGGCTGTGGCTTG GGCTTTTTCCCAACATGGCAGCTGGGCTCTAAGGGCCAGTGTCCCCTGAGACGTGAGAGC CAGGCAGAAGCTGGGTGACCTTTTCCTGCCTCACCTCAGAAGTCACACGGTGTCTTTTCT GGCAAGTTCTGTTTGTTAGAAGCATGTCACCAGGCCAGCCTGTATACTGCGGGGAGAGGA ATGAGACTCATCTGCCTGTGGGAGGGGGGCCCAGGACCTGGCTGACTGCTTGAAACCACA ACCTAAACTGGAATTCGAAGAAAGAACAGAAGCACCTTCAGAAAACATCCTGCAGAGGCA GCTTTCCATGACACATCTAGCCCCAGTCCACTAAGGGCCCAGCCTCAAACTCAGAGCCCC CGACCCAGAGCCTCGGGGCGCAGTGCGTACAGTGTGGCTGTGCACCAAGGCCACCAGCTG TTGGAGACTTTAGCATGGTGTAGCAAGGGATAGAGTGGTACTGCTTGTGCCCAGTGAGGC AAGGCTGCTGGGGCCAGCAAAGGGGTGAGCGGGAGGCCCTGCGGGATGGGTCCCCACGGC CTCTGAGCACCAGCTCCTTGAAGGCCTCATCCTGGCGGTTGCCCTGGTCCCTGGCTACGT GTCAGCACGTGCTGCACAACTGCCCTGTCTGCCTGTAGCCAGGGGTCTTCCTCCTGGGCC CTCTTCCTGCTCTGGGTGGAGCCCTTCTGTTTGACGATTGGGTGGTCAGTGTTCACTTCC TCAGTGTCACATGGGAGCTGCAGGGGGCCCTCCCACAATGGTGGGGCCTCTGTGAGAACT GGGGAGGCTGTTAGAGTACACACTTCGGAGTGCAGGCCTGGACTGAGCCTGGGGCCCACT GACATGGGCACTGAGCTCCCTCCTGGTACCCCCAGGGCCCGCGTAGGGCAGTGCACTGGC TGCGTTGGAGGTTCCGGGGCTCTGTGCTGCTTGTCCAGGTGGCCCTCCCTCCCGGCCCTG CATGAGAGGGTTCCCGTGGGCTGGGAACACTAAGAGAGGTTTAGCCTGGGCCTCAGGGTG GCACTTCCTGGCCCTTGCTGTGGCCTGACCATGTGCTGTACCCCCAGACAGGACAGCTGG AGCCCCTGCTGTCCCGGTTTGCTGAGGAGGAGGAGCTACAGATGACGAGGATGCTACAGC GGATGGATGTCCTGCCCAAGGCGAGCTGTCCCGCTCGGGCAGAGGGCTGGGGCCTTGGGG ACCCGGGACTCATGCAGAAGAGGGCACCTGCCTCACTTGAGCAATAGTTGCTCCCACTCT CCCACCCCCTGCGCCTCTCAGAAAGCCACAGAGATGGGCGTGCGGCTGATGGTGGATGCC GAGCAGACCTACTTCCAGCCGGCCATCAGTCGCCTGACGCTGGAGATGCAGCGGAAGTTC AATGTGGAGAAGCTGCTCATCTTCAACACATACCAGTGCTACCTCAAGGCAAGCCCCCGC TCAGCCCCTCCCTGCCTGCCTTGGGGCTTGGAGACCCCACAGGAGGGGCTCCTCTCATCT GAGCTCCCTGGCATTTGCCTGGTTTTATACTCAGCACCCACCAGGGAGCTCACGGGATGG AGGAGGGAGTGGGCCGTGCCCCCAAGGGGTTGTTGGGGTCTTCTGCAGCACAGCTCACCC AGCGGGCCCCAGGGTCCAGGAAAGAGCCCCTTAGGTGGTGGCCACAGCCAGGCCTGCAGA GGAGGCCGAGGCCAGGGTGACTGCCTCAGAGTAGCAAGAAAGGCCCGCTGCCCGCAGCCT CTCCCCTGGTTCTCAGAACCAGCCAAGAGTGTCTAAGGAGCAAGAGGAAGACAACGATCT TTATGAGGGTCCACTAGATGTACCCGACACCCCAAACTTGGAAGGTGTCACATGACCATT TCCTCCAGCGTGTTTTCAGTTCAGCGCCCCTAAGAACCTCATGAAAGCTTTGGGCCCTCC CCGGAAAGACTCACCCAGGATGTCACATGTATTCAGGGAGCCCATCCTAGCCTGTGGGCC CCAAACTGCTCAGGGATGTTAGCAGGTCCAGAGCTCTCAGGGGTGAGCCGGGTGCCCAGA GGACACAGGGTGGCATGGGGAGGGGGTCCAGGCAGGAGCATGCAGTATGCACCCCTCCTG GTCGGCCTGGGAGTCTGGAGCACATGATGGCTGCTGGCGCCTGGGCTGCCCCCATAGGGC TGTCCACAGGCCACAGAGGAAGCTGCAGATGTCCCCACTGCCATTGCTCCCTGGAGACAG AGGTTGGAGAACCATCCTCACCCACACCTGGTCCAGGTGCCTTGAGCTGCACCTCCCCAG GCAGCTGCTGCCCAGCCCTGGTGACTGCACCTGGGCCCAGGAAGGCCCCATCCTGCTGTC ACTTGGGCTTGCCCAGGCTCCCTGTGACTTTTGGCCCTGGCCCCACAGGATGCCTATGAC AATGTGACCCTGGACATGGAGCTGGCTCACCGTGAGGGCTGGTGTTTTGGGGCCAAGCCG GTGCGGGGCGCATACCTGGCCCAGGAGCGAGCCTGTGCAGCAGAGATCGGATATGAGGAC CCCATCAACCCCATGTACGAGGCCACCAACACCATGTACCACAGGTGCGCAGCTCTCCCA CCCCTCTGTCTTCTCAGGGCAGGGCGCCAAGAAACCAGGCCTGAGGGAGAGGCTTGGAGA CTGAACTTGTCAGCCACATGTGTCCCCAGGTCAGAACTGGTATTTCCTCAGCTTTTATGT CATGTGTTACTTTTTCAAAAGCATTAAGGTTATTCTTTTATAAATATAGAAAAGCCTATT GAACAAAGTAAAATTGGCCACAGTTCCCTGAGCCTGCTCCTGCAGACACTTAGATCCAGA GATGTGCATGAACACTCGGTCCTCCCCACCCTGCAGGCTCAGCCTGTTTGCTTTTCAGTT CACACAGTGGGATCGCCCATCAGCTGTTCTGCACCTGCTTTTACTGCTCTAGTTTCTTGG TGATCTTTTCATGTAGCACAGGCTGGCCAACCATGGGCTTTTCCTCTTAGAGGCGTGTGT GCATGCTGCAAATTCAGAGAGACCCCAAGTGCCTCCTGGTTGGCTCCAGCTCCCCTCCTG CAGCCGCTGTGCATGTCGTGCCACAGCGCACACATCTCCGCCATTGTTGTTTGCGCTGTA GCGTGTCCTCTGCCCGATGTGTTTTTCATTTCGCGTTCCATCTGGGTCCTCTTGTGTGGG CTTGCAGAGTGGTGCTGTCAACAGCCAGCATGGAGTGCCCGGGGCTATGTGTCCAGCATC TTTGATCTTACTGGTTTTCAGTCTGGTTATTGTAACAGGGACTCGCTGGACATACCGTCA CCCTCTGTGGTACACACGGGCCGTCAGGGGTTTTGTGTCTGAGGATGCAGCTGTCCTAGG TTGCTGGAAATGGAATTGCTGGGTCAGAGAGAACGTGCGCGTGGTCAAGCCTGCTGAGTG TCCTCGCTGCAGGAGGGCCGTACTGCTCGGGCTTCCTGGTGCACCGGGATGCCCACCCCC ACTGTGCCCTACAGGGACCTGCTTCCCTTTTATTTCCTTTCAGGGCCAGGGGTGCACTGT ACGATCCCTCCCCAGACGCAAAGCTGTGTCTGTCATGAGGCTTATGGGCATGTTTGGGCT GGTCCCCAGAGTCACGGCATGTTTGATGCAGGGACGCTGTGCCCCAGCTGGTTCTGTGTG AATTGTCTGGTCCCAGACATCGCTTTTGCTTGGTTGCTGGTCTTTCTTTTTCATGAGGTG CTGGGAGGGGCAGATGCTGCTCCTGTTCCTCCTGATCCTGTGGATACCCTTGGAAAGAGC CAGCTCACTGGATGGATCTGGGTCTGCCTTCGGCCTCCTCTCTGTGCAGCTGGTAATGAG AGGTGCAGGCGACCACAGGCCTGGAACTGGGCTCACATGGCACTCTTCACAGGTGCCTGG ACTACGTGCTGGAGGAGCTGAAGCACAACGCCAAGGCCAAGGTGATGGTGGCCTCCCACA ATGAGGACACAGTGCGCTTCGCACTGCGCAGGTAGGTGTGCCCCACCCTGCACCGAAACC CCAACCTGAGCCGTTGTCTCCTCCAGCTGGGGAGAGGTGGCAGCAGTGCAGAGCTCAGGG TGGGCAGAGTGACCTGCTGGGTGGTCGGGCATTTGTGGGGAAAGGCTGGTGAAATGAGCT GGAGGGTGGCTTCAGTGGGCAGGCCCAGGCCCGCTGGCATTCTGGACATCGGACTGTGAG AGCACAATGGCTCTTTGCAGTTACGTTGGTTTTTAAAATTAACTTTTTTTTTTAGATAGA ATCTCGCTGTCACCTATGCTGGAGTGCAGTGGCGTGGTCTCAGCTCACTGCAACCTCCGC TTCCCAAGTTCAAGCAATTCTTCATGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCAT GTGCCACCACGCCTGGCTAATGTTTTGTATTTTTAGCAGAGACAGGGTTTCACCATGTTG GCCAGGCTGGTCTCAAACTCCTGGCCTCAAGTGATCCTCCCTTCTTGGCCTCCCAAAGTG CTGGGATTACAAGCATCAGCCACTGTACCCAGCCTTTTAAAAATTAACATTTGACTTATT AAACTTCACAATTAAAAAACTAAATTTCATCAGGGTATTCGGATGGCTCCTGAAAGGTAC AAATGTAGTGTTGGAGCCCAGTCCCTCAGGAGGCCTCCACAGCGCTGCCTCATTTATAAG CAGTTCCCTTAACATTTTAACTACATCTGGAATGTAACGGTTTGTTTTCTCTTTAAACAT TTGTCCCATGGAGCACAACTTCGGTAGAATTCTAACAAGTCAAATGCAGAAGTTAGGTGA AGTCAGTTCTTGGATACTCCTGTACTGTCACCCTGGTCTTATCTCATGCCTTAGCCCAAG CTATGCACACAATGGGGTCCTAGGTCCCCCTCCCCTCCCAGATTCCGCCTTCCCAGGGAT GGGACCCCCTAGAACCCTCGGAGGCCTGGGCAGTGGCCTCGCTGGCTCTCGCCTTCCTAG GAGCTGAGCAGGAGCTCCACTCTCAGCAGGGCAGGGTGCCCCAGATCCATGACTTGTGGC ACGAAGGGGCTCCCCAAGAAGCTGTGAAGCCACAGGGCCAGGCTTGGGGCCAGGGCTGCA GCCAACAGGACACATCCTGGTGCTGCTCGGGAGCTGACTCACGGCTCTGAAATCATTCAG TGATGTCCACATTCAGCCTTTGTTTACGATGCTGCTACTTTTTCATGGCCTGAAATTGCA GCACACCCATTTTTGTGCCCTGATCCTCTGCTAATTGTCATAGATGTGTCGGTGATGGGA CAGACTCACTTTCTCCACACGCACAGGCCTGGTGGCCAGCACTGCCCAGCACTCGGGAGC TGGTGGGGGTTCCTCTCAGCTTGTAATCAGGACTCCCAGCCTCTGGGTGGACTCTCAATG CTGGTGGACTCACAGCCTGACTAGACACATCACCATCCAACTCCGAAAGGACAGCCCGCC CTCAGCAGGGCTTCCTCTCTCCAGCTTAGCCCCGGCACCTGTCTGTCCCTACTATCTTGC TGTGGCAGTGGCTTCCCCAGTGGACACAGCTTCCCCCTCCCGAGTGGCAGCCGGGCTCTC TCCTCTTGGCCCCAAGGGCCCTGTGCCGGGCTCCCCCAGTACCATGCTCACATCCCTGCC TGAGAGGCTGGACCCCTGCAGGCTTCAGCTCTGCCCCTGCTGTGGGTGGCTGGGGAGCTC TGGGGAGCTGGGGTCCCCTCCCATGGGTAAGTGGTTCTAGACTATGGCAGCTGCTCTGTC CTGTGCTGGGCCCAGACTGCCACACCACCCACCCTCAAACAACCCGAGTTGGCTTTGGCC AAGGAACAAACCAGCCCCAAACTTAGAGGCTGGAACAGTGGCATGTTCTGTAGGCCAGGG GTGGGCTGGCTCAGCTGATGCTTCTGCTGCTGGCTGTGCCTGTGTCCCTCCTGCGTCTGT GATCTGGGGCCGGTGGGGCCTAATGAGCTCATTCATGGCTGGTGTAGTAAAGGAGACTAC ATCCTGGGTCCAGCGTCCCACAGGCCAGCCCAGGCTTGGGCACGTGGTGGGCTCAGGCTT CCAGAAGGCCTGGAGAGGGCAAGCCTCTGCTTGTGTCACCTTTGGTACCTCCCTGTTGGC CAAAGTGCCTCACGAGGCCAAGCCCAGATGCCAGGTGGAGTGAGCCACTGTCCTGTGCTG GGAGGAGCCGCAGTCACATGGCAGAGGCTGTGCACACCAGCAGGGGAGGGGGCAGCCAGC AGTGGCACCCGGCTCTCGGGTTCATGTCCTGGGGTTGAGGAAGACACAAGATGGAAGCCA GGGCGATGTGGCAGAGATGGGTGCCTGCTTTAGACTGGGTGGTCCCGACACTGGAGCCCA GCCCAGGGATGGAGGGAAAGGGCCTCTGGCCGATGGGTACTCTGGGAGCCAGGGCTGGGG CAGATGGAGAACAGGGGAGGTGAGGCCCTCGGGGAGGGAGTGGGAAGCACAGCACAGGCA CTGCCAGTCACCCGGGTGATGCGTGAGCAGCAGGCGGAACTCGCCCCAGGCTGGGGGACC CAGTGCTGTGCAGACCAGCGAAGTCGGGATACCTGGATATAGTAAGCTGGAGATGAAACT TGAGAACTGTCACTGCGTGGATATTTCAAACCCTGAGAATCTTTGTAAGACCCTATACGG GGTGGTGTGAAGACAGGACAGAGTGGAGGCCCCACCCCAGTATCTGAGCAGTGCTAATAG ATGTATAATGCAAGCCACATCTGTAATTTAAAGTTTTTTGGTAGCTACGTTAAAAGATAA AATGAATTTCAAGAATACATTTTAATTAACCCAAAATATACAAAATATTACTTCAAATTG TATTCAAGTTGATTTTAGAAAAGCAATGCGGCATCGTGCATTTTTCTAGAAAGTCTCCTA AATCCCGTGTGTATTTGACATTTGCAACACATCTCGATTCAGAGCAGCCGCCTTCCGAGG ACTCGGGGGCTGGTGGCAGCTGTGTGGGACACAGCAGCTGTGGGGATGGGGCAGAGCAGC AGCCCACAGAGGGCGAGGGCAGGGGCCGGAGACCAGTCATACCACAGAACCCTGGAGGGG CTTGAGGAAGGGATGGCAGGAGAGCTGAGGAGCTTCTCTGCCCTGTGGGGCAGGCTGCTG GGTGGGTCAAAGCCCCAGAAGGGCTGCACCTCCAAGAAGGGAGGTAGAGGAGAGGTGGGA GAGAGGAGGGTAGGGAAGGCAGGAGGGGGGCCTTGTTGGCTAAGGGCTGAGGCCTCCCCA GGGAGAAGAGTAGGGCAGCCCAGCAGAGTCCTGCTGGGAAGAGGCCTCACTGGGAGAGGG CCGGACAGAGCCTATATCCCAACCCCACTTCAAAGGGGATAAAGTCTGGAGAGTGTAGAT GGGGGACCTGGGGTGGCAGGACAGGGTCAGAGCTGGGACAGGGGCCCTGTACCCCTGCAC CGGGGACAGTGTCCTGGAATTAGCCCTCAGCTCCTGGCTTTACCTGCACAGGATGGAGGA GCTGCGCTTGCATCCTACTGTACTTTGGACAGCTGCTAGGCATGTGTGACCAGATCAGCT TCCCGCTGGGTGAATGGGGCCCTCCCTGGTGCGATGGAGTGTGGTGCTGGGCCTGGGCCT CATGGCATTATCTGCCCCCATGCAGGCCAGGCTGGCTACCCTGTGTACAAGTATAGGGCT GTAGGTGCTGTCATACGTGTCCTGCCACGCCCTGGAGAACAGCAGCCTCGTGAAGGGTGC CCGTCGGGAGCGGCAGCTGCTGTGGCTGGAGCTCTTGAGGTGGCTCCGAACTGGCAACCT CTTCCATTGCCCCACCTAGCACCCCCCCGCCCCCGCCCAGGCCATCACCACAGCTGCAGC CAACCCCATCCTCACACAGATTCACCTTTTTTCACCCCACACTTGCAGAGCTGCTGGAGG TGGAGTCAGGTGCCTCCCAGCCCTGCCAATGGTGGGGGCACTCAGGTGTGGGCTGACCCT GATACCTGCCTGGGATAGCCGCTGGTAACTGTTAGGAACTTTCCTCGGATGTGTGGGCCC AAGGCCCCCACCTCTGTGACCCCCATGTCCTTGGACCTAGAGGATTGTCCACCTTCTCCC AAGGCCAGCCCACATAGCCCGAGCCCCTCGGGGAGCAGTGGCCGGGCTGGGGAGGCCTGC CTGGTCAATAAACCACTGTTCCTGCAGCTGAGAGCCCTTTGCCCTCACACAGGGTCAGCT CTGGGCAACAAAGAGGGAGTGGCTCAGACAGGGTCAGAGAAGTCTCATCCCAAACTCTGG CTTCCAGCATCATCTGTTCCTGGAGGCCAGGCCCTGCGCAGACCTGGCCACCCAGGGTGC TGGACACCCCCAACCCTGACTCCACAAGGACCCAGCAGGGCCCATGGGCCCAGGCCAGCA CGGAGGGGACTCATGCAGATGCAGAAGCCAAGCTCCCCAGGTGGCCCTGGCATGCCTCCC CGCAGGGCTCCTCCTCAGGCCATTCCTGCCACCCTGAGTCCTCTATCCCAGTGACCTGCA TCAGGCTCCTCCTAGTGGTTCCCACAGGACACTGACCAGGCTGCGGCAGGCGCATACTTG CACTCCGGGCCTTCTCCCATGCTCTCCAGCCCCCAGCCCCACAAAGCAGGCTGGATGTGG TCCTCATGCCCTGGGGCTTGGATGGGGGCCCCTGTACAGATGGCAGTGACAGCATCCTGG GACCAGGGCTGGGGCAGGAAAGGCTGGAGGGTCCCAGTCTCCAGGCAGGGGGAGGGGTGG TGGGCTGGGCACACAGCCCAGGAAGGGGCCCCCAGGTAGAGGCCTCTGCTGCAACAGAGG TGGGGGCCCTACCAGGGCCCCGGTTATGGCAGAACCTGCAGCTATCACACAAGCCACATT TGGGCCACACTGAGGCATCTTTATTTCTCTGGGTCACACAGAGCTTGGGCCTGGGAGCCT CTTCTGCATGGGGGCTGCAGCTGCCAAGGGAGCGGCTGAGACCAGAACAAGCGCTCGGGT GGGCCCCAGTGCAGGTTGGGATGTGCCCGGTGGAGTAAGGTGTGAGAACCCCCAGCCTCA CTCTCTGCCTGGTCCTGAAGCAGACAGCAGCAGGCTGGCCCAGCCTCCCCTTTATGAGAC TATCCTAGGGTTTGACAGCAAGTCCCAGATGAAGGGTGACAGGCAGCTGGGGTCCACCTG GTCTCTCCTCAGCAGGGGGAACCCCCTGCGGGCAGCTGGGAAGGCAGTGGCCAAAGGTCA AGAAGATGAACTCTGCCCAGACTTCTGCTGCCTGTGGCTATGGTGGGACAGGGCTGCCTT TCTGGTCACACTGGGCAGCAGGCGACTGCCAGGGACCTCCCAGTCCCAGGGCTGCATTCC CTGCCCGGCAGCCCCTCTGCTGCAGCTTTCGGATGAGTTCCAGCAGGTTCATCTGCAGCA TCAGCTCCTGGGATACAGAGGGGCCCCATGAAGGCCAGAGTGACCCCCAGGCCCCTTTCA TCCCGGCCCAAGGGTGCCAGGCAGTGAGCCCACGGGGGCGACCATCCTGTGAAGCCTGGT CATGGCCTGCCAGGACAGGGTGGAATCTGAGCTTCTGCCCCCAACAGGGCCACTCGGAAT CTGAGCCTCTGCCCCCAGCAGGGCCACTCGGAATCTGAGCCTCTGCCCCCAGCAGGGCCA CTCGGAATGGCATGCGGGAGGGGAGCAGAAGAGGGCCCTGTGTGGCACAGCAGGCCTGGA GAGGGTGGGCAGTGCCCATGGCTGGCTGGCTCACCCTCCTGTAGGGAATGGGCCTGGCCT CCGTGCTGGGAATGGCACAGAAATCCTTTCCAGAGCCTCAATCCTTCCACTTCACCCCCA TACCCTGAGCCTGGCTCCCCAGGGACCAGCAGTAAAACCCCAGATGGAATGGGGCCCCGG GGCACTCGGGCTAGGGCAGAGCAGGCCCAGGCACTGACCTGTGGGTTGGTGGTCACGTAG AAGCCAGCCACCCCCGCCTTCTCCAGTGTGCTCTGCTGGTCAGCCACCTTCCGGTCCAGC TCCAGGATGATCTTCTGGTCCATCGCCCGCTGCTCCTCACGGATCCGGTGTTCCACGGCC TGCCATGGGGCCAGGGCGCGGAGGGGGAGAGGTGAGGGCAGCTCTGCCATCAGGGGGCGG GGAGGTGAGGGCAGCTCAAACACATGCTCCTGCTGCCGTCCCCAGTGGCAGCAGCTCTGA CACCACCGGTGCATCCTCACCACACCCAGGCCTCTCCTCGGCCATGTGAACCCCTGAAGG GGGCCCCAAGGGGCTTCCACGAATGCCCTGGCTTGGGACTGCCTTAGGACCACACACACA TCTCCACAGGCACACCCTGCCCCACGGCTTGTTGGTTACATGCCAGTCTCTGGTTGTCCC TGTGCTCCCTGGGAGAAGGATGGGCCCCTTCCTGGCCCCAACACCCTACCATTGCCTTTG TACCCCCCTGAGGCCACCTGCTCTGTGGAGGCCCAAACCCACGAGGAGCTGCTATCCAGG GGAGCACTGGTGGGTTCTCAACCTCCACCCAGCCCCAGCACACTTGGTATCCGCAAAGAG GAGCCACCTGGTGGGACTCACTAGGCCTCCAGTGTCCCACTCAGGCCCAGGACAGCCACC CATGCCTGAGCCCCCACACCCCTTCAGCTCAGCCCAGTCAGCAGAGCCAGGTGGTGCCCA CAGGAGAGCTGGGCCGGCCACCCTGCCTGCGCCCCCAGTACCTCTAGTTCTCGCTGCTGA GCCGCCTGAACCACAGGCAGGTTGTGGGGCCGGCAGGCCTGCTGGGCTTCCTGGTGCTTC TGCCGCAGCGCCTGCCTGAGCACTGGGAGGGATGAGAGCCCGTCAGCAGGCGGTGGGACT CGGCAGCAGCCCTCACCCATGGGCACCTGCCTTGCCAGCCCAGCAGAGCAGCCAGGCCAA AGGGGTGTCTGCAGTGGGGAGGATGCCAGCCTCCTCCATCCTCCCACCCCACTGTCACCC AGGGCCGGGCCCTTATCTGGTGGCAAGTCTCCAGGGGACTCCAGAAGGTGCGGGGCACTG AAAGAGTGGGGTGGGGAAGACTGGCCAGGTGGGGCCAAAGCTGCAGCCCCAGACCCAGGG CAGCTGGAGGAGGCAGGCGGGAGGGAGGCTGCAGTCTGAGGGCTTCAGGGCCCCCATGGC CACAGGCCATGAGACATCTACTCAAGTGTCAGAGACCACCTCAGGCAAAGGCATGGGAAA GGAGCAGGAGGAGCGGGGTCGCTGACCCTGAGCGTGGGGCCTCTGCCTACCAGGGGTGTG GTCGGGCCCTCCACCCTCAGTGCTGCCCTTCAGGGTAGACAGCCCCTCCCTCATTTGCAG CTCCCAGCCCCCGCACAGCGCCAGAGGGAGAAGGGTGGGCAGACCTGGGAGGCCCAAGTT TGGGATGAGCTTGGGGTGCCATAAGCCTGTGTGGCTCTGAGGGGCTCACCCTCAGCCATC AAGTAAAGCTCCACGGTCCTTGCTAAACTGTGCACAAACACAGATGTGTGCAAACTGTCT GGTCTGAAGGGAATGTACAGAGGAAGCGATCATGGGACATAAACCACCATGAAACAGAAC ATGCAGCACCCCACAGCTGGCGCCAGTGACCAGTAAGTGCCAAATGGCTACAGCTCCCCA GGAGGGAGGGGTCCCTGGCCAAAAGAAAAGGTGAAACCCAACACTCTGTTCCTGGGAAGA GCCCTGATGTTGCAGCTCTTCTCTGGTGGCCAGTCCCCAGGGAACTCCAGGGCCCCTGAG GCTCCCCTGCAGGTAGCCTGGCTCCCGCTGTGCCTACAGCCTTCCCCGATCTCCTGTCTC TTCACATTCTGTACACACCTCGACGATGGTATTTACCAAAAGCCCCCCTCAAGACAGCTC CATAACACAGGGAAGCAGGGAAATGTCATCTCCTTCACTCTACTGATCAAACATTGCTAC AAGTGCCAGGTCCACTACCAGATTCCACTAGATAGACAACCAAAAGGCAAGAAGAGAGCC ACGCTGGCTGCCTGGGCAGAAGGGGCCTGGCCTTAGACATGACCAGGGAACACAAAGGAG GCAGGAGCAGCCAGTCGCCCCTGCCAAATCAGCAGGAGAAGCAGAAAGTCAATGCAGGCA TGCCTGCTGCTGGTGGAACCAGGGCCTGGCCCAAACCTTCTGAAGCACAAACAGCATTGG GCCCAGGCAGTTACTAAAACTCCCCATCCTGCTATAAGGAACTATAGCTCTGTGGAAAAA TGGCCGATTCCAGGACTGAGACACGGAGAAAACAAGAAAAGATGAGCCTGAACACATACC TGAACCAGAAAGCAAGAAAGTGCTCACAGAATGATGGGGCGGCATCTAAAGGTGCCTGGA GCGCCCCCAACTGGCCACACATGGCACAGTCTGGGCATTAAGGAATAATGAGCCATGCGC TTGGGATTAGTAAACTACATACATCTTACAGCTCAATAAAAAATTTTAAAAAGACAATCG CCCAGACATTTAGGGGAAGAAGTAATGCCAATTCTACACAAACTCTTCTGGAAAACTGAA GAGGAGAGAACACTTCGAAACTCATCCTATAAGGCTGGCAAAAACCCTGATACCCAAACC AGATATTACATGAAAACTATAGACCACAGTTTATCATGAACATAGACCAAAAAATTCTTA AGATTTTAGCAAATCAAATCCAGCATACATAAAAAGGATACTACAGCACGACCCAGTAGG ATGGAATTCAAGTGACTGTAATTCACTGTAGTAATAGAAAATGATTCACCACGTGATCAT CTCAAGAGCTGCCGAAAAGGGAACTGCCAAAATCCAACATCCATTCTTGATAAAAACACA CCAATCCTGGAGCAGATGGACACTGCCTGCCTTCTGGGGAAGGGGGCCTTGCACTGCTAG GAGAAATTTTTGCCACATTAAACCTGCTTCTAATCAGGCTTCTACCAGCAGTCCAGTGTA CAGGAAACCCAGAGAGTGGAGAAGCAGGTTAAATGCAAGAGGAAGCATTTCGTCTGATAG ATCAAGAGCAGGGCATTCTATAAAATTAGAATGCCCTGGACTCTACAAAAAAGTCAATGC CACAATAAATGTTGGGGACTGTTTCAGAAGCAAAGAGACCAAAGTAACCAATTGTAGTAC ATGAACTTGGACTGGATCCTTACTTATAAAGCAACAATTTGCCACAAGTTAGGTACCATG ATGTGGCCGTGCACTGAAAGTCAGGAGACACCCGGAACAGTTAATTTCTCAGGGGGCTAC CTTCTGCAGCTGTGTAGGAGACCAGTTTTGTTCTAGAGCACTTGCTCCCAATCCAAGGCC AAGGTTGACCCCACCACCCAGGTTGTCTGGAGGCATTCTTGGTTTTCAGGACTAGGGAGG GTACCCCTGCCATCCTGCAATGCGCAAGACAGCATCTCACCAAAAAGACTTACCCAGCCA CAAATGTCAGCATGGCTGGGTTGAAAATCCTGGTCTCCAGGAATGCTGAAGACTCTTGGT GAATGCTATGCTGTCACATGTATATATTTTGGGATGTCAAGAACTAGAGCAAAACGTTAA CTATTGAACTTTAGTGAAGAGCACAAGCGTATGCACTATACTATCTTTTCAGCTTTTCTA AATGTTTTAACTTCTAAAATAAGAAGTTTGGGAAAAATAACAAAGGTGGAAACTGATGGT GCAGGGGGACCTGCCCTATATAATTTGCAGGAAAGGGCTCCCAAGCCCACCAAGCAGGCC TGGCTCAAGTGGGAAGCTCTTCGGGTTTGGGAGGGCGCCACAGGAGACAGTGCTTAGGAA TTTGATGTGCAACTGGAATGAGGCCCTGCGAGGCCTGAGAGGGAACTGACCCTGAAAGAC GCTTGCTGCACAACTGTCAACAGACTTCCAACTACTGAGGGGCAATGCTGCTGCAGTCAG CGCCACCACCAGAGTCTGCCTGCAGGCCAATCAGGCACCGAGGCCTAGGGCCATCCTGTC TGGAAGCGCGTGGGGCACAGGGAAGCCCGAACAGGGAGTTTCTGCTAAGAAGGTGGGTGC TGCGCAACTGAAAACAAAACTGGTTGGACCCACTGATCACCAAAAAGGCGCTGTCTCAGT CTCTGCGCCCGTTGATTTTGTCAATCTTAGAGACCTCTTCCGAACACTTCCAAGGAAGGA GCTGATCTGCACAGATACCAAGAGCTGCCCGGAGGCGCCGACCCGGAGGAGGCGGCACCT CATCCCGCTGCCCCCGCGCACCTCGGTGCTCGTTCTGTAGGCGCAGGCGCTGGTTGTACA GGCTCTTTTCGGTGAGGTGCTGGATCTCCAGTAGCCCCTGCACGATTTCGAACACGGTGC CGTCGAGAAGCGCCAGGGCCAGGTCGCTGAGCGTGGTGTAGGACAGGCGCTGCTGGAAAG AGCTGCGGGTAGGGGGGCGCGGTGAGCCCCGGCGGGAAACGAAGCCGCCTCCGCAGGCCT CCGCCCGCCCCGCCTGCGTACCTGGGCAACTCCTTCACCAGGCTCTGTAGCGCCGACAGC AACTGGTAGTGTCGCTCCTGCTGCCGGGCACCGTCCGCCACCTCCTCCAAGGCGGCCGCG TAGCGCTCCATGGCGCGGACGCCCGCTAGCCGCCGGCGGCGGCGACGAGCTCCCCCAGCT TCACGACATCCCGAGCGCGGCGCGTCCCGCCCCTTTTACGATTGTCCCACGCGCGGCGCG CTCCGCCCCCTTTTACGACAGTCCCGAGAGGGCCTGGCCGCCTGCCCCGCCCTGCCCCGC GCCCCGCCGCCTGCTTATTCAGGAGGCGCGCCCCGCTCCTCTGCGGAGACTCCAAGGAGG GTTTTGCTCAGCCCAGGCTCGCACCTGCCTGCTCCCCGCGCGACATTAAAGGCGAGACTC CGCCGTCTGGATATCGCAGGACCACGGAGAATCCCACCGGCCCTTGCTGAGTCATTGCGG GCCGAGGTCCGGGCTCCGCGCCGGCGCGCCTGCGAGGTGGACTGTCCCGGTCACCGGCCT CCCCAGAGGCAGGGGTCTCGGGCCAGAGCCCACAGGACCTGGGTCTAAGGCACCATCCGC GGCCGAGCAGTGGCGCTGGGGTCTGTCTGCGTCCTCCCCGCCCCCGGGGTGAGACAGACA GGACCGCCCTAGAGCCTCTGGGTCAGGACAGTCGGAGCCAGAGTCCCCATCTGGGCCTCC GTGGCCCCCAGCCCGTGAGCCCAGACTGCGCTCTCCCCGCTCCACAGGCCAGCTGCTGGG CCGGGGCTGGGGGGCTTCTGGTGGCTGGGGCTGAACCGTCTAGGAACAGGCTTTAATAGG AGGGGCTCGGGTGGAGGAATGGCTCCTGCAAAGGCCCTGCCAGAGTCAGGTCAGGTGGGT GGGCGAGAGGACCAGTGTGGCCAGGCTGCGGAAGCCAGCACAGGAGACAAGGGCGGGTGC TGTGCATTACAGAGGCTGCTGGGGGCTTGTCCATAGAGCAGCAGGGGGCACGGAAGGGCT TGGGGACGCAGACACGATCAGACTTGCATTTCATCAGGCACTCTTTAGCTGCCTGCTGGG TGGCTGGGCTGTGTGAGATGACCCTGCTTTGACCAGGTTAGGAATTATGACTGGTTTCCA GAGAGAACATTGGTTAGTAATTGGCTATGGGGAAGAGGGGGCGTTGAGCCAACCAACTGG GGTCCCCCTGAAGTGCCAGCAAAGTGTCTGGTTTTCTCCAGGATCTGTTTCCTCAGTCCC TGGCAGGATTGGCTCCCCTGCACGCCATGTCCTTGTGGTCCCTTCCAGACAGAAACCAGG CATGAATAAAGTGAATAATGACAAGCGCTCAGGCCACTGCCGTGGAGGGCGGTCCACAAC CCAGAGCTGGTCATGCGCCGGGCACCCTGGCCTCCAGGTCCCTCAGAATGGCAGGAGCTG GAGGTGGGACTCGAGATTGTGGGAGCTGAGTCTGTAGTGAGTGACCAGGAGCCAGCTGGG GCTGGGCTTGGTCACTGTCACCTCCCCCGGAGGTGGTGAGTGGGATGATTAGCTGGGTGA AGACGCCTGCCTGTTTCACCAGTGTCCCTGCCCTCAGTCCCCCAGTCCATGAAGAGTGTC TGTGTGGAGACTGGGAGGATATAGGGCAGCCCCACGTGATGATATGTCTGTGGCCAGCTG GGCAACACTGTCGCCAGCTCCGGGGTCTGCCAGGTGGGGTGGGGGCTGTGAGCCACAGCA GGAGCAGGCCAGGCCTGGGTTTGGACACCTGCTCCTCTGCCTTGTCCGGGTGCTGATGCA GGAAGGCCCCTTCTCCAACCCTTGGTGCAACGTCTGACCTGAACCAGCTTCCCCAGCCCA GCAACACTGCTCCCCTAATGTGGATGTCAACCTAAAGAAACAAACTGAACCACAGTTCAT ATAGAGAGTTTGTCTGGGCCAAGGCTGAGAGAGCTGCCCAGGACACACCAAGTTGCCCTG GGAGTGCTTCATTCAGCCTTATTACAAGCAAGTTTTTAAAGACTGAAGGGGACAAGGAGT GGGCTGATATGAAGTCAGCAGGAATTCTGACTGCTTTCCAGAGAGAACCCTTAGTGACTG GCTATAAGGTGTTGGACTACTGGGTAAGAGTTATGGTGTCCAGCGTATGGCTTTTTATGC CTACTTGGTGTCAGTCTGGAACCCACAGAGCAATTGGCTTCAAGAGGTCGTTAGCTCAAG TAGGAGTGGGATGTGACTGCTGTTTCATTCCAGTGCCTCTCTGGGCCAGATAATTAAAGG GAGCTCACATTCCTCAGATAAAAAAGTTCCTTTCGCATTAGGAGAAATACGTAATGTAGA TGATAGGTTCATGGGCACAGCAAACCACCATGGCACGTGTATACCTATGTAACAAACCTG CACGTTCTGCACATGTACCCCAGAACTTAAAAGAATATATATATGAATATATATATATAC TTTATACATATATATATAAACTTCCATTGTGCGCGCGCGCACACACACACACACACACAC ACACACACAAAGTTCCTTTTCTTTTTCATGGACATGAGCAGTATGTCACTCAGGCATGGG GACTCCTGGGCCACCTCTGGGTCTTCAGGGAGGGCCTGGTCTGGAAGGAGGGGTCTCATC CTCCAGAGCCCCAGGGATGGGTGATATATGTGTAGGAATGGGGGAGCTGGTTCGCAGACG TGCGTTTCCCCTGTGTGTGTGGACACGATGGGCCCCATGGCTCTTCCCGTCGGGTGGTAT CCACCGAAGGCCTTTGCTCTGCGGCCACTGTCCACCTCCACTTGGCCTCCCGAGTCCACC AGGCTGGCTGGTGACACCTTGGGTTAGTGTGGGGTGACTGGTGTGTGTTGGGGAGCTGCA GGTCCCCTGTCTCTGACACAGCTGTGTCTGGGCAAACACCAAGGTTTGAAGGAGTGGGTG CCTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCGCGCACGTGTGTCAAAAGGCTTT GCTGCATGCCTTTTGTGTGGCTCTTTGTCACATGTGCCTTTCTGTGTTTCTCTGTGAGTT TTCATGTGTCAGGTTGTATGTCTGTTGTGTGGGCGGAGAAACAGCTCTGCTGTGGGGCCC TGAGCCATTGCAGGATCCCCATAGGCCATGCAGAGGTGCACACAGCACCTGACCTAAGCC TGCTCCTCCCAGAACATCAGGCTGCACCCAGGCCACCGCTCACTCCGTCTGTCTCCCGAG ACTGTTCCTCACTTCCTGCCTCACTGGCCCTGGTGAGGGTGAGGCTCCCTGCCCCCGGCC CTCAAAACAGAGCTGCAGATGAGGACGCCACCCAGCTGCAGTGTCAAGGGGCTCCTCCAC AGTGGGTGTCCCGCAACGCATGCTCTCTGACCCCTTTTGTTTCAGTGTTGTCCTTTTTAG TGTGTGGCACCAGGACAGCAGGGAGCTGCCACCTCAGGCTTCTCTTATTTTCACTCTGTG AGTAATAAGGTCTGAATGAGAGGGGCTCATTGTGTCTTTACTGTCAAATCAGTCAGGCCT TGGCCTTGGCATGTCTTGCGTGTGCTTGACAAATGTATTGCTCATGGGGTCTGGTGGAGG CACAGCACCCTTTTAGAAGACAGGGTCTGTGCGGACCAGTTACGGGTGACATTTCCTAGG CACAGCTCTTGCCTAAGTGGTGTGGGATGGGAGGAGGGCGGGGCTTCCCACTGTCCCACC AGCAAAATGGGCTGTGTGGCCAGCTCCTCAGGACCCGAGAGCATTTGCTCCAGGTTGATA AAATGTTGCGGTGTTGGGTGGCAAGAGTAGGATTGAAACCAGCCACCCAGTTGGTGCTTT GTTACCATCAGAAGTGTTTGCTGAGTCAAAGCAAATGGCCACTCAAAACTGAGTGGGCTG AGTGTGGTGACTCACACCTGTAATCCCAGCACTTTGGGAGGCCAAGATGGGCAGATGGCT TGAGGCCAGGAGTTCAAGACCAGCCTGGGCAACAGGGTGAGAGCCTGTCTCTACAAAAAG TTTAAAAAATTCGCTGGCATAGTGGCATGTGCCTGAAGTCCCAGCTACTTGCCACTTGGG AGACTGAGGTGGGATCACTTGAGCCCAGGAGGTCCAGGCTGCAGTGAGCTGAGATCACAC CACTGTACTGCAGCCTGGGCAACAGAACAAGAATCAAATAATAAATAAGTAAGTAAGTGA GTAAATCCATCCATCCACCCCAGAGTGAGTCAGCTGGGGCTGCTGCATTGCTCCTCCCTC ACCCACAGGATTCCCTGGTTCCAGCCAGGAGACGATGGTCAACAGCCACTGCTGTAGTGG TGCCTAGTTAAAACTCTTTGGTTTCTGTCTCTGTAAAAAAAAGTGTCTAATTGCATTTGG GCAAAAAACTTGCAGTGAAGAGAGAAAAAGAGATGAAAGCAACCTGTGAGGGAGGCTCGG GTTGACGTGCGGCATGTGCTGTTTGAGTGTGATGAGTGTGTCGGTTTGCACTGTAGATGC TCAGGAAGTGAGGATATGTGGAGTGCTGACTGTATACATGATGCCTCCACTGGCTGGCTG AGTAGAGTTTCATGAAACAATGTTGGTGGACTCAGCCCTGAATCCCTCAGGCATTCAGAG AGCAACCTCTGTCTTTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGGCTCGCTCTGTTGC CCAGGCTGGAGTGCAGTGGTGCAATCTCGGCTCGCTGCAACCTCTGCCTTCCGGGTTCAA ATGTCCTGGCTTTAATTTCACAGATTCTTTCTTCTGTTTCTTCTGTTTGATCAAGTCTGC AATTGAAATTCTCTATTGCATTTTCATTTCATTCATTTATTTATTTTTATATATTTTTGA GACAGAGTCTGTGTCACCCAGGCTTGAATGCAGTGGTGCCATCTTGGCTCACTCCAACTT CCACCTCCCGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCTAGTAGCTAGGATTACAGGC ATATGCCACCATGCCTGGCTAATTTTTGTATTTTTAATACAGATGGGGTTTTGGCATGTT GGCCAGGCTGGTCTTGAACTCTTGACCTCAAGTGATCCGCCTGCCTCGGCCTCCCAAAGT GCTGGGATTACAGGAGTCCGCCACGGCACCCAGCTTGCATTTTCATTTTATTCATTGTAT TCTTCAGTTCTAGAATTTCTGTTTGGTTCTTATTATTTCTGTATCTTTATTGAACTTTTA GTTTTGTTCATCTACTATTTTCTTGATATTATTGAGTTGATATATACATTCTAGTAAATT TCACTGAGCTGTCTTAAATCAATTATTTTGAATTGTCAGACAATTTGTAGATCTCTATTT TTGGGGGGTTGATTACAGGAGATTTATGAGTTTATTTTGGTAGTGTCATATTTGCTGATT CTTCATGATCTACAGACTTTCATTAATGTCTATGAAGAAGCAAATACCTCTTCTTTTTTT TTTTTTTTTTTTTTTGAGACAGAGTCTTGCTCTGTCACTCAGCTGGAGTGCAGTGGCGTG ATCTCAGCTCACTGTAACCTCCACCTCCCAGGTTCAAATGATTCTCCTGCCTCAGCCTCC CAAGCAGCTGGGATCACAGGCATGTGCCACCACGCCTGGCTAATTTTTTTGTATTTTTTG TAGAGACAGAGTTTCACCGTGTTGTCCAGGCTGGTCTCAAACTCCTGGCCTCAAGTGGTC TGCCCGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCATGCCCAATCTC TTTCTGTCTTTATAGATTGGTTTCAGCAGGTACAAACCTTTTCCTGCTGGATCCCTTGAC TGGATCACAGTCAAGTGGGCCTGGAGCCATATCACATGGCTGCTGCCTGGTCTGCAGCTG AATCTCTGATTGGCAGGCCTGCTATCAAGGCATAGGTTAGTGATGCAGTTTCTGCTGGAT CCTCAGGAGAACTGGACTGCCTCTGATACCCTGATTGAACAGGACTGGAGCCAGGTCATG GGGCCACTTCTAGTTCTACAGTCAAGTCTTCAGATATCAGGCCTATTACCAAGGGCATGG ACTGGTATAGCTCCCTGTGGGTCCCAGATTGAGCTCCTGCTGGTTTACTAGGTAGGTCCA TGGGAAGACAGGACTGCCTCCAGACCACAGTAGAGCAGGGCTAGAGCCAAGTCACAGGAC AGCTTTGGTGACCACATTTGGGTTCAAGATTGGTGGTCCTCTTATTAGGAGAATGGATGG TATGTCTTTCACCAGGTCCCAGGATGGGCTGGACTGTGCCCAGACGGGCAAAGCAAGACT GGAATGGAGTCACAGGGCTACTTTAGTGTCCATAGCTGACACTGAGATCAGCAGGCCTGT TACCAAGGGCATGTAAAGGCATCACTGAATTCCTGGGCAGGCAAGACTGACTGTGGTAGA GTGGGGCTGAAGCCAGGTCAGGGCTGCTTTAGTTTCTGCAGTCAGGACCATGGTTAGAAG GCCTGTTACTGGGGGCATAAATGGTCATGGTTCCTCCTAGGTGCTTAGTGGATGGGGCTA GTTGCAAGAACATGATCTAGTGGAGCTGGACCCAAGTCCATAGGAGGACAAAGCTGCTTT CAGTCTGCAACTGGGAACCTGTCACTGGTGTGTGGACCTGCCTTCTCAAAGCAGCTCTCC TTGGTTTTGGGCTTTGCTAGAGTTTTGCCACCTCCTGCCTGGATATTAAAACTCTTGCAA AGGCAGTTTTGTCCATGAATGGCTCCAGATCATTGTTTGTGTGGGGAGAGGTGAGTGGAG GGCCTCCTGTTCTGCCATCTTGCTGATGTCACCCTAAGATGATTATTTGAATTCTTTGTC AGGCAATTTGTAGATCTTCATGTCTTTGGAGTCAGCCACTGGAGTTTCATTTTGTTTCTT TGGTGGTGTCATATTTTCTCATGCTTCCTGTTCTTTGAAGACTTAGATTGCTTTCTTCAT GTTTGAAGAAGGAGTCATCTTTTCCACTCTTTACTAACTTCAGGAGAGAAAGACCATCAA TTAGCTAAGCTATAGATTCTGGGGGTCTCTCAGTCCTTTTCTGTGGGTGGTCCTTCCCTT TTAAGGGGGATGTCTTAGGATTTTGTCCCTTGTCTTCATTTCACAAATGAATAAAACAAC CAGACCAGACATAAGTAAAGGAATACAGCACTTGAACAACACCTGAAAAAACAACTAGAC CTAACAGACATACACAGGATATTCTACCCAACAACATAATACACATACTTCTCAAGTATA CATGGGACATTTTCAGGATAGACCATATAACACATCACAAATTAATTCTCAATAGGGGCT GGGTGCAGTGGCTCACGTCTGTAATCCCAGAGTAATTTGGGAGGCTGAGGCGGGTGGATT GCTTGAAGCCAGGAGCTTGACATCAGCCTGGCCAACACGGTGAAACCCCATCTCTACTAA AAATACAAAAATTAGCTGGGCATGGTGGTGCGTGCCTGTGATCCCAGCTTCTTGGGAGGC TGAAGCGTGAGAATTGCTTAGGAGCCCAGGAGGTTGAAGCTGCAGTGAGCAGAGATTGTA CCACTGTACTCCAGCCTGTACTTCATGACAAAGAAAATGTACCATTGTACCACTGACAGA ACGAGACCCTGTCCCAAAAAAGGAAAAAAGCTCAGTAGATTTAAAACGATAGACATCATA CAAAGTGTCTTCTCTGACCACAACAGGATAAAGTTAGAAATCAATAACAGAAGATTTTAA AAAGTTCACAAATTAGTAGAATTTAAACAACACACTCTCAAACAACCAATGGATCAAAGA AATCACAAAGAAATTATAAAATTCTTAAAGACAAATGAAAATGAAAGCACACTATATCCA AACTTATGGGTTGTGGCCAGTTGTGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGA CTGAGGGAGGTGGATAGCTAGAGGTCAGGAGTTCAAGATCAGCCAGGCCAACATGGTGAA ACCCCGTCTCTACTAAAAACACAAAAATTAGCTGGGAGTGGTGGTACGTGCCTGTAGTCC CAGCTACCCAGGAGGCTGAGGCATGAGAATTTCTTGAACCCAGGAGGCAGAGGTTGCACC ACTGAGCTAACACCACTGCACTCCAGCCTGGGTGACAGAATGAGACTCTGTCTCAAAAAA CAAAGAAACAACAAAAAACCACAACTTATGAGTTGTGGTGAAAGGAGTGCTAGGGAGGAA ATTTATAGCTATAAACACATTAAAAAAAGAAACACCTCAATTCAACAACATAAGTTTACA CATTAAGAAACTAGAAAAAGAAGAATGAAACTAAACCCAAAGTTAGCAGAAGGAAGGAAA CAATAGAGATCAGGGCAGAGATAAATGGAAAAGAGAATAGAAAAACAATAAAAAACAAAA CCAAAAGTTGGTTCTTCAAAAAGATTAATAAAACTGACAAGACTACACTTTGGGAGGCCG AGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCAGCTAAAACGGTGAAACCCC GTCTCTACTAAAAATACAAAAAATTAGCCGGGCATAGTGGCGGGCGCCTGTAGTCCCAGC TACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGC CGAGATCCCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAA AAAAAAAAAACTGACAAGACTAAGGAAAAGGGAAACAATCTAAATTACTTAAAACAGAAA TGTATTTGAGAATATCTTTATATATTTCTGTCTGTCTGTCTGTCTGCCTGTCTATGTTTT AGAAACAAGTGTCTGTCTATGTTTTAGAGACAAGGTCTAGCTCCATCGCCCTGGCAACAA TCAGATGCAACCACAATCAGTGGCACAATCGGCTCACTGCAGCCTCGAATTCCTGGGCTC GCCACTATGCCAGCTCTTTTTTTTTTTTTTTTTTTTAAGAGACAGGATCTTGCCATGTTA TCCAGGCTGATCTTGAACTCCTGGCCTCAAGGAATTTTCCCACCTCGGCCTCCCAAATTG TTGGAATTACAGGCATGACCCACCATTCCCAGCCTAGAAAGGATTATAAAAGATTACTAT AAATAATTGTGTGCTCATAAATTGGATAACCCAGATGAAATAGATGAATTCCTAGACACA CAAAACCTACCAAGACTCAATTATAAAGAAACAGAAAGTCAGAATAGACCTAACCTAGTA AGGGAATTGAGTCAGTAATAAGACAATCTCATGACAAAGAAAAGTCCTGGACCTGATAGC TTTACTGGTGAGTTCTGCCAAACGTTTAAAGAAGAACTAACACTGATTCTTTTCAAGCAT TTCCAAAGAGTGGAAGAGGAGTGAATACCTCTTAACTCTTTCTATGAGGCCAGCATTACC CTGATACCAAAGCCAAAAACACTATAAGAAAATAAAACAACAGATCAATATGCCTCTGAT CATTGATGCAAAAACTCTAAAAATACTAGCATACTGGGTGGGGCATGGTGGCTCATGCCT GTAATCCCAGCACTTTGGGAGGCTGAGGTGGGTGGATCACCTGAGGTCAGGAGTTCAAGA CCAGCCTGACCAACATGGAGAAACTCTTGTCTCTACTAAAAATACAAAATTAGCCATGTG TGGTGGTGCATGCCTGTAACCTCAGCTACTCGGGAGGCTGAGACAAGAGAATCGCTTGAA CCCGGGAGGTGGAGGTTGTGGTGAGCCGAGATCATGCCATTGCACTTCAGCCTAGGCAAC AAGAGCAAAACTTTGTCTAAAAAAAAACTATCATACTGAATTCAGCATCATATTAAAAGG ATTATACACCATGACCAAGTGGGATTTATTCCTGGAATGCAACGATGTTTCAATATATAA AAATTGATCCATATAAATTCAGACAATTAATTTTAACTGATGTATAGTTTCACATTATAT GCACATACCACGATTTACTTTTCCATTTTCCTAGTAATGAGCTTTAAGATTATTTCCAAT TTTTAGCTATTACTAATAATGTTTCAACAAACACCTTTGGACATCTCTTCTGCAAATGTG TGTGTATTCTCTTGGAGAAGGTTTACCTTGTGGAATTTCTAGTGTGTATGTGTATGTGTG TGGGTATATATATATACACATATGTATATATTTTCAACTTTACTATATATTTCCAAAAAA CTCTTCAAAGTGGCAGATTTAAACTCCCCTTCGCCGTATATAAATTCACGGTTCCTAAAG CCCTTGGCATAGTTTTGTGTTTTTATAGAAAAGGGCATAAAGTCTTATTGCTGTTGAATT GTACCTTGCTCTTTTCAATTGTCTTTTCACAATAATTCACTATTTATCATCTGTTGTGAA TATTGTGGAGTAGTTCTTAATCATTTTTTCTACTAAACTATTTTATATCATTAATTTGTA GAAATCCTCTTTATATAATGGATATTATACTTTTCAATTATATATTTTGTAAATATTTTA CCCAATTTTCTGGCTTGCATTTAACCTTTATTTAGGATGCCTAGGATTTTTCATTTTAAT ACAGTCTAATTTATTTTTCTTTTCTTCTTCATGCATTCTGGCATTTGATTAATAAGCCAT TTGTTGCCCAACGTCATTAGTTCTCTGTATATACTTTTAAATATCTTAAACATGTAATAT TCATAAGTAATTTCACAAGTAGTTTAAGATTTGAGTCTGTAACCTAAATAGATACAGGAT TTTGTGATATGCTGTAATATACAGATTTTTATTTATATATGGATAAAAATTTATATGTTT TTTCTGACAGCATTTATTGGATAGTATAATTTTTCTACTGACTTTTAGTATCATATCTTT TATTACAAGTCCAATGTATTCAGCATACCTATGGATCTATTTCTTGGTGCTTCCATTTTT TCCCATTGTTTAACGGTGTATCACTGAGCCAAAGCAAACCATTTTAATTATTATAATTAA AACACATTTAAATATTGATAGGCTATTATTGTTCAAAATAACTTTGGTTCTTCCTTTCCG TTGATTACTTTAATATGACTTTTCCAATCAAGTTCTAAAAAAATACTGGTGGTATATTTA TTGAAATAACTTTAATAAAATTAAATGAAAGATCATGTATTTGTTTTCTGAATTAATTCT TAGATACATTAATGTTTTATGTTACCATGAATGTGATATTATAATATAATATTTTTAATT GGTTGCTACTGTTTATAAGAATTTCATTTTCTGTTTACTTTGCCTTCATATCTGAAAACC TTGCTGATTTGATTAGTGCATCCACAAATTTTCTTGGATTTTCTATGGGTAATTACAAAT CTCCACACAATGAGGTTGCAGTGAGCCAAGATCACACCACTGTACTCCAGCCTGGGCGAC AGAGTGAGACACCATCTCACAAAAACACATAAACAAACAAACAGAAACTCCACACAATGA CAACGTATGTGTTTTCTTTTTTTCTTCCTCTTTCTGTAATATTTCTTTGTCCTATCTTAA CTGAACTGGCCAGAAACCCCAGGACAATGATAAATACGAGCAGTGTCAACAGACATCTCA TTCCCTTTCCTAGCTTTTATAAAAAATAACGATTATGCTTCAACATTACATATGGTGGTG TCGATGGTTTTGTTATAGATAAGCTTATCAGGTTAAGAAATTTGTCTGCTTTTCCTAGTT TGGTATAAAGATTTTAATATAAATGAATGTTGTATTTTACCATCTTATTTTTTTCCTACA TCTGCTAAGATAATCCTGTGTTTTCCCTTTTTCAATCTCCTAATGTGGTGAATGACATTA AAATACCTTCTATTGTTAAAATATTCTTGCAACGCTGTATAGAACCAATGCCTTTATTCT GTATTGCTGATGGATTTTTGAAAAATATGTAGGTGGACTTAGTTTTCTAAGGGGAATAGA ATTTCTAATATATTTAAAATATTTTGCATGTATGTTCTGAAGGACATTGGTGTGTCATTT CTATACCATCTGGCTACGAGAGGAGCCGACTGAAAGTCACACTGCCGGAGGAGGGGAGAG GTGCTCTTCCGTTTCTGGTGTCTGTAGCCATCTCCAGTGGTAGCTGCAGTGATAATAATG CTGCGGTGCCGACAGTTCTGGAAGGAGCAACAACAGTGATTTCAGCAGCAGCAGTATTGC GGGATCCCCACGATGGAGCAAGGGAAATAATTCTGGAAGCAATGACAATATCAGCTGTGG CTATAGCAGCTGAGATGTGAGTTCTCACGGTGGCAGCTTCAAGGACAGTAGTGATGGTCC AATGGCGCCCAGACCTAGAAATGCACATTTCCTCAGCACCGGCTCCAGATGCTGAGCTTG GACAGCTGACGCCTTGGATCATCTGCCACTGATCTCTGGTCAACATTTTTATCACCCAAC ACAAAAGAAGCAGAGATTTATCAAGTTACTTAACCTGACCCTTTCATCTTTTGCTACACA TACTCTTGTAATTGATCTCTCCATGAATTGTTTTCTGTTTAAAATATCTAGAATGTTTTC TGCTTCCTGACTTGATTCTAATATTGTATAGGTACTAGAAATGGTTCTAGAAATAGATCT TTATAGATGAGAATCTGGAAGTGGTTTGCTGACCTGTTTCAGTCTGAATGAATTCCTGAC CTTCTTGTCGGGAGGAGACAGAAACCTGATCATCTGTAGTGTACGGTGGTATCATGATTA CTTAAATCATCAAATGTGGTTATTGGGAATGATGTGTTTTTTAAAGTGGTACATGAGAGG TAAAATTGCTATTGTAGTTGACTGTTGCAGTTATAATTTTGTCAACATGGTCTGTAAGAG TGCAATGGCGTCGGCCCGGTGCGGTGGCTCACGCCTGTAATCCCAATACTTTGGGAGGCC AAGGTGGGCCGATCACGAGGTCAGGAGATCCAGACCATCCTGGCTAACACAGTGAGACCC CATCTCTACTAAAAATACAAAAAATTAGCCGTGTGTGGCGGCACACGCCTGTAGTCCCAG CTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCAGAGCTTGCAGTGAG CCGAGATCGCGCCACTGCATTCCAGCCTGGGCGACAGAGCAAGACTCCATCTCAAAAAAA AAAAAAAAAAAAAAAAAAAAGAGTGCAATGGAAAGCTGGTAGAAAATGGACATTGTTTAA AGACCAAAACAACCACACTACTTTGCTAATCCCTATCAGCTAAAGACTAGAGAATATATG AGGCATAGATTTACGTGGTGTTTGACTAAGGCATGCAGAATATAATCTTGACATAGGCTA AGTTTATCAACATAGATACATAGAGATTCTAAAGTTAAAGTCTTAGCCCAAGAAGTTAGA AGTGGTGCTCAAGTTTGTTTGTCTGACAGAAACTGTGAATCCACACTGGCCTGCTTATTT TGAGGTTGCGTTGCCAGAGCTTTCCTAGCATAATAAAGAGAGGTGCATACAAAGGAATAG GAATAGTAGGAGGTGGGGGTAAAAATATCAGGTGTGATCCACATGCCAAGCCGACCCCCA CTGTGTCCCACAGGAAGCCCCAGAAGATGTTTCTCCAAGAAATTAGGATTCGTTTGTTGG GGAGGGCTGCTGGCATGCTTGAAAAGTACTGTGACGGCTGAATTTTGTGGGCTTGGGGAG ATTGCAGGTTCTCTGATTTCAAGGGGAATGATGTGATCCTAGAGTTGCAAAGAACAAGTG ACAGTGGAGGCGCTTATGCTTTGTGACTGCACTAGAGACAAGGAAGACACAACTAGAATA ATGGGGAGCAGGAATGGAGCGGCCAACAGAATATCTGACTGTTAGGGATCTTTGATGAAG GCTGATTCTCAGGGAGTGAACTAGATCAGTGACCAACTATTTGTCTTTATATAACTGGGT AGTGTGGATGGATTCTAATAAAGGGACTACTTACAGCACAGCAGGAAAGTCACAAAGAAA CCAGACAGAAGAGTGTAAGTAGTAAGGGGCCAAGCAGTCATCTGACTAGAGACAGTGCCA GCTTGCCAAAGAAGGCACCAGACAGAAGCTGTGATCTTCAGCAAAGGGACACAGTCTGCC TGTGCTGACCCTGCAGGGGCAGAGGTGGGGGATAAACACACTCTTCTCTCACCTATCTTC TGCCACCCCCTCCATTAGCTGAACCCCAATAAAAGCATGAGGGTAAGGGAGATCTCTGAA GTATCCAATTCAGGTGAGCCTCCTAAGGAACAAAGCAGAATGCAGAAAAATTAAGAGTGG GTCTAGGGAATAAAATAGAGATATGCACCAGAGTATGATGATGTGTCTGGGAAAGAATAT ACAAATACTTTTAAAATTACTAGACAATAAACCTGAGATGACACTAATACAGGTAAATCT CATTTAATGGTAATATATTCCAAGAAATGCATCATTAGGTGATTTTGTGGCTGTGCAGAC ACCAAAGAGTGTACTTTATACAAACCTAGACAGCGTAGCCTACTACATACCTAGGCTATA TCGTACAGCCTATTGCTCCTGGGCTACACACCTGTGCAGCATGTCACTGTGCTGAATACA GTAGGCCAATGGTCCGCAACACCCAAGCCATGGACCAGCACCGGTTCGTGTCCTGTTAGG AACTGGGCACAAAGCAGTAGATGAGTGGCTGGCCAGCCGAGCATTACTGCCTGAGCTCCG CCTCCTGTCAGATCAGCAGCAGCATTAGACCCTCACAGGAGCGCAAACCCTATTGTGAAC TGCCCATAGAAGGGATCTAGGTTATGTTCTCCTTATGAGAATCTAACTAGGCTGGGTGCT GTGGCTCACGCCTGTAATCCCAACACTCTGGGAGGTCGAGGCAGGTGGATCACAAGGTCA AGACAATCCTGGCCAACATGGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCTGGGT GTGGTGGCGCATGCCTGTAGTCCCAGCTACTCGGGGGACTGAGACAGGAGAATCACTTGA ACCCGGGAGGCAGAGGTTGCAGTGAGCCGAGATTGTGCCACTGCACTCCAGCCTGGCAAC AGTGGGAGACTCAGTCTAAAAAAGAAAAAAAAGAAAAAAAAGAATCTAACTAATGCCTGA TGATCTGAGGTGGAAGAGTTTCAGCCCAAAGCCATCCCCACTCCGTGGAAAAATTATCTT TCACAAAACCAGTCCCTGGTGTCAAAACTTTGGGGCCCACTGCTGTAGGCAGTTATATCA CAGTAGTAATATCTAAACATGGAAAAGATACAGTAAAAACATAGTACATTGGGAGACCGA GGGGGGCGGATCACCTGAGGTCAGAAGTTCGAGACCAGCCTGACCAACATGGAGAAACTC CGTCTGTACTAAAAATACAAAATTAGCTGGGCATGCTGGCGTGGGGCTGTAATCCCTGCT ACTCGTGAGGCTGAGGCAGGAGAATCACTTGAACCCCGGAGGCAGAGGTTGCAGTGAGCC AAGATCGTATCATTGAACTCCAGCCTGGGCAACAAAATAAAACTCCTTCTCAAAAAAAAA CAAAGAAAAAAATATATATATATTACAAAATTTAAAGAGAGTGGTACACCAGTAAAGGAC ACTTAGCATGAACAGAAGTTGCAGGACTGGCAGTTGCTCTGATGAGTCAGTGAGTACTTG GTGAGTGAATGCGAAGGCCTAGGATATTACTGTACATAACTATAGACTTTATATGCACTG TACACTTAGGCTACACTAAATGTATTTAAATTTTTTCTTTCTTTAACAAGTTCATCTTAG CTTATCATGACTTTATAAACTTTTAATTTTTTTAATTTTTTGATTCTTTGGTAATAACAC ATCTTAAAACAAAAACACATTGAACAGCTGTACAGAAATACTTTATATCCTTATTTGATA AGCTTCATTTATTTTGATTTTTTATGTTTTAAACATTTTTGTTAAAAACTAAGATACAAA CACACACACTAGCTTAGCCCTGTAAGGGTCAGGATAATCAATATCACTGTCTTCCATCTC GAAATCTTGTCCAAGTGAAAGGTCTTCAGGGTCTTCAGTGGCAATAACATACATGCAGCT GTCATTTTCTATGATAACAAGGCTTTCTTCTGGAAGAACTCGTGGAAGACCTTCCTGAAG CTGTTTTATAGTTAATTTTTTTTAATGAGTAGCAGTACTACACTCTAAAAATATGAATAA AATCTATAGTATTATACATACTGTTGATCCTTGAACAGTGCAAGGGTTAGGGGACCAGCT CCTCTGCAGTTGAAAATCCATATATAATTCTGGGATATCCCCAAACTTAACTTAATATAT GATATATATCATATATAATATATATGATATATATTACATATGATATATATCATATATATT ATATATGATATATATTATATATAATATGTATGATATATATTATACACAATATGTATGATA TATATTATACACAATATGTATGATATATATTATACACAATATGTATGATATATATTATAC ACAATATGTATGATATATATTATATACAATAAGTATGATATATATTATATACAATATGTA TGATATATATTATATACAACATATGATATATAATATACTATATGATAGAGATTATATACA ATATATATGATAGAGATTATATACAATATATATGATACAGATTATATACAATATATATGA TACAGATTATATACAATATATATGATACAGATTATATACAATATATATGATAGATTAGAT ACTATATATGATATAGATTATATACAATATATGATATAGATTATATACAATATATATGAT ATAGACTATATACTATATATGATATAGATTATATACTATATATGATATAAATGGTATATC ATATATGATATAAATGGTATATCATATATGATATATACAATATATCGTATATATGATATA GATGATATATCATCTATAAGATATAGATGATATATCATCTATAAGATATAGATGATATAT CATCTATAAGATATAGATGATATATCATCTATAAGATATAGATGATATATCATACCTGAT ATAGGTGATATATCATATATGATATAGATGATAAATCATATATGATATAGATGATATATA TCATATATGATATAGATGATATATATCATATATGATATATCATATATTATATAATAAATG ATATATATTATATATAATAAAAGCTATATATTACATAATAAATGATATATATTATGTAAA ATATATGATATATATTATATATTATATCTGATATATATTATATATTATATATTATATATT TTATCTGATATATATTATATTTGATATATTATATATTATATTATATATAATTATATTATA TTATATTATATTATATATATTTTATATTATATTATATATAATTATATTATATTATATTAT ATATTATATATAATTTATGATATATATAATGTATTATATATAATTTATGATATATATTAT ATATTATAAATCATATATCATATATATTATGCTATATTATATATAATATAGCATAATATA TCATATATTATATAATATATAATATAATATAATATAATATAATGTAATCATATATAATAT ATAATATATTATATTATAAAATATATTATATTATGATATACTATATATTACATATTACAT TAGATAATATTTTATATAATATATAATATATATTATATATTATACTTTATTATATTACAT ATATAATTATACATTATATTTCATATATAATTTATAATTATATATAATTATATATTATAT TATATAATATTATGTATAATATATTATTAAATATAATATTATATATTATATTAAATATAA TATTATATATTATATTAAATATAATATTATATATTATATTAAATATAATATTATATATAA GGATGCAGGATGTAAAAGGAAATTATATATATGTTATATATTATATATATTATATTATAT ATAATTATATATATATTTGGGGGTGCCCTATTTCCTATCTCATAACTTATTTTAAGAAGC ACAGCATAATAATGTGTGGACTTGGGATTCAGTTTTTGAAACGACACACTGAGCCTTCCA TGACCTTCCTGTACATGTGAAAGCACACCTGTCTGCATGGCAGCATTTGGACCTCACAGT GTGGATTGTGCCTTCACCCTGGAATGTTTATGCCCTATCGCCATGGTGATGTGATTAGGG ATCTGCTGCCCTTGGTCCTAAGTGCCACTATCTGTGCTGAGTTTTTCAAAGGTCAGAGCA GATTGAACCTTTGTGGTTCCATTTTCCCTGATTTTGATTTTTCTTATGGGGAACCTGTGT GGCTGCATTCAAGGTATGTTCATACTGGCCTGTCAAATGCGATCTTTTCAAATTACTAGT TAATGCTTTCAAAATATGTTATTTAAAAAATTAGCCTCTGTATTTTCCATATGCAGTTAT AAATATGTTTCATGGTTAGGTTTTATTCCTCAATTTATATATTTGATTCTTATACCAAGC AGAGTACCTTTGAAATTTTTCTTCATTTAAAAAATATGTATCTTGGCTCAAGCCTGTAAT CCCAGCACTTTGGGAGGCCAAGGCAAGAGGATCACAAGGTGAGGAGATCAAGACCATCCT GGCCAATACGGTGAAACCCTGTCTCTACTAAAAATACAAAAAATTAGCCAGGCATGATGG CAGCTGGTGTAGTCCCAGTGTGAATTGGGATTCAGTTTATTCCCAAATTCCCAAAATATA TATATATATATAATATATATTATATTATATATAATTTTATATATAATATATATTATATTA TATATAATTTTATATATATATATTTGTCGGTGCCCTATTTCCCATCTCATAACTTATTTT AAGAAGCCAGCATAATAATGTGTGGGCTTGGGATTCAGTTTTTGAAACAAAACACTGAGC CTTTGATGACCTTCCTGTACTTGTAAAAGCCCACCTGTCTGCATGGCAGCAGTTGGACCT CACAGTGTGGATTGTGCCTTCACCCTGGAATGTTTATGCCCTATCGCCATGGTGATGGGA TTAGGGATCTCCTGCCCTTGGTCCTAAGTGCCACTATCTGTGCTGAGTTTTTCAAAGGTC AGAGCAGATTGAACCATTGTGGTTTCATTTTCCCTGATTTTGATTTTTCTTATGGGGAAC CTGTCTTGCTGCATTCAAGGTATGTTCATACTGGCCTGTCAAATGCGATCTTTTCAAATT ACTAGTTAATGCTTTCAAAATATGTTATTTAAAAAATTAGCCTCTGTATTTTCCATATGC AGTTATAAATATGTTTCATGATTATGTTTTATTCCTCAATTTATATATTTGATTATTGTA CCAAGCAGAGTATCTTTGAAATTTTTCTTCATTTAAAAAATATGTATCTTGACTCAGGCC TGTAATCCCAGCACTTTGGGAGGCCAAGGCAAGAGGATCACAAGGTGAGGAGATCAAGAC CATCCTGGCCATTACAGTGAAACCCTGTCTCTACTAGAAATACAAAAAATTAGCCAGGCA TGGTGGCAGCTGGTGTAGTCCCAGTGTGAATTGGGATTCAGTTTATTCAGTTTATTCCCA AATTCCCAAATTATATATATATATATATATAATTTCCTTTTACATCCTGCATCCTTCAAC GTTCCATCCCCCACCCCACAGATTAAGTTATTCCCCAGGGGAGAATATGGCAAAGTCTAT TTTAATGCAGTTTTTAACCCAATTAAGAACCTATGAAATCATTACTTTCCAAAACTTTGG AACAAAGCCACAGTAGTATGGATCCGTTGGAGGCTTTTCACACAATAAAATGTACATCTC TTTGTTTTTAACATGTTTTTCCCTTCCTCTCTTCTTTTTTTGTGAAATGTGTATTTACTT TAATATATTTGTAGTAAGTCACTTCCATGCACATATTAATTTTTTAAAGTAATAAGTATG TGTATTGTCTACGTGTGAAAGAAAACACACATTTATTTTTATGCCTTGGAAGTTATCCAG AATCATGGAATTGTCAATCACAGTCAATCACCCAACCTACTCACCTTTCCAGTGTAATCT TAGTCAAATTTTTTTTTTGTTATCCAATGAGATGCAGTATTTCAACTCAGAAAGATAAAT AGAGTGAATTTATAGAGACTATTAACTAAGAACATACAGTTTTATTTATACTCAGAAGCA AGTAGATTATGTACATATATATGAAGATAAAAATTAAAAGGATAATTGTGTAAATTTGCA TGTAGAGAGCTTTGAAAACCTGTTTACTTGTTAATGCTGTTTTGATGTATTGTGTCTTTG TTCTCCCGACCCATCATCCAGAGCTCTCTGCAGGAGCTAAGTGCTCATCAGTTCCATGAT TTGGAAACTGTCTAAGTTTAGAGGCACTTGTATTTGTTAGTAAATAAGGCAAGATGATAT TGTTTCACAGGTTTTAGTGCAGAAGACTGAATAGATAAGCTGCTCCACCCAGTACACTGG TGTTCATTTCATGGTCATCTCATCTGTTAACCATGGATATAAAACATTTATCTTCAATGA TGGGGTTTTACCATGTTGGTCAGGCTGGTCTCGAACTCCTGACCTCAAATGATCCACCCA CCTCCACCTTCCAAACTGCTGGGATTACAGGTGTGAGCCACTATGCCTGACTGATTATTT TCATAACCAAGAAAAGAAATAAATACAATTAATGCTGGTGCATGGTATTAAATCTAGTTT TTAAAACATTCACACATAAACAAGGCAGAACCCTATACCCTCCATGATAAATGCAGTAGC AGTGTATGTGGGTCTGTGGAGGTTGAAAGGGACTTGGTAGATGTCAAGAAGGTAGTGGCA GTCCTGCTGGGCTTTTAAAGGGTCTGAAGAAGTGACAGGATGCTGTGGTTGAATCCTAGC ATGTATTTTAGCATTTGTTCATTTGGAGTTGATTATTTCACGTTGCTTTCATTTGCCATT ACCTGGAAAGCCAAGGGCTCTACTCTCATTTCCTTGCTGCTCTTTCTTTGCCTTCCTTGG TCCGTGAAGAAGATGGTCCAGGAGAAGCTCATTCCATGCTTGTTAACCAGGCACGCCCCT AAGTTCCAGTCCCTGAGTCATTCATGAGTAGCACTGCCAATGAACTGACAGCAATGCTGT GTCCCTCCACATCCCCTAGGTGACTCGAAGAAGCCTTCCAAAAAGCGTGTGAAAAGGAAG CCCTACTCTACTACCAAGGTAAAGTAGCCTGTCTTTGCCTAAGATGCAAATGTTGTTTTC TTGGATCCTTTATTTTTCAGTTGATATCAGCTATGGGAAAATTATCCACTACATTATAGG TGTTAGATAATATTTCCTTGGGGGTGGAGGAGGTGTATTTTACCAACTGACACCTGATTC CAGAGGACGTGCAAAATTGGCAGTGTCAGATAGTACACTGGGTGCTAAGGGATGTTTTCT TCAGGAACAAGCTTTCCACTTTAGATAAGAATTCTGCAATTTCTACTCAAAAATTACCTA GACAGAAACATTCTTCAAGAAAAGCTCCTGTGCTTTCCTAAGGGAACTCTACTCTAGAGT TGGGGCTTTTGACTTGAACCTTATTTCCAGTCTTGGTTACCCAGAGTTTCCAAGTGAACA AAAGACCTGTGTGAGCCATCCATAGCATAGCCTGATTCTCAGAGTGTTTTCCTTCTCTAA TTACAGGTGACTTCAGGGAGCACATTCAATGGTACGTATTCTGGAATCACTCACTGGTTG TTAGAAAAGGATTCTACAGGAAATCTGGAGCTTAACTGCTGGCTTTTGTCTGGAGAGCCT CCATGATCCAAGACATCTGGTGGGAATGAGGATGTAGGGTATAGTAAAAGAAACTGGTTT TCCTGGTGACATACTCTTTTTATCTATGTATAGTTTCTGGGAACATGTTCACATTAGGTT GTGTGTGGGTATGTGTGTATTAGGGCGGGGGTGGGGTGAGGTGGTCTGTGTGCAAGTCTG CATGATTTGCTTGTGAATGTGTGTCTATGTGTGTTTCCCCTACGAAAAAAATGTTGTGTT TACCCAGCACAACTCTCAGTGCCATGTTTCTTAATTTAACAAATCAGACCACATACTTTA CTTACATTAGTTCACACCTCATCATCATCATGCCCATATGTTGTGAGCTTGTTTACTGAG CCCACATGCCAGATGGAGAAACTAAGCCACATAAATAAATGTGCCCTGGTTCACTTGCTG CATAGTGAAGAGTCAAAATGTTTACTCATACGGTGCTAATGTTGAAGGCCTGAACTACAA CCTCTATTTATCAGCCAGTGAAGAGATCACTATTCACCATGCAAGGGAGTTCCAGCACCC TCTATGCCTGGAATTACCCACACCTGCAGAGATCCCAAACGCCATCCCTCACATAAGAGA ACGTCATGATCTCATAATCCAGGTAGCTATGTAGACATCTTCCTGCAGGTGTCACATAGT CCTTAGTGTGAAACCAACATAGAAAGCCCATGTTTCTGATCAAATCACAGGTTCTGAAAC ACTAAGGGAGGCACTAAGTAGGACAATGTGGTGCCTGCGTGTCATAGCTGGGTCTCCTCA AGACATGGATCAAGTCCAGTAAGAATTGGGGAGATGCTTTAGAGTCTTGATGGAGTTATC ACCACAAGCCCTCTGAGCTACACACTTTAGGGATCATGACCATTAAGTACTCAAATTACC ATTTGGTTGTTATCCGGGTATCCGTCGTCCTTGTGGCAACCCTCTTGTGAAGCTGGTGTG GACAGCCTCAGTGCTGGAGCTGTGCCTGCCTTCTGAGTGGACCCTTTCTGTGTTAGCAGG TGGGTACAAGCGTGGGGGTCAGCACACTCAGTGGATTTACACACACAGCGTTGAAGAGTA AGGCTGGGCTTCATTATTTATACATTTTCAATAAATGATGATCTTCATAACATAAAATCA ATGATGTAGTACACTAGAATACTGTCCCTAGTATTGAATCTTGTCTCTCAGCAAAGGGTT GCTTAAAGTCACGTGACAGATTCCATTCAACTGATGGCACATGCTGTAGCAGCAGTTAAA GCAGTCATTTGAAAAGGCTTTTACTATAAACTTACGTGTGAGCCTGAAGTGGGGGATAAA AGAGGCGATTAGCTCCCCTGTGCCATGTTTCTCTTATGTGCGTGGTGGAGGAAAATTACA CAGGAAGGTGATGGAGAGAACAGAGCAAAGGATTGGACAGGTCCATTGAACCCATAAGAC TATGGTGAGGTTAGTGAATGAGATTGGTCATTTTAGGTCAAATTTTACCCAGAGCTGGTG CAGCCACTGCCCATTCTTAGCCAGACCTTATTGCAGGCAGCTCTGATCAATAGTCAAGGA GGCAGTGGGGGTTGTAGACTTTACTCATTAAATCACCAAAGCACCAGCCCACACGGCCAC TTTTCCAGTTAATTGACAGTAGCTTGCACATTCAGGTTTGATCAGTGGAAGGGAAGTTAC TCTTTGCAGACCCATTTTTGACAATCATTTTGCGGTGTCGGAAGGTCTGAGCAGCCTCGG GAGGCAAGCAGTCCCTGGTCCCTCAGTGTAGTCACTGGAGGAGACAGTCACTGAGAGGCA GCTGGCAGGGTGAAGGGAAAGGGGAGGCAGGCCACAGAGATGACAGCCTTTAAGCTGTCA TACTGGGAGGTCAAGGATCTGAAAGAGGAAGGAGAATTCTTTATCATTAAGGACCTGTCC TTATCTCAGGCATTTCCTCCAGAGCACCACCTTTGTCCACCCACACACCTTGGGCTAGGA GGACTGGAGAAAGACAGTGAGGGGTCTCTTGGGTCTCTGGCACAGGGCGTGATGAAGAGA TGGCAGTTTTTCAGGAATCTCTCTCTTTAGGGAACCAAATACATTTCCCATCTCAGGTCC TTCACTCAGCGGGGTTGAGGTTCTGCTCGTCACTTATCATCTCTGAATGTCAGCACCCTC AAGTGTAAAATCTCAGCCACAGCCCCTCCTCTGCACCCCCTGCAGGGCTGATGTTCTCTA TAAACCATAAGGCGTCATGCCCACGGAAAAGCCGAACAGGAAAGCATGCTCCACTGCCCC GGAGCCATCCAAGTTCCCCCTCCATATTCCGCCACTGCTAAGTGTCCAGCTTATTCCTCC TGGCATGTAGTAAACACTTAGAGAACATTACTGAAGTACCAGTCCTCTCTAAGGTTTTCC TGTATTTAGTGATTTTTTAGCCCTGTACTGTGATACTAAGAAGTAGGGCTTAAATAGGGC CTAAAAAGTATTGCTAAAATTACATTATGACAGTGCAGAGAACTGAGGGCAGAGGGAGGA CATGAGCTTGCCAGGTCCACATGGCTTAGTGGAATTTGAATCCGGGCCCCCACTCTGCAC CAGCCCTGCACTCACAGTCATCCTGCTGTGTTCCCCTCTCCAGGAAGGCACTGCCCACGC AGTCTGTCTGATAGAGGTGTTGAGTGCTCACTGAACTCCGTGATCTTCCTGAAACCCAAC TTTGATTCAGTGGGCTCTGCTTGGAAGCCTGTAAAGAAAAGGATCATAAGTTTAAACTTA GAACAGATTATCACTATTTTCCCTCTGGTCTTCTGTCAGCAAGATGTCAACAGCCCTATC TATTGTCAATGCATTAACCAGCATCTTCTCTGATAGAGAATACAAGAAGATATGCTGTGC ACACCAACCAGTGTAGGAGACCTCATGGCTCCCGGGTAAAGAAGAAGAGGTACCCACAAG AAGGTACTGTGGAAGTTCATTAATTAAGTTGATTCAAGAATTGCAGTTGCGGGGAGTATT CAGTGTCCCATATGTAAGAGGAAACTATGAAGAGACTAAGCCATATTTTTTAATGTGTCA GGATTCTAATTTGCCTGGTCAGTAAATATTGCTACCACCACAAAAGTAAATATCTACTTA AAAGTCAATTTTGGTTCATGTTTAATGATAGACAATGTTTCAAGCTAATGTCTAGAACTT ACCTGGTTGTTAAACATAAGCATAGATCTCCCTGAAAGAGTGGTGCTATATTATTATTTT TCAATTAATATATTTCTTTAGAGAGTTTTAAATTGACATAAAAACTGAGCATATGGCCGG GCGTGGTGGCTCACACTTATAATCCCAGCACTTTAGGAGGCCAAGGCAGGCAGATCATCT GAGGTCAGGAGTTGGAGACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAAT CCAAAAAAATTAGCCGGGTGTGGTGGCAGGCGCCTGTAATCCCAGCTACTCAGGAGGCGG AGGCAGGAGAATCGCTTGAACCCAGGAGGCAGAGGTTGCAGTGAGCCAAGATCATGCCAT TGCACTCCAGCCTGGGTGACAAGAGTGAAACTCCATCTCAAAATTAATAAATAAATAAAT AAATAAATAAATAAATAAAAATTGAGTATATAATACAGGAAGTTCCCATATTATTCTGTC TCCTCACCCTCACTTCCTAATTTCACCTATTAGTAACATCTTACATTACTGTGGTACATT TGCTAGAATAATGAGAAAATATTGACACATTATTATCTGAAGTCTACATTTGCATAATGT TCATTCTTTCTGTTATACATATATATGAATTTTGAAATATTTAAAACATTATGTTCACCC TTATGGTCTCATAAAGAAAATGTTCACTTCCCTAAAAATCCTCTCTTCTCATTAATCTCT GTCCTCTTTCTCCAGAAACCTTGGCAACTATTAATATTTTTACTATCGCTTCAGCTTTGC CTTTTCCAGAATGTCATATAGTTGGAATCATATATTATGTAGTTTTTTCAGATGAATTTA TTGCACTAAATTGATGTACGCTTTAGCTGCTTTCATGTCTTTTTTATGCCTTAATGGCAA AAAATGGCACATTAAATCACCAAATAATATTGCATTCAATGAATTTTTGTCTTTTTATTC ACCTGTTGAAGAATTCGGTAGATTTCATGAGAGAAACCATCTGGGCCTGGTGCTTTCTTT TTCGGAATGCTCTTAATGTGAATTCAACTTATTTAATAGACATAAGTTTATTCACATTAG GATTCTAGCGTGACCTTGGGAAGATTGCCTTTCAAGGAATTGATACATTTCACTGAGGTT ATCAGACTGCGGTCATAGAACTGTTCATGATATTCCTTTTAATGCCTAACAGTTCAGTAG AGATGGCTCCTCTTTTATTTCTGAAATTGGTCATTTGTGTTATCTTCTTTTTCTTGGTTA GCCTGCATATCAATTCATTCATTGTAATGAGCATATCAAAGAACCAGCTTTTGGTTTTAT TGATTTTCTGATGATTTCAGTGTTTTAATTTTATTGATTTCTGTGATGTTGTTTATTACT TTTACTTGCTTTCCATTGCATTCCTCTATTTTCTATAGTTCCCTAATTGAAACATGATAT TACTGATTTTAGGTCTTGTGATTTTTAGTATATTGCATCCAATGCTGTAGATTTCCCTCT AAGGACTGCTTTTGCTACATCCAGAAATCTTGCCAAGTCACATTTTCTTTTAATGTAGTT AAAAGTATTTTTAATTTTCTATTGAGACTTCTTAACCCATGAGTTATTTAAAAGTGCATT GCTAATTTGCAAATATTTGGGGATTTTGTGGCTCTTTTACAGTTGTTGATTTTTTGTTGT CAGGTATGTGTTGCAAAAGCAGTCGTCTACCTCATCTTGCCACCACCCAAGATGGCCCAG GATGTGGGCTCTCCCTGAGTGAATCTTTGGCAATCTGCCAACCTGATGTGTTCGGCCTCC TTCTTTAGTCTGAGCTTGCCTTCTGCTTAGAAAGGGCCATTCTCAGTTCTGGCAGGGAGT TTTCCCAACATTGAGAAGGTGGCATTCTTACTCCCCACTGCAGCCTGCACCTCTGACCGG TGGTCAGCAGACAGGACAGAGGTCCTCATTAGACAGAGTTCAGCAGGGTCTCTGACCAAA GTGCATCTTCAGAGTCTGCACCTACCCACTGTGACCACGGGCAGGCTCTGAGTCCTAAAG CAGGAGGAACTGTGCGACCATCCTGATTGGAAATTTGTGAGGATCACCGTGTTACTCAAG TAAGGTCTTTGGAAAGTGTCGTATTACTACTGTTTGTGAACTGCTTGTTGGTGGCCTGGC TGAGCCACACACTTTATGAAAACCAGGACCCCTCAGCTGGTGTGGGTGTCTATGCAGCCT GAGACCCTCATGTGAACAGCCTCGTGGCAGCTGTCTTTGCCCCTTGCCACCATCAGTGCC TCCTTGTTCCTGGGCACTGCTTTCTCTGATGGTGCTCCATTGTTTTCCTGCACCTCAGTG TCTACAGCTGGATGTCTCTTCCGCAATCTAGGCGAGGGGGCATCAACGGCAGTTCTGCTG TGGCACTGCCCTCCTTCTTAGCTTGTCTTGCTCTGTCTTAGGCTCCCTCAAAGATCCCAC CCTTCAGGTTCTTCCACAAGTTTCTTATTGAAATCCGAGCAGAAAACTATGACCAATATG ACCAATCCTATACCCACTGAAGACATGAATGAAGAATTAAAACAATTCTCCATGGACTCT ACCATATAGATCCCTAGAAGTAATTTCTAAAAAAAAAAAAATCAAGGAAAATGTAATAGT TTTCCATAAATTAGAATACCCTACATGTACAATTAAATGAAATGGCTAGTATAGTCTTGA AACCAAAACCAGATAAGGTAAATTAAATTCTGTGATATTTTAAAATACTTTAAATTCTGA CTAATGTGAGTTAATCTCAAAATATGAAATAGTAGATTAACATTGAAAATGCAATAAATA AAATTAGCTACCTCAAGAGTTTAATGGAAAAAAATGTGATTATTGCAATAGATTCAGGAA ATTCATGAATAACATTCACCCTATATTTGTAGGACAACTATCTGATTAACTTTAAGTGAC CTGTTACAACCACTTGAATTAATGCTGCTTTCACAGCATATCTCTTGGCTTGTTAAAAAC CCGACAAGAATTTCCGTAACATTATTTTTAACACCTATATTGGGTGTGAACCCACCATAA AGTTTGCCCACTGAAAAGGTCTACAATTTGATGCTTTATTAAATTGATACTGTGTGCACC CAACACCACGATCTAATTTAAATATGTTTCCCTCACCCAAGTTCTGTCTTGCGCACTGGC AGTTAATCCCCACTCCCATCTCCAGCCCTAAGCAATACTGCTGTGACATTCCATCTCCAT AAATTTCCCACTTGCTTTATAGAAATGGACATATATATATATTTGGAATCTGACTTCCTT CATTTAGCATACTATGTTTGAAGTTAATTGACGTGTTAGCACGTGCTGGTCATGTGTTTT CCTTCATAGTCTGCTGTGTTTATTCATACAAATAGTGTTTATTCATTTATCAGTTAATGG ACATTTAATTGTTTTGTTATTTTCTTTGATGAGTAATGTAGCTTTGAGCATTCACATACA GTCATGTAATGCATAATGACATTTTGGTCAAAAAAAATTTTTTTTTTTCTGAGACCCAGG CTGGAGTGCAGTGGCACAATCTCGGGTCACTGGAACCTCCATCTCCCAGGTTTAAGCAAT TCTCATGCCTCAACCTCCCGAGTAGCTGGGACAACTGGCACACACCACCACGCCTGGATA ATTTTTGTATTTTCAGTAGAGACAGGATTTTGCTGTGTTGGTCAGGCTAGTCTCAAACTC CTAGCCTCAGGTGATCCACCCACCTCTGCCTCCCAAAGTGCTGGGATTATAGGCATGAGC CACCACACCCAGCCTAATTTTTTTAAGAAAGAAGGGAACTATTTTCTAAATTACTTTTGC CAATTTATATTCCTACCATGATGCATAGCACTAATTTCACCGTACAATGTATGGTAGGCC CCAATATGTAAGAAATGATGAAAGTAACACATAAAGATTAGTATAAGACAAATGAGATTA TCGTTGTTGCTATCATCTTTGTCAAATTCTGAAAACAATCTGAGTATATTTTTATATAAA TATGCTTGGCAACATAGCTGAAAAAAGCATTATCAGTTACATTTATCAGTAACAAAGACA TAAATTTGAAGGGGGAAAAACACTTGTACTAACAATGCAATGTCAGAATTAACATAAAAA TTCTGCTGGTCACTTTGGAATATTTAATTGCCTGGGGCAGTGTTTAGTAGACAAATGAGC ATCTATGGAGCACCCAAAGTAGGGGAATCAACAGAACTTGGGTTTCAAAAGTTATCTGGG TTTAGAGCGTGAAACTTTGTTAGAGGACACACACCTTGCATGAGTGAGGTGCCTTGGTGT GTGTGGACGTATCATTATGCTTGGAGGTACAGCATAATGGTGGCTTCCTCCAGAAAGGGA CATTTGGGGTAGATTCATGCCATCTAGACAACACAGCCTGATGTGGCATGGACATGAATG GAGGTGAAATGGTCAGTAGTTGAGAGGATCAGTCCTGACAAGGGCCGAGGTGAAAAACCT GGGAACACCTTCAGGTGCAAAGTCTTCAGTTGAAAAAGGAGGTGGTCACAGGAAATACTG AGATGGGTCAGCAATGCATGGGAGACAGAGTTCTTGGCCCTGCAGGGTGAGTAGTGTGGA TTCTCAAGTTTTCTCCTCTCTCCATTAATTTCTTTCCCAATGCAGATGACTTCCATCATA CAGTCTTCAGCAACCTTGAAAGATTGGACAAGCTTCAGCCCACTCTTGAAGGTAAAGGAA GGCAGCTAACAAGACTGGCATCTGGGCTTGGCTGTGCATGTTTTCTATCGTGGGGAAATA TATGTAACACATTATTTATCATTTGAACCTTTTAACCAAAGTGTGCACTCCGTGGCATTC AATATATTCACAGGGTTGCATAACCAACACCACTATCTACACCCACAATTTTGATGATTT CTTACAAAACCTTGTCCACAATAAGCAATATAGCACCTTCCCCCTATTTCCAGCCCATGG TGATTCCTATCCGACTTTCTCTTGTATGAATTTGACTATTCTAGGCACTTCATGTAATTA CAATTATACAATATATTCCTTTTGTGTCTGGCTTATTTCACTAAGCATAATGTTCTCAAT GTCCACCCATGTTGTATCATCTATCAAAATGATGTTCGTTTTTTACAGATGGATGATGTA GCATTGCATGCAGACCACCTTGCTTTTATTACATTCATTTGTTCACTGATGGTTGGATTA TTTCCACCTTTTGGCTCCTGTGAAAAGTGATGCTACAAACATTAGTATACAAACATCTGT TTGATTTCTGTTCTCTATTCTTTGGGGTGCCTAAGAGTAGAGTTCCTGGGTCCAACAGGG GTTCTATATTTAACCTTCTGAGCCACTGCAGACTGTTTTTCACAGTGGCTGCAACTTTAT CCATTTCTACCATCAATGTATCAGGGTTACAATTTCTTTACGTCCTTGTTCACACTTATT TTCCTTTAAATCATCCTAGTAGGTGTATATTGGTGGCTGCTTGTGTTTTTCATTTGCATT TCCCTAATGACTAATGATCCTGAGCAGCTTTTCCTGTGCTACTATCTGTGGCTGTATCTT CTTTAGGGAAATATATGTTGAAGTCTTTTGCCCATTTTTAAAGAGTTGTCTGATTTTTAT TTAGTTAGTTTGTTGCTGTAGATTTTTGAATATATCTTAAATATATTTAAAAATATTCTA AATTTTAGTCTCTTACAAGATAAATGATTTGCAAATATTTCCCCCTTTGTGTAGAACTTT AGATTCACAAACTTCATTAATTTGTATGAAATCCTCAGCAGTTGACCCCAAACAGATAAG ACTGAAGCAGTATTTTAGGAATAGTTGAAAGTATGATCACCACAAAACATAAGCGTAATC AAATCCTGCAAGCTACATGTAAGGCACAATGACAAATAAGGCAGCAAAGGGCCATCTGGT GATTAGTTCACCACACTTGTTGCAACTGTTTGCACTGCAGAGTTAAAACATACCAGCATT CAACCCATGTCTCCTGTCTTGAAGTAAACTGTCGTATGTTGGCTGGCCTGAACAAGCGTA GATATTCTCCATCCTCAATTAATATGCATGCATGACAAAGAAAAGGAGGCCTGGATGAAA AAATACTGTGTGATTAATAATTATGCTTTAATTAATTTTAAAGGATATAATTTCAGTACT TCTAATTCTCCCATCAGCAGTTATAACAAAGGATTAGTGAATAAATACCATAGACTGTTT TGCCTAGAATTGAATCCAACCTGTCTATTAAACTTTGCTTTTATTCAAGTGCAAAATGCT AAAACACATAATAACTGCAGTGACAGCCACTGTGGATCCTCAGAGGTAAAAGTAGTCTTG GGACATAAATCCTGCAAGTAATATTGTTTTTACAGGTTTAGAAAACCATTTAGCTGGGTT TCAAACCTCACAGTGTGAGCAGTGGGACTCTCATCAAACTATAGCATGTGCTTCAGTACC ATTTGTAGACTGACTCATTCCCATTGCCTTAAGTTGCCATCAGCAAAATGCCAGGGACTC TATTTCTTGCTCCTTAGCTCCTCGTTCTTGCCTGTCTTTCCACGAGGGAGGATTTTCTAG CAGGAGCTCAAGCTGTGCTTTTAATGAAACACATCCACACACACTGTCCTGTTGTCCACA TTAAGCAGAGCTCCCTGAATAACTCATGAACAAAAGCATCTATGACTAACTGTTGCTCTG TGTCCTCCTAGCCTCTGAGGAGTCTCTAGTTCACAAGGACAGAGGAGATGGAGAGAGGCC AGTCAACGTGAGGGTAAGGTTGCCTTGCTTTCTCTGAAATAGAAATGTTCCTTTCTTGTT GTCTTTCTTTTTCAACTGACTTTACATGTGAAAAGATGACAATGTCCATGACAGGTATTA AATGCAGTTTTCTGAGGGGGAGGAAGAAGTGACTCTTAGCAACTGATATGTAATACAAAA TGGCATTTAGCTATGATGGCTTCAGGTTGTAGACTGTATCCTTGGGGTCCTTGTCCTTGG AAGCAATGTCTTCTCCTTGGATTCAGTATTTTGCACTTGCCAACCTACGTGGACCTGAGA GATCCACCATCCAGAAGCTGATGTCTTTTCCAGTGTGTATCCTACCCTTGTTTTGGAGGC CTTGAAGTTGACTACACTTTCTGATCAAGTTTTCAATATTCATTGAGAGAAACACAGCCT TGTGCAAACAATCCACAACATGACATACCCCTCAAAAAGCTTTGTTTCTGTATTGCAGGT GGTGCAGGTGGCCCCTCTGAGGCATGAATCTAGTAAGTATTCTGGAATCACTTACCAAGA AAACAATCTGGATGCCAAGAAAGGTGTGGCATCCTTGCCTGGTTTCAATGTGAAGAGCCA CCCTGATCCTGGGATTGTGATAGGAATAAGTATAGGGGAAGTGTTTTTTTAAAACCTGAA TTCCCCAGGGAAAAATTATGGCCAAATTTTGAGGAAGCAGCTGTGCTCCCTTTTGGGTGG TGCTGAGTTGGGTGCTTGAGGATTGGTGGTGTCTTGTGTGAGGCTGCATCGTGTGGTGTG AATGTGTGTGTTTCTGTACAGGTGAGGCTGTGTGTTTTCTCAGGAGAGATTTCCCACTTA TACAACCCAATCACCAGTGTCCACTTCTAACAATAAAATCCACCCCCGCTCTACTCTCTC TGTACAGTGACTCCTCCACCCTCACCAGAGCCATCCCCGGGTCTGCCTTATTATCCCCAC TGCTCAGGTGGAGAACCTGAAGGGCCAAGGGAGTGGCCCCAGCTCCCGAGTTCCTGAATG AAAAAGTGAAAACACGAACCCAGGAGTGTGGGCCAGTGCTGACGCTGACATGCACTTAGT CATGGGGTGTTCACCACCACACAGGGAGTCCAGCATTCATGTATAAACCCTAAGGCACCG AGCCCAAAAGGCCCCAGGCACTGCCCATCATCATAAAGTGGTCTCCGTGGTCACACAACC CAGGGCAGTTATAGGTTCATCTCCCCACGGACAGGCATAGTCATCAGTGTGTCAAAAGCA CAAAGATCCCCAGGTGTTTGGCTCAGCTCACCGATCCTTTTTTTTTTTTTTTTTAACTTT TAAGTTCAGGGGTACATGTGCAGGATGTGCAGGTTTGCTACATATATAAATGTGTGTCAT GAGAGTTTGTTGTACAGATTATTGCATCACCCATATATTGAGCCTAATATCAGTTATTTT TCCTGATCCACCCCCTCCTCCCACCTCCCACCCTCCAGTAGGCCCCACGCTCACAAATTC TAAGAGGAGTGGGGGACCACAAAGGCCAGTGTGGCCCACTTCAGTTGTGAAGTTAATTTG CTCAGCAACTGGCCAAAGTCTATAAGGATGGGTGATGTATTTTAGTAGATTTAGTAATAC TATCTTCCCAAGCCCTAAAATGCTCAAATCCTGCCAGCCAAAAATGGTGAGGAGGGACAG ATAGGAACTCTGTGTGGCACTTGGTTATTAGCCTGGCTTCCATCCCTTAGTGGCAACTCT CTTGTATATGTGGGTTAAAGACCCTCAGCCTCAAGCCAAGCCTCCTCCATGAGGAGCCAT CTCACTATTGACTGGCTAGTGCCGGGTATGGCCACCAGCCCAACTGAAACAAAATGTTGC TTTAAAACAAGTGTAAATCTCATACATACAACAGGCAAATGCAGAAGCAGTGTGGTCTCG CAAGTTGTAAAGAGGACAGTCGCAATTTTGCTGGACTTCAACCTGGGTAGAAGACACGAG GGAACTCTGTCACTAAATCACGGCAGAGTTCAAGGCCGCTTGTAGACTATTTCGTGTTAT AGAAGGTGGCCTTTAGCTACTAAGCAAAGGCCTCTGTTTCTCATTTCTTTCCTGTTCATC TCCTTGGTCATCCTTCTTCCACAAGGGAAACGAGCCCAAGCAAAAGGCAGTTTCAATATT AATTTGACCGAGGTTTTGTGCAGTTAATTATCATCCAGGTAATCAGGTGCAACCCAGTCT GCCTAGCAGCCCCCCTATCTCTGCTCTGTGTTTTCATTTAATAAACATTTTGGTCTACTT ACTATGTGCTAGATTTTCTCGAGACCAAGTAAATGAGATAGAATCCTTATGTTGGCAGCT AAGTTAGATTTCACATAACTGACAAAAATTAAAATTTCTGATTTCTTGTAAAAATATTTT GTATGTGTGAATGCATACTGAATGTAAAGTGGATAAAAAACTCACACTTGCACTCATGGA AGGCTTTTCATGAATTTGTCAATTTTTATTTTTTATATTTCCCCACTTCACTGGATAATG CATACCTGAACCTGGAAACTGATGCCCACTGCAGAAAGTGTTCTGAGCCACATCCCTTAG CTTCACTAGTGCAGGTCCACCTGGGAGGATGTCCCAGCATCAGCTTGGCCCATGCTGTGA TCAGCCACCTCCATGCACCACACCAAGCAAGCCCCTGGGTGATTCACAGTCTCCACTACC AGGGCACTGACCTTAACTCTGTGTTCTTCTAGCTCCCCATGAGGACACCGTACACAACAT CACTAACGAGGATGCCTCACACGATATCACTAACGAGGACGCTGTCCACGGCATCGCTAA CGAGGCCGCCGACAAGGGCAACGCCAACGAGGACGCCGCCCAGGGCATCGCCAACGAGGA CGCCGCCCACGGAATCGCCAGCGAGGACGCCGCCCAGGGCATCGCCAACGAGGTCGCCGC CCAGGGCATCGCCAACGAGGACGCCGCCCAGGGCATCGCCAACGAGGACGCCGTCCAGGG CATCGCCAACGAGGACGCCGCCCACGGCATCGCCAACGAGGACGCTGCCCACGGCATTGC TAACGAGGATGCCGTGCACGGCATCGCTAATGAGGACTCCGTATACGACATCGCTAATGA GGATGCCATATATGACATCGCTAATGACACCGTACAAGGCACGCTAAAGAGGACGCTGTA CACGACATCGCTAATGAGGACACCATACAAGGCATTGGTAATGAGGACGTTGTATATGAC ATCGCTAACAAGGACACTCTACAAGCCGTCGCTAACAAGGACACTGTACACAACATCGCT AATGACGGCACCGTACAAGACATCACCAATGAGGGCGCTTTATACGACATTGCTAATGAT ACCGACAAGGCACGCTAACGCGGACACTGTACACGACATCGCTAATGAGGACTCCGTATA CCACATCGCTAATGAGGGTGCCGTATATGACATCGCTAATGACACCGTACAAGGCACGCT AACGAGGACGCTGTACACGACATCGCTAATGAGGACACCATACAAGGCATCGGTAATGAG GACGCTGTATACGACATCGCTAACGAGGACACCATACAAGCCGTCGCTAACAAGGACACT GTACACAACATCGCTAATGACGGCACCGTACAAGACATCACCAATGAGGGCGCTTTATAC GACATTGCTAATGATACCGACAAGGCACGCTAACGTGGACGCTGTACACGACATTGCTAA TGAGGACACCGTATAAGACATCGCTAGTAACTATCGCAAGAACAAAAAACCAAACACCGC ATATTCTCACTCATAGGTGGGAATTGAACAATGAGATCACATGGACACAGGAAGGGGAAT ATCACATTCTGGGGACTGTTGTGGGGTGGGGGGAGGGGGGAGGGATAGCATCGGGAGATA TACCTAATGCTAGATGATGAGTTAGTGGGTGCAGCGCACCAGCGTGGCACATGTATACAT ATGTAACTAACCTGCACAATGTGCACATGTACCCTAAAACTTAAAGTATATATAAAAAAA AAAGACATCGCTAGTGAGCACGCTGTATACGACATCGCTAATGAGGACACCATACAAGGC ATCGCTAACGATGACGCTGTACACAACATCACTAATGATGACACCGTATAAGACATCGCT AATTATGACGCTGTATACGACATCGCTAATGACACCGTACGAGGCACGCTAACAAGGATG CTGTACACAACATCGCTAATGAGGACAGTGTACAAGCCATCGCTAATGAGGACACTGTAT ATGACATTGCTAACGAGGACACTGTACAAGGCATTGCTAACGAGGACGCTGTACACAACA TCGCTAATGAGGACACCATATAAGACATCACCAATGAGGATGCTCTATATGACATCGCTA ATGACACCCACAAGGCATGCTAACGAGGACGCTGTAGACGACATTGCTTATAAGGACACC GTACAAGACATCGCTAACGAGGACGCTGTATACGACATCGCTAATGAGGACGTTGTATAT GACATCGCTAATGAGGATGCTTTACAAGACATAGCGAATGAGGTTGCTGTATATGACATC GCTAATGAGGACATTGTATATGACATCGCTAATGAGGACGCTCTATACGACATCACTAAT GAGGACGCTGTATACAACATCGCTAATGAGGACGCTGTATATGGCATCGCTAATGAGGAT GCTGTATACGAATTCGCTAATAAGGACGCTGTATATGACATTGCTAATGAGGACACTGTA CAAGACATCTGTAAAAAAGAAGATGCTGCCAATGTAAGACACTTTTCTTTGTCTTGAACA GAAATGTTACTTTCCTGGCTTCTTTCCAATCAGATGTAGACATGAACATCTGCCAGTGTG CATTATCGATGTCATCTGCAGTTTAATCAAATGTAGACATGAACATCTGCCAATGTGGAC TATTTATGACATCTGCAATTCCCTTGGTGTGGTGCTATTGATTGGCGGCCTCTCACCAAC CCATGCCAGGCACACTGGGGTGTGGTAGATGGCAGCATCCACGATCCACTGCAATGCAGA GGTGTTTCCCTCCACAGCAGTTTTCCCCCATGGATTAAGAGTTGTGAAACTGCCAATCTA GATACACTTTAAAGATAAATTCTGTGGGAAAAGGTCTTGTCTTTTCCACAGGTGTCTTCC GTGCCAGTTTTGGGGGACTTTGACCTTTGACTCAATCACTATACCCCTTCTTATTTTCTC TCTCAAGTTGTCGAGAGACTATCAGATCTGTGTGACGTGTATGGCATCATTTCACCCTCC TAATGTTTTCTTTTCTATAATTGCAGGAGCCATTGACACTGGAGAATGATACCTACCCTG AAATAACTCACTTCCTGAGGAAAAAACGCCATCTCTAGGGTACAGAAACCTGATTCTGGG CTCCTTTTGGGAAGGAGGATTTGGAGTCTGGTGAGAGCAAATGATTTTGCAAGTATAAAA CAATGTCCAGAGAGGCTGTAGGGATATCTGTGAGCCCAGAGGAAACACCAGGGGATCCTG TGCGAAGCACCATGGCTTCAGCTAGGGTGGGAGGAGTGGGTGGGCCTCTCTCTAATGACT TATCCTGGTGTTTGTGTTTCTAAAGATTTGATTGTGGAGAGCATATCTGATGATGGGGAT TTGTAGGTAGGTAACTACTTTCCACGTAAGATCCAATTGGAGAGAGTTCCCAGGGGCCTT CGGGGTATCCATGCTGCTTGGGAGGTTAAGGGAGGGGGCATGAAATCAAAAGGAAACAGG AAATATGTGTCATATTGGATTTGGTCTTTTCCGGGTTTATTGGCATAATAGTTAGAACTG TCTCTCTGGGCTATGAGGGTGCTGTGATATTTAAAGGTGGTCTTTCCCAGAACACCTGGC CTTTTCTTTTCTGCCTCTGCCAAACATCACAGCCTTTGGGTTGGATTAGTCAGCACCCCT TGGGATTGTGCAGAAGAGGTTTGGGGTTGCATCGAGTGTCACCTGTGGTGAACAGAATCT GAGGGACACAACTCTCTCACAGGCACTTCCTTCAACCTGGAGACAGAGTTCTCCTGGTGT GTGCCCAGGGGTGGAGGAGAAATTGACAGTCTGCCTCTGAACTTTCAGGACTTTAAAAAG CACTCATGTTTCCATCCTCACTGTTGACTCCTGGCTTAAAGGGATCTCCCGGGGTGAGTG AGGAGGCGGGATCGGACCCTGGCAGTCTGACGGCAGCACCTGTGTTCCTCTGCACTGGGC CGTGGATGACATTACACACCTTGGTGAGAATCAGGAATTGAGGCTAACCACATCTGAAAT TGAGATGGGCCTTGAGTCATATAAATAGTTTGGAAAAGATGCATTTTACTACGCTATTGA AAGAAACCATTTATTTCTCACTCCAGCAGGATAAATGGTTTTCAGTATCCACTTAACTGC TCATTGACTCTTACTGTAGATGAGGAGGTGGCCAGCAGCCCCTGCCCTCCCCCAGTTGTA GGCCCAAGGTAACCAGCAATTGACTGGATATAATGGAAGAGTGGTGCATTCGGAGGTATC TGTATTAATGGGACCCACATGATATGGATGAGAGCTATTAGGGTGAGAAAAAGCCTGGGA GCACAATGAAATATTTAAATATTAAACAAAACATTGTTGAAATCTCCATTGTACTTTAGT AGTTGAAGTCATTCTTGTGGTCATCACTGCCTTTCCCAAGCATAACAAGCTACTTAATAT CACATGGACCCGTGCCATGAGGAATGATGATCAGTTTGTCAAATGCCAATAAAACAATTG CCTCTATAAGCCACAATGTTTCATCCATATATTTCAATTTCCATGTGTAAGTATAGTTCA AATTTCAGAAATTTATTATTATCTAATAGAATATGCATGGTATATCAATGAGCAATTATC ATACTGTTTCTATTAACAATTATTTGTATGATGAAAAAAGCAGACTCCCATTCTTGGATT TTTCTCAGTTTGCACACATTAGCATGACAGCCCCATTTCCACCTGACATGTGCCAGCAAG AGGCCAGGAACAGAGGCTTTTCTTATTAACTAAGATTTCTAAATGTATTACGTATTCACA TTTAGAAACTCTAAATATCATAAAAGGTTAGCAAGGAAGTTTCCCTTCCACTCTGAACTT CCAAACACCAAGTCAACGTTTTTGTTTGCATATCATCCCTGCAATCTATGTGCAAATAGA AGCATGCACCTGGAATGCAGGCTGATGTGTGATCGTGTTTACACAAAGTCCTCTGCACCT CTGCATATATCACTGGGCAATGCACCTTAGTTATCATTCCACATTTCAAATGTAAATCCA TTGTATTGTTTCAGAGCTATAAAGTACTGCACCCCATGACTATTCCCAAAATTACTTAAG CACCCCGCTATGGGTATCCGTTTGTTCTGTTTCCAGTCTTGCTCTTATAACCAATGCTGT AGTGAACAGCACTGTGTTGAGAAGCGGTGAACGTGGGCATCTTTGTCTTGTTCCCTTCCT CAGGGGGAATGCTTTCAACTCTCCCCCATTCAGGAAAATGTTGGCTGTGGGTTTGTCATA GATAGCTTTTATTACCTTAAGGTATGTCCGTTCTATGCTGATTTTGACGAACGGTTTTAA TCATAAAGAAATGCTGGATTTTGTCAAAGGCTTTTTCTGCATCTATTCAGATTATCATGT GATTTTTGTTTTTAGTTTTATTGATGTGATGTATCACATTTATTGACTTGCGTATGTTAA ACCATCCCTGCATCCCTAGTATGAAACCCACTTGAATCATGGTGGATTATCTTTTTGATA TGCTGTTGGATTCAGTTAGCTTGGTTGTAGCATTTCTTATTATTCCATCTGTGGAATGTA TTGGTTGAAATAATGAAAACATGTTCTATCCTCACTGCTTAGCACTTTGTGTTTCTTTAA TAGCCTTCCCAACAGGGCAACATAAAAGCAGGAGCCCTGCTAGTCTCCCCTTAACCCGGA ATCCCCCCTTCTCCACAGCTCGCTCATTGGACAGGATAGACTGGGCGCCCAGGCTTCAAG GTAAGGACGTGCTCTGTCACCTAGAGGTGCAGTGCTTGGGAAGGCCAACCTTGGAGGGTT GCCTGCCAGCTTTACAGTGACAGAGGTGTTGAGAGGGACTGACCACCAGTGCATAAGGCT GTGCTTTGTTGGTGACATAAAGGATTGTTTCACAGATTGTTGGGGAGGGACAATCCCAAG GCCTCCCCTGGCCCTGGTGCTGGCTCTGCACAAAGGCAATAAGAGAGGGATGCTGGTAAG GGCTGACCTGTTGCTGTGCTGGGGAGGAAGGTGCTGGGCTGAAATTCAGGAGGCTGAGGA TGCAGCAGTCCCATAGGAGGTACATGACCTTCAGGATACATTTTCTTCATTGATGATCAA TGGAAATGAGAAATCACTGACTATTTTTTCTATCATTGGAATCTACTCTCCACTGCTCAT GCTGTTCCTGTCTTTTGGGGAAGATGGAGGATCAATCAGTGTGCGCTGCACTGAGTGGAA GGAAGGAGAACTGTGACAAAAATTAAGGAAGGATGAGAGACGGGAGGGCCCTTCATCCAG CTGCTTGCAGAGTCCTCCTGAGGAGGAAAGCCCCGTGGCTCCCTGGCGAAGGAGCAGTGA GGGCTGCGTGACTCCCACAGTGAAGTGTGTGGTATGTCTGAGGACACCCAGGCTGGTGGT CCATGAGGAGCCAGTGGCAGAGTGAGAAGAAGAAAGGCCAGGAGGGTGGCTGGAGGCCAG GCTCTGAGTCATTCTCCATGTGATGGAAACAGCCGGAGCCCAGTGGGCTTGGAGGTACAG GATGCGGTGGCTGATGACAGAACAATGTGGAGAGAGGCGTCATTTGTCAAATCCTTACTT TGTTCTGGGCATTGTACTAAAAATTCTAATGGCTCATCCCATTTAGGGGCTGAAAGTTGC AGAGGTTTAGGAAGCTCACCCACGATACTGGAGCCCCCATCTCTTGCCCTAGTGCTGTCC ACCTTCTCACCCAGCCACCACCTGTTTCGGGGGAACACACAGAAGTGGTAACCTCTTATG GAGAGGCAAGTAAATTCTGCTGTTTTTGTTATTCACAGAAAAACACTGGCTCGTGTGGGT TGGGAAGGTGAAATACCAGAAGTATTTCATCTGGTTATTTCTACCCATGCAACTCCTATA GTATTGAAATGCATAGGTTAGCATTTTTGGCCAATTTACTCAGCATTCTGGGTTAAAGGC TTTTATTTATTTTATTTATTTATTTATTTATTTTTGAGATGGAGTTTCACTCTTGTTGCC CAAGCTGGAGTGCAATGGTGCGATCCTGGTTCATTGCAACCTCCGCCTCCCAGGTTCAAA CTATTCTCCTGTCTCAGCCTCCCAAGTAGCTGAGATTATAGGCACATGCCACCACACTGG GCTAATTTTTTTGTATTTTTAGTAGAAATGAGATTTCACCATGTTGGTCAGGCTGGTTTC GAACTCCTGACCTCAGGTGATCTGCCCTCCTCGGCCTCTTAAAGTGCTGGGATTACAGGC GTGAGCCACCACGCCTGCCCTAAAGTCTTTTAAAATTCACTTGTATAAGTTGACTTAGTT TTCTTTAACCTTGTAGAAAAATACAAAAATGGCAATCTCTTTTATCACACAAATAATGTC TTTTTAATGGAGTGATTTTTTTCTAATTGAGGTATTATGTACTTTTCATTTACAAATTAT TGTTTACATTTGAAGTGTTTTATGAATTAATATTTAATTGCATAGATGAAGATTACTAGT TATAGGCATTTTACTAACCAATACTCATTAAGCATAGCATGGATTCATATGACATCAAGG AGCTATTTTATTTGGTAAAACGAAAAAGCACAAGAATGAACGAATGCAAGAACTGAAACA GTGGAGACACCTAGAATGACTTGTCTAAGATCTAAATCATTTTGTTGTCTTCCCAGCATA CTTATTATCCTGATCATTGTCATCAGCATTGTTTGGGTCCTTTTAGCACAGATTTCTCAA AATGGGTAACTCCATAACAGTTGGAAGCTTACGAATTCATATAATTTGTAAGAGGTCAAT TTGGAAGTACCTATCTATTTTAAAATTCCAATAACCTGGGAATTTCATCCCATGTCTAGA GTCTTTTATGTAAAATATTTCCACAATTAGGAGAAATATGTGCATGGGGATTTTCTATGT AGCGGTGTTTTGATAGAATAGAAAATTGGGATAAATCAAATTTCCATCACGAAGGAAATA GTAATATGCTGAATAATAATACAGCGAATATTATGCAGGCTTTAAACATCAAAAAAGAGT TCAACTTCTGACTTCCGATGATGGTGTTGAAGCAGGTCACTGCTGGTTTACATTTGATTT TCATGTGGGAACTCTGGAAGTCCGCCTTAGTGATTTTACATGTGGCTAAATTGAGCTAAT GACAAGCCGTTCGAAGTATGGCAAAATGGAACTTTAAAACAGTATCTTGTCAACAACCAA GAGGACCTGTTTCACATAAAGCCCATGCATTCATCTGCCTGTCCATCATTCTGTCTGTCC ACACGGGCATCATTCGTTAGTGGAACTGAGTGCCCACTGTCGAGCTGACAAGCCCATAAC CTCCCTGTTCCTAGTCACACATTAATTCTTCAACAAGTCCCTTTTGATAGATTGTGATTA AGCTTAGCTACTATTTCCAATTGCTTCCCCAAACGTACTTCTCACTGTTCTCCCATCACA CCCTTCAGCCCATCCATGCGGGGTTCCTTTGCTTTTCCCACCTTACACCAAACTCCCTAT TTTTACTCCCACTTTTACCTCCTCTCCAAGACAAAACAAACAAAACTAGCATTTTAAAAC TTAGTTGTAATCTTTCTTCCTTCATGAAAATTTCTCCAACAGCCACTCCCACGGTCCTGT GTGTTCCAGATATTTTAAAATAATGGCTATAAGGCTGAGCACTTCAGGATATGCTGTTTT GCTGTGTGCAGATGGAGGCAGTGGCTGGAGTGAATGAACGGCAACACTTGCTGGCAACCG GCAGAAGCTGAGAGACAGGGAACAGGCTCTCCTCCAGAGCCTCCAGGAGCCAGGCCTTTG GACACCTTGAATGTGGGCTTCTGGGAGACCATGCGTTTCTGTTATAAGCAGCCCAGTCTC TGGCAGTTTTTACGGCTGCCCCGGAACACTCATCTATACCTGTCTGACAAGGTCAAGCTC CAAGGAAGGGATTCTCTACATATCTACATTGTTTGCAGATTTTACAATAATCATTTATTC TTGCATGGCTGATCATTGTTAACCAATACAAATAAAATAATAAAGAAATGACCCACATTT TATGTTGGGAGTTTGATCTGCCATTTATCAAGTATGGAATCTTGAACAAGGGGTTAAACA TCTGAATGTCTCCATCACTTCATCTCTAAAGTGGGGGTGCTCACACCCACTGGGCTCCCC CACCCAGGTTGGTGCCGGACTCTCCCTGGGCCCCCCTGTTCTCTCACCAGCCACATCCAT TCTCCCCCCAGAGGCGCTAGTGACTGTGCGTGGCTTTCCATTCCCACCATGTTTGTCTCT AACCCCAGTGGCAGATCAGTGTAAGAACACAGCTGAGTGCTCCTCGCCTCCTTGCCCCTT CAAGGGCTCCTCACCACCCACCAGATCAGGTGCAAACTTCCAAGCCTTACTGGATCCCCT TCCACATTCTGAGCTCCGCCTGCCTTCCCATCGCTATCCTTCCCCACCTGCCTCCCTGGT AGAGAAAAGCAGAGTGTGTGATGCTGTCTGAATGCTGAGCACGGCCTTTTGCAGCCAGTC CACTGTGTATGCTGCCCCTATCGGAGACCTCCACCTTAACCCTTTCCAGCCTGGAGGCTC CTCCCAGGGCCCCACAACAGAAGTGACTTCCCTTGCCTTTGAATTTCTATAGCACAAGCC CTACTGCCCCCCGTTAAAACTGCAAAGTCCTTTTGTGGAAAATAACTTTATTCATGACTG TGTTTATCACACTATCTTATGGAGAAGAGATGATCAATAAATATTTGTTGAATAAATGAA TAGCAGTTACAAAACACTTGATTCATATGGAATTAATGTTGGTTCTCAAAGTGAAAAATT ACAAACAGCACTGATATTCAGCCAGTATACAAGTCTGGTCACAGCAGTTGTATAATACTG AAATACCCCCTGCCACTGACCTTTGGCCCCCAGATGCCTCCCACTGCCACTGCTCTCCCC ACTGGGAACCCCTGAAGTTCCCACAGGCTCATAACTAAAGGGCTAATGTCTCGCACAGCA GCGAGCACCCAGGACCGAGCAGCCACATGGCCGGGTCTGCTGGTGAAAGCATCCATTCTG ACTGATCAGGACCTGAGGGGCCTCATGGTTACATATTTTGATAATATCCCTAATTATAAA TAAGGCTCAGTTATATAGTTTGAAAACAATGCTTCTCCTCATCGCAAAATCTCTTAGAAG ACTCCGTAGATCCAGGAACGGAAATGGAAAATGACAGCGTGTCAATCTCTGAAGGTTTTG GGCATTTCCATTAGCACTCCATCTTCATGTAAACCAGAAGATATGCAGTTTCCTGCCTAG AGAGAAGAGAAGACACATCAGCACAGCGGCATGAAAGCTTCATCAGAAAACAATGCTTCA TTAATCCGTGACAGGACAAGCGTCAGCAAACTTCCAGGCGGCTGGATTAGGCCTTCATCT ATCCATCACCTTGGAGAGGAACAAAATAGGTGGCCTGGGAAGATAAGCACTATGTTTCTA TTCCTTAATATCTAAAGCGGAGGTTAACAAGCTATGGACACACAAGCCAAACCCAGCCCT CTTGGGGTTTTTTAAATCTACTTTCAACTTTTATTTTAGATTCAGCGGGCACATGTGCAG GTTTGTCACGTGGATATGAGCATACTCCCCAACAGTTGGCCTTTCACCCCTCCCCTCCCT CCCCATCCAGCAGTTCCCAGTTGTTGCCATCTTTAAGTCAATGAGTCCCCATGTTTAGCT CCCATTTATAAGAGAGAACATGCATTATGTTTTGTTTGGTTTTTGCTGGTTTTTTTTTTT TTTTTTAATGGAGTCTTGCCCTGTAGCCCAGGCTAGAGTGCAGTGGCACAATCTTGGCTC ACTGCAACCTCCGCCTCCCAGGTTCAAACGATTCTCCCTCCTCAGCCTCCCGAGTGGCTG GGACTACAGGCGCCCGCCACCACGCCCGGCTAACTTTTTGTATTTTTAGTAGAGACAGGG TTTCACCGTGTTAGCCAGGATGGTCTCAATCTCCTGACCTCATGATCTGCCCACCTCAGT CTCCCAAAGTGCAGGGATTACAGGCGTGAGCCACCGTGCCCAGCCTTTTGTTTATTTTTT GACGAGACGGTTCTTGCTCTGTCACCAGGCTGGAGTGCACTGGCACAATAATAGCTCACC ACAGCCTCGTGCTCCTGGGCTCAACTGACCCTCCTGCCTCAGTTTTAGCTTCCTGAGTAG CTAGGACTACAGGTGTGTACCACCATGCCTAGCTATAATAATTTTTATTTTTTTGTAGAG ATGGAGTCTTGCTTTGTTGCCCAGGCTGGTCTTGAAGTCCTGGCTTGAAGTGATCCTCCT GCCTCGGCCTCCCAAATTGCCGGGATTAAAGGTGGGAGATCGCACCCAGTCTCCAACCCT CTTTTTGCAAGTAAATGTAACTGGACCCCAGCCATGCTCATCTGCCCATGTACTGTCTAC GGCTGCTTTTGCTCTACAGGGCAGAGTTAAGTGGTTGCAACAGACACCGCACAGACCACA AAGTCTGAAGTACTTTCTCTCCAGCCCTTTACAGAGAAAGTCTGCCAACCTCTAATCTCA ATAACAGGGAAATCAATGACAACCACAAAGTGACAAAGACTGGGTGTCTAAGATGGATGC TCAGAATAAACAAGAGAGAAAGATGAAAAGTAGAAGGAGGATTTCAAACGCAAGCTTCAC CTAATCCGTTATTTTTCAAATGACCAGGCCTATCTCTGTAGCTGAAAATCACCTCAAATA AGATCTCTGATATACAGTCTCCAAAAGCTCAGCCAAGAAACTTACAAAGTCTCTCTGCCT TAACTTCATCCACCTTTTTTCTCTCCAGCTTCTCCTCGGTAGTTAATGATTATAAAAATA TTTATTGGCTCATGCCTGTAATCCCAGCACTTTGAGAGGCCGAGGCGGGCAGATCACGAG GTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAATCCCGTCTCTACTAAAAATACA AAAAATTAGCCAGGCGTGGTGGCGGGCGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGC AGGAGAATGGCATGTACCCACAAGGCGGAGCTTGCAACGAGGTGAGATCCCACTACTGCA CTCCAGCCTGGGCGACAGAGCAAGACTCCATCTCAAAACAAACAAACAAACAAACAAAAA AACAGTGTGATGGCCAGGCGCAGTGCTCATGCCTATAATCCAAGCACTTTGGGAGGCTGA AATGGATGGATGGCTTGAGCCCAGTAGTTTGAGACAAGCCTGGCAACATAGCGAGACCTC ATCTCTACAAACATCTTTAAAATATGCCAGGCATGGTGGTGCATGCCTGTAGTCCCAGCT ATTCAGGAAGCTGAGGTGGGAGGATCACCTGTGCCCGGGAGTTCAAGGCTGCAGTGAGCT ATGATCACACCACAGTGCTCCAGCCTGGGCAACAAAGCAAGACTCCATCTCTAAAAATAA AATAAAATTAAAAAAAAAAGATCTTCGCTGTAAAAGAGGTACACTCAAATGCAATAAAAG CATATAAGAAGGCCGGGTGTGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGA GACGGGCGGATCACGAGGTCAGGAGATTGAGACTATCCTGGCTAACGCGGTGAAACCCCA TCTCCTCTAAAAGTACAAAAAAATTAGCTGGGCTAGGTGGCAGGCGCCTGTAGTCCCAGC TACTCAGGAGGCTGAGGCAGGAGAATGGCATAAACCCGGGAGGCAGAGCTTGCAGTGAGC CTAGATAGCACCACTGCCCTCCAGCCTGGGTGACAGAGCGAGACTCCGTCTCAAAAAAAA AAAAAAAAAAGAAAAAGAAAAGAAAAGTTCTTGTGACATTTGTGTATGAAATCAGCCTTC ACTACATGGATAGGACCAGCACGCTTCTGCGGCACGACTCTGCAATCTTACTACATTTTT TTTTACTTTGTATTTTATTTATTCCTTTTGAGACAGAGTCTCACTCTGTCACCCAGGCTG AAGTGCAGCCGAGATCTCGGCTCACTGCAACCTCCACCTCTTGGGTTCAAGCAATTCTCT TGTCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACACGTCAAAACGCCCGGCTAATTTT TGTATTTTTAGTAGAGATGGAGTTTTGCCATATTGGTCAAGCTGGTCTCGAACTCCTGAC GTCAGGTGATCGACCTGTCTTAGCCTCCCAAAGTGCTAGGATTACAGGTGTACATTTATT TATTTATTTGAGATGGAATCTTGCTCTGTATTTATTAATTTATTTATTTGAGATGGAGTC TTGCTCCATCGCCCAGGCTAGAGTGCAGCGGTGCAATCTCGGCTCATTGCAACCTCTGCC TTCCAGGTTCAAGCGATTCTCCTGCCTCAGTGTCCCAAGTAGCTGGGATTACAGGTGCCT GCCACCACAGCTGGCTAATTTTTGTATTTTTAGTAGAGACAGTGTTTCACCATCTTGGCC AGGCTGGTCTCGGGCTCCTGACCTCATGAACCACCTGCCTCAGCCTCCCAAAGTGTTGGG ATTACAGGCCTAAGGCACCATGCTCGGCCATATTTATTTATTTAATTATTTAGAGACAAA GTCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGCGCCATCTCAGCTCACTGCAGCCTCC GCCTCCGAGGTTTAAGCAATTCTCATGCCTCAGCCTCCTGAGTAACTGGGACTACAGATA CTTGCCACCACGCAGGGATTTTTTTTTCTATTTTTTTTGTAGAGACACAGTTTCACCATG TTGGCCAGGCTGGTCTCGAACTCCTGACCTTAGGTGATCTGACAGCCTCGTCCTCTCAAA GCACTGGGATTACAGGCATGAGCCCCTTGCCCGGCCTCTCACTACATTTAAGTGACGCCA TGGCTCATGCCTGTAATCCTAGCACTTTGGGAGGCCAAGGCAGGTGGATCACCTGAGGTC AGGAGTTCGACATGAGCCTGGCCAACATGGGGAAACCCCGTCTCTAGTAAAAATACAAAA ATTAGTCAGGTGTGGTGGTACAAGCCTGTAGGCCCAGCTACTTGGAAGACTGAGGCAGGA GAATCACTTTAAGCGGGAGGCAGAGGTTGCAGTGAGCCAATATCATGCCACTGCACTCCA GCTTGGGTGACAGAGTGAGATACTGTCTCAAAAAAAAAGAAAAAAAGAGAGAAAAACATA TGATGCCAGGGCATCTCGGCCTCAATACCTGGGTGAGCACAGTCATGTCCAGGCCAGGGC TGCTGGTCGAGGTCCGGCCCCATCTCTTCCAGCAGAAAGGGAGTAAGCTTGCAGGGAGGC TGGGGGACAAGATCCCAGGATCTCAGCCTCTGCTCATGGATCAGCTCTGAGACCCCGAGT GAGCTGGGGGTGCTCTGTGCGCATTGGTTTCCCCAGCTGTCAAGTAAAGAGATTGGATGA GGAAGTCTTGTCAAGGTGGAATGATCTCAGATTTGGGGCAGCAGTGAATGATCCCGCTCC CTGGGCCATGCCAGTGGCCCGGCCTCGGCTGAACACAGCCCCAACACTCTGGAATGGGGA TGAGGGGGCAGTCAGCTCTTGCTCCTAGTAAGAGAGATGCAACAGGGCTCTGTGGCTGAG CTGGGTGCCTTGCCTCACACCTGTAATCCCAACCTTTGAGAGGCCGAGGCAGGAGGATTG CTCGAGGCCGGGAATTTTGAGAATAGCCTGGACAACATAGCCAGACCCCATGTCTACAAA ATAATAATAAAACACACAGCTATAGTCCAAGCTACTTGGCAGGCTGAGGCAGGAGGGTCC CTTGAGTCCAGGAATTGGAGGCTGCATTGAGCTATAATCGCACCACTGCACTCCAGCTTG GGTGACAAAGTGAGACCCTGTCTCTAAAAGAAAAAAAAATTGGCCTGTGAGCATGGGCTT GATTTTCAAACAGGACCCGGAGGGTAGGGTAAACGTGTGGGTAAATCTAAATGAATGTTA TTGGTATAAAATTACAGTAGTATAGAAAATGATATCTTGTGGGGTTTAAAATAAATAAAA CATACTGAAATATGTATGGGTACAGTTATATATCTGGGATTTGCACTGAAATAATGTGGG GTAGAGGGAAGCAGGAAAGAGTATACATGAAATGAGCTTGGCCATAAGATTGTTGTTGAA ATTGAATGGATACTCTGGGCTTCATTACACAATTCTCTTTACTCTTACATAGCTCTACAC TCTCAACATAAATAAGAATAAAAACACAAAAAACACACAGATACATCTATGCACACACAC ATATTTAAAATACACAAAAATATTAGCATATAAGTCACTGGGGGTAAATTTAGTTCCTGT TCCAAGGTTCTTGTACTGACTAGGAAGAGGATAGAAGTACTAACTCATAGGCTGGGCGCG GTGGCTCACGCCTGTAATCCCAACACTTTAGGATGCCGAGGTAGGCAGATCTCTTAAGGT CCGGAGTTCAAGACCAGCCTGGCCAACATGGTGAAACCCTGTCTCTACTGAAAAAGAATA CAAAAATTGGCCGGGCATAGTGGTGCACACCTGTGGTCCCAGCTACTCAGGTGACTGAGG CAGGAGAATTGCTTGAACCCAAGAAGTGGAGGTTGCAGTGAACCAAGATTGCTCCACTGC ACTCCAGCCTGGGCAGCAGAGGAAGACTCTCTCTATCTCAACCACAACAAAAAGTACTAG CTCATGTTAGACTTTGATAAGGGAAGGATGCATGTTGTAAGCTCTAAAATAATCCAGTCA TCTTTTAAAATAACTCTAAGACTGCACAGTTATGAAACTAATAGAGAAGGAGGAAATTAA ATAATAAAACTAATAAATCCAAAACAAGATGTGAGAGGAGATAAGAAGAAATAGAATAGG CATGGAAAACAAATTGGTGGTGGGTTTCAACCCAAATAAATCATTAGTTACATTTAAAAG GACAATAAAAATTAAAATAATTGAAAATAAAGTAAAACCCAACTAATGCCTTTTATATAA GGGTACAGAGAGGTGGAAGATCATGAAAAATATGTCATGCATGTACTAACCAAGAAAGCT GTATAACTTTTTTTTTTTTTTTTTTTTTTTTTTTGGAGATAGAGCCTCACTCTGTCTCCC AGGCTGGAGTGCAGTGATGTGATCTTGGCTTACAGCAATCTCTCCCTTCTAGGCTCAAGC GATTCTCCCACCTCAGCATCCCAAGTAGCTGGGACTACAAGTGTGCCAACTTAGAATTAT ATTAGCCACACCCAGCTAATTTTTGTATTTTTTGTAGAGGCAGGGTCTCGCCATGTTGCC CAGGTTGGTCTTGAACTCCTGGGCTTCAGTGATCCACCCACCTCGACCTCCAGCAAAGTG CCAAGATTACAGCCATGAGCCACCATGCCCAGCATAACTATTTTTAATGAAGTAGACTTT AAGAAGAAAAGTATTATTAGAGGTAAGGGACACATCACAGAAAAGAAGAATTTACTAGGA GCCAGGCGCAATGGCTCGTGCCTGTAATTCCAGCACTTTGTGAGGCCAAGGCGGCGGATC ACCTGAGGTTGGGAGTTCAAGACCAGCCTGACCAACATGGAGAAGCCCTGTCTCTACTAA AAATACAAAAATTAGCCAAGCATGGTGGCACATGCCTGTAATCCCAGCTACTCAGGAGGC TGAGGGAGGAGAATTGCTTGGACCCAGGAAGTGGAGGTTGCGGTGAGCTGAGATTGTGCC ATTGCATTCCAGCCTGGGCAACAAGAGCAAAACTCTGTCTCAAAAAAAAAAAAGAAGTTA CTAGCTAGTTTCGGTAATTCTTAACATCCAGGAAACTGGATGTGAAAGTTTTTCAGAGAA ACTAAACCAATAGATTATACATAGAGAGAGACTTATTTAGGAATTGGCTCACATGATTGT GGGGACTAGCAAGTTTTAAAATCTGTAGGGCAAGCCAGCAGGCTATAAATTCAGGTAAGA GTTGATCTCGAAGTCTGGAACCTAAAATCTGTAGAGCAGTCAGCAGGCCAGAAACTCAGG CAGGGTTTGTGTGTTACAGTCTTGAAGCAGAATTCCTGCTTCTCTGGGAAACCTCAGTTT TTGTTCTTAAGGCCTTCAACTGATTGGAGGTGGCCCACCCATATTATGGTGGGTAATCTG TTTTACTTAAAGTCAATTGACTGTCAGTGTTAATCACATCTATGAAATAACCTCCCAGCA AGATATTGACAAGTATTTGACCAAACAACAGGACACCATAGCTTAGCCAAGTTGACACAT AAATTAACCATCAGGAGCAAGTAGAATATCCAAAAAACAACATACTAGGGGTATTATATC TTATATAGCTATTATAATTATATAAAACATATAATTATAGAATGACGATATTAAGATAAC CATTAGAACAAAAATATAAACTTTTCTTTCTTTTTTTTTTTTTTTTTTTGAGACCAAGTC TTGCTCTGTCACCCAGGCTGGAGTGCAGTGGTGCAATCTTGGCTTACTGCAACCTTTGCC TCCTGCGTTCAAGTGATTCTCCTGTCTCAGCCTCCCAAGTATCTGGGATTACAGGCACCT GCTACCATGCCCAGCTAATTTTTGTATTTTTAGTAGAGACATGGTTTCACCATGTTGCCC AGGCTGGTCTCCAACTCCTGACCTCAAGTGAGCCACCCCCCTTGGCCTCCCAAAGTGCTG GGATTACAGGTGTGAGCCACCACACCCAGCCAAAAATCACCTTTTTTACAAGGATCAAAA CAGTCATTATGCTGGAGATGACAGACCTCACTGTCACCATGCTCCTTTTGTATGTCTACT AGGCACGGTGCTGGGTCCACACTCACAGAAACCTTAGGAACTCGCACCCAGGGGCTCCGG CTGTAGCAGAATCCCAAGAATAAAACCTGGTGCTGAAAGAGTAGGAGATGAGGCCGGGTG CCATGACTCACTCCTGTAATGCCAGCACTTTGGGTGGCCAAGGCGGGTGAATCAAGAGAT AGAGACCATCCTGGCCAACACGGTGAAACCCCGTCTCTACTAAAAATACAGAAATTAGCG GGGCGTGGTGGCTGGCACCTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCAT TTGAACCGAGGAAGCAGAGGTTGCAGTAAGCTGAGATCGCGCCACTGCACTCCAGCCTGG TGACAGAGTGAGACACCGTCCCAAAAAAAAAAAAAAAAAAAAAAAGCAGGACACTGAACT CTGGGAGGGCCTCCTGGTGAGAGGTGAGCACAGAGGGGAGAGATGGAGGCAGGAGCATGG GCTTCTGGTGGCCCCAGCAGACCCTGTGGCAGCGTGGCCAGGGTCCTCTGCAGGGAGGAA TCTTGGCCAGGATGACGCTGTAGCAGGCCTCTTCCTGAGGCCTCCAGCCAGCCCGGCCAG GGTCCCAGCGTCCAGTGACCCCTGTTTCACAGCAGCAGCTGGGGCCAGCCCCAGGCTCTC TTCCACTCCCAGCTTCTTAAAACTGGAAGTGGAGAGAGTTGTTTGATAAAACACTGGGGC AAACCACATCCTCTCTTCACCAAGGGAGAGTTCGAGGGGATGCCGGCAGAGGGAGCTTTA GAGTAGAGACCCCTACCCAACCAGTGACCGTCACGCACACAGCAGGGCATGCTATGGAGA CCCCCAGACAGTCACTCGGGGAGACCCAGCAGGTCCAGACTCTTCAGAGATCTGTGGCAG CAGGTCCCCACTCCCAAAAGCCACGTGCCCACGGGTGGTCTCTGGTGCCTGAGACCCCAG TCTCATTTGCATCTTTGCAACTTCGAGTTTAAGTGGGTGTCGCATCTCTGTATGTCCTCC CGAGCAGAGGAGGGGCACAGCCTGGGGTGGCAGCTGGCGTCAAACCCTCAAATCCCCTGA GAGCCACTGGGGAGACTAAGCAGTCCCCAGCCCCCACTTGTCCCTGAGCTGCCATTCTCA GCCCTGTGGGAGGAGACAGAAAGCCCTGAAGAGAAACCAAAGGACCAGGTCAGGAGGGGC TGGGGGGGTGGCATGAGCAATCAGGGCAGGGAAGGATGGACCGATGGGGGAATGGAGGGA AGAAGGAATGAATGAAAAGGTGAATGAATGAACAAAGAGAGAGAACGGCCACTCCTCCCT TGCTTTAGTTTACAAAGTACTGGGATCCTCCCAACAGCCTGCAAGACAGAATTTCTGGGA AGCAGACCAGGTGGCTGGCAGGGAGGGGAGGCTTGCCCTGGCTTTTGTGGGCCCAATGGG AGGCAGGGGGCAAGAAGGGGCATCCTGTGTGTGTCCTCCCTGCAGTGGCAGCAGCACCTT CCTGGAAGAGGGTCAGGAAACACCCGCTGTGGCCCCTCTCCACCACGCCCTCATCCAGGA CACCAAGTATCAGTCACTCAGCTCACGAGACCCAGGCCCTGACTCAGGGAGAGAGGATGT GAGGGGTGGGGCACCGGGCTCCTCAGGACTGAGAGACCTGGGATGTGGCCCCGGGCTGGG TGTAGGGGCAGACTGGCTATGGCAGCATTGTGTGTACCCCAGCAGGCCAGTACCCACGCA GGGAGCCTCCAAACCCCTTCACCCATGACCCTGGGAGAAGACCGCAGCCTTGGAGAATTG GCCTCACTGAAGGGGCCTGCACCGGCCAGCAGGGTCAGGCGGGGCCAGACAGGTTCCCAC CTGGGATATGCAAATGGGCCTCCTGAATCCTGGAGCCAGGTATGGACTCACACACCACCA TTGTCCCCAAGTCCCCATCTGCCCCACGGGCACACCCTGCCGCCTGTTCTGTGCAAGGGC CCTGAGGCTGTCTCCTTGCGCCCAAGACCTGCAGGTGCTGAAGCCCACACACACGGCTCC TGCTTCCTGGGCCAGTGCACGTGCACACACACACACGTGCACACACACACACACACCCAC AAATATACCCACACACAATCACACACATTCACACATACCCACCCCCCATACTCACACACA CATTTACACACACCCACACACCCACACTCACACACTCACAGTCACACACACCCTCACACA GCGAAACACAATCACACACATTCACACCCACCCACACCCCACACACTCACACTCACATAT ACTCACACACACCCACACACACATACACAAACACAATCACACACATTCACACACACCCAC ACTCACACATACACACACCCAAACACGATCACACACATTCACACACACCCACACCCCACA AACACACTCACACATATACCCACACACACTGACACATAATCTCTCACACACACACATGCT CACACACACACGCCTTCTCCAGGAGGGGCTGGCTGCCAAAGGCCACCCAGCTTCCTCCCA CGTCTCACTCACCGTACAATATTTGAGAAGACCTTGGAGTCAGCAGCAACAGCGGCGTGG GCAAAGGCCGGGGGTCAAATGGGGCCTGGTGTCGAGAGAGGACCACAGCCAGCACAATGA CAGCCAGCGCCAGCCCCAGCCCCAGCCCCAGCAGGACCAGGTCACCCATGGCTCCGTAGT CCTGGGCCATGGCTCTGCGGCCCAGAAGGAGAGGGGAGGCCGGTGGGCAGATGGAGGGAC AGATGGGTGGGCAGATGAATGGACAAGAAGATGCATAGATAGACTCACAGGTAATTGGAC AGATGGACAAACAGGTGGGGGCTGAAGACAGACACGAAGATGGATCGACAGACAGGCCAG ATAGCTAGACAAAGAGGACAGTAAGAGAAAGATGGTCAGATAGACAATGGGACAGAGATG GGCTTACAGATGGGCGGACAGACAGACAGGTCTGAACAGCGGGCTGCCAGATGGACAGAT GGGTGAATGGACAGATGGCTGGCAGCTGTGGCGAGCTGCTGCCCTCACCAAGTGCACACT ACGGAGTGGCCAAACTCATGCCTCAACTTCTAGTTTTCAGCTCCTGCTTGTTCCTGGCAG GAGGCCAGGCAGCAAAGCGTCTGAGGGGAGTTTTCTTTGCCTAGAGAAGTCAGCTGCTGT GTTAACTCCCTCACTGCTGGTAGGTCCAAAGGCCCCACCTACCGCCCGCCAGAGCCCATG GTCACACTGTCGCAATGTGCAGGAGAACTTGGTGCGTGCTGCACTGCGGTTGCCAGGTAG GGGCAGGGCTCCCTGGAACCTCCACACCATTCCCCAGGTTCTCAGCAGCTCTGGGAAAGC AGAGCTGGGGCCGCTTAACTCTGCCCTGGATCCGGCAAGGCTGCCCCCCTCCCAGAGTGG AGCCCTGCTCCCCAGCTCCCATCTCTATCCCCTAACCCTCTCCGCATGGCCCAGCCTAGT CAGCATCAAGGTGGAGCTGAACAGAGGCAGAAGGAGGAGGACCCAAGGTGGTGTCACTCA GGACCCGGGTTCAAGTCCTTATGCTTCTGCAGCCTGGCCTGGGTCCCCCAACTCCCCCAG GGTGACCAAGGGCTTTCCAGTCTGCACAGAGGACAGGGGGTCTTGACAGCATCAAATTCT GGTGACTACGAGACACTTACGTGGGAAATGCAGACAGACCATGCCTCTAGCCCTTGGCAC CAGGCACCATCCATCCCTGGGACTTGCTGTCCTGGAAATGCAGCATGGTCCTCCAGGGAG GGGGGCTGTGCCATGTGGGGGCCCCACCCCACCTGCAGCTCTTTCCCACCCTGGCTGCAG GTCTGCTTCCCTGAATCCAAATCCGCTACTACTGTGCTGGCAGCGCAGCCTCTCTGGGGA CACTGGCCTGGCTCTGTTCTCCCCAGGCCTCAGGGTGCCTAAATGGGAGGCAACCAGGGG AGTGAGGACCCACTGAGGGGCTCCGTTGACCAGGCTCAGCAGGGGTGCAGGTGATGTGGG GTGGAATCCTTCCCACATGGCCCCCACAGTCCTCCCCGCTTCCTCCCCAACTGAACACTG CCTGCTCCAGATGTGTACACCTGGAGTCTGGGCCCCTCCATCTGGGCAGCAGAGAAACTG AGGCACAGAGACAGACTGTGTCCTTACAGGGCACACAGCCTGCCAGGCCCCTATGTCCGG CCAGAGCCCCTGGTCAGCCTGGGCTGCAGTGATTGTTTAGAGGTAGGCTGTTCCCACGGC TGCCTCTCACGGTAGGGGGGCCTGTGGACGCCTCCTCCCGCCCCCACCCGACTCCCAAGC CTCAGTGACATTGCTCAACCAGGAGCTGAAGTGCATTCCTGGGCTCCGGCCAGCCCACCC ACCCACCCGCTGCAGTCCTGGAAGCCCAGAGGCCTGGGCAGCAGGAACAGTGGAGACAGC AGTGTGGGGGACGTCCCCCTCCTCTCCCCACCATCCTCGTCAGGCAGAGGCCAGGGTGCA GGGACCACCGGAGCAAAGGCCCAGGGAAATGAATGGGTGTCATTCTGGTCCTGACCCGAG GCACAGCCAGGAAGGTCCCTGTGGGGAAAAGAAAGAGATATCAGACTGTTACTGTGTCTA TGTAGAAAGAAGTAGACGTAAGAGGCTCCATTTTGCTGTGTAGTAAGAAAAATTCTTTTG CCTTGAGATGCCGTTAATCTGTAACCCTAGCCCCAACCCTGTGCTCACAGAAACATGTAC TGTGTCGACTCAAGGTTTAATGGATTCAGGGCTGTGCAGGATGTGCTTTGTTAAACAAAT GCTTGAAGGCAGCATGCTTGTTAAGAGTCATCACCACTCCCTAATCTCAAGTAAGCAGGG ACACAAAACACTGCAGAAGGCCGCAGGGACCTCTGCCTAGGAAAGCCAGGTATTGTCCAA GGTTTCTCCCCAGGTGACAGTCTGAAATATGGCCTCGTGGGAAGGGAAAGACCTGACCGT CCCCCAGCCCGACACCTGTAAAGGGTCTGTGCTGAGGAGGATTAGTAAAAGAGGAAGGCC TCTTTGCAGTTGAGATAAGAGGAAGGCATCTCTCTCCTGATCGTCCCTGGGCAAAGGAAT GTCTCAGTGTTGATTGTATATTCCATCTGCTGAGATAGGAGAAAACTGCCTTAGGGCTGG AGGTGGGACATGCTGGTGGCAATACTGCTCTTTAATGCATTGAGATGTTTATGTATATGC ACATCAAAGCACAGCACCTTTTTCTTAACCTTGTTTATGACACAGAGACATTTGTTCACG TGTTTTCCTGCTGACCCTCTCCCCACTATTACCCTATTGTCCTGCCACATCCCCCTCTCT GAGATGGTAGAGATAATGATCAATAAATACTAGGGAACTCAGAGACTGGTGCCAGCGTGG GGCCTCCATATGCTGAGTGCAGGTCCCCTGGGCCCACTTTTCTTTCTCTATACTTTGTCT CTGTGTCTCTTTCTTTTCTCAGTCTCTTGTCCCAGCTGATGAGAAACACCCACAGGTGTG GAGGGGCAGGCCACCCCTTCAGGTCCCTGAATGTCCTTCCTCAGGAAATGATGGGGGAAG GGGCGATGAGAATGAAGGAGACGATTTAAGTCCCTCACCCCCCGAGGTAGTCCTGGGCTG AGCCCCATGGGACCTAGAGAACCAGGGTGTACCCCACCAGCATGTCGGGTCCAGGAAGCC TCGTGGCCAGCTCCCACTTCTCTTCCTGCTGTGCAACCCAGAGCAAGGCCTGCCCCTCCA GCTTCAGTCTTCTCCCCTGCAAATGGGGCCACGGCCTTTCCTCTCAGGCCAATATAAGGA TTGAGGCCGGGTGCAGTGGCTACCCCTGTAATCCTAGCACTTTGGGAGACTGAGATGGGG GGACTGCTTGAAGTCAGGAGTTAAGACCAGCCTGGTCAACATAGTGAGACCCCATCTCTA TTGGTTTAAATTTTTTTTAAAAAAATTAAATAAATAAAATAAGGATTGAAGAGTGACTTG TACACCAGTTGAGCCCACCTCCATCTCACCCTTGCAGAGCCCCAGAGACACAGCCCTCCA GAGCTCAGACCCAGTGGGACTTGACTCCACAGGCATAAAACCCTGTTTGTCTATGGGCCC TTTGGAATCACCAGGTTTTCGGGGCTCCTGAAGGATAGCCCCGACCTGGCCTCACCTGGC CCCTGGCCCCAGTGCCCCTGGTGATATCCAGGTGCTGGGCTGTGATCACCGCCTCCCACC AGCCCACCTCCACCAGCCCTTCCCAGAACCCTGCTCCAGGTGTTGGAACTGTGCACAGAG GAGGGAGCAGGCCCCGAGGGAGGCCTGGAGGGGCTGCCAATGGTGAAGGCTGCTGTGTCG AGCTGTTTCCTTCCGGACCCACTCCCTCTGGGCTGCGTCCCCGGCTGGTCCAAGCCCTGA TCCCTGGGATCTGGGGACATCTTCCCGTTTGCTGTTCCCTGAGAACCAGGCCTCCCTGTG GAGAGGATCACAAGCTTGGGTTTCACTCTGGGCTTGCTCTTGGGAACCCCCCAGGGGCAT GGCTCTGACCGAGATGTTTTCCTCCAGCCTGTTGCCCAGTCCCCATTCCTCGGACCCTCA GCTTCACCTCCAGTGTCATCGGCAGGGTGAGCTGGACGCCTACGGGTCTGAGAAGGCGCC CGGGTTCCCAGCATCGGCTGGCCACCCTCTGCCTAAGAAAGCGCCAGGGTCGTGACACCC CCTGGTGGCTGATCCTAGGTAGTGTCACTGCCCAGCCCCAGTAAGGGAGGGCCTGGCCCC AAAGTCTGAGGGATCAGGGTGGGAAGGGGCAGGGTTTGGTGTGAACCTTCCCCTGGCCCC CAGCCATGTGCCTTGCTCTCCCCATGCTGAAGATGCTGAGGCTAGTTCCAGTGCCCGCAT TGTGAAGATCTCCGAATCCCACCTCTCTGTTCCTCCCCAGCCAGATGGCTCCATTTCACA CACAATACACTGAGGCCCAGAGAGTGGGGAGACAGGCCAGGGAGGCCACCTGGAGCCTGG CACAGTGGCCTCATTTATTATGCTGCTCTGCTGCTCACAGGGGAAGCCCGTCCCCCAAAG TCCTCTTCCTCATCCTGGTGAGTATCTTGTCCCTGGATTGCTTGTCAGCCTTGTCTGCCT GGAGCAATCAGTAGCCAGCAGGTTCCCCGCCTTTCCTGGAGTCCGAGGCAGCTGCCCAGC CACCAGCCGTGCGGACGATGGCTTGCACCACAGCGATGAAGGTGGATGCGATCTGGGTGT GATGGTGCCGGGTCTCCAGGGCTGCAGTCACTGCCTGGGGGTGGGAGGAGAGGGGAAGCC TGAGCAGGGCTCCAGATGCCACCTGAACCACACCTGTGTGGTCACAGGCCTCAGCCCAGG TGGTGCCATTTCAGGCCAGGTCATCAGGAAGAGCAGGTTGGGGCCTGCTGGGTCTCACTG GAGCAGGGGGCTTGGCTCTCATGTCACAGGGGCTCCAGATGGCCCAGGCACTAGAGAGAG GACACCAACCATTGTCCACTCTGTGATGATCCAGGCCTCCAGCCCAGGATGCCCTGGGAC CCCACACCGTGACTCAGTTTCTCCAACCCCTGGCCCACCTGGTCAATGTTTCTCTCCACT GTCGTGACGTTGGGCAGAAGCTTGTTGTGCAGCCGGGGCTCCTCCACGGCCCTCTTCACG TCATAGCCGAACCAGAGGTTGTAGATGATGGCCTGGGGCATGGGAGTGTGATCAGCGTGG CTTGGGGGCTGTGCAGAGTGGGCAGGGCCAGGGAGAAAAGGGGTGACACATACCAGTGCA GTGTCTGTGGTGATCTGCGTGCCCCCAGCAGCTCCCACCACCATCCGGACCTGGCCGTCC TGGCCCACCATGATCGTCGGGCACATGGACAAGAGCGGCTGCTTCCCTGCGGCCGATGGG AGAAGACAGGGATGCCCGTCAGCTGCCTGCCCAGGACACCCGCCCCTCTCCACCCCAGTC CCCCACCCCCCAGACCTCCACCCCATACCTGGCTGGATGAAATTGGCAGGTGAGGGGGGT GCCCCAAACTCATTGGTGAATGCTGGGAGAGCTGAAGTCGTCCATTCATTATTGAACAGG ATCCCACTGACCGGGGAGCAGACCTTGGAGCCAAAGCTACCGCCCAGCCAGGTCAGACAG CACCCGACCTTGCCTGGCCCAGCCTGGTCCCTATCCACCCACTGAGGCTCAAACATACTC ACTGAGAGGCCCAGGATAAGCTACCAAGGTTGGGCCTCAGTTTCCCACCAGGAAAAGAGG TGATGGAGCCACCTTACTGGATAAGTGGGCAGTCCCTGGGCCACCCGCCCCTGGCCCTTT CCCACCCAGGCGGCCCAGCAGCCCCTACTAGAGGTTGATGGTGCTGGTGGCGGACACAGC ACTGCCGTCCTCTGCGACGACAGACAGGTGAGCAGTGCCCCCGTCATCCGGCGTGTAGAA CTCGGGCTTGTAGTAGGAGATCGGGTGAGTGGTGTGGTCAGAGATCTGGGACCGGAGCTG GGCAGCGAAGAACTCAGAGGTCATGTTGCGGACCACCTGCTGAGACCCCAGAGCTGGCCT GAGGAGGTGGGGAGGGAGGGTGGGGAGGGGGCACAGGTCTCAGAAGGCCCTTGACTGTGA CTCTGACCGCAACCCTCTGGCACCCACAACCTTCCGTGGCTCCCCAGGACCCAAGGGCAG GCCCAAGACCTTGCATGACCAGTCTGACTCCCTGTCTCTGTCGCGTTCCAGCAACTCTGA ATGTCTGTCTGCCTGGTCCTCAGCCTCCAGACCCTTGCCGCATTCAATCACTCATTCTTT CATGCAAAAAATATTTCTACAATTTGCACTGCATGCCTGGCACTGGGGAATCAACAGGGA ACAGACACTTAGGTCCTGCCCTCATGCCAAGAAAAACAAACACACACAGGGAAAGTGCTG AAACCACAGGCCAGGTAAGGGGAATCAAGAGGCATGAGGTATGGGCAGAGTGGTCAGGGA GGGCTTCTCAGAGGAGGCAACGTGTGAAAAGAGCCTGGAAAGTGGCCTAAATGGTCAGTG CAAAGGCCCTGAGGCAGGTGGCATAGGCTGGTGAGCGATAGGCAGAGAGTGAATGGAGTA GGGTGGGGAGAAGAGGATGAAGATGCAGGCTGGGGCCCATCCCACAAGACCTCCTAGGTC CCATAACAACTGGCTTTTGCTCTGTGCCATGCAGGCTTAGGACAGAGGAATGAGCAGGCT GGGGAGTGTTTTCACAGGGTCCCTCTGGCAGCTATGACGGGGATAAGGATAAAGCCCAAA GGGGAGGCTGTGGGTATCAACCAGGCAAGAGATGATGGCCTGGGTGGGAGAAAGAGAAGA AGCCTGTGCGTTCTGTCCATGTGATGAGGTGGGCTCCTCTCAATCTTCACACAGCACAGC TGAGCCTCAAACCCAGCACTCACCCTGACCTCTCACCTCCCCACAAGGTAGGAAAACCTG TTGCCCAGGCTGGAGTGCAGTGGCACGATCCCGGCTCACTACAACCTCTGCCTCCTGGGT TCAAGCGATTCTCCTGCCTCGGCCTCCCAAGTAGCTGGGACTATAGGCGCCCACCACCAC GCCTGGCTAATTTTTGTATTTTTAATAGAGACAGGGTTTCACTGTGTTGGCCAGGCTGGT CTTGAACTCCTGACCTCGGGATCCACCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGG AGTGAGCCACCGCATCCAGCCCCTATTTCTTTTTAAGACACGGTCATGATTCATTGCCCA GGCTGGAGTGCAGTGGCACGATCACGGCTCACTTCAGCCTCACACTCCTGGGCTCAAGCA ATCTTCCTGCCTCAACCTCTTGAGTAGCTGGGACTATAGGAATGCCGCACCACACCTGGC TAATTTAAATTTTTTTTTTTTTTTTTTGTAGAGGTCTCACTATATTGCCTAGGCTGGTCT TGAACTCTTGGGTTCAAGCAGTCCTCCCACCTCAGACTCCCAAAGTGTTGGGATTACAGT TGTGAGCAACCATGCCCAACCAGATTTTATTTTTATTTTTATTTTTGAGACAGGGTCTCA CTGTATCACCCAGGCTAGAGTACAGTGGTATGTTCACGGCTCACTGCAGCCTTGGCTTCC TGGGCTAAGTAATCCTCCCACTTTAGCCTCCCTAGTAGCTGGGACCACAGGCACACACCA CCATGCCCAGCTTTTTATGTTTTTATTTTTTTATTTTTTGTAGAGATGAAGTTTTGCCAT GTTGCTCAGGCTGGTCTGAAACTCCTGGGCTCAAGCGATCTGCCTGCCTCAGCCTCCCAA AATGTGGGGATTTCAGGGGTGAGTCACCACACCTGGCCCAGATTTTATTATTTAAGTTAA CATTTTGTTACCATTGCTTAAAGCCACCTGGTCCTTTTCATCAGATAGTTTCTTACAGCC AGGCACAGTGGCTCACACCTGTAATCCCAGCACTCTGGGAGGCTGAGACAGGTGAATTGC TTGAGCCCTGGAGTCTGAGACCAGCCTAGGCAACACAGTGCAACCATGTCTCTACTAAAA GTTTTGGTCTCGATCTACTGACCCCGTGATCCGCCCGCCTTGGCCTTCCAAAGTGCTGGG ATTACAGGCGTGAGCCACCGCGCCGGCCTAAATATTTTTTTTTTAATTAGCTGGGTGTGA TTGAGGCTGGGCACTGTGGCTCACGCCTGTAATCCTAGCACTCTGGGAGGCAGGTGGATT GCCTGAGCTCAGGAGTTCAAGGCCAGCCTGGGCAACATGGCGAATCCTCGTCTCTACTAA AAATACAAAAAAAAAAAAAAAAAAAAAAAAAAAATTGGCCAGGTGCAGTGGCTCACGCCT GTAATCCCAGCACTTTGGGAGGCCAAGGCGGGTGGATTACCTGAGGTCAAGAGTTCAAGA CCAGCCTGGCCAACATGGTGAAACCCTATCTCTACTAAAAAATACAAAAATTAGCTGGGT GGTGGCGGGTGCCTGTAATACTGGGTACTCGGGAGGCTGAGACAGGAGAATTGCTTGAAC CCAGGAGGTAGAGGTTGCAGTGAACCGAGATCACGCCATTGCACTCCAGCCTGGGCAACA AGAGCGAAACTCCGTCTCAAAAAAAATTAGCTGGGTATGGTGGCATGTGCCTGTAATCCC AGCTGCTTTGGGGGCTGAGGCATGAGAATTGCTTGAACCGGAAGGCAGAGGTTGCAGTGA GCCAAGATCGTGCCACTGCACTCCAGCCTGGGCAACAGAGTAAGATTATCCAAAAAGAAA AAAAAAAATTAGCGGGTGTGGCTACACCTGTAGTCCCAGCTATGTGGGAGGCTGAAGTAG GAGGATCGCTTGAGTCCAGGTGGTGGAGGCTGCAGTGAGCTATGATTGTGTGACCGCACT CCAGCCTGGGTGACAGAGCCAGACCCTGTCTCAAAAAGAAAAGGGTCATGATGCCTCCCC AACAGGATATGGTGGGGACGCTCCAAGACCATGGGAGAAGAGGATACGAGCCGGAGCTAT GGAAGAAGTGAACACGTAAGACTCCAGAGGCAAACTGCCCGTCTAGACAGCTCTGGCCTG CAGAGCCCACCACTCCCAGGCCAGCGCCGTGAGGTCCTGTTGAGCCACCCACCTCACTGT GATCATCCCCAGGTAAACCCTCGGACGTGGGCCCGGTGCCCTCCTTGCTGAGGCTGAAGC CACGTGGCCATGAAAACCTGAGCATCACAAGCCGCTCTCTGCGTTTGTGGTGTGATCAGG GCCTTCTACCACAAGCCATGTGCCCAGGCTGATCCTCTCTAATGTGGAGAGGCGGCTGCG CTGGAAAGGCCTCAGTAAGAGCAGCTGGGCCCTTGCAGACAGACATCACGGTGTGACCCT CGGGACTGGCTCCCTGCGGCCACTGCAGCACAGGTGTTCACATCTCACAGTGGAGGCAGT GGAGACCATGCTCGCCTGCAGGCTCTCGGCGAGGCTCTGAGTCTGGCCCCTTGCGTAGCC CCTGGTGACTCCTTGGCTTCTGGAGCGGAACTCCATCCTTCACATGGCGCTTTCCTCGAG GGCCTGTGTGCGTGTCATCTGTGGCCAAGTTCCCACTTCATAAGGACATCAGTCATATTG GATGGGGTCCCACCCTGCTCCAATGAGACCCCATCTTTTTTTTTTTTTTTTTTTTGAGAC AGAGTCTTGCTCTGTGGCCCAGGAGGGAGTGCAGTGGCACGATCTTGGCTCACTGCAAGC TCCGCCTCCTGGGTTCACGCCATTCCCCTGCCTCAACCTCCCAAGGAGCTGGGACTACAG GCGCTCGCCACTGCACCCGGCTAATTTTTTGTATTGTTAGTGGAGACGGGGTTTCACTGT GTTAGCCAGGATGGTCTTGATCTCCTGACCTTGTGATCCACCCTCCTCGGCCTCCCAAAG TGCTGGGATTACAGGCATGAGCCACCGCGCCCGGCTGAGACCCCATCTTAATCACACCTG CAGTGACCCTGTTTTCAAGTTAAGGCCACATTCTGAGGTATTGAAAATTAGGACTCCAAT ATGAATTCGGGGGGACCCAATTTAACCTGTAATATGTATATCCCAAATGCAATTTTCTCC TTTTTTCTTTTAGACAGTTGCTCTGTCACCCAGGCTGGAGTGTAGCTCTTGGCTCACTGC CTCTGCTTCCCGGGCTCCCGCAATCCTCCACCTCAGCCTCCTGAGTAGCTGGGACCACAG GCGCACACCACCACACCCAGCTAATTTTTGTATTTTTGAGAGAGATGAGGTTATGCCATG TTGCCCAGACTGGTCTTGAACTCCTGAGCTCAAGAGATGCACTCACCTCAACCTCCCAAA GTGCTGGGATTACAGGTATGAGCCAGCATGCCTGGCCTCCAAATGAAATTTTCAGAAAGG CCTTTTCATAATAGTAACGGGAAACAAGTGACATAGGCATAGCCTTGATAAATGTGCCAA AAGCCCATTTGGAAATAGCACATTTGTAGGAGGCCTGAGGCAAGACTTGTGGGATTGGAC GGGAATGCTCTGTTCCTGGGACCAATACCTGATGATGGAGAGTGTCCATTCTTCCCTTTA AAATTTTTATTTTAATATTTTATTTTTAAAAAGGGGTCTTGCTATGTTGCCCTGGCTGGT CTGGAAGTCCTAGGATCAAGTGGTCCTCCCACCTTGGCCTCCCAAAGTGCTGGGATTACA GGTGTGAGCCACTGTGCCCAGCCCATTCTTCCCTTTTAATATATACGCTTCACATACAGG CTTTGTTTTTGCAACTCCTATCAAATCCCAACAATGTATTTGGGAGAATTTGACAAGACA GTTCTAAAGTTTATCTGAAAGAGAAGCAGCAGATCCTAAATGGCTTTCCCCAGGCCCAGC ACTGGCAGATAGCACCTTTCTAACCTTCGGGTGTGAATCCAAGGGTGGTGGTGGAGCTTC AGGACACATGCACCCTCGTGTCCCTCCTCTATGTAAATGGAAGCCTTCAGTGCCAGCTCC TTTAGGTTGTAAGGACTGAACACGCTCACAGAAGTCGTGTGCACAGTGAGGTGGGGGCTG AAATGCAGCAGCTCCTCTCCCTGTGTTCTGTCCCCGCCCCACCTCTCCCCCACCTCAACT TTGACCACTAGGGGACCAATTATAACATGAGATGGCAAGAATAGCTCATGTAAAGCCACC CCACCCCAGTCACCTGGTTGCTGCATAGTGTCACATTCATAGGAGTACAATTTAGTCACT AGTAATCCTTTGCGAATAATACAAACGTCCAGTAATTCTTTGCTGCATAACAGGCCTTGT GCTCAAAAGGTTCTGCAGACCTTGCCTGGGTGCTCTGGCCAAGCTGCAGTCATCCCAGGG CTGGCTGGGGGCTGGGGGTTTGCCGGCATGCCTGCTGGTTGGTGCTGCTGCCAGTCCCAG CTACTCGGGAGGCGGAGGTACGGGGACGGCTTGGTCTCAGGAGGTTAAGGCCGCAGTGAG CCATGATCGTGCTATTGCACTCTGGCCTGGGCAACCAAGCAAGACTCTGTCTCAGAAAAA AAAAAAAAATAAGGGGGTGAGAGGGGCTATAAAGGCTCTGAGGTCCACGCTCTGGAGGCC CACAAAGTCTTTTCTGCATCATCTTGTTTGTCAGAGCAAGACCCAAGGGTGAGCCTAGAT TCCTGAGCTGGGGAAACAGGCTCCACCTACTGGTGGTGAGAACTGTAGACAATCTGTGGT CGTTTTGAGTCCACTATAAGTAACCAAAATACCTTCAGTCTTGCTTGCATTTCTCAACAT TAGTGAAAGGGGACCCAGTGCTTGGTTGTAGCAGGCGTCCTAAACTCTCCTTCTGACCTG CAGAGTTTTCATCTGCAGAATGTCCCCTCCTGGTGCACAGCATTCATGTCCCTTGTCCTC ATCCCTGGTGGGTGCTCTCGCCGCCTCCTTTCTGATCCCATCCTCCTCCTTGTCCTACCA ACCGTCTGTACTCACCCTGTGTATTTAGTTTATATAAATGTTAATGCCTGGCCAGCCGTG GTGGCTCACGCCTGTAATCCCAGCACTCTGGGAGGCTGAGGCGGGCGGATCACCTGAGGT CAGAAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCGTTTCTACTAAAAATACAAA AAATTAGCTGGGCATGGTGGCGAGTGCCTGTAATCCCAACTACTCGGGAGACTGAGGCAG GAGAATTGCTTGAACCCTGGAGGCGGAGGTTGCGGTGAGCTGAGATCATACCATTGCACT CCAGCTTGGGCCACAAGAGTGAAACTCTGTCTCAAAAATAAAAAAATTAAAAATAAATGT TAACACCTGTAGTCCCAGTGCTTTGGGAGGTCAAGGCAGGAGGATCACTTTAGCACAGGA GGTCGAGGCTGCAGTGAGCTGGGATGGCGCTACTGCTCCTCAGCCCGGGCAACAGAGTGA GACTCGATCTCAAAAAAAAAAAAAAAAAAAAAGACCAGCGGGGGTGGTTCATGCCTGTAA TCCCAGCATTTTGGGAGGCCAAGGTGAGTGGATCACTTGAGCCCAGGAGTTTGAGACCAG CCTGGTCAACATAGTGAGACTTCATTTCTACAAAAAAATAATCAGCCATGCTGTAGTCCC AGCTACTGGGGGTGCTGAGGTGGGAGGATTCCTTGAGCCCAGGATTTCAAGACCGCAGTG AGCTAGGATCAAGCCACTGAAGTTCAGCCTGGGTGACAGAGCAAGATCCTTTCTCTCTAA CAAAAAATTAAAATTAAAAAATATTGGCCAGGTGTGGTGGCTCACGCCTGTAATCCCAGT AACTTTGGGAGGCTGAGGCAGGTGGATCATTCGAGGTCAGGAGTTCGAGACCAGCCTGAC CAACATAATGAAACCCTATCTCTACTGAAAATACAAGGATTAGCCAGACGTGGTGGTGGG CGTCTGTAGTCCAGCTACTTGAGAGGCTGAGGCAGGAGAATCACTTGAACCACCACGCCC AACCTAATTTTTTTGTTGTTGTTGTTGTTAGTAGAGGCAGGGTTTCACCATGTTGGCCAG GCTCATCTTGAACTCCTGACCTCAAGTGATCCACCTGCCTCAGCCTCCCAAAGTGCTGGG ATTACAGGTGTGAGCCACCGCGCCCGATCTGAAGACATTTTTGATTGGTTGATTGAGTTG GGGGTCTCACTGTTGCCCAGGCTGGAGTGTGGTGGCATGATTATAGCTCACTGCAGCCTT AAACTCCCAAGCCCAAGAGATCCTCCCAGCTCAGCCTTCTGAGTAACCGGGACTACAGGT GCACACCAGCACACCCAGCTCATTTTAAATTTTTTCTTTTTTTTTGAGATGGATCTTGCT CTGTCACCCAGGCTGGAGTGCAGTGGCGCAAGCTCCGCTCACTGCAAGCTCCGCCTCCCG GGTTTATGCCATTATCCAGCCTCAGCCTCCCGAGTAGCTGGGACTATAGGCACCCGCCAC CACGCCCGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGA TGGCCTCGATCAGGAGATCGTCCATCTTGGCCTTCCAAAGTGCCGGGATTGCAGGCGTGA GCCACCGCGCCCGGCCCCGTAGTTTCTTACATGTTCACAAAGAGACAAACTTAAAAGAGC AAAGCATTACGAACTTTAAGAGGCCAGTGCAGGAGGATCACTTGTAGCTAGAATTTTGAG ACCATCCAGGGCAACAAAGTGAGACTCTGTCCCTACAAAAAAAATTTTTTTTTTTTTTTT TTTTGAGACAGACTCTCGCTCTGTCGCCCAGGCTAGAGTGCAGTGGCGTGATCTTGGCTC ATTGCAACCTCCGCCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCTCCCGGGTAGCTG GGATTACAGGCACTCGCGACCATGCCCAGCTAATTTTGTATTTTTAGTAGAAATGGGGTT TCACCGTGTTAGCCAGGATGGTCTTGATCTCCTGACCTCGTGATCTGCCCTCCTTTGCCT CCCAAAGTGCTGGGATTACAGGCATGAACCACCGTGGCCAGCCTCTTTTACTATTTTTTT TTTTTTTTTTTTAAGATGGAGGCTCACACTGTTGCCCAGGCTGGAGTGCGGTGGCACGAT CTCGGCTCACTGCAACCTCCGCCTCCTGGTTTTATGCGATTCTCCTGCCTCAGCCTCCTG AGTAGCTGGGATTATAGGCCACCATGCCCAGCTAATTTTTTGTATTTTTAGTAGAGGTGG GGTTTTACCATGTTGGCCAGGCTGGTCTCAAACTCCAGACCTCAGGTGATCCACCCACCT TGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAACCACTGTGCTTGGCCTTTACTACTAC ATTTTTTTTTTTTTTTGAGATGGAGCCTCCCTCTGTCACCAGGCTGGAGTGCAGTGGTGC AATCTCGGCTCACTGTAACCTCTGCCTCTCGGGTTCGATTCCCCTGCCTCGGCCTCCCGA GTAGCTGGGACTACAGGCAAGCACCACCATACCCGGCTAACTTTTTTTTTTGTATTTTTA GTAAAGACAAGATTTCACCATCTTGGCCAAGCTGGTCTTGAACTCCTGATCTCATGATCC ACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGTGCCCGGCCGTC TACTACTTCTTAAAGGGTGAGAGGCGGAAGGATCACTTGAGCCCTGAAGTGTGCGACTGC AGTTAGCTTTTATCGTACCACTGCACTCCAGCCAGGGTGACAGCAGGACCCTGACTCTAA AAAAAAAAAAAAAAAGCAAAAAAAAGCATATACTATTAATACTTCCTCCTTACTATAATG TTTACTGTGGCCTTTATCAGACTAGAGAGTGCTTTTATCTCTCCCTTTTGTTAGAGCTTT TAGAATTATCAATAGATGGTCAGGTGAGGTGGCTCAGGCCTGTAATCCCAGCACTTTGGG AGGCCGAGGTGGGTGGATCACGAGGTCAGGAGTTGAAGACCAGCTTGGCCAAGATGGTGA AACCCTGTCTCTACTAAAAATACAAAAAAATTAGCTGGGCGTGGTGGCGAGTGCCTGTAA TGCCAGCTACTCTGGAGGCTGGGGCAGAGAATTGCTTGAACCCAGTAGGCGGAGGTTGCA GTGAGCCAAGATTGTGCCACTGCACTCCAGCCTGGGCAACAGAATGAGACTCTGTCTCAG AAAAAAAAAAAATTATTAATAGATGGTGACCCTCATCTTCATGTTTTCTGCATCTATTGA AGTGCGCCATTGAGATCGTGGAGTTCTTCCCTGTTTGTCCATATGGTGAACAACACTGAT TTTCTGATGTTGAACAATCTCTGGAATAAACTCTGTTTGGTTTCCATGGATTTCTGTTTC CCTTTAGATTTTGTTAGTAATGTATTTTGGATTTTTGCATCCAAGTTCATAATTGTAACT GGACAAATAAAGGCAGATTTTAAAAGGACAAGTTGGGAGGCTAAGGCGGAAGGATCACTT GAGCCTGGGAGATTGAAGCTGCAGCTAGCCTCGATTGCTCCACTGTACTCCAGCCTGGTG ACAGCAGGACCTGCCTCTAAAATAATAATACTTAAAAGGACGAGTTTACCTACAGTCTCA CCAAGCAATGAAAGAGCTTATCTTCCTCCTGTTTCCTTTACGGATCTTGCTCATGTATTT TATCTTAGTTACTTTAGCATAAATGCTAAATCAAATTCTTTTTCACTAGACATCATCATA GCTCTAGGCATACCACATAATACCCAAGCACATTTTAATCATTAAAATTGTTTCCTTTTT TTGTGGCTACTGTAACCACTTCAGCGAACCATTTAGTGTGTAGTGGGTTTAAATTATTTT TTCTCCTTTCTGGGATAAATTTCCCAGGAGAAGGATAATTGGGTCAAAAGCTACGAACAT ATTTATGGGTTCTTTTCCACAAGAGATCGATCAGTTTGCAAGTGCCATCTGCAGTGTGGG GGAACCAATCTTCAACTTGCCTGCATTTGGTATTAGTATTTTATTAACCTTTCATTGATC CAATTACGGCTCTTTGACACAATTTTCATCAGTGATGGACTAAGTGTGATCCGGCCTGAC CCGCCTCCGGCCGGCGTGAGAAGGGGCATGTGTCGGGCTACGCTCGGGCTTCCCCTGCCG CCCATTGTGATCCAGCCCGCTAGGCGCTCCCTGCCGCCCATTGTGACGCCTGCCAGCCGC AGGCTGGGTCCCCGAGGCGGGCGGCATTTAGGCTCGGTCTCCACAGCCATGGCCGCGACG CAGGAGCTGCTGCTGCAGTTGCAGAAGGATAACCGAGATGGTCGCCAGCGGAAGCAGGAG CTAGAGAAGCTGATGCGCGGGCTCGAGGCCGAGAGCGAGAGCCTCAACCAGCGCCTGCAG GACCTGAGCGAGCGGGAGCGGAGGTGCGCGGGGAACGCCGCTCTTCTACCTGGCGGGCGC GCGAGGGTCGGTCCCGCAGGCAGCGCCGCGAGGTGCTTCGCAGAGTACCAGGCTGATCCG CCCGGGCCCGCATCTCTGCTCTAGGCCCTTGGGAACGGGTGATCCACCCAGCGGACCCAG GTGGGGGACTCGGCCAGGACTTCCCAGTCCTCAATCATGAGCCTGCGGCTGGTCCTTCCT GGCGACTGCGGGATCCTGAGCGACCCAGTCCGCCTTGTAGCGCCAACCTCAGTTTCCCTC TGCAGCCTGCTGCGGAGGCGAAGCCAGGCAGCGCAGCCTCTGCAAGGGGAGGCGCGCGAG GCGGCGCGGGAGCGCGCGGAGCGGGTGCGCAGAAGACTGGAGGAGGCGGAGCGCCACAAG GAGGACTTGGTGAGGAAGAGTCCTAGAATGGGGCTGGACCCAGGGTGGGGTGAGGCAGGG CGGGCGGAAGGGGGCGGGATCCGGGGGTGGGGTGAGGTAGGACCGGCGGAGAGGTCGGCA CCGCCCCAGGACCCCGTCCGCAGGAGCAGCACAGCAGGCAGCTGCAGGAGCAGTGGGAGG AGCTGTCGAGTCAGGTACGTGCAGGAGATGGGAGGGCCTGTCTCTTGGTTCCTCTCGGAA GTCCTGCCCTTGTCCTCGCCCTTGTTGTCTCCCCGTCCCTGTCTCCCCTGACACTCGTTC CTCCAGAAGCCCGGTAAACCCCGCCCTTACAAGCCCCGCCCCTAGCTCTTCTACGGAGGG GAACCGCAGAGCCAGAAGAGCACGGAGCAGCAACTCGCAGCCCAATTGGTGACGCTGCAG GTGCTTGAGCGGGACCCTGAGGTCTTTAGTAGGGGCGGAGCAGCAGCGTGAGCGGGGCCG TGACCACCTGGGGGTGTGGCTTAAGGCAGGCCCTGAAGGCGTGGGCGGGGCGGGGGATGT GGGCGGAGCACAATCGCATGGGGGCGGGGCTCTGAGGGCTAGAATAGGGGCGGAGCGCGG AGGGGGCGTGGCCATGACCAGTTGGGGCGTTGCTTACGACTGGTCCTGAGGACGCGGGCG GGGTCATGATCGCCTGGGGGCGGGCACTGAGGGCCGGGGGCGGGGCCCGGAGGCGCAGCG GGTTGCCGGCCTGCGGACCTCCTGACATTCCCTGGGTCCTTCTCAAGAATGAACTGGAGC TGGCGGAGACCAAATGCGCCTTGCAGGAGGAGAAGCTGCAGCAGGTGAGGGCAGAAGCGG GTTCTGTTGGAGGAGGGTAGGCTTTCGAGTGTGGATGGGGAAGGGCCTGTCGCCCCGACG CCGCCGAGTCTAACCCGGGTGTCCACACCCAGGACGCGCTGCAGACAGCGGAGGCCTGGG CCATATTCCAGGAGCAGACCGTAGTCCTGCAGGTGCGGCCCCACTCAGACGCCAAGGTGC CTCCCGCCTCTCCTCCCCCAGACCTGGGGCGGTAAGTCTCCCAACCCACCGCCAGGACGC CTCCCCGAGGCCTCAGTCCGCACTCTCACCCGCTCCAGGAGGTGCAGGTGAAGGTGATGG AGGCTGCGGAGGAGCTGGACGCCTGGCAGAGTGGCCGGGAACTGTAAGGGAGTTGGGCCT GCGGGCGCGGCGGGGCACTGTGGGGCCGGGCTGGGCTCCCACCTGCATGCCTGTCCCCGC AGGTGTGACGGGCAGCTTCGCGGAGTGCAGTACAGCACCGAGTCGCTCATGGAGGAGATG GCCAGGGCGGACCGAGTGAGCGCCTGCGCGGGTCCGGGCGGGGTGGGCTGGAGCGGGACA ACCCTCCCCGTCCCCCCCGCGGTACCGCCTCCCCCTCCTCCTGGAAACCGGGCCGGCGCC GCGGGCGCGGAGGTAGCTGGATGCGGCCCTCTCTCCCCGCAGGAGACGCGGCTGTTCGGC GGCCCTCGCGCGCTGGCCATCAGGTGAGCCGGGCGGTGGGCGCGGCCGCGGTCCCCCACC TGCCCGCCTTTCGCCCCGCAGGCGGTGCGTGCTGGGCGCGCTGCAGGTGCTGCTGACGCT GCCGCTCCTCTTCCTGGGGCTGTCGCTGCTCTGGACGGTGCTGTTGGACCCCGGCGCCGT CTCCGCGTGGCTCTGGAGCCTCACCTCGGAGACGACGCTGCGCCGCCTGCGCTACACGCT GTCCCCGCTGCTGGAGCTGCGCGCTAACGGGCTGCTGCCAACCTAAGTGCAGCGCCCCGC GCCTGGCTCCAGGTGGACTCCAGGGCACCTGGCTTTATTTCTGGTGCACTCCTCTCCTGA GAGTGTAGACCAAGGTTGCCTAATAAACTCAAGGGATGAAGCTCGTGGGTTCGTCGTCTG TCTCCCATGTCATGTAGGAGCTTGACTGGCTTTTCAGCCTCCAAAGATTCCTTCCTTCCT TACAGCTCATGGATTTAGAGCCACTCCCCAGTATTGTAAACAGCATTTTTAGTTTTTCAG GATACATAATTTGCCATATTGCCAGAAACTTGTACATAGCATTTGACACTTTGCCAATCT GACAAGCTGCCTTTTCAAGTATTGTGATTTGAAATTATTTCTACTATATTTGAATTTATT TCTACCATTTGTAACATGTTGATCCTGATTTTTCAATGCTCCCCTTTCCTTCTGTTTATT TGGGAAGTCACACTCTGTATTCTGGTATTCTTACTACTTGAGCTCAGCTCATCGCTGTCA TCTTCCCCTAAACCGCCTTAAAGCATCTTAGGTCTTTTCCCTCTGATCACATGTGCTACT TTTTTTTTTCTTTTCTTTGGAGACGGAGTCTTGCTCTGCGCCCAGGATGGAGTGCAGTGG CATGATCTCGGCTCACTGCAAGCTCTGCCTCCCGGGTTCCAGCGATTCTCCTGCCTCAGC CTCCCAAGTAGCTGGGACTTCAGACGCCCGCCATCATGCCCGTTTAATTTTTTGTATTTT TTGTAGAGACAGAGTTTCACTGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGAT CCGCCCACCTTGGCCTCCCCAAAGTGCTGGGATTACAGGCATGAGCCACCGTGCCTGGCC AACATTTTCTCCAGATATCTTACTGAGTCCTAATTTCCACTGTGGCCAGAGAATACACTC TGCATGATTTTAATTCTATCTCATTTACTGAGACTTGTTTTGTGGCCCAGCACAAAGCTT GTAATGAACACTGAGAAGAAAGTGTATTGTTGCTGTTGACAGAGTGATTTTTTTTTTTTG AGACGGAGTCTCGCTCTGTCGCCAGGCTGGAGTACAGTGGTGTGCAATCTCGGCTCACTC CAACCTCCACCTACTAGGTTCAAGTGATTGTCCTGCCTCAGCCTCCTGAGTAGCTGGGAC TACAGATGCATTCCACCATGCCCAGCTAATTTTTGTATTTTTAGCAGAGACAGGGTTTCA CCGTGTTGGCCAGGATGGTCTTGATCTCTTGACATTGTGATCCGCCTGGCTTGGCCTCCC GAAGTGCTGGGATTACAGGCATGAGCCACCGTGCCAGGCCGACAGAGTGTGTTCTAAATA CCCAGCAGGTCCAGGTGAGTGACAGGCTTGTTCAGGGCTCCTGTGTTTACTTTCTTGTTG GTAGCGCCGTCAATTGCTTATTGTGAAATGCCCCTTCATAAACACCCATACACATTTTGA TTATGGATGGCCAGTCTTTTTTCTATCTGTTATTTTCATCTTACATCTGTCTTCATATGT AAAGTGCTTTGGTTATAATCAGCACATAACTGGGTCTCACTTCTTCAATTCATGTTTGTA ATCCCTGACTTTTAAATTGGGCTGGTAAGTCTATTATGTTTCTCCTTTTCTGCCTTCTTT TGGATTAGTATCTTTTAAAACTCCTTTTAACTTATACGCTGACTCATATTTTTTATTAAG ACAGTCTCACTCTGTCATCCAGTGTGGAGTGCAATGGTGTGATCTTGGCTCACTGCAACC TCCACCTCCCGGGCTCAAGCGATCCACCTCAGCTTCCTGAGTAGCTGGGACTACGGGCTT GTGCCACCATGCCCGGATAATTGTTGTATTTTTTGTAGAGATGGGGTCTTGCCATGTTGC TCAGGCTGTTTTCAAACTGCTGGACTCCAGCAATCCACCCACCTCAGCCTCCGAAAGTGC TGGGATTACACATGTGAGCCACTGTGCCCAGCCTATATTTTTATTTGTACTGCTCAGGAC TCAACTGTGCTTCCTAAAGCTGAATATTTGTGTTTTTCAACTCTGGAAATCCCATTAGAA GTTGATTGGGGCCGGGCACGGTGGCTCCAATCTCAGCACTTTGGGAGGCCGAGGCGAGCA GATCACCTGAAGCCAGGAGTTCAAGATCAGCCTGGCCAACATGACGAAACCCCTTCTCTT CTAAAAGTACAAAAATTAATTAGCTGGGTGTGGTGGCGTGCAGCTGAAATCCCAGCTACT TAGGAGGCTGAGGTAGGAGAATTGCTTGAACCTGGGAGACAGGTTGCAGTGAGCAGAGAT CACGCCACTGCACTCCAGCCTGGGCGACAGAGTGAGACTCCGACTAAAAAAAAAAGAAAA AGTTGACTGGAGCTTTTCATTCAACTCTTCTTTTTTTTTTTTTTTTTGACCGTTACCCAG GCTGGACTGCAGTGGTACCATCATAGCTCACTGGAGTCTTGCTCTTCGGCTCCAGTGATC CTATCTTTCTTACCCTCCTGAGTAGCTGGGACTACAGGCATGCACCACCATGTGGCTAAT TTTAAATTTTTTTGTAGAGACAGGGTCTTCCCGTGTTGCTTATGCTGGTGTTGAACTCCT GGACTCAAGCAATCCTCCTGCTTTGGCCTGCTGAAGTGCTGGGATTAGGAGCCACCCCGC TTGGCTTTCCTTTCCTTCACACTGCATGTTTGGTCATTTCACTATTCTGATCACTGATTT TCTCTTCTTACCTGCTGCTGTTTAGCTGATCTTTTGGAGTTTCTCTTTTGTTTCAGGTCT TTATTCCTATCTGCTTCAGATGCAGGGCCTCGGTGGGTGGGAGTAGATGCTTGCTGGGTC TAGTCACACAAAGATTTTGGAGTTCCAGAGCACAGCACTTGAAAACAAGCAAGAGGGAAA GACTAGAGCAGTGGGAAGATGCTGCGGAGAGCCACAGACAGGCCCCCACGGCAGCCCTGA GGGAGACAGTTTTGGAGCGGGCAGACAGCAGTCAGGACAAACACAATGTGTGTTTTGGTG CACCTATGCCGTGGTGAGATTATGGGAATCTCATGTTTGTTAAGCCACAACCCGTGCTCC TGGGCCCCAGCCTATGAGTGCAGCCAACACTGGGCCCCCCTCTCTAGGGGCAAATCCAGG AACTGCCCTTTGGCTGAAGGTGGACTTAGGACTTGACACAAAACCTCACAGATTCCAACA CAGCACTATTTTGGGTTTTTATTTTGTTGATGTTGGTTAAATCTTATCTCTTTTTTTATA CACAATACTTCATGTACCTATGAAATAAAACAGGTAGGGAATATGTCCAGTGCAAACAGA GGACTCACACCTGTGCATAGACAGCACCATCCACTGATTGTCGCTGCAGTCCACGGCGTT ACTAAGCCTGCGCCACCCACGTGCTGCCCCAGGAGGCGCTACCAGGCTCTTCGGGCCACA GGCCTCTCCTCCACTGCATGTGGCGGCAGGGCGGGTAGGTCGCAGGGCTCCATGATTGTG GGGCAGCTTCAAGGGCACATGGGGCAGAGGCCCTCGAAGGTCCCCTCCTCAGTAGGGGAT GTCATTCTGATAGTACTGGATCATGTTGTAGGTCCGGCTCCTGAAAGGCCAAGAAGAGTG AAGGGAGATTCGAGGAGCCAGCAGGGTCTGGGGTCCCTCCCTGCAGGGAGCCCTACCTGT CCAATCAGCCCCTTATGGCTGCCCAGCACCTGAAGGTACAAATGTCCCAGGCGTGCCCTC CCCCACCCAGTGACCACATCTGCTCCAACCCGGGGGACTCTGAGGCCACCTCATGCTCCT CAGCCAAACCGCTTCCATCCGTGGGAGCCACTGCTCTGCCCCATGGGCCTCGGGCCCCTT GGCTCGTCCTGGCCAGATGCCTCCAAGGGGTCTGTACGCTCTCCTCGCACCTCTGGAGAG CCCCTTCTCGCCCTGCCCCAGCCCCTCTTGAGGTCCTGGCCGCCTGGCTTCTCTGCTTCC TATCAGCAAGAGCTCCTTCCGTGCCCAGCCTCCACAGTGCCCTTGTTAGGGTGACGGACC TGGGCGCCACCTGGGGGCAGGTCTCAGCTTCCTCTCACGCTCCTCCGGCAGTTCCCCATG CAGATGGCCACACCTGGGCCCCTCCTCCTAGTCTGGATGTGGTCTGTCCCATGGCACAGC CTTGGGCTCCCACATGCCCCTCAGGGCCTTACCAACCCCACATGTTTAAGTGCTGCCCCC TTCGGGGATGGCTCACTTGCTGGGATCGCCATCCTCCCTTTGTTCAGGCCGGACCCCTCA AAGCCACCTCTGACTCCCACAGCAGGGAGCCTGTCAGCCCGGTGCTCTGCCTTCAGACCC TGAGCCAGCCCTCCCCAGGGCCCCCTGCACCCATCTCCTCCCACCACAGTAGTGACCAGC AGCCCGGCCTGCACCTCCAGTCACTGCCCACGAAGCAGCCCGAGGCAGGCGCAGGGAAGG AGTGGGCTCTGACTCCTCAGCCCTCCTGGGAGGAGGGAGGCCTGACACCACACTCAGTTC TAATACTCCTGGCCTTGTGTCCCTATTGCTCCTTCGGCCTCACGAGCCCTGCCCAGGTGG GCCCGGCCTCTGCCAAGTGTTCCCCTCGGCACACCACAGCCCCCTAAACCAGCACCCCAC TGCTCCTCAAGAGTTCCTGTCAAGACTTGGGTCTTCATGAGAGGGGCGGCTTGGGGACAG CAGAATCCTCATCGCCTGGTGCAGGCAAGGCCCTGCAGGTGCCCAGTGGCCACCGAGGCA GTGGGAGAAGGCAGGGGGGCGGGGCACTCACCTGTTGCTGAGGAAGCAGCTCTGGATGAC CTTCATGATGAAATTTGCAGCCTCGCGCTCAGTCATGTTGGGGCTAAACCTGTGCCTGGA GAAGAGGCTGTGTCAGGGCTGCCATGGGCAGGGCCGTGCTGGCTCCCTGGCCCAGTGGGA GGAGGGTCTTCCATGGGGACGGACTTCAGCTGAGAGCCATGCCCTGGAAATGTACCTTTG GGGTCCACATGTTGGAAGATGGGGTGCTGTGAAGGCCACACCTGGCCTATCATGGGCCCT GTCCCCTTCCCAGCATCACCTGAGTGGCCCCATGGCATTAGGGGACTAAGCATTGGGGAG CTAAGCTACTGCAGCCCCAGACCTTAGGGTGGAGGTGGGGTGGGCGTAGCATCCTTGACA TAAATAGAGGCCCCTGGGTGGGTCTCTGGTGTGGCCGGCACAAGCAGGGGCCCCTCACAG TTGTGGTCTAGGGGTAGAGCCTCACCTAGGAACCCTGTCTGCTCTGAGGTTCCAAGGAGA TGACAACCACAGTGACAATTACATGAAAGGTACCTATCTTGGATGGAGCCTCAGCTAATG GACAACTGTCCCCCAGATGGCCTGCGTGTCCACCAAGGAACCTACTTCAAGAACTTGATT GTCTGGCCGCGAAAACAGGGCAGGCCCGTGTCCAACATGATAGTGACCAGGGAGACGACC ACATCCATGTAGGGCCTGGGGAGAGACAGGAGGGAGCGGTGGGCAGAGGCCAGCCTAGGT GGTGGCCCTGCCTGTAGTCCTGTGGACTGGCTGATGCCAACAGCCTCAGGTGTGGGCTCC TGCCACCCACCTCGCCTGCCACATCTTGCACATCCCCGAGGCAACTTTCGATCTGCTGCA CTCGGTCACCCGTACTGCCCAGGCAAGGGCTGCCCATACGCACTCTGGACAGGCTGAGTG TCCTGCCCTGTCCCCCACATAAGGCTGCCGGCCATGGCTTCTGCACCTGGGTGGGATGCA GACACGCTGACCTGCCTTTCTCTGCGGGGCAGTGGGGATGAACCCAGGTTGGACTGTGGC CTTGGCCAAGTGACCTGTATATGAAACTGGGACAAAGCCCATCTTTGGCACGTAGCCTGT GGGGTGGCAGGTGCTCAGGCTTTGGTGACAGGGTGGATGGGATGCCCAGAAAGGGAGAGC CCATGGCTGAAGGCGTGGGCAGGATTGTGGGGAAGGTGGTTGGAATTAGATGCCCAGAGC AAGAATTTACTGGCACAGGTGGGCAGACAGAGGTGACCAAAGGACAGGTGTAGGTCAGCA GGTGGCTGCTAGCACCTACCTCACTCTCTGGAACCGGATTCCCTTCATCCTAAAGGGGAT CTCAGAACGTTCCACACACCCCCTCCGCCTCCACCCTGGTCCTCACCCAGGCTCACCGCA CAGCCAGGTAGCCTGGACACACATCTCCATGAACCACTTGAAGGGTGTGGCCTCCATCTT GCCCCCCATGATCATCACCATCTCATCCGTCAGCTTGATGTCGGGTTCCCAGCCGAGATT GCCGCCCGGCGAGCTTTCAAACATGAAGCCAAAGTCTGCAAAACCCCAAAGAGCTGCCTG TGACTGGGTAGGAGCCAGGGCGGGCAAGGACGAGTGGTCTGTTTTGAGGAGTGGAAAAGG ACTCTGCAACAGGAGCACCCCCTCCACCCCCAAAAGGCAGGTTGTGTTTTCTTGGAGACA GTGATGGGGTGGGTGGTGGGGCAGCAGGCAGAGAAAGAGAAGGGAGGAAGTGGAGGAAGG AGCCAAGCTGGGGCACTGAACCTGGACGAGCCCCACTCCGCCCAGCTCCAGCTTCTGACT CAGAGCAATGGCGGCTCTCGCCCCAGCTCCCTGGGGCCGGGGCCAGGCACCCTCTACAGC AGAACAGCTTGGTGGCCGACAGTTCGGACCTCAGAGCTGGACCCTGACACTCCTGGCAGG GTGGTCCTGGGCATTCTCCTCTCTGTGGGGTGGGGATCCCTATCCACCCCTGGGTGCCGG GGTGAAGGGAGAGGAGGGTGGCGCTGTGGCTGGCTGACCGATGTGGATGATATGGCCCTT CTTGTCCAGCATAATGTTGCCGTTGTGTCTGTCCTTGATCTGCAGCAGGAACAGCAGGAG GCTGTAGGCGGCCATGCTTCGGATGAAGTTGTAGCAGGCCTGTGCAGAGAGCGCCCTGGG CTCAAAAAGGCCCTGGGGCCTGTGGGCATTCTCCCTGGTCCCACACCCAGGATCCCTGGG CCTGTGGGCACTCTCCCTGGCTCTGTGCCCCCACTATGGAGGCAGAGCCCGAATCAGCAA GTCAGTCTTCGGCAGCAGGAGTGACGGGCTGTCTGGATGTGGGGGTGCAGGCACTTCCTC CCACACTCAGAACTTAACTTTCTTCTAAGGAGTCCAGCCCAGCTCTACATTCTTTTGACT CCCAAAGTGGCTTACAGATGCCCTGGTGTTTTTTTTTTTAAATGGAGTCTCGCTCTGTCG CCAGGCTGGAGTGCAGTGGCATGATCTTGGCTCACTGCAACCTCTGCCTCCTGGGTTCAA GAGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGCCCACCACCATGCCC AGTTAATTTTTTTACTTTTAGTAGAGATGGGGTTTCACCATGTTGGTCGGGATGGTCTCG ATCTCTTGACCTCATGATCTGCCCGCCTTGGCCTCCCAAAGTGCTGGCATTACAGGCGTG AGCACTGCGCCCGGCCAGGTGCCCTGGTTTTTTTTTTTTTTTTTTTTTTCAGATGGAGTC TCACTCTGTTGCCCAGGCTGGAGTGCAGTGGTGCAATCTCGGCTCACAGCAACCTCTGCC TCCTGGGTTCAAGCGATTCTCCTGCCTCAGCTTCCTGAGTAGCTGGGACTGCAGGCGCGT GCCACTATGCCCAGCTAATTTTTGTATTTTTAGTAGAGACAGCGTTCACCATTTTGGCCA GGATGGTTTTGATCTCTTGACCTCGTGATCTGCCCGCCTCGGCCTCCCAAAGTGCTAGGA TTACAGGCGTGAGCCACCGTGCCCAGCCCAGGTGCCCTGGTTTTAACCCTTAACAAAGGA TGACTGAGGAGAGCAGGGTGTGGGTAGAGGCTGGGTATGGGTGGCTGGGGTAGGGTGGAG CGCACACGACTTTCCCCGGCCTGTGGCCACCCTGGCTACCTGCTGGAAGGCCAGAGTGGA CTCATCCCCGTACTGGCGTGTGAAGTAGTCGTACATGCCAAAGTCTGTCTGGCGGCCCAG CTGGTCCCGGGAGGTGCAGTCGGGGATGCACTCGATCACCCCGCACTAGGAGGAAAGGCC AGTTCTGAGGCCCGCCGGGTGCGAGGTGCCCAGGGCTGCCCTACTGGCTCCACTCAGGGA ACTTACCCCAGGGGCAGTGGCCACCACGCGGTAGGGAAAAACAAAGAGGTCCAGGCCGAC CAGCTGGAAGATGTTCTTGAAGAGGTCGATGATCTGCAGGGCCAGCATGTCCTGGGAAGC CGGGAGGCGCAGGATGCGGTCAGTTGGCGTCCTTGCACCCCAGTAGCTCTTCTGGCTCAT GCAGGGCAAAAGCCGAGAACCCAGAGATGGAGTGAGGTGGGGAGACCACAGCCGAGCAAG AGTCTGGAAGGCCTTCCTTTCTGACCACCGGGGGCTGGACTCAGGCTGCTGGGACCACCA GGCCCTGGGCTGAGCATGAGGCTGAGCTGTCTGGTGGGGTGTGTGAGATAAGGCACCCAA CCCCTGGACAGTTCCCTCAGGTGGGCACTGGCATGGCAGGGACAGTGGGAAAGGGGCTGG AAGGAGAGGCCTCTGGGTTTTGCAGAAGCCCTAATTTACCCCGTGGCACCTGAACCATGT AAGAGAAGGAAAAGATTCACATTTCTGCATATGAGATTGGACTCTGGTGGGCCTGGAGCC TTGGGGAGCAGCAAGCCCAGTCCCCAAGCCTACTTGAGGCCTGACCCTGCTTACCTGCCG GCAGTCGTCTCCCAGTTTGAAGATGGCTGCCTGCCAGGAGATCTTCTGGCCGTCGGCCTC CTGCGTGCTGCACTCATCCTCAGAGTCTGAGCGGCACCGCAGACCTGCCCGCAGGGAGAG AGGCCACTGTTAGCCTGTAGGCAGTGAGAAGCCCTCTGAGGGGGCCAGGGATGAGTCCTC TCACATGCCTGAAAGGAACCCCAGCAATCTTGGGGATCACTTCTTCAAAACCCCAAGGAG GCCAAGGAGGAGCCCAGCAGGGCCCAAGGAAAGCCTTGGAACAGCAGGTGGAATCCAGGG TTGGAGTCTAAGAACATTTAGGCTTAAATGGAACTAAAAACAGAGCCCCATTTCATCTGC AGTGAAGACCCAGGTGTGCCTGAGTCAGTGTTGGGTGCTGTGAGCTGGGGGGCAGGAGGA ATGTGTCTGTACACTGGGGTCCTGGGAGGGCTCGGGGCCAGTGTGCAGACATGGCCATGG GAGGCAGGTCCCTGCAAGGGGTGGCAGCCCCCTCTCCACAGCTGGCCACACCGTCTGTAC TGGGTTGAAGAGTGTCTCCCTAAAATTCATGTTCACCCCAAACCTTAGAATGTGTCCTTA TTTGGAAACAGGGTCTTTGCAGATGTAATTAGGTTAAGACAAGGTCACAGTGGATTAGGA CAGTCCCTAATCCAATGACTGATGTCCTTCAAGGGAGATCATCATGTGAAGAGGGAGGCG GAGATGGGAGTGGAGCATCTGCAGGCCAAGGTGAGCCCAGGACTGCTGGCAACACCACCA GAAGCTGAGGAGGCAAGGTGTGACCTGCCCAGAGCCTTCAGAGGGGGCACGACCCTGATG ATGGACTCCAGCCTCCAGAACTGAGATCGTCCGTACAGTAGCCCCAGGAAACTAACACCC CCCAACGGCCCCGGGCCCCTCAGCCTCAGGAAGGAGCCTGCTGGAGCATGCCAGCTGACT CATGGCAGACAGACCTCTGGGGTGGCACATCTGTCAGTGCATTTGAGCTCCCTGCCATCC CCTAGCGAGGGGTCTGACTGAGGGCAGACAGATGGACAGACATCATCTTTTATGGAGAGC TGTACTAAATTTAACACATGCCAGAAGAAACTGGCATTAGCAAGGAAAAGCACTGAGCTC CCAAACAAAATCAGGGTTCTTTACTGACCTTCTTTTTCAAGTTCACTAACTCCACATCGC TTCACCTTGAACTTGGCCAGATATGGGGCTTTTGCAGCACTGAAAACAACAAAAAGAATG TGGCACCCATGATGCAGCCGAGAAAAACTTCACACGCGGACGCTGTGGAAGCGGGGGATG GGTAGGGTGAGGCGCTCACCTCTACATCGGGGTCCCAGACTTGTAGTCGACGTCCAGCAC AATGGCTTCAGGGTTGCTGGGCAGGGAGCAGCCTGTGCAGGGACAGAGGCAGTCACAGGG AGTGCATGTGTCACCACAGGTGAGCCAGAGTCAGTTTCCAACACGGATGTTACGTGCGTG TCCACCCTGCAGGCACACCCCACCCCTGTGGAGGGGCCTCTGGTCCTTGTCCAGGGCACC ACTAACTTATTATTCTGTGGCCGCTAAGGGTCCGTCCTCCTTGACTCTTCGACTCTTCCA ACCATCGACCAAATGAGTCTGCACCTTCTGGGAAATGCTCTTCCTAGAGGAGCACTGAGC CCCATCCTCACGGATACAGCCCTACTGCTTCTGGGGGTGTGTGTGAGGCCTGTCCTGCCT CCACCTGCCTGAGCAACAGCCTCTGAACAGCCCTCCATTCTTGGAGTCCCTGTTTTTTTT TTTTTTTTTTTTTTTTTGGATAGAGTCTCACTCTATCACCCAGGCTGGAGTGCAGTGGTG CGATCTTGGCTCACTGCAACCTCCGCCTCCCAGGTTCAAGCAATTCTTCTGCCTCAGCCT CCTGAGTAGCTGGGATTATAGGTGCCTGCCACCATGCCTGGCTAATTTTTATATTTTTAG TAGAGATGGAGTTTCGCCATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAAGTGATC CACCCGTCTTGGTCTCCCAAGTGCTGGGATTACAGGTGTGAGCCACCACGCCCGGCCTTC CTTACCCATTTGAACACTACTCTCTCAAAAGCCTTACTTAAATGTCACTATGTCCATGAA CTGTCCTTGGTTCTCCCAGCCTTTAGGACTTTTGTCTAACACGTTTCTTTTTTTCTTTTT CTTTTTGAGACGGAGTCCTACTCTGTCGCCCAGGCTGGAGTGCAGTGGCTCGGTCTCGGC TCACTGCAACCTACGCCTCCCAGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGC TGGAATTACAGGCGCCCGCCACCGCGCCTGGCTAATTTGTGTACTTTTAGTTGAGATGGG GTTTCACCAAGTTGGCCAGGCTGGTCTGGAACTCCTTACCTCAGGTGATCCACCCGCCTT GGCCTCCCAAAGTGCTGGGATTAAGGCATGAGCCACCGTGCCAGGCCAACATTGTTTCTT TATGCTTATTTTATATCTCTGTGAGTTCAAGCTTCTTCAGCTCAGGGACCATGTCTTACT CATCTTTTTAACAGCCACAATACCCAATGTCATGCCTTTTCTTTTCTTGTTTTTGCTTTT TTGAGACAGAGTCTCACTCTGTCACCCAGGCTGGAGTTCAGTGGTGCGATCTCGGCTCAC TGCAACCTCCACCTCCTGGGTTCAAGTGATTCTTGTTCTCAGCCTCCCAAGTAGCTGGGA TTACAGGCACGCGTCACCATGCCTGGTTAATTTTTTTTTTTTGAGACCGAGTGTCGCACT GTCATCCAGGCTGGAGTGCAGTGGCGCAATCTTGGCTCACTGCAAGCTCTGCCTCCCGGG TTCACGCCATTCTCCTGCCTCAGCCTCTCGAGTAGCTGGGACTACAGGCGCCCGCCACCA CACCCGGCTGATTTTTTGTATTTTTGGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATG GTCTTGATCTCCTGACCTCAGGTGATCCACCTGCCTTGACCTCCCAAAGTGCTGGGATTA CAGGCATGAGCCACCGCGCCCGGCCCAGGCTCCCCTTTCTCAGTGAGTGCTCAGTAAATG TGTGAAATAAGAGGACAAGGGAAAGCCTGTTTCTGGCCAAGGAACTGCCAGTCCCCAAGG GGGATGTGTGCTCCTTGAGCCCTGAGAGGTGGGCTTTGAGGGGAGCCAAGCTTGGCCTTG CTGTGGGGTACAGGGAGAGGGGGGCATGCCATCTCCTCCTCTTCCTCACCTGCCCTTCTG CCTGCTCCAGGCTGTGGCTCACACTCAGGCCCCTCTGTGCTCTCCTGCTAGAGCCCTGCT GGCTTCCCTGACCTCTGGGGCAAGACTCAGCCAACACAATCTAGAGCCAGGGGCTGGGGA CTTCTGTGCATGCCCCCTGCAGTACAATGCTTCCAGCTCTTTTTGCCCCTCCCCAGCAGT TGTGGCCTTCCCAATGCGGGAACAGAGGCCCTGCATACATGTCCTGCCCTCTCTGGGAAG CCGTGCTGTTGTGTGAGCTCAATACAATGTGTTGGGAAGAAATGGTTAAACCGGGGTCCT GCAGGGCACTCAAGGACCAGCCGCTGTGAACATCTGCACGGGACAGACAGAGTTTGGAAG ACAAGTTTGTGCCAATAGTTAGAAATGCAGAGAGAGGCCGGGCACAGTGGCTCATGCCTG TAATCCTAGCACTTTGGGAGGCCGAGGCGAGAGGATCACAAGGTCAGGAGATTGAGGCCA TCCTGGCTAACACGGTGAAACCCTGACTCTACTAAAAATACAAAAAAATTAGCCGGGTGT GGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCGACAGAGCAAGACTCCA TCGCAAAAAAAAAAAAAAAGAAATGGAGAGAGAGCTGCCCAGTGAAGTCTGGATTTCCAT CTTCTCTTGAAAAGCTCGGCTTGAATTCCCATGTGGCCACAGCCACAGGCACCCAGGAGC GACTTCCCATGGAGGCGGTGAGGGGGTGGTGCTGATGGGTCCCAGCACACCTGAGCCCTC CCACACTGGGCCCGCCATCCCCTTGTCTGTGTGACCTGCTCCTGTAATGCTGGAGCCTAG GAACCCTCTTCTAGAGTAACAGCTACTGAGGTCAGTGTGAGCCTCCAGAAAAACAGCAGG CAGCAAGGAAGATGAAGGGCACCCACTGACGAGCAGCTCCCGCTCACTAGCGGCAGATTT TGGGGAGGGGCTGGGGTCCTGGGGCACCAGGCACCGTGGGCCCAAGCAGGTGTGGGGCTG GCCAAGCCACTGTGTCCACATCCTGCAGTGCCTGGAGAGGGCAGCAGCCTTCACGCCCCG CCGCCCGCCTGCAGGAAGAGGTTGGTCTGAGCCTCCAGAAGCCACCTGCTCACCTGGCTG CACCGTCACTTCAGACAGGGCCGACAGACAAGCCTTCTTTCTCTGGTCGCCTTTAGGGTA GGGCCTGAGAAACAGGAAAGAAATTCTTGCTTTGTCTCCTGGAGGAACTGGCTACTGGGG GACAGCCCAGGGCCCCTCCTGCCTGGATTGCCCTCTCTGTCCCTCTCTTCCCGGCTCTAG CTGCTCCTAGCGCAGCCAGGCACCACTGGCAGCGATTCTTGCCCTGCATTCCTCATGGAG AATCCCCGACAGCCCCGGAAGGGCTGTTTTCTGGCAGGCTCTGCCCCTGCTGTGTGCGGG GGTGTGTGGCTGGGGGTGGTGGGGGTAATAATGCCTGCCACCTGCTGCCACCCAGGAAAC GCTAGCTCCCGTCCTTCATCTCTTGGGTTAGAAGCTGCCAGCACTGCTATTCTCAGCACC CAGCCCAGAAAAGGGCCTCAAAGTGACCAAGGTCAGAGCAGTGTACGCAATGGAGACAGC CCAGCCGGCGTGCCCAGCATTGATGACTCCAGGGTTTCATCCCTTATGAATGCACAAAGC CACCAACATCCAAGGATGGTGTCAGGTCCACATGAATGGAAGGTCTAGGAAAACGATGAC AGGAGCTGCAAAGGCAGTCTGGTTTTCCTGACACCAAGAGGATCGATCCTTCAGAGTGTG GAGAGGGCAGGGTGACCTCAAATTGTGGAGCCATGTGCTAAATTCCACAAATCTCAGGAA TGAAACTAAGCTGTGTTTCCTGAAACTGGCGTGGCCGTGCATTCTGCCATACCTTCAAGA GCCCTGACTCACCCCAAGCAGTGCTTCCCCACCCACCCTGGCTGTCCATCAGAACCACCA GCAGCCCCGCTGCTAGAGACTCTCATTCATCTGGTTGGGGCCACGCACGGGTATTTTTAA AGCTTCCCAGGCAATTCTGTTAAGCTGCAGGGACTGGAACAAGATTCCAGAAGGTGGTTT CCGAAGCACTGCACTTACTTGATGACAGCCGACACGTTGGTGATCTTGTTAAAGAAATCA AACTCCCGCTGGTAAAAGTCCTTCGCTGGGCCCGACAAGGAGCCTGTGTTCTCCTCTACT AACTGCTCCCGGAGGTCGCCGATGTCAGCTGCCAAGGAAACAAAGAGGCTGAGTCTCTGT GGCTGTGGCAGAGGCCCCTCAGGAATACCAGCCCTGTTTCCCAGGCCCCAGACTGGCGGT GCCCAGAGTATGCTCTGCAGGCTTGGTGAGACCATAACAGCTGCTGTGTGCCCCTTTTGC TGTGTTGACACAGCCGACACTGCTGGGAAGCTAAGCTGGTGCCTAAGCGGAGCTCAAGGT AGGGGCCAAGCGCACAAGTGGACGTGGAATCCTCACTGCTGCACACGCTACACACGCAGG AAGAAAAAAGCCCGTGTCATTTATGAATGTCACAGGGCATAGAATCCTCACTGCTGCACA CGCAGGGAGAAAAAAGCCCGTGTCATTTATGAATGTCACAGGTGAAGCAATGAAAACTAA TTCTTATTAAACTCTACCCTGAATACATGTCTTTTTTTTTTTTTTTAATAGAGGCACACA GTGGGCGGGGCTCTATATTAAATAGTGTCTCTCACTATTCAAATCCCAGGCTGAATTTAA ATTCCTGGGCTCAAGAGATCCTCCTGTCTCAGTCACCTGAGTTGCTGGGACTACAGCTAG TAGGTGGGACTACAGGTGCGCATCACCACATCTGGCTTTTAAAGTATCCTTTGTGATGAA ACGGGATGTGCACGAAGCACTCTTGCCGCATCTAAGGCAGGCGCTGTCCTGAGAAGCACC TGGAGCTTGTCTGATTTTGGAGCCTGGACTAACCTTTTTCACAGACCTTTGTTTTTACTT GAAGAAATGACTGACAGACAACATATAGCTATTCAGACTTAGGGATGTGGCAGATGTTTT CCTGAAAATGAACAAAAAATAAATCTTCCACTCAAAGGAACTGAAACATTTTTTTGGAAA ACTTGGATCTGCTGCTGTGAGCTCAATAGCTTCCCTTAAAAACTCTTTCTGGGCCGGGCG CGGTGGCTCATGCCTGTAATCTCAGCACTTCAGGAGGCTGAGGCGGGCGGATCAGAAGGT CTCAGCCAGGCAATGTGGCCCACACCTGTAATCCCAGCACTTTAGGAGGCCGAGGTGGGC GGATCAGAAGGTCTCAGCCAGGCAATGTGGCCCACACCTGTAATCCCAGCACTTTAGGAG GCTGAGGCGGGCGGATCAGAAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAG CCCCGTCTCTACTAAAAAATAAAAAAATTGGCCGGGCACGGTGGCTCATGCCTGTAATCC TGGCAATTTGGGAGGCTGAGGTGGGCGGATCACGAGGTCAGGAGATTGAGACCATCCTGG CTAACACAGTGAAACCCCATCTCTACTAAAAATACAAAAAATTAGCCGGGTGTGGTAGCG AGCGCCTGTAGTCTCAGCTACTCGGGAGGCTGAAGCAGGAGAATGGTGTGAACCCGGGAG GCGGAGCTTGCAGTGAGCGGAGACTGCGCCACTGCATTCCAGCCTGGGCGACAGAGCGAG ACTCCGTCTCAAAAAAAAAAAATTTAAAAAAAAGTCCGGGTGCGGTGGCTCACGCCTGTA ATACCAGCACTTTGGGAGGCCAAGGCGGGCGGATCACGAGGTCAGGAGGTCGAGACCATC CTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAAAATACAAAAAATTAGGCGGGCGTG GTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCATGTACC CGGGAGGCAGAGGTTGCAGTGAGCCGAGATTGTGCCACTGCACTCCAGCCTGGGCGACAG AGTGAGACTGTCTCTCAAAAAAAAAAGAAAGCCCTCTCTCGTCTCTCAGTCTGTGCTTAG TAATCAGCTAGGCGCTGAGAGAACAGGGCATGGCCTCCTTACAGAGGTTTGCTGCTTCTT TGCGTGGCATGTTTTCATTTGGCATTCCCTTCCCTGGAATGCTCTGCCCTGTCCACCCTG CATGCCGGTGCAGCCTTTCAGACTCAGCTCAACTGTCTCTTTTCCAGAAAACCATCTCTG ATTCCCCAACTCTGGATGGGCCAAGTGTCTGTACCGGAGGACTGCTCCCACAGTTCCCTC AAATTGAAATCACCTGTCCATTTGACTGCCTCCTACAAGACAACAACAGACCATACCCCA GTCATGTAGGTGTGCACCAGCCTCAGCCCAATCCTGGGCCGTCAGCTGCTCAGAGGGGTG TGTGAACGCAGCTGGTGCCAGTGCAGGGAAAGTGCGGTGAGTGGAGTCTGCTCAGCTGCC TCGGGTGCGTGTTTATGCTTCTCAGCTCAGGCCAGAGGAGAAGCAGAGCCAGGCATGCAC CGGCCTCCAATGACCGTGCAACACAGTCAAGGCCAGGGCTCAGGGGGGTCTAACTGCTTT TCCGTCCCTACCTCTGGCACCCTCAGGGCTGCCCACGTTGCACCCCTGTCCTGCCTGCCT CCACCATGAGCAGCTGCACTGTTGAGGGTTACCTGGGGGAACAAGCTGGCTGGCGGTCAG GTACTTCTTATCTGAGAACATGGCGGTCCAAAATTTAATCATGATGCTTATGTCTTCACG CAGCCGCTTCTCTCCTTGAGTAGGAAACTTTGGGGGACAGCTTCAAACAACCATAAGAGG ACAACGGCTGAGTGTCTGGAGTCAGGGACTAGAGGCCAGTCACACAAAGCAGTGAGAAGG TGATGCAGACAATTTCTTAATGACAAAAGGTCAGCCCAAAGCCAGTCATTAGCAAGGATG ATCTGGCCCATTCCCATGGGAAGGTGTGGTGGCTCGGGAGCCCTCTTATGCCCTCCTCCC TCACTGCCCACAGCAGCCCCTACAGCTTTCCACTGAAGCTGCACTTGTGGCAGATCAGGG CAAGGAAGCTGCACTCACACAACAGCACAGCCTATGGGTGCAGAGCCCCAGGCAGTGGGT GTTTGCAATTCTTCGTGCAATAAGCCTTCACATTAACAGGGAAATTAAATGGACGCACTT AATCTCCTAAATCAGTTGAACTGAGGGAAAATTTGCCTACATCTCGCTGGGAGGCTAGCT CTAAGTACACGGGACACATGCACAGGGCTGAAGGGTATTTAAATCCACAGGGCCAGGGAT ACTGAAGGAGGAGGTTGGTGAGCACAACTGGGCAACTGTGTATTCCTGTGAGTGACTTCC TGGGGGTCTGGGGCGATGGAGCACTTGGAGGGGCTGGGCCGTCCCTGGATGCCGCACAGC ACATCTTGGGGGAGTACCTGAAGTAGTCAAAGGCAGTGGAGTAGATCTTCTCGCGAAGCA CATTGCGGATGGTTGCATTTGGAACCACATCGGCATGCAGGAGGGACAGCCCCAGGGTCA GCAGCCTGTGAGGGAGCCCCGGACCCGGTCAGAGCAGGAGCCTGGCCTGGGGCCAAGTTC ACCTTATGGACTCTCTTCCCTGCCCTTCCAGGAGCAGCTCACTGAAATGTGTTCCCCGTC TACAGAAGTACCGTGATACACAGACGCCCCATGACACACTGTACACACCAGGGGCCCTGT GCTCCCCAGGAAGAGGGCCCTCACTTGAAGCGGGGCCCGATGGCCGCCACATGCCGGTTC ATGCTCCCCTTGGCCCCACCGATGTTCAGGGACATGGAGCGCTGCAGCAGGCTGGAGAAG ATCTCCACTTGGTCAGAGCTGCAGTACTTGGCGATCTCAAACCGCTGCACCAGGAACTGA GCGAAAAGAGGAAGACGCTGTGGTGGTGGGGCTGAGCCGGGCTAACCCTGGGGCCGGCAG AGACAGCTGAGGAGGGGTTCTGGGCTGACTGGCCGAGCTTGCCGAAGGCCTTGGGCTCGG CTACCCCCACCCCAGGAACAGTCCTGGCCCCCCAGGTGGAGCACCCCACAGCCTTGGTCG GGGAACAGGCACGTACGTCGATCCAGATGTAGTGGGGGGTCACTTCGGGGGGACAGGGTT TGGGTTGACTTGCTTCCGAGGCAGCCAGGGGGTCTGCTTCCTTTATCTCAGCAGAAAACA GGCCAAATTTCTGCTCCACTGTCATGTGCCAGCCCCTGCCATCTCCCGCATGAACTACAG GTACAGAAGGAATGTTAGCTCCTCTGTGAAACACAAAAAGGGGCTACTGTCAAGTTTTCT CTAATTAAAAAAAGGAACACGTGCTCACTGAATAAGATTTAGAAACCAAGAAAAGTATGA AGAAAAAAATGAAGATCGCCTATAGTCCCACATCCCAGGGTCATCTGTGTCAGTGCTTGG TGTATTTTCTCCAGCCTCTCGAGGCTGCATTCTGTTCAACCCGGTTCACCGTGTGGACAT TGCAGGTGTGAGGTGGCAGCACCCGGGATGTACCTGATGCCCTGACAGGCACTGGGTAGG TGACTCTTCTCTATAACCCTGATAACCCTCTGTGGCTGTGAGCGGGAAGACCCCTCTGTG GGCTGCTGAGCTGGCAAGGGCAGAAGAGGGTTCTGGATCCAGGGCCTGGCTCCTCCTCTC CACCCCACACAGACCCTACCCGATACCTTCATCCCGCTAACCAGATTAGGAAGGCCCATG GAGCCGAGGCGAGCCTGCAGCCACCATGGCCAGGCCTGGGAGCCCAGGGTCAGGCTGCTC CCATGGCCCAGTCATGCTCAGCCAGGTCCCCATTCCGTGGGGGGGCATGAGGGGTGCCCA GAGGGAGCAGCATGGTGCCCGGGAGACACCTGGGTGCTGCAGAGAGTAAGAGGTGCAGCT CACACATGGAAAGCCCCCATGATTTCTGGGTGTTTTTACAAGGAGACCAACAAACAGCAT TTGGCATCTGGCCAAAGAGGATTCTATCCCTCTGTTGTCCCAGCAGCACCAGCTTCTACA AGGGCTCCAAGCGCCTGTGCAGAGGTGGCAACGAGGCCTAGTGAGTAGAACACACACCGC GGCTCCCTCCCCAGTACCCATCCCCTCCCTTCTTCCTCATGAGGTGGATCAGGGTGGCCA CGTGGCCAGTCACAGGGCTCTCAGCCAGGGTCCCTGCTGGCTGAGGCGGCCACTGTTCTC CCCGGCCACTCTAATGAGCCAGGGAGAGTGAACTGGGCAAGGCCCTGGGAACACATGCCA GGGTGAGCTTCATAAGGTCAAGGCAGCTGGCCTCTGCCCTTTGCACTCTGTCCTTGTCTT CCTTCCTGCTGGGAGGCACAGAAGCCATCTGGAGGCTGTGAGGGTGGAGACAAAGGCCAC ATAAGCCCAGGATGGCCAGGAGCCGACAGGAGCCTGGGGCGGGGCTCTGTCTGCTGCTTC CCTCTAAGCTGCCCATGGGGTGAGGACTCCTGGGGGACGAGGCACTACAGCCTGCTTTGT GTTTCCTGCAGACCGATTAGTCTTATGGAAGCAGACAAGGGAGCAAAAAAATGGCGGTGG GGGGGACCCCAGAACTGCCACGGACAAGAAGCCATCGAAGAGACACACTCCCATCTATTT GTTAAACAACCACATCCTGGTGGCTGCCTCTGTGCCAGGGCCTCGTGGACTCTGGGCATG GTGCTCTGAATGAAGACAAACATGACCACACAGTCTATGGTGGGGACCACAGACAACAAG AAATAAGCTACACACACTGAGAAACACTAAGAAACAGCAGTGCGAGTGAGCGTGTGGCCA GGTGGCCAGGGAGAGCCCTGGCAAGGAGGGGACATCTGAGATGAGCCCTGGAGGAGACGC AGGAGTGAGGCGCAAGGCACGGCCATCTGAGAAGAGGGAATGCATGACGCGGCCTATGGT GGGAGCAGCCCAGGAGGGCAGCTTCAAAAGTGAAACTGATGGGATGTGCTGAGAATCATC CGTGGGAAGTGCAAGCAGGACCCCAGCATGGCCACAGCACAGCAAGCGAGGGGAAGAGAG AGGATGGAAGTCAGGTGGGAAGGGGGCTTGCAGGTCACAGAAACAAGCCCACCTTCGAGT CTGTAGGGGGGCTCTGATTCCCCCCTGGCTTGCGCCAACCGCATGTGGAGAAGGGGAAGA TGCCTGGGGTTGGGGAGCAGGAGGCCAGGCCAGGGGGCAGCAGCTGGGATGCTGGGACTT GTCAGATCCTGGATAAACCTCAAAGGTGAAGGTGATGGGATGTGCTGAGAGTCATCAGTG GGATGTGCATAAGAGGGAGTCAAAGATGTCTGAGCAGAGAGAAGGACCTCGCTGGCATCT ACAGTGAAGGGAGGGACCTCGCTGGCATCTACAGTGAATGGAGGGACCTCGCTGGCATCT ATAGTGAAGGGAAGGGTCACGCTGGCATCTACAGTAAAGGGAGGGACCTAGCTGGCATCT ACAGTGACGGGAGGGACCTCGCTCACATCTACAGTAAAGGGAGGGGCCACACTGGCATCT ACAGTAAAGGGAGGGGCCCCACTGGCATCAACAGTGAAGGGAAGAACCACCCTGGCATCA ACAGTGATGGCGGATGGGCGACCAGTGCAGCGGTGTCTGGAGCTGAGGTCGGACCCATTA GTTGGGGCTGCTAATCAGTTCACCATGCAGGCCGTGGGCCAAGCCACTGAAGGGATCCAC CTGGACTAGGAGTGACTGGGCTGGGTAAGCAGTGGAGCTGGCCAGAGTAGCCAGGCAGGG AGGAGGAGCCCTGGGGCATGCCAGTACTGAGAAGGCCCTGAGCTCAAGAAGGTCCCTTTA TTCTGGAGGATGAAAGGGCTACAAAAATTACAAAGGCCTTAAGTCCCTGGGAAAAGCAGT TAGGCTCCCAAAACAGCTGCTTTCAAGACCCAGAACTCTGAAGGAGGCTGTGAGCAGCTT TCCCTGAGCTGATCTGTCCTCCTACCATCTGGGACCTGCTAGTGATATCTGTACTCCAAC TGCCCTGCCCCCAGGGGTCAACCAACCCACTCCTATCTGAGGACCCACTCCTATCTGAGG ACCAGACAGAGAGGCCAGAGGTTCAAGGGCACCTGGACTAAGGAAGTGGAGAGGAGAGAG ACAGATCTGGGCTCCAGAGCCCCAGGTACAATGTGAGGGCTGACACGTGGTCAGGAGCAC CCCCAGTGCCCTGTGAAATGGGAACGGCTACAGCCCCTACTCTTTTTCTTTTTTTTTTGA GACGGAGTTTCGCTCTTGCTGCCCAGGCTGGAGTGCAATGGTGCGATCTTGGCTCACCGC AACCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCTAAGTAGCTGGGATT ACAGGCACATGCCACCACACCCGGCTAATTTTGTATATTTAGTAGAGACAGGGTTTCTCC ATATTGGTCAGGCTGGTCTCGATCTCCCGACCTCAGGTGACCCACCCGCCTCTGCCTCCC AAAGTGCTGGGATTACAGGCGTGAGCCACTGCGCCTGGCTGAGCAGCCCCTACTCTTAAG GCCCACCGAGGTCTGAGGCTGAAGCAGCATATGGAAAGGAATCCTGACCATAGCAGCACT GTGTCCTGACAGTCACAATTTGTGGTGAAACCAGGGCTTTCTGGTGACCCCAGAAAACAT CAGTGCCGGCTCTGCCAGCACCCAGACACCTGGTACCCCGAGACTGGCAAGGGGACTGGA AGAGGGCAGTGGCCTCCAGGCTCCCTCCACACTGCCCAGTGCTGTGTGCACACAAAGGAC CACGGGTCAGACAGATAGAGGCCGAGCTGTGGGGCTGTGTGCCACCCAGAAACTCCGGGG AAAGGGGCCAGTGGCGGTCCCCCCTCCCCTGCGCCTCACCAGAGTGCCAAGGGCCCAGAC ACCATGTGAGCAGCAGCCAGCGGGGCGGGGGGGCCTCCTGAGCCCGTGGCCTTCCTGGGG GTGCATCAATGCCTGGTCACCTGGGGAAGAGAAGACGAACACAGTCACCCAGACAGCCAT CAGCGAAGAAGGGAGGGAAGGGCGATGAAGGGACGCACCTGGGGGTCTCTGGCTGGGGCA CCAGGGGCAGCCCAGCAGGAGACTGTGACCACAGAGCCTGGTAGTTGGATCTGACAGCCA GGAAGAGTAAGGACCAGCGAACAGGTCTAGGGAGTTCGGTGGGTGCAATACCTGCCCCTC TAACACAGGACATTAAGGACACCCAGGGGCCACCTAGGCCAGGGGGCGCTGGTGGCAGAC ACACAATTCTTGGCCTGCGGGTGTGGTAGCAGGAGGCCTGGGCAGGGCCCTTCCCAAGCT GGCCACATTTACATAACAAAAGGTCAAAATTGGAGCCACTTGTGCAAGGGCTATTTATAA GAACCAGTCTCAACCGCTTTTGGCTCCCCCGTTCCTTCCCTTCCTGATCTGCTCTCACCG CCTCCCTGCCCAGAGGGCCCAGCCCCCTAGCCCGCCTGGCCTGACCACATGGCCTTCCCT CTCCAGAGGTTTCACTGCCTAAGGGCTCACCCAAGCTGCAGGCCTGGGCTTTCAATACCG GCCCACCAAAGCCTTGGGCAGCTGCCGCCAGGAAACGAGGGGTTCCCTCCCACCCCCAGA GCTCTGGGTCCTGCAGGAGGGGGGATCCTGAGGTGGTGGGTCCTTGCACCCCACACCTGC CAGCCTGGGCCCCCACGGAATAGAGCCGGGGTCGGGGAAGGGGCGTGCAGCGGCCCGTCC CCGTTTCCCCTTCCCCCACTCCACCCCCAGGGCCAGGGAGGCTCAGCAGGTCCCTCAGGA ATGTGACCAGACCCGGGGTGCCGGCCCAGAGCTGGGAGGCCTAGGCTGCAGCTGGCCAGC TGTCTCTCCAGCCCCTAGTGCAGCTCCCCTCTGGGTGGGGGGTGTGAGGGGAGGGGGTGC ATGGCTGGTCCAGGACCCCACCAAAGGCCCAGAGGGCAGCAGAACCAGCTCTGTTGGTAG GAAGCCCAAGGGCGTCCACAGTTAGCAAGGGTTGGGCCCAGTGGGCTAATGCTGGGATGA GGCCAGTAGGGCAGGGGCCACTGAAACACTGGCGGGAGCAGTCGAAAAACTGGGTAGAGG GTCTCGGTGGCACACTGGAGGCAATCAGATACCACCATCCAGCCCTGTGCCACTTCATCT GCTCCCCAACCACTGCCCCTCAGGACCCAGTCAACCATGGGTAGAGCGCATGCCAAACTC TGCAGCCAGGGGTCACCTGACCTTGCCAGAGGCCAGGCCAGCTACTGGATGCCGCGCAGG GAGTGGGTGGCAGAATAGGACTGGGGGGCACCTGAGTGCCATGGGGGTGGGAACACCCTC CGTCAGTGCCCACTGCCTGGCCCTGTCGGGCACTTTACAAGCCCAAGGCTAGCACTAGGT CTCCCCACCAAGGAAGGATGGCCCCAGGCTGGGTCAGCTGTGATGTAGAACAGGGCAGGG CTCCTCCCTGAACCCTCCACTTGAGTTAGGGGGTGTCAGAGCCACCTGCAGAAAGTTCAC AGCAAGCACAATGCCACCATGCGTGCCATCCACACCAGCTGGGCCAAGTCTGGGCCACAT CTAGGCCGCCACTGTGTCTGCGAGCAGCAATGCAGCCTCACCCCATGGCGGGGCAGGGGG TGGCACACGCCATCCTGCTGCGAAACGCCTACTAAGCACTGCTCCTTCCTTCTCAGCCAC AGAGCTCTGCCCTACCCGCTCACCGCTGGACATGAAACAGCAAGTGCCTATCCTGGAGGG CTCCGCCTGGGGAGTGCCAGGTAACTAGAAACACGGCTCTGGGTGCTGGCCCGGATGCTG GTGCTCAGCCCTGGAAGCAGCTGACTGCAGGCTGCGATGAGAATGCCACCTAGCACCCAG CAGCCCCTGGCAGCAGGCCCGGGCAGCACGCCTATCAGCCCCCAGGCTAGCACGCCCAGC CCTGGCCTACTGCCCATCCAGAAACGCTGGCTCCCAGGACGCCCAGGATCTGAGGCAGTA GGGCAGCAGGCCTGGGCTATCGGGGCCAGCTGGGCATTCGCCTAAGGCTGTGACCCATCC CCTCCCTAGGACAGAGCCTTCAGGGGTCCAGGGGAGAGGCAAGAAGGGTAGCACAGAAAC TATCTCCCAAGTTAGGATCCCTCAGCTCCAGGATTCCCCCTGTAGGTGGACTGGCCCCAG CTGGGAAGGACAAGAGCCCCAGCAGCTGGAGGGAGGGCCAGCTCTCTCTGCGGAATCCTG AAGGTCTGCAGGAAGGAACACAAAGCCTCGGGGTTACCTTCCCAGGCCATCTGTTAGAGC AGACACACGGCCCTGTTTACGGAACAGCCAACATGCCAACTGCTCTCCTTTCCTTCCCCT CCCAGTCCCCGCCACAAAAAAGCCAGAAAGCCATGTCCCCAGACACTGAGGGCAGGCCCA GGGCCCAGGCAGGTGCTAGCGGGCTGGAACAGCACTCAGATACTCCAGCCTCTCAAGCCA CTGGTGGCACATGCCCAAGCTCTGTAGAACTGGGCCAGCCACAGCAGCCCCACCCCCGCT CTGTTCGCCCTCAAAGAACTTACAGTAAGGCAGCTGCTCTGCAAGGCCCAGTCAGAGCCT ACCCGGCCCAGCAAGTCCATTCTGTGGCAAGAAAAACCTTGGGGACACACATACCCAACT GCTAAGTAAGATTATCTCTGGGGAGCGGGATTACAGAAAAACCATAGTATCTGTATATTA TGTTCATTTCTGTAATACTCAACATTTTAAAAAGTATTACCTTTTTTTTTTTTTTTGATG GAATCTCATTCTGTCCCCCAGGCTGGAGTGCCATGGCGATCTGGGCTCACTGCAAACTCT GCCTCCCAGGTTCATGCCATTCTCCTGCCTCAGTCTCCCGAGTAGCTGGGACTACAGCCG CCTGCCACCACGCCCGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTT AGCCAGGATGGTCTCGACCTCCTGACCTTGTGATCCGCCTGCCTCAGCCTCCCAAAGTGC TGGGATTACAGGTGTGAGCCACCGTGCCCAGCCAAAAGTATTACTTTTAAAACCCTAAAA ATACAAAGGGCAGAAATGATGCAGAGACATGGATGAACCTCACAGTCGTGGCACTGAGTG GAAGAAGACTTTTGCGAGAGTACAGACTGCATGACTCCATTTACATCAAGTTCTAGAATA GGCAAAACGGACTGAGGGTAAAAACCAGAGCAGAACAGGCTGTGAGCGACAGGTGAGGAC TGTCCAGGATGGGGCGAGGGACAAGCGCCCACTGCCTGGCCCTGACAGGCCTATCCAGGA CGGGGCATGAGGAACTCTTTGGGGTGAGGGCAGCTCTCTAGCTTGCTGGGAATCTGGGTT AAGGTAAATGAAAATACAATTTTAGGCCCTTAATTGTATGTAAACTTTACTTCCAATGAA ACCTCCAGACAGAGGCTTTCACAGGACATTCTCATCTCAGAAGCTCAGCACGATACCCTG GAAACACCACCAGCTAACCTTTCCTGGGCACTGTTGTATGCCCGACCATGTGCTATGTCA CTGGAGCCGCAGGGCCACCCAGGAGCCTGTGCGTTCTGTCCATGTGATCAGGTGGGCTCC TCTCAATCTTCACACAGCACAGCTGAGCCTCAAACCCAGCACTCACCCTGACCTCTCACC TCCCCACAAGGTAGGAAAACCTGGGGCTGAAAGTGTGCAGGGACATGTCATCTTTAGCCT TGGGCAAAGGCAACCTTTCCCACTGATGATTTCCAGATCACCCAGGGGTATTGGTTCTGC CTCAATTTTCCAACTGGCACAGAGATCAAGGGCGACGGTGAAGCAGATTGGAAGGGCAAA GACGCCACGGTGGGAACAGGTTCCTATCCCTCCCGGCTGCTCTCCAGCTGGGTGGCCCTC AGTGCATCTCCAGAGGGCAGTCAAAAGCCCCAGGCCAAGACCACCCTGCACTCTGGATCC CTCTACTCGCTCACCAAACACAGACCAGCCAAATAACCAACGGGGATGGCAGAGGCCAGG GCACTGTGCACCCCTCGCTGCCTCCTGACACTCGAAGCAGCCCCCCATTGGGGGACCCAC TAGCCCATGATCCTACGCGAGCGAGGTCAGGCCACACCTGCCCGTGTGTGGAGCCTCTGG TCAGTCCACATGGCTGCAGGGGCCTGTGGGGAGGCTGGAACAGGGTTACCTCAAGGCCTT TCTTCTAAGCACCCAGCAAAGGGCAGGAGAGTGTGCTCTAGTCAGCATCTTTCAAAGGCC AGGAGAGAGGAGGTTTTCAGATCAAAGTGATCCCTGCTGAGGCAGAAAGCAGAAAACTCC AGCCCCAATTCTGAGAGGCACTGCCCCAACCCAGCCTGGATACCCACCTTGCCTAGAACC TTGGCCTCCAAGAAGCCAGGCATTCAGAACTGCTCCAGGTGCTGGGCCTCCACATGGCTC CTTCTGGAGGCCATGACTCTGGGAGACAGGAACACATCTCACGCCCATATCCAGGCCCAC AGGCGGGGGTCAAACTCAGGGTCAAACTCATCTCAGCCACATGATAACCCTGCCTTCAGA CTGAAACTTCTGGCTCACGCCCAGCCAGGAGGGAGGCCTGGGGAGAAGGGTGAAGACCAG GAAAGGGCGCCACCCCAAGCAGGCCATCCCGCCTAGCCCTCCTAGGATCCTTCTCATCCA CCTTCCTCCCTGGAAGCTATCCTTTACTACCAAGAAAGGGGCTTCCTGCAGGAGGCCTGG AGCTATGCTGGAGGACTGAGGAGAAGCTCCCCCCAGCCCACTCCCAAGGAGCAGCACCTT AACCACCTCCCAGCCTAAACAAAAGTAACAGTGAAATGAGAACCCTCCAGCTAACGCCAA GACAGCTGAGAGTGAGCAGGGCCGCCCACGGCCCGCCCGAGCTCCACTGTCAGGTGGGAG GGACAAGCATGCCAGGCTGAGAAGCCATGCTAGGCAGGGCAGGGCCACCAGGCAACTTCC ACAACCCAGAGGTCTAAGTACATCTGACCAGGGTCAGAGAGAGCCCCAGACTTGCTGCCA CTGCTCCCAAACTTCAGTCTAAGGTCAGCATCGGCCCTGGTTCCAGACCAACCCCCAACT ATAGTCACCTCATACTTCAGGTGTGGCCGGGACAGATGCAGCTGCAACGGGTGGCCCTGT GTGAGCTCAGCCTCTTCCCAGGAGCCCCTAAGCCATGGGAGAGACTGCCTGGAAGGCACA AGGCAGCCAGGCAGGAGGCAGCCAGTATCCAGAAGTGCCCCAAGGCCCAGCCCCTGGACA CACCAACCAGCCCTGCACAGTCACAAGCCCAGGACTTTGTTCATCCTATCCCCATGGCCT GGAGTCCTCCCTGAGGAAGCCTTGGTAGACAAGAGATAGCTCCAAGGGGCGAAGGCTGCA CCTCGCCCACCGCCACAGCCCTGTATGAGATGGCAGCCTACATGTGGGCAGGTCTGATCT CCTCAATCTTCCCACAGGCCCAGTGAGCGGGGCGTTATCTCTGCAGTTTCAGATAAGAAA TTAAAGGTTCTTGGGGGTTAAGCAACTTGCCCAGGGTCACAAGAGCATGTAGGTAGCAGA GTGAGGACCTAAAGCCTGACTCCAGTGTTGTAGTCTGATGACCCCCCCGCCCAACGCAGG CAGGGCCAGGGATGCCACTCACACATGGCTTCCAACTGCCAAGCTGCTTGGGGGAGCTAG AGGAGGCACCGCCCTCCATGCACTGGTGTGCCAGGAGCAGGGACTCCAGTCTGCACCTCC ACCCTCACTCCCCCACCCCCCAGCACCACCTGCCTATCCACAGCAGGGCCCCACCTGGGT GTATTCTCCCAAACTCACTGTGAGGGCATGGGGTCTGCCCAGCTCCTCCACCAGAAGTGA CAAGGTGGAGCCCCCATGCAGCCTGCTTTGGAGGCCCAGGTTGTGCTTGCTGCTCCTCTG GGAACACCCCCACCCTCCATGACCTCCTTCCCTGTCCCTTCAGGAATGGGCTGGTGGTGG GTGGGGCTCACTGCTCTGGGCAAGCCCAGCCATGTAATCCAGCCCAGACCTGGATGCCGG CCAGCAGCTCCTGGCTCTCCTCCCAGACCATCCTCCTGGAAAGCAACAGGCTCCACAAAT GCCAGGAGCCCAGCTCTCTCCAACGCAGCTTCTACAAAATAAACACTCGGGTTCCACTCA ACACATTGGTGATGCAGCGATTGGGACCTGAGGCTGGCCTCCTCCTCCTCTGCTCCTCAG GCCAGCCCTGTGGCCTAAAGTGAGGGAGGTAGCATCCACCCCAGCCCAGTGACTTTCCCA AGCCCATCCTGAAATGCCCATGGGACCTACTGATGACCTCGTACTGCCCACAGCAGGGCC AAGCCCTGCATGGTCCGAGCCACCCGCCCATGGGTACTGTCCCTTCTGAGGTGAGGAAGC CCGGGCTAACACCCCAGCACAAGGAAAAGGCTGGAATGGGCTGCTGAGAGGAGAGCTGGA CCGAAAGTGCCTCCAAACTGTGCTTCCCCCAGAAGGTACAGCCTCAGTCCCCTCCCTCTC CTCATCACTTCCGGGTTTGGAGCCAGCAAGGAATGAGGCCAGGAAGACCTTGGAGACCTG CAGCTGATTTCCCCTGCTAGTGCTGGCACCAAGGAGGGCCCCAGGCCTGGCACAGTCTGG CTGACAGTTCTGAGACAGGATATTGGAGACAGGCAGGGGAACATTGCTTTAACAAGCAGA ATAACGGCCAGGTGCGGTCTGTAATCCCAGCACTCCGGGAGGCTGACATGGGTGGATCAT GAGGTCAGGAGATCAAGACCATCCTGGCTAACACTGTGAAACCCCATCTCTACTAAAAAT ACAAAAAATTAGCTGGGTGTGGTGGCACGCGCCTGTAGTTCCGCTACTCCGGAGGCTGAG GCAGAATTGCTTGACCCTGGGAGGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACTGCAC TCCAGCCTGGGTGACAGAGTGAGACTCTGTCTCAAAAAAAAGAAGCAGAATAACAATTTA AGGTTGTGATAGTGACTACGAATGCTGTGCTCACCTCCAAACTCCATCCCAACACTACCC TGCCCCCTAAGAGGGGCCTGAGCATGGGGTAAAAGGCTCGCTGACAGCCACAGAGCAGGA GCCCACGCAAGACCACCAGAGGATGTGCAGCTGCTGTTCCTAGTAGGTGAGGAAAAAGCA GGAATCTGGCATCCACACATGTCAGGAGGCAAAACAGGCCCTGTGCTGGTTTCTGCAGTC CTCTCCAGCCCACTTCCCTGCTCTTCTTGCTCACTGGCCATGACCCAAGAAACAGCAGCA GCACCACCAGGGATCTTGTTAGACACGCAGAGGCTCAGACTCCATCCAGCCCTAACCACC AGAACCCTGGGGGCTCAAACACACAGTCAAGTCTGAGAAGGGCTAGGTTAGAAGCTTTTT TTCCTTCTTTTGTTTTTACCTGGACACCACTGATTGGAAAGCTCTCTACGGCCTGAAAGC CAAGCGCTCCACAAGTAGTAAACGCTCAAGGGATGGGCATCAAGTGGAAGGAGAAAGCCC CCAAAACGAAGATACATGTCCCATGTACACAAGCTCACAGGCTGGTGAATACAAACGGTA GACCTAGAATGTGCCACACCAAGGCTGAGCGTGTTACCACAGGTACCAGAATGGTGACCA CGCTGGAGGGTTCCACAAACCCTGATCAGAGAAGTAGGCGTGAGTGGCAGGCAGAGAACA CAGCTCTGTGTGGCAGGGAGGGAGGACAAGCAGTGGCAGCACAGCAGACAACAGGTGGAA ATTCTACATGAAAGGCACTAAATGGGCGAGGAAAGCCTGGGCCGCCCAGGTGGAAGGGAC TATCGTGATCATCTCTCTCACCCTAGGGACACCTTAAAAGGAGGTAACTGAGCCCCAAAG GGAGGCACCCAGCACAGCTGAGTCACAGAGCTGCAGCCATCCAACCCACAGCTCCAGGAG GGACTCTCCAACTTCACAATTTCAAAATCAACCCTAGCAGCTAACAATTTCTTAATGCCA AGTAAAGACTAGAATTAGAAACAAACAGAAACCCTATCATCTTCTACCATCATTACTTCA CAAAAGAAAACATTTCAATGATCAAAGAGCAGGAAGAACTACTGGCAGTCCTGCACCTAT GCAGTTCTCCAGGCCTGGCAAAGGCTCTGAGGCAACCAGCATGAAGGAGCCACTGCACCG TCTGTACGCAACACCGCCTGACCCCCGGGCTGGGACAAGACTGAGATGACATGCTGAAAG GGAATCAGAAAAAACAAAAACACAAAAATTCAACAACAGATACTAAAGACGGGATTTTGA CTAGCCAAACAAAGACTAAAAGAGTATCTCCCAGGGATGAAAGGAAAATGGGGATGTTTT TAAACAAGACCAGGACTGTTCCAGAAACAATAAGAACGTCAAAGCCCATCAGGAAAAAGG TAACACCAAATGTTGGCCAGGCAGTAAGGCAACCAGAAGTCTCACATACTTTATAATTTT TTATTTTTTTGAGAGAGAGTCTTGCTCTGTCACCCAAGCTGGAGTGCAGTGGCGAGATGG CTCACTGCAACCTTCGCCTCCCAGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCGAGTAG CTGGGATTACAGAAGTGCAGTGCGCCACCACCCGCAGATAATTTTTGTATTTTTCGTAGA GATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCCAACTCCTGGCCTCAAGTGATCTGCC CATCTCAGCCTCCCAAAGTACTGAGATTACAGGCATAAGCCACCGCACCTGGCCAGAAAA CTCACATGGTTTAGAAAACCCTTGGATGGCACAATCTAAAACTGTGGGCCCAAGGCCGTG GGTGGTGGCTCACGCCTGTGATCTCAGCACTTTGTGAGGCCAAGGTGGGTGGATCGCCTG AGGTCAGGAGTTTGAGACCAGCCTGGCAAACATGGTGAAACCCCGTCTCTACTAAAAATA CAAAAATTAGTTGGGCGTGGTGGCAGGCTCCTGTAATCCCAGCTATTTGGGAGGCTGACG CAGGAGAATCACTTGAACCCAGGAGGCAGAGCTTGCAGTGAGCTGAGACTGCGCCACTGC ACTCCAGCCTGGGCGACAAGAGCCAACTTCGTCTCTAAATAAATAAAGCAAACAAGCTAG CTAGCTAGCTAGCTGTGGGCCCAAAAACTCACCCAAGGTATACGCCAACAAGAATGCACA CGTATGCACACCAAACAGGCAGGCACACTCATGAAGGCCCAACAATGGAAAACAACTATG CCCACCCTGAGAAGGGACAGCTGCAGGTATAAAAGACTATGAGCCAGCAGTGAACAGGAC AGAACGTTTAGGTTGAACTGTATGAATTTACCTTTTAGGGGTAAAAAAACAGCTGGCTGC TGATAATTTCACATGGTTTGACTTACAAACAACGCAGATGAATCTCTCAAATATACTTTT GAGCAAAGATGCCAGAGCCAAAAAGACTATTACAGTTCCATTTGCATACATTCAAAAGCA GGCAGCACTAATCTTATGGGTGGGGGTTGAGAGTGACTACAAGCTTCAGGATACGTGAGG GGCTTCCAGGGTTGTGTTTCGCACTCTGGATGCTAGTTACAAGGGCATAATCATTTGTGA AGAACTCTTGGAGCTGTGCACACTTCTTACGTAGGCTGTCCTTGAATTCAATACGTGCAC AGAAACCTTCAGAAGGGAAGCAGAGATCAACTTCATCCTGATCAGCAAGTAGACTCAGTC CAGGCAGCAACCTCTCCTGGACCCCCAACCAATAACAACAACACAGGAAGCAAAATATTG GCAGGACAGCTGGCAACGCGGCAATGTCCAGGATGGAGTGCCCATCCTAGAGGAGGGAGC TCACACAAGCCAGCCAGGAGCCCACAGGAAACAGAGAGACTACTGTAAATCCTTATTAAG AGCAGACTCATGTCTGCTATCTAAAAGATAAAAACCTGATAATGTTTTTAAGTTGTCCTA AGGTCCAATTGACTTTTTTTTTTTTTTTTTTTGAGACAGAGTTTTCCTCTTGTTGCCCAG GCTAGAGTGCCATGGTGCAATCTCGGCTCACCGCAACCTCCGCCTCCCGGGTTCAAGCGA CTGTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTCCAAGTGTGCACCACCATACCTGGCT AATTTTGTATTTTTAGTAGAGACGGGGTTTCTCCATGTTGGTCAGGCTGGCCTGGAACTC CCGACCTCAGGTGATCCGCCCGCCTCGGCCTCCCAAAAGTGCTGGGATTATAGGCGTGAG CCACCACACCCAGCCCCAGTTGACACTTCTAAAGGAGAAGGGGTTCTGCAAATCCTGAAA GGCAAGCCCAGGAGAAACAGGAAATCTGGGAGCACGGCAAGCAAGGTTCAAACAGGACAC CAAATAATTCCCAGCAACCGCCTCCAGCAGGTCCTGGTGGCAGCCAGGTAGGGCAGGAGC CAGCTGGGCATATCAAAGGGAGGAAGCCAAGAAGAAAATGGGCGAGACTCTCAGGGATTT GGGGTCAAAGGAAGCCGCTGGGAGAAGGAAACAGCCCACTTGCTCCTTCATAGGTAATCA ACACACAAACCCCCCAGTGCCTCAAATGGCTCAGGAGCTACAGCCAAAGATCAAGGATCA AGTTATTTTACTCCAAGAAAGAAAAAAAAAAGAAAAAGTTATACATTCGGCATCCATTGG AAGTTTTGTGATTGTTTGGAGACAGGGTCTTGCTCTGTGGCCCAGGCTGGAGTGCAGTGG CGCGATCATAGCTCTAATTTATGGGCTCAAGGGATCCTCCCGTCTCAGCCTTTTGAGTAG CTGGAACTAAAGGTGCATGCCACGATGCCCAACCAATTTTTAAAATTTTTTGTAGAGATG AGGTCTCACTATGTTTTCCAGACTGGCCTCAAACTCCTGGGCTCAAGTGATCCTCCTGCC TCAGCCTTCCAAAATGTTGGGATTACAGGTGTGAGCCACTGTGCCAGGCCTTAAAAAGCA ATTTTTACAGATAAAATTGGGCAACTGCTATGGAAAATATTACAATCTATGGTCCTGTGC CTGAAAAGCCTCCAAGTGTGATACTTTATAGAGAAGAGGTCTCTCCTGTCCAGTAGGTAA CTTGAAAGTGTCTAAGAGACAAGTGCCCAAGAACTCAAAGAATGAGAGAGGGATCCATTC AAACTCAGGGAGAAAAATGCTCACAAAGTTTCACTGTACTTCTAAACTGAGTCTCAGTGG TGCATTAAGGAACTGGATTTAGAGACAGAAAACAAAATAAACTGGCACTTCTCTGCCTCA AGAAGCATAGCTTGGGGGTGAAAAGTCAGCTACTTAGAAAGCAGCAAAAACAAAACCCAG AAACCCACCCAAATGCTCACTGCCAGCAGGACAGGTGATTATGTGTGGATGGCGCCACCT TGGACACTGAAATGGAGAATGGGAACGCAGGAACTGCTGCTCCTGGTGTCCCAAGAGTGC AGGACACCCGGCTCCCAGCGAACTGAGCCAAAATCATTCGCAGTTTCAGGATCCAGACAA AGGTGGGAAACCGACAAAAGCCAGCGAATGGCTGACCCGCAATTCAGAAGACTGTTGGCT TCTAGAGGGAAGTGGGAATAAGCCAGGGAGAAGTGCATGGGGTGTCCCAGGAACTTGGGA GGCTATTTCTTATGTGGCCATGAGGGAACAACGGTGTCACGCTTAGTGGGACACCCACGA TTCAACCAATAGTGGGTTTACCACTACTTATCTATTCTTTAAGATTTGTGCACACACATA TATACGTGTAATTTACCTTTGTTTTTTTTTTTTTTTGAGTATTCCTCTGTCGCCCAGGCT GGAGTGCAGTGGCACAATCTCGGCTCACTGCGACCTCTGCTGCCCGGGTTCAAGCGATTG TCCTGCCTCAGCCTCCCGAGTAGCTGGGGATTACAGGCACCCATCACCACACCCAGCTGA TTTTTGTGGTAGAGACAGGGTTTCACCATCTTGGCCAGGCTGGTCTCAAACTCCTGACCT CAAGTGATCCGCCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGGGTGAGCCACCACG CCCGGCCTATTTACACCTTTGTAAATAACTTCTTAGAGTTCAATGGCTAAGTATACTAAG GAAGCTGAGCCTGCTCTGTGCAGAAGTCTCAAAAGAAAGGAAAGTCGGGTGTGGTGGTTC ATGCCTTTAATCTGAGCACTTTGGGAGATTGAGGCAGGAGGATCGCTTGAGCCCAGGAGT TCAAGACCAGCCTGGGCAACAAGGCAAGACCCTGTCTCATTTATAAAACAAACAAACAAA CAAACAAACAAAAAACAGAGAGAGAGAGAGAAAAGAAAGAGAAAAAAGAAAGAGGAAAGA GACCTCCCTGGAAATCCCAATCCAGGGAGAAGCCAAGAGGCTAAAGCTGAGGTGCACTCA CAGAGCACCTCCCTTTCACCACCCGAGAGATCACCTGGTCCACAGCCGTCAAACTCAGCA AGGCCACACACTTGGGCAGCTATGACAACAATGCACAAATGAGGGCTTTGGAGGGCTCAG TTCCCACAAGAGATAACCCAGAAAATATCACCCTGTCCTAATCATACAAAGCAAAAGAAC CTGGGGCCTCCACACCTGGGGTAGCAGGTATGCTTCACTCAACAGAAGTTGTCCAGGAGG GAGAAATCCCAGGCAACCTCCCAAGGTCTACAGGAGAAAGAAGGGCAGAATCAGAGCCAC TCTAAGTGAAAATATCAGCGTTTAGAACACAATCACCCCCCTGCAACCCCTCCAGCCAAC CCTCGCCTAGTCCCTGTACCCTCCAATTCAGAGTCCAATAACCCTGAGTTATCTTTCTCA ACACCCATCTCATCAAGCCACTCCCCTGCTTAAAGCTTTCAATGGGCCGGGCATGGTGGC TCACACCTTTAATCCCAGCACTTTGGGAGGCCCAGGCGGGCAGATCATCTGATGTCTGGA GTTTGAGACCAGCCTGACCAACATGGAGAAACCCCATCTCTATTAAAAATACAAAATTAG CTGGGCATGGTGGCGCATGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATC ACTTGAACCCGGGAGGAGGAAGTTGCAGTGAGCCAAGATCGCGCCATTGCACTCCAGCCT GGGCAACAAGAGTGAAACTCTATCTCAAAATACAAAAAAAAAAAATTAAAAAAATAAATA AAGCTTTCAATAAATTCCTGATGCCTAGCATAAAGTCCATGCTCAGGCCTGGCATGCAAA GCACTTCCCACAGGGGCATCAAGCCCACCTCATCCCTTCCACAGACACACCTGAGAGCTC CTGCAGGCCGTGCCCTACTCTGGGAGGAGCCTGCCCCCTCACGACTCCCTCCTCTGTGAA GCCAGAACTGCCTCTGTTGTCCCCCCGCTTCCAACGTCAGCACCAGCTGCCAAGCTGCTT CCCTCCTGCCTACCCCCACCACCCCATAGACCAGTACCCAAAGTTAGACCCTGTGTCCCA AGCAGCCACAGCAGCAGGCATGCAGCAGGCACTCATAAACATTCATTAGGTGAGCAAATG CTGGTCAGACCCAATGCTGAGAAGAGACAGAGTGAAACAACATGTGAAAGATAAGGAGGC ACGTGGGGAAGATGGAAATGTCCAGTACCCTCCTTGCCACTGGTTCTAAAATTGTGTTGT TTACATTCTGAAATAGTCAGGGCTCCTAACGAGCATCTGCTTATGTGGCTTCTCTATTAC TGTTTTGTGGGGTTTTTTCTGAGACAGGGTCCTGCTCTGTCACCCAAGCTGAAGTGCGGT GGTACAATCATAGCTCACTGCAACCTCGAACTCCTGGCTTCAAGCCATCCTGCAGCCTCA GCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACTGCATCTGGGCTGTTGCTGTATTT GATGTTACCATGTACTACAACAGTGCCCTCTTATTCAGTTTTTTTTTTTTTTTTTCCTGA GACGGAGTCTCGCTCTGTGGCCCAGGCTGGAGTGCAGTGGCGTGATCTCGGCTCACTGCA AGCTCCGCCTCCCGGGTTCACGCCATTCTCCTGCCTCGGCCTCCCGAGTATCTGGGACTA CAAGCGCCCGCCACCGCGCCTGGCTAATTTCTTTTTGTATTTTTAGTAGAGACGGGGTTT CACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCTGCCGCCTCAGCCTCC CAAAGTGCTGAGATTACAGGCGTGAGCCACCGTGCCCGGCCAAAAAAATTTTTTTTAAAG GCTGAGCACAGTGGCTCACGCCTGTAGTCCCAGCACTTTGGGAGGCTGAGGTAGGTGGAT CACTTGAGGTCTGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTA AAAATACAAAAAATTAGCCAAGCGTGGTGGCAGGTGCCTGTAATCCCAACTACTTGGGAG GCTGAAGCAGGAGAATGGCTTTACCCCAGGGGAGGGTGTGGGGTGGAGGTTGCAGTGAGC CAAGATTGTGCCATTGCATTCCAGCCTGGGCAACAAGAGCGAAACTCTGTCTTAAAAAAA AAAATAGGCTGGACGCAGTGGCTCATGCCTGTAATCCTAGCACTTTGGGAGGCCGACGCG GGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCCTGCCCAACAGGGTGAAATACAAAA ATTAGCCAAATGTGTTGGTGCATGCCTGTAATCCCAGCTACTGGGGAGGCTGAGGCAGGA GAATCACTTGAACCTGGGGGAAGCAGAGGTTGCAGTGAGCCAAGATCACGCCATTGCACT CCAGCCTGGACAAGAGCAAAACTCCCTCTCAAAAAAAAAAAACAACAACATTTAAAAAAA TACAAAAATTAGCTGGGCGTGGTGACAGGTGCCTGTAATCCCAGCTACTCAGGAGGCTGA GGCAGCAGAATCCCTTGAACCCAGGAGACGGAGGTTGCAGTAAGGAGAGATTGTGCCACT GCACTCCAGCCTGGGTGATAAGAGTGAGACTCTATCCGAAAAACAAAACAAAAAAAGGGC TGGGCACGGTAGTTCACACTTGCAATCCCAGCACTCTGGGAGGCCGAGGCGGGCGGATCA CGAGGTCAAGAGATCCACACCATCCTGGCCAACAGGTGAAACCCCATCTCTACTAAAAAA TATATATATACATACAAAAATTAGCTAGGTGTGGTGGCGCGCACCTGTAGTCCCAGCTAG CTAGAAGGCCGAGGCAGGCAAATCACTTGAACCCGGGAGGTGGGGGTTGCAGTGAGCAGA GATCGCGCCACTGCACTCTAGCCTGGCGACAGAGCGAGACTGTCTCAAAAAAAAAAAAAA GAAAAGAAAAGAAAAAAAGAAGGATAGCATTGCTTTACACTTTTATAAATCTCTTTAATG TCTAGCCTAATAGAAGAAGCTGGATTCTCATATCTCCTGCATTCAGTCTGTTAGGATATG CGGTTTTGATTAAAGTCTTTAAAGAAATTTGGCCTTACACAGAAAGAAGGAAGGAGTATT TTAATAACTTTTTCAGAAAACTGTCTATTCTTCATTGATATTACACTAAAGCTCGACAAA TGAGTTTCTTAAAAGTTGCTATGGGCCAGGCGCGGTGGCTCACGCCTGCAATCCCAGCAC TTCGGGAGGCTGAGGCAGGTGGATCACTTGAGGTCAGGAGTTCAAGACCAGTCTCGCTAA CGTGGTGAAATCCCGTCTCTACTAAAAATACAAAAATTAGCCAGGTGTGGTGGCAAGCGC CTGTAATCCCAGCTACTCAGGAGGCTGAAGCAGGAGAATTGCGCAACTGCACTCCAGACT AGCAACAGAGCAAGACTCCGCTTCAAGGAAAAACATAAATAAAAGTTGCTATGTAGGCCA GGTACGGTGGCTCACATCTGTAATCCCAGCACTTTGGGAGGCTGAGGTGGGCGATCACAA GGTCAGGAGTTTGAGACCAGCCTGACCAACATGGAGATACCCAGTCTCTACTAAAAATAC AAAAATTAGCCGGGTGTGGTGGCACGTGCCTATAATCTCAGCTACTTGGGAGGCTGAGGC AATAGAATTGCTTGAACCCGGGAAGCAGAGGTTGCAGTGACCCAAGATCGTGCCACTGTA CTCCAGCCTGGGCAACAGAGACTCCATCTCAAAAAAAAAAAAAAAGTTGCTATGTAGGCT GGGCACAGTGGCTCAACACCTGTAATCCCAACACTTTAGGAGGTCGTGGTAGGAGGACTG CCTGAGCCTAGGAGTTCAAGACCAGGAAGATCCCATCTCTGGAAAAAAAAAAAAAAAACA GCTGGGTGTGGTGGCACATGACTGTGGTCCTAACTACTCGGGAGGCTTATGAAGGAGGAT CATTTGAGCCCAGAAGGTGAGGCTGCACTGAGCCGTGATTACATCATCACTGCACTCCAG CCTAGGCAACTGAGGGAGACCACATCGTCAAAAAAAATTAAAAATAAAAAAAAAATTGTA AAGCCTCATGCCGGGCATGGTGGCTAATACCTGTAATCCCAGCACTTCGGGAGGCCAGGG CGGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGCGAAACCCAG ACTTTATTAAAACTAGAAAAAATTACACCAGGCGCAGTGGCTAATGCCTGTAATCCCAGC ACTTCGGGAGGCTGAGGCGGGCGGATCACGAGGTCAGAAGTTCGAGACCAGCCTGACCAA CATGGTGAAACCCCATCTCTACTAAAAATGCAAAAATTAGCTGGGCATGGAGGCGCATGC CTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAGGCGGA GGTTGCAGTGAGCGAGATCGCACCACTGCACTCCAGCCTGGGCGACAGGGAAAGACTCCG TCTCAAAAAAAAAAAAAAAGAAAAAATTAACCAGGCGTGGTGGCAGGCGCCTGTAATCCC AGCTACTCGGGAGGCTGGGGTAGGAGAATCGCTTGAACCCAGGAGGCGGAGGTTGCAGTC AGCGAGATCGCACCACTGCACTCCAGCCTGGGCGACAGGGCAAGACTCCGTCTCAAAAAA AAAAAAAAAAAAGAAAAAAATTAACCAGGCGTGGTGGCGGGTGCCTGTAATCCCAGCTAC TCGGGAGGCTGAGGCACGAGAATGACTTGAACCCAGGAGGTGGAGGTTGCAGTGAACTGA GATTGCGCCACTGCACTCCAGCCTAGGCAACAGCGAGACTCCATCTCAAAAAAAAAAGCC TCATGCATCCTTCCTATTTCATCACACAGAAAAGTAAAAATATGTGTAATTAAAGGTCTA GATTTAATACTCAGTCATCTGTACTGCCTCCCCTGGACATGTGTAGAACTGGTGGGTCTC AGCCCCCAGTGCTGGCTAACAGTGGGGAACACAACTCTGGCAGTGCAAGTGTCCACCCAG GGCAGAGGCCAGTGATTTTTTTTTTTTTTTTTTTTTTTAGATGGAGTCTCGCTCTGTCAC CCAGGCTGGAGTGCAGTGGCAAAATCTTGGCTCACTGCAACCTCCGCCTCCCAGGTTCAA GCCATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGGCGTGCGCCACCACACCC AGCTAATTTTTGTATTTTTAGGAGAGGCGGGGTTTCACCATGTTGGCCAGGCTGATCTGG AACTCCCAACCTCAGGTGATCCACCCGCCTTGGCCTCCTAAAGTGCTGGGATTACAGGCG TGGGCCACCGCATCTGACCTTGTAACTTCTAAATATACTGAAAAAGGTATCGGGGACCCT CAGGGTCTGCAAACAACTTTGAGAATAGCTGACAAGCACTGCCTCAAATATTTCTAACAA CTCGTTTACAGGCAAGGAGAATGAGGCCTGGAGAGCTTAAGAGACTGGCCCACGGAGGAA GCAGGAGTCATCTTTCCACAAAACTACACTTCCCCAAAAGCTGAACTCTTGGACAAGGAG CCCACTGCCAAACCTAAGAGTAACAAATTTCAGCAAATAAAAGGAAGTGCCATTTCCAAT GGCATCCTCGAATACTCTAAGAGGCGGGGCTGGCAGAATCCATCAACAGGCCTCCAGAGG CCTGCTACTAAGAGCCAGCAACGCTGCTGAAAGCGGCACTGCCTCTGCAGGCTGTGTGAC CAGGACCCGGCGGGGGCCCTGCACCTGGAAGCAGAGGGGGTCGGTGGGGGAGGGGTGCTA CCAGGAAGCAAGATGTGCATGCTGCCTGCTGGGGGCATGAAGTCAAAGTCCCCTCCTGCC CAAAGTGGCTAGGCCAGCCCTAGGCTGCCTTATCAGCATCAACACATGCCTGGAATGTGG GACTTTGGCAACAACCCTCTCCCTTCGGCCAGCACGGGGCTGACGCCTGGTCCCTGGGCT CTGGTTCACAGTCAGAGGACAACCACCTTGAAGCACCAGAACCCGCACCCCCAGCCCATC TACCAGAAGACAATCTACCAAGAGTCACCGAGAGGAGCTATCCTGCCTGTGTCCCCACAC CACCAAGGGCCGTGTCTGAGCACCTCGTTTGTCCACACTCAGAGAATCTCAAGAGCTCAG CTCTGGTCCCTGCAATGTCCAGACCATCCCCCACATGGCTCTCCTGGAGGTGTTCGGCAA AGGCACAGGCTGCCGTGGGGCAGAAGATTCCCACATACCAGACAGTAAGGAGCTGCGTGT GACTGGAAGGATGAGGACGACCCAGAGAATGCTCTCTGGCCCCTGGTCCTTGGGGAGGAA TGGGGGACCTGCAGGGGACCACCTTCCCACTCTGGGAGCCAGATTCCCTGGCTTTGTCCA GAGCAGCCAACCCACTACCCCACTCAAAATGAACACCTCAGCTGCCCTCCAGCCCAAGCA CTGCGCAGAGTAAGGAGTCCTCACTCACAAATGTGTCCGAGGCTTTCACACACTTCACCT GGAGCTTCGCCACTCACTTCTTAGATGTACGACTCTGACTTCTCTGAGCCACTTGTACAA CGAGGACTGACAAGATGATGAGGACCACACCGGTGTGTTCTGACAACTGAACCGGGCATA GAATGCCCACTGCCAGGCTGAGCCCAGGAACGAGCCCAGGATGAGTGCTTACCCTCTTCA CCACTACCTTCCTAGCCTTCTGTCAGAACTCCGTGGGGACCATCCTCTTCCCATCTTCTA AGCCAAGTACCCTGGTACAAGCCAGACAGAAACTATAATAGCAGCTGCTTCCATAAAGGG GAAGGGCCATGGGGTCCCGGACAGGGCTGGGGTGAAGGGGCACAAGACCTAGGGCAGGAA TCCACCCCCAGATCACTCACTCAGGACAGGACCACCTGGACCCAGCTACCAAGATGTGCA CCACCCCCTTCCTTCTGAGCCCCTTTTCCCATCTCCTCTACTGCCCCTGGCCAGGTTGCT GTCCCACCTCTAAGAGGCAAGATTTCCTACCCAGGATGCTGACCAAAACCACACCCAGGG GCCTACCCGTCCCCTCACATGATAGGCTGCTCCTAGCTGGCAGGAGCCGTCTTATAGCCT CTCTCCCTCAAATACAAACAAAACCCCAGAACAACCTATTGTTCTGTTCCAGCAAAGGGG GCCAGGCCCAGCCAGGCCCTACCTGAAGCTCTCTGTCTCCCGTCCCCCTTCAGTCCCCTC CCACTCGTCCTTCTCTGCCCCTATTAGTGGCCTCATATACCTTGTCCAGATAACACAGCC CAAGAAACCAACCTCAGGGACAAATCTCTTCTCTTCCAGGGAGAAGAAGACAGAGGTCCC AGGAGTCCCAGGGGCCCCCATTACTGGCCACCTCCGCTCAACCCAAACTGCACTATGTGA GGTCCCGCCAGACTACATACAGGCCAACGAGAGCCCAAGAATGCAGCCAGGAAACCAGGC CAGGCAGACTCCAAAAGCCCGTGCTACGGGGTCAGTAGGACCCGCCTACAGCTGAGGGCT ATAGGCAGCTCTCCAGAGGACCTCCCAGGAGAAGCCAACAGTGCACATGGGCTAGGTTCC TCCTGCTGTTCACCCTCTGTGCCCAGTAACTACCCAGGAACAGGCATGACTGACTCTGCC AACTCATTCACTGGGTAACCGCTGACCCCACCAGGCCCTCGGAGTAAAGCAGTTAGTGAG AACCGCAGGTAAAAGCGCGGGCTACTCATCCCTCCGCCTGTGGATTCTATTATCAACAAA GATGCATCACCTGCTAGAAGAGCGGTTCTCAAACTTGGCTGCACATCAGGTTCACTTCAA AAGCTTCAGGAAAAGCTAGTGCCTTGGGCCCACCCCAGAGACAGGTATAGCTCAAGGTGA GAACCAGAGCACCAGCAGGCAGGCCTCTGCCAGGCAGCATGGGCAACGTGTGCTGGGTGA ATGTGCGGCCTGCACTCATCACCCAGAAGAGCCCCACTGTGTCCACACAAGGACAGGCCA CCGAAGTCCCAGTCCCCACTCTCCCTTCTCACTTACTTTGTGAATGAGAGCTCACCAACA GTTTACCGTCTTCCGAGTGCTGCGGCCTCACCGCCCGACCCGCAGCAGGGGTGAGGGCTT GCAAAGGCCATGCAACAGATCCTCCCCCTCAAAGGGGCTCAGGCCTCAGTGTGGTAATGG GAGCGGCCCCTTCCCACACTTGTCAGAGAACCCACCCACCCGGTGGAATGAAGCCGAAGC CCAAGCCGCAGGGCAACAGCCCGGGACGGCAGGGGACAAGTCTGTACAGCAGCCACACCC CTGCCAGAGCCAGGAGCTCTTCCATTTCCACAGCAGGCCAACTCTCACCTGGAAGCACCC ACCTAACCAAACCCAATCCCTGCCTTCTTTTAGTCACACTGGGAACTGCCACAGGAAGGC TGGGGACTCCACATACTCCCTCCTCACCACACCCCATGTGTGATCACCCTTAAACAGAAA ACATGCACCTAAGATCCTACGATGCGCTGCCCACCTCACAAAAAACCGGTGCCTCTAGGT AAGAGCACCTAGCTGACCCATCCCTGTTCCCTCTCACCATTAGAAGGACCCCCAAGTCCC CCTGTTCTAACACCGAGGCTGCACTCCCACTCTCCACCCACTAACATTACCATAAAAAAT AAATTGAATAGTTAGAAGGAGGGGCTCCCCAAAGCCTGGCCACCTTCCCGAGGGTCCCAA GCAGTTTCTTGGAGTCCCTCTCCCCACTCAGTCCCGCCACACCCCTCTCAGGCAGTCTGG CCCTTCCCTCCTGAACTTTGGCGGGCTGGAACCAGCTCCCAGCCTCTCCTCCCTTATCAA GCTCACTGGGGGCCCACACAGAGCTCCCATCCCCACCAGCTTTTCTCGCTTTGCCACTCC CAGCTAAAACTGGGCTCAAGGCAAGCAAGCCTGCAGTCCCTCAGTCCCTCGGGGCCCAGC CCCTCCTCAGAGCCCTCCCCCAGCCCAGGTCCCTAACCCTTAGGGCAAGGCCCCTCCCAG CAGGAAGCGAACCCTGGCGGTGCCAGGCAGAGTCAAACTGGAAGGGCTGGTTCAGAAGCC CCTAAATGGGAGCCAGGACCTCTCTTAGGGAGGGGGCTTTCAGCCCCATCCTCCCTAGTG CGATCTCAGATCTCTCCAGACACCACACTATATGGGCCTCCACAGGCGGTGGAGTCAAAG GCATTTACCCTCCAGCCCCCAGCAAGCTACTTAACTTCCAAAAGCAGCTCCTTCCCTCCT CCAGAGGAGAACGGGCCGCGTGCTGAACTGCAAGCGCCTCTATCACCCCGCCCAGGCAAG TCTGTGAGCACCAGAAAGCAGGGGCCAACATGACTTGGTCTAAACCAAGGGGGCCAGGCA CTGACTTTGCGCTCAGAAAATGTGGCGGAATGAATAAAAAGCTTGAAAAAGAGCCGCATA CATGCCTGGCTGGCAGTGGGGTCTCCCAAGCGGGCTTCACAGCTCACACAGTACCCGTGG GTCCGCGTCCTCAGTGCTCTCCAGCTGTCTCCACTGCCACCCTCTACCCTACCACTGCCT TTTAAAACCCAGTAGCAGTCACTTCCTGGTTAAAACCCCTCCCCTCCAGGCCAACCCCAA CCCACATGGCATCCAGGGTTCTCAGCCAGCCCACGGCCGGCTCCTCCCACGCCACCGCTA TGCCCTCTGCGCTCCGCTAGATGCCAGCCCTTATCACCCCATCTTATAATCATTTGTTGA GTGTCTGCCTTCCCTAGGCTGAGCTCCAGAAAACAGCGATACATGAATGTAAACTCCAGT TCTGTTCCCATTGTAGAAACGAGGCAGGAGAGTCAATCACTGCTCAAGGTCATGACGGGA GTAAATGGCAGAGCCAGCAATGCACTACAGACTTTGCGACCGACCAAGTCCAGTTCCCCT CTCACTGCCCCACAGGGGTCCGTCCTAGGCCAGGCTCGGAGGAGACAAGAAGGGAGGAAC CCAATGTGTTCTTTCTTCCCGCGGTGAAAGCTGCCTCCCAGGCCAGCAAGAGCAGCCCAG AAGAAGTGCCCCACTCTCCCAGGGATCAGGTACTGGGGGCAGCGGGCAGGACAAGGTATG GGGTGGGGCTGACTGCTCCAGGCCAACAGACCAAGCAATAAGGTTGGAACCACAAGTCCC CTGGGTAAACTGAACTTTATTCCTTCCTCACGGCTCTCACTCTCCAGAACTGCCCCGCCA GCTCTTCTCCAGGGGCTGGCTGCTGATTAAATGGCACTTCCCCACCCCTCAGATCTGACC CCGCAAACAATAAGGACTTGAGGGGAGGCGGCAGGCTATCAGCTCAATAATGCAAAACCC TGTTGCTCCTCGTCCACAACAGCTGACTTCAAGTGGATGGGAGGCTGCGCTTATTAACAA AATGAGAAATCTGATCTACGGAAAGAAAACACTACGTGAGGATTAATCCGCGACTGCAGC TTGTGGAGAAGGCTGGGCTGCTGGCCAAGACCAAGGATCGAGGATGGGATCGTGCCTACC TGTCCCCAGAGCAGGTATCATGCAGCACAACAGGACTCATCACCCCTGCCCCACCTGCGC CTTCTCTTCTCCTTCCAGCAACTTCGACAATCATTTCGGTTTTGCTTTAATTAAAGGCCT GACTCCCTCGGGCTGCTTCTTCCTCTACACAGAGGCAGCAACCAGAGGGAGATTTTTCTT TTCAGGAATCGGTCGTAAAAACTCAGTGACTAATCTACAAGTTCCAACAACTGGCAGAAA AACACCGCAGGGAGAGGTTGCTGGGCACACAGCAGCACTCAGAGCCAACTGACCAGAGAA GCTGGGCCACAGGCACTCTACTACAACCTCCCTCCCCCAGCCCGACACTGGCCTGCCGAC CCACCTGAGAGCTCTGACCTCCCAAGGCAGGCAGCTGGGGAGCCTCTTCCCAGCCTTCCA AGTCTACTGCTCGTCCTGTGGGACCCAGAGATCTTCACAGTCTCAATGGCACAAGACGGA ACGTTCACTTCCAGGCAACAATCCTGGGCCACAGGACTCAGTTCAGCCCCCAAGTATCAG ACTCCCGGCCCTGTTCTTGGTGTTCAGAGCCCACTAAACCAGCAGTCTTGCCACACAGCT TTGTCTCCAGAGGCCAGCTCATCTGCTTTTTTTCACACTACTCCAGCAACCCTGCTCCAC CAAGCACGCACACTCTCCTTGACACAGGGCTCCAGACCAACCACATGGCCTGTGCTCCTG AAAATGCCTGCCTGAAGGCCCAGGGAGTCCAGACACTGGCAGATGAGAATGGACGAAAAG AAGCCAAGCCAGAAGCCAGGAAGCAATAAATGCTAGACCCAGAAAGGCCTTAGGTTTGAG TGACCCAGCTCCATCCCTCCATCCTGGATCCTTGGAGGACAGCCCCAAGTTCAGCCTAGG AGCTCCCAAGGCCCCTCCACTACCTGCCAGCTCTCCGACAGCACAAGAGACACAGCAATC CCACATCAGTCAACAACCCACCGCCACAGGATTCCTAGGGGCAAGGCTCTGCCCCTCCCC CCAGCACATATGTCAGAGGGGTGGCTTCCCTGGGTTACACCCCTCCTGCTGTTGGGGGAG TGGGGGGTCGGGGAACACAGCTTTCCAGATGTGCTTGGCAACTCCTACTAGAAAAGACTA GGGGTGCGGGGGACAGGAGAAGACATGGATAAATAACTTTAACACCGCTCCAACCCATCC GGCTCAGCACTGGGCTCCCTTACACAGGCAGTCCCAGGCAGTTCTGGGTGGGGCCGTTGG GAGCATTATCTGGCCTCATTCCCTCCTCTCAGCAAGTCACTGCCCCCCAGGGAGAGGCAT TCCCCCCCCCCCCCAACCACGCACACACCCAGGCCTGTGTGAATATGAACCATCTTGGAA GATAAACAGAGAGTGACAAGCTGGGGCCCCTCAGCCCCCTCCCTCAGCCAGCCGGTCCCT TCCCCCTGAAAGCCGACCCCCTCCCCCAAGCCCATCTCTCTTCCTGCCAAGCGGCTGCCT ACAGGAGAGGTCTGGGGGCAGGGCAGCGAGCCCACCAGCCGCAGAGCCCGGCCCTCTGGG AGCGGCCCCTCCAGGCCCGCCCCTGCCCCTGCCGTGGCCAGTCTTCCCGCGGGGAGGAGG CAAGAGGAGAGGAGACCGCAGACGGTGCAGGACCGCAGTCCTGGAAATCGCAAAATCCTC TAGCGAGGGGGCGGCCGCGGGCGCAGGGCCGTTTGCATAATGGGAGCCCTCCCGCCTGTC AGGCAGCGCAGCTCGCCCGACGCTGTTCGGATTAGATTGCTAATGAAAAGGCACAAAGAG CCGGCGCCCGCTCGCCCGCCGACCCCCGCTCCGCAGACCCGCGCCGCCTGGGCCTGGCGC GGCCCGGTGGGCTTTGTGCCCCGGGTGCCCCACCGCCCGCGCCCCTCCGGGCATCTGGCC CACAAAGCCCGGGCCTGCCAGGGGTCCCGCGTGCGCCCCAGCCCAGGCCCGCCAGACGCT AGCGCGTCCCCCACGCGCGCAGTCCCACGTCGCCCGGGCGCGCGCGCGTCCACGCCCCTC TCCCCGGGGACGCGCCCAGCCCGCGGCCCCGGCCCCCGCCCGCTCCCCGCGACCCCCGCC CCCCAAGGCCGCCCCTCACCTCGTGTGCGTCGGCGGCGGCGCTCCGCCCGCCGGCCGGCC GGCCCTACCAGCGGCCCTTGTCCTCAGCGCCCGGCTCGCGCCGCACGCGCCCGCCCCGTC CGCCTGCCGCCAGCCCGGCTCGGCTCCCCGCCTAGCGCGCCCCGAGCGCCGCTCACAGCC GCCCGCCCAGCGCCATCTTGGAAGCTTGTGACGTCGGCGCCGCCCGCTCACCCCTGACCC ACATCTGAATGGGCGAGCGGCGGGGCGGGGACAGGGGGCAGCTGGGGGCGTGGCCTGTGA ACAGGGGCGGGGCCTCGGGGGCGGGGCCGGGCCGGACAGCGGTCCCAGCACTAGGCGGGC GGGCTGCCGGGGTCCGGCGCCGTGGGGAAGGGGTGCGCGGGAGGAGACGGGGACCCAGAC CCCGGACATCTAACCGGACTCCGACCTCAAGCGCCAGGGCAGGACCGCGACCTCGCCCCT GAAATACCCGAACCGCATACCGGCCCCCGGGACAGGGACCCTGGCCCCCCCCGACAGGCT GACGCCCACCCCCTCAAACTCTGGTGGACTTACCCCCTTTTAGCCCTACCCTGACCCCTA GGAGCCCCGAATTAGGGACCTCTATCGGCCTACGCGCCCCCTCCCCGACCCCTTTGCGAC CCCTGCTCGACGCTCCCTGCGGTTGCCCGAGGCTCAAAGGCGCAGCCAGCAGTGACTGCA AGCTCGGGGGTCTGGGCTCCTGGGGAAGCCCGGGCTGGTTGGGTGCAAAAGAGAAGGGGC GCCCCTCCCGTGACCCCAGCGCCCCTCGGGCCCCCGCGGGCGCACCCCCGCGAACCCTAC CTGCTCCGAGGGCGCGGAGGACCCAGCACGCTGCGCTCAGCCAGCCCCTTCCGGTGGCCG CAGCCCCTCGCAGGCCCCAGGGGTCAAGCGCCTGCCCGAGCCGGCCCACCAGGACCCGGG CTCCCGGCCGCCATGCAGATAGCCTTCCCAGGGGCTGGGCTGGCCTGAGCCGCCACTGCT TCTCTAGGGAGCTAGTTAATGGACCTCTCTCTACTTTGAACACCAAACAAAGGAACACAC CGCTATCGAGGTCGCCCAAGTCCAAGAGGAGCCCAGGTCTGCCTTACAGGGAAGTTGTGC CCCAGCTCCAGTCAAGATCAAAAACCAGTTTCAATAACCCTTCGCCAAGCTGATGATGCA ACCTTTCTTTCCTAATGGATGTATAGGTTTAATGTCATCCTAACAACATTTCTTAGACTT CACAAAATTCTAAGTTCACCTGGAAAAATAAATAGCCGAAAACAGCTATGGTTTTCCCCC CTTGGGAACAAAAGCAGGCATGAGGGTAGACTTGCCCTAACAGATATCAAAACAAATTGT AAAGCTACCGCAGGCATTTCAGTGTGGTACAGAACAGGAAATAGAATAGAAAGCCTAGAA ACATCCTAGAAATGGTAAGAACACAATAGATGAAAAAGGAAATATCAAACATCTGTGGAA AAAAAAGAGCTATCAAACATCCCTGAAGAAGAACAAGAATATTCCACAAATAGAGCCTAG CAGAGGACTCACTATTTGAAGGAACGACTGAAAAAAAAAGTCAACCATTCTACTAACCCA AGACATATAAATTAAAACTATAGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACT TTGGGAGGCCAAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACAC GGTGACACCCCGTCTGTACTAAAAATACAAAAAATTAGCCAGGCGAGGTGGCGGGCGCTT GTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCCGGGGGGCGGAG CCTGCAGTGAGCCGAGATCGTGCCACTGCACTCCAACCTGCGCGACAGCGAGACTCCGTC TCAAAAAAATAAATAAATAAATAAAATAAAACTATAAGAAGCTGGGTTTTCTCCTAAACA AATTAAAAATCAGTTTCAAGTGATAACCTTCAAACACAAGGTCTGATAGGTAATCACTCT CATATACTTTTGGAAGCACATAAATCACCTCAAAATTCCTGGACATGAGTTTGGCGGTAT GAGCCAAAAGTTGTTTTTTTGGGGTTCTTTTGTCTTTTTTTGTTTTTTGGAGACTCGGAG TCTCACTCTGTTGCCCAGGCTGGAGTGTAGCCTCAAACTCCTGGGATCAAGTGATCCTCC TGCCTTCCAAAGTGCTGGGATTACAGGCATAAGCCAACGCACCTGGCCCCAAAAATATTT TAAAATAATTATACCCTCTCAGACCAGTATTCCAGGATTCTGTTCTGGGGAAATTAGCCA CGTTTGCAGACCACGTACTTATTATCTGTCTCTCTACCCATAAGTAAGTTTGTACCTGTA AGTTTGGAACCCATGCCTAGGAGCACACCTGAGACAACAGGCATTCCACGGTCCCTTGTT TCATCTGCATTTGAAAACTAATGTGGCTGGCTCATGCCTGTAATCCCAACACTTTGGGAG GCCAAGGCCGGCAGATCGCCTGAGGTCAGGACTTTGAGACCACCCTGGCCAACATGGTGA AACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGATATCGCGGTGCATGCCTGTAGTC CCAATTACTCGGGAGGCTGAGACAGGAGAATTGCTTGAATCTGGGAGGCTGAGGTTGCAG TGAGCTGAGATCATGCCATGACACTCCAGCCTGGGCAACAGAGGGAGACTCGGTCTCAAA AAACAACAAAAAAGATAACTAATATATGATAATATTTTTTAAGTGTCATGTATAATAGTG AAAGAAAGCATATACAGGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAG GCGGGAGGATCACCTGAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTAAAACCCC GTTTCTACTGAAAATACAAAATTAGCTGGGCATGGTGGCGTATGCCTGTAATCCCAGCTA CTTGGGAGGCTGAGGCAGGAGAATTGCTTGAACTCCAGAGGCGGAGGTTGCAGTGAGCTG AGATCCTGCCATTGCACTCCAGCCTGGGCAACAGAGCAAGACTCCGTATCAAAAAAAAAA AAGAAAAAAGCATATACAAATTTCTCACAATGGGGTTAAATAGTCCACATAATGGAATGT AGATTAGGCCAGGAGTAGTGGCTCATGCCTGTAATCCCAACACTTTGGGAGGCCGAGGCG GGCAGATCACCTGAGGTCGGGAGTTCGAGACCAGCCTGACCATTATGGTGAAACCCCGTC CCTACTAAAAATACGAAAAGTTAGCTGGGCGTGATTGCGCATGCCTGTAATCCCAGCTAC TCGGGAGGCTGAGGTAGGAGACTTGCTTGAACCCGGGAGGCGGAGATTGCGGTGAGCTGA GATCGCACCATTGCACTCCAGCCTGGGCAACAAAAGTGAAACTCCGTCTCAAAAAAAAAA TTAGCCAAGCATGGTGTCACATGCCTGTAGTCCCAGCCACTCAGCAGGCTGAGGCGCAGC ACGAGAGTTGCTTGAACCTGGGAGGCAGAGGTTGCAGTGAGCCGAGGTGGTGCCACTGCA CTCCAGCCTGGGCGACAGAGTGGGACTCCATTTTAAAAAAATAAATAGGCCAGGCGCGGT GGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCCAGGCAGGTGGATCACGAGGTGAGG AGTTCAACACCAGCCTGGCCAAGATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATT AGCCAGGCATGGTGACGGGCACCTGTAATCCCAGCTATTCGGGAGGCTGAGGCAGAGAAT CGCTTGAACTCAGGAGGCGGAGGTTGCAGTGAGCCCAGATCACACCACTGCACTCCAGTC TGGGCGACAGAGTGAGACTCGGTCTCAGAAAATAAATAAATTAATAAATAAATAAATTAA ATTAGTAGGATGGGCACGGTGGCTCACTCCTGTAATCCCAGCACTTTGGGAGGCCGAGGC GGGTAGATCACCCAAGGTCAGAAGTTTGAGACCAGCCTGGCCAACATGGTAAAAGCCCGT CTCTACTAAAAATACAAGATTAGCCAGGCATGGTGACGTATGCCTGTAATCCCAGCTACT TGGGAGGCTGAGGCAGGAGAATTGCTTGAGCTCCAGAGGCGGAGGTTGCAGTGAGCTGAG ATCCTGCCATTGCACTCCAGCCTGGGTGACAAGAGTGAAACTCCGTCTCCAATAATAATA ATAATAATAATAATAAAGTAAATTTTTAATTAAAAAATAAAATTTTTATTTATTTATTTA TTGAGACAGAGTCTCACTCTGTCATCCAGGCTGGAGTGCAGTGGCATCATCTCAGCTTGC TGCAACCTCTGCCTCCCAGGTTCAAGCAATTCTCTGCCTCAGCCTCCTGAGTAGCTGGGA TTACAGGCGCCCACCACCACACCCAGCTAATTTTTGTATTTCTAGTACAGACGGGGTTTC ACCATCTTGGCAAGGCTGGTCTGGACCTCTTGACCTCATGATCTACCCGCGTTGGCCTCC CAAAGTGCTGGGATTACAGGCGTGAGCCACTGCGCCCAGCCAAAAATAATTTAAAAAAAA AAAAAAAAGAAGAGGCCAGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGAAGAC TGAGGCAGGAGGATCACTTGAGGTCAGGAGTTCGATACCAGCCTGGCCAACATGGTGAAA CCCCATCTCTACTAAAAATACACACAAAAATATTAGCCAGGCATGGTGGCACACCCCTGT AGTCCCAGCTACTCGGGGGACTGAGGCAGGAGGATCACTTGAACCCAGGAGGTGGACGTT TCAGTGAACCAAAATCACACCACTGCACACTCCAGCCTGGTCAACAGAGCTAGACCCTGT CTTAAAAAAAATTGTATTTTTTGTACAAAAAAATTAGCCGGGCCTGGTGGCAGATGCCTG TAGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATGGCGTGAAAACCTGGGAGGCGGAG CTTGCAGTGAGCCAAGATTACGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGATTCCG CCTCAAAAAAAAATTTTTTTTTAATTGCATTTTTTAAATGCATGCAAAAATATTGATATA TGGAAAAGCAACTGAAGAAAATAAATCAAAAATATTTACGGAGGTTCTCTCTGGAAGTTG GGAATTTTTTTTCTACACATGTATACTTTAGCGTGTGTGTTATTTTTATTTTATGAAAGT AACATTTCTAATCGGAACAATTCTAGAGATGCTGAAGCACATTTTATTTTTATTTTATTA TTTATTTTTGAGACAGAGTCTAGCTCTGTTGCCCAGGCTGGAGCACAGTGGCGTGATTAT GGCCCACTGCAGCCTCCACTTCCTAGGCTCAAGTGATTCTCCCAGCTGGGACTACAGGCA TGCACCACCACACCCGGCTAATTTTTTATTTTCTGTAGAGACAGGGTCTCACTATGCTTC CCAGGCTGAACTCAAACTCCCAGACTCAAGCAATTCTCCCACCTCAGCCTCCCAAAGTGC TGGGATTACAAGCATGAGACACCATGCCCGGTAATTTTTTTTTTTTTTTTGTGCAGATGG AGTCTCGCGCTGTCACCCAGGCTGGAGTGCAGAGGCGCGATCTCCGTTCATTGCAACCTC CGACTCCCGGGTTCAAGCGATTCTCCTGCCTCGGCCTCCCAAGTAGCTGGGATTACAAGT GAGCACCGCCACACCAAGCTAATTTTTGTAGTTTTAGTAGAGATGGGGTTTCACCATGTT GCCAGGGTTGTCTCGATCTATTGACCTCGTGATCTGCCCACCTCGGCCTCCCAAAGTGCT GGGATTACCGGCGTGGGCCACCGTGCCCAGCCCATGCCCAGCAATTTTTTAAAGTTAAAT TCTCCCTCCTCCTCCACCCTAATCAGTCCTTTCTGAAGTAACATTAACAGTTTAACTCTC TTCTCTGACATCAATATGTATCTACTCACCTTACAAAAAAGATCCTACTATTTTGAAATC ATAGACATTTTTCTAGTTCATTTCCATAATTCATTCTTTTTTTTTTTTTTCATGTCAGAC GGGTAATGTGCCTATGTCGTAACAAGATTTGAAGGTGGCGGCCGGGCGCGGTGGCTCACG CCTGTAATCGCAGCACTTTGGGAGGCCAAGGCGGGCGGATCACGAGTTCAGGAGATTGAG ACCATCCTGGCTAACACAGCGAAACCCCATCTCTACTGAAAATACAAAAAAATTAGCCGG GCGTGGTGGCGGGTGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATGGCAT GAACCCGGGAGGCGGAGCTTGCAGTGAGCCAAGATCGCGCCACTGCACTCCAGCCTGGGT GACGGAGCAAGACTCCCTCTTAAAAAAAAAAAAAAAAATATTTGAAGGTGGCACATCTTA CATGGGAACGTGTGTGAACACATAATCATCATGCTTATGAACTACAAAAGGATCATAATT CATTCTTTAAGAGCTGACAAATGTTCCAGAGTATGGAGCTTCTGTAATTTATTCACACTT CGTTAACCATGGTTGTTCAGTTTATTTACAGTGTGGTGCTTTTTGTTTTCTTTTTTCTTT GTTCTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCTTGCTCCTGTCACCCAGGCTAGAG CGCAATGGCACAATCTTTGCTCACTGCAACCTCCACCTCCCGGGTTCAAGCAATTCTCCT GCCTCGGCCTCCTAAGTAGCTGGGATTACAGGTATCTGCCACCATGCCTGGCTAATTTTT GTATTTTTCGTAGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTTGAACTCCTGACC TCAAGTGATCCACCCGCCTCAGCCTCCCTAAATGCTGGGATTACAGGTGTGAGCCACCGC ACCCAGCCTACACTGTGGTGCTATTAAAAAATATTTTTAGGCTGGGCACAGTGGCTCACA CCTCTAATCCCAGCACTTTGGGAGGCCGAGGCAGGTGGATCACTTGAGGCCAGGAGTTTG AGACCAGCCTGGTGATCATGAAGAAACCCTGTCTCTACTAAAATATTAGCCAGGCATGGT GTCACATACGGATAATCCCAGCTACTCGGGAGTCTGAGGTGGGAGAATCACTTGAACTAG GGAGGCAGACGTTGCAGTGAGTTGAGATCATGCCACTGCACCCCAGGCTGGACAACAGAG CGAGACCTCGTCTTAAAGTAATTAATTAATTAATTAATTAATTAATTAATATAAATAAAA TAGCAGATTTCAAAAATTTGTTCTCCCTGGCAGCAACCCAGTAAAGCTAGATCTGGGCGG AAGGTAATATGGAAATATAAAAACTACAGATTAGCAGGGATGGGAAGACCCGCCAGAAAT GGGTTCATTTTGTGTCCTCACTCCCTCCGGATTCTCTGTGTGTCTAGTCCAAGATCTAAC CACTATGATCTGCTTTCCTCATCCATTTGGGAATCAGGGCACCACCCCCTTGGGAGGCTT GAATGACATGAGGTTGAGCAGGCAGAGCACGGAACTCAGGCTCTGGTGAGCCCTGGTACA GTGACTATGATTGTTGTGTCATGTAGGAAGGGTCTGTTATTTTACCATTATCTGTTTTCA ATAAAAAACAGAAAGAAAACAAATATACAAAATACCCCCAAACAGTAAATTTCAGCTTCT TACTATGACAGCTAGATTTAGCATTTAACAGTATGCCCAAATTTGAATTTCGGATAAACA ACCAATAATGCTTAATATAAGTATGTCCCGGCCGGGCGCGGTGGCTCACGCCTGTAATCC CAGCCCTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCAAGACCATCCTGG CTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGCGGTGGCG GGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCCAGGA GGCGGAGCCTGCAGTGAGCTGAGATCGTGCCACTGCACTCCAGCCTGGGTGACAGCGAGA CTCCGTCTCAAAAAAAAAAAAAAAAGTATGTCCCATACAATATCACTTCTCACCTTCCCA TATCCCAAGCTCAAGACACCTTATATACGCCATGATTTCTCCTGCCATCTCTGGGCATTC ACACCAGGGCTCCTCCTGTCCAGGCCAGCCTTCTCCCTTCTCCCACCTGTGCCTGGTGAA CAACTCAGTTCAGACATCATCTCTTGTAGGAAGTCTCCTGTGATCCCAGGCTAATCACCC TGATACAGTCCTGAAATACTGCTGAGCAGTTGGCATGAGGGCAGGAAGGCTTGGTCTTGT TCACTCTTGTATCCAGCTTATACATGGCACAAAGTAGGTTCTCAATTAAAAGTCAAATGA GGGGCCAGGTGCAGTGGCTCACGCCTGTAATCCCAGCACTTTAGGAGGCCAAGGCGGGTA GATCACCTGAGGTTGGGAGTTCGAGACCAGCCTGGCCAGCATGGTGAAACCCCATCTCTA CTAAAAGTACAAAAATTAGCCAGACGTGGTGGCAGGCACCTGTAATTCCAGCTACTCGGG AAGCTGAGGCAGGAGAATCACTTGAACCCGGGAGGGAGAGGTTGCAGTGAGCCAAGATTG CGCCATTGCACTCCAGCCTGGGCAACAGAGCAAGACTCCATCTCAAAAAAAAAAAAAAAG TCAAATGAAGTCGGGCACAGTGGTTCGCGCCTGTAATCCCAGCACTTTGGGAGACTGAGG CAGGAGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAGCATATGAAATCCTGT CTCCACTAAAAATACAAAAAAAAAAAAAAAAAAAATAGCTGGGTGTGGTAATGCACGCCT GTAATCCCAGCTATTTGGGAAGCCGAGGCAGGAGAATTGCTTGAACCCGGGACACAGAGG TAACAGTGAGGTGAGATCATGCAACTGCACTCCAGCCTGGGCTACAGAGCAAGACTCTGT CTCAAAAAAAAAAAAAAAAAGTCAAATGAGACCACTCATGGTAGCTGGCGCCTATAATCC CAGCACTCTGGGAAGCCAAGGCGGGAGGATTGCTTGAGTCCAGGAGTTTGATACCAGCCT GGGCAAAACAGTGACATCTCATCTCTACAAAAAATTTTTAAAATTAGCCGGGCATGGTGG CACTCGTCTATAGTACTAGCTACTTGGGAGGCTGAGGTGGGAGGATTGCTTGAGCCCAAG AATTCGAGGTTGCAGTGAGCTATACTTGCGCCATTGTACTATAGCTGGGCAACAGCCAGA CGCGGCCTCAAAAAAAAAAAAATTTTAGCCGGGCTTGGTGGCATGTACCTATAGTCCCAG CTACTCAGAAGCTAAGACAGGAGGATCATTTGAGCCCAGGAATTTGACGCTGCAGTAAGC CAAGGTCACACCACTGCACTCCCGCCTGGGAGACTGAGTGAGACCCTGAGTCTAAAACAA ATAATAATAAAAACGAAAATGAAAGTCAAATGAATGAGGCCAGGGGTGGTGGCTCATGCC TGTAATCCTAGCACTTTGGAAGGCTGAGGTGGGTGGGTCAGCTGAGCCCTGGAGTTACAG ACCAGCCTGGGCAACATGGCGAAACCCTGTCTCTACAAATACAAAAAAGAAAAAAGCCAG GCCCATGGTGGTGAGTGCTTGAAGTCCCAGCTACTCAGGAGGGTGAGGTGGGAGAATAGC TTGAGCTCAGGAGGTCAAGGCTGCAGTGAGCTGAGATTGCATCACTGCAGCCTAGCCATG ATGACAGGGCAAGACCCAGTAGAAAACCAAGGTTAGAGAGGTTAAGTCACCTGCCCAAGG TCCGTAGCTAACCTCAGACTCCAGCCCCTCAGACTGACATGAGCTCAGTTCACCCTCTTA GGAACACCATCTGAAGAATGCATCCCCAGCAAGCCTATTCCCTATAGCAAAGGCCAGGCC TGTGATGGGTGCCCCGCTCCTCACCACACACCTCCCTGTCAAGTCACCACTCCTTCACCC CTAGCAGAGCCAGGCACCTTGAGCCCAAGGGAGGCCACTCATGGAACTGTCATTCAACCA ACAGATGCTGACAGCAATGATGAGGTGGGCACTGGGCTAGTGAACAGCACAGATGTGTTC CCTGGCCTCAGAAGCCCACAGACTAAGGAGAAGACGACAGAATACTCACAAGGGTGCTGG GGCTGGGCAAAAAGGTGCAGGCTGCTTTGTCTGGTCATAAGAGGGGAACTGACTTCCCTG GTCTGAGGAGCCAAGGAAGGGGCCGTGCCCTTGCCATGCCCTCTGCCTGCCATGCCTTGC TGTTCTTCAGTGCCTGCTCTAAGGTCACTCCTAGTGTGGTTTTCTCTGGCTGCTCTTTTT TTTTTTTTTTTTTTTTGAGACAGAGTCTCGCTTTGTCACCCAGGCTGGAGTGCAGTGGCA CCATCTTGGCTCACAGCAACCTCCGCCTCCTGAGTTCAAGTAATTCTCCTGTCTCAGCCT CCTGAGTAGCTGGGACTACAGGTGTGCACCACTACGCCCAGCTAATTTGTGTATTTTTAG TAGAGACGGGGTTTCACCATATTGGTTGGCCAGGATGGTCTCAATCTCTTGACCTCCAGA TCAACTCCCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGCC ACTTTTTTTTTGGAGTCTCGCTCTGTCCCCCAGGCTGGAGTGCAGTGGCGCGATTTCGGC TGACTGCCAGCTCCGCCTCCCGGGTTCACGCCATTCTCCCACCTCAGCCTCCCGAGTAGC TGGGACTACAGGCGCCCGCCACCACGCCTGGCTAATTTTTTGTATTTTTAGTAGAGACGG GGTTTCACCCTGTTAGCCAGGATGGTCTTGATCTCCTGACCTTGTGATCCGCCCGCCTCG GCCTTCCAAAGTGCTGGGATTACAGGCTTGAGCCACCACGCCTGGCCTCTTTTTCTTTTT TGTTGAGACAGATTCTCACTCTGTCGCCCCCAGGCTGGAGTGCAATGGCGCAATCTTGGC TCATTGCAACCTTGGCCTCCTGGGTTCAAGCAATTCTCTGCCTCAAGCCTCCCGAGTAGC TGAGATTACAGGCGCCCACCACCACACCCGGCTCATTTTTGTGTTTTTAGTAGAGACAGG GTTTCACCATCTTGGCCAGACTGGTCTTAAACTCCTGACTTCGTGATCTACCAGCCTCGG CCTCCCAAAGCGCTGGGATTACAGGCTTGAGCCACCGCACCTGGCCATGCTCTTTCTTTT TTATTTGTATTTTTATCTGTTTCTTAGAGACAGGGTCTCACTCTGTCATCCAGGCTGGAG TGCAGTAGTGTAATCATAACCCACTGCAGCCTCAAACTCATGGGCTCAAGGGATCCTCCT GCCTCAGTCTCCCAAGCAGCTGGGACTACAGGTGTGCACCACTATGCCCAGCTATTTTTT TTTTTTTTGAGACAGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGTGCCATCTCG GCTCACTGCAACCTCTGCCTCCTGGGTTCAAGTGATTCTCTGCCTCAACCTCCTCAGTAG CTGGATTACAGGTACCTACCACCATGCCAAGCTAATTTTTGTATTTTTTTTTTTTTTTGA GATGGAGTCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGCGCAATCTCGGCTCACTGCA AGCTCCACCTCTCAGGTTCACGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTA CAGGTGTCCGCCACCATGCCCGGCTAATATTTTGTATTTTTAGTAGAGACGGGGTTTCAC CGTGTTAGCCAGGATGGTCTCAATCTCCTGACCTCGTGATCCGCTGGCCTCGGCCTCCCA AAGTGCTGAGATTACAGGCGTGAGCCACCGTGCTCGGCCTAATTTTTGTATTTTCAGTAG AGACACGGTTTCACCATCTTGGCCAGGCCGGTCCTGAACTCTTGACCTCATGATCCACCG GCCTCGGCCTCCCAAAGTGTTGGGGTTATAGACGTGAGCCACCGTGTCTGGCCTGGCTCC ACTTTCTTAGGGAGCTTTGCTTGCCCTCTGCTTGGGGTAGTTTGTGAATTCAGTGCTCTC TCTCTCCCAGGTCCTGGGTCTCCTGCTCCTGATCAATCTGCCACGTGCCATTTGATCTCA GAGTTGTCTCCACCATTAGACTGGCAGGGTCTGTCTTCTGGGGTCTGTGTGTAGGATACA GTGGCCCCAGGATGGGTAAAGGAGGGGACCCAGTTGTTCTTCTGTGATAGGTGGAGACAA GGGCCAGCAGGGCTGTTTCTCGGTGAAACCCATGGGGAGATAGTCGAACCAGGAACCAAA TCCGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTAGGAGGCCGAGGTGGGC GGATCACCTGAGTTCGGGAGTTGGAGACCAGCCTGACCAATATGGTGAAACCCCATCTCT ACTAAAAAAAAATATATACATATATATACAAAATTAGCCTGGCGTGGTGGCACATGCCTG TAATCCCAGCTACTCGGGAGGCTGAGGCAAGAGAATCACTTGAAACTGGGAGGCAGAGGT TGCGGTGAGCCAAGATCGCACCATTACACTCCAGCCTGGGTGACAGAGACTCAGTCTCCA AAAAAAAAAGTCCAAAAATTAAAAAAAAAAAAAATGCCGAACTCAGTGGCTCACGCCTAT GATCCTACCACTTTCAGAGGCCGAGGCGGGCACGGTGGCGCGTACCTGTAATCCCAGCTA CTCCGGAGGCTGAGGCAGAAGAATCACTTGAACCCGGGAGGCAGAGGTTGCAGTGCGCTG AGATCGTGCCACTACACTCCAGTGTGGTGACAGAGCGAGACTTGGTCTCAAAAAAAAAAA AAAGTCCTGGCTGTGCCACTAGGCCAGACGCCCTAGAACTGACTTCTATGGACCTCAGTT TTCATACCTGTGAAATGGAACTCAGAGCCATATCTACTTCATTGGGTTGTGTGAATGTGA GGTAGTTACAGGATGGAGCAGGCAGAAGGTCAGCACAGCCTCAGTGGTTGGAGGAGTGGG GGTCAAGTGGACAGAGAGGGTACCCCAGGGCCAGAGGTAGAGGCAAGGTGAAGCAATGCT TCATTTCCCTGCTGAGGTTCCTCTCTAGCAGTTTGGAGGTGGGGCAGGAGTGGAGAAAAC ACATCCCCTCCTCAATATGGGCTGAGGAACAGGGTTCTTGAGGCACCAAGAACCTCTTTG GGGCTCTGGCTTGCCTCCTGTAGTGAAAGGGATGGGCTGTGTGCAATGAACTCACACCTG TAATCCCAACACTTTGGGAGACAGAGTCAGGAGGTCCAGAACAGCCTAGGCAACATAGCA AGACCTTGTTTCTACAAAAAACTTTTTAAAAAATTAGCCAGGCGTGGTGGTGGCACCTGT AGTCCCAGCTACTTGGGAGGCTGAGGTGGGAGGATTGCTTGAGCCTGGAGAGGTTGAGGC TGAAGTGAGCTATGATAGCACCACTGTACTCCAACCTGGGCAACAGAGTGAAACCCTGTG TCTAAAATAAAAAGAGGGCCTGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTAGGA GGCCGAGGCAGGCAGATCACTCGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTG AAACTCTGTCTCTACAGAAGTACAAAAATTAGTCGGGCGTGGTGGCAGGCGCCTGTAATC CCAGCTGCTTGGGAGGCTGACCTGGGAGAATCTCTCGAACCTGGGAGGCAGAGGTTGCAG TGAGCTGAGATCACACCATTGCACTGCAGCCTGGGCAACAAGAGCGAAACTCCGTCTCAA AAAAAAAAACAAACAAATAATAAAATAATAATAATAATAATAATAATAATAATAATAATA ATAAAGAGGGTGGGAGTGGACCAAGGCCTAATGGCATGGAATTTGCAAACTTTATCTTCC TAGTTTTCTCTGGGGAGCCTTCCTCCATCACCAGGGCCCAGCAGGGCCTCAGATGGCGGG GGGTGGGGCGGGGGCTACACATATCCCTCATGGCCAAGATGCTAGGAAGCCCCTGAGCTG GGGCAACAGCCTCTGGTCACAGTCATGAGGGAGGCTGGGTGCAGTAGCGCTTATCTTGCC TGACCTTGTGGAGACTGACCACTCCCTCCTTCTGGAAAACGTTGCCCCCTGCCCTCTCCA AGGCCTCCTCCTTCTTACATCCCCTGGTCCTGGCTAGGGCCTCAGCACCCAATTCTACTC CTTGACCCACCACCCTGTGGTGCTTTTGCCGGGCCCCAGCCCACAGCCATCTGCCAAACA CCTCCCCCTGGAGTCCCTGCGGCGTCTCTGACTCAGCAGGTCTGTGCCTGCCCCTGCTGT TCCCCCCACCCAGTGGCTCTGCAGAGCACCCTGTTGCCCAAGCCCTGAGTACTGGCCACT CCCAGGGCCTCGCCTTCCCCCACCACCAGCACCACCAATTTGAATTCCTATCTCTCCCTC TGGCCTCCTCCCTAGACCCAGGCCCTGCCCCAGGGTGGGGGAAGGAATCCTACAGATAAG GGCATTGGTCAAAGGGACACACCTGGGTTCGGGCCCCCAGCTCTGCCCCCAACCTGCTAA ATGACCACACCCCTCTCCAAATCTGAGTTATTCCCTATGTAAAAGGGGGAAATAATTAAA CTAGCTGGGTATGCTGTGCTGAGCAATATGTGAGCTCAGGAAAGGTCAGTTTTCCAGGGG AAGTATGGCCGCTGAGAGCCTCAGTCTAGTGCTTCTCATTCACGAGCTGGTTGGTGCCTT TCCTAAGTTGGAAATGGGTTAGTCCACAAATCCCGTTGCCTGGTCCATGCCAGGTCCAGG TACCTGCATTCAGGGTGCAGGGTGGAGTAAGACAGTCTGAAGGGTGAGGTAGCAGGATGA ATGAGTAATTCCTGGGTGCGTTTTGGGATTATGCCTAAGGGCTGTCCGAGCACGTGGGAG GGGTAGCTAAATCCTGTAAGGGAGGGGAGAATGGGGAAGGCTGCCTGCAAGAGGTGGTGG AGGGTGCTTTCAAAGGATACCTGGGAGTGAGCTGGCTGTAGGCACAGGGCAGCTGGAAGG CCAATTTTAGGGAAGAGGAAATTGAGGCTGAAAGTCAAAAAACAAAGGAACTGCCTGAGG TCTCTCAACTGAGAAGTGAGTGGGCCAGGGTTCACATTCAGCCCCCACTCCATTCTCAGG CTGGGCAGGAAGGTACTTTGCCCTCAGGAGGGCACCTTCGACCCAAAGCACCCTGACCAA GAGGCTGTGCACCGTGGCAACCCTAAGACTGAGGCCCAGGTCCTGGAGTTCCACCAAGGT AGGTACAACCAGCCAGGCCGTCAGTGACAGGAGGATTTACTGGGGCTTCTAGGGACCCCG TGAAAGGGCAAAAAGACGGCACAGAAGCCTGTGGATATCAGATATAGATAGTGAGGGCTA GGGCTTTATGGGGAGGGTGGATGGGCCCTGTGGGCGGGGGATGTTTAGGAGATCTTCCAA ACAGAGCATTCCAGCCTGGGGGAACAACCCATTATAGACCCCACCCAGCACCGAAGACAG GGACAGGTGGGGCTGAGCCTGGAGGCTGGCAGGACCTGTGCGGCCGGCTGCGGTGTCAGC AATGCCACCGTGTGTGGAACCCTCTGTGCCTAGGTTCCTGGAGCCAGCTCCACCTGGCAA TGTGCTTTGTTTCACAAGACTAGAGTTGGCCCATCTTGTTCCGCCTCAGATGGAACACCT GACCTCCTGCACACCCCACCCAGCCTCCAAATTCAACCACCCCAGTAGTGAGACAGGGAG AGTAGGGTGTGACCAGAAGGTGGAATGAGCTTAAAATAGGCTGCAGAGATATCTCTGAGC TTTTAAAAAGTCGTATCTTTCATATCGTGTTCTTGTGAAATCTTCTCCAGATTTTTTACA ATGACTCTGAAATTGATCAGTTAGCCTCATGCTTTCAAAATCCATAGCCGCAGTGAGCGA GAGGGGGCAGGTCAGCTAGAGGTGGAGGGGGCAGATGGGCCAGAGGCGCCTGGGCAGGGG TGGATGGGGCCTGGACAGGCTTAGTGACCCTTGCACCAGAACTCTCCGGACACCGCCCCT TCGCCCACTCGTTAGGCTCCTCTGCCTTCGGGCCGGCTGGGCTGCCCATCCCCCCTCGTT CCCCTCCTGGGCAGGTCTGGACTGGACAGGTTTTGGGCCTCCAGGTCGTGCAGTCAGTCA GGGAAGGGGCCTGGTGCAGGCCACAGCCAGGCATCCACCTCCTTCCCAGCCCACCCCTAC CCCCCAGCAGCCTGCCAACTTGCTAGACCTGTGCGGCCTTCCCTTCTGCTGAAAGTCTCC AGATTCTCAGGGCTCTGCACGGGGAGCCAAGCTGGGGCTTAGAACTGTGCTCCCTTGCCC CCACCCCCGACCTGTGCCCTCATCAGCTTTCTTTTTTTTTCTTTTTTCTTTTTTTTTTTG AGACACAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGTGCGATCTCAGCTCACTGC AACCTCTGCTTCCCGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGAGT ATAGGTGCGTGCCACCACACCCCGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCA CCGTGTTGGCCAGGATGGTCTCCATCTCCTGACTTGGTGATCCACCCGCCTCGGCCTCCC AAAGTGCCGGGATTACAGGCGTGAGCCACCGCGCCCGGCCTATTTTAAATTTTTTTTGTA GAGACAGAGTCTCACCATGTTGCCTAGGCTTGTCTCGAACTCCTGGTCGCAAGTGATCCT CCCCTCTCTGCCTCCGCCGTAGCTGGGATTGCAGGCACAAGCCATGACGCCCCCCTTCTC ATCAGCTGTCTGGGTCTTTCTCGGGGTCCTGACACCAGGCCAAGTGTTCTGCACACCTGA CCTGCTCAAAACAAAACCAGTTCTGCCACAGTCTGGGAAAATGAGGCTCAGAGAGGCCTT GCAGCTGTCTGTGAGCAGGCTGGCATTTGGATCCAGTGACTGGGTTCTGGAGCCCAGTGG CTCTGTGGCCCTGCCTCTCCAGCCTTATGCTTCTTTAAAACTGCCCATGGCCTCTGAAGT TGAGGATTTAGGGGAGCAGGGCTGCCAGAGGGGTGAAGAAGGAGATCTTTCTGTGGGTGG GTGTCAGAATGACCAGGCCTGACACTGGTCCCTGCTCCCCTGGAAGGACAGCCCCTCTCG GTTATGTGGGGGGTGGGCTGCCTAGGGTGCTCTGGGGAGAAGGGAGGGAGAGAATGGGGC TGGTCGGGTGGGGTGTGGATTCCAGGCCTGCTTGGTCAAAAAGATACAGTATTTCCTATT CCCCCTTACACTGGGCAGGCAGGTGTGGCCCAGGCAGCTCTGCCAGGTGTGCAGCCCCTG GAGAATGACCCTAAAGTGCACCTAGGCTGCCCCCTGGCGGCAGCATGAGGTCCTCCTCCC TGGACTTGTAACCCTCCCAGGGGGAGGAGGTCCACACCAGCTGATGGGTTGGCACCTGTC CCCAGTCCCTTTCTTCTTTCCTGTGCCACTGCTCCCAGAGTTGCCCAAAACACACGATCA GGTGCAGTGGCTTGAACGAAACCCTGTCTCTACGGAAAATACAAAAATTAGCCAGGCATG TTGGCTCATGCCTACAGTCCCAGCTACTTGGGAGGTTTAGGTGGGAGGATCGACAGCCCA GGAAGTCAAGGCCGCAGTGAGCAGTGAGCTGTGGTTGCGCCACTGTACTCCATACTGTAG CCTGGGTGACAGAGTGAGACCTTGTCTTTTTTTTTTTTTTTTTTTTGAGACGGAGTCTCA CTGTGTCATCCAGGCTGGAGTGCAGTGGCGCGATCGGCTCACTGCAAGCTCCGCCTCCCG GATTCATGCTATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTATAGGCGCCCCTCAG CCTCCTGAGTAGCTGGGACTACAGGCGCCCGCTACCACGCCTGGCTAATTTTTTTGTATT TTTAGTAGAGACGGGGTTTCACTGTGTTAGCCAGGATGGTCTCGATCTGCTGACCTCGTG ATCCACCCGCCTCGGCTTCCCAAAGTGCTAGGATTACAGGCGTGAGCCACTGAGCCTGGC CGAGACCTTGTCTTAATTAAAAAAAAAAAAAAAAAGATAAAAACCTAGGTAACATGCTGG GGGGTGATGAGGGTTATGGAGGAAAATAAAACAGGTGGGGGCTGGAGTGAAGAGTGGGGG TGCCCCTCATAACTCTGACAAAGACTACAACGCAGCTGTCAGAAAAAAAGCTGATTTGAG GCAATGGTCAGGGAAAGTTGTGAAATTAAGTAAAAAGTGATGGAACAGGCTAGGTGGGAA GTGGTGGCTCACACCAATAATCCAAGTGCTTTGAAAGGCCAAGCAGGAGGATCTCTTGAG GCCAGGTGTTTGAGATCAGCCTGGGCAACATAGCAAGACCCTGTCTTTACAAAAAATGTA AAAATTAGCTGGAGGCTAGGCTCAGTGGCTCATGCCTGTAGGGAGGCCAAGGTAGGTGAA TTGCTTGAGCCCAGGAATTTGAGACCAGCTTGGGCAACATGGCAAAATTCCATCTGTACA AAAAAATTGCCAGGCATGGTGGCTCATGCCTGTAATCCTAACACTTAGGGAGGCCAAAGT GGGCAGATTACCCGAGGTCAGGAGTTTGAGACCACCCTGGCCAACATGGTGAAACCTCGT CTCTACTAAAAATACAAAAATTAGCTGGGCGTGGTGGCGCATGCCTGTAATCCCAGCTAC TCGGGAGGCTGAAGCAAGAGAATTGCTTGAGCCTGGGAGACAGAGGTTGCAGTGAGCCTA GATCGTGCTACTGCACTCCAGCCTGTGTGACAGAGCAAGACTATGTTTCAAAAAAAAAAG AAAAATTAGCTAGGTGTGGTGACATGCACCTGTAGTCCCAGTTACTCAGGAGGCTGAGGT GAGAGGATCACTTGAGCCCAGGAAGTTGAGGCTATGATCGTGCTACTGCATTCCAGCCTG GGTGACAGCCAGATTCTGTCTCAAAAATCAAAACACAACAGGGCACAGTGGCTCACTTTG GCCTTCCAAAGTGCTGGGATTACAGGTCTGCACCACCATGCCTGGCCTGAGAGTGCATGA ATTAAAGTTTCTATTCTCTTGCTAGTCTGTCATTGCTTCCCTAGTGTGTGGGAACCATAC TTGTCCCCACTTCTCAGTCAGAGAGCAGCCTGCCAACCTCCAAGTCTCTTGCCCCTGACC CAAAAGGTCCCAGGCTGTCCTCTCGGCAGAAGCAGCTGTCTTGTGGCTTACTGTGCTCAG ACTCCATTTCTAGGCTGTGGGCTCATCAGGGATCTAGTGCTTTGAAAAAGTGTAAAGGAG AGCAGCAGTATTATTCACAATAGCTAAAAGGTAGAAACGACCAAAGTATCCATTGACAGA TGAATGGATAAACAAAATGCATTACATACACACAATGGACTATTATTCAGCCTTAAAAAG GAAGGAGGCCAGGTGTGGTGGCTCACACCTATAATCCCAGCGCTTTGGGAGACTGAGGCA GGCGAATCACTTGAAGCCAGGAGTTGGAAACCAGCACAGACAGCATGGGGAAACCCCCTC TCTACTGAAAATACAAAAGTTAGCCAGGTGTGGTGGCACATGCCTATAATCCCAGCTACT CTGGAGGCCAAGGCATGAGAATCGCTTGAACCTAGGAGGTGGAGGTTGCAGTGAGCCAAG ATCATGCCACTGCACTCCAGCCCGGGTGACAGAGTGAGACACTGTCTCAAACAAACAAAC AAAAAAAAAGAAACGTGTGGTGGAAGAAGGGAAGAAAATTGGCCAGGCACACTCTGGTAG GGGATGGTTGGATGATGAAATACAGACAATAGAAAAACCTAGATAAAGACCTGGCGTAAT GGCTCATGCTTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGAAGGATCTCTTGAGGCCA GGAGTTTGAGACCATCCTGGTAAATATAGTGAGACCCCAGTCTCTTAAAAAATAATAATT GGCTAGGCGCGATGGCTCACGCCTATAATCCCAGCACTTTGGGAGGCCAAGGCGGGCGGA TCACCTGAGGTCGGGAGTTCGATACCAGTCTGGCCAACATGGAGAAACCCCATCTCTGCT AAAAATACAAAATTAGCTGGGCGCGGTGGTGTGTGCCTGTAATCCCAGCTACTCGGGAGG CTAAGGCAGGAGAATCGCTTGAACCTGCAAGGTGAGGTTGCAGTAAGCTGAGATTGTGCC GTTGCACTCCAGCCCAGGCAACAAGAGTGAAACTCCATCTCCAAAAAATAAAAATAAAAA TAAAAATAAAAAAGAAATAGGCCGGGTGCAGTGGCTCACGCCTATAATCCTAGCACTTTG GGAGGCCAAGGTGGGGGCGGGGTGGATCACTTGAGGTCAGAAGTTCGAGACCAGCCTGGC CAACATGGTGAAACCCAATCTCCACTAAAAACACAAAAAATTAGCCGAGCATGGTGGTGG GCACCTGTAATCCCAGCTACTCGAGAGGCTGAGGCAGGAGAATGGCTTGAACCTGGGAGG CGGAGGTTGCAGTGAGCCAAGATCACCCCACTGTACTCCAGCCTGGGTGACAGAGTGAAA CTGTCTCAAAAAATAAATAAATAAATAAATAAAACTTTTAAAAAGTAAGAAGAAGAAGAA AAAAAATATATGGAAATTAAAAAACAAGAAAAAAATAATAGGCCAGGTGCAGTGGCTCAT GCCTGTAATCCCAGCACTTTGAGAGGCCGAGGTGGGCGGATCATGATGTCAGGAGTTCAA GACCAGCCTGGCCAACATAGTGAGACCTCGTCGCTACTAAAAATAGAAAAAAATTAGCCA GGCGTGGTGGCGGGCACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCACT TGAACCCATGAGGCGGAGGTTGCAGTGAGCCGAGATTACGCCATTGCACTCCAGCCCGGG TGACAGTGCGAGACTCCATCAAAAAAAAAAATAAATAAAATAAATAAATATTAATAATAA TTTTAAACAATTAAAAAATATGGGATTTTTTTGAGACAGAGTCTCACTCTGTCGCCCAGG CTGGAGTGCAGTGGCATGATGTCAGCTCACTGCAACCTCCGCCTCCTGGGTTCAAGTGAT TCTTCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACGCGCCACCACGCCCAGCTA ATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATATTGGCCAAGCTGGTCTGGAACTC CTAACCTTGTGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGAACTATAGGTGTGAGCCA CTGCACCCGGCCAAAAAATATGTTTTTTAAAATAATAGAGATGAGGCCGGGTGTGGTGGC TCACACCTGTAATCTCAGCACTTTGGGAGGCCAAGGTGGGTGGATCACTTGAGGTCAGGA GTTCGAGACCAGTCTGGGCAACATGGTGAAACCCTGCCTCTACTAAAAATACAAACCTGA GCTGGGCATGGTGACGCATGCCTCTAGTTCCAGCTACTCGAGAGGCTGAAGCAAGAGAAT CGCTTGAACCCGGGAGGCGGAGACTGCAGTGAGCCAAGATAGCACCACTGCACTCCAGCC TGGGAGACAGAGCAAGACCCTGTCTCAAAAAATAAAATAAATAAAATAAAATAAAATAAA ATAAAATAAAATAAAATAAAATAAAATAAAATAAAAAATAAAATAAAATAAAATAAAATA AAATAAATAAAATAGATGAAGGCCAGGTGCAGTGGCTCACGCCTGTAATCCCAACACTTT GGGAGGCCAAGGCAGGCAGATCACCTGAGTTCAGGAGTTAGAGAGCATCCTGGCCAACAT GGTGAAACCCTGTCTGTACTAAAAATACAAAAAAATTAGCCAGGCATTGTGGCGGGCGGC CAATAATCCTGGCTACTTGGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGAGGCGGA GGTTGCAGTGAGCCAAGATGGTACTACTGCACTCCAGCCTGGGTGACAGAGCAAGACTCT GTCTCAAAATAATAATAATAGGCCGGGCACGGTGGCTCACACCTGTAATCCCAGCACTTT GGGAGGCCGAGGCGGGCAGATCACAAGGTCAGGAGATCGAGACCATACTGGCTAACATGG TGAAACCCTGTCTCTACTAAAAATACAAAAAATTAGCCGGGTGTGCTGGTGGGCGCCTGT AGTCCCAGCTACTTGGGAGGCTGAGGCAGGACAATGGCGTGAACCCGGTAGGCGGAGCTT GCAGTGAGCCAAGATCGTGCCATTGCACTCCAGCCTGGGCGACAGAGCGAAACTCCACCT CAAAAAATAATAATAAAATAAAATAAAAATAAAAATAATAATAATAGAGATGAGGTTTTG CCATGTTACCCATGCTGGTCTCCAACTCCTTGGCTCAAGTGATCCATCCACCTTGGCCTT CCAAAGTGCTGGAACTACAGGCTTGTGCCACCACCCTGGCCTTAAACAATTATTCATGAA AGGAAGGAAATTCTGACACATGCTGCAACATGGATGAAGCTCAAGGACATTATGCTAAGG GAAGGAAGCCAAAGACCTGCCAGCCGAGGTCCCAGTCCGTCAAGGGCTCCAGTGAGCGAG CAGGATTGAGCAGGTCCCTGGGCTGAGCAGTGGGAACTCTGCTTTGCTGTGAGTGTGGCA CGGGTGGCGGCAGACTGTGGAGTGCAGGCTCTGTGGAGCAACTGGACACTCTGTTGAACG AAGTGCAAGGTGGTAGGTTTTTGCTTTTTTTTTTTTTTCTGAGACAGAGTCTCACTCTGT CTCCTAGGCTGGAGTGCAGTGGCATGATCTCGACTCATTGCAACCTCTGCCTCCCGGGTT CAAGCAATTCTCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCGCGAGCCACCACAC CCAGATAATTTTTAAAATATTTTTGGAAGAGACGGGGTTTTACCATCTTGGCCAGGCTGG TCTCGAACTCCTGACCTCGTGATCCACCTGCTTCAGCCTCCCAAAGTGCTGGGATTACAG GCATGAGCCACCGCGCCCGGCCACAAGGTGGTAGTCTTTCTCAAGGACGCTGGGGCAACC CCATTGCGTCTTCACCAACACCCTGGTACAGATCCTGCAGCTGCAGTCTACCCCGCATAC AACATGTTCAATGATCCCTAGCTAGACAAGGCATTGAAAACTATTCCATAGGCCGGGTGC AGTGGCTCCTACCTGAAATCCCAGCAGTTTTGGAGCAAGGCGGGTAGATCATCTGAGGTC AGGAGTTCGAGACCAGTCTGGCCAACATAGCGAAACACCGTCTCTTCTAAAATACAAAAA ATTAGCTGGGTATGGTGGTGCATGCCTGTAATCCCAGCTACTTGGGAGGCTGAGACATGA GAATCACTTGCACCAGGAGGCGGAGGTTGCAATGAGCCGAGATCACACTACTGTACTCCA GCTTGGGTGACAGAGTGAGACTCTGTCTCAAAAAAAAAGAAAAAAAAAATTAGCTGGGCA TGGTGGCATGCACCTGCAGGCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAA CCCAGGAGGCTGCAGTGAGCCAAGATCGAACCACTGCACCACAGCCTGAGTGACAGAGTG AGACTCCATCTCAAAACAACAACAACAACAAAAATCAACTATTCCTTGGGAGGCAGAGGC AGGAGGATGGCTTCAGGCCAGGAGTTGGAGCCAAGCTAGGCAACATAGCGAGAATTCCTC ATCTCTACACACACACACACACACACACACACACACACACACACATTAATTAGCTGGGTG TGGTGGCACATGCCTCCAGATACTCAGGAGGCTGAGCGGGAAGGATGGTTTGAGCCCTGG AGGTTGAGGCTGCAGTGAGCCATGATCACGCCATGACAGAATGAGGACCAGGTGGAAGAA CTGAAAACGCTGGGGATCCACACATCCGCTCCATCCTTTCAGATGGAAAGAAATACCAAG ACTCAAAAAAATGAGGGTGCCCAGGTCCTCACTGAGCAGAGACTCACTGCTAAAAAAAAG CCTTACCTATTTGGGTTTTCACTAGTAAGCAGTTGGTTTGTAAGCAGTTGGTGATTTTAG TTTGTCTGGGTTTCAGCCATGAATATTCTATTGTAAACTTAATTATAACAACTGCACTGT AATAATTCAATGTCCTATTATGATGTTGTTATAGACAAAATTTGCCTTTACATTGTCATT TATTTTATTTTATTTTTCTTTTGAGACAGGGTCTCACTCTGTCACCCAGGCTGGAGTGCA GTAGCTCAATCTTGGCTCACTGAAACCACTGCTTCCCAGGCTCAAGCGATTCTCCCACCT CACCTTCTCGAGTAGTTGGGACCATAGGTGTGAACCACCATATCAGGCCAATTTTTGTAT TTTTAGTAGATACGGGGTTTCAACATGTTGCCCAGGCTGGTCTTGAATTCCTGTGCTCAA GCGATCCACTTGCCTCGCCTCCCAAAGTGCTGAGATTACAAGTATGAGCCATTGACATTT AATCTTCCTTCCTTCCTTCTTTCCTTCCTTCCTTCCTTCCTTCCTTTTCTTTTTTTGAGA TGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCACGATCTCGGCTCACTGCAAG CTCCACCTCCTGGGTTCAAGCATTTCTCCTGCCTCAGCCTCCGGAGTAGCTGGGACTATA GGCGCAGGCCACCATGCCCAGTGATTTTTTATTTTTATTTTTTTTGAGACGGAGTCTCAC TCTGTCACCAGCCTGGAGTGCAGTGGCGCATTCTTGGCTCACTGCAACCTCCACCTCCAG GGTTCAAGCAATTCTCTGTTTCAGCCTCCCGAGTAGCTGGGATTACAGGCACCCACCACC ACACCCAGCTAATTTTTGTATTTTTAGTAGGGATGGGGTTTCACCATGTTGGTCAGGCTG GTCTTGAACTCCTGACCTCGTGATCCACCAACCTCGGCCTCCCAAAGTTCTGGGATTACA GGCGTGAGCCACCGCACCTGGCCTACTTTTTTAAACTTAAATTTTAAAAAAAATTAAGTT TATTTTTAATGTGATTTATTTATTTATTTATTTTTTAGATGGAGTCTTTCTCTGTCGCCC AGGCTGGGGTGCAGTGGCACTATCTTGGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGT GATTCTTCTGCTTAGCCTCCCAAGTAGCTGGGGCTACAGGTGCATGTCACCACGTCCAGC TAATTTTTTTGTATTTTTAGTAGAGACAGTGTTTCACTGTATTGGTCAGGCTGGTCTTGA ACTCCTGACCTTGTGATCCGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGTGTGA GCCATGGCACCCGGCCTATTTATTTATTTATTTTTTAATTTTTTTTGAGACGGAGTCTCG CTCTGTCACCCAGGCTGGAGTGCAGTGGTGCAGTCTCGGCTCACTGCAAGCTCCACCTCC CGGGTTCATGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGCCCGCC ACCACGCCCAGCTAATTTTTTTGTATTTTTAGTAGAGATGGGGTTTCACCGTGTTAGCCA GGATGGTCTCGATCTCCTGACCTCGTGATCTGCCCACCTTGGCCTCCCAAAGTGCTGGGA TTACAGGCGTGAGCCACCGCGCCCGGCCTCTTTCAATCATTTCTGATGACACCGGAGAAG GCAGTGTCCTTTCCTTATTGTCTCTTTTTTTTTTTTTACCTGGGTATTCTGGAAGTGTCT TTCCTCTGACAGTGTTGTTGGACGTGGGGAAACAAAAGGGTATCAAAGAGAGGGAGAGAA GGATAAAGACCACCCCTCATTCCCGCCCTCCCAGAGGGGCAGCCTGGGGAGTCTATGGAG GAGGTGACTTTGAGCCATGATGTGACCTTAGATGAGGGGCCTCTCTGAGCCTCAGGAAGC TCTTCTGCAAAATGGGGCAAATAAAGAAATGCCCTCTGTGTGCAGTGGCTCAGGCCTGTA ATCCCAGCACTTCGGGAGGCCAAGGCAGGAGAATCACTTGAGCGCAGGAGTTCCAGACCA GCCTGGGCAACATAGTGGGACCCCATCTCTACCAAAAAATTTAAAAATTAGCCAGGCATC AGGTGTGGTGGCTCAGGCCTGTAATCCCAGCACTTTGGGAGGTTGAGGCAGGCAGATTGC TTGAGCCCAGGAGTTTGAGACCAGCCTGGGCAACACAGTGAGACCCCTGCTTCTACACAC ACACCAAATTAGCCAGGTGTGGTGGTGCACATCTGTAGTCCCAGCTACTTGGGAGGCTGA GGTGAGAGAATCATTTGAGCCCTGAAGCTCAAGACTACAGTGAGCTGTGATTGTGCCACT GCCCTCCAGCCTGGGCAACAGACCAAGACTCTGTCTCAAAAAAAGGAAAGGTCCAGGCGC GGTGGCTCATGCTTGTAATCCCAGCACTTTGGGAGGCTGAGGTGGGCGGATCATGAGGTC AGGAGATTGAGACCATCCTAGCTAACACAGTGAAACCCCGTCTCTAGTAAAAATACAAAA AATTAGCCAGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGG AGAATGGCGTGAACCCGGGAGGCAGAGCTTGCAGTGAACTGAGATTGCACCACTGCACTC CAGCCTGGGCAACAGAGCAAGACTCCATCTCAAAAAAAAAAAAAAAAAAAAGTGGAAAAG AAGGCCGGACGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCA GATCACGAGGTCAGGAGTTTGAGACTAGTCTGGCCAACATAGTGAAACACTGTCTCTACT AAAAATACAAAAAATTAGCCAGGTGTAGTGGTGTGCACCTGTAATCCCAGCTACTCGGGA GGCTAAGGCAGGATAATTGTGTGAACCCGGGAGACAGAGGTTGTGGTGAGCCGAGATTGT GCCATTGCACTCCAGCCTGGGTGACAGTGCGAGACTCTGTCTCCAAATAAAAAAAAAAAA GGAAAAGAAATTCCATCCCAAGGTGATCCCTCACAGGAAGTGACTCAGAAAAGCAGTGAG CTACGAAAGCTATCTGAATGAAGCAAGATCGTCATGTGGGAGACACTGACTACCCACTCC TAGCAAAGGGCTCAGCAGCACACGGCCTCCCTGCGGGGATGCCCTCGGGGAAGATGTGGC CCAGAGGAGTTTTTTGGGCCTTGCTCCTCAGTCCTGGCTCTTAGTAGGACCCTCTGCAGC CAAACTAGCATATCCTGAGCCAGGCTGACCTTAAAAAGTAAGCATTCAGGGCCTAGGGAC CTTGGGCAAAGCAGAGAAGCTGGTGTCAGATGAGGTGCAGCAGGTACAGTAGGATAAGGT GTTCTCAGGTCGCCAGTGCAGCCCCAAGATGAGCCTGCAGTATTTTCCTTACATGATCTG GTCCTACTGTGGGCAGCGCTGCTGCCCAGAGCCTGAGAGGATTATGAAAACATGGCAACG GAAGTGAGGCCAGGGGACACAGCATGGGGGCATGAGGAGCAGGGGACAGCGGGTGGGGCT AGAGGAGAGGGAGGTTTAGGGAGGCCTCTGCTGCTGTGACTGTGAGCCCCAGCCAATGAT GACGTGGCCACCGCACAATCTGAGTTCTGGGAACCATGTGATGGAATGTGTTCTGGGAAT AGACTGCAGCCAGGAAAAAAAAAGCAGAAGGGTGCACCCTCCCTTCCTCAGCCAGGAAAA CATTTCTGTAGCCCTTGCGGACCCAATGCGCAGAAGTGAGAGGGAGGAAAGCCAGGCTCA TGTTTCCTTGGTTGTGCTTCAACTTCTAAAATATGTTTTTTCTATGAACATTTTAATGAG GGGTTGGACATAAGACAGAAAAATAACATTCTGAGAGCTCGTTTGGAGGTTCTAGCAGGG GAGCGCAGCTACTCTTATACTCTTGACTGAAGACCGATCGTCCTCTATCGGGGATGGTCA TCCTCTTCCATTGAGCACACAGCTTCTGGAGGGACGCACATGGAGTGGTGAGGGAGGAAG GGGATACCCGCCTAGTCAGCCAGATCAGCCGAATCAACCCTGATGATCAATGGGGTGACA CATGTCGCAGCCAGATCACCCTCACATCCCAAAAATAACACTCAGATTGCTTTTTCTTTT TTAAGATGTAGACTTTTCTTGAGACTTTCGTACAAGGCAATGGACAGATAACCTTAACCT CCCAGAGAAAACACACATTGGCCACAGAAAAAGGCGCTTCTCCGTATACCTGCCATAGTT ATCAGCAACTTTGAAATGGCTTCTCAGACTCTCAGGGGTTGGCTTGTTGTGTTTGCTTTT GCAAATAACTCAGGCTTAAGTCCTGCTACTTTCAGAGCCTAGGGGACGCTTAAGGACTAC CATCAGTTTAACACAGTGGCACTTGGAGCACATGCTACTTATGGTGGTGAGGAGGGGTGC TGGTGTGAGGGTCTGAACGCTTGCTTCTCCTCTGGCAGGGTGTGAGCAGCCAGAGGCCAC TACCTGGGCAAGGATGCTTGCTCAGTGTCCATGGAGGGACATGTGATCCAGCAGGTGGGC AGGAACCAGGCCCCTGTGGCCGCCCAACTCTCCATAATAGAATCCTTGGTCATCCATGGG CCCGTAAACCATGACCACGTCTCCTGCCCTCAGCGCCAGCCTGCCCTTCCCCTGGCCCCC CATTTGCCCATCCCCAGGATCATAGTCCAGAGCTGCTATCATGATCTTTGGAGTCCACAG TGGGAGTCTCTTGGAGTTCCCCTGGAGCACCAGGGAGGAACCCTGGGGGATGGTGAGCCC CTGAAAGTCCTCGAGGTGGGCCACAGAAGGCAGGTTCCCTTGGGCCGGAGAACGCCACCT CCTATCAGTCTGCTCTGTCCCCACCTCCATCTCAGCCACTAGGCGCCCGGGGATATTGCC CACTTGCCTGTTGCACTCGCTGAGGTAGAAATCATGGGTGTCCTGAGAGCCCCACACTCT TAGCAACTGCCTTTTCTGGAAGACCAGCTCCTCCTCTGCAGCCTTGAGGTTGGCAGACAT CACCAGGGGGTTGTAATCAGAGAGGGCCACAAAGACCCTGGCTGGAGTGTTGGCCCCCGT CCCCAGCTGTTGGGGGCCACCCCTGGGCATCTTGATGACTTTGGCGGATGGAGCTGGACA CAGTGCCGAGCTGGGCTCATGGAGCTGGCACCCTCTCTTCACCCCCAGAGCTTGTCCTTG CCTGCTGTGGGGCTCAGGCTCCCTCCTCTCCTCTCGCCTCTCTGTGCCCCACAGATCCAA GCACAGTGCCTCCTGCTCCTCCTTCAAAACGTTATGGAAGTCAGATGCATACTGTTGGCT GGCGCCCAGCTGGGGAGGTGTGAACCCTTGGGCATCTTGCTTTTGCCGAAGTACCCTCTC AAGGGCAGCCTTTTCCTGACACGGTTCTTTCCTGGGCCCACACTCGGTGCGTAGATGGAT GAATCCTGGAGCAGGGCTTTTGCTGGTGCCCATGTGCTGGGAGCAATTTTCCTTCTCCCC AGGCTGGTCACTGACTGAAGGTGGCCTGTGGTTCTGGGGACTCTTCTGAAAGAGCAGGTC CTTTCTACAGCCCTCCCAGGCCTCTGCAAGCTCCTGGGCTTGGCTGCCAGCCCCTGAACT CGGACATTCTCCTTCTGAGCCCAGGTTGGACACTGGGGATTGCCTCCTTGGGGGTTCTTC AAAGAATGCTTCTAGGAACTTGGCCTGGGGCTCCCCGCAGCTTCCAGGGTTGTGGGGGCT GGCCTTGGCACTCGGAGGAGCCAGTGACAGCTTCTGGGGGCAGACGGGGAAGGTGACTCT GTAGGTGGATGGGTCGCCACAAGTGTAGCTAAAGGGTGGAGTCTCTGGCCATCGGTGACA CATGAAGAAGTCCTCGGGGATCTGAGCAGGCACTGAATCCAGGGACTCACCACAGAGTGA CATGGTTCTCACTGAGACCTTCTGCCACGTGAGGGGCACCTGTAGCTGGGAGAATTCCAA TAGGGTGCTCCCAGCAGTGGCATCGGCGACCTCACAAACCTTAAGCCCATCTGCATACAC AGCATAACCGGTGACCTGGACTCCATTGGAGGACCCAGCTGAGTCAATGGTCACAGGGAG CCAGCTGACCACCAGGACACCTGGCGAGGCATGGCGCTCCACCAGCACATCCAGCGGTGG GTAGGGAGGTCCTGCCAAGAGTGTGTCGAAGGTGACGGTGGAGGACATAGTTCCCCAATA CACCTGCAGCAAGTCCCGTGGCAGCCGCACCTCCACCCGCGCCCGGTAGTGCGTGCCGGG GCACAGGCCCTGGAAGGTGTAGCAGCTCACGCCCGCTGGGGTCAGGGCATGCTCTCGGTC ATCAAGATATACCACATGGGGGTGGCGGTGGCTGCTGTAGACCCAGGTGATGTTGGCTGA TGTGGCTGTGACATTCTGCAGGTGTAGCTGCATGGGAGCCATACGGAGCACCCCTTTGGT CCCCAGAAGGGGTCTGGAGAGGCCCTGCTTCCCCATGCTCTGCAGCAAGCCCAGCTGGCA GGCTTCCGTCTTGGTATCCAGGATCTCTGTTGCTACTTCTGTCTTGGAGCCCACCCTGAC CATCTGGCACAGCCCTCTGTCCACCACTCCCTGGGCTTTCCCAGATAATAAGCTGTCTTC CTCCAGAGCTTCATCCTGCCCCGCTGGGAGTTGACTGGGGCCAAGATCAGGGGATTTGGC AGGCAGGCAGCCTGGGATGTAGCTGTCCGGAATCTGCTCCACGAAGTTGGAGGGCACCAG CCCCCGCCGGCCATCCTCAAGCTCCCCCTCATAGAAGCCATCCTCATCCATGTCCCCGAA GATATATATGTAGTCCCCAGCTGTGAGGGGCAGCTCACCCTCAGGGTGATCATTGGGCCC CTCAAATGGGTTGTAGTTATACTGAGCCATGAAGATCTTGAGCTTGGGGGCAGCAGGAGC CGCCGAGCCCCCCATTTCCAGGGCCAGGGACACGCTGTCAGGCTCCAGGTCATCCACCTC ACTGGCTGTGTCCCTGTCCAGAGTAGGGGAGGACGGCACGGTGGCCCACATCGACCCCTC AGAGGAGGAGTTTGACTGGGAGCTGGTTTTCTTGGACAGAGGTTGGCTGGCAGGGACTGT CTCTGAAACTTGGGGGACACTGGCTGACTCCCCAAGGGCAACTGGGCTCTCCTCGAGGGA GGCTTCCCTTTTCTCCTGTATGTCTCTGTTGGGCTGTGACACTGCGTAGTCTTCAGGCCG TGCGTGGAACTTGGGCCTTCTGCTACCTTTAACTTGGGACTCCGGCATTTGACTGGTCTC CCAGGGCTGTTCAGGAGGGTGACCAGGCCGACACTGGAGCGCCTGTATCTCCTTCCAGGA AGGATGGAGGTCACACAGGGCCTTCTGCGGGTCGCGTTGCAGCTGCTGCTGCCTGTCCTG CCCGCGCGCCGCCTGCTGCAGCAGCTGTTCGGCGATCAAGCCGGAGGCATCGCGCTCTTG ACACGCGCGGCCCAGGTGGCCGCGCACTTCGCTATTCTCAGCCTCCACCTTCCGCACCCA GTCGGTCTTGGCCTGCAGCCGCCCATTCTCCTCCGCCAGCCAGGCGTTTTTGAGCAGCGC CGCCTGCAGCTGTGTCTCGGCCTCCTCGCCACGTCGCCGCGCCGGGGCCGCCTGCGCGCC CAGCTCCTGGCACTCGCGCCGCCGCTGGTCCAGCTCGCGCTCCAGCGCCAGCATCTGCCG TCGCACCTCCTCGCAGGTTGCGCTCTGGCCGCCCGCCTCGGGCCAAGCGCCACCGTTGCC CTGCTGAAGCATCAACTGCCTCTGCAGGCGCAGCACTTCCCGCTGGGACTCGCGCTGCAG GCGGTCTAAGTCTCTGACGTTGAGCCACTGGGTGCAGGGGCCGCCCCTTCCAGTATGCAG GTCGCTGCAGGGACCCGAGGCGACACGCGCCTGCAGGAGGTGGCACTCCTGCCGCAGCTC TTCGATCTGCTTGTCCTTGGCCAGCGGCGCGCTCGCCTGCTCCGACAGGTCCCGAGCGCG CTGGCGGGCAAAGACTTGGCACAGCTCCAGGCCGGCGCAACTCTCGCCGGGTATAGGCGC GCTCACGGCCCGGAGGTTGGTCTCCTGCAGCTTGCGGGCGCGGTCAGCTAGGCGCCGCGC GAGCCCGGTCAGCTCCGCGCGCTTTACCTTGAGCCGCTTCACCTTCTCGTCTGCCTGGTA GGGGCAGCCTGCACGCCGCAGCTGCTTGTTCTCCGCTTGCAGGGTATAACAGCGGCGCGC CAGCACCCACAACGCCTTGGCCAGCAGCCAGTTCAGCTTTATCAGCTCGTGGTGGCCCAG GCCCGGGGGTGAGGGAGTCAGGACTTCGCAGGGCTGGCTCTCAGAGCCTTCTCCTCCCCG CGGGTTGCTGAGTTTCCTGTGGGCTGACGGTGGTGGTGGCGGCGGGAGTGGCGATGGTGG CGGCGAGAGCCTCCGGGAGCCAGGGGTGGAGGTGTCTGGTGAGGAGGGCCGCTCCTCGGA TTTGGAGGCCTTGGGGAGGCTCCGGGTGCTGTCAAGCGAGCGGGAACACGCAGGTGCGAA GCAATCAAGGGAGCTGGCGTGCGTGGAGAGCAGGCCGTCTGGGGAGCTGGAGCACGCAGA GGACACCAGGCCTAGCGAGCGGGAGCGCACACCGACTTCAGCACTCAGGCTGTCTAGAGA TCCGAGTTGGCAGGCGGGTTTGGGGGCGTGGCAAGAGCCGCTGGAGGTCTGGGGCAGCGG TTCTTCCAAGGAATGCACAGCTTGGGGGTCCGGTGATCCCGACAAAGCCGGGTGCCCGCG GAAGTGCGCCAGGATGTACTTGAGGAAGAGCTGGCGCTCCACCTCCAGCGCCGCCTGCAG ATAGCGGATGCGCGCCGCCTGCTCGCCGTCAGTCTGCCAGCGAAGCTGCGCCAACACTTC CTGCAGGCGACAGCGGCACTGCGCCGCGGAAACCTCGGACGCCCCCGGGCGGCTACAGTG GCCGCGGTTCACCAGCTCCTCGGCCAGCTGGCGCTGCAGCTCCCGGGCTTGGCGCACCAC GCCATCGCGCTCCCGGTGCAGCAGCTGCTGCAGCTGCCGCTGCTCGGCCTCCTTCCAGCG CAGCAGCTGCCGGATCTCGGCCTCGCGTTCCCGCTGCATCTCCTCTTGCAGCTGCCGCAA CTCCCGGCTGCGCTGTGCCTCCCACTTGGAGCGCAGACGGTCAGCCAGCTGTCGCCGCTC CCGCTCGGCCTCCTCACGCAGCTGGCGCTCCCGGGCAGCGAAGCGCCGCCGTTCCGCCCG CCAGCCTGCCCGCTCCGCCTCCAGCTCCGCCCGTAGCTTCTCCAGCTCCCGCCTCTGGTT CTCCAGCACTGCCGCCGCCGGGCTGGAGCAGCCCGGCTTCTTGGGCGACGCGCCCAAGGG GCTGGGCGAGTCCTTGGCCATTGCGGCCGCGGCCCGGCTGGTCTCCAGACTTCGCCTGGC AGGCCTCAGCTCGCCAACGGCCGCCGCCGACCGCCCGGCGCGCCCCTTCCGGCGCCCCCT GGGGGTCTCCGCGTGCCCCTTCCGCCTCTTGGCATGTTCCCCCGCCTCCTGCCGCGGATG TCTGGCCCAGGCGCCTCTCCACCTGGCAAGGCCAGGGCTGGACTCCCACACCCCTGCTCA AACTGGCCCATGTCCCTTCCAGGATGCACGGGGGCTTTTCCGGTCCCTACACTTTTCCTG CTTCTAGCACCAAGTCCCTTGTGACCCTCCCCAGGCACCCTCCCAGGCACTGTGCAGTCC CAAGCAGGCCGCGGTGCCCCGAGGGAAGTCTGGGCTGCTGCTCCTGTACTTGGAGAAAGG GGCAGGGTGTGTGTGTTAAGGCCACCCATGGGGTGTGGGCATACCTGGTCGTGGGTTCAG GTGGTGTATTTGGGTCAGGTGGCTGGAAAATGCAGCAGGATAGGCCTCAACTTGGCTAGA CCTGGACTCCCCAGGACCCTTCAGCCCCCTAACAGCCCTGATCACCCAACTGGTCATCCA CCTTTTGAGGAAAGGATGTGCCTGATACACATTTGTGTTACTGCGCAGAGTCGGCTCAGA GGAGGTGTAAGCGAAAAGACCCTCCCCAGCAAGGTGGCATAGGGACCCAGGGCCAGATAG GCATGAGGGGCACTGCCCAAACTGGCCATCTGGCCTGTCTACCTCCCACATCCAGGTACC ACCTGGTGACAAACACTGGAGCCCCAGTGTCTGGGCTACTCATTGGCATCTTAGGTAGAG GCCTCAGGATGGGCCAGGAGTGAGGGTGAGGCTTCAGCAGAAATGTTGGAGGCATCCTGA GCTGCAGAAGAAACACACACACACACACACACACACACACACACACACACACAGCTGCAG AAGAAACATACACACACACACATACTGAGCTGAAGGAATACACACATACACTGAGCTGCA GAAGAAACATACATACACACACTGAACTGAAGAAATACACACACACACACACACACACAC ACACACTGCCTCTCTGACTGGTGGCCACCTGTGTGGCTTGGGAGCTAAGAAGCCCTGCTT CAGTCTGGCTCCTCTTTGCCCTTCCTGTCTGCTCCTGGTGCCAAGTGGCTTTTGGGAGAC ATGGATGGACACCTGATACCTGGCAGTCTGCATCGAGACATTGATTTTTTTTTTTTTGAG ATGGGGCCCCACTCTGTCGCCAGGCTGGAGTGCAGTGGCGCAATCTTGGCTCATTGCAAC CTCTACCTTCCACGTTCAAGAGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACA GGCTTGAGCCACCATGCCCAGCTAATTTTTTGTACTTTTTAGTAGAGACAGGGTTTCAGT ATGTTGGCCAGGATGGTCTTGAACTTCTGACCTCAAGTGATCCACCCGCCTCGGCCTCTT AAAGTGCTGCGATTACAGGCATGAGCCACTGTGCCCGACAAGACCTTGATATTGAAGTCA CATCAGAGGCCCCTTTGCTTCTTGGTCTGTCGTGTCTCCCTGTCCCCAGGAAGCACCTGG GACCTCTCACAACTGCTGTGTTCACTCAGCTTATGGCCCTCCTTTACAACACTCAAGTTT TTCAAATCAGTTTCCCCGGAACCCACAGAACATGCCTTTAGGCAGTGTCAAGTAACAAGA ACATCGGGAATTATGGCTCCAGATGTTCTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCT CGCTCTGTCGCCCAGGCTGGAATGCAGTGGCGCAATCTCTGCTCACTGCAAGCTCCGCCT CCTGAGTTCACGCCATTCTCCTGCCTCAGCCTCTCAAGTAGCTGGGACTACAGGTGCCCG CCACCATGCCTGGCTAATTTTTTTTGTATTTTCAGTAGAGATGGTCTCGATCTCCTGACC TTGTGATCTGCCCGCCTCAGCCTCACAAAGTGCTGGGATTACAGGCGTGAGTCACGGCGC ATGGCCCTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTGTGAGA CAGAGTTTTGCTCTGTCACCAGGCTGGCATGCAGTGGCACGATTTCGGCTCACTGCAACC TCCAACTCCCTCGTTCAAGTGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAG GCATGCGCCACCACGCCCAGCTAATTTTTGTATTTTTAATAGAGACAGGGTTTCATCATG TTGGCCAGGATGATTTCGATCTCCTGACCTTGTGATCCGCCCACTTTGGCCTCCCAAAAT GGTGGGATTACAGGCGTGAGCCACCGTGCCCAGCCCAGATGTTCTTTATGAGAGGTGAGG CAGATGTAGTGTGTATGTGAGAGGAAGGGGAGGTGAGATGCAGGGATGGCCTTAGCCTTG AACTGGTGCTGGGCTCCTGGGCATGGTAGCCACAGGGAGGACTATCTTGAGAGAATCTTC ATCACAGCCCTGTTTTACGGAAGAGAACCTGAGGCTCAGTGAGAGCCACTGGCTTGCCAG GGCACATGGGCAACTGAGGCAGAGCTGGTGGGTGGGGTTTCACAGCCTGGATGCCCTGCT GCAGTCTCCAGTAGGGCCTCAACTGTCAGGTCAGGAGGGCAGGCTGGGCCGGGCATGGTG GCACAGACCTGTAATCCCAGCCCTTTGGGTGACCAGGGTGGGAGGATCACTTGAGCCCAG GAACTCAAGACCTGCCTGAGCATCATAGAGAGACCCTGTCTCTACAAAAAAAATTTTAGC TGGGCGTGCTGGTGCATGCCTGTAGTGCCAGCTACCCAGGAGGCTGAGATGGGAGGGTCA CTTAAGCCCGGGAGTTTGAGACTAGCCTGGGCAACAGAGTGAGCGAGACCCAGTCTGCAT TTCCAGCTGAGATGAAATGATTGCTTGAGCCTGCAAGGTGAGGTCAAAGTCGCAGTGAGC TGTGATGGCACCACTGCACTCCAGCCTGGGCAACAGAGCAAGACTGTCTCAAAAAAAAAA AAAAAAAAAAAAAGCCTGGTGCGGTGGCTCACTCCTGTAATCCCAGCACTTTGGGAGGCC AAGGCAGGCGTATCACGAGGTCAGGAGATAGAGTCCATCCTGGCTAACATGGTGAAACCC CGTCTCTACTAAAAATACAAAAAATTAGCCATTAGCCAGGCGTGGTGGCGGGCACCTGTA GTCTCACTTATTTGGGAGGCCGAGGCAGGAGAATGACATGAACTCAGGAGGTGGAGCTTG CAGTGGGCCGAGATCCCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCATCTC AAAAATAAAAATTAAAATTAAAATTAAAAAAAGAGAGTAGGCTGTCACCAAGAAAAAGGA CACTGGGGTGGATGGGTCTCTGGGAACCCCACAGTTACCATCCAGCAGCTGCCCCAAAGT ATGTCAAAAGCATTCGGGAATGTTGGTTGAGTGGATGAGCAAAGCCAGGCTTCCCCAGGG CTGGGTTTCTGCCATCCCTGTTCTCATCATGTTGTGCAGTGATTGTCCAGTGACCTGCCT GTCACCCCCATCAACTCTGATCTTGGGGAGAGCAGAAGCCATGTTTTCCCTTGTCCCTAT TGCATCCGTGCATGTGACTTAGCACATCACTGGTGCCCAAGACATATGAGAATGAGTTGC AGGGAGGCAGGAGTCCCTGCATGAGCTCACCATGCTGCTGCCTTCTCCAGGTTCAACTTT CTCACCTATTAAATGAAAGAGATGGTTTCTTTTTTTTTTCTTTGAGATGGAGTCTCACTC TGTCACCCAGGCTGGAGTGCAGTGGCATGATCTCGGCTCACTGCAATCTTCACCTCCTGG GTTCACGTGATTCTCCTGCCTCAGCCTCTCAAATAGCTGGGATTACAAACACCTGCCACC ATGCCTAGCTGATTTTCATAGTTTTAGTAGAGACAAGGTTTCGCCATGTTGGCCAGGCTG GTCTTGAACTCCTGACCTCAAGTGATCCACAGGCCTCAGCCTCCCAAAGTGCTGGGATTA CAGGCATGAGCCACCATGCCCAGCCTGAAAGTGGTGGTTTCAAACTGGGTTCTTTAGGGC TCCAGGGTTCCTTGGAAATATAGAACTGCTGGGCACACACCTATACTCCCAGCTACTTGG GAGGCTGAGGCAGGAGGCTTGCTTGGATCCAGGACTTCTGGATTGTAATGTACTATACTG ATGGGGTGTCTGCACTAAACTTGGCATAAATTGGTGACTTCCTGGGAGCAGGAAACCACC AGCTTGTCTAAGGAGAGGTGAACTGGCCCAGGTTAGAAAAGGAGCAGGTTGGCCGGGCTC GGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGCAGGTCACCTGAGG TCAGGAGTTCGGGACCCGCCTGGCTAACATGGTGAAAACCCGTCTCTACTAAAAATGCAA AAATTAGCTAGGCATTGTGGCGCATGCATGCCTAATCCCAGCTACGCGGCAGGCTGAGGC AGGAGAATTGCTTGAATCTGGGAGGTGGAGGTTGCAGCAAGCTGAGATTGCACCACTGCA CTCCAGCCTGGGCGATAGAGTGAGATTCCATCTCAAAAAAAAAAAAAAAAAAAAAAGAAA GAAAGAAAAAGGAAAACGAAAAGGAGCAGGTCAAAACTCCTGTGCTGGCTGGGCGCAGTG GCTCACGCCTGTAATCCCAGCACTTTGGGCAGTTCAAGACCAGCCTGACCAATATAGTGA AACCCCATCTCTACTAAAAATACAAAAAATTAGCTGGGTGTGGTGGCAGGCGCCTGTAGA CTCAGCTTCTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGCGAGGCAGAGGTTGCA GTGAGCCAAGATCATGCCATTGCACTCTAGCCTGGGTGACAGTGCAAGACTCCATCTCAA AAAAAAAAAAAATTTTTTTTTTTGTAGGCTGGGCGCCGTGACTCATGCCTGTAATCCCAG CACTTTAGGAGGCCAAGGTGGGTGGATCACTTGAGATCAGGAGTTCAAGACCAGCCTGGC CAACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAATTAGCTGGGCATGGTGGCAGG TGCCTGTAATCCCAGCTACTCGGGAGGCCGAGGCAGGAGAATTGCTTGAACCCAGGAGGT GGAGGTTGCAGTGAGCTGAGATCACGCCATTGTACTCCAGCCTGGGCAACAGAGTGAGAG TCTGAGTCAAAAAAAAAAGAAAAAAAATTAACAACAGGCCAGATGTGACGGCTCATGCCT GTAATCCCACCACTATGGGAGGCTGAGACGGGCAGATCACTTGAGCTCAGGAGTTCAAGA CCAGCCTAGACAACACGGCAAGGCTGCATCTCTATAAAAAAATTACAAAATTTAGCCTGG CATGGTGGTGCACGCCTGTAGTCCCAGCTATTTGGGAGGCTGAGATGGAAGGATACCTTG AGCCTGGGAGGCAGAGGTTACAGTGGACTGAGATTGTGCCACTCCACTTCCACCTGGACG AGAGAGCCAGTCCCTGTTTCAAACAACCAAAAAAACAAAAAACCCACCCCCCCAAAATAA AAACAATAATAATAATAAAAATAAATAAAACAGAAATACCATAGGCCTTTTGGGAGGAGG TATGCAGGAAGCGGATGGGGAGAGACAGTGCCCCTACTTGCTTCAATGGGCAGATCTACT CTTTTTTTTTGAGACGGAGTCTCGCTCTGTCACCCAGGCTGGAGTGCAGTGGTGCAATCT TGGCTCACTGCAACCTCCGCCTCACGGGTTTATGCCATTCTCCTGCCTCAGCCTCCCGAG TAGCTGGGACTACAGGCGCCCGCCACCGAGCCCGGCTAATTTTTGTATTTTTTAGTAGAG ATGGGGTTTCACCATGCCAGCCAAGATGGTCTCGATCTCCTGACCTCGTGATCCACCCTC TTCGGCCTTCCAAAGTGCTGGGATTACAGGTGTGAGCCACAGCACCCGGCCGGCAGATCT ACTCTTAAGTGTCACTTATCAGATTCGAAGAAGGGTTTTGGTTTAATAAAGCGTTATGCT CCTTGAAACAAGTTTCAGAACCCCTGGGTTAGAGGACCTCTTGCCCATTGTGTGCTCACA CACACACGACACACACATACCCCATCCCAGTACGTAATGACTTCCCTGACCCGGAGAGAA GGCGATATGTCCCCCTTACATCTCAGTTGGCTCCACATCGCCAGCTCTGTATTCCACTTT CCCTCTCTCTACAACCCCAGTCTATGACTTTTGGGCCATGGAGTCTGGGTTCCTGGGCAA CCTGGTTCACATCCTTCTCCTGCCGGCATGGCCTGGCTCCATTTCTGTTCGGCTACCAAA GCCCCAAGCTTTGTGGAGCCTGCCTGGCCTTGAGGAGGGGAATGGGCTCCATCCTGAGAG CCTGCAGGAGGGTTTAAGCTTCCCATCCCCCATTGCTACAGTAGCTCCCCCAGCTTTTCA TTCCATATCCTTTATTTTTATTTATTAAAAACTTAATTTTTGGCCGGGCGCGGTGGCTCA CGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGATCACGAGGTCAGGAGATCG AGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATAGCCAG GCGTGGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGGAGGAGAATGGCGT GAACCCGGGAGGCAGAGCTTGCAGTGAGCCAAGATAGCACCACTGCACTCCAGCCTGGCG ACAAAGTGAGACTCCGTCTCAAAAAAAAAAAAAAACAAAAAACACTTAATTTTTTTTTTT GTAGAGACAGGGTCTCCCTATGTTGCCTGGGCTGGTCTCAAACTCTTAATCTCGAGTGAT CCTCCCACCTCAGCCTCCCAGAGGGATGGGATTACAGGCATGAGCCACCATACCCAGCCA CATATTTTTTATTTTTGTTTCTATGTGTTTTCTGTCTGTTCTTTGCTGTCTCCTCCATGT CTAGAACAGTGCCTGGCACATGATAGGTGCTCAGTAAATACAGGACTGAAGAAGACAGAG GCAGACAGGCCAGATGTGGCTGCTGTCCTTAGGGAATTGACAGTCCATGATCTATGAGAA TGAGCTGCAGACTGGGAGGCAGGTGTCCCAGCATGGGCTCACCAAGCTCCTACCCTCTCC CGGTCCAGCTTTCTCACCTATGAAATAAAAGTGAAGCTGGACATGGTGGCCCACGCCTCT AATCTCAGCAGTTTGGGAGGCCAAGGTGGGTGGATCACTTGAGATCAGGAGATTGAGACC AGCCTGGCTAACATGGTGAAACCCCATCTCTACTAAAAAACACAAAAATTAGCCGGGCGT GGTGGCAGTTGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATTGCTTTAAC CTAGGAAGTGGAGGTTACAGTGAGCTGAGGTTGTGCCACTGCACTCCAGCCTGGGTGACA GAGCAAGACTCTGTCTCAAAATAAATAAATAAATAAACAAACAAACAATAAATGAAAGTG ATGGATTACATGAATGGTTTTCAAACTGGGTTCCTCAGGGCTCAGTTTGGGGGAGAATGC AAAGGTTAAATACAAACAACAATGGAATGTAGTAACATTGGCGCTTGGGCGAGTGCTCTG GGTGAGGAATGTGGTGCTCATGGGGAGAAGAGCCGCATCAGAGCAGGCTTCCTGCATGAG GCAAGGAGGAAACACAAGAGCTGAGAGCTGAAGGAGAGGGAGTGAGGGCTGCCAGAGGAG CTGGGAGGCGATTTCAGGCAGTGGGAACTGGAGCTGCAAACCCCCAGCCTCCTGCAGTCT GGCTATCTGTACCAGCCCTCACCCATGCCCACCACCCTCAGGGCACTCACATCCCTTCTT GCACTGATATTTTCCCGTCCTATCATGACCCTCGTTTGAGGGTGCAGGGAGGACTGGAGC TCAGAGAGGGAGGGTCACCTGGCCAGACCCTAACCCGCTGAGTTCCAGTGGAGTTCCTTC CATCCCCCAGCACTGGATGGGGCTGGATACCTGGAGAGGGACCGTGCTGCTCTTCCTGGG CCCTGCAGCCTGCCTGGCTGGTCCACATCTTCCTGGGCCAGGAGGGCAGTATGGTGCGAG CTTCTAGCTCTCTGGCTGGGGCCCCCTGGCCTACTGGGCTGGCTTCCTGCCCTGGTGGCC CTGGCCCAGCTCCCCAGCTCATGTGCCTCTGCAGCGAGTGGTCAGCCCCTTCCCATCTGC ACTTTACAAGCCTCATCGCTGAATCCTCCCCATAGCCAGAGAGCCTGGCACTCTCCTCTC CAGAGAAGGAAACTGTCCAGGGTCACACAGGGACTTATGGGCAGGTCTGTGTGACCTGAG TCTGGGCTCTGAACTGGTGCTTGCAGGGTCTTGCCCTCTGGCTCTGGCAGCCTGAGTGTT ACTGTAATCCCTGAGCCTTTGAACAAGCAGGCACTGCTTCCTGAGATCCCCATTGCTGCT GTCAGCGTCTCTGTTTCCCAGCTCCACACTGCTGCCAGGACAGACGTATCTTCTGTCTTG TAGCATCTCCATTTCTCTTCCCTTTAGATCTATTTTTTTTTTTTTGAGATGGAGTCTCGT TCTATCATCCAGGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGTAACTTCTGTCTCCT GGATTCAAGCGATTCTTCTGCCTCAGCCTTCCAAGTAGCTGGGATTACAGGTGCATGCCA CCACGCCTGGCTAATTTTTGTATTTTTAGCAGAGACGAGGTTTCACCACGTTGGCCAGGC TGGTCTCAAACTCCTGACCTCAGGTGATCTGCCCACCTCAGCCTCCCAAAGTGCTGAGAT TACAGGCATGAGCCACCGCTCCCGGGCTGGATCTATTTCAATCACCCTCCTTCCTCTGCA GCCTGAAACTTGAGGCTTTGCCCACTTCCCAAAGGCACGAGTAGCAATGACCATTTGTTC TGTCATTTGGCCATAGCTCTAGGTCAGAGGACAGTCCTTCATCAAATGTTTGTTAAGCAT GCAATCTAGACAGGGGAGGACAAGGGCCAAGTAATGTGTTAATTTCTTCATTCACTAAGC ACAGAGAATGGAGTAGAAGGAAGGTGGAAAGGAGAAGGAAGGAAAAGAGAGAAGAAGGAG GATAGAAAGGAAGGAAGGTGGGAGGGAGAAAGGAAAGAAAGAGGGAGTCCTTTAGGTTAT CTAAGTCCCCTCAGGACTTGGTCTGGCCCTCACAGTTCAAGAACAGACTTGGGCTTGAGC CTCTGACCAGCCAGATGACTGAGAAGCTGAGGAACAGCTTGAGCCTCAGTTTTTTCCTCT GTAAATGGGGTTGATAGCAGTTCTGACCCAGCGGGCTGTTGGGGGAGTCACAGAGATGCT GGGTGCTGCCTTTGCACTCAGAAGGGTCCAGGCTAGGTGTGGTGGCTAACACCTGTAATC CCAGTACTTTGGGAGGCTGAGGTGGGAGAATTTCTTGAGTCCATGAGTTACAGACCAGCC TGGGTAATATAGAGACAAACTGTCTCTATAAATTTTTTTCCCAAAAATTAGCCAGGCATG GAGGTGTGCACCTGTAGTTCTAGCTGCTTGAGAGTCTGAAGTGGGAGGATAACTTGAGTA CAGGAGGTTGAGGCTGCACTGAGCCATGATCACACCACTATACTCCAGCCTGGGTGACAG AGCCAGACCCTATCTCAAAAAAAAAAAAAAAAAAAAAATCAAAGGGGTCCAGCAGCATCT TCCTGTCAGCTTACCTAAGGGAGCATGATAAATTGAGTCCCTGCCACGATGGACATGGGG AGTGTAGTGGGAGGGAATGGCAAAGGCACCACAGGCAGAGGGAACAGCCTGGGCATAGTG GGGGGTGTGATGTGGCACAGCACTGATTGTGGGAAGCGAGGGATGTGGGAGATGAGGCTT CAGGTAGTCAGAGGGAGGTTGAGGCAAAGCCTCACCTGCATGCTCTAGATGAGGTCCAAT TCCTGGTACCCAGCTCCTAGCCTAGTAATGGGCCACTTCTCCCAGGGCCACAGTTTTCTC TCCTATCAAATGGAACATGTTAGCACTGCTCTATGGTGCCAATGAAGGGTATAAGTGTCC AGTGCCCATTTCCTGCCCCAGCAAAGGCACTTTCTTTTCCTTGTCCTCTTGCCTACTGGG GAACTCCTTCCATCCCTGGTTCCTCCCCCAGCTTCTGCGAGGCCCCTCCTCTGAGCCCCC ACACCTCATCAAACCCTCAACACCGTCTGGTGTTTTCCTTGGTGCCTCTTCAAGATGCTG TGAGCTCCTTGGGGGCAGGTGGTGCCTGTGTACCTCTTTTTTTTTTCTTTTTTTTTTTTT TTTGAGACGGAGTCTCGCTCTGTCACCCAGGCTGGAATGCAGTGGCGCGATCTCGGCTCA CTGAAAGCTCCACCTCCCGGGTTCACGCCATTCTCCTGCCTCAGTCTCCTGAGTAGCTGG TACTACAGGGGCCCGCCACTGCGCCCGGCTAATTTTTTCTTTTTTTCTTTTTTTTTTTTT TTGAGACGGAGTCTCGCTCTGTCTCCCAGGCTGGAGTGCAGTGGCACAATCTCCGCTCAC TGCAAACTCCGCCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGG ACCACAAGCACCCGCCACCACGCCCAGCTAATTTTTTGTGTTTTTAGTAGAGACGGGGTT TCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCATGATCTGCCTGCCTCGGCCT CCCAAAGTGCTAGGATTACAGGCGTGAGCCACCACGCCTGGCCTTTTTTTTTTTTTTGTA TTTTTAGTAGAGATGGGCTTTCCCTGTGGTCTTGATCTCCTGACCTCGTGATCCACCTGC CTCGGCCTCCCAAGGTGCTGGGATTACAGGCGTGAGCCACTGCGCCTGGCCCTGTGTATC TGTTTTTCTGGACCGCCATCATGTTCAGGAAGGCATCGAATATTTTGTCCGCGACTGCAA TCACACACTGCATCATCCCTGAAACGAGGTTTCCACGGATAAGGTGCAGCAACAGAGAGA GAATCCTAGCAACTTTCAATCATTTTCTTTTGAAATGGTTTTAGACTTACAGAGAGTGGC AAAGACAGCACAGAGCTCCCATATACCCTTCACCTAGCTTCCCTGGATTTTAACATCTGC AGTAACCAGGCTGATGTGTCAAAACTGAGTCAGATATTGATATAATCCTATTACCTAAAC TTCAGACTTCGTCTGGATTTCCCAGGTCTCCCACACATGCCCCGCTTCTGTTCCAGCATC CAACCCAGGACACCAGGTGGCATTTAATCTCTGTGTCTCCTACTCCCTTCCAATATGTGG TGACTCCCCAGTCTGTCAATGTCTTTTGTGACTTTGACACTTTTGAAGAACAGTTATTTT GTTGAATGTCCCCTAATTTAGGTTTGTCTGATGCTTGCCCATGATAAGACTAGGGTTACA GGATTTGAGGGAGAACTCCATAGAGTGGATGTGCCCTTTCTTTTTTTCTTTATCATTTTT TTGAGATTGAAACTCGCTCTGTCGCCCAGGCTGGAGTGCGGTGGCACGATCTTGGCTCAC TGCAACCTCTGCCTCCTGGGTTCACGCCATTCTCCTGCCTCAGCCTCCTGAGCAGCTGAG ACTACAGGCGCCCGCCACCACGCCCGGCTAATTTTTTGTATTTTTAGTAGAGACTGGGTT TCACCACATTGGCCAGGATGGTCTTGATCTCCTGACATCATCGTGATCCGCCCGCCTTGG CCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGTAACCAGCCTGCCCTGCGTAATT TTTGTTGTCGATGATCCAAAGTGCACCCACTCGCCATCTCTGGTAGCTGGGAGGAGCAGG TTTTTGGGTGAAGGTGAAAGAGCCGGAGTTGGGGATGTGTGTGGCTGGGGGGTACAGGTG CGACCACTTTGCAGTCCACTCCTGTGAGTAGGGCATTCCTGAGGGAGAGAAGGCCTGGTT AGCCCTGGGGCTCCTTCAAGGCCTGGCCCCAGGCCTTGATACCTAGAGCAGCCTGACACA TGTGTGGATGGACTGCCAGGCGGTCCCCTTAGGAGGGGCTGTTCTAGCAGGGACCCTGGC TTGGGTGACCACCACCTCCTGCCACACTCCAGGATCCCTGTCCCTGCTGGAAGCCGGGCC CAGGGCCATGCCAGCCGTGAGGGGTTCCTCATGAGCCTCCTCCCCGGGACCCAGGCCCCA TGCAGCTGCCCCCTCCCACCTGCCTGACCCCCCTGGCCTGCACTTCCCTCCATCCGAATC CTCAATCTCCGCCTGGAATTCCCTTCCCTCCTCAACCCCACCTGGAAACTCTCCTCCTCC TCCCACACAGCTGGGACTCCCTTTCTCCCACCTCTGGGGGCCCAAAGCTCTGGATGCCAC CCTAGCCCTCAGCTGGCCTCACCTGTCTCCTCATAGCCCCACAGCTTGATGGTGTCTGTG TGGGCAGCGCCTCAACGTGCCAGGTCAGGCTGAGGTTGCCTGAGGTGTTGGCGGTGCCGT AGTATTGCCAATGCATCTCGTTCATCAACTCACTCTTCTCCTTCATCAACACTTTGTTGG GGTGCACTGGGCAGGGGAGGTGAGGGGTGAGTGAGGGCTGGGCCCAGGGAAGCAGGGAGG AGGCCCTGAGGCTTCAAGGGCTGTGGGAGCCTCCAGGGCCAATGCATGGCAGCAGCTGGG TGATGGTCTCTCCGGCCCTCATGGCAGAAAAGGACGCTGAGGCCGGAGAGGCAGGGGCCT GCCCTCCCTCCACTGCCCACGCCTCTAGCCTCCACCTGGCTTCTCCCCTGCCCTGGTGTG GCGGAGCTCAGTGGATAGGCACTGGCCCACCTCTCTAGGGACCCCTGCAGCTCCCACTCA CTCTGGGCTGGGACCGTGGTCTGGATGCCAGGGAGTGGAAAGGAGCTGAAGGAACTCTCG CAGGCGGGCAGGCCTCCCAAGGGATTGGTTCAGCCAGAGAGGCAGGACACGGACGGGCCA AGTCTCGGTGTGCATAGATTAGCGCTGGGGAAAGTCCCCGTGGGGGCAGGCTGTGGGCAG GGAGGAGGGCTCACCAGCCAGCCAAGTGCCCGCACGAGGGAAGGAGTGGCCATCGTCCAG TGAGATGGTGAAGGGGACGTGGCCGCTCTCATAGAGCAGGGGCGACACGAAGTGCACTTG CCCGGAGGAGTCCACATGGCCGAGGGTCTGGATGCTCTCCTGTGGCATGCAAACCTGTGG GCCTGGTGGTGGTAGGAGGTGCTGGGCCACTGCAGGCTGAGCATCTCGACAGTCTGGGCC CCCAACCTCCCTGGGGAACCCATCCAACCAGCTGTCCTGTGCCCTCCTGCCTGTCCTGGG TCTGAGGGGCTCCATTCAGCCTGCCAGTGGGTCTGAGCCCTCAGCATGCACAGGGATGGG GGCGTGGGGCCCCAGTGCCCGGCCTCCTGGGCCTCCCAACCTGCAGATCACACTGGTGTC TGTGGGGCTGGACATCTTGAAGTGCCGCACCACAAAGTCCTTGCCGCCCATCATGGAGAC TGAGTAGGGCAATATCTCCAGGCAGAAGTCCTGGAAATCCAAGCAGCAGGTACCAAGGCC GGAGCACGTCGGGTGGCAGGAACATGGCCTGTCCAGGGCGCCACAGCGCATGGAGCAGCT CTCTTGGGCATCTGGAGGAGAGGATGCTGAGGGCCTGGCCTGGCGGGTGCTGGACTGGCT GCCCCATGTGCAGGGCCTGTCTCTGTTTCTCCCACAGCCCATTAGACAGCAGCATCCCCA GGAAAATGCTGGCCGGGAACTGCACAGGCCACCACGTTGATATGGTAGAAAGTCACAGGG CCCGTGGGGATGGGATGAATCTTAGCATAGACCATCTGGCTGTTGCCCTGACTGATGGGC TAGAATGCAGCGAGAAGAGGCAAAAGGCTGCACCCGCTCAGCCCTGCAGGGTCAGGAGAA GCCCCCTGGGCATGACTGCCCTTCAGCCACCAAGTCCTCTATCCCACAGGCGGGGGTCCT CAGAGGGCCACGGTAGCTGGAGCCCTCGACCTCTCTTGGCTCCCCAGGGTGTCTGTGAAC AGTGCGCCTTCTGGGGGCCCTGAAGACTGCAGAAGAACCCCTTTTGCTTGCCTGGACTGG GGTGGGGGATACAGCTGCCAAGAGTCCCATTCGAGGCCTGGTAGGGAATGGTGCAGGGTC TGAACTTGACCCTTCTCACTCTCCCAGGGGCTGCAATTGAGACCCCAAAGAGAGCTCCGC CCTCAGGGCAGAAGGCCAGGGCTGCTCCCCTCCTTTCTGGGGGGAGGAGGGACATTTCAA GCAGGTGTCCCAGCAGTGGGGAAAGGGAAGTGGGGGTGCACACTGCGAGGGGCTGTGGGT GCTGGGCCCAAAGCATCTCCTATGGGCCATGACACCCACGGCAGGGCCACATTTCCAGGG ACTGGCATGGAGGAAGAGTTGCAGGGAGGGACTGGGGCAGGTGCGTGCCCAGCTGGGGGC CCTGAAGGGAGACAGGTCCCATCCCCGGGCTCCAGAGATGCCACCACCCAGGGAAGCCTT GGCTGTCCCAGCCACACCTAATCCTGCCGCTCAGAGTTTGCATAATTTAATCAGTGTTCA CCTCCCTTCTTGAGCAGATGAGGAAACTGAAGCTCAGATGCGGACAGGCAAGGCCGAGCC AGGCCCTTGGTGGCTTCTGGGAGCCCCTGCAGCCAGGGCGGGTGGTAAGGGCTGTGGCCT GGGACTGGTCACTTGGGCCCTGCCACCTGGGCTGGACGTCCAGACAGGCGGGTGGTCCTC TCCCAGCTGCAGCTTCTCAAGGTGTCCAAAAGGGACCCTTGCCACACTTGCAGGGAGTCA GAGCTGGCCCCAGGGCATGAGCTTAGGCCATCTGGTCTGTGATCTGCCCCAGCGCTTGCG GCCAGGCCAGCCCATTGTTCCCCAGCCTGTTCTCTCTCTTGCCTGGGTGGTGCCTGCTGG GAACCACAGGGTGTTGGCAGCCACCCATGGCTCCAGAGAGGTGGGCCCCTACTGAAAGTG TTAGAGGCATGTGCTGGGGGACTTGGCTCCTGAGTCCCCACACAGAGCTGTTCCAGGGAA GCCACACCCGCATTTGATGTCTTCCCACCTCCCAAAGACATTTGGTGAGAGGCCCCCAAG ACCTTCAGCTTCTCAGGACTGTTTCCAGAGGGGAGAAAGTCCCTTAACCCACAACGGTGC TCAGGGTACCTCTGCAGGTGAGTGTCCTGTAAGTGGATGCTCAGGCGCTGAGCCACGGAG AGCCTGTCTCCTGGCCCCAGGCTGGCGAGGGCATTCATCCCAGGCCCAGGGCTGAGGTTG CTCCCTACAGCACTGGGTGCAGGTGTCCATCCTCCTGTAGGTGAGGCTCCGGGTGGTTCG TCTGAGACAGTTCCCAGGAACTGGAGGAGGGAGAGCTGCTGCTTCCTGTTTCTGGCCTGG GAACCCTTGCTGTGCGTGTCTGGAGACGATGGAAGTCCGGTAGGGGCTAGGTGTGCAGTC TGGGAAGCTCCATACCACAGGCCCTGCCGTCAGCTGAGTAACTGAGGCTGGCTCATCTCA GCCCCCCAAAGGTGGGTTCAGCCAGACCCATATGGGGCCGCCAAGCCTCTGGTTCCAAAA GGGCAGTGCCCAGCCCTTTAGGCCTTTCTTCTTGGGTGGTGGGTGTGTCCAGCCCATGAG GGGCACAGGGCAGTGAGGAGGGTCCCCATCTCCATCCTGGACAGAGAGCCTGCTCCATGG GAGGTCCCAGGGGCCAGGAGGGCCCCTGTGGTGCTGGGAGGGGAAGAGAGTTGTGAAGGG TGGGTTTGGGTCCCTGGAACCTGTGCATCCCCAAGACAGATGGGCCTGGAGCCTTCGGGG GCGGGGCCAGCTCTAGGGGCTTGTTTAATGCTCTGTGGTGGCTGCCTTAAAATACTTAAT CATTTTTGGTCAGGCACGGTGGCTCAGGCCTGTAATCCCAGCACTTCGGGAGGCCTAGGT GGGCGGATCACTTGAGGTCAGGAGTTCAAGACCAGCCTGGCTAACATGGTGAAACTCCGT CTCTACTAAAAATACAAAAATTAGCTGGGCGTGGGGACATGTCCCTGCCCCCTGCCCCTT GTGCCCGCCCTCTCCCTCCCCCTGCCCCTTGTGCACACCCTCTGTACCCCCCTTCCCGCC CCAGCCTGTGTTTGCAGTGCTACAGGGTGGCTGCAGCCTTCGCCTCCCACAGCTCCTCAA GGTCAGAGGGCTGGGAACCCCTGGGATGTGGCTGGGATGAGGACCCTGCCCCTCGTCAGG CATTTCCAGGCAAGGGGCACAGAGACACAGCCAGCCCAGCAGCTGAGCCCCTCCTATCAG GCCAGGTGGGAAACTGAGGCATGAACACAACCCCAGACTGGAAATAACAAAGCCATCATG TACTGTGTGCCTACTGAATGCCAAGCCCTATGCCCTATTCTTTAATGTCATCTTTAATGA GATGTCTTCACTCAGTTCACCACATGATAGTAGACAGGTATTTATGATGCGCCTACTGCA TGCTGGGCACTTTTCTGGGCCATGAGGACTCAGGGAGGTCCCTGCTCTGGTAGAAGTCAA ACCCGACAGGAGACAGAGACTAGGGCCCCACACACCAGGAGGAAGAACCCAGTGCAGAGA GTGCCAGCAGGAAGCCACAGGGGCAGGGAAGGCGCTCCTGGGAGGGTGGGTGGCTGCGTC CTGAGGCTGAGCAGGGGGAGCCGGCCTAGGGACAGGATGTGCGCAGGCGTGGAGCAGGAA GTTCCTGGAATGATGGGCAAGAGGGAGCAGGTCACTGGGGGCAGGTCTAAATACTGGGGG CTGCATTGGGATAGCAGAGGCCTCGGACAGGCCCGCTCAGCATGTAGACTCCCCTCCCTG GCCCCTTCCCTAGGATGACAGTGGAGGTCCATCCCCAGGTGGTGGTGAGGACACTCCTGA CACAGAGACCCCAGCCCAGCTGGCAGGGGAAGGGGAGCCCTCAAGGACCCCTGTAGGTTG GGGCATGTCCCGCCAGGCGGCCCTCCATGGTGCCCAGCAGCAGCCCTGCTTGCGGACCCT CTTTAGTCCCCACTGTTAAAAAAATAATTCAGTGAAAATTGACACTAGCTGGGCATGGTG GTAGGTGCCTATAATCCCAGCTACTCGGGAGGCTGAGACGGGAGAATCGCTTGAACCAGG GAGTCGGAGGTTGCAGTGAGCCGAGATGGCACCACTGCATTCCAGCCTGGTGACAGAGTG AGACTCCGTCTCAAAAAAAAAAAAAAAAAAAGAAAAGAAAAAAAGAAAAATGACACTTGC CAAAGCGCCGTAGGGAAGACTGTCCAGGACCGTGTCACTAGGCATAAGGGACCACGGCGA TGAGATTCTGCAGTGGGTAAGAGACTGGGCTCAGCCTCGAAGACAGCATGGGCAAGTGGG ATCGGACCGTCAAGGAGCAGGGGGAAGACTATCCAGGACCGTGTCACTAGGCATAAGGGA CCATGGCGATGAGATTCTGCAGTGGGTAAGAGACTGGGCTCAGCCTCGAAGACAGCATGG GCAAGTGGGATCGGACAGTCAAGGAGCAGGGTGGGGGGCAGTGCATGGAAAGTGACTAAG AGGAAGCATCAGCAGCCAGGGATTCTGGCTTAACTCCCCTAACAGAATTCTTGCTGAAGG CCGGCCAGGATGGCCAGACCGCACCTGGGGGACAGAGGGGGAACCCGATCAGATGTCAAG GACGGGGGGCTTCTCGCTAAACTGACTTTGCAGAATTCTTGCTAAAACTAGAGTTTATAA GGAAGTGCTCAGATGTGCCTAGGAGAAGGCTCAGGAGCCTAAATAAAGCTTGACCAAGCA GAGAATCTTTGTCACCAGCTTGGTGAGAGTGAGGCCCAGAGTGGGTCAGGGCTTACCTGC TCAGGTTGTACAACTGGGAGGGGTCCGTCAGGAGCCTGGAGTCTTCCCACTGGGGGCTGA GTCTGGGCCCTCAGGGCACATATAGGTAGATGCTGTCAGGCCCGACACCCCATGCAGTGC AGACTGGGATGCTGGTGCCTGTGGCACAGACTGGTCAGACCCACCCCAGGCTAGACATTC CTTGCAGGGAGAGCTGAGGCTGTTGATGCCAGGAACAGACAAACAAGGTCATCACCAGGT AATGAGCTCGTTACCCACATCAGGCAAGGCCATTGCTGGGTAATCATTTGGCCTCCTGCA GAGCCACCTGCCCTGGGACGGTTCCCAGAGATCTGGCTAGCCAGTGCCACAGGAGGCCGG GGCACAGTTCCAGGGCCTAGTGGGCAAGGCAGGGGTATTCAGGGACAAGGTGCACCTGAG TCCCCACAGGAAGCCCCAGACCCTGCAGTAAGACCTGGAACAAGTCATTCTCCTCTCTAA GCTCTCAAAGAAGAGGTCAGCTCAGGGGTCGGGGAGGCAAGGTCTCCTAGAGTAGACTAA GCTGGCCGCTGCCATGCTAACACACACCCTCCCAGCCAGCCGCTGGTCCTGACAGATGAG GAAACTGAGGCTAGGTGTTTTCATGAGGCCTCTGAACCCAGAACCCAGTACCCAGTGTCC TGAGCAAAGCCCAAGCCAGACCTGCCAGCTTATGTGTACAGAGGCCCCACTATGCAGCCT GCACTCCTGGGGCCTAAAAGGACCATCACCTCCCCTTCTAAGAGGCTGGGCACAGGGGCT GGGTCCCAGCCAGGCCACCCTTCCTCCTCCTCCAAAGGCCCAGCAGCAGCTGCTCCCAGA GCAAGCAGCACAGTCGCAGCAAAGGCTGGACAAGTGTCCTGTGCTCTGTGGTCTGAGCAG AGGGCCAGGGGCCGTGACCCACATGGCCCCTCCAGGAGCAGCTCCAAGATGAGCCTTCCT GTCCCCACCAGTGTGTCCGGGACCTCAGGGTGGATCAGCCGCACCTGCCGGCTGCTCCTG TTCCTTGCTCTTCCTGGGGCTCCCCACCACCTTTGGGAGGCAGCTCCAAGACCCTGCTGC CTCCCTTGCTTACCCTCAGGCCCCTGCTGGGTGTGCCCCCTCCCCACACAGACCCCTGAG CCAGCTTCGAGGACTCTGCCTGCATCTCCACGTCCACTCTGATGACTGTGAAGCTACAGT CACACTCTCTGCCTCCACCCCCGCCCTGCCGGGTCCCTGCAGCAGCTCTCAGAGGCCCCC ACAGCCCCTCCCACACACAGAGCTCGCCTGGCCCTGCCGTCTGAGCGCTCCCCTGGCTCC CAACCGCCCTTGGGCTCTGGGACATAAAACCTCCAGCCAAGGTGGCTGCCAAGTTTTATT TAATAGCCATAGAGCACAAAAGGGATGACAATTAAGTCACACGGAGAGACTGCCCCATCG GGACCCCTGGCCAAGCTAAGCAGAGGAGCCCTGGGGTTTCCTCTGACCCCCCACTGTGAA TGCATTACTGACGGGCAAGGTGGCCAGTTGCCCACCCAGCCTGCACGTCTGCATGCACAG AGCTACAGCCTGGGCCCAGGGACAGCCCCGGGAGCCGGTGGCTTCCCACAGGCACAGCCA GCCTCCCCACCCCCACCATGCCTGGCCCCCGGCCCAGGGCACACTGAGGGGAACAGGAGG GGAAGGAGCAGGCCCTGCACTGGAGCCTCAGTCAGCTCCTCTGTGCCAGGGGCAGCTGGA GCTGGCTGAATTTGAGGAACTTCCCGGAGGAACTGGAGAGCAGACCTGTGTGCGCGCCCC GCTCTGCCACGTTCCCTGAGGGACACACTGCTTTGACCAGCCCCCACCCCATCTCCTCTC CGCCAGTCCCCGGTGTCCACCCCAGCCCCGCTACCGATGAAGCTCAAGCCAGGGTCCCCA TCACCTGCTTCACGGCCATCCCTGCACTTGAGCTCAGGGGCTCAGGGTTGTGCTGACACG CCTGCCTGTCCACCCCTCACCGACACCTCTGCAGTCCTCCTCAGGGCCCAACAGGGTCCC CAGGACCCCGCTCCCATTCTGCCCTTGCTCCTGCCAACCTGTCTTCTCTATGCCCATGAC TTTGGGACCCTCTGCCATAAGTACCAGCCCCCACATCTGTAGCCTCTGCAGGAACAAGCC CTAATCTGGACTCATTGCAATGGCTCAGGTTCTCTTCCAGCCTGTCCATAGACCTCCAGG CAGAGATCACAAAACTAACAGCTCTTTCTGCAAATCTGGACTCTCTCCTGCACCTTCGCC ACCATCCAGGCCTCCCTGTCACTCCTCCTGGGGACCTCAGAGATCTCTTCATTTCACAGG TGAAGACTTGGCTCAGAGAGGGCAGAGCCCCTGGGTCACACAGCCCGTCTCCAGTAGGGC TGGGGAAAAGAACCCAGGGCGCTCTTGTTGGCAGTGTTTTAATGACTGGGGGGAGGGAGA GAATGGCTAATGAGGCTCTAGGGATCAGCAGAGCCAGCTGCTGGAGGTCCCAGAGGACGG GTGACTCAGCAGGAATGGGGGAGTCTGAGGGGTTGACAGAGTGCCCAATTCTCACCTCCT CCTGCACCTTCCAGGGAGACGAGCTGCAGGGAGTGATTGAGTGGATGAACATGACTAGAG GGACCCAGTCTTCTCCTCAGCAGCTGCTTGAGGACAGTATTAGCGGTCCCCTCAATCCCC CCCCCAACACATGCACACATGCACTGATCTGCAGGGGCCAGACCCCAGTGGACCCGGGGA CACAGCTTCCAGGGGCTGTCTTGCCATGTCCCTTCCCAGACCTGCCTGGATTCATGCCAG GCCACCCTCCACAATTTGCTGAGGGAGGCCCCAGGGTCCAGGGCACCCAGAGTGCTAACT AGCCCCTGGGGGTACAGGGAGAACAGCATGCTGTGTGGGGGATACTACTCTGCCCATACC TAGGGCCCTGTGGCGGCCACAGGCAGCACTGCTTATGCAGAGGCACTGGGGTGAGAAGGA GTAGGGTGCAGTAGTGAGGGCTCCACAGAGGGTGGAAGGGCTGCAGGGGAAGGCAGACCA GCAGGGATGCCATGAGGGCCTCAGATGCCAGGCTGCAGAGGTGGAGGGGCTGGCCAGCTC CTCTCCCCAGCCTCCTACTGTAAAATGGGAGAGGATCCCAGCCCTGCTGATTCATCCACT CACTCCATGGCAAGACAGCCACTGACAGCTGTGTCCTGGGGCCCACCCGGGCCTGGCCTC TGTGCAGGCTAGCACTCAGCATGCTTAGTGCCACACTCGGCCCAGGAGAAATGACCCAGC CCTGGGCTCAGAGACCCCACAGTGGGTGGATGGAAAACAGAGTCATTTAAATACAACTGT GAAGGGTGCTGTGGAGGAGGGGCTCGAGGTCCCCAGTGTGGGATGGGGTGGTCATGGAAC ATTTCCCTATCTAAGTGGGAAAAGTGAGAGCCACCAGGAAAATGGCAGGGGTTGGGGGGA GAGGAACTGTGCAAAGACCCAGAAGGATGAAGAAGACTGTTGGATTAGAGGAGGTTAAAG ACACAGACAGGCTTGGACATGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAA GACAGGAGGATCACTTGAGGCCAGGAGTTCAAGACCAACCTGGGAAACACAGTGAGACCC TATCACTACAAAATAAATAAATAAATAAAATAAAATAAATAAAAATAAAAATAAAAAAAG CCGGGCATGGTGGTGCACGCTTGTAGTCCCATCTACTCAGCAGACTGAGGTGGGAGGATC GCTTGAGCCCAGGAGATGGAGGCTGCAGTGAGCTATGATTGCACCACTGCACTCCAGCCT GGGCAACACAGCAAGATCCTGTTTATAAAAACAAAACAAAACAAAAAACCCAGCTTGAGA TGGCCAGGGATTTCCAGAGGCCCTAGGCGGGCCACCTTGGAGAGTAAGGGAGACCCTGAA GAGCTCGCATGGAAGGACCAGCAGCTTGGGGCTGGAGATGCCACCTGGATGGATTCCTCC AGCTGTGTGGACTAGGGGACAGGGTGAGGAGATAATGGCCTGGACTACATGGAAGGACGT CGCCCTGAGCCATTGAAAAGGGCTGTGGGGTCCATGACCACTCCTGGCCTTCCCTTGACT ATGATGGAGACAGTAGCTTCTAGTGTAAAATGTGAGCTCAGTTCCTGACTCGTGTCTAGC GGGTGGTGCCTGGTCTTTGGCTGGCGATGCTCCATGTCACCTATGCCCAGAAACTGGGCC ATGCCCTTTGTGAGGACCTGCCCACAACGTCGTTGTTCCCGCAGGGTCCCCGGGGAACCG GGCTGCCCGGGACCCAGCCCTGCCAGGGTTCCCTGCGGCCCAGGCCGGAACCCCCAGGGC TTTGGTCGTTCCCAAAGCCGTCCCGCGGGCCCCCTCCACGGTGCGTCGCCTGGGCCCCAG CGATGGCTAGGCTGCAGCCTGGGGCTCCCGGCCATCCCGGGCGCTGGCGGCGGTGGAGGC AGCAGGGCGGATGGCGACGCCCGGCCGCCCGTGGGCCCAGGCGCGTAGTGCGTACAGAGC CAGCGAGGTGCTGCGGCGCGTCACGGGCCGCCGGCGGGACCCGGGGCCGCAATCCAATGG GCCGGGCCGGGAAGACGCCCGAGCCCCGGGCCGGCTGGCTCGCCTGCTCCGCCAGCTCCG GGCCGAAGCGGCTTCGCGGTCCGAGGTGCCGCGGCTGCTGAAGCTGGTGGAGCGTGCGGG GGCCGGGGCGGCGGGCGCGGGCGAGAGGACCGGCGCGCACAGCCGCGGCTCCGTGTGCTC AGTATGCGGGGAGCCCCGCGGCAGGGCCACCTACCCGGCGGGGGTCCTGGAGGTCAGCGA GCGGCGGCTGCAGGAGGGACTCGCGGCAGTGCGCGAGGAGCTGGGCGCCGAGATTGAGGC GCTGCGCGCGGAGCTTCGAGCGGAGCTGGACGCTCTGCGCGCGCTGCTGCCGCCGCCGCC GCCGTCCCCGCCTGCCCGCCGCGAGCCCCGCGCCGTCCCCCGCGCCGCGCCCCGCGGCCC GACCCTGCTGCGGACGCTCGGCACCGTGAGCGCCCTGGTCGCCGCCTCCAGGCCCGCAGA CGACGCCCCGGACGGCCCAGCAGAATGCGGAGCGCACCGAGCCCCGGCCAGGAAGAACCA CAAGAAGATGCCAGTGCCGCCTGGGGCCCCGCAAGGTGGCGGGGACTGAGGGCGGCCGCA CAAGGGCAGCCTAGGCGAGGTGCGGAAGGCGTCGCGCTGGCTACTCTGGTACCCCCAGGA CGGGGCAAGTGAGCAGATCGGTCCCCCTCGTGTAGCGTGGCTCTGAGTCAGAAGGGTGCC CGGGTGCCGCCAGTTAGGGCTCCGGTACTGGAGGGAGGGGGTGGGCGCGGGACGGGCGGA GGGTGGGCCACGTGCGTCTGGGGAGTGCGGATGGGAGCCGGGGTCCTTGCGAGAGACTGA GTCCGGCTAGAGAACAGGGTGGAGCCCCTTTGGACCTTAGAGCTGGGCCTTTGGGCCTTG GGTCTGGGTCAGCCTTTGGGCCAGGGCTGGGTCAGCCTCCAGGGGAACAGCCGAGACTCC TCTTCCTTGGGGTGACTGCACGTTCTTTTTCTTTCAAGGTCTCAGTGACGAGGCAGGGTC TCCACGCACTGGAATAGTGTCAAACCACACACAAGGGCACAGAACCCAGGGACACAGGGC GGCTGAGACACGCAGCTGGTGATGGCCCTCAGGACGGTGTCAACCAGTATGTAATCACAT TCCACACGGCACCAATTGGACAGCGGCCTCCGGTCAGCCTCACTCTGACTGCAACCCCCT CTCACTGGACAGCATCACCTGGACACAGAGCCTCACCTAGTCTGTGTCCCGTGGACACAA CTTTGCTTTGGACAGTGACACCAAGAATACTCACCCCTTTACTTAGCCAGACCACAGGCA GCCACCGGGACACAAACATCTCTCCAGAGTCACCCTCCACACAGACTCCACAGCAGAACT CCCAGGGAGGCACTGGGGCATGGCTTCAGAGCACCAGGCAGCGCTCCGCGGTGCCCAGCA CCCGCTCAGCCAGGGCAGCCTGCAGCGGAGCCTGGAGCCAGCAGCTTCTCATCTCTTGGC CTCGGGAAATGTAGCTGGAGTCATCATTTAGCAGAGCACGGTGTCCCTGGGTTGGCCACC CAGCTTAGTTTTAAAATAAAATAATGTAATCTTGGCCTAATGGTGGAATTTCTGACTGCT AGATGTTCTCCTTCCATCTGACCAGGGGATTCAGACACAATGTATAGGATGAGGGTGGGG GCCCTTCTCCCTCTGAGAGGAACCTGGAGAGGCCCGGGTTCTGCTGAGGTAGGAGAGGGT CACTCAGAGGGCTCCCACGGAACAGACAGGCACACTGAGGCCTCAGTGGGATTAGGGCAG GAAATGCCCCTCTTGCAGTGGGGGACCGCGCCGGGTCACGCACCCACCCCCTCCAGGCCA CATTAGGCTAAGGTCACTCTGTGCATTATTTCATTATTTCACACGGAGGGTCGCCCACGG TTGACAGAACCACAAGCTGAGCCCCAGGCGCATCAGAGCGCAGTGCACACCTGCTAGGGT GGGGGAAGAGCGGGGGGCTGCTGCGGGGACCGAGGCTCTGGGGGAGGCCAAGACTGTGTG CCCCTTAAGAGGCTGCTGTGGGCCAGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTT GGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCTGGCTAACACGG TGAAACCCCGTTTCTACTAAAAATACAAAAAATTAGCCGGGCGTGATGGCGGGCACCTGT AGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATGGCATGAACCCGGGAGGCGGAGCTT GCAGTGAGCTGATTCCACCACTGCACTCCAGCCTGGGCGACACAGCAAGACCCCGTCTCA AAAAAAAAAAGAGGCTGCTGTGGGCGGAAGTTACCCGTGAGCGGTATGGGGTGAAAGTGG GAGGGTCCAGCCTGGGGCACGGTGTGGAACAGGGTCATCTGAGGGTGCAGGGCAAAGAGA TCAAAACGGGGTGGGAGAGGGGCAGCGCCTGCGGGAGGAGCCAGGCAAGGCCCGGGGAGG GGTGTGGGGGGCGAGGGGGGACGGTGGCTCCACCTGCACCCACCGGATGGGTATGGGGTC CTCGAAGACGGTCCAGAGCACCGTGGGCTCGCAGTCAGGCGGGGTCAGTGACCCAGCGAA GCGACAGTAGCTCGAGGTGTTGGGCCGCATCGACGCCAGCAGGAAGGTGGACATCAGATT CACTGAGAGCCCCAGGCGGCAGAGGGAGGGGGATTGGAACCAGCCTCCCCGTGCCCTGCT GGTGCCATCCCCAGCAAACCCCCCGCCCCTGAGCCGGAAAACGACCCGGTGGCTCCTCAC CTGGCTCAGGCACCTTCCTCAAGCCCGACACTATGGCGCAGAAGTTGGTGTTGCTACAGT CCTGCTCCTGGGAGGGGCTCCAAGTGCAAGGGGTTCTGAGTGGGGGAGTCTCTGAAGAGG GAACTCAGCTGGGATACTCTAAGCAGGAGGCTCCCAGTGGCAGGAGGCATCTGGCATCTG GGAAATGGGGTGGAGGGTTCTCGCCGCCACTGGCCCTGCCGATTCAGGCATTCCCACCGC CCTATCCTCCCACAGATCCTATAGACCCTAGAGATCCCCCCATGGACTCCACAGGCCTAG CCTGTCCACTTCCAGGATCCCCATGGACCCCTTTTTTGGAAACATACATAGAATCACAGC CCTGCCTTCACCGCCAGCAGCACCTCCAGCAGCAGGGCCTGCACTCGAGCCCATCACCGT GGCGTGGTGCCTCCTCCATGCTCTGGTACTTTGTGTTACTGTGGACCACATGCATCTGGG GGTAGGCACAGATGTGGGAGATACCCAGCTGCCCCCACCTTGCTCTCTCTCATCTCCGTG GGAGCACTCCGGCCACCCCACCTTGTGCCCAAGTCTACCTCCATAGCCTGGCGCTGCCTG TCCAGGCTGTGCTCTGAGTCTGCTCGCCTCGGGCCCCCCCCCCGCCCCCAGCTGATGTGC AGCCGCAGTGCGCGGTAGACAGGCAACAGCAGCCCAGCCTCCCAAATCTCCAGGTGGTTC TGAGGATCAGTGTCCATTCAGAGTAGCAATGAAGGGGAGGAGGCACACGACACCCTCTCT ACCTCACCACCTGCCCACCTTGTGCCATTAGAGCACAGGGTACGAGCAGAGGCCCTTCCT CAGGCCAGGCTGCTCCCCAGTGGGGCCCCATTGCCTCCATGGAATGGAACCCATGGTGTC CATGGCTCCTGCTGGTCAGCAGAGCAGAGTTCCACAGACTGCAGGGGAAACCCTCCCCCA CACACCCAGCATGGCATGGAAGAAGGCAGAGGGCAGCCCTCCTGGGGTGGGGCTCTGAGC CCAGCTGTGTGTGGTCAGGGATCCTGGCCCTGCCAGAGACCCTGACTCCTGGTCCTCAGG GGCCATGGCTGGTTGTGATTTGTAGGGTGGGACCTGGCACCTACACAGAGGGCGCATCTG GACTGCTGTGTGAGGATGCTCCTTTCCTGTGAAGCTAGCCCCTGTGGAGGGTTTTTGGCC CTGTAGGATGTCTCAGCTCCCGTGTGTAGGGGGTCCTGGTCCCTGTGTACACTCAGGAGC TCTCATGCCCTGTGTGGGGCTCCTGCTCCTGCCTAGGAGGGGTCCTGACCACAGGGTGCT GACCTGTGTGGCTGTCATTCTCCAGGGTCCAAGGGCCTGGAGGTGCTGAGTCATAGCCTC GGAAGATGAAGGGCCCTAGGGTAGAGTTCCGCCGGACCCTGTGAAAGTCAATGTCGATGA GGGACTGGCCTGGGCCCCCACAGGCAGGGGCCAGCTTCTTCCAGTGGGTGGGGACTGCTG GAACAAGGACTGGGGCCTTTGGGGTGGGGGTGGGGTCAGGGAGGCAGCCTGGGGGTGCAG CAGAGAGTGGCCCCCATACACCCAGAGGATAGCCCAGCCTCAGGAAACCCAGCTCCCTGC TTCCCCCACACCTGAACAGGTCACAGCATCCCCCTTCTCTTGTACCCTGCATCTGGCCTA TCCATCTAGTCTCCCGGTTGGCTGCCCCATGGTGAGATGAAGGCAAAAGAGTCTCTGGCA CACAGTTGCACACCCACACTGGGACCTCTATCACCTGCACACACTTGGGCTGGCCACAGT AACAGGCACACCAGCCTTGACTGACTCGGGACCGGCGATCACAGCAGGCCCAGACTCACA CACAGGGGGACAGTCACGCACTTGCACCTGATGCCCAGTCAGTCTACTGGGGCAGACGAC TACAGACAGAAACCTGCAAAGGCCACAGAGTGCAGGGTGCAGACTGTCTCTTGGCCACTC TTGCACCACTGGCACATGCTCTGGTGGTCCCAGGACACAGACTGATCTCACTCAGCCTCA CATGACACTCTGTCCTCACCACACTTGGGGTCCTGGGAGTCGTAGCACCAGGCACCTATA GAGACAGTGGGCAGGAGGCTGAGCTGAGAGACCAGTCATCTGGGTCCTCTCCAAGCCCCA TCCCATCAGCTGGGGTCCAAGAATGAAGTAGGCCAGGGGCTTAGGCCAGGGGAGGCCAGT GAAGAACCTTCTCTCCTGTCACCCCTACCCCAGTATTTTCTCTCCCCAGTCCCTCCCCAC TCCCAGTGGCAAGACCTAAGGGGTGGTGGAGCATGGCAAGAGGGTAGATTGGAGACCAGA CGGGAAGAGTCCTGGTGCTCACTCTCAGAGTCTGCGCCACACCAGCTGCACTGCAAGGCG AGCATGATGCCAGGGGCGTGCGTGGCTCAGTGGCAGCCGCAGCTAGGAAGGACTAGAAAG TGGCTAGACCTCGGAGCCAAGATGCTGGCCTGGCTACTACATATTTATTCATTAGCTAGC TGGGCTAGGGGCGTGGCTATTGAGGAGAGGATGGGAGGGGTCGGTGCCCAGGCAGGGCCA CCAGGAAGAGCGCGAGCGCTCCAAACTCACCCAGGTGTGCTGTGGTCCCAGGGCTGCACT GATGCCAGCAACAATCGCTGAGGTTAATCCTGCAGGGGAGGGGTGACCTATTATTATCCC CACTTACAGTCCAGGAAACAGAGGCTGGGGACCAGAATGACACACCAAAGCCACCAGCAA GAAAGGTCAAATAGTGAGGGCACCTTGAACCCCAGCGCTCATGACTTTCGGGGCAAGGAT GGGAGGAAGGTGTAGGATGTCTCTTGCCCCACTGTGGGCCTGGAACTGCCACCCCATCCC AAGACCACAGCCTCCCTCCTGCCCAGGCCCCCCTGCGGATCCAGGCCAAGACGGAGCCCT GCAGGAGGTCAGAGGAGGGGACTTCTTTGCACCCTGCGCCTCTTTCCAATCGCCAGAGGG CAGGGCCCTACCCACATTGGGGATGGGAAGTCAAATGCAGAAGTTAGGTGAAGTCAGTTC TTGGATACTCCTGTACTGTCACCCTGGTCTTATCTCATGCCTTAGCCCAAGCTGTGCACA CAATGGGGTCCTAGGTCCCCCTCACCTCCCAGATTCCACCTTCCCAGGGATGGGACCCCC TAGAACCCTCGGGGGCCTGGGCAGTGGCCTTGCTGGCTCTTGCCTTCCTAGGAGCTGAGC AGGAGCTCCACTCTCAGCAGGGCAGTTCACTGCAGCCTCTGCTTCCTCAGCTCAAGCCAT CCTCCCACCTCAGCCTCCTGAGTAGCTGGGAATGCAGACACACACCACCACGCCTGGCAA ATTTTTGTATTTTTAGTAGAAACAGGGTTTCACCATGTTGCCCAGGCTGGTCTTGAATGC TTGAGCTCAAGCGATCTACCCACCTCGGGCTCCCAAAGTGTGGGAGCCAAGATGGGAACC CAAGCATACGGCCCCAATGCTGAGGCTCTGAACTACTGACCTGCCCTCAGCACTCAGCCT TGGGATCATGAGTCACTGTGCAAGGGAGTTCCAACATCTGCATGTATGTCTGGAATGATC TGAGCCTGCAGAGTTCCTACACACTGGCCACATTATAGGGTGGTGTCTGTGGTCACACAG CTCAGGTCAGGTATTTATTAGTACATGAATAGCTTAGCTGTGTCATAGTCTTTATGTGAA AGGCACATAAAAGGCACTTTGGCAGGCTCAAAGTGTGGGGATTATAGGTGTCAGCCACCG TGCCTAGCCCACTGGATGACTTATGATATCATATGTGACATTGTGACATCATGTGAGTCA GGGATGTACCCCGTTCTCAGCTGCTATATGCTATGTTACAGTGACAGAATGGGAATGAAG AATGTGTCCCACTCTCTCAGCTGTTGTATTATATCATACAAGGTGCAGTGACTAAGTGTG TCAGCTGTGTCCTCATCCTACATAGCATATGAGAGTGTGTGACGGGAGATAGGATGCAGA CCTGAGAAGCATTAAACACCTAGGCAATAAAGGTGCCAGCATCAGCTGAGAGCGCAGGTA GACCTCAGTCACATTTGTTACTGTGTAACTAAAAATACAAAATTAGCTGGGTGAGGTGGT GCACGCCTGTGGTCCCCACTGCTCAGGCGCCTGAGGCAGGAGAATTGCTTGAACCCAGGA GGCAGAGGTTGCAGTGAGCTGAGATTACACCACTGCACTCCAGCCTTGGGGACAGAGTGA GACTGTATCTCAAAAAAATAAAATAAAATAATCTAGGACAACCAAGAGAAGGACTCAGGC TCACCTTACTCTATGTCACATGTGATATATAACACTTGAGGGGGACACACACTTGTCACT TTGTATCACTATGTCCTATATGATAGCCAATGATAACCCAAGAGGGGGAGGCAGCCTTGT TCACACCATGTCACTTATGATATTATATGACATCAAGGAGCCCAGACCTCAGTTGCATTA TGTCACTATGGCACATACAGTATCATGCAACAACTGACAGAAGGGTTGTGGGCCTGATTC ACAGTGTATGTGAGTCACAAATGTCACTTATGACACCATACAGCAGGTGAGAGGGGATAG TCATTATTCACCTCGGGTCACATTGTCACACATGATACCATATGATAGCAGAGGGGGATG CAGAACTGAGTTGTATTATGTCGCTATGTAACCTGTGATAGATGATAGCTGATGCGGGTG AAGACCAAAGTTACATTTTGTACCTACATTATATGAAATATACAACATGCACGAGGGAGG TGCAGACACAAATCACACTGTGTGCTGTATCCCATATATGTGTTTAATTTCTGTGTTTAA GAGAGCTGGGTTCATGAATGAACGTAGTCACTGCACCTTGTATGATATAATACAACAGCT GAGAGAGTGGGACACATTCCTCATTCCCATTCTGTCAGTGATTGACCACACCTTGTGTTA ACTGTATGAGCCACTGTGTTTGGCCGTGTATAGCGCACGTGGCACATTGTTCACTGATAC ATATTTGCTCCGTACTGAAGTGAGGCAGATGCATGAGTGATTGAGAATAATGCTACCTTG GTGAAGCATTCCATTTAGAAGCATGAGGTTCAGGTACTACCATCTTCTGTGATACATATT TAAATGTTAAGTTTAACAGTTAGGTCTACCATGAGTGAATGAGCCTTGCATTATCTTTGA GAATCGATGTGTTTAGCAGGGTGTGGCTCATCTAAGATAGCCTTCTTTAATGCATGTTCA CTATCTGTGTTTATTAGAAATAGGTTTGTGAATGACTTAGCATAGTGTTTTCCCTATGAA ACCCTGTTTATTTATTTATTTATTTATTTTGAGATGGAGTTTTGCTCTTGCAACCCAGGC TGGAGTGCAATGGTGTGGTCTCGGATCACTGCAACCTCTGCCTCCCAGGTTCAAGCGATT CTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACGGGCGTCTGCCACCACACCCAGCTAA TTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAGCTCC TGACCTCAGGTGATCCTCCTGCCTCAGCCTCCCAAAGTGCTGGAATTACAGGCGTGAGCC ACCACACCCGGCCAAAACCCTGTTTTAAGAAGTGTGTGGTGCAGGTACTACATTCTTTGT TGTTCCAAGTTCAAACCCTGTGTTTAACAGAGATAGGTGCAAGATTGCCTAGGTGTAGCG TAGACTCTTGGAAATACTGTGGTTAGCATTCTGTGTTTTCTGTAGCATAGTTTCAGTGAT GCCTGTTTAGATAGTTTATCAGATCTAGGTACTAGTGTTATGTCAGCGAAACACTGTTTA GCTGCACGCGGTGCATGTAATACTTTGTTCATTGATATTTGTTTAAATCAATCAGTGTTT ACCAGAACTAGATATGTGAGTTACTGAGAATAGCCTAATCTCTGAAAATGTTTTCCAGCC TATGGTGCATGTAATATACTCTTCTATGGTACCTCTTTCCAATGTTTTTATTGTGGCAAA ATATACATAACAGATTATACCATTTTTAAGTGTAGAATTTAGTGGTAGTAAGTATAGAGA GAACCTTAAAAGCAGCAAGAGAAAAGTGACTTCTCATGTGCAAGGGAGCCTCTAGAAGAT TATCAGTGGATATTTCAGCAGAAACCCTGCAGGCCAGAAGGTGGTGGGATGATACATTGA AAGTACTGAAAGAGTAAAGCCTGCCAACTGAGAATACTATATTTGACAAAACTGTCCTTC AAAAGTGAAGGAGAAATTAAGACATTCCCAGATAAATAAAAGCTGAAGGAGTTTATTACC ACTAGACCTGACCAATAAGAAATAATAAAGGGGCCAGGCACAGTGGCTCATGTCTGTAAT CCCAGCACTTTCGGGGGCCGAGGCAGGTGGATCACCTGAGGTCAGGTGTTCAAGACCAGC TTGACCAACATGGCGAAACCCCATCTCTAATAAAAATACAAAAATTAGCTGGGTGTGGTG GCACGCACCTGTAATCCTAGCTACTCAGGAGGCTGAGGCAGGAGAATCACTTGTACCCGG GAGGTAGAGGTTGCAGTGAGCTGAGATCACACCACTGCACTCCAGTCTGGGCAACGGAGC GAGACTTCATCTCAAAAAAAAAAAAAGAAACAATAAAGGGAGTCCTTCAAGTTGATATGA AGGGATACTAGACAGTTGCTCAGCCTGATGAAAATATAAAGGTAAATACATAAAGATTAA AACCTGTATTATTGTAATTTTTGCTAATAACTCAATTTTTAATACTTTAATGGAATTTAA AGGACAAAAGCATAATAATTATAAATCTATGTTAATGGATACAAAGTATATAAAGTTTAA TTTGTGACATCAGTAACTTAATAGTGGGGCAAAGATGTAAAGAGTAGAGGTTTTGTATGT GATTGAAGTTATCTGTTTAAAATAATAAGATAACTTTAAGATGTTCCATGTAATTCCCAT AGTAACCACAAAGAAAATACCTACAGAATATATGCAAAAGGAAATGAGAAGGAAATCAGA GCATAGCACTACAAAAAATCAACTATAACACAAAGGAAGACAATAAGGGAGGAAAGAAGG ACAAAAAGGCTCTAAACATACAGAAAACAATGACCAAAATGGTGGTAGTAAGTCCTTCCT AATCAATAATTTCATTAAATGTGCATAGGTTAAACTCCCAATAAAAAGACATAAATCGGC TGATTGGATTTAAAAAAACGGGATTCAACTATATATGCTGTCTACAAGAGACTCATTTGA AATCCAATACAAATGGGTTGAAAAATGAGAGAATGGAGAATATTCCGTGCAAAAAGTAAC CAAAACAGATCAGGAATGGCTGTACAAGTTATGGCTGTACAAGTTGTTGGGTTTTATGTT ACTGAAGAATGAACAGAGATGAGTAAGTGGAGGTGTTATGTAAAGGCATACTGTACTCAA AATCTGAAGACCTGCAGCAGATTTAAATTCCAGCTCTTATTATAACTTTTTAAAAGATTG TGAAAATATCAAAATATAGATGAATCAAGTTTTAATATACTGTATGATGGGTGGATGAGG CTGTCCATTGTACCATTTGTTTGAATTCTCAGGCATGGTTTGGCAGTGCAAGAACTCTGT AACGTTAACAAATTCAATAAAAAGTAAATATATGGAAAAAAAAGTATAGACAAAATAGAT TTTAAGTAAAAAACTATTACAAGAGAGGGTTCTGGGAAGAAAGTGGAGTAGGAAGCACTG GGAATTCATCTCCCCACCTAGAAAATAATCACACTGGCAGAATCTGCCTGATATAACTAT TTTGGAACTCTAGACTCTATCAAAGGAGGCTTGTAACCTCCAAATGAAGGCTTAAACTAT AATTTTTACTTAATTTTGGTCAATTTCAGCTCTTAGCTCAGCAGTGGCTACCCAATCCCC ATGCCCCAGCTTCACGGCAAGAAGCTTTGCATGTGTTCCTGAAGCAGCTTGTACCAAGCT TGTGGGAACAATCATGGGCAATAAGCACTCTGTCCTCCAAGTGTTAGCATCTGCGTTCTG GTTGTTGATTGCTACTTTTGATTATGGAAGGGCAAACACAGAGGCTGGCAGCCATTATTG CTCACAACTCCCCACTCCACTGCTGCAAGCCCTTACCAGACTGAAGCAACTTCTAGGAGA TAGAAAAGGCCAGAACCCCATTTCCCTTCCCCTTCATTGTTCTCTTTTCCTTTTTTGGGA GCCAAACATTAAAGACTAGGACACTCAAAAGCAATGGCATACCCAGAGGAAATTAAAGTT ACCACACATCCTTGGAGAGAGGAGTGTGTGCCCAGGGAAAGGAGCAGCTTCAGACCTGAG AAGACCTCAAGCTTACAACTCAGGTTGATCCTCAGCATGGAGACAACCTACAACAATGTA AAACATAACAAAACCCCAAAACAGCAAACCCTGAGGAAGAGGAGAGTCTCATCTCCAGAG TTACTGCATTATTATATTCAAGTGTCCAGTTTTCAATACAAAACACAAGGCATACAAAAA ACAAGAAAATATGGTATTTCAAAGGAAAAACAACAACAGAAACTGTTCCAGAGAAAGACC AGATGGTAACCTACTACACAAATACTTTAAAACAACTTTCTTAAAGATGGTCAAAGAACT GAAGGAAGATGTGGAGAAAGTCAGTAAAATTATGTGTAAACAAAATGGGAATATCAATAA AGAGATAGAAAACCTAAAAAGAAAAAAAATAAATAAATTCTGGAACTAAAAGTGTAGTGA TTGAAATAAAAGTTCACCAGAGGGATTCAAAAGCAGATTTGAGCAACAGAAGAAAGATTC AGTGAATTTGAAGATGGGACACTTGAAATGATCAAGTCAGAGGAACAGAAATAAAGATGA TTGTTGAATAGCAAGCAGACATTGTGGAAGTCCCAAAAGAAGAGAGGGAAAGGGGCAGAG AGATCATTTGAAGAAATAATGGCTGAGGCTGAACACGGTGACTCACACCTGTAATCCCAG CACTTTGGGAGGCCAAGGTGGGTGGATCACAAGGTTAGGAGTTCGAGACCAGCCTGGCCA ATATGGTGAAACCCCGTCTCTACTAAAAACACAAAATTAGCCAGGTGTGGTGGTGCATGC CTGTAATCCCAGCTACTTGGGAGGATGAGGCAAGAGAATCGCTTGAACCCAGGAGGCAGA TGTTGCAGTGAGCCGAGATTGTGCCATTGCCCTCCAGCCTGGGCAACAAGAGTGAAACTC CGTTTAAAAAAAAAAAAATTAGCTGGGCGTGGTGGCACGTACCTGTAGTCCCAGCTACTC AGGAGGCTGAGGCAGAAGAATCGCTTGAATCCAGGAGGCAGAGGTTGCAGTGAGCCAAGA TCACACCACTGTACTCTAGCCTGGGCAACAGAGCGAGACTCTGTCTCAAAAAAGAAAAAA AAAAAGCTGAACACTTCCCAAATTTGATGAAAGACATGAAAATAAATATCCAGAAAACTC AATGGACTCCAAGTAGGATGAAAAAAAAAAAAAAGACTCATACTGAGACATTATAATTAG CCAGTAGGGCCTCTTGAAAGCACCAAGAGAGAAGCAACTAGTCACATGCTAGGAATATAT AATAGGATTATAAGTAGATTTCTCATCAGACACTTTGGAGAACAGAAGACAATGGGATGA CATATGTAAAGGGCTAAAAGAAAAACAACCACTACCTCTCAACCAAGAATCCTATATCCA GCAAAACTGTCCTTCAAAAGTGAGGAAGAAATTGGGAAATCCCCAAATAAACCAAAGTTG AGAAGTTTGCTACCATTAGACCTGCCCTGCAAGAAATCTTAAAGAGAATCATGCAGGTTG AAAAGAAAGAACACTAGATAGTAACTCAAAGCCATATGAAGAAATAAAGATGCCAGTAAA AGTAAATATATGGGAAAATATTAAATCTAGTATTATCGTAACTTTGGTTTAAAACTCCAT GTTTTGCTTTCTACATAATTTAATAGACAAATGCATTAAAAACAATTATTAGTTTATGTT TATGGACACACAATGTACAAAAATGTAATTTTGTGACATTGATAACTGAAAGAGGAGTGG CAAAACTGTGAGGAGAGTTTTTGCATATTATTGAAATATAGCTGGTATGAATTCAAGTTA GAGTGCTATAACTTTAGAATGTTAAGTGTAATCCCTATGGTAACCACAAATAAAACATTA TATAACATAAAAAAGTAAATGAGAAGGGAATTAAAACACTTCGCTACAAAAAATCAACTA AATACAAATGAGATCATGCAGGAAATGGACAAAAATGCTGTAAGGCATATAGAAAATGTA TAGCAAAATGCCAGAAGTAAGTCCCCCCTTATCAGTAATTACTTTATTACTTTTTAAACC ATTTTGTTGAGGAATGATTTACATAAAAACTGTACATATTTAACGTACACATCTTGATAA ATTTACACCATAAAACCATTATCATCAAGCCTATAAACATATCCATCACCTTTTAAAATT TCCTTCTGCCTCTTTATTATTATTATTGTATAAAAAAAAGTTTTTAATGGCCAGGTGCGG TGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCAGGCGGATCACCTGAGATC AGGAGTTGGAGAGCAGCCTGGCTAAGATGGCAAAACCCCATCTCTACTATAAATACAAAA ATTAGCTGGGCGTGGTGGCGGGTACCTATAATCTGAGCAAAGTACTGGGAGGCTGAGGTG GGAGAATCTCTTGAACCTGGGAGGCGGAGGTTGCAGTGAGCCGAGACAGCACCATTGCAC TCCAGCCTGAGCAACAAGAGTGAAAATCTGTCTAAAAAAAAATAATAATAATAAAAGTTT TTAATTAAACAATTTAAGAATATAGTAACATTTTCTGTGGGTACTATGCTGTATAGCTCT CCAGAACTTACTTATCTTGCATAACTGAAATTTGTACACTTTAACCATCAACTTCCCATT TCCTTCTCTTCCCCAGCTCCCAGCAACCACCATTCTGTTCTCTTCTTCTGAGTTTGACGT CTTTAGGTTCCACACATAAGTGAGATTGTACAATATTTCACTTTCTGTGTCTGGCTTATT TTACTTAACATAATGCCCTCCAGTCCATCTATGCATAGAAATGCAACTGATTTTTGGATG TTGACTCTGTATCTTGCTACTTTATTGAATTTATTACTTCTAACAGTCTTTTAGTGAAGT CTTTACAGTTTTCTATACATAAAAATATGTCATCTATGGAGACCATTTTACTTCCATTCT TATTTCTTTACTTGCTTAATTGTTCTGGCTAGGACTTCCAGTCCTATTTTGAGGAGAAAT GGTGAGAGTAGGAATTCTTGTCTTGTTCTTCATCTTCGAGGAAAAACGTTCAGTCTTTCA CTGTTGAGTATGTTACATGTGGTCTTCATTATGTTGAGGTACATTCCTTCTGCACCTAAT TTGCTCAGTTTGTTGTTTTTTTTAATCATGAAAGGATGTTGAATTTTATCAAGTGCTTTT TAATAATAAAAATAAAGGATTTTTATCGTTTATTGTGTTGATGTGGTGTATCACATTTAG TGACTCTTGTATGTTAAAGCATCCTTGCATGCCAGAGATAAATCCCACTTGATCCTGGTG AATAATTCTTTTTTTGTTTTTTATTTATTTATTATGTATTTATTTTTTTGAGATGGAGTC TCGCTCTGTCATCCAGGCCTGAGTGCAGTGGCGCGATCTCGGCTCACTACAAGCTCCGCC TCCCAAGTTCACGCCATTCTCCTGCCTCAGCCTCCCTAGTAGCTGGGACCACAGGCACCC GCCAGCATGCTCAGCTAATTTTTTTTTTTTTTGGATTTTTGGTAGAGACTGGATTCATGG TGAATAATTCTTTTAATGCACTGTTGAATTTGGTTTACTAGTATTTCATTGAGGATTTTT GCATCTATGTTAACCAGGGATATTGACCTATAGTTTTCTTATTTTGTAGTGTTCTTATCT GGCATTGATATCAGGATAATGCTGGCCTCTTAGAATGAGTTTCAATGTGTTCCCTTTTCT TCAATTTTTTTGGAAGAGTTTGAGAAGGATTGTTATTAATTCTTTAAATGTTTGTTGAAA TTGACCAGTAAAACCATTTGGTCCTGGGATTTTCTTTGTTGGGAGATTTTTCATTACTGG TTTAATCTCTACTTATTTTTTCTGTTTTTTTTTTTTTTTTTAATTATACTTTAAGTTCTG GGGTAGCCGTGCAGAAAGTGCAGGTTTATCACTTAGGTATACATGTGCCATGGTGGTTTG CTGCACCCATCAACCCATCATTTACATTAGGTACTTCTCCTAATACTATCTCTCCCCTTG CCCCCATCCCCCAACAGGCCTCAGTGTGTGATATTCCCTGCCCTGTGTCCAAGTGTTCTG ATTGTTCAACTCCCACTTATGAGTGAGAACATGCGGAGTTTGGTTTTCCGTTCCTGTAGT TTTCAGATGTGCTCTGGCCTCCAAGGACCATGAAGCCAGGCGGTGGTGGGGGGGTGTCCT CTGTATAAAAAGTGCTGTCCCAGGAACTTCCTGACGGACACTTTGGGGCATGTGAGCGAT TCCTGGGGAGGGCACCTCGGCCTTCCTAAGGCTACCCCTGCAGCCAGTGCTGGCCATTCT CACCAGCAACCAACCAAAAAACATCAGAGTCCCTGTAAACCTGGTTGTGATGATAAAAAC CAAATGTTTTCTAATCTAAGACTTGTATGCAGAACACAGAAAACTGCAGTTAAGGAACAC CCTACAAAATTGACCAACAGTATTTTCCAAAAACATTTTTCATCACTTCAAATAATGTAC AAAATCTACAAAAATCATATTTACCAGGACACATCTGTTAAATAAAAGCATTGTTTCGTG TTGGTATACATATACACAAGACTATGTATAACAGACTGTTTCCCCTCCCTGCAACCACAG AACCATCACACACAGGCACAGATACACGTCGGCTATGCCGCTTTCCACGAATGCATGGAA CCCAGGACGCGAACCCACAGCTCGAGGTCTTATACCTTCACTACTGAGCTGCCGCCACTG CAGCAGCAACACCTCTGCGGAGGTGTCGCTGAAGTCACTGGTGTCCCTGCCCAAGGTGTC CTGGTCCTGGTCAACTCTACTGATTGACCCTTCGTGGATACCTCAGGTCTAAAATCCTTT CCTCCGAGCCAGAGCTCTTCCTGTTGTGCAAACTCAGCCCCGTCTGTACCTTCCTGCTTG GCCGGTGATCGCAGTCCTTCTTCGCCAGCAAGTGTGGGCTTCCAAAGACAGGGCTGGGCC TGGCCTGGGACTCCCTGAAGGCCGAGAAGGACAGGCCCTGCAGAGGCAGCCCCAGGTAGG GGCTGAAGAGGCCGGTCTCCCTGCCCCCCCAGGAGACACCCTTTCCAAAGTGGAAGAGCT GGGTGGACAGCAGCGGGGAGAAGCCCGCAAGGGGCAGGCTGGGCAGGCCCCGGGCGGGGC CCTCGGACCCCTTGTCTCTCTCTCCCAATGCCGCCCCCTCCACGCCGCATCTCTACCTTT GGAGCGCAGTGCCCATGGGCTGGGCAGCCGACTGTGGTGGGCAAAGTCACTCCAGGGCGG GGCGCGGTTGGCCTGGGCTCCAGCATCCCTCTCAGCTCCCGGGCTGGGGGTCAGGAAGCT CCGGTTCCTGCAGTCCACGTGAAAGGTCCCCCTGGCTCCTCCTGCCTCGGTGCCCGCTGT GGCACAGCTGGGCCGCTGACCAAAAGCACTTTTGGCAGCAGGCTCAGCCCCTCGTGGGCT CTGGTCTTTGTAAACGTAAACATTTCTCTTGCTCACTGAGAGACCTTGGCGGAGACACTG CCGCTGGTCCTCCTGGGTGGCGCATGATCCCCTCCGCCGGATCAGTGGGGAGCCCCTTCC CACCCTGGCTGAGCAGGCACGCTGCGCCCTGTGCTCCTGCGCTCCCTGCGCCTCGATGCC CTTTAGCCAAATGTGGGACCCCCCGGCCCTCTGGCTCCGTGTGCACATGCCAGGCAGTGG GGCCGGCTCCTTCCTGCAAGGTACCTCTGGCCTGGCTGGGCCCCCTGTCCCGAGAGCGGT GGGGCCCTCTGCCTGAACTTCTGAACTGCTCACCGACTCCTTGGCCTTTTCCACCAAAAA CTTCCTAATCTCCAGTTCGATGCTATCGTCGCTGTCCACTGAACTGCTGTTGTCAGACAA GGAGCCAGGGCTGGGAGCTGGGCCCTGGGACTCTCTGGGGAATAGATTCTCTTCGGAGGC GGAGGCAGAAGCGGGTCTCCTCACCAGGAAGGCCGGGGCTTCGTCCTCTCGGCCCTGCTG CCAAAGACACTGGGGGGTTTCTTCCTGGAGCCCTCGCAGCTGTCTCTCTTGGACTTAGGC AGCTCTTCAGCACCTGCAGGTGCCTGTTTTTCCACTCTCTCAGGAGCCTGCCCAGCTGCT CCTGGAAGTGCGTCTGGGAGGTGCTGAACCTGACCTTCTTCCTGCACACAGCCCTGGGCT CCCTGGACCTCTTCTTGAGCTTTCACTTGGACCTTAACAAGTCCTTGATGGCTGTGTCCA GGTCCTTGTCACTGTCCAGGGAACTGCTTTTGTCTTCGGAGCTCTTCTTCTTGTCTAGGT GCCTTGCCTCGTCTGTCTTACCCTGGCCCTGTGACGTGCGAGTGTCACCGGGCACCCTAG CGGCGCCCTCTCCTCCCGGGGCCTCGCTGGCTGTGCCCTGGATGGAAAGGTCCCGCCCCT CATGGCCCGGCCCGGCTCTCCCCTGGCTGCGGTCGGCATCCTGGCCACCCTCTTTCCCCA CCACCCGCATGTTCTTGGGAGTGGATGGCCTCACTTGGCAGCCGCCTCTATGCTTCCTTT TGCAGCCAGGAGTGGGTCCAGTGTTTTAGAGAGAGGGGCCTTGGGGCTGCCGGTCTGGCT GCTGAGGCCAGGTGGTGAAAGTGGGCCCTGGGCAGCCTGAGGGCAGCTCTCACCTCTGGC CAGCAAACTTCTAGACTGCACCTTGAGGGCCAAAAACGTCCAGATTTCCTGCTCAATGCT GTCGTCGCTGTCCACGGAGCTACTGTCACCATCAGAGCGGGAAGGCACGTTGGGGGAGTA GAAGAGTGGGCTTGCGGACAGGGACCCATCGCTGCCCTCCATAGGGCCGGCAGGATCGTC TTGAAAATGTCCAGGACTGCTTCTACACACATCAGCTCAGCGGAGGTGTCTGCCTGGCAA GAGGACCATTCCACAAACTTGCTCCTGGAAGCCGGGCTCGTTGGAGGTGGAGCTTTGGTT TCCTTTGGGATCTTGGGGGAATGGTCAGCGTCCAGATCCCCTGGACCAGGGTCCGTGGTC TTGGTGGGCACTGGCTTCTTCTTGCTGGGTGTTTTCCTGTGGGTCTCTGGCAAGGCACTT TTTGTGGCGCTGCTTGTGCTGTGTGCGGGAGGGGCAGGTGCTCTTTCCTCTTGGAGCTGG ACCCTCTGGGGCGGGTCCCCGTCGGCCTCCTTGCGTGTTTTCTGCACCTGGTACAGCTGG ATGGCCTCCTCAATGCCGTCGTCGCTGCTGGAGTCGGACGCCTCGGGCGCCTGTACGGCG CTCGTGACTCGCTTTCCCCTCCTTGCGGTGCTGGCGTTCCTTTTAATCCCACTTTTATTC TGTACTGCTTCTGAAGGGCGGTGGGGGTTGCTGGCTTTGTGCTGCCCTCCTTCTCCTGCG TGGTCGTGACCTTGGACCTGAGGCTTCTGGGCTGCACGTTTGTCTTTGCTAACCGGGGGA GGTCTGCAGAAGGCGAACTCCTTCTGGACGCCCATCAGGCCCTGCCGGTGCACCACCTTT GTAGCCGGCTCTTGGTGGGATTTCGAGAGTGACTTCGCCGAATTTTCATGTGTGTCTGGT TTCTTCTCCACTGACCCATCACATTTTTGGTTCTCATGCTGTCTTTTCTCATTCAGAAAC TGTTCGATTTCTGCCCTGATGCTCTGCTCAAAGGAGTCTGCTCTGCTCATGCTGACTGGG GAGGCAGAGCCCTGGTCCTTGCTGGATCCCACCTGGCTGCCAGGGCCACACCACCTGAGC CAGGTACAAGTTTTGGGGAACACAGGGCAGTTGGGCACTGCTGTGAGCCAGTTCCGGCTT ACATCTACTGCCTCCGCCTGCAGCCCTGGAAGGCTGTGCATGGCTGGGCCCCGCTGGCCC CGGGCTGTGCGGCTCCACTCTTTACCTTCAGGTACTCCTGGATGGCCTCCTCAATGTCCC GGTCCACGGAATCGTCACTGTCTGAATCTAGCACCAATGGGCCAAAGTCTGCAGTTTCCT CCTCCCCCACGGGGTCAAAGTCAGCAACAAGACCACAGGCAGCCAACGCAGGCAGCTCCT TGTGCATGGTGGGCTTGGCAGCAGGCCTGGCGTCGTGGCATCCCTCTGCCCCCTCTGCGC AGTGCGCTCATCGCTGGTGCCCCTAGCAGCCCTGTCGCTCTGCAGCGTGCTGATGAGCAT CTGCACCCGGGTGCTCACCGACATGCTCTCCACGCCCTTGTCAGCCTCCGAGAAGCACCC GGGGAACCTAAAGCTCCCTGGTGGGACAGAGGCCACCCATTTGCGCTGGAGAGCAACCAC TGGAGGAGCATTCATGAGAAACATTCTGGCAGATGGGGAGCGACGCGCAGAGGGGCGACA CTTTATTTCTCTGCAGGCTTCACATCCTCCAAAGATTGGCAAGCAGTACCCGTGAAATAA CTTTAAACCTGCAAATGCTTCTTTGCAGGTTTAAAAGGATGACTATAAACTATGACGTCA TGCCTAGATTCATTCTTGACCCAACCAACAAGCTCTTGACATTCTCTGAGTCCAGGTTGA CTGTGATGAAAGGCAGCTAGTGTTCCCAAATGGCCCAGGGATCAGGTCTTCATCGCTCCA CTCAGAGGGAAGCATCCTCTCTCTGCTTTTTAAATAGACTTTTGACTGGGGCTCCAGCAG CGCGGGGCGCGCAGACCTGGAGTTGCATGGAGGCCAGAGCCACGACACCCGCCTGGGGAA CGGAGCAGCCCCAGGCGCTGATCCCCGTCCACCTGCCCCACGGAGCCCTCGCCGCCCGCT TGCCACTGCCTGCATGGCCCTCCTGTCCCCGGCCCCCCAGCCCTCCTTTCCCCAGCTCCC CCACCCTCCTGTCCCCGGCACCCCAGCTTCCCAGCCCCCGAAACCGCCCCCCCACCTCGA CCCGGCCCATGCCGCAAGTCGCCCGCTGCGCGGACCCGGCCTCCGCCCGCCTCCTGCGTC CTGGGGGAGGCGGCTGCCGGGGGTGGTGGGGGAGGGAGAGGGGGAAGAGGCCGCCCTCCG CCCGGGTGCGGGGAGGGGGCGCAGGGGTGTCCGGCCAGGCCCCCCGCCTCCCCGCCTCCC CGCAGCAGCTGCCCCGCTCCCGGGCCGCCTAATACTTTTACATTTTAACTTTTATACTAC AGTGAAAAGTGATTTACACACCACCACTGCAATATTACAGTGTTATGAATGTGACTATAT ACTTACCTTTCCCTGTGAACTTTTTTTTTTGAGACAGAGTCTCGCTCTGTCGCCCAGGCT GGAGGGCAGTGTCCATGATCTCGGCTCGCTGCAAGCTCTGCCTCCCGGGTTCAAGTCATT CTCCTGCCTCGGCCTCCCGAGTAGCTGGGACTACAGGCACCCGCCACCACGCCTGGCTAA TTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGCATGATCCCCCTCTT CTGACCTTGTGATCCACCCGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCA CTGCGCCCGGCCTACCTGTGAACTTAATATCTTAATGTTTTAATGTTGCTAATCAGAATC CTTTTATTTCAACTTGAAAAACTGCCTTATAAGGCAGGTGCAGTGGTGATGAACTCCCTT AGAATTTTTTTGGTGGGAGTCTGGGAAAGACCTTATCATCTCTTTTTCATTTCTGAAGGA CAGCTTTACAAGTTGTGGTCTTCCTGATTGGCAGTTTTTTTCTTCCAATACATTGAATAT AGCATCCTATTCTCTCCTGGCTTATAAGGTTGCTGCTGAGAAATCCACTGATAGCCTTAT TGAAGTTTCCTTGTATGTGATGAATTCCTTTCTTCTTGCTGCTTTTGAAAGTCTCTGTCT TTGACTTTTGATATTTTAATTATAATACATCTTGGTATTATGGTCTTTGGGCTGGCCTTT TTTGGGGCCTCTGAACTTCATGTGTCTGGAAGCCCACTTGCCTCTAAGAATTTGGAAAGT TTTTACCCATTATGTCTTCAAATATACTTTCAGGCCTTTTCTATCTTTTTTACTTCTAGA AAGTCCATAATATGTTTGACCCACTTCATGGTGGTGTCCTATAAATCCCAAAGGTTTTTA CTTTATAAACTTTTTTTTCTTTCTGGTCTTCTGACGGGATATTTCAAATGTCCTGTCTTT AATTTCACAGATTCTTTCTTCTGTTTGATCAAGTCTGCAATTGAAATTCTCTATTGCATT TTCATTTCATTCATTTATTTATTTTTATATATTTTTGAGACAGAGTCTGTGTCACCCAGG CTTGAATGCAGTGGTGCCATCTTGGCTCACTCCAACTTCCACCTCCCGGTTCAAGCGATT CTCCTGCCTCAGCCTCCCTAGTAGCTAGGATTACAGGCATATGCCACCATGCCTGGCTAA TTTTTGTATTTTTAATACAGATGGGGTTTTGGCATGTTGGCCAGGCTGGTCTTGAACTCT TGCCATCAAGTGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTCCGCC ACGGCACCCAGCTTGCATTTTCATTTTATTCATTGTATTCTTCAGTTCTAGAATTTCTGT TTGGTTCTTATTATTTCTGTATCTTTATTGAACTTTTAGTTTTGTTCATCTACTATTTTC TTGATATTATTGAGTTGATATATACATTCTAGTAAATTTCACTGAGCTATCTTAATTATT TTGAATTGTCAGGCAATTTGTAGATCTCTATTTTTGGGGGGTTGATTACTGGAGATTTAT GAGTTTATTTTGGTAGTGTCATATTTGCTGATTCTTCATGATCTACAAACTTTCATTAAT GTCTATGAAGAAGCAAATACCTCTTCTTTTCTTTTTTTTTTTTTTTTGAGACAGAGTCTT GCTCTGTCACCCAGCTGGAGTGCAGTGGCGTGATCTCAGCTCACTGTAACCTCCACCTCC CAGGTTCAAATGATTCTCCTGCCTCAGCCTCCCAAGCAGCTGGGATCACAGGCATGTGCC ACCACGCCTGGCCAATTTTTTTGTATTTTTTGTAGAGACAGAGTTTCACCGTGTTGTCCA GGCTGGTCTCAAACTCCTGGCCTCAAGTGGTCTGCCCGCCTTGGCCTCCCAAAGTGCTGG GATTACAGTTGTGAGCCACCATGCCCAATCTCTTTCTGTCTTTATAGATTGGTTTCAGCA GGTACAAACCTTTTCCTGCTGGATCCCTTGACTGGATCACAGTCAAGTGGGCCTGGAGCC ATATCACATGGCTGCTGCCTGGTCTGCAGCTGAATCTCTGATTGGCAGGCCGCTATCAAG GCATAGGTTGGTGATGCAGTTTCTGCTGGATCCTCAGGAGAACTGGACTGCCTCTGATAC CCTGATTGAACAGGACTGGAGCCAGGTCATGGGGCCACTTCTAGTTCTACAGTCAAGTCT TCAGATATCAGGCCTATTACCAAGGGCATGGACTGGTATAGCTCCCTGTGGGTCCCAGAT TGAGCTCCTGCTGGCTTACTAGGTAGGTCCATGGGAAGACAGGACTGCCTCCAGACCACA GTAGAGCAGGGCTAGAGCCAAGTCACAGGACAGCTTTGGTGACCACATTTGGGTTCAAGA TTGGTGGTCCTCTTATTAGGAGAATGGATGGTATGTCTTTCACCAGGTCCCAGGATGGGC TGGACTGTGCCCAGACTGTGGCAAAGCAAGACTGGAATGGAGTCACAGGGCTACTTTAGT GTCCATAGCTGAGACTGAGATCAGCAGGCCTGTTACCAAGGGCATGTAAAGGCATCACTG AATTCCTGGGCAGGCACGACTGACTGTGGTAGAGTGGGGCTGAAGCCAGGTCAGGGCTGC TTTAGTTTCTGCAGTCAGGACCATGGTTAGAAGGCCTGTTACTGGGGGCATAAATGGTCA TGGTTCCTCCTAGGTGCTTAGTGGATGGGGCTAGTTGCAAGACCATGATCTAGTGGAGCT GGACCCAAGTCCATAGGAGGACAAAGCTGCTTTCAGTCTGCAACTGGGAACCTGTCACTG GTGTGTGGACCTGCCTTCTCAAAGCAGCTCTCCTTGGTTTTGGGCTTTGCTAGAGTTTTG CCACCTCCTGCCTGGATATTAAAACTCTTGCAAAGGCAGTTTTGTCCATGAATGGCTGCC AGATCATTGTTTGTGTGGGGAGAGGTGAGTGGAGGGCCTCCTGTTCTGCCATCTTGCTGA TGTCACCCTAAGATGATTATTTGAATTCTTTGTCAGGCAATTTGTAGATCTTCATGTCTT TGGAGTCAGCCACTGGAGTTTCATTTTGTTTCTTTGGTGGTGTCATATTTTCTCATGCTT CCTGTTCTTTGAAGACTTAGATTGCTTTCTTCATGTTTGAAGAAGGAGTCATCTTTTCCA CTCTTTACTAACTTCAGGAGAGAAAGACCATCAATTAGCTAAGCTATAGATTCTGGGGGT CTCTCAGTCCTTTTCTGTGGGTGGTCCTTCCCTTTCAAGGGGGATGTCTTAGGATTTTGT CCCTTGTCTTCATTTCACAAATGAATAAAACAACCAGACCAGACATAAGTAAGGAAATAC AGCACTTGAACAACACCTGAAAAAACAACTAGACCTAACAGACATACACAGGATATTCTA CCCAACAACATAATACACATACTTCTCAAGTATACATGGGACATTTTCAGGATAGACCAC ATAACACATCACAAATTAATTCTCAATAGGGGCTGGGTGCAGTGGCTCACATCTGTAATC ACAGAGTAATTTGGGAGGCTGAGGCGGGTGGATTGCTTGAAGCCAGGAGCTTGACATCAG CCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATACAAAAATTAGCTGGGCGTGGT GGTGTGTGCCTGTGATCCCAGCTTCTTGGGAGGCTGAAGCGTGAGAATTGCTTAGGAGCC CAGGAGGTTGAAGCTGTAGTGAGCAGAGATTGTACCACTGTACTCCAGCCTGTACTTCAT GACAAAGAAAATGTACCATTGTACCACTGACAGAATGAGACCCTGTCCCAAAAAAGGAAA AAAGCTCAGTAGATTTAAAATGATAGACATCATACAAAGTGTCTTCTCTGACCACAACAG GATAAAGTTAGAAATCAATAACAGAAGATTTTAAAAAGTTCACAAATTAGTAGAATTTAA ACAACACACTCTCAAACAACCAATGGATCAAAGAAATCACAAAGAAATTATAAAATTCTT AAAGACAAATGAAAATGAAAGCACACTATATCCAAACTTATGGGTTGTGGCCAGTTGTGG TGGCTCACACCTGTAATCCCAGCACTTTGGGAGACTGAGGGAGGTGGATAGCTAGAAGTC AGGAGTTCAAGATCAGCCAGGCCAACATGGCGAAACCCTGTCTCTACTAAAAACACAAAA ATTAGCTGGGAGTGGTGGTACGTGCCTGTAGTCCCAGCTACCCAGCAGGCTGAGGCATGA GAATTTCTTGAACCCAGGAGGCAGAGGTTGCACCACTGAGCTAACACCACTGCACTCCAG CCTGGGTGACAGAATGAGACTCTGTCTCAAAAAACAAAGAAACAACAAAAAAACACAACT TATGAGTTGTGGTGAAAGGAGTGCTAAGGAGGAAATTTATAGCTATAAACACATTAAAAA AAGAAACACCTCAATTCAACAACATAAGTTTACACATTAAGAAACTAGAAAAAGAAGAAT AAAACTAAACCCAAAGTTAGCAGAAGGAAGGAAACAATAGAGATCAGGGCAGAGATAAAT GGAAAAGAGAATAGAAAAACAATAAAAAACAAAACCAAAAGTTGGTTCTTCAAAAAGATT AATAAAACTGACAAGACTAAGGAAAAGGGAAACAATCTAAATTACTTAAAACAGAAATGT ATTTGAGAATATCTTTATATATTTCTGTCTGTCTGTCTGTCTGTCTGTCTGCCTGTCTAT GTTTTAGAGACAAGGTCTAGCTCCATTGCCCTGGCAACAATCAGATGCAACCACAATCAG TGGCACAATCGGCTCACTGCAGCCTTGAATTCCTGGGCTCGCCACTATGCCAGCTCTTTT TTTTTTTTTTTTTAAGAGACAGGATCTTGCCATGTTATCCAGGCTGATCTTGAACTCCTG GCCTCAAGGAATTTTCCCACCTCGGCCTCCCAAATTGTTGGATTACAGGCATGACCCACC ATTCCCAGCCTAGAAAGGATTATAAAAGATTACTATAAATAATTGTGTGCTCATAAATTG GATAACCCAGATGAAATAGATGAATTCCGCGNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN 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TGCACTCTGTCCTCCCTGGTAGGTACAGGCTTTTCCCCACCACCATGGTGCAGCCATGCC TGCCCCCAACACCACTGGTGCAGCTCCATCCTCCACGCAGTGGTGCAGCCCCATCCCAGC GCCCATTCGAGCTGCTGTCCCATTGCAGGGGGCATTGTGACAGCTGAGGACCTGAACAAC TACCGTGCTGAGCTGATCGAGCACCCGCTGAACATCAGCCTGGGAGACGCGGTGCTGTAC ATGCCCAGTGCGCGGCTCAGCGGGCCCGTGCTGGCCCTCATCCTCAACATCCTCAAAGGT GAGTGGTCGCACCACAGCCGTGTGGTAGGACCCATGACACTGCCTCTCTCTCCCCACGCC CCACCCCTCCTGCATCTCTGCTCGCCCCCCATGCCACGTCTTTCCATCACTGAGCTCCTG AGGTGTGTCCCTGCGTCACAGCTCACCATGTCCTGAAGGAGGCAGTGCAGAGCAACAGGG CTGAAGCGGGCAATGCTCAAGGGTTGGAGGAGGAACAGGAGTCATCAGGAGGGAGAGAGG TGCAGGAGCTCAGGGCTGCAGGGCCGGTCCAGAGGGTACCCCGGTCCAGTGGGTGACCCT GCCACTTGGTCATTGAATGGCCAGAGCTGATGCGTGACGTGAGGTCCAGGCTCAGGAGCC CTCACCTTACTTCACTCTCACCACAGCCTTCTGAAGCAGCTGCTGCTATTGGTTATTAAA CGCTCCTTGAGGTGGGCAAAGTGGCTCAGGCAGGTGTTAACCCTCTGAGCCCCTCAGAGC CTCTGGGGCTCAGCAACATGCACCTGGCTCTGATCAACCAGGGTACAACTTCTCCCGGGA GAGCGTGGAGACCCCCGAGCAGAAGGGCCTGACGTACCACCGCATCGTAGAGGCTTTCCG GTTTGCCTACGCCAAGAGGACCCTGCTTGGGGACCCCAAGTTTGTGGATGTGACTGAGGT AAGGGGCAGGGGCTGGCTCACTGTGGGTGTGGGGCCTGCCGTAGAGGCATCAGGTGGGCT CCCCAGGGTGGCTACAGCCTCACATATGCTTTATGAATCCATTCCTGCCACAGAATTTGA TTGCGGGCCTACTGTGTGCTCGGATGGACTCGTGGGTGACCCCCAGTCTTGGCTTCTGCC CCACAGAACTGACAGTGTGGGGAATTAGTGGCCACCCTCCTACCTCAGGTCCTTTGCACA TGCTGTGGTTCTTGAGTGCTCAGTGCTGAGATGAGGAATGCATGGGGGCATTGCAGCCCT CGGGCATGGTGAGATGGATGGGTGAAAGGGAGAGGGCCAGGTGAACAGAGACCTCGGCCA CCCACTCCCTGTCACTCCAAACTAACGCTTCCCAGATGCCACCCTCAGCCCTGCACCACC TGACCCACTCACCCAGTAGTTGCCCCAGCTCCATGCTGGGTCCCTATAAGACCCTGTCAT ATCCCTTCCCGCTGAGGATCCTCACATCCCTCCTTACCTACTTGGGTCCTTGGCATTCCT GGGCGGATCTGCAGACCCCCCCACACTGACCAGTGACCTCCCAGGAGGGGCGTCAGCTGC CCGGGTGGTGTCTTCTCTTTCCCTGTGAGCATTCTGCACCTCTGACTCCCGCTGCAGCCA GTGACCTGGTGTCTTGTCTCTCTAAGGGGACAGAGCCACTGCAGCGTGTCCCTCTGCCCT CCTTTTTGGCTAAGGCCAGGTCCTTCATCTACTCGCTGGCTCGGGGTGCTGTTCCTACAA TGCTCCTGTCTGCCTCTGATGATTTATTTCTTTATCACGGATTATTCCCAAAAAAAAGGC AGCTGTTATTGCTCTAGAGACTTCTATCTGCTGCCTCCTTCTGTCCTTTGCTCCTCTTAG AGCAAACATGGCTGGGCTATGTCCTCTCTCCCTTCAGGGTATCCCCTCCCCTGCTCTATC CCCATGCCACCAGATCGCCATGTCCAGCCTCAGTTTCCCCATCAGGCCCCACTCAGCAGC ATCTCACACAGCTCACCACACTCTCCTCGAGTTTTCATTTTGCAAATTTTCGCACCTACA AAAATGTAAAAAAAACCCAAAACTGCCCAAGCATCAATGTTCCCTTTTCCTGGATTCTGC AGTGTGGACATTTCTGCCGGATTTCCTAACTCTCTCCGTCTCCACCCACATCTATTGGGA TTCGTGTTTTTCTGAGGGATTCCACAGTAGGTTACTGATGTCGCACCTCAGGGTGTGTCT CAAAAGTGAGACTTGAACATAGCACAGCAGGATGTCGGGGTGACACAACCTGCTGTCCCT TCTCTTTACCTACAGTAGGCTCCCTTGGCTGTTTTGTTTTTGTTGTGTTTTTGTTTGTAC ACAGAGTCTCTGTCATCCAGGTTGGAGTGCAGTGGTGTGATCTTGGCTCACTGCAGCCTC AACCTCCAGGGCTCAAGCAATCCTCCCACTTCAGCCCCCTGAGCAGCTGGGACCAGAGGC ACGTGCCACCAAACCTGGATAATTTTTGTATTTTTTTTAGAGACAGGGTCTTGCCCAGAC TGGTCTCAAACTCCTGGGATCAAACTGTCTTCCCACCTCAGCCTCCCAAAGCGCTGGGAT TACAGGTGTGTTTTTTCTTTGAGACGGAGTCTCGCTCTGTTGCCCAGGCTGGAGTGCAGT GGCGCTATCTCGGCTCACTGCAAGCTCCGCCTCCTGGGTTCACGCCATTCTCCTGCCTCA GCCTCCCGAGTAGCTGGGACTACAGGCGCCCGACACTGCGCCCAGCTAATTTTTTGTATT TTTAGTAGAGACGGGGTTTCACCGTGATCGCGATCTCCTGACCTCGTGATCCGCCTGCCT CGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACCGTGCCTGGCCTGTTTTGTTGA TTTTTAAAGCCCAGGGCAGTAGTCTTGGAAAATGTCCCACATCGTGGATTTGCCTTTCTG TTTCCTTGAGGGCAGATTCAGACAGAACACCTTTCCCTGGGATTGGTCAGCTAATCCGTG GGGTGTTGCTGAAACTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTTGAGACG GAGTCTCGCTCTGTCCCCCAGGCTGTCCCCCAGGCTGGAGTGCAGTGGCGGGATCTCGGC TCACTGCAAGCTCCGCCTCCCAGGTTCACGCCATTCTCCTGCCTCAGCCCCCCAAGTAGC TGGGACTACAGGTGCCCACCACCGCGCCCAGCTAATTTTTTGTATTTTTAGTAGAGACGG GGTTTCACTGTGTTAGCCAGGATGGTCTCGATCTCCTGACGTCATGATCCGCCCTCCCAA AGTGCTGGGATTACAGGCCTCGGCCTCCCAAAGTGCTGGGATTACAAGCGTGAGCCACCG CGCCCGGTGTTGAAACTCTCTTGAGGCATTTTCTTCGGCCTTCGGGTCCATCCTCTCTGT CTCCTCTTTGACCTCCTCATCTCCTCCTCGCCACTGCCTTGGGGACCTTGGCCAGGCTCA TGGCATCCAGCAGCCACTCAATGTCAATACCTCCATGTTCATCTCTCAGCCCGCCCTTGC CCGTGAACCCATGCTTATTTATTTATTTTTTTTATACGTGCACCCATGCTCTTTGGGTCT GATGAAGTATCCAAAATCAAGCTCCTGACCATCCCCAAACCGGCCCCTTCTGCCGGCCTC GCTAGTCGGCACCATGCTTGATTCTTCTCTTTCTCCCACCCAGGCCATCATCTCTTGCCT GGTTGATACCCACAGCCTCCCCTTTGGGCTTTATCCTTATCCCCGTCATAGCTGCCAGAG GGACCCTGTGAAAACACTCCCCAGCCTCCTCATTCTTCTGCCCTAAGCCTGCATGGCACA GAGCAAAAGCCAGTTGTTATGGGACCTAGGAGGTCCTGTGGGATGGGCCCCAGCCTGCAT CTTCATCCTCTTCTCCCCACCCCACTCCATTCACTCTCTGCCTATCGCTCACCAGCCTAT ACCACCTGCCTCAGGGCCTTTGCACTGACCATTTAGGCAACATTCCAGGCTCTTTTCACA CGTTGCCTCCTCTGAGAAGCCCTCCCTGACCACTCTGCCCATACCTCATGCCTCTTGATT CCCCTTACCTGGCCTGTGGTTTCAGCACTTTCCCTGTGTGTGTTTGTTTTTCTTGGCATG AGGGCAGGACTTAAGTGTCTGTTCCCTGTTGATTCCCCAGTGCCAGGCATGCAGTGCAAA TTCTAGAAATATTTTTTGCATGAAAGAATGAGTGATTGAATGCGGCAAGGGTCTGGAGGC TGAGGACCAGGCAGACAGACATTCAGAGTTGCTGGAACGCGACAGAGACAGGGAGTCAGA CTGGTCATGCAAGGTCCTGGGCCTGCCCTTGGGTCCTGGGGAGCCACGGAAGGTTGTGGG TGCCAGAGGGTTGCGGTCAGAGTCACAGTCAAGGGCCTTCTGAGACCTGTGCCCCCTCCC CACCTCCTCAGGCCAGCTCTGGGGTCTCAGCAGGTGGTCCGCAACATGACCTCTGAGTTC TTCGCTGCCCAGCTCCGGTCCCAGATCTCTGACCACACCACTCACCCGATCTCCTACGAC AAGCCCGAGTTCTACACGCCGGATGACGGGGGCACTGCTCACCTGTCTGTCGTCGCAGAG GACGGCAGTGCTGTGTCCGCCACCAGCACCATCAACCTCTAGTAGGGGCTGCTGGGCCGC CTGGGTGGGAAAGGGCCAGGGGCGGGTGGCCCAGGGACTGCCCACTTATCCAGTAAGGTG GCTCCATCACCTCTTTTCCTGGTGGGAAACTGAGGCCCAACCTTGGTAGCTTATCCTGGG CCTCTCAGTGAGTATGTTTGAGCCTCAGTGGGTGGATAGGGACCAGGCTGGGCCAGGCAA GGTCGGGTGCTGTCTGACCTGGCTGGGCGGTAGCTTTGGCTCCAAGGTCTGCTCCCCGGT CAGTGGGATCCTGTTCAATAATGAATGGACGACTTCAGCTCTCCCAGCATCACCAATGAG TTTGGGGCACCCCCCTCACCTGCCAATTTCATCCAGCCAGGTATGGGGTGGAGGTCCGGG GGGTGGGGGACTGGGGTGGAGAGGGGCGGGTGTCCTGGGCAGGCAGCTGACGGGCATCCC TGTCTTCTCCCATCGGCCGCAGGGAAGCAGCCGCTCTTGTCCATGTGCCCGACGATCATG GTGGGCCAGGACGGCCAGGTCCGGATGGTGGTGGGAGCTGCTGGGGGCACGCAGATCACC ACAGACACTGCACTGGTATGTGTCACCCCTTTTCTCCCTGGCCCTGCCCACTCTGCACAG CCCCCAAGCCACGCTGATCACACTCCCATGCCCCAGGCCATCATCTACAACCTCTGGTTC GGCTATGACGTGAAGAGGGCCGTGGAGGAGCCCCGGCTGCACAACAAGCTTCTGCCCAAC GTCACGACAGTGGAGAGAAACATTGACCAGGTGGGCCGGGGGTTGGAGAAACTGAGTCAC GGTGTGGGGTCCCAGGGCATCCTGGGCTGGAGGCCTGGATCATCACAGAGTGGACAATGG TTGGTGTCCTCTCTCTAGTGCCTGGGCCATCTGGAGCCCCTGTGACATGAGGGCCAAGCC CCCTGCTCCAGTGAGACCCAGCAGGCCCCAACCTGCTCTTCCTGATGACCTGGCCTGAAA TGGCACCACCTGGGCTGAGGCCTGTGACCACACAGGTGTGGTTCAGGTGGCATCTGGAGC CCTGCTCAGGCTTCCCCTCTCCTCCCACCCCCAGGCAGTGACTGCAGCCCTGGAGACCCG GCACCATCACACCCAGATCGCGTCCACCTTCATCGCTGTGGTGCAAGCCATCGTCCGCAC GGCTGGTGGCTGGGCAGCTGCCTCGGACTCCAGGAAAGGCGGGGAACCTGCTGGCTACTG AGTGCTCCAGGCAGACAAGGCTGACAAGCAATCCAGGGACAAGATACTCACCAGGATGAG GAAGAGGACTTTGGGGGACGGGCTTCCCCTGTGAGCAGCAGAGCAGCATAATAAATGAGG CCACTGTGCCAGGCTCCAGGTGGCCTCCCTGGCCTGTCTCCCCACTCTCTGGGCCTCAGT GTTTTGTGTGTGAAATGGAGCCATCTGGCTGGGGAGGAACAGAGAGGTGGGATTCGGAGA TCTTCACAATGCGGACACTGGAACTAGCCTCAGCATCTTCAGCATGGGGAGAGCCAGGCA CATGGCTGGGGGCCAGGGGAAGGTTCACACCAAACCCTGCCCCTTCCCACCCTGATCCCT CAGACTTTGGGGCCAGGCCCTCCCTTACTGGGGCTGGGCAGTGACACTACCTAGGATCAG CCACCAGGGGGTGTCACGACCCTGGCGCTTTCTTAGGCAGAGGGTGGCCAGCCGATGCTG GGAACCCGGGCGCCTTCTCAGACCCGTAGGCGTCCAGCTCACCCTGCCGATGACACTGGA GGTGAAGCTGAGGGTCCGAGGAATGGGGACTGGGCAACAGGCTGGAGGAAAACATCTCGG TCAGAGCCATGCCCCTGGGGGGTTCCCAAGAGCAAGCCCAGAGTGAAACCCAAGCTTGTG ATCCTCTCCAGAGGGAGGCCTGGTTCTCAGGGAACAGCAAACGGGAAGATGTCCCCAGAT CCCAGGGATCAGGGCTTGGACCAGCCGGGGACGCAGCCCAGAGGGAGTGGGTCCGGAAGG AAACAGCTCGACACAGCAGCCTTCACCATCGGCAGCCCCTCCAGGCCTCCCTCGGGGCCT GCTCCCTCCTCTGTGCACAGTTCCAACACCTGGAGCAGGGTTCTGGGAAGGGCTGGTGGA GGTGGGCTGGTGGGAGGCGGTGATCACAGCCCAGCACCTGGATATCACCAGGGGCACTGG GGCCAGGGGCCAGGTGAGGCCAGGTCGGGGCTATCCTTCAGGAGCCCCGAAAACCTGGTG ATTCCAAAGGGCCCATAGACAAACAGGGTTTTATGCCTGTGGAGTCAAGTCCCACTGGGT CTGAGCTCTGGAGGGCTGTGTCTCTGGGGCTCTGCAAGGGTGAGATGGAGGTGGGCTCAA CTGGTGTACAAGTCACTCTTCAATCCTTATTTTATTTATTTAATTTTTTTAAAAAAAATT TAAACCAATAGAGATGGGGTCTCACTATGTTGACCAGGCTGGTCTTAACTCCTGACTTCA AGCAGTCCCCCCATCTCAGTCTCCCAAAGTGCTAGGATTACAGGGGTAGCCACTGCACCC GGCCTCAATCCTTATATTGGCCTGAGAGGAAAGGCCGTGGCCCCATTTGCAGGGGAGAAG ACTGAAGCTGGAGGGGCAGGCCTTGCTCTGGGTTGCACAGCAGGAAGAGAAGTGGGAGCT GGCCACGAGGCTTCCTGGACCCGAAATGCTGGTGGGGTACACCCTGGTTCTCTAGGTCCC ATGGGGCTCAGCCCAGGACTACCTCGGGGGGTGAGGGACTTAAATCGTCTCCTTCATTCT CATCGCCCCTTCCCCCATCATTTCCTGAGGAAGGACATTCAGGGACCTGAAGGGGTGGCC TGCCCCTCCCCACCTGTGGGTGTTTCTCATCAGCTGGGACAAGAGACTGAGAAAAGAAAG AGACACAGAGACAAAGTATAGAGAAAGAAAAGTGGGCCCAGGGGACCTGCACTCAGCATA CGGAGGCCCCACGCTGGCACCAGTCTCTGAGTTCCCTAGTATTTATTGATCATTATCTCT ACCATCTCAGAGAGGGGGATGTGGCAGGACAATAGGGTAATAGTGGGGAGAGGGTCAGCA GGAAAACACGTGAACAAATGTCTCTGTGTCATAAACAAGGTTAAGAAAAAGGTGCTGTGC TTTGATGTGCATATACATAAACATCTCAATGCATTAAAGAGCAGTATTGCCACCAGCATG TCCCACCTCCAGCCCTAAGGCAGTTTTCTCCTATCTCAGCAGATGGAATATACAATCAAC ACTGAGACATTCCTTTGCCCAGGGACGATCAGGAGAGAGATGCCTTCCTCTTATCTCAAC TGCAAAGAGGCCTTCCTCTTTTACTAATCCTCCTCAGCACAGACCCTTTACAGGTGTCGG GCTGGGGGACGGTCAGGTCTTTCCCTTCCCACGAGGCCATATTTCAGACTGTCACCTGGG GAGAAACCTTGGACAATACCTGGCTTTCCTAGGCAGAGGTCCCTGCGGCCTTCTGCAGTG TTTTGTGTCCCTGCTTACTTGAGATTAGGGAGTGGTGATGACTCTTAACAAGCATGCTGC CTTCAAGCATTTGTTTAACAAAGCACATCCTGCACAGCCCTGAATCCATTAAACCTTGAG TCGACACAGTACATGTTTCTGTGAGCACAGGGTTGGGGCTAGGGTTACAGATTAACGGCA TCTCAAGGCAAAAGAATTTTTCTTACTACACAACAAAATGGAGCCTCTTACGTCTACTTC TTTCTACATAGACACAGTAACAGTCTGATATCTCTTTCTTTTCCCCACAGGGACCTTCCT GGCTGTGCCTCGGGTCAGGACCAGAATGACACCCATTCATTTCCCTGGGCCTTTGCTCGG GCGGTCCCTGCACCCTGGCCTCTGCCTGACGAGGATGGTGGGGAGAGGAGGGGGACGTCC CCCACACTGCTGTCTCCACTGTTCCTGCTGCCCAGGCCTCTGGGCTTCCAGGACTGCAGC GGGTCGGTGGGTGGGCTGGCCTGAGCCCAGGAATGCACTTCAGCTCCTGGTTGAGCAATG TCACTGAGGCTTGGGAGTCGGGTGGGGGCGGGAGGAGGCGTCCGCAGGCCCCCCTACCGT GAGAGGCAGCCGTGGGAACAGCCTACCTCTAAACAATCACTGCAGCCCAGGCTGGCCAGG GGCTCTGGCCGGACATAGGGGCCTGGCAGGCTGTGTGCCCTGTAAGGACACAGTCTGTCT CTGTGCCTCAGTTTCTCTGCTGCCCAGATGGAGGGGCCCAGACTCCAGGTGTAGACATCT GGAGCAGGCAGTGTTCAGTTGGGGAGGAAGCGGGGAGGACTGTGGGGGCCATGTGGGAAG GATTCCACCCCACATCACCTGCACCCCTGCTGAGCCTGGTCAACGGAGCCCCTCAGTGGG TCCTCACTCCCCTGGTTGCCTCCCATTTAGGCACCCTGAGGCCTGGGGAGAACAGAGCCA GGCCAGTGTCCCCAGAGAGGCTGCGCTGCCAGCACAGTAGTAGCGGATTTGGATTCAGGG AAGCAGACCTGCAGCCAGGGTGGGAAAGAGCTGCAGGTGGGGTGGGGCCCCCACATGGCA CAGCCCCCCTCCCTGGAGGACCATGCTGCATTTCCAGGACAGCAAGTCCCAGGGATGGAT GGTGCCTGGTGCCAAGGGCTAGAGGCATGGTCTGTCTGCATTTCCCACGTAAGTGTCTCG TAGTCACTAGCATTTGATGCTGTCAAGACCCCCTGTCCTCTGTGCAGACTGGGAAGCCCT TGGTCACCCTGGGGGAGTTGGGGGACCCAGGCCAGGCTGCAGAAGCATAAGGACTTGAAC CCGGGTCCTGAGTGACACCACCTTGGGTCCTCCTCCTTCTGCCTCTGTTCAGCTCCACCT TGATGCTGACTAGGCTGGGCCATGCGGAGAGGGTTAGGGGATAGAGATGGGAGCTGGGGA GCAGGGCTCCACTCTGGGAGGGGGGCAGCCTTGCCGGATCCAGGGCAGAGTTAAGCGGCC CCAGCTCTGCTTTCCCAGAGCTGCTGAGAACCTGGGGAATGGTGTGGAGGTTCCAGGGAG CCCTGCCCCTACCTGGCAACCGCAGTGCAGCACGCACCAAGTTCTCCTGCACATTGCGAC AGTGTGACCATGGGCTCTGGTGGGCGGTAGGTCGGGCCTTTGGACCTACCAGCAGTGAGG GAGTTAACACAGCAGCTGACTTCTCTAGGCAAAGAAAACTCCCCTCAGACGCTTTGCTGC CTGGCCTCCTGCCAGGAACAAGCAGGAGCTGAAAACTAGAAGTTGAGGCATGAGTTTGGC CACTCCGTAGTGTGCACTTGGTGAGGGCAGCAGCTCGCCACAGCTGCCAGCCATCTGTCC ATTCACCCATCTGTCCATCTGGCAGCCCGCTGTTCAGACCTGTCTGTCTGTCCGCCCATC TGTAAGCCCATCTCTGTCCCATTGTCTATCTGACCATCTTTCTCTTACTGTCCTCTTTGT CTAGCTATCTGGCCTGTCTGTCGATCCATCTTCGTGTCTGTCTTCAGCCCCCACCTGTTT GTCCATCTGTCCAATTACCTGTGAGTCTATCTATGCATCTTCTTGTCCATTCATCTGCCC ACCCATCTGTCCCTCCATCTGCCCACCGGCCTCCCCTCTCCTTCTGGGCCGCAGAGCCAT GGCCCAGGACTACGGAGCCATGGGTGACCTGGTCCTGCTGGGGCTGGGGCTGGGGCTGGC GCTGGCTGTCATTGTGCTGGCTGTGGTCCTCTCTCGACACCAGGCCCCATTTGACCCCCG GCCTTTGCCCACACCGCTGTTGCTGCTGACTCCAAGGTCTTCTCAAATATTGTACGGTGA GTGAGACGTGGGAGGAAGCTGGGTGGCCTTTGGCAGCCAGCCCCTCCTGGAGAAGGCGTG TGTGTGTGAGCATGTGTGTGTGTGAGAGATTATGTGTGAGTGTGTGTGGGTATATGTGTG AGTGTGTTTGTGGGGTGTGGGTGTGTGTGAATGTGTGTGATCGTGTTTGGGTGTGTGTAT GTGTGAGTGTGGGTGTGTGTGAATGTGTGTGATTGTGTTTGTGTATGTGTGTGTGGGTGT GTGTGAGTATATGTGAGTGTGAGTGTGTGGGGGTGTGGGTGGGTGTGAATGTGTGTGATT GTGTTTCGCTGTGTGAGGGTGTGTGTGACTGTGAGTGTGTGAGTGTGGGTGTGTGGGTGT GTGTAAATGTGTGAGTGTGAGTATGGGGGGTGGGTATGTGTGAATGTGTGTGATTGTGTG TGGGTATATTTGTGGGTGTGTGTGTGTGTGCACGTGTGTGTGTGTGCACGTGCACTGGCC CAGGAAGCAGGAGCCGTGTGTGTGGGCTTCAGCACCTGCAGGGCTTGAGCGCAAGGAGAC AGCCTCAGGGCCCTTGCACAGAACAGGCGGCAGGGTGTGCCCGTGGGGCAGATGGGGACT TGGGGACAATGGTGGTGTGTGAGTCCATACCTGGCTCCAGGATTCAGGAGGCCCATTTGC ATATCCCAGGTGGGAACCTGTCTGGCCCCGCCTGACCCTGCTGGCCGGTGCAGGCCCCTT CAGTGAGGCCAATTCTCCAAGGCTGCGGTCTTCTCCCAGGGTCATGGGTGAAGGGGTTTG GAGGCTCCCTGCGTGGGTACTGGCCTGCTGGGGTACACACAATGCTGCCATAGCCAGTCT GCCCCTACACCCAGCCCGGGGCCACATCTCAGGTCTCTCAGTCCTGAGGAGCCCGGTGCC CCACCCCTCACATCCTCTCTCCCTGAGTCAGGGCCTGGGTCTCGTGAGCTGAGTGACTGA TACTTGGTGTCCTGGATGAGGGCGTGGTGGAGAGGGGCCACAGCGGGTGTTTCCTGACCC TCTTCCAGGAAGGTGCTGCTGCCGCTGCAGGGAGGACACACACAGGATGCCCCTTCTTGC CCCCTGCCTCCCATTGGGCCCACAAAAGCCAGGGCAAGCCTCCCCTCCCTGCCAGCCACC TGGTCTGCTTCCCAGAAATTCTGTCTTGCAGGCTGTTGGGAGGATCCCAGTACTTTGTAA ACTAAAGCAAGGGAGGAGTGGCCGTTCTCTCTCTTTGTTCATTCATTCACCTTTTCATTC ATTCCTTCTTCCCTCCATTCCCCCATCTGTCCATCCTTCCCTGCCCTGATTGCTCATGCC ACCCCCCCAGCCCCTCCTGACCTGGTCCTTTGGTTTCTCTTCAGGGCTTTCTGTCTCCTC CCACAGGGCTGAGAATGGCAGCTCAGGGACAAGTGGGGGCTGGGGACTGCTTAGTCTCCC CAGTGGCTCTCAGGGGATTTGAGGGTTTGACGTCAGCTGCCACCCCAGGCTGTGCCCCTC CTCTGCTCGGGAGGACATACAGAGATGCGACACCCACTTAAACTCGAAGTTGCAAAGATG CAAATGAGACTGGGGTCTCAGGCACCAGAGACCACCCGTGGGCACGTGGCTTTTGGGAGT GGGGACCTGCTGCCACAGATCTCTGAAGAGTCTGGACCTGCTGGGTCTCCCCGAGTGACT GTCTGGGGGTCTCCATAGCATGCCCTGCTGTGTGCGTGACGGTCACTGGTTGGGTAGGGG TCTCTACTCTAAAGCTCCCTCTGCCGGCATCCCCTCGAACTCTCCCTTGGTGAAGAGAGG ATGTGGTTTGCCCCAGTGTTTTATCAAACAACTCTCTCCACTTCCAGTTTTAAGAAGCTG GGAGTGGAAGAGAGCCTGGGGCTGGCCCCAGCTGCTGCTGTGAAACAGGGGTCACTGGAC GCTGGGACCCTGGCCGGGCTGGCTGGAGGCCTCAGGAAGAGGCCTGCTACAGCGTCATCC TGGCCAAGATTCCTCCCTGCAGAGGACCCTGGCCACGCTGCCACAGGGTCTGCTGGGGCC ACCAGAAGCCCATGCTCCTGCCTCCATCTCTCCCCTCTGTGCTCACCTCTCACCAGGAGG CCCTCCCAGAGTTCAGTGTCCTGCTTTTTTTTTTTTTTTTTTTTTTTTTTGTGACGGTGT CTCACTCTGTCACCAGGCTGGAGTGCAGTGGCGCGATCTCAGCTTACTGCAACCTCTGCT TCCTCGGTTCAAATGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGGTGCCA GCCACCACGCCCCGCTAATTTCTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCC CAGGATGGTCTCTATCTCTTGATTCACCCGCCTTGGCCACCCAAAGTGCTGGCATTACAG GAGTGAGTCATGGCACCCGGCCTCATCTCCTACTCTTTCAGCACCAGGTTTTATTCTTGG GATTCTGCTACAGCCGGAGCCCCTGGGTGCGAGTTCCTAAGGTTTCTGTGAGTGTGGACC CAGCACCGTGCCTAGTAGACATACAAAAGGAGCATGGTGACAGTGAGGTCTGTCATCTCC AGCATAATGACTGTTTTGATCCTTGTAAAAAAGGTGATTTTTGGCTGGGTGTGGTGGCTC ACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGGGGGTGGCTCACTTGAGGTCAGGAGT TGGAGACCAGCCTGGGCAACATGGTGAAACCATGTCTCTACTAAAAATACAAAAATTAGC TGGGCATGGTAGCAGGTGCCTGTAATCCCAGATACTTGGGAGGCTGAGACAGGAGAATCA CTTGAACGCAGGAGGCAAAGGTTGCAGTAAGCCAAGATTGCACCACTGCACTCCAGCCTG GGTGACAGAGCAAGACTTGGTCTCAAAAAAAAAAAAAAAAAAGAAAGAAAAGTTTATATT TTTGTTCTAATGCTTATCTTAATATCGTCATTCTATAATTATATGTTTTATATAATTATA ATAGCTATATAAGATATAATACCCCTAGTATGTTGTTTTTTGGATATTCTACTTGCTCCT GATGGTTAATTTATGTGTCAACTTGGCTAAGCTATGGTGTCCTGTTGTTTGGTCAAATAC TTGTCAATATCTTGCTGGGAGGTTATTTCATAGATGTGATTAACACTGACAGTCAATTGA CTTTAAGTAAAACAGATTACCCACCATAATATGGGTGGGCCACCTCCAATCAGTTGAAGG CCTTAAGAACAAAAACTGAGGTTTCCCAGAGAAGCAGGAATTCTGCTTCAAGACTGTAAC ACACAAACCCTACCTGAGTTTCTGGCCTGCTGACTGCTCTACAGATTTTAGGTTCCAGAC TTCGAGATCAACTCTTACCTGAATTTATAGCCTGCTGGCTTGCCCTACAGATTTTAAAAC TTGCTAGTCCCCACAATCATGTGAGCCAATTCCTAAATAAGTCTCTCTCTATGTATAATC TATTGGTTTAGTTTCTCTGAAAAACTTTCACATTCCAGTTTCCTGGATGTTAAGAATTAC CGAAACTAGCTAGTAACTTCTTTTTTTTTTTTTTTTTGAGACAGAGTTTTGCTCTTGTTG CCCAGGCTGGAATGCAATGGCACAATCTCAGCTCACCGCAACCTCCACTTCCTGGGTCCA AGCAATTCTCCTCCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCATGTGCCACCATGCT TGGCTAATTTTTGTATTTTTAGTAGAGACAGGGCTTCTCCATGTTGGTCAGGCTGGTCTT GAACTCCCAACCTCAGGTGATCCGCCGCCTTGGCCTCACAAAGTGCTGGAATTACAGGCA CGAGCCATTGCGCCTGGCTCCTAGTAAATTCTTCTTTTCTGTGATGTGTCCCTTACCTCT AATAATACTTTTCTTCTTAAAGTCTACTTCATTAAAAATAGTTATGCTGGGCATGGTGGC TCATGGCTGTAATCTTGGCACTTTGCTGGAGGTCGAGGTGGGTGGATCACTGAAGCCCAG GAGTTCAAGACCAACCTGGGCAACATGGCGAGACCCTGCCTCTACAAAAAATACAAAAAT TAGCTGGGTGTGGCTAGTATAATTCTAAGTTGGCACACTTGTAGTCCCAGCTACTTGGGA TGCTGAGGTGGGAGAATCGCTTGAGCCTAGAAGGGAGAGATTGCTGTAAGCCAAGATCAC ATCACTGCACTCCAGCCTGGGAGACAGAGTGAGGCTCTATCTCCAAAAAAAAAAAAAAAA AAAAAAAAAAAAAGTTATACAGCTTTCTTGGTTAGTACATGCATGACATATTTTTCATGA TCTTCCACCTCTCTGTACCCTTATATAAAAGGCATTAGTTGGGTTTTACTTTATTTTCAA TTATTTTAATTTTTATTGTCCTTTTAAATGTAACTAATGATTTATTTGGGTTGAAACCCA CCACCAATTTGTTTTCCATGCCTATTCTATTTCTTCTTATCTCCTCTCACATCTTGTTTT GGATTTATTATTTTTATTATTTAATTTCCTCCTTCTCTATTAGTTTCATAACTGTGCAGT CTTAGAGTTCTTTTAAAAGATGACTGGATTATTTTAGAGCTTACAACATGCATCCTTCCC TTATCAAAGTCTAACATGAGCTAGTACTTTTTGTTGTGGTTGAGATAGAGAGAGTCTTCC TCTGCTGCCCAGGCTGGAGTGCAGTGGAGCAATCTTGGTTCACTGCAACCTCCACTTCTT GGGTTCAAGCAATTCTCCTGCCTCAGTCACCTGAGTAGCTGGGACCACAGGTGTGCACCA CTATGCCCGGCCAATTTTTGTATTCTTTTTCAGTAGAGACAGGGTTTCACCATGTTGGCC AGGCTGGTCTTGAACTCCGGACCTTAAGAGATCTGCCTACCTCGGCATCCTAAAGTGTTG GGATTACAGGCGTGAGCCACCGCACCCAGCCTATGAGTTAGTACTTCTATCCTCTTCCTA GTCAGTACAAGAACCTTGGAACAGGAACTAAATTTACCCCCAGTGACTTATATGCTAATA TTTTTGTGTATTTTAAATATGTGTGTGTGCATAGATGTATCTGTGTGTTTTTTGTGTTTT TATTCTTATTTATGTTGAGAGTGTAGAGCTATGTAAGAGTAAAGAGAATTGTGTAATGAA GCCCCGAGTATCCATTCAATTTCAACAACAATCTTATGGCCAAGCTCATTTCATGTATAC TCTTTCCTGCTTCCCTCTACCCCACATTATTTCCGTGCAAATCCCAGATATATAACTGTA CCCATACATATTTCAGTATGTTTTATTTCTTTTAAACCCCACAAGATATCATTTTCTATA CTACTGTAATTTTATACCAATAACATTCATTTAGATTTACCCACACGTTTACCCTACCCT CCGGGTCCTGTTTGAAAATCAAGCCCATGCTCACAGGCCAATTTTTTTTTCTTTTAGAGA CAGGGTCTCACTTTGTCACCCAAGCTGGAGTGCAGTGGTGCGATTATAGCTCAATGCAGC CTCCAATTCCTGGACTCAAGGGACCCTCCTGCCTCAGCCTGCCAAGTAGCTTGGACTATA GCTGTATGTTTTATTATTATTTTGTAGACATGGGGTCTGGCTATGTTGTCCAGGCTATTC TCAAAATTCCCGGCCTCGAGCAATCCTCCTGCCTCGGCCTCTCAAAGGTTGGGATTACAG GTGTGAGGCAAGGCACCCAGCTCAGCCACAGAGCCCTGTTGCATCTCTCTTACTAGGAGC AAGAGCTGACTGCCCCCTCATCCCCATTCCAGAGTGTTGGGGCTGTGTTGAGCCGAGGCC AGGCCACTGGCATGGCCCAAGGAACGGGATCATTCACTGCTGCCCCAAATCTGACATCAT TCCACCTTGACAAGACTTCCTCATCCAATCCCTTTACTTGACAGCTGGGGAAACCAACGC GCACAGAGCACCCCCAGCTCACTCGGGGTCTCAGAGCTGATCCATGAGCAGAGGCTGAGA TCCTGGGATCTTGTCCCCCAGCCGCCCTGCAAGCTTACTCCCTTTCTGCTGGAAGAGATG GGGCCGGACCTCAACCAGCAGCCCTGGCCTGGACATGACTGTGCTCACCCAGGTATTGAG GCCGAGATGCCCCGGCATCATATGTTTTTCTCTTATTTTTTCTTTTTTTTTGAGACAGTA TCTCACTCTGTCACCCAAGCTGGAGTGCAGTGGCATGATATTGGCTCACTGCAACCTCTG CCTCCCGCTTAAAGTGATTCTCCTGCCTCAGTCTTCCAAGTAGCTGGGCCTACAGGCTTG TACCACCACGCCTGACTAATTTTTGTATTTTTACTAGAGACAGGGTTTCCCCATGTTGGC CAGGCTCGTGTCGAACTCCTGACCTCAGGTGATCCACCTGCCTTGGCCTCCCAAAGTGCT AGGATTACAGGCATGAGCCATGGCATCACTTAAACGTAGTGAGAGGCCGGGCAAGGGGCT CATGCCTGTAATCCCAGTGCTTTGAGAGGACGAGGCTGTCAGATCACCTAAGGTCAGGAG TTCGAGACCAGCCTGGCCAACATGGTGAAACTGTGTCTCTACAAAAAAATAGAAAAAAAA AATCCCTGCGTGGTGGCAAGTATCTGTAGTCCCAGTTACTCAGGAGTCTGAGGCATGAGA ATTGCTTAAACCTCGGAGGCGGAGGCTGCAGTGAGCTGAGATGGCGCCACTGCACTCCAG CCTGGGTGACAGAGCAAGACTTTGTCTCTAAATAATTAAATAAATAAATATGGCCGAGCA TGGTGCCTTAGGCCTGTAATCCCAACATTTTGGGAGGCTGAGGCAGGTGGTTCATGAGGT CAGGAGCCCGAGACCAGCCTGGCCAAGATGGTGAAACACTGTCTCTACTAAAAATACAAA AATTAGCCAGCTGTGGTGGCAGGCACCTGTAATCCCAGCTACTTGGGACACTGAGGCAGG AGAATCGCTTGAACCTGGAAGGCAGAGGTTGCAATGAGCCGAGATTGCACCGCTGCACTC TAGCCTGGGCGATGGAGCAAGACTCCATCTCAACTAAATAAATTAATAAATACAGAGCAA GATTCCATCTCAAATAAATAAATAAATGTACACCTGTAATCCTAGCACTTTGGGAGGCTA AGACAGGTCGATCACCTGACGTCAGGAGTTCGAGACCAGCCTGACCAATATGGCAAAACT CCATCTCTACTAAAAATACAAAAATTAGCTGGGCGTTTTGACGTGTGCCTGTAGTCCCAG CTACTTGGGAGGCTGAGACAAGAGAATTGCTTGAACCCAAGAGGTGGAGGTTGCAGTGAG CCGAGATCTCGGCTGCACTTCAGCCTGGGTGACAGAGTGAGACTCTGTCTCAAAAGGAAT AAATAAAATACAAAGTAAAAAAAAATGTAGTAAGATTGCAGAGTCGTGCCGCAGAAGCGT GCTGGTCCTATCCATGTAGTGAAGGCTGATTTCATACACAAATGTCACAAGAACTTTTCT TTTCTTTTTCTTTTTTTTTTTTTTTGAGACGGAGTCTCACTCTGTCACCCAGGCTGGAGT GCAGTGGTGCGATCTAGGCTCACTGCAAGCTCTGCCTCCCGGGTTTATGCCATTCTCCTG CCTCAGCCTCCTGAGTAGCTGGGACTACAGGCGCCTGCCACCTAGCCCAGCTAATTTTTT TGTACTTTTAGAGGAGATGGGGTTTCACCGCGTTAGCCAGGATAGTCTCAATCTCCTGAC CTCGTGATCCGCCCGTCTCGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACCACA CCCGGCCTTCTTATATGCTTTTATTGCATTTGAGCGTACCTCTTTTACAGCGAAGATCTT TTTTTTTAATTTTATTTTATTTTTAGAGATGGAGTCTTGCTTTGTTGCCCAGGCTGGAGC ACTGTGGTGTGATCATAGCTCACTGCAGCCTTGAACTCCCGGGCACAGGTGATCCTCCCA CCTCAGCCTCCTGAATAGCTGGGACTACAGGCATGCACCACCATGCCTGGCATATTTTAA AGATGTTTGTAGACATGAGGTCTCGCTATGTTGCCAGGCTTGTCTCAAACTACTGGGCTC AAGCCATCCATCCATTTCAGCCTCCCAAAGTGCTTGGATTATAGGCATGAGCACTGCGCC TGGCCATCACACTGTTTTTTTGGTTGTTTGTTTGTTTGTTTGTTTTGAGATGGAGTCTTG CTCTGTCGCTCAGGCTGGAGTGCAGTAGTGGGATCTCACCTCATTGCAAGCTCCGCCTTG TGGGTTCACGCCATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCGCCCGCC ACCACGCCTGGCTAATTTTTTGTATTTTTAGTAGAGACGGGATTTCACCGTGTTAGCCAG GATGGTCTCGATCTCCTGACCTCGTGATCTGCCCGCCTCGGCCTCTCAAAGTGCTGGGAT TACAGGCATGAGCCAATAAATATTTTTATAATCATTAACTACCGAGGAGAAGCTGGAGAG AAAAAAGGTGGATGAAGTTAAGGCAGAGAGACTTTGTAAGTTTCTTGGCTGAGCTTTTGG AGACTGTATATCAGAGATCCTATTTGAGGTGATTTTCAGCTACAGAGATAGGCCTGGTCA TTTGAAAAATAACGGATTAGGTGAAGCTTGCGTTTGAAATCCTCCTTCTACTTTTCATCT TTCTCTCTTGTTTATTCTGAGCATCCATCTTAGACACCCAATCTTTGTCACTTTGTGGTT GTCATTGATTTCCCTGTTATTGAGATTAGAGGTTGGCAGACTTTCTCTGTAAAGGGCTGG AGAGAAAGTACTTCAGACTTTGTGGTCTGTGCGGTGTCTGTTGCAACCACTTAACTCTGC CCTGTAGAGCAAAAGCAGCCGCAGACAGTACATGGGCAGATGAGCATGGCTGGGGTCCAG TTACATTTACTTGCAAAAAGAGGGTTGGAGACTGGGTGCGATCTCCCACCTTTAATCCCG GCACTTTGGGAGGCCGAGGCAGGAGGATCACTTCAAGCCAGGAGTTCAAGACCAGCCTGG GCAACAAAGCAAGACTCCATCTCTACAAAAAAATAAAAATTATTATAGCTAGGCATGGTG GTACACACCCGTAGTCCTAGCTACTCAGGAAGCTAAAACTGAGGCAGGAGGGTCAGTTGA GCCCAGGAGCACGAGGCTGTGGTGAGCTATTATTGTGCCAGTGCACTCCAGCCTGGTGAC AGAGCAAGAACGGTCTCGTCAAAAAATAAACAAAAGGCTGGGCACGGTGGCTCCCGCCTG TAATCCCTGCACTTTGGGAGACTGAGGTGGGCAGATCATGAGGTCAGGAGATTGAGACCA TCCTGGCTAACACGGTGAAACCCTGTCTCTACTAAAAATACAAAAAGTTAGCCGGGCGTG GTGGCGGGCGCCTGTAGTCCCAGCCACTCGGGAGGCTGAGGAGGGAGAATCGTTTGAACC TGGGAGGCGGAGGTTGCAGTGAGCCAAGATTGTGCCACTGCACTCTAGCCTGGGCTACAG GGCAAGACTCCATTAAAAAAAAAAAAAAAAAACCAGCAAAAACCAAACAAAACATAATGC ATGTTCTCTCTTATAAATGGGAGCTAAACATGGGGACTCATTGACTTAAAGATGGCAACA ACTGGGAACTGCTGGATGGGGAGGGAGGGGAGGGGTGAAAGGCCAACTGTTGGGGAGTAT GCTCATATCCACGTGACAAACCTGCACATGTGCCCGCTGAATCTAAAATAAAAGTTGAAA GTAGATTTAAAAAACCCCAAGAGGGCTGGGTTTGGCTTGTGTGTCCATAGCTTGTTAACC TCCGCTTTAGATATTAACGAATAGAAACATAGTGCTTAACTTCCCAGGCCACCTATTTTG TTCCTCTCCAAGGTGATGGATAGATGAAGGCCTTATCCAGCCGCCTGGAAGTTTGCTGAC GCTTGTCCTGTCACGGATTAATGAAGCATTGTTTTCTGATGAAGGTTTCATGCCGCTGTG CTGATGTGTCTTCTCTTCTCTCTAGGCAGGAAACTGCATATCTTCTGGTTTACATGAAGA TGGAGTGCTAATGGAAATGCCCAAAACCTTCAGAGATTGACACGCTGTCATTTTCCATTT CCGTTCCTGGATCTACGGAGTCTTCTAAGAGATTTTGCAATGAGGAGAAGCATTGTTTTC AAACTATATAACTGAGCCTTATTTATAATTAGGGATATTATCAAAATATGTAACCATGAG GCCCCTCAGGTCCTGATCAGTCAGAATGGATGCTTTCACCAGCAGACCCGGCCATGTGGC TGCTCGGTCCTGGGTGCTCGCTGCTGTGCGAGACATTAGCCCTTTAGTTATGAGCCTGTG GGAACTTCAGGGGTTCCCAGTGGGGAGAGCAGTGGCAGTGGGAGGCATCTGGGGGCCAAA GGTCAGTGGCGGGGGGTATTTCAGTATTATACAACTGCTGTGACCAGACTTGTATACTGG CTGAATATCAGTGCTGTTTGTAATTTTTCACTTTGAGAACCAACATTAATTCCATATGAA TCAAGTGTTTTGTAACTGCTATTCATTTATTCAGCAAATATTTATTGATCATCTCTTCTC CATAAGATAGTGTGATAAACACAGTCATGAATAAAGTTATTTTCCACAAAAGGACTTTGC AGTTTTAACGGGGGGCAGTAGGGCTTGTGCTATAGAAATTCAAAGGCAAGGGAAGTCACT TCTGTTGTGGGGCCCTGGGAGGAGCCTCCAGGCTGGAAAGGCTTAAGGTGGAGGTCTCCG ATAGGGGCAGCGTACACAGTGGACTGGCTGCAAAAGGCCGTGCTCAGCATTCAGACAGCA TCACACACTCCGCTTTTCTCTACCAGGGAGGCAGGTGGGGAAGGATAGCGATGGGAAGGC AGGCGGAGCTCAGAATGTGGAAGGGGATCCAGTAAGGCTTGGAAGTTTGCACCTGATCTG GTGGGTGGTGAGGAGCCCTTGAAGGGGCAAGGAGGCGAGGAGCACTCAGCTGTGTTCTTA CACTGATCTGCCACTGGGGTTAGAGACAAACGTGGTGGGAATGGAAAGCCACGCACAGTC ACTAGCGCCTCTGGGGGGAGAATGGATGTGGCTGGTGAGAGAACAGGGGGGCCCAGGGAG AGTCCGGCACCAACCTGGGCGGGGGAGCCCAGTGGGTGTGAGCACCCCCACTTTAGAGAT GAAGTGATGGAGACATTCAGATGTTTAACCCCTTGTTCAAGATTCCATACTTGATAAATG GCAGATCAAACTCCCAACATAAAATGTGGGTCATTTCTTTATTATTTTATTTGTATTGGT TAACAATGATCAGCCATGCAAGAATAAATGATTATTGTAAAATCTGCAAACAATGTAGAT ATGTAGAGAGTCCCTTCCTTGGAGCTTGACCTTGTCAGACAGGTATAGATGAGTGTTCCG GGGCAGCCGTAAAAACTGCCAGAGACTGGGCTGCTTATAACAGAAACGCATGGTCTCCCA GAAGCCCACATTCAAGGTGTCCAAAGGCCTGGCTCCTGGAGGCTCTGGAGGAGAGCCTGT TCCCTGTCTCTCAGCTTCTGCCGGTTGCCAGCAAGTGTTGCCGTTCATTCACTCCAGCCA CTGCCTCCATCTGCACACAGCAAAACAGCGTATCCTGAAGTGCTCAACCTTATAGCCATT ATTTTAAAATATCCGGAACACACAGGACCGTGGGAGTGGCTGTTGGAGAAATTTTCATGA AGGAAGAAAGATTACAACTAAGTTTTAAAATGCTAGTTTTGTTTGTTTTGTCTTGGAGAG GAGGTAAAAGTGGGAGTAAAAATAGGGAGTTTGGTGTAAGGTGGGAAAAGCAAAGGAACC CCGCATGGATGGGCTGAAGGGTGTGATGGGAGAACAGTGAGAAGTACGTTTGGGGAAGCA ATTGGAAATAGTAGCTAAGCTTAATCACAATCTATCAAAAGGGACTTGTTGAAGAATTAA TGTGTGACTAGGAACAGGGAGGTTATGGGCTTGTCAGCTCGACAGCGGGCACTCAGTTCC ACTAACGAATGATGCCCGTGTGGACAGACAGAATGATGGACAGGCAGATGAATGCGTGGG CTTTATGTGAAACAGGTCCTCTTGGTTGTTGACAAGATACTGTTTTAAAGTTCCATTTTG CCATACTTCGAACAGCTTGTCATTAGCTCAATTTAGCCACATGTAAAATCACTAAGGCGG ACTTCCAGAGTTCCCACATGAAAATCAAATGTAAACCAGCAGTGACCTGCTTCAACACCA TCATCGGAAGTCAGAAGTTGAACTCTTTTTTGATGTTTAAAGCCTGCATAATATTCGCTG TATTATTATTCAGCATATTACTATTTCCTTCGTGATGGAAATTTGGTTTATCCCAATTTT CTATTCCATCAAAACACCGCTACATAGAAAATCCCCATGCACATATTTCTCCTAATTGTG GAAATATTTTACATAAAAGACTCTAGACATGGGATGAAATTCCCAGGTTATTGGAATTTT AAAATAGATAGGTACTTCCAAATTGACCTCTTACAAATTATATGAATTCGTAAGCTTCCA ACTGTTATGGAGTTACCCATTTTGAGAAATCTGTGCTAAAAGGACCCAAACAATGCTGAT GACAATGATCAGGATAATAAGTACGCTGGGAAGACAACAAAATGATTTAGATCTTAGACA AGTCATTCTAGGTGTCTCCACTGTTTCAGTTCTTGCGTTCGTTCATTCTTGTGCTTTTTC GTTTTACCAAATAAAATAGCTCCTTGATGTCATAGGAATCCACGCTATGCTTAATGAGTA TTGGTTAGTAAAATGCCTATAACTAGTAATCTTCATCTATGCAATTAAATATTAATTCAT AAAACACTTCAAATGTAAACAATAATTAGTAAATGAAAAGTACATAATACCTCAATTAGA AAAAAATCACTCCATTAAAAAGACATTATTTGTGTGATAAAAGAGATTGCTATTTTTGTA TTTTTCTACAAGGTTAAAGAAAACTAAGTCAACTTATACAAGTGAATTTTAAAAGACTTT AGGGCAGGCGTGGTGGCTCACACCTGTAATCCCAGCACTTTAAGAGGCCGAGGAGGGCAG ATCACCTGAGGTCAGGAGTTCGAAACCAGCCTGACCAACATGGTGAAATCTCATTTCTAC TAAAAATACAAAAAAATTAGCCCAGTGTGGTGGCATGTGCCTATAATCTCAGCTACTTGG GAGGCTGAGACAGGAGAATAGTTTGAACCTGGGAGGCGGAGGTTGCAATGAACCAGGATC GCACCATTGCACTCCAGCTTGGGCAACAAGAGTGAAACTCCATCTCAAAAATAAATAAAT AAATAAATAAATAAAATAAATAAAAGCCTTTAACCCAGAATGCTGAGTAAATTGGCCAAA AATGCTAACCTATGCATTTCAATACTATAGGAGTCGCATGGGTAGAAATAACCAGATGAA ATACTTCTGGTATTTCACCTTCCCAACCCACACGAGCCAGTGTTTTTCTGTGAATAACAA AAACAGCAGAATTTACTTGCCTCACCATAAGAGGTTACCACTTCTGTGTGTTCCCCCGAA ACAGGTGGTGGCTGGGTGAGAAGGTGGACAGCACTAGGGCAGGAGATGGGGGCTCCAGTA TCGTGGGTGAGCTTCCTAAACCTCTGCAACTTTCAGCCCCTAAATGGGATGAGCCATCAG AATTTTTAGCACAATGCCCAGAACAAAGTAAGGATTTGACAAATGACGCCTCTCTCCACA TTGTTCTGTCATCAGCCACCGCATCCTGTACCTCCAAGTCCACTGGGCTCCGGCTGTTTC CATCACATGGAGAATGACTCAGAGCCTGGCCTCCAGCCACCCTCCTGGCCTTTCTGCTTC TCACTCTGCCACTGGCTCCTCATGGACCACCAGCCTGGGTGTCCTCAGACATACCACACA CTTCACTGTGGGAGTCACGCAGCCCTCACTGCTCCTTCGCCAGGGAGCCACGGGGCTTTC CTCCTCAGGAGGACTCTGCAAGCAGCTGGATGAAGGGCCCTCCCGTCTCTCATCCTTCCT TAATTTTTGTCACAGTTCTCCTTCCTTCCACTCAGTGCAGCGCACACTGATTGATCCTCC ATCTTCCCCAAAAGACAGGAACAGCATGAGCAGTGGAGAGTAGATTCCAATGATAGAAAA AATAGTCAGTGATTTCTCATTTCCATTGATCATCAATGAAGAAAATGTATCCTGAAGGTC ATGTACCTCCTATGGGACTGCTGCATCCTCAGCCTCCTGAATTTCAGCCCAGCACCTTCC TCCCCAGCACAGCAACAGGTCAGCCCTTACCAGCATCCCTCTCTTATTGCCTTTGTGCAG AGCCAGCACCAGGGCCAGGGGAGGCCTTGGGATTGTCCCTCCCCAACAATCTGTGAAACA ATCCTTTATGTCACCAACAAAGCACAGCCTTCTGCACTGGTGGTCAGTCCCTCTCAACAC CTCTGTCACTGTAAAGCTGGCAGGCAACCCTCCAAGGTTGGCCTTCCCAAGCACTGCACC TCTAGGTGACAGAGCACGTCCTTACCTTGAAGCCTGGGCGCCCAGTCTATCCTGTCCAAT GAGCGAGCTGTGGAGAAGGGGGGATTCCGGGTTAAGGGGAGACTAGCAGGGCTCCTGCTT TTATGTTGCCCTGTTGGGAAGGCTATTAAAGAAACACAAAGTGCTAAGCAGTGAGGATAG AACATGTTTTCATTATTTCAACCAATACATTCCACAGATGGAATAATAAGAAATGCTACA ACCAAGCTAACTGAATCCAACAGCATATCAAAAAGATAATCCACCATGATTCAAGTGGGT TTCATACTAGGGATGCAGGGATGGTTTAACATACGCAAGTCAATAAATGTGATACATCAC ATCAATAAAACTAAAAACAAAAATCACATGATAATCTGAATAGATGCAGAAAAAGCCTTT GACAAAATCCAGCATTTCTTTATGATTAAAACCGTTCGTCACAATCAGCATAGAACGGAC ATACCTTAAGGTAATAAAAGCTATCTATGACAAACCCACAGCCAACATTTTCCTGAATGG GGGAGAGTTGAAAGCATTCCCCCTGAGGAAGGGAACAAGACAAAGATGCCCACGTTCACC GCTTCTCAACACAGTGCTGTTCACTACAGCATTGGTTATAAGAGCAAGACTGGAAACAGA ACAAATGGATACCCATAGCGGGGTGCTTAAGTAATTTTGGGAATAGTCATGGGGTGCAGT ACTTTATAGCTCTGAAACAATACAATGGATTTACATTTGAAATGTGGAATGATAACTAAG GTGCATTGCCCAGTGATATATGCAGAGGTGCAGAGGACTTTGTGTAAACACGATCACACA TCAGCCTGCATTCCAGGTGCATGCTTCTATTTGCACATAGATTGCAGGGATGATATGCAA ACAAAAATGTTGACTTGGTGTTTGGAAGTTCAGAGTGGAAGGGAAACTTCCTTGCTAACC TTTTATGATATTTAGAGTTTCTAAATGTGAATACGTAATACATTTAGAAATCTTAGTTAA TAAGAAAAGCCTCTGTTCCTGGCCTCTTGCTGGCACATGTCAGGTGGAAATGGGGCTGTC ATGCTAATGTGTGCAAACTGAGAAAAATCCAAGAATGGGAGTCTGCTTTTTTCATCATAC AAATAATTGTTAATAGAAACAGTATGATAATTGCTCATTGATATACCATGCATATTCTAT TAGATAATAATAAATTTCTGAAATTTGAACTATACTTACACATGGAAATTGAAATATATG GATGAAACATTGTGGCTTATATAGGCAATTGTTTTATTGGCATTTTACAAACTGATCATC ATTCCTCATGGCACGGGTCCATGTGATATTAAGTAGCTTGTTATGCTTGGGAAAGGCAGT GATGACCACAAGAATGACTTCAACTACTAAAGTACAATGGAGATTTCAACAATGTTTTGT TTAATATTTAAATATTTCATTGTGCTCCCAGGCTTTTTCTCACCCTAATAGCTCTCATCC ATATCATGTGGGTCCCATTAATACAGATACCTCCGAATGCACCACTCTTCCATTATATCC AGTCAATTGCTGGTTACCTTGGGCCTACAACTGGGGGAGGGCAGGGGCTGCTGGCCACCT CCTCATCTACAGTAAGAGTCAATGAGCAGTTAAGTGGATACTGAAAACCATTTATCCTGC TGGAGTGAGAAATAAATGGTTTCTTTCAATAGCGTAGTAAAATGCATCTTTTCCAAACTA TTTATATGACTCAAGGCCCATCTCAATTTCAGATGTGGTTAGCCTCAATTCCTGATTCTC ACCAAGGTGTGTAATGTCATCCACGGCCCAGTGCAGAGGAACACAGGTGCTGCCGTCAGA CTGCCAGGGTCCAATCCCGCCTCCTCACTCACCCCGGGAGATCCCTTTAAGCCAGGAGTC AACAGTGAGGATGGAAACATGAGTGCTTTTTAAAGTCCTGAAAGTTCAGAGGCAGACTGT CAATTTCTCCTCCACCCCTGGGCACACACCAGGAGAACTCTGTCTCCAGGTTGAAGGAAG TGCCTGTGAGAGAGTTGTGTCCCTCAGATTCTGTTCACCACAGGTGACACTCGATGCAAC CCCAAACCTCTTCTGCACAATCCCAAGGGGTGCTGACTAATCCAACCCAAAGGCTGTGAT GTTTGGCAGAGGCAGAAAAGAAAAGGCCAGGTGTTCTGGGAAAGACCACCTTTAAATATC ACAGCACCCTCATAGCCCAGAGAGACAGTTCTAACTATTATGCCAATAAACCCGGAAAAG ACCAAATCCAATATGACACATATTTCCTGTTTCCTTTTGATTTCATGCCCCCTCCCTTAA CCTCCCAAGCAGCATGGATACCCCGAAGGCCCCTGGGAACTCTCTCCAATTGGATCTTAC GTGGAAAGTAGTTACCTACCTACAAATCCCCATCATCAGATATGCTCTCCACAATCAAAT CTTTAGAAACACAAACACCAGGATAAGTCATTAGAGAGAGGCCCACCCACTCCTCCCACC CTAGCTGAAGCCATGGTGCTTCGCACAGGATCCCCTGGTGTTTCCTCTGGGCTCACAGAT ATCCCTACAGCCTCTCTGGACATTGTTTTATACTTGCAAAATCATTTGCTCTCACCAGAC TCCAAATCCTCCTTCCCAAAAGGAGCCCAGAATCAGGTTTCTGTACCCTAGAGATGGCGT TTTTTCCTCAGGAAGTGAGTTATTTCAGGGTAGGTATCATTCTCCAGTGTCAATGGCTCC TGCAATTATAGAAAAGAAAACATTAGGAGGGTGAAATGATGCCATACACGTCACACAGAT CTGATAGTCTCTCGACAACTTGAGAGAGAAAATAAGAAGGGGTATAGTGATTGAGTCAAA GGTCAAAGTCCCCCAAAACTGGCACGGAAGACACCTGTGGAAAAGACAAGACCTTTTCCC ACAGAATTTATCTTTAAAGTGTATCTAGATTGGCAGTTTCACAACTCTTAATCCATGGGG GAAAACTGCTGTGGAGGGAAACACCTCTGCATTGCAGTGGATCGTGGATGCTGCCATCTA CCACACCCCAGTGTGCCTGGCATGGGTTGGTGAGAGGCTGCCAATCAATAGCACCACACC AAGGGAATTGCAGATGTCATAAATAGTCCACATTGGCAGATGTTCATGTCTACATTTGAT TAAACTGCAGATGACATCCATAATGCACACTGGCAGATGTTCATGTCTACATCTGATTGG AAAGAAGCCAGGAAAGTAACATTTCTGTTCAAGACAAAGAAAAGTGTCTTACATTGGCAG CATCTTCTTTTTTACAGATGTCTTGTACAGTGTCCTCATTAGCAATGTCATATACAGCGT CCTTATTAGCGAATTCGTATACAGCATCCTCATTAGCGATGCCATATACAGCGTCCTCAT TAGCGATGTTGTATACAGCGTCCTCATTAGTGATGTCGTATAGAGCGTCCTCATTAGCGA TGTCATATACAATGTCCTCATTAGCGATGTCATATACAGCAACCTCATTCGCTATGTCTT GTAAAGCATCCTCATTAGCGATGTCATATACAACGTCCTCATTAGCGATGTCGTATACAG CGTCCTCGTTAGTGATGTCTTGTACGGTGTCCTTATAAGCAATGTCGTCTACAGCGTCCT CGTTAGCATGCCTTGTGGGTGTCATTAGCGATGTCATATACAGCATCCTCATTGGTGATG TCTTATATGGTGTCATTAGCGATGTTGTGTACAGCGTCCTCGTTAGCAATGCCTTGTACA GTGTCCTCGTTAGCAATGTCATATACAGTGTCCTCATTAGCGATGGCTTATACACTGTCC TCATTAGCGATGTTGTGTACAGCATCCTTGTTAGTGTGCCTCGTACGGTGTCATTAGCGA TGTCGTATACAGCGTCATAATTAGCGATGTCTTATACGGTGTCATCATTAGTGATGTTGT GTACAGCGTCATCGTTAGCGATGCCTTGTATGGTGTCCTCATTAGCGATGTCGTATACAG CGTGCTCACTAGCGATGTCTTTTTTTTTTTATATATACTTTAAGTTTTAGGGTACATGTG CACATTGTGCAGGTTAGTTACATATGTATACATGTGCCACGCTGGTGCGCTGCACCCACT AACTCATCATCTAGCATTAGGTATATCTCCCGATGCTATCCCTCCCCCCCCCACCCCACA AGAGTCCCCAGAGTGTGATATTCCCCTTCCTGTGTCCATGTGATCTCATTGTTCAATTCC CACCTATGAGTGAGAATATGCGGTGTTTGGTTTTTTGTTCTTGCGATAGTTACTAGCGAT GTCTTATACGGTGTCCTCATTAGCAATGTCGTGTACAGCGTCCACGTTAGCGTGCCTTGT CGGTATCATTAGCAATGTCGTATAAAGCGCCCTCATTGGTGATGTCTTGTACGGTGCCGT CATTAGCGATGTTGTGTACAGTGTCCTTGTTAGTGACGGCTTGTATGGTGTCCTCGTTAG CGATGTCGTATACAGCGTCCTCATTACCGATGCCTTGTATGGTGTCCTCATTAGCGATGT CGTGTACAGCGTCCTCGTTAGCGTGCCTTGTACGGTGTCATTAGCGATGTCATATACGGC ACCCTCATTAGCGATGTCGTATACGGAGTCCTCATTAGCGATGTCGTGTACAGCGTCCAC GATAGCGTGCCTTGTCGGTATCATTAGCAATGTCGTATAAAGCGCCCTCATTGGTGATGT CTTGTACGGTGCCATCATTAGCGATGTTGTGTACAGTGTCCTTGTTAGCGACGGCTTGTA TGGTGTCCTCGTTAGCGATGTCGTATACAGCGTCCTCATTACCGATGCCTTGTATGGTGT CCTCATTAGCGATGTCGTGTACAGCGTCCTCGTTAGCGTGCCTTGTACGGTGTCATTAGC GATGTCATATACGGCACCCTCATTAGCGATGTCGTATACGGAGTCCTCATTAGCAATGTC GTGTACAGTGTCCACGTTAGCGTGCCTTGTCGGTGCCATTAGCAATGTCGTATAAAGCGC CCTCATTGGTGATGTCTTGTATGGTGCCGTCATTAGCGATGTTGTGTACAGTGTCCTTGT TAGCGACGGCTTGTAGAGTGTCCTTGTTAGCGATGTCATATACAACGTCCTCATTACCAA TGCCTTGTATGGTGTCCTCATTAGCGATGTCGTGTACAGCGTCCTCGTTAGTGTGCCTTG TACGGTGTCATTAGCGATGTCATATATGGCATCCTCATTAGCGATGTCGTATACGGAGTC CTCATTAGCGATGCCGTGCACGGCATCCTCGTTAGCAATGCCGTGGGCAGCATCCTCGTT GGCGATGCCGTGGGCGGCGTCCTCCTTGGCGATGCCCTGGGCGGCGTCCTCGTTGGCGAT GCCCTGGGCGGCGACCTCGTTGGCGATGCCCTGGGCGGCGTCCTCGCTGGCGATTCCGTG GGCGGCGTCCTCGTTGGCGATGCCCTGGGCGGCGTCCTCGTTGGCGATGCCCTTGTCGGC GGCCTCGTTAGCGATGCCGTGGACAGCGTCCTCGTTAGTGATATCGTGTGAGGCATCCTC GTTAGTGATGTTGTGTACGGTGTCCTCATGGGGAGCTAGAAGAACACAGAGTTAAGGTCA GTGCCCTGGTAGTGGAGACTGTGAATCACCCAGGGGCTTGCTTGGTGTGGTGCATGGAGG TGGCTGATCACAGCATGGGCCAAGCTGATGCTGGGACATCCTCCCAGGTGGACCTGCACT AGTGAAGCTAAGGGATGTGGCTCAGAACACTTTCTGCAGTGGGCATCAGTTTCCAGGTTC AGGTATGCATTATCCAGTGAAGTGGGGAAATATAAAAAATAAAAATTGACAAATTCATGA AAAGCCTTCCATGAGTGCAAGTGTGAGTTTTTTATCCACTTTACATTCAGTATGCATTCA CACATACAAAATATTTTTACAAGAAATCAGAAATTTTAATTTTTGTCAGTTATGCGAAAT CTAACTTAGCTGCCAACATAAAGATTCTATCTCATTTACTTGGTCTCGAGAAAATCTAGC ACATAGTAAGTAGACCAAAATGTTCATTAAATGAAAACACAGAGCAGAGATAGGGGGGCT GCTAGGCAGACTGGGTTGCACCTGATTACCTGGATGATAATTAACTGCACAAAACCTCGG TCAAATTAATATTGAAACTGCCTTTTGCTTGGGCTCGTTTCCCTTGCGGAAGAAGGATGA CCAAGGAGATGAACAGGAAAGAAATGAGAAACAGAGGCCTTTGCCTAGTAGCTAAAGGCC ACCTTCTATAACACGAAATAGCCTTGAACTCTGCCGTGATTTAGTGACAGAGTTCCCTCG TGTCTTCTACCCAGGTTGAAGTCCAGCAAAATTGCGACTGTCCTCTTTACAACTTGCGAG ACCACACTGCTTCTGCATTTGCCTGTTGTATGTATGAGATTTACACTTGTTTTAAAGCAA CATTTTGTTTCAGTTGGGCTGGTGGCCATACCCGGCACTAGCCAGTCAATAGTGAGATGG CTCCTCATGGAGGAGGCTTGGCTTGAGGCTGAGGGTCTTTAACCCACATATACAAGAGAG TTGCCACTAAGGGATGGAAGCCAGGCTAATAACCAAGTGCCACACAGAGTTCCTATCTGT CCCTCCTCACCATTTTTGGCTGGCAGGATTTGAGCATTTTAGGGCTTGGGAAGATAGTAT TACTAAATCTACTAAAATACATCACCCATCCTTATAGACTTTGGCCAGTTGCTGAGCAAA TTAACTTCACAACTGAAGTGGGCCACACTGGCCTTTGTGGTCCCCCACTCCTCTTAGAAT TTGTGAGCGTGGGGCCTACTGGAGGGTGGGAGGTGGGAGGAGGGGGTGGATCAGGAAAAA TAACTGATATTAGGCTCAATATATGGGTGATGCAATAATCTGTACAACAAACTCTCATGA CACACATTTATATATGTAGCAAACCTGCACATCCTGCACATGTACCCCTGAACTGAAAAG TTAAATAAAAAAAAAAAAAGGATCGGTGAGCTGAGCCAAACACCTGGGGATCTTTGTGCT TTTGACACACTGATGACTATGCCTGTCCGTGGGGAGATGAACCCATAACTGCCCTGGGTT GTGTGACCACGGAGGCCACTTTATGATGATGGGCAGTGTCTGGGGCCTTTTGGGCTCGGT GCCTTAGGGTTTATACATGAATGCTGGACTCCCTGTGTGGTGGTGAACACCCCATGACTA AGTGCATGTCAGCGTCAGCACTGGCCCACACTCCTGGGTTCGTGTTTTCACTTTTTCATT CAGGAACTCGGGAGCTGGGGCCACTCCCTTGGCCCTTCAGGTTCTCCACCTGAGCAGTGG GGATAATAAGGCAGACCCGGGGATGGCTCTGGTGAGGGTGGAGGAGTCACTGTACAGAGA GAGTAGAGCGGGGGTGGATTTTATTGTTAGAAGTGGACACTGGTGATTGGGTTGTATAAA TGGGAAATCTCTCCTGAGAAAACACACAGCCTCACCTGTACAGAAACACACACATTCACA CCACACGATGCAGCCTCACACAAGACACCACCAATCCTCAAGCACCCAACTCAGCACCAC CCAAAAGGGAGCACAGCTGCTTCCTCAAAATTTGGCCATAATTTTTCCCTGGGGAATTCA GGTTTTAAAAAAACACTTCCCCTATACTTATTCCTATCACAATCCCAGGATCAGGGTGGC TCTTCACATTGAAACCAGGCAAGGATGCCACACCTTTCTTGGCATCCAGATTGTTTTCTT GGTAAGTGATTCCAGAATACTTACTAGATTCACGCCTCAGAGGGGCCACCTGCACCACCT GCAATACAGAAACAAAGCTTTTTGAGGGGTATGTCATGTTGTGGATTGTTTGCACAAGGC TGTGTTTCTCTCAATGAATATTGAAAACTTGATCAGAAAGTGTAGTCAACTTCAAGGCCT CCAAAACAAGGGTAGGATACACACTGGAAAAGACATCAGCTTCTGGATGGCGGATCTCTC AGGTCCACGTAGGTTGGCAAGTGCAAAATACTGAATCCAAGGAGAAGACATTGCTTCCAA GGACAAGGACCCCAAGGATACAGTCTACAACCTGAAGCCATCATAGCTAAATGCCATTTT GGATTACATATCAGTTGCTAAGAGTCACTTCTTCCTCCCCCTCAGAAAACTGCATTTAAT ACCTGTCATGGACATTGTCATCTTTTCACATGTAAAGTCAGTTGAAAAAGAAAGACAACA AGAAAGGAACATTTCTATTTCAGAGAAAGCAAGGCAACCTTACCTTCACGTTGACTGGCC TCTCTCCATCTCCTCTGTCCTTGTGAACTAGAGACTCCTCAGAGGCTAGGAGGACACAGA GCAACAGTTAGTCATAGATGCTTTTGTTCATGAGCTATTCAGGGAGCTCTGCTTAATGTG GACAACAGGACAGTGTGTGTGGATGTGTTTCATTAAAAGCACAGCTTGAGCTCCTGCTAG AAAATCCTCCCTCGTGGAAAGACAGGCAAGAACGAGGAGCTAAGGAGCAAGAAATAGAGT CCCTGGCATTTTGCTGATGGCAACTTAAGGCAATGGGAATGAGTCAGTCTACAAATGGTA CTGAAGCACATGCTATAGTTTGATGAGAGTCCCACTGCTCACACTGTGAGGTTTGAAACC CAGCTAAATGGTTTTCTAAACCTGTAAAAACAATATTACTTGCAGGATTTATGTCCCAAG ACTACTTTTACCTCTGAGGATCCACAGTGGCTGTCACTGCAGTTATTATGTGTTTTAGCA TTTTGCACTTGAATAAAAGCAAAGTTTAATAGACAGATTGGATTCAATTCTAGGCAAAAC AGTCTATGGTATTTATTCACTAATCCTTTGTTATAACTGCTGATGGGAGAATTAGAAGTA CTGAAATTATATCCTTTAAAATTAATTAAAGCATAATTATTAATCACACAATATTTTTTC ATCCAGGCCTCCTTTTCTTTGTCATGCATGCATATTAATTGAGGATGGAGAATATCTACG CTTGTTCAGGCCAGCCAACATACGACAGTTTACTTCAAGACAGGAGACATGGGTTGAATG CTGGTATGTTTTAACTCTGCAGCGCAAACAGTTGCAACAAGTGTGGTGAACTAATCACCA GATGGCCCTTTGCTGCCTTATTTGTCATTGTGCCTTACATGTAGCTTGCAGGATTTGATT ACGCTTATGTTTTGTGGTGATCATACTTTCAACTATTCCTAAAATACTGCTTCAGTCTTA TCTGTTTGGGGTCAACTGCTGAGGATTTCATACAAATTAATGAAGTTTGTGAATCTAAAG TTCTACACAAAGGGGGAAATATTTGCAAATCATTTATCTTGTAAGAGACTAAAATTTAGA ATATTTTTAAATATATTTAAGATATATTCAAAAATCTACAGCAACAAACTAACTAAATAA AAATCAGACAACTCTTTAAAAATGGGCAAAAGACTTCAACATATATTTCCCTAAAGAAGA TATAGCCACAAATAGTAGCACAGGAAAAGCTGCTCAGGATCATTAGTCATTAGGGAAATG CAAATGAAAAACACAAGCAGCCACCAATATACACCTACTAGGATGATTTAAAGGAAAATA AGTGTGAACAAGGATGTAAAGAAATTGTAACCCTGATACATTGATGGTAGAAATGGATAA AGTTGCAGCCACTGTGAAAAACAGTCTGCAGTGGCTCAGAAGGTTAAATATAGAACCCCT GTTGGACCCAGGAACTCTACTCTTAGGCACCCCAAAGAATAGAGAACAGAAATCAAACAG ATGTTTGTATACTAATGTTTGTAGCATCACTTTTCACAGGAGCCAAAAGGTGGAAATAAT CCAACCATCAGTGAACAAATGAATGTAATAAAAGCAAGGTGGTCTGCATGCAATGCTACA TCATCCATCTGTAAAAAACGAACATCATTTTGATAGATGATACAACATGGGTGGACATTG AGAACATTATGCTTAGTGAAATAAGCCAGACACAAAAGGAATATATTGTATAATTGTAAT TACATGAAGTGCCTAGAATAGTCAAATTCATACAAGAGAAAGTCGGATAGGAATCACCAT GGGCTGGAAATAGGGGGAAGGCGCTATATTGCTTATTGTGGACAAGGTTTTGTAAGAAAT CATCAAAATTGTGGGTGTAGATAGTGGTGTTGGTTATGCAACCCTGTGAATATATTGAAT GCCACGGAGTGCACACTTTGGTTAAAAGGTTCAAATGATAAATAATGTGTTATATATATT TCCCCACGATAGAAAACATGCACAGCCAAGCCCAGATGCCAGTCTTGTTAGCTGCCTTCC TTTACCTTCAAGAGTGGGCTGAAGCTTGTCCAATCTTTCAAGGTTGCTGAAGACTGTATG ATGGAAGTCATCTGCATTGGGAAAGAAATTAATGGAGAGAGGAGAAAACTTGAGAATCCA CACTACTCACCCTGCAGGGCCAAGAACTCTGTCTCCCATGCATTGCTGACCCATCTCAGT ATTTCCTGTGACCACCTCCTTTTTCAACTGAAGACTTTGCACCTGAAGGGGTTCCCAGGT TTTTCACCTCGGCCCTTGTCAGGACTGATCCTCTCAACTACTGACCATTTCACCTCCATT CATGTCCATGCCACATCAGGCTGTGTTGTCTAGATGGCATGAATCTACCCCAAATGTCCC TTTCTGGAGGAAGCCACCATTATGCTGTACCTCCAAGCATAATGATACGTCCACACACAC CAAGGCACCTCACTCATGCAAGGTGTGTGTCCTCTAACAAAGTTTCACGCTCTAAACCCA GATAACTTTTGAAACCCAAGTTCTGTTGATTCCCCTACTTTGGGTGCTCCATAGATGCTC ATTTGTCTACTAAACACTGCCCCAGGCAATTAAATATTCCAAAGTGACCAGCAGAATTTT TATGTTAATTCTGACATTGCATTGTTAGTACAAGTGTTTTTCCCCCTTCAAATTTATGTC TTTGTTACTGATAAATGTAACTGATAATGCTTTTTTCAGCTATGTTGCCAAGCATATTTA TATAAAAATATACTCAGATTGTTTTCAGAATTTGACAAAGATGATAGCAACAACGATAAT CTCATTTGTCTTATACTAATCTTTATGTGTTACTTTCATCATTTCTTACATATTGGGGCC TACCATACATTGTACGGTGAAATTAGTGCTATGCATCATGGTAGGAATATAAATTGGCAA AAGTAATTTAGAAAATAGTTCCCTTCTTTCTTAAAAAAATTAGGCTGGGTGTGGTGGCTC ATGCCTATAATCCCAGCACTTTGGGAGGCAGAGGTGGGTGGATCACCTGAGGCTAGGAGT TTGAGACTAGCCTGACCAACACAGCAAAATCCTGTCTCTACTGAAAATACAAAAATTATC CAGGTGTGGTGGTGTGTGCCAGTTGTCCCAGCTACTCGGGAGGTTGAGGCACGAGAATTG CTTAAACCTGGGAGATGGAGGTTCCAGTGAGCCGAGATTGTGCCACTGCACTCCAGCCTG GGTCTCAGAAAAAAAAAAATTTTTTTTGACCAAAATGTCATTATGCATTACATGACTGTA TATGAATGCTCAAAGCTACATTACTCATCAAAGAAAATAACAAAACAATTAAATGTCCAT TAACTGATAAATGAATAAACACTATCTGTATGAATAAACACAGCAGACTATGAAGGAAAA CACATGACCAGCACGTGCTAACACGTCAATTAACTTCAAACATAGTATGCTAAATGAGGG AAGTCAGATTCCAAATATATATATATGTCCATTTCTATAAAGCAAGTGGGAAATTTATGG AGATGGAATGTCACAGCAGTATTGCTTAGGGCTGGAGATGGGAGTGGGGATTAACTGCCA GTGCGCAAGAGAGAACTTGGGTGAGGGAAACATATTTAAATTAGATCGTGGTGTTGGGTG CACACAGTATCAATTTAATAAAGCATCAAATTCTAGACCTTTTCAGTGGGCAAACTTTAT GGTGGGTTCACACCCAATATAGGTGTTAAAAATAATGTTACGGAAATTCTTGTCGGGTTT TTAACAAGCCAAGAGATATGCTGTGAAAGCAGCATTAATTCAAGTGGTTGTCACAGGTCA CTTAAAGTTAATCAGATAGTTGTCCTACAAATATAGGGTGAATGTTATTCATGAATTTCC TGAATCTATTGCAATAATCACATTTTTTTCCATTAAACTCTTGAGGTAGCTAATTTTATT TATTGCATTTTCAATGTTAATCTACTATTTCATATGTTGAGATTAACTCACATTAGTCAG AATTTAAAGTATTTTAAAATATCACAGAATTTAATTTACCTTATCTGGTTTTGGTTTCAA GACTATAGTAGCCATTTCATTTAATTGTACATGTAGGGTATTCTAATTTATGGAAAACTA TTACATTTTCCTTGATTTTTTTTTTTTTAGAAATTACTTCTAGGGATCTATATGGTAGAG TCCATGGAGAATTGTTTTAATTCTTCATTCATGTCTTCAGTGGGTATAGGATTGGTCATA TTGGTCATAGTTTTCTGCTCGGATTTCAATAAGAAACTTGTGGAAGAACCTGAAGGGTGG GATCTTTGAGGGAGCCTAAGACAGAGCAAGACAAGCTAAGAAGGAGGGCAGTGCCACAGC AGAACTGCCATTGATGCCCCCTCGCCTAGATTGCGGAAGAGACATCCAGCTGTAGACACT GAGGTGCAGGAAAACAATGGAGCACCATCAGAGAAAGCAGTGCCCAGGAACAAGGAGGCA CTGATGGTGGCAAGGGGCAAAGACAGCTGCCACGAGGCTGTTCACATGAGGGTCTCAGGC TGCATAGACACCCACACCAGCTGAGGGGTCCTGGTTTTCATAAAGTGTGTGGCTCAGCCA GGCCACCAACAAGCAGTTCACAAACAGTAGTAATACGACACTTTCCAAAGACCTTACTTG AGTAACACGGTGATCCTCACAAATTTCCAATCAGGATGGTCGCACAGTTCCTCCTGCTTT AGGACTCAGAGCCTGCCCGTGGTCACAGTGGGTAGGTGCAGACTCTGAAGATGCACTTTG GTCAGAGACCCTGCTGAACTCTGTCTAATGAGGACCTCTGTCCTGTCTGCTGACCACCGG TCAGAGGTGCAGGCTGCAGTGGGGAGTAAGAATGCCACCTTCTCAATGTTGGGAAAACTC CCTGCCAGAACTGAGAATGGCCCTTTCTAAGCAGAAGGCAAGCTCAGACTAAAGAAGGAG GCCGAACACATCAGGTTGGCAGATTGCCAAAGATTCACTCAGGGAGAGCCCACATCCTGG GCCATCTTGGGTGGTGGCAAGATGAGGTAGACGACTGCTTTTGCAACACACACCTGACAA CAAAAAATCAACAACTGTAAAAGAGCCACAAAATCCCCAAATATTTGCAAATTAGCAATG CACTTTTAAATAACTGTTGGGTTAAAGAAGAAGTCTCAATAGAAAATTAAAAGTACTTTT AACTACATTAAAAGAAAATGTGACTTGGCAAGATTTCTGGATGTAGCAAAAGCAGTCCTT AGAGGGAAATCTATAGCATTGGATGCAATATACTAAAAATCACAAGACCTAAAATCAGTA ATATCATGTTTCAATTAGGGAACTATAGAAAATAGAGGAATGCAATGGAAAGCAAGTAAA AGTAATAAACAACATCACAGAAATCAATAAAATTAAAACACTGAAATCATCAGAAAATCA ATAAAACCAAAAGCTGGTTCTTTGATATGCTCATTACAATGAATGAATTGATATGCAGGC TAACCAAGAAAAAGAAGATAACACAAATGACCAATTTCAGAAATAAAAGAGGAGCCATCT CTACTGAACTGTTAGGCATTAAAAGGAATATCATGAACAGTTCTATGAGCGCAGTTTGAT AACCTCAGTGAAATGTATCAATTCCTTGAAAGGCAATCTTCCCAAGGTCATGCTAGGATC CTAATTTGAATAAACTTATGTCTATTAAATAAGTTGAATTCACATTAAGAGCATTCCGAA AAAGAAAGCACCAGGCCCAGATGGTTTCTCTCATGAAATCTACCGAATTCTTCAACAGGT GAATAAAAAGACAAAAATTCATTTAATGCAATATTATTTGGTGATTTAATGTGCCATTTT TTGCCATTAAGGCATAAAAAAGACATGAAAGCAGCTAAAGCGTACATCAATTTAGTGCAA TAAATTCATCTGAAAAAACTACATAATATATGATTCCAACTATATGACATTCTGGAAAAG GCAAAGCTGAAGCGATAGTAAAAATATTAATAGTTGCCAAGGTTTCTGGAGAAAGAGGAC AGAGATTAATGAGAAGAGAGGATTTTTAGGGAAGTGAACATTTTCTTTATGAGACCATAA GGGTGAACATAATGTTTTAAATATTTCAAAATTCATATATATGTATAACAGAAAGAATGA ACATTATGCAAATGCAGACTTCAGATAATAATGTGTCAATATTTTCTCATTATTCTAGCA AATGTACCACAGTAATGTAAGATGTTACTAATAGGTGAAATTAGGAAGTGAGGGTGAGGA GACAGAATAATATGGGAACTTCGTGTATTATATACTCAATTTTTATTTATTTATTTATTT ATTTATTTATTTATTTATTTTGAGATGGAGTTTCACTCTTGTCACCCAGGCTGGAGTGCA ATGGCATGATCTTGGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGCGATTCTCCTGCCT CAGCCTTCTGAGTAGCTGGGATTACAGGCGCCTGCCACCACACCCGGCTAATTTTTTTGG ATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCCAACTCCTGACCTC AGATGATCCGCCTGCCTTGGCCTCCTAAAGTGCTGGGATTATAAGTGTGAGCCACCACAC ACGGCCATATGCTCAGTTTTTATGTCAATTTAAAACTCTCTAAAGAAATATATTAATTGA AAAATAATAATATAGCACCACTCTTTCAGGGAGATCTATGCTTATGTTTAACAACCAGGT AAGTTCTAGACATTAGCTTGAAGCATTGTCTATCATTAAACATGAACCAAAATTGACTTT TAAGTAGATATTTACTTTTGTGGTGGTAGCAATATTTACTGACCAGGCAAATTAGAATCC TGACACATTAAAAAATATGGCTTAGTCTCTTCATAGTTTCCTCTTACATATGGGACACTG AATACTCCCCGCAACTGCAATTCTTGAATCAACTTAATTAATGAACTTCCACAGTACCTT CTTGTGGGTACCTCTTCTTCTTTACCCGGGAGCCATGAGGTCTCCTACACTGGTTGGTGT GCACAGCATATCTTCTTGTATTCTCTATCAGAGAAGATGCTGGTTAATGCATTGACAATA GATAGGGCTGTTGACATCTTGCTGACAGAAGACCAGAGGGAAAATAGTGATAATCTGTTC TAAGTTTAAACTTATGATCCTTTTTTTACAGGCTTCCAAGCAGAGCCCACTGAATCAAAG TTGGGTTTCAGGAAGATCACGGAGTTCAGTGAGCACTCAACACCTCTATCAGACAGACTG CGTGGGCAGTGCCTTCCTGGAGAGGGGAACACAGCCGGATGACTGTGAGTGCAGGGCTGG TGCAGAGTGGGGGCCCGGATTCAAATTCCACTAAGCCATGTGGACCTGGCAAGCTCATGT CCTCCCTCTGCCCTCAGTTCTCTGCACTGTCATAATGTAATTTTAGCAATACTTTTTAGG CCCTATTTAAGCCCTACTTCTTAGTATCACAGTACAGGGCTAAAAAATCACTAAATACAG GAAAACCTTAGAGAGGACTGGTACTTCAGTAATGTTCTCTAAGTGTTTACTACATGCCAG GAGGAATAAGCTGGACACTTAGCAGTGGCGGAATATGGAGGGGGAACTTGGATGGCTCCG GGGCAGTGGAGCATGCTTTCCTGTTCGGCTTTTCCGTGGGCATGACGCCTTATGGTTTAT GGAGAACATCAGCCCTGCAGGGGGTGCAGAGGAGGGGCTGTGGCTGAGATTTTACACTTG AGGGTGCTGACATTCAGAGATGATAAGTGACGAGCAGAACCTCAACCCCGCTGAGTGAAG GACCTGAGATGGGAAATGTATTTGGTTCCCTAGAGAGAGAGATTCCTGAAAAACTGCCAC CTCTTCATCACGCCCTGTGCCACAGACCCAAGAGACCCCTCACTGTCTTTCTCCAGTCCT CCTAGCCCAAGGTGTGTGGGTGGACAAAGGTGGTGCTCTGGAGGAAATGCCTGAGATAAG GACAGGTCCTTAATGATAAAGAATTCTCCTTCCTCTTTCAGATCCTTGACCTCCCAGTAT GACAGCTTAAAGGCTGTCATCTCTGTGGCCTGCCTCCCCTTGCCCTTCACCCTGCCAGCT GCCTCTCAGTGACTGTCTCCTCCAGTGACTACACTGAGGGACCAGGGACTGCTTGCCTCC CGAGGCTGCTCACACCTTCTGACACCGCAAAATGATTGTCAAAAATGGGTCTGCAAAGAG TAACTTCCCTTCCACTGATCAAACCTGAATATGCAAGCTACTGTCAATTAACTGGAAAAG TGGCCGTGTGGGCTGGTGCTTTGGTGATTTAATGAATTAAGTCTGCAAGCCCCACTGCCT CCTTGACTATTGATCAGAGCTGCCTGCAATAAGGTCTGGCTAAGAATGGGCAGTGGCTGC ACCAGCTCTGGGTAAAATTTGACCTAAAATGACCAATCTCATTCACTAACCTCACCATAG TCTTATGGGTTCAATGGACCTGTCCAATCCTTTGCTCTGTTCTCTCCATCACCTTCCTGT GTAATTTTCCTCCACCACGCACATAATAGAAACATGGCACAGGGGAGCTAATCGCCTCTT TTATCCCCCACTTCAGGCTCACACATAAGTTTATAGTAAAAGCCTTTTCAAATGACTGCT TTAACTGCTGCTACAGCATGTGTCATCAGTTGAATGGAATCTGTCACGTGACTTTAAGCA ACCCTTTGCTGAGAGACAAGATTCAATACTAGGGACAGTATTCTAGTGTACTACATCATT GATTTTATGTTATGAAGATCATCATTTATTGAAAATGTATAAATAATGAAGCCCAGCCTT ACTCTTCAACGCTGTGTGTGTAAATCCACTGAGTGTGCTGACCCCCACGCTTGTACCCAC CTGCTAACACAGAAAGGGTCCACTCAGAAGGGAGGCACAGCTCCAGCACTGAGGCTGTCC ACACCAGCTTCACAAGAGGGTTGCCACAAGGACGACGGATACCCGGATAACAACCAAATG GTAATTTGAGTACTTAATGGTCATGATCCCTAAAGTGTGTAGCTCAGAGGGCTTGTGGTG ATAACTCCATCAAGACTCTAAAGCATCTCCCCAATTCTTACTGGACTTGATCCATGTCTT GAGGAGACCCAGCTATGACACGCAGGCACCACGTTGTCCTACTTAGTGCCTCCCTTAGTG TTTCAGAACCTGTGATTTGATCAGAAACATGGGCTTTCTATGTTGGTTTCACACTAAGGA CTATGTGACACCTGCAGGAAGATGTCTACATAGCTACCTGGATTATGAGATCATGAGGCT GTCTTATGTGAGGGATGGCGTTTGGGATCTCTGCAGGCGTGGGTAATTCCAGGCATAGAG GGTGCTGGAACTCCCTTGCATGGTGAATAGTGATCTCTTCACTGGCTGATAAATAGAGGT TGTAGTTCAGGCCTTCAACATTAGCACCGTATGAGGAAACATTTTGACTCTTCACTATGC AGCAAGTGAACCAGAGCACATTTATTTATGTGGCTTAGTTTCTCCATCTGGCATGTGGGC TCAATAAACAAGCTCACAACATATGGGCATGATGATGATGAGGTGTGAACTAATGTAAGT AAAGTATGTGGTCTGATTTGTTAAATTAAGAAAAATGGCACTGAGAGTTGTGCTGGGTAA ACACAACATTTTTTTCCTGGGGGAAACACAGATAGACACACATTCACAAGCAAATCATGC AGACTTGCACACAGACCACCTCACCCCACCCCCGCCCTAATACACACATACCCACACACA ACCTAATGTGAACATGTTCCCAGAAACTATACATAGATAAAAAGAGTATGTCACCAGGAA AACCAGTTTCTTTTACTATACCCTACATCCTCATTCCCACCAGATGTCTTGGATCATGGA GGCTCTCCGGACAAAAGCCAGCAGTTAAGCTCCAGATTTCCTGTAGAATCCTTTTCTAAC AACCAGTGAGTGATTCCAGAATACGTACCATTGAATGTGCTCCCTGAAGTCACCTGTAAT TAGAGAAGGAAAACACTCTGAGAATCAGGCTATGCTATGGATGGCTCACACAGGTCTTTT GTTCACTTGGAAACTCTGGGTAACCAAGATTGGAAATAAGGTTCAAGTCAAAAGCCCCAA CTCTAGAGTAGAGTTCCCTTAGGAAAGCACAGGAGCTTTTCTTGAAGAATGTTTCTGTCT AGGTAATTTTTGAGTAGCAATTGCAGAATTCTTATCTAAAGTGGAAAGCTTGTTCCTGAA GAAAACATCCCTTAACACCCAGTGTACTATCTGACACTGCCAATTTTGCACGTCCTCTGG AATCAGGTGTCAGTTGGTAAAATACACCTCCTCCATCCCCAAGGAAATATTATCTAACAC CTATAATGTAGTGGAGAATTTTCCCATAGCTGATATCAACTGAAAAATAAAGGATCCAAG AAAACAACATTTACATCTTAGGCAAAGACAGGCTACTTTACCTTGGTAGTAGAGTAGGGC TTCCTTTTCACACGCTTTTTGGAAGGCTTCTTCGAGTCACCTAGGGGATGTGGAGGGACA CAGCATGGCTGTCAGTTCATTGGCAGTGCTACTCATGAATGACTCAGGGACTGGAACTTA GGGGCGTGCCTGGTTAACAAGCATGGAATGAGCTTCTCCTGGACCATCTTCTTCACGGAC CAAGGAAGGCAAAGAAAGAGCAGCAAGGAAATGAGAGTAGAGCCCTTGGCTTTCCAGGTA ATGGCAAATGAAAGCAACGTGAAATAATCAACTCCAAATGAACAAATGCTAAAACACATG CTACGATTCAACCACAGCATCCTGTCACTTCTTCAGACCCTTTAAAAGCCCAGCAGGACT GCCACTACCTTCTTGACATCGACCAAGTCCCTTTCAACCTCCACAGACCCACATACACTG CTACTGCATTTATCATGGAGGGTATAGGGTTCTGCCCTGTTTATGTGTGAATTTTTTAAA AACTAGATTTAATACCATGCACCAGCATTAATTGTATTTATTTCTTTTCTTGGTTATGAA AATAATCAGTCAGGCATAGTGGCTCACACCTGTAATCCCAGCAGTTTGGAAGGTGGAGGT GGGTGGATCATTTGAGGTCAGGAGTTCGAGACCAGCCTGACCAACATGGTAAAACCCCAT CATTGAAGATAAATGTTTTATATCCATGGTTAACAGATGAGATGACCATGAAATGAACAC CAGTGTACTGGGTGGAGCAGCTTATCTATTCAGTCTTCGGCACTAAAACCTGTGAAACAA TATCATCTTGCCTTATTTACTAACAAATACAAGTGCCTCTAAACTTAGACAGTTTCCAAG TCATGGAACTGATGAGCACTTAGCTCCTGCAGAGAGCTCTGGATGATGGGTCGGGAGAAC AAAGACACAATACATCAAAACAGCATTCACAAGTAAACAGGTTTTCAAAGCCCTCTACAT GCAAATTTACACAATTATCCTTTTAATTTTTATCTTCATATATATGTACATAATCTACTT GCTTCTGAGTATAAATCAAACTGTATGTTCTTAGTTAATAGTCTCTATAAATTCACTCTA TTTATCTTTCTGAGTTGAAATACTGCATCTCATTGGATAACAAAAAAAAAAATTTGACTA AGATTACACTGGAAAGGTGAGTAGGTTGGGTGATTGACTGTGATTGACAATTCCATGATT CTGGATAACTTCCAAAGCATAAAAATAAATGTGTGTTTTCTTTCACACGTAGACAATACA CATGCTTATTACTTTAAAAAATTAATATGTGCATGGAAGTGACTTACTACAAATATATTA AAGTAAATACACATTTCACAAAAAAAGAAGAGAGGAAGGGAAAAACATGTTAAAAACAAA GAGAGTTACATTTTATTGTGTGAAAAGCCTCCAACGGATCCTTACTACTGTGGCTTTGTT CCAAAGTTTTGGAAAGTAATGATTTCATAGGTTCTTAATTGGGTTAAAAACAGCATTAAA ATAGACTCTGCCATATTCTCCCCTGGGGAATAACTTAATCTGTGGGGTGGGGAATGGAAC GTTGAAGGATGCAGGATGTAAAAGGAAATTATATATATATTATATATATTATATATTATA TGATATATATAATATATATATTATTTATTATATATATTATATGTTATATATAATATATAT AAATAATATATAATGTACATAATATATAATTATATAATATATATTATATATTATATATTA TATACATAATATATAATTATATAATGTATAATATATATTATATATAATATAATATATATT ATCTATAATATATATAATATATTATATATATTATATATTATATTTAATATATTATATATA TTATATATATAATATATTATATATAATATATATAATATATTATATATAATATATTAAATA CATTATATATATATTTTATATATATATATATTTTTTATATATATATATATAATATATATA TATAATTTGGGAATTTGGGAATAAACTGAATCCCAATTCACACTGGGACTACACCAGCTG CCACCATGCCTGGCTAATTTTTTGTATTTGTAGTAGAGACAGGGTTTCACTGTATTGGCC AGGATGGTCTTGATCTCCTCACCTTGTGATCCTCTTGCCTTGGCCTCCCAAAGTGCTGGG ATTATAGGCCTGAGTCAAGATACATATTTTTTAAATGAAGAAAAATTTGAAAGATACTCT GCTTGGTACAATAATCAAATATATAAATTGAGGAATAAAACATAATCATGAAACATATTT ATAACTGCATATGGAAAATACAGAGGCTAATTTTTTAAATAACATATTTTGAAAGCATTA ACTAGTAATTTGAAAAGATCGCATTTGACAGGCCAGTATGAACATACCTTGAATGCAGCC ACACAGGTTCCCCATAAGAAAAATCAAAATCAGGGAAAATGAAACCACAATGGTTCAATC TGCTCTGACCTTTGAAAAACTCAGCACAGGTAGTGGCACTTACGACCAAGGGCAGGAGAT CCCTAATCCCATCACCATGGCGATAGGGCATAAACATTCCAGGGTGAAGGCACAATCCAC ACTGTGAGGTCCAACTGCTGCCATGCAGACAGGTGGGCTTTTACAAGTACAGGAAGGTCA TCAAAGGCTCAGTGTTTTGTTTCAAAAACTGAATCCCAAGCCCACACATTATTATGCTGG CTTCTTAAAATAAGTTGTGAGATGGGAAGTAGGGCACCCACAAATATATATATATATAAT TATATATAATATAATATATAGTATATATATAATATATTTAATATATTATATATATATATA ATTTGGGAATTTGGGAATAAACTGAATCCCAATTCACACTGGGACTACACCAGCTGCCAC CATGCCTGGCTAATTGTTTGTATTTGTAGTAGAGACAGGGTTTCACTGTATTGGCCAGGA TGGTCTTGATCTCCTCTCCTTGTGATCCTCTTGCCTTGGCCTCCCAAAGTGCTGGGATTA CAGGCCTGAGTCAAGATACATATTTTTTAAATGAAGAAAAATTTCAAAGTTACTCTGCTT GGTACAATAATCAAATATATAAATTGAGGAATAAAACATAATCATGAAACATATTTATAA CTGCATATGGAAAATACAGAGGCTAATTTTTTAAATAACATATTTTGAAAGCATTAACTA GTAATTTGAAAAGATCGCATTTGACAGGCCAGTATGAACATACCTTGAATGCAGCCACAC AGGTTCCCCATAAGAAAAATCAGAATCAGGGAAAATGAAACCACAATGGTTCAATCTGCT CTGACCTTTGAAAAACTCAGCACAGATACTGGCACTTAGGAGCAAGGGCAGGAGATCCCT AATCCCATCACCATGGCGATAGGGCATAAACATTCCAGGGTGAAGGCACAATCCACACTG TGAGGTCCAACTGCTGCCGTGCAGACAGGAGGGCTTTTACAAGTACAGGAAGGTCATCAA AGGCTCAGTGTTTTCTTTCAAAAACTGAATCCCAAGCCCACACATTATTATGCTGGCTTC TTAAAATAAGTTATGAGATGGGAAATAGGGCACCCACAAATATATATATATATAATTATA TACAATATAATATATATAATATATATAACATATATATAATTTCCTTTTACATCCTGCATC CTTATATTATATATAATATTATGTATAATATAATATGTATTATTATATATAATATAATAT ATATGAATATATATAATTATATAATATAATATGTAATTCTATATAATTCTATATAATATA ATATATATTATATATAAATATATATAATATACTATATTATATATAAGTATACATAATATA TTATATATACGTATACATAATATAGTATATTATATAAACATATATATAATATATTATATA TACGTATATATAATATATTATATATACGTATATATAATATATTATATTATATATACGTAT ATATAATATTATATATACGTATATATAATATATTATATTATATATACGTATATATAATAT TATATATATATTATATATAAGTATATATAATAATGTAATATATTATATATAAGTGTATAT AATGTAATATATAATATAATATAATAATATATTATAATATATTATATATTATAACATAAT ATAATATAATATACATTATAATATAATATATAATATTATATATAATATAATATATAATAT AAAATAATATAATATATAATATAATATATAAAATAATAATTAATATTTTAAATTAATTAT TATTAATTAATATTAATTAATATTAAAAATAATATAAAATATAATTACTATAATATAATC TATAATTATTATATATAATATAATATACAATTATATATAATATTATATATAATAAAATAT ATATAATTATGTGTATTTATTACATATAATATAATACATAAATTATATATAATTATTACA TATAATATAATATATAATTATATATAATTATTACATATAATATAATATATAATTAGATAT AATTATTACATATGATATAATATATAATTATATATAATTATTACATATGATATAATATAT ATAATTATATATAATTATATAATTATATATACAAGATAATAATATATAATATATAATATA TATTACATAATATTTTATATAATATATTATACTATATATATTATATAATATTTTATATAA AATATTATATATTATATATAATATAATATTTTATATAAAATATTATATATTATATATAAT ATAATATTTTATACAATATAATATATAATATATATTATATTATCATATGTTATACAATAA TATGTTATAGTATAATATATATTATAATCTAATATATTATATATTATTATATATTATATT ATATATAACATTATATAGTATTATATATTTTATAATAATAATATAATATATAATATTATA TATTATATATAATATATAATATATTATGACATATTATATATAATATATAATATATTATGA ATATATTATATATAATATATAATATATTGTGATATATATTATATATTATATAATATATTA TGATATATATTATATATAATATAATATAATTATATGTAATATAATATTATATATCTTATA TATCACATATTATATATTTTATATCATATACTATATATTATATCATACATTACATATAAT GTATGTCACATATTATATGTAATATATGTCACATATATGTAATATATGTCATATATTATA TATAATATATATCATATATTATATATGTCATTTATTCTGTAATATGTATCATACATATTA CATAATATGTATTATGGTGTCTGGTGGTCCCAGGACACAGACTGATGTCACTCAGCCTCA CATGACACTCTGTCCTCACCACACTTGGGGTCCTGGGAGTCGTAGCACCAGGCACCTATA GAGACAGTGGGCAGGAGGCTGAGCTGAGAGACCAGTCATCTGGGTCCTCTCCAAGCCCCA TCCCATCAACTGGGGTCCAAGAATGAAGTAGGCCAGGGGCTTAGGCCAGGGGAGGCCAGT GAAGAACCTTCTCTCATGTCACCCCTACCCCAGTATTTTCTCTCCCCAGTCCCTCCCCAC TCCCAGTGGCAAGACCTAAGGGGTGGTGGAGCATGGCAAGAGGGTAGATTGGAGACCAGA CGGGAAGAGTCCTGGTGCTCACTCTCAGAGTCTGCACCACACCAGCTGCACCGCAAGGCG AGCATGATGCCAGGGGCGTGCGTGGCTCAGTGGCAGCCGCAGCTAGGAAGGACTAGAAAG TGGCTAGACCGCGGAGCCAAGATGCTGGCCTGGCTACTACATATTTATTCATTAGCTAGC TGGGCTAGGGGCGTGGCTATTGAGGAGAGGATGGGAGGGGTCGGTGCCCAGGCAGGGCCA CCAGGAAGAGCGGGAGCGCTCCAAACTCACCCAGGTGTGCTCTGGTCCCAGGGCTGCACT GATGCCAGCAACAATCGCTGAGGTTAATCCTGCAGGGGAGGGGTGACCTATTATTATCCC CACTTACAGTCCAGGAAACAGAGGCTGGGGACCAGAATGACACACCAAAGCCACCAGCAA GAAAGGTCAAATAGTGAGGGCACCTTGAACCCCAGCGCCCATGACTTTCGGGGCAAGGAT GGGAGGAAGGTGTAGGATGTCTCTTGCCCCGCTGTGGGCCTGGAACTGCCACCCCATCCC AAGACCATAGCCTCCCTCCTGCCCAGGCCCCCCTGCGGATCCAGGCCAAGACGGAGCCCT GCAGGAGGTCAGAGGAGGGGACTTCTTTGCACCCTGCGCCTCTTTCCAATCGCCAGAGGG CAGGGCCCTACCCACATTGGGGATGGGAAGTCAAATGCAGAAGTTAGGTGAAGTCAGTTC TTGGATACTCCTGTACTGTCACCCTGGTCTTATCTCATGCCTTAGCCCAAGCTGTGCACA CAATGGGGTCCTAGGTCCCCCTCACCTCCCAGATTCCACCTTCCCAGGGATGGGACCCCC TGGAACCCTCGGGGGCCTGGGCAGTGGCCTTGCTGGCTCTTGCCTTCCTAGGAGCTGAGC AGGAGCTCCACTCTCAGCAGGGCAGTTCACTGCAGCCTCTGCTTCCTCAGCTCAAGCCAT CCTCCCACCTCAGCCTCCTGAGTAGCTGGGAATGCAGACACACACCACCACGCCTGGCAA ATTTTTGTATTTTTAGTAGAAACAGGGTTTCACCATGTTGCCCAGGCTGGTCTCGAATGC CTGAGCTCAAGCGATCTACCCACCTCGGGCTCCCAAAGTGTGGGAGCCAAGATGGGAACC CAAGCATACGGCCCCAATGCTGAGGCTCTGAACTACTGACCTGCCCTCAGCACTCAGCCT TGGGATCATGAGTCACTGTGCAAGGGAGTTCCAACATCTGCATGTATGTCTGGAATGATC TGAGCCTGCAGAGTTCCTACACACTGGCCACATTATAGGGTGGTGTCCGTGGTCACACAG CTCAGGGCAGGTATTTATTAGTACATGAATAGCTTAGCTGTGTCATAGTCTTTATGTGAA AGGCACAAAAAAAGGCACTTTGGCAGGCTGAAAGTGTGGGGATTATAGGTGTCAGCCACC GTGCCTAGCCCACTGGATGACTTATGATATCATATGTGACATTGTGACATCATGTGAGTC AGGGATGTACCCCGTTCTCAGCTGCTATATGCTATGTTACACTGACAGAATGGGAATGAA GAATGTGTCCCACTCTCTCAGCTGTTGTATTATATCATACAAGGTGCAGTGACTAAGTGT GTCAGCTGTGTCCTCATCCTACATAGCATATGAGAGTGTGTGACGGGAGATAGGATGCAG ACCTGAGAAGCATTAAACACCTAGGCAATAAAGGTGCCAGCATCAGCTGAGAGCGCAGGT AGACCTCAGTCACATTTGTTACTGTGTAACTAAAAATACAAAATTAGCTGGGTGAGGTGG TGCACGCCTGTGGTCCCCACTGCTCAGGCACCTGAGGCAGGAGAATTGCTTGAACCCAGG AGGCAGAGGTTGCAGTGAGCTGAGATTGCACCACTGCACTCCAGCCTTGGGGACAGAGTG AGACTGTATCTCAAAAAAATAAAATAAAATAATCTAGGACAACCAAGAGAAGGACTCAGG CTCACCTTACTCCATGTCACATGTGATATATAACACTTGAGGGGGACACACACTTGTCAC TTTGTATCACTATGTCCTATATGATAGCCAATGATAACCCAAGAGGGGGAGGCAGCCTTG TTCACACCATGTCACTTATGATATTATATGACATCAAGGAGCCCAGACCTCAGTTGCATT ATGTCACTATGGCACATACAGTATCATGCAACAACTGACAGAAGGGTTGCAGGCCTGATT CACAGTGTATGTGAGTCACAAATGTCACTTATGACACCATACAGCAGGTGAGAGGGGATA GTCATTACTCACCTCGGGTCACATTGTCACACATGATACCATATGATAGCAGAGGGGGAT GCAGAACTGAGTTGTATTATGTCGCTATGTAACCTGTGATAGATGATAGCTGATGCGGGT GAAGACCAAAGTTACATTTTGTACCTACATTATATGAAATATACAACATGCACGAGGGAG ATGCAGACACAAATCACACTGTGTGCTGTATCCCATATATGTGTTTAATTTCTGTGTTTA AGAGAGCTGGGTTCATGAATGAACTTAGTCACTGCACCTTGTATGATATAATACAACAGC TGAGAGAGTGGGACACATTCCTCATTCCCATTCTGTCAGTGATTGACCACACCTTGTGTT AACTGTATGAGCCACTGTGTTTGGCCGTGTATAGCGCACGTGGCACATTGCTCACTGATA CATATTTGCTCCGTACTGAAGTGAGGCAGATGCATGAGTGATTGAGAATAATGCTGCCTT GGTGAAGCATTCCATTTAGAAGCATGAGGTTCAGGTACTACCATCTTCTGTGATACATAT TTAAATGTTAAGTTTAACAGTTAGGTCTACCATGAGTGAATGAGCCTTGCATTATCTTTG AGAATCGATGTGTTTAGCAGGGTGTGGCTCATCTAAGATAGCCTTCTTTAATGCATGTTC ACTATCTGTGTTTATTAGAAATAGGTTTGTGAATGACTTAGCATAGTGTTTTCCCTATGA AACCCTGTTTATTTATTTATTTATTTATTTTGAGATGGAGTTTTGCTCTTGCAACCCAGG CTGGAGTGCAATGGTGTGGTCTCGGATCACTGCAACCTCTGCCTCCCAGGTTCAAGCGAT TCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACGGGCGTCTGCCACCACACCCAGCTA ATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAGCTC CTGACCTCAGGTGATCCTCCTGCCTCAGCCTCCCAAAGTGCTGGAATTACAGGCGTGAGC CACCACACCCGGCCAAAACCCTGTTTTAAGAAGTGTGTGGTGCAGGTACTACATTCTTTG TTGTTCCAAGTTCAAACCCTGTGTTTAACAGAGATAGGTGCAAGATTGCCTAGGTGTAGC GTAGACTCTTGGAAATACTGTGGTTAGCATTCTGTGTTTTCTGTAGTATAGTTTCAGTGA TGCCTGTTTAGATAGTTTATCAGATCTAGGTACTAGTGTTATGTCAGCGAAACACTGTTT AGCTGCACGCGGTGCATGTAATACTTTGTTCATTGATATTTGTTTAAATCAATCAGTGTT TACCAGAACTAGATATGTGAGTTACTGAGAATAGCCTAATCTCTGAAAATGTTTTCCAGC CTATGGTGCATGTAATATACTCTTCTATGGTACCTCTTTCCGATGTTTTTATTGTGGCAA AATATACATAACAGATTATACCATTTTTAAGTGTAGAATTTAGTGGTAGTAAGTATAGAG AGAACCTTAAAAGCAGCAAGAGAAAAGTGACTTCTCATGTGCAAGGGAGCCTCTAGAAGA TTATCAGTGGATATTTCAGCAGAAACCCTGCAGGCCAGAAGGTGGTGGGATGATACATTG AAAGTACTGAAAGAGTAAAGCCTGCCAACTGAGAATACTATATTTGACAAAACTGTCCTT CAAAAGTGAAGGAGAAATTAAGACATTCCCAGATAAATAAAAGCTGAAGGAGTTTATTAC CACTAGACCTGACCAATAAGAAATAATAAAGGGGCCGGGCACAGTGGCTCATGTCTGTAA TCCCAGCACTTTCGGGAGCCGAGGCAGGTGGATCACCTGAGGTCAGGTGTTCAAGACCAG CTTGACCAACATGGCGAAACCCCATCTCTAATAAAAATACAAAAATTAGCTGGGTGTGGT GGCACGCACCTGTAATCCTAGCTACTCAGGAGGCTGAGGCAGGAGAATCACTTGTACCCG GGAGGTAGAGGTTGCAGTGAGCTGAGATCACACCACTGCACTCCAGTCTGGGCAACGGAG CGAGACTTCATCTCAAAAAAAAAAAAAAGAAACAATAAAGGGAGTCCTTCAAGTTGATAT GAAGGGATACTAGACAGTTGCTCAGCCTGATGAAAATATAAAGGTAAATACATAAAGATT AAAACCTGTATTATTGTAATTTTTGCTAATAACTCAATTTTTAATACTTTAATGGAATTT AAAGGACAAAAGCATAATAATTATAAATCTATGTTAATGGATACAAAGTATATAAAGTTT AATTTGTGACATCAGTAACTTAATAGTGGGGCAAAGATGTAAAGAGTAGAGGTTTTGTAT GTGATTGAAGTTATCTGTTTAAAATAATAAGATAACTTTAAGATGTTCCATGTAATTCCC ATAGTAACCACAAAGAAAATACCTACAGAATATATGCAAAAGGAAATGAGAAGGAAATCA GAGCATAGCACTACAAAAAATCAACTATAACACAAAGGAAGACAATAAGGGAGGAAAGAA GGACAAAAAGGCTCTAAACATACAGAAAACAATGACCAAAATGGTGGTAGTAAGTCCTTC CTAATCAATAATTTCATTAAATGTGCATAGGTTAAACTCCCAATAAAAAGACATAAATTG GCTGATTGGATTTAAAAAAACGGGATTCAACTATATATGCTGTCTACAAGAGACTCATTT GAAATCCAATACAAATGGGTTGAAAAATGAGAGAATGGAGAATATTCCGTGCAAAAAGTA ACCAAAACAGATCAGGAATGGCTGTACAAGTTATGGCTGTACAAGTTGTTGGGGTTTATG TTACTGAAGAATGAACAGAGATGAGTAAGTGGAGGTGTTATGTAAAGGCATACTGTACTC AAAATCTGAAGACCTGCAGCAGATTTAAATTCCAGCTCTTATTATAACTTTTTAAAAGAT TGTGAAAATATCAAAATATAGATGAATCAAGTTTTAATATACTGTATGATGGGTGGATGA GGCTGTCCATTGTACCATTTGTTTGAATTCTCAGGCATGGTTTGGCAGTGCAAGAACTCT GTAACGTTAACAAATTCAATAAAAAGTAAATATATGGAAAAAAAAGTATAGACAAAATAG ATTTTAAGTAAAAAACTATTACAAGAGAGGGTTCTGGGAAGAAAGTGGAGTAGGAAGCAC TGGGAATTCATCTCCCCACCTAGAAAATAATCACACTGGCAGAATCTGCCTGATATAACT ATTTTGGAACTCTAGACTCTATCAAAGGAGGCTTGTAACCTCCAAATGAAGGCTTAAACT ATAATTTTTACTTAATTTTGGTCAATTTCAGCTCTTAGCTCAGCAGTGGCTACCCAATCC CCATGCCCCAGCTTCACGGCAAGAAGCTTTGCATGTGTTCCTGAAGCAGCTTGTACCAAG CTTGTGGGAACAATCATGGGCAATAAGCACTCTGTCCTCCAAGTGTTAGCATCTGCGTTC TGGTTGTTGATTGCTACTTTTGATTATGGAAGGGCAAACACAGAGGCCGGCAGCCATTAT TGCTCACAACTCCCCACTCCACTGCTGCAAGCCCTTACCAGACTGAAGCAACTTCTAGGA GATAGAAAAGGCCAGAACCCCATTTCCCTTCCCCTTCATTGTTCTCTTTTCCTTTTTTGG GAGCCAAACATTAAAGACTAGGACACTCAAAAGCAATGGCATACCCAGAGGAAATTAAAG TTACCACACATCCTTGGAGAGAGGAGTGTGTGCCCAGGGAAAGGAGCAGCTTCAGACCTG AGAAGACCTCAAGCTTACAACTCAGGTTGATCCTCAGCATGGAGACAACCTACAACAATG TAAAACATAACAAAACCCTAAAACAGCAAACCCTGAGGAAGAGGAGAGTCTCATCTCCAG AGTTACTGCATTATTATATTCAAGTGTCCAGTTTTCAATACAAAACACAAGGCATACAAA AAACAAGAAAATATGGTATTTCAAAGGAAAAACAACAACAGAAACTGTTCCAGAGAAAGA CCAGATGGTAACCTACTACACAAATACTTTAAAACAACTTTCTTAAAGATGGTCAAAGAA CTGAAGGAAGATGTGGAGAAAGTCAGTAAAATTATGTGTAAACAAAATGGGAATATCAAT AAAGAGATAGAAAACCTAAAAAGAAAAAAAATAAATAAATTCTGGAACTAAAAGTGTAGT GATTGAAATAAAAGTTCACCAGAGGGATTCAAAAGCAGATTTGAGCAACAGAAGAAAGAT TCAGTGAATTTGAAGATGGGACACTTGAAATGATCAAGTCAGAGGAACAGAAATAAAGAC GATTGTTGAATAGCAAGCAGACATTGTGGAAGTCCCAAAAGAAGAGAGGGAAAGGGGCAG AGAGATCATTTGAAGAAATAATGGCTGAGGCTGAACACGGTGACTCACACCTGTAATCCC AGCACTTTGGGAGGCCAAGGTGGGTGGATCACAAGGTTAGGAGTTCGAGACCAGCCTGGC CAATATGGTGAAACCCCGTCTCTACTAAAAACACAAAATTAGCCAGGTGTGGTGGTGCAT GCCTGTAATCCCAGCTACTTGGGAGGATGAGGCAAGAGAATCGCTTGAACCCAGGAGGCA GATGTTGCAGTGAGCCGAGATTGTGCCATTGCCCTCCAGCCTGGGCAACAAGAGTGAAAC TCCGTTTAAAAAAAAAAAAATTAGCTGGGCGTGGTGGCACGTACCTGTAGTCCCAGCTAC TCAGGAGGCTGAGGCAGAAGAATCGCTTGAATCCAGGAGGCAGAGGTTGCAGTGAGCCAA GATCACACCACTGTACTCTAGCCTGGGCAACAGAGCGAGACTCTGTCTCAAAAAAGAAAA AAAAAAAGCTGAACACTTCCCAAATTTGATGAAAGACATGAAAATAAATATCCAGAAAAC TCAATGGACTCCAAGTAGGATGAAAAAAAAAAGACTCATACTGAGACATTATAATTAGCC AGTAGGGCCTCTTGAAAGCACCAAGAGAGAAGCAACTAGTCACATGCTAGGAATATATAA TAGGATTATAAGTAGATTTCTCATCAGACACTTTGGAGAACAGAAGACAATGGGATGACA TATGTAAAGGGCTAAAAGAAAAACAACCACTACCTCTCAACCAAGAATCCTATATCCAGC AAAACTGTCCTTCAAAAGTGAGGAAGAAATTGGGAAATCCCCAAATAAACCAAAGTTGAG AAGTTTGCTACCATTAGACCTGCCCTGCAAGAAATCTTAAAGAGAATCATGCAGGTTGAA AAGAAAGAACACTAGATAGTAACTCAAAGCCATATGAAGAAATAAAGATGCCAGTAAAAG TAAATATATGGGAAAATATTAAATCTAGTATTATCGTAACTTTGGTTTAAAACTCCATGT TTTGCTTTCTACATAATTTAATAGACAAATGCATTAAAAACAATTATTAGTTTATGTTTA TGGACACACAATGTACAAAAATGTAATTTTGTGACATTGATAACTGAAAGAGGAGTGGCA AAACTGTGAGGAGAATTTTTGCATATTATTGAAATATAGCTGGTATGAATTCAAGTTAGA GTGCTATAACTTTAGAATGTTAAGTGTAATCCCTATGGTAACCACAAATAAAACATTATA TAACATAAAAAAGTAAATGAGAAGGGAATTAAAACACTTCGCTACAAAAAATCAACTAAA TACAAATGAGATCATGCAGGAAATGGACAAAAATGCTGTAAGGCATATAGAAAATGTATA GCAAAATGCCAGAAGTAAGTCCCCCCTTATCAGTAATTACTTTATTACTTTTTAAACCAT TTTGTTGAGGAATGATTTACATAAAAACTGTACATATTTAACGTACACATCTTGATAAAT TTACACCATAAAACCATTATCATCAAGCCTATAAACATATCCATCACCTTTTAAAATTTC CTTCTGCCTCTTTATTATTATTATTGTATAAAAAAAATGTTTTTAATGGCCAGGTGCGGT GGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCAGGCGGATCACCTGAGATCA GGAGTTGGAGAGCAGCCTGGCTAAGATGGCAAAACCCCATCTCTACTATAAATACAAAAA TTAGCTGGGCGTGGTGGCGGGTACCTATAATCTGAGCAAAGTACTGGGAGGCTGAGGTGG GAGAATCTCTTGAACCTGGGAGGTGGAGGTTGCAGTGAGCCGAGACAGCACCATTGCACT CCAGCCTTAGCAACAAGAGTGAAAATCTGTCTAAAAAAAAATAATAATAATAAAAGTTTT TAATTAAACAATTTAAGAATATAGTAACATTTTCTGTGGGTACTATGCTGTATAGCTCTC CAGAACTTACTTATCTTGCATAACTGAAATTTGTACACTTTAACCATCAACTTCCCATTT CCTTCTCTTCCCCAGCTCCCAGCAACCACCATTCTGTTCTCTTCTTCTGAGTTTGACGTC TTTAGGTTCCACACATAAGTGAGATTGTACAACATTTCACTTTCTGTGTCTGGCTTATTT TACTTAACATAATGCCCTCCAGTCCATCTATGCATAGAAATGCAACTGATTTTTGGATGT TGACTCTGTATCTTGCTACTTTATTGAATTTATTACTTCTAACAGTCTTTTAGTGAAGTC TTTACAGTTTTCTATACATAAAAATATGTCATCTATGGAGACCATTTTACTTCCATTCTT ATTTCTTTACTTGCTTAATTGTTCTGGCTAGGACTTCCAGTCCTATTTTGAGGAGAAATG GTGAGAGTAGGAATTCTTGTCTTGTTCTTCATCTTCGAGGAAAAACGTTCAGTCTTTCAC TGTTGAGTATGTTACATGTGGTCTTCATTATGTTGAGGTACATTCCTTCTGCACCTAATT TGCTCAGTTTGTTGTTTTTTTTAATCATGAAAGGATGTTGAATTTTATCAAGTGCTTTTT AATAATAAAAATAAAGGATTTTTATCGTTTATTGTGTTGATGTGGTGTATCACATTTAGT GACTCTTGTATGTTAAAGCATCCTTGCATGCCAGAGATAAATCCCACTTGATCCTGGTGA ATAATTCTTTTTTTGTTTTTTATTTATTTATTATTTATTTATTTTTTTGAGATGGAGTCT CGCTCTGTCATCCAGGCCTGAGTGCAGTGGCGCGATCTCGGCTCACTACAAGCTCCGCCT CCCAAGTTCACGCCATTCTCCTGCCTCAGCCTCCCTAGTAGCTGGGACCACAGGCACCCG CCAGCATGCTCAGCTAATTTTTTTTTTTTTTGGATTTTTGGTAGAGACTGGATTCATGGT GAATAATTCTTTTAATGCACTGTTGAATTTGGTTTACTAGTATTTCATTGAGGATTTTTG CATCTATGTTAACCAGGGATATTGACCTATAGTTTTCTTATTTTGTAGTGTTCTTATCTG GCATTGATATCAGGATAATGCTGGCCTCTTAGAATGAGTTTCAACGTGTTCCCTTTTCTT CAATTTTTTTGGAAGAGTTTGAGAAGGATTGTTATTAATTCTTTAAATGTTTGTTGAAAT TGACCAGTAAAACCATTTGGTCCTGGGATTTTCTTTGTTGGGAGATTTTTCATTACTGGT TTAATCTCTACTTATTTTTTCCTGTTTTTTTTTTTTTTTTAATTATACTTTAAGTTCTGG GGTAGCCGTGCAGAAAGTGCAGGTTTATTACTTAGGTATACATGTGCCATGGTGGTTTGC TGCACCCATCAACCCATCATTTACATTAGGTACTTCTCCTAATACTATCTCTCCCCTTGC CCCCATCCCCCAACAGGCCTCAGTGTGTGATATTCCCTGCCCTGTGTCCAAGTGTTCTCA TTGTTCAACTCCCACTTATGAGTGAGAACATGCGGAGTTTGGTTTTCCGTTCCTGTAGTT TTCAGATGTGCTCTGGCCTCCAAGGACCATGAAGCCAGGCGGTGGTGGGGGGGTGTCCTC TGTATAAAAAGTGCTGTCCCAGGAACTTCCTGACGGACACTTTGGGGCATGTGAGCGATT CCTGGGGAGGGCACCTCGGCCTTCCTAAGGCTACCCCTGCAGCCAGCGCTGGCCATTCTC ACCAGCAACCAACCAAAAAACATCAGAGTCCCTGTAAACCTGGTTGTGATGATAAAAACC AAATGTTTTCTAATCTAAGACTTGTATGCAGAACACAGAAAACTGCAGTTAAGGAACACC CTACAAAATTGACCAACAGTATTTTCCAAAAACATTTTTCATCACTTTAAATAATGTACA AAATCTACAAAAATCATATTTACCAGGACACATCTGTTAAATAAAAGCATTGTTTCGTGT TGGTATACATATATACAAGACTATGTATAACAGACTGTTTCCCCTCCCTGCAACCACAGA ACCATCACACACAGGCACAGATACACGTCAGCTATGCCGCTTTCCACGAATGCATGGAAC CCAGGACGCGAACCCACAGCTCGAGGTCTTATACCTTCACTACTGAGCTGCCGCCACTGC AGCAGCAACACCTCTGCGGAGGTGTTGCTGAAATCACTGGTGTCCCTGCCCAAGGTGTCC TGGTCCTGGTCAACTCTACTGATTGACCCTTCGTGGATACCTCAGGTCTAAAATCCTTTC CTCCGAGCCAGAGCTCTTCCTGTTGTGCAAACTCAGCCCCGTCTGTACCTTCCTGCTTGG CCGGTGATCGCAGTCCTTCTTCGCCAGCAAGTGTGGGCTTCCAAAGACAAGGCTGGGCCT GGCCTGGGACTCCCTGAAGGCCGAGAAGGACAGGCCCTGCAGAGGCAGCCCCAGGTAGGG GCTGAAGAGGCCGGTCTCCCTGCCCCCCCAGGAGACACCCTTTCCAAAGTGGAAGAGCTG GGTGGACAGCAGCGGGGAGAAGCCCGCAAGGGGCAGGCTGGGCAGGCCCCGGGCGGGGCC CTCGGACCCCTTGTCTCTCTCTCCCAATGCCGCCCCCTCCACGCCGCATCTCTACCTTTG GAGCGCAGTGCCCATGGGCTGGGCAGCCGACTGTGGTGGGCAAAGTCACTCCAGGGCGGG GCGCGGTTGGCCTGGGCTCCAGCATCCCTCTCAGCTCCCGGGCTGGGGGTCAGGAAGCTC CGGTTCCTGCAGTCCACGTGAAAGGTCCCCCTGGCTCCTCCTGCCTCGGTGCCCGCTGTG GCACAGCTGGGCCGCTGACCAAAAGCACTTTTGGCAGCAGGCTCAGCCCCTCGTGGGCTC TGGTCTTTGTAAACGTAAACACTTCTCTTGCTCACTGAGAGACCTTGGCGGAGACACTGC CGCTGGTCCTCCTGGGTGGCGCATGATCCCCTCCGCCGGATCAGTGGGGAGCCCCTTCCC ACCCTGGCTGAGCAGGCACGCTGCGCCCTGTGCTCCTGCGCTCCCTGCGCCTCGATGCCC TTTAGCCAAATGTGGGACCCCCCGGCCCTCTGGCTCCGTGTGCACATGCCAGGCAGTGGG GCCGGCTCCTTCCTGCAAGGTACCTCTGGCCTGGCTGGGCCCCCTGTCCCGAGAGCGGTG GGGCCCTCTGCCTGAACTTCTGAACTGCTCACCGACTCCTTGGCCTTTTCCACCAAAAAC TTCCTAATCTCCAGTTCGATGCTATCGTCGCTGTCCACTGAACTGCTGTTGTCAGACAAG GAGCCAGGGCTGGGAGCTGGGCCCTGGGACTCTCTGGGGAATAGATTCTCTTCGGAGGCG GAGGCAGAAGCGGGTCTCCTCACCAGGAAGGCCGGGGCTTCGTCCTCTCGGCCCTGCTGC CAAAGACACTGGGGGGTTTCTTCCTGGAGCCCTCGCAGCTGTCTCTCTTGGACTTAGGCA GCTCTTCAGCACCTGCAGGTGCCTGTTTTTCCACTCTCTCAGGAGCCTGCCCAGCCGCTC CTGGAAGTGCGTCTGGGAGGTGCTGAACCTGACCTTCTTCCTGCACACAGCCCTGGGCTC CCTGGACCTCTTCTTGAGCTTTCACTTGGACCTTAACAAGTCCTTGATGGCTGTGTCCAG GTCCTTGTCACTGTCCAGGGAACTGCTTTTGTCTTCGGAGCTCTTCTTCTTGTCTAGGTG CCTTGCCTCGTCTGTCTTACCCTGGCCCTGTGACGTGCGAGTGTCACCGGGCACCCTAGC GGCGCCCTCTCCTCCCGGGGCCTCGCTGGCTGTGCCCTGGATGGAAAGGTCCCACCCTAG CGGCGCCCTCCCATCCCGGGGCCTCGCTGGCTGTGCCCTGGATGGAAAGGTCCCGCCCCT CATGGCCGGGCCCGGCTCTCCCCTGGCTGCGGTCGGCATCCTGGCCACCCTCTTTCCCCA CCACCCGCATGTTCTTGGGAGTGGATGGCCTCACTTGGCAGCCGCCTCTATGCTTCCTTT TGCAGCCAGGAGTGGGTCCAGTGTTTTAGAGAGAGGGGCCTTGGGGCTGCCGGTCTGGCT GCTGAGGCCAGGTGGTGAAAGTGGGCCCTGGGCAGCCTGAGGGCAGCTCTCACCTCTGGC CAGCAAACTTCTAGACTGCACCTTGAGGGCCAAAAACGTCCAGATTTCCTGCTCAATGCT GTCGTCGCTGTCCACGGAGCTACTGTCACCATCAGAGCGGGAAGGCACGTTGGGGGAGTA GAAGAGTGGGCTTGCGGACAGGGACCCATCGCTGCCCTCCATAGGGCCGGCAGGATCGTC TTGAAAATGTCCAGGACTGCTTCTACACACATCAGCTCAGCGGAGGTGTCTGCCTGGCAA GAGGACCATTCCACAAACTTGCTCCTGGAAGCCGGGCTCGTTGGAGGTGGAGCTTTGGTT TCCTTTGGGATCTTGGGGGAATGGTCAGCGTCCAGATCCCCTGGACCAGGGTCCGTGGTC TTGGTGGGCACTGGCTTCTTCTTGCTGGGTGTTTTCCTGTGGGTCTCTGGCAAGGCACTT TTTGTGGCGCTGCTTGTGCTGTGTGCGGGAGGGGCAGGTGCTCTTTCCTCTTGGAGCTGG ACCCTCTGGGGCGGGTCCCCGTCGGCCTCCTTGTGTGTTTTCTGCACCTGGTACAGCTGG ATGGCCTCCTCAATGCCGTCGTCGCTGCTGGAGTCGGACGCCTCGGGAGCCTGTACGGCG CTCGTGACTCGCTTTCCCCTCCTTGCGGTGCTGGCGCTCCTTTTAATCCCACTTTTATTC TGTACTGCTTCTGAAGGGCGGTGGGGGTTGCTGGCTTTGTGCTGCCCTCCTTCTCCTGCG TGGTCGTGGTCGTGACCTTGGACCTGAGGCTTCTGGGCTGCACGTTTGTCTTTGCTAACC GGGGGAGGTCTGCAGAAGGCGAACTCCTTCTGGACGCCCATCAGGCCCTGCCGGTGCACC ACCTTTGTAGCCGGCTCTTGGTGGGATTTCGAGAGTGACTTCGCCGAATTTTCATGTGTG TCTGGTTTCTTCTCCACTGACCCATCACATTTTTGGGTCTCATGCTGTCTTTTCTCATTC AGAAACTGTTCTATTTCTGCCCTGATGCTCTGCTCAAAGGAGTCTGCTCTGCTCATGCTG ACTGGGGAGGCAGAGCCCTGGTCCTTGCTGGATCCCACCTGGCTGCCAGGGCCACACCAC CTGAGCCAGGTACAAGTTTTGGGGAACACAGGGCAGTTGGGCACTGCTGTGAGCCAGTTC CCGCTTACATCTACTGCCTCCGCCCGCAGCCCTGGAAGGCTGTGCATGGCTGGGCCCCGC TGGCCCCGGGCTGTGCCGCTCCACTCTTTACCTTCAGGTACTCCTGGATGGCCTCCTCAA TGTCCCGGTCCACGGAATCGTCACTGTCTGAATCTAGCACCAATGGGCCAAAGTCTGCAG TTTCCTCCTCCCCCACGGGGTCAAAGTCAGCAACAAGACCACAGGCAGCCAACGCAGGCA GCTCCTTGTGCATGGTGGGCTTGGCAGCAGGCCTGGCGTCGTGGCATCCCTCTGCCCCCT CTGCGCAGTGCGCTCATCGCTGGTGCCCCTAGCAGCCCTGTCGCTCTGCAGCGTGCTGAT GAGCATCTGCACCCGGGTGCTCACCGACATGCTCTCCACGCCCTTGTCAGCCTCCGAGAA GCACCCGGGGAACCTAAAGCTCCCTGGCGGGACAGAGGCCTCCCATTTGGGCTGGAGAGC AACCACTGGAGGAGCATTCATGAGAAACATTCTGGCAGATGGGGAGCGACGCGCAGAGGG GCGACACTTTATTTCTCTGCAGGCTTCACATCCTCCAAAGATTGGCAAGCAGTACCCGTG AAATAACTTTAAACCTGCAAATGCTTCTTTGCAGGTTTAAAAGGATGACTATAAACTATG ACGTCATGCCTAGATTCATTCTTGACCCAACCAACAAGCTCTTGACATTCTCTGAGTCCA GGTTGACTGTGATGAAAGGCAGCTAGTGTTCCCAAATGGCCCAGGGATCAGGTCTTCATC GCTCCACTCAGAGGGAAGCATCCTCTCTCTGCTTTTTAAATAGACTTTTGACTGGGGCTC CAGCAGCGCGGGGCGCGCAGACCTGGAGTTGCATGGAGGCCAGAGCCACGACACCCGCCT GGGGAACGGAGCAGCCCCAGGCGCTGATCCCCGTCCACCTGCCCCACGGAGCCCTCGCCG CCCGCTTGCCACTGCCTGCATGGCCCTCCTGTCCCCGGCCCCCCAGCCCTCCTTTCCCCA GCTCCCCCACCCTCCTGTCCCCGGCACCCCAGCTTCCCAGCCCCCGAAACCGCCCCCCCA CCTCGACCCGGCCCATGCCGCAAGTCGCCCGCTGCGCGGACCCGGCCTCCGCCCGCCTCC TGCGTCCTGGGGGAGGCGGCTGCCGGGGGTGGTGGGGGAGGGGGAGGGGGAAGAGGCCGC CCTCCGCCCGGGTGCGGGGAGGGGGCGCAGGGGTGTCCGGCCAGGCCCCCCGCCTCCCCG CCTCCCCGCAGCAGCTGCCCCGCTCCCGGGCCGCCTAATACTTTTACATTTTAACTTTTA TACTACAGTGAAAAGTGATTTACACACCACCACTGCAATATTACAGTGTTATGAATGTGA CTATATACTTACCTTTCCCTGTGAACTTTTTTTTTTGAGACAGAGTCTCGCTCTGTCGCC CAGGCTGGAGGGCAGTGTCCATGATCTCGGCTCACTGCAAGCTCTGCCTCCCGGGTTCAA GTCATTCTCCTGCCTCCGCCTCCCGAGTAGCTGGGACTACAGGCACCCGCCACCACGCCT GGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGCATGATCCC CCTCTTCTGACCTTGTGATCCACCCGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGT GAGCCACTGCGCCCGGCCTACCTGTGAACTTAATATCTTAATGTTTTAATGTTGCTAATC AGAATCCTTTTATTTCAACTTGAAAAACTGCCTTATAAGGCAGGTGCAGTGGTGATGAAC TCCCTTAGAATTTTTTTGGTGGGAGTCTGGGAAAGACCTTATCATCTCTTTTTCATTTCT GAAGGACAGCTTTACAAGTTGTGGTCTTCCTGATTGGCAGTTTTTTTCTTCCAATACATT GAATATAGCATCCTATTCTCTCCTGGCTTATAAGGTTTCTGCTGAGAAATCCACTGATAG CCTTATTGAAGTTTCCTTGTATGTGATGAATTCCTTTCTTCTTGCTGCTTTTGAAAGTCT CTGTCTTTGACTTTTGATATTTTAATTATAATACATCTTGGTATTATGGTCTTTGGGCTG GCCTTTTTTGGGGCCTCTGAACTTCATGTGTCTGGAAGCCCACTTGCCTCTAAGAATTTG GAAAGTTTTTACCCATTATGTCTTCAAATATACTTTCAGGCCTTTTCTATCTTTTTTACT TCTAGGAAGTCCATAATATGTTTGACCCACTTCATGGTGGTGTCCTATAAATCCCAAAGG TTTTTACTTATAAACTTTTTTTTCTTTCTGGTCTTCTGACGGGATATTTCAAATGTCCTG TCTTTAATTTCACAGATTCTTTCTTCTGTTTGATCAAGTCTGCAATTGAAATTCTCTATT GCATTTTCATTTCATTCATTTATTTATTTTTATATATTTTTGAGACAGAGTCTGTGTCAC CCAGGCTTGAATGCAGTGGTGCCATCTTGGCTTACTCCAACTTCCACCTCCCGGTTCAAG CGATTCTCCTGCCTCAGCCTCCCTAGTAGCTAGGATTACAGGCATATGCCACCATGCCTG GCTAATTTTTGTATTTTTAATACAGATGGGGTTTTGGCATGTTGGCCAGGCTGGTCTTGA ACTCTTGACCTCAAGTGATCCGCCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGT CCGCCACGGCACCCAGCTTGCATTTTCATTTTATTCATTGTATTCTTCAGTTCTAGAATT TCTGTTTGGTTCTTATTATTTCTGTATCTTTATTGAACTTTTAGCTTTGCTCATCTACTA TTTTCTTGATATTATTGAGTTGATATATACATTCTAGTAAATTTCACTGAGCTATCTTAA TTATTTTGAATTGTCAGGCAATTTGTAGATCTCTATTTTTGGGGGGTTGATTACTGGAGA TTTATGAGTTTATTTTGGTAGTGTCATATTTGCTGATTCTTCATGATCTACAGACTTTCA TTAATGTCTATGAAGAAGCAAATACCTCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTGA GACAGAGTCTTGCTCTGTCACCCAGCTGGAGTGCAGTGGTGTGATCTCAGCTCACTGTAA CCTCCACCTCCCAGGTTCAAATGATTCTCCTGCCTCAGCCTCCCAAGCAGCTGGGATCAC AGGCATGTGCCACCACGCCTGGCTAATTTTTTTGTATTTTTTGTAGAGACAGAGTTTCAC CGTGTTGTCCAGGCTGGTCTCAAACTCCTGGCCTCAAGTGGTCTGCCCGCCTTGGCCTCC CAAAGTGCTGGGATTACAGGTGTGAGCCACCATGCCCAATCTCTTTCTGTCTTTATAGAT TGGTTTCAGCAGGTACAAACCTTTTCCTGCTGGATCCCTTGACTGGATCACAGTCAAGTG GGCCTGGAGCCATATTACATGGCTGCTGCCTGGTCTGCAGCTGAATCTCTGATTGGCAGG CCGCTATCAAGGCATAGGTTGGTGATGCAGTTTCTGCTGGATCCTCAGGAGAACTGGACT GCCTCTGATACCCTGATTGAACAGGACTGGAGCCAGGTCATGGGGCCACTTCTAGTTCTA CAGTCAAGTCTTCAGATATCAGGCCTATTACCAAGGGCATGGACTGGTATAGCTCCCTGT GGGTCCCAGATTGAGCTCCTGCTGGTTTACTAGGTAGGTCCATGGGAAGACAGGACTGCC TCCAGACCACAGTAGAGCAGGGCTAGAGCCAAGTCACAGGACAGCTTTGGTGACCACATT TGAGTTCAAGATTGGTGGTCCTCTTATTAGGAGAATGGATGGTATGTCTTTCACCAGGTC CCAGGATGGGCTGGACTGTGCCCAGACTGTGGCAAAGCAAGACTGGAATGGAGTCACAGG GCTACTTTAGTGTCCATAGCTGAGACTGAGATCAGCAGGCCTGTTACCAAGGGTATGTAA AGGCATCACTGAATTCCTGGGCAGGCATGACTGACTGTGGTAGAGTGGGGCTGAAGCCAG GTCAGGGCTGCTTTAGTTTCTGCAGTCAGGACCATGGTTAGAAGGCCTGTTACTGGGGGC ATAAATGGTCATGGTTCCTCCTAGGTGCTTAGTGGATGGGGCTAGTTGCAAGACCATGAT CTAGTGGAGCTGGACCCAAGTCCATAGGAGGACAAAGCTGCTTTCAGTCTGCAACTGGTA ACCTGTCACTGGTGTGTGGACCTGCCTTCTCAAAGCAGCTCTCCTTGGTTTTGGGCTTTG CTAGAGTTTTGCCACCTCCTGCCTGGATATTAAAACTCTTGCAAAGGCAGTTTTGTCCAT GAATGGCTGCCAGATCATTGTTTGTGTGGGGAGAGGTGAGTGGAGGGCCTCCTGTTCTGC CATCTTGCTGATGTCACCCTAAGATGATTATTTGAATTCTTTGTCAGGCAATTTGTAGAT CTTCATGTCTTTGGAGTCAGCCACTGGAGTTTCATTTTGTTTCTTTGGTGGTGTCATATT TTCTCATGCTTCCTGTTCTTTGAAGACTTAGATTGCTTTCTTCATGTTTGAAGAAGGAGT CATCTTTTCCACTCTTTACTAACTTCAGGAGAGAAAGACCATCAATTAGCTAAGCTATAG ATTCTGGGGGTCTCTCAGTCCTTTTCTGTGGGTGGTCCTTCCCTTTTAAGGGGGATGTCT TAGGATTTTGTCCCTTGTCTTCATTTCACAAATGAATAAAACAACCAGACCAGACATAAG TAAGGAAATACAGCACTTGAACAACACCTGAAAAAACAACTAGACCTAACAGACATACAC AGGATATTCTACCCAACAACATAATACACATACTTCTCAAGTATACATGGGACATTTTCA GGATAGACCATATAACACATCACAAATTAATTCTCAATAGGGGCTGGGTGCAGTGGCTCA CATCTGTAATCCCAGAGTAATTTGGGAGGCTGAGGCGGGTGGATTGCTTGAAGCCAGGAG CTTGACATCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATACAAAAATTAG CTGGGCGTGGTGGTGCGTGCCTGTGATCCCAGCTTCTTGGGAGGCTGAAGCGTGAGAATT GCTTAGGAGCCCAGGAGGTTGAAGCTGCAGTGAGCAGAGATTGTACCACTGTACTCCAGC CTGTACTTCATGACAAAGAAAATGTACCATTGTACCACTGACAGAACGAGACCCTGTCCC AAAAAAGGAAAAAAGCTCAGTAGATTTAAAACGATAGACATCATACAAAGTGTCTTCTCT GACCACAACAGGATAAAGTTAGAAATCAATAACAGAAGATTTAAAAAAGTTCACAAATTA GTAGAATTTAAACAACACACTCTCAAACAACCAATGGATCAAAGAAATCACAAAGAAATT ATAAAATTCTTAAAGACAAATGAAAATGAAAGCACACTATATCCAAACTTATGGGCTGTG GCCAGTTGTGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGACTGAGGGAGGTGGAT AGCTAGAGGTCAGGAGTTCAATTTCAGCCAGGCCAACATGGTGAAACCCTGTCTCTACTA AAAACACAAAAATTAGCTGGGAGTGGTGGTACGTGCCTGTAGTCCCAGCTACCCAGGAGG CTGAGGCATGAAAATTTCTTGAACCCAGGAGGCAGAGGTTGCACCACTGAGCTAACACCA CTGCACTCCAGCCTGGGTGACAGAATGAGACTCTGTCTCAAAAAACAAAGAAACAACAAA AAAACACAACTTATGAGTTGTGGTGAAAGGAGTGCTAAGGAGGAAATTTATAGCTATAAA CACATTAAAAAAAGAAACACCTCAATTCAACAACATAAGTTTACACATTAAGAAACTAGA AAAAGAAGAATAAAACTAAACCCAAAGTTAGCAGAAGGAAGGAAACAATAGAGATCAGGG CAGAGATAAATGGAAAAGAGAATAGAAAAACAATAAAAAACAAAACCAAAAGTTGGTTCT TCAAAAAGATTAATAAAACTGACAAGACTAAGGAAAAGGGAAACAATCTAAATTACTTAA AACAGAAATGTATTTGAGAATATCTTTATATATTTCTGTCTGTCTGTCTGTCTGTCTGCC TGTCTATGTTTTAGAGACAAGGTCTAGCTCTATTGCCCTGGCAACAATCAGATGCAACCA GAATCAGTGGCACAATCGGCTCACTGCAGCCTTGAATTCCTGGGCTCGCCACTATGCCAG CTCTTTTTTTTTTTTTTTTTTTTAAGAGACAGGATCTTGCCATGTTATCCAGGCTGATCT TGAACTCCTGGCCTCAAGGAATTTTCCCACCTCGGCCTCCCAAATTGTTGGATTACAGGC ATGACCCACCATTCCCAGCCTAGAAAGGATTATAAAAGATTACTATAAATAATTGTGTGC TCATAAATTGGATAACCCAGATGAAATAGATGAATTCCTAGACACACAAAACCTACCAAG ACTCAATTATAAAGAAACAGAAAGTCAGAATAGACCTAACCTAGTAAGGGAATTGAGTCA GTAATAAGACAATCTCATGACAAAGAAAAGTCCTGGACCTGATAGCTTTACTGGTGAGTT CTGCCAAACGTTTAAAGAAGAACTAACACTGATTCTTTTCAAGCATTTCCAAAGAGTGGA AGAGGAGTGAATACCTCTTAACTCTTTCTATGAGGCCAGCATTACCCTGATACCAAAGCC AAAAACACTGTAAGAAAATAAAACAACAGATCAATATGCCTCTGATCATTGATGCAAAAA CTCTAAAAATGCTAGTATACTGGGTGGGGCACGGTGGCTCATGCCTGTAATCCCAGCACT TTGGGAGGCTGAGGTGGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGACCAAC ATAGAGAAACTCTTGTCTCTACTAAAAATACAAAATTAGCCATGTGTGGTGGTGCATGCC TGTAACCTCAGCTACTCGGGAGGCTGAGACAAGAGAATCGCTTCAACCCGGGAGGTGGAG GTTGTGGTGAGCCGAGATCATGCCATTGTACTTCAGCCTAGGCAACAAGAGCAAAACTTT GTCTAAAAAAAAACTATCATACTGAATTCAGCATCATATTAAAAGGATTATACACCATGA CCAAGTGGGATTTATTCCTGGAATGCAACGATGTTTCAATATATAAAAATTGATCCATAT AAATTCAGACAATTAATTTTAACTGATGTATAGTTTTACATTATATGCACATACCACGAT TCACTTTTCCATTTTCCTAGTAATGAGCTTTAAGATTATTTCCAATTTTTAGCTATTACC AATAATGTTTCAACAAACACCTTTGGACATCTCTTCTGCAAATGTGTGTGTATTCTCTTG GAGAAGGTTTACCCTGTGGAATTTCTAGTGTGTATGTGTATGTGTGTGGGTATATATATA TACACATATGTATATATTTTCAACTTTACTATATATTTCCAAAAAACTCTTCAAAGTGGC AGATTTAAACTCCCCTTCGCCGTGTATAAATTCACGGTTCCTAAAGCCCTTGGCATAGTT TTGTGTTTTTATAGAAAAGGGCATAAAGTCTTATTGCTGTTGAATTGTACCTTGCTCTTT TCAATTGTCTTTTCACAATAATTCACTATTTATCATCTGTTGTGAATATTGTGTAGTAGT TCTTAATCATTTTTTCTACTATATTATTTATTTTATATCATGAATTTGTAGAAATCCTCT TTATATAATGGATATTATACTTTTCAATTATATATTTTGTAAATATTTTACCCAATTTTC TGGCTTGCATTTAACCTTTATTTAGGATGCCTAGGATTTTTCATTTTAATACAGTCTAAT TTATTTTTCTTTTCTTTTCTTCTTCATGCATTCTGGCATTTGATTAATAAGCCATTTGTT GCCCAACGTCATTAGTTCTCTGTATATACTTTTAAATATCTTAAACATGTAATATTCATA AGTAATTTCACAAGTAGTTTAAGATTTGAGTCTTTAACCTAAATAGATACAGGATTTTGT GATATGCTGTAATATACAGATTTTTATTTATATATGGATAAAAATTTATATGTTTTTTCT GACATCATTTATTGAATAGTATAATTTTTCTACTGACTTTTAGTATCATATCTTTTATTA CAAGTCCAATGTATTCAGCATATCTATGGATCTATTTCTTGGTGCTTCCATTTTTTCCCA TTGTTTAACGGTGTATCACTGAGCCAAAGCAAACCATTTTAATTATTATAATTAAAACAC ATTTAAATATTGATAGGCTATTATTGTTCAGAATAACTTTGGTTCTTCCTTTCCATTGAT TATTTTAATATGACTTTTCCAATCAAGTTCTAAAAAAATACTGGTGGTATATTTATTGAA ATAACTTTAATAAAATTAAATGAAAGATCATGTGTTTTCTGAATTAATTCTTAGATACGT TAATGTTTTATGTTACCATGAATGTGATATTATAATATAATATTTTTAATTGGTTGCTAC TGTTTATAAGAATTTCATTTTCTCTTTACTTTGCCTTCATATCTGAAAACCTTGCTGATT TGATTAGTGCATCCACAAATTTTCTTGGATTTTCTATGGGTAATTACAAATCTCCACACA ATGAGGTTGCAGTGAGCCAAGATCACACCACTGTACTCCAGCCTGGGCGACAGAGTGAGA CACCATCTCACAAAAACACATAAACAAACAAACAGAAACTCCACACAATGACAACGTATG TGTTTTCTTTTTTTCTTCCTCTTTCTATAATATTTCTTTGTCCTATCTTAACTGAACTGG CCAGAAACCCCAGGACAATGATAAATACGAGCAGTGTCAACAGACATCTCATTCCCTTTC CTAGCTTTTATAAAAAATAACGATTATGCTTCAACATTACATATGGTGGTGTCGATGGTT TTGTTATAGATAAGCTTATCAGGTTAAGAAATTTGTCTGCTTTTCCTAGTTTGGTATAAA GATTTTAATATAAATGAATGTTGTATTTTACCATCTTATTTTTTTTCCTACATCTGCTAA GGTAATCCTGTGTTTTCCCTTTTTCAATCTCCTAATGTGGTGAATGACATTAAAATACCT TCTATTGTTAAAATATTCTTGCAACGCTGTATAGAACCAATGCCTTTATTCTGTATTGCT GATGGATTTTTGAAAAATATGTAGGTGGACTTAGTTTTCTAAGGGGAATAGAATTTCTAG TATATTTAAACTATTTTGCATGTATGTTCTGAAGGACATTGGTGTGTCATTTCTATACCA TCTGGCTACGAGAGGAGCCGACTGAAAGTCACACTGCCGGAGGAGGGGAGAGGTGCTCTT CCGTTTCTGGTGTCTGTAGCCATCTCCAGTGGTAGCTGCAGTGATAATAATGCTGCGGTG CCGACAGTTCTGGAAGGAGCAACAACAGTGATTTCAGCAGCAGCAGTATTGCGGGATCCC CACGATGGAGCAAGGGAAATAATTCTGGAAGCAATGACAATATCAGCTGTGGCTATAGCA GCTGAGATGTGAGTTCTCACGGTGGCAGCTTCAAGGACAGTAGTGATGGTCCAATGGCGC CCAGACCTAGAAATGCACATTTCCTCAGCACCGGCTCCAGATGCTGAGCTTGGACAGCTG ACGCCTTGGATCATCTGCCACTGATCTCTGGTCAACATTTTTATCACCCAACACAAAAGA AGCAGAGATTTATCAAGTTACTTAAACAACCTGACCCTTTCATCTTTTGCTACACATACT CTTGTAATTGATCTCTCCATGAATTGTTTTCTGTTTAAAATATCTAGAATGTTTTCTGCT TCCTGACTTGATTCTAATATTGTATAGGTACTAGAAATGGTTCTAGAAATAGATCTTTAT AGATGAGAATCTGGAAGTGGTTTGCTGACCTGTTTCAGTCTGAATGAATTCCTGACCTTG TTGTCGGGAGGAGACAGAAACCTGATCATCTGTAGTGTACGGTGGTATCATGATTACTTA AATCATCAAATGTGGTTATTGGGAATGATGTGTTTTTTAAAGTGGTACATGAGAGGTAAA ATTGCTATTGTAGTTGACTGTTGCAGTTATAATTTTGTCAACATGGTCTGTAAGAGTGCA ATGGATTCGGCCCGGTGTGGTGGCTCACGCCTGTAATCCCAATACTTTGGGAGGCCAAGG TGGGCCGATCACGAGGTCAGGAGATCGAGACCATCTTGGCTAACACAGTGAGACCCCATC TCTACTAAAAATACAAAAAATTAGCCATGTGTGGCGGCACACGCCTGTAGTCCCAGCTAC TCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCAGAGCTTGCAGTGAGCCGA GATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGCAAGACTCCATCTCAAAAAAAAAAA AAAAAAAAAAAAGAGTGCAATGGAAAGCTGGTAGAAAATGGACATTGTTTAAAGACCAAA ACAACCACACTACTTTGCTAATCCCTATCAGCTAAAGCCTAGAGAATATATGAGGTATAG ATTTACATGGTGTTTGACTAAGGCATGCAGAATATAATCTTGACATAGGCTAAGTTTATC AACATAGATACATAGAGATTCTGAAGTTAAAGTCTTAGCCCAAGAAGTTAGAAGGGGTGC TCAAGTTTGTTTGTCTGACAGAAACTGTGAATCCACACTGGCCTGCTTATTTTGAGGTTG TGTTGCCAGAGCTTTCCTAGCATAATAAAGAGAGGTGCATACAAAGGAATAGGAATAGTA GGAGGTGGGGGTAAAAATATCAGGTGTGATCCACATGCCAAGCCGACCCGCACTGTGTCC CACAGGAAGCCCAGAAGATGTTTCTCTAAGAAATTAAGGATTCGTTTGTTGGGGAGGGCT GCTGGCATGCTTGAAAAGTACTGTGATGGCTGAATTTTGTGGGCTTGGGGAGATTGCAGA TTCTCTGATTTCAAGGGGAATGATGTGATCCTAGAGTTGCAAAGAACAAGTGACAGTGGA GGCGCTTATGCTTTGTGACTGCACTAGAGACAAGGAAGACACAACTAGAATAATGGGGAG CAGGAATGGAGCGGCCAACAGAATATGTGACTGTTAGGGATCTTTGATGAAGGCTGATTC TCAGGGAGTGAACTACATCAGTGACCAACTATTTGTCTTTATATAACTGGGTAATGTGGA TGGATTCTAATAAAGGGACTACTTACAGCACAGCAGGAAAGTCACAAAGAAACCAGACAG AAGAGTGTAAGTAGTAAGGGGCCAAGCAGTCACCTGACTAGAGACAGTGCCAGCTTGCCA AGAAGGCACCAGACAGAAGCTGTGATCTTCAGCAAAGGGACACAGTCTGCCTGTGCTGAC CCTGCAGGGGCAGAGGTGGGGGATAAACACACTCTTCTCTCACCTATCTTCTGCCACCCC CTCCATTAGCTGAACCCCAATAAAAGCATGAGGGTAAGGGAGATCTCTGAAGTATCCAAT TCAGGTGAGCCTCCTAAGGAACAAAGCAGAATGCAGAAAAATTAAGAGTGGGTCTAGGGA ATAAAATAGAGATATGCACCAGAGTATGATGATGTGTCTGGGAAAGAATATACAAATACT TTTAAAATTACTAGACAATAAACCTGAGATGACACTAATACAGGTAAATCTCATTTAATG GTAATATATTCCAAGAAATGCATCATTAGGTGATTCTGTGGCTGTGCAGACACCAAAGAG TGTACTTTATACAAACCTAGACAGCGTAGCCTACTACATACCTAGGCTATATCGTACAGC CTATTGCTCCTGGGCTACACACCTGTGCAGCATGTCACTGTGCTGAATACAGTAGGCCAA TGGTCCCCAACACCCAAGCCATGGACCAGCACCGGTTCGTGTCCTGTTAGGAACTGGGCA CAAAGCAGGAGATGAGTGGCTGGCCAGCCGAACATTACTGCCTGAGCTCCGCCTCCTGTC AGATCAGCAGCAGCATTAGACCTTCACAGGAGCACAAACCCTATTGTGAACTGCCCATAG AAGGGATCTAGGTTATGTTCTCCTTATGAGAATCTAACTAGGCTGGGTGCTGTGGCTCAC GCCTGTAATCCCAACACTCTGGGAGGTCGAGGCAGGTGGATCACAAGGTCAAGACAATCC TGGCCAACATGGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCTGGGTGTGGTGGCG CATGCCTGTAGTCCCAGCTACTCGGGGGACTGAGACAGGAGAATCACTTGAACCCGGGAG GCAGAGGTTGCAGTGAGCCGAGATTGTGCCACTGCACTCCAGCCTGGCAACAGTGGGAGA CTCAGTCTAAAAAAGAAAAAAAAAGAAAAAAAGAATCTAACTAATGCCTGATGATCTGAG GTGGAAGAGTTTCAGCCCAAAGCCATCCCCACTCCGTGGAAAAATTATCTTTCACAAAAC CAGTCCCTGGTGTCAAAACTTTGGGGCCCACTGCTGTAGGCAGTTATATCACAGTAGTAA TATCTAAACATGGAAAAGATACAGTAAAAACACAGTACATTGGGAGACCGAGGGGGGCGG ATCACCTGAGGTTAGAAGTTCGAGACCAGCCTGACCAACATGGAGAAACTCCGTCTGTAC TAAAAATACAAAATTAGCTGGGCGTGCTGGCGTGGGGCTGTAATCCCTGCTACTCGTGAG GCTGAGGCAGGAGAATCACTTGAACCCCGGAGGCAGAGGTTGCAGTGAGCCAAGATCGTA TCATTGAACTCCAGCCTGGGCAACAAAATAAAACTCCTTCTCAAAAAAAACAAAGAAAAA AATATATATATATTACAAAATTTAAAGAGAGTGGTACACCAGTAAAGGACATTTAGCATG AACAGAGGTTGCAGGACTGGCAGATGCTCTGATGAGTCAGTGAGTACCTGGTGAGTGAAT GCGAAGGCCTAGGATATTACTGTACATAACTATAGACTTTATATGCACTGTACACTTAGG CTACACTAAATGTATTTAAATTTTTTCTTTCTTTAACAAGTTCATCTTAGCTTATCATAA CTTTATAAACTTTTAATTTTTTTAATTTTTTGATTCTTTGATAATAACACATCTTAAAAC AAAAACACATTGAACAGCTGTACAGAAATACTTTATATCCTTATTTGATAAGCTTCATTT ATTTTGATTTTTTATGTTTTAAACATTTTTGTTAAAAACTAAGATACAAACACACACACT AGCTTAGTCCTGTAAGGGTCAGGATAATCAATATCACTGTCTTCCATCTCGAAATCTTGT CCAAGTGAAAGGTCTTCAGGGTCTTCAGTGGCAATAACATACATGCAGCTGTCATTCTCT ATGATAACAAGGCTTTCTTCTGGAAGAACTCGTGGAAGACCTTCCTGAAGCTGTTTCATA GTTAATTTTTTTTAATGAGTAGTAGTACTACACTCTAAAATATGAATAAAATCTGTAGTA TTATACATACTGTTGATCCTTGAACAGTGCAAGGGTTAGGGGACCAGCTCCTGTGCAGTT GAAAATCCATATATAATTCTGGGCTATCCCCAAACTTAACTTAATATGATATATATCATA TATAATATATATGATATATATCGTATATATTACATATGATATATATCATATATAATATAT ACGATATATATCATACATAATATGTATGATATATATTATACACAATATGTATGATATATA TTACATACAATAAGTATGATATATATTATATACAATATGTATGATATATATTATATACAA TATATGATATATATTATATACTATATGATATAGATTATATACAATATATATGATAGAGAT TATATACAATATATATGATACAGATTAAATACAATATATATGATACAGATTATATACAAT ATATATGATACAGATTATATACAACATATATGATAGATTATATACAATATATATGATATA GATTATATACAATATATATGATATAGATTATATACAATATATATGATATAGATTATATAC AATATATATGATATAGATTATATACTACATATGATATACATGATATATCATATATATGAT ATACATGGTATATCATATATGATATATACGATATATCGCATATATGATATAGATGATATA TCGTATATATGACATAGATGATATATCATACCTGATATAGATGATAAATAATATATGATA TAGATGATATATATCATATATGATATATCATATATTATATAATAAATGATATATATTATA TATAATAAAAGATATATATTACATAATAAATGATATATATTATGTAAAATATATGATATA TATTATATATTATATCTGATATATATTATATATTATATATTTTATCTGATATATATTATA TATTATATCTGATATATTATATATAATTATATTATATTATATTATATATTATATATTTTA TATTATATGATATATAATTATATTATATTATATTATATTATATATATTATATTATATATA ATTATATTATATTATATATTATATATTATATTATATATAATTATATTATATGATATTATA TATTATATATAATTTATGATATATATTATGTATTATATATAATTTATGATATATATAATA TATTATTTATCATATATCATATATATTATGGTATATTATATATAATTGTATTATATATAA TAATATATAATATACCATAATATATCATATATTATATAATATATAATATAATATAATATA ATGTAATCATATATAATATATAATATATTATATTATAAAATATATTATATTATGATATAC TATATATTACATGTTACATTATATAATATTTTGTATAATATATAATATATATTATCTATT ATACTTTATTATATTACATATATAATTATACATTATTATATTTCATATATAATTATATAA TTATACATAATTATATATATTATATTATATATAATGTTATGTATAACAATATATATTATA CATAATATTATATATTATATTAAATATGATATTATATATAAGGATGCAGGATGTAAAAGG AAATTATATATATGTTATATATTATATATATTATATTATACATAATATATATATATATTT GGGGGTGCCCTATTTCCTATCTCATAACTTATTTTAAGAAGCACAGCATAATAATGTGTG GACTTGGGATTCAGTTTTTGAAATGAAACACTGAGCCTTCGATGACCTTCCTGTACATGT GAAAGCACACCTGTCTGCATGGCAGCAGTTGGACCTCACAGTGTGGATTGTGCCTTCACC CTGGAATGTTTATGCCCTATCGCCATGGTGATGGGATTAGGGATCTGCTGCCCTTGGTCC TAAGTGCCACTATCTGTGCTGAGTTTTTCAAAGGTCAGAGCAGATTGAACCTTTGTGGTT TCATTTTCCCTGATTTTGATTTTTCTTATGGGGAACCTGTGTGGCTGCATTCAAGGTATG TTCATACTGGCCTGTCAAATGCGATCTTTTCAAATTACTAGTTAATGCTTTCAAAATATG TTATTTAAAAAATTAGCCTCTGTATTTTCCATATGCAGTTATAAATATGTTTCATGGTTA GGTTTTATTCCTCAATTTATATATTTGATTATTGTACCAAGCAGAGTACCTTTGAAATTT TTCTTCATTTAAAAAATATGTATCTTGGCTCAAGCCTGTAATCCCAGCACTTTGGGAGGC CAAGGCAAGAGGATCACAAGGTGAGGAGATGAAGACCATCCTGGCCAATGTGGCGAAACC CTATCTCTACTAAAAATACAAAAAATTAGCCAGGCATGGTGGCAGCTGGTGTAGTCCCAG TGTGGTGTAGCCCCAGCTACCTGGGAGGCTGAAGCAGGACAATCGCTTCCACCCGTGAGG CAGAGGTTGCAGTGAGCCAAGATGGCACCATTGCATTCCAGCCTGTGCAACAGAACAAGA ATCTGTCTAAAAAAAATTATATATATATAATGTATATTATATGTACTATATATTACATAA AACATATAATATATAATATATATAATATATATAAAATATAATATACATTATATATAAATA ATATATATTATATGTCACGTTATGTAATATCTATGATACATATTACATAATAAAAGACAT ATATAATATATGATATATATTACATATAATATATGATATATATTACATATAATATATGAT ATATATTACATATAATATATGATATATATTACATATAATATATGATATATATTACATATA ATATGTGACATATATTACATACAATATGTGACATATATTATATGTAATATATGATATATA ATATATAATATATGGTATATAATATATAATATATGATATATAATATATGATATATAATAT ATAATATATGATATATAATGTTATATTACATATAATTATATTATATTATATTATATATAT CATAATATATTATATAATATATAATATATATCATAATATATTATATAATATATAATATAT ATCATAATATATTATGTAATATATATAATATATCATTTTATATATTATATATAATATGTC ATAATATATTATATAATATTATATATAATATATCATAATATATTATATATTATATTATTA TAAAATATATAATACTATATAATGTTATATATAATATAATATTATATTAGATTATAATAT ATAATATTATATTAGATTATAATATATAATATTAGATTATAATATATAATATATTAGATT ATAATATACATTATATTATAATATATTATTGTATAATATGTTATAATATAATATATATTA TATATTATATTATACATAATATTATATTATATATAATATATATAATATTTTATACAACAT ATTATATATAATATCTTATATAATATATTATACAATATATTATATTGTATAATATATTAT ATATATTATGTATTATTATCTTGTATATATAATTATATATAATTATATAATTATATATAT TATATTATAGGTAATAATTATATATAATTATATATTATATTATATGTAATAATTATATAT AAGTATATATTATATTATATGTAATAATTATATATAATTTATGTATTATATTATATGTAA TAAATACACATAATTATATATATTTTATTATATATATTATGTATAATTGTATATTATATT ATATATAATAATTATAGGTTATATTAGATATATTAATTGTATTTTATATTATTTTTAATA TTAGTTAATATTAATTAATAATAATTAATTTAAATATTAATTATTATTTTATATATTATA TTATATATTATGTTATATATTATATTATATATTATATTATATTATATATTATATTATATT ATATATTATATTATATATTATATTATATATAATATAGATAATTATATATTATATATCATT ATATATAATTACATATATTATATTATATTGCTATATATATTCATATATATTATATTATAT ATAATAATACCTATTATATTATACATAATATTATATATAATATAAGGATGCAGGATGTAA AAGGAAATTATATATATGTTATATATATTATATATATTATGTCATATATAATTATATATA TATATTTTTTTGGGTGCCCTATTTCCCATCTCATAACTTATTTTAAGAAGCCAGCATAAT AATGTGTGGGCTTGGGATTCAGTTTTTGAAACAAAACACTGAGCCTTTGATGACCTTCCT GTAGTTGTAAAAGCCCTCACCTGTCTGCATGGCAGCAGTTGGACCTCACAGTGTGGATTG TGCCTTCACCCTGGAATGTTTATGCCCTATCGCCATGGTGATGGGATTAGGGATCTCCTG CCCTTGGTCCTAAGTGCCACTATCTGTGCTGAGTTTTTCAAACGTCAGAGCAGATTGAAC CATTGTGGTTTCATTTTCCCTGATTTTGATTTTTCTTATGGGGAACCTGTGTGGCTGCAT TCAAGGTATGTTCTTACTGGCCTGTCAAATGCGATCTTTTCAAATTACTAGTTAATACTT TCAAAATATATTATTTAAAAAATTAGCCTCTGTATTTTCCATATGCAGTTATAAATATGT TTCATGATTATGTTTTATTCCTCAATTTATATATTTGATTATTGTACCAAGCAGAGTATC TTTGAAATTTTTCTTCATTTAAAAAATATGTGTCTTGACTCAGGCCTGTAATCCCAGCAC TTTGGGAGCCCAAGGCAAGAGGATCACAAGGTGAGGAGATCAAGACCATCCTGGCCAATA CAGTGAAACCCTGTCTCTACTACAAATAGAAAAAATTAGCCAGGCATGGTGGCAGCTGGT GTAGTCCCAGTGTGAATTGGGATTCAGTTTATTCCCAAATTCCCAAATTTTATATATATA TATATATAAAATATATTAAATATATTATATATATACTATATATTATATTATATATAATTA TATATATATATATTTTTGGGTGCCCTATTTCCCATCTCATAACTTATTTTAAGAAGCCAG CATAATAATGTGTGGGCTTGGGATTCAGTTTTTGAAACAAAACACTGAGCCTTTGATGAC CTTCCTGTAGTTGTAAAAGCCCACCTGTCTGCATGGCAGCAGTTGGACCTCACAGTGTGG ATTGTGCCTTCACCCTGGAATGTTTATGCCCTATCACCATGGTGATGGGATTAGGGATCT CCTGCCCTTGGTCCTACGTGCCACTATCTGTGCTGAGTTTTTCAAAGGTCAGAGCAGATT GAACCATTGTGGTTTCATTTTCCCTGATTTTGATTTTTCTTATGGGGAACCTGTGTGGCT GCATTCAAGGTATGTTCATACTGGCCTGTCAAATGCGATCTTTTCAAATTACTAGTTAAT GCTTTCAAAATATGTTATTTAAAAAATTAGCCTCTGTATTTTCCATATGCAGTTATAAAT ATGTTTCATGATTATGTTTTATTCCTCAATTTATATATTTGATTACTGTACCAAGCAGAG TATCTTTGAAATTTTTCTTCATTTAAAAAATATGTATCTTGACTCAGGCCTGTAATCCCA GCACTTTGGGAGGCCAAGGCAAGAGGATCACAAGGTGAGGAGATCAAGACCATCCTGGCG AATACAGTGAAACCCTGTCTCTACTACAAATACAATCAATTAGCCAGGCATGGTGGCAGC TGGTGTAGTCCCAGTGTGAATTGGGAGTCCGTTTATTCCCAAATTCCCAAATTTTATATA TATATATATATAATATATATAATATATAATATATTATATATATTTTATATAATATATAAT ATATATAACATATATATTATATATAATATATACATTATATGTAATATATTATATATATTT TATATTATATATAAAATATATATACTATATATAATATATATAGTATATATAATATATATT ATATATAATATATATAGTATATATAATATATATTATATATAATATATATAGTATATATAA TATATATTATATATAATATGTATAATATTATATATTATATATATTATATATATTATATAT TATATATTATGTATATAATATATATTATTTATATATTATATATAATATATAATATATAAT ATATAATATATAATTATATATTATATATATTATATATTATATAATATATAATAAATAATA TATATTATATATATAATATATATGATATATAATTATATATTATATATTATATAATATATA ATATATAATAAATAATTATATATCATATATATAATATATATAATATATATAAATAATATA TATTATATACATAATATAATTATCATATATTATATATATGTCATATATAATATATGTCAT AATATAATATATGTCATAATATATATAATATAATATATGTCATAATATATATATAATTTC CTTTTACATCCTGCATCCTTCAACCTGCATCCTTCAACGTTCCATCCCCCACCCCACAGA TTAAGTTATTCCCCTGGGGAGAATATGGCGAAGTCTATTTTAATGCTGTTTTTAACCCAA TTAAGAACCTATGAAATCATTACTTTCCAAAACTTTGGAACAAAGCCACAGTAGTAAGGA TCCGTTGGAGGCTTTTCACACAATAAAATGTAACTCTCTTTGTTTTTAACATGTTTTTCC CTTCCTCTCTTCTTTTTTTGTGAAATGTGTATTTACTTTAATATATTTGTAGTAAGTCAC TTCCATGCACATATTAATTTTTTAAAGTAATAAGCATGTGTATTGTCTACGTGTGAAAGA AAACACACATTTATTTTTATGCTTTGGAAGTTATCCAGAATCATGGAATTGTCAATCACA GTCAATCACCCAACCTACTCACCTTTCCAGTGTAATCTTAGTCAAATTTTTTTTTTTGTT ATCCAATGAGATGCAGTATTTCAACTCAGAAAGATAAATAGAGTGAATTTATAGAGACTA TTAACTAAGAACATACAGTTTGATTTATACTCAGAAGCAAGTAGATTATGTACATATATA TGAAGATAAAAATTAAAAGGATAATTGTGTAAATTTGCATGTAGAGGGCTTTGAAAACCT GTTTACTTGTGAATGCTGTTTTGATGTATTGTGTCTTTGTTCTCCCGACCCATCATCCAG AGCTCTCTGCAGGAGCTAAGTGCTCATCAGTTCCATGACTTGGAAACTGTCTAAGTTTAG AGGCACTTGTATTTGTTAGTAAATAAGGCAAGATGATATTGTTTCACAGGTTTTAGTGCC GAAGACTGAATAGATAAGCTGCTCCACCCAGTACACTGGTGTTCATTTCATGGTCATCTC ATCTGTTAACCATGGATATAAAACATTTATCTTCAATGATGGGGTTTTACCATGTTGGTC AGGCTGGTCTCGAACTCCTGACCTCAAATGATCCACCCACCTCCACCTTCCAAACTGCTG GGATTACAGGTGTGAGCCACTATGCCTGACTGATTATTTTCATAACCAAGAAAAGAAATA AATACAATTAATGCTGGTGCATGGTATTAAATCTAGTTTTTAAAAAATTCACACATAAAC AGGGCAGAACCCTATACCCTCCATGATAAATGCAGTAGCAGTGTATGTGGGTCTGTGGAG GTTGAAAGGGACTTGGTCGATGTCAAGAAGGTAGTGGCAGTCCTGCTGGGCTTTTAAAGG GTCTGAAGAAGTGACAGGATGCTGTGGTTGAATCGTAGCATGTGTTTTAGCATTTGTTCA TTTGGAGTTGATTATTTCACGTTGCTTTCATTTGCCATTACCTGGAAAGCCAAGGGCTCT ACTCTCATTTCCTTGCTGCTCTTTCTTTGCCTTCCTTGGTCCGTGAAGAAGATGGTCCAG GAGAAGCTCATTCCATGCTTGTTAACCAGGCACGCCCCTAAGTTCCAGTCCCTGAGTCAT TCATGAGTAGCACTGCCAATGAACTGACAGCCATGCTGTGTCCCTCCACATCCCCTAGGT GACTCGAAGAAGCCTTCCAAAAAGCGTGTGAAAAGGAAGCCCTACTCTACCACCAAGGTA AAGTAGCCTGTCTTTGCCTAAGATGTAAATGTTGTTTTCTTGGATCCTTTATTTTTCAGT TGATATCAGCTATGGGAAAATTCTCCACTACATTATAGGTGTTAGATAATATTTCCTTGG GGATGGAGGAGGTGTATTTTACCAACTGACACCTGATTCCAGAGGACGTGCAAAATTGGC AGTGTCAGATAGTACACTGGGTGTTAAGGGATGTTTTCTTCAGGAACAAGCTTTCCACTT TAGATAAGAATTCTGCAATTGCTACTCAAAAATTACCTAGACAGAAACATTCTTCAACAA AAGCTCCTGTGCTTTCCTAAGGCAACTCTACTCTAGAGTTGGGGCTTTTGACTTGAACCT TATTTCCAGTCTTGGTTACCCAGAGTTTCCAAGTGAACAAAAGACCTGTGTGAGCCATCC ATAGCATAGCCTGATTCTCAGAGTGTTTTCCTTCTCTAATTACAGGTGACTTCAGGGAGC ACATTCAATGGTACGTATTCTGGAATCACTCACTGGTTGTTAGAAAAGGATTCTACAGGA AATCTGGAGCTTAACTGCTGGCTTTTGTCTGGAGAGCCTCCATGATCCAAGACATCTGGT GGGAATGAGGATGTAGGGTATAGTAAAAGAAACTGGTTTTCCTGGTGACATACTCTTTTT ATCTATGTATAGTTTCTGGGAACATGTTCACATTAGGTTGTGTGTGGGTATGTGTGTATT AGGGCGGGGGTGGGGTGAGGTGGTCTGTGTGCAAGTCTGCATGATTTGCTTGTGAATGTG TGTCTATGTGTGTTTCCCCTAGGAAAAAAATGTTGTGTTTACCCAGCACAACTCTCAGTG CCATGTTTCTTAATTTAACAAATCAGACCACATACTTTACTTACATTAGTTCACACCTCA TCATCATCATGCCCATATGTTGTGAGCTTGTTTATTGAGCCCACATGCCAGATGGAGAAA CTAAGCCACATAAATAAATGTGCCCTGGTTCACTTGCTGCATAGTGAAGAGTCAAAATGT TTCCTCATACGGTGCTAATGTTGAAGGCCTGAACTACAACCACTATTTATCAGCCAGTGA AGAGATCACTATTCACCATGCAAGGGAGTTCCAGCACCCTCTATGCCTGGAATTACCCAC GCCTGCAGAGATCCCAAACGCCATCCCTCACATAAGACAGCCTCATGATCTCATAATCCA GGTAGCTATGTAGACATCTTCCTGCAGGTGTCACATAGTCCTTAGTGTGAAACCAACATA GAAAGCCCATGTTTCTGATCAAATCACAGGTTCTGAAACACTAAGGGAGGCACTAAGTAG GACAATGTGGTGCCTGCGTGTCATAGCTGGGTCTCCTCAAGACATGGATCAAGTCCAGTA AGAATTGGGGAGATGCTTTAGAGTCTTGATGGAGTTATCACCACAAGCCCTCTGAGCTAC ACACTTTAGGGATCATGACCATTAAGTACTCAAATTACCATTTGGTTGTTATCCGGGTAT CCGTCGTCCTTGTGGCAACCCTCTTGTGAAGCTGGTGTGGACAGCCTCAGTGCTGGAGCT GTGCCTGCCTTCTGAGTGGACCCTTTCTGTGTTAGCAGGTGGGTACAAGCGTGGGGGTCA GCACACTCAGTGGATTTACACACACAGCGTTGAAGAGTAAGGCTGGGCTTCATTATTTAT ACATTTTCAATAAATGATGATCTTCATAACATAAAATCAATGATGTAGTACACTAGAATA CTGTCCCTAGTATTGAATCTTGTCTCTCAGCAAAGGGTTGCTTAAAGTCACGTGACAGAT TCCATTCAACTGATGACACATGCTGTAGCAGCAGTTAAAGCAGTCATTTGAAAAGGCTTT TACTATAAACTTACGTGTGAGCCTGAAGTGGGGGATAAAAGAGGCGATTAGCTCCCCTGT GCCATGTTTCTCTTATGTGCGTGGTGGAGGAAAATTACACAGGAAGGTGATGGAGAGAAC AGAGCAAAGGATTGGACAGGTCCATTGAACCCATAAGACTATGGTGAGGTTAGTGAATGA GACTGGTCATTTTAGGTCAAATTTTACCCAGAGCTGGTGCAGCCACTGCCCATTCTTAGC CAGACCTTATTGCAGGCAGCTCTGATCAATAGTCAAGGAGGCAGTGGGGGTTGCAGACTT AATTCATTAAATCACCAAAGCACCAGCCCACACGGCCACTTTTCCAGTTAATTCACAGTA GCTTGCATATTCAGGTTTGATCAGTGGAAGGGAAGTTACTCTTTGCAGACCCATCTTTTG ACAATCATTTTGCAGTGTCGGAAGGTCTGAGCAGCCTCGGGAGGCAAGCAGTCCCTGGTC CCTCAGTGTAGTCACTGGAGGAGACAGTCACTGAGAGGCAGCTGGCAGGGTGAAGGGAAA GGGGAGGCAGGCCACAGAGATGACAGCCTTTAAGCTGTCATACTGGGAGGTCAAGGATCT GAAAGAGGAAGGAGAATTCTTTATCATTAAGGACCTGTCCTTATCTCAGGCATTTCCTCC AGAGCATCACCTTTGTCCACCCACACACCTTGGGCTAGGAGGACTGGAGAAAAACAGTGA GGGGTCTCTTGGGTCTCTGGCACAGGGCGTGATGAAGAGGTGGCAGTTTTTCAGGAATCT CTCTCTCTAGGGAACCAAATACATTTCCCATCTCAGGTCCTTCACTCAGCGGGGTTGAGG TTCTGCTCGTCACTTATCATCTCTGAATGTCAGCACCCTCAAGTGTAAAATCTCAGTCAC AGCCCCTCCTCTGCACCCCCTGCAGGGCTGATGTTCTCCATAAACCATAAGGCATCATGC CCACGGAAAAGCCGAACAGGAAAGCATGCTCCACTGCCCCGGAGCCATCCAAGTTCCCCC TCCATATTCCGCCACTGCTGAGTGTCCAGCTTATTCCTCCTGGCCTGTAGTAAACACTTA GAGAACATTACTGAAGTACCAGTCCTCTCTAAGGTTTTCCTGTATTTAGTGATTTTTTAG CCCTGTACTGTGATACTAAGAAGTAGGGCCTAAATAGGGCCTAAAAAGTATTGCTAAAAT TACATTATGACAGTGCAGAGAACTGAGGGCAGAGGGAGGACATGAGCTTGCCAGGTCCAC ATGGCTTAGTGGAATTTGAATCCGGGCCCCTACTCTGCACCAGCCCTGCACTCACAGTCA TCCTGCTGTATTCTCCTCTCCAGGAAGGCACTGCCCACGCAGTCTGTCTGATAGAGGTGT TGAGTGCTCACTGAACTCCGTGATCTTCCTGAAACCCAACTTTGATTCAGTGGGCTCTGC TTGGAAGCCTGTAAAGAAAAGGATCATAAGTTTAAACTTAGAACAGATTATCACTATTTT CCCTCTGGTCCTCTGTCAGCAAGATGTCAACAGCCCTATCTATTGTAAATGCATTAACCA GCATCTTCTCTGATAGAGAATATAAGAAGATATGCTGTGCACACCAACCAGTGTAGGAGA CCTCATGGCTCCCGGGTAAAGAAGAAGAGGTACCCACAAGAAGGTACTGTGGAAGTTCAT TAATTAAGTTGATTCAAGAATTGCAGTTGCGGGGAGTATTCAGTGTCCCATATGTAAGAG GAAACTATGAAGAGACTAAGCCATATTTTTTAATGTGTCAGGATTCTAATTTGCCTGGTC AGTAAATATTGCTACCACCACAAAAGTAAATATCTACTTAAAAGTCAATTTTGGTTCATG TTTAATGATAGACAATGTTTCAAGCTAATGTCTAGAACTTACCTGGTTGTTAAACATAAG CATAGATCTCCCTGAAAGAGTGGTGCTATATTATTATTTTTCAATTAATATATTTCTTTA GAGAGTTTTAAATTGACATAAAAACTGAGCATATGGCCGTGTGTGGTGGCTCACACTTAT AATCCCAGCACTTTAGGAGGCCAAGGCAGGCGGATCATCTGAGGTCAGGAGTTGGAGACC AGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATCCAAAAAAATTAGCCGGGTG TGGTGGCAGGCGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAA CCCGGGAGGCAGAGGTTGCAGTGAGCCAAGATCATGCCATTGCACTCCAGCCTGGGTGAC AAGAGTGAAACTCCATCTCAAAATAAATAAATAAATAAATAAATAAATAAATAAATAAAT AAAAATTGAGTATATAATACACGAAGTTCCCATATTATTCTGTCTCCTCACCCTCACTTC CTAATTTCACCTATTAGTAACATCTTACATTACTGTGGTACATTTGCTAGAATAATGAGA AAATATTGACACATTATTATCTGAAGTCTGCATTTGCATAATGTTCATTCTTTCTGTTAT ACATATATATGAATTTTGAAATATTTAAAACATTATGTTCACCCTTATGGTCTCATAAAG AAAATGTTCACTTCCCTAAAAATCCTCTCTTCTCATTAATCTCTGTCCTCTTTCTCCAGA AACCTTGGCAACTATTAATATTTTTACTATCGCTTCAGCTTTGCCTTTTCCAGAATGTCA TATAGTTGGAATCATATATTATGTAGTTTTTTCAGATGAATTTATTGCACTAAATTGATG TACGCTTTAGCTGCTTTCATGTCTTTTTTATGCCTTAATGGCAAAAAATGGCACATTAAA TCACCAAATAATATTGCATTAAATGAATTTTTGTCTTTTTATTCACCTGTTGAAGAATTC GGTAGATTTCATGAGAGAAACCATCTGGGCCTGGTGCTTTCTTTTTCGGAATGCTCTTAA TGTGAATTCAACTTATTTAATAGACATAAGTTTATTCAAATTAGGATCCTAGCATGACCT TGGGAAGATTGCCTTTCAAGGAATTGATACATTTCACTGAGGTTATCAAACTGCGCTCAT AGAACTGTTCATGATATTCCTTTTAATGCCTAACAGTTCAGTAGAGATGGCTCCTCTTTT ATTTCTGAAATTGGTCATTTGTGTTATCTTCTTTTTCTTGGTTAGCCTGCATATCAATTC ATTCATTGTAATGAGCATATCAAAGAACCAGCTTTTGGTTTTATTGATTTTCTGATGATT TCAGTGTTTTAATTTTATTGATTTCTGTGATGTTGTTTATTACTTTTACTTGCTTTCCAT TGCATTCCTCTATTTTCTATAGTTCCCTAATTGAAACATGATATTACTGATTTTAGGTCT TGTGATTTTTAGTATATTGCATCCAATGCTATAGATTTCCCTCTAAGGACTGCTTTTGCT ACATCCAGAAATCTTGCCAAGTCACATTTTCTTTTAATGTAGTTAAAAGTACTTTTAATT TTCTATTGAGACTTCTTCTTTAACCCAACAGTTATTTAAAAGTGCATTGCTAATTTGCAA ATATTTGGGGATTTTGTGGCTCTTTTACAGTTGTTGATTTTTTGTTGTCAGGTGTGTGTT GCAAAAGCAGTCGTCTACCTCATCTTGCCACCACCCAAGATGGCCCAGGATGTGGGCTCT CCCTGAGTGAATCTTTGGCAATCTGCCAACCTGATGTGTTCGGCCTCCTTCTTTAGTCTG AGCTTGCCTTCTGCTTAGAAAGGGCCATTCTCAGTTCTGGCAGGGAGTTTTCCCAACATT GAGAAGGTGGCATTCTTACTCCCCACTGCAGCCTGCACCTCTGACCGGTGGTCAGCAGAC AGGACAGAGGTCCTCATTAGACAGAGTTCAGCAGGGTCTCTGACCAAAGTGCATCTTCAG AGTCTGCACCTACCCACTGTGACCACGGGCAGGCTCTGAGTCCTAAAGCAGGAGGAACTG TGCGACCATCCTGATTGGAAATTTGTGAGGATCACCGTGTTACTCAAGTAAGGTCTTTGG AAAGTGTCGTATTACTACTGTTTGTGAACTGCTTGTTGGTGGCCTGGCTGAGCCACACAC TTTATGAAAACCAGGACCCCTCAGCTGGTGTGGGTGTCTATGCAGCCTGAGACCCTCATG TGAACAGCCTCGTGGCAGCTGTCTTTGCCCCTTGCCACCATCAGTGCCTCCTTGTTCCTG GGCACTGCTTTCTCTGATGGTGCTCCATTGTTTTCCTGCACCTCAGTGTCTACAGCTGGA TGTCTCTTCCGCAATCTAGGCGAGGGGGCATCAATGGCAGTTCTGCTGTGGCACTGCCCT CCTTCTTAGCTTGTCTTGCTCTGTCTTAGGCTCCCTCAAAGATCCCACCCTTCAGGTTCT TCCACAAGTTTCTTATTGAAATCCGAGCAGAAAACTATGACCAATATGACCAATCCTATA CCCACTGAAGACATGAATGAAGAATTAAAACAATTCTCCATGGACTCTACCATATAGATC CCTAGAAGTAATTTCTAAAAAAAAAAAAAAATCAAGGAAGATGTAATAGTTTTCCATAAA TTAGAATACCCTACATGTACAATTAAATGAAATGGCTAGTATAGTCTTGAAACCAAAACC AGATAAGGTAAATTAAATTCTGTGATATTTTAAAATACTGTAAATTCTGACTAATGTGAG TTAATCTCAATATATGAAATAGTAGATTAACATTGAAAATGCAATAAATAAAATTAGCTA CCTCAAGAGTTTAATGGAAAAAAATGTGATTATTGCAATAGATTCAGGAAATTCATGAAT AACATTCACCCTATATTTGTAGGACAACTATCTAACTTTAAGTGACCTGTGACAACCATT TGAATTAATGCTGCTTTCACAGCATATCTCTTGGCTTGTTAAAAACCCGACAAGAATTTC CGTAACATTAATTTATTTTTAACACCTATATTGGGTGTGAACCCACCATAAAGTTTGCCC ACTGAAAAGGTCTACAATTTGATGCTTTATTAAATTGATACTGTGTGCACCCATCACCAC GATCTAATTTAAATATGTTTCCCTCACCCAAGTTCTCTCTTGCGCACTGGCAGTTAATCC CCACTCCCATCTCCAGCCCTAAGCAATACTGCTGTGACATTCCATCTCCATAAATTTCCC ATTTGCTTAATAGAAATGGACATATATATATATTTGGAATCTGACTTCCTTCATTTAGCA TACTATGTTTGAAGTTAATTGACGTGTTAGCACGTGCTGGTCATGTGTTTTCCTTCATAG TCTGCTGTGTTTACTCATACAGATAGTGTTTATTCATTTATCAGTTAATGGACATTTAAT TGTTTTGTTATTTTCTTTGATGAGTAATGTAGCTTTGAGCATTCATATACAGTCATGTAA TGCATAATGACATTTTGGTCAAAAAAATTTTTTTTTTTCTGAGACCCAGGCTGGAGTGCA GTGGCACAATCTCGGCTCACTGGAACCTCCACCTCCCAGGTTTAAGCAATTCTCGTGCCT CAACCTCCCGAGTAGCTGGGACAACTGGCACACGCCACCATGCCTGGATAATTTTTGTAT TTTCAGGAGAGACAGGATTTTGCTGTGTTGGTCAGGCTAGTCTCAAACTCCTAGCCTCAG GTGATCCACCCATCTCTGCCTCCCAAAGTGCTGGGATTATAGGCATGAGCCACCACACCC AGCCTAATTTTTTTAAGAAAGAAGGGAACTATTTTCTAAATTACTTTTGCCAATTTATAT TTCTACCATGATGCATAGCACTAATTTCACCGTACAATGTATGGTAGGCCCCAATATGTA AGAAATGATGAAAGTAACACATAAAGATTGGTATAAAACAAATAAGATTATCATTGTTGC TATCATCTTTGTCAAATTCTGAAAACAATCTGAGTATATTTTTATATAAATATGCTTGGC AACATAGCTGAAAAACGCATTATCAGTTACATTTATCAGTAACAAAGACATAAATTTGAA GGGGGAAAAACACTTGTACTAACAACGCAATGTCAGAATTAACATAAAAATTCTGCTGGT CACTTTGGAATATTTAATTGCCTGGGGCAGTGTTTAGTAGACAAATGAGCATCTATGGAG CACCCAAAGTAGGGGAATCAACAGAACTTGGGTTTCAAAAGTTATCTGGGTTTAGAGCGT GAAACTTTGTTAGAGGACACACACCTTGCATGAGCGAGGTGCCTTGGTGTGTGTGGACGT ACCATTATGCTTGGAGGTACAGCATAATGGTGGCTTCCTCCAGAAAGGGACATTTGGGGT GGATTCATTCCATCTAGACAACACAGCCTGATGTGGCATGGACATGAATGGAGGTGAAAT GGTCAGTAGTTGAGAGGATCAGTCCTGACAAGGGCCGAGGTGAAAAACCTGGGAACCCCT TCAGGTGCAAAGTCTTCAGTTGAAAAAGGAGGTGGTCACAGGAAATACTGAGACAGGACA GCAAAGCATGGGAGACAGAGTTCTTGGCCCTGCAGGGTGAGTACTGTGGATTCTCAAATT TTCTCCTCTCTCCATTAATTTCTTTCCCAATGCAGATGACTTCCATCATACAGTCTTCAG CAATCTTGAAAGATTGGACAAGCTTCAGCCCACTCTTGAAGGTAAAGGAAGGCAGCTAAC AAGACTGGCATCTGGGCTTGGCTGTGCGTGTTTTCTATCGTGGGGAAATATATATAACAC AATATTTATCATTTGAACCTTTTAACCAAAGTGTGCACTCCATGGCATTCAATATATTCA CAGGGTTGCATAACCAGCACCACTATCTACACCCACAATTTTGATGATTTCTTACGAAAC CTTGTCCACAATAAGCAATATAGCACCTTCCCCCTATTTCCAGCCCATGGTGATTCCTAT CCCACTTTCTCTTGTATGAATTTGACTATTCTAGGCACTTCATGTAATTACAATTATACA ATATATTCCTTTTGTGTCTGGCTTATTTCACTAAGCATAATGTTCTCAATGTCCACCCAT GTTGTAGCATCTATCAAAATGATGTTCGTTTTTTACAGATGGATGATGTAGCATTGCATG CAGACCACTTTGCTTTTATTACATTCATTTGTTCACTGATGGTTGGATTATTTCCACCTT TTGGCTCCTGTGAAAAGTGATGCTACAAACATTAGTATACAAACATCTGTTTGATTTCCG TTCTCTATTCTTTGGGGTGCCTAAGAGTAGAGTTCCTGGGTCCAACAGGGGTTCTATATT TAACCTTCTGAGCCACTGCAGACTGTTTTTCACAGTGGCTGCAACTTTATCCATTTCTAC CATCAATGTATCAGGGTTACAATTTCTTTACGTCCTTCTTCACACTTATTTTCCTTTAAA TCATCCTAGTAGGTGTATATTGGTGGCTGCTTGTGTTTTTCATTTGCATTTCCCTAATGA CTAATGATCCTGAGCAGCTTTTCCTGTGCTACTATCTGTGGCTGTATCTTCTTTAGGGAA ATATATGTTGAAGTCTTTTGCCCATTTTTAAAGAGTTGTCTGATTTTTATTTAGTTAGTT TGTTGTTGTAGATTTTTGAATATATCTTAAATATATTTAAAATATATTCTAAATTTTAGT CTCTTACAAGATAAATGATTTGCAAATATTTCCACCTTTGTGTAGAACTTTAGATTCACA AACTTCATTAATTTGTATGAAATCCTCGGCAGTTGACCCCAAACAGATAAGACTGAAGCA GTATTTTAGGAATAGTTGAAAGTATGATCACCACAAAACATAAGCGTAATCAAATCCTGC AAGCTACATGTAAGGCACAATGACAAATAAGGCAGCAAAGGGCCATCTGGTGTTTAGTTC ACCACACTTGTTGCAACTGTTTGCGCTGCAGAGTTAAAACACACCAGCATTCAACCCATG TCTCCTCTCTTGAAGTAAACTGTCGTATGTTGGCTGGCCTGAACAAGCGTAGATATTCTC CATCCTCAATTAATATGCATGCATGACAAAGAAAAGGAGGCCTGGATGAAAAAATATTGT GTGATTAATAATTATGCTTTAATTAATTTTAAAAGATATAATTTCAGTACTTCTAATTCT CCCATCAGCAGTTATAACAAAGGATTAGTGAATAAATACCATAGACTGTTTTGCCTAGAA TCGAATCCAATCTGTCTATTAAACTTTGCTTTTATTCAAGTGCAAAATGCTAAAACACAT AATAACTGCAGTGACAGCCACTGTGGATCCTCAGAGGTAAAAGTAGTCTTGGGACATAAA TCCTGCAAGCTAATATCGTTTTTACAGGGTTTAGAAAACCATTTAGCTGGGTTTCAAACC TCACAGTGTGAGCAGTGGGACTCTCATCAAACTATAGCATGTGCTTCAGTACCATTTGTA GACTGACTCATTCCCATTGCCTTAAGTTGCCATCAGCAAAATGCCAGGGACTCTATTTCT TGCTCCTTAGCTCCTCGTTCTTGCCTGTCTTTCCACCAGGGAAGATTTTCTAGCAGGAGC TCAAGCTGTGCTTTTAATGAAACACATCCACACACACTGTCCTGTTGTCCACATTGAGCA GAGCTCCCTGAATAACTCATGAACAAAAGCATCTATGACTAACTGTTGCTCTGTGTCCTC TTAGCCTCTGAGGAGTCTCTAGTTCACAAGGACAGAGGAGATGGAGAGAGGCCAGTCAAC GTGAGGGTAAGGTTGCCTTGCTTTCTCTGAAATAGAAATGTTCCTTTCTTGGTGTCTTTC TTTTTCAACTGACTTTACATGTGAAAAAATGACAATGTCCATGACAGGTATTAAATGCAG TTTTCTGAGGGGGAGGAAGAAGTGACTCTTAGCAACTGATATGTAATCCAAAATGGCATT TAGCTATGACGGCTTCAGGTTGTGGACTGTATCCTTGGGGTCCTTGTCCTTGGAAGCAAT GTCTTCTCCTTGGATTCAGTATTTTGCACTTGCCAACCTACGTGGACCTGAGAGATACAC CGTCCAGAAGCTGATGCCTTTTCCAGTGTGTATCCTACCCTTGTTTTGGAGGCCTTGAAG TTGACTACACTTTCTGATCAAGTTTTCAATATTCATTGAGAGAAACACAGCCTTGTGCAA ACAATCCACAACGTGACATACCCCTCAAAAAGCTTTGTTTCTGTATTGCAGGTGGTGCAG GTGGCCCCTCTGAGGCATGAATCTAGTAAGTATTCTGGAATCACTTGCCAAGAAAACAAT CTGGATGCCAAGAAAGGTGTGGCATCCTTGCCTGGTTTCAATGTGAAGAGCCACCCTGAT CCTGGGATTGTGATAGGAATAAGTATAGGGGAAGTGTTTTTTTAAAACCTGAATTCCCCA GGGAAAAATTATGGCCAAATTTTGAGGAAGCAGCTGTGCTCCCTTTTGGGTGGTGCTGAG TTGGGTGCTTGAGGATTGGTGGTGTCTTGTGTGAGGCTGCATCGTGTGGTGTGAATGTGT GTGTTTCTGTACAGGTGAGGCTGTGTGTTTTCTCAGGAGAGATTTCCCACTTATACAACC CAATCACCAGTGTCCACTTCTAACAATAAAATCCACCCCCGCTCTACTCTCTCTGTACAG TGACTCCTCCACCCTCACCAGAGCCATCCCCGGGTCTGCCTTATTATCCCCACTGCTCAG GTGGAGAAACTGAAGGGCCAAGGGAGTGGCCCCAGCTCCCGAGTTCCTGAATGAAAAAGT GAAAACACGAACCCAGGAGTGTGGGCCAGTGCTGACGCTGACATGGCACTTAGTCATGGG GTGTTCACCACCACACAGGGAGTCCAACATTCATGTATAAGCCCTAAAGCACCGAGCCCA AAAGGCCCCAGACACTGCCCATCATCATAAAGTGGGCTCCGTGGTCACACAACCCAAGGC AGTTATAGGCTCATCTCCCCACAGACAGGCATAGTCATCAGTGTGTCAAAAGCACAAAGA TCCCCAGGTGTTTGGCTCAGCTCACCAATCCTTTTTTTTTTTTTTTTTTAACTTTTAAGT TCAGGGGTACATGGGCAGGATGTGCAGGTTTGCTACATATATAAATGTGTGTCATGAGAG TTTGTTGTACAGATTATTGCATCACCCATATATTGAGCCTAATATCAGTTATTTTTCCTG ATCCTCCCCCTCCTCCCACCTCCCACCCTCCAGTAGGCCCCACGCTCACAAATTCTAAGA GGAGTGGGGGACCACAAAGGCCAGTGTGGCCCACTTCAGTTGTGAAGTTAATTTGCTCAG CAACTGGCCAAAGTCTATAAGGATGGGTGATGTATTTTAGTAGATTTAGTAATACTATCT TCCCAAGCCCTAAAATGCTCAAATCCTGCCAGCCAAAAATGGTGAGGAGGGACAGATAGG AACTCTGTGTGGCACTTGGTTATTAGCCTGGCTTCCATCCCTTAGTGGCAACTCTCTTGT ATATGTGGGTTAAAGACCCTCAGCCTCAAGCCAAGCCTCCTCCATGAGGAGCCATCTCAC TATTGACCGGCTAGTGCCGGGTATGGCCACCAGCCCAACTGAAACAAAATGTTGCTTTAA AACACGTGTAAATCTCATACATACAACAGGCAAATGCAGAAGCAGTGTGGTCTCGCAAGT TGTAAAGAGGACAGTCGCAATTTTGCTGGACTTCAACCTGGGTAGAAGACATGAGGGAAC TCTGTCACTGAATCACGGCAGAGTTCAAGGCCACTTGCAGACTATTTCATGTTATAGAAG GTGGCCTTTAGCTACTAAGCAAAGGCCTCTGTTTCTCATTTCTTTCCTGTTCATCTTCTT CGTCATCCTTCTTCCGCAAGGGAAACGAGCCCAAGCAAAAGACAGTTTCAATATTAATTT GACCGAGGTTTTGTGCAGTTAATTATCATCCAGGTAATCAGGTGCAACCCAGTCTGCCTA GCAGCCCCCCTATCTCTGCTCTGTGTTTTCATTTAATAAACATTTTGGTCTACTTACTAT GTGCTAGATTTTCTCGAGACCAAGTAAATGAGATAGAATCTTTATGTTGGCAGCTAAGTT AGATTTAACATAACTGACAAAAATTAAAATTTCTGATTTCTTGTAAAAATATTTTGTATG TGTGAATGCATACTGAATGTAAAGTGGATAAAAAAAATCACACTTGCACTCATGGAAGGC TTTTCATGAATTTGTCAATTTCTATTTTTTTATATTTCCCCATTTCACCGGATAATACAT ACCTGAACCTGGAAAATGATTCCCACAGCAGAAAGTGTTCTGAGCCACATCCCTTAGCTT CACTAGTGCAGGTCCACCTGGGAGTATGTCCCACCATCAGCTTGACCCATGCTGTAATCA GCCACCTCCATGCATCACACCAAGCAAGCCCCTGGGTGATTCACAGTCTCCAGCACCAGG GCACTGACCTTAACTCTGTGTTCTTCTAGCTCCCTATGAGGACACTGTACACGACATCGC TAATGAGGACGCTGTATATGACATTGCTACTGAGGACGCCATATATGACATCGCTAACGA GGACGCCGTCCAGGGCATCGCAAAGGAGGACGCCGCCCAGGGCATCGCTAACGAGGAAGC CGTCCACGACATCGCTAACGAGGATGCCGCCCACGTAATCGCTAACGAGGACGCCGCCCA GAGCATCGCTAACGAGGACGCGGTCCACGACATCGCTAACGAGGATGCCACCCAGGGCAT CGTTAATGAGGACGCCGCCCACAATATCGCTAATGAGGACGCCGCCCACGGCGTCGCTAA CGAGGTCGCCGCCCAGGGCGTCGCTAACGAGGACGCCGTCCACTGCATCGCTAACGAGGT CGCCGCCCAGGGCGTCCTTAAAGAGGACGCCGCCCAGGACATCGCAAACGAGGACGCCGC CCAGGGCATCGCTAACGAGGACGCCGCCCACGGCATCGCAAACGAGGACGCAGCCCAGGA CATCGCAAACGAGGACGCCGCCCAGGACATCGCAAACGAGGACGCCGCCCAGGGCATCTC TAAGGAGGACGCCGTCCAGGGCATCGCGAACGAGGATGCCGCCCAGGGCATCGCTAAGGA GGACCCCGTCCAGGGCGTCGCTAACGAGGACCCCGTCCAGGGCGTCAATAACGAGGACGC CGTCCAGGGCATCGCTAAGGAGGACCCCGTCCAGGGCGTCGCTAACGAGGACCCCGTCCA GGGCGTCGCTAACGAGGACGCCGTCCAGGGCATCGCTAACGAGGACGCCGTCCACGGCGT CGCTAAAGAGGTCGCCGCCCACGGCGTCGCTAACGAGGATGCCGCCCACGCGATCGCTAA GCCGGACGCCGCCCGCGGCATCGCTAACGAGGTTGCCGCCCAGGGCATCGCTACCGAGGA CGTCGCCGACGGCATCGCTAACGAGGACGCCGCCCACGGCGTCGCTAACGAGGTCGCCGC CCAGGGCGTCGCTAACGAGGACGCCGTCGAGGGCGTCGCTAACGAGGACGCCGCCCAGGG CGTCGCTAAAGAGGTCACCGCCCACGGCGTCGCTAACGAGGACGCCGTCCAGGGTGTCGT TAACGAGGACGCCCTCCACGGCGTCGTCGCTAACGAGGATGCCGCCCAAGCGATCGCTAA GCCGGACGCCGCCCATGGCATCGCTAACGAGGTCACCGCGCAGGGCATCGCTAACGAGGT CGCCATCCACAGCATCGCTATCGAGGACACCACACCGTCCAGGGCGTGGCTAACGAGGTC GCTGCCCAGGGCATCGCTACCGAGGATGTCGCCGACGGCATCGCTGAGGACGCCGCCCAT GGCATCACTAACAAGGAGGCCGCCCAGGCCATCGCTAAGCAGGATGCCGCCCACGGCATC ACTAACGAGGACGCCGTCCAGGGCGTCGCTAACGAGGTCGCCGCCCAGGGCGTCGCTAAC GAGGACGCCGCCCACGGCGTCGCTAACGAGGTCGCCGCCCACGGCGTCGCTAACGAGGAC GCCGCCCAGGGCGTCGCTAACGAGGTCGCCGCCCAGGGCGTCGCTAACGAGGACGCCGCC CAGGGCGTCGCTAACGAGGACGCCGTCCAGGGCGTCGCTAACGAGGTCGAAGTCCAGGGC ATCGCACACAAGGACGCCATCCAGGGCATCGCTAACGAGGATGCCGCCCACGGCATCGCT AACGAGGTCGCCGCCCACGGCATCGCAAACGAGGACGCTGCCCAGGGCATCGCGAACGAG GACGCCGCCCAGGGCATCGCTAACGAGGTCGCCGCCCACGGCATCTCTAAGGAGGACACC GTCCAGGGCATCGCGAACGAGGACGCCGCCCAGGGCATCGCTAAAGAGAACCCCGTCCAG GGCGTCGCTAACGAGGACCCTGTCCAGGGCGTCGCTAACGAGGTTGAAGTCCAGGGCGTC GCACACGAGGACGCCGTCCAGGGCATCGCTAACGAGGACGCCGCCCACGGCATCGCGAAC GAGGTCGCTGCCCAGGGCATCGCGAACGAGGACGCCGCCCAGGACATCGCAAAGGAGGAT GCCGCCCAGGACATCGCAAACGAGGACGCCGCCCAGGGCATCTCTAAGGAGGACGCCGTC CAGGGCATCGCGAACGAGGACGCCGCCCAGGGCATCGCTAAGGAGGACCCCGTCCAGGGC GTCGCTAACGAGGACGCCGTCCAGGGCGTCGCTAACGAGGTTGAAGTCCAGGGCGTCGCA CACGAGGACGCCGTCCAGGGCATCGCTAACGAGGACGCCGCCCACGGCATCACTAACGAG GTCGCCGCCCACGGCATCGCGAACGAGGACGCCGCCCAGGGCATAGCACATGAGGATGCC GTCCAGGGCATCGCTAACGAGGACGCCGTCCACGGCGTCGCTAAAGAGGTCGCCGCCCAC GGCGTCGCTAACGAGGATGCCGCCCACGCGATCGCTAAGCCGGATGCCGCCCACGGCATC GCTAACGAGGTCGCCACCCAGGGCATCGCTACCGAGGATGTCGCCGACGGCATCGCTAAT GAGGACGCCGCCCACGGCGTCGCTAAAGAGGTCACCGCCCACGGCGTCGCTAACGAGGAC GCCGTCCAGGGCGTCGTTAACGAGGACGCCCTCCACGGCGTCGTCGCTAACGAGGATGCC GCCCAAGCGATCGCTAAGCCGGACGCCGCCCATGGCATCGCTAACGAGGTCGCCGCCCAC GGCATCGCGAACGAGGACGCCGCCCAGGGCATCGCTAACGAGGTCGCCGCCCACGGCATC TCTAAGGAGGACGCTGTCCAGGGCATCGCGAACGAGGACGCCGCCCAGGGCATCGCGAAC GAGGACGCCGCCCAGGGCATCGCACACGAGGACGCCGTCCAGGGCATCGCTAACGAGGAC GCCGTCCACGGCGTCGCTAAAGAGGTCGCCGCCCACGGCGTCGCTAACAAGGATGCCGCC CACGCGATCGCTAAGCCGGACGCCGCCCGCGGCATCGCTAACGAGGTTGCCGCCCAGGGC ATCGCTACCGAGGATGTCGCCGACGGCATCGCTAACAAGGACGCCGTCCACGGCGTCGCT AACGAGGTCGCCGCCCACGGCGTCGCTAACGAGGATGCCGCCCACGCGATCGCTAAGCCG GATGCCGCCCACGGCATCGCTAACGAGGTCGCCACCCAGGGCATCGCTACCGAGGATGTC GCCGACGGCATCGCTAACGAGGACGCCGCCCACGGCGTCGCTAACGAGGTCGCCGCCCAC GGCGTCGCTATCGAGGACGCCGTCCAGGGCGTCGCTAACGAGGACGCCGTCCACGGCGTC GCTAACGAGGACGCCGCCCAGGGCGTCGCTAAAGAGGTCACCGCCCACGGCGTCGCTAAC GAGGACGCCGTCCAGGGCGTCGTTAATGAGGACGCCCTCCACAGCGTCTTCGCTAACGAG GATGCCGCCCAAGCGATCGCTAAGCCGGACGCCGCCCATGGCATCGCTAACGAGGTCGCC GCCCACGGCATCGGGAACGAGGACGCTGCCCAGGGCATCGCGAACGAGGACGCCGCCCAG GGCATCGCTAACGAGGTCGCCGCCCACGGCATCTCTAAGGAGGACGCCGTCCAGGGCATC GCGAACGAGGACGCCGCCCAGGGCATCGCGAACGAGGACGCCGCCCAGGGCATCGCACAC GAGGACGCCGTCCAGGGCATTGCTAACGAGGACGCCGTCCACGGCGTCGCTAAAGAGGTC GCCGCCCACGGCGTCGCTAACGAGGACCCTGTCCAGGGCGTCGCTAACGAGGACGTCGTC CAGGGCGTCGCTAACGAGGTTGAAGTCCAGGGCATCGCACACGAGGACGCCGTCCAGGGC ATCGCTAACGAGGACGCCGCCCACGGCATCGCTAACGAGGTCGCCGCCCACGGCGTCGCG AACGAGGACGCCGCCCAGGGCGTCGCTAACGAGGACGCCGCCCAGGGCGTCGCTAACGAG GACGCCGTCCACGGCGTCGCTAACGAGGACGCCGCCCAGGGCGTCGCTAAAGAGGTCACC GCCCACGGCGTCACTAACGAGGACGCCGTCCAGGGCGTCGTTAACCAGGAGGCCCTCCAT GGCGTCGTCGCTAACGAGGATGCCGCCCAAGCGATCGCTAAGCCGGACAGCGCCCATGGC ATCGCTAACGAGGTCGCCGCCCAGGGCATCGCTAACGAGGTCGCCGTCCACAGCATCGCT ATCGAGGACACCACACCGTCCAGGGCGTGGCTAACGAGGTCGCTGCCCAGGGCATCGCTA CCGAGGATGTCGCCGACGGCATCGCTGAGGACGCCGCCCATGGCATCACTAACGAGGAGG CCGCCCAGGCCATCGCTAAGCAGGATGCCGCCCACAGCATCACTAACGAGGATGCCGTCC AGGGCGTCGCTAACGAGGACGCCGCCCACGGCGTCGCTAACGAGGACGCCGCCCAGGGCA TCGCTAACGAGGTCGCCACCCATGGCATCTCTAAGGAGGACGCCGTCCAGGGCATCGCGA ACGAGGACGCCGCCCAGGGCATCGCGAACGAGGACGCCGCCCAGGGCATCGCACACGAGG ACGCCGTCCAGGGCATTGCTAACGAGGACGCCGTCCACGGCGTCGCTAAAGAGGTTGCCG CCCACGGCGTCGCTAACGAGGACCCCGTCCAGGGCGTCGCTAACGAGGACGCCGTCCAGG GCGTCGCTAACGAGGTTGAAGTCCAGGGTGTCGCACACGAGGACGCCGTCCAGGGCATCG CTAACGAGGACGCCGCCCACGGCATCGCTAACGAGGTCGCTGCCCATGGCATCGCGAACG AGGATGCCGCCCAGGGCATCGCGAACGAGGACGCCGCCCAGGGCATCGCACACGAGGACG CCGTCCAGGGCATCGCTAACGAGGACGCCGTCCACGGCATCGCTAAAGAGGTCGCCGCCC ACGGCGTCGCTAACGAGGATGCCGCCCACGCGATCGCTAAGCCGGACGCCGCCCGCGGCA TCGCTAACGAGGTCGCCACCCAGGGCATCGCTACCCAGGATGTCGCCGACGGCATCGCTA ACGAGGACGCCGCCCACGGCGTCGCTAACGAGGTCGCCGCCCACGGCATCGCTAACGAGG TCGCCGTCCACTGCGTCGCTATCGAGGACGCCGCCCAGGGCGTCGCTAAAGAGGTCACCG CCCACGGCGTCGCTAACGAGGACGCCGTCCAGGGCGTCGTTAACGAGTACGCCCTCCACA GCGTCGTCGCTAACGAGGATGCCGCCCAAGCAATCGCTAAGCCGGACGCCGCCCATGGCA TCGCTAACGAGGTCGCCGCCCACAGCATCGCGAACGAGGACGCTGCCCAGGGCATCGCGA ACGAGGACGCCGCCCAGGGCATCGCTAACGAGGTCGCCGCCCACGGCATCTCTAAGGAGG ACGCCGTCCAGGGCATCGCGAACGAGGACGCCGCCCAGGGCATCGCGAACGAGGACGCCG CCCAGGGCATCGCACACGAGGACGCCGTCCAGGGCATTGCTAACGAGGACGCCGTCCACG GCGTCGCTAAAGAGGTCGCCGCCCACGGCGTCGCTAACGAGGACCCCGTCCAGGGCGTCG CTAACGAGGACGCCGTCCAGGGCGTCGCTAACGAGGTTGAAGTCCAGGGCGTCGCACACG AGGACGCCGTCCAGGGCATCGCTAACGAGGACGCCGCCCACGGCATCACTAACGAGGTCG CCGCCCACGGCATCGCGAACGAGGACGCCGCCCAGGGCATCGCGAACGAGGACGCCGCCC AGGGCATCGCACACGAGGACGCCGTCCAGGGCATCGCTAACGAGGACGCCGTCCACAGCG TCGCTAAAGAGGTCGCCGCCCATGGCGTCGCTAACGAGGATGCCGCCCACGCGATCGCTA AGCCGGACGCCGCCCGCGGCATCGCTAACGAGGTTGCCGCCCAGGGCATCGCTACCGAGG ATGTCGCCGACGGCATCGCTAACGAGGACGCCGCCCACGGCGTCGCTAACGAGGTCGCCG CCCACGGCATCTCTAAGGAGGACGCCGTCCAGGGCATCGCGAACGAGGACGCCGCCCAGG GCATCGCTAAGGAGGACCCCATCCAGGGCGTCGCTAACGAGGTTGAAGTCCAGGGCGTCG CACACGAGGACGCCGTCCAGGGCATCGCTAACGAGGACGCCGCCCACGGCATCGCCAACG AGGACGCTGCCCAGGGCATCGCGAACGAGGACGCCGCCCAGGACATCGCAAACGAGGACG CCGCCCAGGGCATCGCTAAGGAGGACCCCGTCCAGGGCGTCGCTAACGAGGACCCCGTCC AGGGCGTCGCTAACGAGGACGCCGTCCAGGGCGTCGCTAACGAGGTTGAAGTCCAGGGCG TCGCACACGAGGACGCCGTCCAGGGCATCGCTAACGAGGACGCCGCCCACGGCATCGCTA ACGAGGTCGCCGCCCACGGCATCGCGAACGAGGACGCCGCCCAGGGCATCGCGAACGAGG ACGCCGCCCAGGGCATTGCACACGAGGACGCCGTCCAGGGCATCGCTAACGAGGACGCCG TCCACGGCGTCGCTAAAGAGGTCGCCGCCCACGGCGTCGCTAACGAGGATGCCGCCCACG CGATCGCTAAGCCGGACGCCGCCCGCGGCATCGCTAACGAGGTCGCCGCCCAGGGCATCG CTACCGAGGATGTCGCCGACGGCATCGCTAACGAGGACGCCGCCCACGGCGTCGCTAACG AGGATGCCGCCCAAGCGATCGCTAAGCCGGACGCCGCCCATGGCATCGCTAATGAGGTCG CCGCCCAGGGCATCGCTAACGAGGTCGCCGTCCACAGCATCGCTATCGAGGACACCACAC CGTCCAGGGCGTGGCTAACGAAGTCGCTGCCCAGGGCATTGCTACCGAGGATGTCGCCGA CGGCATCGCTGAGGACGCCGTCCAGGGCATCACTAACGAGGAGGCCGCCCAGGCCATCGC TAAGCAGGACGCCGCCCACGGCATCACTAACGAGGACGCCGCCCAGGGCGTCACTAACGA GGTCGCCGCCCAGGGCGTCGCTAACGAGGACGCCGCCCACGGCGTCGCTAACGAGGTCGC CGCCCACGGCGTCGCTAACGAGGACGCCGCCCAGGGCGTCGCTAACGAGGTCGCCGCCCA GGGCGTCGCTAACGAGGACGCCGTCCACGGCGTCGCTAACGAGGACGCCGTCCAGGGCGT CGCTAACGAGGTCGAAGTCCAGGGCATCGCACACAAGGACGCCATCCAGGGCATCGCTAA CGAGGACGCCGCCCAGGGCGTCGCTAACGAGGACGCCGCCCAGGGCGTCGCTAACGAGGT CGCCGCCCACGGCGTCGCTAACGAGGATGCCGCCCACGGCGTCGCTAACGAGGTCGCCGC CCAGGGCGTCGCTAACGAGGACGCCGCCCAGGGCGTCGCTAACGAGGTCGCCGTCCACGG CGTCGCTAACGAGGACGCCGCCCGGCACATGGCTAAGGAGGACGCCGCCCAGGACATCGC TAACGAGGACGCCGCCCACGGCATCGCTAACGAGGACGCCGTACACGGCATCGCTAACGA GGACTCCGTATACGGCATCGCTAATGAGGATGCCGTATATGACATCGCTAATGACACCGT ACAAGGCACGCTAACGAGGACACTGTACAGGACATCGCTAATGAGGACACCATACAAGGC ATCGGTAATGAGGACGCTGTATACGACATCGCTAACGAGGACACCCTACAAGCCGTCGCT AACAAGGACACTGTACACAACATCGCTAATGAGGGCACCATACAAGACATCACCAATGAG GGCGCTTTATACGACATTGCTAATGATACCGACAAGGCACGCTAACGCGGACACTGTACA CGACATCGCTAATGAGGACACCGTATAAGACATCGCTAGTAACTATCGCAAGAACAAAAA ACCAAACACTGCATATTCTCACTCATAGGTGGGAATTGAACAATGAGATCACATGGACAC AGGAAGGGGAATATCACACTCTGGGGACTGTTGTGGGGTGGGGGGGGGAGGGATAGCATC GGGAGATATACCTAATGCTAGATGACGAGTTAGTGCGTGCAGCACACCAGCATGGCACAT GTATACATATGTAACTAGCCTGCACAATGTGCACATGTACCCTAAAACTTAAAGTATAAT AAAAAAAAAAAGACATTGCTAGTGAGCACACTGTATACGACATCGCTAATGAGGATGCTA TATATGACATCGCTGATGAGGACGTTGTACACGACATCACTAATGAGGACACCATACAGA TGGCTTGAGCCCAGTAGTTTGAGACAAGCCTGGCAACACAGCGAGACCTCATCTCTACAA ACATTTTTTAAAAATATGCCAAGCATTGTGGCGCATGCCTGTAGTCCTGGCTATTCAGGA GGCTGAGGTGGGAGGATCACCTGTGCCCAGGAGTTCAAGGCTGCAGTGAGCTATGATCAC ACCACAATGCTCCAGCCTGGGCAACAAAGCAAGACTCCATCTCTAAAAATAAAATTAAAT TAAAAAAAAAGATCTTCGATGTAAAAGAGGTATGCTCAAATGCAATAAAATCATATAAGA AGGCCGGGTGTGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCAGGTGG ATCATGAGGTCAAGAGACTGAGACCATCCTGGCCAACATCGTGAAACACCGTCTCTACTA AAAATACAAAAAACAATCAGCTGGGCGTGGTGGCACACACCTGTAGTCCCAGCTATTCAG GAGGCTGAGGCAGGAGAATCGCTTGAACCTGGGAGGCGGAGGTTGCAGTGAGCCAAGATT GTGCCACTGCACTCCAGCCCGGCAACAGAGCGAGACTCCGTCAAAAAAAAGAAAAAAAGA AAACCATACAAGAAGCCCCTACGAATCTGGGTGAATCAGCAAAGGCTTCACAGGATAGGA AAAGGCCATGAGTATGAAAACATGAGTGGGGGGTTGGCCTAGATGGTAAACGAGGAGAGG ACAATACGGCCAGGAAGTGCATGTGCACAGCAGACGGGAGATGGAGGGCACGGCTGATTG GCAGCTACCTGTACTTTAATATTCCTGGAGTGTGACGTGTGAGGCAAACATGGGGTGGTG GGGTGAGTGCTGGAGATGAGGCAGACAGTGAGCCATCAGCATGTTAGTAACAGGGGAATC CATGGGCGTTTCATGCAGCTCCAAGGACCCTATGTGAGCTCATCAGACTCCAATTACCCA AATCTGTTTCTTACCAGTGGTAGTTAGGATTTCCGTAGGTACACTGGATGTCTTCCCAGG ACACCTGGAAGCCAGAAACAAATGGGGTCAAGCAGCCAGCTGACACAAGCACCCACAGAG GACAGGACACTATAATGTGCCCCTCATCCTCCAGACAAGCCCACCCCTAGCAACTCCCAA GAGCCTGGAGCAACAGCAGCAGGATGTCTTGTGCAGTGAAGTATGTTTAGCGCCCTTGTC CCTGTGTTTAAACTTGGTCATGCTTTTCCCAGAGGCCTAACTTACATATTTCTAACTCTC CTTGCTCTCTAGACATTTCCCACAAAGCAGGAAGCTGCAGAGCATGAGCCTAGAGCACCA AAAACTTAGAAGTGAAGTGAATTGAACCCTTGCAGAGGTGCAAACCACATGGGGACTTCA GACCGAAAAAGGCTTGAACCCTCATCAAAGGCCCCACACCCAGACAGCAGGATCACAGTG TGTCTTGATCTCAGAGACCCAGGCAGCCCTGAGCCCAGCATTTGCTAGAGGTGGCCAATC CAGGGCAGGAGGCAATTGTGGATGATGTTTCTTACCAAGCAAATATTGGAGTCATTGAAG CAGAACCATTTTCCATCCACAGCATTCCGGATGTAGACACAGTAATGACCGGAGTCTGCC ATTCCCACGTGCGCAATCACAGCAAAAAGCTCATACTGCCCTCCAGACTGGAAAGAGACA GGCACGAGACAGTTCACCCACCACCACAAACACGACTGCCTCACACATTTATACCTGTCC ACCCGAGGCCCGACTCTGGGACTTTAGAATATTAGAAAACAGTCCTGACCCACGGCCAGT GGGCACCCTCCAGGAGGGGCTTTCCTGAGTGTGGATCCTGAACTGTGAAGGCTCAGCAGC TTGGCAGGGCAGAGCACACAAGAGATGGAAGGCATGAGGTCATGAGAGAGGCCCCCAAAG CCAAGGCAAGATTAGAACTTTCCCCTGGGAGGGGTGGGGATCATCAGAACACAGTTCTGC TTCTCCACAAGCCCAGGAATGACCCTAACCTGAGCCACCGTACCCAGCCAAAAAAGTCTT TTGTAGGGGGAGGGGAGACAGAGGGCAAGGGCGGCACACGCAAAGCTGAGTCACCCACAG TGGAGGCTGAAATTATCCCTATTTCCATTTGCAGACTTTGTGCAGAACTTTACAAAATGC AGGGCAATATCTGTATCTAAATATTAGTTTACAAGAAATAAAAGGGTTATAGATGATGTT AAAAGATATCAACCAGCCAGTGCTGGCATATGAAAAAAATATTCAACCAAATCAACTGCA AGGAAAAGAAAAGGAGCGGAAATCTAAAAGGAACTTAAGAAAAGAAAGAAACTTAACCAA ATGCAAGTGTAGGCGTTGTTTGGATCTAGAGTCAAACAAACCACATAGATTACACAACAG TCAGGAAGTCTGAACGTTAACCAGATATTTGATGCTATTAAGGAATTGCAAGATGGAGAT GATTATCTTTTAAGTGCTTATTCTTTAGTGATACATAATGAAGTGTTTATAAATGAAATA GTATGATGTCTGGGATTTGCTTCAAAAAAATCCAGTACAGGGTTGGGTGAAACAAGAGGG TGGGTTATAGGAAAAGCAAGACCTGCCATGCGTTTTGGTAACTGTTACAGCCAAGGATTC ACTGTCTAACTCTTCCTTCACTACACACTTTGGTATCTATTTGTAATGTTCCATAACAAA GTCTTTTTTGTTGTTGTTGCAAAGATGGAGTCTTGCTTTGTTGTCCAGCTGGGCTCGACT CCTGGACTCCAGCAATCCTCTTGGCCCCGCAAAGAGCTGGGATTACAGGTGTGACCCACT GCATCCAGCCAAAAAAGTCTTTTTTAAGTGAAAAACAATTTTAAATGTACTTCATCGGGT AGAATCTGCAGGTAGAAATAAAAAGAACTAGAAGGAAAGAAAAAGAGAATCCTATGAAAT GTATACGGCCTAAGTAGGGAAAGAAATCATGTTATAATAGGAGCTATTGTAACGTATTAT CAGGTCTTAATTTTCTAAATGTCATGGTTCTAGAGCATAAATAGCCAAACAGACCAATAA AACAGAAGAGACACTCCAAAAATAGACCCGTACATATAGAAATACAAAATTTGTTATGTA GCATTTCAAATCTATGGAAAAAAAGATATTTGGGTCCTTTTTTTTTTTTTTTTTTTTTTT TTTTAAGTTTTTCTTGTTTTGTTTTTAGAGACGGGGTCTTGCGCTGTCACCCAGGCTGGA GTACAATGATAGATCATAGCTCACTGCAGCCTCAAACTCCTGGGCTCAAGTGATCCTCCA ACTCAGCCTCCCAAGTAGCTGGGACTAAAGGTTTGTGCTACCACACCCAGCTAATTTTTT TTTTTTTTTTGAGAGACGGGGGTCTCATTATGTTGCTGAAGCTGGTCTCGAACTCCTGGG CTCAAGCAATCCTCCTGCCTCAGCCTTTTGAGGTGTGGGATCACAGGCATGAGCCACATC ACCTGGCCTTGTGTCTCTTTTTTTCTCAGAATCTCTTGATAAGTTATTATCTCAGGACAA GTGAATTAAACTTTACTCTGATTAAATGTGGTCCAGTCAACTGGCTACTTTATGATTATC CTCAAAGTGGGCACAGGCCATCTGAGGAAGAAAGTTTGGTTGGTACTATGAACCCACAAT GACATGTTTGCCTATGCATTTCTATGTATTCCACATTATTTCCAAAGAAGCCTACTGAAT TCTGGATTCTGTCCCTCAGTGCTGTGAGGCATATGAACACAAATCCAATATTTAGAAAGA AGAAAGGCTTTTGAGGCTGGGGACAGTCTCTGAGGAACAGGTGGCTCCTAGCAGACTGGG GGTGAAGGAAGGGAAAACAGAAAGGACATGGCATCTGGGGAGAGGCAGGATGGCAAGGAG GTCGGGATCATCCCACCTGCTCCTCAGCATCACAAGACTCTCGCTTCATTGGAAGGATCT GGCTGAAATCCAAGCTCTGGGGGAAGTACAGGGAGTGGCAGATCTTTCTCGTCTGTGAAT TCCTGATGGAGAATCGCATGAGGTGGATTGTCAGGGTCTGGGGCAAATGGGTCAGCTTCA AGACCTGAAAAATCAAAATCACTCTGGAGATGAAGTCCACTTGGAATGAGAGAGTCAGAG AAATGTCAGGGCTAGCAACGCGTTCTTCCCAAAAAGAGGGGAGCAGGGCGACATCATGGA GTTCAAGGCTCCTGTTCACTGAATCTGCTGAGCTCCTCTGAGAATAATGCAAAACTTGGC TATTCTACCACCATGTGGCCCCCCACAAGAGGCACAAGGGCACAGGGCTCTGCACAGGTA CCAGGACCCAGGGAGCCACCACGTCTGCTCCAGGGAGCTGGTCTCGCCCCGAGGGAAATG CCCTGCCCGCTGCTCTCAGGCCCCCTCCTGCAGTCCCTCTCAGTGATGCGACATGAGAAG CCACATCCACGGCTCCAGATATCAGAACCACAGCGTCCCAGATACTAGCCAGACCCAGAT TCCCTTTCTCACCCATCATGGGATGTCCTCGGGGCCTCTGATGATTCTATCAGGAAATCC ACGGGGGGCAACCCCAGACTAGTGAAGGACAGATGACCCCTGAACAGCACAGGTTTGGAC TGTATGGGTCCGCTTATACACAAATTGTTGTAAATAAATACAGTCTGCCCTCCCCCATAG GCACGAGTTCCACATCTGCAACCAAATGCTCATCAAAAATAAAGTATCCCAGATGCACAA CCGCATAGATGAGGGACTGACTTTTCCTATACGTGGGTTCCACAGGGCCGACTGGGTGTG ACTTGAGTGTGTGTGGATTTTGGTAAAAGCAGGGGTCCTGGAACAAATCCCCCATATGCA CCGAGGGACAGCTGAGTCTGATTTAGGGAATGAGTACCTGTTTCCCACGGGTCTTCTTCC CACAGTTCTCACAGAAGCATTTGCTTTTGCTTGATAACTCCCTGGGCTGGAAGAAGCAGT GCAGGGCGTCCTCCTGTCCCACCCAAGAGAACAGGGAAAAAGCAGCGTTGGTATTTCCCA CCTAGAATACTATATTCGTCTGTTCTCACACTGCTGTGAGACAATAGCTGAGACTGGGTA ATTTATAAGAAAAGAAGTTTGATTGGCCCATGGTTCTGCAGGCCGACCGGAAGCATGGCA GCATCTGCTTCTAGGGAGGCCGCAGGAAGCTCCCAATCATGGCGGAAGGCCAAGCAGGAG CAGGCGTCTTACCTGGGAGGAGCAGGTGCAAGAGACCAAGTGGGGAGCTGGAGTGCAGGA GCGCGATCTCAGCTCACCGCAACCTCCACCTCCCGGGTTCAAGCGATTCTCCTGCCTCAG CCTCCTAAGTAGCTGGGACTACAGGCTGCACCACCACGCCCAGCTAATTTTTTATGTTTT TAGTAGAGACAGGGTTTTACCATGTTGGCCAGGCTGGTCTCAAATTCCTGACCTCAGGTG ATCCACCCACCTCAGCCTCCCAAAGTGCTGGGATTATGGGCGTGAGTTACCACACCCAGC CTGGCTCCACACTTTTAAACAACCAGATCTCATGAGAACTCACTGTCACAAAGACAGTAC CACGGGGGATGGTGCCAAACCATTCACGAGAAACACTCCCCCATGATCCAATCTCCTTCC ACCAGGCTTTACCTCCAACACTGGGGATTACAATTGAACAGGAGATTTTTGGGCAGGGTC ACAGACCCAAACTGTATCAGGTACAGCCTCCCCACACTTTGCAGCTGAGGGTTTCAGCTT TTCTTCTGGTTCTCCTCATATCCCCCTTAATGCCTGGCCTGGTGTGGTCCACTTGGCAGG ATCTCAAAGGGCACCTACTGACTGGAGGAACAAAACAGGAGGGGGCTCCTATTAAGGGAG CAGTATGAGGGAAGTGGAGGTAGGAGGGAAAAACATTTTTGCCAAAACAAAGGAGAAATT GGCCAGCGCCGTGAAAATGAAATAAGTGACTTTCCAGAAGCCAGAGATAGATCAAAGAGG CAAGGGGGAATTGGCCACACTGTGCTGTGTGGTGTGATGGTTTGAGGGTTTGTCCTCCCC AAAACCCAGGTTGAAACTGAATCCCCAGTGTGGCTGTATGGAAAGGTGGGGACTTTGAGA GATAATTGGGGAGGGCAAGGTGTCTCATGCCTGTAATCCCAAGGAGGCTGAGGCAGGGGG ATCACTTGAAGTCAGGAGTTTCAGATCAGCCTGGCCAACATGGTGAAACCCCATCTCTAC TAAAAATACAGAAATTAGCCGGGCATGGTGGTGGGCACCTGTAATCTCAGCTACTTGGGA GGCTGAGGCAGGAGAATCGCTTAACACCGGAGGCGGAGGTTGCAGTGAGCTGAGATTGTG CCACTGCACTTCAGTCTGGGTGACAGAGGAAGAATCTGTCTCCAAAATAAAAGGTAGGGG GGTTATTGCGTCATGAGGGCACAGCCCTCATAAATGAATTAATCCACTCATGGATTAATG GGTTAATCGGTTATCCTGGGAGTGGGACTGGTGGCTTCATATGAAGAGGAAGAGAGACTT GAGCCGGCACATTAGCATGCTCAGCCCCCTCGCCATGTGATGCCCTGCACTGCCTCGCCT CAGGACTCTGAGAGTCCCCACCAGCAAGCAGGTCCTCACCAGGTGCAGCCCCTCAACCTT GACTTCCCAGCCTCCAGAACTGTAAGAAATAAATTACCTAGTCTCAGATATTGAGTTATA ACAACAGAAAATGGACTAACACATACAGGTGCAGCAAATACCAAGAGAATCCCCTTACCA GTGTCTTCAGGGGCTTTGAGTCCACATCAAAAAAAGAAAGTCGGAGGGTGAGCATGCTGC TGTTTCTGCTACTCTCCATGGCACAGTCAAGGCAAATCAAGGAGTCCTTCATCCGGATCA TATACAGGGCCTGCAGTCTCTCCACCTGCAAGAGGGAGAGCGGAGAGGTGAGATGGGAAC ACCACCATAAGCCAGGTCAGGCCAACTGCCAGAAGGTGGAGCCAAGAGACAGAAGCTCCT CACAAAGGTGCAAAATAATACCAGTAACAATATTATGAAACTGTGATAAGTGTTTTATGT GGATTAACTCTTTTTTTTTTTTTTTTGAGACAGAGTCTCGCTCTGTCACCCAGGCTGGAG TGCAGTGGCGCGATCTCGGCTCACTGCAAGCTCCACTTCCCGGGTTCACGCCATTCTCCC ACCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGCCCGCCACCACGCCCGGCTAATTTTT TGGTATTTTTAGTAGAGACAGGGTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGA CCTCGTGATCTGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGGGATTAACTCTTT TATTCCTCTACACACTACGGGAACAACAGCTGAATGAATGAAATGAGCAGCATCTTAAGC TTCCGAGTGCTCTGGTTCTAGGTTCTTACCAAGTGCACATCAGCGATCTGGTCCTTAATC AGGTTCCAGAGTTTGAGGTACAGTTGGGCAGCATCATGTTGGACAAACACTGGCCACAGA GAAAAGGAACAGCCAATTAGTGAGGTCTTCAGAGTCACTTCTCATGCCCTGGTACTTAGA CACACATGAACACTGATCACTTTGTCTCTTCCTAGCCCTCCACCCCACTGCAAGGCTAAG TCGGCACGACAGAAGAAGTGATCAATAAACAAACAGTAAGTTTGGTATTGATTTTTGAGA ACGCCTCTTAATACTTAAGCTCCAGTGCCAGATGTAGTGGCTCATGCCTATAATCTTAAC ACTTCGGGAGGCCAAGGTGGGAGGATCGCTTGAACTCAGGAGTTTGAGACCAGCCTGGGC AACATAGCAAGACTCCGTCTCGATAAAAAATTTAAAAAAGTACTTGAGAGTAGCGGTGCA CGCCTGTAGTCAGAGCTACTTGGGAGGCTGAAGCAGGAGGATCACTGGAGCCCAGGATGT TGAGGCTACAGTGAGCTGTGATGTCACCACTGCACTCAGCCTGGGGAATGAGATGTCCAC AGGGACTTTGAAAAGCACTGATACATTCCTGAGGATCTAGAAGGCCAGGCGCATGCCCAA GTAAGACCTGAGAGGGCTCCGATCCCTCCCCTCTGGCTGACCACAAGGCCACATGCAAGA AAGCAATGAAGGCGAATACAGAATCATGAACTGCCCAGAAACGATAAAACAGCAACAACA ACAAACCCTGCTGTGGGGATCTCTGATGTCCAGTGTTGCCACCTTACATTATCTAAAATG TTCATTTTCCAACAAAAGAGACAAGACCTACAATGAAACAGAAAAGCATGGCCACCCAGG AAAACAGCAATCGTAGAACCTTCCACTGAGAAGCCCTAGATGTTGGCGTTACTAAACAAA GACTTTAGCCGTTATAATATGTTCAAAGAACTAAAGAAAACCATGTCAAAAGATGTAAAA GAAAGTGTGAAAATGCTGCCTCACCAAATAAAAAGTATCAATAAAGAAAAAACTATAAAA GAAGAACCAAGCAGAAATTCTTGAGTTGAGGAGAACCATAACTGAAATGAAAAATGTACT AGAGGAGGTCAATAGCAGATTAGAGCTGGCAAAAGAAAGAATCCATAAACCTGGAGCTAG GTTGGTTGAGGTTCTCCAGACTGAGGACGGAAGGAAAGCTGAACACAGTACCTATGCAGC GAAAAGCTGAGGAGCTGCTCACTGCGTGTAGAGCTCAACTAACTCTGATACAAAAAAAAA TGTGATACAAAAGGTTTTATACACAAACGTGAACTTATTCTGAAGCCAGCCTGGGGCAAG GAGCAAGACACATCCTGCCTTTTAATGTGCCATTTCTCCTTTGGAGGAGAGAGGGGCATT TTCATAAGGTGTGTTGTGGGGGTGGGGGGAGTGAGCAATGGCGGTTTTCTACCGGGCAGT TATCAAGTTCCCAGCAGCTGAGTTGGCGCCTTTCCGGGCAGAAAAGTGGCCGAGTGGGCA TGCTTTCTCCATGCCTTCCTAGTGGGTGAAAGACAAACCCCTAGAGGGTGGAAGTTTCGA AGCCACCCCCTGGCAGGGAGAGTTCCCTGGTGGGTATGCTTTGGGCTGCAAATCTACTGC CAAGTCTCAGGAAAGATCAGCTGGAAGATCTTGCCCTGTTCTCAATCAAACCTCCATCAG CCTACCTCCATTTACAGATTCTTCCTTTTGCCCAGCAGGGAATGTCTGGAGAGGGGTAGG CGAAAGGTGATATTTGCATTTCTAAAGGGCTAACAGGAAACAGGAAACACGGCCGCGGTG AGGGGTGAGGGATGGGAAGAGGAGAAGTGAAAAGAAAGTAATAAGAAAACTAATATAACT ATCTCTTAGAAAATGGGAGTACTCAGTTACACCCAGATGGGTTCACTAGTGAACGATGGG TTCTACCAAACATTTATGGGAAAAATTATACCAATTCCCTACAACCTCTTCCAGAGGACA AAAGCAGAGAGAACACTTCCTAACTCATTCTCTGAGGCCAGCATTATCCTAACACCAAAA CCAGACAGACATTGTAAGAAAAAAACCTAAAAACCAGTATCTCCCGTGAACATAAATGCA AAATTCCTCAACAAAATATTAGTGAATCAAATTCAACTATGTATAAAAAGAATCACAGAG GGTGACACAAGCGAGATGGTAGAGAAGGAGGCTCCTGGTGCTCCCCTTCTCCCATGGACA CACGGTAAAACTCTCATCTACACACAGATCAACTCCCTCTGAAAGAAACCCAGAAACCAG CTGAGAGAGGCCTGCACAGGGGGCAATGGGGAGAACACCCACATCGCAACCGGTTGGAAA AGCTGAGACACACTCGAACCACAAGCCCCACCCCAGACACAGTACCTTACTGTGAGGAAG GAATCCCTACTCCCAGCTTTCTCCTGGAGAGGGGAGGGTTTGGACCACACATAGGCAGCC CCAGCTTTTGCAGTTCCCACTCAAGGGCTTGGCTGCTGGGTCACCTAGCTCTGGGAGCAG ACATGGGTTAGCAGTTATGAATCCTCTGCGACCACAGAGGACAAAGAGGCAGTTTTAAAC GGGCACGTGAGCACTTCCAGCAGCTGTGCTCAGCACAGAGCAAACAGGCAGAAATGCCAG GCTCCCAGTTCCTCCCTGGAAGGATATGTCTGCGGGCTCTCCTAGCTGTTGCCCGAGGGA TGGGCTTCTATTAATAACTGGCCTGCACCTGGAGCCAATGAGGCAGGTAAACAATAGATT TCCAGGAGTCTGGACAGGCCTGTGGGCAAGTCCCATGCCTTCTCCCACCCCCTGCTGTAT GGTAAAATCAGGTCTCTAACTTCACACATTGAGTACACATTTACCCCTCTCAAATGAAGG TCTAGCTCCTCAACCACCGAGCTTTGGGAGCTGACAAGGCTCTGTATTTGTAAGTCCCAC AGGGGCACAGAGACCAAAGAACATCCCCCCAGGCCCAGTGCAGCGTGAACAGGCAAAAAG GCCCGGCTCCTACTTTCTCCCTAGAAGGAGTTTGTCTGCACAGTGCAGCTGCTGCCCTGG GGTTTCTATGCATCTGGGAGCTGACAGAGCAGGAAACCAGTGCTCCTCTGAGACCCTGAA CAGGCAGGTGAGCACCTCCACAGCTGCTCCCACTGGTTTGCTCAACAGATAGCGTCAAGC TTCCAACTAACCTGTCCGTCTCTAAGCAGAGAACAGCCAGCATTTGCTAGGCCCCTGGGG GTGACAAAGAGTAAAACAATGCATGAAGGAGTGTGCAGTTTAAACTTGAGTGCAGGCACT TGCCACAGATCCTCTCTCCAGCATTTTGCAGAGCGAGTGGCAGATAAACTCATGCTCCCA GCTTTTCCCTGAGGATAGAATAAACTGGAACACACATTTAATGCCCCAACGTCTCCAGCT GCACCTCAAGGGGCTGGTTTCTATCTCCCCTGTCTGGGGCACTGACAGGACATGACACAT TCTAATCTCCTGAGGGCCGCTAAGAACATAGATGACAGTTTGGACAACACAAAAGGTCAA GAGGTGCTCCGAGTGTCTGGCAGGGCTAATTGGTAAGTTCGTCTCTTATACAAGGCCCGT GGGACAAGACTGGGAGAGGTATTTTTCTTTTTTTTTTTTGAGACGGAGTCTCGCTCTGTG GCCCAGGCTGGAGTGCAGTGGTTCGATCTCGGCTCACTGCAAACTCCACCTCCCAGGTTC ACGCCATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACCCGCCACCACGC CCAGCTAATTTTTTGTATTTTTAGTAGAGACAGGGTTTCACCGTGTTAGCCAGGATGGTC TCCATCTCCTGACCTCATGATCTACCCGCCTCAGCCTCCCAAAGTGCTGGGATTACAGGT GTGAGCCATGATGCCCAGCCTGGGAGAGGTATTTTTCTTAACTAAAGTACAGAAAATAAC AGAGAATCAAGAACAATGAAGAAACAGAGAAATCTGTTCCAAGCAAAAGAACAAGATAAA TCTCCAGAAAATGCCCCCGGTGAAATGAAGATAAGTGACTTACCTTACAGACAATTCAAA ATAATGATCATGAAGATGCTTACCATGTCTAGGACAGCAATGCATGAACAAAGTAAGAAT TTCAGCAAAGAGACAGAAAAAAGTTCAAAAGAAATAATAGAGCTGAAGAATACAATAACT GAAATAAGAGCTCAAAAGAGAAGCTCAACAGACTAAATCAAGCAGAAGAAAGGATTAGCA AACTAGAAGATAGGTCACTGGAAATCATTCAGAGGAGCAAAAATGAAAAAGAATGAAAAA GAGTGAAGATATCTTAAGAGAGTTAGGGGACATCAAGCCGACCAAAATAGGCATTATCAG AGTCCCAGAAGGAGGAGAGTGAGAAGAGATGGAAAGCTTATCCAAAGAAATAACAGTCAC GAGTTGCAGATCAACATTTCAGTCAATGGCAGACCGCATGAGCACAGTGGCCATAGCATG CAGCCTAGCTGTGTAGTAGGCTGTCCCATCTAAGTTTGTGTAAGTGCACTCCATGATGTC CACACAATGACAAATTCACCTTCTGATGCATTTTCAGAACGTATCCCTGTTGGTAAGCAA CGCATACCTATAATGACTGAAAACTTCCCAACCCAGGGAAGGAAATACAAATCCACATCC AGGAAGCCCAAAGGACACCAATAAGACTGATCTAAAGCAACCCACATCAAGACACATCAT AATCAATTTCAAAAGTTAAAGACAAAGAATTCTGAAAGCAGCAAGAGAAAAGCAATGTAT TATACACAAGGGAAAACCCGTAAGTGTAGGATTTCTTCTGGGGGTGATGAAAATGTTATG AAATTAGATTGTGATGATGGAGGCACAACTCTGTGAATACACTACAAACCTTTGAATTGT ATGTTTTTAAATGGATGAATTTTACAGTATGTGATGCACATCCCAATATAGCTATCTAAA AAAAAAACCTGACCTATTTGTGAATTATTTTGTAGAACATGTTTTCTCTTTTTTATTCCT TTATTCTGTTTTCCTGCAGTTCCCTAACTGTGGTGGAATAAGCTTGGAGGAGATGCCTTC ACCTACTCTACTGAGAGGCAGAGAAAGGTTAGGTATGGAAGTAAAATGTCCATAAATAAT ATCAATTCTGCACCTCCAAAGATGTGTGCCTCTTACACAAATGTCTCTATAATCTGAGGG GTCCAGGCTACCTAACTTGCTTCTTGTTGCTTCACTGCCCATTAGTTCCTCTCTTCACTC TCTAAGCTTCCCATCCCCTTTGGATCAGCAGCCAGGAACCCAGTGGGAGGGTATCTTACG GGGCACGTTGTACTTCTGCAGGCAGTAGGCCAGCTCCAGGGGCCACACTGCTTTCTGCCG GCTGTCCTGCATCTTCTCCAGCAGCAGAAGCATCTGGAAAGGGACGCTTCTCCTCTGCTC GTCAGCTCCCCTGGGCACCGTGATCCTGCCACACAAGAACAGCCAGAAACCTCCTTACAT GTAGTTCTCAAAGCTAGGTGCACACTGGAATCACCTGGGAGAAATTTTAAATCTTCATAT CCAGGCCACTCCCATACAAATTAAACCAGAATTCTGGGTCCGGGGGGCGGGGCGCGGGCA GTTATCAGAATTTTTCAGTTCCTCAGATTATTTCAATGTGTGGTCAAGGTTGAGAAGCAC CATGTGAGTGAATCTGCTCTACAGGGGACCCTCTAGAGAGGGGGATCAGCATGACTGCTG CCTTGGCCCCTCTGCACCCTGCCCAGCAATCGTGCTAGAGCAGACGGCAGATGCACGAGG TCCTGGCTCCACATCTCGGGCAAGCAGCAAAATGTCCAGCCTAAAGCTCTCCAGACTGCA GAGGCACATGCTGGGTTAGGTCCTGGGAAGAAAGGCAACATGAAAGGCTGTCCGTGTGGT TTGGCAATGATTCATTTAATCACAGGCATTTTTCGGATGGGAAAAAGGAGAGAGCACAGG GCAGAAAAGGAGTGATAGAGAGAGACAGAGGGAGAGAGTGCATTGTGTGAGTGTTGGTGG AAGGAGGGGTGTGTACTCCCCACTGACTTACTCCTTCTCCGACTTTGGTCTAGTCCCAAC TCTGTCTTTTAACCAAGTCAGTGGCTGTCAATCCTGGCTGCATTTAAAATCACCCAAAGA GGGCTTTTAAAAAGTACCAATACCTGGCCGGGCCCAGTGGCTCATGCCTGTAATCACAGA ACTTTGGGAAGCTGAGGCGGGCAGATCACCTGAGGTCAGGAGATTGAGACCAGCCTGGCC AACATGGTGAAAACTCGTATCTACTAAACATACAAAAATTATCCAGGTGTGGTGGCGGGC GCCTGTAATCCCAGCTACTTGAGAGGCTGAGGCAGGAGAATCCCTTGAACCTGGGAGGCG GAGGTTCCACTGAGCCGAGATTGCACCATCCCTCTCCAGCCTGGGGACAGAGTGAGGCTT TAGCTCAAAAAAAAAAAAAAAAAAGTACCAATACCTGGGCCGCACCCCAGACCAACCTAA GAAGTTTTAGAAATAATGTGTAGGCCAGAATATGCAGAAGCCGATCTTTCCTCTGCCAGC TTTTGAGAGAAGATCTCAAAAAGTCCAAGTCACTGTGAATAACTGAATATATTAAAAATG AAATAATACAGGCCGGGTGCAGTGGCTCACGCCTGTAATTCCAGCACTTTGGGAGGCTGA GGTGGGTGGATCACGAGGTCAGGAGATCAAGACCATCCTGGCTAACACGGTGAAACCCCG TCTCTACTAAAAATACAAAAAAAATTAGCCAGGCATGGTGGCGGGCTAGTCCCAGCTACT CGGGAGGCTGAAGCAGGAGAATCGCTTGAACCCGGGGGGCAGAGCTTGCAGTGAGCCAAG ATCGCGCCACTGCCCTCCAGCCTGGGCAACAGAGCAAGACTCCATCTCAAAAAAAAAGAA AAAGAAATAATACAAACGCTTATCAGCCTGAAGAACAGTCTTACCTCTTCAATATCCTGG CGAAGTCCACATTCATTACAAACACCTGAATCAAGGAGTTAAGGCAGCAGGTCTGTCCAA TGTTGTGTAAACCAACCAGGCCTATAAGGGGAAGAGAAAAAAATGCTGAGGGCAAGGCCT AGGTAAAGAAGTTGACAAGGCTGGGCATGGTGGCTCACGCCTGCAATTCCAGTACTTTGG GAGGCCGAGGTGGGCGGATCACCTGAGGTCGGGAGTTCGAGACCAGCCTGGCCAACATGG TAAAACGTGTCTCTACAAAAAAATCCAAAAATCAGCCGGGTGCGGTGGCTCACGCCTGTA ATCCCAGCACTTTGGGAGGCCAAGGCAGGTGGATCACCTGAGGTCGGGAGTTCGAGACCA GCCTGATCAACATGGTGAAACCCCGTCTCTACTAAAAAATACAAAACTAGCTGGGCATGG TGGCACATGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGTAGGAGAATCATTTGAAACC AGGAGGTGAAGGTTGTGGTGAGCCGAGATCACACCATTGCACTCCTTTCTGGGCAACAAT AGTGAAACTCCATCAAAAAAAAAAAGAAGAAGAGAACTTACAGCAGGGTGTGGCGTTTCA CACCTGTAATCCTAGCACTTTGAAAGGCCAAGGTGGGCAGATCACCTGAGGTCAGGAGTT CAAGACCAGCCTGGGCAACAGGGCAAAACCCTGTCTCTACTAAAAATACAAAAGCTAGCC AGGCATGGTGGCAGGTGTCTATAATCCCAGCTTCTTGGGAGGCTAAGGCAGAAGAATCAT TGGACCTAATTTATTGATTTTTTTGAACTTGGAATATGGTGTCATGACAAAATCATGCCA CTGCACTCAATCCTGGATGACAGAGCAAGACTCTGTCTCAAAAAAAAAAAGGTTACTTTT TATTTCCTGAAAAGCTTGACTTCCTGACAATGAAAGTCCTAAGGAGCGAGTGGCAGAAGA CAGCTGGCAGCTGGCAGTCATTGGTGGGGCAAAGGCAGAGATACCACTGCTGACTCTACT GGGCCTCGGGCCTCAGCGCTCATGGGAAGGGACCCTCTTTCAAGCTTTTGGAGGGGTAAG GGAGGGCCAGGTAACAGTGCCTACATCTTAGGCACCGAAAACAACTGGAGCTGCTTCATG CAGTCAAGAATCCCCTGAAGAAGGAAGGCAGTTACCTCCCTATTCTCCAGATCTCTTCAG CTGGCCTGGCTCTCATCCTTTCCAAGACCTGGCTGTTTGCTTTGCATTTAGTTATATGAG CTAATTCATATGCTTCTAATAAATGCCTTTCATCCTTCTCCAAAGTTAGCCCTAGAGTAT ATTGCTGTTGTTTACAATCAAAAAGCCCCAGAGGTGTACTCTCCAATTCTCTTAATAACC TCATTTTTCTGTCATTACCCCTGTTTTAGTACAAGTAGCTGAGCTTCAGAGAAGTTAAAA GATGTGCCCAAGGTCGCACAGCTAATGAGTGTCACAGTCAGAATCTGAACCCAAGTCTGT CTGATGTGGGGTCATGACAAGAGCATGGGTTTTCTTGGGAGAAAAAAATGACTTCAAATC CCCACTTCCCGCATAAAAATGCACAATGGGTGTAGCAATGCCGAAGCCACAGAGCTATTG AGGATGGATGGAGGTGACGCATAGGAAAGCACATGGCCTAGGGTATGTATTCCCAACACT TTGGGAGGCCGAGGTGGGCAGATCACCTGAGGCCGGGAGTTCAAGACCAACCTGACCAAC ATGGAGAAACCCTGTCTCTACTAAAAATACAAGATAAGCTGAGTGTGGTGGTGCATGCCT GTAATCCCAACTACTTGGGAGGCTGAGGCAGGAGAATAGCTTGAACCCAGGAGGCAGAGG TTGTGGTGAACCAAGATCATGCCATTGCAGTCCAGACTGGGTGAAAAGAACAAAACTCCA TCTCAAAAAAAAAAAAAAAAGAATTAAGAAAAAATATTTGAACACTTCTTTTTATCCCTA ATTTATTCATGTTTTTGAACTTGGAATAGGAACACTACTGCCAACAAAAGTAATTAGAGA ATATAGATGTTAAATCTATTCTCTAAATGAATTATTGCTTATTTTAAAATTGCATATTAA TTAATTACAGGAAATTTGGAAAGTAAGAGACATATGAAGAAAAAAAGGTAAAAATGATTT GTAATCCCTCTGAGAGTTACCCTTTAATGCCATTTTTGGTATATTTCCTCTCAGTTCTTT CCCCCTCCACCACCAGTATGTGTGAGTAGTTTATGTTTTATAAAGCTGGGTTCATACAAT ATGATTTCTTGGCTGGTGTGGTGGCTCATGCCTGTAATTCTAGCACTTTGGGAGGCTGAG GCAGGCATATTGCTTGAGCCCAGGAGTTCCAGACCAGCCTAGGCAACACAGTGAGACCCC ATCTTTACAAAAAGTAAAAAATTAGGTGGTGGATATGGTGGCACTTGCCTGTAGTCCCAG CTACTTGGGAGGCTGAGACCAGAGGATCACTTGAACGGAGGAGGAGTCTGCAGTGAGCCT AGATTGTGCCACTGCACTCCAGCCTGGGTGACAGAGTGAGACCCTGTCTCAAAAAACCCA AAAAAGATTTCTCTATAATTTCTTTTTTTATTCAGTAAAATATTGTAGTTTTCTCTATCT TGATTAAACAGTCTAAAATGGTTTTAATGTCACCATAGTATTCCATTTTATAAATTTGTC AAAATTTATTTAACCCTTATTGTTACATTTCCATTTTTTAAAATAGTGCTGCAGCTGTTG ATTTGAAAAGATACTTTTTTCAGGTAGTTGACAACCAAAATATCCCCTTGATGGGTTCCA GCATACCCCATGCCTCAGTATCACACAGTATACCTTTGTAACTAACCTGCATGGTACCCC CGATTCTAAAATAAAAGTTGAAAAAAAATCATCTAGTGTTTTAAAACAAATTATGTATTA TTTTTGGGCCAAGCGTGGTGGCTCACACCTGTAATCCCAGTTCTTTCAGAGGTTGAGGTG GGAGGATCACACAAGAGGATCCACTGAGACCAGGAGTTTGAGACCAGCCTGGGCAACATA GTGAGACCCCCATCTCTACAGTAATTAAAAAAAAAATTAGCTGGGCATGATGGCATGCAC TTGCAGTCCCAGCTACTTAGGAGGCTGAGGTGAGAGGATTGCTTGAGCCCAGAAGTTTGA GGTTACAGGGAGCTATGATGATGCCACTGCACTCCAGCCTGGGTGACAGAGCGAGACTCT GTCTCAAAAAAATATACTATTTTAAATTTATAAATGACAATTGAATTACATTTATATAGT ACAATGTGATGTGTGATGTATGTATACTGTTAAAGAGAAAAGCCTTTGACTAAATTTGAC AGAGTTTAGTTGAACAGAAAAAAGATTCATGAACCAGGTAGCACTGAGAACCAAAAGTGG TTCAGAGATCTCTGCTCCACAGGTGGGCAGGGAATATTTATAGCCAGAAAACGTAAGTTT CATTGAGAAATAGCCTGTTGGTTACAGCTCTGCAGTTGCCTTGTTTGAACATGTTTTTGG CAGCTTGCAGCCTGTGAAGGGCTGAGAGCTCAGCTGCTGTGATTGGCTGAGACTGGGCTA CTTATTCCAAGGGCATATTCTCAGGTTAGGTTGCAGTTTGTTTTGTTTACCTATGTGAAC CCCCAATAGCTGAGACAGGTCTCAGTTAATTTAGAAGGTTTATTTGCCAAGGTTGGTGAC ATGCACCCATAACACAGCCTCAGGAGGTCCTGACATGTGCCCAAGGTGGTCAGAGCACAG TTTGGTTTTATACATTTTAAGGAGACATGAGACATCAATCAACATATGTAAGATGAACAT TGGTTCGGTCCAGAAAGGCAGGACAGCTCGAAGCAAAGGTGGGACAACTCGAGGTGGGGA GGGGGCTTCCAAGTCTTAGATAGATAAGAGACAAATGGTTGCATTCTTTTGAGTTTCTGA TGAGCCTCTCCAAAGGAGGCAATCAGATATGCATTTATCTCAGTGAGCAGAGGGGAGACT TTCAATAGAATGGGAGGCAGGTTTGCCCAAAGCAGTTCCCAGCTTGACTTTTCCCTTTAG CTTCTTGGGGCCCCAAGATTTATTGTCCTTTCACATCTATTAAGTTAGATTACAGTGTGC TATATATGGAGGCAGCTTTATTTTATTTTAATTAATTAATTAATTTATTTGTTGGTGATG TTGATTTCTCTTGTTTTAAAATCAACATTATAGGCTGGGCATGGTGGCTCATATCTGTAA TCCCTGCACTTTGGGAGGCCGAGGCGGGTGGATCACTTGAGGTCAGAAGTTCAAGACCAG CATGGCCAACTTGGCAAAACTCTGTGCCTACTAAAATATATGAAGATTAGCTGGTCACGG TGATGCACACCTGAAATTCCAGCTACTCAGGAAGCTGAGGCAGGAGAATCACTTGAACCC AGGAGGCAGAGGTTGCAGTGAGCTGAGATTGCACGATTGCACTCCAGCCTGGGTGATAGA GTGAGACTCCAACTCAAAAATAAATAAATAAATAAATTAATTAATTAAATAAAATCAACT TTACGGAGAAAAATTTACATTCAAGAAAATACGCCCATTTTAAGTGTCCAGCTGGATGAA TTTTCAGAAATGTGTGTACCCTCATGACCACTTCCTCAATCATGATACAGAACATTCCCA TAACCTGGAAATCCCTGTGGCCCTTTGCTGGCAGTTTCCCCACCCAGCCCCAGGCAACCC CTGATCTGCCCTCTGTCACTGTAGACTAGTTTTGCCTTTTCTAGAATCTCATATAGAGGG AGTCATGCAGTATGTACTCTCTTGTAGCTAATGTTTTTGAGGTTCCTTGGTGTTACTGCA TTGTTTTCTATTGCTGAGTAGTATTCCGTTCTACTCAGTACCATAATTTGCTCATCTACT CTTCTGTGGCTGGACTTTCCAGTTTGGCGCTAATATGAATTGTGGAGGCAGTTTTAGACC AAATTTAACTTAACAATACAATATGAAAAGATTAAGTCAAGCTAATGAACATACCTGTTA CCTCACTTACTTGGCATTCTTTTAAGTTGATGCACTAGAAATTTTCTCTCTTAGACCGGG CACAGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGGGGATCATGAG ATCAGGAGATCGACACCATCCTGGCTAACACAGTGAGACCCCGCCTCTACTAAAAATACA AAAAATTAGCTGGGCGTGGTGGCAGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGC AGGAGAATGGCATGAACCCAGGAGGCAGAGCTTGCAGTGAGCCGAGATCACACCACTGCA CTCCAGCCTGAAAGACAGAGACTCCGTCTCAAAAAAAAAAAAATTTCCTTAGTTATTTTG AAATATTCATTATTATTGACTATAGTCTCCTGCTGTGCAATAGACCTCAAAACTTTTTTC TCCTGCTTGGCTGAAACTTTGTACCTTTTGAAGAGGAAGTCTCCATTCCCTTGTTCTGCC ACCTCGCCAGCTCTGGTAACCATTATTCTTCTACTGAATGAATTCAACTTTTTTTTTTTT TTTTTGAGATGTCTCACTCTGTCACCCAGGCTGGAGTGCAGTGGCACAATCTCAGCTCAC TGCAACCTCCGTCTCCCAGGTTTAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGG ATTACAGGCGCCTGCCACCATGCCCAGCTAATGTTTGTATTTTTAGTAGAGACAGGGTTT CACCATGTTCGCCAGGCTGGTCTCAAACTGCTGACCTCAAGTGATCCACCTGCCTCAGCT TCCCAAAATGCTGGGATTACCAGCATGAGCCAGGGCGCCTGGCCCAAATTCAACTTTTTT TTTTTTTTTATGAGTGAGCAGCAGCAAGATTTATTGCGAAGAGCGAAAGAACAAAGCTCC CACAGTGTGGAAGGGGACCCGAGTGGGTAGCTCCCAAATTCAACTTTTAAAAGATTCCAC GGGCATGTGAGATTATGTGGTATTTGTCTTTCTGTGGTATTTGTATTTCACTTAGCAGTA TGTCCTCTAGATTTATTCGTGTCATTCCCAATGACAGAGTTTTGTTCTTTTTTATTTTTT TGGAGATGGAGTTTCATTCTTGTTGCCCAGGCTAGAGTGTAATGGCATGATCTCGGCTCA CCACAACCTCCACCTCCCGGGTTCAAGTGATTCTCCTGCTTCAGCCTCCCAAGTAGCTGG AATTACAGGAATGTGCCACCACACCTGGCTAATTTTGTATTTTTTTTTAATAGAGATGGG GCTTCACCATGTTGGTCAGGCTGGTCTTGAACTGCCGACCTCAGGTGATCTGCCCACCTG GGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGCTCCCGGCTGAGTTTTGTTCT TTTTAAGGCTGAATAGTACTTCGTTGTATGCGTGCACCGCCTTTTCTGTACGCATTCATC TTAGGCTGATTGACTTAGGCAGATTCCACATCTTGGCTACTGTAAATGGGGCCGCAGTGA ACATGGGAGTGCAGACATCCCTGAACACACTGATTTCAATTCCCTTGGATATATTCCCAG AAGTGGGATTGCTGGCTCACATGATAGTTCTATGTTTAGTGTTTTGAGGAACCTCCATGC CATTTTCCATCGCGGCTGTACTACACACCGGCAGTATACTAGGATTCCCCTTTCTCCACA TCCTGGCCAAAACTTACCTTCCATCTTTTTCATAAAACTTGTTCTGATGAGAGGGAGATG TTATGTCACGGTGGTTTTAATTTGCATTTTCCTAAAGATTTAGGTTGCCACTGGTCAGCT CCAGGCCCCAGAGCTTCTTCAACTGAAGAAGTACTTGCTGTCTATAGTAAGCATCTCAAA CTTTAACGTGCATGTGGATCACCTGGGAATCTTGTCAAATGCAGCTTCCAGGTAAGTGGG TCTGCAGTGGGGCCTGATATTTTGCCTTTTGAACAAACTCCCAGATAATACTGATGTTAC TGGTCCCAGGACCTGCCTTTGAGGTAGCATTTTTTCATGAACCTATTGGCTGTTTGTATG TCTACTTTTGAAAAATGTGTTTGGTTCCTTTATTCATTCTTACATTGGCTTATTGGTTCT TTTGCTGTTGAGTTGATTGAGTTCCTCATATATTTTGGATATTAGCCCCTTTTCTAATGT ATGGTTAGCAGATATTTTTTCTCACGACGTGGGTTATCTCTTTGTTACTTGTTCCTTTTG CTGTGCAGAAGCTTTTCAGTTTGATGCCATCCTGTTTCTATTTTTGCTTTTATTGCCTGT GCTTTTGGGGACATACCCAAAAAATCATTGCCCAGATCAGTGTCATGGAGCTTTTCCTCT GTGTTTTCTTATAGTAATTTTACAGTTTCCGGCATAATGTTAAAGTCTTTGATCCATTTT GGGTTGATTTTGGGGCATGGTGTAAGATAAGGGTCTAATTTTATTTTCTGCATATGAATA GCCACTTTTCCCAGCACCATTTATTGAATAGAGTGTTCTTACCCCTTGGCGTGTTCTTTG TGCCTTTGTCAAAAGTCAATAGACAGTCAGCTGGGCACGGTGGCTCACGCCTGTAATCCC GCACTTTGGGAGGCCAAGGCAGGTGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGG CCAACATGGTGAAACCCCATCTCTAATAAAAATACAAAAAATTAGCCGGGTGTGGTGGCA GGCACCTGTAATCTCAGCTACTCGGGAGGCTAAGGCAGGACAATCGCATGAACCCAGGAG GCAGAGGTTGTAGTGAGCCGAGATCACTCCACTGCACTCCGTCCAGCCTGGGCAACATTC CTTCTGTGTTCTGGGATATATATTCTTGAGGGTATTCCCATCTGTGGATAGTTGCCAGTT AGATTTCTGTGGTGGGGAAGTGGAGCTAGAGTATTCTTTTCCTTTTTTTTTGAGACAGCA TCTCACTCTGTCATCCACGCTGCAGTGCAGTGGCATTAACATGGCTTCATTGATCTCTTG AGCTCAAGTGATCCTCCCACCTGAGTCTCCTGAAAAGCTAGGGCTACAGGCATGCATCAG CATGCCCGGCTAATTTTTAATTGTTTTGTAGACATGGGTTCTTGCTGTGTTGCCTAGGTT GGTCTCGAACTCCTGGACTCAAGCAGTCCTCCTGCCTTGGCCTCCCAAAGTGCTGGGATT ACAGGCGTGAGCCACTGCTCCTGGTCTGTTTCTTTTGTTTAGTATTTTTGTTTGCTTTGT TTCATGAGCTGCTATTTTGACTACAGTTTTAATATTTTTAGCAATTTTTTGGATATGCAG AAATTTAATTCTTATTCATCAAATATATTTTAGTATTTTGTGTGTTTTTTAGATTTCCTT TTTTAGGAGTCTTTCCAACCATAAACTGCCAAATAGAAATACACACACACACATACACAG AGGCTATGAATAGACAACTAATTGAAACAGAAAAAAATTGATTAATTAAAGCACCTATGG GATTTTTCTCCAGCCAAATTAGCATTTTTATTTAAAAAAATCTTTTAGTGTTATTTAGCC TACATAGGAAGAGGCTTACTTATGTAGTTGATAAGAGTGTGAATTGATCAACTTTTTGGA AAATAATCTGCCAGTATAATTAAAATTATAATTAAAATGTATCATAGGTCAGTTACACCA CTTTGAGAATCCTATACTACAGAAAAGAAGAAATATAAAACAAAAACATCAGTGGGATCC ATGTATATTGATGATTATTGTAGTCATCAGTGGTGGGAATTTTTAAAAACAACCTTAATA TCCATCTAATAGGGAATAAAGAAATCAGAACTTATCTACAATGCGCATTACTACACAGAA ATTCAAATGATACATTTGATCTACATTAGCTGGAATTAGTAATGTCCATGTACTCTAATG GGAGAAAGAAAATTACTAAATAATGTAAGATTTGAGCACGTTCTTTAAACAAAACAAATC AATAAAGCCCTAAAAGGACATACTATTGAAAGCAAGTCACAAAATCCAGAAATGGCAGGG AAAATGTTCAACAATTTGACTATGTAAAAATGAAAGTTTCTTCAAAACAAAGATAATATA AACAAAATAAAAGTTATGATACAGACCTTAAGAAACAAACTTGCTAATTATATGGAATTA ATAAATGTGCAGAATATATAAAGAATATCATTACATGAATTAGAAAACTAGTAAATCATA ATGAAATTCTCAGCGAGAAATACAATTGGCCAGTAAACATATGCAAAAATGCACAATCTC TCTAATAAGTGGAGAAATCCAAATTAAAAATGGAAATATTTCTTAATCATAAAATTGGCC AAGGTTAGCATTGGTCATTTCCTCTGTAACTAAGGGTGTGTGGAAATAGGAACTCTCATA TACTGATGGTGGGAGTATAATATCATTAAATTTTTTTGAAACACAGATTTTTAATTTTTT ATCAAAATATGGCCAGGTGCAGTGGCTCACGCCTGCAATCCCAGCACTGTGGGAGGCCAA GGCAGGCAGATCATGAGGTCAGGAGATCGAGACCATCCTGGCTAACATGGTGAAACCCCG TGTTATTTGTAAAAATACAAAAACAAAATTAGCTGGGTGTGGTGGTGGGCACCTGTAGTC CCAGCTACTTGAGAGGCTAAGGCAGGAGAATGGCATGAACCTGGGAGGCTGAGCTTGCAG TGAGCCAAGATCACACCACTGCACTCCAGCCTGGGCAACAGAACGAGACTCTGTCTCAAA AAAATAATAATAATATTAAAAATTTATACTTTGCCTCAGTATTTTATTTTTAGAAATCAA TCATAAAAGAAAAAACCCACCTAACTAAGGTACCAAAAAGATGAATATACAGGTATGCCC AGGATAGTGTAGGTAACGGTAACAAATAACTGAATCCAACATAAAAATTCCACAGTGAAA AAGTGACCAAATTATGGTCAGATACCCTGTTGAATAATGATGCAACTATAAATAAGAATG AGAGAGACCTATATGCACTCATAAGCAATGATCACCACATTCTATTTTATATGGAAAAGC CAGTTAGGGAACAACACACAAAGCAGGATTACGTTATGTAAAAACATGGGCATGTCTATC TGTGTGTAAGTGGATGTAGACGACTGAGTGGGCTGGGAGCTGGCGGTGACACATTTCATT CTACAGACCTAAGCAGTATTTGAATTTTTATAACAATAGCATATGTGTTTCTTATGTGAT TTACTAGAAACAATTTAATTTCTTCAGTTGTAAAAGGTGTATTTTAAACCTATATAAAGG TACATGGAGATCGTCATTCAGAATAACAAAATAAAAAGCAATATAAAGACACAAATAGAT ATAGTACAATATTAAATACTACAATATTCAAGATGATATTAAAATTATATATATCTACAT CTATATGCATGTAAGGATCTCAGTAAATGATATTGTCTAAAATTAAACTGTGGTAATTTT TGCAGACCTCTGTGAATATATTAGAAACCATTGAATTGTGTGTACACTGTGTGTGGGTGA AATTTATTGTATTTGAATTATATCTCAAAGTTCTGTTAAAAAATGAATGGCTTGATATTC TAGTGCTAAGGGATGGTTTTATTTAAAACATAAACTAATAGTTTCCAGCAGAGTCCCTTA AAAATATTACAGCAGGGATTTCCAGATCACACTCACTCCCACCTTTTTAGAATTTTTGAC TATTTTCCAAAGTATTGAATTCAATGAGGTGCAGGTGGAAGAAAGCGTTTACAAAGTCAG TGGGTTACGGGAGAGCTGGTGTGGGTTGAGGCAGAGTGGGGAGAGAGGCTCCTTTCAAAT GGAAGCCTCTGGAACCCACAGACGGCAAGCGTAAGGCAGGGCAATCTTCTGGAGACCTAC CAGAGAAAGGCTTTAGACCCAACACTCCCATTCACAAACATGGACAGGGGCATCTCAGTG TTTCCTTAACTTCACTCTTTCTATATTGACACAAGGAAATAATTAAAAGGGGGTAATTCT GGTTTAGTTTATTTCATTAAATTATCAACAAACAACTTTTGAAACAAAAACTATTAAAGT GGAACTCAATAAAATTACTAGGTTTAATAATCTGTATCTGTGACTCAGATACAGATCAGG CAAACCAGCTGTGGATGAAGCTCCCAGCTGTGGATGATGCCCCCAGACATGGATGAAGCC CCCAGCTGTGGATGATGCCCCCAGATGTAGATGAAGCCACAAATGATGCCTGTGTGGACA GACAGAATGATGGACAGGCAGATGAATGTGAGGGTTTTATGTGAAACAAGTCCACTTGGT TGTTGACAAGATGCTGTTTTAAAGTTTCATTTTGCCATACTTTGAATAGCTTTTCATTAG CTAAATTTAGCCACATGTAAAATCACTAAGGCAGACACCTGGAGTTCCCATATGAAAATC AAATGTAAACCAGCAGTGACCTACTTCAATGTGATCATCGGAAGTCAGAAGTTGAGCTCA TTTTTGATGTTTAAAGCCTGCATAATATTCACTGTGTTATTATTCAGTGTATTACTATTT CCTTCATGATGGAAATTTGGTTTGCCCCAACTTTCTATTCTATCAAAACACTGCTACATA GAAAATCCCCATGCACATATTTCTCCTAATTGTGGAAATATTTTACATAAAAGACTCTAG ACATGGGATGAAATTCCCAGGTTATTGGAATTTTAAAATAGATAGGTACTCCCAAATTGC CGTCTTACAAATTATATGAATTCGTAAGCTTCCAACTGTTATGGAGTTACCCATTTTGAG AAATCTGTGCTAAAAGGACCCAAACAATGCTGATGACAATGATCAGGATAATAAGTACGC TGGGAAGACAACAAAATGATTTAAATCTTAGACAAGTCATTCTAGGTGTCTCCACTGTTT CAGTTCTTGCATTCATTCTTGTGGTATCTTTTCCCTTTTACCAATAAAAAAGCTCCCTGA CATCACATTGTGGCAGTCCCCATGGTTTGCCGCAGTTACTGCGGGACTGAACGAAGGAGG ACGAATGAAGAAATGAAAACCAAGGAAAAAAGGAGCTGTTTAAAGAAGGGTCCAGGGAAG AAGAAGAGGGCTCCCAGCTTCTAGTGAGCAAGGGCAGCAGCCCTGAGCTTCTACAGCCCT TCATATTTATTGAGTAGAAAGAGCAGGGAGCAGGAGGTAATGATTGGTCAGCTTCTCAAT TGATCACAGGTTCACATTATTGCTAACAGGTTTCAGATGTGCCTAATCTCAAGAAACGCC GCGCCTGGGGCATGACTGCCCTCAGCATTCCCTCTGGGTGGCAGACGCAGTTTGCCAACA TTCTGCATTCATGAGAACAGTTTACTGTTTACTCATATAACCTCCAGTGGTACACCGAGT TGATCATGACCCTCCCTCTTTCGGCCTGCAACATCACATGAATCCAATGAGTATTGGTTA GTAAAATGCCTATGACTAGTCATCTTCATCTATGCAATTAAATATTAATTCATCAAACAC TTCAAATGTAAGCAATTAATAATTAGTGAATGAAAAATACATAATACATCAATTAGAAAA AAACTCTATTAAAAAGACATTTGTGTGATAAAAGAGATTGCCATTTTTGTATTTTTCTAC AAAGTTAAAGAAAACTAAGTCAGCTTATATAAGTGAATTTTAAAAGCCTTTAGGCCAGGC ATGGTGGTTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGTAGGCAGATCACCTGA AGTCAGGAGTTCGAGACCAGCTTGGCCAACATAGTGAAACCCATCTCTACCAAAAATACA AAAATTAGCCAGGCGTGGTGGCAGGTGCCTGTAGTTCCAGCTACTAGGGAGGCTGAGGCA GGAGAATTGCTTGAACCCGGGAGACGGAGATTGCAGTGAGCCGAGATGGTACCACTGCAC TCCAGCCTGGGCAACAGAGTGAGACTCCATCTCAGGAAAAAAAAAAAAGGGCATATATAT CCATTATTTCACTGGGCCTTCACACAGCCCTGCTAGTCAACCCTCGAGAATTGAGGAATC AGAGAACTTAGGAACAAGAACATGACCTGGGACTTCTTATGTAAGTGAGAATCTTAGGCT AAATTGTCTTGCGGTAAATGCCTTTCCCATCCTCAGAAGTCTATAGTGAGCAGAAAGCAG TAACAGCATCCTCCCTTCCACTCTTCCCTTCAGGCTGAACACATCCCTGTCCCTACAGCC TTTGCAGAGACACAGGTGACAGAGCACTCAATATAGAATCCCTGGGCTCACACTGGCTTT CCTACAGACACAGGGGATCCATGAGGCCCACAGTGACATACACATGTACACAAGGATGCA CGTACTGACACTGATGAGCCCTGGAAGTCAACACAGAGTGGCATACAGAAGAACACATGC AGGCACACACACAGCCACAACTGGACCTGCGAGGTCTACAGCCCCACAAATGAAAAGAGG TGCATACCATCTCACAACAAACTAGAACTTTGAGAGAACCACACACACACACAGGTCTAC AAATTCACCTGAACAAGACGCCGTCCATAGTCACTGGCGTAAGGCATCTACTCACTATCA CACAGAACACACTCAGGCAAGCAGACATAGATGATACATCCACAATCTACAGAGATGAAC TGCAGAGTTACATGCACAGATATGCCAGTCATACATTCAGATCTGTGTAAACAGTGAGGC ACACACACACATACACATGTGCACTTATACAGAAAGACTCAGACCCAGCCAGGCATGGTG GCTCAGGCCTGTAATCCTAGCACTTTGGGAGGTCGAGGCGGGTGGATCACGAGGTCAAGA GATTGAGACCATCCTGGCTAACATGGTGAAACCCCATCTCTACTAAAATTACAAAAAAAT TAGCCGGGTGTGATGGCGGGCACCTGTAGTCCCAGCTACTCAGAAGGCAGAGATTGCAGT GAGCTGAGATTGCGCCACTGCACTCCAGCCTGGACAACAGAGCGCGACTCCATCTCAAAA ACAAAAAAAGAAAGACTCAGACCTGACCTGTTAGTCAACAGCCAGATGCCAACAGATAAT CACATTAGACTCATCAGCAAGCACACCGTGCACACAAGTTCCACTCCCATGTACAGGTTC CTGTCCCATGTACACAAGCCTGCACACAAACACGTATCCAGACTCCAGAACTGTTGAGAC AGTGCCCACATACACACTCATAGAGTCACCTGTGCACAGGTGCACGTACACATTGTTTCA CAGAGGCACACCAGGACAGAAACACATAAGAAATGTGAGGCAAAAGCGGACCCACACCTA CACCTGTAAGATACACAGAGAAACAAACACATACACAAACTTACACATAGACTCATGCAC TGTGTAATCAACAGTTCTATACCCCACCAAGGCTCTTGTCCTGGTGTGTCCAGAATTGGT GGGTTCTTGGTTTCACTGACTTCAAGAATGAAGCCACAGACCCTCGTGGTGAGTGTCACA GTTCTTAAACGCAGCGTGCGCGGAGTTTCTTCCTTCTGGTGCGTTCGTGGTCTCGCTAGC TTCAGAAGTGAAGCTACAGACCTTCGCAGTGTTAACAGCTCATAAAGGCAGCGTGGACCC AAAGAGCTAGACACAAAAGTTCTCCACGTCCCCACTAGATTAGCTAGATACAGAGTGTCC ACTGGTGCATTCACAAACCCTGAGCTAGACACAGAGTGCTGATTGGTGCATTTACAAACC TTGGGCTAGATACAGAGTGCCAATTGGTGTATTTACAATCCCTTAGCTAGACATAAAGGT TCTCCAAGTCCCCACCAGAGTAGCTAGATACAGTGTTGATTGATGCATTCACAAACCCTG AGCTAGACACAGAGTGCTGATTGGTGTGTTTACAAACCTTGAGCTAGATACAGAGTGCCG ATTGGTGTATTTTCAACCCCTTAGCTAGACATAAAGGTTCTCCAAGTCCCCAGCAGAGTA GCTAGATACAGTGTCGATTGATGCATTCACAAACCCTGAGCTAGACACAGGGTGCTGATT GGTGTGTTTACAAACCTTAAGCTAGACACAGAGTGCCCACTGGTGTATTTACAATCCCTT AGCTAGACATAAAGGTTCTCCAAGTCCCCACCAGACTCAGAAGCCCAGCTGGCTTCACCC AGTGGATCCCGCACCGGGGCCGCAGGTGGAGCTGCCTTCCAGTCCCGCGCCGTGCGCCCG CACTCCTCAGCCCTTGGGTGGTCGATGGGACTGGGCGCCGTGGAGCAGGGGGCGGCGCTT GTCGGGGAGGCTTGGGCCGCGCAGGAGCCCACGGCGGTGGGTAGGCTCAGGCATGGCGGG CTGCAGGTCCCGAGCCCTGCCCCGCGGGGAGGCAACGAAGGCCCGGCGAGAAGTCGAGCA CAGCAGCTGCTGGCCCAGGTGCTAAGCCCCTCACTGCCCGGGTCCGGCTGGCAGCTCCGA GTGCGGGGCCCGCTGAGCCCACGCCCACCCGGAACTCGTGCTGGCCCGCAAGCGCTGCGC GCAGTCCCGGTTCCCGCCCGCGCCTCTCCCTCCACACCTCTCGGCAAGCTGAGGGAGCCG GCTCCGGCCGCAGCCAGCCCAGGAAGGGGCTCCCACAGTGCAGCGGTGGGCTGAAGGGCT CCTCAAGCGCGGCCAGAGTGGGCGCCAAGGCCGACTAGGCGCCGAGAGCGAGCAAGGGCT GTGAGGACTGCCAGCACGCTGTCCCCTCTCACTGGGTTGCTATTAAAAATGGGCGCTGAA TATAAAAATTAACCGGGCGTGGTGGTGCATGTCTGTAAGTCCCAGCTACTCCGGAGGCTG AGGTGGGAGAATAGTTTGAGCCCGAGAGACTGAGGCTACGGTGAGCCGTGATTGTGCAAC TGCACTCCAGCCTGGGCGACAGAGTGAGACCCAGTCTCAAAACAAAATCAAACACGGGCG CTGCCCTGGGCAGGGGAGGTCTGGGCACAGCTGTGCTGATGTGGGCAGTGGACAGGTGTC ACGTGAGAGCCTGTGGCCAAGCCTAAGCTGCGGTTAAGGTGGGAGGCTGAAGGGTTTGGA GTGTGCACCACCCCGGACCACCTCTCAGTAGGAATGTGGCAATGCACTTCGGTGCTGGAT TCCTACTTCTGGCTGTGTGATTTGCGACACGTTACCCCACGCACCTGTACCACAACTTCC TCATCTCCCAAATGGGATGACAGTAGTTAGCAAACCTCCCTGGACTGCTGTGAGCATTCT GCCAGAAAAATGTACTCCAAGCTCTTGAAGTAGGCCTGGCGTTTGGTTGGTGCTCAGTAC ATATTCAATGTTTCTGTCGCTGGCGTCATCACTGTGTTCATGGCAGAGCTCTGTGCCGCC AGGACTGGGCCGCCACCCAGACCTTCCTTTCCCGCCCCAGGGGTGAACTGTGGACAGGAC CCTCTGGGGGGACTAGGCCAAGGCTCTCCCACCTCTGGGGACCCGCGCCCATGGGCCTCA GATATGTGGCTCGAGAAGGGGGAGTGGGGGCAGGGCCCCTGGAAAACCCCTCGAGGCCAG ACCCACCACTCCCAATATCCGTAAGACCAGGGGCTTGAGTCTTTCCTGCAGACAGATGTG GGCAGGAACCCACCCGACTCCCAGCTACGTGGGGACGTGGACGAACTAACAGGACAAATT CTAGTTCCTGGACACCGCCCTCCCAGTATCCTCGCAATTAGACACCCATGCAGCGGCACT ACACTGATCCGCACACGGAGATACACAGCGACATCCGCCCGCAAGAGCATGGTGGGTGCC GGAGCTCCCGGCTGTGGGCCCAGGAACTACATTTCCTAGAAGGATGTGCTAGATACTCGT TCGCGTCGGGACGCAAGCACCGGCCCGAACTCGCTCTAGGAAATGGAGTCTGACGCCTGC GCGGCGCAAACGCTCCCGGGAGGTGTAGTTTGCGCCTATTTCGCGCAGGCGCGCTTTCCC GCAGCGGCCGCCTGCTGCTCTTTGTGGCAGTCGCAGTCCTTTTGTGGGAGTCCGGTCTGT CCACTTGCCGGTCCCTCAGACCGTCGGCGGTCTCTGTCCGCTTCGGGACCTGTCCGCTGG TCGCTCCGCGTCCGATGGCTCCTGGCCGCGGAACCTTAGGCCTGGCCCTGGTCTCCGAGC GCGGGTTCGCCGGGAGGAGCGTGTGGCGGGGGTGTGCCGGGGCGTGAGTGCGCCGAGCAT GGGGCTGAGCCTGGTGTGGGGAGTGGGTATCTGCGGAGCCGGCCTGAACCCCACCTCAGC CGGGCGCGGGGAGGGGGCTCCGTGCGTGTGATCGTGCAGCTGTGAGCGCGTGGCCGCCCC GCGGGGCTCCGCTGCAGGCCCCTCAGCCCCAGGAGCAGTACTCGCTCTTCAGGGCCTGCC CTGGATCCTGGAGGCTACACAGCTGCCCACTCCTCCTGGGGAGGCTGCCGTGGAGGCCAT GGAGATCCCTGCCCCGGAGCCCGAGAAGACAGGTACAGCTTCACTCTTGTAGTCAGTATG TCTGTGGATTTGCACTTGAGGATATTGACACTCAGAGAGATCAGGCCAGTTTCCCAGAAG CATCCAGCATCTTTTTTTTTTTTTTTAAATGGAATCTCACTCTGTCACCCAGGCGGGAGT GGTGCAATGGCTTGATCTCCGCTCACTGCAACCTCTGCTTCCCGGGTTCAAACTATTCTC CCGCCTCAACCTCCCAAGTAGTTGGGATTGCAGGCGCGCGCCACCACGCCCGGCTAATGT TTGTATTTTTAGTAGAGACGGGGTTTTGCCATGTTTCCCAGGCTGGTCTTAAGCTCAAGC GATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCATCGCTCCTGG CCTCCTGCTTCTTTCTTCCCCGCGCCCAATCATTTGTTCCCACTCAGACGTGCTTGATTG GGGGTAGCAGCTCTTTTCGTCATGAATTGGAGTGTCTGAGACACAGGCAGATTATTCCTC GATGCTGTTTCTGTTGGTGCTAGCTACTGGGGCTCGTTCCCCACCTGGCCTTGCTGGCAG GTTCTGCCTCCTCTCTGATTCTCACTTGTGCCACGACAGGTGGGCTTTAAGCTCAGCCGT GAGCCTGGTAGCCGTGACCTTGACTCATTTCTCCTTCGCCAGCTCTTTCCTCTCAGGATC CTGCTCTTTCCCTGAAAGAGAATCTCGAGGATATATCGGGTTGGGGTCTTCCCGAAGCCA GGTCCAAGGTGAGTGGCTGTGTGTTCTTCCTTCTTTATGAAGAGGGTTGGCACATTCCCT CCCCAGCCCTGGATCGTCAGCCTTCCACAGACCTTCACCCGGGTACCTGCTGGCCAGTCC TCAGGAAGCTCTGGGCAGTGGGAACAGCTCCAAGTGAAGAGTCCAGATGTTGCCCGAGGT GTGCCCTCGAGATGTGCTCTATTGAATGTCTCGCCCAGTCCAATTCAGTGGGTCAGTTGC TGGGGATCTCTCTGCCCAGCGCTGTGCTGGGAGCTCTGAGGAGTGGTTTTAGTGGAATCT GAGAAAGTAGTTGACGGCTCACATGGGGTCGCTTTGCCTATCCACGTGCTGGCCAGGGGT TGGTGCAGGATGTTTGCACGGTTGGTTTACTGGGACTGGAATCAGACAGGGGCACGTGGA GCAGCTCAGGAAGGGCTGGATTGCAGCACTTGGTTAAGAGTGGCGGTAGGGGACACATCC CTGTGTGCTTTCCTCTGTACCCAGCCCCTGGCATGGGGCCTTCATGGAGAGTCCTGGAGA TTTAGAGAATCTGGGGGCTCAGCAAGGAATTGGCCCTGGGAACTTCTGCAGCCAAATGGG ACCCAGATCCCTCCTTTTACAGACTGTGAACTCTGTTCCTCATTTTGTTTTTCTACCGCA ACCACCTCACCCCATGGTTGTATTTAGAAGGGTGCAGTTGACAAAGAGAGCACAGTAGAG TGAAAGATGTGGAGGAATAAGGGGTCTTTGGGTTTTTGTTTTTTTTTTAGCATTTTGTGA TTTTGTCAGTCTGCCACTCAAAAGAGGTGATGGCAGATTATAGCCAGCTAGAAACAGCTG CAAAACGTGTATTGTGAGCTGTGCCTCACTTCTGTAGTGTGTCACAATGCTTTATAGTCC TCAAGAAACTTGACAGTCAAATGCAATGCATGTTCCTTGCTTAGGGGATAGATCCAAAAA AATTGATAAAGGGCAGTTTGGGACAAGAGGAAATTTGAAAATGGGCTGTATCTTAGGTCA TATTGTATCAATATGAAATTTATTGCATGTGATAATAGTACTGTGATTATGTAAGAGAAT ATCCACAGGAGATACAAGCTGAAGACTGTAGAGTGAGACATCATGATGTCTGCAACTTAC CTTCAAATGGTTCAGCAAAAAAAAATGTGTGTGCATGTATGTATGTCTGTATGTGTCTGT GCAGCTCCGTCTAGGTGGTAGGGCCCTCATGCATGTGTTATTCCTATTCCTAAAATATTA GCTTGTTAGATCTTTGAATAACCAGTTGACTCTCCTGAGTACACATCAGCTTTGGCATTT TCTCTTGACAGTCTCATTCTCACTCACAGTTCAAGTTGTTCTGTCTCCTGGGACATCTTG TGTGGTCTGTTCTGACTTCTCTGCTAGTGTCATTACCACATCAGCATCTGATTCCAATGC ATACTGTCTGGGTTTTTTATTCCTTTCCCTGCAGAATGATGACACATTAGACATGGACTT CTCCCAGTGGCCAGAAATACCCATCAAAAAGGCCTGTGCGGGCCTCAGACCCTACCCTAC TCTTGCTGGTTTTGCTCCCCACACTGAAGCCATGTAGCCTCTGACAGCCTCACTGGCCAA CACCATATTCTGATACCTGTGGAGCTATTTTTTTGTTTTTCTTTCTTTTTCTTTTTTTTT TTTTGAGACAGAGTCTTGCTCTGTCGCCCAGGCTGGAGTGCAGTGGCACAATCATAGCTC ACTGCAGCCTTCAACTCCTGGGCTCAAGCAAATCCCCCACCTCGGCCTCCTAAGTAGTTG GGACCAACGTGTGTGCCCCCACACCTGGCTTGGAGCCTTGTTTCAGCCCATTTTCTCCTT CCCTCGGCTATCGTCATCCTTCATGCCTGCATGCCTCAGTTAGGGGTGTGTTCTCAGCAT TTGTCCTCATCTCTACCTCCTCATCTGAAATCCAGTGTTTTGGGGGGTAAGGTTTTATTG AGGTGTAATTTATATACAGCACAGTTGTACAATTTTAAGTATACAATTTGATGAGGTTGA GCAAACGTATACAGTTGTGTAACCAGCACCACAATCATGATATGGGACGTTTCCATGACC CAAAAATGTCCTCATGCCCCTGTGCATTCAGTGGTCTCCCCTCTCCCCTTGGTAACCACT ATAGTTTTGCTGTAGATTTTCCTCTTTAAGGATTTCATATGGAATTTATATTATGTGGTC TTTTGTCTCTGCTTTATTTCGCTTAGCATATGCTTTTGAGTTTATCCATATTGTTGTGTG AATCAGTAGTTTGTTCCTTTTTATTGGTGAGTAATATTCCATTTGTATTAGTCTGCTAGG GTTGCCATACAAAGTATGGCAGGAGGGCTTAAATGACAGAAATGTATTTCCTCACAGTTC CAGAGGCAAGAAATCTGAAATCAAGGTGTCAGCAGGGTTGATTTCTTCTGAGGCTTCTCC TTGGCTTGTAGATGGCCACCTTTTCCCCGCATCTTCACATGGTCTTACCTCTTCTATGTC TGTGTCCAAATTTCCTCTTCTTATGAGGACACCAGTTATATTGGATCAAGGCCCACTGCA TTGAATTCATTTTAACTGAATTACTTCTTTAAAGAACCTCTCTCCAGGCCGGGTGCAGTG GCTCACGCCTGTAACCCCAGCATTTTGGGAGGCTGAGGTGGGTGGAACACTTGAGGTCAG GAGTTCAAGACCAGCCGGCCAACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAATT TAGCTGGGCGTTAGTTCTCGCCTGTAGTCCTAGCTACTCAGGAGGCTGAGGCAGGAGAAT CTCTTGAACCCAGGAGGCAGAGGTTGCAGTGAGCTGAGATTGCACCACTGTACTCCAGCC TGGGTGACAGAGTGAGACCATGTCTCAAAAAAAAAAAAAAAAAAAAAAAACCTCTCTCGG AGTTACCAAGCATTGAGACATAAGAATTTGAGGTGAGGGGTATACATTTCATCCCATTGC TCCATTGGATGGAGCAAACTGCTCCGCAGTTTGCTTATCCATTCACCAGCCGATGGACAT TTAGGTTACTCATAGGCTTTTGCTATTACAAATAAACCCGCTATAAATATTTGAGTACAC GTATGGCTGTGCATTTTAATTTCTCTTGCATAAATACCTAGGTGTGAAATTGCTGAGCCA TATAGTAAATATATGTTTGTCAATTTTATTGATCTTTTCAAAAAACAATTTTTTTGTTTT ATTGATTTTTCTCTATTTTCTGTTTTCCATTTCAGAGATTTCTGATCCTTATTATCTCCT TGGTGTCTTAAGATTGAAACTTACTTTACTTACTTTAGTGTCTTAAGATTGAAACATAGA TATTGATTTGATACCTTTTTTCCTCATATAGCATTCAGCGTTGTAAATTTCTAAGCACTG CCTCAACTATATCCTACAACTTTTGTTATGTCACATTTTCACTTTTATTCAGCTCAAAAT AGCTTCTAATTTCCCTAGTGATGTATTCTTCAGCCATATGTTATTTCAGAATGTGTTGTT TCATTTCCTAATATTTAGGGCATTTTCCAGATAACTTTCTATTATGGATTTCTGATTTAA TTCTATTGTCATCAAAGAATGAACTTTGTGTGATTTAAAGTCTTTCAAGTCTACCAAGAT TTATTTTATTTCCCAGAGTGTGGTTTATCTTGGTAAATGTTCCACATGCCCTTGAAAAAA TATGTATTATGCCTTTGTTGGTTAGAGTGTTATGTAAATATCAGTTAAGTCAAGTTGAAT GATGGTGTTTTTTAATTGTTCTGTCAGTTTTTGCATTATGTATTTTGAAGCTCTTTTAGT AGATACATTTATATTTAGGATTGCTATGAGTTCTTAATGAATTAACTGTTTTATCATTAT GTAATATTCTCTTTGTCTCTGATAGTCCTCTCTCTGAAATCTACTTTGCTATTACTATAG GCACTTGAGCTTTTTTTTTTTAAATTAGTATTTGCAGGGTTTATCTTTTTCTATCCTATC ACTTTTAACCTATCTTTGTATGTAAAGTGGGTTTCTGTAGCTTGCTTTTTGAATTCAGTT TGACAATCTCCACCTTTTAATTGGAGTGTTTAAACTATTCGCATTTAGTGTAATTATTAA TATGGTTGGATTTACATGTCCTTTTTTTTTTTTTCTTTTGACAGAGTTTGGCTCTTGTCA CCCAGGCTGGAGTGCAGTGGCGCAATCTTGGCTCACTGCAACCTCCACCTCTTGGGTTCA AGCAATTCTCCTGACCTCAGGTGATCTGCCACCTTGGCCTCCCAAGTGCTGGGATTATAG GCGTGAGCCACCCCACCCGGCCTTGCTATTAGTTTTCTATGTCTTATGTCTTTTTTGTTT CTCATTTCCTCCCTTAATGACTTCTTTTGAGGTAAACACATACTGTTCTAATATACTCTT AATTCCTCTGATTTCATTAATATTTTTCACCTTTTAGTTATTTGATTGGATGCTGGACAT TGTGAATTTTACTTTGTTGAGTAGGTTTTTGGAATTCCTTGTTTCTGGGATTTGTTCTGG GATGCAGTGAAATTACTTGGCATCTATTTGATCCTTTCAAGGATCAAAGCAAGTCTGGAG TTGGGTGGTGGCCAGGATGCCCAGGCCCAGGGGGAGCAGGCAGGAAGGATTGGAAGGCAG TCAGGTGAGGACTGCAGGTAGCCTCTCTTAGTCCCTTGGGGCACAGAACTTGCTGATATA TACAAGGTTTCCTTCCTTAGAGTGTAATAACAGTTTCTAGTCTGGGATCGGATCCTTTTG CGTTACTGAGCTTTGAATAGAGTTGCTGGGATTTTTGTACTATTAATATTCTCTCAAATT TTTAAGTTTGAAATTCTTGACTCCAGGAAGCATAGCATCTCACCCAGGACTTAGAGCCTG ATACAACAGCCATGTACAGCCCCTGGGTCCCACCGCAAGTTGCCAGGGCCCTGTGGTGTC TATTCAGACCTGCTTTCCCAATCTAGAAGGTGGTGTGGGAATGCATGCCAATTCCCAGCC CCCCTCCCTGCCCCTGGTCCCCGAATTCAGTCATGGTCAGTGTCTACAGCACAACCTTCT GGCATGGGAGAAGGTCACCTGAGGTTAACCCTTTACTTGTAGGGTCCTTAATCAATCTCT GGCTCCTTTGGAACCATGGACACAGAATCAGTCCTGGGTGAGAACCTATATGTCATTTCA GGAATCGGTGAGTTTCAAGGATGTGGCTGTGGACTTCACCCAGGAGGAGTGGGGTCAACT AGACTCCCCTCAGAGGGCCTTGTACCGGGATGTGATGTTGGAGAACTACCAGAACCTTCT TGCCCTAGGTAAAATCCCCCCAGCCCCAGGCTGGGCGTCGGCTGTCAGCCTTCTTCTACA TGGTAGATATTAAGGAGAATCCCTTCAGTGCCGGCCCTGGTGCCAGATATATCATGGGGT GGGAAGCAAGGCCTGTGCCTTGGGGCACTCATGGCCAGGGGCCTCGGCCACGCCAGAAGT CCCCAAGGGACAGCGTGGGAGCTGGTAGCATCCTTGCTTGGTGTTTCCCCTGGTCCTGGG ATAGGGGCAGGGAGAGAAGAGAAGAAGGTGGGACCCTGGAGCCCAGCTGGCTCTGGAACA GTCCTCAAGCAATGAAGTAAGGACGTCAGCACACGTGGGGTCTTCAGAGTATACACTGGG ATTTGGGTTCCAGTCTGAGACCCTCACTGCTTTTGGCCCAGAGGACCTCCTGTTCCCAGA GAGGGTGTCCACTTCACAGGCATTGTCCTTGTTAGGGGGCGGCCTGTAAGGTCGACTGGG CCTGGAGAGCTGCAGCATGCCCAGCCCACTTTCTCTCCACGAGCAGGACCTCCACTGCAC AAGCCAGATGTGATCTCTCATCTGGAACGAGGCGAGGAGCCATGGAGCATGCAGAGGGAA GTCCCCAGAGGGCCCTGTCCAGGTGAGCAGAGGCACAGGTGGAAGGGTGCCAGCCCCAGC ACCCCTGGTGGCACCTCCTCCTATGGCCCCTATTTTCCTCCCTCAGTTTGCCCCGGCTCC ATCTCCCCTTTTCAGGTCCCCCGCCAGACCCTCCTGCCTGCCTCCCTTCAGCACGTACTG AGCACTGCCTGTGTGCTGAGACCTGTTCTGGGATACAACAGGGAACAAACCTGGCCACTG GGACCAAATGGTCACCTGCTGATGGGGATGGGGAGGCAGGAATAGAAATATTGAGCCATT GGTGTTGGCTGGGCAGCCGGGGGTGTGGGATTGGGGGCAGGGGGCCACCAGGGAAGGGGC AGCTGGCGTTGGTCAGTGTCAGGGTCAGCATGAGCATCAGCATCAGGGCCAGCGTCAGCG TCAGGGTCAGAGCCAGCATCAGTGTCAGGGCCAGCGTGAGCATCAGCATCAGGGCCAGTG TGAGCATCAGTGTCAGGGCCATCATCAGCGTCAGGGTCAGAGTCAGCATCAGCATTAGGG TCAGCATCAGCGTCAGGGTCAGGGTCAACGTCAGCGAGGGCTTCCAGGCTGTGGCCAAGA TGGCAGCCCTCCGTAAGGGAAAGGGGACTGGGGTGGGTGTTGAGCAGAAGGTAGTCTGCT TTTCTTCTCCAACATTCTTTTTGTAGACTTCTCTTCTGAGTTGGTTGATAAATAAGTGGT AAATTCGGCTTAATTTATTGGACAGTGACTGCTGAGCATGTTAGGTGTCTAGAAAGCTCG AGAAACACCTTTCTGTGGGAAAGGCAGCCGATAAGCTGTGGAGCACAGAACACGGGGGTC GGGGAAGGCAGCCCCTCCTCCATCCCCCTCCACTGCCACATACCACCCAGCCCCAACCTG CAACAGTGGCTGCAGCCCCAGCTTTCTGGTCTTTATTTCTCACCCCAGCCTCTGATGCTT GCTTTGTCCTCTTTAACAAGATTACAGGTTTCAAAAAATTCATATTTTTGTTTGATTCAT ATATGAATATACTGAAAATGAGGCTGAAAGGTTACTGAAATCCTGCATAATCTTTCCACC CAGAGATGACGGTTCTCTAATAACTGACCATGTCACTGATTCCTCTGGGCCCTTCCTCTG TGTGCGGATAGACTTCTAAAGCTACATGTGGCCAGGCGTGATGGCTCACACCTGTAATCC CAACACTTTGGGAGGCCGAGGCAGGCAGATCACCTGAGGTCAGGAGTTCGAGACCAGCCT GACCAACATAGAGAAACCCTGTCTCTACTAAAATTACAAAATTAGCTGGGCATGGTGGTG CATGCCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCAGGAG GCAGAGGTTGCGGTGAGCTGAGATCGTGCCATTGCACTCCAGTCTGGGTAACAAGAGTGA AATTCCATCTCAAAAAAAAAAAAAAAAAAAAATTAGCTGGGCGTGGTGGTGAGCACCTGT AATCCCAGCTACTCTGGAGGCTTAGGCAGGAGAATTGCTTGAACCCGGGAGGCAGAGGTT GCAGTGAGCCATGATCGCACCATTGCACTCCAGCCTGTGTGACAGAGCTAAACTCCATCT CAAACAAATAAATAAATAAAGCCTTATGCACCCTCGGAAGCACAGCACTCACGCCGGAGT CTGGCTACCACACTGGCCATGGCATAGTGAGCGTCCTCACGTGATGTTTACTGATTGCAC AGTATCCCTTAGATGAATGTGCCCTGCTGTGTTGTTGGCCCTATTGCTGCCAAAACAACT TTTTTCTGGCCTGGGCCTCTTCTCTCTGCTCTGTGCAACTTCCCTAGACTCTTACCTCTC ACTTGGTGTGGATGTGGTCTACACTATAGCAACACCAGCTGAGCTGCAGCCCTGTGTCAC TGACCATCTCGTGGGCCTCTGCTTGCATGTCTAAAACCAGACTTGGAACTGGCCCTTCCT CCTCCTTTATAAAAGCTTCCACCATCCACCCAGGCTTGGGTGTTTGCTGAGGTCTCATCC TCTCCCCCATTTCCTCTCTTGCCACCTTCAGTCAACAGCTAGGGCCATCAGTTTTAGCAC CTAAATGGCTCTTAGATCTGCTGCTTCCTGTTCATTTTCACAGCCTTGGTTCCAGTCTGA AGTTGCCATTTTTCACCTGAATTATTCAGATGGCCTCCCTAACTGGCCTCCCTAACTGCT GGCTCCAACCACCTTACACACCACCAAAGCCACCTTCAAGACAAACCATTATGCCAGGCA CAGGGACTCACACATCTAATCCCAGCACTTTGGGAGGCCAAGGCAGGAGGACCACTTGAG CCCAGGAGTTGGAGGCTGCAGCGAGCTGTGATTGCACCCCTGCACTGCAGCCTGGACAAC AGAGCGAGACTGTGTCCCCGCCCTCTGCTTCCCCCGCCCCCCGTCAAGACACCATATGAT CACATCACCCTCTGTGCAAAAGGCCTTTCTTCCCCGGCTGCCCTCTGAGTCAGGCATCTG GTCACCTCGAACCCATCCACCTTGCCAGGGTCACCTCTCAGCCTTCCTGCCTCTGCGTGT CTTGTCTTCATCTTACTGTCTCACCTCTGAGCTTCTGCACATGCTGTTCACTCTCCACCT GGCTACCTGCCATGTGTGCCATGGCCAGTTCCACTTGCCCTGCTCTTCTGCATGTGTTGG GGCCACAGGCAGCACCACCATGCAGCTCTGCCCGCTGCACTAGACTGCTCCCTTTTAAGC CTGGGGAGGACAGAATTAGAAATAAAATCTCCTGCCAGCTCAGAAAACCTTGCTATAGAG ATAGAAAAGAAAGAAAGCCTTCTTATTGAATAAGTGTTTAACCAGATGTGGTGTACAGGA GAGAAAGAAACCTCCCCCTTTGACAGAGGAGACACATCCAACCCAGCACACAGGGCAACT GTCTTCACCTAAAGGAAGAGTACAGCTTTTTGGGTTTTGTTTGTTTGTTTGTTTTTTGAG ACAGGGTATCCCTCTATTGCCCAGGCTGGGGTGCGGTTGCATGATCATAGCTCGAAGCCT CAACCTCCCTAGGCTCAGGTGATCCTCCCACCTCAGCCTCCTGAGTAGCTGGGACTATAG ACAGGTACCATCATTCCTGGCTAATTTTGTATTTTTTGTAGAGGGGTCTCACTGTGTTAC CCAGGCTGGTCTTGATCTGGGCTCAAGTGATCCGCCACCTTGACCTCCCAAAGTGCTGGG ATTACAGGTATGAGCCACCGCGCCCAGCCTCTTGTAGCTTTTTGACAAGCAGGAAGTTAC ATCTTGGACTCGACTCTTGGACTAGGCTCATTTTGCTTCTGAAAAGGTTTGCATGCGTCT CGAGACAGAGAAAGCATTTGCAATTCCCAGTGTTCTAAAGGGAAGCGCTGGGGTGTGGGG AGCCCTTTTTTCCTTTTGGCACCACAGACAATTTTTAATTCATTTGTTAGTCCTAGCTAC TCGGGAGGCTGAGGCCGGAGAATGGCGTGAACCTGGGAGGCGGAGCTTGCAGTGAGCCGA GATCGCGCCACTGCACTCCGGCCTGGGCGACAGAGCGAGACTCGGTCTCAAAACAAACAA ACAAACAAAAAACATGTATTTGTCCTGTGTATTGCGAGCACCTGCCATGGTATTGATACA GAGCAGTCGGTCAGCCGACGTGTCCGTGAATGAGTGTAATCCTCATCAACAGCTCCCAAG CTTGCCCCTTTCTCAGCCCCCTTGGTGCTTGTAGCTGTTCCTTCCGGGATTCCTTTGGCT TCTCCGCCTCTCCCGGTCTTTGCTCTCTGGTAGCTCCTTCCGTGTTCTTCCTCTCCCTTC TTCCCGCTCTTGCTTGCCCATCTCATCCTGGGCGCTCCCTTCTCTCTGCCTGCACCTTCC AGCCTTGGCCCTGGTGGCCTCTCCTCTCTTTTCTGGGCTTATCTGCATCTCCAGGCCAAT TCTCAAATCTGAATTCTAGGCCAGGTTTCCGCTTGCTCACAGAAAATCATTTTCAATATC TTACAGAATGGGAGCTGAAGGCGGTGCCCTCTCAACAGCAGGGCATTTGCAAAGAAGAAC CGGCCCAGGAGCCCATCATGGAGCGGCCCCTCGGCGGGGCGCAGGCGTGGGGGCGCCAGG CAGGTGCTCTGCAGAGGAGTCAGGCTGCGCCCTGGGCGCCCGCACCTGCCATGGTCTGGG ACGTCCCTGTAGAGGAATTCCCCCTCAGGTGTCCCCTCTTCGCCCAGCAACGCGTTCCCG AGGGGGGACCCTTGCTGGACACACGCAAGAACGTCCAGGCCACTGAGGGCAGAACCAAGG CCCCCGCGAGACTGTGTGCAGGGGAAAACGCCTCCACGCCAAGTGAGCCAGAAAAGTTCC CCCAGGTGCGCCGGCAGCGCGGGGCGGGCGCCGGGGAGGGCGAGTTCGTGTGCGGCGAGT GCGGGAAGGCGTTCCGCCAGAGCTCCTCCCTCACGCTGCACCGGCGCTGGCACAGCCGGG AGAAGGCTTACAAGTGCGATGAATGCGGCAAGGCCTTCACCTGGAGCACCAACCTTCTGG AGCACCGGCGCATCCACACCGGCGAGAAGCCCTTCTTCTGCGGCGAGTGCGGGAAGGCCT TCAGCTGCCACTCGTCCCTCAACGTGCACCAGCGCATCCACACGGGCGAGCGGCCCTACA AGTGCAGCGCCTGCGAGAAGGCCTTCAGCTGCAGCTCGCTGCTCAGCATGCACCTGCGGG TGCACACCGGCGAGAAGCCCTACCGGTGCGGCGAGTGCGGCAAGGCCTTCAACCAGCGTA CACACCTCACACGCCACCACCGCATCCACACGGGCGAGAAGCCCTACCAGTGCGGCTCCT GCGGCAAGGCCTTCACCTGCCACTCATCCCTCACCGTGCATGAGAAGATCCACAGCGGGG ACAAGCCGTTCAAGTGCAGCGACTGCGAGAAGGCCTTCAACAGCCGCTCGCGCCTCACCC TCCACCAGAGGACGCACACGGGCGAGAAGCCCTTCAAGTGCGCCGACTGCGGGAAGGGCT TCAGCTGCCACGCGTACCTGCTCGTGCACCGGCGCATCCACAGCGGCGAGAAGCCCTTCA AGTGCAACGAGTGCGGCAAAGCCTTCAGCTCCCACGCCTACCTCATCGTGCACCGGCGCA TCCACACAGGCGAGAAGCCCTTCGACTGCAGCCAGTGTTGGAAGGCCTTCAGCTGCCACT CGTCCCTCATCGTGCACCAGCGCATCCACACCGGTGAGAAGCCCTACAAGTGCAGCGAGT GCGGCAGAGCCTTCAGCCAGAACCACTGTCTCATTAAACATCAGAAAATCCACTCCGGGG AGAAGTCGTTTAAGTGTGAGAAATGTGGGGAGATGTTCAACTGGAGCTCGCACCTCACTG AGCACCAGAGGCTGCACAGCGAGGGGAAGCCCTTGGCCATCCAGTTCAACAAACACCTGC TCAGCACATACTACGTGCCTGGCAGCCTGCTGGGTGCAGGGGATGCTGGACTGAGGGACG TGGATCCCATCGACGCGCTGGATGTGGCAAAGCTCTTGTGCGTGGTTCCCCCCAGAGCTG GCAGGAATTTCTCCCTGGGGAGCAAACCTCGAAACTAACATGATGTGCTTTGGTGTCAGT AGCTGCTTTCTGAGCTACTCAACAAGGAAAGCACCCTGGTCCTCCCTGGCTCCTAGATCC AGACCACCTTCCTCCAGGTGTGGGAGCCTTGCCTTATCACCCCCATCAGGTCTGCATGCC AGGGTGCCTCCTCTAGTTAAAGTCAGTCACCTCCCCAGAAGGGCCACACTCCAGGAGGAG TGTTGAGAGTCATTTGAGGTAGTCTTGCCACCTGTTTTCCTTGATGGGCCTGGAAGTTGT TGACAAGGGGAAAGATCTTTCTTGCCAATAAAAAGAAGGGATATCGTTGGGTGCCATGGC TCACACCTGTAATCTTAACACTGTGGGAGGCCAAGGCAAGGGGATCACTTGAGCCCAGGA GTCTAGGACCAGCCTGGACAACATGGTGAGACCTCGTCTCTACAAAAAATGCAAAAATTA GCCAGATGTGGCGGCATGTCCCTGTGGTCCCAGCTACTCAGGAGGCTGAGGTGGGAGGAT CATTTGAGCCTAGGAGGTCAAGGCTGCAGTGAGCCATGATTCACAGCACTGCACTGCAGC CTGGGTGACAAAGCAAGACCCTGTGTGAAATTAAAAGGAGGTATATCAACTGTTGTATCC TCGGACGGGCTCCTGACACGGCTTTAATCAGGAGTTTCCTCCATAAACTATTATTTTCAG AATAATAATAAAACAAGAATTATGACTAGCATCACTTTCCAGTGAACATTATTATATTGC TAGAGAGGAGAATAATCTTGGGTGGTGGGCATTTGGAAAAAGTGAATTTCCTGGACTTAA CTCATGTAAATAGCTCTACTGCAGAGCTGTGTGTTTAGTGACAGTGCAGTCAGGGGCATT CCCACAGCTGTCACAGCACGGCCCAGCATCATTGTAGCCAGATCCTAACATGCCAACATC ACCTCTTGCCATTTAGCCCCTAGTGAGAAATTGGGAGCTACCAGGCAGGTGCCCAGTGCA TTCAGGGAAGATGGGCACAACATCAGGATGGGTGTGTCTGGAAGCTCCTCTTCACTGACC AGGGCTGGGCAGGGCCACCCTGGGCTGGTGAGGCTGCCCTGCAGGGCTTCTCACTATGTA GCACCAGTCACCAGCCCAGGTCACAGAAGAAGCCCCTCCCAGCACCCACTGGAGAGGGGA AGCTGAAGCCCAGGGAGACGGGCTCCATGGTGGTCCCACATGGAGCTGTTTTGCAAACCC TGGAAAAGGCGGCTCTCCCTGTCCCAACACTCTTCAGAGACAGGAAGACAGAGTTAATCT TGAATGAATGTTATTTCTACTTAGTCCTAAGCAAGAATAAGCTGCCCTCTTGGTGATCAT ACTTTATACGGAGTGCTATCGTTTATGAAATGCTTTCAGTATGCTTTTTAAATTATGCAG GCAAAATAACCACACTTCAGAAAATGTGGACTTCTGAAAAATAAAATCCCCATAATTCTT AATAAAACTGCCTTTTGCACTTTGATACATTACCCTCTGCTTTGTTCATGTAGAACTTTG CATTATTTTCAACACAGTGTATCTATAGTTTTTATCTTTTTTCCCTCTTTAGCTTTGTAG CAGATTTTTTTTTCAAGCATCTAAACTGTCCTCGTTATACTTCTTTTTTTTTTAAATTAT ACTGTAAGTTCTAGGGTACGTATGCACAACGTGCAGGTTAGTTACATATGTATACATGTG TCATGTTGGTGTGCTGCACCCAGTAACTCGTCATTTAACATTAGGTATATCTCCAAATGC TATCCCTCCCGCCTCCCCCCACCCCACAACAGGCCCTGGTGTGTGATGTTCCCCTTCCTG TGTCCATTGTTCAATTCCAACCTATGAGTGAGAACATGCGATATTTGGTTTTTTGTCCTT GCGATAGTGTGCTGAGAATGATGGTTTCCAGCTTCATCCATGTCCTTGTTATACTTCTAA ACAGAGGCACAGTGTTCTCTCTGGTCTCTTAGATACTCTTTTGTTGGATGTTGAAGTTAT TTCCATGGTGCAGTGGTGTGATCACAGCTCACTGAAACCTCAACCTCCCTGGCTCAGGTG ATCCTCCCACCTCAACCTCCTGAGTGGTTGGGACTTTGGGACTACAGGTGTGCGCCACCA CGCCCAGCTAAATTTTTGGGGGGGGCAGGGGGTCTCGCTGTGTTGCTCAGGCTGGTATTG AACTCCTGGTCTCAAGCGATCCTCCCACCTCAGCCTCTCAAAGTGTTGGGGTTACAGGCA TGAGCCACTGCACCCAGTCTATTGATCTTTCTTTTTGAAAAATATTTGAATATGCCCTTT GTCCCTCTTTATATTGGAGTCTTGATGTTTTTCTTTTATGTATGTAAAAACTTTTCCCTG ATTATGAATATTATATGTATACTGCAGAAAATTTTTAAAAATCACAAAATCATAATAAAA ATGACCCTCAGGTTCCAGCTCAGTGGCTCATGCCTGTACTTTGAGAGGCCAAGGCAAGAT TGCTTGAGCCCAGGAGTTCAAGACCAGCCTGGGCAACATAGGGAGATCCCATCTCTACAA AAAATACAAAAAAAAAAAAAAAATTAGCTGGGCATGGCGGTGCGTGCCTGTAGTCACAGC TACTCTGGAGGCTATGGTGGGAGGATCACTTGAGCCCAGGAGGTCAAGGCTGTAGTGAGT CATAATGGCACCACTGCACTCCAGCCTGGGTGACAGAGCAAGACTGTCTCAAAACAAACA CCTTCAGAATCAGTTCATGGATCATTACCATTATTAACATTTAGATGCGTTTTCTTGCAG TTGTTTTCTATGCATGTATACATATCTTTTTTCTTTTAAAAGAAAATTAGGCTGGGTGCG GTGGCTCACGCCTGTAATCCCAGCACTTTGGGAAGCCGAGGTGGGCAGATCACCTGAGGT CAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCCCTGTCTCTACTAAAAATACAA AAATTAGCCGGGTATGGTGGCCGGTGCCTGTAATCACAGCTAGTCAGGAGCCTGAGGCGG GAGAATCGCTTGAACCTGGGAGGTGGAGGTTGCAGTGAGGCCAGATTGTGCCATTGCACT CCAGCCTGGTCAACAAACAAGAGCGAAACCCTGTCTCAGAAAAAAAAAAAATTAGATTCA TATTCTACATATTATTTTTATCATTTTCCATTTAACATTTTATGATAAGCATTTTCTATG TTATAAAATATTTTCAGACATAATTGGCCACATCATTTTCTGTCTTACAGTTGTATCTTA ATTTATCTATTCCCTTACTATTGAACATCAAGATTGTTTGTGCCTATAAATAATTATTTA ACATAGGCCTCTGTTCCCATCTTTGATCATTTCCTCTGAGTACGTTCTGAGAAATGCAGT TAACTGGTGAAGGTTATGAACACTGAAGCTCGTTGGTGTTCCTGGGTGGAATCTGATCTG GTGGACCTGCTGTGAGCTGTTCCCATCGGCTCTGACATGACGCTGTGAGCTGTTCCTCCC TGGACGAACATGTTCCTCCCATGACGCTGTCACCACTCCCTGGCCAGCATGGGGTCGTTC CGGCCTTGATTTTTATATCTGCCATTTTGAGGCTTTAAGAATGTTATTTGTCCCTGCTTG TTGGCCGTTTGTTTTTCTCTTCATATGTGTGGTCTGTTCTTGTCTTTTGTCCACTATTCT CTTTGGTTTATTTCACACACACACATACACAGAGAGACACACAGATGCATAAATGACATA CACATACACACATAAACAGATGCATAAGCACAGACACACACACACACATATATATCCCCT TGGCATGTACATTTTCTGTCTTTGTTACAGCATTTTTAATGCCCAGAGGTTTTCTTTTTC TTTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTTTATGAGATGGAGTCTCCCTCTGTTGCC CAGGCTGGAGTGCAGTGGTGTGATCTCGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACG CCATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACCTGCCACCACGCCCA GCTAATTTTTTGTATTTTTAGTAGAGGCGGGGTTTCACCGTGTTAGCCAGGATGGTCTCG ATCTCCTGACCTTGTGATCCGCCTGCCTCGGCCTGCCAAAGTGCTGGGATTACAGGCGTG AGCCACCGTGCCCGGCCAAAAAGGACTCTTAAAACTCAACAATAAGAACATGAACGGCTG GGCACAGCCGTTCATGACACAGATGGCTGTGTCATGGGCCATAGAATAATTTAAAAAACA ATAAAAGTCTCAGCTGGGCACAGTGGCTCATGCCTATAAACACAGCATTTTGGGAGGCTG AGGTGGGAAGATCACTTGAGGCCAGGAGCTCGAGACCAACATGGACAACATAGCAAGACT CCATCTCTACCAAAAAAACAAAAAAAATTAGTTGGGCATGGTAATATGCACCTGTAATCC CAGCTGCTTGGAGACTTAGGCAGGAGGATTGCTTGAGCCCAGGAGTTCAAGTCTAGTGAG CTATGATTGTGCCACTGCACTTTAGCCTGGGCGACAGAGCAAGACCTTGTCTCAAAAAAA AAATTCTATTTTTTAATAAATGGACCTGAATATTTTACCGTAGGTCTTCCCAAAGGGTCC AGGGATGCCATCACCCGATTGCATAGCTAGAAAGCGGATGGACTTTTTAGTAAGTGTGGG TTCTAGTAAGTGTTGCCGCACTGAAGAACATCTCTGGCCAGGCGCGGTGGCTCACGCCTG TAATCCCAGCACTTTGGGAGGCCAAGGTGGGTGGATCACTTGAGGTCAGGAGTTCAAGAC CATTCTGGCCAACATGGTGAAACACTTCCTCTACTAAAAGTACAAAAATTAGCTGGGCAT GGTGGTGGGTGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAAC CCAGGAGGTGGAGCTTGCAGTGAGCCGAGATCATGCCACCGCACTCCAGCCTGGGCAACA GAGCAAGACTCCGTCTCGAAAAACTAAAAAAGAAGAAGAACATCTCTGCTCTCAGCTTAG GTCCTGGGCCCCTAAAGTCAGCGAGGCAGCCATTGTCCTAGGGTGATACGATGACATGGA TTTGGGACTCCCTGGAGGAGGCCACACACACACAAAATGCAACTCTACAAGGGGAGGGAA CAGAAACTGAAACAGGTATCTGCAATGTGCCTGGATCCGAATTTCAAAATGTGTTGAAGA ATTATGGATTTACACCATGAGAAAAGCACTGGTTGGCTTTTGTGCCTGCTTCACTTTGGC TCTGAGTTAACTTCAAACACTGCCAAACACAATGCTAGCACATGGTGACCCTCAAACTAA TAATTTATAAGATGGGATCCACTTCCCCGGTGGAAGTGGCCACATGCTGCCCTCTCCCCA CCCCCACGGCATACTTGGGCATGACCTGCAGCCTTTTGTTGCCATCCAGGTTCGTGTTCA TTCTGCTGGTGCAGTAAATCAACCACTATGACATGGTTTTGCAAAAAAGAAAAGATTTAT TCACAAGGGCACTGATCGGGGAGGTGGATCCTTTTTAAGTCCTTAAGTCTTTCGTCCCCC GAAGATAAGTAACAGCTTCCTTCTGTGTGAGCATGGCTGGAGTTCATGGCATTTCACAAG ACACGTGTACAGAAGAGGGTAGCATTAGCATTTTCCAAAGGCGGAGTTTTTGGCCCTCCA GTGTCAAAAGGCCACCTTTCGGGCACTTGTGCAGGCCCAATTGAAGGGTTGGTGGTCTCA ACCAGTTTGAACTGGACAGGAGCTGGCCCAAGTTCCTGAAAAACAACTGAAGAAGCCGGC ACCATGGTGACTTATGCATGTTATCTATACAGTAGCCAGGGAAGGTTAAGTTTCAGCATT CAGCGGCAAGGCTTTCAGCTACTGTGGCCCTTAAGCTTCACAGAAAAAGGAGAAAAAAAA ATTAAAAACCAAATGACCCAAAAGCAAGCAGAGCAGGCAGACCTGGCCAAATTAACCCTT CAGTTTCACTTTCCCTTGCTCTGTTGGGCATTTGTGGCTTGGAAAATGGCCCCCTCCTGC CGCAGACAGACCCTTTACTGCTGACACCAATCATTACATTGATCTGTCGTGGTTCAGTGC ACCACTGCACTCCAGCCTGGGCAACAGAGTGAGACCCTGTCAAAAAGGAAAGGAAAGGAT AGGAAAAGGGAAGAGAAGAGAAAGGGGAAGGGGCAAGGGAAGGGGAAGGGGAAGGGGAAG GGGAGGAGGGAGGGAAAGAAAGGAAGAAAATAAAAGAAAGAAAAGAGAAAAGAAAAGAAA AAGAAAAAAAGAAAAGAAAAGAAAATGCACAGTGAGTTAGATTTGGCCTGGAGGCTGTAG CTTGCCAACTCCTCCTCTGGATTCATGGATTAGAGTTTAAGGAAGAGATGGAGAAGTATG GCAGAAGAACTGGAGTGAGGCCGAACCCCGTGGTGTTCTAGAAGAAAGGCTCTTGACACA AGCATCTGGCAGTTATTTTTCAGGGGAGAACCTACAAATTATAAGGCTGTGCAGCTCCCC TTACTAGTCAAGTGATTACACTTTTGTTTTCTTTTATTTTTTTTTAGAGACAGGATCTCC CTCTGTCACCCAGGCTGGAGTGTGGTGCCGCAGTCACAGCTCACTGCAGCCTCGAACTTG TGGGCTCAAGTGATCCTGCCTCAGCCTCCTGAGTAGCTGGGACTACAGACACACTCCACC ATGCCTGGGTAATTGTTTCTAGTTTTGTAGAGATGAGGTCTCCTTGTGTTGCCCAGAATG GTCTTGAACTCCTCGCCTCAAGCAATCCTCCCACCTTGGCCTCCCAAAGTGCTGGGATTA TAGGTATGAACCACCATGCCCGGCCTCATGTGACTACTCCTACAGAGGCCCTCGATGGCC TGGAATGATTGATTCCAGGACCCTGTCTTCTCATCCCAAAGCACCAAACCCTACTGACAA GGAAAGAGCCCCCATTTCAGAACTTCCATGGCCACCCCCCAACCCTGGGATTGGCAACTT TGCAACCTGGGCTTCTCCGATAATCAGATTTACGGTAAGACCCCAAAGAGGTCCAGGCGC TTCTAAAACAAGTTAGACAGAGAATCCAAACCCAAATCCTCAAGATGTGAGCCCACATCA CATCCCCCAAAATAGAGCATCCGTGACCACTCCAGCCTTGCTAGGGACACAGCATACTCC TTGACCCTCACCAAAGGACAATTGCTCTCACAAACACGGGGATCCACTGGTTCCCACGCA CTAAGCAAACTTTTATGGCCTTAATTGACACTGTTTCCCCAGAGACCCCACTAAATGTAA GGGAGGAACCCTCTCTGTTCCCTGGGACATAAGATTGAAGGCAAACAAGTCCGCCTTAGC CTGATCACTGGTGCAATAGCTTTGCAGAATATTCCAGTGGTCTCAGTTCTTTCTGCTCTT GAATTTCTATCACTGGGACAGATGCTCCAATTGGAATAAAACCCACACTTTGTCCCTTAC TGTTGGTCTTGGCAGATGAGACCCTGAGGACCTGCCCAAGCCATTAAAATCATTAATATG ACCCAAAATAAATTAAAACAGGGACTTCAAGGATTGAGAGAAGGGGTGACCATCCCCTCT GTTTCTCCATTTAACAGCCCAATTTGACGTGTTCTTCAAATGGAAAAGAACAAATGACAT CTCACAGTCGATGCTGAAACCTTAACCCAGGGGGCCATGGGGGCCCCAGCCCCTGCAGAC TGAAAGGATGGAGCCCATTCAGTCTTTTGCTGGCCGATATTTTGCTATTTTTGCTTCCTT GCAGTGTTCCACAGTACCTAATTCAACAGGCTCTCAGCCTCTACTCCCATTCTATTTATT GTGGTAAAATATATAACAAAATTTACCGTTTTAACCTTTTTCTTTCTTTTTTTTTTTTTT TGAAACAGGGTCTCACTCTGTCACCCAGGCTGGAGTACAGTGGTGCCATTGCAGATCACT GCAGCCTCAACCTCCTGGGTTCAAGTGATCCTCCCACCTCAGCCTCCTGAGTAATTAGGA TTACAGGCTTAAGCCACCATGCCCAGCCTACTTTAATCATTTTTTAATTTTAATTTTATT TATTTTTTTAAATTTTACTTTAAGTTCTGAGATTCATGTGCAGAACGTGCAGGTTTGTTA CATAGGTATACACATGTCATAGTGGTTTGCCGCAGCTGTCAACCCGTCATCTAGGTTTTA AGCCCTGCATGCCTTAGGTATTTCTCCTAATGCTGCCCCTCCCGCAGCCCCCCATCCTCA GACAGGCCCCGGTGTGTGATACTCCCCTCCCTGTGTCCATGTGTTCTCATTGCTCAACTG CCACTTATAAGTGAGAACATGCAGTGTTTGGTTTTCTGTTCCTGTGTTAGTTTGCTGAAA ATGATGGCTTCCAGCTTCATCCGTGTCCCTGCAAAAGACATGAGTTCATTATTTTTTATG GCTGCATAGTATTCCATGGTGTATATATGCCACATTTTCTTTATCCAGTCTATCACTGAT GGGCATTTGGGTTGGTTCCAAGTCTTTGCTATTGTAAATAGTGCTGCAATAAACATATGT GTACAGGTGTCTTTACAGTAGAATGATTTATAATCCTTTGGGTATGTACCCAGTAATGGG ATCACTGGGTCAAATGGTATTTCTGGTTCTAGATCCTTGAGGAATCGCCACAATGTCTTC CACAATGGCTGAACTAATTTACACTCCCACCAACAGTGTAAAAGCGTTCCTATTTCTCCA CAGCCTTGCCAGCATCTGTTGTTTTCTGACTTTTTAATAATCGCCATTCTAACTGGTGTG AGATGGTATCTCATTGTGGTTTTGATTTGCATGTCTCTAATGACCAGTGATGATGAGCGT TTCTTCATATGTTTGTTGGCTACATAAATATCTTCTTTTGAGAAGTGTCTGTTCATATCC TTTGCCCACTTTTTGATGGGGTTGTTTTTTTCTTGTAAATTTGTTTAAGTTCCTTGTAGA TTCTGGATATTAGACCTTTGTCAGATGGGTAGCTTGCAAAAGTTTTCTCCCATTCTGTAG GTTGCCTGTTCACTCTGATGATAGTTTCATTTGCTGTGCAGAAGCTCTTTAGTTTGATTA GATTCCATGCGTCAATTTTGGCTTTTGTTGCCATTGCTTTTGGTGTTTTAGTCATGAAGT CTTTGCCCATGCTTATGTTCTGAATGGTATTGCCTAGGTTTTCTTCTAGGGTTTTTACGG TTTTTGGGTTTTACATTTAAGTCTTTAATCTATTTTGAGTTAATTTTTGTATAAGGTATA AGGAAGGGGTCCAGTTTCTGTTTTCTGCTTGTGGCTAGCCGGTTTTCCCAGCACCATTAT TAAACAGGGAATCCTTTTTCCATTTCTTGTTTTTGTCAGGTTTGTCAAAGATCAGATGGT TGTAGATATGTGGTGTTATTTCTGAGGTCCCTGTTCTGTTCCATTGGTCTATATATCTGT TTAAAAATATGGAACACTTTGGCTGGGTGCGGTGGCTCACACCTGTAATCCCAGCACTTT GGGAGGCCGAGGCGGGTGGATCATGAGGTCAGGAGATCAAGACCATGGTGAAACCCCGTC TCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCGGGCGCCTGTAGTCCCAGCTAC TCGGGAGGCTGAGGCAGGAGAATGGCATGACCCTCGGAGGCAGAGCTTGCAGTGAGCCGA GATTGTGCTACTGCACTCCAGCCTGGGTGACAGAGCGAGACTCCGTCTCAAAATAAATAA ATAAATAAATAATAAAAAATAAAAATAAAAATATGGAACGCTTCACACATTTGCGTGTCA CCCTTGGGCAGAGGCCATGAGAAACTTCTATCGTTCTGATTTTAGTATATGTGCTGCCGA AGCGAGCACTATTTTAATCATTTTTAAGTGCACGGTTTAGTGACATTAAGTACATTCACT GCCATCTATTTCTAGAATCTTTTCATCACCCGAAACTGAAACTCTGCATTCCTATTAAAC ACTAATTCCCATACCTCCCAGCCCGTGGTAACCACGGTTCCACCTTCTGTTTCTACAGAA TTTTTTTTCTTTTTTTTTTTTTTTTTTGAGATGGAGTCTTGCTCTGTCGCCCAGGCTGGA GTGCAGTGGCGTGATCTCGGCTCACTGCAACCTCTGCCTCCCGGGTTCAAACGATTCTCC TGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACACATCACCATGCCCTGCTAATTTT TTTGTTTTGTTTTGTTTTTGTTCATTTGTTTTCTTTGAGGCAAAGTTTTGCTCGTCACCC AGGCTGGAGTGCAGTGCCGTGGCAGTGGCGCAATCTTGGTTCACCGCTACTCTGTCTCCC GGGTCCAAGTGATTCTCTTCCCTCAGCCTCCCAAGTAGCTAGGATTACAGGCACACACGA CCACATACGGCTAATTTTTGTATTTTTGGTAAATTTTGTATTTTTGGTATTTTTGCCAGG CTGGTGTCAAACTCCTGACCTCAGGTGGTTCACTCACCTCCGCCTCCCAAAGTGCTGGGA TTACAGGCGTGAGGCACCTCGCCCCGCCTGTCTCTACGAATTTGACTCCTCTAGATACTC CACATACATGGAACTGCACAGTATTTGTCGCTGACTTCTGGCTGTCCTCACCTGGCTATG CCTACTTTGGGGGATAATCTGGGCCTGGGACACCACATCCCCGTAGCAGGCCCCACCGGG CCGCACAAACAGAACTGCGCCGAAGTTCCCCCAGGCTGTTACCCCCACTTGTTTAGGCTC TTCAAAGATGCTACCGGCCGGGCGTGGTGGCTCATGCCTGTAATCCCAGCACTTCGGTGG GCCGAGGTGGTCAGATCACTTGAGGTCAGGAGATCGAGACCAGCCTGGCCAACATGGTGA AACCCCGTCTCTACTAAAAATACAAAAATTAGTCGGGCTTGGTGGTACGCGCTTGTAATC CCAGCTACTCTGTAGGCTAAGGCAGGAGAATCGCTTGAACCTGGGAGGCAGAGGTTGCAG TGAGCCAAGATTGCGCCACTGCACTCCAGCCTGGGCGATAAAGTGGATACTCTGTTTCAA AAGAAAAAAAAAAGGTGGCACCGGCCCGGTGCGGTGGCTCACACCTGTAATCTCAGCACT TTCGGAGGCTGAGATGGAAGGATGTCTTGGGCCCGGGGGATGGAGGCTGCAGTGAGCGGT TATTGAGTGACTGCGTCCAGCCTGGGCCTCAGAGTGAGGCCGTGCCTCAAAAAGAAGAAA AAAAAAAAAAAAAAAAGCAGCAGCTACCCATCCAGCCGCCAGATGGCAGCACGATTCCAT GGCCCCCACTAGCCGCTGGCTGCCTGGTGCCGGATGCAGTCCGAAAGGCGGTTCTGAGCT CCAAGGGCCGGCGCCGCGCGTGCCTGCGGTCCCAGCTGCTCGGGAGGCCCAGGCAGGAGA ATCGCGTGACCGGGAGTTCTGCGCTGCAGTGCGCTGTGCCGATCCAGGTCCTCACTGAAG CCGGCATCAGTATGGTGGCCTCTGCCACCAGGCCGCCTAGGGAGGGGCGAGGCAGCCCCA GTTGGAAACGGAGCGCGTCAGTAGTGGCATCGCGCCTGGGAATAGCCTCTGCGCTCCAGC CGGGTCGACCTGCCGAGACTCCGCCTCTAAAACCAGCAAATACCGGGAGGCTGAGGGACG AGGACCGCTGGAGCCCGGGAGGCGGAGGTTGCAGTGAGTAGCTGGGATTACAGGCAGGCG CCACCACGCCCGGGTAATTTTTGTGTTATTAGTAGAGACGGGGTTCCACCATGTTGACCA GGCAGGTCTTGAACTCCTGACCTCAAGTGATCCACCCTCCTTGGCCTCCCAAAGTGCTGG GATTACAGGCGTGAGCCATTGCGCCTGGCCAAATAAGTAATTTAAGCAATTTTTTTTGTT TCAGAAAAAAAATTAACTCATAAAGGAGCCCCCCCGACACACACACACACACACACACAC ACACACACACACACACACACACGGGAAGCAGCAGTGTAATTGGAATGCCCACTGCTGAGG CTCTCGAATGAGTGTCATGTGTGCACATTTTGCCCCTCACAATGCACGTCAGACTATTAA TAGGAAGCAGCCGCTGGAATACCTCTGGTGGGGTGTGTGCAGCAGGCAGCTTTGGAATTA CGGTATCCCCAATGTGACAGCAGGACAATGGCTTCTTCACTGGCACTGTCCAACCACAGT GCTGTGGTTATGTCTCGAGGTCCATTATTGGGCGAAGGTCACTGCAGACTCAGGGAGGCC CAGGAGCTCACCCCAACCAGCTTCTTTACCAGGTGAGTGACCTCAGCCCTGCAGCTGGAC TCATGGAGACAATGACCTCACCCGAAGATAGATGACCCCTGTCCACCGTCCACACAACAA CTGAACTGGCCCCAGGTTGACTCCTCTACACCCTGCTTCCTACTGTACCCCTGTCAAAGG CCTTCCTCCCACCCTTCCCACTCCTCTTCCTCCCTGGCAGCCCAACCACCACAAATTCAC TAACTTGTTTTCAACTCTTACAGGCTGCCCTCCTAACCCTAGCCCTGACCTCTGCCCTCC TAACCCCAGCCCTGACCCCACCGCCCTCTCAGGAGGCTTTACCAATCCTCTGCATGTTAG CCCCACCCAAGGAACAGGCCTATTTTCAAGCATACTAACTGTTGGGTGTGTCGCTTACCT ATGCATATGACAGCCAATCCAACCTACCTAAATGACAGCACAGCTCCATCCATGGATGTG GCTGATCACATCTCCAAACCATCTTCCCACTGGGGACAAATTCTTGGATTTTTTAGAGAC AGGTTCTCACTCTGTCACCCAGGCTGGAGTACAGTGGTGAGCCTCAGACTCCTGGGCTCA AGGGATCCTCCCACCTCAACCTCTGGAGTAGCTGGGACTACAGGTGCACACCACCACCGT GCCTGGCTAAATTTTTAATTTTTTTTTTTTTTTTTTTTTTTTTAGAGATGGGGGTTGCTA TGTTTTCCAGGCTGGTTTTGAACTCCTATCCTCAAGTGATCCTCCCACCTCAGCCTCCCA AAGCACTGGGATTATAGATGTGAGCCACCAATCTTAGCCCAAATTTTTAACTAAACTCAA TTAAACAAATGAACAAACAGAAAAGACTCTCAAACCACACACTGTCGTCTCTCACCCAAC AGAGACAAGACGTCTATAAACCACCAATCTGTTTAAAGAGACCACCAAATGTTCAGACCA TGAAACTGAAAGAATCAAAATAAAGTTGTCACTATGCCACCAACGGACAAAAGCCAGGAG CACACAACCAGGGGTCATACTCACTGAAGGTCCCCCACCCTGATGGCCAGTCAGGGGCGG CTTGCGGGTCTCAGAGAGGGCTCACCACCTGCTTGCAGTCATTGGGGATGGGAAGGTAAG TCCAACCCATGCCAGGTCAAGGCACAAAGACTGTGGGGGGAATGCACCAGACACATAAAG AGATGACTTTATTCAGGCTACTGCTATGGGAAGAAAACTCACTAATGAGGAAGGAGGGGC CTGGTAAATAAGAGACTCATGTGGAAGGCTGGAGGCGGGGTCTTAGAATGGACAAGGGCA AAGTGCTCCTTCTGGTCTCACTATTTCTCAGAACGCAAAGGAAGAGGCAATTTCTCAGTG AGCACCATTTCCCTGGAACCCAGGGCTGAGATGAATTTCAGTACTGCCAGCGGCCTCCGC GTTCTCCTGTGTGTGTAGGAGTTTGTGTGTTCCAGTGTGCGTGTTCCCACTGTGTCTTGG TGTTTCTGTGTGCCTGGTGGCATGTCTGTGTGTGTATCTGCAGGGTGAAAGACCACAGAT ACTATCCGAATTCCCGTGCTCAAGTGCGCCTCCTGCCTTGGCTTTCCTGAGTGTGGGGAT GACAGGCGTGAGCCACCACGCTGGGCCTCATGATCTGTTTCTAGTGCAGTGAGTGCATCT TAGGGTGGGTGCCCGCCTCCATGCTTAGTAGGGCATGGATGCTGTGTATGCCATCTGTCG GTAGGATGGTTACACTTCTGTGTGTTGGCTGATGTGTGTGTCTCAGTATTTAGTCGGCTC TGGTGGCACATGTGTGTGTTGCAGGTTGTATAAGTTGCTGTTTCTCTGTGTCTGGTGGAA GGTGTCTATGGTTGCAGAGTGAGGGTCTCTTGGTGTTTCTGTGTGTCAGAGGGTGTGTGT GTGTGTGTGTGTGTGTGTTTTGCAGGATGAGGTGTGTTCTGTGAGTGTGATGTGGGGGGT GCAGGGTGCATATCTGTGTCTACATTTCTTTTTTTTCTTCTTCTTCTTTTTTTTGAGACA GAGTCTCGCTCTGTCGCCCAGGCTGGAGCGCAGTGATGCGATCTCCACTCACTGCAAACA CTGCCTCCCAGGTTTAAGCAATTCTCCCAGCTCAGCCTCCCAAGTAGCTGAGATTACAGG CGCATGCCACCACGCCCGGCTAATTTTTTGTATTTTTAGTAGAGATGCGGTCTCACCATG TTGGCCAGGCTGGTCTTAAACTCCTGATCTCAAGTGATCTGCCTGCCTCGGCCTCCCAAA GTGCTGGGATTAACAGGCGTGAGTCACTGCACCCAGCCTGTCTGCATTTCTGTCTGGTGG TGAGCTATGTGCTGCAGGTAGTGTGTTTAATGTGTTTCTCTCATGTTTCCGTGTATCTGG TGGAAGATGTGTGTCTGCAGGATGAGTGTCTCTTTTGTCGGTGTATCATGGATTGTGTGT TTGTGTGTGTTGCAGGGAATGTGTTTGTCTCAGTATCTGAGTGTGTCGGGTCGTGTGCAG GTGTGTGTTTTTTTGTGTGTTTGTTTGTTTGAAACAGAGTCTCACTCTGTCGCCAGGCTG GAGTGCAGTGGTGTGATCTCAGCTCACCTGCAACCTCCGCCTCCCGGGTTCAATCGATTC TTCTGCCTCAGCCTCCTGAGTGGCTGGGATTACAGGCGTCTGCCACCACACCCAGCTAAT TTTTTGTATTTTTAGTGGAGACAGGGTTTCACCATGTTGGCCAGGCTAGTCTTGAACTCC TGATCTCAAGTGAACCGCCTGCCTCGTCCTCCCAAAGTGCTAGAATTACAGGCGTGAGCC ACCGTACCCGGCCGCACGTGTATTTTGTATGATATATATCTGCTTCTGACTTTGGTGAAG TGTGTGTGTCGCAGGGTTGTGTCTATCTTGTGGTTTCTATGTGGCCTATGGTGTGTGTGC TGCAATGAGTCTCTGTCTTGGTGTTTCTGTGTCTGGTGATACATGTTGTATGTTGCAGAG TGAGTCTTTTTTTCCATTTTTATGTGCGTGGTGTGGGTTGAGTGTCCTCCATAGTAGTGG GCTGGCTGTGTGTGTGTCACAGCGCGTCTGTCTCTGTGAGCGGTGGGTTGTGTGTGTGTG TTGGAGAGTGAGTGTTTCTGTCTGATGGAGTGTCGTGTGTGTGTGTGTGTTGGCAGGTGA GTGGCTGTGTTCCTCTGGGCCCAGTGCTGTGTGGGCGTCTGTTGCAGTGTGTGTGCCGTC TCTGTGTTTCTGAGATTCTGCTGGCTTTGTGTGTGCATGTCTGCGTTTCGGGCTGTGTGT CTGTGTTGGTGTTTCTGAGTCTAGAGGAGCGTGCATGCGGCGTAGGGTTAGTGTCTGCAG GCCTCTGTGTGCTGTGGCCTGCGTCTGCTTGTAACCACCAGGCTGGGCCTGGTCCCCACA TTCCTCCCACTCCATTGTGCTCTGTGCTACAAAAAATATTTGCGTTTTCCACCACTCATA ATTTTTCCTCCTCAATCGCTACCAGATGTGGGTCCTGTGGTCCCCATCCCCATGGCAACG GAGGTTCCAGCAGCCGCGGTTCCCTTTTGTGGCGCCTGCCCTGGGAGCTGGCACTGCTGT GAGCCCCGAGGGGGTGGGGAGAAGCGGGTGGTGGGAGGCAGGAGGCTGGAGGGGTCCCAG CAGCTCCCTTCCCAGCAGCCACTGGAACCCTCCTGGGGGGAAGGGAAGGCTGGTGGTAGG TTGGGGGGGGTCCCCTGTTTTGTGAGGCACCCACAGGTAGAGTCTTCGACACCTAGGGGG CTTCCCAGAGAAGGGCAAGGAGAGGTCCCCAGAAAGGCCTGGTCACTGGGACATCCCTGT TCTCTCCAAATCGTGCTGGTCCAGGCTGGGGGAGGAGAAATAGCAAGAGCTGAGGGCTTG GGGGTTCAGGGAACAGCAGTGTGGCATCCTCATCGTTCTGGGGGGTGGGCACCGCCTAGA CAAGAATGTGGTCTGAGCCCCTGCACTGCCCACCCCTCACCCACACGCAGGCACTGCCCT GCCCTCGCGGAGCCCGGCTGGGCATCTGCCCTCTTGGCCTTAACCCCAGCAGCCCGCATG GCGCAGTGCCCAAACAGAGCAGAGGCTTTCCAGCCACAGGGCCATGGCTGGGAGGGTGCC CAGCTGGCTCCAGGGAAGCAGTATGGCCCAGAGGCAGGGATCCCACATTCCACGTGCTGC CTGTTTGTGTCCTGCCAGCTGCTCTGGACGGCCGACCCAGGGCAGGACGCAGTCTCCACT CAGATGGCTGGGCCTTCTGAGATGGGTGACCCCTCTAAGATGGGTGACCAAAGTTGACCT CCGTGGGGCATCTGGTCAGAGGATGAGAGTGTGCCCAGGACGAGAGCCCCAAGTTGCCAG CGTGAGACCAATGAGTGCTGTGTGGCGGCTGCAGGGTGATCAGAAGATCCTGGCAGCAGG GGAAGACTTTGAGCTGGGCATCAAAAGATGAGATGCAGGAGAACAAGAAGAGGCCTGCCG GGCAGAGGCAGGAGGTGGGAGGAGAACAGACAGGCAGGAGCTGGGGGAACTTAGTGTGGG GACAGCAGGGGCCATGGAAGGTGAGGGTCTGCGGCTTAGGAGGGTCACTTAGGAGGCCTG CCCGGGAGGGTAGGCGGGTCAGGAGCAGCGAGGCCGTAGGGCACAGATGGTCACAGATAG CCAGGCAGCCGGGTCTGGCTTGGGGGCAGGGGATCTGCCCCGGGCGCTCCCTCCGTCTCC CTCAACCTTCCGCAGTAGCAGGCGGGGCTGGCAGGCAGGAGAGTTTCCGGCTGAGGACAA GGAACCCGTTGAGGCAGACAGCAGGGCTCTGTGCAGCAGGAGACCGGCTTGTTTGTGCTG GAGGAGGCCCGGCTGGAGTAAGCGCCCGCGGCCCCCTGGCCGCAGCCTCTGCACCCCCCG CAAGCTGCCACCAACAGCTCTGAGGCCCAAGTTTAACAGCAGCGGCCAGGGGAAAAGGCC GGGATCCGAGGCAGGCCCATCGCCTGTCCCTTTCAGTCAGGGACTCTGCCTGATCTGCCC CCACCCAACCTTCCTCCCCGTGACCAGTCCAGAATGGGGCAGGCCAGATCGGGGCCGCAT AAACTTGATAAACAAACGAAGCCTGTGGGTTGACACAGCCCACTTCCACCATGGTCCCGG GAGACCCACGCCTGGGCTCCAGGGCTCCCCGCTGAGATAAGGGGTGGGGGAGAGTGCAGG ACAGGTCGTCACATAAAGTTACTTCTGGGGGGCATCTTTGGCCGAAGCCTGCAAACAGGA AGTGATCAGAGCATGAGGGGAAAGGCATTTTATTAAGAAAGCTTTGGCCAAGCCCCCGCC GGGAGGAGCCCATCCTGGGGCACCGGCCGAGGGGGGAGCAGCCCTGCTGGGGGCCTATAA ATACATCTCCTCAGGCCACTAGAGTCGCCCCTGGAGGTCCCGCGGCGTGTGGGGCAGTGG GAGGCCATCAAAACCAGCTGACAGCTGAGGGGCCTGGGCCCGGGGCGCCAGGGAAGGGAA GGGGTGGGACGGACTAAGCCCCTGAGGAGCGGAGACCAGCCCTCCTCCCCAGGCTGGGGT CAACCAGATGCCCGGGAGCCTGGCCAGTGGAAGTAGCCCCGGGCTCTATTGGGACGGCCA AGGGAGGGAGCCTCCAGCCAGAGGCACCAGGCCCTAGCGGGGGAGAGGCAGCCCGGGCAG AAGTGCCTCTGGCTGCAACCAATGAGAAGGTAGCCCTAACCAAGTCCACTCCTGGCCAGT AAGAGGCGGTCTTTAAGCGGAACCCTCCCATCTTTGGCCAATGAGACGCTGTCTGGTCGG AGCGCTCCATAACTCGGCCAATGGGGAGGGAGTCGCCCGCTAAGCGCCTGTCAGGCCTCG ACCAACGGGATGGGCCCTTCCCGCCAGAGCACACTGCTCCAATCCAGGAGCGGCTGCAGG ACCTGAGCCAATGAGACGCAACCTCCGCTAGCCGCGCGGTGCCCGGCCAATAGGAGGCCG CCCGTGCCCGGTAGCGTGGGAGGTGTGGGGTGGCGGGCGGCGCTGCGAAGCTGAGGGAGC TGCGCGCGGACGAGCCACAGCCTGCTACAGGGTGGGTGCGCCCGCCCTGCCCAGCTCGCC CGCCGCCTCCCGGCTCTTCTTGCGCGGCGGCTTCTGGATGGGGGTCTTCTTCCGGGGGGT CTCAGGCGCGGGCAAGTCGGTCGCCGCCTTCTCAGGCCCCGGGGTTTCGGTCGCTGGGGG CGCGCGGGCGGGCGAGGCTGGCGGCACGGAGCGCTTGGCTTTCTGTGGGGGCGCCGGCTT TTCCCTGGGGCCCTGCGCGCCCAGGGCCACCTCGGCGCGGCCCAGGCTGCGAGTCTTGCC GCGCACGTCAGCGGCCAACATGGAGGCAGCCTCGGCGCGCTTCGCGGGGGACCGCCGGCG TCCGTGGGCTCCAAGAGGCCGGGGCGCGGCGCGCGCTCGGGCCCTGGGCGGCGAGGGCGC AGCGAGGGCTGTCGCCTCCTCGGGCAGCCCCGGGGGGCGCGGCGTTGGGTCGCGGGTCCG GGGGCTGCCGTGTTCGACCGTATGCGCCGATTTGTCGCTGGGCGTCCGTTTCCTCTTGCT GGGGCTGGGCGCGGCCTCGGAGCCTGGCGGCGGTGGTGAGGGCGCACGGCCAGCTGCAGC GGCGCTGTGCTTGCCGTGGATCCAGGACACCTTAGGCTTGGTGGCGGGGTCAGGTGGCGG CGGTTTCCTGCGCTCGGGCGATGGCGACAGCGACAGGCCCCCAATCTCGCCCCGGGCCCG GGCCGGCCGGGCCTCGCGTCGGGCCACGCGCGCGTACAGAGCCCCTCCGGGCCCCTCCAC GCTGGACGCCGACCGCTCGCTGTCGGAGGACGCGGGGAGGGGTATCGCCTCCTCGGCGGA GGCTGGCGTGGTCAAGGGCACAGGGGACGGCGCCGGCGCCTCGGCAGGGACAGTGGGGAC TTCGGGGTCCCGGCTCTCCGCTGGTGCCTCTGGCAAGGGAAGAGCAGGGCGGTCACAGCC TTCAGGAATACCTTTAGGGGCTCCAACCTCCAGGCGCAAACCTTCACCTTTCCTCCTCTA CCCACGCCCTTGGCACTGTGTCACCCTGGCATCTAGACGTGCCTGGCACAGAGCACCAAC CACACATGTGTACTTCAGAGCCTCAGGCTATGCGGCTGACTCTGAACTCAGGCTGTGCAA GGAGTGATGGGCGCATTCCCGCGACTGAAGCCCAACCTTGGCCTGGCCTGGCAGCCCTGC CCCAACACCCCTCCAGCCTTGGGCGCCTGCATCATCGAATTTGAGGCTGGAACATCCCCA CAGGGGTTCCATTATCCTTCTTTAAGAGAGAAGGCTTCTAGCTGGGCGTGGTGGCTCACG CCTGTAATCCCAGCACTTTGGGAGGCTGCGGTCACCTGAGGTCAGGAGTTTGAGACCAGC CTGGCCAATATGGTGAAACCCTGTCTCTACTAAAAATACAAAAATTAGCTGGGCGTGGTC GTGGGCGCCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATTGCTTGAACCCAG GAGGCAGAGGTTGCAGTGACTGGAGATCATGCCACTGCACTCCAGCCTGGGTGACAGAGG CAGACTCCGTCTCAAAAAAAAAAAAAGAGCGATAAGGCTTCTGAGGCTCTGAGTTGGTCA CCTGCCCGTGGCATCTGGGGGCACAGTCAAGGGAGGAGACAGGGCAGAATCCCAGACCAT CAGGCTCCTTTCTGCTATCTGTGGATCAAAGGCTGTGTGTGGATCCAACTCCCATACTCT GTGGATCCCTTCCTCCTGTCTCCTCTGCCACCCCCTACCTAAGCCCAGAAACTTCTCTCT GCCTACTTTCCCCCAGGAGCAACCCTGGGCTGTGGCCTACCCATCTCCCCACACCTGTCT CCGGTACTGAGCAGCAGAGTCCAGCTCTATGGCCTCAGCTCTGTGGGCAACCCTCAGGTA CCAGCATGGCCAAGGCCTTCCTCTCCATGCTGCCTGCCCTTCTCCTGTGTCCCACCCAGA ACACAGCTTTTCCATGGGAACATTTCACTGGGCTGGAGTGATGCCCAGGTAGGGCCTTAC TTACCCTCATGGGGTACACAGTACACAGGGCCTTCATCAGTGGTGTCAAACGAGGAGAAG GAGGCCCGAGAGGACCAGGATGGTGAGGGCTGCTCCAGCCCTGAGGGTGGCTCCAGGAAG CTGCAGTTGAGTGTGTTATCCAGGTCGTGGTGGGCCACTGGGGAAGGATGAGGCAGAGCT GCCAGGGGTCCACTCTGCCCCAGCCGGTGCCCTCATTAGCCCCTGCTGTGACAAATGTTG ATGGGGTGACCAAGACCAGCCCTATTCTTGGACTGCAGGGGGCACAGGCAACTCAACAGG TACAGTGACACTGGGCCAGGACTGGAGAATGGGTAGAGGTGAACCCAGAAAAGAAACATG GGCACAACATGTGTTAAGGTGCAAGGCAAGTGTGTGCATCCAGGGAGCTGGAAAAGGTTG GGCTGACGTGTTGAGAAAAGGCCAGAAAGGTGGGAGACGGGGCTGAAGAAGACACCTGGC TGGGACAGGCCACCAAGGCCTCCAAGCTCTGTTTTAGGCCCTGCCTTGTGCGAATAAGGA AGCCTTGGAGCACTTCTGAGCCCAAATGCGCAGTGGCAGGGCTGCCCTTTGGCTCAGCCC ATCTCAGCAAGCTGCACCCTAGGCCTGGCCTCCAGAGATGCAGACCCCACAACAGGGGGC CCCAGAAGCTGGCTAGCCTGGCCTCCATTCCACAGAGGGATGTGGGGTCCACTCCCCACA CCTATGTCCCCAGATGCCATGCTGCTAGGTGGGCCAAGGGGTTTTCTCTCTCTTGCTTGC TTGCTTTTTCTTTTCTTTCGACAGTTTTGGGGTTTCTCCTCCCTCCCTCCCTCCCTCCCT CCCTTCCTTCTCTTTCTTCTTTTCTCTTCTCTTCTCTTCTCCTCTCCTCTCCTCTCTTCT CTTCTCTTTTTTCTTGACAGTTTTGCTCTTGTTGCCCAGGCTGGAGTGCAATGGTGCGAT CTCGGCTCACTGCAACCTCTGCCTCCCTGCAACCTCTGCCTCCCAGGTTCAAGTGATTCT CTTGCCTCAGCCTCCAGAGTAGCTGGGATTATAGGCATGCGCCACCACACCCGGCTACTT TCGTATTTTTAGTAGAGACCGGGTTTCTCCATGTTGGTCAGGCTGGTCTTGAACTCCAGG CCTCAGGTGATCCACCTGCCTCGGCCTCCCAAAGTGCTGAGATTACAGGCATGAGCCACC ATGCCCGGCTTGGCTAAGGGGTTTTCTCAGCTGTTTCCTTCATCTCCCAAGAGTCTGAGC CCCCTTCCCCTCCATCTTTAGAAATAAGTGGGAAAGAGAGGAAGAGAGGGTCCACCCAGG TGCAAGTCAGCATGGAGAGAAGCGGCACACCCCACCCCCCTATCCTGTTGGTGGGTGGGG ACCAGCCTGCCCACTTTCCAGGAGGATCTCTGGGTACTGCTGGGGATGTAGGCAGGAGGG TGGGTCCTCTCCGTGGGGCAGGGCACTCCCGTGGCCACGCATTCCCCCAGCAGCTCTGGC CGGGATATACAGCATTCTCCTACAGACTGGGGGAGCCTGCGGGACCCAGATGTCCACACA CCCCTGATGCGATCACTCTCAGGGCCAACGTTTCTGACATATGCCCACATCAGCCGGCGC ACCTAGGACTCCTGCCTTCCTACCTTGCCTCGCCCTCAGCAGCTGCCACACCTCTTACCT TCCCTCTGGTCGCACCTCCTCGCGCCCACGCACATCAACACTCAAGGTCCCCCATTTCCT CACTGAGATCTGGACCCCCTCACACCCATTTCCCAGCAGCTTCCTGCGTCGAGAGGTGTT TCTCCCAGATACCCCGCGCTGTCATCCTTACCTACGACTTTGGGTAGTTTCTGCCTCCGG AGCGGGATCCGGGGCAGCTTCATGCTGATGCGACTGAAGCGCCCGCATAGTCGGTGCGGC GCCTTCTTCCTCCCAAGCGAAAGCTCCCTGCGGGGGCGGGGTCTGAGCGGAGGGGCGGGG CCGGGGCGGGGCCCAGGGGCGATTAGATCTCGGCCGGAGCCAAGCACAGAAGGGGCGGGG CCACGTCCGGGGCAGGGGCGCGGAAGGGTTGGATCTGGGTCCGGTGGCACCCAGAGGGTG CGGCCTGAACCCAGGCGAAAGCGGGGCAGTATCTGGGCTCACCGGCGCGTAGGGTCCTTG CCGCGGCAAGCGCAGCAGCAGCCGAGCAGCGAGAGCAGCAGGCAGACGAGCAGGACGAGC AGCGCGCCCGCGCCCATCACGCCCTTGCGCTGGTTGGTTTCTGTAGGGGGTTATGGGGTC AGCGCGGTTTCTGGCACCCCCTGCATTCCTTAACGGGACGCCCCTCATCCACTTACCTAG GTGGCAGGCACCAGTGACCGGGTCGCACGTGTCCTCGTGGCAGCTGCAGGCCTGAGCACA GTCCGCGCCGTGGAGTCCGGGCGGGCACGTCACGTTACAGCTGCCGGGACATAGGGTCAA GGCATGCCACAGCCGCCCCCCTAGGATGCCCCCTACCCTCACCCCTCACCCGCGGCCAGG GCCCAGGGTCCAGGGTCCCAGAACCGGGCTCTCTCTCGCTGCATTCGTCGTCTAGGGATT GAAGCCCCGCCCCGCCCGTGGCACATATTGGGTAGTGGATGTTTTGGAAACGAATGTTGC ACAACACTAGTGGGGGGGCCTGGGGGATAGGACCCGTGGTGGCCGCGCCGCAGCGCCCAC TGCTGGGGCTGACTCGGCTAGGTGGGAAGAGGGGCTGCTGAACCAGACCCTTGGCTGTAA GTGGAGGTCCCTGGTCCTTCTGTACCAACTCGGGGTCACCTTAAGAGCCCACTGACACAG GCTGGACTGGTCACCAGATCCTGCGCTGAACGTGAGCTCCCCGACATCCAGGCAGGAAAC TGCCCCCAAAGAGCCTACTGTCACTCCCTAGCTGGGGTAAGGGACCAAGGCTGAGGTGAT AACCCAGCTCAGGGTGGAGAGGCCACCTCGTCGGCTGGACCCCCCTCTGTGGCAGGTACA AGCCCCCAACCCCCTCCCGGTCCCGGGGCACTCACTGGGGCCCGTGGACGCCAGGGCTGC ACAGGCAGCGCCCCGACTGGAAGTCGCAGTGTCCGCTGCCGCAGTCGGCGCACACGAAGG CGCAGTCCTCGCCGTAAGTGCCATTGCTACACTTGGTCTCGCACCTTGGAAAGAGGGGAG GAGGCGTCAGCAGAAATGGAGGCAAACGGACCACAGCGCCCTCGACATTGGGGCCTTTGT TAGGGAGGCAGGGCGGGGGACTGCGTGTCTGGGCCGACGCAGGCAGGGCTGCTCACCGGT CGCCGATCCAGCCCGCGTTGCAGCGCGTACACTTGCCGGTGACATGGTTACAGGCATGCC CGTCGCGGCATGGCGGACAGCGGTGGCTGCAGCCCTCGCCATAGAAACCGGTGGCGCAAG GCTGGTCGCACTTGGTTCCGTTCCAGCCGGGCTCGCACGTCAAGCAGCGGCCCTCGGCCA CCGTGCACGGCTGCTGGCCCTTGCACTGGCCACACCTGGGGGAGGGGTCGGAGGCTAGGG AAGGCTGGGACCCGCCTCACCCCTGTCCCCATCGGCGGCCCGCCCTTCCACCTCCTCCCT CCCTGGCCGAGAGACCGCGCTTACCGGCGGCGACAGCCCAAGCCGTAGAAGCCGGCGGGG CACGGCTCGCGACAGTACTTGCCGCGGTAGCCCGGCTCGCAGGCACACGTGCCGTCCACA GGGTGGCAGCGGCCGCGGAAGCACTGGCAGTAGCGATCGCAGCGCGCGCCGAACGTACGC TCGCGGCACTGACAGCGGCCGCTCTGCTGCTCGCAGGGAGACGAGTTGCAGGCGCACTGG TTGTTGCAGCTGCGGCCCCACCAGCCTGCGTGGCACAGGCAGGCGCCGGTCTGTGGGTCG CAGCGCGACGTGGCGCTGCAGTAGCACGCGCTGGCGCACTGCGCGCCCCACCAGCCGGGC TCACAGCGGCACGCGCCGCTCCGCGGGTGGCACGTGCCGTGCTGGCACTGGCACGCATGC TCGCAGCGCGCGCCCCAGCGCCGCGCGTGACAAGTACACTGGCCTGTCACGTCCTCGCAC TGCCCGTGTGGGTGGCAGCTACACAGCTCCTTGCAGTCGGGGCCCCAGAACTGGCGCGGG CACTCTGCAGGGGAGGAGCGGAGGGGGTGGAAGGCCCCGGTGCTTGAGTAGGTGCCCCCA GCCAGCCGGAACCTGCCCGCGTCCCGCACTCCAGCCGCCACCTCTGGGGACCCGCCCCGA AGGCGGATCCGACCCCGCCCCACCTTGGACCCCGCCCCATCTCTCCAGGATTCCGCCCCA CCGACCAATACCGGCCCGACCCCACGCTCACTGGTGTCGCAGTTGGCACCGAAGTAGCCG TGGCGGCAGCGGCACTCGCCAGGCCTCACGCACACCTCGTTCTCTGAGCACGTGGAGTTG CCTTCGCACACCGCTGTGGACGAGACAGGCCAGAGCTGCTGCGCGTCCTAGCCCCGCCCC CTCCCCCGCCCCAGGTCCCCGGGATGACCCACTCACCAATCCCACACTCGTCCCCTTGCT GCCTCCAGCCAGCGCAGCACGTGGGCACCTGGGAGCTGCGAGCAGAGGGAGGACATCTAA GCCCGATGCCCCTCCCCCAGCCCCCATCTGCTCCGGGCTCCTCCGCAGCCTCCGCACAGC CTCCCTGCCTCTGCAGTTGCGCTCCTGTCCTAGGGGGGTGGTTAATGAGGTCCTGCCAGG CAGTCGTCTGGCGGTGGAGGATTGCCACAAACAGACCTCACCGCCAAGAACTTTCCAGCC CAGAACCACCCACCCCATCTCACCTAACTCCTTTGTGTCCTCCTCCCACAGCCATTAGGC CTGAGAAAAGTGGCCTTGGAGTCCCAGGAAGGACAGCAGTGAGGCAGCAGCCAGGGAGAA GACCATGGGCCTCAGGACCTGCCCAAATGTGAGGGTCTAGGGCATCCCTCCAAGTGGAGG GCCCAGCCTAATGGGTGTCATGGATCCAGATGTCATGTGATGTGACATGATGAGGACGGC ACTAGGACTAGCTGGGCCACAGGGACACACCTAGATGCCCAGTTGGGGTTGAGCTCCAGT GGGGAGGGGCAGCCTCTGGACAGGGGGACGGGGGGTGGCATGTAGACACAGTGAGGTGAC CACTTGGCAGAGGGGCTTCTGGAGGGAGGTGGGGTGAGGCCCACCCAGCCACTAGGGAAG GAGCAGGCACATGGGGAGTCCTCCACGTGCAGGAGGAAAGTCAAACTGTGGCAAAAGGGC TCAGGGACCAGGGTGGAGGAAGGAGCTATTAGAGCATCTGTTTATGCCTTTGTTGTTGGT TTTGAAACAGTCTCGCTCTGTTGCCCAGGCTGGAGTGCAGTGGCACGATCTCGGCTCACT GCAACTTCCACCTCCCAGGTTCAAGCAATTCTCCTGCCTCAAACCTCTCAAGTAGCTGGA ATTACAGGCACGTGCCACCACACATGGCTAATTTTTGTATTTTTACTAGAGACAGGGTTT CGCCATGTTGGCCAGGCTGGTCTTGAACTCTGACCTCAGGTGATCCGCCCGCCTCGGCCT CCCAAAGTGCTGGGATTACAGGCATGAGCCAGCGTGCCCGGCCTGTTTATGCTGATTGTC TTCCACTTGTTCCATTTGATTTGGCTTTACAGAGTACACAATGCTACCGCAGCACTTCAG GAGAGCACAACCTCTAAAACCCTGGGTGGATGGGGTGTGTGAGCACAAAGGTTTGGAGGC CCTGCCTGAGTGAAGCCATGATGGCTAGAGAGGGTTCGTAGGGGATGAGCTGGGGTTAGC CCACATTCAGGGGCAGAGGAGGCTGAGAAGGAAGAGCCACCAAGGCTGCGGGATCCTAGG TTCCCAGCCGACCCAGAACTTCGCCTCGATCCTAAAGGCATGTAACAGCCATTCGCACTT TAGAGAAAAGGAGCACGCTTCTTCACAAGCAGGCTTGGATTCAGTATCTGAATCTAGAAC CGGAAGGTGGTGGGGGGCGGAGTTTGGGAGGAAGCCTGAAGACCCTAGCGGGATGGGATA GACTGGCCCTACACCATGCATTTCAGGATAGTACATGCCTGGCATGCTCCTGCCCAGGCT TCCGAGGATGCCCCAGCCATTCCCCAGCTAGACAGAATACCACCCCTTGTACAGAGCACC TAGGCCTCGAGGCCCCTCCTCCTGAACAAGGCTTGGTTCTCCTTCAGGCCAAAGATGGGA CCTGGCGAACAGGACCATAAAATCTCAAACTTTGGGGAATTGGTCCAGTGTATTTTCCAC AATAAAACTGGAAACCAGAGACAGGAATACAAAGGCCTGGCTTTTCCTGGCCCCTGCCCC CACTGTCTCCACCCACCCAGCCCTGACCCAGGGGGCACCAAGGTGCCCATCTGCAGGGAG ACCCAGGTCTGCTGGCCATCACCCACCCACTTGTGTCTGCTTCTGAACCATGTTCACTGG ATGGAGAGCCTCTGCAGGTGTCCCTCCTGTGGGGAGCTTTGCCAGGTGGCCTGCTGGAAT TGCCCCGGAAGCACTAACCCCCTTCTGCCCTGGACATAAGGCTCTGTGGCTAGGACCCTC GTTTTAGGGCCACACTCAGCTGGGTTTGATGTCTGGGTTTGCCACTTGCTGTGTGACCTC AGGCATGCAGCTTAAGCTCTTTGAGCTTCAGGCTCCTTAGCTATATAGTAGGGCTCTACT GTAGAAAGATTAAGAGAGCCAAGATGTTTTTTTGCCGGGCGTGCTGGCATGTACCTATAA TCCCAGCTACTCAGGAAGCTGAGGCAAGAGGATTGCTTGAGCCCAAGGAGTTTGAGGCTG CAGTGAGCTGTGATGGCACCACTGCTCTCCAGCCTGGGCAACAGAGTGAAACCCTGTCTC AAAAAGAGAGATAGAGATAGGAGAGAGAGAGAGACAGAGAAAGACGACAGAGAGAAAGAG AGCGCGCGCCAAGACCTATGAAGTGGTTTGGCACATACTAAGCACAAAGGATCCTGAGCA CAGGCTGCAGTGCCTCTGAGTGCACTCGGTCATCTCTGGTTTAGCGGGCACCCAGGCCTA ACCCCAGCAGAGCTAATGCCCACCAGCAGGACAGAGTCCATTTGGGTTCACTGAGAACAA GATGCTTTCCTTCTTGGAAGGGCTGGCAGAAAAGGCCGGGTGGTGGTGGCAAGCCCACTC TCTGAAGATCTTACAAAGACGGGTGGGGAAAGATGGCCTGCTGCAGGGCTGCTCCCACGC TTGCCCTGTAAGTCGGCAAGACCCTCAGAAGCCAGGCCATAGGCTGGGCCCAGGCCCCTC AGCAAGCAAGACATGCCGTAGGCTTCTCCCTCGTAGAGCATTAGTTCTGTGATTCAAACC CACTGTGAGTTCTTACATCTTCAAGTTGGCTCATGGGGTCCATACCTGTCCATGTTCTCC CCCAAATCCAGGCAGAAAAGCTTCTCTGCCTGCCACACCCTTCAGACCCCACACCAGCCC GACCCTCCTGCTGCCCCGCCTGGACACCTCTCCTCTGGTTTCTGTCCCCATCCTCAGATC TCTCTTGTGTCCACACTAGCTCCCTCGGGAAGCTCACTGAACACTGACAACACCTTGGAC CCTCCAGCAGGGCTCTCCCCATCAATGCTAGACTTGCAAATCCAACTGCCCAATAGATGA TGTCACCAGAAGGAGACCTCATGGGCCTCATGCCCAACCAGCCCAAACCTGAACTCTTGA CCTCCTTGCACCCTGAGTCTTCCCTGCCTCAGATAACCCTGCCTGTCCTTTCAGGCTGGG AAGCTGAGACCTGGAGAAGCATCTCTGACTCTTCTTTCTTGTCTACACCAGATCCATTGA GAAAAAAGACGCTGAGCTCTAGCTTCTAAACGATATCTAAAATGGGATCACTTCCTCCTT CCCTATCCCAGCCTTGTGAGAGCCACCCTCCATCTCCCTGGCTTTTTGTAACTGGCCCCA GCTTCCACCCCCTGCCCCCCAGGGTGCGAAGTGTTTGCCACCTGGCAGCCAATCCTGGTC AGATCCCACCCTTTCTCTGTACAAAGCCCCCAGGGGGTCCCATGGCCCTCAGAAGAAAAG CCCAAGTCTTCTGAGCAGTGGCAGCCCACGGTGGCCTGCCCACCCTCACTCTCTGGCAGC TCCTCCGCCAGCCCCTCCTGGACTCTGCCCAGCCTCCTGGATCCTTCCTGTACTTCCACA GGCCCTCTGCCTTGGGAACCTTGCCCCAGACACCCACAGGGCCTCCGTGCTCTTCTGCAG GCCTGTGCTCACTCACCACCTTCTCAGCCGTTCTCCTCAGAACACCCTAAAGGAAACAGC ACTCCCCAACCCAACACCTCCCAGCTCTTGCTAGATGTCTAGACACACTTGCTTTTCTGG ACTGTGTCTCCTCTGCCACAGGGTCAGGGTCTCCACTGCAGCTTCCCTACACCAATACAG GCCTGGCACAGTGTAGGTGCATAGGATTTTACTGGCTTTGCACTCTCCTCTCCCCATCCT CCTGCCTCCCAGGAAAGGGAAGGGCTGATGGAAGCCTTGCAAGATGTGGGAGGACAACAA AGACATGACCCAGACCCCAGGTGGGCAGTGCCCAGAGTGGCTGTCCTTCCCATCGACCCT TGCCACTCATGACCACAGCGATCTCCACTTAGCCCCATGAGGGGGTTGTGATCACTATCC CATTTTACAGAGGTGGAGACAGGCCCAGAGAGGTCAAATGACTTGCCCAGGTCCACACAG CTGGGTAGGGTGGCACCAGGATGGGAACCCAAACCCAGGCTCGGTTCCTGGAGCAGGCGC CTCCTGGGAGGTCTGCAGACCAGGGGAGCGGGAGCAAAGGGCTCTCAGGCTCCGGACAGC TAGAGCCTGGCACCGGACCCCCATTCCTGGTCCCGGGCCTGTCTGGGAAAGAGGGCTGGA GTGAGTAGACAGAGACCACGGCGATCGCGCCCTCCCCCTCTCCAGGCACCGCCCCGCCCG CCGCCGCCGCTGCATTCCTGGGGCCGGGGAGGCCCGGGAGTCCGCGGCGGCGGCATCGAT GCGAAGCAGATGGCGGGCCAGGCCGGCCCCCGCCCCGGAGGCGGGGCTCGCAGCGGGGAG GGGTCTGCGCGCCTCCGGACCAGAGTTCGGAAACCCCGCGCTGCCCCCTCGCAGCCCCTT GTCTCTGGCGCCGAGCCCCTGGACTCGGCTTGCCCCGGGTCCGTGGCTCAGGCGCCCCGA CGCGACCCAGCTCGGAGCCCGGCGGCCGCCTCTGCGGCTGCCAGCCCGTGCGGCGTCTCC AATCCGCGCCAAGGCGCCCCCACACGCCCGCTCCTCACCCCTGCGTGGAGATGCCCTTGA CCTTGTGGCCTTTGGCCAGCCCACCGGCTCCCCCGACCCCAGCTCCTAACTCCTGGCGTC CGGGGCTCACCTCTCTGCCCTCCGTGCAAACTCCCGGACTCGCCCCCCCACCGGCCGGGC CTGTCGCCTGCCTGTCCCACACTGGCTTCCCCAGCCGGCGCCCCACATTTGCGCGGCCCC TTCTTGCACCCAGAATCCCCACCTGGTTGTCGCCAGGGTTCCCACATCCTCCGGTCCCCT GTGCCTGTCATAGTCCTTCCGCCTCCCGCTTCTGGTCCACTTCGAGCGCCGGCTCCTCGA CGCTCGCCTGGGTCACCACCCTCCCACCCGCGCCCGCTCTAAGACGCGAATCTGTCTTTC CAAGTCTGGCTTGCTCCAGGACCTCCCCGGCTAGCACCGCTCCTCCTCCCCTGACCTTCC TCCTCACTCCCACTCCCGGCTCCAGTCGGACTAGTGCGGGGCTTTAGCACATGCAGTTCC TTTGGCTTGAATGCCCTTCCCCAGCTTTTCCCCGTGGACTTCACAACTCGGTCAAGGGTC CCTTTTTCCAGGTCTTTCCTGATATCCCGGACAACGCGTGTCATCCCCTGGGTGCCCACG GCACTCCATCCTCCCCTCAGGTGACTTCTCGTCCAGGGTTCCCTGGGATGCACTGTCGTG GCCCATGGCCGAGGCTGGGCCTGGAACGGAGCCGCGCTGGCTAGGGAGCCGAAGGGGCCC ACACTTGGCGTAGGAAGGTTCCGGTAGCCCCCTCCCAATGCCCGGCGCCGCCACACCACT GCCCAGAGCGTCCCTCTCGCCCCCTCCCCCAGCCAGGCCGGCTCCTACCCGGGAGCACGG CACACGTTGCGGCCGCGAGGGTTCAGTTCCTGAGGCGCCACGGTGTCCGGCAGCATCCAG AGCAGCAGCAGCAGCAGCAGCGACGGCAGCAGCGGTGACGGCGGCCCCCCGGCTCCCCGG CGCCGCGCCGGCCCGGCCCCCCGGGGCCCTGCGCCCTCCATGAGGCGCGGGGCAGGCGCG GGGCGGGCACGGGCGCGGGTGCGGCCGCAGCGAGAGCGGCCGGAAGCGGAGTGCGAGGCC GGGCGGGGGGCGGCAGGAGGGGCGCCGAGCCGGGCAGGAAATTCCACATCGGCTCCCTGA TCCCGGGCCAGCCGCGGCCGGCCAGGCGGCAGCGCCCGCCCCGCGCTCCAGACCCCACCC TCCGGCCTAGCCACCGCCCCTCCGCGCAGCCCCCGCCCCCATTTGGCCGCTCGCAAACTT GGGGGCGAACTTGGAGGAGGCCCGCCGAGCGCTGGTGGAGGGAGGGTCCGCCCGAGGAAT CCCCGGTTCCCGCGCCTTTCCCTCCGCTCCCTAGACATCTACCGAAGCAGCCGCCCCTGC GCGCCCACTGCGTAGGCCAGGCCCGGGACGGGAAGGCGGATCGGGGACACGGACGCGACT CCGCAAGCCCACCCCCGCTCCCAGCGCCTCGGAACCGGCCAAGGGAGTGGGGCCCGCAGA CAGCCCAGGTCGAGGCCGAAAGCCAACTCCACAGCACCCCACCGCGAAGTCCTTGTAGTG CTGGGGTGTGCGGTCAGCTCCCCCTGCGCACTCCTGCGCACTCAGAGCTCGCAAGGGGCG GACCGAGCAGGATGCTTGAGGCCTGTGGTAGACTCGGTCCGGACCAGAGGCCTGGGGGAA GGGGTCTCCGTAGGGACGGATGGGAGAGATACAGAGGAAGTAGAATGGCCAGGCTGTGGA CTGCGGTAGGAAGTAGAGGTAAAGACAGAAGGAGACCCCCGGGATGGAAACCCTGCAGTC CTAGTTGAGGAGTGAAGGGGGCTGGGGGAGCCTGGGCGGTGGATTCTGCTGGCTGTCGGA TTTCCAGGAGAGGCTGGGGCTAGAGAGTGGGAACTCCTGCATCAGGTAAGGTGGGTAACA GCACAGAGCAGAAGGAAGGTGTGGGAAGGGAGCAAGACAGAGGGACAGGCCCTAGGGAGC CAGCGGGGAAGCTGGACTTGGACTGGGAGGCAGCAGGGTGCGGGTCCCCGGGGGGCAACT GGGGCAGACCGACTCTGGAAGTTGGGCAGGGAGCACAAATGTCCCGTGTGTAGCCAGGTC CTAGTGAGGATGTTTGTCTTTTGGGGACTTGAGAGCTGGCAGAAGGCCTAAGCAGGGAGC GGGCACCTCCGTTCTTTGGGGAGCAGGCCACATGCGGCCCACAGGCTCACTGAGGGAGGG CAGGCAGACTCTGTCCAGGTGTGGCCAGATAGCCCCGAAGGGTGGGGCTGGAGGAGGGGG CTTGGAGGAGGAGCCACTGAAAGGCCCAAGGCCATGGGCATCACCAGGCTGGCACAGGCA GAGAGTGGGATGGGGAGGAGCAGGCCCCTCACTCAGCCATGCTGGTGCAGGAGGGAGGTG GGGTGGGCTGAAAGAGAAGTGGAACTAGTGTGGACACGAATGCAACTACCTGGGGACAAA TAAACAGGCCAACCACTTCTGGGCAGCTCAGCCTGGGCACAGCCCTGACCTCAGCAATGC CCTCCCAGTGCTGGGGCCCCTCCCACGGAGGATCCGGATTGCGGCTCCCAATAGAGGCCA GCAGAGGTCCCAGTGGCTGCAAGGTCTGTCCCAGCCCAGCTCCTGGCTTTAGAGGCACTG ACCCCCTGGGCTGGAGGGGACACTCACCTCAGCTGGATGGAGGTCATGAATAGGCATATC AAATTCCATCACCTTTCTCCTTCCCCCAGACCTGTCCAGGGAGGGCACCACCTGGATGGT GCCCCCCAATCCCCCAGGACCTCCTAAATGTCTTAGATCTCTGCCCACCCCTGTCTGCCA CCCAGAGTAGTCTTTTTAATCTCACATGTGGCTATCCACCACTGCTCTCAGGATGAGGAC CATCCCCAGTCTCACCTCACGACCCGGTACATACATCCCTTGCAGTAGATGGCAACTTTG GGTGTCATGGGCAGAGCCCTCATTTCTCCCAACCACCACTGCACCCCTAGGGGAAGACTG AGAAGTATGGGAGGAATGAAGGATCATCCACATGGCACTAGGATGTAGAAGGAACCCACA GACAATTGACCACCACGCAGGCAAACTGCTGGAGCCCGGAGCCCTGGCCTGCTGGTTTTG GCCAGGATGGCTGCCAGAAGGAAGTGGACTTTGAAGGCTGAGGAGGGTTTTGCATGAAAG CAGGGACCAAGAGCCTAGCCACACTGGAGAGCACAAGTTGGGCTGCCTCAGCTCCGGGGT ATCAGGTGTAACCCTTAGAAACCCAGCTTAGCCCCAAGGCTGACAGTGGCAGGGCCAGGC TGCGCCCAACTCCAAGGCACTCTCCACCCCTCCCCAGGCTGTGCAGGACCTCAGTGAGCC CTTCCTGCCCCTGCAGACCAGAGGCCTCCTGCAAACCTGTGGCTGTGGCTGGTGGCTCAG GTGAGATGTTGCCAGCCAGCATGTTGTCAGGACGCACTGGAGAGTGGTGGGGCATGTCTG GTGCTCCTCCTTTCCAGAAACCTCTGAGGTCTGCAGGAGCTATTGCCCTGGCCTGCCAGG TGGTAGCAGAGACACAGGAAGTGCTGGGTCTGAGACCCACAGAGTGGGGTTCCTACTCTG AACCCATGCTCTTGCGCCTCACCCTCAGGAGAAAGGGAGATGACACTGGAGTGTATAGCC ATCCCGCCCCACTAGGGCTCAACACTGGGTCTGCCACCCCAAGGCAATGTCATGTTTCCT CAGGGGTACTCAGGGCCCAGGACAGTGAGGAAGGTCTTCAGGTTACACACACACCCACAT ACCACAGCCCTGGGGGCACAGGCCAACTCTGAGGGCTTTGTCTCTACCCTCAAGAATCCT TGGGGTATCCATTTAGGAGAGCCCAGAGTGACTGATAGTCCCCCGTCCCCATCCCCTCTC CTCCCATCAAGGAGCACACACTGCCCCCTTCTCTTCCGGGACAGGATGCCAGCAGGAACA CATGGCTGTCCCCATGCTCTGCCCAGTCAACTGGGAAAGGATGGGGCCTTGGCAAGTTAC AGCCCTGTCTGCGGGATGGCTCTGGGCACAAGGCTCATAGTAGGAGTCTGCAGGTGCAGA CCCTATGAGGGCATCAGGATGGAAGAAAGCTTCGTGTGGTGCAGCCCTGCAGTGAGGCTG GTGTGGGCTCACAGCCCATCCATGCAGGGTGCAGGCTAAGCCTGGCAGTCTCAAGAGGGC AAGAAAAGCATGGGGACAGGTGAACAGGGGAGGGTCCCAGCTGCTGTGCCAGTGTGGTCC TTGAGACTGGGTGCCTAAGGGTTACCTGGCAAACCTGTAGCACATGCAGCTCCCCTCCCC AGCACACTCAGCTTCAGGAGGTTGGGAGGAGCCGGGCACAGGCCAGAGGGGCCCCCCCCA CCGACCCCCTGATGCTGCCCCGCTCCCAGGTGCTGAAGCTCTGGTTCCCTGACCCTCAGG CTGAGAGACTCCTAACGAGCTGGGCTGAAACTCTGCGTGCTGCCCTGGTTGGGAGACAAG TTCCACACCAGAGAAGACACTCAAGTGGCTGCACAACCACCTGTCACCAGACCAGGGCGA GTTCCAGCTTTCCCACTGAGTGCCCTGTTAGCTTGTGGAGTTTACTGTGCTTCCCTCTCA GGCAGGGTCTTCCCAGTGCCACTTGGCTTCTGGCCTGGGCTTCCACAGCCTCTCCCAGAT GGCAGCTCTGACCCCAGAAGCCACGCTTCCTGGGGACGCAGCTTGGGTGCACTGTGGAAA GGAACTGAGGCTGTGGCTACAGAAAAACAGTTAACACCTTCTGTAAGATGCTTTATTTCA TTGACCAACAACATGGGGTCTGAAAACCCAGCGGGAGGGGTCTTTTTATCACAGAGCCAG TCCCAGGCGAGCTGATGCATCTCTGCTCCTCTGCCCCTCAGGAGCTCTCATCCTCCAACC CCAGCTGCCCCCACAGCCCCACCCCATTCACAGAAAGAGGGCTACCACGTGCCTCAGCCC CCCTGCCCAGGCTGCCAGCTCCCAGGTCCTTTTGGAGAAGGACTGATCTAGGCAGGGAGG AGAGAAGGCCAACCCCTCCAGGGCTCACTGAGGAAGGCCAAAGCCTTTCAGAAGCAGTTC CTGCAGTGACGTAATCCACAGCCTGGGATCTGCATGGCCCTGAGATGCCTGCGGCAGGCT GGCCAAGGGGCTGGTGTGAAGAAAGAGGGCAGGGCCCATAAGCTGTGGCCAACAGGGGCA GGGGCCCTGCCTGGAGTAAAGTGCTCTGGCCTAGGCTGCGTGGGTTTCACTGCCCTGCAG CCCCAGCCTCCCTTCCCTCTGATGCCAGGCACAGGGAGCCTAGTCCTCACTGGAGTTGTC AAACTCCTCCCAGTCAGACACACTCATCACCTCAGAGGCAAAGTCCGGGTCGGCCTGGCT GCGGTCAGGGGTCCCGCGGGGCGGCTCAAGGAGCAGGGAGCGGGGCAGGGTGAGCACACA GGCCGCCAGGCCTGAGATGGAGTTGTCCAGCTGGGGCCCTTCCTCCCAGCAGTCCTTCTC CACATCGTAAATGTGCACGTAGCCTGTGCGGCTGCCGCGGTTGTGTGAGCGGCCACCTAA CACATAGATCCTGTTGTCCAGCACAGCAATGCCAGGCTCACCGTGCCCAGCAGGGAGTGG GCAGACAGATGACCACTGTCCAGACGTGCAGCTGTAGCAGGCCACCTGCAAAGCCAAGGC TAGAATAAAGCCCAGCACCCACTGGGACGAGGCTGCCAGCTCCCCCACAAGCCCACTGAC CAAGGTGGCAACAGTTACCCACCATTCTGACAGGTCAGGCCCCCTGGAAGGATGGCACAG GGCCAGTGTTGAAACATCCTGTCCATGGGCCAGGCTGCTGCTTTCTCACCCAGGGATGGC CGAGTCTGCAGGAGGCTGTCTTTTGGGCCCTCCCCTAGAATGATCCTATTCTCTCTGGCT TTGCTCCAGCCCAGCCCTTCCTCTAGGCCACCCGGCCCTAAGCCAGTCTCTGCCCCCGAG TGTGTGGCTGTGCCCCCATCAGCATGTGGTCATTCCCCATGTCTGAGGGCACTGCTTCTC TCCCCTCCAGGAAACGAAGCTGCCTGAGAGACATGGACTCCACTGAAATGTCTTCAACCT CCTTCTGCAGCACTCAACACAGGCAGTAGGACAGGGGGAGGAGACAGAAAGTGGAAGTAA TTCCTTGCGGAGTAGTCAACATCAATATTCTCTTGTCTTCAAAATTGTCCAAACAGTAAA CAAAGACATGGAAAAATGTTCAACCTACTAGTAATTCAAGAAACGCAAAATCAAAACATC AGAGACAAATTTTTCAACTAGCAACCACTGCAGAGAAGGAAAATCACAAAACCCAATGCT GTTGAGGGTGTGGTAAAGCCATACTACTATGTATCTGTCTGACATGCCAGAAAAGGAATT TGGCAATAAGTATTAAGAGCCATAAAAATCTTACCTTCTTGACTGATTAGCCGTTTTTGG TTCTCTAGCTATGGTGGGCAGAATAGTGGCCCCCAAAGATGTCTAGTTCCTAATCTCCAG AATATGCTACCTTAAGCAGCCAAAGGGGCTTTGCAGATAGGATCAAGGGTACAAGAATAC AAAAAAAAAAAGGATTAAGGGTGCAGACCCTGAGATGGGGCAGTTTTCCTGGATTTTCCA GGTGATCCATCCAATTGCATGAGCCCTTAAAAGTGGAAGGGGAGAGGCCGGGCGTGGTGG CTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCTGATCACAAGGTCAAGAG ATCAAGACCATCCTGGCCAACATGGTGAAACCTCGTCTCTACTAAAAATACAAAAATCAG CTGGGCATGGTGGCACATGCCTGTAACCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATC ACTTGAACCCAGGAGGCAAAGGGTGCAGTGAGCCAAGAGCACCACTGCACTCCAGCCTGG CGACAGAGCGAGACTCTGTCTCAAACAAACAAACAAAAAAAGTGGAAGGGGAGGCCGGGG GTGGTGGCTCACGCCTGTAATCCTAGCACTTTGGGAGGCTGAGGTGCGTGGATCACCTGA GGTCAGGAGTTTGAGACCAGCCTGGCCGACATGGCAAAACCCCGTCTCTACTAAAAATAG AAAAATTAGCTAGGTGTGGTGGCGAACACCTGTAGTCCTCCTAGCTACTCCGGAGGCTGA AGCAGGAGAATCGCTTAAACCCAGGAGGCAGAGGTTGCAGTGAGCTGAGACTGCACCAGT GCCCCCCAGCCTGGGTGACAAAGCAAGACTCCATCTCATAAAAAAAAAAAAAAGAAAAAA AGGAAGGGTAAAGCCAGACAAGAGAGCTGAGAAGTGAGGAGGACTCAACCTGCCATTGCT GGCTTTGAAGACGAAGTGTCCCCAAGCCAGGAAAAGTGGGGACCTCTAGAAGCCAAGAAG GCCCTCAACTGACAGCTAGCAAGGAAATGGGACTGCAACTGCCAGGGACTGAATTCTTCT AACCAGAGTGAGCAGGGAGGCAGATCCCCACTAGGGTCTCCACAAAGGAAAGATGCCCTG ACCGCACCCTGGTTTTAGCCTGGCAAGGCCCATGTCAGAGGACTTATGAATGCAGAATAA ATTTATCTTAAGTTGCTATGTTTGTGGTGATAGGTTACTGAAGCAATAGAAAACTAATGT GCTGGCCCAGAGGATCTCACAAGCAGAGGACCTGTTAAAAGCCAGATCGCCAAGACCCAC CCTCAGGGCTTCCGAATCAGTGGGTCTGAGGTGCCTCGTAACACAGCGTTCCCAGGTGAT GCAGACTCTGTTAGTCTGAGAATCACACTTTGAGAACTGTTGCATGAACCTAAGGAAATA ATCCAAAATACCTGAAAAGCTCAACAAAGCAACTATTACTTGTCATGTTATTTGGAATAG TTAAAACTTAGACACAAGAGGGGATGATGAAGATTGAGTGTTAATCTGGAAAATGCCCAG GACAAAAGTCACTGAGAGAAGCAGAATGGGATATGGGATGCTCTTTTTTGTTTGTTTGTT TGTTTGTTTGAGACAAGGTCTCGCTCTGTTGCCCAGGCTGGAGTGCAGTGGCACCATCAC GGTTCACTGCAGCCTCAACCTCCTGGGCTCAGTTGATTCTCCCTCAGCCTCCCAAGTAGC TGGGACCACAGGTGCAAGCCACCATGCCTGGCTAATTTTTTTTTGTATTTTTAGTAGAGA TGGAGTTTCACCATGTTGCCCAGGCTGGTATCAGTTCCTGGACTCAAGCCATCCACCTGC CTTGGCTTCCCAAAATGCAGGGATTACGGGTGTGAGCCACTGTGCCCAGCTCAGAATGGG ATGCTCTTTGACTACAATCAGGCAGAAAACATACACGGTCAATGCTGAAAATGGTCATAC TGAAAAACAGTACTGCAGGGGCCTGATTATGAATGCTTTTCCTCACCCTTCTCTATTTTT TTTTTTTTTTTGAGATGGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAGTCGTGTGATC TCAGCTCACTGCAACCTCTGCCTCCCGGGTTCCAGCGATTCTCCTGCCTCAGTCTTCCCA AGTAACTGGGATTACAGGCGCCCATCACCGCACCTGGCTAATTTTTGTATTTTTAGTAGA GATGGAGTTTCACCATCTTGGCCAAGCTGGTCTTGAACTCCTGACCTCGTGATCCACCTG CCTCAACCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGCCACCCATCT CTATTTTTTAAAGTTCCGGCAATGTGGTTATATTAACTTTATAATAAAAGTTTTGTTCTG TTGTTTTGTTTTATTTTGTTTTGAATAGAGACAGGGTCTCATTCTGTCACCTAGGCTGGG GAACAGCGGTGTGATCATAGCTCAGTGAAGCCTCCAACTCTGGGCTGAAACAATCCTCCC ACTTCAGCCTCCTGAGTAGCTGGGACTACAGGTATGTGCTGCCAGCCTGGATCTAAAAGT TTTGTTGTTGTTTTTTTTTGTTTTTTTTGAGACAAGGCCTGGCTCTGTCGCCCAGGCTGG AGTGCAGTGGCGTGATCTCAGCTTACTGAAACCTCTGCCTCCTGGGCTCAAGCCATCCCC CAACCTCAGCCTCCCAAGTAGCTGAGAGTACAGGCATGCAACCACACCTGGCTAATTTTT GTATTTTTGTAGAGATGGGGTTTTGCCATGATGCCCAGGCTGGTCTCAAGCTCCTGAGCT CAAGTGATCCTCTCGCCTTGGCTTCCCAAACTGCTTGGATTACAGGCATGTGCCACCACA TCCGGCCTAAAAGTTTTTAAGAGTAATAAGCAAAGGTAGATGTGTATGTGTGTGATACTG TCATGGTGACATTTGTCCAAACCTATAGAATGTGCCAAGAGTGAACACTGTGGACTCTGG TTGATGGTGATGCATCAATGCAGTTTCAACAACTGTGACACATCCACCCCTCTGGAGCGA GAGGTCTGCAGTGGGGAGGCTATATGTGTATGGGGGGAAAAGGGGGTGTATGGAAACTGT ACCTTCCACTTAATTTTGCTGTGAACCTAAAACTGCTCTAAAAAATAGTCTATTTTTAAA AGGCACATGATTCAATTACATTTTCCATCAATAACAACTGAGAGGCTTGGGAATGATGAC GGGTGTGGACTGCCCCGGGCCCCACTCACCTGGTGCACGTCCCTCCTGTATCCGGCATCG TTGTTGCTGCCCCCGATCACATACAGCTTGTTGAGGAGGGTTGCCATGCCGTGCCAGGCG CGCCGCACAGGCCCATCAGCCAGTGTGTGCCAAGTGTTGCTGCCTGGATCGTAGCAGTGT GTCTCTTTCAGGTAATCCTCCCCTCTGCGGCCGCAGGTGATATACATCTTCCCCTCCAGC GTCGCGCCTGCGTGGGCATACACCTGTGTGGAGCCACCAGGAGAAATGGCGTGAGAGGGC AGTGAGGAAGGGTGGCTGGGTACTCTGTCAAGGCCAATCACCACCCCACACCTGCCTGAC AACGCTGTGAGGCTTCACCACTTCCCACTCACATACAGACAAGAATCCTCCCTCGATTCT CATGGGACATACTGTCCTAGTCATTGACCTCTAACCTCTTCCCAATAGCGGATACATCTA TAGACTCAGAAATGAAATGTAGCCTTTGTCTTTCCTTTGCTCCTAAAGTTAACATCTGCA TGGGGGAACTCCAAGAAAATAAAAGCATTTTTTCCAAGCCAGTCTGGAAGAAGCCACAGA ACCCTGGGGACCGCTGACCCACCCTCAGTGGCCTCCACCCCTCCCCCACTCCCACTGGAC ATGCAATCCTCCGGGCCTGCTGACCCTGCTTTTAAAATGTTTGTAAATAATATCTGACAC TAAACTCATGATGTGGGGAGAAAAATACACATGGTTCTTGATTCTGGTCCTGAACCTTCT GTGGGCTTTTTACCCTCCCTCTGTTGGGCTATCTCCATAGGGCCCAGCCTGCCCCCATCA TGGGCACCTCTTACCCTGGCCCTACCACTTCCGGAACCCACCCACTCACACCCAGCAGGC CAACAGAAGGCCCCAAGGCAAGGCCCTGGGTGTTTTGAGGGTAGAGGAGCCCCTCAGGGT GTGGAGGCTCCAGAGCTATGTTACTGGGCAAGTCACTAACACTGAGGCCATGCGGGTGGC AAAAACACAAGAGTCTAGAGAGGTGGACGGAGCAGTGGGGAGGAAGAACACACACCAAGG GTTTGGTTTGGGTCTTCTCCTATCACCAGTAGGGGTCTGGGGAGGTTTTTCTTTTTTTTT TTTTTTTTGAGACGGAGTCTCGCTCTGTCACCAGGCCGGAGTGCAGTGGCACGATCTCGG CTCACTGCAATCTCCGCCTCCCGGGTTCGAATGATTCTCCTGCCTCAGCTTCCCGAGTAG CTGGGATTATAGGCGCCCGCCACCATACCCAGCTAACTTTTGTATTTTTAATAGAGACAG GGTTTCACCATGTTGGCCAGGATGGTCTCGATCTCCTGACCTCGTGACCCACCTGCCTCG GCCTTCCAAAGTGCTAGGATTATAGGCATGAGCCACCACGCCCAGCCGAGGTTTTTCTTT TTAATTAAACTTTTCATTTTGAGATAAGTGTAGTCACGTGTAGTTGGAAGTAAAAATACA GAGGTCTCACGTGCCTTCTTTCCCCCATGGTAGGAGCCTGTGTCACTATATCACAGGGTC ACCAGGATGCTGACGTGGACACAGCCACAGAACTGTCCTATCACCACCAGGATCCCGAGG GGCTGCCCTTTAGGGTGACATCCACTCACCTCCCTTACCCTAATCCCTTCCTTAAGCCCT GGAAACCACCAATCTCTTCTCTATTTCTACAGTTTTGTAACTTCAAGAGTGTTGTCTGAA TTAAATCAGTATACAACCTTTTGACATTGGCTTTCTACCCCAACCCAGGTTCTACCAGGT TGTTGCATGTACAGGTTGAGTACCTCCAAAATCTGAGACTTTTTGAGCACAAACACGGCA CTCAAAGGAAATGCATTTCAGACTTTTGGATAAGAAATGCTGAACCAGTAACTATAATGC AAAGATTCCAAAATCTGAAAAAATCTGAAATCCAAAACACTTCTGGTCCCCAGCATTTCA GATAAAGGGTACTCAACATGTACCAATAGTTGGTTCCTTTTCTATTGCTCAGTAGTATTC CATGGGTATCTACCACAGCTTGTTTAACCATTCACTGTTGAAAACACCTGATTTGTTTCC AGTTTCTGGCTATTACCAATAAAGCTACTATTAACATTCATGTACAGATAAGTTATTTTT TATTTTTTTGAGACAGGGTCTTGGTCTATTGCCCATGCTGGAATATAGTGGCACAATCTT GGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGCAATTCTTGTGCCTCAGCCTCCGGAGT AGCTGGGATTACAGGCACGCATCACCATGCCTGGCTAACGTATACAGAGTAGTTTTCATT TCTCTGGGATAAATACCCAGGATGGTAACTGCCAGATCTGTTTTCCAGAGTGGCTGTACC ATTTCACATTCCTATCAGCAATCCATGAGCATAGTTTCCCTGCACAGTCACCAGTATTTC GTATTTTCACTACTTTTTATTTTAGTCATCCTGAGGTAGTTCTCTCACTGTGGCTTTAAT TTGCATTTCCCTGATAGCTAATAATGTTACATATCTCCTTATATGCTTATTTGCCATCTA TACATCCTCTTCAGTGAAATGTTTCTTCAACTCTTTTGCCTTCTGTTTTTTGTTTTGTTT TGTTTTGTTTTGTTTGTTTTTTTTTGAGACAGAGTCTTGCCGTGTCGCCCAGGCTGGAGT GCAGTGGCACAATCTCAGCTCACTGCACCCTCTGCCTCCTGGGTTCACACCATTCTCCTG CCTCAGCCTCCCAAGTAGCTGGGACTATAGACGTCCGCCACCACATCCAGCTAATTTTTT GTATTTTTAGTAGAGATGGGGTTTCACCGTGTTAGCCAGGATGGTCTCAATCTCCTGACC TCGTGATCCACCCACCTTGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCGTGC CCGGCTTCTTTTGCCCTCTTTTTAATGGGGTTATTTGCAGTATTTTGCCAAGGTTTTTTG TTTTTGAGATGGAGTCTCACTCTGTCGCCCAGGCTGGAGTGCAAAGGCACGGTCTCGGCT CACTGCAACCTCTGCCTCCCAGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCAAGTAGCT GGGATTACAGGCGTGTGCCACCGCGCCCAACTAATTTTTGTAGTTTTAGTAGAGATGGGG TTTCACCATGTTGGCCAGGCTGGTCTCTGGTCTCGAACTCCTGACCTCGTGATCCGCTCA CTTCAGCCTTTCAAAGTGCTGGGATTACAGGTGTGAGCCACCACACCTGGCCGTTTATTG AGTTTTAAGAGTTGTTTATATATGTATTTATTTAATACTAGTCCTTTGTCAGATAGGTGG TTTGCAAATATTTTTTAATTTTGATGAAGTCCAGCTTATCAATTTTTCTTTTTATGGCTC ATGCTTTTGGGGTCAAGTCTAAGAATTCCTTGCCTAGCCCTAGCTAGATCCTCAAGATTT TCTTCTGGTCATCACGTCGCGTGGCCGCAGGGAGCAGACCCGGACAGCTCCAGAGCCTCC GGGCCGGGGCGGCGGCGGCGACGCTTCGGCTCCTCCTGAGCCACCTGCTGGACCCGCACC CCACTCCATCCCCACAGGCTGGGGACAGGCCCTGGCGCGGCTGTGTGGGATCAGAAGCAG AGTTGCAGAATCCAAGGACCTATTTTTGTTCTTTCTCCGCACTGCTTTATGGGAGGCATT ATGGCCCCCAAAGACATAATGACAAATACTCATGCTAAATCAATCCTCAGTTCAATGAAC TCCGTTGGGAAGAGCAATACCTTCTGTGATGTGACATTGAGTAGAGCAGAAAGACTTTCC TGCCCATGAGATTGTGCTGGCTGCCTGTAGTGATTACTTCTGTGCCATGTTCACTAGTGA CCTTTATAGAAGGGGAAACCCTATGTTGACATCCAAGGTTTGACTGCCTCTACCATGGAA ATTTTATTGGACTTTGTGTACACGGAAACAGTACATGTGACATGGAGAATGTACAGGAAC TGCTTCCTGCAGCCTGTCTGCTTCAGTTGAAAGGTGTGAAACAAGCCTGCTGTGAGTTCT TAGAAAGTCAGTTGGACCCTTCTAATTGCCTGGGTATTAGGGATTTTGCTGAAACCCACA ATTGTGTTGACCTGACACAAGCAGCTGAGGTTTTTAGCCAGAAGAATTTTCCTGAAGTGG TACAGCATGAAGAGTTCATTCTTCTGAGGCAAGGAGAGGTGGAAAAGCTAATCAAGTGCG ATGAAATTCAGGTGGATCCTGAAGAACCAGTCTTTGAGGCTGTCATCAGTTGGGTGAAGC ATGCCAAGAAAGAGCAGAAAGACTCCTTGCCCAGCCTGCTACAGTATATGCAGATGCCCC TGCTAACCCTCAGGTATATCACAGATGTAATAGATGCTGAGCCTTTCATCCACTATGGTT TACAATGCAGGGATCTGGTTGATGAAGCAAAGAAGTTTCATCTGAGGCCTGAACTTCAGA GTCAGATGCAGGGACCCAGGACAAGGGCTCGCCTAGGAGCCAATGAAGTGCTTTTGGTGG CTGGGGGCTTCGGAAGCCAGCAGTCTCCCATCGATGCAGTAGAGAAATATGACCCCAAGA CTCAGGAGTGGAGCTTTTTGCCAAGCATCACTTGTAAGAGATGTTATGTGGCCTCAGTGT CCCTACATGACCAGATCTACGTCATTGGTGGCTGTGATGGTCGTTCCAGCCTTAGTTCAG TGGAATGTCTAGACTACACAGCAGATGAGGATGGGGTCTGGTATTCTGTGGCCCCTATGA ATGTCTGATGAGGTCTTGCTGGAGCCACCACCCTGGGAGATTTGATCTATGTCTCTGGAG GCTTTGATGGAAGCAGGCGTCACACCAGTATGGAGCGGTATGATCCAAACATTGACCAGA GGAGCATGCTGGGAGATATGCAGACAGCCCGGGAAGGTGCCGGACTCGTAGTGGCCAGTG GAGTGATCCACTGTCTAGGAGGATATGACGGCTTGAATATCTTAAATTCAGTTGAGAAAT ATGACCCTCATACAGGACACTGGACTAATGTTACACCAATGGCCACCAAGCGTTCTGGTG CAGGAGTAGCCCTGCTGAATGACCGTATTTATGTGGTGGGGAGATTTGATGGTACAGCCC ACCTTTCTTCCGTTGAAGCATACAACATTCGCACTGATTCCCGGACAACTGCCACCAGTA TGACCACTCCACGATGCTATGTAGGGGCCACAGTGCTTCGGGGGAGACTCTATGCAATTG CAGGATATGAAGGTAAGTCCCTGCTAAGTAGCATTGAATGTTACAACCCTATCATCAACA GCTGGGAAGTCGTGACATCCATGGGAACCCAGCGCTGTGATGCTGGTGTGTGTGTTCTCC ACAAGAAGTGACCATTGTTGGAGCACCATCCAGAGCTAGTGACCAGTCCAGTGGACAGTT AGTGGGAGAATCAAGAATCCTTTCTAGAATGTCTGTTTCTCACTATGTGCACAGGGTGAT TACAGGCACCAGTGCAGTGATGATTGTACTTATTTGACACATACTCCCCCTCGTCCTGGT TGTTGTTCCTGAGAAGGGTGGGTAACAGATACTCCAGGGAAAAGAATGCACATTGAATGG ATGTGAGAGACCACATTACCTCTCCCACTACTTTGGGGAGCACTTTCCTGTCATTTCTAA CTTACCACGTGCTTGGTGTACTATATGTATGTTGTGCCTCATATGTTGCAAAGAACTAAG GTGAGTATAGCCTACTAGATGTGAGCAATATCCAGCCTAGATGATTGGAAAGATACCAAT TTAAGTAAACTTGGTAAAATCCAAGTCTTTTTTTTTTCCAGGAACAAATACATTTTCTAA TCTACAGGTAGCTAGGGGCAACACAGTTCCATTCTAAAGGGAAACAAAAGGGAGAGCCCC ACAAAACTTTGGGGGCAAGGGAGAGATACTCATCTGACACTTCTTTTGGAGGTCAGGGTT TGTATATCAGAATTGAAGTTAGAATCAGTGAATTAAACTGAATTTGATGGAATGTGAGTG AACCTAGAACAGCACTGAAGTATTACATAACCTGGAAGACTGAGAAGGGTATATTCTTTG AATGATCTTTTTATTTCCCCAAGGTCTTTCACACTGGAGACAGCATAAAAGAGTGAACCA ATGTTGGGATGAGAGAAGATGACATAAATGTGGGAGTTCAGTATAACTGGGGATAAACTA GAAGTACCTGTGATTTTACAGTCATCTTATTGCCTGCCAGGGGTCATCTAGCCATGGCAG TGTTAACCTTGAATGGGGGTGAAAGCCTTTCTTTGTTGAATCAAATACTACTACACTATT ACACTTCCACACTATTTATTTGGGGATGGACTGGGAGTGACAGTAGCCTAGTAGTTCAGC TACCTGATTACTGCCCCATTCTTTTAGAAGCACACTTCTGCCAAGGAGTGGTTTGTACTG CTGTGATTGGTACATTTAGTCTTTTTTCTGCTATAAGTTTTCCTTACCTGTCCTTTAGTG TAGATTTTATTCATTACAGGACAGAATAATCAAGGACAACCAAAATCCTTTTGTTAGTTT CAGTACCTCAGCTATCAACATTTCTGAGCTATCATTCAATGTTCCTCTGTGTCATGGAGT GAAATTCTTGTTTTATGGGTATTGGGAGTGTGGGAATGTGATAACCTAAACAACCTTTGC TCTGAAATTCCATTTTTCCCTCTTTCCCTGAAGTGTACTGACCTGTACTACAGAGTTAAT TTCTTTTGTATTTTTTTAAGAAAATATTAAAAATCAATGGTCTCAAAAAAAAAAGATTTT CTTCTTCTTTTTTTCTAGAAGTTTTATGGTTTTACATTTAACTCCATAAACCATTTTGAA TTCATTTTGGTATATGGTATGAGACTTGGGTTGATATCCATTTTTTTGCCGACGCATGTC TAATTGCTCCAGTACTATTTGCTGAAAGATATCTTTCCTCCACTCAATTGTTTCTGCACC TTTGTCCAAAATCATGTGTAACATGACTGGGCATATTTACTCAGGTCTGTGGGTTTTCCA TTCTGTTTCACTGATCTATGTGTCTATCACTGTATCCATACCACAGCCTTGATTACTGTA GCTATGTAGTAATCTTGAAATTGGGCAGACTCCTCCCATCTTATCGTTCTTTTTCAAAAT TATTTGAGCTATTCTAGTTCCTTTGCCTTTATATATACATTTCAGAATAATCTCATTTAC ATCTAATGTCACAAGTAAAATCAAATCAAAATTAAAAAATTTATTTTATTTATTTTGAAA TTTATTTTATTATTATTTTTTGAGACAGTCTCACTCTGTCGCCCAGGCTGGAGTGCAGTG GCGCAATCTCGGCTCATTGCAACCTCTGCCTCCTGGGTTCAATCCATTCTTGTGCCTCAG CCTCCCCAGTAGCTGGGATTACAGGTGTGTGCCACCACACCCAGCTAATATTTGTATTTT TAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTTGAACTCCTGACCTCAAGCG ATACACCATCCTCGGCCTCCCAAAGTGCTGGGATTACAGACGTGAGCCACTGCGACCAGC AATAAAAAAAAATTTTAGGCTGGATGTGATGGCTCACTTCTCTAATCCCTGCACTTTGGC CGAGGCAGACAGATCCCTTGAGACCAGGGGTTCAAGAACAGCCTGGGCAATATGGTGAAA CCTTCATGATACTAAAAATATAAATATTAGCTGGGCATGGTGGTGCTCACCTGTAATCCC AGCTACTTGGGAGGCTGAGGCAGGAGGATCACTTGAACCAGGGAGGCAGAGGTTGCAGTG AGCCGGGGTCATGCCACTGCACTCCAGCCTGGGTGACAGAGCGAGACTCTGTCTCAAAAA AAATAAAAATAAAAAAATTTTTAAAAAGCTTATTGTGCATGTAAAAGAAGAGTTCATGAA CCAGGAAACCCCAAACTGGAAGTGGTATGAAGCCCCAATTCACAGCAGTTACAACACAGT TTATAAAGCATGAATGAGGAAGAATATTGTTCCCTACTGTGCATGTGGAGCTGCATAAGC TTTCTTTGTAAAGCTATCACACTGAGGGAGGAAAGCTTAAGTTTCAGTTACATGGCTACA GGCAGTTGGCCTAGGGGTATATCCAAACCTCAGCTTCAATTTTCATTTTCCCTTAATAGT ACAAAAAACCTTCCTGAAATTTTGATAGGAATACTAGTATTCCTGTATTAATACTATATC AATATGAGGAATTCTGTATTAATATATGTATTATTAATTGGGTTGGGAAAGAACTGACAT TCTTACTATTCTTACTCTTCCAATCCATGGACTTGGTATGTCTCTCCATCTATTCACATC TTCTTTTATTTCTTTCATCGGCTTTTTGTAATATTCAGTGTACCAATCCTGTGAGGTTTT TTGTTTGTTTGTTTTGAGACGGAGTCTTGGCAGAGTCTTGCTCTGTTGCCCAGGCTGGAA TGCAGTGGTGTGATCTCAGCTCACTACAACCTCTGCCTCCCGGGTTCACGCCATTCTCCT GCCTCAGCCTCCTGAGTAGCTGGGACTATAGGCGCCTGCCACCATGCTCAGCTAATTTTT GTATTTTTAGTAGAGATGGGGTTTTACCATGTTGGCCAGGCTGGTCTTGAACTCCTGACC TCAGGTGATCCCCCCCGCCTGGGCCTCCCAAAGTGCTGGGATTACAGACGTGAGCCACCG CGCCCTGCCAACATGACCTATCCTTTTTCATTCCCCAACATCTGATCCATCAGCAAGCCT GATCAGTTTACCTCCACGCTACACTCTGAACCCTCCCATCTCCCTCCAGCTCCACAGCTG TGTGCCAGCACAGGCTACTGCTGTCTTCAGCCCAGACCACGCAATGACCCAGGGCCAGCC TCGCTTGCTCTGCTCTTGATCCTTGGGCTTCCTTCTTCTACAAGCATGCCCCATGCTTCC TCTCCTGCTCAGGAAAAGCTGTGAGCCCCTCTCCTGCCCTGGAAGGTCCTTCTGCTAGAC TAGGCAGATGGTTAGATATGAGCAGGGAAGTGGACTCCAGGTTGGCCTCAGCCCCTAGCA TGGCCAGAGGGACTATGACCTCTGAGACATGTTAACCACACCTGGGGTTTCAAAAGTCCC AACCCCTCTTTCAGTTAAGCTTATCCAGCTCAAGTAGGAACTAACCCCACAAGGAACTTT CCACCCCCGGGAGCCTGTGTTGTATAGTTTGCCCCAGTTTTGCTTGCTTCGAAAGTAACC TTTTGTTCACTGCAGTGGTAAAAAACACACCTCCTGGGTGAAGATTTAAGATGCTAATGA GACATGCGATAAATACACTAGCACGTAGAGCCACAGTGCATGCGCTGCTAGAAGACCACC CAAACAAAACATGCTTATGAGTAACACCTCTTCCCGCCCATTTATGAATAATCATGTAAG CCTCCTGTAAGGGAGGTTTCCCATGCCAGTTGGGGCGATCTTATTCTGGAGCAGCCTGCT CTGACTCGGCCTTCAGAATGTACCTTCACTTTTGCAATAAACTGCCTTACACTTATCTTC GTTAATGCATGTCTCTTGCTTAAAGTCTTAACCAAGAAGACAAGAACCGAGGACACATTC CCAGTAACATTTCCTCCCTCTGTAAATGGCAGGTCTAATTGACATCTCCTCAGATGGACA TTCCCCAAAAAGTCCCTCTAGATTCATCCTACCAGAGCCCCCTCCCTGGAAATCCTCCAG TTCAGCCCCCTCTTCATTTCACTTACCATAATTGTAACCACTTTACTGATCTGTTTTCTG GTTAAGAGTGGTTTCTCCTACAAGGCTGTAAACACCATGGAGTGGGGAGTCTTTCTCATT TCTTGTCACCACTGGATTCCCAGGACTTGGTACATGGTGCTTAGTAGTTGCGGAATGAAT GCGTGCATGAAGGAACACCGGCAGCCACGATGCAGCTGGTTGAAAGCCAAGGCTGCCCCT CTGTGGCCAAGCTGGAGAACTGCTGCTTCCCCAGCTGCCGCAGACAAAGAAACTGCTAAG CCCCTCCCACAACACCCACCTAGGAGGCTTCCCCCAACACCCTTCCTGGAACAGAACCCA AGAGCCCTCCTTGTCCACTTGGTCTCAAGGACCTCCTGATCTTCTCCATGCAAAATCTGC TCCTTTCCCCAGGATGGGGGTACGGAGTGTCCTTCCAAAGAGGCTTCCTTCTCCCCAAAG AAGCTGAGAGCAAGGAGCAGGCCACAGCTTGCAGCTCACAGCTGCCCAAGAGACAGAAAT CTGTGTGGGCTGTGTGAATGACCTGGATAAAGAAGAGCTGGATCCATTGAGGAGACACTA GCCCTGACCCCTGAAGTCAAAGTCTAAGTCCCTGGGGGAATTGGGGGTCATGAGAGATAC TGGAAAGAGGCTTACAAAGGCCAGAGCTCTTGCTAAACTCCCTGAGGCAGTGCAGGTAGA ACCTGCCCCCCATACCATCCTCTGCCTCTCAGGTGCTCGGTTATCCCCAAAGGTGTACCT GGCCCATAGCAGGTGCTCCACAAATGCTATGCCAGTACTAGCGGGGTGCCCTGCTAGGGT ACCCCTAGTACTGCTAGGGTATGCCCTGTTCTCGGAGGCTGAGACCCAGGAAGTAGGGGG CCAGCAAGGCCTGTTCCTTCCCTGGGATCCCATGTCTGGTTGTTACACCAGGCCTGAGTA CCCCAAAGTCGTAAGATAAAAGAGAGGCACCTCCCTAAAACATCATTTTTCCATAATGAA CATAATTTTAAACATCTTCAAATTGGAATAAACCAAGGTATTATATAGGATGCACGTGAT TTTTTTTTCAAGTTGTCCCAATATTCAAACATAGGATTTAATATAAAAAATAAAACCTTA GAGTGTTTCTTGGCTCTGGAAGGCGCCACCCTTCAGGGGACCTAGAGGGAAGAGGTCTGA GGATAGGCCAGTGAGCTCTCAGGGTCCCTTCCTGTTTAACATCTCTGGAAGTGTCCCAGC TCGTCTCAGCTGCACAAAGAGCAAATGTTCTATCCACATTTGAACTACAAGGTTTAAGCA GCTTGCCAGGGCCAAGGTGGTGTGACAAGGTCTCAGCATGTAAGACTCACAAGTGGGGCA CAATTCTGAGCAATGGGGTCTGGATCCTTGATGAGCCTCCCCAGGTGGACTGGCTGAACC TCATGGGGCCTCATCAATGCATTCAATGCAGGGTCTCTGGAGAAGCATGGATTCTGCTCA CTGAGCCTGTTTCTCTGACCTGGGACAGGCCTTGACACCTAGGCCTCTCACTCTTTGCAC ATCCTGGGGAAGGAAATTAGGCAGGAGAAGGCCCCTCTTCGAGGGCTTCCTTACCTCCCT CTTGAGTGGGGCCACGTATGCCCAGGAGTTGGTGGCAGGGTCGTAGCGCTCCACAGCATT CAGGTCATTGTGGTAGTCACGGCCCGCCACAGCGTAGATGTACCTGCCTACAACACACAC GGACAGGTCGGCGTGCTCCTGCTGCAGGGACTGGATCTGGAACCAGCGGTTGTGCCGTGG GTCATACCTGTGCCGAGACATGAGGAAAGCACAGCACTGACTAGGCAGGGAGGCTGCTCC GCAGGCTTGCACCTCTTGTTCTGGTCCCATTTCTGCTTTGCCTCAGCCCAGCCTGAACCC TCCCCAACTACCCCACATACCAGTGCACACCGATCTACAGCCAGGCCCACAGGCTGTTAC ACACCTGAGCACAGGTGCTCCCCACCACACAGCATGGAAGGAATGGCCAGGAGCCTCCTG TGTTTCCACCTAACTGTCCACCTGGGCTGTCCAATGGCTCTTTTTTTTTTTTTTTTTTTT TTTTTGTGGAGAGAGGTCTCTCTGTCCCCTAGGCTGAAGTGCAGTGGCACAATCATGGCT CACTGTAGCCTCGACCTCCTGGGCTTAAGTGATCCTACCACCTCAGTCTCCTAAGTAGCT GGGACTACAGGTGCGCACCACAACGCCCGCTATTTTTTTTTTTTTTTTTTTTTTTGATAG AGACAAGGTCTTGACACCTGGGATATGGACTTAGGCAATCCTCCTGCCTCAGCCTCAATG GCTGTTCGTACAGCCACTTCCATCTCACTTACCTCATCTCATCATCCACATAGCCTTGCT CCCACTCAGGAGGGCCTCCAAATACAGCCTCTCCACTGGATGGGTGCCATCAGCCCCCTC ATTGGCCTCCAAGCCTTGAACTTCCCCCAGCACTCCATCAGGAGTGGTCTTCTCAGGCAC GCCCTGCACACAGCACCCATCATACCCTCAGCTTTGTCAACAGCCGCACACACAAGGCTC TGGTCATCGTACTTTCACCTCCTCCCACCATGCTGCCAGCCCCGCGTTTCCTGACCTCCC CTCCACTCAAGGTCTCCCTCTCTGGGAAAGTGGAAAGGGTTAACTCCCACCCTGGACTGC AGGCTCTGTAAGGGCAGGGTGTGTGAGATTCCAGGGACCCCCATGGGAGAAAAGGCACTA GACTGGTTGCGGCAGGCCCACGAGGGTGGAGGAGTCTGGCCAGGACCCAAGGAGGGCTCC TGGGCTGAGCACCAACAGGGAGCATGGCCTGCAATGTGGAGCAGAGATGATCACAGAGCC ATGAGGGGCCAGGACCCAGTGAGCTCAAAGCCAGTATCTGAAGTGTCCTTGCCCAACAGC CCACAGATAGTTACAGCCTCTTTCAGACAAGTGTTTTTATTTCTGCAGAGGAAGACCTAT TCCAAAGAGAACAAGTCTAACTTCAACTGGCTCACAAGAGAGTAACCAAAGTTTTCTTTG GCCTGTGCCCTATGCTGGTTAGTGAAAAGATCAGTTTCATCCTTTGCTTCCTGCAGCCCC TGCAGTGTGGCCAGCCCCTCCCCGCCGCCTACCAGGAATGCAGCAAACAGGAGCCTCAGA AGGCAGGGCCAACAAGAGAGGGAACACACACCTGGGTCTCCCTGACCTGGTGTGAATCCT GGGCTCTTCCCTGTCTAGCTGGGTGTGCCTGGGCATCAGTTTTAATCTCTTGGGATTCGC TCAGTTCTGCACCTGTCCAATGAGGTGATATCACCTGCCTAGAAAAGCTATCAAGAAGAA TCATAAAAGACGAGCATGAAAAGTGTCCTTGTTAACTTGGGGGTTCTGATGGATCATGGT TCATTTCTATATTCCTAGACCTCAGTGTAGCCTCTGGCTCAGGACAGATTATGTTCTGTC TGCTGAATGAATTAAACAAAGGAGTAAGATTACCAGCAATGAATCATTCATTCACTCTAC CAACATTCCCAGGTCTCCCACTGTGGATGGGGCACAGTGCTCAGCACTACATCCAGAGGG AGCCTCAAGAGGTCAGGGCCCTGCCTTCCCCTGCAGAATGGAAACAGGCTAACCAGAGCA AATGAGTGCTTAATATAATGACAGACACTGTGAAAGAAAGAAAGGGAGGTAAGAAGATGA GTGACCTGCTGGTCAGCGGGGCCTGGGGAGGGCAGAGCTCCCTTGCAGTGATCAGGAAAG GCCTCCTGAGAAGGCAGGGTCTGAGACCTGCATGACCTGGGGCACAAGCCAGAAGAAGGT CTGGCAGGGGACCAGCAGTCCAGGATTGGGGCACAGCACTCACAGGGGCCCCGTCAACTG GCTGGGATGGTGCTGTTCACTGCCTTTCCTGTGCGCCTTCCTCCTTTATATACAAGTCTC TCCTCTCTTGTGTTCAGTCTGGCTCAATCACCATCTGCACCCCAAGTGTCTGCAACCAGA GTGAGATAGGACCCTCCATGTGCTCCTGTTTGCTCCTTCGAGTCTATTGTCCTCACAGCT TTTTAAGCTGAGTTCATGTGGATTTGTGGCTTCAAATTTAAAGACACACACAGGGCCGGG CGTGGTGGCTCACGCCTGTAATCCCAGAACTTTGGGAGGCTGAGGCGGGTGGATCACGAG GTCAGGAGTTCAAGACCAGACTGTCCAATATGGTGAAACCCCGTCTCCACTAAAAATATA AAAAAATTAGCCAGGCATGGTGGTGTGTGGCTGTAGTTCCAGCTACTCGGGAGGCTGAGG CAGGAGAATCACTTGAACCCGGGAGGCAGAAGTTGCAGTGAGCTGAGATCATGCCATTGC ACTCCAGCCTGGGCGACAGAGTGAAACTCCATCCCCCAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAGACACCAACAGTGATACCATATGTCAATAATTTAGACATAGCCAAA GTTACACTGATTTCTTATTTCCCTGTTGTGTCCTGTATCCCTCTTCTTCTTTATTGAATT AATTTTCTACTTTATGAGAATAAGTCCTCAAGTAATATTTTTCTAATAAAAATAAGCTTT ATTTTTGCATATAAAGATATAAACAATTTTACTTATTGCTATGGACTAGATGTTTGGATT CCCCCAAAACCCCTATGTTGAAGGCCTAACCCCCATTGTGATGGTAGTGGGAGGCATGGC CTTTGGGAGGTGATCAGGTCATGAGGGTGGAGCCCCATGATGGGCTTAGTGCCCGAGGCT ACAGCTCTTTCTCTCCACCATGTGAGAACACAGACGGAAAGGACCTTACCGGGACTAAAC TGGCCAATACCATGATCTTGGACTTCCCAGTATCCAGACAGTGAGAAATAGAGGTATGTT GTTTAAGTCACTCCATCTATAGTATTTGTTATAGCAGCTGAAACTGACTAGGAGAGTCAC TTTAACTCAGATTTGTATCATCCTATTGTAAACTCAGACATGAACCCTAGAGTTGGTACA AAAATATTCTGGAATTCTTGTTAAATTCATTAGACTGTGGCCAGGCACGGTGGCTCACGC CTGTAATCCCAGCACTTTGGGAGGCTAAGGCGGGAGGATCATGAGGTCAGGAGATCAAGA CCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCAGGC GTGGTGGTGGGCGCTTGTAGTCCCACCTACTCGGGAGGCTGAGGCAGGAGAATGGTGTGA ACCCAGGAGGTGGAGCTTGCAGTGAGCTGAGATCACGCCACTGCACTCCAGCCTGGGTGA CAGAGAGACTCCAACTCAAAAAAAAAAAAAAAAAAAAAAAAATTCAGTAGACTGTTTTTC TTCTATATTTCCATGACTTAAGTATTTTCATGACTATTAATGTTTTAATGTCTATTAAAT TTTTTAAATAAATTAAATGTACTGACCGGGCACGGTGGGTGGCTCATGCCTGTAATCCCA GCACTTTGGGAGGCTGAGGCAGGCGGATCACCTGAAGTCAGGAGTTCAAGACCAACTTGG TCAACATGGTGAAACTCCATCTCTACTAAAAATACAAAAAAATGGCCGGGCGTGGTGGCT CACACCTGTAATCCCAGCACTTTGGGAGGCCCAGGTGGGCAGATCACCTGAGGTCAGGAG TTCAAGACCAGCTTGGCCAACATGGTGAAACCCTATCTCTACTAAAAATACAAAATTAGC CAGGCATGGTGGCGTACACCGGTAATCTCAGCCACTCGGGAGGCTGAGGCAGGAGAATTG CTTGAACCTCAGAGGCGGAGGTTGTGGTGAGCCAAGATTGCACCATTGCACTCCAGCCTG GGCAGAAAACAGCGAGACTCCATCTCAAAACAAAAAAACAAAAAAACAAAAAAATTAGCT GGGCGTGGTGGTCGGCGCCTGTAATCCCAGCTATTCAGGAGGCTGAGGCAGGAGAATGGC TTGAACCCGGGAGGCAGAGGTTGCAGTGAGCCGAGATCGTGCCACTGCACTCCAGCCTGG GCGACAGAACGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAATTAAATTTACTTTAAAAT ATTAGCAGCCATGAAATGCCGAAGGAAAAATTACATTATTATTTAAAAAATTTTTTTTAG AGACAGGGTCTCACTCTGTTGCCCAGGCTACAGTACAGGGGCATGATCATAGTTCACCGA AGTCTAAAACTCCTGGACTCAAGCGATCCTCCTGCCTCAGCCTTCCAAGTAGCCAGGGCT GCAAGCACGTACCATCAGGCCCAGCTAATTTTTTTTTTTTTTTTTTGGTAGAGACGAGGT CTCGCTTTGTTGCCCAGGCTGATCTTGAACTCCTGGGCTCAAAAGATCCTGCTGCCTCAG CATCCCAAAGTGCTGGGATTATTGACATGATCTGCACATAGCCTAAATTATTATTTTAAG TAGGGTCAGGGCACATAGCAACATGTAACCAGCTACTACTAGGTATACCATGTAGTTGGT GTTCAGTAATCCACAGGGCTATGGCTATGGGAAAGGTACAAGGAATGATGCAGATAATAT AGATGCAAAGAAAGAAGGAATGAAAGAGAAAAAGTAGTATATTGGAGACAAACACCATTT GCTCCAAGAGAGGAAAGGTTTTTAAATATGCGGTTTCTGCACCAATGCTTTAAGATATAA CCTGGTAAGTAATAACTCCTCAAAATGCAAAGCCTAATTTACTGCAAAATCTTGTTTCCT TATATTCTAAAAGCCTATAGCTTTTAGAATTCACCAGTTCTAAGGCTGATGAAATGAGAA AGCAGAGAAAGAACCAAATCTTAGAATATTCAAATTAAGGATTCATCTGGAAGTTTTGAG TCCACTGGAAGGTTAATGATGCCTTCATTCTGGAGAGATGATACATCCCTGGGTTAACCA GTAGGGCTGTGGTCCTTCCTGCCACCTCCACTCTGTGGATACCTACTCTCTCAAGGCAGG TCTTGCCGACCCCACTTGCCATTGTGATCCTACTCTCAGACAGGAGTTTGGGACTGGTTA TTGGTTACAGGGTCTAATATTAGCATTACCCAGAGTTCAGCTTTGGTCTCACACCCCTTC ACAAGGGGAAATTCAGACTCCTACTTTAAGTGAAAGTGAGATCAATGGCCCATAGCAGGT GGACAGAGGACAGGGGCAGGCAGAATCCCAAGCTGTCACCCCCCTAGGGACAGCTCTGTG GAAACATCCAGCATTACTGCCAAGCACACAGCTGTCTCTACCTTCTGCTAAGGGCCAGAA GCCAAAACACTCTGGAGAGATTGGCTTTGCAGGTCTGTAGGTTACTCTCAGCAGGGTGGT CTGGCCTTGGCCCCCCAACTGGGAAACCGAGCCCCCTAAAAAAGAAGGTTTAAGGTTTGG CCAAAGGGAAGTCTAAACCTAGTGTGGCCACACCCACTGGGGGCACAACTTTGTTTAGAG AGGAAATGAAGGTAGCTGTTTCCTTTTTTCTGAAACCTGCCCTTCCTCATATTTAACCAC AATGTGTTATTGCCAACCTCTGAGTCTCATCAGCTTAACAAAGGAGATCACTTCCTGGGG CCAGCAGTAGAGAGGTCGCTGAGAAATGGCCTTCTCCTCCTTGGCCAGGATCACTATGCT TGATTCCAAGTGGCATGGCCAGGCCAGGCCCTACCCCACCCTGACCCCAGAAAATAGAAA CATGAACATGAACTCAGTCAGCATCTAGCTCATGGCTGCTGGAAATAGTTGGCAATCTCT CTCTCTCCAACACAGCTCCATCTTTCAGCATGAAGGGTAGGGAGATTTCTCACATGCACC AAGGTAAGGTGGAGGGGTCCCTGGAAATCTAATGGTAGCTGAGTCTTGTGGGCCTGAACC ACTGTCCCTTGCTCTGCCCAGAAATGGATTTGTGAGATCACAGCCTAAGTGACTGATGGA GCCAGGCGCATCCTACGGACCACAGCAGTCCACAAAACTCTCTCTTGGGAAAACTGCCTC TTCCCAGGGGAAATTGTGCCAAGTGCGTGGCAGACTGCAGGGCAGAGCGGAAATGTAGCA TGAGGCTCGGTGATGCTTGTTGGCCTTTACCACGCCCCTGGGCTGAAGACCGCATGCTGC TCAGGCACAGCAGCAGGAAGCCTGTTCCACCATTAGCAAGCAGGTAAGAGAGAAGAGCAA AGCTGACTGGTGAAGGGAAACTGAAACAACATGCATCAAAGCTCAGAGAGAGCATGATGG AGGGCGAGGCTGCTCAGCTGGCACTGCTCCTAGGTCCTCTCCATCCCAGTCCGACTGCCT TTCCTGCTGTGGGCTCCTCTCCCCTCCCGTCTCCTCCTGCACAATGCACTCGCAACCTAG CCTACCCCAAAGAGGCTGAGCAAGGGTCCAGGGTAAGTGGGTTTCTGAGGAGTAGGGGAC ATGCAGGAAAGAAGGCGATCCACATGTCCCCTTCTCTTATCTCACTGCTGTCCACCTTCT CTGAATTCACCTAGACTTCATTCCACAATGGCAAGTGATGCTCATGTGAAAGGCTGGGGG GAACCTGACCAGCTCTCTCAGCCCATGACTTCCACACCCTCTCATCACCTGAAGACAAAG GGACCAGCTAGCACCCTGTCCTACCTCCAGCATCGGGACTCTGCTCGAAATCCTTGGACA TTGTTGTCCCCTCCAATCAAGTATACGAAGTTGTTGAGCACCGCGATGCCCTGGTTGGAC ATGCGGGGGGCCAGGGAGGCAGTGAAGTGCTTCCACTCTCCCAGTAAGGGGTTTAGATAC TTGGCCTGGTCGCTGAGGACAGTGGACGGCGTGGAGTGAATGCCCCCGAAGCCCACAACG CACTGGAAGTCCGACCGCAGCTCCGTTTGCGGGCTCTGCAGGCTGGGCTGTAGGCTCTCG TTCCGGTGGTACATGAGGGCGCTGGCCACTGTGTCCCTCAAAGGGCTGGGGTCCAGCTTG TCATGCAGCCGCTGCAGGACCTCAGCTTCCATCAGCGGAAACCGCACTGTCTCAAGGAGC TTTGGGGGCTCGTGCAGCGAGATCTGGTCAGCCTGCACCTGCTCCAGGCTATAATGGTAG AGAAGGGCCCCCTCATATACCTCGGTCTCGCAGGAGACCTCCAGGCGATTGCTGCTGAGG AGGGAGTAGACCTTCTCCAATGGAAGCTGGCGGTACTTGTCAGTCCGAGAGAAGGCCACA AAGTTTTTGAGGATATAGGTGTCCAGTTGCTCAGTCAGGCGGCTCAAGTCAAACAGCTCT GCCAGCCGGTAGACATCGAGAATGTTCTCTTCGTCCACCCAGGACATGAGGAAATCACAG CAGAAATGGATAATTTCTGGGATCTGCAGAGAGAATGACACCCATTCAAGCCTGGGCTGG TGAACAGCATCTGGGGGTGGGAGAGAGAATGATGGCAGAAATACGGGCTGTTGTGGGCCA GGCAAAGCAGAAGGGAAGTCCTCCTTAATTGTCCCCGACCTTTTCTGACACACTGGAGAC TGGGGCTTACTGCAGACTAGGTTTAAGTCCTGGTTTGGAAACATACTGGGTGACAGGATA TACAGCATGTTTTTTTTTGGTTTTGTTTTGCTTTTCTTTGAGACAAAGTTTCGCTCTTGT TGCCCAGAATGGAGTGCAGTGGCAGAATCTCGGCTCACTAACACCTCCCCCTCCCGGGTT GAAATGATTCTCCTGCCTCAGCCTCCCGTACAGAATGTTTTTAACCTCTCTAAGCCTCAG TCTCCTCATCTATAAAACGGGGATCATTAAAGTGAAGAGGAACATGATGAAAATGAGAGA GCACATAGGAGTGGACCCTTGATATATGTCAGTTCCCTCTTAGATCAAAAGTACTGGCTC GGCCGGGTGCGGTGGCTCATGCCTATAATCCCAGCACTTTGGGAGGACAAGGTGGGTGTA TCACGAGGTCAGGAGTTTGAGACCAGCCTGACCAACATGGTGAAACACCGTCTCTACTAG AAATACAAAAATTAGCTGGGCGTGGTGGCGCGCGCCTGTAATCCCAGCTACTCAGGAGGC TGAGGCAGGAGAATCGCTGGAACCCAGGAGGCGGAGGTTGCAGTGAGCCGAGATCGCGCC ATTGCACTCCAACCTGGGTGACAGAGCGAGACTCCGTCTAAAAAAAAAAAAAAAAAAAAA AAAAGAAGTACTGGCTCTCCAGAAAAGCAGGAAGCAGCACAAATGGTCACTGTGTCATAT ATCTGTGGTTTGGAAAGGACCTAAGACCATCCCCATCCCTACCCTAGGATTCTCTGGAAG AGCCTGCCTCTCCAAAAGCGCTGGGACCAGAAACAAACCTGGCTTTTTCCCCCGCCAGAA CATCCACCCTGGTTCCCTAGAAGTGCTCAGAGCCTCTCAGACACCTAACCAATACCCACC CAGGAACCTGATTTAGAATTATTTTTCTTTTTTCTTTTTAAAATTTGAGACAGGGTCTTG CTCTGTTGCCCAGGCTGCAGTGCCATGGCAAGATCATAGCTCACCGAAGTCTCAAGCTTC TGGGCTCAAGTGATCCCTGACTCAGGGTTCTTGAGGTTCCCAGCTGGGTCAAGACCCACT TGACTCCAGTAAGTGCAGGGGCTCCCAGGTGGCCATATCAACCCTTCCCAGCCATGGGGA AGCACTAGTGGTTTGAAAGGACGGCAAGAGAGAGAATGACACTGGAGGGAGGTGGGAACA AGGCCACAACCAGCAGGCTGTTAAAGGCCACCCTGTGAACAGACAACCAAATCTTGGGAG AATAGGAGACAAAGGGAAGGGCCCTAGGGAAAACTGCCTGGGAGAACAAACCAGGTGTTG ATAAATTATTTAGGAAAGCAGGATTTTAGAGTTTCAGTGGGGCCCCCCAGTGCCCACCTC CTTTCTTTCCATGTTCAGATTGAAAATGACGAGTACCTTGACCTCTCTGTGACCCAGACA GCTGCAGGTTCTCCCAGCAGGCTTGAACCCAAACCAGGCCCTTGAACATTCCCAGGCACT GATAAAGGCATCAAGGTTGTTACCCAAAACACTGAAGGAAAAACTGGTTCTGGCCCTGAG ACAAATTCCTTAAACCCTCATAGAAACTCCATACACTAACCCACTCTGTGGGCAAGCCTG GGTAAAGCCTCTCTTTTCCCTTGTCTGTCTTGAGGACATGCTACATCACTCTATACATAA GTTCCCCTAATAAACGCTCTGGCCAGATCACCCTGGTGTTTGATGCTTCTTTCTTTGGAA TCCCAAAACGGCTCCATTTCAGGATGCTGTGGGGTCCCCTCTTGTGGGAACCCCCTTGCT ACCAACTCCAGCCATGTGTTCAATGCAATAAATCAGTTTCTCTAAAAATGGGAATACCTG GCTTATCCAGGATCATCAATAAATTAACCATTACACATGGAATAATTCTGCTGGTATTTA TGTATTTATTTATTTATTATTTTGAGATGGGGTCTCACTCTGTTGCCCAGGCTGGAGTGC AGTGGCACGATCTCGGCTCACTGCAAGCTCCGCCTCCCGGGTTCACGCCATTCTCCCACC TCAGCCTCCCAAGTAGCTGGGACAACAGACGCCCGCCACCACGCCCGGCTAATTTTTTGT ATTTTTAGTAGAGACGGGGTTTCATCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTC GTGATCTGCCCGCCTAGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACGGCGCCC GGCCAAGGTATTTAAAGCTTATCAATAAACACTAACTATAACTTTTTTAAAATTCAAAAG ACATCTAATATTGCTTGCAAGGTGACATGCTAAGAGCTGGATGAGACCTACCAAATGTCA GGCAGGTATGTGCCCTACATGAGGTATCCCCATGGTGCTTTGCTAGGCCCTGACCAGCTT CACTTGACCTTCTTTTTTCCCTTTTTGCCAGTCTTCTGCTTTGCCACTTCTTCCCAAACT ACTAGGTTTTCCTAAAATTCTGGTTTGTCATGGGCCAGGGTCCTGAAGACCAAGTTTTGG GCAGAATGTGAGCACAAACAGCCCTGAAGAAGCCTGGGCAGCAGCAGCTCCTTGAGAGGA CCCTGCTGACTCTCCTTTCAGTCCTATTTTTAGAGCAGTGGGCCTTCTGTTCAGTGAAAT CTCACAGAGGAACCAGCCTGGGCTCAGGATGTGCAAAAGCCCCATACGAGGCTCACAGAA GGGACTGCATTTGCTTGGGGTAACCCAATAGGACAGTGTTACTGGGAATCAGACTCAGGC CTCTGGGGACACCGTGAAGAATCTGGTCCCAGGCCTACTGCAGGGCACAACACATGGGGG AAGCCTCCAAAAGTCACTAGCTATGTGAACATACATGATTCTACAGGCCTATGGACTTGA CATGGTTATGCCAAGTAAAACCTGCTATAAAACAATATTGTGAAGAAGCCTGTTTTTTTT TGTTTGTTTGTTTTTTGTTTTGAGATAAGCTGGCATGTAGTGGTGTGGTGATCACAGCTT ACTGAAGCCTCCACCTCCTGGGCTCAAGTGATTCTCCCACCTCAGCCTCCCGAGTAGCTG GAACTACAGGTGCATGCCACCATGCCCGGCTATTTTTAAAGTGCTTCTGTAGAGGTCTCG CTTTGTTGCCCAGGCTGATCTCAAACTCCTGGCCTCAAGCAATCCTCTCACCTTGGCCTC CAAAAGTGCTGGGATTACAGGCATGAACCTCCAAGCCTGGCCCAGAAGCCTGATATGTTA AAAAACTAAGATAGGCTGCTGCCTCTGAGATGACCTCTGAGGCACTGGGGGAGATGACCC AGGGCTGTCGGGAGCACTGGCCAAACCAACAGAGAGAGACTGCAGGCCTCCAAGCTGACT GGTGAGAGAGGGAAAGTGATGTTAGAAAGATGATCTGGTTTCAGTGTGTGTGATCACTGC AGGGAGAAGAAAGAAATGGCAGAAACATACAGATGGAGGCCAGGCAAAAGGGAAGGAAAT GGAAACACAGAACAGAGATGGCTACAAGCAATACCGAGAAGGATGACCTGTCCTCACATG GACGCAGGGAATTAAGAAGGTGGACGGGCTCAAGGAGCCACTGAGGTTGAGCCCAGGGAA CTGGCAGGATGGTCACAGGACACACAGAGCAGGGGGACTGACAGGGGAGCTCCGGGAGAG TGGCAGGTGTGGAGGAGGAGATAGGACAGCTGGACCTGAGCCATGCTGGGGGAGATGTTT CAGAGGCAGCTGAATCAGGAGTTAACTGCTGCCAGGGTGGAGAATTGGATGAGGGTTCCC AAAGCAACAATGACAGGGATGTGGGGACAGAAGAGATCTCTAAATAAAAGAACAGGGGGA AATTCTTTTGTTGTAAAAGCCAACTCCATCTACTCACCATGCAGGTATGGTGAGACAAGT CAGTCATCTCAGATCAACCCACCCAGCATCCAGGTCCTATCCACCAATCTGAGAACAGGG CACAGGGAGGCTGCCAGGGCCCTGTCACATGGGGTGGCACCTCCACATGCCCCCGAACAG GTTGGTTTTCCTGTGACTTGAGTTGTTTTTTTTTAATTGCGTCAAACCTGCTCTCATGGG AAGAAAGGTCAGAATGATGAACTTAGCATAGCATTTTGTTTCACTTTTAAGAAATGTGGG AAGAGGTGACTGTCAACTCCTATCAGGCCTCTGCCTGATAGCTCAAAGGCACCTGCAGGC CCAGTCCCGTGTAACTGCACCCACACCACTGAGAATAATCATCCAAAACATAAGTCACAC TGTCAAAGCCTCAGAAAACTTTTTGAATTCTGCAGGAAAACAAGTACTTAAAGTTCAATA ACAGGTTGGGTACAGAGGCTCACACCTGTAATCCCAGAACTTTGGGAAGCCAAGGTGGGA GTATCACCTGAGGCCAGGAGCTTGAGACCAGCCTGGGCAACATAACAAGGCCCCATTCCT ATAAAAAAATATATATATATATAATATCAGCTAGGCACAATGGCACGCATCTTTAGTCCC AGCTACTCAGAAGGCTGAGGCGGGAGGACCACTTGAGCCTAGGAGGTGGAGGCTACAGTG AACTATGATCATGCCATTGCATTACAGCCTGGGTAACAGAGTGAGACCCTGTCTTTAAAA AAAAAAAAAAAATAGGCCAGGCGCAGTGGCTCACACCTGTAATCGCAACACTTTGGAAGG CTGAGGTGGATCACTTGAGGCCAGGGGTTCAAGACTAGCCTAAGCAACAAAGCAAGCCTC TATCTCTATAAAAATAATTTAAAAATTAGCCAGGTGTAGTGGCACATGCCTATAGGTCCT AGCTATTTGGGAGGCTGAGGCAGAAGGATTGCTGGAGCCCAGGAGGTTGAGGCTGCAGTG AGCTGTGATCGTGCCACTGCACTCCAGCCTGGGCAAGAGAGAGAGAGCTTGTCTCCAATA AATTGAATGAGTGGATGGATGGATGGGTGGATGGATGGATGGATGGATGGATGGATGGAT GCATGCATGGATGGATGGATGGATGGATGGATGCCTGCCACCATGGATGGATGCATGCAT GGATGGATGGATAGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGGATGCAT GCATGCATGGATGGATGGATGGATGCGTGCATGCATGGATGGATGGATGGATGGACAGAC AAATAGATAGATAAAGTTCAGTAACAAAACACATAAGTTTTCAAGAGACTGAAGTGGCCC AGTGAGGGGACAGGCAGGAAGGGCCCTGGCAGCAATGCTGGTGGATGCTCCCCATTGCCA GACCCCAGAGCACACAGGACGTCCACATGCAGCCTCGGGTCAGCCTACCTGCTTATGTGC AGGATGCTTTCAATGACAGGGAGACTCAGACAGTGGGGAAACAACCAGCAATAGAGAAGA GGGCAAAGGCAGAGGCCTGGGGTGTCTAGAGCGGCTCTGCTTGAGCTCCAGGAACAACTC CAGAGTCTTCCCCACTTCATGGCCTTCGTCACTGCTTGGTCTCCTCACATTCCTGACCCA TCTTCAAGCCAATGCTAGTGCCCGGCAGGCATCATGGGCATCTCAGGGAACCCCACCTGG GTGTGGGTCTGCCTTGCTAGATGAGACATGCCTCACCTGAAGCTGGCAGGCAGCCACCAG TGTCTCTTGTACATTGCTCAGGCTGAGCTCCAGCTCGGAGGTGTATATGAAATGTAGGAT TTGGCACATAGCATTGTAGGACACACCGTGGATCAGGACCTCTTCCTGTTCCATCTCCTT CAATCCCCCAGCAAACATTCCTCTGCAAAGTGAAGACACAAGAAAATGGAGTTATACCAA CAGGGACTTAAGCCCATGTTGCCAAGAGTGACAGCATTCAGAGAAGAACCTGGCGCTGGT GGCAGGGCCCTGGTAGTGGGCTGACCCACTCTGTGCTCTGAGTGAACTGTGTGCATGCAT GAGCCATCCCACAAAAGGATCCTGACGAAGTCTGGGCAGCCATCCTGGAAATTGTCCTGT TTCCCTTTTCATGGGCCCAAGGAGTCCCATGTGAGCTTCGGGAGGTGAGGCCACAAGCAG CTTGCTTCTGCTGTGTTCATCAGTGCAGTTCCTGGATCGGAGCAGTAGCAACTCCAGAGC ACATTTCAAAGCAATGAATTAATTAATGGGAAACTAATAAAAGTCATTTCATTCAATGTA TATTTATGGAGTTCCTACCAAATGCCAAGTATCATTCAAAGAGCAGAAGGAGGCCAGGCA CGGTGACTCACACCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGCAAATCACTTCAG GTCAGGAATTCGAGACCAGCCTGGCCCACATGGTGAAACCGCGTCTCTACTAAAAATACA AATATTAGCCGGGCATGATGTTGTGCATCTGTAGTCCCAGCTACTTTGGTGGCTGCGGCA CAAGAATTGATTGAACCTGGGAGACGGAGGGTGCCATGAGCCAAGATTGCACCACTGCAC TCCAGCCTGGGCAACAAAGCAAGACTGTCTCAAAAAGCAAAACAAAACAAAAAACAAAGA GCAAAAGGAGTTATAGTGAAGTAAACTGGTAAAATAGCTGGGCGCGGTGGCTCACGCCTC TAATCCCAACCCTCTGGGAGGCCAAGGCAGGCAGATCCCTTGAGCCTAGGAGTTCAAGAC CAGCCTAGGCAACACGGTGAAATCCTGTCTCTACAAAAATTACAAAAATTGGCCGGGCGT GGTGGCTCACGCCTATAATCTCAGCACTTTGGGAGGGCAAGGCGGGCAGATCACCTGAGG TCAGGAGTTCAAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAA AAATTAGCTGGGTGTGGTGGCGGGCGCCTATCATCCCAGCTACTCAGGAGGCTGAGGCAG GAGAATCGCTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCCGAGATCACACCACTGCGAT CCAGCCTGGGTGACACAGCAAGACTCTGTCTCAAAAAACATATATACATATATAAATAAA TGCTAAGAAGGAACCCAGCAGGGCATCTGACCTGAGAGTCACTGGTTAGAAGAGGGGTAG GTGGAATGATGAGAAGGAGCAGGGAAAGCCCCATCTGAGGAACTGAGACCTAAGGATAAG GCTGAGAGGGTGTAGGCAGCCGGGGAGGGAGTTCCAGGTGAAGGAGACAGGTGTACAAGT TCTGGGGCAAGGCAGAGCTTGGCATGCAAGGCAGCCAAGATGATGACGAAGTGAGCAGAA AGTGGGATGGGCAGGCAGGCCTTGGAGCCAGGTCCCCCTACCTCTAGGGTAGGTACAGTC CAGATTGAAATCTGAGCATTATGGGATGCTCTTGAGGAATTTACCCACAGAGTGATGTGA ACTGACTGGCTTTCCGACTCAGGTGCTGGAGGAGGAGCCCCTGGAAGACCAATTAGGCCT GAGATTCCTGTGGGCCTGGAGGCCAAGGGTGACCCTGGCCCTGGGGAGGGCATTGCAGCT AGAAAGCAGTAGCTGAGACTACCCTGGGACACGGCACAGAGAACCTACTGACAGACTGAA TCTAGGAGGACCTGGGAGGACTCAAGGCCCTCTGCTGGGCCGAAGAGGACCCTTTACTGA TGGAAGAAAATCTGGACAGCTGGGAGTCCCGTGATTGTGTGATCAGTGTGAGATACCTGT GAAACCTCCAAGTGTAGACACTGATTAGGTGTTTGGAGGTAAGAGGCTGGCAACTGGGGC TGAAGTTTAAATATGACAGCAACATGAAGATTATAAGAGAAGCTATTTTTACATTTTATT TATTTTTTTGACTAGGTAATACATATACATGCTACCAGATTTAAAATACAGTATAGGTTA GGCCGGGCACAGTGGCTCACGCCTGTAATCCCAGGACTTTGGGAGGCCGAGGCAGGCCGA TCATCTGAGGTTGGGAGTTCGACACCAGCCTGACCAACATGGAGAAACCCCATCTCTACC AAAAATACAAAATTAGCTGGTCATGGTGGCACATGCCTGTAATCCCAGCTACTCAGGAGG CTGAGGCAGGAGAATTGCTTGAACCCAAGAGGCGGAGGTTGCAGTGGGCCAAGATCGTGC CATTGCACTCCAGCCTGGGCAACAAGAGTGAAACTCTGTCACACACACAAAAAAAATACA GTATAGGTTGTGGTGAAAGTCCCCTTGCCTCTGCTCCCAGACCATCAGTCAGTTCCCCTC CCAGGGCCACACTATCCTCAGGGCAGTCATATTCCACATCTCACTTGGCTTTTTTCTTTT TTTTGAGACGAAGTCTCACTCTGTTGCCTGGGCTGGAGTGCAGTGGTGCGATCTTGGCTC AGTGCAACCTCCGCCTCCTGAGTTCAACTGATTCTCCTGCCTTGGCCTCCCAAGTAGCTG GGACTACAGGTGCCCCCCACCACGCCCAGCTAATTTTTTGTATTTTTAGTAGAGACCGGG TTTCACCGTGTTAGCCAGAATGGTCTTGATCTCCTGACCTCGTGATCTGCCTGCCTCGGC CTCCCAAAGGGCTGGGATTACAGGCGTGAGCCACTGCACCCAGCCCGGTTTTTACTTAAC AATATAGCTTGGAGACTGATCCATGCCCATGTACGCAAGACTGCCTCACTCTTTGCAAAG GCTGTCTCACTTTTTTTATTTTATTTTATTTTTTTAAGATGGAGTTTCGCTCTTGTCGCC CAGGCTGGAGAGCAATGGTGCAATCTCGGCTCACTGCAACCTCCGCCTCCCGGGTTCAAG CAATTCTCCAAACTCAGCCTCCCGGGTAGCTGGGATTACAGTCCTGCGCCACCATGCCCG GCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCACGTTGGCCAGGCTGGTCTCAA ACTCCTGACCTCAGGTGATCCACCCGCCTCAGCCTCCCACAGTGCTGGGATTACAGGCAT GAGCCACCGCGCCCAGCCAGCTGTCTCACTTCTTTAACCCATTTCTACAGGGGTATATTT AGGTTACTTGTAATTTCCAGTTATTTCACATGATGAACATCCTCACCCTTACAACATGCT GAGTATGCATATGCCAGGATTAAGTCCCACAAATACAGTTGCTGGGTCGAAGGGTTTACA TACATTTATACTTTGATAGTATTGTCAAGTTGCTGTTTTTATATCATTCCTGATAGCAGG GCCTGGTGCCTGCACCCTCTACCCCCAACCCAGGAAGACACCAGCTCTAACTCACCACCA CAAAAGGGAAAAACCTTATCACATAAGGTACAGAAAATCTCCTTATATGTTAAAGAGCCA TTTCCATTTTCTTTTCTGAACAGTTCAGATTCTTAGTCAGTTTTCTGTTAGACTGTCTTA TCAATTTGAAGAAATCTGCCCTTTCTTTGATGAGGAGTTTCCCCGATCTTTTGATTTTGT ATTGCTTTTTTGCCATGCAGAAATTTGTCTTTTTTTTTTTTTTTTTTAAACAAGTCTAGT CAACTGCAGTAGTGAGAAGGTGAAAAAGTAGAACCTGGAGTTGGATCTGTGACTGTGAAC AATCAACTGAGGTAACTCACACTACCTTCGGACCAACCTATCTTCGTTTTCAATGTTGTC AAATTTACCATTTACTTGCCTCTGAGTTTTGTGTCAAACTTAGAAAGGCTTTTTCTTTCT GAGATTATTCAGAAAACATTTCCGATATTTTCCTCTAGTGTTTTTATTATTGAATCTTTC ACATTTAAAACTGTTGATCCACCTGTAATTTATTTTGGTATAAGGTATGAAGCAAGGGTC CAACTTTGTTTTTCTAGATGGCAACTCTGTCGTCTTCCCTCCTTCCTCTAATTTTTTTTT TTTTTTAAGACGAAGTCTCACTCTGTTGCCCAAGCTGGAGTGCAGCAGCGCGATCTCGGC TCACCACAACCTCCACTTCCCGGGTTCAGGAGATTCTCCTGCCTCCCAGCCTCCCGAGTG GCTGGGATTACAGGCATGTACCACCACACCCGGCTAATTTTGTATTTTCAGTAGAGATGG GGTTATCACTATGTTGGCCAGGCTGGTCTTGAACTCCTGACCTTGTAATCCGCCTGCCTT GGGCTCCCAAAGTGCTGGGATTACAGGCGTGAGTCACCGCACCCAGCCCCTTCCTCTAAT TTTTGAGGCTATCTTTATCACATAGGACAGGAATAGTCTCCTTTCGTTACTTTTGTTTTC GACAATTTCCTGGCCACTGCTAGCTTTTTACATTTGAATTTTAGAATCAAGTTTCTGTAA TTGCCCCAAATAAACCCTCTTGGTATTTTAGGGGGTTATGTTAAACATCTAGGTTAATGT AGAGATAAATAGCATTTTTATAATGGACACAGTGGATCTTTTTAACTGTCCAGCCATCCT CTGGGTCCATTTGGAAGTCTTATAAGTCATCTTCCTACAGATCTGGCACATATTTGTTAC ATTTATTCCCAGACATTTCACTCCATGCATGGCCAAGCCCTCGGGGCACTTTCCCATGCT CTTTACAGTGGTCTGTCTGGCTCTCCTGGACTTTCCAGGACTGTGCTCGTGCATGTACTT CTGTCTGATTCTTCTGCTGGCACCTCTGGGACTATATTGGCAGCAGTGGAGATGGCAACA TTTCAAGCACTGCCCGCCCTGAGCAGGGTGTGGGTGCTTGGATTGACACAGATTTTTTTT TTTTTTTTTTTTTTTTTTTGAGACGAAGTCTTGCTGTTGCCCAGGCTGGAGTGCAGTGGC GCGATCTCGGCTCACTGCAGGTTCCGCCCCCCGGGGTTCACACCATTCTCCTGCCTCAGC CTCCCGAGTAGCTGGGACTACAGGTGCCCGCCACGTCGCCCGGCTAATTTTTTGTATTTT TAGTAGAGACGGGGTTTCACCATGTTAGCCAGGATGGTCTCGATCTCCTGACCTCATGAT CCGCCCGCCTCGGCCTCCCAAAGTGCTGGTATTACAGGCGTGAGCCACCACGCCCGGCCT TTTTTTTTTTTTGTGACAGAGTCTCGCTCTTGTTGCCCAGGCTGGAGTGCAATGGCGCAA TCTCGGCTCACTGCAACCTCCGCCTACCACGTTCAAGCGATTCTCCTGCCTCAGCCTCCC AAGTAGCTGGAATTATAGGCATGTGCCACCACGCCTGGCTATTTTGTATTTTTAGTAGAG ACAGGGTTTCACCATGTTGGTCAGACTGGTCTCGAACTCCTGACCTGATGTGATCCACCC ACCTCGGCCTCCCATAGTGCTGGATTACAGGTGTGAGCCACCACGTCTGGCCGGACACAA ACTTTTACCATTTTAAGAAAAAAAAAAATCTTATAATCCTTTCGGGGGCCAAGGCAGGAA GACTGCTTGAGGCCAGGACTTTAAGATTAGACTGGGCAGCCAGGCACCATGGCTCACACC TGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACTTGAGGCCAAGAGTTCGAG ACCAGTCTGGCCAACATGGTGAAACCTCATCTCTACTAAAAGTACAAAAATTAGCTGCAT GGTGGTGCACACCTGTAATCTCAGCTACTTGGGTGGTTGAGGCATGAGAATCACTTGAAC CTGGGAGGCGGAGGTTGCAGTGAGCTGATATCGCACCACTACTCTGTGACAGAGCAACAC TGTGCCTCAAAAGAAAAAGAAAAAGAAGGGAAAAAAAAAGACCAGATGGGGCAACACAGA AAGACCCCATCTCTTAAAAAAAATTAGCTGGGCATGGTGGCACGCATCCATAATTCCATC AATTTGAAATGCTGAGGTGGGAGTGTTGCTTGAGCCCAGTATTTTGAGGCTGCAGTGAGC TATGATGACACCATTGCCCAGCCTGGGCAAAAAAGTGAGACTCTGTCTCTTAAAAAACAA AACAACACAAAAAAGTTCACTGCATGGATCATGAATGGAGGGAGGAGAAAGAAGATATGA GACATTGTGGGTGCAATTAGGAAAATCTGAACGTGGACTCAATGTCAGATGAAATTATAG AATGATTACACATTTCTTTTTCATTTTTATGTTTTTAGAGACAGGGTTGCTACGTTGCTC AAGTTGGACTTAAACTCCTGTGCTCAAGTGATCCTCCCACCTCAGCCTCCCAAATAGCTG GGACTAAAAGCACTCGCCATGGAACATGGCTCAATCATAAATTTTCTTATATCATGATAG TATTACAGGTATGTCAAAGAATCTCCTCATCCTTAGAGAGGCGAACTGAAGTTGTTAGAG ATAAATTGTCATGATATCTATAACTTACTCTGAAGTGAATATGCAAAATGTTAACTGATG AATTTAAGCCAAGAGCACAGTTGTTCACTATACTTAACTTTCAACTTTTCTGTAGGATTT AAATATTTCAAAATAAAAATCAGAGAGAAAAAAGAGCCCAAAAGAAAAATAAAATATATA GTCACTCCTTTTTATTGTTTTTCTTTTTCACCAAAAATTTTACCAAATTTAATTTTTTTT TTTTTTTCTGAGGCAGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAATGGCGCAATCT CGGCTCACCACAACCTCCAGCTCCCAGGTTCAAGCAATTATCCTACCTCAGTCTCCCGAG TAGCTGGGATTACAGGCATGCGCCACCACACCCGGCTAATTTTGTATTTTTAGAAGAGAT GGGGTTTCTCCATGTTGGCCAGGCTGGTCTTGAACTCCCGACCTCAGGTGATCCACCCTC TTCAGCCTCCCAAAGTGCTGGGATTGAGCCACCAAGCCCGCCCCAAACTTAATTCTTTTT TTTTTTTTTTTTTTTTTTTGAGATGGAGTCTTGCTTTGTCACCCAGGCTGGAGTGCAGTG GCATGATCTCGGTTCACTGCAAGCTCCGCCTCCCAGGTTCACGCCTTTCTCCTGTCTCAG CCTCCCGAGTAGCTGGGACTATAGGCGCCCGCCACCACGCCTAGCTAATTTTTTGTATTT TTAGTAGAGACGGGGTTTCACAGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGA TCCTCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCATGCCCAGCC TCCCAAACTTAATTCTTTAAACATTCATTTTTGGGGCTGGGCGCGGTGGCTCATGCCTGT ATCCCAGCACTTTGGGAGGCCGAGGTGGGCGGATCACCTGAGGTCAGGAGTTCGAGACCA ACTTGGCCAACATGGTGAAACCCCGTCTCTAGTAAAAATACAAAAATTAGCTGGGTGTGG TGGCAGGCGCCTGTAATTCCAGCTACTTGGGAGGCTGAGGAACAAGAATTGCTTGAACCT GGGAGGTGGAGGTTGCAGTGAGCCGAGATGATGCCACTGCACTCCAGCCTGGGCGACAGA GTGAGATTCTGTCTCAAAAAAAAAAAATTCATTTTTGGCTCACGGCAGCCTCAACCTCCT GGGCTCAAGTGATGCTTCTGCCTCAACCTCCCAAGTAGTTGGGACTATAGGCATGCACCA TCATGCCTGGCCAATTTTTAAAAAAATATATTAAAAAAAAATTATTACTACTTTTCTGTA AGTCAGGAATTATGTCAAAATAAAGACTTAAAAGTTTTAAAAAGTTGTTCACACCTCATA TCCATTAGGATGACCACTATAAAAAAACAACAAAACAAACAAACAAAAAGTAGGCCGGAT ATGGTGGTTCACATCTATAATCTCATGTCCATAATCCCAGCACTTTGGGAGGCTGAGGTG GGAGGATTGCTTGAGCCCAGGAGTTTGAGACCAGCCTGGGAAACATAGTAAGATTCTGTC TCTACAAATAATTTTTTTAAAAAACTGGCCAGGCATGGTGGTGCATGCCTATAGTCCCAG CTACTTGGGAGGCTGAGGCAGAAGCATTGCTTGAGCCCAGGAGGTGGAGCCATGATGGTG CCCCTGAACTCCAGCCTAGGTGACAGAGCAAAATCCTGTCTCAAAAAACAAAACAAACAA AAAACCAGAAAATAACAAGCTTTAGCAAGGATGTGGAGAAACTATAACCCTTGTGCACTG TTGGTGGGAATGAAAAGTGGTGCAACTGCTATGTTCCTCAGAAAATTGTCAATAGAATGA TCTAGCAATTCCACTTCTGGGAATATAGCCAAAGAACTGAAAGCAGAGTCTCAGACATTT GCACACCCATGTTCATAGCAGCATTATACACAATAGACAAGAGATGGAAGCAAACCAAGT ATCCCTTGATAGGTGAATAGACAAACAAAATGTGGTATATCCATAAAATGGCACACTATT CAGCCTTCAAAATGAAGGAAATCAGGTCACATGTTATAACACACATGAACCTTAAGGACA CGATGCCAAATGAAATAAGCAAGTGATAAAGGGCTAATGCTGTATGATTCTACTTATGAG GTACTTAGAATAGTTTAATTCATAGAGACAGAAAGTAGAATGGTAGCTGCCAGGGCCTGG GGGTAAAGGAGAAAAGGGAGTTGTGGTTTAGTGGGTATAGAGTTTCAGATTTGTAAGATA AAAAAGTTCTGGAGATCTGTTTCGTAATAATGTGAATAATCGTAATATTACTGAACTAGA TGGTAAGTGTTATTTGTGTTTATTATTTTAGAACCACAATTTTAAAAAATTGTTCACCAA GTACCCAGTAGCAGGGGAATGACCAAATAAATAGAGGACATCGACAGCACGGAACACTCC ACAGCACAGAGAGTGAGGGCCATCTCTACTGCTGGTGCCTGGGACCTCCAGGATGTCACA GGTACAGAATGGCAAACCAGTATGCTGCCTCTGGTGTGAGATGGGGGAGAAAAATATTTC TTGGGTCAGCTTGTTCAGGTCCTCGATCCCTTGTCCAAACTCTTTAAATCAAGTGTATTT TAGAATTCAGAAGGTTCCAACGTGAGAAATACTGTACACATGCCACATAGGATCTTATAT CCCACGGGATCTGGGTCGGTGCCTCCTCTTCAAACACCTTAATATGCCCCCCAAAAAAGT ATAAAGACTCACACTAGGATAAAAAATATAATTAGCCTCGAAAATAATTTTGAGTTTTGG GTGGGTTCCGTGGCTTAATGAGTTTACGCCAAACTTTTTTTTTTTTTTTTTTTTTTGAGA CGGAGTCTTGCTCTGTCGCCCAGGCTGGAATGCAGTGGCGCGATCTCGGCTCACTGCGAG CTCCGCCTCCTGGGTTCACGCCATTCTCCTGCCTCAACCTCCCGAGTAGCTGGGACTACA GGCACCCGCCACCATGCCCGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCA TGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCGCCTGCCTCGGCCTCCCAAA GTACTGGGATTACACGCGTTGGCCACCGCGCCCGGCCTACGCCAAACATTTCTTTAAGAC GTTTGGCTGGGCCAAAGTTAGCTGAGTTATTTCAGTTGATTGTTCATAGTCAGTAACAGA TCAAACTCATTCGTCTCTGTCCCCCCTTCTCACTACTGCACTTAACTAGTCTAAAAAATA ATAATAGATAAAAATAGGCCGGGCACGGTGGCTCACGCCTATAATCCCAGCACTTTGGGA GGCCAAGGTGGGTGGATCACCTGTGGTCAGGAGTTCAAGACCAGCCTGGCCGACATGGTG AAACCCTGTCTCTTTCAAAAAATACAAAAATTAGCCGGGTGTGGTGGCATGCACCTGTAC TCACAGCTACTCAGGAAGCTGAGGTGGGAGAATCACTTGAACCTGGTAGGCGGAGGTTGC AGTGAGCAGGGATCATGCCACTGCACTCCCGCCCGGGCGACAGAGAGAGACTCCATCTCA AAATAAAAAAAAATTAAAAAAAAATTTTTTTTTCTTTTGTAAACGGAGTCTTGCTCTGTC GCGCAAGCTGGACTGTAGTGGGACCATCTTGGCTTGGCTCGGCTCACTGCAGCTTCAACC TCCAGGCTCAAGCAATCCTCTCACCTCAGCCTCCCAAGTAGTGGGGACTACAGGTGTGTG CCACTGTGCCCAGCTAATATTTGTATTTTTTGTAGAGATAGGGTTTTGCCATGTTGCCTA GGCTAATCTCAAACTCCAGGACTGAACTGATCTGCCCACCCCAGTCTCCCAAGGTGCTAG GGTTATAGGTGTGAGCCACCCTGCCCAGCCCCAAAAGTTTTTTGGTTTTTGTTTGTTTTT GAGAGATGGACCCTTGCTATGGTGTCCAGGCTGCAGTGCAGTGACATGATCCCACTACTC AACAGCATGGGAGTTTTGACCTGGTCCATTTCCATCCTGGGCCGGTTCACACCTCTTTTT AGGCAACCCTGCTCCAGGAAGGTCACCATATTGATGCCAAATTTAGTGTGGATACCCTGT TGGCATGGCACACTGCAACCCAGAACTTCTGGGCTCAAGCAATCCTCCGGCCTATTCTCC CAAGTAGCTGAGACTATAGGTTTGAGCCACCACGCCCTGCTCAAAAAACTTTTTAACTGT GAGAATGGATGCTGAATGTTATCACATGCCTGCTCACCATGTCTGCAGATATGCTATATA GACACGTATGCAGATATGTTGGGTTGTCATGAATTGATGGAATGCCATGATTCCCTGGCA TGTAACTAGTAAGACAGATTCCCTAGCATGAAACCAATTTTTTTTTTTTGAGACAAGGTC TTGCTGTGTCACCCAGGCTGGAGTGCAGTGGCACACTCACAACTCATGGCTCACTGTAGC CTTGACCTCCCCAGGTTTAAGTGATCCTCCCACCTCGGCCTCCCCAGAAGCTGAGACTAC AGGCATGCCTGGCTAATTTATGTTTTGTTTTGTTTGTTTGTTTTTCTTTTTTTTTTTGAG ACGGAGTCTCACTCTGTCGCCCAGGCTGGTGTGCAGTGGTGCGATCTCGGCTCACTGCAA CCTCCGCCTCCCAGGTTCAAGCGGTACTTCTGCCTCAGCCTCCCAAGTAGCTGGGATTAC AGGCATGCACCACAACACCTGGCTAATTTTTGTATTTTTTTTTGACCTCTGAACTTTTTA TTGGCCTCCTGCTCCCCAAAGGATACCCTGCTTCTGGTGGCTTAATGTCTCAGAACTTTG GTGTCATTGGTCTCAGACACCACTTTGCCATCCATTATCTGGCAGGTGGTGGTCTTTTGG ATGGTTTGCATGGAGTTGCTGCTGTCCAGGGCATCACCAAGACTGAAGTCCTCGCCATCT TCCAGCAGGCGGCAGTAGGTGGTGATCTTAGCCTCCAGCTTGACCTTAACGTTCACCAGG GCGTCCTACTCCTAGGCCTGGCACTGTCCCTCTGCCCGGATTTGTGCCACCTCTGACTCC AGGTGCAGCAGGATCCCATTGAGCTGCTCCATCTACAGGGCATAGAAGGCCTCCACCTCC CTGAGGCTGTTCTACAAGCTGGCCTTCAGATTTCTCAAGGAGTCCAGGTTGATCTCCAAG GACTGGACGTACGTCTCAACTCCGTGATTGTCATCTCAGCAGTTCCAACCTCAGTGGACT GCATGGTGACCACTGTGGTGCTCTCCTCAATCTGCTGAGACCAGTACTTGTCCAGCTCCT GTCGTTTCTTCCGAGCCAGCTCATCATATTGAGCCTGGATGTCTGCCACGATCTTGGCAA GGTCCTGAGATTTGGGGGCATCTACCTCCACAGTCAACCCAGAGCTGGCAATCTGGTCTT GTAGGCCTTTTACTTCCTCTTCATGGTTCTTCTTCATGAAGAGCAGCTCCTCCTTGAGAG CTTCGATCTCTGTCTCCAGCTACAGCTGAGGGACATTGGTGTCATCAATCACTTTGCGAA GCCCATGTATGTCGCTCTCCACAGACTAGTGCATGGGCAGTTCTGTCTCATACTTGACTC TAAAGTCATCAGCAGCAAGACGGGCATTGTCGATCTACAGAAAGATACGGGCGTTGTCCA CAGTATTTGCAAAGATCTGAGCCCTCAGGTCCTCGATGGTCTTGAAGTAATGCCTCCAGT CTCTGATCTGGGTCCCTTCTTCTCCAAGTGCTTCTGGATTTTGCTCTCCAGCTTCCGGTT CTCGGTCTCCAGGCTCCTCGCTCTGTCCAGGTAGGAGGCCAGGCGGTCATTCAGGCTTTG TATGGTCTCCTTCTCATTCTGGATGCTTCCCATTCCTGCCAGATCCCCAGCCATCCCCGC GGCCAGGCCCCCAGACCCCATGCAGCCCTGGAAGCTGGTGGAGCGGGACACGAAGATCCA GGAACCAGAGCCCTCGGTGCCTGCATAGACGCTGGCCACGCTGCTGACCAGCCAGGCGCC ATAGCTGGGAGCCTGGACAGAGCCCAGGGACCAGTAGTTGGTGGAGAAGGTAGAGCGAGT GGTGAAGCTCATGCTGTCCGGGGAGGAGAGCAAGAGGACAGGACTCAGGCTTTGCTGATG ACCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCGCCATGTTGGCCAGGCTGGTCTCC AACTCTGGACCTCAGGTGATCCACCCGCCTCAGCCTCCCAAAGTGCTAGGATTACAGGCA TGAGCCACCGTGCCCAGCTTTGTTTGTTTTTTGCTTTTTGAGGCAGAATCTCACTCTCTC ATCCAGTCTGGAGTGCAGTGACGCGATCTCAGCTCACTACAACCTCCACCTCCCAGGCTC AAGCGATTCTTGAGCCTCACCCTCCTGAGTAGCTAGAATTACAGGTGTGCACCACTACGC CCAGCTAATTTTTGTATTTTTAGTAGAGATGGGTTTTTGCCATGTTGGCCACACTGGTCT TGAACTCCTGGCCTCAAGTGATCTGCCGGCCTTGGGCACCCATTGTGCTGGGATTACATG CATGAGCCACTGTGACCAGCTCTAATTTTTGTATTTTTTGTAAACATGGGGTTTCGTCAC GTTGCCCAGGCTGGTCTCGAACTCTTGGGCTCAAACAATCTGCCCACCTTGGCCTTGCAA AACGCTGGGATTATAGGTGTGAGCCACTGTGCTCAGCCTTTTTTTTTTTTTTTTTACGAG AAAAGAGGTCTCTTACTTTGTCATCCAAGCGAGAGTGCAGTGGCATGATCATGGCTCACT GCAGCCTCAACCTCTCAAGCTCAAGCTATCCTCCCTCCTCAGCCTCCCAAGTAGCTGGGA CTATAGGCCACGCCACCATGCCCAGCTAATTAAAAAAAATTTTTTTTTCTTTTTCTGAAA CTGCTCTGAAAAAATTTAAATGGCATTGGAGTCTTTTCATAAAAGGCACACCTACCCTGG AAAACATCTGGGCTTTGTGTTTTGTGGGGAATACAACTTTCTCTCTCTTTTGGGGATAAA GTGCTATTAATTTTTTAAAAATAAAATCATCAATTGTATCCAAACTTTTAAAGTAACAGA CATTGAGTTCAGCTAAGCAGTCTTTTATAATTCACAGATGGAATGAAAACTAAGGGAGGG ACGAGGTGACCCATGGAGGTTGTATAAAAAGGACAGAGCCTTTGGGCTCTTCAGACTATA AGGGTCCAGGAGGAGAGGAGAAACCACCTCAGAAGCATACAAGGTTAAAGCATACAAGGT GGGTGTAGGGTCAGGGGAGCCAAAGTAAGTGTGCAATGAAAAGTTATTAACAAAAGTCAA AAAAAGAAAATGCAGTCTGGCCATCCAATTCCTAAACCATGTACAGAGTACACAGAAAGA GCACAGGAGGCTTCCTGCAGCAGGGCAGTACAGCTCTCTGCCCTTCAGGAAAACTGAGAT TCCTCCACCTGGGAAACAGCAAGGGACAATTAGGGAAAGCTCTCCAGAGGAGGAGGCATC TCAGGTGGGCCAAAGAAGAAAAATTTCCACAGGACAGAAAAGGCAGAGAAGAGGCCAGGC ATGGTGGCTCACGTCTGTAATCCGAACACTTTGGGAGGCCAAGGCAGGCGGATCACCTGA GGTCAGGAGTTGGAGACCAGCCTGGCCAACATGGCAAAACCCCGCCTCTACTAAAAATAC AAAAATTAGCCGGGTTTGGTGGCACACGTCTGTAATCCCAGCTACTCAGGAGGCTGAGGC AGGAGAATCACTTGAACCCGGGAGGCAGAGGTTGCAGTGAGCCGAGATTGCCCCACTGCA CTTCAGCCTGGGGGACAGGGCGAGACTCCATCTCAAAACAAACAAACAAAAGGCTGGGCG TGGTGTGGCTCACACCTGTCATCCCAGCACTTTGGGAGGCCAAGGAGGGCAAATCACAAG GTCAGGAGTTCGAGATCAGCCTGGCCAACATGGTGAAACCCTGTCTCTACTAAAAATACA AAAAATTAGCCGGGCGTGGTGGCGCACACCTGTAGTCCCAGCTACTCGAGAGGCTGAGGC AGGAGAATTGCTTGAACCCGGAGGCAGAGGTTGCAGTGAGCCGAGATCACACCACTGCAC TCCAGCCTGGGTGACAGAGTGAGATTCCGTCTCAAAAAAAAAAAAAAAAAAAGAAATTTA TGTTCTTTGTAAAATAATTCAGACAATGCAGTAAACAGAAGAAATTCAGCCTCCCTCTCC CTTTGCCCTTTGCTGAGTCCCCACGGTGCAGCAAGATCCTTCCAGATCTTCATGGTTGGG ATCATCTTCCACCAGGTCCTGAGGTTTTTCACTCCCTATCAGTAGACACACCACACTGCC CAGTTTGCAAGTAGCTCCATTGGATAACACAGAAAGGCTCTGTCCTGCACCATGTCTCAG CACTGGGTGCATCGTCCCACAGGAATGACACCTAGACACCCAACATACAACTGCAGGGCC CTAAACTATGGGCCAGCCCACCCCTAAATGCCCTTCTCTCTACCCTTGCCCATATCAAAT ATTACAAGCCTCTCACACATATTTACCAGTCATACTGGGAAAAAAATAGTCCTTTATCAT TTTAACATTTCACAAGTAACTAAAACAAATAATAAACATGCTAACACTTGGGAATGGTCT ATTTCTTTTCTTTTTTTTTTTTTTTGAGACGGAGTTTCATTCTTGTTGCCCAGGCTGGAA TGCAGTGGCGCAATCTCGGCTCACTGCAACCTCCGCCTCCCGGGTTCAAGCAATTCTCCT GCTTCAGCCTCCTGAGTAGCTGGGATTACAGGCATGTGCTACCACGCCCAGCTAATTTTG TATTTTTAGTAGAGACAGGGTATTGCCATGTGGGTCAGGCTGGTCTCCAACTCCTGACCT CAAGTTATCCGCCCTCCTTGGCCTCCCGAAGTGCTGGAATTATAGGAGTGAGCCACCAAG CCTGGCCTCTTTCTAGGCTCACTGCAACCTCCGCCTCCTGGGTTCAAGTGATTCTCCTGC CTCAGCCTCCCAAGTAACTGGGATTACAGGCATGTGCCACCACGCCCCGCTAGTTTTTGT ATTTTTAGCAGAGACGGAGTTTCACTATGTTGGCCAGGCTGGTCTCGAACTACTAATCTC AGGTGATCCACCCGCCTCGGCCTCCCAAAGTTCTAGGATTACAGGCGTGAGCCACTACAT CCAGCCTCTTTTTTGTTTTTTTGAAACAGGGTCTTGCTCTGTCACCCAGGCTGGAATGCA GTGGCACAATCTCGGCTCACTGCAACCTCCGCATCCTGTGTTCAAGCAACTCTCCTGCCT CAGCCTCCCAAGTAGCTGGGATTACAGGCGCCCACCACTACACCCAGCTAATTTTTTTTT TTTTTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTAGTCTCAAACTCCTGGCC TCAAGTGATCCACCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCGT GACTGGCCTCTATTTCTTATTTATACATACACACACACACCCCCCTGTGAAATGACTCTG GGCTCTTCCAATAATGTTGCCACTCTCCCGCTCCCCTTCCCAATGGAAGTGGAGGGATGC AGGGGGTGTTAGTGATGCATTGTAATCATCCCGGAGAGCAGGACACCTCTCATAAGAGGT TTACCCAAGGGGGGCTGTGGAAAGGGCTAGAAATTGCTCCTGGAGGACGTGGAAAGCCAC ATAAGGTTTTTGACAGATTAGCGAAAGGCTGCCTACACTGTCATCTTCAGAAAGAGTGGT ATGACACTAAATCAAGAAGGGCTGAGAGGGTGAGCCCAGGGGATCCCATAGGCTCCACAG CCTCTGGCCATCCTCTCCTATACAGGTCCTAAGTCCCCTGCAGGGGGCCCTCAGCTCCTC TACCCTACCCACTGTCCTGTATGTTATCTTAATGCTTATTAAAGGCCTCAGCTGAATTTC CCTGGTGAACTGAAAGAATTCTGCTGCTCCTGGACACATCTGCACCAAGCCACCACTGGG TCACATTCTCCAAAACTCCTTTTGGAGACAGAAGGCACTTAAACCATACATATTTTGCTT GATAGTTCCTAATTAAAAAAATAGAGAGAGATAAAGAGAAAGAGAGAGAGAGAGAACTAC AACTAAGCCTTGAAGACAAAATGATTGAACAGAATCAGAGCTAGAATGGAACACCTCAGG CTTGGAGTCCCATGACAACCTTGCTGGGGACCACGAGTGGCCCTCAGTCCTTCTCAGGCT CATGCCACCTATGTGAAATAAGGGTGGTGACACCCACCTCCTTGAGTGCAGAGCAGTGCA GACAACAGGGCAAATGGTGTATTGATCTGCTATCTGTGTACATAAATTTATTCATTTAAG CCATGGGTATTGTTTGAATATGTGAAAACCATGTCAGGTGCTAGGGATAAGAGACAGATT CCAACCCAGTGGAAAAGACAGATAAGCCAGTGGTTCCTCACACTGTGCCAAGTGCTGGGA TGGGGTCACGAAGGAGGCCATAAGAAACCCCAGAGGGGCCACAGAGCTCACTGGGAGGGC CAGAGGCTAACACCTGAGGGATGAGAGGTGTGAGCCAGAAGGTGATGCATACACAGCAAT AATTACTGACGGAGGGGAGGAATGGTAAGGGAGGGGAGGAGAGAAAGGAGGAAAGAAAAG AGAAAAGAAAAGAGAGAGAAAAGGAAAAGAAAAGGAAGAGAAGAGAGGGAAGAGAAGAGG AGGCAAAGCCCAAGAAACAGGATCACTGAATGAAAAATGAACTCTAAGACCCCCTTCTGT CTCTCACACTTGTTGACTAAGTCATAGCCACAAGGAGTAGGGGAGGCTCTGAGGTGAGCA GGAGACCAGCCACTGTCACCGCCAGTACCTTTACTAGCAGAGCCAGGATTACCTTTGTTA TTCTTCTTACAAACAGCTCTAGTGAACTCACCTGAAGTAATCGCAGGACGCAGCCAGCAG GATGCGATGGGCCTCGATGTGTCTGCCCTCCACCACCAGCACAACATCGAAGAGGATTCC GCTGTCCCGGAGAGCCAGCAGCCCTCGGAGCAGAGCCTGGGAGTGCTGTGCGCTGCGGTA GGTGTTGTTCACGCAGTGTGGGTGTGAGGGCTGTGCAGGCAACTTGCAGAGCTGGGTGAA CTCCTGCTCCTCTGCCATCCTGACAGCCACAAGATCCCTTACAACTGTGGCCTGACAGTT GGCAAAACAGGGGACAGGGGTGAGCAGGCAGGCATCAGCCCCACCACACACAGACTGGCC AGCTCTGTTGCTCCCCTTGCTCCTGTGCCTCCCTCAGGCTCACCTGTTTTCCCTCTATTT CCAAATCCCCTAATTCCTTCTCCCCTCCCACCTCTAGTCCAACTTCCTGATGGCCTTCTT CCTGATGACCTGTGTGCCATCATGCCCAAGACTCTTCATCTCATTGTATCTTCTTCAGCG GCAAAGTCAAACAGAAGGGTTTTGGGAAACAGACTGACCTGGGTTGGAGCCCAACTCCAC AGGGACTTACAACATGACCAAACTGAGCCTCGAGCTCCCCAATTTTAAAAAGAGAGATAA AAGTACCTACCCCATTGGGGTATTCTAAAATTGGTAAAACAATGAACATGAAGCATAAGC ACACACCAGGTTCTCAAGAGCTCTTCCTCCCTCCCATTGAATGAACAAAATTGGGTTCTT CCTCATAGTAATGGATGCATAAAAATGGCAAGACCTGAGTAGTGGTCAGGAGACACACAG CCAGGAGTCTGGGAGGTGGGTGCCAAGGGGGTGGCATTTCCAGGGCCTACTTTCCTTTAC TGAAATTCTGAGAGTCAAGCACAGTAACTGTGATTGAACCTGATGCATTTAAATTCATCT GCATTGCCTTTTAAATTAAAGATTATCTTCTAATAATTGAGTCAGTACGTTCAAAAACCG CAAACTGATTTGAATTGGAGCCTCTAAAACCTAATCAGTAAAATGAACCTACTGCAAATG CACTTTTGCCATGGAGCAACCAAAAGTGACTGTGGATAGCCAGCTGTTCAGGTCAGGGAG TGGCCAGTCCCACACTGTGAGGCATGGCCAAGCTGCAGGCTCTGTAGGCACAGCACAGTC AGCTACAAGATGTGAACATCTGTCTTCTCCACCTCACTTCATCCTACTTTCCTCTCCCCA AAATATGTTTATTCTCCAATTACCTCCAGTCATGCATCACTTAATGTTGGGGATACATTC TGAGACTTATATCGCTGGAGGCAATTTCCTCAGTGTGTGAACATCAAAGGGTACACCTAC ACAAACCTAGGTGGTAGAGACTACTACACATCTAGGCCATAAGGTACAGCCAATTGCTCC TAAGCTACATACTTGTACAGCATGTTACTGTAGTTCTGAATGAAAAAAACACACTGTTTT TCCTCTCCTCTTATACCACAACAACAAACACAGGAGACTTCTGTGACCAAATGTGTGAGG GTTTTTCCCCATTAACAAGCAAGCAATCAATTCTGCCACAGATACCAGCTGGGTGTCTTC TAATTCAGCAGTCCCCAATCTTTTTGGCACTAGGGACCACTTTCTTGGAAGACAATTTTT CCACGGGGTCGGGGAATGGTTTCGGGATCAAACTGTTCCACCTCAGATTATCAGGCATTA GCTAGATTCTCTTTCGGAGCACACAATCTGGACCCCTCACATGCATGGTTCACAACAGGG TTTGTGCTCCTATGAGAATCTGATGCCACCTCTGATCTGACAGGAGGTGGAGCTCAGGTG GTAATGCTGGATCACCCTCCACTCACCTCCTGTTGTGCCACCTGGTTCCTAACCAGCCAC AGACTGATACAGGTCTGAGGCCTGGGGGTTGCGGACCCCTGCTCTAATTCAACTCTAACA CTACCTGGAGACAGTGTCTGATCCCACAGGTTGAGGGCTGAGTCTCCCAGGTTGTTTCAC CTGTGCTTCTGACCAATAGGCTATAAATTGGGTTCCCAAGATCCCCTTCTTGGGTTCAAC TAATTTGTTAGAGCGACTCACAGAACTCAGGAAAATACTCATGTCTACTGGTTTGTTATA AAGGGTATTGCAAAGGATACAGATGAAGAGATGCATGGGGCCAGACATGTGGGAAGTGGT GCAGAGCTTCCAGGCCCTCTCCAGGAACATCACTCTTTAGGAGCCTCCATGTGTTCAACT ATCCAGAAGTTTTCCAAACCCAGTCATTCAGAATTTTTATGGAAGCTTCATTATGTAGGC ATGACTGATTAAATCACTGGCCATTGGTGATGAACTTAATCTTCAGCCTGTCTCCCCTCT CCGGAGGTTGAGAGGTGAGGCTGAAAGTCCCAACCCTCTTATGCCTGCCTTGGCCTTTCC TGTCACCAGCCCCCATCCTGAAGCTACCTAGGGGCTGCTGGCCAGCATACAAAAAGGCAT CACTCTGGAAAGTCCAAAGATTTTAGAAGTTGTATGCCAAGAAATGAGACAAGACCAAAT ATATATTTCACAATATCACAGTACTGAATACTATAGGCAACTGTAACACAATGGAATTTG TGTATCTAAACACAGACAAGGTACAGTAAAAATATGGGACCACCATCCTTCATGTGGTCT GGTCTGTCATTGACGGAAACATCACTGTGTGGCACATGATTATATATCTTTAAAAGAAAA AATGAAAAATATATGCTGATATAAAACTTGCCAAAAAGAAGAGGTCTCTCTCTTCACTAC TGAGGCTCAGGGCCACCCCCAACCCCATCCCCTGGATGCTCTACTGCTTCCCATCAAACA CACTCAGTTATCTGTGCCTGCCCCTGAGTCTGCCTCCAGGACCTGGGACCTCTATCCAGC CACCCACCCATTCCTTTCCCCCTCCTGGGCTGGCTCATCTTTCTCACCAAGGCCCCAGAG ATGGCCACAGCCAAGGTGTATACCCCATGACCACCTCTCACTTAGGATTCTCTTGCCTGG AAACTCTGCTCTATGATAATTCTCCCTTTTCTGACCTCTCTCTTTCATCCCACACACCCT GCTCGAGCCCATCCTTCTTTCCCAGTGGTTCACATTCCCAGTTTCACCTACAGCTCAGTG CCTAATCTAGCTAGTAGCCCTCCAACTTGTCTAATTTTACTATCAAAGCTACCATTTCCA TCTTGGAAACCTCACTGTTTTAATATTTTTCCTTTCTAACCCATGTGCCCTATGAACCTA CTAGTTCTGATTCCTTCACCTCTCCTCCATCTCAGACAGCCACCCTCCACTCCTCCAGGG CCACCACTCTGGGTCAGACCACAGCAGTGATGCCCCTACCAGTTTCCCTACCTCTTGTCT CATCCTCCCCAGGCTGCTCTCCTTCAAGCCTTACCTACTCAGCCCCATCAGCAGTCCTTA GTCCAAAAGGGATACCTGTCATCCCCTGAACACCTTTAGTCACCCATCTAGGCCATTCTG TCTCTCATAGTCCCATATTGGTCAAGGTCTAGCTCGATTACTTCTCCCTCTATCATTGTA CAGAGAACATGACAAATATGCCCCACCATTCAAACCTGCCCTTTTTTTTTTTTTTAGACG GAGTCTCGCTCTGTCACCCAGGCTAGAGTGCAGTGGTGTGATCTTGGCTCACTGCAACCT CTGCCTCTCAGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCCAGTAGCTGGGATTACAGG CGTGCACCACCACACCCAGCTAATTTTCTTGCATTTTTAGCAGAAATGGGGTTTCACCAT GTTGGTCAGGCTGGTCTCGCATTCCTGACCTCAGGTGATCCATCTGCCTTGACCTCCCAA AGTGCTGGGATTACAGGCATGAGCCACCGCGCCCGGCCCAAACCTGCCTATTTACCCACG ACAAGGAGATTCCGGCTTCTTGAGGGGTGAGGTTTTATCTCACAAACATCTCAAAAGCCC CCATGGCTGGCTGTACCACTTGCTAATGACAGACTCTGGGCACTTGTCTCATATACCCAC GCCTCAGCTTTCTCATCACCACCATGACCACCGCAGAGTCAAGAGGGCCTCCTTCTCTGA GGGTGACCTCCACAACTACCCCAATAGACATCAAGCCCCCAGGTGGATCCTCACCTTCCG TGAGCATAGTAGTGCCTCTGCTCTCTTGGGAAGGGATAAATGGCAGCTACTCCCTAAACC CATACTGCAAAAACAAGTACGGTAAAACAGAAAGACATCTTCCATGGGCAGGCAAAGCAC ATTTACCAAGTCCAATTTAAGAAACTTAGCTAGGGGCTAAGGGGGATATGCTGAAAAATG AATCAATTCTCTGCTGGGAAGGAGCACAGCACAGGGTGCAACATTTAAAACATTTAAAAC CACATCTATACGGTTTTAAATCACCAAAACGTCCACCCTCAGGAGAACAGATGTCATGGC ACAGTGATCTGACAGAACATTAGGCAACAGTCAAAATGCGCAAAGTGGCCGGGCGCAGTG GCTCACGCCTGTAATCCCAACACTTCGGGAGGCTGAGGCGGATGAATCACGAGGTCAGGA GTTCAAGACCAGCCTGGCCAACATAGTGAAACCCCATCTCTACTAAAAATACAAAAAAAT TAGCCGGGCATGGTGGTAGGTGCCTATAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGA ATCACGCCACTGCATTCCAGCCTGCATGACAGTGCGAGACTCCGTCTCAAAAACAAAAAA ACAAACAAAAAAACAATGTGCAAAGTAGGCTGGGCACAGTGGCTCATGCCTGTAATCCCA GCACTTTGGGAGGCCAACGTGGGAGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGG CCAACATGAGGAAGCCCCATCTCTACTAAAAATACAAAAATTAGTGGGGCGTGGTGGCGC ATGCCTGTTATCCCAGCTACTGGGAAGGCTGAGGAAGGGGGAAGCTGAGGCAGGAGAATC GATTGAACCCAGGAGGTGGAGGTTGCAGTGAGCCAAGATTGTGCCATTGCACTCCAGCCT GGGCGACAGAGCCAGACTTTGCCCCCCCCCCAAAAAAAAAAGTGCAAAGTAGGCTGGGCA TGGTGGCTCATGCCTGTAATCCCAGTGCCTTGGGAGGTCCAGGTAGGTGGAGTTCAAGAC CACCTTGGGCAATATAGCGCGACCCTTTCTCTACAAAAAATTTAAAAATTAACTAGGCAT GGTGGTGTACACCTGTAGTCCTGGCTATCTGGGGTGAGACAGCTTGGGGTTCACTGCAGT CTCAAGCTCCCAGGTTCAAGTGATCCTCCCACCTCAGCTTCCTCAGAAGCTGGAACAACA GGAGCTCACCACTGCGCCCGGCTAATTTTTCAAATTTTCTTTGTAGAGAAGAGGTTTCAC TATATGGTGTAGGCTAGAGTGCAGTGGTAGGGTCTTGGCTCACTGCAGCCTTCATCTCCT GGGTTCAAGCGATTCTCCTACCTCAGCCTCCCGAGTAGCTGGGATTACAAGCACGTGTCA CCACACCCAGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGC TAGTCTCTCCTGACCTCAGGTGATCTGCCAGCCTTGGCCTCCCAAAGTGCTGGGATTACA GGCATGAGCCACCGCGCCCGCCCCCAGTCTCATATCCCCAATTTTAGTCATCACAAGCCA AGTTCCCCGTTTTGTACTGTTCGAAGTTTCAGTGGGGAGAAGAAAGGAAGCTTAATGTGC CACATAGCTACAAACTGAAGGATACAAGATGAATTTTTATGAAGTGATGGATGGCAGAGT CAAAATCATTTCAGAGGAACTTTCAGTTCGGTACGATTTTACTGAGTAGATACCCTCAGC CTTCTGTAGGGCCCCCGAACGAGCAGTATCCCCTCAAAGAAACTCAATCCTTGGCAGCAT GAAGCAAGGTCGGCCACAAAGGCAAAGGGGGCGGCTGCGGTAGAGGAGAGGGAGTGGGTG CTGATTCCCAGCGGGGCAGCCTTCCAAAGGGTGCGCTCTGGAAGAATCTCCCAGGGAGCC GTCTTGGAGGCACTCGGCCCCGCGGAGGGCCGGAGCGAGGGCGCCCAGTGAGGAGCCCAG GCGGAAGGTGGCGGTGCCCCGCTGCCCGCCCCAGATCCACTCGGCTCGGCCCGCACCGGA TCTAAAATGGTCAGAACTGGCAGCCGACGCTTCGCGCTCTGGTGATTTTGCACTGACAGC GCGATCTCCGGCTCCTCTCAGGAAACCGGGAGAGCTGGCAAGGCTGGGCTCCTACGGCTG CAGTCCCCGGGCCCGATCGAGGGAACTGAGGCTCGTCAGGCCGGCCCCAAATCGCCCGCC TGTTGCAAGGCCGCCCGCTCCAGCGCGGAGGGTCGCGGCCAGGAAGGCCGCATTCGGACC TGCCGCAGGCAACAGGGCCCGCCCGGGTGCCAGGAGGCCGGCGCGCCAGCAGCCGCGCTG AGGAGGCCTCGCACCCTGGCCCCTGGCCGAGCGCCAGATCCCGGCCCCTACGCGCCCGCG CCCGGGGATCCCGCCGGCCGCGTCCCCGCCACGTCAGGCCGCGGGCTCTCCTCAGGTCGC CGCCCCCGAGCCTCCGGCCCGCGCCCGCCCCCGCGCCCCTCGGACTCCGCGCCGCTAGCT TGTCCCCGGCCCAGCGATGCCTGCTTGCCGCCCGACCCCCGCGTTCGCTGCCGCCCGGCC CGTTCGGCTCACGCCTCGCAGCTGACCTGGTGCCGCCGCCAGCCGAGGCCGCACTCGCAG CTGGAGGGAGGCGGGAGGCGGGAGGCGGGAGGCGCGAGGCGGGAGGCTGGGCGAGAGGAG CCGGCGGGGCGGGGGCCTGGGCCGTCACGTGGGTGCGCCGCGCAGGCGCCGGGCGGGCCA GACGTAGGCCCGGGGGTTCCCGGCCTGGCCGGGTAGCAGCGCGGGGGCGGCCCTGGGGCG GGGAGACAGGGGGCGGCTCCACTCCGGCGCTCTGCGGAGGACAGCAGCGATCGCCAAGGC TCAGTGAGTCCCCGCACTAGCGCGGCACCTGGCACTTGGCGGGGCCGAACCAACGAATGA GCGAATTTGCCGAGAGTCGCCCCGTCTGAATCCATTGACTCTTTCCTTGCCTCGGTCCTA CCCTCATTCCTCGACTCACTTCCTCCTGCCCTTCCTCACTATCTCCCTCCCCGCTCATAT CCTCCCTCTCCTTTCCTCATTCCCTTCTTCACTCTCTTCTCTCCTCTCTTACTCCCCCCC TCTCATTCCCTCCCTCCTTCACTCTCTCATTCCTTCCCTTCCTATCTTACCCACCATTCC CTTCCTCCCTCACTCATTCACTCCCTCCCTGTCTCCCCCTCTCATCCCCTCCCTCTCTCA TTCCCTTTCCCTCCCTCCTTAACTCTCGTTCTCTCCTCCCTCTCTCATTCCTTCCCTCCC TGTCTCATTTCCCCCCTTCTTTCCCTCCCTCCCTCTCCTCTTCTCCCATTGCCTCCCTCC CTCTTTGATTCCCTTCTTTCCTCCTCTCCTCCACTTGTTCCCTTCGTCACGCGCTCATTC CCTCCCTCCCGCGGCCTGCTCCTTCCCCGCTCCCTCCGCGGTCGGTGCCGCTGACCGTAG CGCGCCCCCTGCGGGATATCGGGGAGAATCGCTTAACACCGCCAGAGTGAACAGGTGCTA GTTTGTGTGCCCGTTCCTTTACTGAGATTTTGCAGTAAATCTAATTCCCAGTCTTAAAGC CTGGTCCCAGAGGTCCGTGCGCGGGTCTTGGCATTCATTGTTCTCCACCTCACCTTCGAG CAGGTCCCATCCTCCGAGGCGTATCCGTAATGGCTGCGCCCAGAGCTTGGAGCCACAGAG CCTCCTGATGGGATAAGGTTGGAGTGTCCCACACTGTGGTTTTGAGAGGGACTGTGAAGG GTGGAGAGAGTGGTAGGGGGAGGATGTGGCTGGAGATGGCTCATGGTTAAAAAGAAAGGT GGGGCCGGGCTTGGTGGCTGGCGCCTGTAATCCCAACACTTTGGGAGGCCGAGACGGGCG GATCACGAGGTCAGGAGATGGAAACCATCCTGGCCAACATGGTGAAACCCCATCTCTACC AAAACACACACAAAAAAACTAGCCGGTCGTGGTGGCGCGCACCTGTAGTCCCAGCTACTC GGGAGCCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCAGAGGTTGCAGTGAGCCGAGA TCACGCGCCACTGCACTCCAGCCTGGTGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAA AAAAAAAAAAAAAAAAATTGGTGGAAGGGGAATGTAAAAGGAGGAAAATACACGGAAGAG AATTGCTGTCCTGGCTGAGTCCAGAGAGATAACTGAGGGTCCCAGACAAGGATCAAGAGA ACGGGATTGGCCTCCAGAGGCAGAGGTTCCAAATGGGAGTGGGCTTCCTCCTAGAAAGAC TTTCTGGAGGAGACCCCCCTACTGTGTAACAGAGGAGGACTTTGGGATTAAGAAAAGCAT TCCAGGAAGCCGACAGTGTCAGCAAACGTGGAGGTGAGATCCTTCAAAGTGAGTGGTGTG GAGGTTTCCAGAATTTTCTGAGCCTGAAGGGAAGGTTGGAGAGCAGACCCTGCCCTTTGG AGGCTTGACTTAGCCCTGAGGGCACCCTGTAGCCAGGGTGGGCAGATGCCAATATGGTAG AGACGAAGACTGAGTAGGGAGCCAGCCACAGTGCTGTGGTCTCAGGCAGGGAGTGAAGAC CAGAGTGGAGCAGGCTAGAAACCTGGGAAGGAAGCAGGTTCCCCAGTATAAGCCCAGTGA TGTGTGAAGAATGAGCCCAGGAGATGGGTGGGGAAGTAGGCCCACCCTGCCTACAGGGGA GCCAGAGCAAGAGCAGGTCTGGGGGAAGATGAGGCCCCCCTTGGCTCCAGGTAGGGGAAG ACTGACCTCCAAGGCCCAATGGGACCAGGTGTACGGGTGGACCCAGAGAGGAGAGGAAAA GGAATCCTGGGGCAGCAACAAGAAGGCCACTGTGGTCTGAATGTCGGTACCCCCAAAATT CATATGTTGGAACTGAATCCTCAGTGCGATACTATTATAAAGTAGGGCCTTTGGGAGGGG TTTAGGTCATGAGGGTGAAGTCCTCACCATGGGATCAGTGTATAAAAGAGGTTCGAAGGA GCTCATCCCCCACTTTTGCCAGGTGAGGACACTGTAATAAGCACCATCTTCAAGGCAGAG AATCGGCTGGGCGCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAAGCCGAGGTGGG AGGATCATGAGGTCAGGAGATCAAGACCATCCTGGCTAACATGGTGAAACCCCATCTCTA CTAAAAATACAAAAAATTAGCCAGGCATGGTGGTGGGCGCCTGTAGTCCCAGCTACTTGG GAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCAGAGCTTGCAGTGAGCTGAGATC GCGCCACTGCACTCCAGCCTGGGTGACAGAGCAAGACTCCGTCTCAAAAAAAAAAAGCAG AGAACGGACCCCTCACTAGACACTGGGGCTTTTTTCTGAAGGCTACTGTGTCATATAAAA TTTATATTAAATAAATTGATATGCTTTTCTCTTGTTAATCTGTCCTTTGTTAGAGGGGTG TCAACCAGGAACCTTGTGATGCGTGAGGAAAAAATATTACCTTTTCCCCCTACTCTTTCT GGCACCCAACAAACAAGTGGGGTGGCAAAGACACCCCACTTGGGACAACTGGCAAAAGGT AAGCATTCTTACCAAGGTTAACTGTCCCATATCACTGCCTGTAGTACTCAGCTGAGAGTG GAAGGTAAAAACTTCTTGTCCCTTCCTTTCCAAATTCAAATTAGCAGAAGAAAACATTGT GTCTGGATTGTGACTCTTGCTTAAATTTGGTTGAGGGGTAACTGTTTGTTATTGATCCTT TCCTCCCAGGCACAGCTACCTCTTTCCTGTTTGTTTTATTTGTGTCCTGAGAGCTTGGCT TTATGACCAGTGAGAATATTCTCCCTGATCTCTGAATAGCCAGTGGGTGCAAGTGACAGC TTGCTTTAGGACAGCTTGCGTTAGGGTTGCAAGTAACAGCTTGCATTATTACAAACTCCT CTGTCCCGGTCAGAAAAAGAGAAAGTTTGGTTTTTGTTTGTTTGTTTGTTTTTTGTTTTG TTTTAATAGTCTTGCTCTTTCGCCCAGGCTGGAGTGCAGTGGCATGGTGGCATGATCTTG GCTCACTGCAACTGCTGCATCCTGGGCTCAACTGATTCTTCTGCCTCAGCCTCCTGAGTA GCTGGGACTACAGGCACACACTACCACGTCCAGGTAATTTTTTTTGTATTTTTAGTAAAG AGGGGGATTCATCATGTTGCGCAGGGTGATTTTTTTTTTTTTTTTTTAATTCTTTTCTGA GACGGAGTCTTGCTCTGTCACCCAGGCTGGAGTGCAATGGCGGGATCTCGGCTCAGCCTC CTGAGTAGCTGGGATTACAGGCACCTGCCACTACGCCAGCTAATTTCTGTATTTTTAGTA GAGACAGGGTTTCACCATGTTAGTCAGGCTGGTCTCGAACTCCTGACCTCGGGTGATCTG CCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACCGTGGCTGGCCTCTT TTATTTTTAAGATACAGGGTCTCACCCAAGCTGGTCAGGAACTCCTGGCCTCAGTCCACC TGTCTCAGCCTCCCAAAGTGCTAGGATTACAGGCATGAGGGACCACGCCCGGCCAAAGAG AAGGTTCTGAACCTTCTCTTTTTCTGACCATGACAGAGGAGTTTGTAAGTGGGCCAAGTC TATAAGGGGTTTTTGTTGTCTCAGTATTCATTGGTGTGATAGATGAGCCAGTCCATGACT GGAGACGTGACAGTGTTCTGTACAAGATGACATCCTTAGTCACCTGTGTCAACAAAGGTG CCTTCTGGCTAGGCACGGTGGCTCACGCCTATAATCCTAGCACTTTAGGAGGCTGAGCCA GGCAAATCACGAGGTCAGGAGTTAGAGACCAGCCTGACCAACATGGTGAAACCCTGTCTC TACTAAAAATACAAAAAAATTAGCCAGGTGTGGTGGCGCATGCCTGTAATCCTAGCTATT CGGGAGGCTGAGGCAGGAGAATGGGTTGAATCTAGGAGGCAGAGGTTGCAGTGAGCCAAG ATCGCGCCACTGTACTCTAGCCTGGGTGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAA AAAACCCAAAGGTGCCTTCTTTCTTTTGACTGTCTTTGCAAGGGGTCTGGATTTGAGAGT GCTGAATCTTTGTGCACCCTCTTTGGAGTGTGTCTTTCTCTCTCTCTCTCTCTCTTTTTT AAGAGACAGAGTCTCACCCAGACTGATCAGGAACTCCTGGCCTCAGGTGGTCCTCCCACC TTGACTTCCCAGAGTGCTGAGATTACAGGCATGAGCCATTGTGCCCAGCCTTGAGGATGT CTCTTTTGTCCATGGTTAAGTCATAAAATGCTTATTGGTCTTGGTTCTGAGTTGCTTGGT AGGTACCTTTGGTTTACAAGAAGAAAAGTGGTGGGTGGGCAGGGTGGTAGTAGGGAAGAG TGTTCAGTACTGCCAGGAATATGTACTATCTGTTCTGGCTGAAAACTGACAATGAGATAT TTGAAAGGATTTTTTTTTTTTTTACAGCTCTATGATCAGAAGATGGCATAAGTGGGGCTG GGCACAGTGGCTCAGTGCCCAGCTGGGATTACGCCTGTAATACCAGCACTTTGGGAGGCC AAGGTGAGTGGATCACGAGGTCAGGAGTTGAAGACCAGCCTGGCCAACATGGAGAAACCC CGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGCGGTGGCAGGTGCCTGTAATCCCAG CTACTCGGGAGGCTGAGGCAGGAGAATAGCTTGAACTCAGAGGGCGGAGGTTGCAGTGAG CCAAGGTTGCGCCACTGCACTCCAGCCTGGGCGACAGAGTGAGACTCCGTCTCTAAAAAA CAAAAAAGAAGATGGCGTAATCAGAAGTTGATATTCTGTCTATTGGATCCTGCTTCTCCC CTGGGGTCTTCTTGGTTCCCTGAAGCCCCTCTTCCGGAGTCCCTGCCCTCTATCGGCTCT GCCTCCTGCTTCTTTTCTGTATTGCCATGATTTTTGCTAAGGATAAAGGACAACTTCATT GGCTTCTTTGGGAAACTTGCAATCTCCCCAAACAGGCCCCTCTGAGATTTCTCCTTTCCA TTTGCTTCTGCTCCCCCTTCCTCCTTTTGCCACCTTCGATATTTTCTCCAGTTTCCTTGA TTCCTTTGATACGTCCCCATTCAGGCCACTCTGGCCCTCCATCCAACCTGCCAGAACTTT TTCCACTTCAGCACTTCAGTCACTTAAACATTAGGGCTCCCTGCCCTAAAGGGACTCAAA ATCCTGCAAAAACAACCACCTAAGGATAAAAAGAGAAAAAATGAAACTGTTTGGAAACTG GGCAAATGAATACTCTTTTAAAAGTCTTTTTCTGGGGGCCAGGTGTGGTGGCTCACGCCT GTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCAGATCACAAGGTCAGGAGTTCAAGACC AGCCTGGCCAATATGGTGAAACCCCGTCTCTCCTAATGATACAAAAAAAATTAGCCAGGT GTGGTGGCATTCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGAAGGAGAATCGCTTGA ACCCGGGAGGCGGAGGTTGCAGTGAGCCAAGATTGCACCACTGCACCAGCCTGGCGACAG AGCAAGAAGACTCCATCTCGAAAAATATATATGTATGTATATATACGTATTTACGTGTAT ATATGTATGCATACATATATATATATATAGAGAGAGAGAGAAACCAGTGAGTTTGTATTA CTGTGTTTTACTGACTCACGACTACCATTTTGGAATGAAAGCTATAAGATCTTTGTGTTT TTACGTTTATGTGTTTATATTTGTGTATATCTATGTATGTATGTTTGAATATTATATATA ATATTTTTTCTATCTCCATATTCAGTATTACTAACTGAATTTTTTTTTTTTTTTGAGATG GAGTCTCACTCTGTCACCAGGCTGGAGTGCAGTGGCATGATCTCGGCTCACTGCAACCTC CGCCTCCCAGGTTCAAACAATTCTCCTGCCTCAGTCTCCTGAGTAGCTGGGACTACAGGT GCGAGCCACCATGCCCAGCTAATTTTTGTATTTTTAGTAGAGACGAGGTTTCACCATGTT GGCCAGCATGGCCTCTATCTCTTGACCTAGTGATCCACCCACCTCGGCCTCCCAAAGTCC TGGGATTACAGGCATGAGTCACCACGCCCAGCCTAAATTTTTTAAATCCCTTAAAGGAGT TATATTCAAATTGTACCCTCCTTTGAAGATGTCTCTTTTGTCTGTGGTGTCTTAAAAGGA TTATTGGTCTTAGAGGGAGGTAAATAAGCATTCATACAAGCTAAATATTACCAAAAATTT AAGAAAAATAGAAACTAATCCAACTGCTTTTTAGTTTACATGATCTGGAATATTCTTTAG TAAATAAAGCTAGTTTGAAAATTGATGGACTACGCCGGGTGCAGTGGCTCAGGCCTGTAG TCCCAGCACTTTGGGAGGCCAAGGCGGGCGGATCACGAGGTCAGGAGATCAAGACCATCC TGACTAACACGGTGAAACCCTGTCTCTACTAAAAATACAAAAAATTAGCCAGGTGTGGCA GCATGGGCCTGTAGTCCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCA GGAGGCGGAGCTTGCAGTGAGCTGAGATCACGCCACTGTACTCCAGCCTGGGCAACAAAG CAAGACTCCGTTTCAAAAAAGAAAATTGATGGACTAGTCAAACAAGTTTTATGTTATCAC TACTGAATGGTAAAGATAATAAAACTATCAATCCAACCTAAGAGCAAAATGTGCAATAAA AATGAATTGCGGCTGGGCTCACCCCTGTAGTCCCAGCAGTTTGGGAGGCCGAGGCGGGCG GATCACCTGAGGTCAGGAGTTTAAGACCAGCCTGGCCAATATGGTGAAACCTGGTCTCTA CTAAAAATAAAAAGTAAGGCCAGGTGTGGTGGCGGGACCTACCTGTAATCCCAGCTACTT GGGAGGCTGGGGCAGGAGAATCGCTTGAACCTGGGAGGTGGAGGTTGCAGTGAGCCGAGA TCGTGCCACTGCACTCCAGCCTGGGAGACAGTGCGAGACTCCATCTCAAAAAAAAAAAAA AAAATTGGGCACGGTGGTACAAGCCTGTAATCCCAGCTACTTGGGAGTCTGAGGCAGGAG AATTGCTTCAACCTGGGAGGCGGAGGTTGCAATGAGCCAAGATCGTGCTGCTGCACTCCA GCCCAGGCAACAGAGTGAGACTTTGTCTCAAAAAAAAAATGAATTGCATGGCTTGGCGCG GTGGCCCATGCCTGTAATCCAGCACTTTGGGAGACTGAGGCAGGTGGATATTTGAGGTCA GGAGTTCGAGACCAGCCTAGCCAACATGGTGAAACCCCATCTCTACTAAAAATACAAAAA ATTAGCCAAGTATGGTGGCGTGCGCCTGTAATCCCAGTTACCTGGGAGGCTGAGGCAGAA TTGCTTGAACCCAGGAGCTGGAGGTTGCAATGAGCCAAGATTGCACCACTGCACTCCAGC CTGGGCAACAGAGTGAGACTCCATCTCAAAAAAAAAAAAAAAAGAAACAATAACCACTCC CTCCCTACCTCCACATACATAAGTGATTACAGGCTCCAGGCCAAACACACAGAGGAGTTA CATCTGGCTGGTGCTAGGATGGAAGGTGGGGCAGGTCACCACCTCCCAGGGACTGAGTGC CAAAGCAAGGAGGAAGATTGGAGGTGAGGCTGTATGGAGACAGCCACAGGCTCACAGAGC TTGTGCTGAGCTCTCTAGGGGGAAGCTTGATCTGCCTGGGAAGCTTCCAGCTCCTACCTC CTGCAGAGCCCAGAATGGTGTCCAGGAAATATGTGGCCATTGAGTGGAACCAGCCTTGGC ATGGTAGCCTGCAGGAACCCGCAGGCTAGAGACATGGCCACATGAGCAGTGAGGATCTCC CCCGAGATGAGGATCCCCAGAACATAGTGTGGGATGCAGGTCTGGCCTCTGCATACCCTC GGCTTCCTCCATCACTTATTCTCAGACATCTAAACCACTAACATCCAGACTGGGGATCCC AGGAGGGTCTACAGTGTCAAGAGAGGGCAAGTCCACTTCAGGATCAACCTTCCTGTGGCC CCTTACTTTCCTTGGTCCACCTTTCCAGGTCACCCATCCAGACTGTCACCAGGGATCACT GCCCTGGGCACCAACCCAATGCGGGGACAAGTAGAAAAAGAGCTCTGGGGAAAATACCAA AGGGAATAGTCACTCAATTTTACTGTGATGATAAACTCCTGGCAAAGTTCCAGGCCTCTC TCTGTCTGTGTCTGTGAATTATGTGTCTGTGTCTCCATCAACCATAGTGCAGAAGGGGGA GGGTTGTCCTAAGTATCCTATTAACATGTTTGTTTGTTTGTTTGTTTGAGACAGAGTTTC GCTCTTGTTGCCCAGGCTGCAGTGCAATGGCATAATCTCAGCTCACTGCAACCTCCACCT CCTGGGTTCAAGTGATTCTCGTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCACACA CCACCATGCCTGGCTACTTTTGTATTTTTAGTAGAGACGGGGTTTCACTATGTTGGCCAA GCTGGTCTCGAACTCCTGACCTCAGGTGATCCACCTGCCTCAGCTTCCCAAAGTGCTGGG ATTATAGGCGTGAGCCACCATGCCCAGCCAGAACACAGGACTTTCAAAACTTAAACCTGG GGCTGGGCACGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGA TCACCTGTCAGGAGTTCAAGACCAGCCTGGGCAACATGGTGAAACCCCATCTTTACTAAA AATAAAATAAAATAAAATACAAAAATGAGTCGGGCGTGGTGGCGCACTGTAGTCCCAGCT ACTGGGGAGGCTGAGGCACGAGAATCACTTGAACCCCGGATGGCGGGGACTGCAGTAAGC GATCACGCCACTGCACTCAAGCCTGGCAGACCTAGCGAGACTCTGTCTCAAAAAAAAAGA AAACAAGGCTGGGCGTGGTGGCTCACGCCTGTAATCCCAACACTTTGGGAGGCCAAGGCG GGTGGATCGCCAGGTCAGGAGATTGAGAACATCCTGGCCAACATGGTGAAACCCTGTCTC CACCAAAAATACAAAAATTAGCCAGATGTGGTGCCAGGTGCTTGTAGTCCCAGCTACTCA GGAGGCTGAGGCAGGAGAATCACTTGAACCTGAGAGGCGGAGGATGCAGTGAGCTGAGAT TGCGCCACTGCACTCCAGCTTGGGCGACAGAGTGAGACTCTGTCTCAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAACCAAGGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCAAT TTGGGAGGCCGAGGCAGGCGGATCATGAGGTCAGGAGATCGAGACCATCCTGGCTAACAC GGTGAAACCCTGTCTCTACTAAAAATACAAAAAAATTAGCTGGGCGTGGTGGCGGGCGCC TGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAG CTTGCAGTGAGCCAAGATTGTGCCACTGCACTCCAGCCTGGGCAACAGAGCAAGACTCCA TCTCAAAAAACAAACAAACAAACAACAACAACAAAATAAAACAAGGAAAAAAACACTAAA ACCTTGGATGTCCTGGGCAACCCCGGTCAAGATGATCACTCTAGATAGAGTTTTTAAAAA TTAGTTTTCAGCCTGGCCAAGATGGTGAAACCCTGTCTCTACTAAGGATACAAAAAATTA GCTGGGCTTCGTAGCAGGCACCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAAT CACTTGAACCCGGGAGGCAGAGGTTGCAGTGAGCCGAGATGTCACCACAGCACTCCAGTC TGGGCAACAGGGCGAGACTCCATCTCAAAAAAAAAAAATTAGTTTTGCCACCGGGCACGG TGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTAGGTGGATCACCTGAGGTC AGGAGTTCGAGACCAGCCTGGCCAACGTGGTGAAACCCTGTCTCTACTAAAAATACAAAA ATTAGCCAGGCATGGTGGGGTACGCTTGTAATCCCAGCTACTGGGGAAGCTGAGGTGGGA GAATTGCGTGAACCCGGGAGACAGAGGTTGCAGTGAGCCGAGATGGCACCACTGCACTCC AGCCTGGCCAACAAGGGCTAAATTCTATCCCCCCCCCCTCAAAAAAAACAATAACGATTT TGGGAGTATTAGAGCAGAAAACGTTTGAAAACATGAACAGCTCTCACATCAACACAAGAC ATGAAACTCTGTTGGCAAGAGAGGCCTCTTTGGGGAGAGTGCTCACTCCTCTCCCACCCA CTCTCTACCACCTTCAACCTCCTGTCTGGGGCACAGTAACAAGGGGTGTAAGGGGTGTAC AGTGCCTAGCAGGGGCCTGGCACTTAGGCTTCAGTAGGTGCCAGTCCACAGTCTCCTGAG GCCCATGTTGGTTGGTGTCCACCTGGTGCTTATTGACCACTTGGGCTCCAGATTTTCCTC ACGTCACCTCCCTCCCCAGGGTAGCAGATAAAGGCCCCATAGTTCCAAGGAGGCAGAACT TTTACATTCCTAAAAGCATTCTCAGGCCCCCATAACCCCTCATCCCACCTTCAGCTCGCT CACCTTCACCTATCAAATTCGTTTGACATCTGGCCTCCAGCAACTAGCTAGGCTGAGTTC TCCATGAAGGGGACGTGGCTGCTGGTCAAAAGGGAAAGCCCATGTCTAAAAATTAATTAA CGTGGTTTAGGGGCCTCACATTCCCAACCCCCAAATACAGCCATTGTGAGATGTGAGATT CAAAAAATCGAAGAGTGAAGCGGTGATGAGAGTCCATCTGGTCGCTCTTCGGATGCGGCG CTAGCAGGCAGTTGCATGAAAGCAGATCCCAGATCACGTTGCACTGGCACACAGTAGGTA CCCACACATATTTGATAAATAAACACTCTCTCCCTAGCATTACTATAGATAGCTGGCAGC TTGGCCTGGCATCCCGGAGGGGTCTGGATCTCAGGGACTAGATAAGGCCAGCACACTGAA GAGTTGCAGAAAATGCAGGCGGTCAGGCGCAGGTCTGCTGCGGGAGCACCTGCCGTGTCC GACAGCTGCACTGGAAGAGGGGCGTGTGTCTCCCAGCCGGGCGGAACCGAGACCGCTTCG TCCCCGGCGGGGAACTTCATTTCCCACAAGCCTGTGCGCGAGACTCCATTTCCCACAAGC CCTCCTTCACTTCCGGCTCGGCCGAGGCACTGCGGACGGCTTCCGGGTTTGGGCCTGGCT CTGTGACTGAGGCGGCGGCGGTGGCGGCCAAGCGGGATACGGGCGGCGGGAGCTGGGGAA CAGGCATGGACGTTTCCGGGCAAGAGACCGACTGGCGGAGCACCGCCTTCCGGCAGAAGC TGGTCAGTCAAATGTGAGTAGTGGTCGGGGCAGGGGGCTGGATTGTGGGACCTTCCTCCT TAGCGTGGCGGGCGAGGCCAGGGCCGGACCCGTGAGAAACCTACGGCGCCGGGAGACAGA AGGCGCCGGGGACTTGCGTGGGTCCCAGGGCTCGGGCCCCCCCGTCTGTCCCCTCCGTTC CCGACATGGACTGCTCGTTTGCTTTCCTGGGGCCGAGCTCTGCAAACCAACGAGCCCTGG CTTATGAATCATCGTCTTGAGCTTTCTCATTCGTCATTTGTGTGCTTTGGGTGCAGACTT TCCCCCGCTTTTTGAACCACACTGAATAGGGAAAGGGAGAGAAGCAGCCGGCTGTAGTGG GTTTAGCAGGGCTGGGTGCTTCATGGTTTCTCTTCCAGAAAAGCGCATCAACGGTGAAAG AAAGTGATGGGAGGAGGGTGGATGTAAGATGAGATTGGGAAGGTAGTTGTTTGGGGTGTG GGCGGTGGGACGGAGGATCTGAGGGTGACCCCCCGAAGGAATGTGGACTTTGCCGAAGGA TGGCAGGAAGCCGCTGTCAGGAAGCGATCGTCGTTTCTGGAGGAGAGCGTGAAGAGCTGG GGTCAGTGGCCCCGCTCAGAGGAACTCTAGCCCCTCACCACCGACTGCTTGTTTCTGTTA GCAGTTTTTTTCATGCGCGTCCGTGTGAAGAGACCACCAAACAGGCTTTGTGTGAGCAAT AAAGCTGTTTATTTCACCTGGGTGCAGGTGGGCTGAGTCCGAAAAGAGAGTCAGCGAAGG GAGATAGGGGTGGGGTCGTTTTATAGGATTTGGGAAGGTAATGGAAAATTAGTCAATTGT TCTCTGGTGGGCAGGGATGGATCTCACAAAGTACATTCTCAAGGGTGGGGAGAATTACAA AGAACCTTCTTAAGGGTGGGGGAGATTACAAAGTACATTGATCAGTTAGGGTGGGGCAGG AACAAATCACAATGGTGGAATGTCATCAGTTACGGCTGTTTTTACTTCTTTTGTGGATCT TCAGTTACTTCAGGCCATCTGGATGTATACGTGCAAGTCACAGGGGATGCGATGGCCTGG CCTGGGCTCAGAGGCCTGACAGTTTTTAACTGAGCTTATTGGCGTGTTGTGCTCGCCACA GATTTGCCTGAGACACAGCCCCTTTCCTGAGGGATGGTAAAGGAACCGTCACAGCGCAGT CTTGTCAGTCTGTAATGTGTATACTGCAGGGGTTTCTTGCGAAGCGGCGGTTAACTTGAG CAGACCAGGGGCAGTCTAGTGAGTAGGTGACGAATAAATTGAGTCCTAGAAAGACAAGTA GGAGTGTGAATAGCCACCATTCCAAGCTGAGGAGTTAGGAAACATGAGCATTTACTGCGT GGAGGGAGGATCTTGTAGGCTGAGAGTTCTGGGGCTCGAGGAGTAAGAGAGCCCAGGAGA TGATGGAATGAGGGTGAGCAGGTACTAGCACTTTAGTGGGGGTGTTGTGCCAAGGAATTG AGCCTGTGTTTGGCATGTGGCATTCTTTGGAGTAGTGAAATGCCAGTGTTGGGAATATGC CTAGAGTCCCTGTCCCTGGAGATGGATGCAGGGCAGAGAAGAGAGACCAGTTAAGCTATT AAGAGAGTTTGAACATTTGGAGGGAGTAGGGAACCTGTCCTTTTTCATCCTTATATTCTC ACTGCTAGCTCACTGCCTGCCACATTGCTCCGTAAAATGATTGTGGCATGACCTAGTCGG GCGGGAGATTATAAAGGCCTGCATTAGACAGTGGTAGAAACTGAAAGAGGAAGACAGATT CCAGAGGAGGGGGAACGTAGACAATTCTTGATGACTAACTGGATGGAGGGGGGTGGGGTG TGGGGTGAAGCATTGTGGAAGGAAGGAAGCCGGGCTGAATGCTGGGAAACCTTTCAGTAC AGTGGAAAGAGTAGGGACTGTAGAGTTAGTAGCTATGGGCTTTGGGAAGAATAGCCTTGG GATATTTCTGGGGATATTGCACAATCAGTGAACAGTGTTTTACTGATAAAATATAGTTAT CTGTAGGTGAAAACTTGAGAGATATCCCACTTCAGGGTTCCTGCAAGAAAAGTCCCCAGG GCAAGGGTTCTTCTCTGGGAAAGCCCAGAATTAAGAACTGGAAGGGTGAGTTGTCATCAA AGAAAATGGGGGTAGGGGAAGTGTTAAGAGGGCTGGGAGGGTGATTGAAGAGTTTATGGG TGTTGAGTGACTGGATACTTCATAACTCAGCTTTATTTTGCTTCCGGGAGAATTGAGTTC TTTATTTTTTTCGATATTGCTGTTTGTGTGTTTTTCTGTTGCCGCTCTAACAAATTACCA CAAGTTTAGCATCTTAAAACAACACACCTTTAGCCAGGTGTGGTGGCTCACGCCTGTAAT CCCAGCACTTTGGGAGACTGAGGCAGGCGGATCACGAGGTCAAGAGACTGAGACCCATCC TGGCCAACACGGTGAAACCCTATCTCTACTAAAAATCCAAAAATTAGCTGGGTGTGATGG CGCACACCTGTAGTCCCAGCTACTCAAGAGTCTGAGGCAGGAGAATTGCTTGAACCCGGG AGGCGGAGGTTGCAGTGAGCCGAGATCGCACCACTGCTATCCCGCCTAGTGACAGAGCGA GACTCCATCTCAAAAACAAACAAACAAAAAACACACATTTATCATTTGGTAGTTTCTGTA GGTCAGAGGTCCAAGTAGGTTCCACTAGGTCTCCTGCTATCAAGGCCTATCAAGGCTGAA ATCAAGACGTTGGCAGGGTGCATTTCTTACTGAATGCCTTGGGGTGGAACTTGCTTGCAA GCCCATTTATGCTGTTGGCCATATTCAGTTCCTTGAGCTTGTAGGACTGAGGTGCCCTCT TTCCTTACTGGCTGTTAGCCAGGAGTCAGTCTTTGTTCCCAGAAGCTGCCCGAATTCCTT CTCATGCTTTTTATGTGTCTCCCTCCAGCGAGGGCCAGTTCTTCTGCCACATCTCTCTGA TTTCAGCTAGAGAAAATATCCTGCTTTTCAGGGCTCAGATTAGATTTAGTGCACACAGAG AAGCCAGGATAATCTCTTTATTTTAAAGTTTGTAATTTTAATTACATCTGCAGAATTCCT TTTGACATGTAATGTAGCATATTCACAAGTTCTAGGGATTAGGCACGAGATGGGCATTCT CCTACCACAGCCGGGAAGAGCAGTTTCAGAGGTTGCCTTCCCTAGGTGATGTGGGTGTTC TCAGTTCTCAAATTTGTCTGTTTTCTCTCTAGGGTTCACTTTCTCTCTAGGGTTCACGTT GAAAGTCGTGATTCTTGGTGCCTTGATTTATTGTGCTCATAGGGTTAAAAAAAGATTTGC ATTAGAATTCTAGACATACCTTTAACTCTTAACTTCCTCCCATGTTTTTACACTGTTGTT TAAATTTGGCCATTTTGGGAAATGCTTTATACCCAAGTAATTTATAGTTGATGAAGGGAT AGAGAAAGTGTCTTGCTTGTGGCACCGTGACTCACACCTGTAATCCCAGCACCCTGGGAG GCTGAGATCTGGGACTACTTGAGTCCAGGAGTTTGAGACCAGCCTAGGTAACATAAACAA ATACAACAATCAGCTGGGTGTGGTGGCGTGCACTCGTAGTCCCAGCTACTCTGGAGGCTG AGGTGGGGGGATCGCTTGAGCCTGGGAGGTCGAGGCTGCAGTGAGCTACGTTCACACCAC TGCACTCCAACCTGGGCCACAGAGCAAGACCCTTCTCAAAAAAAGAAAAAAAAAAAGCAT TCTGAGTTTTGACAGAGAAGAAAACATTTGATGGTTACTTATCTGTCTCTACCAAGAGCC TAGGGAACAATGCCCCAGAGCATGAGCTGGTATATAGTCTAGTCTCCTGGCTCCCACTGA GAAAAAGGGATTTTGAGTTGAGGCTGGCTATGACTAGACAGCCATTGCAGGGACTCTCCA GTTCAGCCTCTTAGCCCTCCACTGCCTCCACCTCATCTGGGCCCATTCTGGGCTGTGGTC TCCTGCTCTCTTCCCAGTCCCATCCTTCTTCCCCCAACAGCACAAGTGAGGGTGAGGCCT AAAGTGTAGCCACTGCCCTGCTGCAGAACAATCAGTGACTTTTCTTAGCATTAGAATAAA ACTCCAGATGTCCCACCCTCCCAGCCCCTCACCCTGACCTGGTATAGTCATCTCTTCTCC TACCCCCACCGTGTTCTGACCTCTCTGGTCTTTCAGCTTCTTGAACATTCTAAGCTTTTT TTTTTTTTTTTTGGAGACAAGATCTTGCTCTGTGGCCCAGGTTAGAGTGCAGTGGTACGA TGATAGCTCACTGCAGCCTTGACCTCCCAGGGTCAAACGATCCTCCCACTGTAGCCTTCT GAGTAGCTGGGACTACAGGCACACACCACCACGCCTGACTGATTTTTATATTTTTTGTAG AGATGAGGTTTCACCATGTTGCCCAGACTGGTCTTGAACTCCTGGGTTCAGATGATCCTC CCTACTAGCCTTCCAAAGCACTGGGATTACAGATGTGAGCTACTATGCCTGGCCCCTGAG CTCTTTCTTTTTAACCAATTTATTATCAAGTGTTTAAGGTATTGAAAAATGAGTAGACTC CTCCCCACCACTGTATTCATGACTAGCTCCTTCTTTTAGGTGTCTGCTCAGTATCAGATA CTCAGAGGATCCTTCCCTGCTTGATCTAAAGTTGGTCACCGCCTCTGATATGTTTTATCT CAGCCCTTTTTAGTTTCCCATAGGACCTCATACCTATGAAGCTTGGTACCTGGTATACAA AACACTTGTTGAGGAGTGGAATAGTATTTTCTTGGGGGCTGTGTGAGTCACCTCTTTTTT TTTTTTTTTTTTTTTTGAGGTGGAGTCTCGCTCTGTTGCCCAGGCTGGAGTGCAGTGTCA CAATCTCAGCTCACTGCAAGCTCCGCCTCCCAGGTTCACGCCATTCTCCTGCCTCAGCCT CCTGAGTAGCTGGGACTACAGGCGCCCGCCACCACGCCCAGCTAATTTTTTTTTTTTTTG AGACAGAGTCTCACTCTGGAGTGCAATGGTGTGATCTCGGTTCCTTCAACCTCTGCCTCC CGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAATAGCTGGGGTTACAGGCATGCGCC ACCAGGCCCAGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGTCAGG CTGGTCTCGAACTCCTGACCTCGTGATCCACCCGCCTCGGCCTCCCAAAGTGCTGAGATT ACAGACATGAGCCACCACGCCCGGCCGGGTCACCTCTTGTATTACAGTGGTATTCTTTAT TTTTTTATTTTTTTAAGAGACAGGGTCTTGCTTTGTCACTTGGGCTGGAGTGCAGTTGGT ATGATCATAGCTCACTGCAGCCTCAAACTCCTGGACTCAAGTGATTCTCCAGTGCAGTGG TATTCTTTACCTGAACCAGTGTACATTGTGAAGCTCTGGAGCCCCTGCTTTCTCCACTTG TTACACTTGTTAGCCTTTCTTTTTCTTTCTTTTTTTTTTTTTTTGAGACGGAGTCTCGCC CTGTGGCCCAGGCTGGAGTGCATTGGTGCGATCTCAGCTCACTGCAACCTCTGCCTCCCG GGTTCAAGCAGTTCTCTGCCTCAGCCTCCAGAGTAGCTGGGATTACAGGCACCCGCCACC ACTCCTGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATCTTGGTCAGGCTG GTCTTGAACTCCCAACCTCATGACCCACCTGCCTCGGCCTCCCAAAGTGTTGGGATTACA GGCGTGAGCCACTGCACCTGGCCTAGCCTTTCTTAATCGTGTCCCTCATCAATGAAATGG TAGAAAGCCAATCTGTTTTACAGGTGTGGTAGAAGGGAAAAATGAAGAGACCTCTGTACA AGCCTTTGCAGAGGTCTTGGGACTCAGTGGTGAGCTCCCTGCCACCACCTCCTGTGCCCT TGCAGGAGTAGGAGATCTATAGTCTTGGAGAATAAGGGTGATACCCCAATACCCCAAGCC TGGTTAGGGAAATTGATGACCCTTATTGGAGCCTGTGGGGAGGTAGTTGGGAAATTGTTA GTAGCTTCATGTTGGCATTTTCTTTTTTGAAACAGAATCTTGCTCTCTCCTCCAGGTTGG AGTGCAGTGGCATGATCTTGGCCCACTGCAACCTCTGGCTCCCAGGCTTAAGCGATCCTC CCACCTCAGCATTCCCAAGTAGCTGGTACTACAGACGCATGCCACCAGGCCCAGCTAATT TTTGTATTTTGTGTAAGAGACAGGGTTTCACCATGTCTCCCAGGCTGGTCTTGATCTCCT GGGCTCAAGTGATCCACCTGCCTCAGCCTCCCAAAGGGCTAGGATTACAGGTGTGAGCCA CCGTGCTTGCCTTTGTTGACTCTTTATTGAACATCTACTCTCCCTGCAGAAACTGCAGGA TAAGCCACCCTTAGAACCAATTTAACTGGCCTTTTTGGGGCTTGGACCTTCCTCATGGGA TTGCTGGGTTAATGTTGAGCCTGTCTCTGGGCTATAGGCTCTCAGAGAACAAGAGTGCAT CTGCAATGACTAGTGCTGCACCTGTCATACAGGTTTGTAGAAAATATTTAAGCTGTGAAT GAACAAAAGGACCTTATCAGCTATTTTCTTTCAATTCTTTTTGGTTTTTCTGTGATCCTA TTCTTGAGACTTTTATCCCTCCCACCCCTCTGTACAGCAGATTGCACATATAAAGATGAA ATAAATAGATGTCCATTGGGCAAAACAATACAGAGAGGAGGTGACACAGCTGGGGAAGGA CAGTCCAGTATCTGATGAAGTGCCAGAAGGGGCTTAGGGGGAGCATGGAATGGAGCTGGA ACTGCCAGGTCAGCCACCTCTTCTAGTGAAGCCTCCAGAGAGACGTGAGCTCCAAGTAAG ACAACACAAGGAACAAAACATACAACTGAACAGCAACTTCCAAGGGTCTGGAGAGGTCTT CAGAAGGATAGGGAGTGCCCTTAAACTTGCAAGCCCAGATTCTGCCTGTGGGTCATGGTC GTGCCATAGGCAGAAGCCAGTGAGTCAGCCTTGGCGGAGCTTTTTCTGTGGCTTTTCCCA CTCACAGCTCTGTGCCCTTCTGGTGCAGCTCCAGCCCTTGTTTTCCAGCTTCATAGCCTG GTTGTCACATAAATGGCTGCACTGGCTTACAGGTGTCTGTGTATAACCTATTGCTAGAAT GTCCATTCCTTGAGGCTAGGGGCCCTGTCTGTCCCTCTCTTCACTTTCATAGCCCCAGTG CCCCATAGGGCTGCTCCTGATGGGCAAATAGGAAGTGGTTGTTGAGCCTCTAGGCCAGTC GTGACAGTGACCTGAGGGGACTTGAGCCCTCAGTCATCTGCTCAGGAGTTCAGGGCTTTC TCAACAGCAGCTGACGTGCGGCAGGCATGCTATGCCAAGCCCTGTTGCAGGAGTTGACAG ATGTTGACTCATCCCCTCTTCATGCTTTCTTTGTCAGTAGATGTACATGTTACTCACATT TATAAGATGAGGAAAATGAGACCTAGAGGAGAGGCCACTGCTGCTTAGAGTTACTCTACA TGTTGTAAATCAGGGGTGTCCAGTCTTTTGGCTTCCCTGGGCCATGTAGGAAGAAGAATT GTTTTGGGCCACACATAAGATACACTAACACTAATGATAGCTGACGAGCTAAAAAAAAAA AATTGCCAAAAAATCTCATAATAAAGTTTACGAAGGCTGGGCGTAGTGGCTCACGCCTAT AATCCTAGCACTTTGGGAGGCTAAGGTGGGTGGATTGCTTGAGGTCAGGAGTTTGAGACC AGTCTGGGCAACATGGTGAAACCCCATTTCTACTAAAAATACAAAAATTAGCTGGGCATG GTGGCGGGCACCTGTAGTCCCGGTGGGGAGGCTGAGGCAGGAGAATCGCTTAAACCCGGG AGGCAGAGGTTGCAATGAGCCAAGGTCGCGCCACTGCACTCCAGCCTGGCGACAGAGTGA GACTCAGTCTCAAAAAAAAAAAATATATAGACCAACCTGGCCAAGATAATAAAACCTCGT CTCTACTAAAAATACAAAAATTAGCCAGGCATAGTGGTGCACACCTGTAGTCCCAGCTAC TTGGGAGGCTGAGGTAGGAGAATCGCTTGAACCTGAGAGGCGGAGGTTGCAGTGAGTCAA GATCACGCCACTTCACTCTAGCCGGTGACAGAGCAAGACTCTGTCTCAAAACAAACAAAA AAAATTAAAAATAAATAAGTAGGTTGGGCGCGGTGGCTCACACCTGTAATCCCAGCACTT TGGGAGGCTGAGGCGGGCGGATCACCTGAGGTCAGGAGTTCTAGACCAGCCTCAACATGG AGAAACCCTGTCTCTACTAAAAATACAAAATTAGCTGGGCATGGTGGTACATGCCTGTAA TCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATTGCTTGAACCTGGGAGGCGGAGGTTGC AGTGAGCCGAGATCACGCCATTGCACTCCAGCCTGGGCAACAAGAGCGAAACTCCGTCTC AATAAATAATAATAATAATAATAATAATAAATAAGTAAATAAATGAATGGTTGCTGAACC ACAGCAGGACAAGACAGACAAACCCCTTTTATTTGTTTTTGTTTTTTGGACCCTAACAGC AGAGGAAGAGCCAGCCAGCAAGCCCCTTTTGATCTTTGGCCTCTGCCAACCAGTACCCCC CAGACCTTAACCCCCAGCCCTACCAGGTCCCTTTTGTAAGTGACCCCACATGCCTACCAC AGTTGCAGGCCCTGACATCCACCCTGACCAGAGGTCTTCCAGCTATTACTGGTGTTTGTG ATGCTGTGGCTGTGGCCAGTCAAGGACCCATGCTGGGCATCCCGAGGTAGGAAGGACCAT GACCACGTGATTATCTCCCTGCCACTATCATGGCAGGGCTGTGGGGTACTAGCCTCCTTT AAAGCATTCCTTAGGACAGTAATTTCTGTTAAATATCTGGAGGGCAAACATTTTAAGGAA TTTTAATGAATTTTTAAAAATTCAAAATGAAAAATTTAAATCATAAAACCAAATAGAAGA AAGACCCCTATGTCCATATACGCACCACTCTGCTTCAGTGATTATCAACTCTTGTCTCAT GTACTTGACCTCCACACAATTATTATTACTATTTTTTGAGAAAGGGTCTTGTTATGTTGC CCAGGCTGGAGTGCAGCAGTACAATCATAGCTCACTGCAGCCAACTCCTGGGCTTAAGTG ATCTTCCTGCCTTAGCTTCCCAAGTAGCTGGGACTAAGGGTAGGCGCCACCATGCTGGCT AATTTTCTTATTTTTTTTTTTTTTGGTAGAGACCAGGTCTTGGTTTGTTGCCCAGGCTGG TCTTGAACTCTTGGCCTCAAGTGAGCCTCCTGCCTTTGCCTCCCAAAGTGTTGGATTACA GGCATGAGCCACTACGCCTGGCCATCCCCAGTTATTTTGATGCCCAGACTGTTTTAGTGG CTGCCCACTGTCTGTGGATTCTGTTCTTAGTCTTGATCCTTCGTGCAGTGTCTGCACCTG CCTGCCAGTTCCTTGACCTGTGCTCCATCATACCAAGCTGGCAGTGGATCCTCAGACAAT CTCAGTTCTTACTCCCTGCTCTGTGTCCAGTTTGTTTGCCTACCTAAAAGATCCTTCCTT TTCGTGGCTGAGCAGTGCAGCCGTTCTCCAAGGAGACTGGGCCTTCTGCTGCCCCCACCA CCCTGCTCTTTTCACTGCCCCCACCCCGCTGCTCTTTTCACAGGCTTGGTGAGGAGTTTC CAGGTTGCACTTGGTTTGTTTGAAGGCTTATCTCCTCCTAGTCTGAACTCGTCAGGGCAG GAGCTTGAATCAATCATTTCCTGCCTCTCAGGACTGCTGTAGCAGGGGGATTCCTAGATC TGTCCAGTGCCAGTTTCCTGAGTACTGCTGCAGGCCCATTTCAGCTGCTATGGTGATGGG GGCTGTCCTGAGGGGACATTCACCTCAGGTAGAGATGGCAGAAACACCACAGGAAGATGC AACCAGTCAGCTCAGCTGGGGGCACTTAAATTAGATCCATTTTTTTTCTGGAAGGCCTTT TGGCAATACTGTTTATCCCAGTACCTTAAAAATGTCGATTTCCTCCAAGGGCGCAATCAG GAAAGTATTCAAAGCCTGATGGTGATGAGGGTGCTCATTGCCGCATCATTGTCTCAGGAG TGGGATTTGTGAAACAGACCAACAGTGGGAGCTTGGTTAGAGTTGTAGCTGCAGGTGATG GGCTCTGTGCTGCTGTTGACATTGACGTGAGGGACTGCCATAATGCGTCAACATCGACAA GCGCTTGTTACCAAGCAGGATGCGTGGTACAGGCTGAGTTTTATTAAACAGATGTCTTTA ACTGGTATGAATGATTTTAATTTTTTCTTTGTTTGAGATGGAGTCTCGCCCTGTCGCCCA GGCTTGCGTGCCTGGTGACTTCTCTCACCTGGGCCCTCAATGCCAGACCAGCCTTCCCAG GATGATTCCATCCTTTCCATCCCTGCAAGGGAGCTTGAGGCCCTTTCCCTTTCAGTTTGA TACTGTCCTTCCAAGTTACCTTTCTTCATGTTGGCACGAGAGCTTTGTGCCACACCATGC CCTTTCTGGATGCTGTAGTGTCTGGCTCTTCCTCTGTCTCTGTGACCCTCTTCCCTGGTG AAGCTTTGACCTTGGCTTCATCCCCTCCAGATGACCCCTGATGGTTAGCTCAGCCTGTGG TGGCTCCAGAGCCCTCATTGAGTGCATCCTGACACGAACTGAACAGATTAACTATTTTTT GCCTTGCCAACAGCATTGGAAATTCCTTGAGTGTGGGACCTGGACTCATAGCTCTATGTC TCTTATAGTTCTTAGCACAATATCTTGGCCTAGATGAAGTACATAATAATTATATGTAGG GTTGTGGAAAGCAGTGCTGGCTTTAATTAAAGCCTGCCATGGTTTTGTCCATCAGTGCTG CCTAAACCGTTCTGAACTTCTCATTTTGAACTTTTTTCCCTCACAGCTTGTGATGGTTGA TTTTCTTTTTGAGACATTCATTTATTTGTTTGTTTATTTGGCAGGGTCTCACTCTGTCAC TCAGGCTGAAGTGCAGTGGCGTGATTGTGGCTCACTGCAACCTCCACCTCCTGGGCTCAA GCGATCCTCCCACGTCAGCCTCCCAAGTAGCTGGGACCACAGGTGTGTGCCTCCATGCCC GGCTAATTTGTGTAATTTTTTGTAGAGTAAGGGTTTTGCCATGTTGTCCAGGCTGGTCTC CAACTCCTGGGCTCAAGCGATCTGCTGCCTCCGCCTCCCAAAGTGCTGGGATTACAGGTG TGAGCCACTGTGCCTGGCCCTGAGACATTTATTTAAAGACAAAGGGGAGTACAGGCTAGA GAACAGAGGGGAATAGAATAGCAGCCAAGGCATAATTACCTCTAAACCATCACCCCATAA GGGAGATGTTCGCCTCAACTTTAGTAATGACTCTCTTTCTAGTCTAGGTGCAGGGCTGTT AATAGAGGCCCTGTGATTGGGTTAAGGGCTTGAGGTACAATAACTCTGTTATTTAGGGCA GTGAGTCCTTGGAGAAGATGCTTTACCTTCTTTCCAAGCCCCATGTCTCCTTATTTTGAG GGTGTTTTTTTTTTTTTTTCTTGAGACAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAGT GGCCTGACCTCGGCTCACTGGCCTGACCTTGGCTCACTGCAACCTCCGCCTTCCGAGTTC AAGTGATTCTTGTGCCTCAGCCTTCTGAGTGGCTGGGATTACAGGCCTGCACCACCACAC CTGGCTAATTTTTGTATTTTTATTAGAGATGGGGCTTCACCATATTGGCCAAGCTGGTCT CAAACTTCTGACCTCAGGTGATCTGCCTGCCGCATCCTCCCACAGTGCTGGGATTACAGG CGTGAGCCATTGCATCCGGCCTGAGGTTTTTATTTTGAGAATATTTCAAATGTACAGAGA GTTGCAAGGACAGTAAAAGCACTCAGGCATCCTCTTCCCTGGGATTCACCAGCTCATTCT CACTTTTACCATCTTCCCATACTCCCTGGGGGCCTCCTCCTTCTGGGCTTCGTTTCCTTT TATTTGTTTCTGTCTTTTTAATTTTAATTTTTTTTTAACTAGCCCTTCTCTTTGGCAGGA TTTGTTCACTTTTAATCCTTGGCCCCAGCAGCTTCTCTGAGCTCTGCCTATGAATCTGGG GGAGAGATAGGGGCTCACACAAGAGTGAAGTGTGAGCTGGAGGGCTGAAGGGGAATGAGG GACATCTGGGCCAAAGGCAGGGGACCAGCAAGGCCCCCACCCCTGGAACTTCTGTTCTTC CCAGCCTACGTACCCCTTGCTTCTTCTTATGTTGGTGGGCTCTGGGGGCTGGGTGAAAGC AGTGCTTGTAGGTTAAGAAGTGACTCATCCCACGTGCCCGCTGCCCTTCTCTTCTGTTCT TTGCTCTTTCCAAGCTTCCTTTCAGTCCTCCTCATCAGACATGACCTTTGAGGAAGTGAT GAGTCTGCTATGTGTGAGACAGAGCCCCTGTTGTGAAGTAGAACTCAGGGTTCGTCCTCA GAAGCCTCGGTGGGTCACCATACGTCCTGCAAGTGCTGTCAGTCTGCTTTGATTCGTGTG AGGGAGAAGTCCCTGCCAAGTGCTCTGTAGTAGGCATTCAGGCCAACCAAAGAACAGGGC AGGGTCCTGAGGCACAGGCTCAGAGCTCTTGCTTATTTCATGGGGTGTTTAAATTGAGGT ATAATCCCTATCACAGTGAACGACACGCATACTTCCAATCTTGAGTGTGCCACCAGAGAA CTGTTGATACTCCCCAGATCTGCACCCAGCCAGGGCAAACTGTGCACCACTCTTCTGACT TCCATGGCCAGCCGAGACATTTGGAATCTCTGTGCAATCACACTCTGTGCCATCTTTTTT TCGAGACAGGGTCTCGCCCTGTTGCCCAGGGTAGAGTGCAGTGGTACAATCTCGGCTAAC TGCAGTCTCGACCTCCTGTGGGCTCAAGCAATCTTCCCACCTCAGCCTCCCAAGTAGCTG GTACTACAGGTGCTCACCACCATGCCCTGCTAATTTTTGTGTTTTTTGTAGAGATGGGGT TTTGCCATGTTTCCCAGGCTGGTCTCGAACTCCTGGGCTCAAGCAATCCACCTGCTTCAG CCTCCCAGAGTGCTGGGATTACAGTCCTCTGTGCAATTGTGTGCATCATGTGTGAAATTT ATCCTCATTTATCCTCACAGGCAGCAGTTGTTCTATTTTTTTTTTTTTTTTTTTTTTTTT TGAGATGGAGTCTCACTCTGTTACCCAGGCTGGAGTGCAGTAGCACAATCTTGGCTCCCT GCAACCTCTGCCTCCTAGGTTCCGGCAGTTCTCCTGCCTCAGCCTCCCGATTAGCTGGGA TTACAGGCACGTACCACCATGCCTAGCTAATTTTTATATTTTTAGTAGAGACTGGGTTTT GCCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAGGGGATCCACCCACCTCGGCCT CCCAAAGTGCTGGGATTACAGGCATGAGCCACCATGCCCAGCCTGTGTTTTTGTTTTTTT TGAGACTGAGTCTCACTCTGTCGCCTAGGCTGGAGTACAGTGGCGTGATCTTGGCTCACT GCAAGCTCTGCCTCCCGGGTTCACACCATTCTCCTGAGTAGCTGGGACTACAGGCGCCCA TCACCACACCCGGCTAATTTTTTTTTTTTTTTTGTATTTTTTTTAGTAGAGATGGGGTTT CACCATGTTAGCCAGGATGGTCTCCATCTCCTGACCCCGTGATCCCCCTACCTCGGCCTC CCAAAATGCTGGGATTACAGGCGTGAGCCACTGCGCCTGGCCTTATTTATTTATTTATTT ATTTATTTATTTATTTATTTATGTATTTCTGAGACAGAGTCTTTCTCTGTTGCCTAGGCT GGAGTGTGGTGGCTCAATCTCACCTCACTGCAGCCTCTGCCTCCAGGGTTCACATGATTC TCCCACCTCGGCCTCCAGAGTAGCTGGGACTACAGGCGTGCACCACCATGCCTGGCTAAT TTTTGTATTTTTAGTAGAGATGGGGTTTCGCCATGTTGGGCAGGCTGGTGTTGAACTCCT GACCTGACCTCAGGTGATCCACCCGCCTTGGCCTCCCAAAATACTGGGATTATAGGCGTG AGCCACCGTGACCAGCCTGTTCTTGTTTATGACGTGTTATTCCATGGAGGACTCTGTTCC AGATAACTCTTTTATTTTGTGCCTAATGAACTTTGGTGTTAGTTCCAGATGTTGGCTACA TGAAGGCAACCCCAGGAATGGCTTTGGTCATGTCTTCTGGTAAATATATGCACTTAATTC TCTGGGCTGTGCACCTAGAAGTTAAATAAAGGACTGGGTCAGAGGATGTGTCTCTTAGCA TGTTCTGCTGAACAGTGTTCCCAAGTGGTACCAGTTTACGTTCCCACCAGCAAAATGTGA GGGAGTTCCACTTGCTGGACATCCTTGCCAACACCTGGTATTGTCAAGGAACCTGTCTGT AACAGGCACTCTCGTTGATTTTGAATGCACGCTGAAATTTACTGGGTGCAGTGACAGTAG GAAGGGAAAGGATGATTAAAAAAAGAAAAAAGGAATGGAAAGGCTGATCTAGGAGGGAGG AAGCTCATAAGGAAATAAGTGGTAGGATTTGGTGATTCCATGTGTGAATGCTCGTATTCC GTGTGCATGCGTATATGAGCTTGCAGGAGAAAGGGAGGAGGGAGGCACTGGTATGCACTC CACCAGTTCTCCTGCCACAGCCTCTCTACTGATCTGAGGGCTGGGTGTAAGGTGATGATG TGGGGAGTGGGTGTGGGTGAAATTTCCAACAGTCAGTGGGAGATGGGGGAAGCAAACCTG GGAAGAGCAGCCTGCTGCAGGAGCAAGTGGGTGGGCCGTCCTGGAGGCTGGATGAAACCT GTCTGCCTAGGTGGGGATGGCAGAAGTGGCAGAGCAGAGAACTCCAGAGCAGGGCTGTGG TGGGCAGAGCAGATGTGAAGGAAGACTGTGGGATGAGAGGATGGGGCACGGTGGAGCCCC ATCAGCCTCCAGTCACAGCAGCCCCGGTCCCGTCTCTCCCTCCTGTTTTGGTCAGTACCT TCAGGACTTGTGGGTGTAGTACTTCGTTCCATTCCGGATAACATTGTAGAAGACTGAAAA ACAGGAAGGTGTTTTCCATACTCTCTCCAGAGCTGGCTGTTACTGACATATTGATATATT TCCACCAGTCTTGTGTGTGCTTATAGCTTGATATGTATGCATTTTAAATATGTAAATATA TATACACATGTATTTTTAAAGTGAAACTAGGCTGAGACTGTGAGTTCAGTTTTGAGTTAT AGGTTTCCCATGGCTTAGGTTGTGTTCTCTCCCTGGATGGAGTCTGGCTGGGACCTGCAT GCTCACAGCCCAGGTTCACATGAGGTGTGGATCATGCACAATCATCCCACGGGCTTGAGA TAAGCATTTGACTTCCTTTTTTTTTAAAAAAAAAGTTCATTATGGACTTTTTCAAGTGTT CCCAGAAGTAGAGATAATAGAAAAGTGGTCTCCCAACTACTTTGAGAGTTAGCAACATAT GGCCAGTGTTGCTTCACCCACCCTTCCTCATCACCTCCTGGACTATTTTAAATCACATCC CAGACACCAGATGATTTCAACTGTGAGTACTTGAGTGCAGATCTCTAAAAGATCGAGCTT CCCCACCCCCACCAAATACCACAGAGCCATGTGAGATCAGCATTTGGGTTGGCAGATCTC CTCCTTGCCACATGCTTGCCTGGTGGTGAATGGGACACTTTCAGGCGAGACCTAGGGGAG CCCCAGGCCAGCACCAGGACAGAGATCTCGAGGCCTTTCCAACTCTGCTGGTTCTTCAGA AGTCAAACCAACAGATTGGAGTATGTGAAGGAAGCTCGTGTATTCTTCTGAACTAGCTTA GACAACAGTACACAGTTTGAAGGGCAGCAGCCTTTTCTGCAGGTTAGTTCTTTCAATGAT CTTGTTTACTGGGAGTTGAGAGACCTTGAGGATAGAGGCAAGAAAGCGTCATCAAGCAGC CCAGAGGGGTGTACAGTGTTTGAAGTCTCCTGTATTGGTATGATTCGAGATTTTCATTCT GCAGGACCAAAATACCCATTCCTACATGTGGGTGGAGAGTTAAATTCTGAAGTATATATT TTTATCTTGTTAAAAAAACACTTTTATGTTTCTTTAAAAAGTAAAAGAACCAGAATGTAG AAGTTTCAGTAAAGGAAAAAGTTACAGTTTGTTTGGGATACAGTTTGACAAATGTGCTCC CAGCTGCCTGAGAGATAGAGAGGCCAGATGTTTGCTTTGGTTACATTGATGACTGAACAA TCGGTATCTCATCATTTCCTGCCTTAGAGATGAAAGCACCTGGGCACTGGGGCAGTAAAT TGGTTGCAGGATTGGAAGAAGTCCAAGGACAGTGTGGGATGCCACCCTGTAGCTGTCTAG AGACCTAGTGAAGGTCAGAAGGCTGGGTGAGAACAAGCCCTGCACTGATGGAGGACAGGG AAGGGGCTTTTCTGGCTGGGAGTGTGGTGAGGCATTTGAGCTGCAGCCAGAAGGCACCTC TCAGATTGGTGGGGCTTGGGGTGGGGGTGGGAGGAACTGCTTTGCCTGTGCCGTTGGCCT CACTCATCCTGAGGTCCCTTCAAACTCCATCCATACTCTGACTGTGGGGCCACTTGCAGA AGGAATATGTGGCAGGTTTTTTTTGTTTTGTTTTGTTTTTTGTTTTGAGACGGAGTCTAG CTCTGTCACCCAGGCTGGAGTGCAGTGGCACGATCTCGGCTGACTGCAACCTCCGCCTCC CGGGTTCAGGCGATTCTCCTGCCTCAGCTTTTGGAGTCGCTGGGCTTACAGGCGCCTGCC ACCATGCCCAGCTAACTTTTGTAATTTTAGTAGAGACGGGATTTAGTAGAGACGGGATTT CGAACTCCTGACCTCGTGATCCGCCTGCCTTGGCCTCCCAAAGTCCTGGGATTATAAGTG TGAGCCACTGTTCCCTGGCCTGTGGCAATTTTTTGAGACGCTCCTGGGGGTCCAAATTGC TAATAGAGGAGAAACTTACTGTCCCTGTGTTGACTGAGACAGGAAGGTTTAGATAGGTGA AAGTGGTCTGGGCGCAGTTGCTCACACCTATAATCCAGCACTTTGGGAGGCCAAGGTGGG AGAATCGCTTGATGCCAGGAGTTCAAGACTAGCTTGGGCAACATAGTGAGATCCTGTCTC TATAAAAAATAATTTTAAAAAATTAGCTAGGCCTGTAGTCCCAGCTACTCGGGAGACTGA GGAGGGAGGATTGCTTGAGCCTAGGAGTCGGAGGCTGCAGTGAGCTGATTGCGCCACCAT ACTCCACCCTGGGCAACACAGAGAGCCTGTCTCAAAAACAAAAGAAAGGTGAACATGAGT TTTTTTGTTTTTTTTTTGACATAGTTTTGCCCTGTCCCACAGGCGTGGGCCACCACGCCC AGCTAATTTTTGTATTTTTAGTGGAGATGGGGTTTCGCCATGTTGGCCAGGCTGGTCTTG AACTCCTGGCCTCTCAAGTGATCCACCCACCTCGGTCTCCCAAAGTTTTGGGATTACAGA CATGAGCCATTGAGCCTGGCCAAACATGACCTTTCTCTTTTTTTTTTTTTTTTTTTTTTT GAGACGGAGTTTCACTCTTGTTGCCCAGGCTGGAGTACAGTGGCACGATCTCAGCTCACT GCAAACTCCACCTCCCGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCTGAGTAGCAGAGA TTACAGGCATGCACCACCACGCCTGGCTAATTTTGTATTTTTAGTAGAGACGGGGTTTCT CCATGTTGGTCAGGGTGGTCTCAAACTCCCCACCTCAGGTGATCCACCTATCTCAGCCTC CCGAAGTGTTGGGATTACAGGTGTGAGCCCCTGCACCCGGCCACGTGACCCTTCTTAATA GCTATTGTTCGCTGAGTATTTCTCTACGACACTCTTTATGTGCATTTTGTCATTTAATTT TAATTTTAATTTTCTTTTTTTTTTTGAGACAGGGTCTTACTCTTTCACCCAGGCTGTAGT GTAGTGGCACGATTTTGGCTCGCTGCAGCTTCCGCCTCCTGGGCTCAAGAAATCCTCCCA CCTCAGCCCCCGAGTTTCTGGGACTACAGGCATGCACCACTATGACTGGCTAAGTTTTTG TATTTTTTGTAGAGATGGGGTTTCACTATATTGCCCAGGCCAATCTTGAATCTTGAATTC CTGGGCTCAAGCATGAGCCACCACACTCGGCCTCATTTAATTTTTAATTACTTTAAATTT TATCAACATAATATATCACATAAACTGTAAAAAGACAAGAAACTACTGTGGTTCCCATTC TGCCAGTTCTCCCCAGAGGTAGCAACTGCCTTTACATCTGAGCTTTCTTCCGGCATATAC CTCTGATTCTAGGTAATAGGCACATGCTGCTTTTGCAACTTGGCGTTTTACAAATTATGT TGGCTTGCTCTGTAGCTCCCTTCCATCGCCATTATCTCTGCTTCTCCTCCCTCATTCTTC CAGTATTCTACTTTCTGCATAAATCGGTAATGAATGTTTACATTTTCTGCCTACACAAAT GATATTCACAGCTGAGCATAGTGGTGTACTGCAATTGCTTGTTATTTTTTTCCTAGGAAA GAATTGCCTTTTCAACACTATGCCCAGTTTTCTGTGTATCTATTCTTATTCTTCCTTTAT TTTCCGGTTGCCTCTCAGTACAGTTATCTTCAGGCTCAGTGCTAGTGAGCTGTCAGTTTC AGTTCAAACCCCTGGCCTCAAGTGATCCATCCACCTCGGCCTCCCAAAGGAGGTGGGCGG TGACCCTCTTGGACCTCCCTTCTGGAACCCTCTAGGCAGTGGCCGCAGGTGTGGTGTGAC AGCCCTCAGCGTGCTTGTCTGCCCAGACTGGGACTGTTGTCTTCCTTTGTTGGGTCCCTT GTTTTCTATATCCTGTGTCTCATCCTTTGTGATTTATTCCCTCATTCTGCCAGCTTCCTG AGAAGGGACTCCTGGGAATTAACTTTCTGGAGACCATGCCAGTTTGAGTGCATCTAGGCC CCCTGGTGGGACATGCTAACTGGGGAATTTAAAATGATGTTACACTGCGCTGCCCTTGTG CTGGCTTGTGCTTCTGTCTCCTGAGGTGCTAGCTCTCAGCCTCATATGACTTGTCTTGAT CCCTAAACCCAAGTCCTGCTCTACTTGGGAAGTTCCTTTGAGTCATTTCAGGGGAATCTC ACTCCCTACACTTCCTGTGCTGGAGTGTTTAATGTTCACTCTTTGGGCCTCTAGTTTTAT CTTTTATCTCCTGTTTTCTATCTCTTTTACTTTTAGCTCTCCTCTCTAAGTGATTTCCTC AATTTTTTCTTTGACCTTTCACTGCTAACATGCTTTTATTTCCAAGAACTCCTTCTTTTT CCTTTTTTGTAATCTGACTTTTCCCTTTTTATAGCACCCATTCCTGTTAAATGGATACAA TGTAGTATAATATTTATATATATTATAATAAATTCTACCGTTTTTTTTGATAAATTTTCA CTTTATTGCCCAGGCTGGAATGCAGTGATGTGTTCATGGCTCACTATGGCCTCGAACTCC TGGGCTCAAGTGATCCTCCCACCTCAGCCTCCCAAGTAGCTGGGACTGCAGGCATGCACC ACCACTCCTGGCAAATATTTTTGTTTTTCGTAGAGATGGGATCTTGTGATGTTGCCCAGG CTGGTCTCAAACTCCTGACTTCAGCTGGACGCAGTGGCTCACACCTGTAATCCTAGCACT TTTGGGAGGCCGAGGGCAGGCGGATCACAAGGTCAGGAGTTCGAGACTAGCCTGGTCAAT ATGGTGAAACCCCATCTCTACTAAAAATCAAAAATTAGCTGGGCATGGTGGCGCGCGCCT GTAGTCCCAGCTACTCGGGAGGCTGAGGCAGAAGAATCACTTGAACGTGGGAGGCAGAGG TTGCAGTGAGCCGAGATCGTGCCACTGGACTCCAGCCTGGGCGACAGAGCTAGACTCCGT CTCAAAAAAAAAAAAAAAAAAAATCAAACTCCTGACCTCAAGTCATCCTCTCGCCTCAGC CTCTGAAAGTACTGAGATTACAGGTGCGAACCACTGCACCTGGCCTAATAAATTCTACAT TTCCTTCTTCTTGCTGAATTTCTGTTACCGCTAAATTACTCTTTTCTTGTGGGGTCTTTA TTGGCCATGTTATGGCGTCTGGCTGCCCTTGGCTGGTTGCTCACTCTGAGAGGGACCCTC CAAAGCCTAGGAGTCTCTTCCAGCACGTGGAGCTTGTCGACTGGGAACTTCATTGAAGGC TCATCTGGCTGCAGATTCTGAGTCCTTCCTCCAGAACAATGGTCCCCAACCTTTTTAGCA CCAGGGACCGGTTTCACAGAAGACAATTTTTCTACAGACCGGCAGATGAGGATGGTTTCA GGATGAAATTGTTCCACCTCAGATCATCAGGCATTAGATTCTCATAAGGAGCATGCGACC TGGATCCCTCACATGCACAGTTTACAGTGGGGTTCGTGCTCCTATGAGAATCTGATGCCA CCACTTATCTGACAGGAGGCAGAGCTCAGGCAGTAATGCTTGCACACCCTCTGCTCACCT ACTGCTGTGAGGCCTGGTTCCTAACAGCCCACAGACCTGTACTGGTCTGTGGCCCAGGGC TTGGGGACCCTGCCCTAGAGCATGGTCTTCCAGACACTGGGCTGTGGGTGAAGACCTGGC TGCCAGCATGCTGCTCACTGCGATGGAGGAGGCTGATGGGCCAGTGGGTCTCGGCAGTTA GCGTGCATCGATCTAGTCACTTAGACCAGAGCACAGATGACCACACAACCCACCTCTCTC AGTGTCCTACCTGTGCCTTTTCAACTGGGTGGGTTGGCCCCCTAGTCCAGTGACTATCCG TTTCATGCTTTTCCAGAGAGTAAACCTCCTGTGGTCACCTGGTGCTGGAGTAGCTACCCA CAGTGTAAAGTTGGGGAGTGAGGGATGCAGGGATATCTTTCACCCTCTGTCCCAGAGCAC TTGGTGCAGCCTGGCCTCAACCTGGCAAAGACCTAGGGCTGTCACTGGGTGGGCGCACCC CCTCCTCACTGTTACTTAGGACTCTGGTTTCTTGGTCCTCTTGCTGTGTACGTTACCACT GGGCCATCAGCTTTCCAGCTTCTAAAAGGCTGTTGAGGCCACCATTTCCATTCTTCCTGG TTTTTTTTTCTGTGGTTTTAGTCAGGTTTTAAGATGGAAGGGAATTAAATGTGCAGGCTT TGTTCCTCATTTCAACTTGGAGGTTCATATCTGTTATTTATTTATTTATTTAGAGACAGA GTCTCACTCTGTTACCCAGGCTGGAGTGCAGTGGTGCTACCTTGGCTCACTGCAACCTCC ACCTCCCTGGTTCAAGCGATTCTCTTGCCTCAGCTTCCCAAGTAGCTGGGATTATGGCCG CACACCACCATGCCCAGCTAATTTTTATATTTACGTATATATTTTTTTGAGACAGAGTTT TACTCTTGTCACCCAGGCTGGAGTGCAATGGCTTGATCTTGGCTCACTGCAACCTTCACC TCCCAGGTTCAAGTGATTCTCCTGCCTCGGCCTCCCGAGTAACTGGGATTATAATGCACG CCACCATGCCTGGCTAATTTTTGTATTTTTATTTTTTATTTTTGAGACAGAGTTTCACTC TTGTTGCTCAGGCTGGAGTGCAGTGGTGGGATCTTGGCTCATTGCAACCTTTGCCTCCCG GGTTCAAGCCATCCTCCTACCTCAGCCTCCCGAGTAGCTGGGATTGCAGCGCCCACCACC TTGCCCTTTTGTATTTTTAGTAGGGATGGGGTTTCACCATGTTGGCCAGTCTGGTCTCGA ACTCCGGACCTCAGGTGATCCACCTGCCTTGGCCTCCCAAAGTGCTGGGATTACAGGCGT GAGCCACTGTGCTCAGCTAATTTTTGTATTTTTAGTAGAGACGTTTCACCATGTTGGCCA GGCTGGTCTCGAACTCCTGACCTCAAGTGATCCACCTACCTCCGCCTCCCAGAGTGCTGG GATTACAGGCGTGAACCACCACACCTAGTCCAAATCTTTTTTTTAATATGGGAAAATCTT TTTTCCTTTATAGTGTCTGCTTCTGGTGACATGCTTAGGAAACCATCATCCTGATATTGT ATAACTCTCCTTCTGTTGTCTTGTGGTCCTCTGTGATTTAGGTTTTTAAACACATAGGGT AAAGGGTCCCACATACAAGCTGTGGGCTCCTGTCCCAGGTGGGCAAGCAGGCTGCACACA CAGGCAGCAATTTACTGGGAGGCTTCTTGGGTGGGGTGAAGTGGAGCTGCCAAAAATTTA TTCTGCCCTATGAGAGTTCACTTGATTGATAGATGTTGTGTTTCTCAAAATTAGAAAATT AGAAGAAACTCACAGAAGTTTCCTTCGAGCAGAACATAAATCGGCTGAGAGGAATCATCC TGCCCCCCTGAGGTCAGGAGGGTGAATCTGTGCCCCAGGGAGGAACGCACCTTCCTGCTT TAGACACCAGCAGAGGACAGAGAAGCTGGCTGTCCTGGGAATTCATGCCCTCATTCAGCA ATGGGAAAGCAGTTGGACCAGGGAAGGGGGCCTGGCAGTGTGGCAGGGGGAGCAGTGGTA AAGAGGGTGGGGTCAGGGGAGAGGCCTCACTGAGAGGGGAGAGGGTCCTTGGTGGGAACC AGGGCGGTTGTGGAAGGAAGAGTCAGGGCGTGGAGCAGAGCAGAGCGGGAGTTGGGGTGG GGAGGCCAGTTCATGCCTCATCAACATGCCTGTGTGAGTGTCCTTCTTGCGGGCGGTGGA GCTGGCCTCAGGGTACACTGCAGGTGACTCAGAGCTCCAATGGGAGGGTGGCTCTGCCTG AGGGAGCTGCACATGCTCATTGGGCCATCGTCTGCTGAGGAGGGGGCAACAATAACCTCA ACACATGGCGGAGGTGTTTTGGGATGCATCCTATTGCACATTTGGGCAGGTTGTTGCCTG ACTTATTTCTTTAACGCATACCTAGCACTTCCTGTATGTCCAGTGCCTTATAATATTAAC TACTATCATCCTCAGGGCTACCCGTGAGGTCGGCACTGTCCCCACCCTTTGTGCAAAGGA AGGATAGGGAGGCGCTGCCAGATGGGTTGAGGGCAGACCGGGGCAGCTTGCCTCAGGCCT CCCTGGCCTTAGCAGGCTTTAAGTTTGATGCCCTGTGCTCCTTCCCTAGGAAGCTCAGTT TGCCAGTTTTTTGCTTTATCTTAGTCTCATTCAGATTACAAGGACTGGGACTGGACTCCT GTTGCCAGTTTCTCAATTTGGAATAGGAAGGAGGTGAGGAACAACAGGCCCATGCTGGGC AGAAGCCCTGCAGACCAGGCCAACAGTGGGCCTCTCCTCACTGCCTGGCCCTGTCTGTGC ACCTTCGTCCTGGCCACAGCCTCTCAGAGCCCCTCCTTTATGAAGAGGTTACGCTTTAGG GGAGGGAGTGCCTCTCCCAAGACCCAGAGCTGGTGAGTGGCTAGGTTGGAATTTGAGCCC AGGTCCCTCCTCCCCTGTCTGCTTCCTGCCAGGCCCAGCTGCCTGCCCCAGCACACGCCT TCTCCCAGGCACCCTGGCCTCCACTGCCAGGACGTGCCTGTTTGAGGCCTGGATTCCCCC ATGGCTGCCACGCTGATTCTCCGTTCTGCACGTCGTGTCCCATCAAAATGCATGACAGTA CCACTCCTTGTGTTAAAAAGAGCCAGTTATTGTGCCTGGCAGTGATGACTGCCCACTGCA CACAAAAGTGGACGCCGTTGTCTCTGCTCTCAGGGTGTTCTTAGTATAGTGGGGCTGGCA CACAGAAACCAGAGATTCCTCCAGGAAGTGCCACAGACATGGGTGAGGTGGCTACAGGGT TTCTGTGCAGCCGACAGCAGGGGTGCCTGGGTGGAAGCAGGGTGGGAGGCCCTAACAGGC CTGATGGGATAGTCAGGCAATGTGCCAGGTAGTAGGTCATCATCCCTGGGGCCCTGGGGA GAGTGTGTGGGGCAGGCGCAGGAGCAGGCTCCTCAGTGGGCTCCTGGACAGGCTGCCTGC TCTGCTGGTGAGGAGCGAGGCTCAAAGCAAGCAGGGGCCTGGGTTGCTTCCCACACTCAG TGGCAGTTGCTCACTGGCAGTCATGTGACCTGGGGAGCTGGCGAGGGCGTGGGGGTAGGA ATGGGAACGGGAGACAGAGGCCCTTTGGGAGGAATTGCCACAGATGCAGTGGGAGTTGGG GCAAAGATTTGAACTTTGGAGAATTAAAGGTGGCAGTCAGAAAGTGAAGGGAGTTCCCAA GTTTCTGGGATGGGCACTTGGTGCTGGGAGATGAAGACACGAAGAGCTTTTCTTGATGTA TCCCTGCTGGCTGCTCAGGCAGGGTCGGGCAATGCCATTAAGACACACTGTGTTCTGGGA ACAGTTCTGGACAGTAAGGACATCCTCAGACCCCTTAGTGGGGGAGCTCCAAGGGCCTGT AATGATGAATCGGTCCCCTGGGAGGCTTCAGTCTAAGCTAGATACCAGTTGAACCCTGAC TGAGTACACAGACATAGCTATGGCAACCAGCTAGACCATGGCCAAAGGCAGAGCATTGGA GCCCAGTTCACAGCAAGGGACTGTGTGCTCTGAGCCGGTAACAGTCCTTGTGTTCAGAGG TCTGGCTGGTCGCAACCAGGGAGTGGCCCCCAGCCCCCTTCCTCAGCTAGAGCGAGCACC ACCCAGGCAGCCTTGGGTTCTTTGGTGGCCGCCTCATAGGCCTGTACTGTTTGTGAACCT GTTGCTTATGTTGGACAGGACTGTGGCAGGACAGCCATCAAGGGTCTTCCCTTGTGAGCT GACACCAGTCTGCTCTTTGTCCTCCTCTGGCTGGACAGGCCCTCGTCTTCCCGGCTTGCT TGTGTGGGGGAAGAGCTCACTGTGACATGTGGGTTCTCTGTTCTTTGTCTCAGGCTGTGC TCCTTACTCCATGTTGGGGGCTGTCTCCTTGTGTGATAGTTTCCTTTCATTTCGTGACAG TGGCTCGAAGGGTTTTGTTTTCAGGAGACAGAGACTGAGGAGTAGCCATTCGGGTCTGGC TGCTCAGGAAGGCATGCTGGGGATGGCAGCAGAGCCCAGCTCAGGGAGATCAGAAGGGGG CTTTGGGAGCAGCCAGTGCTGTTCTGAAGGTGGGCCTGGGTCCTGCCTGCAGCCTCCCGC AGCGGGTGTGTTAGGTCTAAGGGAGGAAGGAAGGGGACTTGACCTCAGCAGGAGTGTGAA ATGGGTACCCTCCTGGAGTCAATGTCCAGGCAGGGGATGCCTCTGAAAAGCGCCAGGCCA GCTGGAGAGGAAAGGGCAGGCATCTGCAGGGAAGTGTGGGGGATTAGCTGCAGAGTTCCT GGGTTCTTTTTCGCTTTGTCTCTATTTTGTGACACTTGATCTTCAAACTAGAAACAGGAG CAAAGACATTTTCAAAAATACAGTTGTCAGAACTGCAAAACCACACAGGGTCTGTTACTG GCCACCAAACCTGCTGGGCCTCCCTGGGTGGATGCTGTGGGAGAGATGTGGGAGTGACAT GGCACAGTGCTGCTGTTCTGAGCGGTAGCCTTGCTGGAGGAGCGCCTTGCCGGAGGAGCA CCTTGCTCCTTGGCCCCACTCTGACCCTCCGAAGCTCATGTGGAACTGGACATTTACCCC AACTCGGTTTCCTGGTGTTGGCCTGTCAAGACTTGCCTGTTTCTGGCCTCCAGCTTGGTC CATGCTCCTCAGGCTCTGAGTAGCTGACGGTGCCCACAGTGTCCATGTCATCTGTGACAT TACCCAGGACAGGACAGGGCCCGTCTGCCAGTACATCCCCTGGTGTATGGTGCCACCTTG CCTTCAGCTCACCAAGCATATTTCTTCAACCTGGTCATCCCACCACCTCAGAGCTATTGC ACTAAGCTGTGTTTCTAGATCTTGTCTCTCTGACTGTGCCAGGTGTCTCTGCTGCACCAA GACAGTGTCTGTCTGGGGCCTGCCAGTCTGCTGACACTCTCAAAAACGGCCAGTAGCAGC TCAAAAGGACTGCTTTTGTATCAAAGCCTGGCTTTGCTCTCTGATCGCTGCTGGTGGGCA CAGAGCCCATCTGTGTAACTGGTTGTCACTCCCCAGACTCTTCTGCACCCTTCTTCCTCT CTGACTCCTGGCCACATCTGCCACCCACAGCCTCCCCACTCCTTACACAGCCAGCCTCCA GGCCGTCCTGCTTGGCCTGCAAATGCTCCCCTGCTGTTTCTACTGCTCTTCCCTGAGTCT CCCTATTGGTCCCAGTGCTTTGGGAGGCCAAGGTGGAGGATCACTTAAGGCCAGGAGTTT GAGACCAGCCTGGGCAACAAAGCAAGACCCTGTCTCTACAAAAAAAGTAAAAAATAAAAA AATTAGCCAGGCGTGGTGGCGCATGCCTGTAGTCCCAGCTATTTGGGAGGCTGAGGTGAG AGGATACTTGAGCCCAGGAGTTTGAAGCTGCAGTGAGCTGTGTTTGCCCCACTGCGCTGC ACCTGTGAGAGAGTGAGACATTGTCTCAAAAAACAAAAGAAGAAAAAGAAAAAAGGAAGG TGGGAAGAACAAGTATGCCTTTGAAGGGCATACCTGCCTTGAGGGGCACAGATGAGGCAA CCTTCAGAAAACAGGCCTGTGCAGCCACTTCTATCAGAAATGCTCACTGACTTCTTGGGG CCTAGCAGCTATCGTCTCAGCCTCTGGGTGTGGAGACCCTGCCCCATGCTAACCTGAGAG CATGAGTACAGGGTCCTCCCAGCCCACCCATGCCTCGCTTCCCCAGGACTCTGACCCAGT GCCGCGCGTGAGTTGGTTGTCATTATCCTCCTGGATTGTTAACTCTCTCATCTTTTGTGG CCCTGGTGACCTTTCTTACTTGTCTGTTCCACACCTGGATGCCTTGTGAGCAGAAACCCC TTGCTGCCTCCTCGCCTCCCTTCAGGCTTCAGGAAGCAGACAGGGAGGTAGGAACTCCAA GAGTGCAGGAGTCTCATTCTGTCGCCCAGGCTGGAGTGCAGTAGTGCCATCTTGGCTCAC TGCAACCTCTGCCTCCAGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGG ACCACAGGTGCCTGCCACCACACCCGACTAATTTTTGTATTTTTAGTGGAGACAGGGTTT CACCACGTTAGCCAGGCTGGTCTCAAACTCCTGACCTCAGGTGATCCACCTGCCTCGGCC TCCCAAAGTGCTGGGATTACAGACCTGAGCCATCGTGCCCAGCCCAAGAGTGCTTTTTGA ATCTAGCAGGGTGGTAGGTCAATGTCATTTTGTTTTTCTTTTTACATTAAGACAGATGTA TGCACCTCTCAGCATTAAACCTCCTTTCTGTGATGTCTTTAAGAATCACAGTCATATTGC TGTAGCCCCGTCTGTACATTCCTTCAGTTTCCTGAGGTTGGCTGGTTGCTGTTTGGAGTG GTCAGAATGCTTTCTAGCACGAGCTGGACTGGGCCTATTCAATTCACTCAGAGGCAAGGC CCCAGAGCCCTGACATGTTTGCTTGTTTCTTTTTTCTTTTGCTTTCTTTCTTTTTTTTTT TTTTGAGACGGAGTCTCGCACTGTTGCCCAGGCTGAAGTGCAGTGGCGCAATCTCCGCTC ACTGCCAGCTCCGCCTCCCGGGTTCATGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTG GGACTACAGGCGCCTGCCACCATGCCCAGCTAATTTTTTGTATTTTTAGTAGAGACAGGG TTTCACTGTGTTAGCCAGGATGGTCGTGCTCGCCTGACCTCGTGATCTGCCCGCCTTGGC CTCCCAAAGTGCTGGGACTACAGGCGTGAGCCACCGTGCTCAGCCTGCTTCTTTCTTTCT TTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTTTCGCTCTGTCACCCAGGCTGGAGTGCA GTGGCGCAATCTCGGCTCACTGCAACCTCCACCTCCCGGTTCAAGCGATTCTCCTGCCTC AGCCTCCTTTGTAGCTGAGATTACAGGCATGCGCAACCACACCCTGCTAATTTTGTATTT TTAGTAGAGACAGGGTTTCACCATGTTGGTCAGGTTGGTCTTGAACTCCTGACCTCAGAT GATCCACCCGCCTCGGCCTCCCAAAGTGTTGGGATTACAGGCGTGTGGCACTGCACCTGG CCACTTGTTTCTTTATCAGCAAGCCCAGGGTGGGAGGCCAGGCAAGGAAGAGACCCATCC CACTGGCAAGAAGGCAGGCAGTGAGCACACCAGAGCAGTCCACCCCTGGGAGCCACAGGC CAGGCCTTTCTAACAGATTCACACTTGTTTTTGTCTTTCTGTGTGTGAGTGTGGGAGGTG GGGGTGGGGGTGGGTGGGGCAGCATGTGCTGCATTCGAGGGGTCTCCACCTTAACTGATG TACATGGCTCTTTATCCCCGTGGAGCAGCAGTGTGATGAAAAACTCTTCATAGGATTCTA TCTCTTAGTACAAAATTGTTGGTAAATGGGTTTTTAAAGATATTTTTCACATTTATGTGG TCTTTCTCTTCTTGTCTCTAGCTAGAAGTGAAGTTGGAGCTTGTCTCTTTGATCCCACAT ATAGGACCTGCATTGTCCCACATAGGGACAGACCCTTGCCTTGGGAGGGACTGAGAGATG ACTTCATGTGGCCCCTTCATTTTATAGGAGTAATCGAGTCTAGAACCAGGGTGCTACCAT CTGGCAGAGTTCATGGGGAACGCAGGCCACTCAGAGAGCAGGAAATAGAGGCCAGCTGGC CACCCAGTAGTCTAATGCGTACTTCCTTGATCCTGAACTGGTTGTGAGCCATCCCTAGGG GCAACCCCTCCCCCGCCCCGTCCCTTCATGCCAGGGCTCTCTCTTTGCTTCATGGCGACC ACCTTTCCTGTCCCTTTTTTCTGATCACCCTGCTCAGTGGCCTGTCATTGCTCTCTGTTG AGTGCCCCTTCCCATATGCCTCTCCTGGACCTCAGCCAGGCTGGTGTGCTGGCGATAGCA CCTTGTCACCAGGGGCAGCTGCTATGCAGGGCCTGTTGGCCAGGGCCCTGCAGCGGTGGA GTCACTGGTGTGTGCAAACGTCTCTTCTCCTTTTGTGTTTCAGCGAGGATGCCATGAGGA AAGCTGGTGTGGCACACAGTAAATCCAGCAAGGATATGGAGAGCCATGTTTTCCTGAAGG CCAAGACCCGGGTAAGGCCCTAGTAAGTGGAGCCACTCTGGCAGAGAGACTTGGAGAGGA GAGCATCACAGACTCTGGAACCCCTCTGTTGGGTGTCCTAGGGTGTAGGGGTGGGGTGGT TGCTCATCGATTGATCACAGGCACAGTTGGTTTTGCCTGCAGCAGTCTGGGTCCTCCTGT GTGTCCATGGTACAGGGAGGGCACTTGGGTTGCAGGCCCAGCCTCTGGCCCCAGACCTCA CTGCCGTCACCTTCCATCTGTGGTCAGCCCCTGTGAATCCTACAGGAAGCTTGCTCAGGC CCCTAGCTGGTCCTGTGGAAGGGATCAATGCCGTGAGCAAGAACCTGGTTTGTCCTCAGC CCCTCCTAGGGGTCCTTACCTAAGCCCTACAGGGCCCTGAGGCATGGGCCCAGAAGCATT CCTGTCCCCTTTTGCTAAGTGAAGAAGGTGATGCTTATCTAGAACTCGCCACAGGTGGAG TGGAGAAGAAGGATTTCCACTATATGTGGGCTTTCTCCCTGGGTGGGGCACCACACTGAG GGCTTTATGTGACCACCTCCTTTAATCTGTGTGTGCTCCCTGGGACAGAACCGTTTTACT AGAAATGAACAAAACACATATTCACTTGTGTGCACGTGTGTGTGCAAGGCAGAGCTGCAA GAGCACAGGGACTGAGGTGGGGTAGAGTGGGCTCTGTACTATGGACACGTGTGACTGAAG TGGGATAGAGTGGGCTCTGTATTATGGATACGTGTCACTGAGGTGGGGTAGAGTGGGCTC TGTACTACGGATACGTTTGATAAGCTTTTTCTTTTTCTTTAAGAGCTTTATACGGTTCAC CCTTTAAAGTTTACATTTCAGTGGTCTTTAGGATATTCACAGAGTTGTGTGGCCATCACC AAAATCTAATTTTGGAACATTCATTAATAAATAAAAAGAGGTGCAGCTTCCCTGTGTTGC CCAGGCTGGTTTCAAACTTTTGGCCCTACGCCATCCTCTTGCCTAGGCCTCCCAAAGTGC TGGGGTCACAGGTGCGAGCCACCAAGCCTGGCCTAATTTTAGAACTTTTTTTTTTGAGAC AGGGTCTCACTCTGTACCCAGGCTGGAGTGCAGTGGCGTGATCTCAGCCTGCTGCAGCCT CAATCTCCCGGGCTTAGGCGATCCTCCTTTCTCAGTCTTCTGAGTAGCTGGGACTACAGG CATGTGCCACCACACCCGGCTAAGTTTTTTATTTTTTGTGGAGACGAGGTCCCACTATGT TGCCCAGGCTAGTCTTGAACTCCTGATCTCAAGCTATCCCCACGCCTTGCCCTCCCTAAA TGCTGGGATTACAAGTGTGAGCCACCATGCCCAGCCTTCAACATTTTTTAAAGAAACTTT ATACCTGTTAGCAGTCACACTCCCTTTTCCTTCAACCCTCAGCTACCACTTATACTTTCT ACTGCTGTAGATTTGCCTATTCGGAATTTTCCATATAAATATAATTGTACAACATATGGG TTTTTTTTGTTTGTGCGTGTGTGTGTGTGACAGTTTTCCTCTTGTGCCCAGGTTGGAGTG CAGTGGCACAATCTCAGCTCACTGCAACCTCCGCCTACCAGGTTCAAGCGATTCTTCCGC CTCAGCCTCCCAAGTAGCCAGGATTACAGGTGCGCACCACCACACCCGACTAATTTTTTG TATTTTTAGTAGAGACTGGGTTTCATCATGTTGGCCAGGCTGGTCTCAAACTCCTGAGCT CAGGTGATCCGCCCGCCTCAGCCTCCCAAAGTGCAGGGATTACAGGCATGAGCCACCGCA CCCAGCCACGTACAATATATGTTTTTATTTTGCAGCTGGCTTATTTCACTTAGCATAATA TTTTCAAGGTTCAAGGTCCATGAACCATGTTTGGTATATACTTTATTCCTCTTTTGTTTT TTTTAGATGGAGTCTCGCTCTGTCACCCAGGCTGGAGTGCAATGGCACGATCTCGGCTCA CTGCAACCTCCACCCACTGGGTTCAAGCAAGTCTCCTGTCTCAGCCTCCTTAGTAGCTGG GATTACAGGTGTGTGCCACCATGCCTGGCTAATTTTCGTATTTTTAGTAGAGATGGGTTT TGCATTGGTCTGGGGCTATGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAGGTGATCC ACCTGCCTTGGCCTCCCAAAGTACTGGGATTACAGACGTGAGCCACCGTGCCTGGCCAAC TTTATTCCTTTTTATTGTCAAATATTCCATTTCTGGATGGCCATATCACATATTTTATAG CCATTCGTTAGTTGGTGGACATTTACATAGTTTCTACTTTTTGGTTATTATGAGTAAGCA GAACAATAGTTAATGTATAAATTTTTGTGTGGGCTGTGTTTTCATTTCTCTTGGGTATAT ACCTAGGGGTAGAGTTGCTGGGTCATACCCTACCTCTGTATTTGACCTTTTGAGGAATTG CCAGATGTTTCCCAAAGCAACTGTACTATTTTAAATTCTCACCAGCAGCTTAAATGAGGT TTCAATTTCTCCTCATCCTCTCCAATACTTGTTATTGTCTATTTTAATTGTAGCCATTAT AATGGGTGTGATTTGCATTGTTTTGATCTGCATTTGCCTAGTGACTAATGATGTTGAGCA TCTTTTTCATGTGCTTTTTGGCCATTTGTATATTTTCTTTGGAGTAATGTCTATTCAGAT CCTCTGTCCATTTTTAAATTGGGTTGTTAGTCTTTTTATTGTTGGGTTGTAATAGTTCTT TATGCATTTGGATATAAGTCTGTTATTAAATATATAATTTGCAAATATATAGGGGGGATT TTATTTATTTATTTTTATTTTTGTTTTTTTGAGACAGTCTCGCTCTGTCGGCCAGGCTGG AGTGCAGTGGAAGTGGAGGGATGCAGGGGGTGTTAGTGATGCATTGTAATCATCCCGGAG AGCAGGACACCTCTCATAAGAGGTTTACCCAAGGGGGGCTGTGGAAAGGGCTAGAAATTG CTCCTGGAGGACGTGGAAAGCCACATAAGGTTTTTGACAGATTAGGGAAAAGGCTGTGCC ACTCTTTCTGAAGATGACAGTGTAGGCAGCCTTTCCATTTTGTGCCAAAAATAAAATTTT AAATACATAAATCACAGTAGTTTCTAGGACAACTGGGTAACTACATACAAAATATTGAGG TTGAGCCCCAACTTCATACCCTATACAAAACTTAACTCAAAATAAAAAACACCAGCCAGG CATGGTGGCGCGTGCCTGTAGTCCTGCCTAATCGACAGGCTGAGGCTGGAGGGTCACTTG ATCCCAGGAGTTTGAGATTACAGTAAGCTATGATTGTACCAACATGTTCCAGCCTGGGTG ACAGAGTGAGACTCTGTCTCTAAAAAACTTTTTAAAAAATTAACTCAAAATGGGTCAAAA ACCTAAATTTACAAGCTAAAACTATAAATATCTTAGAAGAAAGCAGAGGCGTACATCTTC ATGACCTTGGATTAGGCAGTAGTTTCTTAGATATGATGCCAAAAAAGCACAAGCTACAAA AAACGTAGATAAATTTGACTTCATCAAAATTAAAAACTTGTACATCATAGGACACTGTCA TAAGAGGGAAAAACAACCCATGGGATGGGAGAAAATATTTGCAAATCACATATCAGGTAA GGGTTTAATATCCAGAATACATAAAGAACTCCTACAACTCAACAACAACAACAACAACAA CAACAAAAAGCAACTAAAAAATAGGCAAAATACAGCCAGGCGCGGTGGCTCACACCTGTA ATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACGAGGTCAGGAGATTGAGACCATC CTGGCTAACATGGTGAAACCCTGTCTCTACTTAAAATACAAAAAAAGGAAAAAAAATTAG CCAGGCGTGGTGGTGGGCGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATG GCGTGAACCCGGGAGGTGGAGCTTGCAGTGAGCCAAGATCATGCCACTGCACTCCAGACT GGGTGACAGAGTGAGACTCCGTCTCAAAAAATAAATACATAAATAAAATAAATAAAAATA AATGAAATAAAAAATAGGCAAAATACTTGAATAAACCTATCTCCAAAGAAGAGATACAAA TGGCCAACAACCATGTGAAAAGATGCTCAACATCACTAGTCATTAGGGGATTGCAAATAA AAACCACAATGAGATGCCACTTCATTTCCATTATAATGGCTATGATTTTTAAAAAAGACA ATAATAAGCGTTGGCAAAGATGTAGAGAAATTGGAATTCTCTTTCTTTTTGAGACAGTCT CGCTCTGTCGCCCAGGCTAGAGTGCAGTGGCACAATCTCGTCTCACTACAGCCTTCGCCT CCCAAATTCCGGTGATTTTCCTGCCTCAGCCTCCTGGGTAGCTGGGACTATAGGCACACA CCATCACACCCAGCTAATTTTTTTTTTTTTTTTTTGAGATGGAGTCTCGCTCTGTCGCCA GGCTAGAGTGCAGTGGCGGGATCTCGGCTCAACGCAACCTCCGCCTCCCAGGTTCAAACA ATTACCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCATGTGCCACCATGCCCAGC TAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGATGGTCTCGATC TCTTGACTTTGTGATCCACCCACCTAGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGC CGCTGCACCCAGCCAATGCCCGGCTAACTTTTGTATTTTTAGTAGAGACAGGGTCTCATC ATGTTGGCCATGCTGGTCTTGAACTCCTGACCTCAGGTGGTCTACCCATCTTGGCCTCCC CAAAGTGCTGGGATTACAGGGGTGAGCCACCGTGCCTGGCAGAGAAATTAGAATTCTCAT ACATTGCTGGAGAGAATGTGAAATGGTGCTGTGGAAAACAGTATGACAGTTCATCAAAAA ATTGAATGTAGAGTTACCATACGACCCAGAAGTTTCACTCCCAGGTATATATCCAAGAGA AAAGAAAACATGTCCACACAAAAATATATACACAAATGTTTATAGCATCATTATAACATC ATAAATAGCCAAAAATGGAAACAACCCAAATGTCTACCAGCTGATGAATGGATAAACAGA ATGTAGAATATCCATACAATGAATATCATGTGGCCACACGAAGCAATGAAGGGCTGATAC ATGCTACAACATGGATGAAGCTAAAAACATTACATAAATGAAGCAGTCACAAAGGATGAC ATGTTGCATTTTTATAAAATGTCCAGAGAATACGAAGATCTATAGAGATAGTGTATTAGT TTCCCATGGCTGCCATAACAAGTTACCATAAAGTTGGAGGCTCCTATCTCTGGGTCCATG GCTCTGACTTCTGAGCCCATGTCTCTGCCCTCACTTTCTTAAAGGGTAGCACATGTTTGA GGCTGCGTAGTTTTATCAGCCTATTCCTGCTTCTAAAATTTTGGGAGTTCTTCTTTGATT TTGCCTAGTCTATAAACTTTCTACTCCAAGCTGGCAGTGTTTCTGCTGGTAGAACATTCT CAAAATCCTTGTGGAACTTCTACATATATGATGGAATTCATAGTATTAGATGAGGTTTCT CCACAGATCTTTCCTAGATAATTTCATCTCTCTTCCTCACTTCTGTGAGATGATTGAGGG GATTCATGAGTCCCATGTTTAATCTCTTTGGCACTAGAAAAATTTGGAAGATATGCCACA CCCTTGGCATTCTCTACAGAACACACTTTCCTAACAGTAAATCTCCTAGTTTTAGCATTT TGGTTTTTTCCAATCTAAATAGCCTGAGAATTTCCCAAATCATTAAGTCTAGATTCCTTT CTGCTTAACAGTTCTTCCCTCAATTTCTCTCTCTTCTTTGTGTGTGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTATTTTTAAAATTCTTTTTATTGTACCCTTAATTTATCTCTT TCATGTTGCTTTTTTTTTTTTTTTTTTTTTTTTTTGGAGACGGAGTCTCACTCTCTCGCC CAGGCTGGAGTGCAGTGGCGTGATCTCAGGTCATTGCAACCTCCGCCTCCTGGGTTCAGG TGATTATCCTGCCTCAGCCTCCCAAGTGGCTGGGATTATAGGTGCCCACCACCACACCCA GCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAGGCTGGTCTTGA GCTCCTGACCTCAAGTGATCTGCCTGCCTTGGCCTCCCAAAGTGCTGGGATTATAGACGT GAGCCACCATGCCCGGCCTCCTCTTGCATTTTACTACAAGCAGAAAGAAGAAACCAAGCT GAGTCCTTTTTTTTATTTTTTTGAGACTGAGTCTCACTCTGTCGCCCAGGCTGGAGTGCA GTAGCGCGATCTCGGCTCATTGCAACCCCCACCTCCCAGGTTCAAGTGATTCTCCTGCCT CAGCCTCCCAAGTAGCTGGGATTACAGGTGCCCACCACCACGCCCGGCTAATTTTTGTAT TTTTAGTAAAGATGGGATTTCACCATGTTGGCCAGGCTGGTCTTGAACTCCTGACCTCAG GTGATCTGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTATAGGCATGAGTCACTGTGCCC GGCCCAAGCTGAGTCTTGAATGCTTTGATTGGAAGTCTGCTCAGCTAAATGTGCAAACTT ATTGCTTATGATTCTGCCTTCCACACAGCTACAGGACACAATCCAGCTAAACTTTTTGCC ATTATATTACAAAGATCACCTTTGCTCTAGTTTCCAGTAACATGTTCCTTGTTTCCTTTG TTTCTTTAGAATCACCTTTAATTTTCATGTTTCTAACAACATTCTGTTTTTGATAGTATA TGTATTCTTGAAGATAATACAGACTTTCTGTATCAAGTTCGTCATTTCCTTTAAGGCAGA GTCTGGGTGACAGTCTTTAACATCCGCATTTCTACCCACAGCCTGTTCAAGGAAACCTAG GCCTTTTCTATCATGCTTCTCACAATTCTACTAGCCCTCCTTTGTTATTGAGTTACAAAG CCACTGCCATATTTTTAGGCACCCTACTCCTGGTAACAAAATTCGTATGTTTCCTGTGGC TGCTATAACACGTTACTACAAGCTTGGTGGCTTAAAAGAACAGAAATTTAGGCCGGGTGC GGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCAGATCACAAGATC AGGAGATCGCAACCATCCTGGCTAACATGGTGAAACCCCCGTCTCTACTAAAAATACAAA ACAACTAGCCAGGCGTGGTGGCGGGCGCCTGGAGTCCCAGCTACTCGGGAGGCTGAGGCA GGAGAATGGCTGAACCCGGGAGGTGGAGCTTGCAGTGAGCCGAGATCGCGGCACTGCACA CCAGCCTGGGTGACAGAGTGAGACTCTGTCTCAAAAAAACAAACAAACAAACAAATTTAA TCTCACAGTTTTCAAGGCCAGATGTTCAAAGTCAGTACCATGGGTAAAAATCGCAGTGGC AGCAGGGAGGCTATAGGAGAGGAACCTGTTTCTTGCATCTTTTAGCTTTTGGTCACTGCT GGTACTCCTTGGCTTGTGGCTGTATCACTCCAGTCTTCAAGTCCAGCATCACGAGGTAAA TACATTCTCTGTATTCCATCTTCACGTCGCCTTCTCTGTGTGTGTGAACTTTCCTTCTGC CTCCCTCTTAGGAGGATACATGCAATTGCACTTGGGGTCCACCCGCATAACCCAGGATAG CCCTCCATCTCAAGATGTTTAACTTCATCATATCTGCAAAGACCTTTTTCTCAAATAAGG TAACATTTACAGGTTCCAGGGAATAGGACCTGATTTAAGGACTGTTATTTAGCCTGCCAC AGACAGAAAGTTGATTATTGATTGCCAAGGGTGCTGTAGGGAAATGGAGTTTTCATTTGG AGTGAAGATGATGTTCTAAAATTAGGCCAGGCACGGTGGCCCATGCCTGTAATCCCAACA CTTTGGAGGCCAAGGCCAGTGGATCACTTGAGGCCAAGAGTTTGAGACCAGCCTGGCCAA CATGGTGAAATCCTGCCTCTACTAAAAATACAAAAATCAGTAGGGGGTGACAGCGCATAC CTGTAGTCCCAGCTACTTGGGAGGGTGAGGCAGGAGAATCACTTGAACCCAGGAAGTGGA GGTTGCAGTGAGCTGAGATGGTGCCACTTCAGCCTGTGTGACAGAGCGAGACTCCACCTC AAAAAAAAAAAAAAAAAAAAGAAACAAAATGCTTTAAAATTGATTTTGGTAATGCTGCAG AACTCTGAATGTAGTACCAAAAACCATTGAATGGCATGTTTTAAATGGGTGAATTGTATC TCAGCCAGCCTATTACATATATTTTTAATGGAACGAGTGAAAATGATGAATAACAGGTCT GTTTTCCCTTGGTGGCCAGGTCAGGGCCGTGTGGTTTCCTTGGGTGGGGGGCACTGCTGG GGCTGCCTTGTCCCTACTTGGTTTCTGATGGTGTGTCCTGTGGGCTGGGGCCAAGGTCCC ATGGGGCTATGGATACTATCATGGAACCACTCTTTCTGGAATTTGATGGTATTTCAAAAT CTATTAGGAAAACTCTGAGACATGAAAGCACAGAGGCGTCATATTCCTCTAATCTGGAGT CAGCAGTTGCTGTGGTTTGGTGACTTTTCCCTTGGCTGTCTCTGTGTCCCTCCTGAAGTG TCGAGGGCACATCCCCACCCTCAGGGTTGTATCTCTGCTGAGCATCTCCACACTGGCCTG TGCTAGGGCCCCACCTGACAATCACATGGAGTCACTATTGTCATCAATGGGAAGGCCTCT TTCAAGTTTCAACGCTTTTTTCCTAATAGTTGGCTTCTTTGAATCAGGATCCAAACGAGG GTCTTTTCTCCCCTCTGGCCCCTCCCAGCCTGGTGAAGGAGCAGCCCAGCTGTCCTGCAG CCCTCCCCATGGGCTCTGATGTCTCCCAAGCTGCTGATTCATCCTGCCTGCCCCCTCCTT CTTATGAACTGGGTGCTAAATCGGAGAGCAGAGAGGGTGCCGTGGTCTTTTAGCAAAAAT ATATCCGAAGTATTGCCTTAATTTGTATTTCTGGGCATCTTGATATTCCCATAAGACCAC AATTTGGAGGTTTGGAGAGCAACAACAGCCTGACCCTTGTAAAGCTCCTATTATCTTTCC GATGGTTTCATCATTTAATTAGGGCTTGTAGACTAATTCTGGCTTTTCTCCCACATTCGT TACATGGAGTTTTTTTTGTTTTTTTTTTTTTGTCAAGAAAGGTTTTCTTTCGTCAGCCAG GGCTAGATGATTGTTTGAAACCCTGTGTGTTCAGGACCTCCAGGAATGCTGTTTTTCTGA TCTTTCCAGTCTAAGGAGTCCCCCCAAGGGCCACAGATGGGTTCTCTTTGAGTGACATTT TGAGCTCAGGGCTGTGTATCTGGGGTGTTCTGGTCCATTGCTGTCATTCATTTTGATGCT TAGATGGTCCCATCCTCATCTAGCAGGAGCTCCTGTGTCTCTTGGGGCCCTCACACAACC TCTTTAGTTTTTCTTAGTCTGCTGGCATTTAGACCCAGATTGGCTGCTCCTCTAGGGAGT GAACCCTGGGGACTTCTGCCGGTAAAGTGGCATTTGGAGCATGCTACGTGGGCGCTGGTC TGTTCATTGCTTTGGGCCTTTCAGTGAACAGAACTAAGAAAAAACATGTTTTTGAAATAA CAGCAACAACAAAAACCCATAATTTTACACTAATATTTCTAATTCAAATTTAAACTTATA TATAGGGTTTTTACTTCTTTCATTTTAAACATGTGTCTTTTCTGTTACTATGAATATCTT GGTTTCTAATATTATTAGCATACTTGGTTGCTTTACTTTAAAATCATTTCAGAATAAACA GTTTATATTACCACTGATAACAAAAGGTAGACTAAACATTTAAGATTACCTTGAAGTTCT AATTGTCCTTAAAATGTATCCCTTTTATTGAGAACATTTTGCTTTTTGTTTCATTTTTTT AAAAAGCCTCTCAAAAGATTTCTTTTCTCTATATGATTCTGCCACCAACTTAATATTCAG CTCATTTGTTTTAGTTTATTTGGAATGTTTAGGAATTGGACCTTTTTCTTTAGATTTGCT ATACTTTTCATTTTAGAAAAATATTTTAGGCTGGCTACAGTGACTCACACCTGTAATCCC AACACTTTTGGAGGTTGGGGACTGAGCAAAAGACAAGCCGGCCAACATGGCAAAACCGTG TCTCTCCAAAAAATACAAAAAATTGGCCAGGCGTGGTGGCTCACGCTTGTAATGTCAGCA CTTTGGGAGGCTGAGGTGGGCGGATCAGAAGGTCAGGAGATCGAGACCATCCTGGCTAAC ACAGTGAAACCCCGTCTCCACTAAAAAATACAAAAAATTAGCCAGACGTGGTGGCAGGCG CCTGTAGTGCCAGCTACTTGGGAGGCTGAGGCAGGAGAATAGCTTGCACCACCCGGGAGG CGGAGCTTGCAGTGAACTGAGATTGCACCACTGCACTCCAGCTTGGGCCACAGAGTGAGA CTCCATCTAAAAAATAAAAATAAATAAATAAATAAATTAGCTGGACATGGTACTGCATGC ATGTAGTCCCAGCTGCTCAGGAGGCTGAGGTGGGGAGGCTCACTTGAGCCCAGGAAGTTG AAACTGCAGTGAGCGATGATCGCGTCACTGCACTCCAGCCAGGGCGACAGAGTGAGACCC CATCTCAAAAAATATATATATTTTTTAGAGAAATAAAATTCACTGAGTTGCCCCTGAGTT GCCCAGGCTGAACTCCTGGGTGTAAGCAATACTCCTACTTTATCCTTCCAAGTAGCTGGG ATTACAAGACTGTGCCACTGTGCCTAGCTTTGATTTGTTTTTTTTGTTTTTTGTTTTTTG TTTTGAGACAGAGTTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGTGGCGTGATCGCGGC TCACTGCAACCTCTGCCTCCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCAAGTAGC TGGGATTACCGGCATGCGCCACCAGGCCTGGCTAATTTTTTTTGTATTTTTAGTAGAGAT GGAGTTTCACCATGTTGGCCAGGCTGGTCTTGAATTCCTGTCCTCAGGTGATCCTCCTGT CTTGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCAAGGTGCCCGACTTGATTTGGC TTTATCTTCTCAATAGTAAAAGATTTATGTGCTTCTGAAGTGAAAACCCATAACACTGTG GATTTAGAGAGGGCACATTTCCAGCCCAGTTCCTGATACTTGCTGGGTTGTCGATTTTCA ATTTTGGTGTATCTTTCCAGTGTTTCTCATGGCAGATTTAAGCAAATATGTGTGTTTACG TTCCACAGGTCTTATATAAAAGGTGTATGTCATGCAGTGTATGTGGCACGGAGCACGTGT TTCTATCCTTTCTCCTTCCTTGTGTTCACTCCACAATATGTTGTGAGGCACTTGTGCAAG ATTTCCACATGTCAAGCAAGTGTGGAAATTCTATGAAGCTTTTAACTTTTTGAGGTGGGG TCTCACTCTGTCGCCCAAGCTGGAGTGCAGTGGAGTGGTCACAGCTCATTGCAGCATTGA TCTCCCAGGCTCAAGCCTCAGCCTCCCACTTTGAGGCCACTGGCGTGTGCTACCACACCA GGGTAATGTTTGTTTGTTTGTTTGTTTTGTAGAGATGGTGTCTTACTATGTTGCCCAGGC TGGTCTTAAACTCCTGGCCTCAAGGGACCCTCCTGCCTTGGCTTCCCAAAATGTTGGAAT AATAGATGTGAGCCTCCACGATTGGCCTCTATGGAGTTTTTTAAGGTTTCTGTTTTCTTA TCTGTAAACTGGATGTGTTAGTAGTACCTGCCTCTGGGTGGTTGGGAGAAATCAAGGCGA TAATGCATGTGAATTGATGACTCCTGTGTCTGGCACTGTAAAGTGCCAAGTGCTAGCTGT TACTGTCATGATGTTCATTGTTTATGGTTTGTAATACTCAGCCTCAGGTACAGCTGGACA CTTATATTTTGATAGCCAGCAGGGGAGCTGGAGCAGACCAAGTGAAGATAGGGAGTTTCC TGACTCTTTAGCCCTTTTTTCTGCAGCAGTTCTAGGGGAGTTGGTGTGCTTTGGAGCAGA AAGGGGCATAATAATCTGTCCATCTGGATGGGGGTGGGGGTGGATGGTGAGGAGTCAGGA GTGAAAGAGCCCAGCCAGGGGGATCAGAGGCGCAGTTTCAGATCCAGGGCTTTCTCCCCT GGAAAGGAGTTGTTGTGGTTGACCCATTGCGTTCAGCCCCTAGATGTTCCTAGATAAACC TGCCTTTGGGGGAATGTAGGAAGTATCATCGCCCACAAGAAAGCTCCTTGGCTGGCAGCA TGACAGCTTCCTGTGCTTGTTTCTCACATCAGGCGTCCCCTCCCTCCCCGCTCCCCACTA AGCCTCCTCACGCTGGTCTGGTAGCAGAGCTGGAGCAGATGGCAGTGGCTGCGCCTACTG TGGGTCAGGAGTGGTGCTGGGCACTGCAGGATGATGATGCCTGCCCACTGCATTCCAGGT GCTGGGCTGGCAGTGGGTCTTGCTCAGCCCTGGTGTTATGCAGAAGGGCTTTGGTGACAT GGTCCTGAGCCTTGTTTGAGTGTCTTATGTCTAAGTAAAAGTATCTTGTTGCTTTTGAAA CCTACTTCCCCTTGTTTTACCTTTTATGGATGTGGAGAAAACAAATTCATTTAAAGTATC GCAGACCTAAAAAATGGTCCAGGGCCTTAGGAGCTGTGAGGTGTTGACCTGGCTGGCCCC CAGGCCCTTCTCCCCTCATACATGGCCACCTTGCTTCTGTTCCTGAGTGTCCTCAGGTTC CCACACCTCCATTGCCCCCATTCTCATACCACTGTCCTTCAGGGACAGGCTGCCTGTGGG CCCCCTTCACACGCACATCAGCTTTCCTATCAGTAATTGATGGGGATAGCCGGAAGCTGG GCGTGTTGACAGAACTGCCTTGGTGTCCCCTTGTTCTGGCCAGGCCTCGCTGCTTATCCC AGCTTTTCCTCTCCCCTGGGCCTTTCTTCTTCATGTTGTCCTGTGCTAATTCCCCGAGCT GAAAAAAAGAAGTGGGGTAGAGTTTGGAGATAGATGTGGGATGATAAAGATTTCAAAAGT GTCTTTGTATTTTTACACTCATCTGAGACCACTTTTCATCATCCTTCCCTCTGCCCTGCC ATCTTATGAGTGTAGTGGATGGACTCTGAGTGTCACAGTGACTCAGGAGGAAGGTTGGGT GAGCAGGGCACGGAGCCGGGGCTTTGCCCCTTCCAGGCTGCCCTGCCAGAAGGATGGGCA CTTCAGCAGAAGACAAGGATGCTTCTCTGAAATAAGACTTTACCTCAGACATCCGTGTAT TGACCACAAGACCAACTCACAGTAGTGATTTCCCTGAAACACTTGGGAGTGAGTGTCCTT GGGGCCAAAGAGGACAATGGGTGGGAGGCAGCCCCTGCCAGACAATCTCAGAAACACAGC AGCTTGCATATAGCGGGCCTTGGCAGCCAGTGGCCTTCACCTCACCTGAGAGTGGGCTTC ACCCTCTGCCTTGCCTGTGCGTAAATGGAGATGAACACAGATTCTCCCGGAAAGCGCAGC CAGGGGAGGGCCGCCCAGATTTTCAGCAGATACTGCGTCATCAGTGAAAAGGCAGCAACA GGCTGGGCTGAGAAATGAAAGCCCTTCTTTACAGATAGTGCAAAGACACAGCGGCCCACC CACGGCTGCCTCTGACAGGCCTTGTGCCCTCAGGCCAGCGCCTCATCACCCCCAACCTGC CTGTCCCCTTTGTGATGAAGAGAGGGCACTCAAGATGGCTCCCAGCTCTCTCAGCTGAGT GCCTTTCCATCAGGGGAGAGGAGTGGCCGTTGGAGGCTCTCTGTCCCCTTCTTGTTTCTT TGTGCCTGTTATTGGCCAGCCTAGCTGAGCACTGACGGCTCTCTTCCGAGAGGCGGATTC CAGAGGAGGCCGAGCAAGCGTCTGAATCTGCCTTGCAGGATGGGCTTCAGCTCGGTGCCA GCAGGCGAGGGGGCGGGAAGGGGGAGTCCGATGACTGCGCTTCTTCATCTGGTATTAAAC TGCTTCCTGTTTTTACTTTTAGGACGAATACCTTTCTCTCGTGGCCAGGCTCATTATCCA TTTTCGAGACATTCGTAAGTAAGATTTTGCATTTCTGGGTTGTTGAGATCTCTGTGAGAG GCCAGCCCTGACGCTGCCTCGGCAGAGCTTTCTGGGCAGGCCGTGGTGCCTGAGTCAGAA GGTCTGTGTCACCTCACTTGCTTCTGGGAAGTTCTTCACTGCCTTGCATTTGACTCCAGA TCCCTCCATCCTCCCAGAGCCTTGGCCTCAAAAATGCTGATTCTAGCATCATGGAAATGC TGTCCTCAAAGTGGTCTAAACGGGTTGCTGCTTCACTTGCTCACTTAATCTCCCTTTTCA TAGGGCTGTTGTTTTTACTTCTGGGAAGTTCTGTTTACCCTGGAACAGAAACTCTCTTCC CTAAAAGTTGATTTTATTGACCCATGGAGGCCAGAGACACTTAGGCATATTTTCCCTCCA GACTAGAAGCTTCTGAGGAGGACCTCCTGAGTCTGCACCCTGGCTCCCTGCTGTGCTGAG GGCCCCCGTGTTAACCTCACGTTGTGCCTCCTCTGATTCAGAGGGCCCAGTGTGGTTCTG TCAGCCAGGCAGTGGCCCCAGCTCTACAGAAATGAGTTGTCATTGCATCCTAGGGCCAGG GTCTTCGTGCTTGTGTGTGTTACGTGGAAGTATGTGGACACCAAGTGTTCCTGGATGGCC ACAGCCTGCGAAGGAAACTGGGGCCAGCAGCTGCTCTGTGTTTTCAGCCAACAATGGCTC CTGCCCACTGCCGCTGCATAACCACCAGAGGCAGGCTTCTCTTGACACAGGCCTGTCGTT GGAGCATGTGCCTGGCGAGTCCTATTTCTATTCCCCTGTGGGTTAGGGACAGGCAGCTGT ACCTTCAGTGTGTTGCTGGGTCAAAGGGAGACATTGAGCACTCCAGAGCCTGGTAGGTTT TGCTTCTTTCCACACTAACTTTTCACCAGAATGGCCAGCAAAGTGAACTTCACTGATCTC ACTCTCTGTGGTGTTGGAAAGTCACTGGGATGTGTAAGAGGAGAAGGAGCAGCCCAGATG ATCTGGCCCAGATGCGCTGGCAGACTCCGCATGTTCTGCTTTCAGGGACAGTGGCCTGGC TGGTGCACACAGTCCTGTGGGGCTCATGAGGTCATGGGAAGCAGCCGAGCAGGAGGCTCA GGGCCCCATGACTGCTACAGAAGTTCTCCGCTGGGCACTGGGGTCAGCTGGACTGGCTTC CAGCTGGCCGTGGACAGCCTCAGTTTCCTCATCTAGGTAACAGAGACACTGCCTCACCAG AGTGGGCTGCAAAGGGAGGCAGTGTCAAGTCTTGGCACCACTCCAGCCCCGCCGTGGGTT CCTGTAGTGTTCTATTATTTGTTTGCTCAGGGCTGAGCTGAGACTCTGGACCTGGGAAAA AGGAAAGCCAGAGTAGGCCCAGGGTCCCCAACCCCAGAGTGCTTCAGCCTTGGGGGCAGC TCCCTAAGGCCCCAGGTTGACCCTGGAGCTACTCTGCACCCTCTGCCCCCAGGTCTCTTG TGGGGTCAGCGCTGCCAAGAGTAGAAACACTTCCCCCAGTAGTGGGGAGTACTAGGAGCT CCGTGGGGATTAAGGCTTGGGCAAATGCCTACAGAACTGACCTACCCATGGAAATATGTG GTTATATAAAACTATGTTTACTTTCGTTTTTTTGTTTGTGTTTTCATATAGATAACAAGA AATCTCAAGCTTCCGTCAGTGGTAAGAGTTTTCCCTTTTGAGTTAAAGTTTCTCCCAACT TTGGATTTGAGGTGGCAAGAATGACATAGTGCATGCCTCATGTGCCTGGGTTAACCTGTC ACTAGAGGAGAATGCTTGCGGAGAGAACTCTGGAGGGAGGAGGCTGACCAGCTGTGCCCT GACCTCTCCAGCCTTCGTCTTCCTGCAGACTGTGAAGTCGCATGATCCTCCCCATCCCGT TTCACACAAATGCTGCCTTTGCCCAGCAAAAAGATAAACACTCACAAAGCTATTAAGACA TTATCCAAAAGTGTTGAGCTCTCTGGTAGAGAATTATTATGCTGTTTTCAGTGTAATAAT ATGTAGTAGCATTTCTGAGAAAATGCTGTGAATTCACTTTTTTAAGTTACAAAGTTTCTT TGTAAATTTAGAAATCTGGCTGGGTGCTGTGGCTCATGCCTGTAATCCCAACACTGTGGG AGGCTGAGGCAGGCAGATCAGTTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGT AAAACCCCATCTCTACTAAAAATACAAAAATAGCCGGGTGTGGTGGCATGCGTTGTAGTC CCAACTACTCAGGAGGCTGAGGCATGAGAATCGCTTGAACCTGGGAGGCTGAGGTTGCAG TGAGTTGAGATCGTGCCACTGCACTCCAACCTGGATGACTGAGTGAGACTCCATCTCAAA AAAAAAGTTTAGAAATCCATATTTTTAAATTTCAGCGTTTATCACATGAGGGGAATTCTG TCAATTCAGCCTTGGTCTCTGCTGACTAAGCTGCAAGGGAAGGGGTTTAACAGATCCAGG CTGGCCAACTGCATTCTGCCATTGGGAAGGGAGTGCACTGACCTCACAGGCTGCACGCCA CTGTCTGCCCTGCCCTAGACAGTAGAGCTGTACATGCCAGCTGTCTCTGCCCATGTGGGT CTCACCTCTCAGCCAGGACAGGGATTCTTGGCCAACTGGGGCTGAGCGGCAGCAAGGGCA TGTGGCCAGAACATCTGGGCCCCTTGGCATTGGAGCATGGGGTCCTGGGTACCACCCAGT TGTGTGGCATGGCGGTCCTGCCAGGACATACCTGTCTGTGGGTAGCTGTTTGCTGTGAAG TCCACACTGTTGTGACAATGGCATCCTTGTCCTTGGTTGTGGCATTGCTCACTGAGCTGC TGACCTGGTGGGCTTGGGACATTTCTCCTCAGTGCTCTGTGGAGCCCTCCTCTGCACCCC TAAGCTGTTCTGGCATGGTGGCCCTGCACACAGGGGCCCAGGCTGAGTTGGACTCTGCAA CAGCACGAGTGGAGCTGTGTGTGCCTGTGGACTTGTGCCCTCCCTGGGAGAGCGTCCCCT GGCCACTGTGTTACCGCTTGCTCAGAAGGGCCCATCGTGCTTTGTACGCTCACCCAGCAG GAGGGCTGGACAGCCAGGAGAGGCAGGGGTTGCCACCTGCCCTCAAGGCCTCAGCCCATC TTTAGTGTATCTGCAGGCATCAGAGAGGTCATTTGTCCCTTAACATTAGGACTCTGGTCC AGGCCAGGCTAGAGGTATGGGTCATGCAGTGACCAACACACCTGGCGTCCTAGCCATTCA TATTTGGGAGTCTCCAGGAGCCTAGTCTCTTACTGCTTGGGGCTGTGAGGGGATTGAGCC TGTAGGTAGGCGAGATCTGTGCTCTGTGAGCCTTACGCCCTTTGAGCCATGGTCAGTCTG GTAGGCCCTTTCCTGAGAAGCTCTGCCCTTGTGTTCCCACAGATCCTATGAATGCACTCC AGAGCCTGACTGGCGGACCTGCTGCGGGAGCCGCTGGAATTGGCATGCCTCCTCGGGGCC CGGGACAGTCTCTGGGCGGGATGGGTAGCCTTGGTGCCATGGGACAGCCAATGTCTCTCT CAGGGCAGCCGCCTCCTGGGACCTCGGGGATGGCCCCTCACAGCATGGCTGTCGTGTCTA CGGCAACTCCACAGAGTGAGTACCACACTTCTTGGAGGATTTGCCGCTTTCCTTGCAAAC GACAACACATTTTATTCCTGTGCTTATGATGGTATCAAGAAAAGCATGGAGAAGCTCCAA ATCGCTCTGGTTTTTTTGTTTTTTAAATCTTTGTTGTATGTGGAGAGAAAAGGCTTCCAA TTTTCGTTGAGCTCCCAGAAGGGTTAAGTGATGCTGGGCCCTCCTCCTCCTCCTAGCCCT GGGCAGGAGGGTGGGGATGGCCTGAGACTCCATCCAAAGGTTTGTGTTTTGTGGCTGAAA TAGATACAGTGTGTTTCAGAGGGTTCTCAGCTACAAGGCAGGGCTGAGTGCCAGCAACCA CAACTCCACTGTCTGCCCGAGCTCTGGGCACCATCTCTTCTAGTGCTGCCCTTGCCCCTT GGTCACCATGGGTGTGGCTTGCAGCCATATTATCTTCTGGCTTCCTTTCTGTGAAGGAGA CTCTGTGTCCTCTCCACACGGCTGCTGTGGTGGGAAACTGGGCTGTGATCAGGTGTTATA GGTGAACCAAAGCTGCTCTAAAGGGGAGGCAAAGAACCTCCCTCTTCTTTCCAGGACCTG TCTTTTGAGACGGGGTCTCGCTCTGTGGGCCACATTGGAGTGCAGTGGCACAATCACGGC TCACTGCAGCCTGGACATCCTGGGCTCAGGTGATCCTCACACCACAGCCTCCAAAGTAGC TGGGACTATAGGCTCACAGCACCGCGCCCGGCTAATATTTTGTATTTTTTGTAGAGATGG GACTTCACCATGTTGCCCAGGCTTATCTTGAACTCCTGGGCTCAAGCGATCTGCTCACCT CGGCCTCCCAAAGTGTTGGGATTACAGGCGTGAGCCAACGCGCCTGCCCTTAGGACCTAT CTTGATGTATCCTTGAGTCCCATCAACATTTCTTAGAGTGTGCTGTGCCCACTGCAAAGT GCTTTAGTGTTTTTTTCCCCTTTCATGTAACTTTTTATTTTTAAAATAGGAGTCACAGGA AGTTGTGGAATAGCATAGAGAGTTCCCACATGCCCTTTACCCAGCTCTCCCCAATAATAG TATCTTACATAGAACATTGTCAAAACCAAGAAATTAATGTTGAGGCAGTGTAATCACCTC AGTTAGAGACTTAGCTTGGACTTCATCAGTTTTTTTTTTTTTTTTGAGACAGAGTTTCAC TCTTGTTGCCCAGGCTGGAGTGCAGTGGTGTGATCTCAGCTCACTGCAACTTCTGCCTTC CGAGTTCAAGCAGTTCTCTTGCCTCAGCCTCCGACTACCTGGGACTACAGGCGCCTGCCA CCATGCCCAGCTAATTTTATATTTTTAGTGGAGATGGGGTTTCACCATGTTGGCCAGGCT GGTCTCAAACTCCTGACCTCAAGTGATCTTCTCTCCTGAGCCTCCCAAAGTGCTGGGATT ACAGACGTGAGCCATCACGCCCAGCCGACTTCATCAGTTTTTTTACACTCATTCTTGGGC TACATATATGGGTATGTGTAGGTCTGTGAGATTTTACCACGTGTCAAATCAAGTAGCCAT CACCACAATTGGCATGCGGAGCCGTTCCATTACCACCAACCAACTCTCTTGTGTTACCTC TCAGAGATCGCGCCCTCCCTCCAGCCTTAAGCAGTGACAATTGCTGATCTGTTGTGTGTC TCTGTAGTTCTGTCCCTTTGTGAATGTTGTATAAATGGAATCATACCATATGTGACTTTT TGAGATTGGCTTTTTAAATTCAATCTGATACCCTTGAGACCCAGCCAGGTTTTATCAGTA ATTCATTCCTTTTCATCACTAAGTAGTGTTCCATGGAACGGATTTTCCACAGTTTGCTTA TTCATTCACCCATGAAGGACATTTGGGTTGTTTCCTGCTTTTGCTTACTATTATGCAAAC AAAGCTGCTGTGGATATCTGTGTACAGGCTTCAGTGCTTCAATATATTTTTGTCACACAT CCCTCATGATAACCCTATGAAGAGGGCAGCATCGCCCCCTCTGCTTTGTCAGTGTAGCAG CTGCTAAGGAGAACTGGAGTCACACCCCAGGCCAAGAGCTGGGATCGAAGCTGCTGCCTG ACCCCATCCTCACACTCCTACCCTATCCTGTGTCAGGTTTCCTCTTCTTTGGGGCCATAG GCATCCTCATTTCTACCCACTCCTGCTCTGGAATCTTCCTCCATCCCAAGCCACCTTCCC TTCTCATACCTTAATTCTCCTCTTACTTCCTGTTGTCTAACTCTCAGCACGTGCTGGTTT CCTCATCCTAGAACAATCTGCCTGGTTCTTCGTCCCAAGCTCTCATTACCTCACTATTTT TAGCACATCAGATAAGTACACCAGCTGGGTTTAGGGAGGACCCACAGAAGTGCCGGCTCA AGTTCCTTGTCACGTTCTACTCTTGTGTACTTGGTGCATTTTTTTTTTAATCATGAGCTT ATACTTGATAATAAATTGTTTTTAAATGTATGAGTTTAGAATGCTCACATGAGTTGCCTT GTCATCCCACTTCAGAGACTTTGTCTTAAACCAGTTCTTTGTCAGAAGTTCAGTGGTGAA GCTTGATATCCTGCCCACGCCCTCCCCAGTCTGGCCTTGGCCCCCACATCCTGTTTTTAG CTTCCAAGGGGACTGGTCTTCTGTGTGGCACTAGTTGAAAGTTACAGTGGGGGCTGGGCA CGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGGGCGGATCACGAGGT CAGGAGATCGAGACCATCCTGGCTAACACGGTGAGACCCCGTCTCTACTAAAAATACAAA AAATTAGCCGGGCGTAGTGGTGGGTGCCTGTAGTCCCAGCTACTCGGGAGACTAAGGCAG GAGAATGGCATGAACCTGGGAGGCGGAGCTTGCAGGGGGCGAGATCGTGCCACTGCACTC CAGCCTGGGCAACAGAGCGAGACTCTGTCTCAAAAGAAAAAAGAAAAAGTTACAGTGGAT TCTCTACTTCAGGATCTTGTTACCACCTTTGTCAGAGGCCAACTCTCCCTGCCCTTCACC ACTTGCCTCCCTGGTCTCCCTGAAACTAGTAGTGTTGTCTGATGGGCATTTGATACCCCT TCCTGACACCTTTCCCAAAGACTACTGCATGATTAAGGCCCTTTCAGATCTTCATCTAGT GCCACCAAGAGGACAGAGCCTGGGTCAGAGGTTTTGCCAGGGACTCTCAAAGGGTAGGCC AGGGGACATTGTGATAGTTTTCAGGTGACCCAGGTTAGTCGGAAAAGGCTGAAACTGCTG GTTGTTCCTATGGCTGGGGCTGCAGTACAGAGTTGGAAGCAAAGCTTTTCACTCTGCGCA TTTTTGTTTCTCATGAGTTTTATATGACGTGCATGTAAAGCCGACTTTAAAAATAATTTC TACTGCCAGTTGTGTGATGGACTGGGGTGCTGAGGACATGTTCTCCTCCTGGATAACTAG GTTCTGGTTAGGATGCACTTTGGAATGCTGGCCTGCAGCAGTTGGGGAGGCTTTGTCCTC CTGAGCCTCAGCTGCTGCAAGGTGGGGGAAGAAAGCTTGGGATTCCTGTAGTCAGCCAGG ATTTGTGCTGGGTCCTGTGTCACCCCCCATACCCCCCAGAAGCAGGTGCAGATTGGAGCA GGTGCGGATTGGAGGAACATGTCCCCAGTGTAAGACCTGTGACAGTTGCACATTAAGGTC AAGCAATCAGCATACAAAGATCACCAGCCTTGAAGGGAAGGAGAGACACCATGAAGGTGA ACAGAAAAAATAGACACATGTAGGCATGCCCAGTGACCACACACACAGGAACTGTTAGTT ATGGGCCATGGGGTATATAAAGAAATAAAGTGGAGAGCCGGGCACAGTGGCTCACGACTA CACTCCTGGCTACTCGGGAGGCTAAGGCAGGGGGATTGCTTGAGGCCAGGAGTTTGAGGC TATGGTGAACTATCATTGCACCACTGCACTCCAGCCTGGGGGATAGAACGAGACACCATC TCTGAAAAAATAGAAAACAAAAAGGAAAAGAAAGAGCAGAAATTCAAATGAAAAAGCATC ATCAGGAGCAATGAAGCAGATTTGAAACGGAACCAAATGAAAGCTGTAGAAATGAGCAAT ATAATTATTGAACGAAGAAAGTGGATGAATGAACAGCGCATTAGATACAGCTGAAGAAAG AATTTGTGTAATAGAAGCTACATTTGAAGAAATTATCTAGAATGTAGCACAGAGAAGAGC ACATGGAAAATGTGAAAGCAAGGGTAAGAAATGAGGATAAGATCAGAAATTCTAATTGGC ATCCAAGCAAAGCCCCGGAAGGATCAAATGGAGAGAATGGAAGAAAACAATATGTGAGGG GATAACTACTGAGAAAAGCCCATCTGGCCCCAACCAAGTTAGCCATCTCATCTGCTGTGA GACTGCAGAGCACCAAAGGCCAGAGGAATGTCTTAGAAATAGGCAGTTCCTGCAAAGGAG CAATGAGTACACTCAGCAGTAGAGTCAGAAAGGTAATAGAATATTGTTAAAGCATTGAGA ATAGAGCTGCCAGTCACATTTGTATGCCAGCAAGACTCAAGGATGTGGAAAGCAAGCCAT TTTTAGATAAACCTAGCCTTAGAGACCATCACTAAAAGATTTTCTAAAGGATATATCTTT TTTTATCCTTTTTCCAATAAGCCTTTTGCACTCTTAGATATATTCTTTTGTTTATCCTTT CCTCTTCCCTCTCAGGTGATACAAGGTAAAATGGGAAAAGAAACGTTGAGCATAGGTGAT AGTAAATGTGTATAAATCTAAAGCAGTGATTCTCACCCAGGGTGACATTCCACCACAGGG GATATTTGACAATGTCTGGAGACATTTTGGGATGTCACTACTGTGGGGGTGCTGCTGGTG CGTAATGGGTGGAGACCAGAGGTGCTGCTAACCATCTTACAAATACAGGTCAGTCCCCCC AACAACATAGAATCATCTGGGTCAAGTGTCAGTAGTGAAAAGGTTGAAAATCGCTGGTGT AAATAAAAATTTACCTCTGTAAAATAAAACAGTCAGGCTAACCTCCATACAGACTTGCCA CATTCCAAGCACTATTTCATTTCTTTTCTATTATTATTATTATTATTATTAAGATGGAAT CTCGCTCTGTCAACAGGCTGGAGTGCAGTGGTGCAATGTCGGCCCACTGCAACCTCGACC TCCCAGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCGCGC GCCACTACGCCCAGCTAATTTTTGTATCTTTAGTAGAGACGGGGTTTCACCATGTTGGCC AGGATGTTCTCGATCTCCAGACCTTGTGATCTGCCCTCCTTGGCCTCCCAAAGTGCTGGG ATTACAGGCATGAGCCTCCGCGCCTGGCCTATTATTATTATTTATAGAGCTAGGGTCTCC TTATGTTGCCCAGGCTGGTCTTGAATTGCTGGGCTCAGGCGATCTGCTCTGCCTTGGCTA TCCAAAGTGTGAGATTACGGGCATGAGCCACAGCACCCAACCCCAGGGACTATTTTGAGT GTTTTACTTACATAATAGATTATTTAATTCTTAGTCCAGTGTGTGGAGTAGGTACTATTA TTGCATTTTCTAACAGGGAAAACTAAGGCAATGAGCAGTGACAGAGTTGAGGTTTTGAGT CCAGGCAGCCTAGCTTGGAGGCTGTATCCCTAGCCTCAAATCCATCCAGCCTCTTAATAG AATGTCTGCTGTGAAGAGTTAAAATAGATCAACAAAATATGAAATAAATGCAAGTCAGCA GGGAGATAGAGCGTGAATATCGGTTCTGTCGGGTACATATAAAACCTACTTTAAAAAGTA GTAAATTTGGGAAATGCTAAAATATTGATGAGGTTTATACTTTAAGTTAAATATGCATGA TAAAATTGAAAAATTATTGCTAAAACAATGGAAATAATTCATAAAATTTAAGATCAATAG AAAAAATAGGATAAGGCAGAAATCCAGATAATACAAAGTTCAATCCAATCATCCAACAAA AATGCAATCAAAAAAGACAAAGAAGCTTAGAAAAGGAAAGACAAAGGCCGGGCGCGGTGG CTCAGGCCTGTAATCGCAGCACTTTGGGAGGCTCAGTCAGGCAGATCACGAGGTCAGGAG ATTGAGGCGATCCTGGCCAACATGGTGAAACGCCGTCTCTCCTAAAAATACAAAAAGTTA GCTGGGCATGGTGGCGCATGCCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAAT CGCTAGAACCCGGGAGGCGGAGGTTGCAGTGAACCGAGATCACGCCACTGCACTCCAGTC TGGCGACAGAGTGAGATTCCATCTCAAAAAAAAAAAGGAAAGGAAAGACAAATATTAATA GAAAATAAGTTTAAAAATATAAGTAATCATAATAAATGTGAGTGGACTAAACTTTTCTGC TGAAAGATAGGAAATGTGAGAACAAATGGAAATGAAACAGCACAGTTGTTTTATTGTTAG AAGAAACACACACCTACAATAAAACATGCGGAAGGATTTAAAAATAAAAGATGAAGAAAG GCCAAGTGCAGTGGCTCACGTCTGTAATCCCAGCACTTGGAGAAGCTGAGGTGGGAGGAT CACTTGAGCCCAGGAGTTCAAGACCAACCTCGGCAAAATAGGAGACCCCCATCTCTACAT AAAATTTAAAAATTACCTGTAGGTACACTTGAGATGCTGAGGTAAGAGGATCACTTGAGC CCAGGAGGTCAAGGCTGCAGTGAGCCATGATTGCACTATTGCATTCCAGCCTGGGCTGCA GTGAGACCCAATCTCAATCAATCAGTCAATAAAATTAGCTGAGTGTGGTGGTACACACCT GTAGTCCCATCTACTTGTGAGGCTGAGGTGAGAGGATCGCTTGCTTGAGCCTGGAACGCT GAGTCTGCAATGAGCCACGATGGAGACACTGCATTCCAGCCTGGGTGACAGAGCAAAATC CCGTCTCAAAAAAGAAACAAACAAAAAAAAGGATGAAGAAAGATGTATTGGGCAATTACT TCAATTACATCTCTTTGTTATTGATAAACAGACCAAAAAATTCATTTACAGAAAATTTGA ACAATGTAATGAACAACTTAATTTAAGCATATGATGGAACAATATGTTTATAAAAATAAA TCATAAAGCAAGCTTCAACAATTTCAGAAAATTTAAAAGTATTGTCATACTTTTTGTTGT TGTTGTTTTGAGACAGAGTCTCACTCTGTCACCCAGGCTGGAGTGCAGTGGCGCGATCTC GGCTCACTGCAACCTCTGCCTCCCGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCTGAGT AGCTGGGATTACAGGCACGTGCCACCATGCCCGCTAATTTTTGTATTTTTAGTAGAGATG GGTTTTTGCCATGTTGGCCAAGCTGGTCTCAAACTCGACCTCAAGTGATCCTCCTGCCTG AGCCTCCCGAAGTGCTGGGATTAGAGGTGAGAACCACCGCGACCCGCCGGTCATACATGT TTTATGAATGTCAAATAAAGTTAGAAATCTGTCACAAAATAAATTAAAAATTTTTTACAC TTGGCCAGGCACAGTGACCCACGCCTATAATCTCAACACTTTGTGAGGTCGAGGCAGGAG GATCACTTGAACCCAGGAGTTTGAGATCAGCCTGGGCAACAAAGTGAGACTGTCTCTATA AAAATTTTACAAATTAGTTGGGTGTGGTGGCATGCACCTGTAGTTCCAGCTACTCGGGAG GCTGAGGCAGGAGGGTCACTTAAGCCCAGGAGGTCAAGGGTGCAGTGAGCCACGATGGTA CCACTTCAGTCCAGACTGGGCAACAGAGTGAGACCCCACCTCAAAAAAAAAAAATCTTAC ACCCATCAGAATTGCTAAAATGAATAGTGACAGCCTCAAGTGTTGGTGACAGAGAAACTG AATCACTCATATATTGCTGGTGAGAATGTAAAGTGGTTTTACCTTCTTTCAAAATGAAAT ATGCAACTCAGCAGTTACACTCTTGGGTATTTTTCCTAGAGAAATGAAAACCTCGCATAA AAACCCATGCACGAATTTTAACAGCAGCTTTATTTGTGGTAGCCCAAATCTACAGTTACC CAAATGTCCTTCATGTAGACGGTTAAACACACTGTGAGGTGCTTGTACCATGGAATACCC TGGCAGTACTGTTGCTAGAACTACAACCTGTATGCTGAATGTAAAAAGCCGATCCCAAAA AGGTTCATACTGTATGGTTCCATTTTTGAAATATTATGTATAATTTTGAAATGACAAAAT TTTAGAAATGGAGGATGCTGCAGTGGTTGCCAGAGTTTGCGGGTCAGGGTTGCTGAGAGA GGTGGCAACACGAGGGATCCTTGTGGTGTGGGGACTGTGTAGTGTATTGACTGGTGGTGA CTGAACCCACCCAGGTGGTTAAATTGTCCAGAACTTAGTACACACACCCACACAAGTGAG CACACATCGTGGTTGTGATACCTTGTAAGTTTGTAGAATGTTACCATTGGAGGAAACTGG GCAAAGTATACAAAGGATCTCTCTATATTTCTTTAATACAGCTTTATTGAGACATAATTC TCATTAGCATACAATTCACCCATCTGAAGTGCTCCACTCGGTAGGTTTTAGTGTAATCCA CAGAGTTGTGCAGCCATCAGCACAATTTTAGAACATTTTTATGTTCACCCCAAAAGAAAC CTCACCTCAGAAAATCTTGTAATTGACTGTTCATATAAGCAGTTTTCATAATAGCCACAA AGTAGAAGCAGCGCAAATGTTCATCACAGGAAATCGGTGTAGTAGAATATTATTTGGCGA TAAAAATAACACAGAAAAACTGATAACATGCCACGACATGGGCGAACTCGTAAATATTAT GCTAAAGAAGCTAGTCACAAAAGAGCACATGTTCGATGCCATTCGTACAAAGAGTCCGGA AGTGGCCAATCCATAGAGACAGAGAGGAGATTACTGATTGCCAGAGTCTAGGCTTCTTAT TTATCCAAAAGACTTAGTTGTCCCTTTTCTTTTGTCTTTGGTTATTATAGAGTAACTCAT GATAGGAAATCCCAAAATCAACACAAATGCTACTTCGTATTCTATCTTTCTGTCTGTGGT AAATGGAACGTTCAGATTCCAGCGGCAGCCGTGGCAGTGGGGCTTTTGCTGGCTGTTTTG TCCCTTGCTGTGCAGCCCTGCAGCGTTTCTGGGAATCTGCCCTGTGGACTGACTGGCGAC TCTGGTCTTTTCTCAGCCCAGCTGCAGCTCCAGCAGGTGGCGCTGCAGCAGCAGCAGCAA CAGCAGCAGTTCCAGCAGCAGCAGCAGGCGGCGCTACAGCAGCAGCAGCAGCAGCAGCAA CAGCAGCAGTTCCAGGCTCAGCAGAGTGCCATGCAGCAGCAGTTCCAAGCAGTAGTGCAG CAGCAGCAGCAGCTCCAGCAGCAGCAGCAGCAGCAGCAGCATCTAATTAAATTGCATCAT CAAAATCAGCAACAGGTACCAGGTCCCCTGTTCCTGCTCTTGGCCTCCCTCCTGCAGCGT GAGCCCTGGGCTGGCATCAGCCACAATGCTGGGTGCAGTGCCCGGAGCCAGCCGAGGGTT CTCTTGACACGTGTGCCTGCCCTTTTCCATGGGCTTCTCAAAAAGTCTGGGACAAGGCCG GGCCCAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGCGGATCATC AGGAGTTCAAGACCAGCCTGGCCAGCATGGTGAAACCCCATCTCTACTAAAAATACAAAA AATTAGCTGGACATGGTGGCGTACACCTATAATCCCAGCTATTTGAGAGGCTGAGGCAGG AGAATCGCTAGAACCTGGGAGGCGGAGGTTGCAGTGAGCCGAGATCGCGCCGCTGCGCTC CAGCCTGGGCAGCAGTGAGACTCCGTCTCAAAAAAAATAAAAATAAAAAAGGAAAACAAA AGTCTGGGACAGGCAGGAGAAACAGGCCCTGACTCACTAGGAAGGTTCTGTGATCGAGCC CTTCCATTTGGGCTTGGGCATCTGGCGGTGGGCCTCCTGTGTAGTTCAGGGGCTGCGAGG GATTCAAACAAGAGTGAGCTGAAGCAAGAAGTGAGACGAACACTCTGAGGCTTGGGACTA CTTGAGCCATTGTGGGCCATTTTGGCTGAGGACACATGTGCCTCCTGTTTACCTCTGTGT CTTGGGTTCTGGGCTCAGTGTTAGTCGACCTCCAGCTGGTGTTGCATGAGGTGATGGTGA TGTCATGTTAGCAGCACTGCTTCTCAGAAGTTGTTCAGCTTCCCATGGTTTCCATAGAGA GTCTAGACGTGTTAAACATCTGCCCTCACAGCAGGTCTTGGTTGGGAGAGGAAAGCCCCA AAGGGCCCCACCACCCTCCCACCCCTTCAGGCCTCAGCTAGCTGGGCTTCTCTGGATCTT GGCACCCCTCTCCCAGAGCTGACAGTGCATCTGCAGCCACACTTTTCCTTCTCCTGCTGA CAAGCAGACCTTGACCGTCACCGTGAGGCTCATGCCACTGACCTATAGGGGCAAAGCAAG CATCGTTGCAGAGGCTTCAGAAATCTGCAGTTGCCCCAGCTTTTATCCTAGCTTCCATGG AGCTGTACCTATTCCTTGTCATCTTTTGCTGAAGTTTCTAGGTATGCCCTTTGAACCATC CTAGTGGAATGGTAGACCAGAGCCCAGGCACACACTTGGTGCCTCCCCGGTGTCAGGAAG GCAGCTGGTGTTGGCCTCGGGGGGCCCAGAGCCTTTCTGTGGGAGATGTGCCCCTCCCCG TGTGAGTGAGAGGTCAGCCTGTCAGCCCAGGAAGGCCTTTTTTTTTTTTTTTTTTTTGAG ACTTTTTTTTGCCTCTTACTCTGTTGCCCAGGCTGGAGTGCAGTGGTGTGATCTCGGCTC ACTGCAACCTCTGCCCCACCAGTTTAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTG GGATTACAGGTGTGAGCCACCATGCCTGGCTAATTTTTGTATTATTAGTAGAGACGGGGT TTCGCCAGGTTGTCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCTGCCCGCCTCGG CCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGCACCCAGCCAGGAAGGCTTTCTT GATGGCTCTGCAGATGGCCACCTGGGCTTCCTGAGGCCCAGACTGTCACAGGGAGGAGCT GGTGCCACAGAGGCAGATGAGTGATAACCGAGTGCTGCTTGTTCTGTCTCTAGATACAGC AGCAGCAACAGCAGCTGCAGCGAATAGCACAGCTGCAGCTCCAACAACAGCAACAGCAGC AGCAGCAGCAGCAGCAGCAGCAGCAGCAGGCTTTGCAGGCCCAGCCACCAATTCAGCAGC CACCGATGCAGCAGCCACAGCCTCCGCCCTCCCAGGCTCTGCCCCAGCAGCTGCAGCAGA TGCATCACACACAGCACCACCAGCCGCCACCACAGCCCCAGCAGCCTCCAGTTGCTCAGA ACCAACCATCACAACTCCCGCCACAGTCGCAGACCCAGCCTTTGGTGTCACAGGCGCAAG CTCTCCCTGGACAAATGTTGTATACCCAACCACCACTGAAATTTGTGAGTACCTGTGGCC CACAGTGGAGCACATGCAGCCCGTGGCTCTGTCAGCAGTAGTTTCTAGGCTCTTTGGCTA GAGATAGCATATCCTATTCTTCAAGTGCTGAGACTTCAGGCAGCCCCCACCCCTTGCCAG CCCTGCCGACTCTAGCATGGCTCAGCAGGGAAGCATGTGTTTTGCATTTTGTGTTCTGAG AGCTGCTTCTGCACCCCGCACAGGGTACTGCTGCTGCTACTGGGGCACCAAGTCGCATTC CAAGCAGTGTGGCTGATTGCAGCAGTTCTGTGGGCCCTTTCAGGGTGTGAGCACTTCGAC TGATGAGCACCACATGAAAGGATGTGCCAGCTCCGTCAGTGTCTGGTGGTTTGCCAACTA CTCAGAGTTAATGTTCTAAGCAAATATCTTTTTTTTTCCTTTACACTCTGCCAAGTCTCA GGAGAACAAAGTTCATTACTATATTTTTGAGAGGAGTATTAATATAAAATACTGTTATTC CTAGTTTCATTTTGTATCTCTGCACAAAGCCAGTCTACTCAGTGACAGGCACAGAGGACA GAGTATGATCACCAGGATGAAGGTAGCTGCATCAAGGGGCCATTTGGACAACTCTTTGGC TCCCTTGGTTTTTGTGGCCTTTTCTGTTTGTGATTCATGAGTGGAATGGGGCCTGAGGCA AGTGTGGGGGTGAACAAGAGGCCTCATAGGCAGACTGGAAGTCTAGGAGAGTTGTGAGGA GCAGATATGCTCACTCATCTGTCACCTGGGAGTCGACACACTGCCTGACCCCGGGGACTG TGCAGACTGAAACCCTTGTGTGCTGCATGGTGGAATGTGCCCTGGCAGGGTTTGCCCTAG TGGCTTGGAGATGTCTAAGGAAGAGCCCGCTGCATGTTGGAATGTGCCCTGGCAGGGTTT GCCCTGGTGACTTGGAGATGTCTAAGGAAGAGCCCCCTGCATGGTGGCGCGGGCCTGTCT CGTCCTTCTGGAGGCCCCTCCATGCAGTGGCTCCACAGTTGCCCCTTTTGCCCAAGTTTG CCCCCCACCCCCACTTTTTTCTTTTGAAGACAGGGTCTCTGTTTTCCAGGCTGGAGTGCA CTGATGTGATCAGACCTCCCTGCTCAAGCGATTCTCCCACCTCAGCCTCCCAAGTAGCTG GGGCTGCAGGTGTGCATCCCCACACCTAGCCAAATTTTTGTATATTTTGTAGATATGGGG TTTGGCCGTGTTGCCCAGGCTAGTCTTGATCTCCTGGGCTTAAGCGATCCACTGGCCTTG GCCTCCCAGAATGCTGAGAATACAGACAGGAGCTACTACGCCCTGCCCTCTGCTGGCCGG CTAACCCAGCTTTCCTTCAAGGAATCAGGCTGAGTGAAGCTCTTGTGGACCATCAGCATG CTTGGCCCAGAGGATGAGCAGGCTAGGCTGTAAGCTCTGCATTGGAAGTCGTCAGTGCTT GTGATAGCTGGTGCTGAGGGATGTGTGGAGACATCCCAGTGGGAGGGCCACATTGCCACT TGCCAGAATTGGATTTGAGCTAATGGGATCCTAGATGGCCAGTAAGGTGACATCACAGCA CATGAGGTCATGAAAGTCCCATTCCTCACCTCAAGAAAGGCTCCATTTCCTAAGCTTCCA CAGGAAGACTAGGCTCTGCTCTCTGACCCAGGTGGTTCCAAATCACATTCTCTCTTTCTC CCTCAAGGTCCGAGCTCCGATGGTGGTGCAGCAGCCCCCAGTGCAGCCCCAGGTGCAGCA GCAGCAGACAGCAGTACAGACAGCTCAGGCTGCCCAGATGGTGGCTCCCGGAGTCCAGGT GAGGGCCTGGGGGTGGAGGGCTCCATAGTCATCAGCAGGTGCATGTTCACCTGCGCATGT AGTGCTACAGTGAGGGTTCTGGTTGGATTAGGGGCTGGGGGCTAAGTTGGTAAAGCAGGG GCTTCTCTCCCCCTTGCACTCTGCAAGACACCCCTGGAGTCCTTGGGGTGGGCAGCTGTG CTTGGTCATGTCTAGGACCCACCAGCCGCATAGTGCGTGTCGTTTCCAGCTTTGCTCGGG CATCCTTCATTATCCCTCTTCCTTGCCACTCTGAGTGACTCTGGGAACATGTATCAGAGA GTAGAAGGCTGCAGTTTTCTCCTCACCTCTCATCTTCCATTTGAGAGTCTGAGTACAGAG CTGACCAGGGTGGAGGGCAGCTTTCTAGACACTCGGAAGCAGATGGGAGCCACATGCAGC AGGAGCGTGGGAGAAGGCATCCTGGTGCTCGCCTGCACTCAGGGACTCCGGCGCTGTGTG GTGAGAAGCGTGCCACTCCCACATTGCTGAGCTTGCTGGTGTGTCACATGTGTGGAATGT GTGTGTGTCTCTGTGTGTCACGTGTTAGTGGAATGCAGATGGCCTTGTCTGGCCTCCTAG TGTCCTGGGATTGGAGGCCCAGGGTTTCTGTACCTTTTCCTATAACCCCTCCTCTCCTTC TTGTGGGGAGACGTACAGTCTGGGGCTTCAGTTTTTCCTCTTTTCACATGATGGTGGAAA CACTGTTGGTCCTGACTCGGAAGGGGCGTTGGCTTTAGAAAGAGTCAGAATGTGCTAGTT GATGTGGTAAGACTGTGGCTGGGTCCCAGCCCACAGCTGGCAGTGCGCCTGCACTTTGCT GTCGTCTTAAGGGTACCTGGCCTTGTGGTTTTCTGCAGGCCTGCTGGGCGCAGCGCCTGG GTGCACAGTGCCTCGTGGCAGGCTTCATCTCACGCTTGGGGGGTGCTCAGTAATGTCCTT CATGGTGCCTGCTACCTCTTTTAAGTCCCTGGGAAGCTGCCTGAGGAGGGAATCTGAGGT ATTTGGCTGCAGCTGAGGTTAGCATCCTACCTTGGCAGGGCACCAAATGACTTGGGGCCA GGGTCCCCATGTCACATTCTTACTTAGATGGAGCCCTGTATGAAACACACCACAGAACTC ACATTTACTTACAGCTGGGGGAGGCTGACCACTTCGTGTGATGAGGTTGGAGGTCTCTGC CACCTCCCTGTGTGTCTCGGCATGTCCTCAGGCCTCGTGGGGGGGCTCCCTTGTCCTGGT CTCATGCAGAGGAAGGACGTGGTGTGTTCAGAGTCAGCTTAGTGGTGGCACATAGTGACT GCCCACCCAGTGGAAGCTGCTACCTTTGCCTCTTTGGACCATCGATCCTCTGTTCAGGAC CTCACTTCCTGGAAACTTTTTTTATTTTTTGAGATGGAGTCTTGCTCTGTTGCCAGGCTG GAGTACAGTGGCACGATCTTGGCTCACTGCAACCTCTGCCTCCTGGGTTCAAGTGATTCT CCTGCCTCAGGTTCCCGAGTAGCTGGAACTACAGGCGCGTGCCATCACACCTGGCTAATT TTTGTATTTTTAGTAGAGACATGGTTTCATCATGTTGGCCAGGATGGTCTCCATCTCTTG ACCTCGTGATCTGCCGGCCTTGGCCTCCCAAAGTGCTGAGATTACAGGCATGAGCCAATG CGCCCGGCTGGAAACTTCTGTACCGCAGGTCAAGCCCAGCAGGAAAGTGAGAACTGCTCT TTTCTTTATTTCTTTCTGTCTTTTTTTTTTTTTTTTGAGACAGAGTCTGACTCTGTCATC CAGGCTGGATTGCAGTTTTGCGATCTCGGCTCACTGCAACTTCTGCCTCCTGGGTTCAGG CGATTCTTGTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGTTGCACACCACCACACCTG GCTAATTTTTGTATTTTTAGTAGAGATAGGGTTTCACCATGTTGGCCAGGCTGGTTCGAA CTCCTGACCTCAAGTGATTCACCCGCCTCAGCCTCCCAAAGTGCTGGGATTATAGGCATG AGCCATCGCACCCAGCCTAGAACTTCTCTTCTCTTCTCTTTTTTCTCTTTTCTTTTCTTT CTTTCTTTCTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACAGAGTCTTGCTCTC TCACCTAGGCTAGAGTGGCTGGAGTGCAGTGGTGCAATCTTGGCTCACTGCAACCTCTGT CTCCCAGGTTCAAGTGATTGTCCTGCCTTTGCTCCCAAGTAGCTGGGACTACAGGCATGC GCCACTATATCCAGCTAATTTTTTTTTCCTCTTGTATTTTTAGTAGAGATGGGCCAGGCT GGTCTTAAGCTCCTGACTTCAAGTGATCCACCTGCCTCAGCCTCCCTAAGTGTTGGGATT ACAGGCGTGAGCCACTGTGCCCAGCCGAACTTTCCTTTTCTTTCCATTTCTTGGTATTTG GAAAGTGAATAGCCATGGAGCAGCCAGTGTTTTCAGCTGGTGCAGGCCCAGCTGGTATGT TCGTTAGGCTTCTAGCTCAAGGAAGTGTCCACTCTGCAGTCTCACTGGCATCCCATCACC GTCCAGAAGCAGGATGCCAGCTGTGTGTGCTCATGGCCCATGCTGAGCCTGTCCTTGGGC CACTGGTCTTCCTGTCGGTTTCTCCACCCCTTGTTTCAGGTGGAGGTCCCCGCCCGACTG GCACAGGAAGGAGGATGGAGAGTGGTTACCACTGTGGTTCGTTGCTGAGTGAGTTTGATG TTTTAAGGATCATTGCCCACCTGCCCTGTGGCACCATGTGCTGCCATGCAGGGGATTCCC AAGTGCTAGTGGCAGAGGAGCCTTGTAGTTTCTCAAAGTCCAGCAGTGGGTAGGGCGTAT CTATGAGGTGCTTCTCAGTCCAGATGTGGCACCTTGAAATTCTGTAACCCGCATTCTGCT GGTTTTCTCTCAGAGCTTGGTCTTGCCTTGTTGCAAGCAGTGCAGTTTCTTGGGGGCTAA TTGGGCACCCTCACTGCCTTGTTCCACCAATAGCAGCGCCCCCTGCTGGGGGATGGGGAG GGTTGGGCACAGCAAGCCCTCTGAGGGGCCATCTGGATTCATGATCTTCTTTTGAAACTT GAATTGGCACAGTGACCAGCTGAGGCTACGGTACTCTGATCTAAAAACTTTAAAAGTATG TGTGGTAACTGTAGAACCCAGAAAGTGGAGAGGAACAGAAGTAAGAGAACCATTCCATGA GAGAGAAGGTCTGGTGTCCTTTCTGGTTGTTTCCCATAGTCAAGGTCATGCTATCCTGGG GTTTTTTGTGCAGCATCTTCCACATCAATAGGGGATGAAATGTTCTCCTTTAATTAGAAA GCCCTGGTCGGCACGAAGGAACTCTTGTCATACCCCTCTGTGAATGTCTCTTGCCATCCT CAGTTACCATCTTGTTCTTGGACCTTGTAATAATTTCCGATTTTTTGCTATTACCAGTGA CACATATCTTGATGTCTTAGATTGTTCCTGGAGCTGATGGAGGTGAGTGAATGGTTCACT TTGAGGGCCTTCAGATCTGATCAGCCACCCTCTAACCAGGAGGTCAGAGATGCAGATTGG AGCTCCCCTTGTGTTGTTCACACAGTGGACATCAGGACGTGGTGACCACGTGTTGCAGCT TTGTATGTATGAGCTGCCGCGTGCTGCGGAAACAGGCCTCTTGAGTTCATGAATGAAATT CTGGGATTGGGGGCCCCCATGCAGGCAGCCCTCCATGCCCTGTCTCTCAAGCCCATCTTA GAGCCGAAGAGAGGCGCCCTGTCAGGCCCTTGCAGTACTGTGGTGTTGGATGTGGTAAAG GGTAGCCTGGGACTGAGTATTCTAGAAAAATTGCTCAGCCTTGTGCTGTGCACACCTGGT TCTCTTTCCAGTGTTATTTATGTAGCTGAGAAGGGCAATGTCACCCACAAGGGGATTTAG CAGGACACAGCAGACTTCTTTTTCTCCCCTATAAAACCAAGACATGCAGGCACAGTGGCT CATGCCTGTAATCCCAACGTTTTGGGAAGCCAAGGTGAGAGGATCACTTGAGACCAGGAG TTCAAAACTATCCTGGGCAACACAGCGAGACCCTGTCTCTACAAAAAAATAAAAATAAAA AGTTAGCTGGGCGGAGTGGTGCGTGTCTGCAGTCCCTGCTACTCGGGAGGCCGAGTTGGG AGGATCGTGCAAGCCCAGGGGTTTGAGGCTGCAGTGAGCTGTGATGACAGCATTACACTC TAGCCTGGGAGACAGAGCAAAACTCTTGTCTCTTTAAAAAAGCAAGAAACAGGACAGAAT GCATCTCATTTTCTTTTTTTTTTTTTTTTTAAAGACAGAGTCTCTTGCTCTGTCACCCAG GCTGGAGTGCAATGGTACAGTCTCAGCTCACTGCAGTCTCCACCTCCTGGGTTCAAGGGA TTCTCCTGCCTCAGCCTCCCAAGTACCTGGGATTACAGGCATGTGCCACCATGCCTGGCT AATTTTGTATTTTTAGTAGAGACGGGGTTTTGTCATGTTGGCCAGGCTGGTCTTGAACTC CTCGCCTTAAGAGATGTGCCCGCTTTGGCCTCCCAAAGTGCTGGAATTACAGGCGTAAGC CACTGTGCCTGGCCATGCATCTCATTTTCAAACAGTCCCTATACCTTTGTCATTTAACGT TCTATTCTTTTAGTATGTGAAAACCAAACTCTAATTTACAAGTCACAAAGAATTATCCAA GACAGACTCCCATTGTGGACATGTATGTGGCATTGCTGATTCACAGCTACTGTACTGAAG CCTTGGCCACCCTGGAGGACCCTCTCTCTTTTTGCTGGACCCTTCTGCACAGTGACTGCA GTAGGGAGAGTCAGACCTGGGGGTGGGGGAGAGGTTTTTTGGATTCCTCTCTGGGTAGAT GTGTGTTGGGAAGTAGTGAAATGTTGATAGCTGCCTGGCTCATCTGATCCAGTAAACATA ACTGGTGCGTTTGGGTTGAAAGAAATTGGGCTTTCCTTATCAGTTCTCAGTTATAAGAAC ATCAGTCAGCAGTACATTAGACTTAAGCTTTGCATTCCTTGCGTTTTTTTGTTTGTTTTT CTCTTCCTGGAAAAAAGTTTGCTTCTCTCATACCATCTGACTTACTTCCAGGCTTTTCTC CCTTGTGGAACGAGTGCCGTTGAGCCCTGCTGCACTCTCAGACGGGCTCCTCCGAAGTGC CGCAGGTGGTGGTAAATCGACTCTCACCCACTGGGGTCGCTCCTTCGTGTCTCCCCCCGG TCGGTTCATCTGTTGCTCTGGCTGCAGGAGGAACGAGTGAGCTTCTGGTCGGCGTCTGCC ATGCCGTGTCACCCCGGCTTCTGGCACCTCCTGTGCGTGCCCAGGATTGTGAATGTGGGC CGTGTGTGTGAGGCCACGGGTCTCCCTGCAGCCACTCTCCTGCTGGAGCTCTGTTACTGG CACCTGTCGCTGCCTGCACCGAAGGCTGGCAGCACCTCCTGGAGCTTGGGACCCAGAGCA CAGCCTCCCACCATGAGATGTGTTGTTTTTCTGTGGATCAGTCCTCCTTTCTTTCTGAGC CTGGCGTGTTTTGTTCTAGTTTGTTACCGTCCTAAGTGCCTGTAGGCCCTGCTCTCCAGG GACGAGACTCGGGCTCTACCCCCAACTCAGAACCCAGAGCAAGAGTGGTCGGGCCCGGGC CCACAACAGTGCTCAGCTGTCCTGCTGCCTTTGTAGTTCAAGAAGTGTCCATTGATGAGG GGAATGGTCCTGGCTCATGCTGGAGTTCCTGACTCGCATCCCTGTGGAGATGAACTTCCT CGTCAGGGCGGAGGCCTGCCAAGCAGTCCCCCCAGGCTTCTCTTGCTCACCTTTGCCCAT TTTTATTACGAAAGAAAACCAGTTCCTTGATAGATACCAGGACCATCAGCCTCAGGCCTG GAGGAGGAGAGGAGGATGATTTGGGTTCGGGCTGTAAGAGGTGTGCCACTGAGAAGGAGG GATGCTGTGAGCAGGCTTAACTGAGCTCATGGTTCAGTGGGAGTTGAGTGTTCTCATCAC AGGCTTTGGTGGAATGTACTCTTGACATCTGTCCCCAGGAGCCTGGTCTCCAGAAACACC AGCTCAGGCCCTCAAGGTCTGGCTCTGATGGTTCTGTGGGCTATAGGATTCTGATCTGTT AGCGAGGTGTGTTCAGAAGTGTGTTGAGGACACCAGTGCAGGAGAGCAACCAGTAGAACA GAAAGGTCTGGAAGCAGCATTCTTGGCAAATCTTCTAGATTCCCAATGCCCAGACAGACC TGGAGGTGCTGTGGGCTTGAACATGTGGGTGGCCTCCCCTCCCAGGCTGCCCCGAGCTGC CCAAGCTTTCCTTGCCCTGGTGCTCCTTCTTGCAGAGGCTACACGTGCCCTCTCCACCTG CCCAGGCACTGAGTTTCTTTGTTGCGATCACCTTGTCTGTTGTCCCTCTGTCCTCAAAGA TGATCACGGAAGCCTTGGCCCAAGGTGGGATGCACATAAGAGCCCGGTTCCCGCCTACCA CCGCTGTGTCCGCCATCCCGTCAAGCTCCATCCCTTTGGGCAGACAGCCCATGGCACAGG TATTTACGGGGCAGCGCCCAGGGCACGGCTGGGAGCTCGGGGCGTCCTCTGAGCGCCTTT GTAAAGCGCACCTGTCATTATCATCGCCGGTGACTTCTTTTATGGTGGCTTTCAGAGTGC CAAACCCACAGTCCCTTTTTTTTTTTTTTTAAATGAAGCCAAGCCCAGCTTGTGCTTGTT GAGAGGATATGAATAAATATCTGGGTCTTTTCAGCTTAAAATAGTTATACTGTACGCATT TTCATGGTAAAAATAAAACAGCAGCTAAGCAAAAGTTTTGCTTTCTAGAGCTGTGCTATC TAATAGTGTAGCCACTAAGCATGTGTGGCTGTTTACATTTAAATTGACTAAAATTAGGCC GGGCACAGGGGCTTATGGCTATAATCTCAACACCTCAGGAGGCCGAGATGGGAGGATCAC TTGAGGCCAGGAGTTTGAGACCAGCCTGGTTCACATAGCGACATACTATCTCCAATTAAT TATATACATTTTTTAATGGAAAATATATATATAAATGTATATATATTTTAAAAACAAAAA AAAAACCTAAAAGTCAACTAAAATTAAATAAAATGAAAAAATGCAGTTCTTCAGTCGCAC CAAATAAATTTTAAGCCCTCATTTGCCCACAGGGAAGCTAGTGGCTGCTGCAGTGGACAG CACAGACTCCAGAACGTGGCCAATGTGGCCAAAGTGGTATTGGGCCATGCTGTTGTAGAG TTTGATGTGTAGAGTGTGTAATTTGTAACACAGATCCCCTTTCTAGGTAGAGGTAGACTT CTGTGTACATTATGCTTTTCAGAGACAGGAATTCCCGGCTTCTTTTATTGTATGAAATTT GGAATTACTCCATGGCATAGTCATTTTTTTAACAAATAGTTACCACATGCATTCTGTGTG CCAGGCACTATGCTAAGATTCTGAGAATAAAATTATGCTGAAGTTCTCTCATCATACAGG CTTTCCATTTTAGTGGGTTTGTCGTGAAAACATCAAATTTGCCAAAAGAACTTTACAATT CAGTTTCTGTTTTAGGGGAAATGCCTCTATAATCCTAGCAGACAGTGGTCCCTGTCCATC CCTGAACGCCCAGGTGCAGGCACTGCCACCACCCTGGCTTGTGCAGTGGCGTCTCTGCAG CTCTGTTCTACACAGGAAGAAGTGGAGGCTGAGCCAGGATGGCCCTTGGTCCATGTGGCC CCAAGCCTGGTGCCACACACCACACACTGCCAAGAAAGTGGCCTGGGCCCCCACTGGGGC ATGTTTAGCTTTTGCCTTCCCTGCATACTAACCCCCTGGGTTACCCCAGATGTGCCATTC ACATAGTTACTGACTGGAACTTAGTTTCTTTGGCAGGGACTTCCTTTTTAGTAGTTGTTG AATTACAGCGGGAGGAAGTGCCTGGAATCTGCTGGTAGAAAGGCTTTGGTGAAAGAGCTC TCCAGAGGAGCGGAAGTTGGGGGTCAGGAATTAGGTTGTGTGGCTGTGGTGGAAAGGAGG CCAGTGTGGGCAGAAGCCAGGGTGCAGTTCCCCATGGCCTGGAGGCTGTGAGTTCCACGA CCTGCTCACATGCTGCTGGTGTCTCCTTGACTAGACCCCTTTGGGTTCTGCACTCTTGTC TGCTAAAACTGCTTCTCCTCCTTTCCCATAGCCTCTGGCACAGTATCCACTGGGTTCACA GAATCAGGTTGGGTTGCTGTATGTGGAGGAATGACTTTAGTAGCAGCATGTAGGACTGAT TTTTATAAACACAAAAAGTCCCCAATGTTTTCAGTGTGCGATATGTGCCAGGCCTTGAGC TAAGCTCTTTGTGTACAGAGCCTCACTGTTTCTTGTTGTAGCAGGTCATAGTTTGAGCAT TTAGCCCCTTTTACAGACTAAGAGTGCCCCCAGGGAGCCACCAAGTCAGGATCGAGGGCT CAAGTGAAACCCTTATGCCTTGGCCTCTCAGCAGGATTCTTCTGGAAACTCCTCACTCCC CTTCCTGCTGGGCTGATGACCTGGCTTCTCCTTCTTGTTTACTGGGCACTCCTGGGTGGG CTCCCTCCATGCCCCCACAAGACCAGGCTCTGTCCTGGGCTTTGGCTTGCCCATACTCTG TTGTGGGCTGGCCACTCCCCACACCCAGCTGCTGGTGTTTCCACCTAGACTTCTCACAGG CAGCCCCTCCCTCCCTCCCTCCCTCCTTCCCACCAGTTCCCAAACCCACTCAGTCTTTCC CTCCCCAGCCTTCCCTGGCCCTTAGATTAAGTCTGCCCTGAGTTATGCCAGCTAATAGGT CTCTCTCTCTCTAGAAGCTTCCAACCCCCAAAGGCAAGGAGGTAAGGTGGAGCAGAGGAA GGATAGAGAAATATATGATGAAGCTGAAAAAGTTATTGCCTTGGAATAAATGCCATGGAG AATTCTGGTTTGTCCTCATTGCACTGTGTGGGGTGAGAGCGCTCTCATGGGAAGATGCTG GCTAACTGCACACAGATTTAATCACCAGGCCCCGCCCATTCGCACTGGCAGTGAGAGGCA CACCCCTGAACCTTTGGGGTTGTTCACTGAGGACAAACGCGGCAGCATTTAGAAAGTATT CCTCGTCCCGGCAGTAGACAAAAGCCACAGGATTTATGGAAAGAGGAAGGAAGGCACAGA ACTGGGGCAAGGTTCTGGTTTTGTTCTGTTATTTTGTTGTCATTGTTACTGTTTGTTTTT CTTTTTTTGAGACAGAGTCTCGCACTTGTCCCCCAGGCAGGAGTGCAATGGCGCACTCCT GGCTCACTGCAACCTCCACCTCCCAGCTTCAAGCGATTCTCCTGCCTCAGCCTCTCGAGT AGCTGGGACTACAGGCGCCTGCCACCACATCTGGCTAATTTTTGTATTTTTAGTAGAGAT GGGGTTTCACCATGTTGGCCAGGCTGGTCTTGAACTCCTAACCTTAGGTGATCCGCCCGC CTCGGTCTCCAAAACTGTTGAGATTACAGGTGTGAACCACTGCGCCCAGCCTTGTACTGT GTTTTTAAAGCAGGTTTGTTCATGCTTGTTAGCTGCAAGAAAGGAAACTGGAAGGAGTTG AGGGAGGGAGAAAAGCGAATAGAGGGACACCCTGGAGTGGAAGCCTCCCTACTGCCCAGG CAGCTGCTCTGCAGCCCTCTCTGGTCCTGGCCTTCCCCGCAGAGGGCCCTCCCCAGCTCA GCCCTCTACGTCCGGCTGCAGTACCAGGCTCTTGGTACAGTCTCCCACACCTGGCTGTGG GCCAGCCTTGGGGACGTGAGGCATGCTGGGCCCCGTGTACCAAGGGCATTCACCTGACCA CACCAGGTGGCTCCAGAGGTGTGCCTCTGCTTTTGTGTCAGTATCTTTCAATCCAGTCCC TTCCGAGAAAAAGAGAGCTTATCTCAGGAACGTATCTGTTGATCTCACCAGGAATTGCCC CACTGTGTCCCATCTGAGCTCTCAGGGGCCAGAACCTGCTGTCTCCAGTGTGTCCAGATG GCCACTCTGTGGCCACTGTCCCTCCAGCCACTTGCCCTCAGCAGTCAGCAGGACACAGTC TGGAGACTACACTGCCCATTTGCTCTTGCTCTTTCCTTGTGGCAACAAATTAGGTCCAAG AAAACAGGTGGAGCTGAAAGAGGGAATGTACGTGCCCCATGGGCTCTTCTCCAGAGGGCA GGGCCCAGGCTCCCTCCCTCTGTCTTGACGGCTCATGGCTGCATCTTCCTGTTACGGTGG GCATGCCTCACGGACTCAGGCTACCAGGTCAAGCAGGATTCTGTGCTGGGTGCATTCTGG ACACAGTGCTCAGCACGGGGGCCCAGGGGGTGGCATGGCCCATGCCACAATCCTCCCTTG GCAGATGTAAGCAGGCAGTGTCTTTCTCACTAGGGGCTTGTCCTTTGACTAGAGAGGCAC TGTTGCCTCCCTGTTCCCTGCTCCAATCCCAGACCTGACATGAGACCAAGGACTGGGAGG TTTCTTTCTCGCCTCTCTGGCTGCCTCCCCAGAGCCTAGAGCCTGTCTTTGGATGAGTCT TTGCAGGGGCTGCTTCTCATCCCAGGTGCTGGACTTGGACTGCCCACCACTGACACCTGG GCACAGGGCCTGGCTTCCTGCTCTCGTGCTCTTGGAAATGAATGGGTGCCCTTCATTTGT TTTTCTTCCCACTAGAAATCTCCCTGCCTCCCATCCTCTGGGCTCTGCTGGCTGCCAGCT GCCAGGGTGAGACCCGTGTTCCACTGCACCCTGGGGAGTGCCTCCAGGGGACACACTGAG TGCCGGAGCCAGGAGCAAGGAACGGTTCCCAGTATGTGGGGGCGGGGGTGGGGATCGCGC AGTGCCGCCAGCCTTGCCTGAGGGTTCCTCTGTGAGATGGAGCTCCCTGGACCAGCCTTT TTGCCCTCCCACCTCCTCTCTGGCCCACTCGGTGTCCACCTGCCTTCAGCTCCAGGCTGC TGTGTGGGTCCCTTTCCCAGAAATGCAGTTCAGGCCATTGTTGCTGGGGTGGTGGTGTTG CTGGGGTGTTGATGTTTAGCGTGCCTGAGCTCCCTTCATCACTAGGATGACCTTGAGCAA GTCACTTCCCTCTGAGCTCCCTGTTCCCAACCTGTGACACACAGGTGACAGGTGGTGTGC ACTCAGAAACTCCTTCCCTAGGATGTGTGACATTCTTGTCCTGGGGCCTTTGCTAGCTGT GACACACCGGCGCCCGACCTCTCTGGCTGCCTCCCTAATGCCAGTTGTGTCTTCAGATGA GTCTTTGGGGGGCTGCTTCTCATCCCAGGTGCTGGACTTGGACTGTCCACCACTGACACC TGGGCACAGGGCCTGGCTTTCTGCTCTTGTGCTCTTGGAAATGAAAGGGTGCCCCTCGTT TGTTTTTCTTCCCACTAGAAATCTCCCTGTCTCCCATCCTGTAGGCTCTGCTGGCCACCA GCTGCCCTGACCTGGGGCAGCTGAAGAACTGAGATAGACCTAAGTTCTGGGTGGGTGCTT GGAGGCCCCACGGGCGTGGCTGGTGAGATTGCCGCTTCAGGGCCAAAGTGGGTGCCGGGC TTCCTGAGGTTCGAGAGCAGATGAGAACTCTTGAGTGGCAATGAGGGCTGTGTGGTGGCC TTCTCTGCTTGGCAGCCTCTGCCCAGCCTGCAGCTGCCTTGCTCCTCAGAGAAGCGCTGA GCTTTAGGCCACAGAAAAGAGCTGACCATCTTCTAGAAGGGACAGCTGCTCAGGCACAGG CGCCTCTCTGGAAGAGGCAGTCAGGTAGAGGCAAGGGGAGGGGCCCTGACCTTCAAGCAG GAAGAGAGGTGAGCTTGTGAGACAGCTTTCTGGGGTGCAGGTAGAGAGTAGCAGCAAGTC AAGCATGGGTCAGAAAAGGCAGAGTTGGGCTGAGCATGGGGCCCAGAGTCTTTGCTTCCC ACTCCTCGGTGCCGTCTGTGGTTCTCTGTATGGAATTTTCTGGCCTTGGGATTTTGTTGA CACCTGGATAAGTCTAGAACATGTGTTCCGGTGTCTGGGACATGCCCTTACTCCTCTTCA GCTAGGGCTCTGTGCACCCCAGCACCGTGAATGCACAGGACTTTGGTGGGAGAAATCTCT GTACTTGTTCAACTCAGCATTTAGCAAATGAAGTCACATCAGTCCCCGTTTCTGACCTTG CTAACCTTTGCGAGAATCACCGCGAGCCATTTTGCTCCATTAGGTAGAGTGATGAGGCCC GGCAGGCCGAGGCTGGGGCTCCTGCACTCTGTCCCTCCTCTCCCAGGCAGAGGTGTCTTG ACCACAAAGCAGTGGGAACAGTCCATTCCATCGGTTGATGTCCCACTGGGCCAGTGGCTT TGGATAGGGACCTCTGACCCAGCTGTGCTGGAAGTACCCTGTTGTGGTCACGCTCTGTGC CTTAGCCCCCCACCCCCTGGTCTCCCAGCACACGTGGTTCAGGAACGGCCTGCACCCAGC ATCCCTTGCAGCACTGTCAGGCACAGCCCATGGAACAGGCAGGACAGAAAGAGCATTTCG CTCCAGGAGCTTGGTCCACGTGGTGCTGAAAGGGCAGTGCCACCCTGCCCTCTGCTGCCT GGTGGGGTGGTGAGGAGGGCAGCATTCTGTTTGTGGGCTCTGGGCCTGGTCCCCAATTTA AGGAGCTTAGAAAGCAGCTGTCCTATCCCAGTACTCACAGCACTGTGGTGGAAGTGCTTG GGGGGTGGTGTGAGCCGTGGCCCGAGACAGGCGTTCATGGCCAGAGTGCCCCAGGAGAAG CACTGAGTTGGTCTACAGAAATGGATGGTCCAGGGGCCTGGCTCCTAGGCAGACTGTGAG GGGATGCTTGGCATGCAGGGGGTTGACTAGGGTGTGGGAAATGTAGCAGGGCCTTCAGGG TTCCCCCAGTGTGACAAGGCATCCCTCTTTGCTGAGAGTCCCTCTAGGGGAGGCCAGACG CCCAGGGCTGGCAGGCAGCTGTTCTCCCCATCAAAGCAGCCATCTGTGCTCCACCCCAGG AAGGTGTGGGTGTTTTACATATGCTTATCCCTGATGCCTGGGTGGTGTGGTTTCATCCCT AGAGTTTACGGATGAAGACAGTGGTGTCCCTGGTGCAGACACTGGGGTGATTGATCTTAT TTGTGTCGTGGCCAGCAAGGCCTGGGGAGCAGAGCAACAGCCTTGCTTTGCATACACACA GGGCCTCAGAGGACTCAGGTGCACACCTGCATGGCAGAACCAGGCTCTGGGCCCAGGTGT CTGACACTGAGTAGCTCGCCTCTCCCAGGGGCGCTGGTGTGGGCCGATGCAAGTGCCACC ATGGGTGTGTGTGTGGCTCCCTGGGCCACCTCACTCCAATTAAGCAGGTCCAGGTGGGAC AGCCTGGGACCAGCCCTGCAGCAGCATGTGTTAACCACAACAGATGGCTGTAGCCACCAG GCTTGTCCTTGGTGCAGTCAGCCTGGGGGCTGCGGGGGCTTGTGGAAGCATGTGGGAAGA CGGTCCCACTGGTCAGCAGAGCAGGGTCCTCACCGACCCACAGAGGAGCATGTCACAGGA CAGGCAGGCGTGTCAGGGAAGGCCAGGCTAGTGCCAGCCAAGGGCATTGCCTCCCAGACT GCCCTTTTCCTCACACTCCCACGACACAGCCCTCAAAGCCTGTGCCCCTGTGGGTCCCTG GCCAAGGAGCAAGGGCTGCCCTGCCTGGGCCCCCCCCGCCAGCCCCTTGGTCACCTCATG CTGTGTGCCAGGCTGGGGGAGTGTGCCTGTGTGTATGTCCAGGTGGCATTTGGATGAAGA CACAGGTGTGTGGTGAGGCTGTGCGGGAGGATGGGCCTATGCGTGCGGTGGGCAGGTGGT GTGGAGGCAGGCGGGCGGGCGTGTGGCAGCGCGCCGGCTGAGCCCCTCCACTTCCAGGTC AGCCAGAGCAGCCTCCCCATGCTGTCCTCGCCGTCACCGGGCCAGCAGGTGCAGACCCCG CAGTCGATGCCCCCTCCCCCCCAGCCGTCCCCGCAGCCCGGCCAGCCCAGCTCACAGCCC AACTCCAACGTCAGGTAGGCCTGGCCTGGGGTGCCCCTCCCCACCTGGCCCTCGAGGCTG GCCCTGCCTGGACCCCGGCTCACATGTTTCTCTCACTTGGCTGCAGCTCTGGCCCTGCCC CATCTCCCAGTAGCTTCCTGCCCAGCCCCTCACCGCAGCCCTCCCAGAGCCCAGTGACGG CGCGGACCCCACAGAACTTCAGTGTCCCCTCACCTGGACCTTTAAACACACCTGGTAAGT TGGGCCTGAGGTGCTAAGGTCACTCCTCACCTTTATGAGGCCTCAGCTCATACTGGGTGT GCGAGCTCTGGGGCCCTCAGAGCTCAAGTTCCCCACCCGAGGGTCGAGGGCTGTGGCCTC ACCCGCCTGTGTCCTGCAGTGAACCCCAGCTCTGTCATGAGCCCAGCTGGCTCCAGCCAG GCTGAGGAGCAGCAGTACCTGGACAAGCTGAAGCAGCTGTCGAAGTACATCGAGCCCCTG CGCCGCATGATCAACAAGATCGACAAGAACGAAGGTAGGCTGCAGCCAGGGCAGGGGCCT GCACCCTGGGGACACCACCAGGCTTGTGTCTTAGTGTGTACCCTCTTCTGTCCCAGACAG AAAAAAGGACCTGAGTAAGATGAAGAGCCTTCTGGACATTCTGACAGACCCCTCGAAGCG GTGAGCTTTGCCCACAGCCCACGGAGGGTCCACAAGGGCACAGATAGCCCAGCCATGGAT GGGCACTTGGTGATGATGTGGGTTTAACAAAGGCACCAGGCAGCTCTTTGGACCCTGGCC AGAGGCCTCCAAGGCTCCACTCTGGTGTGTGCTGGGGCTTCAAGCCCAGGCTTCATCTTG GCCCATGCCCAGCCTTTGCCCTATTCCTGGCTGCTGCTGTGGCCCTCATGCTGGGCCATC ACAGCCAGACCTCATCCAGTCAGCAGCCAGGGGCCCAGCTTGCAGTGGCCTGACCATCAG CTGGCCCAGATGGGCCTGAGCCTGACCTGGAGTTCTGCCCCTGACTTGCTGGTGACCATG GGCAGGCGACTGCATCTCAGTTACCCCATTTGTAGCCCACGTGTGTTGTCATGAAAATGA AACAAGGTGGCGCTGGTGAGAAGCAGGTGGAAGGCAGGGCTGCTGGCCACAGGCTGCTGT GAGGATCAGAGCTCGGGCAGTACCTCCAAACTGCATGGGCATTGGCATATACCTCCCTTT CACCTCAACTCAGGCTATCCCCCAACCCCTGTCTAGAAGGGTCTCTCTGTTCTGCTTGTC TGGATAGAATGCCAGTCACTATTGGGTGGTCCTCCAGGTCTTCATGGGTTGATCCTTTGT TAGGGTTATAGGGATGAGTGGGGCAGGGACTGGCCTACATGACAATGAGGCATTCACAGC CTGATCAGGGGCCTGGCCACTGTCCCTTCTCTTGACCCATCACCTCACTCTGCCAACCAG GGACAAGCAGCTCCTGGGGTTGAGGGCTGCCTGCCTTCAACTCTGGGTCTGGGAACAGCT GGTCAACTGGTAGGAGCTCCTGCAGAGGTTTCTGATGGCTGAGGCCCAGTGGCAGTAGTT GGGATCCTGAGCCTGAGAACTGCCATGTCTTCCACTCTTTCAGCCCAAGTCCTCTCTCTC TGCAGGTGTCCCCTGAAGACCTTGCAAAAGTGTGAGATCGCCCTGGAGAAACTCAAGAAT GACATGGCGGTGGTGAGTGGGATGCCAGACACCCCTAGGGGAACCAGGGCTCTCCTAAGA GCTCCTGGGAGTGCTGCTGAGAGGGCCTTCAAGGTCAGGGCATCTGGGCGGGGGCCGGGC CTGACCTTGGACCCTGCCCACGAGGCTTCCTGGCCAGGTCTGCTGTGCTGGGTGGGAACA TGGGAGAAGTCACCCTCTGTCTACGGCCCCCGTGGGTGCCTCCTTCACCCATTTCAGAGG AGGCAGGGACTGTAGGGAGGATAGGCTGTGGGGGTGGGGGATTATTCCCAGGATCAGCCA ACATTGTCTGCACAAGGGTGGAGGCTGTGAGAGAGGCCTCGGGAATCTGACTGTGAGTGA CCATGGGCCTGGGGTGTGAAGGCCCCCTAAATGGGGAACCCTCGGGTCCCGGGCTGCTGA CCGTGCCCATCCTGTCTCCAGCCCACTCCCCCACCGCCCCCGGTGCCACCGACCAAACAG CAGTACCTATGCCAGCCGCTCCTGGATGCCGTCCTGGCCAACATCCGCTCACCTGTCTTC AACCATTCCCTGTACCGCACATTCGTTCCAGCCATGACCGCCATTCACGGCCCACCCATC ACGTATGTCCAGCTGGGCTGGGCTTTGCGGAGGGCGGCCAGCCCTGGGCCGCGTGTGCCA GGTGTGGTCACCATGCCGCCTCCCCAGGGCCCCAGTGGTGTGCACCCGGAAGCGCAGGCT TGAGGATGATGAGCGGCAGAGCATCCCCAGTGTGCTCCAGGGTGAGGTGGCCAGGCTGGA CCCCAAGTTCCTGGTAAACCTGGACCCTTCTCACTGCAGCAACAATGGCACTGTCCACCT GATCTGCAAGCTGGGTGAGTGTCCAGAGGGCCGGGACTGGTGTGGGAAAGCAGGCCCTGA CCGCAGCCCCAGGACTCTGCCATCCAAGCCAGATGGCACAGCGCCCAGAACCCACCCTGT GTTCACGCCCCGCCAGGCTGTCTGCTCTATCCTCACACACACCGGGCTCCAGACAGCCTG GCCATGCCTCAGTCCCCACTGCCAGAGCCCGCACACTCTCACCTCCCCAACACAGATGTG GCTTTGCTTGCCTGGGCCCTAGGGAGGTGGTAGGCAGGACCTCTGGGCACCCATCAATGC AGAGCACTCCAGGCTTCACGTTTGGAAATCTGAGAGTCAGAGAGACCTCCTCCAGGCTTT GCCACAAGCTTCACCAGAACCTCCCTGGGTGTGGAGTCCTGTTCCAGAGCAGGGCTGTGA GGCAGGGCAGGCCGGGGCTTGTCCAGGTCACAGATAGAGCCTTCTGTGTTGCAGATGACA AGGACCTCCCAAGTGTGCCACCACTGGAGCTCAGTGTGCCCGCTGACTATCCTGCCCAAA GCCCGCTGTGGATAGACCGGCAGTGGCAGTACGGTAGGTAGACCCAGAGGAGCTGTCTGG GGACCCAGGGCAAGCAGGGGTCATTGTGGAAAACCAGGCTTCCACTGCAGCAGGGACAGC CTCTGTGTGCTCCTGCCCCTCCCCAGCTCAGGCCCCTCCCTCCCCTCTTCTGGCTCCTCC TGCACAGACAGCAGCGGGTTTTCCAGTGGTCGCCTGAGAGAAAAGGGTCTCTGCTCCCAC TGTGTCCCTTCGCCCCTTCAACTTGTGTCACGTCAGCTGTCCAGGGTGGTTGTGTTCAGT GGGTGAGGGGTGAGTAAGCCCATGGCTCAGGGACAGTCCCCGCTCTTCCCAGAGGCCACT AGCTTCCCAGAGGCCACTACCTTGGCAGGGCTGCCAGGTTCCACTGGTAGCTTGGGCACA CAGGGCCCAATCTGGGTGCTGTGGCTCCAGAGAGAACCACGGTGGCAGGGGGCAGTCTCT GAGCCCAGAGCTGGCACACAGCAGAGCTGGCACACAGCAGAGCTGGCACACAGTAGAGCC AGGCTGGGTCCCCAAGGTTGTTAGAGGCAGAGCCCTAGATCCTGTTCAGATTTGGTTCTC ACTGACGATGAGGCTCTGGGAGGAGAGCCCAAAGGCCGGGCAGCGTGCAGGACACATCAC CTCCTGGTGCTTCGGCCCGCGCCTGGGGCTACCCCTTCCCAGAGCACTGCCGGGTGTGCC AGGAGCGAGCGCAGCGCCGGCCGCCTTAGGTTCACGCCCACTGCTCTGTTGCAGACGCCA ACCCCTTCCTCCAGTCGGTGCACCGCTGCATGACCTCCAGGCTGCTGCAGCTCCCGGACA AGCACTCGGTCACCGCCTTGCTCAACACCTGGGCCCAGAGCGTCCACCAGGCCTGCCTCT CAGCCGCCTAGCCAAGACTGCAGGGATGGCCCGCAGCCTCATCGGGGCCAAGGACACACG CCTCCTGTCAGACACTTCTAGGTGTTGGCTTCCTTAGAGAGCCTGGGGTTAGGTTAGCTT TCCTGCTTTTATCTTCTGCCTTGGGGACCTGCCAAACGAAATCCCACACCTGTACAGAAC TGGGATAGGCGCAGTGGAGCGGGTTGCTTGGGGGGCGTTGGCCGACTTCTTAGAGAAGGC CCTCCATGTGACTTCCTCCCAGGAGCCAGATGCGATCCTCAGGCTGCTCTCACCGTGGCC TGTCCACGGTCCAGGTCCATCTCAGCAGCGTGAGGGTGCACTCAGGGTGTTGTTAGAGCG TCTCGTGTGTGCTAGACGCACCCCTACTCGTTCCTATAGAACACAGAGGACATAGGAAAC CCTTAAAACACACATGGGATTCTCTGGTCACAGTTTTGGGTTCAGGCTATGCTGCTTTGG GCAGGTGGAGCACCCCCCGAGGAAGCCTGCAAGTCCAGGGCACAGGCTGCCTTTTGGAGG GAGGGCTGGCCCATAGGTGCTGCTGGCTCCCCGCCACCAGCTGGGCCTCAGCCCTCACGG CATTCCTGCTGAGCACCGTGGGGCACCCAGGGAGCAGGGGCGTCAGGGATCCTGCTGCCG GCACCCCTGTGCCGCTGGCATGAGGGCCGTGTCCCCACTGTGAAGGATGAAGAGCAAGGC CCTCAGGACCCGTGTCCTCAGAGCACCACACACTGAGCACCCAGAGACAGCGGGCCTGGC AGCGGGCCGGGCCATGCAGGGAGCGCCTCCCTATGTTGCCTGCCACTCTGGGCACCGGCC AGCACCCTCTGGTGAGAAGAGGTCCCCCCTTTTTATGTGCACTACCCCACCATCTGTGAT TATAATAAATTTATTATTCCTGTGTTTGTCAGTTGTTCATCACTGTGTCCCCTTCTCCAT GAATCTGGGCTCGCTGTCCTGTCAGGATGCTGTCAGCCTTGGGGCTGCCTGGGGCCTCTG CCTCCCTTCCAGGAGGGACTTGAGGTGCTGCAGCTGAAAGGCAGGGGCAGGGGTGTGGTG GGGCCTGCGGTGGCACCAGGCTGCCCAGGGCCATCCCCACCACCCAGGGCCTCACCTCTT GGGCCTGCAGAAGGTTGGTATTCCACAGCTGGTGGATGACCACTTACACTGCTGAAGTGT GGTGGGCTTTCGCAGGTGTGGGGACAGGATCATCGGGGAGTGCCATCATCTGCCTGTCCC TGTGCTGGCTGCCCACCCAACAGAAAGCCCTGGCGTTAGAGGCCTCTGCTTCCCTGTCTC TGAAAGTCATTACAGTAGCCAGAAATGACCTGCCCAGGTGGGTCTGAGCATCCAGGCAGC ATACTTCCCCCAGGCAGTGCCCAGGCCAGTGTGGGGAACAGGGCCTGGCTCCCTTCTCCC ACTGACCAGAGTGCTCTGGGTGCCAGATGGGGGTCTCGGACTGGGGTCTGTCCCTTCCTC CCCTCTGAAGGTGCCCTGAGGGTCAGATGTGCTTCAGTGGGTGACCAGCTAGCTGCCAGG CTGCCCTTAGTCAGGACCCTATACCCAGTGGGGCTTCTTGCTTCAGGGCTGGTCTCAGGG GACGTGCAGAGATGCAGGAGCCTGTCCTGCTGGTTGCACCGCTCAAAATGTGCACTCTGC AATGTCTCAGGGCAGCCCGGCAACTCGGGGAGTACCAGAGCCTGGTCCTGCTCTCCTAGT CCCCTCCAAAGGCAGGCTGTGGCCAGGGGCAGCAGGCACTGACCAGCTCCATTCTAATCA GGCCCAGGAACACAACTCACTTTTCTTGGTAAAAAGATTACTTTGTTTTCTTTTCTCTGA GAAAGTGGTTTAGGGGCTGTTGCTGCCCCCTCTTGTGGGCTCAGGTCAGCACAGGTAATC TGCAGGACTCCAGGGGAGGGGAGGAAGACAGCGCCCACCCAATGGACAGAGTGTGGACAG AGCGTGGCCATTGTCTGCTTTCATGAGCCCTGGAGACAGGATCCTAGAGCAAGGGACACA GCAGTTTGCTGACTGCAGTGTGGGAGCCTAGTAAATGTGGTCCTGGGGCCCTTAGGGTGC AGGCAAACCTTACTTGGACTGCCCTTGCACCCCAGGAACTTTGTCCAGCTTGCTGTTGTG AAGCAGTGGCTCCTCCTCAAGGTCTGCCTTCTGGGGTACCTTAGAGTACCCCAGTGCTTT AGAGTCTTGTTTCTTGAAGGAACCGGCTGGGGCCTGTCCTTTCCTTGAGAGTTGGCTGCA GGTCAGACCCGACAGGGCCAGTCACTGCAGGCACCAACAGCCGCTGCCCAAGTCTTTCAG GATCAGCTGTGTGTGCCCAAGAAGCCCCTGCCTCAGTTGCCTCCTCTGTAAAGGGAAGCC TGCCTTGGAGGGTTCTAGGAGGAATAAATGAGGTTCTGAGGATGAAGCCCATGGGTGGTG CCTGTGAACATTCTAGAACAGTCAATATAGTGGAAGCTGGCTCTGTTTTGGGTAAAATAA AACTGCATGCGAGGCCTGGGACTAAGCCTGCATGGGTTTCCTCTGCAAGGACAACGCCCA TGGCACGCCTGGGCTCATCACAGGAACAATAGGGGCCAGGGTTGGGGCCAAGGAAGATGC CTGGTCTCGAGCCACTCCCCGCTCACTCTGCAGAAGATGCCATGAGCCCAGGGCTCCGTG GGCAGTGCCATCCCTCCTGTATCCTGTCTGCTTGTGGAGGGGGACGTGGGCCAAGCAGGG CCTCTGCCAGGCAGCCAGTGTACGTCCTTCCCCCTAGAAGGCCACCCAAGGAGGCCCCTT CTGACCGCTTGCTGTGCTGGCTTGGGGTGCCTGCCATGAGGCCTTGCTGCCCTGCGTTGA CAGCCCCTGTGACTGGCCCCCAGTCCTGCCCACCCCACCCTAGGCCCCTCCTGGTGGTTT CAGCTTACAGGGCTCCCTTCAGCTGCCCGTTCACCCCCAAATCCTTGAGGTTGGCCCAGC AGAGGACTTTCAGCTCCCTGGTCAGCACCTCTAGTGACCACCCAGGCTGGGGTCCCTGTG GGGCACTTGCCCCTGTGAGCACATGGAGGACACCAGCCTGCTCACCTGACACCATTTAAT TCGAGGTGGAGTTGACAGGCTGGAAGTTGCCACCCGCACACCTGCCTGAGGGAGAAGCCC TCCGTCTGCCTGCAGGCCTGTGTCCTCCCTGCTCTACCCACACTCCACTGAGGCAGCCCC AGACCCACCGCGCGCCCAAGGGAGCAGAGGCCCCGGAAAGGTTGTTTCCTGAGGGGCCAT CCAGAAAGCGGGAGGGCTTGGGACCCATGGAGCAGCAGGTCCGGAGGGAGAGCATGGCCT CTTCCCGTGTCCTGTCAGGGTCCCCAGCACTGCAGGGCCACACTTTGCCCAGAACAGCTC ACTGGAGTTGCCACCGCAGCTAGGGAAGGGCCTTCACTCAATCCCATTCTCTGTGTCTCC CTAGAAGGGAGAGCCCTGGACAATGCCCCAGCTGGGATGTTCCCAGGCTGGTCCCATTCA CCAGTCAGATGTCCTCCTGATGTCAGCCCCGGCCCCAAAGGTAATAGAGTGAGCTCAAGA ACTCAACCAGAATCCTGGGCTGGGTGGGGGCCACCATTGGGATGGGGAAGTGCCTTGGAC AAGTGGCAGGGAAGCAGGACTCTGGTTACTCCCCCCACCCCCATCCCCACAGTTGTTGCT GAGATGGCAGGAAGGAATCCCTGGATGGGGTGTGCCCGGAGCCCTTCTCCCAGCGTATCA GGAATCAGAGCCACAAGGCTGGAGATCCTAGGCCCAGCCACAGCAGCAATCTGAGGCAAA GACCCAGCAGGGTAGGGGCTGTCCCAGAGTCCCCTCCCATTTCAGGAGCTCTGCTTGGAG GAGGGAAGGTCTGGGAAGCACCCTGTGCCCACCATCACCACTGCACTGACCACAGCCCTT GGGCTTCCTGAGAGCAGCACGTGCTGTGCTTGGCTGTATAACATCGTCCAGCCATGGCTA GGGGCAGCAGCATCTGTCCAGGTCCCTCAGAACCTGTCCTCACTGGGTTGGCATACAGAG GCCAGTGCAGCCAGGTGGGCAAGGACAGTCTGGAGGGGCTCCCCGGGGTGGCAGGAGCTT CCCATCAGGCACGAAAGGGCCCAAGTCTCACCTTTCTCCTGTGACTCATTCATTATGAGC TTGTAATGGGGATCTGAAATCAAGCGCATGCTCTTCTCATCACTTGTGAAGCCACAGCCA TTCTAAGAGAAGCAGCTTGGGCTGTCCACTACCCCCTGCTGAACCCCGAGAGTCCTGGCT CGCCACTGGCTGACTGGGCCCTGCCCCACAGGTCCCGCTTTCCACCACACCTTTGCTGTG GGCATGTGAGGGCAGGAATGACCCTGCCTGTTCCCTAGGCAGAGCCCACCCAGCACATCA GCCTCATCCTCCCATCTGACTACAAGACGCCCATGGTGCCAGGCAGGACCTGGGCCCCAG GGGTACAGGGGTGAACTCTGGGAGCCCCTGGCCATGGATGAGCATGGTCAGTGTCATGAT CCTGTGGGCATCAGGGACTGGGGAGAGGCTCAAGAAGACATGGGGAAGGGGGATCCTGAC ATAACAGATGCCTGGTGATGGAATACTTAGCACCTAGGTAAGGCAGCACCAGGGAGCGGG GAGGGAGATGAGGCTGCAGGCACTGGTGCCCTCGCTGGAGCCTCTGCTGAAGCAGGGAGG GAGGAGCGGAGCCACATCCTGGCGGGCCCTCTTTCTGCTCTGGTGGGCAGTGGGGGTGAG GAGCTGCTAAGGGTGCAGTTAGGAAGGCCCAGCCCGAGGACAGTGTGGGCACAGGCGAAG CCGGGGGCATGCCTGCTCCTGATCCGGCATGTTTAGTTGGAGAAAATTCACTCAGTTGTG CACCGGCACTGGGCATGTTTTCCTTCTACGAAGTTTTTAACAGATGTGTACCTGACCCTG CCTGGAGAGGGGAGGGCTGGCAGGGTCAGCGTTTGAAATGATGCTGCCCCCGGCGGGACA GGCAGACAAGGAGGGCACCGCCCCGGGCCTAGGCGGGTCTCCGTCCTGGGGCAGGGGAAG AGCTCAGCATGGCAGGACCTAGCGTGGGCTTGCTGCCCTGTGTCCTCCCTGAGCCCAGGC AGGTGCTGGAGTTCATGTGCTGCGTTTCTGCTCTGCGGGCCGCCGCCGTGTTCAGGCGCT TGGGGAGGCAGGGCAGGCCTAGGGCCCTGTGGCTGGGCTCACCCTTGTTCTCCCACTTCA GATGCTCGATCTTCTAGTGGAGCTTGGCCAGCAACTCCAAGGACTGCTGCTGTAGGAACT GCAGGCCCAGGTCCAAGTCCAGGATCTGCCTCTGCTGGTCGATCTGGACACCCACAGACC GGCGGCACTGCGGGGGGTGCGACGTAACCTGAGATCCCGGGGACCAGCTGGGAGGCTAGC TCGCACGGCTCATGTCGCCACCCCCAACTACACAGCCGCTGCCACTGCACCCTGGCTCAG TGGCCATGCTGCCCGTCACAATGGAAAGGGGCGTGGCTGAGGAGCAGGGTGGGCAGCTGG GGCAGGGCCTGTGGCTGGCTCTTCCTGGGTGAGTACAGTCTGATTTAAAAGGCACCCAGG GCTGGGCACGGTGGCTCATGCCTATAATCCCAGCACTTTGGGAGGCCCAGGTGGGCGGAT TACTTGAGGTCAGGAGTTTGAGACCAGCCTGGCTAACATGGTGAAACCCCTTCTCTACTA AAAATACAAAAATTAGCTGAACGTGGTGGCACGTGCCTGTAGTCCAAGCTACTTGGGAGG CTGAGGCCGGAGAATGGCTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCAGAGATCGCGC CACTGCACTCCAGCCTGGGTGACAGAGGGAGACTCTGTCTCAAAAAAGAAAAAATAAAAT AATAAAACGCATTCAGGCATTCTCTACAGCCTGGGCAACATAGTGAGACCCTGTCTCTAT AAAAAGTAAAAGAAAAAAAATTAGCCGGGCTTGGTGCTGTGCTCCTGTAGTCCCAGCTAC TTGGGAGGCTGAGGCAGGAGGACTGCTGGAATCCCGGAGGTCAAGGCCGCAGTGAGCTAT GATGGCACCACTGAACTCCAGGCTAGGTGACACAGTGAGATGCTGCCTAAAAGAAAAAAA AAAAAAGAAATATGTCTTTCTTTACAAGGCCTGCCCTAAAAAAAAAAAAAAAAAAAAATG TTCGCCAGAGCCAGGCCAACTGTGATTTTACCAGATGCCAACATACCCAACCCAAGGGAG GAAGGGAACATACCCAATCCCAGCCTGTTCTAGCTCCTCTTCCCCACATCTTCCTGCCAC ATCAATAGGGCTCCTGTATAATAACAGGGGATACAAGTGAAAGAACAGCCCATCTCAGAA CTTATTTGAGAAATGTCTAGGAAACCCTAAAGACAACAGGGAGACAAAAACTAAGACACC AGAGGAAGTTTATGCCTCTGACACTTACAGTTAGAACAAACAGCACACATGGCCCAACCC CTAACCAGAGCAAAATAAAACCTCACACAGGCCGGGCGCTGTGGCTCACCCCTGTAATCC CAGCACTTTGGGAGGCCGAGGCGGGTGGATCACGAGGTCAGGAGTTCAAGACCACCCTGG CCAAGATGGTGAAACCCCATCTACTAAAAATACAAAAATTAGCGGGCGTGGTGGCAGGCA CCTGTAATCCCAGCTACTTGGGAGGGTGAGGCAGAGAATTGCTTGAAGCCAGGAGGTAGA GCTTGCAGTGAGCGGAGATTGCGGCACCGCACTCCAGCCTGGGTGACAAAGTGAGACTCT GTCTCAAAAAACAAAAACAAACAAACAAAAACTTCACACAGAAAGCCCATTTATCACGTA CCCCGTACAGCATGTCCAGCTCCCCACAAAAAATTTCAAGGCATAATAAAAACCAAAAAA TGCAGTTTGAAGAGACAGCAAGCATCAGAGCCAGACTCAAATATGGCAGAGATGTCGGAA TTATCAGACCAAGAAATGGAATACAACTATGATTAATATGCTAAGGGCTCTAATGGAAAA GTGGACAACATGAGATTACAGACAGAAAATGGAAACAGGGATAGACACTCTGAGAAAGAA TCTAAAGAATATGTGAAGGCCGGGCACAGTGGCTTATGCCTGTAATCCCAGCACTTTGGG AGGCTGAGGCAGCGGATCACTTGAAGTCAGGAGTTCGAAGACCAGCCCAGTTCGAGACCA GATGGCAAAACCCCATCTCTACTAAAAAATACAAAAATTAGCCAGGTGTGGTGGCATGAA CCTGTAATTCCAGCTATTCAGGAGGCTGAGGTGGGAGGACTGCTTGGACCCAGGAGGTGG AGGTTGCAGTGAGCTGAGATTGTGCCACTGCCCTCAAATCTGGGTGACAGAGTGAGACAC CACCAAAAAAAAGAAAAGAAAAGAAAGGAAACAAGGAGAGGAAAGAAAGGAAAGAAAGAA AGAAAGAAAAGAAAGAAAGAAAGAGAAAGAGAAAATAAAGAAAAAGAAAGAAAGAAGGAG AATATGTGAGAAGTCAGCCAGGTAGTGACTCATGACTGTAATCCCAACACTTTGGGAGGC TGAGGTGGGTGGATCACTTGAGGTCAGCAGTTCAAGACCAGCCTGGGCAACATGGTGATA ACCCATCTCTACTAAAAATAAAAAAATTAGCTGGATGTGGTGTCATGCACCTGTAATCCC AGCTACTCAGGAGGCTGAGGCAGGAGAATTGCTTGAACCTGGGAGGTGGAGGTTGCAGTA AGCTGAGATTATGCCACTGCACTCCAGCCTGGGCAGTAAGACTCCGTCTCAAAAAACAAA AAGAAAATATAAGAAGTCAAAAACACAACAGAATGAAAGATCAGGGCTGGCAGCAGTGGC TCATGCCTATAATCCCAGCACTTTGGGAGGCCAAGTTGGGTGGATCACCTGAGGTTAGGA GTTGGAGACCAGCCTGGCCAACATGGAGAAACCCCGTCTCTACTCAAAATACAAAAATTA GCTGGGTGTGGTGGCGCATGCCTGTAATCCCAACTACACAGAAGGATGAGGCAGGAGAAT TGCTTGAACCTGGGAGGTGGAGGATGTAGTGAGCCGAGATCATGCCACTGCACTCCAGCC TGGGTGACAGAAGGAGACTCTGTCTCAAAAAAGAAAGAAAGAAAGGTCAGGTGCATGGCT CATGCCTGTAATCCCAGCACTTTGAGAGGCCAAAGTAGAAGGATCGTTTGAGCCCAGGAG TTGGAGACCAGCCTGGGCAATATAGCCAGACCCTGTCTCTACAAAAAATTAAAAAAATTG CCAGCATGGTGGTGCACATCTGCAGTCCCTGCTACTCAGGAGGCCAAGGCTGCAGTGAGC TGTGATTGTACTACTGCACTCTGGCCTGGGTAACAGAGAGAGACCCTGTCTCAAAAATGG GGTAAAATGGTATAGGAAATTTGGATCTAAAAAAAAAAAAAAGGAAGTGCATTAGATAAG AAATAAATGAAGATAAAATAGAATCTTTTTTTTGGTGGGGGTGGGGAACGGAGTTTCACT CTTGTTGCCCAGGCTGGAGTGCAATGGCACGATCTTGGCTCACCACAACATCCGCCTACT GGGTTCAGGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGATTACAGGCATGTGCCA CCACGTCCAGCTAATTTTGTATTTTTATTAGAGTCGGGGGTTACTCCACGTTGGTCAGGC TGGTCTTGAACTCCTAACCTCAGGCGATCTGCCTGCCTCGGCCTCCCAAAGTGCTGGAAT TACAGGCATGAGCCACTGCACCTGGCCAGAATCTTGTATTGTTCTTATTTTTAGTTGAAC AGATGGCAGGTTATTCAAAATAATTATATCAACATGTATTCAGTAATTATAGCTTATGGA TAGCTGGAATGAATGGCAGCAATGTTATAAGGGATGGGAAGGAGGATATCCTGAGTGGAA AGAAAGTCACCCAGGCTGGAGTGAAGTGGCACAATTATAGCCCACTTGCAGCCTGCAACT CCTGGGCTCAAGCCATCCTTCTGCTTCAGCCTCCCTGGTAACTGGGATTACAGACACACA CCACCATGCCTGGCTAATTTTTAAATTTTTTGTCTCGCTGTGTTGCCCAGGCTGGTCTCA AACTCCTGGGCTCAAGAAATCCTCCTGCCTTAGCCTCCCAAAGTGTTGAGATTACAGGCG TGAGCCACTGTGCCTGGTTTATGGTTGCTTTTGAATGTCCTAGTCCTACATAAGTGGCTT CCAAGAAGGGAAAAAGAAAAATGCAGGCAGGAAAAGGTGCTGATCCATAAAAACCTCTGG AAGGTATGTCTCTAGGGAGGGGGTGAGTGTTGAGGAGGGATGTTGCAAACATGGTAGTGG AGATGGCAAGAGTGGCTGCCTGCCTCTTCGTCTGCACTTCCACAATCTGAAGCAGCCATC ATCAATCGGAACACAGATCCCCAGTATTTGGAGGATGGGGCTCTTATTGCCTGCTGTGGC TCCTGCAAGCTATAGGGAAGCTACCCCAGAAGTATAGGCATGGCTGCTTGCACCACCAAT TTAAATGCTAATTACTTCTGAAAACACCCTCACACACATGCCCAGAAATAATGTGTAACC AGATATGTGTGCATCCAGTGGCCCAGACAAGTTGACATATAGAATTAACCATCACAAGTC CACCCCTTGTCAACCTGGTACCTACACACATCTCCTTAAACCATACTTAACCTCCAAATA AACACAATAACAAAGTCAGTGTTAGCAGTGGAAGGTGTCTGAGTTAGTGGCAGCGAATCC GTATGGGTCTGCAGCAACCTCAATTCTTGCCTCCTCAGAAAAAAGAATTCAACTGGCCGG GCATGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCAGACCACGA GGTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCTCGTCTTTACTAAAAATAC AAAAATTAGCCGGGCATGGTGGTGGGTGCCTGTTGTCCCAGCTACATGAGAGACTGAGGC AGGAGAATGGCAGGAACCCAGGAGGCGGAGCTTGCAGTGAGCCGAGATCGCGCCACTGCA CTCCAGCCTGGGCGACAGAGCAAGACTCCGTCTCAAAAAAAAAAAGAATTCAACTGAGGG GCATAAGGCAGAAAAAGAGACCGAGGCAAGTTTTGGAGGAGGAGTGAAAGTTTATTTAAA AAAGCTTTACAACAGGAAAGAAAGTATTCTTGGAAGAGACCTAAACAGGCACATAAAGGT CAAGTGCGGTGTTTAACCTTGATTCTAGGACTTTATAGACTGACCCCTTTCCCAAGATTC TTCCCCTAGGGTGGGCTGCCTACATGCACAGTGTCCTCCTTACCCTTGGGAGATGAGCAC ACGCAGTGTGTTTAGGAAACTGTACGCATGCCCATCTGAAGATTTCTTCCGTCCCAGAAG GTCATACTCTGCCATTGTCTCTTAATGCACATGGCCGGGGAAATTGCTTCTCCCTGGTGC CTGCATTCAATTAACACTTTAATGAAACAGGTGTGACCCATCAGGAACTGGCCTCTCCCT GATGCCAGCTGCCAATTTATCACTTTCATTTTTAATTTATTTTTTAGAAGGAGTCTCACT CTGTCACCCAGACTGGAGAGCAGTGGCATGATCTCGGCTCACTGTAACCTCTGCCTCCCA GGTTCAAGTGATTCTCCTGCCTCAGCCTCCCGAGTGGCTGGGATTAGAGGCATGCACCAA CATGCCTGGCTAATTTTTGTATTTTTAGTAGAGATGGGCTTTCACCATGTTGGCCAGGCT GGTCTCGAACCCCTGATGTCAAGTGACCCACCCACCTTGACCTCACAAAGTGCTAGGATT ACAGGCATGAGACACCATGCCCAGCCCTGGCTTTTTTTTTTTTTTTTTTTTTTTTTGAGA CGGAGTCTCGCTCTGTGGCCCAGGCTGTAGTGCAGTGGCATGATCTCGCCTCGCTGCAAG CTCCGCCTCCTGGGTTCACGCCATTCTCCTGCCTCAGCCTCCAGAGCAGCTGGGACCACA GGCGCCCACCACCACGCCCAGCTAATTTTTTGTATTTTTAGTAGAGACGGGATTTCACTG TGTTAGCCAGGATGGTCTCGATCTGACCTCATGATCCGCCCGCCTTGGCCTCCCAAAGTG CTGGGATTACAGGCGTGAGCCACTGTGCCCAGCCGCTAATTTTTTATTTTTTATTGTTTG TAGAGAGGGGGTCTCCCTATGTTGCCTAGGCTGTTTTGTTTTTTTTTTTAAACAGAGACA GGGTCTTGCTATGTTGCCCAGGCTGGTCTTGAACTCCTGGGCTCAAGCAGTCCTCCCATC TTGGTTTTCCAAAGTGCTAGGATTACAGGCATGAACCACCAGGCCCGGTCTCCACTGTGA ATCTTTTTTTTTTTTTTTTGAGACTGAGTTTCACTCTTGTTGCCCAGGCTGGAGTGCAGT GGCACCATCTCGGCTCACCGCAACCTCCGCCTCCTGGGTTCAAGCAATTCTGCTGCCTCA GCCTCCTGAGTAGCTCAGATTACAGGCATGCACCACTACGCCCAGCTAATTTTTTGTATT TTTAATAGATACAGGTTTTCACCATGCTACCCAGGATAGTCTTGATCTCCTGACCTCATG ATCCACCTGCCTCAGCCTCCCAAAGTGCTGGGATTACAGATGTGAGCCACCGCACCCGGC CGCTAATGTTTTATTTTTTATTGTTTGTAGAGACAGGGTCTCCCCATGTTGCCTAGGCTG TTTTTTGTTTTGTTTTGTTTTGTTTAACAGAGACAGGATCTTGCTATGTTGCCAAGGCTG GTCTTGAACTCCTGGGTTCAAGCAGTCCTCCCATCTTGGTCTCCCACCCAAAATGCTAGG ATAGGCGTGAACCGCCAGGCCCGGTCTCCATTGTGAATCTTTTTTTTTTCTTTTTTTTTT TTTGAGACTGTGTTTTGCTCTTGTTGCCCAGGCGGGAGTACAGTGGTGCGATCTCGGCTC ACCGCAACCTCCGTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTTCCTGAGT AGCTGGGATTACAGGCATGCGCCACCATGCCTGGCTAATTTTGTATTTTTAGTAGAGATG GGGTTTCCCCATGTTGGTCAAGCTGATCACGAACTCCCGACCTCAGATGATCCACCCGCC TCGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCTACCGCGCCCGGTTTACCATTTTG AATCTTAAGAGGACAAAGTCTGGTTCTCTAGAAGGCCCGAGTAGCTTTTCCCCTGAGCAT CTTGAGAAAAAAGTGCTCTGAGCACTCCTTGGGAAGTCCGAGACAGCACATGCAGCCAGG TGCTCCCTGGCTGCTCTCCCAGAGGTCAGCACAGCTGGAGGCAGCTGGAGGGAGGAGGCT GCAGAGGCGCTGAAGCCAGGAAGACCCAACAGTGGAGAGAGAAGTTGCCTGGTGACTGAC CCTCGGCTTGACCCTGTGCTCCCCTGGGCTGAGCTGGGTGTCAAAAGGCCTCCACCCTCC AAAGTGCCAGCCTCCTGCTGTGGCTGGAAAGCATTTGCTCCTCCTCCTCTTCTCACTTCT TTGCATTCTTCTGATCTCTTAAGGGCGTACAAAGATTCACATGGATATTAAAGTCAATGG TGATTTTCATAGCTGGAATACTCGTTGGGAGAAAGTAGGAATTCTTTTTTTTTTTTTTTT CTCCCAAGCTCAAGCGATCCTCCCACCTCAGCCTTCTGAGTAGCTGGGACCACAGGTGTG AGCCTCCATGATGGCCTGGCTAATTTTTTATATTTTTAGTAGAGATGGGGTTTCACCATG TTGCTCAGGCTGGTCTCAAACTCCTGAGCTCAGGCGATCCACCTGCCTCGGCTTCCCAAA GTGCTGGGATTACAGGCGTGAGCCACTGTGCCTGGCCAAAAGTAGAAATTCTTGATTGGG AAATAATTTTATATATTTCGGCCCCTTCTCACTTGGGAAAATTCTGCTAAGAATTCTGGT TTTGAGAGTCAAGGGAAGCTAGCTTAATTTTTAAAGTGAAGGCTACATAAGAGTCCTCTC TCCAGTGTGGTGGCTCACACCTATAAACCCAGCGCTTCGGGCAAATCACTTGAGCCCAGG AGTTCAAGACCAGCCTGGCAATGTAGTGAGACCCCTATCTCTACAAAAAAATTTAAAAAT TAGCAGGGTGTGGTGGTGCGTGCCTGTAGTCCTGGCTACTTGGGTGGGATGCTGATGTGG GAGGATTGCTTGAGCCCAGGATTTGGAGGTTGCAGCGAGCCATGATGGTGCATTGCACTC CAGCCTGGGCAACATGGCAAGACCTCGTCTCAAAAAAATACAAAGAGTCCTCTCTATGTT GGAAATGTCAGAGTTATTGGCCATTTAGGGGATCTAACTTGCTCTTTGATGTCCAAGAGG AAAGGTATAGAGTTCTCCTGATTACACAGGGCCTCTGCTTGTGGGTCGCCTGCATGAGGC GATCAGAATCTGAGTCACCTGGGATCATGTGGGTGCTGGTACATCCTAGAGATAGGATGT GTTGGTCATGGAAGAGAGTGGGCCACAAATGGGTTCCAGGGGAATAGCGTTGCCTGGTGC AAGGAGGGGGATGTGGAGGTGCAGGGAGCAGGGGTGGTGTCTGGAGAGCCTGAAGCTGTG CTGCCAGAGGCTCCAGGCCATGAGGTCTGAGAAGCATCTAGATTTGGCCTGTTCACAGCA GGGAAGTGTGGTCCAGTGGCAAAAAGTTGAGGGCATCCTAGGAGTTGAGGAGGTGGTGGC GGTTAGAGTAAACCACTCTGGAGGCGTCCGGGGTGGAGAGACGAGAATGTGCAAGAACTC CTGCCTCTAGCCTGGCCTCCTCAGGACCCCGGCCACCAGCCTCCCTCCTAGAAGTCCCCA GAGCTTCCTCCTCCTTGTGCCCCAGAACTGGTTCCTGGGTTGCTCCAACCCCAGACCTTC CCTGGAAGTCCCCCCCAGTGCCTCCTGGCTGGGAACCCCCAACTGCATTTTGCTTCTGCT CTCGTGCCAGTAGTCCCCTTTTCTCTCCAGTGTCCAGGCCCCAACCACCAAGACCCTGCC TCCTCACTTGGCTTTGCTGTTCTTAGTTCTCTACTCAGTAGGTTGTGTCTGCCTCCATTC CTGGCCAGACGCTGCCCTGGGGCTGAGTGCACAGTCCCCAGCCACTCCCGAGATCCTCAG AGCTTCTGGAAGCCACAGACATGGTGGCATATGTTTCAGGTCGTCAGCTTGTTTTTTTTT GGAGACAGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCGGTGACGTGATCATTCACTGCA GCCTTGACCTTCTGAGCCCGGGTGATCCTCCCACCTCAGCCTGTCAAGTAGCTGAGACCA CATGTGTGAGCCACCACACCTAGCTTTTTGAATCTTTTTTTTTTTTTTTTTTGAGACGGA GTCTCACTCTGTCACCCAGGCTGGAGTGCAGTGGCACAATCTTGGCTTACTGCAACCTCC AGCTCCCAGGTTCAAGTGATTCTCCTGCCTCTCCCGAGTAGCTGGGATTACAGGTAGGTG GCACCACACCTAGCTAATTTTTATGTCTTTAGTAGAGACAAAGTTTCACCATGTTGGCCA GGCTGATCTTGAACTCCTGACCTCAGGTGATCTGCCTGTCTTGACCTCCCAAAGTGCTGG GATTACAGGCGTGGGCCACGCGCCCAGCCAACTTTTTGAATCTTTTGTAGGGATGGGGTC TTGCTGTGTTGCCCAGGCTGGGCTTAAACTCCTGGACTCAAGTGATCCTCCCTGCCTTGG TTTCTCAAACTTCTGGGATTACAGGCCAATCTACTCAACGTTGATATTGAGTAGAAGGCA AAATTCACATGGCTCCGGCCCAGCCCCTTTCTGTCTCAGGCACTTCAGTGTTAATATGCG CCTTGGTTTTGCTGCTGCCGAGTCTACTGTGCCCCTGGGCAAGGAAAGCAAAGGAAACCA GAGGGCCAGGCCCGTGGACACTGGGGTGAGGGGCCAACAGGGTGCCCAGCAGTTGTTTCC ACAGCCCCCACGTGGTCAGGGACCCTGTATGGGTGAACCGCCTGGATACTGAGCCTAGGC GGCGGAAAACTCAAAAGCAAGGGCAGGTTTATTCAGATCCCTGGTGGAGTTACAAGCTTG GTTTGGTTTTTGGTCACAGGCCCTAAAATACCTGTATTCTGAGTTTACATAAAAATACAA AGCACTGGCCGGGCGCGCTGGCTCACACCTGTAAGCCCAGCACTTTGGGAGGCTGAGGTG GGTGGCTAACATGAGGTCAGGAATTTGAAACCAGCCTGACTAACATGGTGAAACCCCATC TCTACTAAAAATACAAAAATTAGCTGGGCATGGTGGCGCATGCCTGTAATCCCAGCTATT CGGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGAGGCGGAGGTTGCAGTGAGCCATG ATCGCACCGCCGCACTGCAGCCTGGGCTACAGAGCAAGACTCTGTCTCAAAAAAAAAAAA AAAAAAATCACCACCGAGACGCTGTTCTCCCTGGTGTGTCATCTGTGGGGTGAATGAAGG TGTGAGATACCCCGTGGTGGTTTCTGTCAGTGAATCTGTAGTTGTCATATATTTTGAATA CTTCAATATCATCGAACAGTTGCTGTGTCTTTAAGCACTAGATTTTATTGAAGCTACCTT CTTCCCTTTTTAGTCCCATTTCCCCTAGATGCATCCACGACTGCAGGAACCCCCCTGCCC CCACCAGGAGCCCAAAGATAACCATGTTCCCACCTGGAGTGGCCTCCCACCTCCAATGCC ATCAGGGCTGGTGCAGCTTCCCCACCTGGTCAGCAACATCATGGCCAAGGAGTAGAGGGC GTAGCCCAGGGTGAACAGCAGTGCACAGGTGAGGGGCCTGATGCAGAACAGAGAGGCCAT GAAGAAAGCCAGCAGTAGCCAGGGCAGTGTCAGGTAGCTGCCTAGGAAGTGCAGGTTGAG CCTGTGGCCGCTGGCCAGCGTGGGCTCTGTGAGGTGGTAGCAACAGAAGCTGGCAAGGAA GTCATCTCAGGAACACAGAGCCGCCATCTGGTCTGCAAACTGAAGCGCAGGCAGTGGCAC GGGGGCATGGGGTGGTGGCACCACAGGCCTCAGGACAGCTGCACCCCACCCCCACAGGCC ACCCTGAGACTGCACCCCAGGCAGGAGCACCCAGAACCCTCTCTGAAGAGGCCAGGCCAG CCCTCCTGCCCACTCACTCTGTGTTTGATGGCAGATGACAGCCGGAAGGATGCTCAGACC AAGCTGGTAATCTCATAACTCCAGAGGGGCTGCAGCTCCGGGTCCCCCTGGTACTCAATT TCAAACCTTTGCAGCCTGTTGATGATCTAGAAACCCAGGCTATAGGGTTGGGCCAGAAAA CATGGCCCTGGCCAGGTGCAGTGGCTCATGCCTGTAATCCCAGCACTCCGGGAGGCCGAG GCGGGCGGATCACAAGGTCGGGAGTTAGAGACCAGCCTGATCGATATGATGAAACCCCGT CTCTATTAAAAATACAAAAATTGGCCAGGTGCAGTGGCTCACACCTGTAATCCCAGCACT CTGGGAGGCTGAGGCAGGTGGATCACGAGGTCGGGAGATCAGGACCATCCTGGCTAACAC TGCGAAACCCCGTCTCTACTAAAAAAAATACAAAAAAAAATTAGCTGGGCTTGGTGGCGG GCACCTGTAGTTCCAGCTACTCAGGAGGCTGAGGCAGGAGAATAGCGTGAACCCAGGAGG TGGAGCTTGCAGTGAGCCGAGATTGCGCCACTGCACTCCAGCCTGGGCTACAGAGCAAGA CTCCGTCTCAAAAAAAAAAAAAAGTTAGCCAGGCATGGTGGCATGTGCCTGTAATCCCAG CTATTCAGGAGGCTGAGGTGGGAGAATCGCTTGAACCCAGGAGGTGGAAGTTTCAGTGAG CCAAGATCATGCCACTGCACTCCAGCCTGGGCAAGAGAGGGAAACCTTGTTTCAAAAAAA AGAAAGAAGAGAAAAGGAAACATGGCCCCTGCACACAACTCAGAAGAGCCAAGCCATCTC TGCTCACAGGGGAGGAGGGAGCTGGGGACACGTCAGGGCCCAACCCATGCACCTCTGGGA CTTGGGCCTCTCACCTGGTACTGGCCCAGGGGCGTGGGGATGAGTCTGTCCTCACCCGCG ATGCAGTCCGGAGCTGCTTCTTTCCATTCTCATCCTGGATGGTGTCCAAGGCAAGTGTGA GAGACTCTGCGTGAGCTGCACTTCACTGAGCTACCAGAGAGAGAGAAGCTCCTCTGCCCT CAGTGTCAAGGCAAAGGAACACCAGTCTTCACCCCACCAGCCTCTAACAGTGGCAACGAG AGCACCCGGACATGACACTCGGGGTGTGGGCCTGCCTCCCACAACACCGGGTTCATCTGC ATGGGGCCTGGGACGGATGCATGCTGACCATGGGCAAAGGGGGAGTGGATCCTTCGGGGA AGGGGACCCTAACGGCCGTGGAACAGGCCCCATTGCTCATACTCGGAATATCTGGCGCAG GTACGCCAGGGCCTCCTCCAGGCACTCGTCCATCTTCTGGACGCTGTCCTGCCCCATCTC GCCCAGGTCATGGCTGAATAGGAGCCGTTGGTGTCTGTGGGGCCAAAGCCCAGCCACAAC AGGAAGGAGTGGCCAGCTGGAGTCTCCACACACTGGTCCGAGATGGACCTGGCCGTGTGC TTGGCCTGCGTGATGAGCTGAGTAAGGTGCAGGACCTGCAAGGGAGGCGAGGGCAGGTCA CCGGCAGGATACGCCTGTACTGCCACCCAGGCAGGCTGGCTGGGGGCCCTAAGATGGGTG CACAGCCAGATCACTGAGTGACGGGAGGAGCAGCTTCTGCTGGAGGACTTTTAAAGACTA AAGAATGATCTTCTAAATGTGAAATTTCCCAACACTGCCTTCGTTATCAGAAACAAATCA GACTTTACTGAAACGGGGAAAGGAGATGTGCGTCAACTCTCCGCCCAACCTCCCTCCCTC CCGCCAGAACACAGCACTTCCCTGCCTCTGCTCAGCCTTGTGGCTCCAGGGGTCCTGTGA AGGGGCACCGCCAACCCACTCACCAGGGTGTAAGCCTCGGGCCCGAACATCGGGTGTACT TGCAGTCCTGGCCCTCCAGGCCATAGATGTGACTCTTCACATTGAAGGAGGCATCAGTGA CCACTGGTGGCCACTATGACAGGATGCTGCTGGCGAATGTGCAGGCTGTGAAGAGTCGGT GCTGGCGGTAGGGGGATGACGAGCTCCGGCTCCGGGAACAGCTGCTCGAACTGGAAGGCA GGAGACGAACCTAGCGCCCACTTCCGGGGGCACAGAGAACCTGGTTGCTGCCCACAGGTT TTCAGGATGCCTCCGAGGTATCTGGGGAGCCATGACCCAGGGGTGTCCTCCTGAGGTCCT GCCCAGCAGTGTCTCCCATGTAGCAGGTGGCCGACATGGGGAGGGCAAATGAGGGAGCTG GTGGCTCACAGAGCCCCCGACAGTGTCTGCAGGAGGGCAAACACCAGGAGGAGTGACAGA AGCCAATGTCCAACCCAGCCAAGACAAACGCATGCCAGAGACCAAGACCGCCTCCCAAGG ATGCTGCCACGAGTTGCCAGGGAGAAACTGTGGTGCCATGGGCCCGCAGCTTGGTCTGTG CAGACCCCCTCTGTGGGTATACCTGCGGTCTTCCACCCAGTGCCTGGTGCCTGGTGGCTG CAGGGCAGGCAGGGTCCAGCCGGTCTCAGCCTTAACTGTGCAGTGTTATGGATGACAGCT CAGGAGACTGTTGGCAGGCAGGGAGGGCAGGACACAGGCAGGTGAGACCCAGGCCCTCTG AGGTTGCTGCATCCTTAGCAGGCCTGAGGCCCCTTTCCAAGAGGACTGAAGCCACCATGG CAGAGGCCAGCCCAGAGCCTGAGGGGGCTGAGGGAGGATCCCCCAGAGGGTCCAAAGGGG AAGGCCTCGGGCAAGGAAAGGAGAGGTCAGTGGCCACGCCAGGTGCCATGGAAGCCTGGG GGGGTCTGCTGTGAGGATCAAGCAGGAGATGAGGGGAAGGTGGCCACTGGTCTGCTCTGT GCTGGTAAGGAAGGGTTAAGGAACAGTTTGGGGGTCACCATGAAAATTTGTCTGGGCCAG AAGGGAGAGGGGACAGGTCACCCACTGCTTCCATGAGAAACCCATTCTAGGGCCTGGCGA AGTTGAGCCTAGGAAGGGCTGGTCTGTGCTCCACTCAGCAGCAGCTGGGAGAGGGCCAGG GCCAGGCTGACCCCAGCACAGGCCGCTGTCTTGGCCTCCACCCACTGGAGCAACATTTGC CCAGGGGAGCCAGTGGCATGTGAGGAGAGGGGGCCAGGGCTCACCATTTCTCAGACACAC TGGGGCTGGGAGTTGCTGCTGGCAGCCTGCTTGTCAGCTGCATACCACCATGGCTGCAGG TAGCTCAGCCACACCTCCAGGACCTGTGGGGGAGGTGTGTGCTGAAGGCCCAGTGGCTGG GGCCAAATCCTCTGATGGCCTGGAACCCAGGTACCCAGGTGGCAGCTAAGTTGGCTCTTC CCACGTCCCCCAGAGGGCGCAGGAGAGACGCGCACAGCCCTGTGAGATTTGGTTCTCTCA CTGCTCTTCAGAAGCCAGGGTCATGGGCGGGCTCTTCCCGGCCCACGGGACCCAGTGCTA CCTGGTTGCGTAAGAGTAGAAACCTGTCTGGCACCAGACAGGTGGGATGGGGCAGGGCTT TGCTGTGCCTGAGGGTTGCCTTTGGAAACTGAAACAGAAAGAACAACGTGGCCCTCCAAG GCCCCCTCCTCTCCAAGGTCTCTGTGACAACACTTGTTCTCCTTCATCCACCCCAGGGGA CCTCCCTCATTGGGGCACACAGAAGACACAGGCACACATGTGATGGGGCCGACACTCCTA GCTCTGAACAATGCGTCCAAGGGCCAGTGGCCAAAGCAATGCCGCAGGAAGAGGTAGAGT TTCTGCTGGACTAACCTCGGGACAGCGGCCTGCAGAGGAGGGGAGGGTCACCAGCCGCTT GCACAGTAAGGTGGCCCTGCTCACAGATGAGCCCGGGGTTCCCATTAGTGAGGGTTGGCT CTGCTGGGAGAGAGCACTAGGCACTTCTGAGTGGTAAGAGCTGGTTTCAGCCCCATTCTT CAGAGGAAGAAACCGAGGTTAGGGAGCATCCCGAGGACACAGCATGCAGGCGGCCAAGCC ATTGCATCTCCTGCACACACCTCACCTCAGCCCCACACAAAACAGCCCACTCTGCCAGCC CCGATGGGCCTGGGAGTCAGGACCCGCCCCAACCCTTCTCCTGACGGGTGGGGAGGAGCA AGAGACGGCACCGTGATGGGGCCTTTTTGGCCAGTCTGGAGAGAGAGGCTCTGGGAGCCA GAGATCAGCCGGCAGCCCCACCAGGCTAGCTGAGAAGGCACTTGCGTAGCCCTCCAGAGG CCTCACGTGCTCTGTGAGCAGGAAGGGATTCCCCTCACCCTGCCCCCCATGCCCCGGGGA GAGGTCGCCGTGGGCAACACCGGAGGAGGGGAACGAGGAGGAGACAGCAGCGGCTGGAAG CCGCAAGGGCCACCCACTGTTTGAACTCCCCCAGCGGGCTGGTGGTGTGGGAGTGGACGA AAGGTGAGACCTGCTCTGGCTTCAGGCTGTCGGCAAAGGCGTGCAGGTGCTTCAGTGGCA GGCGCACCACCAGCATGTGCCCCTTGGTGGGCATGAACGACTCCTAGGTGGAGGAAGAGG AGTCAGGGCCTGGGAAGGGCTGCAGCACGTGGCCTCCAGGTCTGACCACAGCCCGCCCTG GGACAGCTGATCTGCCTCCACACAGGAGGCACGAGCCAGGCAAAGAGTGGGGCCACCACT GTGCCTCACACCCAGCGGGTGTGCCTTGCACTGCTTGGTCTGAACTGCGACCAGCTCGTG GTAGGGAAGTGCTTGGCCCAGCGCCCACACAGCCTACCTGGGGAGAACTCGCTGAGACAG ACGCATCTGCTTCTGCACCGCTGCACACCCGCGGTGGGTGGCACAGGGCTATGGCCCGCC AGACTGTGCCCTTGGCCATTGCTGCCTATCTTGATCCTCATGGCAGGAGGAGGCCGATGT CACCCCCAAGCTCTGGATCCAGGGTCTCCCAGGGAAGATGGCTAAGGCCAGGAATTCGAG ATTCGCTTGGGCAACATAGGGAGGCCCTGTCTCTACAAAAATTTTAAAAAGTTAGCTGGG CATGGTGGTGTGCACCTGTAGTCCTAGTGACTCAGGAGGCTAGGGTGGGAAGATCGCTTG AGCCCAGGTGTTCTTGGTGCAGTGAGCTAGGATCGTGCCACTGCACTCCAGCCTGGGTGA CAGAGTAAGACCCTGTTCAAAAAATAAGAACCTTCACTTTTCTCCCCAATTCCTTCCATC TGCTCCTCATTCTGGACACAGCTGGATGAGGGCTCACCCTGGGAGAAGCGCTGGGCATCA ATTCCTCTGTTGCCCACAGACCTGTGGTCAGGAAAACCCCAAGACAAGAGCACAGTCAAG CTAACAGGCCAAGCTTTTGATTTACAATGTACAAGCAGGATCCTGGAGACCTACACCCAA GAGGAAAGCCTTTTTGCTGGCTGTGGGCTTTCTCTGGTTGCTCAGCATTGAGAGGGCAGG GGGGAGCATCAAGAGTGGATGAGGGCAGGAGAAGGGGGCACTGAGGGTGGGCCCCTGAGT CCGAGTGGACATGCTCGCCTCTCCATGCTTACCCCCAGCCCTTTGGGCCTCAACACATCT GTGATGCTGAGACTGGTGCAGGGCCCGGCCTCTGGGTATATCCTGGGATGCTGCAGAACC CACATGGACCACTGCTCACATCACTGCCCACTCAAGTCTGCCCGTCAGGAAAATGTCCCC ACAGGTCCAGGAGACACACCCCACAGATCAGTGGCTGAGGCTGGGAATGGAGGTATTTTC TTCTTCTTCTTTTTCTTTTTTTTTTTTTTTTTGAGACAGGGTCTCACTCTGTCACCCAGG CTGGAGTGCAGCAGCACAATGTCGGTTCACTGTAACCTCTGCCTCCTGGGCTCAAGGGAT CCTCCCACCAAAGCCTCCCGAGTAGCGGAGACCAGAGGCACATACCAACATACCCAGATA ATTTTTCTATTTTTTTGTAGAGGCAGGGGTCTCATCATGTTTCCCAGGCTGGTCTCAAAC TCCTGAGCTCAAGCAATCTGCCTTCCTTGGCCTCCCAAAGTTCTGGGATTCCAAGTTTGA GCCACCGTACCTGGCTATTTTCTTCTATCTCTATCTTTAAGTATTTAGGCAATGAATTTG CATTGCTTTGGAAGGAAGGAGAAAACCAGTACCTATACGGCCCGACTAGAATGGTACTAG TGGGGTGGGAAGTTAGACCAGGGTCCTTCTCCTCCAGTCTCACCCCTGGCCCTCCCTCTG ATCCTGGCCTGGGATCCTGAGCCATGACATCCTCCCTGCAGCTCACGGGAGGGGACAGCA TCCACATAGGAACTGGGAACCTGCCTTCCCCTGCAGGTTCACTTACCCAACATCACCTGG TCCTTCCAGAGGGTCTGGAGGGGCCAGTGTCTGCCCCTGCCCCCAGCAGTCACATGAAAG CCTCTCAGGCTATTTTCCCACAGATTTGCTGAGCCAAACTTTTCCCTCAGCTCTGTACCC CTCCTGGGCCACATTCCCATGTGTAGTCCCTGGCCACAGTCACACCTGCTTGCTTCTCAA AGATGTGTACCCAAAGCCCCGCATTTCCCACTGGGCCCAGCCTCACAGCACCTCAGCTCA CTCCTCCAGGGTCTCCCTGGTCACCTGTCCTGCCATGGGGGTTGCTAGATGCTTCCTGGA CACTGCCCTGGCCCAAGCCCTCATGTCCCTCTTCTCCAGTGGCTCCTGCGGAACAGGGAC GCCATCACATGCAGTGGTTGCCATCCCAACCCACCGGCACTTGGGGCCCACATGTTTTTT TCATCTTGCGCTCAAATTCCCATTCAATCACGTGTGCTCCTTCGCAGGGTGAACGGGACA GACCCCTGGCTGTGGGCACAGCTCGTCTCCCCGGGCTCTCACAGACTCACTAACTCTGTC GTCCCCGAAATCCCCCATGGGCCTGAACTCTGCTTCACTGACCTCAAATCCATCCATACG CTTGTCATTTGTGCCTAGGCAGACCACAACCTCTATGAGGTCACTACCTATTCTCATCCA CCTCCACAGGTGTCTACGAGTGGCCAGGGCTCCCTAGGTGAAGGACTGACAGCCTCTGTG AGCTATAGGGCCAAGCCCCAGGTCCTCCCCAACCCCCTTGCCTCACACACAGTCTGGGTC ATGGAACCTCCAAGCTGGATGACAGTTCTGAACAGGGCAGCACCCACATTACCGTAAGTC CCAGGCCTCTGTGTCTTCAGCTCCTTCCTTAGCACTGGGCCCTGCAGAGTGGGGGTGAAC CGGGCCACTGCAGAGGCCTCACGACTCCAGCTCTTCAAACCAGGGCCGAGCACAGAGCCT CCAACACCAGAGAACTTGTGACACCTCCACCACTGGTGGGGAGTCTGGCCAGCCCAGGCC ACATGCCTCATTCTGACACACAGCCACACCCCACTCCCACAAGCCAGGCTAGTCTGCAAA GCCAGGGGCCAGGCCAGGCTGGAGGCTGATCTCTGCCTCCCTCAACACAGGGGCTTCACT CTGCCTGAAACTAGCCTCTGAGTCACCTGCAGCTGGGCCCAGCCAGCCCCTTGCTTTGCC AGGCGCAGACACGCACTCCGGGCAGGGGAAGGGAATGAACGAGCACAGCCAGTACTTGGT AGGTGTGGAGGGCCTGGAGGTTGGGCTGCCGGGGGCTGTGGTGGGCACTGGAGACACAGA GTCGGTAGTGCAGCACCTCCAGCTGAGAGAGTGAACAGAGAGAAGCCGGAAGAGGCAGAA GAGAAAAGCATGAGAGGACAGGGTGGTGAAGGAGAGACATGGTACCGGACATGGGAGGGG GAAGTGGAGAAAGAAAGCAATGAGAACGAGCCCAGAAGTGAGGAGAAAAAAACAACAAAA GCATGGTCAGTGACCATCCAAACAACTGCAGTCCCAGCAGCTGTGACCAGCGCAGCACCC TCCTCGGCAGCCAGGCCAGAGCTGTGACAGCACTCTCCTCAGCCTTGGAGGCTGGGGGCG GATAGACCCTCCAGGTCACCTGGGTGGCCTCTCAATGTCGCTGGGGCCTAAAGGCCTCTC CAGCTCATTGGAAGGGCCCAACTCACTGCCCTTCCAGATGGGACCCGCTCTCCCCAGTGA CTCTGAGGCCACACCGTGTGTGCCATGTGCACACCACAGCCCTTCAAGTACCCAGGGCCT TCTCTCTGGGACATGCTGTCAGCCACCAAGGCCTCAAGATCCTGAGGCCCAGAAAATAAA CTGGCAAAGCATGTTCTAGGAGGAGGACTCTGCCTAGAGAAGACACATATGTAATTGACT GGATTTCCCTGGGTTGTTTTGAGAAATTTATCAAATTTGTATATGACATGATCAATTCCT ATGTCTAAAACCCAATCAATGTCCCCTCCATGAGTGGATCTCTCTGAGAGCTGAGGATGA GGACAGGTCCCCAAGGGTAGCACAGGCTCAGGCCCCCAGGCCCTGCCCTCCGCACTCCCC CAGCCTGGAGGTCAGCCGAGGTGGTGTGTGCATCAGCTCTGACTCCAGGAGGCAACCTCT CCTACCGCATCAGGAACTCCCACCCTCCAGATATGGAGGGCTCCCAAGGGCAGGAGCCCC CGAGGACCTGGCCACAGCCACACACACCCAGAGGCAACAGGAAACTGAGGCTAAGCTGGC CCCCAGCCAGGACCCTCCAATCTACCACCCTGCAGCGCTGCAGCCTCCGAAGCACCCCTT CGGCCCAGCATAGCCAGAGCTGGCCTGGTGCATATCCTGCCCCGTGAGCGCCATGCCTCC TGCCCAAGTAGCACGACCACTGGGCTCCACACAGGAGACATCAGAACACACCTGCTAGAT GTCACAACACGCAGAGCAGGGCGGGAAGGAGCAGGGCAGAGAGATGTTGCCCTTCACAGC CTTCCTCTGTGCCAGACCTAGGGCTCAGGCAGGAAACAGACACACAGTTCTCCAGTGCGT CTCTGCCCTTGCCTAAGGAACACGGTGGCTTGTGGGGCCATTGGCCATCTTTATGGCCCA CGGGGCCACCAGGGAGCAAGCATGCACCTCGGATTCAACCAGGGTGTGGAGGGCAACCCT GGGAGCCTCACTTCCTGCATCTGAAAATAAGAGCTAGCAGAGACAGGCAGGGATTTCTGT ATGGCAGGAGCAGCATAGGCCTGGGCAAGCGGCAAAGGGCCAGGGGCCTTGGGGGCCCCG ACCTCCACAGCTGCCTTTCTCTGACCCCCACCCACCCAGTTTTCTCACGTTTTGTTTTTT GCCTCAACTGGAAGAGGCAAAAAGTGGAGCATCTCTGGGCACTTCGTCCCACCACTGCAA GTCATTCTGAGCAGGACTCACGCAACCCAAGGTCTGGCCAAGAGGAAGCGAACATTGAGA AGAGGGGAAAGGCACCTCCTGCCCTCTGCAGGGACGACGCCTTGCCAGTTCCCCTGACCC ATGAGGACACAGAATCCTCACAGGTGACAGCCCAGAAGAGCCTTTCTGGCTTGCCCTTGA AAGTAGGTGTGTTTCCACTCACTCAGTAGGCAGAACCAGCAGATAGGAAGGTGACACAGT GCAGCGGCCCCTGCCCCTGCCAGGTCAAGCCACATGCAAGAGCGCTGAGCAAGTGTTCTA GGGCGCCAGGGAGAAGCGGGGCCCTAGGAACCAACCAGCCCCCCAGGCACCAGCCAGGCA TATGCAGTAAGTCTGCTCTGGAAGCGGGAAGGGCACTGCAGGCTCTTTGTGACTCAGGAG GCCCAAGATGGGGTCCCCAACTTTTCCTTGTGCTCCTTCCAGGTGATGCTGAGGGTGAGG ATTTGGGGGAACCTATGCTCTGAGAGAGCCTTCACATGGCAAAGCATGGGGCCACAGAAC TGGCTGCAGAGGCTGTGGCAGAAACAGACCCACAGCTCTTCTCCAGAACCGGAGGAAAAG TGACAGGAGGCTCTTCTGAAGTTTAACTCCATCACCCACAGATCCTTCATTCTGGATGAA GCCTTCTGGAACTTTGTCAGCAATAAAGGCAAAGTCTCCCATGCATAGACGCCACGGGCA CCGGACTACAGGATGTGTGCCCAGGGCATGCTGCCTAGAGAGAGAGTGGGGAGAGGCGAG GCGGAGGGGGAGGAAGCAGGCCAGGCTGCTTGTAGGAGGGCGCCAGGCCCCACACACCAT GAGTATGGCGGCTTGCTCATGCTCTGGAGGAGGTGGGGGAGTGGGGGGCAGACAAAGGAG GCTAACTATGGTGCCACTATGGTGGGCATGGGTCTGCCCTGGGCCATCACTTCATGGGAA AGCACAAAGGCACTGCTGATGGTGGAGGAGACAGACGAGAACGCGGCTCCCAGAGGCATT GCAGCTCCCCCACCCCCACAGCAAAAGTGCCACAACCTCATCTGTTCCTGTTGCCAGGGA GGGGGCTCCGGAAGCTGCACAGGAAAGGCCCTGGAGAGGCCAGACCTCCAGGCCTCAGTG TCCACATATCAGGGTGCAATGCCACCTGCTCCTCATCGGGAACTAGAGAACTATGCCCAC TTCTTATTCAGCTTCTGAATGAAGGGTGGGTGGTGGATTTCTGCTTCCCACCATCCGGTC TGTGAAAACTCACTCAAATGCAGGATAAGAAGCAGCAGGCTGAGCATGGTCAGAAGCAGG AGGTGGGAAGAAAAACTATGGAAACCTACCTTGGCATGAGGGGAACTGCATTTTTTGGTA CATCTCCAGAGAATAGTGTTGAAGCCACATTTCGACAAAAACCTGCAAAAGAGCATTACG TAGTCAAATCATTCTCGATAACTGCCAAGACCATTCAGTGGGGAAAGGACAGTGTCTCCC ATAAGCAGAGCTGGGAACACAGAACACCCTCATGCAGAACAGTGAAGCTGGGCCCTGCCC TCACACCATCGACAAAAACTAACTCAAAGTGGGTAAGCGGCCTGAGGATAAGAACTAAGA CCATAAACTTACAGAAGAAAACATGGGAGAAAGCTTCATAACACTGGATTTGGCAATGAT TTATTAGATATGATGCCCAAATCACAAGCAACAAGCAAAAAAATACAGCATCAAAAGATA CTACTGATAGAGTGAAAAGAACCAGCGGGCATGATGGCTCAGGCCTGTCATCCCAGTGCT TTGGGAGGCCGAGGCAGGTGGATGGCTTGAGGCCGGGAGTTCAAGACCAGCCTGGGCAAC ATAGCAAAAAGTTGTCTCCACAAAAAGCACAAAAATTTGCTGGGTGTGGTGGGGCACAGC CATAGTCCCAGCTACTGGGAGTCTGAGGTGGGAGGATCGCCTGAGCCCAGGAATTTGAGC TGTAGTGAGCCAAGATCATGCCACTGCACTCCAGCCTGGGAAACAGAGCGAGACCCTGTC TCAAAATAAATAAATAAATAAATAATAAAATAAAAATAGAAGCAACTCCAGTGTTCACTG AGAGATGAATAGATAAACACAACATGGTACATCCGCACAAGGGAACACTATGCAGCCTTA AAAAGGAAGCAAATTCTGACACATGCTGTAACATGGATTAATCTCGAATCCATTATGCTA AGTGAAGCATGCCAGTCACACACAAAGAAGTACTGTGTGCTACAGAAAAATGCTTTAGAT ATGAGATCTAGGAGTCAAACATATAGAAACAGAAAGCAGAATAGTGGTTGCTAGGGGCTG CGTGGGGAATTAATTGTTCAGTTTCTTTTTTTTTATTAAAAGAGATGGGGAGCCAGGCAT GGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGTGCATCATGAAGTC AGAAATTCAAGACCAGCCTGGCCAAGATGGTGAAACCCCGTCTTTACTAAAAATATAAAA ATTAGCTAGGCACCGTGGCAGGTGCCTGTAATCCCAGCTACTCAAGAGGCTGAGGCAGGA GAATCACTTGAACCCGGGAGGCAGGGATTGCAGTGAGCTGAGATCGCACCACTGCACTCC AGCCTAAGCGACAGAGTGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAGA GAGAGAGAGACGGGGTGTCACTATGTTGGCGAGGCTGGTCTCAAACTCCTGGCCTCAAGC AATCTTTCCCACCTTGGTCTCCCAAAGTGCTAGGATTACAGGAGTGAGCTCTCATGCCTG GTCGGTTCAGTTTCAGTTTTGCAAGATTCTGGAGATTGGAAAGGAAGCAAATTCTGACAC ATGCTGTAACACGGATTAATCTTGAATCCATTATGCTAACTGAAGCATGCCAGTCACACA CAAAGAAGTACTGTGTGCTACAGATAACAACAACGTGAATGTATTTAATACTACTGGCTG TACACTTGGAAATGGTTAAGACAGTAAATTTTATGTTATGTATATTTTACAATTACAGTT TTTTTCTTAATTAAAAAAACGAAAAGCATTAATGCTTTGGTCCAGGAAACAGCAGAACCT TCCAGTTCTGTCCTGTTGTAAGCTTGTGTCATGGTCTCCTGCCCTCTTCACAGCCAGCAC ATCCCATGGTCCACACAGCCAAGAGCCATCTGGGTATCATGGTCCCCTCTGGGGACCATG CCCCTCCCAGGGACAATGGTCCCTATCATTCTAGCACCCGAGGTTGTTGTGAAATGGTGT GGAGGCTTCAAAAAGCACGGCACCCCACATATTAGGAGTTATGTACAAGGTCGGAGACAG AATCCTATCTTATCTACTTCCCAGGTGTCAACGGCGAGGCTGTGGCTACCAAGTCAGTCC TAGGATCCAGTGTCAGGAGCAGCACCTGCCAAATCTCTCCAAGTTATGCTCAGAAGACAC AGCCAGGACCTGGTGGATAGCACTCAGGGAGTGAGCCAGTCCACACCATCGTGAGAGGCT GGAGCCCCCCAGGACAGACAAGGGACTCTGTCACCTACTATTTGGATCCCCTGAATCAAC AGAAGGCCTGTGGAGGCTGCACAAACCCCTCACCATTCCGTGTCACAAAAGTGCTACTGG GGCAACGCTTAATATCCACATGATGGTAATCATAGGAAGCATCTCCAGCAACACGAGAGA CGGACACCAAGAGACCGCAACAATTATATCACAATGGGCAAATGAAACGACTCCGATGGA CAGGGGATCCTGGGATGAAAAACACCATGAATGACAAGTAAGTTAGGTGGGTGGGCAGAC TTATATTCATGTTTTCTAAATGTCATACCAGGAAAATGCCAACAATTTAGAATCAGCCCT GGGCTAAGCGGCCGCCTCTGGGACTCTGACCCATATAGTGGAAGATGGCTGACCAGTTTC TTTTGAAACAGACGTTGAGACCAGCTCAGCTCTCACCCGGAGCAGAGTTTCTGACCTCCA GATCTCCTGGGAGGCGGGGTCTGCATTCACAGACATCTGATGAGAGAAGTGTCGCTTCAG GGGGCTAGTGTGCTGGAGGCTACAGGAAGCAAAGGGCATGGCTGGTGTCCTGAGGGAGAC ACGGAAACAGGCCCAAGGTAGGAGCTCCTGCTGGCCAGGCCAGGGGCGATCTGGACATCA AAATCAATGACAGCAGGGACGTATTACAACTTGTTCAATTGCACAATGCACAAACCTCAC CCCTGCCCCAAATCCACACTGATACACCTGAATAAACAAATAGCAAAGAGAAAAGTCTTC CTTCCCGCAGAGCGACCACTGACCCAATACAGGAAGAATGAAGTTAGGGAACCACCACTT GGCAACTGCCCTATTAATAGCTTATTTGGTCAAGAATCAAGTGAGGCCAGGCATGGGACC TGCGTGAGACGCCTGAAATCTCAGCACTTTGGGAAACCAAGGCAGGAGGATCTCTTGAGG CCAGGCACTTGAGACCAGCCTGGGCAACACAGTGAGACCCCGTCTCTACAAAAACATTAA AACATGAAATAGCTGGGCACGGTGGGGTGTGCCTGTAGTCCTAACTACTTAGAAGGCTGA GAAGGGAAAATGGCTCCCTCCTTGGGCCTAGGAGTTGAGGCTGCAGTGACCTATGATGGC ACCAACTGCACTCCAGCCTGGGGTGACAGAGCGAGACTCTGTTTCTAAAAAAAATAAGAT GTCCAGCTTGGGCAACATGGGCAAAACCCATCTCTATTAAAAAAAAAAAAAAGCATTCAG ATGTGGTGATGCATGCCTGTAGTCCCAGATACTTGGGAGGCTGAGGCAGGAGGATCACTT GAGCCTAAGAGATTGAGGCTGCCGTGAGCCTAGATCATGCCACTGTACTTCAGCCTGGGG TGAGAGAACAAGATCCTGTCTTAAAGTAAAATAAAATCAATTTGAAAAAAGGAAAGAATC GACTTCTCTGTGTATGGAGTAGCCCTTCTTCTATTCCTTTACTTTCTTAATAAACTTGCT TTCACTTAAAAAAAAAGAGAAAAGAATCAACAGTTGATGTTAATACTAGTGATAATACAT AGTCTCTATACATCTTCCCGGTAAATCATCATTACCAATTTTTCTTTTTTTTGAGACAGG GTCTCATTCTGTTGCCTGGGCTGGAGTGCAGTGCTGTGATCACAGCTCACTGCAACCTCA ACCTCCTAGACCCGGGCCTCCCAAGTAGCTGGGACCACAGTGCACACCACCATGCCTGCA TGCCTGGCTAATGTTTTTCTTTCTTTGTTTTTTGTTTTTGTTTTTTTTTTGAGACAGACT CTCACTCTGTTACCAGGCTGGAGTGCAGTGGCTCCATCTCGGCTTACTGCAATCTCTGCC TCCCAAGTTCAAGCGATTCTCCTGCCTCAGCCTCGTTAGTAGCTGGGACTACAGGCATGC ACCACCACGCCTGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTGTCACCATGTTTCT CAGCCTGGTCTTGAACTCCTGACCTCATGATCTGCCCGCCTCTGCCTCCTAAAGTGCTGG GATTACAGGCGTGCACCACCACACCCAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTT TCACCGTGTTGACCAGGATGGTCTCGATCTCCTGACATCGTGATATGCCTGCCTTGGCCT CCCAAAGTGCTGGGATTACAGGCATGAGCCATTGTGCCTGGCCAATATTTTTCTTTTTAG TACAAACAAGGTCTTGCTATGTTGTCCAGGCACTCATTACTAATTTTTTTTTTTTTGAGA TGGGCTCTCACTCTGTAGCCCAGGCTGGAGTGCAACAGCATGATCTTGGCTCAATGCAAC CTCTGCCTCCTGGGCTTGAGTGATCCCCCCACCTCAGCCTCTAAGTAGCTGGGATTACAG GCGCATGCCACCACGCCAGGCTAATTTTAAAATTTTTGGTAGAGATAGGATTTTGCTATG TTGCCCAGGCTGGTCTCAAACTCCTGAGCTCAGGTGATCCACCCACCTTGGCCTCCCTAA GTGCTGGGATTCCAGGCGTGAACCACTGCACTCAGCCTCATTACTATTTAATCTGGGCAC GAAGTACTTATTTTACAGTGGACAAACCTGGCCGACACCCTTTCCACCCAGTGATAAAGC TAGCATCACCAGTGATGGGGCAAAGGGGCACTGCAGGCCTCCTGATGAGACACCTGGCAA GGACCAGGTGTCCCCGCTGCAGTGCTCCTGCCAACAAAGCCCAAGCTATGCCTAATCACC AGCAAATACAAGACAGCCCAAGGGGTGGCCACATGCTTTGGAGGGGTCAAGGTCAGGAAA GACAGACAGGCTGCAGAACCATTCCGGACTGAAGGGGACTAGAAACATCACTGCATGCAG TGGGTGAGCCTGTGCTGAAACCCAGCCAGGTAACGGGCATGAGTGGGGTAGCTGGTGGTG AACCCCGAGGTCTGATGACTAGGTGTAGCCTGGTGGCAATACTAACTTCCTGGTGTGGTG GCATGTGCTGGCTTGCACATGCCAGCAGATCTGGGGTGGGCCAGCATCTGCAGAAACGGG CCTGTATGCAGCCTCCTGGGGAAGGCAATGGGGCAGCAAGATTAAGGAGGGTCAGAGGAG CACACGCACTGTGACTATATAAGATAATGCATTCACTTTTAGGAAATACACTGACATATT AAGAGGCCAAGGGGTGTCCTACCTGTAGTTTATTCTTATTGGCTTAGGGGAGAAAAAACA TATCATAGAAAGGGGATAAAGCAGATGCAGCAAAGTGTTAATTTGGAAATCTGGATGAAG GGTCTATGAACTTTTTTTTTTTTTCTGAAACAGGGTCTTCTTGTGTCACCCCAGGCTAGA GTGCAGTGGCGCAATCATAGCTAACTATAGCCTTGAAGAGTGCACAAGAGATCCTCCCAC CTCAGTCTCCTGAGTAGCTTGGTCCACAGTTGCACGTCACCATACTCAGCTGATTTTATT TTTTGTAGAAATGAGGTCTCCCTATGTTGCCCACACTGGTCTCAAATTCCTGGCCTCAAG TGATCTTTCTGCCTTGGCCTCCCTAAGTGTTGGGATTACAGGAGTAAGCCACCATGCCCA GTCTGCAAGTTCTTTTTATTATGCGTGGAAACTTTTCTGTAAGTCTGAAGTTATTTCAAA ATAAAAAGTTAGCTAGGTGTGGTGGCCTGCGCCTGTAATCCCAGCTACTTGGGAAGCTGA GGCAGGTTCACCCTCCCAATCCGTGCAGTATGGCATCAGGCTCAGCCTGTTTGGTCTGGC ACAACAGGCTCTTTGGCCCAGGGCCTCCCCCTCTCAAGACCTGCTCCTGGGGCCAATAAA GGATTGTGGAAGCTAGGAGTTCAAGACCCACCAGGGCAACACAGCAAGCCTCCCCTGTCA TATCTCTACAAAAAAAAAATGTTTTAAAATTATCTGGCTGTGGTGATGCGTGCCTGTAGT CACTGAGGTGAGAGGATGACTTGAGCCCAGGAATTTAAGGCTGCAGTGAGTGACGACTGT GCCGCTGCACTCCAGCCTGTGTGACAGAGTAAGACCCTGTCTTTAAAAAGACATAATGAC TGAATGTGGTGGCTCATGCCTGCAATCCCTGCACCTTGGGAGGCTGAGGCAGGAGGATCA TTTGAGCCCAGGAGTTCTAGACCAGCCTGGGCAACATAGCGAGACCCATCTTACAAAAAA TACAAGCTGGATGTGGTGTTGCACACCTGTAATCCCAGCTACTCAGGAGACTGAGACAGG AACATCACTTGAACCTGGGAGGTGGAGGTTGCAGTGAGCTGTGATCACAATACTGCCCTC CAGCCTGGGCAATTGAGCAAGACTCTGTTTCCAATATATATATGTGTGTGTATATATACA CATATATATATTATACACACACATATATATAACATAAATGTGTATATATACACACATTAT ATATATATAATTTCTAAAAGAAAAAATTAAATTACAAAAAAACTCAAAAGCTGGGCATAA AAACCACTTGGCAACTGAATTGTACATCTGAGAGGGTGTGAATCCACCTCACCTCCAAGT CCTCCACAGCATCAGCAACATTTCAGGGTCACTGACCACTGCCGCCACCCAGGCAGCCTC TCCTCCTTGCTGCTCCCTTTCTCCCCTCCACCCTCTGGGGCATCTGTCCACACATGTGAA TTGTAGGTAGAGGCCTGTCCTGGCTTGCACATGCCAGCAGATCTGGGGTGGGCCAGCATC TGCAGGAAGGGGCCTGCACGCAGCCTCCCAGGGAAGGCGATGGGGCAGCAAGATTAAGGA GGGTCAGAGGAGCACAGGCAGCTGCTGCCTGTGCAGAGGGTCTCCTGGGAGGTCCCCAGA GGGTCTCCTGGGAAGTCAGCCAAGAGGCCCTGGGTTAGGGCCCACTGAGACCCCAGCACA GCTAGGCACAAGAGCCCCTGGTTCACACTTCCCACCTGTGCAGTCACCAACAGGCTCAGC TTGTTTGGACTGACACACAGGCTCTTTGGCCCAGGGCCTCCCCTTCAAGACCTGGTCCTG GGGCCCAGAGAGATTTCCATTTTTTTTTCCCCCATAGAACAAGCCTTCTGCCATGCTCAC CACCTCAGACACTGCATGTGGGGAGGGCTGCTTCATGCAGAACACAGCCCATTCCCATGC AGGCCGTGGAGGCCTCCAGAGACCTGATAGCTTCAGTGATGGCCACAGACATCGAAAACT AAAGGACAGATCACCCTCCTCACCCCCACCCTCCAGTCTGTGCTAAGAAAGCTGGAGGTG GGGAATGAGGCATAAGACAAATTTGGGAACCCTGGACAAAGGGTGGCACCATTGTCCTGG TGGGTGATTCAACTGACCAGGCCACTCCCTTATGGAAGGGCCAGGTCTTCACAATCAGGG TAGCCTTACCTGGGAGCAGGTGAGGGACTGGCCCATCCTGGGCTGGAGGACAGTGAGGGT GGTGGCAGGCTGCCTTTGGTGGGCAGGAACCATGACAGGTACCTGTCCACCAGGAGGAAA TAGGTACAGTCTGAAATATGGACATGGAGAGACACAGGGAGTGGCTGGAAAACCAAGCTA GTTCTTAGCATGGGGTCTGGGCACTGCCTGGCCCCAGAGCACTGGCAAGTGCAGGGAAGC CCCGGGCCATGCCCCCACCCCTCCCAGCAAGAGGCATCCCTTCCCTACCTTTAGTGTGTT GAGGCTCAAGGCAAAGAAGCATATGTAATACTCGAACGGATCTGATGGCAGCGTCAAGGG CAAATACACAGGGTTGCTGGGAAACCCCCAGTGTTGAGAGCTGAGCCACCCACAACCTTC CTACCTCAAGGCCACCAAAGGATACTCAAGGCCAGGTTCAGGCCAAGGCCCTTAGTTGGG CGGAACTGGATCTTGTGTTACAGAGGACTGTCAAGGACGCACTCCTGGATGGATGCCTTC ACGAGACCCTGCAGAGAGAGGCAGCAAGGCTTGTGGGCAGGCACTGGCGTGTAGTGCCCG GTACGCAGTGCCCCACATGCACAGCAGCCCTTTGGGAAAGCTGCTGGGGAAGAGACCCGC ACACAGAACTCACCTGTGGGGACAGTGCCCTGCTCCTGAGCAGTGCCTCAACAAGAGTCT TTGCTCAGAACTTCCACCATCTCATAAAAAGCTGCAGACAGGCCAGGTATGGTGGCTCAC GCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCACAAGGTCAGGAGTTCCA GACCAACTTGGCCAATATAGTGAAACCCCATCTCTACTAAAAAAAAAATACAAAAATTAG CCGGGCATGGTGGCATGCATCTGTAGTCCCAGTTACTTGGGAGGCTGAGGCTGAAGAATT GCTTGAACCCAGGAGGCGGAGGTTGTGGTGAGCCGAGATCGTGCCACTGCACTCCAGCCT GGCAACAGAGTGAGACTTTGCCTAAAAAAAAAAAAAAGCTGCAGATGGCAAGGGTAGCAG CCATGCAATACCCATCTCCTTTGCCTCTCATACCCCCGGAGCAAATCACTCCTTAATTTG CCCTCAGAGGACCGGAGGAGACTGCCCGTCACCACTGGGCTGTGCAAGGTCCACTGCCCG AGCACCACTCTCACCACTGTGGTTTACTTATGGGCAGGTAGGAGATGGGAAAGTCAAACT TAAGTCTTCAGCTTGAAGCTTATAAACCAACTTCATCATTGGGCCGCTGAACATGAAATT GAACAAACAAAAACAGGGTATATGAACTGCAAATTATCTTGCTTTGATTGTATCTTAACC TTAAGAAATTGAGAGTGACTTAAAAGTGGAATCACCATGAGGAAGCACCCTGCCTCTCTC GGGTGTCAAACCTACCTTCCCAGGGTCGAGAAATTCCATCACCATGCTGTACTTCACAGG ATTCACGCGCCTCTGTAAGCAGCGCAGGTTCCAGCCCACAAGGACACCATCCAGGCTGCC GAAAATGCTACCAGCCATGGGGAGATGGCGTGCAGCTCCTGAAACAGTGTGCGGTGAGGC TGCAGGCAGGACCAGCACCCACTCCTGCCCCAGCTCCAGGCCTGCACCACCTGCTGCCCA GCTCCCTCTGAGGAGGCCACTCCCTGGCTGAGACCCATGGGATCAGCCCTGCGCTCAAAG GAACCCTGGTGCGGTGATGCCAATGAGAAGAGCAGCTTTGGGTTCTTTCACTGGGGTACG TGGCTTACCAAGCAGTGCTGCTTTAGACGTGGGCCAGTTAAGTAAGTCAGCCTCTGATCA AAATACATTTTTTGTGTGTGATAAAAGCCTAAATTAAGGGAAAAAAAAAACAAAAAAATG AAAAAATGAAAAATAAAATAAAATAAACAAAATACCTATTCTTTTGAGACAGGGTCTCAT TCTGTTGCCCTGTCTGGAGTACACTGGCGCAATGCACAGCTCACTGCAGCCTTGGCCTCC TGGCCTCAAGCAATCCTCCTGCCTTAGCCTCCTGAGTAGCTGGGACTGTGGGCACACACC ACCACGCTCAGCTAATTTTTAGATTTCTTGTAGAGATAGGGTCCCAGTATGTTGCCCAGA CTGGTCTCAAACTCCTGGCCTTAAGTGATCCTCCCACCTCAGCCTCCCAAAGTGTTGAGA TTACAGGCATGAGCCACCACACCAAAAGATTTTTTTTTTAATGTCAGAAACTTCTGATGG ATGCGAAGGATGCTCTATTAGAAAGGAAAGCAACACCCTTCCCAGAGTGAAGCCCTTAAA ATCTAACTGACCTGTTGTCATTTGGTATTAACTCCTTGTCTAGGCCAGCTTCCTGGGCTT GACAATTCCAGGTGTGATTTAAATACATATTTTTAGCATTTACATCAGGGTTCCTCAACG TTGATGCTATTGACATTTTGGGCTGGATTCTTCTTTGTTGCAGGGAGCTGTCCTGTGCTT TGCAGGATGTTTCCAGCATCCCTGGCCCCTACTTACTAGATGCCAGTAGCACACACTCCC CATCACCCCCAGTTGTGATAATCAAAAATGTCTCTGATATTGCCAAGTGTCCCCTGAGGG ACAGAGCACCTGTGGTTAAGAACTCCTGATCTGCAAGTTCACATATTTAAATGTGTCCAT AAACATGACGGTCATGATAATGGGCCAGGACTCTGTGCCAGGCAGTGCTTGGAGTGGGGG TGGTGGGGGGAATTAGGAATTATAATAAAGTTTTTAAAGGAATGTGTAAGACATGCTCCA AAGAGAGATGTCAGCAGAGCTATACCTCTGCTGGAAAATCCTCAATGACTTTAACCAAGT CTTGGCATCACTGTGCAAAGGGCTTATTTACAGAGTCAGCTTTCAGGGTAGCCTAGAAGG TAGAACAAAGTGAAAAAATCATGAGGACATTCACTGCAGCTTTTAGTGATACTCTGGCTT GAGTCAAACAGAAATCCAAGTGATAGGTTATGAGTCAGACACATAACCACTGGCGTGATT CCCCAGCCCCTCATGTGTGTCAGTGGCCCAAAGGATGCTACTAGGAGAGGCTGTGGTTCT ATGCACTCTGGACCAGGAACTCTCAGGCTTGCTTCCCAGTACCAACTCAGCGTGCTGCTC ATCATCAGAACAGGACTGACTTCCACCAATCCATAAGTAGGGGTTGGCCCCAGGGTCAGC CCCAGAGCCAGAAGAGGATCTCGATTGTGTAAGGGCACCTCTCACAGACCACAGAGTACT ACTGAGTTCGTACGCATCAAATGGAAATTTCCCACCTGATGGACCATCATGATTACACAG TTGTGATCATCAGTAAGAATGCCCAACAAAATCAGGGCTACAGCATCCTCTCTTAACTGA GCTGTCTTTTATTCAGCAGACAGTGAACCTACTAAACCAATGACCTTAAAACACAGGGCA GGGCCGGGCGTGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAAGTGGGCAA ATCACAAGGTCAGGAGTTCAAGACTAGCCTGGCCAAAATGGTGAAACCTTGTCTCTATTA AAAAAACACAAAAAAATTAGCCGGGCTTGGTGGTGCATGCCTGTAATTCCAGCTACTCTG GAGGCTGAGGCAGGAGAATCGCTTGAACCTGAGAGGCAGAGGTTGCAGTGAACTGAGATC GCACCACTGCACTCCAGCCTGGGCGACAGAGAGAAACTCCGTCTCAAAAAAAAAAAAAAT AAAACAAAGCAAAACAACAACAACAAAACACACACACACACACACAAGGCAGGTAAAAAT TATAGATGGCTTGCACCAAGTAAACACTAAACATAAAGGCAATGGAGAGGCCAGAAAAGA CTCAGGACAAGGACTAATAAAATCTACCCACAGTCGTGGGATATACGGAAAGCATCCTGA CTACAAGGATAAAAATAGCGTCTGTGCAAATGGACTCACATCAGGAGAAACTGCTAGAAT CTGTCTACCTCCAAATGCCTTGTCTACCTCCAAATGAGGGTGGATTTCACAGGATTTGAG ACAAGAGATGGAGACCTGAGTTGTCTGGCCATGTGGCAGACCTGGTGGCTCTAGTGGGGT GCCCTGGGGAGGAACGTCCTTCCACATACATATTTACAAAAATACCTCCTAAGAACTAAA ATTAGGAACTTTCATCACCAATACTTGCTCATACCTAACTTCTCGCTTCTCAGTATCATT TTTTCTTTTTTTTTTTTTTGAGTCTTACTGTGTTGCTCAGGCTGGTCTTAAAACTCCTGG GCTCAAGTAATCCTCCTTCCTCGGTGTCCTTAGTAGTTAGAACTACAGATACGGACCATT ACACCCAGTTCTCAGCACCATGTTTAATAGTTAAAGTGCTAACCTAGCACCCCCTCAGAA CATAAGAGTGTCTCCATTTTTCACTAATGACAATTATGATTATCAGAATGTTTCATGTCT GTTTAAGAACTTACAGATCATGAACACTCTGATGTACATCATTCTCATTTGCTCCTCCCC CTCCTCCTCCTCATACCCCCTTGGGAAGTAGAGAAAATAGAACTTGAATGAATTTGTCTT GTATGGGAAAACCCAGAGCCCAGAGCCCAGCGGTTGAGTAACCCCAAGGCCCTGTAGTAA TAAGGGGGCACAGTCAAGCTTAAGGAGTCTGGCATTGCTTCCATCACACGCTCCTAAAAG ACAACCACTAGAGAATTCTTCAAGTCAATGGGAAGAACATTCAAGTCCCACAGCTACAGA GCTGTGGCCACACTGACACTCATCCAGACAGCTGATATTTACCAGTAGAAAGCTGGGCCG CTGCAGGTGAGGGAACGCCATAAAAGCCTCCAGTGGAGAGTTGAAAATGGCCTTCTTAGC AAACCACTGTGGAAAAACAGAGACAAAATCTCAACATCTGCAGCACAGCAGAATCTTTTT ATTTTTCTAGAGACAGGGTCTCATTCTGTTGCCCAGGAGAGAATGCAATAGCACGATCAT AGCTCACTGCAACCTCAAACTCCTGGGTTCAAGCAATCCTTCCCCCTGAGCCTCCTGAGT AGCTGGAACTACAGGCACACACCACCATGCCTGGCTAATTTTCTTCTTATTTTCTTTTTT GTAGAGACAGGGTCTCGCTATGTTGCCCAGGCTAGTTTCAAATTCCTGGCCTCCACTGGC CCTCCTGCCTTGGCCTCTTAAGTGCTGGGATTACAGGTGTAAACCACTGCACCTGGCCCT AACAAAATATTACATAGTTTCACAATGCCCAGGAGAACATTTGTGTAACTCAACATGCAT TACATTATCGATTCTCATGTCTTCTTTATTTATTTGTAGAGACAGGGTCTTGCTTTCCCA CTCAGGCTGGAGTGCAGTGGTATGATCATAGCTCACTGCAACCTCAGACTCCTAAGCTCA AGCGATCCTCCTGCTTCAGCCTCCCAAATAGCTGGGACTACAGGCACACACCACCAGGCC CTGCTAATTTTTATATTTGTTGTAGAGATGTGGTTACATCATGTTGCCCAGGCTGGTCTT CAGCTCCAGGGCTCAAGTGATCTACTTGCCTCAGCCTCCCAAAGTACTGGGATTACAGGC GTGAGCCACCACGCCCGGCCAGCAATTCCCACATTTTACAGAAACACCATTCGCATGCAG CCCACAGTGGCCAGCTTCAGAGCCAAGTCTGTGGTACTGAATCCTTGGTAATGCTTGTAC AGCACTTGCTGTATGCCAGGACTTGCTCTATACACCACACACACTGTCAGTCCCCAGGAG GGGAATGTGGTATGTGATGCTTCATAAACACATTGAACTCTTTCTGCCAAGGAACAGCCA TTAACACCTTGGGGTTACTTGCAACTCACCTGTGGACTGTTGGTCTGAGCTATTGTCCAT CACTTGCAACTCAGTGTACACAGGTTCATCAGAGCTGGTCCCAAAAGCAAGTCCAAGCGC TGGAGAGCATGAGTGGGAAATCTCACTTCCACCTCGGATGGAGAAAAACTTCAGCAAGAT TGTCATTTTATCTTTCCGACCCAGATGGACTCCATCTTGACTCAAAGACAACCAGAACAT CACCAAGCATCTCCTACAGGGCAGGCTTGGTACTAGGTGGTTTTATTTAGATTATTTCAT GGAATGTTCAGGAGAATTGCTCCCTCTTCACTCCTGAATTCTTGATGCAGTTCTTCCACT TTCGCCCTTTCTTCTCGTCTCTGTCTCATTCAGCTCCCTCATTACAAACCTTCCAACTGG CTGGATGGCGCAGTTGCTCAACCAGCAGAGAGCACTGGGGTAGCCCCAAATAAATGTTCA TGTCATCTCCAAATTACCAGCACTATTCCCAGGTCCCAATTCAAGACAAAACCTTATCAG CCTCATCAGTGAGAGCACGTTTTCACCGAGCTGAGTAAAAACTTAGGTACAAGACCATTC AGTGGGGAAAACACTCATTTCTTTGAAGAATGGTGCTGGGAGAACTGGATATCCACATGC AAAAGGATGAAGCTGGACCCCTACATCACACCATATACAAAAATTAAGTCACAATAGGTC AAAGCCCTAAGTATAAACACTAAAACTGTTAAACTCTAAACAAAAAACATGGAATTACAT TTTCAAGATCTTGGATTTGGCAATGGATTCATTCTTATTTGGTTTTTAACTTCATTGTGA GCCAATTTAGCATCCAAAATTCCAGTAGGTTTGGTAAAAATTGACAAGCTGATTCTAAAA TTATAGCAAAAATGTAAAGGGCAGGCCGGGTGCAGTGGCTCACACCTGTAATCCCAGCAC TTTGGGAGGCTGAAGCAGGTGGATCACTTGAGGTCAGTAGTTTGAGACCAGCCTGGCTAA CATGGTGAAACCCCATCTCTGCTAAAAAAAAAAAGAAAACACCAAAAATTATCCGGGCAT GGTGGAGTGTGCTGTACCTGTAATCTCAACTACTTGGGAGACTCAGGCAGGAGAATCACT TGAAGCCGGGAGGCGGAAGTTGCAGTGGGCCAAGATCATGATACTGCACTCCAGCCTGGG TGATAGAGTGACTCTGTCGAAAGAAAGAAAGAAGGAAAGAAAAAAAAGAAAAAGAAGAAA GAGAGAGAGAGAGAGAGACAGACAGACAGACAGAAAGAAAGAAAGAAAGAAAGAAAGAAA AGAAAGAAAGAGAAAGAGAAAGAGAGAAAGAAAGAGAGAGGGAGGGAGGGAGGAAGGAAG GAGAAAAAAATGTAAAGGACAAAGAAAATCTAGACTCTTCTTAAAGGACAACTCACCTCA TCCTGGTCATCTTGGCGTCTTCATGATTATGAGCATGAGGACTTCTAGAATTCTCTCTCT CTGTCCCCTGTCCTCTCTAGGATTCCACAGTCTTCCCAGGTGACCAATACTTGGGATCCA CCTTTCAGGCAAGGTCCTCTTCTTACCCATGGGGCTTACATATCTAATCCAGTGGTTCTT ACCCAGAGCTGTGCACTGGCACCAGGACACCCCAGGCACACTTGGGGCAGCCCCTAGAGA TTCGGTTGGTCTAGTTTGGAGTCTTCTTCCACAAACGCGACTGTGATTGATTTACATCTG TGACTGAATCCCTAGTGAAGATGCCTAATTGCAGCACAGTGACCTCAGTGACGCTAGCCC TAAGCATAGCCACCCAGAAGAACAGGGCATTTGGCCAAGCGAGAGTGGTGTTCTGAGGAG GGTCCTGGTCACACACCTCCATCTGTCTCCACACCTGTCTGGCCATCTGGGCCCAGCTAA TCGGAGTCCAGTACCTCTTTTCTTGGTCTCAGGCCCACACAGCTTCACTCTTCGCATGTA AGCCATGCCCCTCTGAATTTCTGATGCCACTATCTTATCTCCAGTGCTGGAGTTACTACT TCTTTCAGGGAACATCACAATTCTTTCCGCATTTGAAACTTCCCACTTTCAAAAGTTTTG TCTTTCTCAGTGTCACCTCCTCTGTCTAAGGGTAGGGCCCAGGTGTGAGAAGCAGGGAAC TAAGAGAACCATCCTGACTCTCGGGGAGCTGTTCTGCCTTGGCCCGTTGCTGTGAGGACA CACGTCCATGGTACTGAGACAGCCAACAGCCCTGGGAACCAGAAACCTCACCCTCCTATG AGCAGGTCATGGCAGGTAGACACAAGCCTTGTTCTGGGGGCCAGGCTAAGAATGTCCCAT GGATTGAACAGTAGAGACATTGACCCTGTTTGAGGTGTGAATTCTAGATTTGAGCTCCAG CAGTGCAGTTAGAAGCAGCCGACCTGCCTCTAGTTCCTAAGCCCTCGTACCCATCACACT GTTCTACAGAAGTGGCCACTTGATCCCCTAAAGCTGTGCCCTCAATGGACACTTTGTTCA CATTTAAGTGAATATGATCCTTTTAAAAATTAAAAATACATATTTTAAAGATAGTCTTAC CCTGTCACCCAAGACGGAGTGAAGTGGGATGAACACAGGTCACTGCAGCCTCAATTCCTG AAGTTGAGGAGTCAACTGCCTCTGTCTCCAAAGTAGCTGAGACTACAGGCGTACACCACA CCTGGTTTCATTCAGGTGAATATAATCTTTGCTGCCAGCATCTCACCCTTTGATAGTAAT CACATCCTCTTAAGAGGGCTTTGGAAACAATTGGCCTTGGCTAGCCAATCCCAGGTTTTC GCTTCCATGAGGGTCTCCAGCTTCACACAATATTGTGCCCATATCTGTAGCGGCCTTGGT TTTTAATTGCAAACAACAGCATCCACCCACATCAGTGTTGGTAAAAAATAAATTGCTTTT TTTTTTTTTTTTGAGACAGACTCTCGCTGTCGCCCAGGCTGGAGTGCAGTGGTGTGATCT CGGCTCACTGCAAGCTCTGCCTCCCAGGTTCACGACATTCTCCTGCCTCAGCCTCCCAAG TAGCTGGGACTACAGGTGCCCACCACCATGCCCGGCTAATTTTTTGTATTTTTAGTAGAG ACGGGGCTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTTGTGATCCACCCGC CTCAGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCACGCCCGGCCAAGGTATTG CTTCTTTAAGAATGGAGCTGTTAATACTGCTGGTACCAGAAACATTAAAAAAGAAAAATC CTGCATGAGGGCCAGAGGAGGAGTACAGGAGAGTGAGATGGAGTGGAATCCTCCAGTGGG TGATAGATGGATGGACAGACAGATGAATAGACAGACGAACAGATGAACAGACGGACAGAC ATGAATAAACAGGTGGACAGACAGATGAATAGATGAACAGACAGATGGACAACTGGATAG ATGGACAGATGAAACTAATAATCTGAGAAATCAGAAAAGCTTCATGAAAAAAGTGGGACT GAGCTGTGTCTCCACGGATAGATATAAAAGCAGAGGACTCTCCACTTGAGTCGAGAATGA CCCAGTGTCCTGATCCAGAGAGGAAGCCAGCCTGGCTTGACTGGGAAATTTGTGGGAGGA CTCAGAGGCCCTTAAAATGAGGCCAGGTGAGGTTGGGCTGATCCGAGCCAGCTCAGGACT CCTCTGCCACACAGCACAGCTGCCTTAGGGGACACATTACTCAGGGAGTTGCTGGGACCT ACTGGGCCCAGCATTGCCACCAGCACCAACAGCTTCAGAGAGGGGGGACACACACTTGGG GTGACTCCAAGACTGTGGGTGGCACCTGCCTCAAACAGGGGACAGGCACAGGGACACCTC TCGGGGTCTGGCACCCCCACGCACTGTGCCTAGGTCCCAACAACGCCCACTGCAGCCCTG TGCCCATCATGCCCAGAAGGTTTCCGCTTCAGCCTGGCCCCTGTACTGGCCCCAGGAATT TGGACCCAAGCCTCAGTCACTGGGTAAACAGCAGTGGGAACCAGCTCATTACTCTAGGTA AGTGGCTCTTACAACCTTCCCCAGCCAGTTCCACCCTCTGTTGTCTCTGGAAAATATGTT TTCTCTCCCTGGGGTGGCTTCTCCTCTGCCCTCCCAGCCTTAATCACTGACCCCTACCTT TCTCTATGGGTCCTGGGGGAGGTGGGTTAGTCTTGAGGTAACCAGCAGAAGGGCCCCAGG TTCCAACAGCCAGACGCAGCCTGGTCCCGGGGCCTGGGCTGGGTTTAGGCAAGGTCAGAG TTCCTTCACCTCTTTCAGGGCAGGCACCCGAGGTGCGGGGCAAAGGCCAGTTCTGACTGG CACACTGCAGTAGCATCAGAGACACCCCCCGGACCCCAGGGTCTAGGCTGATGGCTGGAT GCCCATCCAGCCTGGGAAGGCCACACGGGGGCCTGGGGACAAAGGGGTCACCATGAGGTG ACATCAATGCAGGTGCAGAGAGGGCTCTGGGTCTAGGCTGCAGCTCTCTGGCCTCTGCTG GGTCATGAGGACACAGGGACAAAAAAAGAAGATGGGTCAGATGGGGCAAGATGGCCAGAG CCCAGCCCTCCCAGAATAGTCATCAGAGAGGAGCAGATCCCTTAGGGCAGAGACATATTT GTCCCTGGAGCCCCTTCACCCCTGGGGCCTGGCGTCTCACTGTCCATGGGTCAGTCTCCC ACCTTCCTCAAAGGGCACGTTAGACTCAGGAGGTGACAAGAGGGGAGCGAATGGGGGGTG CAGAGGACTCTACGGCAGCCAGCTGAAGTCTAGAGTTGTCAGAGTCCGTGGAGGCAGGCA TGGGGGGCTGCTGTGTCCCGTGGTCCAGGGGAGCAGCCCCAACACCACAGTGGAGGTGAA GGGTCCTATGGTTGGGGTGGTGGGGACAAGGGAGGTGAAGAGCGGTGGAGGAGCCCCGGG GCTTGTCTGGGTGCAGCCCACCCTTCATCAGGAAAGCTGAATGGGATGGGCTGGGGCAAA GCCTGGTGCCCCAGGGGACAGGAAGCTCCAGGCCCCACCAGGCTTGGGCCTTTCCACACT CTGCCAGGATAGTCCTGTGGGCTGGGCGGGGACGTGCAAATTCCAAACTCAGACTCCAGA GACCAGAGAGGAGGGAGCACAGCCTGCCCTGGGTACACACAGGGAAACAGAGGCTGCAGA GGAGGGCTGGGCCAGGGCTCCTAGAAAAGGTGACTTGGGAAGAGCTCCTAGGAAGGTGCG GGCTGGCTGCTCTGCAGAGGTCTTGAGTGAAAAGGAGGGGAATGAGGAGGGAAGAGGCAG CCCCGGGTGGACTGGACAGCCATGCCGTGAACCTCACAGAGACTTTAGACAGAGAGGGGG CTCTACAACACCCCAGTACTCCCTCTGCCTCTCTTGCCCCCTCCTCTGTCCACACAGGTC TGCCCAAGGCCGCCCCTTTGGACACTCTGAGGAACTCCAAGCCAACAAGATGCCTCTCAT TAGTGACTTCTACCCTGATGCCGTGATGGTGGCCTGGAAGGCAGAGGGCACCACCATCAC CCTGGGCATGAAGACTGCCACACCCTCCAAACAGAGAAACACAAGTACACGGTTAGCAGC TACCTGAACCTGACGTCTGACCAGTGGAGGTCCCCCAGCAGCTATGTTCTTAGGCCCCTC ACCCCACCCACAGCAGCCAGGAGCTGCAGGATCCCAGGGCAGGGGTCTCCCCTCCCACCC CAAGGCATCCAGCCCTTCTCCTTGCACCCAATAAACCCTCAGTAAATATCCTCATTGTTA ATCAGAAATTCTGCTCCCTGTCTTCATTTCTTACCTTTCATATAATTTGACACTTCCCCC AGGTTCTCAGTGGGGGATGGGGGAATCCTGACACTCAGTGGGAAAATAGCTTGTGGGAGA GGCTCCCAGGCTCCCAGGGGCATCTGCTGGAGAAACAGGCCAGGCAAGGAGCAATCTGAT CACTGAAGACCAGTCCCTCTGCCCTCTCCCTCCTCCAATTCCCCGCTGCAGCACTCCCTC CCCCAACCCCCCGCCACTCCCTGCCTCCTTTCTGGATGGAGCTGTCCCTGGCTGGGCCTT CAGATATGCCCTCTGTCCTTGCCCTAGTAAAGACACTCTTCCCACCTACGACCTCTTTTT CCTCAGCCTGGAAGTAACACTCTGGGCCTGGAGTTCCTCTGCCCGTGGCCCTGGCCCCTG GAATCCCCTCCTCCCTCTCTGCCCAACTCCCCACCCCTGAGAGCTGGACTGTCCAGAATA CTCCAGCACCTTCAAATTCATGAGCTGTTAAATTTGGGGCCCACCTCGATTCTTCACCTG CCAGCAGGTTCACAGGTGTAAGAAAAATCGGAGGAAGGCAAGAAGGAAACACACACAAAA GGCTTGCAGGAGGCTCAGGACACTTGCCAAAGATAGGCTCTAAGCTCCCCAGGAACCAGA GCAAGAAGGGTAACAGGAGCTCCATCATTTCTATGAGATTTAACAAAGCCTTTGCTTGAG GGAAGTTGGTGTTTTCAATGCTGAGGCCAGGCATCTCCTCAGAACTAACAGTGGGGATGT GTTTTTTGTCTGTTTGTTTTGTTTCGTTTTGTTTTGAGACGGAGTCTCACTCTGTCGCCC AGGCTGGAGTGCACTGGTGCAATCTCGGCTCACTGCAAGCTCCGCCTCCCAGGTTCACGC CATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACCACAGGCGCCCACCACCATGTCCGG CTAATTTTTTGTATTTTTTTTTAGTAGAGGTGGGGTTTCACCGTGTTAGCCAGGATGGTC TTGATCTCTTGACCTCGTGATCTGCCCACCTTGGCCTCCCAAAGTGCTGGGATTACAGGT GTGAACCACCACTCCAGGCAGTGGGGATGTGTTTTACCCAATCCCTTAGTATTGATTGGA TTTGGGCTCCGACACCTGCACAGGTGACTGCCTGGCAGCCCCAGAACACGGAGATGTTCT CAAGTGTGTGGCACAGAAAGTCACTGGCTGCCTTTGGGGGGCATTCTTTTTGGGAAACAC CTCTCCATTGGATCCTGGGCCTCAGGTGGAAAACCAGGTCCTGCCTCGGGACTTTCTCTG TGAGGCTCCCTGGGTCTGGGGCTCCAGCCATTCTCCGAGAGGAAGGTGCCAGGAGAGAGA GCAAGGCCCAGCCCCACCTAGTCTTTCCTGCATGGTCCCTGCTCTGGTAGAGACAGGGTT TGGCCTGGCTGGGTTCCACACCTGCTGAAGGATGCACAGATGGTAGATGCCCAGTCTGAC CCTGGGTCTACCCAGGGCTAACCCCCTTCCCTGAGGCCAGACTGAGGAGGCTGAGCCTGC CCAGGTCCTGCAGGTGTTCAGGGCAGCACAGACAGTGGGTGCCCAGGCCCCTGACCCAAG GTCACCCTTCCCTCTTGAGGCTGTGCTAGGCAGGGCCAGGAGACAGTACTGAACACACCA ATCCCCTTGTGGCTCTGTGGGGTTGGGGACCTACACTCCTCAGTCTACATCGAGCCGGTG TAGGAGACAGGCAGAAGCAGGAGGGTCCTCGGAGTCCACAAAGCAGAGCAGGGGCAGGTG CTCGTGTGCCCATTTCACCGAAGAGAGAACTGAGGCTCCACGGGAAGGGAAGTGACCTGC CCGGGTCAACATGGTGAGCTGCTGCCTGACCCACCCCCAGCCTCTTTCCACAGCTCCTCA GTCCTTCATTCATTCACTCACACACTCATTCAGCAAACCCTCAGTGAGCACAGCTGTGGG CCAGGTGCTGGTGCAAGACCAGGACCACCTGCCCTGGAGAAGCTCATGAGCTGGAGGACA ACAGGGAGACACCACTGCACCCCCCTGTCCTCAAGTCCCCAAGGTTGGAGCTTGGCCCAG GGAGCGCCTGGGGCAGCAGGGGCATCTCCCCCTACTTAGCAGAAGATTTAACTGAAAGAA CCATCTGACGGAGCCCCAGCTGTCATCCCTGGGCCACAGTGGGAACATCACAGTCCCTCA TCAACCCTCACAGTGACCCTGAGGCCCAGGGCAGCCATGGTCCACCTCACATAGGGCAGG CCGACCATGAGGAATGCACCACGGTTGCCTCATAGCGACCCTGAGCTCTAGAGCTGCCAT TGCCCACCTTATAGAGGGGATGCTGACCAGGAGGGAGGCACCAACCAGGGCCACCCAGGC TCCGGCCAGCCTGAGAGTGGCCCAGCTGGCCCTGTGCCTGCACTGCCCTGCACCTGCTGC CCTGCCCAGCTCAGACCCTGGTCATATCCTCAGGAGTGATGGCCCCACAGCACCAGGGCA GGGTAGGTCCACACTGGGAGAGCCCCTAAAGGTACAGAGAAGAGCACTGGAGTCCAGGGA GGAGGGATTGTCCCGGCCCTGGTCCTGCCTCGCTGTGACCAGGGCTGAGGCATTTGCTCT AAATCAGATGGGGGAGGGGCATCTGTGGCTTCCTCTACAGCCAAAACCAGAGCCCCAAAT AGCCCCGGTGGGTATGGAGGGGCCTTGGGGGAGGGGAGGAAATGGGAGGGGAGGTGGCTG CTTGGGGCTCTGGGTGAGGGCTAGGAGCTCCTACTGAGCTAAGCGGATGACTCTCCTATC TGACCTGGCCCCCCATGGGGCCTGCAAAAGCACCAAGAGAGCATCCCCAGCCAGGCCTGT GGAGGGGCCCTTGACCCCTCATACTCACTGAGCCCAGTGGTGGGAAGAGGGGGCTGGTCC CCGGCCAGTATTCTCCTGAGGACACCGACACTCACTGGTAACAAGCAAACCAGACCCAGA TGCTCCAAAAAGAGGCCACTGGATCCCAGCAAGCACCCACTCCCCCCAACACGAAGGTCC AAAACCAAGCTTCAGCCAACTAGAATGTGGGAACTTCCCGGGAGGTAGAGGAAACAAGTC ACTGTCCACTTGCAATGCCCACCGGTTAGTGACGCAGCACAATGACAGTCACACCTCTCA GAGGGGGCTCTCCCTCGACTCTCCTTCCTGACACATCAACAAGGACAGCCTTACCAAGGG CCACACCTGGGCAGTCACCATGGGGTTCGTGGTCTCACGTGGGCCCTGCTTAAAGCCCCT GGGGATAAGCTCACTCCAGCTCATGAGAGGCCGGGGGTGACCCTGACCCCATTTTCCGAT CAGCGATAGCACAGCACATGTGGGGACCAATGAGAAATGAGTCTGGGGAGACCACGGGCT TCAGGGAGAATGTTTCGATGAGGCAAGTACAAGAGCCAGTGAGGGACTGGGTGACTTCTG TGGCCTGTGTCACAGAGCTGGGGCCATCAGATGCCCCATGTCCACTCTGAGGACAGAGTA GCCCATGGTTAGGATGACCCACCAGCCAGGCCTGGCCAGGGCACCCACAGGAGACAGGAA GGCTGTGAGGGCCCGGGGGCACGGAGCAGCTCTGAAAGGAAGGGGTGCTGGGTCCCGGGG CTCCTGGGCAGAGTGGTTGAAGAAGGTGAGACCCCAGACCAGCCAGATGGGCCCACTTCT CAGGGGACCCTGGTCAGACAAGGGAGCACCTCCTGCTCATGGAACACAGCAAGGGGCAAG GTAGGCACAGCCAGGCTCGGAGAACTCGTATTTGGGGGAGATGTAAATCAACCCAATAGA CAAGGGAGCGATGTGGTGTAGGAGATGGGGTTTGTGCTGGGGAGAAATAAGGCAGAGAGT GGGGAGTAGAGAGCACCTGTCTGGGGTCTCAGGAGGAGCGATGTCCCAAGACTGGAGGCG GCCAGGGGCTGAGGAGCCCTGACTGGGCCAGGCCTGTGGCTGGAGCTGGAGGCTCGGGTT CAGCCTCAGTTTCCCCTCTGTGAAGCTAGGTTCATAATAATGGAAATTGACTTTCTGGGT GGCTGGAGAGACATTCAGATGGAAAATGGATATTCTATTTTGTCTCTGTCCTTGGTTGTT GTCATCTCAGGCTGTTCCAGCCCCAGTTAGCCTCGGGTCAGCCCCCTGCCCATGTTGGTC TCCCCATGAGCTCAGGCACCTCATCCCCAGCACAGCTGGCCTGGAGAAACCAAGATTCCC TGAGGCTTAAAATAGCTGGAAAGCCAGCCCAGCTGCAGGTGCCTCCCTGTAGGATAAAAT GTAGGAGAAGATCTTCAAGAGCTAGGACTAGGCAAAATTTTCTTAGACTTGACACCAAAA ACATAGTCTACAAAAGGAAAAATTGCTAAGCTAAGTTTCATCAAAATTTAAAAATTTGTT CTGCAAAACACCTTGTTAAAAAGATGACAAGTTACAGACTAGGAGAAAATATTTTCAAAC AACATATATGAACAAAGCACTGTTATCTTGAGTATATAAGGAACTTGCAAACTCCACACA AAAAATCAAACAATCCAATTAGAAGATGGGCAAAAGACACGAGCAGACATTTCACCAGAG ATGATACACAATGGACAAATAAGCCCATGAAAAGATCTTCAACTTCACTAGCCATTTGGG AAATGCAAATTAAAACCACAATGAAATATAATTTACACACATCAGAGTGGCTAAAATAAA AAATAGTGACAAGACAAAACTCTGCTGAAGATATAAGTGTGAATCACTCATATATTGCTG GTGGGCATATAAAATACCACAGCCACTGCAAAAGTGTGGCAGTCTCTTTTTTCTCTTTAG AGACAGGGTCTCCCTATATTGCCCAGGCTGGACTCAAATTCCTGGGCTAAAGTGATCCTC TCGCCTCAGCCTCCAGAGTAGCTGGGACTAAAGGCACATGCTACTGTTGATGTTTGGCAG TTTCTTATACGACTGAACATGGAACTACCGTATGACCTAGCAATTACATTCTGACTCATC TATCCCAGAGAAATGACAACCTGTGCTCATACAAACAACCGTACGTGAATGTTCATTACA GCTTTAATAGCAATAGCCAAAAATCTAGAAATAACTCAGCCATCCATCAGCAGGTGAACA GTTAAACTGTGCTACATCCATCTTATGGAATAGCACTCAGCCATCCAAAGGAATCAACTC TTGATACACTCAACCAGCTCAACCCTCCCCAGAACGCACCTGGGTGGGAAGGTGCACAGC AGATTGGGCCACTAGATTTACCCAGTGTGGAGGAAACAGGTACCTCCTCCTAGATTTTGT TCTTGGCAGAGATAAACCAGTGCTTCTCAGTCATTTTTGCTTTTTACTAGCCATGGGAGG TATTTTCCAGTAAAGGCTTCTATGACCAAATGCATTTGAAGGAATGTTCCATATGCTCTC CCACACGCCTTCAATTTCCTGAGAGTGACAAGAATACCAGAATAGCCAAGGTTCTAAGAT ATCCTTCAGGATACCTGTTTAACTTAAACTTCCCCAAACTATTTGATTATGAGACTCTTT TCAAGCATAAACATTTTCCTTCCAGGAACATTTGTATTCTTATAAGAAAATATTAGAAAG CATTCCAGGTGACTTACCTAATGGGAGAACCTCAGGCGCCAAGCCAGATGAGATAAAAGT TTCAAATAGTGCTGATAAGGTTTTCTTGGTTCAGCTGCCTCCCTCCAACTCAGCTGCTGT CACACTTCTAGGCAGTTTTGACTGGACTGTAAATTCCTTCTGGCTGGGGCCATGTCTCAG CACCTTGTGCAGGAGTGATACAAACTAATGCTTCATAATAAATACTTACTCACTGACTTT CTCTTTATTCATCCCAGGTGAGCATGTTTTCCATCCCCTTCAATCTCCACACTCACGGGC AAAAGTGAGTTGTGGGTCTTGGAACTCTCAAGAAGAGGAGGAGATCTTCAATTTGCATAT GCAGAATCCAAACAAGTCATCTTGAGTCCCCACAAGTAGGTGGGCTTATTTCTCACATAA CAAAATCGCCCCTCCTTTGCAGCCTCTACACCACTGCAGATCAAGCCCAGCTAAACCTTG CTGTGCTATCCTGAAGGTATTGCTCCCAGATCCCGGCTGTCAGCTCCTGTCCAGCATCAC AGGGCAAGTCCAGAGACATGATCATGTAATTGACCAGGGATGACAAGTGGGTCCTCTCAG TGGACAGGTGTCTGCAGTAGGTAAGCCCTGTCAGGAGAGGAAAGAAAAGCATCATCATCT ACCACCTGATACCCTTTGCCCTGTCTTATGGAGTCCCCAAAGTGCTGTCCCACACTCCCA TCTTTATTTTTTTATTTTTCTGAGACGGTCTCGCTGTCACCCAGGCTGGAGTGCAGTGGC GTGATCATGGCTCACTTCAGCCTAAGCCTCCCGTGCTCGAGCAATCCTCCCATCTCAGCC TCCTGAGTAGCTGGGACTACAGGCATGTGCCATCATGCTCAGCTACTTTTTAACCCCTCC TTTTCTTCACAAGGAAGGCAAGGCTTGATGTCACTTGCTGGCTGAATGGCTTAATAGCTC TACAACCCTAGGGAAGTCTTATCACCTTGCTCAGCCACAGTCAGGGTGATGCCATCCTCC TCTGCTGGAAGCCAAAACATTTAATTGATAGGCTGGAGGCAGAGAAGGGACAGGAAAGAA GGTGGCCAATGACTCTGTGGCCTTGCTGTGCCTTTGTCTGCCTCCAATCCATACACAGAG AAAGACACTGTGAGATGTGGACAACTTTCTTTCAAATATCAGCTGAATACAGGTATTCAT CCAAAAGAAAGGAAATCAGTACATCAAACAGTTATCTGCATGCCCATGTTTACTGCAGCA CCATTAACGATAGTACAAAATCAACCTATGTGTGGAGGAATAGTACAGAATCAACCTAAG TGTCCATCAATGAATTGACAGTTAAAGGAAATGTGGTATATGTATGCAATGGAATATTAC TCAGCCATGAAAAAGGATGAAATCCTGTCACGTGCAGTAACATGAATGGAACTGAAAATC ATTATGTTAAGTAAAAAAAGTGAGGCACAGAAAGACAAATATCACATGTTCTCAGTCATA CATGGAAGCTAAAAAAGTATTTCTCATGAATGCGGAGAGTAGACTGGTGGTTACCAGTGG CTGGGAAGGGAAGAGTAGGAGGGGAATGAAGAGAAGTTGGTTAATGGTACAAAAATCCAG TTAGATGGAATTAAGTTCTTGTATATTATAGTAGGAAAAATATAATTAACAGTAATTTAT TGTATATCTCAAAATAGCTAGAAGAACTGTAAAGTTCCCAACATGAAGAAAACATAAATG TTTGAGGTGATGGATGTTCCAATCACCTCAAACGTGATAGATCAATACGCAGTGGATCTA TCAAAATACTACATGCACCCCCAAAATATGTACAACTACAGTATATCTATTACAAATTTT TTTAAAAAGAGGGCCGGCCGGGCACAGTGGCTCACGCCTGTAATCCCAGTGCTTTAAGAG GCTGAGGTGGGTGGATTGCTTGAACCCAGGAGTTCTGGACTAGCCTGGGCAACACAGTGA AACCCAGACTCTACAAAAAATAAGCAAACTTAGCTGGGTGTGGTGGCGTGTGCCTGCAGT CTCAGCTACTTGGGAGGCTAAGGTGGGAGGATTGCTTAAGCCTAGGAGGTCAAGGCTGCA GTGAGCCAAGATCTTGTCTCTAAAAAATAAAATCGGCTGGGCGTGGTGGCTCACGCCTGT AATCCCAGCACTTTGGGAGGCCGAGGCCGGTGGATTGCCTGAGGTCAGCCATCCTGGCCA ACATGGTGAAACCCCGTCTCTACCAAAAACACAAAAATTAGCCGGGCATGGTGGCACACC CCTGTAATCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATTGCTTGAGCCTGGGAGGCAG AGGTTGCAGTGAGCCGAGATCGCGCCACTGCACTCCAGCCTGATTGACAGAATGAAACTC TGTCTCAAAATAAATAAATAAATAAATAAAACAAAATCAGATAAAAATAAAACAAAATTG CAGCTGGATTAAAAAGGCCCCAGTAAGTCAGGAACCAGGGAGAGAGATACTGAGGGGGCA GCTTGCTTTCAATGATGGTCCTGCCTCAAAAGCACACCTACCAATGTGCCACCCCTTATC TGTCACACCCTGTCACCTGGAGGGTGTTAGGCAAAGTGCCTCAGCAAACGCCAGCCCGTG CTGTTTGCTCTGGCTGTGCCCTTTCCTTCTGGCGTTCCCCAGCCATTTCATCCTCAATTC CCAGTGAGAACCCTGGACATTTTGCAGCCTGGGGACTAAATTCTAACAGGAGGGTCAATC ATAGCAATGATGATGATGATGATGACCACGATGATCTAATAGCTAATACTTACACAGTAC ACACTATGTGCCAGGGAGCTGTTAGCAGAGTTTGCATAACTCAGTCTCGCCACAACCCAA GAAAGCAGGCGCTACTGTTATTTCCATCTTATGGATGGGGTAAGTTGAGGCCCAGACTGG TTATGTCACTTGCACAAGTTACATAACTGGAATCTGAAACCAGATCACCTGGCTCCAAAA CCTGGGCTCTCAACCACCATCAGCCATGAGCCACATCATCTGATGACATTATTCCAGACC CGTATCAACCCCCAACCCTTCCTGCAGGAAGATATTTCATTTGGGTCCAAAAATACTTGC CCAATGCCTGCTCCACGCCAGCCCCTGCATTAGGCAAAAACGTGGATTGTAACGGAACAG GCAAAACCTTTGTCCTAGTAGAGTCACGCTCTCCCCGCCTCTGCAGCACTCAGGTGAGCT GCACTGTGGTTTCACAGGCCCATGCAGGTGTGTGTATAGCTGCAGGTTTTATTTCCCTAG CCTCACAGCCTCAAGAGGAGAGATAAAATGTGTATATTCAGGGATGGAAAACACACACAC ATTTTCTCTCTCCCATCAAGGCTGTGAGGCTACGGAAATAAAACCTGCTAAGAATAAATA TACGGAGAAGAAAAAACAAAGCCTTGGAGTCAGGCCAGAGTCTGAGTCCCAGCTCTGCTG CTTGGTAGCTGTGTGACCTTGGGCAAGTCACTTTGTCTCTCTGTGCCTTAGTTTCCTCAT CTGTTAAGTGGGAACAATCAGAATACAGATACTAGGAATTTAATTTTTAAGTTTTGGGGG AGGCATAACGTCACTTTACTCTTCTGCTTCTGCCCATGACAGAAGGAAACTGACATTTAC TGAGCACCTACAGTATGCAAGACTAGCTGCCAGGGGCTTTCTACATGCTCCTCACTTAAT CCTGAAAACAAACCCTCCACTGTGGCACCTGGGGGTACAATGGTGATTTAATGAAACCTC TGTCCCCAGGGGTCCCCATTGTTCACTGCCCTCTCTTAGGACTCAGGCATAAAATTTATC CCCAATCTGTAGATGATGAAACTGAGGCTCAGAACGGTTCATGTAAATTCACTGAAACCA GGCATTCAGGTAATAGAGGAGGAGAGGTTCAAGCTCCAAAGCCAGTATCTCCTGGTATCG CTGACATAGAGTAGGCACTGATAACTGCCCCTCGGAGACTACACGGCAACAAAGACCTAT GTACATCCATAGAAGGCTCTGCAGAGGATCTGGTACTTCATATTGTCACAAGAGAAGTTT CTGGGGAGCCCTGCAGGCAGAGAAGGAGGGAGATGCTTAGAATAGATTCAAACACAGCCC AGGTTTTTTGGAGAGGAGCACAACTTGTTATCTCTAGAGGGTAACTGAAATCAGCCAGTC ATGAGGAGTGCAGGGAGGGTGCTATTACCTCCACCTTTACAACATCACCTCTTCCACTAC CAGTGACCCAGTTTGCCCTTGAGAAATCACTCCGTCCACAACTTTCTGTCTCCGGTTTCT AAAGGGATGACTCTACCCATTTATAGGACCAAAGCTGGAGTCATAGTAATGGAAGCGGAG ATTGTCATATGACCCAACCTGAGCCAATGAGAATTAGGCTTGGGACTTTCATTGTAACTA CCAGGAAAAAGTCTTTTTCTGCTTCAGTTTCTAAACCAGTGGGATACAGGACAGTAGCTC CTAGGAGCTATCTTTGCCACAACATAAAAAGGGCCTGCCTGTGGATGGAACCATCACAGA GGGAAGCAGAGCCAAGAGCTGAAGAAACAGAGATTCCCGTCTCGAGCATCTGAGCCTGAA GCCACTATGACTCTAAGCTTCTGTTATGCCAACAATTTTTTTTACCTGAACCAGTCTGAA GTGGGTTTCTATCACTTGCAGAAAGAGTTCTGATTTCTTTTTTTTTTTTTTTTTACACAG AGCCTCGCTCTGTCGCCCAGGCTGAAGGGCAGTGGCGCGATCTCGGCTCACTGCAAGCTC CACCTCCCAGGTTCACGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGT GCCCGCCACCACGCCTGGCTAATGTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGT TAGCCAGGATGGTCTCGATCTCTGACCTTATGATCTGCCTGCCTCGGCCTCCCAAAGTGC TGGGATTACAGGCATGAGCCACTGCACCCGGCCGAAAGAGTTCTGATTTCTAAAGTGACC CAGCTGAACCAAGAGCCAAACTGGGATTCAGTATCTGGCTGTTTGCTAAGTAGGTGATGC CTCAAGGATTCTCCCTGCATGGGAATTTGTGGCAACTTTTTTGGCTGAAAGAGGCAAGAG GAGTAAGGTACCAGCATTGCTGGATAAATCCAGGACTGCTGAAGGGAGTGTTGTACGCTC TTGCAATGAACAGAATGGAAGAATATCGGCATATTTATTCTCAGCACATCCCAAAGGACA TCCAGAAAAATCCAGGGAGACTGGGGTGGGGGAAACATAGCCGAAGTCCTCTAGTTAGAC ATGTGAACTCCAAATATGAGAGACAGGTCTCAGTTAATTTAGAAAGTTTATTTTGCCAAG GTTGAGGATGTGCGCCCACGGTACCGCCTCAGGAGGCTCTGACGACATGTGCCCACGGTG GTCAGAGCAGTTTGGTTTTATACATTTTAGGGAGATATGAGACACCAATCACCGATCAAT GTATGTAAGATGAGCATTGGTACTGTTCAGAAAGGCAAGACAACTTGAAGTGGGGAGGAG GCTTCCAGGTCATAGGTAGATAAGAGACAAACAGTTGCATTCTTTTGAGTTTCTGATGAG CGTCTCCAAAGGGGGCAATCAGATATGCATTTATCTCAGTGAGCAGAGGGGAGACTTTCA ATAGAATGGGAGGCAGGTTTGCCTTAAGCTGTTCCCAGCTTGACTTTTCCCTTTAGCTTA GTGGTTTGGGGCCCTGAGATTTACTTTCCTTTTACAGGCACATGGAGGCATTGAATACTG AAGTGTCCCACTTTGTGCCCTCAGGTTGGTGAGGCTTACAGTTGCTCAGGTTACATGAGA ATAGGGTCACATAAACAGCTGTCCCACTGTGGGCTTGGTTTTCTGCAAGGTACTGAGTTC CCCATTATGCCAGGTGTGTAAGCAGACGCTGTAACAGGAGAGAGGGCAGTAAGTTATGGC AGCATTTTTTTTCTTTTTTCTTTTCTTTCTTTTTTTTCTTTTTGAGATGGAGTTTTGCTG TTGTTGCCCAGGCTGAAGTGCAATGGCGTGACCTTGGCTCACCACAACCTCCGCCTCCCA GGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGTATTACAGGCATGCGCCAC CACACCCAGCTAATTTTGTAGGTTACAGCAGCATTTCTCACTGGTCCATGGACCATCAGC CTAACAGTTACTTGCAGTTGCTCCAACATGTGTATTCCTGGGCCCTGCACAGACTTAATC AGAACTTCTATGAAGTAGTCTAGGAGCTGGCATGTTTTGCTACCTAGCCCAGGGGATTCT GATGCACAATAAGAGAATATCCGGTAACATTAGCACTTCTCAATCTTAAAAGTACACACA TGGTCTTGGGACTACGTTAAACACAAATTCTGATTCAGAAGGTTTGGTATAAGAGACTGA CCATCTGCCCGGTGCTGATGCTGGTTCACAGGCCACAGTCTGAGGAGCCAGGAGCTTAGA TGATGACTTCAACAATGTAATGCCAGCCCTCACACCGTACACTCCTATGGCTGAAGACTG GACCACACACTGGTACAGAAGACCAGCAAGGACAATAGGGTTCCATTCTACCAGGCAAGG CCAGGAGGAAACAAAACCAGTCTCATGAACTGGCTGAATAGTTCCAAAGAGACAAAAGCT CTATGGGCAAAGAGAGAGATGCGCCTGAGATTTAAGTTTACATGATAAAATGGTAACTGT TATTTACTGAACAGTACTGTGTGCAGGTGGCATATTCTGGCCACTTTAAACCATTATCTG AGCTGGCATTTACCATAGCCATATAAAGTAAGCACCAGAATCTCCACTGTGTGGAGGAGC AAACTGAGATGAATGTGGTTGACATTAAATAATATTTGAGATTATTCTGCCTTCTAGGCT CATGGTAGGGCTGCACTTCCTGGCTCCTTGTGGATGGGGCCAATGAGCTGTGGGTGGGTA TGACATGCTAATTTTGGGTGGGGCCCTTTTATTGTGGGTTCAAGCCCCTCCAATGCTACC TTTCCCTGTCACTGTGACTGGCAACATTCAAATGATGGCT hisat2-2.2.1/example/reference/22_20-21M.snp000066400000000000000000005076271370663763200201400ustar00rootroot00000000000000rs73387790 single 22:20000001-21000000 145 A rs55902548 single 22:20000001-21000000 427 T rs114690707 single 22:20000001-21000000 770 C rs147349046 single 22:20000001-21000000 809 T rs1978233 single 22:20000001-21000000 949 G rs2079702 single 22:20000001-21000000 1005 A rs139570132 single 22:20000001-21000000 1158 T rs5993894 single 22:20000001-21000000 1332 T rs71788814 deletion 22:20000001-21000000 1587 2 rs111598545 single 22:20000001-21000000 1821 G rs73877109 single 22:20000001-21000000 2230 T rs9605047 single 22:20000001-21000000 2847 T rs62223726 single 22:20000001-21000000 3058 T rs75213299 single 22:20000001-21000000 3151 T rs2531714 single 22:20000001-21000000 3265 G rs2531698 single 22:20000001-21000000 3333 A rs10678141 insertion 22:20000001-21000000 3490 AG rs2531715 single 22:20000001-21000000 3730 T rs2531699 single 22:20000001-21000000 3785 A rs113101099 single 22:20000001-21000000 4162 A rs551721384 deletion 22:20000001-21000000 4216 5 rs2286480 single 22:20000001-21000000 4310 T rs141779732 single 22:20000001-21000000 4429 T rs2531716 single 22:20000001-21000000 4703 T rs2531717 single 22:20000001-21000000 4936 G rs9606210 single 22:20000001-21000000 5257 T rs3788322 single 22:20000001-21000000 6039 T rs2531700 single 22:20000001-21000000 6143 C rs5748501 single 22:20000001-21000000 6353 C rs5748502 single 22:20000001-21000000 6549 C rs2531701 single 22:20000001-21000000 6573 A rs74731343 single 22:20000001-21000000 6710 T rs1858770 single 22:20000001-21000000 6991 C rs115149055 single 22:20000001-21000000 7391 T rs7288996 single 22:20000001-21000000 7598 A rs182897799 single 22:20000001-21000000 7969 G rs73387800 single 22:20000001-21000000 8172 C rs10427828 single 22:20000001-21000000 8227 A rs73387801 single 22:20000001-21000000 8487 T rs737986 single 22:20000001-21000000 8710 T rs111664354 single 22:20000001-21000000 8834 C rs56270168 single 22:20000001-21000000 8887 T rs737985 single 22:20000001-21000000 9077 T rs4819854 single 22:20000001-21000000 9927 C rs2518827 single 22:20000001-21000000 10729 G rs56345620 single 22:20000001-21000000 10818 C rs4819855 single 22:20000001-21000000 10849 A rs55725292 single 22:20000001-21000000 10922 A rs9605049 single 22:20000001-21000000 11158 T rs145679795 single 22:20000001-21000000 11317 G rs1476771 single 22:20000001-21000000 11473 G rs5746852 single 22:20000001-21000000 11836 C rs2518828 single 22:20000001-21000000 11988 A rs113192851 single 22:20000001-21000000 12021 T rs2518829 single 22:20000001-21000000 12285 G rs189209957 single 22:20000001-21000000 12863 T rs9754500 single 22:20000001-21000000 12956 A rs114176759 single 22:20000001-21000000 12998 A rs2531719 single 22:20000001-21000000 13221 G rs8142163 single 22:20000001-21000000 13288 A rs112567206 single 22:20000001-21000000 13681 T rs12165568 single 22:20000001-21000000 13776 T rs2531720 single 22:20000001-21000000 13802 C rs9606211 single 22:20000001-21000000 14031 C rs41326345 single 22:20000001-21000000 14060 G rs5748504 single 22:20000001-21000000 14151 G rs80254054 single 22:20000001-21000000 14197 G rs2531705 single 22:20000001-21000000 14403 C rs2531706 single 22:20000001-21000000 14472 C rs5748505 single 22:20000001-21000000 15015 T rs73389717 single 22:20000001-21000000 15227 C rs4629255 single 22:20000001-21000000 15390 A rs2520732 single 22:20000001-21000000 15510 T rs9606212 single 22:20000001-21000000 15614 A rs5748507 single 22:20000001-21000000 15740 T rs2531707 single 22:20000001-21000000 15973 C rs7290592 single 22:20000001-21000000 16011 G rs5748508 single 22:20000001-21000000 16491 A rs9606213 single 22:20000001-21000000 16970 A rs2531721 single 22:20000001-21000000 17267 G rs73389721 single 22:20000001-21000000 17349 C rs2531708 single 22:20000001-21000000 17444 C rs2531722 single 22:20000001-21000000 17552 A rs556782134 deletion 22:20000001-21000000 17673 1 rs72195121 deletion 22:20000001-21000000 17857 4 rs9605050 single 22:20000001-21000000 18102 T rs200431361 deletion 22:20000001-21000000 18131 3 rs143984612 single 22:20000001-21000000 18691 A rs572490569 insertion 22:20000001-21000000 18720 A rs9606219 single 22:20000001-21000000 19177 T rs2078749 single 22:20000001-21000000 19767 G rs887205 single 22:20000001-21000000 20072 G rs2008591 single 22:20000001-21000000 20228 T rs76656759 single 22:20000001-21000000 20408 G rs117194710 single 22:20000001-21000000 20600 A rs117194710 single 22:20000001-21000000 20600 T rs112637156 single 22:20000001-21000000 20767 G rs79885628 single 22:20000001-21000000 20882 C rs114783249 single 22:20000001-21000000 21229 T rs142921548 single 22:20000001-21000000 21659 A rs2531709 single 22:20000001-21000000 21695 G rs72211264 insertion 22:20000001-21000000 21730 AC rs6518597 single 22:20000001-21000000 21862 T rs6518598 single 22:20000001-21000000 21937 T rs75566222 single 22:20000001-21000000 21984 A rs78880579 single 22:20000001-21000000 22093 A rs2520733 single 22:20000001-21000000 22497 G rs62222174 single 22:20000001-21000000 22745 T rs73150855 single 22:20000001-21000000 22839 T rs4819858 single 22:20000001-21000000 23009 C rs2531710 single 22:20000001-21000000 23086 T rs2531711 single 22:20000001-21000000 23302 G rs2531712 single 22:20000001-21000000 23443 T rs5748510 single 22:20000001-21000000 23631 A rs2520734 single 22:20000001-21000000 23730 T rs2531713 single 22:20000001-21000000 24173 G rs74628130 single 22:20000001-21000000 24900 T rs112148654 deletion 22:20000001-21000000 25176 1 rs9617858 single 22:20000001-21000000 25310 T rs73150856 single 22:20000001-21000000 25651 T rs9606221 single 22:20000001-21000000 25766 T rs117607297 single 22:20000001-21000000 25852 A rs11703292 single 22:20000001-21000000 25950 T rs62222175 single 22:20000001-21000000 26206 A rs71691029 deletion 22:20000001-21000000 26448 1 rs7288316 single 22:20000001-21000000 26524 G rs116356549 single 22:20000001-21000000 26557 A rs11361191 deletion 22:20000001-21000000 26623 1 rs115650955 single 22:20000001-21000000 26676 A rs114089161 single 22:20000001-21000000 26757 T rs393947 single 22:20000001-21000000 26916 C rs16982922 single 22:20000001-21000000 27269 A rs114114568 single 22:20000001-21000000 27321 T rs148174824 single 22:20000001-21000000 27456 C rs143084532 single 22:20000001-21000000 27603 C rs389471 single 22:20000001-21000000 28540 A rs7364127 single 22:20000001-21000000 29082 T rs695326 single 22:20000001-21000000 29259 C rs79343076 deletion 22:20000001-21000000 29411 1 rs58838155 single 22:20000001-21000000 29538 G rs374225 single 22:20000001-21000000 29877 C rs114456898 single 22:20000001-21000000 29953 T rs77937532 single 22:20000001-21000000 30040 A rs148393048 single 22:20000001-21000000 30173 C rs113697305 single 22:20000001-21000000 30656 T rs409519 single 22:20000001-21000000 30748 A rs446388 single 22:20000001-21000000 30979 T rs45548038 single 22:20000001-21000000 31015 C rs12627769 single 22:20000001-21000000 31101 A rs17817665 single 22:20000001-21000000 31663 A rs77046301 single 22:20000001-21000000 31849 G rs140487675 single 22:20000001-21000000 31908 T rs455049 single 22:20000001-21000000 31963 T rs62222177 single 22:20000001-21000000 32006 G rs138004901 deletion 22:20000001-21000000 32054 4 rs397701 single 22:20000001-21000000 32326 C rs17210273 single 22:20000001-21000000 32575 A rs62222178 single 22:20000001-21000000 32679 G rs375897671 deletion 22:20000001-21000000 32795 1 rs111315345 single 22:20000001-21000000 32833 C rs570570040 deletion 22:20000001-21000000 33022 2 rs143413803 single 22:20000001-21000000 33734 A rs114166464 single 22:20000001-21000000 33756 G rs74752779 single 22:20000001-21000000 34313 C rs4819860 single 22:20000001-21000000 34390 T rs148446823 single 22:20000001-21000000 34818 T rs118008587 single 22:20000001-21000000 35027 T rs76173340 single 22:20000001-21000000 35150 T rs79375034 single 22:20000001-21000000 35263 A rs368880 single 22:20000001-21000000 35689 T rs78882292 single 22:20000001-21000000 36513 A rs80146538 single 22:20000001-21000000 36694 G rs138276244 single 22:20000001-21000000 37107 A rs405342 single 22:20000001-21000000 37314 A rs115571782 single 22:20000001-21000000 37506 T rs147134283 single 22:20000001-21000000 37697 T rs111797664 single 22:20000001-21000000 37819 T rs695500 single 22:20000001-21000000 37890 G rs114758499 single 22:20000001-21000000 38296 A rs114758499 single 22:20000001-21000000 38296 T rs79393192 single 22:20000001-21000000 38491 G rs76309774 single 22:20000001-21000000 38721 G rs114226275 single 22:20000001-21000000 38755 C rs174875 single 22:20000001-21000000 39154 A rs189306965 single 22:20000001-21000000 39634 T rs115084231 single 22:20000001-21000000 39717 A rs3804046 single 22:20000001-21000000 39866 T rs77399579 single 22:20000001-21000000 40219 T rs28635282 single 22:20000001-21000000 40539 A rs17817689 single 22:20000001-21000000 40601 T rs695727 insertion 22:20000001-21000000 40821 G rs16982925 single 22:20000001-21000000 41231 G rs366148 single 22:20000001-21000000 41608 T rs115208925 single 22:20000001-21000000 41703 C rs73877127 single 22:20000001-21000000 41735 C rs429965 single 22:20000001-21000000 42074 G rs57180287 single 22:20000001-21000000 42113 A rs758376 single 22:20000001-21000000 42562 A rs61371564 single 22:20000001-21000000 42847 G rs75170603 single 22:20000001-21000000 43100 G rs16982930 single 22:20000001-21000000 43358 C rs113449283 single 22:20000001-21000000 43699 G rs75011475 single 22:20000001-21000000 44040 A rs62222179 single 22:20000001-21000000 44932 A rs73877128 single 22:20000001-21000000 45382 A rs440042 single 22:20000001-21000000 45410 G rs201660291 insertion 22:20000001-21000000 45552 TA rs5748514 single 22:20000001-21000000 45738 A rs59609897 single 22:20000001-21000000 45882 C rs61095784 single 22:20000001-21000000 45966 A rs8140658 single 22:20000001-21000000 46041 G rs148529668 single 22:20000001-21000000 46149 A rs5748515 single 22:20000001-21000000 46343 G rs399162 single 22:20000001-21000000 46686 A rs140134708 single 22:20000001-21000000 47097 A rs58308907 single 22:20000001-21000000 47586 C rs142549904 single 22:20000001-21000000 47765 A rs111276581 single 22:20000001-21000000 47814 C rs111323873 single 22:20000001-21000000 47850 T rs76518635 single 22:20000001-21000000 47972 T rs75418691 single 22:20000001-21000000 48082 C rs113509411 single 22:20000001-21000000 48446 A rs73877129 single 22:20000001-21000000 48646 C rs73389740 single 22:20000001-21000000 48862 T rs61737483 single 22:20000001-21000000 49149 A rs61737483 single 22:20000001-21000000 49149 C rs2286481 single 22:20000001-21000000 49243 T rs2286482 single 22:20000001-21000000 49284 A rs410655 single 22:20000001-21000000 49339 C rs149412137 insertion 22:20000001-21000000 49414 GGCCCA rs426894 single 22:20000001-21000000 49453 G rs2286484 single 22:20000001-21000000 49494 A rs417981 single 22:20000001-21000000 49934 C rs74276823 single 22:20000001-21000000 50018 T rs419135 single 22:20000001-21000000 50400 C rs111669330 single 22:20000001-21000000 50699 T rs73389745 single 22:20000001-21000000 50956 T rs73389746 single 22:20000001-21000000 50978 A rs432909 single 22:20000001-21000000 51465 A rs115297588 single 22:20000001-21000000 51927 T rs75305197 single 22:20000001-21000000 52049 T rs16982614 single 22:20000001-21000000 52088 G rs34082990 single 22:20000001-21000000 52142 T rs3804045 single 22:20000001-21000000 52301 A rs17818431 single 22:20000001-21000000 52419 A rs3804044 single 22:20000001-21000000 52441 G rs3804043 single 22:20000001-21000000 52480 A rs415520 single 22:20000001-21000000 52531 G rs442277 single 22:20000001-21000000 52565 A rs367450 single 22:20000001-21000000 52781 T rs140840130 single 22:20000001-21000000 53008 T rs11555967 single 22:20000001-21000000 53153 T rs1054215 single 22:20000001-21000000 53553 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hisat2-2.2.1/extract_exons.py000077700000000000000000000000001370663763200226702hisat2_extract_exons.pyustar00rootroot00000000000000hisat2-2.2.1/extract_splice_sites.py000077700000000000000000000000001370663763200255542hisat2_extract_splice_sites.pyustar00rootroot00000000000000hisat2-2.2.1/fast_mutex.h000077500000000000000000000207021370663763200152570ustar00rootroot00000000000000/* -*- mode: c++; tab-width: 2; indent-tabs-mode: nil; -*- Copyright (c) 2010-2012 Marcus Geelnard This software is provided 'as-is', without any express or implied warranty. In no event will the authors be held liable for any damages arising from the use of this software. Permission is granted to anyone to use this software for any purpose, including commercial applications, and to alter it and redistribute it freely, subject to the following restrictions: 1. The origin of this software must not be misrepresented; you must not claim that you wrote the original software. If you use this software in a product, an acknowledgment in the product documentation would be appreciated but is not required. 2. Altered source versions must be plainly marked as such, and must not be misrepresented as being the original software. 3. This notice may not be removed or altered from any source distribution. ***************************************************************************** Copyright (c) 2016 Nigel Dyer This version has been modified from the original, whose original Copyright notice is reproduced above. The permissions continue as above. The software has been modified in three ways: a) mHandle is now a mutable pointer variable which is passed to the copy in the new =operator. This allows it to be pushed into a vector or equivalent It is not intended to allow multiple copies of a mutex to be created b) The code for 64bit MSC builds uses InterlockedExchange which provides more efficient access to the xchg assembler instruction than implementing the code as a function in a separate .asm file. MSC does not support in line assembler for 64 bit builds. c) The NO_FAST_MUTEX_ASM option has been introduced */ #ifndef _FAST_MUTEX_H_ #define _FAST_MUTEX_H_ /// @file // Which platform are we on? #if !defined(_TTHREAD_PLATFORM_DEFINED_) #if defined(_WIN32) || defined(__WIN32__) || defined(__WINDOWS__) #define _TTHREAD_WIN32_ #else #define _TTHREAD_POSIX_ #endif #define _TTHREAD_PLATFORM_DEFINED_ #endif // Check if we can support the assembly language level implementation (otherwise // revert to the system API) #if !defined NO_FAST_MUTEX_ASM && ((defined(__GNUC__) && (defined(__i386__) || defined(__x86_64__))) || \ (defined(_MSC_VER) && (defined(_M_IX86) || defined(_M_X64))) || \ (defined(__GNUC__) && (defined(__ppc__)))) #define _FAST_MUTEX_ASM_ #else #define _FAST_MUTEX_SYS_ #endif #if defined(_TTHREAD_WIN32_) #ifndef WIN32_LEAN_AND_MEAN #define WIN32_LEAN_AND_MEAN #define __UNDEF_LEAN_AND_MEAN #endif #include #ifdef __UNDEF_LEAN_AND_MEAN #undef WIN32_LEAN_AND_MEAN #undef __UNDEF_LEAN_AND_MEAN #endif #else #ifdef _FAST_MUTEX_ASM_ #include #else #include #endif #endif namespace tthread { /// Fast mutex class. /// This is a mutual exclusion object for synchronizing access to shared /// memory areas for several threads. It is similar to the tthread::mutex class, /// but instead of using system level functions, it is implemented as an atomic /// spin lock with very low CPU overhead. /// /// The \c fast_mutex class is NOT compatible with the \c condition_variable /// class (however, it IS compatible with the \c lock_guard class). It should /// also be noted that the \c fast_mutex class typically does not provide /// as accurate thread scheduling as a the standard \c mutex class does. /// /// Because of the limitations of the class, it should only be used in /// situations where the mutex needs to be locked/unlocked very frequently. /// /// @note The "fast" version of this class relies on inline assembler language, /// which is currently only supported for 32/64-bit Intel x86/AMD64 and /// PowerPC architectures on a limited number of compilers (GNU g++ and MS /// Visual C++). /// For other architectures/compilers, system functions are used instead. class fast_mutex { public: /// Constructor. #if defined(_FAST_MUTEX_ASM_) fast_mutex() : mLock(0) {} #else fast_mutex() { #if defined(_TTHREAD_WIN32_) mHandle = new CRITICAL_SECTION(); InitializeCriticalSection(mHandle); #elif defined(_TTHREAD_POSIX_) mHandle = new pthread_mutex_t(); pthread_mutex_init(mHandle, NULL); #endif } #endif #if !defined(_FAST_MUTEX_ASM_) /// Destructor. ~fast_mutex() { if (mHandle) { #if defined(_TTHREAD_WIN32_) DeleteCriticalSection(mHandle); #elif defined(_TTHREAD_POSIX_) pthread_mutex_destroy(mHandle); #endif delete mHandle; mHandle = 0; } } /// The handle is passed from the source to the desitination /// Used primarily when mutexes are pushed onto a List either on their /// own or as a member variable of another classs fast_mutex & operator = (const fast_mutex& fm) { mHandle = fm.mHandle; fm.mHandle = 0; return *this; } #endif /// Lock the mutex. /// The method will block the calling thread until a lock on the mutex can /// be obtained. The mutex remains locked until \c unlock() is called. /// @see lock_guard inline void lock() { #if defined(_FAST_MUTEX_ASM_) bool gotLock; do { gotLock = try_lock(); if(!gotLock) { #if defined(_TTHREAD_WIN32_) Sleep(0); #elif defined(_TTHREAD_POSIX_) sched_yield(); #endif } } while(!gotLock); #else #if defined(_TTHREAD_WIN32_) EnterCriticalSection(mHandle); #elif defined(_TTHREAD_POSIX_) pthread_mutex_lock(mHandle); #endif #endif } /// Try to lock the mutex. /// The method will try to lock the mutex. If it fails, the function will /// return immediately (non-blocking). /// @return \c true if the lock was acquired, or \c false if the lock could /// not be acquired. inline bool try_lock() { #if defined(_FAST_MUTEX_ASM_) int oldLock; #if defined(__GNUC__) && (defined(__i386__) || defined(__x86_64__)) asm volatile ( "movl $1,%%eax\n\t" "xchg %%eax,%0\n\t" "movl %%eax,%1\n\t" : "=m" (mLock), "=m" (oldLock) : : "%eax", "memory" ); #elif defined(_MSC_VER) #if defined(_M_IX86) int *ptrLock = &mLock; __asm { mov eax, 1 mov ecx, ptrLock xchg eax, [ecx] mov oldLock, eax } #elif defined(_M_X64) oldLock = InterlockedExchange(&mLock, 1); #endif #elif defined(__GNUC__) && (defined(__ppc__)) int newLock = 1; asm volatile ( "\n1:\n\t" "lwarx %0,0,%1\n\t" "cmpwi 0,%0,0\n\t" "bne- 2f\n\t" "stwcx. %2,0,%1\n\t" "bne- 1b\n\t" "isync\n" "2:\n\t" : "=&r" (oldLock) : "r" (&mLock), "r" (newLock) : "cr0", "memory" ); #endif return (oldLock == 0); #else #if defined(_TTHREAD_WIN32_) return TryEnterCriticalSection(mHandle) ? true : false; #elif defined(_TTHREAD_POSIX_) return (pthread_mutex_trylock(mHandle) == 0) ? true : false; #endif #endif } /// Unlock the mutex. /// If any threads are waiting for the lock on this mutex, one of them will /// be unblocked. inline void unlock() { #if defined(_FAST_MUTEX_ASM_) #if defined(__GNUC__) && (defined(__i386__) || defined(__x86_64__)) asm volatile ( "movl $0,%%eax\n\t" "xchg %%eax,%0\n\t" : "=m" (mLock) : : "%eax", "memory" ); #elif defined(_MSC_VER) #if defined(_M_IX86) int *ptrLock = &mLock; __asm { mov eax,0 mov ecx,ptrLock xchg eax,[ecx] } #elif defined(_M_X64) InterlockedExchange(&mLock, 0); #endif #elif defined(__GNUC__) && (defined(__ppc__)) asm volatile ( "sync\n\t" // Replace with lwsync where possible? : : : "memory" ); mLock = 0; #endif #else #if defined(_TTHREAD_WIN32_) LeaveCriticalSection(mHandle); #elif defined(_TTHREAD_POSIX_) pthread_mutex_unlock(mHandle); #endif #endif } private: #if defined(_FAST_MUTEX_ASM_) #if defined(_M_X64) && defined (_MSC_VER) long mLock; #else int mLock; #endif #else #if defined(_TTHREAD_WIN32_) mutable CRITICAL_SECTION * mHandle; #elif defined(_TTHREAD_POSIX_) mutable pthread_mutex_t * mHandle; #endif #endif }; } #endif // _FAST_MUTEX_H_ hisat2-2.2.1/filebuf.h000066400000000000000000000370611370663763200145170ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef FILEBUF_H_ #define FILEBUF_H_ #include #include #include #include #include #include #include #include "assert_helpers.h" /** * Simple, fast helper for determining if a character is a newline. */ static inline bool isnewline(int c) { return c == '\r' || c == '\n'; } /** * Simple, fast helper for determining if a character is a non-newline * whitespace character. */ static inline bool isspace_notnl(int c) { return isspace(c) && !isnewline(c); } /** * Simple wrapper for a FILE*, istream or ifstream that reads it in chunks * using fread and keeps those chunks in a buffer. It also services calls to * get(), peek() and gets() from the buffer, reading in additional chunks when * necessary. * * Helper functions do things like parse strings, numbers, and FASTA records. * * */ class FileBuf { public: FileBuf() { init(); } FileBuf(FILE *in) { init(); _in = in; assert(_in != NULL); } FileBuf(std::ifstream *inf) { init(); _inf = inf; assert(_inf != NULL); } FileBuf(std::istream *ins) { init(); _ins = ins; assert(_ins != NULL); } /** * Return true iff there is a stream ready to read. */ bool isOpen() { return _in != NULL || _inf != NULL || _ins != NULL; } /** * Close the input stream (if that's possible) */ void close() { if(_in != NULL && _in != stdin) { fclose(_in); } else if(_inf != NULL) { _inf->close(); } else { // can't close _ins } } /** * Get the next character of input and advance. */ int get() { assert(_in != NULL || _inf != NULL || _ins != NULL); int c = peek(); if(c != -1) { _cur++; if(_lastn_cur < LASTN_BUF_SZ) _lastn_buf[_lastn_cur++] = c; } return c; } /** * Return true iff all input is exhausted. */ bool eof() { return (_cur == _buf_sz) && _done; } /** * Initialize the buffer with a new C-style file. */ void newFile(FILE *in) { _in = in; _inf = NULL; _ins = NULL; _cur = BUF_SZ; _buf_sz = BUF_SZ; _done = false; } /** * Initialize the buffer with a new ifstream. */ void newFile(std::ifstream *__inf) { _in = NULL; _inf = __inf; _ins = NULL; _cur = BUF_SZ; _buf_sz = BUF_SZ; _done = false; } /** * Initialize the buffer with a new istream. */ void newFile(std::istream *__ins) { _in = NULL; _inf = NULL; _ins = __ins; _cur = BUF_SZ; _buf_sz = BUF_SZ; _done = false; } /** * Restore state as though we just started reading the input * stream. */ void reset() { if(_inf != NULL) { _inf->clear(); _inf->seekg(0, std::ios::beg); } else if(_ins != NULL) { _ins->clear(); _ins->seekg(0, std::ios::beg); } else { rewind(_in); } _cur = BUF_SZ; _buf_sz = BUF_SZ; _done = false; } /** * Peek at the next character of the input stream without * advancing. Typically we can simple read it from the buffer. * Occasionally we'll need to read in a new buffer's worth of data. */ int peek() { assert(_in != NULL || _inf != NULL || _ins != NULL); assert_leq(_cur, _buf_sz); if(_cur == _buf_sz) { if(_done) { // We already exhausted the input stream return -1; } // Read a new buffer's worth of data else { // Get the next chunk if(_inf != NULL) { _inf->read((char*)_buf, BUF_SZ); _buf_sz = _inf->gcount(); } else if(_ins != NULL) { _ins->read((char*)_buf, BUF_SZ); _buf_sz = _ins->gcount(); } else { assert(_in != NULL); _buf_sz = fread(_buf, 1, BUF_SZ, _in); } _cur = 0; if(_buf_sz == 0) { // Exhausted, and we have nothing to return to the // caller _done = true; return -1; } else if(_buf_sz < BUF_SZ) { // Exhausted _done = true; } } } return (int)_buf[_cur]; } /** * Store a string of characters from the input file into 'buf', * until we see a newline, EOF, or until 'len' characters have been * read. */ size_t gets(char *buf, size_t len) { size_t stored = 0; while(true) { int c = get(); if(c == -1) { // End-of-file buf[stored] = '\0'; return stored; } if(stored == len-1 || isnewline(c)) { // End of string buf[stored] = '\0'; // Skip over all end-of-line characters int pc = peek(); while(isnewline(pc)) { get(); // discard pc = peek(); } // Next get() will be after all newline characters return stored; } buf[stored++] = (char)c; } } /** * Store a string of characters from the input file into 'buf', * until we see a newline, EOF, or until 'len' characters have been * read. */ size_t get(char *buf, size_t len) { size_t stored = 0; for(size_t i = 0; i < len; i++) { int c = get(); if(c == -1) return i; buf[stored++] = (char)c; } return len; } static const size_t LASTN_BUF_SZ = 8 * 1024; /** * Keep get()ing characters until a non-whitespace character (or * -1) is reached, and return it. */ int getPastWhitespace() { int c; while(isspace(c = get()) && c != -1); return c; } /** * Keep get()ing characters until a we've passed over the next * string of newline characters (\r's and \n's) or -1 is reached, * and return it. */ int getPastNewline() { int c = get(); while(!isnewline(c) && c != -1) c = get(); while(isnewline(c)) c = get(); assert_neq(c, '\r'); assert_neq(c, '\n'); return c; } /** * Keep get()ing characters until a we've passed over the next * string of newline characters (\r's and \n's) or -1 is reached, * and return it. */ int peekPastNewline() { int c = peek(); while(!isnewline(c) && c != -1) c = get(); while(isnewline(c)) c = get(); assert_neq(c, '\r'); assert_neq(c, '\n'); return c; } /** * Keep peek()ing then get()ing characters until the next return * from peek() is just after the last newline of the line. */ int peekUptoNewline() { int c = peek(); while(!isnewline(c) && c != -1) { get(); c = peek(); } while(isnewline(c)) { get(); c = peek(); } assert_neq(c, '\r'); assert_neq(c, '\n'); return c; } /** * Parse a FASTA record. Append name characters to 'name' and and append * all sequence characters to 'seq'. If gotCaret is true, assuming the * file cursor has already moved just past the starting '>' character. */ template void parseFastaRecord( TNameStr& name, TSeqStr& seq, bool gotCaret = false) { int c; if(!gotCaret) { // Skip over caret and non-newline whitespace c = peek(); while(isspace_notnl(c) || c == '>') { get(); c = peek(); } } else { // Skip over non-newline whitespace c = peek(); while(isspace_notnl(c)) { get(); c = peek(); } } size_t namecur = 0, seqcur = 0; // c is the first character of the fasta name record, or is the first // newline character if the name record is empty while(!isnewline(c) && c != -1) { name[namecur++] = c; get(); c = peek(); } // sequence consists of all the non-whitespace characters between here // and the next caret while(true) { // skip over whitespace while(isspace(c)) { get(); c = peek(); } // if we see caret or EOF, break if(c == '>' || c == -1) break; // append and continue seq[seqcur++] = c; get(); c = peek(); } } /** * Parse a FASTA record and return its length. If gotCaret is true, * assuming the file cursor has already moved just past the starting '>' * character. */ void parseFastaRecordLength( size_t& nameLen, size_t& seqLen, bool gotCaret = false) { int c; nameLen = seqLen = 0; if(!gotCaret) { // Skip over caret and non-newline whitespace c = peek(); while(isspace_notnl(c) || c == '>') { get(); c = peek(); } } else { // Skip over non-newline whitespace c = peek(); while(isspace_notnl(c)) { get(); c = peek(); } } // c is the first character of the fasta name record, or is the first // newline character if the name record is empty while(!isnewline(c) && c != -1) { nameLen++; get(); c = peek(); } // sequence consists of all the non-whitespace characters between here // and the next caret while(true) { // skip over whitespace while(isspace(c)) { get(); c = peek(); } // if we see caret or EOF, break if(c == '>' || c == -1) break; // append and continue seqLen++; get(); c = peek(); } } /** * Reset to the beginning of the last-N-chars buffer. */ void resetLastN() { _lastn_cur = 0; } /** * Copy the last several characters in the last-N-chars buffer * (since the last reset) into the provided buffer. */ size_t copyLastN(char *buf) { memcpy(buf, _lastn_buf, _lastn_cur); return _lastn_cur; } /** * Get const pointer to the last-N-chars buffer. */ const char *lastN() const { return _lastn_buf; } /** * Get current size of the last-N-chars buffer. */ size_t lastNLen() const { return _lastn_cur; } private: void init() { _in = NULL; _inf = NULL; _ins = NULL; _cur = _buf_sz = BUF_SZ; _done = false; _lastn_cur = 0; // no need to clear _buf[] } static const size_t BUF_SZ = 256 * 1024; FILE *_in; std::ifstream *_inf; std::istream *_ins; size_t _cur; size_t _buf_sz; bool _done; uint8_t _buf[BUF_SZ]; // (large) input buffer size_t _lastn_cur; char _lastn_buf[LASTN_BUF_SZ]; // buffer of the last N chars dispensed }; /** * Wrapper for a buffered output stream that writes bitpairs. */ class BitpairOutFileBuf { public: /** * Open a new output stream to a file with given name. */ BitpairOutFileBuf(const char *in) : bpPtr_(0), cur_(0) { assert(in != NULL); out_ = fopen(in, "wb"); if(out_ == NULL) { std::cerr << "Error: Could not open bitpair-output file " << in << std::endl; throw 1; } memset(buf_, 0, BUF_SZ); } /** * Write a single bitpair into the buf. Flush the buffer if it's * full. */ void write(int bp) { assert_lt(bp, 4); assert_geq(bp, 0); buf_[cur_] |= (bp << bpPtr_); if(bpPtr_ == 6) { bpPtr_ = 0; cur_++; if(cur_ == BUF_SZ) { // Flush the buffer if(!fwrite((const void *)buf_, BUF_SZ, 1, out_)) { std::cerr << "Error writing to the reference index file (.4.ebwt)" << std::endl; throw 1; } // Reset to beginning of the buffer cur_ = 0; } // Initialize next octet to 0 buf_[cur_] = 0; } else { bpPtr_ += 2; } } /** * Write any remaining bitpairs and then close the input */ void close() { if(cur_ > 0 || bpPtr_ > 0) { if(bpPtr_ == 0) cur_--; if(!fwrite((const void *)buf_, cur_ + 1, 1, out_)) { std::cerr << "Error writing to the reference index file (.4.ebwt)" << std::endl; throw 1; } } fclose(out_); } private: static const size_t BUF_SZ = 128 * 1024; FILE *out_; int bpPtr_; size_t cur_; char buf_[BUF_SZ]; // (large) input buffer }; /** * Wrapper for a buffered output stream that writes characters and * other data types. This class is *not* synchronized; the caller is * responsible for synchronization. */ class OutFileBuf { public: /** * Open a new output stream to a file with given name. */ OutFileBuf(const std::string& out, bool binary = false) : name_(out.c_str()), cur_(0), closed_(false) { out_ = fopen(out.c_str(), binary ? "wb" : "w"); if(out_ == NULL) { std::cerr << "Error: Could not open alignment output file " << out.c_str() << std::endl; throw 1; } if(setvbuf(out_, NULL, _IOFBF, 10* 1024* 1024)) std::cerr << "Warning: Could not allocate the proper buffer size for output file stream. " << std::endl; } /** * Open a new output stream to a file with given name. */ OutFileBuf(const char *out, bool binary = false) : name_(out), cur_(0), closed_(false) { assert(out != NULL); out_ = fopen(out, binary ? "wb" : "w"); if(out_ == NULL) { std::cerr << "Error: Could not open alignment output file " << out << std::endl; throw 1; } } /** * Open a new output stream to standard out. */ OutFileBuf() : name_("cout"), cur_(0), closed_(false) { out_ = stdout; } /** * Close buffer when object is destroyed. */ ~OutFileBuf() { close(); } /** * Open a new output stream to a file with given name. */ void setFile(const char *out, bool binary = false) { assert(out != NULL); out_ = fopen(out, binary ? "wb" : "w"); if(out_ == NULL) { std::cerr << "Error: Could not open alignment output file " << out << std::endl; throw 1; } reset(); } /** * Write a single character into the write buffer and, if * necessary, flush. */ void write(char c) { assert(!closed_); if(cur_ == BUF_SZ) flush(); buf_[cur_++] = c; } /** * Write a c++ string to the write buffer and, if necessary, flush. */ void writeString(const std::string& s) { assert(!closed_); size_t slen = s.length(); if(cur_ + slen > BUF_SZ) { if(cur_ > 0) flush(); if(slen >= BUF_SZ) { fwrite(s.c_str(), slen, 1, out_); } else { memcpy(&buf_[cur_], s.data(), slen); assert_eq(0, cur_); cur_ = slen; } } else { memcpy(&buf_[cur_], s.data(), slen); cur_ += slen; } assert_leq(cur_, BUF_SZ); } /** * Write a c++ string to the write buffer and, if necessary, flush. */ template void writeString(const T& s) { assert(!closed_); size_t slen = s.length(); if(cur_ + slen > BUF_SZ) { if(cur_ > 0) flush(); if(slen >= BUF_SZ) { fwrite(s.toZBuf(), slen, 1, out_); } else { memcpy(&buf_[cur_], s.toZBuf(), slen); assert_eq(0, cur_); cur_ = slen; } } else { memcpy(&buf_[cur_], s.toZBuf(), slen); cur_ += slen; } assert_leq(cur_, BUF_SZ); } /** * Write a c++ string to the write buffer and, if necessary, flush. */ void writeChars(const char * s, size_t len) { assert(!closed_); if(cur_ + len > BUF_SZ) { if(cur_ > 0) flush(); if(len >= BUF_SZ) { fwrite(s, len, 1, out_); } else { memcpy(&buf_[cur_], s, len); assert_eq(0, cur_); cur_ = len; } } else { memcpy(&buf_[cur_], s, len); cur_ += len; } assert_leq(cur_, BUF_SZ); } /** * Write a 0-terminated C string to the output stream. */ void writeChars(const char * s) { writeChars(s, strlen(s)); } /** * Write any remaining bitpairs and then close the input */ void close() { if(closed_) return; if(cur_ > 0) flush(); closed_ = true; if(out_ != stdout) { fclose(out_); } } /** * Reset so that the next write is as though it's the first. */ void reset() { cur_ = 0; closed_ = false; } void flush() { if(!fwrite((const void *)buf_, cur_, 1, out_)) { std::cerr << "Error while flushing and closing output" << std::endl; throw 1; } cur_ = 0; } /** * Return true iff this stream is closed. */ bool closed() const { return closed_; } /** * Return the filename. */ const char *name() { return name_; } private: static const size_t BUF_SZ = 16 * 1024; const char *name_; FILE *out_; size_t cur_; char buf_[BUF_SZ]; // (large) input buffer bool closed_; }; #endif /*ndef FILEBUF_H_*/ hisat2-2.2.1/formats.h000066400000000000000000000022701370663763200145500ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef FORMATS_H_ #define FORMATS_H_ #include /** * File-format constants and names */ enum file_format { FASTA = 1, FASTA_CONT, FASTQ, TAB_MATE5, TAB_MATE6, RAW, CMDLINE, QSEQ, SRA_FASTA, SRA_FASTQ }; static const std::string file_format_names[] = { "Invalid!", "FASTA", "FASTA sampling", "FASTQ", "Tabbed mated", "Raw", "Command line", "Chain file", "Random", "Qseq", "SRA_FASTA", "SRA_FASTQ" }; #endif /*FORMATS_H_*/ hisat2-2.2.1/gbwt_graph.h000066400000000000000000003156161370663763200152340ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim , Joe Paggi * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef GBWT_GRAPH_H_ #define GBWT_GRAPH_H_ #include #include #include #include #include #include #include #include #include "alt.h" #include "radix_sort.h" // Reference: // Jouni Sirén, Niko Välimäki, and Veli Mäkinen: Indexing Graphs for Path Queries with Applications in Genome Research. // IEEE/ACM Transactions on Computational Biology and Bioinformatics 11(2):375-388, 2014. // http://ieeexplore.ieee.org/xpl/articleDetails.jsp?arnumber=6698337 //-------------------------------------------------------------------------- struct NongraphException : public exception { const char* what () const throw () { return "Nongraph exception"; } }; struct ExplosionException : public exception { const char* what () const throw () { return "Explosion exception"; } }; template class PathGraph; // Note: I wrote the following codes based on Siren's work, gcsa (see the reference above). template class RefGraph { friend class PathGraph; public: struct Node { char label; // ACGT + Y(head) + Z(tail) index_t value; // location in a whole genome Node() { reset(); } Node(char label_, index_t value_) : label(label_), value(value_) {} void reset() { label = 0; value = 0; } bool write(ofstream& f_out, bool bigEndian) const { writeIndex(f_out, value, bigEndian); writeU16(f_out, label, bigEndian); return true; } bool read(ifstream& f_in, bool bigEndian) { value = readIndex(f_in, bigEndian); label = (char)readU16(f_in, bigEndian); return true; } bool operator== (const Node& o) const { if(value != o.value) return false; if(label != o.label) return false; return true; } bool operator< (const Node& o) const { if(value != o.value) return value < o.value; return label < o.label; } }; struct Edge { index_t from; // from Node index_t to; // to Node Edge() {} Edge(index_t from_, index_t to_) : from(from_), to(to_) {} bool write(ofstream& f_out, bool bigEndian) const { writeIndex(f_out, from, bigEndian); writeIndex(f_out, to, bigEndian); return true; } bool read(ifstream& f_in, bool bigEndian) { from = readIndex(f_in, bigEndian); to = readIndex(f_in, bigEndian); return true; } bool operator< (const Edge& o) const { if(from != o.from) return from < o.from; return to < o.to; } }; static index_t EdgeTo (Edge& a) { return a.to; } struct EdgeFromCmp { bool operator() (const Edge& a, const Edge& b) const { return a.from < b.from; } }; struct EdgeToCmp { bool operator() (const Edge& a, const Edge& b) const { return a.to < b.to; }; }; public: RefGraph(const SString& s, const EList& szs, const EList >& alts, const EList >& haplotypes, const string& out_fname, int nthreads_, bool verbose); bool repOk() { return true; } void write(const string& fname, bool bigEndian) { ofstream rg_file(fname.c_str(), ios::binary); if(!rg_file.good()) { cerr << "Could not open file for writing a reference graph: \"" << fname << "\"" << endl; throw 1; } writeIndex(rg_file, (index_t)nodes.size(), bigEndian); for(index_t i = 0; i < nodes.size(); i++) { nodes[i].write(rg_file, bigEndian); } writeIndex(rg_file, (index_t)edges.size(), bigEndian); for(index_t i = 0; i < edges.size(); i++) { edges[i].write(rg_file, bigEndian); } rg_file.close(); } void nullify() { nodes.nullify(); edges.nullify(); } void read(const string& fname, bool bigEndian) { ifstream rg_file(fname.c_str(), ios::binary); if(!rg_file.good()) { cerr << "Could not open file for reading a reference graph: \"" << fname << "\"" << endl; throw 1; } index_t num_nodes = readIndex(rg_file, bigEndian); nodes.resizeNoCopyExact(num_nodes); for(index_t i = 0; i < num_nodes; i++) { nodes[i].read(rg_file, bigEndian); } index_t num_edges = readIndex(rg_file, bigEndian); edges.resizeNoCopyExact(num_edges); for(index_t i = 0; i < num_edges; i++) { edges[i].read(rg_file, bigEndian); } rg_file.close(); } private: static bool isReverseDeterministic(EList& nodes, EList& edges); static void reverseDeterminize(EList& nodes, EList& edges, index_t& lastNode, index_t lastNode_add = 0); static void sortEdgesFrom(EList& edges) { std::sort(edges.begin(), edges.end(), EdgeFromCmp()); } static void sortEdgesTo(EList& edges) { std::sort(edges.begin(), edges.end(), EdgeToCmp()); } // Return edge ranges [begin, end) static pair findEdges(const EList& edges, index_t node, bool from); static pair findEdgesFrom(const EList& edges, index_t node) { return findEdges(edges, node, true); } static pair findEdgesTo(const EList& edges, index_t node) { return findEdges(edges, node, false); } static pair getNextEdgeRange(const EList& sep_edges, pair range, bool from) { if(range.second >= sep_edges.size()) { return pair(0, 0); } range.first = range.second; range.second++; if(from) { while(range.second < sep_edges.size() && sep_edges[range.second].from == sep_edges[range.first].from) { range.second++; } } else { while(range.second < sep_edges.size() && sep_edges[range.second].to == sep_edges[range.first].to) { range.second++; } } return range; } private: struct ThreadParam { // input index_t thread_id; RefGraph* refGraph; const SString* s; const EList >* alts; const EList >* haplotypes; string out_fname; bool bigEndian; // output index_t num_nodes; index_t num_edges; index_t lastNode; bool multipleHeadNodes; }; static void buildGraph_worker(void* vp); private: EList szs; EList tmp_szs; EList nodes; EList edges; index_t lastNode; // Z int nthreads; #ifndef NDEBUG bool debug; #endif private: // Following composite nodes and edges are used to reverse-determinize an automaton. struct CompositeNodeIDs { index_t id; EList add_ids; CompositeNodeIDs() { id = (index_t)INDEX_MAX; } bool operator<(const CompositeNodeIDs& o) const { if(id != o.id) return id < o.id; if(add_ids.size() != o.add_ids.size()) return add_ids.size() < o.add_ids.size(); for(index_t i = 0; i < add_ids.size(); i++) { assert_lt(i, o.add_ids.size()); if(add_ids[i] != o.add_ids[i]) return add_ids[i] < o.add_ids[i]; } return false; } index_t size() const { if(id == (index_t)INDEX_MAX) return 0; return (index_t)add_ids.size() + 1; } index_t getID(index_t i) const { if(i == 0) return id; else { i -= 1; assert_lt(i, add_ids.size()); return add_ids[i]; } } void push_back(index_t node_id) { if(id == (index_t)INDEX_MAX) id = node_id; else add_ids.push_back(node_id); } }; struct CompositeNode { CompositeNodeIDs nodes; index_t id; char label; index_t value; CompositeNode() { reset(); } CompositeNode(char label_, index_t node_id) : id(0), label(label_) { nodes.push_back(node_id); } Node getNode() const { return Node(label, value); } void reset() { nodes.id = (index_t)INDEX_MAX; nodes.add_ids.clear(); id = 0; label = 0; value = 0; } }; struct CompositeEdge { index_t from; index_t to; CompositeEdge() : from(0), to(0) {} CompositeEdge(index_t from_, index_t to_) : from(from_), to(to_) {} Edge getEdge(const EList& nodes) const { assert_lt(from, nodes.size()); const CompositeNode& from_node = nodes[from]; assert_lt(to, nodes.size()); const CompositeNode& to_node = nodes[to]; return Edge(from_node.id, to_node.id); } bool operator < (const CompositeEdge& o) const { return from < o.from; } }; struct TempNodeLabelCmp { TempNodeLabelCmp(const EList& nodes_) : nodes(nodes_) {} bool operator() (index_t a, index_t b) const { assert_lt(a, nodes.size()); assert_lt(b, nodes.size()); return nodes[a].label < nodes[b].label; } const EList& nodes; }; }; /** * Load reference sequence file and alt information. * Construct a reference graph */ template RefGraph::RefGraph(const SString& s, const EList& szs, const EList >& alts, const EList >& haplotypes, const string& out_fname, int nthreads_, bool verbose) : lastNode(0), nthreads(nthreads_) { const bool bigEndian = false; assert_gt(nthreads, 0); assert_gt(szs.size(), 0); index_t jlen = (index_t)s.length(); #ifndef NDEBUG debug = (jlen <= 20); #endif // a memory-efficient way to create a population graph with known ALTs bool frag_automaton = jlen >= (1 << 16); if(frag_automaton) { { EList > alt_ranges; // each range inclusive for(index_t i = 0; i < alts.size(); i++) { const ALT& alt = alts[i]; index_t left_relax = 128, right_relax = 128; pair range; range.first = alt.pos > left_relax ? alt.pos - left_relax - 1 : 0; if(alt.type == ALT_SNP_SGL) { range.second = alt.pos + 1; } else if(alt.type == ALT_SNP_DEL) { assert_gt(alt.len, 0); range.second = alt.pos + alt.len; } else if(alt.type == ALT_SNP_INS) { assert_gt(alt.len, 0); range.second = alt.pos; } else if (alt.type == ALT_SPLICESITE) { assert_lt(alt.left, alt.right); range.second = alt.right + 1; } else { assert(alt.exon()); continue; } range.second += right_relax; if(alt_ranges.empty() || alt_ranges.back().second + 1 < range.first) { alt_ranges.push_back(range); } else { assert_leq(alt_ranges.back().first, range.first); if(alt_ranges.back().second < range.second) { alt_ranges.back().second = range.second; } } } index_t chunk_size = 1 << 20; index_t pos = 0, range_idx = 0; for(index_t i = 0; i < szs.size(); i++) { if(szs[i].len == 0) continue; if(szs[i].len <= chunk_size) { tmp_szs.push_back(szs[i]); pos += szs[i].len; } else { index_t num_chunks = (szs[i].len + chunk_size - 1) / chunk_size; assert_gt(num_chunks, 1); index_t modified_chunk_size = szs[i].len / num_chunks; index_t after_pos = pos + szs[i].len; ASSERT_ONLY(index_t sum_len = 0); while(pos < after_pos) { index_t target_pos = pos + modified_chunk_size; if(target_pos < after_pos) { for(; range_idx < alt_ranges.size(); range_idx++) { if(target_pos < alt_ranges[range_idx].first) break; } pair alt_free_range; if(range_idx == 0) { alt_free_range.first = 0; } else { alt_free_range.first = alt_ranges[range_idx - 1].second + 1; if(alt_free_range.first >= jlen) { alt_free_range.first = jlen - 1; } } if(range_idx == alt_ranges.size()) { alt_free_range.second = jlen - 1; } else { alt_free_range.second = alt_ranges[range_idx].first - 1; } assert_leq(alt_free_range.first, alt_free_range.second); if(target_pos < alt_free_range.first) target_pos = alt_free_range.first; if(target_pos > after_pos) target_pos = after_pos; } else { target_pos = after_pos; } tmp_szs.expand(); tmp_szs.back().len = target_pos - pos; tmp_szs.back().off = 0; pos = target_pos; ASSERT_ONLY(sum_len += tmp_szs.back().len); } assert_eq(pos, after_pos); assert_eq(sum_len, szs[i].len); } } #ifndef NDEBUG index_t modified_jlen = 0; for(index_t i = 0; i < tmp_szs.size(); i++) { modified_jlen += tmp_szs[i].len; } assert_eq(modified_jlen, jlen); #endif } if(nthreads > (int)tmp_szs.size()) { nthreads = (int)tmp_szs.size(); } assert_gt(nthreads, 0); AutoArray threads(nthreads); EList threadParams; for(index_t i = 0; i < (index_t)nthreads; i++) { threadParams.expand(); threadParams.back().thread_id = i; threadParams.back().refGraph = this; threadParams.back().s = &s; threadParams.back().alts = &alts; threadParams.back().haplotypes = &haplotypes; threadParams.back().out_fname = out_fname; threadParams.back().bigEndian = bigEndian; threadParams.back().num_nodes = 0; threadParams.back().num_edges = 0; threadParams.back().lastNode = 0; threadParams.back().multipleHeadNodes = false; if(nthreads == 1) { buildGraph_worker((void*)&threadParams.back()); } else { threads[i] = new tthread::thread(buildGraph_worker, (void*)&threadParams.back()); } } if(nthreads > 1) { for(index_t i = 0; i < (index_t)nthreads; i++) threads[i]->join(); } index_t num_nodes = 0, num_edges = 0; for(index_t i = 0; i < threadParams.size(); i++) { num_nodes += threadParams[i].num_nodes; num_edges += threadParams[i].num_edges; // Make room for edges spanning graphs by different threads if(i > 0) { num_edges += 16; } } nodes.resizeExact(num_nodes); nodes.clear(); edges.resizeExact(num_edges); edges.clear(); // Read all the nodes and edges EList tail_nodes; bool multipleHeadNodes = false; for(index_t i = 0; i < threadParams.size(); i++) { if(threadParams[i].multipleHeadNodes) multipleHeadNodes = true; std::ostringstream number; number << i; const string rg_fname = out_fname + "." + number.str() + ".rf"; ifstream rg_in_file(rg_fname.c_str(), ios::binary); if(!rg_in_file.good()) { cerr << "Could not open file for reading a reference graph: \"" << rg_fname << "\"" << endl; throw 1; } index_t curr_num_nodes = (index_t)nodes.size(); ASSERT_ONLY(index_t curr_num_edges = (index_t)edges.size()); ASSERT_ONLY(index_t num_spanning_edges = 0); // Read nodes to be connected to last nodes in a previous thread if(i > 0) { assert_gt(tail_nodes.size(), 0) index_t num_head_nodes = readIndex(rg_in_file, bigEndian); for(index_t j = 0; j < num_head_nodes; j++) { index_t head_node = readIndex(rg_in_file, bigEndian); for(index_t k = 0; k < tail_nodes.size(); k++) { edges.expand(); edges.back().from = tail_nodes[k]; edges.back().to = head_node + curr_num_nodes; ASSERT_ONLY(num_spanning_edges++); } } } while(!rg_in_file.eof()) { index_t tmp_num_nodes = readIndex(rg_in_file, bigEndian); for(index_t j = 0; j < tmp_num_nodes; j++) { nodes.expand(); nodes.back().read(rg_in_file, bigEndian); } index_t tmp_num_edges = readIndex(rg_in_file, bigEndian); for(index_t j = 0; j < tmp_num_edges; j++) { edges.expand(); edges.back().read(rg_in_file, bigEndian); edges.back().from += curr_num_nodes; edges.back().to += curr_num_nodes; } if(nodes.size() >= curr_num_nodes + threadParams[i].num_nodes) { assert_eq(nodes.size(), curr_num_nodes + threadParams[i].num_nodes); assert_eq(edges.size(), curr_num_edges + num_spanning_edges + threadParams[i].num_edges); // Read last nodes in this thread tail_nodes.clear(); if(i + 1 < (index_t)nthreads) { index_t num_tail_nodes = readIndex(rg_in_file, bigEndian); for(index_t j = 0; j < num_tail_nodes; j++) { index_t tail_node = readIndex(rg_in_file, bigEndian); tail_nodes.push_back(tail_node + curr_num_nodes); } } break; } } rg_in_file.close(); std::remove(rg_fname.c_str()); if(i + 1 == (index_t)nthreads) { lastNode = threadParams.back().lastNode + curr_num_nodes; assert_lt(lastNode, nodes.size()); assert_eq(nodes[lastNode].label, 'Z'); } } if(s.length() + 2 == nodes.size() && nodes.size() == edges.size() + 1) { cerr << "Warning: no variants or splice sites in this graph" << endl; throw NongraphException(); } if(multipleHeadNodes) { if(!isReverseDeterministic(nodes, edges)) { if(verbose) cerr << "\tis not reverse-deterministic, so reverse-determinize..." << endl; reverseDeterminize(nodes, edges, lastNode); } } assert(isReverseDeterministic(nodes, edges)); } else { // this is memory-consuming, but simple to implement index_t num_predicted_nodes = (index_t)(jlen * 1.2); nodes.reserveExact(num_predicted_nodes); edges.reserveExact(num_predicted_nodes); // Created head node nodes.expand(); nodes.back().label = 'Y'; nodes.back().value = 0; // Create nodes and edges corresponding to a reference genome for(size_t i = 0; i < s.length(); i++) { nodes.expand(); nodes.back().label = "ACGT"[(int)s[i]]; nodes.back().value = (index_t)i; assert_geq(nodes.size(), 2); edges.expand(); edges.back().from = (index_t)nodes.size() - 2; edges.back().to = (index_t)nodes.size() - 1; } // Create tail node nodes.expand(); nodes.back().label = 'Z'; nodes.back().value = (index_t)s.length(); lastNode = (index_t)nodes.size() - 1; edges.expand(); edges.back().from = (index_t)nodes.size() - 2; edges.back().to = (index_t)nodes.size() - 1; // Create nodes and edges for haplotypes for(index_t i = 0; i < haplotypes.size(); i++) { const Haplotype& haplotype = haplotypes[i]; const EList& snpIDs = haplotype.alts; assert_gt(snpIDs.size(), 0); assert_lt(haplotype.right, s.length()); bool pass = true; for(index_t s = 0; s < snpIDs.size(); s++) { index_t snpID = snpIDs[s]; assert_lt(snpID, alts.size()); const ALT& snp = alts[snpID]; assert(snp.snp()); if(s + 1 >= snpIDs.size()) break; index_t snpID2 = snpIDs[s+1]; assert_lt(snpID2, alts.size()); const ALT& snp2 = alts[snpID2]; assert(snp2.snp()); if(snp.type == ALT_SNP_INS) { if(snp.pos > snp2.pos) { pass = false; break; } } else if(snp.type == ALT_SNP_DEL) { if(snp2.type == ALT_SNP_DEL) { if(snp.pos + snp.len >= snp2.pos) { pass = false; break; } } else { if(snp.pos + snp.len - 1 >= snp2.pos) { pass = false; break; } } } else { if(snp.pos >= snp2.pos) { pass = false; break; } } } if(!pass) continue; index_t prev_ALT_type = ALT_NONE; index_t ID_i = 0; for(index_t j = haplotype.left; j <= haplotype.right; j++) { if(prev_ALT_type == ALT_SNP_INS) j--; const ALT* altp = (ID_i < snpIDs.size() ? &(alts[snpIDs[ID_i]]) : NULL); assert(altp == NULL || altp->pos >= j); if(altp != NULL && altp->pos == j) { const ALT& alt = *altp; assert_lt(alt.pos, s.length()); assert(alt.snp()); if(alt.type == ALT_SNP_SGL) { assert_eq(alt.len, 1); nodes.expand(); assert_lt(alt.seq, 4); assert_neq(alt.seq & 0x3, s[alt.pos]); nodes.back().label = "ACGT"[alt.seq]; nodes.back().value = alt.pos; if(prev_ALT_type != ALT_SNP_DEL) { edges.expand(); if(j == haplotype.left) { edges.back().from = alt.pos; } else { assert_gt(nodes.size(), 2); edges.back().from = (index_t)nodes.size() - 2; } edges.back().to = (index_t)nodes.size() - 1; } if(j == haplotype.right) { edges.expand(); edges.back().from = (index_t)nodes.size() - 1; edges.back().to = alt.pos + 2; } } else if(alt.type == ALT_SNP_DEL) { assert_gt(alt.len, 0); assert_leq(alt.pos + alt.len, s.length()); edges.expand(); if(j == haplotype.left) { edges.back().from = alt.pos; } else { edges.back().from = (index_t)nodes.size() - 1; } j += (alt.len - 1); assert_leq(j, haplotype.right); if(j == haplotype.right) { edges.back().to = alt.pos + alt.len + 1; } else { edges.back().to = (index_t)nodes.size(); } } else { assert_eq(alt.type, ALT_SNP_INS) assert_gt(alt.len, 0); for(size_t k = 0; k < alt.len; k++) { uint64_t bp = alt.seq >> ((alt.len - k - 1) << 1); bp &= 0x3; char ch = "ACGT"[bp]; nodes.expand(); nodes.back().label = ch; nodes.back().value = (index_t)INDEX_MAX; if(prev_ALT_type == ALT_SNP_DEL && k == 0) continue; edges.expand(); edges.back().from = ((k == 0 && j == haplotype.left) ? alt.pos : (index_t)nodes.size() - 2); edges.back().to = (index_t)nodes.size() - 1; } if(j == haplotype.right) { edges.expand(); edges.back().from = (index_t)nodes.size() - 1; edges.back().to = alt.pos + 1; } } ID_i++; prev_ALT_type = alt.type; } else { int nt = s[j]; assert_lt(nt, 4); nodes.expand(); nodes.back().label = "ACGT"[nt]; nodes.back().value = j; if(prev_ALT_type != ALT_SNP_DEL) { edges.expand(); if(j == haplotype.left && prev_ALT_type == ALT_NONE) { edges.back().from = j; } else { edges.back().from = (index_t)nodes.size() - 2; } edges.back().to = (index_t)nodes.size() - 1; } if(j == haplotype.right) { edges.expand(); edges.back().from = (index_t)nodes.size() - 1; edges.back().to = j + 2; } prev_ALT_type = ALT_SNP_SGL; } } } // Create nodes and edges for splice sites for(size_t i = 0; i < alts.size(); i++) { const ALT& alt = alts[i]; if(alt.pos >= s.length()) break; if(alt.type != ALT_SPLICESITE) continue; if(alt.excluded) continue; assert_lt(alt.left, alt.right); edges.expand(); edges.back().from = alt.left; edges.back().to = alt.right + 2; } if(s.length() + 2 == nodes.size() && nodes.size() == edges.size() + 1) { throw NongraphException(); } if(!isReverseDeterministic(nodes, edges)) { if(verbose) cerr << "\tis not reverse-deterministic, so reverse-determinize..." << endl; reverseDeterminize(nodes, edges, lastNode); assert(isReverseDeterministic(nodes, edges)); } } #ifndef NDEBUG if(debug) { cout << "num nodes: " << nodes.size() << endl; for(index_t i = 0; i < nodes.size(); i++) { const Node& n = nodes[i]; cout << i << "\t" << n.label << "\t" << n.value << endl; } sort(edges.begin(), edges.end()); cout << "num edges: " << edges.size() << endl; for(index_t i = 0; i < edges.size(); i++) { const Edge& e = edges[i]; cout << i << "\t" << e.from << " --> " << e.to << endl; } } #endif } template pair RefGraph::findEdges(const EList& edges, index_t node, bool from) { pair range(0, 0); assert_gt(edges.size(), 0); // Find lower bound index_t low = 0, high = (index_t)edges.size() - 1; index_t temp; while(low < high) { index_t mid = low + (high - low) / 2; temp = (from ? edges[mid].from : edges[mid].to); if(node == temp) { high = mid; } else if(node < temp) { if(mid == 0) { return pair(0, 0); } high = mid - 1; } else { low = mid + 1; } } temp = (from ? edges[low].from : edges[low].to); if(node == temp) { range.first = low; } else { return range; } // Find upper bound high = (index_t)edges.size() - 1; while(low < high) { index_t mid = low + (high - low + 1) / 2; temp = (from ? edges[mid].from : edges[mid].to); if(node == temp) { low = mid; } else { assert_lt(node, temp); high = mid - 1; } } #ifndef NDEBUG temp = (from ? edges[high].from : edges[high].to); assert_eq(node, temp); #endif range.second = high + 1; return range; } template void RefGraph::buildGraph_worker(void* vp) { ThreadParam* threadParam = (ThreadParam*)vp; RefGraph& refGraph = *(threadParam->refGraph); const SString& s = *(threadParam->s); index_t jlen = (index_t)s.length(); const EList >& alts = *(threadParam->alts); const EList >& haplotypes = *(threadParam->haplotypes); EList nodes; EList edges; const EList& tmp_szs = refGraph.tmp_szs; index_t thread_id = threadParam->thread_id; index_t nthreads = refGraph.nthreads; std::ostringstream number; number << thread_id; const string rg_fname = threadParam->out_fname + "." + number.str() + ".rf"; ofstream rg_out_file(rg_fname.c_str(), ios::binary); if(!rg_out_file.good()) { cerr << "Could not open file for writing a reference graph: \"" << rg_fname << "\"" << endl; throw 1; } #ifndef NDEBUG set snp_set; #endif const bool bigEndian = threadParam->bigEndian; index_t& lastNode = threadParam->lastNode; index_t& num_nodes = threadParam->num_nodes; index_t& num_edges = threadParam->num_edges; index_t szs_idx = 0, szs_idx_end = (index_t)tmp_szs.size(); if(threadParam->thread_id != 0) { szs_idx = (index_t)((tmp_szs.size() / nthreads) * thread_id); } if(thread_id + 1 < nthreads) { szs_idx_end = (index_t)((tmp_szs.size() / nthreads) * (thread_id + 1)); } index_t curr_pos = 0; for(index_t i = 0; i < szs_idx; i++) { curr_pos += tmp_szs[i].len; } EList prev_tail_nodes; index_t alt_idx = 0, haplotype_idx = 0; for(; szs_idx < szs_idx_end; szs_idx++) { index_t curr_len = tmp_szs[szs_idx].len; if(curr_len <= 0) continue; index_t num_predicted_nodes = (index_t)(curr_len * 1.2); nodes.resizeExact(num_predicted_nodes); nodes.clear(); edges.resizeExact(num_predicted_nodes); edges.clear(); // Created head node nodes.expand(); nodes.back().label = 'Y'; nodes.back().value = 0; // Create nodes and edges corresponding to a reference genome assert_leq(curr_pos + curr_len, s.length()); for(size_t i = curr_pos; i < curr_pos + curr_len; i++) { nodes.expand(); nodes.back().label = "ACGT"[(int)s[i]]; nodes.back().value = (index_t)i; assert_geq(nodes.size(), 2); edges.expand(); edges.back().from = (index_t)nodes.size() - 2; edges.back().to = (index_t)nodes.size() - 1; } // Create tail node nodes.expand(); nodes.back().label = 'Z'; nodes.back().value = (index_t)s.length(); lastNode = (index_t)nodes.size() - 1; edges.expand(); edges.back().from = (index_t)nodes.size() - 2; edges.back().to = (index_t)nodes.size() - 1; ASSERT_ONLY(index_t backbone_nodes = (index_t)nodes.size()); // Create nodes and edges for haplotypes for(; haplotype_idx < haplotypes.size(); haplotype_idx++) { const Haplotype& haplotype = haplotypes[haplotype_idx]; if(haplotype.left < curr_pos) continue; if(haplotype.right >= curr_pos + curr_len) break; const EList& snpIDs = haplotype.alts; assert_gt(snpIDs.size(), 0); bool pass = true; for(index_t s = 0; s < snpIDs.size(); s++) { index_t snpID = snpIDs[s]; assert_lt(snpID, alts.size()); const ALT& snp = alts[snpID]; assert(snp.snp()); if(s + 1 >= snpIDs.size()) break; index_t snpID2 = snpIDs[s+1]; assert_lt(snpID2, alts.size()); const ALT& snp2 = alts[snpID2]; assert(snp2.snp()); if(snp.type == ALT_SNP_INS) { if(snp.pos > snp2.pos) { pass = false; break; } } else if(snp.type == ALT_SNP_DEL) { if(snp2.type == ALT_SNP_DEL) { if(snp.pos + snp.len >= snp2.pos) { pass = false; break; } } else { if(snp.pos + snp.len - 1 >= snp2.pos) { pass = false; break; } } } else { if(snp.pos >= snp2.pos) { pass = false; break; } } } if(!pass) continue; index_t prev_ALT_type = ALT_NONE; index_t ID_i = 0; for(index_t j = haplotype.left; j <= haplotype.right; j++) { if(prev_ALT_type == ALT_SNP_INS) j--; const ALT* altp = (ID_i < snpIDs.size() ? &(alts[snpIDs[ID_i]]) : NULL); assert(altp == NULL || altp->pos >= j); if(altp != NULL && altp->pos == j) { const ALT& alt = *altp; assert_lt(alt.pos, s.length()); assert(alt.snp()); if(alt.type == ALT_SNP_SGL) { assert_eq(alt.len, 1); nodes.expand(); assert_lt(alt.seq, 4); assert_neq(alt.seq & 0x3, s[alt.pos]); nodes.back().label = "ACGT"[alt.seq]; nodes.back().value = alt.pos; if(prev_ALT_type != ALT_SNP_DEL) { edges.expand(); if(j == haplotype.left) { edges.back().from = alt.pos - curr_pos; assert_lt(edges.back().from, backbone_nodes); } else { assert_gt(nodes.size(), 2); edges.back().from = (index_t)nodes.size() - 2; } edges.back().to = (index_t)nodes.size() - 1; } if(j == haplotype.right) { edges.expand(); edges.back().from = (index_t)nodes.size() - 1; edges.back().to = alt.pos - curr_pos + 2; assert_lt(edges.back().to, backbone_nodes); } } else if(alt.type == ALT_SNP_DEL) { assert_gt(alt.len, 0); assert_leq(alt.pos - curr_pos + alt.len, s.length()); edges.expand(); if(j == haplotype.left) { edges.back().from = alt.pos - curr_pos; assert_lt(edges.back().from, backbone_nodes); } else { edges.back().from = (index_t)nodes.size() - 1; } j += (alt.len - 1); assert_leq(j, haplotype.right); if(j == haplotype.right) { edges.back().to = alt.pos - curr_pos + alt.len + 1; assert_lt(edges.back().to, backbone_nodes); } else { edges.back().to = (index_t)nodes.size(); } } else { assert_eq(alt.type, ALT_SNP_INS) assert_gt(alt.len, 0); for(size_t k = 0; k < alt.len; k++) { uint64_t bp = alt.seq >> ((alt.len - k - 1) << 1); bp &= 0x3; char ch = "ACGT"[bp]; nodes.expand(); nodes.back().label = ch; nodes.back().value = (index_t)INDEX_MAX; if(prev_ALT_type == ALT_SNP_DEL && k == 0) continue; edges.expand(); edges.back().from = ((k == 0 && j == haplotype.left) ? alt.pos - curr_pos : (index_t)nodes.size() - 2); edges.back().to = (index_t)nodes.size() - 1; } if(j == haplotype.right) { edges.expand(); edges.back().from = (index_t)nodes.size() - 1; edges.back().to = alt.pos - curr_pos + 1; } } #ifndef NDEBUG snp_set.insert(snpIDs[ID_i]); #endif ID_i++; prev_ALT_type = alt.type; } else { int nt = s[j]; assert_lt(nt, 4); nodes.expand(); nodes.back().label = "ACGT"[nt]; nodes.back().value = j; if(prev_ALT_type != ALT_SNP_DEL) { edges.expand(); if(j == haplotype.left && prev_ALT_type == ALT_NONE) { edges.back().from = j - curr_pos; assert_lt(edges.back().from, backbone_nodes); } else { edges.back().from = (index_t)nodes.size() - 2; } edges.back().to = (index_t)nodes.size() - 1; } if(j == haplotype.right) { edges.expand(); edges.back().from = (index_t)nodes.size() - 1; edges.back().to = j - curr_pos + 2; assert_lt(edges.back().to, backbone_nodes); } prev_ALT_type = ALT_SNP_SGL; } } } // Create nodes and edges for splice sites for(; alt_idx < alts.size(); alt_idx++) { const ALT& alt = alts[alt_idx]; if(alt.pos < curr_pos) continue; if(alt.pos >= curr_pos + curr_len) break; if(!alt.splicesite()) continue; if(alt.excluded) continue; assert_lt(alt.left, alt.right); edges.expand(); edges.back().from = alt.left - curr_pos; edges.back().to = alt.right - curr_pos + 2; assert_lt(edges.back().from, backbone_nodes); assert_lt(edges.back().to, backbone_nodes); } #ifndef NDEBUG if(refGraph.debug) { cerr << "Nodes:" << endl; for(size_t i = 0; i < nodes.size(); i++) { const Node& node = nodes[i]; cerr << "\t" << i << "\t" << node.label << "\t" << node.value << endl; } cerr << endl; cerr << "Edges: " << endl; for(size_t i = 0; i < edges.size(); i++) { const Edge& edge = edges[i]; cerr << "\t" << i << "\t" << edge.from << " --> " << edge.to << endl; } cerr << endl; } #endif if(!isReverseDeterministic(nodes, edges)) { reverseDeterminize(nodes, edges, lastNode, curr_pos > 0 ? curr_pos + 1 : 0); assert(isReverseDeterministic(nodes, edges)); } // Identify head index_t head_node = (index_t)nodes.size(); for(index_t i = 0; i < nodes.size(); i++) { if(nodes[i].label == 'Y') { head_node = i; break; } } assert_lt(head_node, nodes.size()); index_t tail_node = lastNode; assert_lt(tail_node, nodes.size()); // Update edges const index_t invalid = (index_t)INDEX_MAX; bool head_off = curr_pos > 0, tail_off = curr_pos + curr_len < jlen; for(index_t i = 0; i < edges.size(); i++) { index_t from = edges[i].from; from = from + num_nodes; if(head_off && edges[i].from > head_node) from -= 1; if(tail_off && edges[i].from > tail_node) from -= 1; if(head_off && edges[i].from == head_node) { edges[i].from = invalid; } else { edges[i].from = from; } index_t to = edges[i].to; to = to + num_nodes; if(head_off && edges[i].to > head_node) to -= 1; if(tail_off && edges[i].to > tail_node) to -= 1; if(tail_off && edges[i].to == tail_node) { edges[i].to = invalid; } else { edges[i].to = to; } } head_node = tail_node = invalid; // Also update lastNode if(!tail_off) { lastNode += num_nodes; if(head_off) lastNode -= 1; } // Connect head nodes with tail nodes in the previous automaton index_t num_head_nodes = 0; index_t tmp_num_edges = (index_t)edges.size(); if(head_off) { EList nodes_to_head; for(index_t i = 0; i < tmp_num_edges; i++) { if(edges[i].from == head_node) { num_head_nodes++; if(prev_tail_nodes.size() > 0) { for(index_t j = 0; j < prev_tail_nodes.size(); j++) { edges.expand(); edges.back().from = prev_tail_nodes[j]; edges.back().to = edges[i].to; assert_lt(edges.back().from, edges.back().to); } } else { nodes_to_head.push_back(edges[i].to); } } } if(nodes_to_head.size() > 0) { assert_gt(thread_id, 0); assert_eq(prev_tail_nodes.size(), 0); writeIndex(rg_out_file, (index_t)nodes_to_head.size(), bigEndian); for(index_t i = 0; i < nodes_to_head.size(); i++) { writeIndex(rg_out_file, nodes_to_head[i], bigEndian); } } } // Need to check if it's reverse-deterministic if(num_head_nodes > 1) { threadParam->multipleHeadNodes = true; } // List tail nodes prev_tail_nodes.clear(); if(tail_off) { for(index_t i = 0; i < tmp_num_edges; i++) { if(edges[i].to == tail_node) { prev_tail_nodes.push_back(edges[i].from); } } } // Write nodes and edges index_t tmp_num_nodes = (index_t)nodes.size(); assert_gt(tmp_num_nodes, 2); if(head_off) tmp_num_nodes--; if(tail_off) tmp_num_nodes--; writeIndex(rg_out_file, tmp_num_nodes, bigEndian); ASSERT_ONLY(index_t num_nodes_written = 0); for(index_t i = 0; i < nodes.size(); i++) { if(head_off && nodes[i].label == 'Y') continue; if(tail_off && nodes[i].label == 'Z') continue; nodes[i].write(rg_out_file, bigEndian); ASSERT_ONLY(num_nodes_written++); } assert_eq(tmp_num_nodes, num_nodes_written); tmp_num_edges = (index_t)edges.size(); assert_geq(tmp_num_edges, num_head_nodes + prev_tail_nodes.size()); if(head_off) tmp_num_edges -= num_head_nodes; if(tail_off) tmp_num_edges -= prev_tail_nodes.size(); writeIndex(rg_out_file, tmp_num_edges, bigEndian); ASSERT_ONLY(index_t num_edges_written = 0); for(index_t i = 0; i < edges.size(); i++) { if(head_off && edges[i].from == head_node) continue; if(tail_off && edges[i].to == tail_node) continue; edges[i].write(rg_out_file, bigEndian); ASSERT_ONLY(num_edges_written++); } assert_eq(tmp_num_edges, num_edges_written); // Clear nodes and edges nodes.clear(); edges.clear(); curr_pos += curr_len; num_nodes += tmp_num_nodes; num_edges += tmp_num_edges; } if(nthreads > 1 && thread_id + 1 < (index_t)nthreads && prev_tail_nodes.size() > 0) { writeIndex(rg_out_file, (index_t)prev_tail_nodes.size(), bigEndian); for(index_t i = 0; i < prev_tail_nodes.size(); i++) { writeIndex(rg_out_file, prev_tail_nodes[i], bigEndian); } } // Close out file handle rg_out_file.close(); } template bool RefGraph::isReverseDeterministic(EList& nodes, EList& edges) { if(edges.size() <= 0) return true; // Sort edges by "to" nodes sortEdgesTo(edges); index_t curr_to = (index_t)INDEX_MAX; EList seen; seen.resize(5); seen.fillZero(); for(index_t i = 0; i < edges.size(); i++) { index_t from = edges[i].from; assert_lt(from, nodes.size()); char nt = nodes[from].label; assert(nt == 'A' || nt == 'C' || nt == 'G' || nt == 'T' || nt == 'Y'); if(nt == 'Y') nt = 4; else nt = asc2dna[(int)nt]; assert_lt(nt, seen.size()); if(curr_to != edges[i].to) { curr_to = edges[i].to; seen.fillZero(); seen[nt] = true; } else { if(seen[nt]) { return false; } seen[nt] = true; } } return true; } template void RefGraph::reverseDeterminize(EList& nodes, EList& edges, index_t& lastNode, index_t lastNode_add) { EList cnodes; cnodes.ensure(nodes.size()); map cnode_map; deque active_cnodes; EList cedges; cedges.ensure(edges.size()); // Start from the final node ('Z') assert_lt(lastNode, nodes.size()); const Node& last_node = nodes[lastNode]; cnodes.expand(); cnodes.back().reset(); cnodes.back().label = last_node.label; cnodes.back().value = last_node.value; cnodes.back().nodes.push_back(lastNode); active_cnodes.push_back(0); cnode_map[cnodes.back().nodes] = 0; sortEdgesTo(edges); index_t firstNode = 0; // Y -> ... -> Z EList predecessors; while(!active_cnodes.empty()) { index_t cnode_id = active_cnodes.front(); active_cnodes.pop_front(); assert_lt(cnode_id, cnodes.size()); // Find predecessors of this composite node predecessors.clear(); for(size_t i = 0; i < cnodes[cnode_id].nodes.size(); i++) { index_t node_id = cnodes[cnode_id].nodes.getID((index_t)i); pair edge_range = findEdgesTo(edges, node_id); assert_leq(edge_range.first, edge_range.second); assert_leq(edge_range.second, edges.size()); for(index_t j = edge_range.first; j < edge_range.second; j++) { assert_eq(edges[j].to, node_id); predecessors.push_back(edges[j].from); } } if(predecessors.size() >= 2) { // Remove redundant nodes predecessors.sort(); index_t new_size = (index_t)(unique(predecessors.begin(), predecessors.end()) - predecessors.begin()); predecessors.resize(new_size); // Create composite nodes by labels stable_sort(predecessors.begin(), predecessors.end(), TempNodeLabelCmp(nodes)); } for(size_t i = 0; i < predecessors.size();) { index_t node_id = predecessors[i]; assert_lt(node_id, nodes.size()); const Node& node = nodes[node_id]; i++; cnodes.expand(); cnodes.back().reset(); cnodes.back().label = node.label; cnodes.back().value = node.value; cnodes.back().nodes.push_back(node_id); if(node.label == 'Y' && firstNode == 0) { firstNode = (index_t)cnodes.size() - 1; } while(i < predecessors.size()) { index_t next_node_id = predecessors[i]; assert_lt(next_node_id, nodes.size()); const Node& next_node = nodes[next_node_id]; if(next_node.label != node.label) break; cnodes.back().nodes.push_back(next_node_id); if(next_node.value != (index_t)INDEX_MAX) { if(cnodes.back().value == (index_t)INDEX_MAX) { cnodes.back().value = next_node.value; } else { cnodes.back().value = max(cnodes.back().value, next_node.value); } } i++; } // Create edges from this new composite node to current composite node typename map::iterator existing = cnode_map.find(cnodes.back().nodes); if(existing == cnode_map.end()) { cnode_map[cnodes.back().nodes] = (index_t)cnodes.size() - 1; active_cnodes.push_back((index_t)cnodes.size() - 1); cedges.push_back(CompositeEdge((index_t)cnodes.size() - 1, cnode_id)); } else { cnodes.pop_back(); cedges.push_back(CompositeEdge((*existing).second, cnode_id)); } // Increment indegree cnodes[cnode_id].id++; } } // Interchange from and to for(index_t i = 0; i < cedges.size(); i++) { index_t tmp = cedges[i].from; cedges[i].from = cedges[i].to; cedges[i].to = tmp; } sort(cedges.begin(), cedges.end()); active_cnodes.push_back(0); while(!active_cnodes.empty()) { index_t cnode_id = active_cnodes.front(); active_cnodes.pop_front(); assert_lt(cnode_id, cnodes.size()); const CompositeNode& cnode = cnodes[cnode_id]; index_t i = (index_t)cedges.bsearchLoBound(CompositeEdge(cnode_id, 0)); while(i < cedges.size()) { assert_geq(cedges[i].from, cnode_id); if(cedges[i].from != cnode_id) break; index_t predecessor_cnode_id = cedges[i].to; assert_lt(predecessor_cnode_id, cnodes.size()); CompositeNode& predecessor_cnode = cnodes[predecessor_cnode_id]; if(cnode.value == predecessor_cnode.value + 1) { active_cnodes.push_back(predecessor_cnode_id); break; } i++; } } // Restore from and to by interchanging them for(index_t i = 0; i < cedges.size(); i++) { index_t tmp = cedges[i].from; cedges[i].from = cedges[i].to; cedges[i].to = tmp; } // Create new nodes lastNode = 0; // Invalidate lastNode nodes.resizeExact(cnodes.size()); nodes.clear(); assert_neq(firstNode, 0); assert_lt(firstNode, cnodes.size()); CompositeNode& first_node = cnodes[firstNode]; first_node.id = 0; nodes.expand(); nodes.back() = first_node.getNode(); active_cnodes.push_back(firstNode); sort(cedges.begin(), cedges.end()); while(!active_cnodes.empty()) { index_t cnode_id = active_cnodes.front(); active_cnodes.pop_front(); assert_lt(cnode_id, cnodes.size()); index_t i = (index_t)cedges.bsearchLoBound(CompositeEdge(cnode_id, 0)); while(i < cedges.size()) { assert_geq(cedges[i].from, cnode_id); if(cedges[i].from != cnode_id) break; index_t successor_cnode_id = cedges[i].to; assert_lt(successor_cnode_id, cnodes.size()); CompositeNode& successor_cnode = cnodes[successor_cnode_id]; assert_gt(successor_cnode.id, 0); successor_cnode.id--; if(successor_cnode.id == 0) { active_cnodes.push_back(successor_cnode_id); successor_cnode.id = (index_t)nodes.size(); nodes.expand(); nodes.back() = successor_cnode.getNode(); if(nodes.back().label == 'Z') { assert_eq(lastNode, 0); assert_gt(nodes.size(), 1); lastNode = (index_t)nodes.size() - 1; } } i++; } } // Create new edges edges.resizeExact(cedges.size()); edges.clear(); for(index_t i = 0; i < cedges.size(); i++) { const CompositeEdge& edge = cedges[i]; edges.expand(); edges.back() = edge.getEdge(cnodes); } sortEdgesFrom(edges); #if 0 #ifndef NDEBUG if(debug) { cerr << "Nodes:" << endl; for(size_t i = 0; i < nodes.size(); i++) { const Node& node = nodes[i]; cerr << "\t" << i << "\t" << node.label << "\t" << node.value << (node.backbone ? "\tbackbone" : "") << endl; } cerr << endl; cerr << "Edges: " << endl; for(size_t i = 0; i < edges.size(); i++) { const Edge& edge = edges[i]; cerr << "\t" << i << "\t" << edge.from << " --> " << edge.to << endl; } cerr << endl; } #endif #endif } template class PathGraph { public: struct PathNode { index_t from; index_t to; pair key; void setSorted() { to = (index_t)INDEX_MAX; } bool isSorted() const { return to == (index_t)INDEX_MAX; } index_t value() const { return to; } index_t outdegree() const { return key.first; } bool operator< (const PathNode& o) const { return key < o.key; }; }; static inline index_t PathNodeFrom (PathNode& a) { return a.from; } static inline index_t PathNodeKey (PathNode& a) { return a.key.first; } struct PathNodeKeySecondCmp { bool operator() (const PathNode& a, const PathNode& b) const { return a.key.second < b.key.second; } }; struct PathNodeFromCmp { bool operator() (const PathNode& a, const PathNode& b) const { return a.from < b.from; } }; struct PathNodeToCmp { bool operator() (const PathNode& a, const PathNode& b) const { return a.to < b.to; } }; struct PathEdge { index_t from; union { index_t to; index_t ranking; }; char label; PathEdge() { reset(); } PathEdge(index_t from_, index_t ranking_, char label_) : from(from_), ranking(ranking_), label(label_) {} void reset() { from = 0; ranking = 0; label = 0; } bool operator< (const PathEdge& o) const { return label < o.label || (label == o.label && ranking < o.ranking); }; }; static inline index_t PathEdgeTo (PathEdge& a) { return a.to; } struct PathEdgeFromCmp { bool operator() (const PathEdge& a, const PathEdge& b) const { return a.from < b.from || (a.from == b.from && a.to < b.to); } }; struct PathEdgeToCmp { bool operator() (const PathEdge& a, const PathEdge& b) const { return a.to < b.to || (a.to == b.to && a.from < b.from); } }; public: // Create a new graph in which paths are represented using nodes PathGraph( RefGraph& parent, const string& base_fname, size_t max_num_nodes_ = std::numeric_limits::max(), int nthreads_ = 1, bool verbose_ = false); ~PathGraph() {} void printInfo(); bool generateEdges(RefGraph& parent); index_t getNumNodes() const { return (index_t)nodes.size(); } index_t getNumEdges() const { return (index_t)edges.size(); } bool isSorted() const { return sorted; } bool nextRow(int& gbwtChar, int& F, int& M, index_t& pos) { if(report_node_idx >= nodes.size()) return false; bool firstOutEdge = false; if(report_edge_range.first >= report_edge_range.second) { report_edge_range = getEdges(report_node_idx, false /* from? */); firstOutEdge = true; if(report_node_idx == 0) { report_M = pair(0, 0); } } assert_lt(report_edge_range.first, report_edge_range.second); assert_lt(report_edge_range.first, edges.size()); const PathEdge& edge = edges[report_edge_range.first]; gbwtChar = edge.label; assert_lt(report_node_idx, nodes.size()); F = (firstOutEdge ? 1 : 0); report_edge_range.first++; if(report_edge_range.first >= report_edge_range.second) { report_node_idx++; } assert_lt(report_M.first, nodes.size()); pos = nodes[report_M.first].to; M = (report_M.second == 0 ? 1 : 0); report_M.second++; if(report_M.second >= nodes[report_M.first].key.first) { report_M.first++; report_M.second = 0; } return true; } index_t nextFLocation() { if(report_F_node_idx >= nodes.size()) return (index_t)INDEX_MAX; index_t ret = report_F_location; pair edge_range = getEdges(report_F_node_idx, false /* from? */); report_F_node_idx++; assert_lt(edge_range.first, edge_range.second); report_F_location += (edge_range.second - edge_range.first); return ret; } private: void makeFromRef(RefGraph& base); void generationOne(); void earlyGeneration(); void firstPruneGeneration(); void lateGeneration(); void mergeUpdateRank(); pair nextMaximalSet(pair range); pair getEdges(index_t node, bool by_from); // Create index first. struct CreateNewNodesParams { PathNode* st; PathNode* en; PathNode* curr; index_t* sub_temp_nodes; PathGraph* graph; }; static void createNewNodesCounter(void* vp); static void createNewNodesMaker(void* vp); void createNewNodes(); struct GenEdgesParams { typename RefGraph::Edge* st; typename RefGraph::Edge* en; EList* label_index; EList* nodes; EList* edges; EList::Node>* ref_nodes; }; static void generateEdgesCounter(void* vp); static void generateEdgesMaker(void* vp); private: int nthreads; bool verbose; EList from_table; EList past_nodes; EList nodes; EList edges; index_t ranks; index_t max_from; //number of nodes in RefGraph index_t temp_nodes; // Total number of nodes created before sorting. index_t generation; // Sorted by paths of length 2^generation. bool sorted; // For reporting GBWT char, F, and M values index_t report_node_idx; pair report_edge_range; pair report_M; // For reporting location in F corresponding to 1 bit in M index_t report_F_node_idx; index_t report_F_location; size_t max_num_nodes; // following variables are for debugging purposes #ifndef NDEBUG bool debug; #endif EList bwt_string; EList F_array; EList M_array; EList bwt_counts; // brute-force implementations index_t select(const EList& array, index_t p, char c) { if(p <= 0) return 0; for(index_t i = 0; i < array.size(); i++) { if(array[i] == c) { assert_gt(p, 0); p--; if(p == 0) return i; } } return (index_t)array.size(); } index_t select1(const EList& array, index_t p) { return select(array, p, 1); } index_t rank(const EList& array, index_t p, char c) { index_t count = 0; assert_lt(p, array.size()); for(index_t i = 0; i <= p; i++) { if(array[i] == c) count++; } return count; } index_t rank1(const EList& array, index_t p) { return rank(array, p, 1); } // for debugging purposes #ifndef NDEBUG public: EList > ftab; #endif }; //creates prefix-sorted PathGraph Nodes given a reverse determinized RefGraph //outputs nodes sorted by their from attribute template PathGraph::PathGraph( RefGraph& base, const string& base_fname, size_t max_num_nodes_, int nthreads_, bool verbose_) : nthreads(nthreads_), verbose(verbose_), ranks(0), temp_nodes(0), generation(0), sorted(false), report_node_idx(0), report_edge_range(pair(0, 0)), report_M(pair(0, 0)), report_F_node_idx(0), report_F_location(0), max_num_nodes(max_num_nodes_) { #ifndef NDEBUG debug = base.nodes.size() <= 20; #endif // Fill nodes with a PathNode for each edge in base.edges. // Set max_from. makeFromRef(base); // Write RefGraph into a file const bool file_rf = base.nodes.size() > (1 << 22); const bool bigEndian = false; const string rf_fname = base_fname + ".rf"; if(file_rf) { base.write(rf_fname, bigEndian); base.nullify(); } // In the first generation the nodes enter, not quite sorted by from. // We use a counting sort to sort the nodes, otherwise same as early generation. generationOne(); // In early generations no nodes become sorted. // Therefore, we skip the pruning step and leave the // nodes sorted by from. while(generation < 3) { earlyGeneration(); } // On the first generation we perform a pruning step, // we are forced to sort the entire list of nodes by rank // in order to perform pruning step. firstPruneGeneration(); // In later generations, most nodes are already sorted, so we // perform a more expensive random access join with nodes in rank order // in return for avoiding having to sort by rank in order to prune nodes. while(!isSorted()) { lateGeneration(); } // In the generateEdges method it is convenient to begin with nodes sorted by from. // We perform this action here, while we still have past_nodes allocated to avoid // an in-place sort. nodes.resizeNoCopyExact(past_nodes.size()); radix_sort_copy(past_nodes.begin(), past_nodes.end(), nodes.ptr(), &PathNodeFrom, max_from, nthreads); past_nodes.nullify(); from_table.nullify(); if(file_rf) { base.read(rf_fname, bigEndian); std::remove(rf_fname.c_str()); } } //make original unsorted PathNodes given a RefGraph template void PathGraph::makeFromRef(RefGraph& base) { // Create a path node per edge with a key set to from node's label temp_nodes = (index_t)base.edges.size() + 1; max_from = 0; nodes.reserveExact(temp_nodes); for(index_t i = 0; i < base.edges.size(); i++) { const typename RefGraph::Edge& e = base.edges[i]; nodes.expand(); nodes.back().from = e.from; if(e.from > max_from) max_from = e.from; nodes.back().to = e.to; switch(base.nodes[e.from].label) { case 'A': nodes.back().key = pair(0, 0); break; case 'C': nodes.back().key = pair(1, 0); break; case 'G': nodes.back().key = pair(2, 0); break; case 'T': nodes.back().key = pair(3, 0); break; case 'Y': nodes.back().key = pair(4, 0); break; default: assert(false); throw 1; } } // Final node. assert_lt(base.lastNode, base.nodes.size()); assert_eq(base.nodes[base.lastNode].label, 'Z'); nodes.expand(); nodes.back().from = nodes.back().to = base.lastNode; if(base.lastNode > max_from) max_from = base.lastNode; nodes.back().key = pair(5, 0); printInfo(); } template void PathGraph::generationOne() { //nodes enter almost sorted by from //this is only generation method that whose // incoming nodes are in the nodes EList generation++; //Sort nodes by from using counting sort //Copy into past_nodes in the process //first count number with each from value for(PathNode* node = nodes.begin(); node != nodes.end(); node++) { nodes[node->from].key.second++; } //convert into an index index_t tot = nodes[0].key.second; nodes[0].key.second = 0; for(index_t i = 1; i < max_from + 2; i++) { tot += nodes[i].key.second; nodes[i].key.second = tot - nodes[i].key.second; } // use past_nodes as from_table past_nodes.resizeExact(nodes.size()); for(PathNode* node = nodes.begin(); node != nodes.end(); node++) { past_nodes[nodes[node->from].key.second++] = *node; } //reset index for(index_t i = max_from + 1; i > 0; i--) { past_nodes[i].key.second = nodes[i - 1].key.second; } past_nodes[0].key.second = 0; //Now query direct-access table createNewNodes(); printInfo(); past_nodes.swap(nodes); } template void PathGraph::earlyGeneration() { //past_nodes enter sorted by from //do not yet need to perform pruning step generation++; for(index_t i = 0; i < past_nodes.size(); i++) { past_nodes[past_nodes[i].from + 1].key.second = i + 1; } createNewNodes(); printInfo(); past_nodes.swap(nodes); } template void PathGraph::firstPruneGeneration() { //past_nodes enter sorted by from //first generation where we need to perform pruning step // results in us needing to sort entirety of nodes after they are made generation++; //here past_nodes is already sorted by .from // first count where to start each from value time_t start = time(0); //Build from_index for(index_t i = 0; i < past_nodes.size(); i++) { past_nodes[past_nodes[i].from + 1].key.second = i + 1; } if(verbose) cerr << "BUILT FROM_INDEX: " << time(0) - start << endl; start = time(0); // Now query against direct-access table createNewNodes(); past_nodes.resizeNoCopyExact(nodes.size()); if(verbose) cerr << "RESIZE NODES: " << time(0) - start << endl; start = time(0); //max_rank always corresponds to repeated Z's // Z is mapped to 0x101 // therefore max rank = 101101101101101101101101 = (101) 8 times index_t max_rank = 11983725; radix_sort_copy, index_t>(nodes.begin(), nodes.end(), past_nodes.ptr(), &PathNodeKey, max_rank, nthreads); if(verbose) cerr << "SORT NODES: " << time(0) - start << endl; start = time(0); nodes.swap(past_nodes); mergeUpdateRank(); if(verbose) cerr << "MERGE, UPDATE RANK: " << time(0) - start << endl; start = time(0); printInfo(); past_nodes.swap(nodes); } template void PathGraph::lateGeneration() { //past_nodes enter sorted by rank //build direct-access table sorted by from, //but query with original nodes sorted by rank //since nodes we query with are sorted by rank, // the nodes produced are automatically sorted by key.first // therefore we only need to sort clusters with same key.first generation++; time_t overall = time(0); time_t indiv = time(0); assert_gt(nthreads, 0); assert_neq(past_nodes.size(), ranks); from_table.resizeNoCopy(past_nodes.size()); if(verbose) cerr << "ALLOCATE FROM_TABLE: " << time(0) - indiv << endl; indiv = time(0); radix_sort_copy(past_nodes.begin(), past_nodes.end(), from_table.ptr(), &PathNodeFrom, max_from, nthreads); if(verbose) cerr << "BUILD TABLE: " << time(0) - indiv << endl; indiv = time(0); //Build from_index index_t from_table_size = from_table.size(); for(index_t i = 0; i < from_table_size; i++) { if(from_table[i].from + 1 >= from_table.size()) { from_table.resize(from_table[i].from + 2); } from_table[from_table[i].from + 1].key.second = i + 1; } if(verbose) cerr << "BUILD INDEX: " << time(0) - indiv << endl; createNewNodes(); indiv = time(0); mergeUpdateRank(); if(from_table_size != from_table.size()) { assert_lt(from_table_size, from_table.size()); from_table.resize(from_table_size); } if(verbose) cerr << "MERGEUPDATERANK: " << time(0) - indiv << endl; if(verbose) cerr << "TOTAL TIME: " << time(0) - overall << endl; if(ranks >= (index_t)max_num_nodes) { throw ExplosionException(); } printInfo(); past_nodes.swap(nodes); } //----------------------------------------------------------------------------------------------- template void PathGraph::createNewNodesCounter(void* vp) { CreateNewNodesParams* params = (CreateNewNodesParams*)vp; PathNode* st = params->st; PathNode* en = params->en; PathGraph& graph = *(params->graph); size_t count = 0; if(graph.generation > 4) { for(PathNode* node = st; node != en; node++) { if(node->isSorted()) { count++; } else { count += graph.from_table[node->to + 1].key.second - graph.from_table[node->to].key.second; } } } else { for(PathNode* node = st; node != en; node++) { count += graph.past_nodes[node->to + 1].key.second - graph.past_nodes[node->to].key.second; } } *(params->sub_temp_nodes) = (index_t)count; //check for overflow if(count > (index_t)-1) { cerr << "exceeded integer bounds, remove adjacent SNPs, use haplotypes, or switch to a large index (--large-index)" << endl; throw 1; } } template void PathGraph::createNewNodesMaker(void* vp) { CreateNewNodesParams* params = (CreateNewNodesParams*)vp; PathNode* st = params->st; PathNode* en = params->en; PathNode* curr = params->curr; PathGraph& graph = *(params->graph); if(graph.generation > 4) { for(PathNode* node = st; node != en; node++) { if(node->isSorted()) { *curr++ = *node; } else { for(index_t j = graph.from_table[node->to].key.second; j < graph.from_table[node->to + 1].key.second; j++) { curr->from = node->from; curr->to = graph.from_table[j].to; (curr++)->key = pair(node->key.first, graph.from_table[j].key.first); } } } } else if(graph.generation == 4) { for(PathNode* node = st; node != en; node++) { for(index_t j = graph.past_nodes[node->to].key.second; j < graph.past_nodes[node->to + 1].key.second; j++) { curr->from = node->from; curr->to = graph.past_nodes[j].to; (curr++)->key = pair(node->key.first, graph.past_nodes[j].key.first); } } } else { for(PathNode* node = st; node != en; node++) { for(index_t j = graph.past_nodes[node->to].key.second; j < graph.past_nodes[node->to + 1].key.second; j++) { curr->from = node->from; curr->to = graph.past_nodes[j].to; index_t bit_shift = 1 << (graph.generation - 1); bit_shift = (bit_shift << 1) + bit_shift; (curr++)->key = pair((node->key.first << bit_shift) + graph.past_nodes[j].key.first, 0); } } } } template void PathGraph::createNewNodes() { time_t indiv = time(0); AutoArray threads(nthreads); EList params; params.resizeExact(nthreads); EList sub_temp_nodes; sub_temp_nodes.resizeExact(nthreads); sub_temp_nodes.fillZero(); PathNode* st = past_nodes.begin(); PathNode* en = st + past_nodes.size() / nthreads; for(int i = 0; i < nthreads; i++) { params[i].sub_temp_nodes = &sub_temp_nodes[i]; params[i].st = st; params[i].en = en; params[i].graph = this; if(nthreads == 1) { createNewNodesCounter((void*)¶ms[0]); } else { threads[i] = new tthread::thread(&createNewNodesCounter, (void*)¶ms[i]); } st = en; if(i + 2 == nthreads) { en = past_nodes.end(); } else { en = st + past_nodes.size() / nthreads; } } if(nthreads > 1) { for(int i = 0; i < nthreads; i++) threads[i]->join(); } if(verbose) cerr << "COUNTED NEW NODES: " << time(0) - indiv << endl; indiv = time(0); //update all label indexes temp_nodes = 0; for(int i = 0; i < nthreads; i++) { // done to check if we exceed index_t range size_t val = (size_t)temp_nodes + (size_t)sub_temp_nodes[i]; if(val > (index_t)-1) { cerr << "exceeded integer bounds, remove adjacent SNPs, use haplotypes, or switch to a large index (--large-index)" << endl; throw 1; } temp_nodes = (index_t)val; } if(verbose) cerr << "COUNTED TEMP NODES: " << time(0) - indiv << endl; indiv = time(0); nodes.resizeNoCopyExact(temp_nodes); if(verbose) cerr << "RESIZED NODES: " << time(0) - indiv << endl; indiv = time(0); temp_nodes = 0; for(int i = 0; i < nthreads; i++) { params[i].curr = nodes.begin() + temp_nodes; temp_nodes += sub_temp_nodes[i]; } if(verbose) cerr << "RESIZED NODES: " << time(0) - indiv << endl; indiv = time(0); //make new nodes for(int i = 0; i < nthreads; i++) { if(nthreads == 1) { createNewNodesMaker((void*)¶ms[0]); } else { threads[i] = new tthread::thread(&createNewNodesMaker, (void*)¶ms[i]); } } if(nthreads > 1) { for(int i = 0; i < nthreads; i++) threads[i]->join(); } if(verbose) cerr << "MADE NEW NODES: " << time(0) - indiv << endl; indiv = time(0); } //------------------------------------------------------------------------------------ template void PathGraph::mergeUpdateRank() { if(generation == 4) { // Merge equivalent nodes index_t curr = 0; pair range(0, 0); // Empty range while(true) { range = nextMaximalSet(range); if(range.first >= range.second) break; nodes[curr] = nodes[range.first]; curr++; } nodes.resize(curr); // Set nodes that become sorted as sorted PathNode* candidate = &nodes.front(); pair key = candidate->key; ranks = 1; for(index_t i = 1; i < nodes.size(); i++) { if(nodes[i].key != key) { if(candidate != NULL) { candidate->setSorted(); } candidate = &nodes[i]; key = candidate->key; ranks++; } else { candidate = NULL; } } if(candidate != NULL) { candidate->setSorted(); } ranks = 0; key = nodes.front().key; for(index_t i = 0; i < nodes.size(); i++) { PathNode& node = nodes[i]; if(node.key != key) { key = node.key; ranks++; } node.key = pair(ranks, 0); } ranks++; } else { PathNode* block_start = nodes.begin(); PathNode* curr = nodes.begin(); PathNode* node = nodes.begin(); ranks = 0; do { node++; if(node == nodes.end() || node->key.first != block_start->key.first) { if(node - block_start == 1) { block_start->key.first = ranks++; *curr++ = *block_start; } else { sort(block_start, node, PathNodeKeySecondCmp()); while(block_start != node) { //extend shift while share same key index_t shift = 1; while(block_start + shift != node && block_start->key == (block_start + shift)->key) { shift++; } //check if all share .from //if they share same from, then they are a mergable set bool merge = true; for(PathNode* n = block_start; n != (block_start + shift); n++) { if(n->from != block_start->from) { merge = false; break; } } //if not mergable, just write all to array if(!merge) { for(PathNode* n = block_start; n != (block_start + shift); n++) { n->key.first = ranks; *curr++ = *n; } ranks++; } else if(curr == nodes.begin() || !(curr - 1)->isSorted() || (curr - 1)->from != block_start->from) { block_start->setSorted(); block_start->key.first = ranks++; *curr++ = *block_start; } block_start += shift; } // if we are at the last node or the last node is mergable into the previous node, we are done if(node == nodes.end()) break; if(node + 1 == nodes.end()) { assert(curr >= nodes.begin() + 1); if((curr - 1)->isSorted() && node->from == (curr - 1)->from) break; } // check if we can safely merge the node immediately following the unsorted cluster into the previous node // must be that: // 1) node is not itself part of an unsorted cluster // 2) the previous node is sorted // 3) the nodes share the same from attribute assert(node + 1 < nodes.end()); if(node->key.first != (node + 1)->key.first) { assert(curr >= nodes.begin() + 1); if((curr - 1)->isSorted() && node->from == (curr - 1)->from) node++; } } block_start = node; } } while(node != nodes.end()); nodes.resizeExact((index_t)(curr - nodes.begin())); } // if all nodes have unique rank we are done! if(ranks == nodes.size()) sorted = true; } // Returns the next maximal mergeable set of PathNodes. // A set of PathNodes sharing adjacent keys is mergeable, if each of the // PathNodes begins in the same GraphNode, and no other PathNode shares // the key. If the maximal set is empty, returns the next PathNode. template pair PathGraph::nextMaximalSet(pair range) { if(range.second >= nodes.size()) { return pair(0, 0); } range.first = range.second; range.second = range.first + 1; if(range.first > 0 && nodes[range.first - 1].key == nodes[range.first].key) { return range; } for(index_t i = range.second; i < nodes.size(); i++) { if(nodes[i - 1].key != nodes[i].key) { range.second = i; } if(nodes[i].from != nodes[range.first].from) { return range; } } range.second = (index_t)nodes.size(); return range; } //----------------------------------------------------------------------------------------- template void PathGraph::printInfo() { if(verbose) { cerr << "Generation " << generation << " (" << temp_nodes << " -> " << nodes.size() << " nodes, " << ranks << " ranks)" << endl; } } //------------------------------------------------------------------------------------------ template void PathGraph::generateEdgesCounter(void* vp) { GenEdgesParams* params = (GenEdgesParams*)vp; typename RefGraph::Edge* st = params->st; typename RefGraph::Edge* en = params->en; EList& label_index = *(params->label_index); EList::Node>& ref_nodes = *(params->ref_nodes); EList& nodes = *(params->nodes); //first count edges, fill out label_index for(typename RefGraph::Edge* edge = st; edge != en; edge++) { char curr_label = ref_nodes[edge->from].label; int curr_label_index; switch(curr_label) { case 'A': curr_label_index = 0; break; case 'C': curr_label_index = 1; break; case 'G': curr_label_index = 2; break; case 'T': curr_label_index = 3; break; case 'Y': curr_label_index = 4; break; case 'Z': curr_label_index = 5; break; default: assert(false); throw 1; } assert_lt(edge->to + 1, nodes.size()); assert_lt(nodes[edge->to].key.second, nodes[edge->to + 1].key.second); label_index[curr_label_index] += nodes[edge->to + 1].key.second - nodes[edge->to].key.second; } } template void PathGraph::generateEdgesMaker(void* vp) { GenEdgesParams* params = (GenEdgesParams*)vp; typename RefGraph::Edge* st = params->st; typename RefGraph::Edge* en = params->en; EList& label_index = *(params->label_index); EList::Node>& ref_nodes = *(params->ref_nodes); EList& edges = *(params->edges); EList& nodes = *(params->nodes); for(typename RefGraph::Edge* edge = st; edge != en; edge++) { char curr_label = ref_nodes[edge->from].label; int curr_label_index; switch(curr_label) { case 'A': curr_label_index = 0; break; case 'C': curr_label_index = 1; break; case 'G': curr_label_index = 2; break; case 'T': curr_label_index = 3; break; case 'Y': curr_label_index = 4; break; case 'Z': curr_label_index = 5; break; default: assert(false); throw 1; } for(index_t j = nodes[edge->to].key.second; j < nodes[edge->to + 1].key.second; j++) { edges[label_index[curr_label_index]++] = PathEdge(edge->from, nodes[j].key.first, curr_label); } } } template bool PathGraph::generateEdges(RefGraph& base) { //entering we have: // nodes - sorted by from // edges - empty // base.nodes - almost sorted by from/to // base.edges - almost sorted by from/to //need to join: // nodes.from -> base.nodes[] // nodes.from -> base.edges.to // nodes.from -> edges.from if(!sorted) return false; time_t indiv = time(0); time_t overall = time(0); //replace nodes.to with genomic position //fast because both roughly ordered by from for(PathNode* node = nodes.begin(); node != nodes.end(); node++) { node->to = base.nodes[node->from].value; } if(verbose) cerr << "NODE.TO -> GENOME POS: " << time(0) - indiv << endl; indiv = time(0); // build an index for nodes index_t node_size = nodes.size(); for(index_t i = 0; i < node_size; i++) { // very rare case where the number of prefix-sorted nodes is smaller than the number of the initial nodes // , which could happen with a very small graph and a variant as follows // ATAGAGCAGTTCTGAAAAACACTTTTTGTTGAATCTGCAAG(T)GGACATTTGGATAGATTTGAAGATTTCGTTGGAAACGGGAATATCTTCATATCAAATG // (G) // where G(T) and G(G) will be combined as there is no other node that intervene those two path nodes. if(nodes[i].from + 1 >= nodes.size()) { nodes.resize(nodes[i].from + 2); } nodes[nodes[i].from + 1].key.second = i + 1; } if(verbose) cerr << "BUILD FROM_INDEX " << time(0) - indiv << endl; indiv = time(0); // Now join nodes.from to edges.to // fast because base.edges roughly sorted by to //count number of edges AutoArray threads(nthreads); EList params; params.resizeExact(nthreads); ELList label_index; label_index.resize(nthreads); typename RefGraph::Edge* st = base.edges.begin(); typename RefGraph::Edge* en = st + base.edges.size() / nthreads; for(int i = 0; i < nthreads; i++) { label_index[i].resizeExact(6); label_index[i].fillZero(); params[i].label_index = &label_index[i]; params[i].st = st; params[i].en = en; params[i].nodes = &nodes; params[i].edges = &edges; params[i].ref_nodes = &base.nodes; if(nthreads == 1) { generateEdgesCounter((void*)¶ms[0]); } else { threads[i] = new tthread::thread(&generateEdgesCounter, (void*)¶ms[i]); } st = en; if(i + 2 == nthreads) { en = base.edges.end(); } else { en = st + base.edges.size() / nthreads; } } if(nthreads > 1) { for(int i = 0; i < nthreads; i++) threads[i]->join(); } if(verbose) cerr << "COUNTED NEW EDGES: " << time(0) - indiv << endl; indiv = time(0); //update all label indexes index_t tot = label_index[0][0]; label_index[0][0] = 0; for(int i = 1; i < nthreads; i++) { tot += label_index[i][0]; label_index[i][0] = tot - label_index[i][0]; } for(int j = 1; j < 6; j++) { for(int i = 0; i < nthreads; i++) { tot += label_index[i][j]; label_index[i][j] = tot - label_index[i][j]; } } edges.resizeExact(tot); //make new edges for(int i = 0; i < nthreads; i++) { if(nthreads == 1) { generateEdgesMaker((void*)¶ms[0]); } else { threads[i] = new tthread::thread(&generateEdgesMaker, (void*)¶ms[i]); } } if(nthreads > 1) { for(int i = 0; i < nthreads; i++) { threads[i]->join(); } } base.nullify(); // delete unused nodes if(node_size != nodes.size()) { assert_lt(node_size, nodes.size()); nodes.resize(node_size); } if(verbose) cerr << "MADE NEW EDGES: " << time(0) - indiv << endl; indiv = time(0); EList& index = label_index[nthreads - 1]; EList temp_edges; temp_edges.resizeExact(edges.size()); radix_sort_copy, index_t>(edges.begin() , edges.begin() + index[0], temp_edges.ptr(), &PathEdgeTo, (index_t)nodes.size(), nthreads); radix_sort_copy, index_t>(edges.begin() + index[0], edges.begin() + index[1], temp_edges.ptr() + index[0], &PathEdgeTo, (index_t)nodes.size(), nthreads); radix_sort_copy, index_t>(edges.begin() + index[1], edges.begin() + index[2], temp_edges.ptr() + index[1], &PathEdgeTo, (index_t)nodes.size(), nthreads); radix_sort_copy, index_t>(edges.begin() + index[2], edges.begin() + index[3], temp_edges.ptr() + index[2], &PathEdgeTo, (index_t)nodes.size(), nthreads); for(index_t i = index[3]; i < edges.size(); i++) { temp_edges[i] = edges[i]; } sort(temp_edges.begin() + index[3], temp_edges.begin() + index[4]); sort(temp_edges.begin() + index[4], temp_edges.begin() + index[5]); edges.xfer(temp_edges); if(verbose) cerr << "SORTED NEW EDGES: " << time(0) - indiv << endl; indiv = time(0); EList past_nodes; past_nodes.resizeExact(nodes.size()); radix_sort_copy, index_t>(nodes.begin(), nodes.end(), past_nodes.ptr(), &PathNodeKey, ranks, nthreads); nodes.xfer(past_nodes); if(verbose) cerr << "RE-SORTED NODES: " << time(0) - indiv << endl; indiv = time(0); #ifndef NDEBUG if(debug) { cerr << "just after creating path edges" << endl; cerr << "Ref edges" << endl; for(size_t i = 0; i < base.edges.size(); i++) { const typename RefGraph::Edge& edge = base.edges[i]; cerr << "\t" << i << "\t" << edge.from << " --> " << edge.to << endl; } cerr << "Path nodes" << endl; for(size_t i = 0; i < nodes.size(); i++) { const PathNode& node = nodes[i]; cerr << "\t" << i << "\t(" << node.key.first << ", " << node.key.second << ")\t" << node.from << " --> " << node.to << endl; } cerr << "Path edges" << endl; for(size_t i = 0; i < edges.size(); i++) { const PathEdge& edge = edges[i]; cerr << "\t" << i << "\tfrom: " << edge.from << "\tranking: " << edge.ranking << "\t" << edge.label << endl; } } #endif #ifndef NDEBUG // Switch char array[x][y]; to char** array; if(debug) { cerr << "after sorting nodes by ranking and edges by label and ranking" << endl; cerr << "Path nodes" << endl; for(size_t i = 0; i < nodes.size(); i++) { const PathNode& node = nodes[i]; cerr << "\t" << i << "\t(" << node.key.first << ", " << node.key.second << ")\t" << node.from << " --> " << node.to << endl; } cerr << "Path edges" << endl; for(size_t i = 0; i < edges.size(); i++) { const PathEdge& edge = edges[i]; cerr << "\t" << i << "\tfrom: " << edge.from << "\tranking: " << edge.ranking << "\t" << edge.label << endl; } } #endif // Sets PathNode.to = GraphNode.value and PathNode.key.first to outdegree // Replaces (from.from, to) with (from, to) { PathNode* node = nodes.begin(); node->key.first = 0; PathEdge* edge = edges.begin(); while(node != nodes.end() && edge != edges.end()) { if(edge->from == node->from) { edge->from = (index_t)(node - nodes.begin()); edge++; node->key.first++; } else { node++; node->key.first = 0; } } } if(verbose) cerr << "PROCESS EDGES: " << time(0) - indiv << endl; indiv = time(0); // Remove 'Y' node assert_gt(nodes.size(), 2); nodes.back().key.first = nodes[nodes.size() - 2].key.first; nodes[nodes.size() - 2] = nodes.back(); nodes.pop_back(); // Adjust edges accordingly for(size_t i = 0; i < edges.size(); i++) { PathEdge& edge = edges[i]; if(edge.label == 'Y') { edge.label = 'Z'; } else if(edge.ranking >= nodes.size()) { assert_eq(edge.ranking, nodes.size()); edge.ranking -= 1; } } if(verbose) cerr << "REMOVE Y: " << time(0) - indiv << endl; indiv = time(0); #ifndef NDEBUG if(debug) { cerr << "Path nodes" << endl; for(size_t i = 0; i < nodes.size(); i++) { const PathNode& node = nodes[i]; cerr << "\t" << i << "\t(" << node.key.first << ", " << node.key.second << ")\t" << node.from << " --> " << node.to << endl; } cerr << "Path edges" << endl; for(size_t i = 0; i < edges.size(); i++) { const PathEdge& edge = edges[i]; cerr << "\t" << i << "\tfrom: " << edge.from << "\tranking: " << edge.ranking << "\t" << edge.label << endl; } } #endif temp_edges.resizeExact(edges.size()); radix_sort_copy(edges.begin(), edges.end(), temp_edges.ptr(), &PathEdgeTo, (index_t)nodes.size(), nthreads); edges.xfer(temp_edges); for(index_t i = 0; i < edges.size(); i++) { nodes[edges[i].ranking].key.second = i + 1; } if(verbose) cerr << "SORT, Make index: " << time(0) - indiv << endl; if(verbose) cerr << "TOTAL: " << time(0) - overall << endl; return true; //----------------------------------------------------------------------------------------------------- bwt_string.clear(); F_array.clear(); M_array.clear(); bwt_counts.resizeExact(5); bwt_counts.fillZero(); for(index_t node = 0; node < nodes.size(); node++) { pair edge_range = getEdges(node, false /* from? */); for(index_t i = edge_range.first; i < edge_range.second; i++) { assert_lt(i, edges.size()); char label = edges[i].label; if(label == 'Y') { label = 'Z'; } bwt_string.push_back(label); F_array.push_back(i == edge_range.first ? 1 : 0); if(label != 'Z') { char nt = asc2dna[(int)label]; assert_lt(nt + 1, bwt_counts.size()); bwt_counts[nt + 1]++; } } for(index_t i = 0; i < nodes[node].key.first; i++) { M_array.push_back(i == 0 ? 1 : 0); } } assert_gt(bwt_string.size(), 0); assert_eq(bwt_string.size(), F_array.size()); assert_eq(bwt_string.size(), M_array.size()); for(size_t i = 0; i < bwt_counts.size(); i++) { if(i > 0) bwt_counts[i] += bwt_counts[i - 1]; } #ifndef NDEBUG if(debug) { cerr << "Path nodes (final)" << endl; for(size_t i = 0; i < nodes.size(); i++) { const PathNode& node = nodes[i]; cerr << "\t" << i << "\t(" << node.key.first << ", " << node.key.second << ")\t" << node.from << " --> " << node.to << endl; } cerr << "Path edges (final)" << endl; for(size_t i = 0; i < edges.size(); i++) { const PathEdge& edge = edges[i]; cerr << "\t" << i << "\tfrom: " << edge.from << "\tranking: " << edge.ranking << "\t" << edge.label << endl; } cerr << "i\tBWT\tF\tM" << endl; for(index_t i = 0; i < bwt_string.size(); i++) { cerr << i << "\t" << bwt_string[i] << "\t" // BWT char << (int)F_array[i] << "\t" // F bit value << (int)M_array[i] << endl; // M bit value } for(size_t i = 0; i < bwt_counts.size(); i++) { cerr << i << "\t" << bwt_counts[i] << endl; } } #endif // Test searches, based on paper_example #if 1 EList queries; EList answers; # if 1 # if 1 queries.push_back("GACGT"); answers.push_back(9); queries.push_back("GATGT"); answers.push_back(9); queries.push_back("GACT"); answers.push_back(9); queries.push_back("ATGT"); answers.push_back(4); queries.push_back("GTAC"); answers.push_back(10); queries.push_back("ACTG"); answers.push_back(3); # else // rs55902548, at 402, ref, alt, unknown alt queries.push_back("GGCAGCTCCCATGGGTACACACTGGGCCCAGAACTGGGATGGAGGATGCA"); // queries.push_back("GGCAGCTCCCATGGGTACACACTGGTCCCAGAACTGGGATGGAGGATGCA"); // queries.push_back("GGCAGCTCCCATGGGTACACACTGGACCCAGAACTGGGATGGAGGATGCA"); // rs5759268, at 926787, ref, alt, unknown alt // queries.push_back("AAATTGCTCAGCCTTGTGCTGTGCACACCTGGTTCTCTTTCCAGTGTTAT"); // queries.push_back("AAATTGCTCAGCCTTGTGCTGTGCATACCTGGTTCTCTTTCCAGTGTTAT"); // queries.push_back("AAATTGCTCAGCCTTGTGCTGTGCAGACCTGGTTCTCTTTCCAGTGTTAT"); # endif for(size_t q = 0; q < queries.size(); q++) { const string& query = queries[q]; assert_gt(query.length(), 0); index_t top = 0, bot = edges.size(); index_t node_top = 0, node_bot = 0; cerr << "Aligning " << query << endl; index_t i = 0; for(; i < query.length(); i++) { if(top >= bot) break; int nt = query[query.length() - i - 1]; nt = asc2dna[nt]; assert_lt(nt, 4); cerr << "\t" << i << "\tBWT range: [" << top << ", " << bot << ")" << endl; top = bwt_counts[(int)nt] + (top <= 0 ? 0 : rank(bwt_string, top - 1, "ACGT"[nt])); bot = bwt_counts[(int)nt] + rank(bwt_string, bot - 1, "ACGT"[nt]); cerr << "\t\tLF BWT range: [" << top << ", " << bot << ")" << endl; node_top = rank1(M_array, top) - 1; node_bot = rank1(M_array, bot - 1); cerr << "\t\tnode range: [" << node_top << ", " << node_bot << ")" << endl; top = select1(F_array, node_top + 1); bot = select1(F_array, node_bot + 1); } cerr << "\t" << i << "\tBWT range: [" << top << ", " << bot << ")" << endl; // assert_eq(top, answers[q]); cerr << "finished... "; if(node_top < node_bot && node_top < nodes.size()) { index_t pos = nodes[node_top].to; index_t gpos = pos; const EList& szs = base.szs; for(index_t i = 0; i < szs.size(); i++) { gpos += szs[i].off; if(pos < szs[i].len) break; pos -= szs[i].len; } cerr << "being aligned at " << gpos; } cerr << endl << endl; } # endif // See inconsistencies between F and M arraystimy thread # if 0 cerr << endl << endl; EList tmp_F; for(index_t i = 0; i < F_array.size(); i++) { if(F_array[i] == 1) tmp_F.push_back(i); } EList tmp_M; for(index_t i = 0; i < M_array.size(); i++) { if(M_array[i] == 1) tmp_M.push_back(i); } index_t max_diff = 0; assert_eq(tmp_F.size(), tmp_M.size()); for(index_t i = 0; i < tmp_F.size(); i++) { index_t diff = (tmp_F[i] >= tmp_M[i] ? tmp_F[i] - tmp_M[i] : tmp_M[i] - tmp_F[i]); if(diff > max_diff) { max_diff = diff; cerr << i << "\tdiff: " << max_diff << "\t" << (tmp_F[i] >= tmp_M[i] ? "+" : "-") << endl; } } cerr << "Final: " << tmp_F.back() << " vs. " << tmp_M.back() << endl; # endif #endif return true; } //-------------------------------------------------------------------------- template pair PathGraph::getEdges(index_t node, bool by_from) { if(node >= nodes.size()) { cerr << "Error: Trying to get edges " << (by_from ? "from " : "to ") << node << endl; } if(nodes[node].key.second == 0) { return pair(0, 0); } if(node == 0) { return pair(0, nodes[node].key.second); } else { return pair(nodes[node - 1].key.second, nodes[node].key.second); } } #endif /*GBWT_GRAPH_H_*/ hisat2-2.2.1/gfm.cpp000066400000000000000000000044301370663763200142010ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include "gfm.h" using namespace std; #ifdef BOWTIE_64BIT_INDEX const std::string gfm_ext("ht2l"); #else const std::string gfm_ext("ht2"); #endif // BOWTIE_64BIT_INDEX string gLastIOErrMsg; /** * Try to find the Bowtie index specified by the user. First try the * exact path given by the user. Then try the user-provided string * appended onto the path of the "indexes" subdirectory below this * executable, then try the provided string appended onto * "$HISAT2_INDEXES/". */ string adjustEbwtBase(const string& cmdline, const string& gfmFileBase, bool verbose) { string str = gfmFileBase; ifstream in; if(verbose) cout << "Trying " << str.c_str() << endl; in.open((str + ".1." + gfm_ext).c_str(), ios_base::in | ios::binary); if(!in.is_open()) { if(verbose) cout << " didn't work" << endl; in.close(); if(getenv("HISAT2_INDEXES") != NULL) { str = string(getenv("HISAT2_INDEXES")) + "/" + gfmFileBase; if(verbose) cout << "Trying " << str.c_str() << endl; in.open((str + ".1." + gfm_ext).c_str(), ios_base::in | ios::binary); if(!in.is_open()) { if(verbose) cout << " didn't work" << endl; in.close(); } else { if(verbose) cout << " worked" << endl; } } } if(!in.is_open()) { cerr << "Could not locate a HISAT2 index corresponding to basename \"" << gfmFileBase.c_str() << "\"" << endl; throw 1; } return str; } hisat2-2.2.1/gfm.h000066400000000000000000007705441370663763200136660ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef GFM_H_ #define GFM_H_ #include #include #include #include #include #include #include #include #include #include #include #include #ifdef BOWTIE_MM #include #include #endif #include "shmem.h" #include "alphabet.h" #include "assert_helpers.h" #include "bitpack.h" #include "blockwise_sa.h" #include "endian_swap.h" #include "word_io.h" #include "random_source.h" #include "ref_read.h" #include "threading.h" #include "str_util.h" #include "mm.h" #include "timer.h" #include "reference.h" #include "search_globals.h" #include "ds.h" #include "random_source.h" #include "mem_ids.h" #include "btypes.h" #include "tokenize.h" #include "repeat.h" #include "repeat_kmer.h" #ifdef POPCNT_CAPABILITY #include "processor_support.h" #endif #include "gbwt_graph.h" using namespace std; // From ccnt_lut.cpp, automatically generated by gen_lookup_tables.pl extern uint8_t cCntLUT_4[4][4][256]; extern uint8_t cCntLUT_4_rev[4][4][256]; extern uint8_t cCntBIT[8][256]; static const uint64_t c_table[4] = { 0xffffffffffffffff, 0xaaaaaaaaaaaaaaaa, 0x5555555555555555, 0x0000000000000000 }; #ifndef VMSG_NL #define VMSG_NL(...) \ if(this->verbose()) { \ stringstream tmp; \ tmp << __VA_ARGS__ << endl; \ this->verbose(tmp.str()); \ } #endif #ifndef VMSG #define VMSG(...) \ if(this->verbose()) { \ stringstream tmp; \ tmp << __VA_ARGS__; \ this->verbose(tmp.str()); \ } #endif /** * Flags describing type of Ebwt. */ enum GFM_FLAGS { GFM_ENTIRE_REV = 4 // true -> reverse Ebwt is the whole // concatenated string reversed, rather than // each stretch reversed }; /** * Extended Burrows-Wheeler transform header. This together with the * actual data arrays and other text-specific parameters defined in * class Ebwt constitute the entire Ebwt. */ template class GFMParams { public: GFMParams() { } GFMParams( index_t len, index_t gbwtLen, index_t numNodes, int32_t lineRate, int32_t offRate, int32_t ftabChars, index_t eftabLen, bool entireReverse) { init(len, gbwtLen, numNodes, lineRate, offRate, ftabChars, eftabLen, entireReverse); } GFMParams(const GFMParams& gh) { init(gh._len, gh._gbwtLen, gh._numNodes, gh._lineRate, gh._offRate, gh._ftabChars, gh._eftabLen, gh._entireReverse); } void init( index_t len, index_t gbwtLen, index_t numNodes, int32_t lineRate, int32_t offRate, int32_t ftabChars, index_t eftabLen, bool entireReverse) { _entireReverse = entireReverse; _linearFM = (len + 1 == gbwtLen || gbwtLen == 0); _len = len; _gbwtLen = (gbwtLen == 0 ? len + 1 : gbwtLen); _numNodes = (numNodes == 0 ? len + 1 : numNodes); if(_linearFM) { _sz = (len+3)/4; _gbwtSz = _gbwtLen/4 + 1; } else { _sz = (len+1)/2; _gbwtSz = _gbwtLen/2 + 1; } _lineRate = lineRate; _origOffRate = offRate; _offRate = offRate; _offMask = std::numeric_limits::max() << _offRate; _ftabChars = ftabChars; _eftabLen = eftabLen; _eftabSz = _eftabLen*sizeof(index_t); _ftabLen = (1 << (_ftabChars*2))+1; _ftabSz = _ftabLen*sizeof(index_t); _offsLen = (_numNodes + (1 << _offRate) - 1) >> _offRate; _offsSz = _offsLen*sizeof(index_t); _lineSz = 1 << _lineRate; _sideSz = _lineSz * 1 /* lines per side */; if(_linearFM) { _sideGbwtSz = _sideSz - (sizeof(index_t) * 4); _sideGbwtLen = _sideGbwtSz << 2; } else { _sideGbwtSz = _sideSz - (sizeof(index_t) * 6); _sideGbwtLen = _sideGbwtSz << 1; } _numSides = (_gbwtSz+(_sideGbwtSz)-1)/(_sideGbwtSz); _numLines = _numSides * 1 /* lines per side */; _gbwtTotLen = _numSides * _sideSz; _gbwtTotSz = _gbwtTotLen; assert(repOk()); } index_t len() const { return _len; } index_t lenNucs() const { return _len; } index_t gbwtLen() const { return _gbwtLen; } index_t sz() const { return _sz; } index_t gbwtSz() const { return _gbwtSz; } int32_t lineRate() const { return _lineRate; } int32_t origOffRate() const { return _origOffRate; } int32_t offRate() const { return _offRate; } index_t offMask() const { return _offMask; } int32_t ftabChars() const { return _ftabChars; } index_t eftabLen() const { return _eftabLen; } index_t eftabSz() const { return _eftabSz; } index_t ftabLen() const { return _ftabLen; } index_t ftabSz() const { return _ftabSz; } index_t offsLen() const { return _offsLen; } index_t offsSz() const { return _offsSz; } index_t lineSz() const { return _lineSz; } index_t sideSz() const { return _sideSz; } index_t sideGbtSz() const { return _sideGbwtSz; } index_t sideGbwtLen() const { return _sideGbwtLen; } index_t numSides() const { return _numSides; } index_t numLines() const { return _numLines; } index_t gbwtTotLen() const { return _gbwtTotLen; } index_t gbwtTotSz() const { return _gbwtTotSz; } bool entireReverse() const { return _entireReverse; } bool linearFM() const { return _linearFM; } index_t numNodes() const { return _numNodes; } /** * Set a new suffix-array sampling rate, which involves updating * rate, mask, sample length, and sample size. */ void setOffRate(int __offRate) { _offRate = __offRate; _offMask = std::numeric_limits::max() << _offRate; _offsLen = (_gbwtLen + (1 << _offRate) - 1) >> _offRate; _offsSz = _offsLen * sizeof(index_t); } #ifndef NDEBUG /// Check that this EbwtParams is internally consistent bool repOk() const { // assert_gt(_len, 0); assert_gt(_lineRate, 3); assert_geq(_offRate, 0); assert_leq(_ftabChars, 16); assert_geq(_ftabChars, 1); assert_lt(_lineRate, 32); assert_lt(_ftabChars, 32); assert_eq(0, _gbwtTotSz % _lineSz); return true; } #endif /** * Pretty-print the header contents to the given output stream. */ void print(ostream& out) const { out << "Headers:" << endl << " len: " << _len << endl << " gbwtLen: " << _gbwtLen << endl << " nodes: " << _numNodes << endl << " sz: " << _sz << endl << " gbwtSz: " << _gbwtSz << endl << " lineRate: " << _lineRate << endl << " offRate: " << _offRate << endl << " offMask: 0x" << hex << _offMask << dec << endl << " ftabChars: " << _ftabChars << endl << " eftabLen: " << _eftabLen << endl << " eftabSz: " << _eftabSz << endl << " ftabLen: " << _ftabLen << endl << " ftabSz: " << _ftabSz << endl << " offsLen: " << _offsLen << endl << " offsSz: " << _offsSz << endl << " lineSz: " << _lineSz << endl << " sideSz: " << _sideSz << endl << " sideGbwtSz: " << _sideGbwtSz << endl << " sideGbwtLen: " << _sideGbwtLen << endl << " numSides: " << _numSides << endl << " numLines: " << _numLines << endl << " gbwtTotLen: " << _gbwtTotLen << endl << " gbwtTotSz: " << _gbwtTotSz << endl << " reverse: " << _entireReverse << endl << " linearFM: " << (_linearFM ? "Yes" : "No") << endl; } index_t _len; index_t _gbwtLen; index_t _sz; index_t _gbwtSz; int32_t _lineRate; int32_t _origOffRate; int32_t _offRate; index_t _offMask; int32_t _ftabChars; index_t _eftabLen; index_t _eftabSz; index_t _ftabLen; index_t _ftabSz; index_t _offsLen; index_t _offsSz; index_t _lineSz; index_t _sideSz; index_t _sideGbwtSz; index_t _sideGbwtLen; index_t _numSides; index_t _numLines; index_t _gbwtTotLen; index_t _gbwtTotSz; bool _entireReverse; bool _linearFM; index_t _numNodes; }; /** * Exception to throw when a file-realted error occurs. */ class GFMFileOpenException : public std::runtime_error { public: GFMFileOpenException(const std::string& msg = "") : std::runtime_error(msg) { } }; /** * Calculate size of file with given name. */ static inline int64_t fileSize(const char* name) { std::ifstream f; f.open(name, std::ios_base::binary | std::ios_base::in); if (!f.good() || f.eof() || !f.is_open()) { return 0; } f.seekg(0, std::ios_base::beg); std::ifstream::pos_type begin_pos = f.tellg(); f.seekg(0, std::ios_base::end); return static_cast(f.tellg() - begin_pos); } /** * Encapsulates a location in the gbwt text in terms of the side it * occurs in and its offset within the side. */ template struct SideLocus { SideLocus() : _sideByteOff(0), _sideNum(0), _charOff(0), _by(-1), _bp(-1) { } /** * Construct from row and other relevant information about the Ebwt. */ SideLocus(index_t row, const GFMParams& ep, const uint8_t* ebwt) { initFromRow(row, ep, ebwt); } /** * Init two SideLocus objects from a top/bot pair, using the result * from one call to initFromRow to possibly avoid a second call. */ static void initFromTopBot( index_t top, index_t bot, const GFMParams& gp, const uint8_t* gfm, SideLocus& ltop, SideLocus& lbot) { const index_t sideGbwtLen = gp._sideGbwtLen; assert_gt(bot, top); ltop.initFromRow(top, gp, gfm); index_t spread = bot - top; // Many cache misses on the following lines if(ltop._charOff + spread < sideGbwtLen) { lbot._charOff = ltop._charOff + spread; lbot._sideNum = ltop._sideNum; lbot._sideByteOff = ltop._sideByteOff; lbot._by = lbot._charOff >> 2; assert_lt(lbot._by, (int)gp._sideGbwtSz); lbot._bp = lbot._charOff & 0x3; } else { lbot.initFromRow(bot, gp, gfm); } } /** * Calculate SideLocus based on a row and other relevant * information about the shape of the Ebwt. */ void initFromRow( index_t row, const GFMParams& gp, const uint8_t* gfm) { const index_t sideSz = gp._sideSz; // Side length is hard-coded for now; this allows the compiler // to do clever things to accelerate / and %. _sideNum = row / gp._sideGbwtLen; assert_lt(_sideNum, gp._numSides); _charOff = row % gp._sideGbwtLen; _sideByteOff = _sideNum * sideSz; assert_leq(row, gp._gbwtLen); assert_leq(_sideByteOff + sideSz, gp._gbwtTotSz); // Tons of cache misses on the next line _by = _charOff >> 2; // byte within side assert_lt(_by, (int)gp._sideGbwtSz); _bp = _charOff & 0x3; // bit-pair within byte } /** * Init two SideLocus objects from a top/bot pair, using the result * from one call to initFromRow to possibly avoid a second call. */ static void initFromTopBot_bit( index_t top, index_t bot, const GFMParams& gp, const uint8_t* gfm, SideLocus& ltop, SideLocus& lbot) { const index_t sideGbwtLen = gp._sideGbwtLen; // assert_gt(bot, top); ltop.initFromRow_bit(top, gp, gfm); index_t spread = bot - top; // Many cache misses on the following lines if(ltop._charOff + spread < sideGbwtLen) { lbot._charOff = ltop._charOff + spread; lbot._sideNum = ltop._sideNum; lbot._sideByteOff = ltop._sideByteOff; lbot._by = lbot._charOff >> 3; assert_lt(lbot._by, (int)gp._sideGbwtSz); lbot._bp = lbot._charOff & 0x7; } else { lbot.initFromRow_bit(bot, gp, gfm); } } /** * Calculate SideLocus based on a row and other relevant * information about the shape of the Ebwt. */ void initFromRow_bit( index_t row, const GFMParams& gp, const uint8_t* gfm) { const index_t sideSz = gp._sideSz; // Side length is hard-coded for now; this allows the compiler // to do clever things to accelerate / and %. _sideNum = row / gp._sideGbwtLen; assert_lt(_sideNum, gp._numSides); _charOff = row % gp._sideGbwtLen; _sideByteOff = _sideNum * sideSz; assert_lt(row, gp._gbwtLen); assert_leq(_sideByteOff + sideSz, gp._gbwtTotSz); // Tons of cache misses on the next line _by = _charOff >> 3; // byte within side assert_lt(_by, (int)gp._sideGbwtSz); _bp = _charOff & 0x7; // bit-pair within byte } /** * Transform this SideLocus to refer to the next side (i.e. the one * corresponding to the next side downstream). Set all cursors to * point to the beginning of the side. */ void nextSide(const GFMParams& gp) { assert(valid()); _sideByteOff += gp.sideSz(); _sideNum++; _by = _bp = _charOff = 0; assert(valid()); } /** * Return true iff this is an initialized SideLocus */ bool valid() const { if(_bp != -1) { return true; } return false; } /** * Convert locus to BW row it corresponds to. */ index_t toBWRow(const GFMParams& gp) const; #ifndef NDEBUG /** * Check that SideLocus is internally consistent and consistent * with the (provided) EbwtParams. */ bool repOk(const GFMParams& gp) const { ASSERT_ONLY(index_t row = toBWRow(gp)); assert_leq(row, gp._gbwtLen); assert_range(-1, 3, _bp); assert_range(0, (int)gp._sideGbwtSz, _by); return true; } #endif /// Make this look like an invalid SideLocus void invalidate() { _bp = -1; } /** * Return a read-only pointer to the beginning of the top side. */ const uint8_t *side(const uint8_t* gbwt) const { return gbwt + _sideByteOff; } /** * Return a read-only pointer to the beginning of the top side. */ const uint8_t *next_side(const GFMParams& gp, const uint8_t* gbwt) const { if(_sideByteOff + gp._sideSz < gp._ebwtTotSz) { return gbwt + _sideByteOff + gp._sideSz; } else { return NULL; } } index_t _sideByteOff; // offset of top side within ebwt[] index_t _sideNum; // index of side index_t _charOff; // character offset within side int32_t _by; // byte within side (not adjusted for bw sides) int32_t _bp; // bitpair within byte (not adjusted for bw sides) }; /** * Convert locus to BW row it corresponds to. */ template inline index_t SideLocus::toBWRow(const GFMParams& gp) const { return _sideNum * (gp._sideGbwtSz << (gp.linearFM() ? 2 : 1)) + _charOff; } #ifdef POPCNT_CAPABILITY // wrapping of "struct" struct USE_POPCNT_GENERIC { #endif // Use this standard bit-bashing population count inline static int pop64(uint64_t x) { // Lots of cache misses on following lines (>10K) x = x - ((x >> 1) & 0x5555555555555555llu); x = (x & 0x3333333333333333llu) + ((x >> 2) & 0x3333333333333333llu); x = (x + (x >> 4)) & 0x0F0F0F0F0F0F0F0Fllu; x = x + (x >> 8); x = x + (x >> 16); x = x + (x >> 32); return (int)(x & 0x3Fllu); } #ifdef POPCNT_CAPABILITY // wrapping a "struct" }; #endif #ifdef POPCNT_CAPABILITY struct USE_POPCNT_INSTRUCTION { inline static int pop64(uint64_t x) { int64_t count; #ifdef USING_MSC_COMPILER count = __popcnt64(x); #else asm ("popcntq %[x],%[count]\n": [count] "=&r" (count): [x] "r" (x)); #endif return (int)count; } }; #endif /** * Tricky-bit-bashing bitpair counting for given two-bit value (0-3) * within a 64-bit argument. */ #ifdef POPCNT_CAPABILITY template #endif inline static int countInU64(int c, uint64_t dw) { uint64_t c0 = c_table[c]; uint64_t x0 = dw ^ c0; uint64_t x1 = (x0 >> 1); uint64_t x2 = x1 & (0x5555555555555555); uint64_t x3 = x0 & x2; #ifdef POPCNT_CAPABILITY uint64_t tmp = Operation().pop64(x3); #else uint64_t tmp = pop64(x3); #endif return (int) tmp; } #ifdef POPCNT_CAPABILITY // wrapping of "struct" struct USE_POPCNT_GENERIC_BITS { // Use this standard bit-bashing population count inline static uint64_t pop64(uint64_t x) { #else // Use this standard bit-bashing population count inline static uint64_t pop6464(uint64_t x) { #endif x -= (x >> 1) & 0x5555555555555555ULL; x = (x & 0x3333333333333333ULL) + ((x >> 2) & 0x3333333333333333ULL); x = (x + (x >> 4)) & 0x0f0f0f0f0f0f0f0fULL; return int((x * 0x0101010101010101ULL) >> 56); } #ifdef POPCNT_CAPABILITY // wrapping a "struct" }; #endif /** * Tricky-bit-bashing bitpair counting for given two-bit value (0-3) * within a 64-bit argument. */ #ifdef POPCNT_CAPABILITY template #endif inline static int countInU64_bits(uint64_t dw) { #ifdef POPCNT_CAPABILITY uint64_t tmp = Operation().pop64(dw); #else uint64_t tmp = pop6464(dw); #endif return (int) tmp; } // Forward declarations for Ebwt class class GFMSearchParams; /** * Extended Burrows-Wheeler transform data. * * An Ebwt may be transferred to and from RAM with calls to * evictFromMemory() and loadIntoMemory(). By default, a newly-created * Ebwt is not loaded into memory; if the user would like to use a * newly-created Ebwt to answer queries, they must first call * loadIntoMemory(). */ template class GFM { public: #define GFM_INITS \ _toBigEndian(currentlyBigEndian()), \ _overrideOffRate(overrideOffRate), \ _verbose(verbose), \ _passMemExc(passMemExc), \ _sanity(sanityCheck), \ fw_(fw), \ _in1(NULL), \ _in2(NULL), \ _nPat(0), \ _nFrag(0), \ _plen(EBWT_CAT), \ _rstarts(EBWT_CAT), \ _fchr(EBWT_CAT), \ _ftab(EBWT_CAT), \ _eftab(EBWT_CAT), \ _offs(EBWT_CAT), \ _gfm(EBWT_CAT), \ _useMm(false), \ useShmem_(false), \ _refnames(EBWT_CAT), \ mmFile1_(NULL), \ mmFile2_(NULL), \ _nthreads(1) /// Construct a GFM from the given input file GFM(const string& in, ALTDB* altdb, RepeatDB* repeatdb, EList* readLens, int needEntireReverse, bool fw, int32_t overrideOffRate, // = -1, int32_t offRatePlus, // = -1, bool useMm, // = false, bool useShmem, // = false, bool mmSweep, // = false, bool loadNames, // = false, bool loadSASamp, // = true, bool loadFtab, // = true, bool loadRstarts, // = true, bool loadSpliceSites, // = true, bool verbose, // = false, bool startVerbose, // = false, bool passMemExc, // = false, bool sanityCheck, // = false) bool useHaplotype, // = false bool skipLoading = false) : GFM_INITS { assert(!useMm || !useShmem); #ifdef POPCNT_CAPABILITY ProcessorSupport ps; _usePOPCNTinstruction = ps.POPCNTenabled(); #endif packed_ = false; _useMm = useMm; useShmem_ = useShmem; _in1Str = in + ".1." + gfm_ext; _in2Str = in + ".2." + gfm_ext; if(skipLoading) return; if(repeatdb == NULL) { readIntoMemory( fw ? -1 : needEntireReverse, // need REF_READ_REVERSE loadSASamp, // load the SA sample portion? loadFtab, // load the ftab & eftab? loadRstarts, // load the rstarts array? true, // stop after loading the header portion? &_gh, // params mmSweep, // mmSweep loadNames, // loadNames startVerbose); // startVerbose // If the offRate has been overridden, reflect that in the // _eh._offRate field if(offRatePlus > 0 && _overrideOffRate == -1) { _overrideOffRate = _gh._offRate + offRatePlus; } if(_overrideOffRate > _gh._offRate) { _gh.setOffRate(_overrideOffRate); assert_eq(_overrideOffRate, _gh._offRate); } } // Read ALTs EList >& alts = altdb->alts(); EList >& haplotypes = altdb->haplotypes(); EList& altnames = altdb->altnames(); alts.clear(); altnames.clear(); string in7Str = in + ".7." + gfm_ext; string in8Str = in + ".8." + gfm_ext; // open alts if(verbose || startVerbose) cerr << "Opening \"" << in7Str.c_str() << "\"" << endl; ifstream in7(in7Str.c_str(), ios::binary); if(!in7.good()) { cerr << "Could not open index file " << in7Str.c_str() << endl; } EList to_alti; index_t to_alti_far = 0; readI32(in7, this->toBe()); index_t numAlts = readIndex(in7, this->toBe()); // open altnames if(verbose || startVerbose) cerr << "Opening \"" << in8Str.c_str() << "\"" << endl; ifstream in8(in8Str.c_str(), ios::binary); if(!in8.good()) { cerr << "Could not open index file " << in8Str.c_str() << endl; } readI32(in8, this->toBe()); index_t numAltnames = readIndex(in8, this->toBe()); assert_eq(numAlts, numAltnames); if(numAlts > 0) { alts.resizeExact(numAlts); alts.clear(); to_alti.resizeExact(numAlts); to_alti.clear(); while(!in7.eof() && !in8.eof()) { alts.expand(); alts.back().read(in7, this->toBe()); to_alti.push_back(to_alti_far); to_alti_far++; altnames.expand(); in8 >> altnames.back(); if(!loadSpliceSites) { if(alts.back().splicesite()) { alts.pop_back(); assert_gt(numAlts, 0); altnames.pop_back(); assert_gt(numAltnames, 0); numAlts--; numAltnames--; to_alti.back() = std::numeric_limits::max(); to_alti_far--; } } if(alts.size() == numAlts) break; } } assert_eq(alts.size(), numAlts); assert_eq(to_alti_far, numAlts); assert_eq(alts.size(), altnames.size()); // Check if it hits the end of file, and this routine is needed for backward compatibility if(in7.peek() != std::ifstream::traits_type::eof()) { index_t numHaplotypes = readIndex(in7, this->toBe()); if(numHaplotypes > 0) { haplotypes.resizeExact(numHaplotypes); haplotypes.clear(); while(!in7.eof()) { haplotypes.expand(); haplotypes.back().read(in7, this->toBe()); Haplotype& ht = haplotypes.back(); for(index_t h = 0; h < ht.alts.size(); h++) { ht.alts[h] = to_alti[ht.alts[h]]; } if(haplotypes.size() == numHaplotypes) break; } } if(!useHaplotype) { haplotypes.nullify(); } } // Read repeats _repeat = false; if(repeatdb != NULL) { _repeat = true; // Number of repeat groups in the index index_t numRepeatIndex = readIndex(in7, this->toBe()); assert_gt(numRepeatIndex, 0); EList > repeatLens; repeatLens.resizeExact(numRepeatIndex); for(size_t k = 0; k < numRepeatIndex; k++) { repeatLens[k].first = readIndex(in7, this->toBe()); repeatLens[k].second = readIndex(in7, this->toBe()); } if (readLens != NULL && !readLens->empty()) { // Load subset of repeat groups. size_t k = 0; size_t k2 = 0; _repeatIncluded.resizeExact(numRepeatIndex); _repeatIncluded.fillZero(); while(k < numRepeatIndex && k2 < readLens->size()) { if (repeatLens[k].first >= (*readLens)[k2]) { _repeatIncluded[k] = true; k2++; } else { k++; } } // at least last repeat group is included _repeatIncluded[numRepeatIndex - 1] = true; _repeatLens.clear(); for(size_t i = 0; i < numRepeatIndex; i++) { if (_repeatIncluded[i]) { _repeatLens.push_back(repeatLens[i]); } } } else { // Load all repeat groups _repeatLens = repeatLens; _repeatIncluded.resizeExact(numRepeatIndex); _repeatIncluded.fill(true); } repeatdb->read(in7, this->toBe(), _repeatIncluded); index_t numKmertables = readIndex(in7, this->toBe()); EList filePos; filePos.resizeExact(numKmertables); for(size_t k = 0; k < numKmertables; k++) { filePos[k] = readIndex(in7, this->toBe()); } for(size_t k = 0; k < numKmertables; k++) { if(!_repeatIncluded[k]) continue; if(k > 0) { in7.seekg(filePos[k-1]); } _repeat_kmertables.expand(); _repeat_kmertables.back().read(in7, this->toBe()); } in7.seekg(filePos.back()); } in7.close(); in8.close(); // Sort SNPs and Splice Sites based on positions index_t nalts = (index_t)alts.size(); for(index_t s = 0; s < nalts; s++) { ALT alt = alts[s]; if(alt.snp()) altdb->setSNPs(true); if(alt.exon()) altdb->setExons(true); if(alt.splicesite()) { altdb->setSpliceSites(true); alts.push_back(alt); alts.back().left = alt.right; alts.back().right = alt.left; altnames.push_back("ssr"); } else if(alt.deletion()) { alts.push_back(alt); alts.back().pos = alt.pos + alt.len - 1; alts.back().reversed = true; string altname = altnames[s]; altnames.push_back(altname); } } if(alts.size() > 1 && alts.size() > nalts) { assert_eq(alts.size(), altnames.size()); EList, index_t> > buf; buf.resize(alts.size()); EList buf2; buf2.resize(alts.size()); for(size_t i = 0; i < alts.size(); i++) { buf[i].first = alts[i]; buf[i].second = (index_t)i; buf2[i] = altnames[i]; } buf.sort(); for(size_t i = 0; i < alts.size(); i++) { alts[i] = buf[i].first; altnames[i] = buf2[buf[i].second]; if(buf[i].second < numAlts) { to_alti[buf[i].second] = i; } } } if(useHaplotype) { EList& haplotype_maxrights = altdb->haplotype_maxrights(); haplotype_maxrights.resizeExact(haplotypes.size()); for(index_t h = 0; h < haplotypes.size(); h++) { Haplotype& ht = haplotypes[h]; for(index_t h2 = 0; h2 < ht.alts.size(); h2++) { ht.alts[h2] = to_alti[ht.alts[h2]]; } if(h == 0) { haplotype_maxrights[h] = ht.right; } else { haplotype_maxrights[h] = std::max(haplotype_maxrights[h - 1], ht.right); } } } assert(repeatdb != NULL || repOk()); } /// Construct an Ebwt from the given header parameters and string /// vector, optionally using a blockwise suffix sorter with the /// given 'bmax' and 'dcv' parameters. The string vector is /// ultimately joined and the joined string is passed to buildToDisk(). GFM( bool packed, int needEntireReverse, int32_t lineRate, int32_t offRate, int32_t ftabChars, const string& file, // base filename for GFM files bool fw, int dcv, EList& szs, index_t sztot, const RefReadInParams& refparams, uint32_t seed, int32_t overrideOffRate = -1, bool verbose = false, bool passMemExc = false, bool sanityCheck = false) : GFM_INITS, _gh( joinedLen(szs), 0, 0, lineRate, offRate, ftabChars, 0, refparams.reverse == REF_READ_REVERSE) { #ifdef POPCNT_CAPABILITY ProcessorSupport ps; _usePOPCNTinstruction = ps.POPCNTenabled(); #endif packed_ = packed; } /// Construct an Ebwt from the given header parameters and string /// vector, optionally using a blockwise suffix sorter with the /// given 'bmax' and 'dcv' parameters. The string vector is /// ultimately joined and the joined string is passed to buildToDisk(). template GFM( TStr& s, bool packed, int needEntireReverse, int32_t lineRate, int32_t offRate, int32_t ftabChars, int nthreads, const string& snpfile, const string& htfile, const string& ssfile, const string& exonfile, const string& svfile, const string& repeatfile, const string& outfile, // base filename for GFM files bool fw, bool useBlockwise, index_t bmax, index_t bmaxSqrtMult, index_t bmaxDivN, int dcv, EList& is, EList& szs, index_t sztot, const RefReadInParams& refparams, EList* parent_szs, EList* parent_refnames, uint32_t seed, int32_t overrideOffRate = -1, bool verbose = false, bool passMemExc = false, bool sanityCheck = false) : GFM_INITS, _gh( joinedLen(szs), 0, 0, lineRate, offRate, ftabChars, 0, refparams.reverse == REF_READ_REVERSE) { assert_gt(nthreads, 0); _nthreads = nthreads; #ifdef POPCNT_CAPABILITY ProcessorSupport ps; _usePOPCNTinstruction = ps.POPCNTenabled(); #endif _in1Str = outfile + ".1." + gfm_ext; _in2Str = outfile + ".2." + gfm_ext; packed_ = packed; // Open output files ofstream fout1(_in1Str.c_str(), ios::binary); if(!fout1.good()) { cerr << "Could not open index file for writing: \"" << _in1Str.c_str() << "\"" << endl << "Please make sure the directory exists and that permissions allow writing by" << endl << "HISAT2." << endl; throw 1; } ofstream fout2(_in2Str.c_str(), ios::binary); if(!fout2.good()) { cerr << "Could not open index file for writing: \"" << _in2Str.c_str() << "\"" << endl << "Please make sure the directory exists and that permissions allow writing by" << endl << "HISAT2." << endl; throw 1; } // Build initFromVector( s, snpfile, htfile, ssfile, exonfile, svfile, repeatfile, is, szs, sztot, refparams, fout1, fout2, outfile, useBlockwise, bmax, bmaxSqrtMult, bmaxDivN, dcv, parent_szs, parent_refnames, seed, verbose); // Close output files fout1.flush(); int64_t tellpSz1 = (int64_t)fout1.tellp(); VMSG_NL("Wrote " << fout1.tellp() << " bytes to primary GFM file: " << _in1Str.c_str()); fout1.close(); bool err = false; if(tellpSz1 > fileSize(_in1Str.c_str())) { err = true; cerr << "Index is corrupt: File size for " << _in1Str.c_str() << " should have been " << tellpSz1 << " but is actually " << fileSize(_in1Str.c_str()) << "." << endl; } fout2.flush(); int64_t tellpSz2 = (int64_t)fout2.tellp(); VMSG_NL("Wrote " << fout2.tellp() << " bytes to secondary GFM file: " << _in2Str.c_str()); fout2.close(); if(tellpSz2 > fileSize(_in2Str.c_str())) { err = true; cerr << "Index is corrupt: File size for " << _in2Str.c_str() << " should have been " << tellpSz2 << " but is actually " << fileSize(_in2Str.c_str()) << "." << endl; } if(err) { cerr << "Please check if there is a problem with the disk or if disk is full." << endl; throw 1; } // Reopen as input streams VMSG_NL("Re-opening _in1 and _in2 as input streams"); if(_sanity) { VMSG_NL("Sanity-checking Bt2"); assert(!isInMemory()); readIntoMemory( fw ? -1 : needEntireReverse, // 1 -> need the reverse to be reverse-of-concat true, // load SA sample (_offs[])? true, // load ftab (_ftab[] & _eftab[])? true, // load r-starts (_rstarts[])? false, // just load header? NULL, // Params object to fill false, // mm sweep? true, // load names? false); // verbose startup? // sanityCheckAll(refparams.reverse); evictFromMemory(); assert(!isInMemory()); } VMSG_NL("Returning from GFM constructor"); } /** * Static constructor for a pair of forward/reverse indexes for the * given reference string. */ template static pair fromString( const char* str, bool packed, int reverse, bool bigEndian, int32_t lineRate, int32_t offRate, int32_t ftabChars, const string& file, bool useBlockwise, index_t bmax, index_t bmaxSqrtMult, index_t bmaxDivN, int dcv, uint32_t seed, bool verbose, bool autoMem, bool sanity) { EList strs(EBWT_CAT); strs.push_back(std::string(str)); return fromStrings( strs, packed, reverse, bigEndian, lineRate, offRate, ftabChars, file, useBlockwise, bmax, bmaxSqrtMult, bmaxDivN, dcv, seed, verbose, autoMem, sanity); } /** * Static constructor for a pair of forward/reverse indexes for the * given list of reference strings. */ template static pair fromStrings( const EList& strs, bool packed, int reverse, bool bigEndian, int32_t lineRate, int32_t offRate, int32_t ftabChars, const string& file, bool useBlockwise, index_t bmax, index_t bmaxSqrtMult, index_t bmaxDivN, int dcv, uint32_t seed, bool verbose, bool autoMem, bool sanity) { assert(!strs.empty()); EList is(EBWT_CAT); RefReadInParams refparams(false /* color */, REF_READ_FORWARD, false, false); // Adapt sequence strings to stringstreams open for input auto_ptr ss(new stringstream()); for(index_t i = 0; i < strs.size(); i++) { (*ss) << ">" << i << endl << strs[i] << endl; } auto_ptr fb(new FileBuf(ss.get())); assert(!fb->eof()); assert(fb->get() == '>'); ASSERT_ONLY(fb->reset()); assert(!fb->eof()); is.push_back(fb.get()); // Vector for the ordered list of "records" comprising the input // sequences. A record represents a stretch of unambiguous // characters in one of the input sequences. EList szs(EBWT_CAT); std::pair sztot; sztot = BitPairReference::szsFromFasta(is, file, bigEndian, refparams, szs, sanity); // Construct Ebwt from input strings and parameters GFM *gfmFw = new GFM( TStr(), packed, -1, // fw lineRate, offRate, // suffix-array sampling rate ftabChars, // number of chars in initial arrow-pair calc file, // basename for .?.ebwt files true, // fw? useBlockwise, // useBlockwise bmax, // block size for blockwise SA builder bmaxSqrtMult, // block size as multiplier of sqrt(len) bmaxDivN, // block size as divisor of len dcv, // difference-cover period is, // list of input streams szs, // list of reference sizes sztot.first, // total size of all unambiguous ref chars refparams, // reference read-in parameters seed, // pseudo-random number generator seed -1, // override offRate verbose, // be talkative autoMem, // pass exceptions up to the toplevel so that we can adjust memory settings automatically sanity); // verify results and internal consistency refparams.reverse = reverse; szs.clear(); sztot = BitPairReference::szsFromFasta(is, file, bigEndian, refparams, szs, sanity); // Construct Ebwt from input strings and parameters GFM *gfmBw = new GFM( TStr(), packed, reverse == REF_READ_REVERSE, lineRate, offRate, // suffix-array sampling rate ftabChars, // number of chars in initial arrow-pair calc file + ".rev",// basename for .?.ebwt files false, // fw? useBlockwise, // useBlockwise bmax, // block size for blockwise SA builder bmaxSqrtMult, // block size as multiplier of sqrt(len) bmaxDivN, // block size as divisor of len dcv, // difference-cover period is, // list of input streams szs, // list of reference sizes sztot.first, // total size of all unambiguous ref chars refparams, // reference read-in parameters seed, // pseudo-random number generator seed -1, // override offRate verbose, // be talkative autoMem, // pass exceptions up to the toplevel so that we can adjust memory settings automatically sanity); // verify results and internal consistency return make_pair(gfmFw, gfmBw); } /// Return true iff the Ebwt is packed bool isPacked() { return packed_; } /** * Write the rstarts array given the szs array for the reference. */ void szsToDisk(const EList& szs, ostream& os, int reverse); bool checkPosToSzs(const EList& szs, index_t start_idx, index_t pos) { assert(szs[start_idx].first); for(index_t i = start_idx; i < szs.size(); i++) { if((i != start_idx) && (szs[i].first)) { // span to next chr return false; } if(pos < szs[i].off) { return false; } else { pos -= szs[i].off; if(pos < szs[i].len) { return true; } pos -= szs[i].len; } } assert(false); return false; } /** * Helper for the constructors above. Takes a vector of text * strings and joins them into a single string with a call to * joinToDisk, which does a join (with padding) and writes some of * the resulting data directly to disk rather than keep it in * memory. It then constructs a suffix-array producer (what kind * depends on 'useBlockwise') for the resulting sequence. The * suffix-array producer can then be used to obtain chunks of the * joined string's suffix array. */ template void initFromVector(TStr& s, const string& snpfile, const string& htfile, const string& ssfile, const string& exonfile, const string& svfile, const string& repeatfile, EList& is, EList& szs, index_t sztot, const RefReadInParams& refparams, ofstream& out1, ofstream& out2, const string& outfile, bool useBlockwise, index_t bmax, index_t bmaxSqrtMult, index_t bmaxDivN, int dcv, EList* parent_szs, EList* parent_refnames, uint32_t seed, bool verbose) { // Compose text strings into single string VMSG_NL("Calculating joined length"); index_t jlen; jlen = joinedLen(szs); _repeat = (parent_szs != NULL); assert_geq(jlen, sztot); VMSG_NL("Writing header"); writeFromMemory(true, out1, out2); try { VMSG_NL("Reserving space for joined string"); s.resize(jlen); VMSG_NL("Joining reference sequences"); if(refparams.reverse == REF_READ_REVERSE) { { Timer timer(cerr, " Time to join reference sequences: ", _verbose); joinToDisk(is, szs, sztot, refparams, s, out1, out2); } { Timer timer(cerr, " Time to reverse reference sequence: ", _verbose); EList tmp(EBWT_CAT); s.reverse(); reverseRefRecords(szs, tmp, false, verbose); szsToDisk(tmp, out1, refparams.reverse); } } else { Timer timer(cerr, " Time to join reference sequences: ", _verbose); joinToDisk(is, szs, sztot, refparams, s, out1, out2); szsToDisk(szs, out1, refparams.reverse); } { Timer timer(cerr, " Time to read SNPs and splice sites: ", _verbose); _alts.clear(); _altnames.clear(); EList > chr_szs; index_t tmp_len = 0; for(index_t i = 0; i < szs.size(); i++) { if(szs[i].first) { chr_szs.expand(); chr_szs.back().first = tmp_len; chr_szs.back().second = i; } tmp_len += (index_t)szs[i].len; } // Write SNPs into 7.ht2 and 8.ht2 string file7 = outfile + ".7." + gfm_ext; string file8 = outfile + ".8." + gfm_ext; // Open output stream for the '.7.gfm_ext' file which will // hold SNPs (except IDs). ofstream fout7(file7.c_str(), ios::binary); if(!fout7.good()) { cerr << "Could not open index file for writing: \"" << file7.c_str() << "\"" << endl << "Please make sure the directory exists and that permissions allow writing by" << endl << "HISAT2." << endl; throw 1; } // Open output stream for the '.8.gfm_ext' file which will // hold SNP IDs. ofstream fout8(file8.c_str(), ios::binary); if(!fout8.good()) { cerr << "Could not open index file for writing: \"" << file8.c_str() << "\"" << endl << "Please make sure the directory exists and that permissions allow writing by" << endl << "HISAT2." << endl; throw 1; } writeIndex(fout7, 1, this->toBe()); // endianness sentinel writeIndex(fout8, 1, this->toBe()); // endianness sentinel for(index_t i = 0; i < _refnames.size(); i++) { _refnames_nospace.push_back(""); for(index_t j = 0; j < _refnames[i].size(); j++) { char c = _refnames[i][j]; if(c == ' ') break; _refnames_nospace.back().push_back(c); } } map snpID2num; if(snpfile != "") { ifstream snp_file(snpfile.c_str(), ios::in); if(!snp_file.is_open()) { cerr << "Error: could not open " << snpfile.c_str() << endl; throw 1; } while(!snp_file.eof()) { // rs73387790 single 22:20000001-21000000 145 A string snp_id; snp_file >> snp_id; if(snp_id.empty() || snp_id[0] == '#') { string line; getline(snp_file, line); continue; } string type, chr; index_t genome_pos; char snp_ch = '\0'; string ins_seq; index_t del_len = 0; snp_file >> type >> chr >> genome_pos; if(type == "single") { snp_file >> snp_ch; } else if(type == "deletion") { snp_file >> del_len; } else if(type == "insertion") { snp_file >> ins_seq; } index_t chr_idx = 0; for(; chr_idx < _refnames_nospace.size(); chr_idx++) { if(chr == _refnames_nospace[chr_idx]) break; } if(chr_idx >= _refnames_nospace.size()) { continue; } assert_eq(chr_szs.size(), _refnames_nospace.size()); assert_lt(chr_idx, chr_szs.size()); pair tmp_pair = chr_szs[chr_idx]; const index_t sofar_len = tmp_pair.first; const index_t szs_idx = tmp_pair.second; bool involve_Ns = false; index_t pos = genome_pos; index_t add_pos = 0; assert(szs[szs_idx].first); for(index_t i = szs_idx; i < szs.size(); i++) { if(i != szs_idx && szs[i].first) { break; } if(pos < szs[i].off) { involve_Ns = true; break; } else { pos -= szs[i].off; if(pos == 0) { if(type == "deletion" || type == "insertion") { involve_Ns = true; break; } } if(pos < szs[i].len) { break; } else { pos -= szs[i].len; add_pos += szs[i].len; } } } if(involve_Ns) { continue; } pos = sofar_len + add_pos + pos; if(chr_idx + 1 < chr_szs.size()) { if(pos >= chr_szs[chr_idx + 1].first) { continue; } } else { if(pos >= jlen){ continue; } } _alts.expand(); ALT& snp = _alts.back(); snp.pos = pos; if(type == "single") { snp.type = ALT_SNP_SGL; snp_ch = toupper(snp_ch); if(snp_ch != 'A' && snp_ch != 'C' && snp_ch != 'G' && snp_ch != 'T') { _alts.pop_back(); continue; } uint64_t bp = asc2dna[(int)snp_ch]; assert_lt(bp, 4); if((int)bp == s[pos]) { cerr << "Warning: single type should have a different base than " << "ACGTN"[(int)s[pos]] << " (" << snp_id << ") at " << genome_pos << " on " << chr << endl; _alts.pop_back(); continue; // throw 1; } snp.len = 1; snp.seq = bp; } else if(type == "deletion") { snp.type = ALT_SNP_DEL; snp.len = del_len; snp.seq = 0; snp.reversed = false; } else if(type == "insertion") { snp.type = ALT_SNP_INS; snp.len = (index_t)ins_seq.size(); if(snp.len > sizeof(snp.seq) * 4) { _alts.pop_back(); continue; } snp.seq = 0; bool failed = false; for(size_t i = 0; i < ins_seq.size(); i++) { char ch = toupper(ins_seq[i]); if(ch != 'A' && ch != 'C' && ch != 'G' && ch != 'T') { failed = true; break; } uint64_t bp = asc2dna[(int)ch]; assert_lt(bp, 4); snp.seq = (snp.seq << 2) | bp; } if(failed) { _alts.pop_back(); continue; } } else { cerr << "Error: unknown snp type " << type << endl; throw 1; } _altnames.push_back(snp_id); assert_eq(_alts.size(), _altnames.size()); snpID2num[snp_id] = (index_t)_alts.size() - 1; } snp_file.close(); assert_eq(_alts.size(), _altnames.size()); } _haplotypes.clear(); if(_alts.size() > 0 && htfile != "") { ifstream ht_file(htfile.c_str(), ios::in); if(!ht_file.is_open()) { cerr << "Error: could not open "<< htfile.c_str() << endl; throw 1; } while(!ht_file.eof()) { // ht66 A*01:01:01:01 371 533 66,69,72,75,76,77,84,88,90,92,95 string ht_id; ht_file >> ht_id; if(ht_id.empty() || ht_id[0] == '#') { string line; getline(ht_file, line); continue; } string chr, alt_list; index_t left, right; // inclusive [left, right] ht_file >> chr >> left >> right >> alt_list; assert_leq(left, right); index_t chr_idx = 0; for(; chr_idx < _refnames_nospace.size(); chr_idx++) { if(chr == _refnames_nospace[chr_idx]) break; } if(chr_idx >= _refnames_nospace.size()) { continue; } assert_eq(chr_szs.size(), _refnames_nospace.size()); assert_lt(chr_idx, chr_szs.size()); pair tmp_pair = chr_szs[chr_idx]; const index_t sofar_len = tmp_pair.first; const index_t szs_idx = tmp_pair.second; bool inside_Ns = false; index_t add_pos = 0; assert(szs[szs_idx].first); for(index_t i = szs_idx; i < szs.size(); i++) { if(i != szs_idx && szs[i].first) break; if(left < szs[i].off) { inside_Ns = true; break; } else { left -= szs[i].off; right -= szs[i].off; if(left < szs[i].len) { if(right >= szs[i].len) { inside_Ns = true; } break; } else { left -= szs[i].len; right -= szs[i].len; add_pos += szs[i].len; } } } if(inside_Ns) { continue; } left = sofar_len + add_pos + left; right = sofar_len + add_pos + right; if(chr_idx + 1 < chr_szs.size()) { if(right >= chr_szs[chr_idx + 1].first) { continue; } } else { if(right >= jlen) { continue; } } _haplotypes.expand(); _haplotypes.back().left = left; _haplotypes.back().right = right; EList alts; tokenize(alt_list, ",", alts); assert_gt(alts.size(), 0); _haplotypes.back().alts.clear(); for(size_t i = 0; i < alts.size(); i++) { const string& alt = alts[i]; if(snpID2num.find(alt) != snpID2num.end()) { _haplotypes.back().alts.push_back(snpID2num[alt]); } } if(_haplotypes.back().alts.size() <= 0) { _haplotypes.pop_back(); } } _haplotypes.sort(); ht_file.close(); } else { for(index_t a = 0; a < _alts.size(); a++) { const ALT& alt = _alts[a]; if(!alt.snp()) continue; _haplotypes.expand(); _haplotypes.back().left = alt.pos; if(alt.deletion()) { _haplotypes.back().right = alt.pos + alt.len - 1; } else { _haplotypes.back().right = alt.pos; } _haplotypes.back().alts.clear(); _haplotypes.back().alts.push_back(a); } } if(ssfile != "") { ifstream ss_file(ssfile.c_str(), ios::in); if(!ss_file.is_open()) { cerr << "Error: could not open " << ssfile.c_str() << endl; throw 1; } map ss_seq; while(!ss_file.eof()) { // 22 16062315 16062810 + string chr; ss_file >> chr; if(chr.empty() || chr[0] == '#') { string line; getline(ss_file, line); continue; } index_t left, right; char strand; ss_file >> left >> right >> strand; // Convert exonic position to intronic position left += 1; right -= 1; if(left >= right) continue; index_t chr_idx = 0; for(; chr_idx < _refnames_nospace.size(); chr_idx++) { if(chr == _refnames_nospace[chr_idx]) break; } if(chr_idx >= _refnames_nospace.size()) continue; assert_eq(chr_szs.size(), _refnames_nospace.size()); assert_lt(chr_idx, chr_szs.size()); pair tmp_pair = chr_szs[chr_idx]; const index_t sofar_len = tmp_pair.first; const index_t szs_idx = tmp_pair.second; // check whether ambiguous base is in exon's last and first base if(!checkPosToSzs(szs, szs_idx, left - 1) || !checkPosToSzs(szs, szs_idx, right + 1)) { //cerr << "Skip ss. " << chr << ", " << left - 1 << ", " << right + 1 << endl; continue; } bool inside_Ns = false; index_t add_pos = 0; assert(szs[szs_idx].first); for(index_t i = szs_idx; i < szs.size(); i++) { if(i != szs_idx && szs[i].first) break; if(left < szs[i].off) { inside_Ns = true; break; } else { left -= szs[i].off; right -= szs[i].off; if(left < szs[i].len) { if(right >= szs[i].len) { inside_Ns = true; } break; } else { left -= szs[i].len; right -= szs[i].len; add_pos += szs[i].len; } } } if(inside_Ns) continue; left = sofar_len + add_pos + left; right = sofar_len + add_pos + right; if(chr_idx + 1 < chr_szs.size()) { if(right >= chr_szs[chr_idx + 1].first) continue; } else { if(right >= jlen) continue; } // Avoid splice sites in repetitive sequences // Otherwise, it will likely explode due to an exponential number of combinations index_t seqlen = 16; assert_leq(seqlen, 16); if(left >= seqlen && right + 1 + seqlen <= s.length()) { uint64_t seq = 0; for(index_t si = left - seqlen; si < left; si++) { seq = seq << 2 | s[si]; } for(index_t si = right + 1; si < right + 1 + seqlen; si++) { seq = seq << 2 | s[si]; } if(_alts.size() > 0) { if(_alts.back().left == left && _alts.back().right == right) continue; } if(ss_seq.find(seq) == ss_seq.end()) ss_seq[seq] = 1; else ss_seq[seq]++; } _alts.expand(); ALT& alt = _alts.back(); alt.type = ALT_SPLICESITE; alt.left = left; alt.right = right; alt.fw = (strand == '+' ? true : false); alt.excluded = false; _altnames.push_back("ss"); } ss_file.close(); assert_eq(_alts.size(), _altnames.size()); for(size_t i = 0; i < _alts.size(); i++) { ALT& alt = _alts[i]; if(!alt.splicesite()) continue; index_t seqlen = 16; assert_leq(seqlen, 16); if(alt.left >= seqlen && alt.right + 1 + seqlen <= s.length()) { uint64_t seq = 0; for(index_t si = alt.left - seqlen; si < alt.left; si++) { seq = seq << 2 | s[si]; } for(index_t si = alt.right + 1; si < alt.right + 1 + seqlen; si++) { seq = seq << 2 | s[si]; } assert(ss_seq.find(seq) != ss_seq.end()); alt.excluded = ss_seq[seq] > 1; } } } if(exonfile != "") { ifstream exon_file(exonfile.c_str(), ios::in); if(!exon_file.is_open()) { cerr << "Error: could not open " << ssfile.c_str() << endl; throw 1; } while(!exon_file.eof()) { // 22 16062156 16062315 + string chr; exon_file >> chr; if(chr.empty() || chr[0] == '#') { string line; getline(exon_file, line); continue; } index_t left, right; char strand; exon_file >> left >> right >> strand; // Convert exonic position to intronic position left += 1; right -= 1; if(left >= right) continue; index_t chr_idx = 0; for(; chr_idx < _refnames_nospace.size(); chr_idx++) { if(chr == _refnames_nospace[chr_idx]) break; } if(chr_idx >= _refnames_nospace.size()) continue; assert_eq(chr_szs.size(), _refnames_nospace.size()); assert_lt(chr_idx, chr_szs.size()); pair tmp_pair = chr_szs[chr_idx]; const index_t sofar_len = tmp_pair.first; const index_t szs_idx = tmp_pair.second; bool inside_Ns = false; index_t add_pos = 0; assert(szs[szs_idx].first); for(index_t i = szs_idx; i < szs.size(); i++) { if(i != szs_idx && szs[i].first) break; if(left < szs[i].off) { inside_Ns = true; break; } else { left -= szs[i].off; right -= szs[i].off; if(left < szs[i].len) { if(right >= szs[i].len) { inside_Ns = true; } break; } else { left -= szs[i].len; right -= szs[i].len; add_pos += szs[i].len; } } } if(inside_Ns) continue; left = sofar_len + add_pos + left; right = sofar_len + add_pos + right; if(chr_idx + 1 < chr_szs.size()) { if(right >= chr_szs[chr_idx + 1].first) continue; } else { if(right >= jlen) continue; } _alts.expand(); ALT& alt = _alts.back(); alt.type = ALT_EXON; alt.left = left; alt.right = right; alt.fw = (strand == '+' ? true : false); _altnames.push_back("exon"); } exon_file.close(); } // Todo - implement structural variations if(svfile != "") { cerr << "Warning: SV option is not implemented " << svfile.c_str() << endl; } // Sort SNPs and Splice Sites based on positions if(_alts.size() > 1) { assert_eq(_alts.size(), _altnames.size()); EList, index_t> > buf; buf.resize(_alts.size()); EList buf2; buf2.resize(_alts.size()); for(size_t i = 0; i < _alts.size(); i++) { buf[i].first = _alts[i]; buf[i].second = (index_t)i; buf2[i] = _altnames[i]; } buf.sort(); for(size_t i = 0; i < _alts.size(); i++) { _alts[i] = buf[i].first; _altnames[i] = buf2[buf[i].second]; } EList buf3; buf3.resize(_alts.size()); for(size_t i = 0; i < buf3.size(); i++) { index_t before = buf[i].second; assert_lt(before, buf3.size()); buf3[before] = (index_t)i; } for(size_t h = 0; h < _haplotypes.size(); h++) { EList& alts = _haplotypes[h].alts; for(size_t a = 0; a < alts.size(); a++) { index_t before = alts[a]; assert_lt(before, buf3.size()); alts[a] = buf3[before]; } } #ifndef NDEBUG for(size_t i = 0; i < _alts.size(); i++) { if(i + 1 < _alts.size()) { assert(_alts[i] < _alts[i+1]); } const ALT& alt = _alts[i]; if(alt.snp()) { assert(_altnames[i] != ""); } else if(alt.splicesite()) { assert(_altnames[i] == "ss"); } else if(alt.exon()) { assert(_altnames[i] == "exon"); } else { assert(false); } } #endif } writeIndex(fout7, (index_t)_alts.size(), this->toBe()); writeIndex(fout8, (index_t)_alts.size(), this->toBe()); for(index_t i = 0; i < _alts.size(); i++) { _alts[i].write(fout7, this->toBe()); fout8 << _altnames[i] << endl; } writeIndex(fout7, (index_t)_haplotypes.size(), this->toBe()); for(index_t i = 0; i < _haplotypes.size(); i++) { _haplotypes[i].write(fout7, this->toBe()); } EList >& repeats = _repeatdb.repeats(); if(_repeat) { ifstream repeat_file(repeatfile.c_str(), ios::in); if(!repeat_file.is_open()) { cerr << "Error: could not open " << ssfile.c_str() << endl; throw 1; } if(parent_szs == NULL) { throw 1; } if(parent_refnames == NULL) { throw 1; } EList > parent_chr_szs; index_t tmp_len = 0; for(index_t i = 0; i < parent_szs->size(); i++) { if((*parent_szs)[i].first) { parent_chr_szs.expand(); parent_chr_szs.back().first = tmp_len; parent_chr_szs.back().second = i; } tmp_len += (index_t)(*parent_szs)[i].len; } index_t parent_jlen = joinedLen(*parent_szs); string prev_repName = ""; while(!repeat_file.eof()) { // >rep1*0 rep 0 100 470 0 // 20_rep:26622650:+ 20_rep:26628088:+ 20_rep:26632508:+ 20_rep:26635636:+ // 20_rep:26669936:+ 20_rep:26672654:+ 20_rep:26675373:+ 20_rep:26678095:+ string repName, repAlleleName; repeat_file >> repAlleleName; if(repAlleleName.empty()) // Reached the end of file break; if(repAlleleName[0] != '>') { cerr << "Error: the file format is not correct" << endl; throw 1; } repAlleleName = repAlleleName.substr(1); // Remove '>' index_t alleleID = 0; size_t star_pos = repAlleleName.find('*'); if(star_pos >= repAlleleName.length()) { repName = repAlleleName; } else { repName = repAlleleName.substr(0, star_pos); string strID = repAlleleName.substr(star_pos + 1); istringstream(strID) >> alleleID; } string refRepName; index_t repPos, repLen; repeat_file >> refRepName >> repPos >> repLen; index_t rep_idx = 0; for(; rep_idx < _refnames_nospace.size(); rep_idx++) { if(refRepName == _refnames_nospace[rep_idx]) break; } if(rep_idx >= _refnames_nospace.size()) { cerr << "Error: " << refRepName << " is not found in " << endl; throw 1; } if(repeats.size() == 0 || repeats.back().repID != rep_idx || repeats.back().repName != repName) { if(repeats.size() > 0) { repeats.back().positions.sort(); } repeats.expand(); repeats.back().init(repName, rep_idx, repPos, repLen); } // update repPos and repLen if(repPos < repeats.back().repPos) { repeats.back().repLen += (repeats.back().repPos - repPos); repeats.back().repPos = repPos; } if(repPos + repLen > repeats.back().repPos + repeats.back().repLen) { repeats.back().repLen = repPos + repLen - repeats.back().repPos; } size_t baseOff = 0; if(repeats.size() > 1 && repeats[repeats.size() - 2].repID == rep_idx) { baseOff = repeats[repeats.size() - 2].repPos + repeats[repeats.size() - 2].repLen; } index_t numCoords, numAlts; repeat_file >> numCoords >> numAlts; EList snpIDs; EList snpStrIDs; if(numAlts > 0) { string snpStrID; repeat_file >> snpStrID; tokenize(snpStrID, ",", snpStrIDs); if(snpStrIDs.size() != numAlts) { assert(false); cerr << "Error: the number of SNPs (" << snpIDs.size() << ", " << snpStrID << ") does not equal to " << numAlts << endl; throw 1; } for(index_t i = 0; i < snpStrIDs.size(); i++) { if(snpID2num.find(snpStrIDs[i]) == snpID2num.end()) { cerr << "Error: " << snpStrIDs[i] << " is not found" << endl; throw 1; } index_t numID = snpID2num[snpStrIDs[i]]; snpIDs.push_back(numID); } } EList >& positions = repeats.back().positions; size_t sofar_numCoords = positions.size(); while(positions.size() - sofar_numCoords < numCoords) { string chr_pos; repeat_file >> chr_pos; size_t colon_pos = chr_pos.find(':'); if(colon_pos + 1 >= chr_pos.length()) { cerr << "Error: : is not found in " << chr_pos << endl; throw 1; } string chr = chr_pos.substr(0, colon_pos); string strPos = chr_pos.substr(colon_pos + 1, chr_pos.length() - colon_pos - 3); bool repfw = (chr_pos[chr_pos.length() - 1] == '+'); index_t pos = 0; istringstream(strPos) >> pos; index_t chr_idx = 0; for(; chr_idx < parent_refnames->size(); chr_idx++) { if(chr == (*parent_refnames)[chr_idx]) break; } if(chr_idx >= parent_refnames->size()) { cerr << "Error: " << chr << " is not found in " << endl; throw 1; } assert_eq(parent_chr_szs.size(), parent_refnames->size()); assert_lt(chr_idx, parent_chr_szs.size()); positions.expand(); positions.back().tid = chr_idx; positions.back().toff = pos; positions.back().fw = repfw; positions.back().alleleID = alleleID; pair tmp_pair = parent_chr_szs[chr_idx]; const index_t sofar_len = tmp_pair.first; const index_t szs_idx = tmp_pair.second; bool involve_Ns = false; index_t add_pos = 0; assert((*parent_szs)[szs_idx].first); for(index_t i = szs_idx; i < parent_szs->size(); i++) { if(i != szs_idx && (*parent_szs)[i].first) { break; } if(pos < (*parent_szs)[i].off) { involve_Ns = true; break; } else { pos -= (*parent_szs)[i].off; if(pos < (*parent_szs)[i].len) { break; } else { pos -= (*parent_szs)[i].len; add_pos += (*parent_szs)[i].len; } } } if(involve_Ns) { assert(false); throw 1; } pos = sofar_len + add_pos + pos; if(chr_idx + 1 < parent_chr_szs.size()) { if(pos >= parent_chr_szs[chr_idx + 1].first) { assert(false); throw 1; } } else { if(pos >= parent_jlen){ assert(false); throw 1; } } positions.back().joinedOff = pos; } repeats.back().alleles.expand(); assert_geq(repPos, baseOff); repeats.back().alleles.back().init(repPos - baseOff, repLen); } if(repeats.size() > 0) { repeats.back().positions.sort(); } repeat_file.close(); index_t total_repeat_len = 0; for(size_t r = 0; r + 1 < repeats.size(); r++) { if(repeats[r].repID != repeats[r+1].repID) { index_t repeat_len = repeats[r].repPos + repeats[r].repLen; total_repeat_len += repeat_len; } } index_t repeat_len = repeats.back().repPos + repeats.back().repLen; total_repeat_len += repeat_len; if(total_repeat_len != s.length()) { cerr << "Error: repeat length (" << repeats.back().repPos + repeats.back().repLen; cerr << ") does not match sequence length (" << s.length() << ")" << endl; throw 1; } _repeatLens.resizeExact(szs.size()); for(size_t i = 0; i < _repeatLens.size(); i++) { _repeatLens[i].first = numeric_limits::max(); _repeatLens[i].second = 0; } for(size_t i = 0; i < repeats.size(); i++) { index_t id = repeats[i].repID; index_t len = repeats[i].repLen; assert_lt(id, _repeatLens.size()); if(_repeatLens[id].first > len) { _repeatLens[id].first = len; } if(_repeatLens[id].second < len) { _repeatLens[id].second = len; } } writeIndex(fout7, _repeatLens.size(), this->toBe()); for(size_t i = 0; i < _repeatLens.size(); i++) { writeIndex(fout7, _repeatLens[i].first, this->toBe()); writeIndex(fout7, _repeatLens[i].second, this->toBe()); } _repeatdb.write(fout7, this->toBe()); writeIndex(fout7, chr_szs.size(), this->toBe()); // number of repeat indexes EList seqs; EList tableFilePos; streampos filepos = fout7.tellp(); for(size_t i = 0; i < chr_szs.size(); i++) { writeIndex(fout7, 0, this->toBe()); } for(size_t i = 0; i < repeats.size(); i++) { const Repeat& repeat = repeats[i]; assert_lt(repeat.repID, chr_szs.size()); index_t template_len = 0; if(repeat.repID + 1 < chr_szs.size()) { template_len = chr_szs[repeat.repID + 1].first - chr_szs[repeat.repID].first; } else { template_len = s.length() - chr_szs[repeat.repID].first; } assert_leq(repeat.repPos + repeat.repLen, template_len); index_t pos = chr_szs[repeat.repID].first + repeat.repPos; assert_leq(pos + repeat.repLen, s.length()); seqs.expand(); seqs.back().clear(); for(index_t j = 0; j < repeat.repLen; j++) { int c = s[pos + j]; assert_range(0, 3, c); seqs.back().push_back("ACGT"[c]); } if(i + 1 == repeats.size() || repeats[i].repID != repeats[i+1].repID) { const size_t w = RB_Minimizer::default_w, k = RB_Minimizer::default_k; RB_KmerTable kmer_table; kmer_table.build(seqs, w, k); kmer_table.write(fout7, this->toBe()); seqs.clear(); tableFilePos.push_back(fout7.tellp()); } } assert_eq(tableFilePos.size(), chr_szs.size()); streampos origpos = fout7.tellp(); fout7.seekp(filepos); for(size_t i = 0; i < tableFilePos.size(); i++) { writeIndex(fout7, tableFilePos[i], this->toBe()); } fout7.seekp(origpos); } fout7.close(); fout8.close(); } // Joined reference sequence now in 's' } catch(bad_alloc& e) { // If we throw an allocation exception in the try block, // that means that the joined version of the reference // string itself is too larger to fit in memory. The only // alternatives are to tell the user to give us more memory // or to try again with a packed representation of the // reference (if we haven't tried that already). cerr << "Could not allocate space for a joined string of " << jlen << " elements." << endl; if(!isPacked() && _passMemExc) { // Pass the exception up so that we can retry using a // packed string representation throw e; } // There's no point passing this exception on. The fact // that we couldn't allocate the joined string means that // --bmax is irrelevant - the user should re-run with // ebwt-build-packed if(isPacked()) { cerr << "Please try running bowtie-build on a computer with more memory." << endl; } else { cerr << "Please try running bowtie-build in packed mode (-p/--packed) or in automatic" << endl << "mode (-a/--auto), or try again on a computer with more memory." << endl; } if(sizeof(void*) == 4) { cerr << "If this computer has more than 4 GB of memory, try using a 64-bit executable;" << endl << "this executable is 32-bit." << endl; } throw 1; } // Succesfully obtained joined reference string assert_geq(s.length(), jlen); if(bmax != (index_t)OFF_MASK) { // VMSG_NL("bmax according to bmax setting: " << bmax); } else if(bmaxSqrtMult != (index_t)OFF_MASK) { bmax *= bmaxSqrtMult; // VMSG_NL("bmax according to bmaxSqrtMult setting: " << bmax); } else if(bmaxDivN != (index_t)OFF_MASK) { bmax = max(jlen / (bmaxDivN * _nthreads), 1); // VMSG_NL("bmax according to bmaxDivN setting: " << bmax); } else { bmax = (uint32_t)sqrt(s.length()); // VMSG_NL("bmax defaulted to: " << bmax); } int iter = 0; bool first = true; streampos out1pos = out1.tellp(); streampos out2pos = out2.tellp(); if(!_repeat) { // Look for bmax/dcv parameters that work. while(true) { if(!first && bmax < 40 && _passMemExc) { cerr << "Could not find approrpiate bmax/dcv settings for building this index." << endl; if(!isPacked()) { // Throw an exception exception so that we can // retry using a packed string representation throw bad_alloc(); } else { cerr << "Already tried a packed string representation." << endl; } cerr << "Please try indexing this reference on a computer with more memory." << endl; if(sizeof(void*) == 4) { cerr << "If this computer has more than 4 GB of memory, try using a 64-bit executable;" << endl << "this executable is 32-bit." << endl; } throw 1; } if(!first) { out1.seekp(out1pos); out2.seekp(out2pos); } if(dcv > 4096) dcv = 4096; if((iter % 6) == 5 && dcv < 4096 && dcv != 0) { dcv <<= 1; // double difference-cover period } else { bmax -= (bmax >> 2); // reduce by 25% } iter++; try { if(_alts.empty()) { VMSG("Using parameters --bmax " << bmax); if(dcv == 0) { VMSG_NL(" and *no difference cover*"); } else { VMSG_NL(" --dcv " << dcv); } { VMSG_NL(" Doing ahead-of-time memory usage test"); // Make a quick-and-dirty attempt to force a bad_alloc iff // we would have thrown one eventually as part of // constructing the DifferenceCoverSample dcv <<= 1; index_t sz = (index_t)DifferenceCoverSample::simulateAllocs(s, dcv >> 1); if(_nthreads > 1) sz *= (_nthreads + 1); AutoArray tmp(sz, EBWT_CAT); dcv >>= 1; // Likewise with the KarkkainenBlockwiseSA sz = (index_t)KarkkainenBlockwiseSA::simulateAllocs(s, bmax); AutoArray tmp2(sz, EBWT_CAT); // Now throw in the 'ftab' and 'isaSample' structures // that we'll eventually allocate in buildToDisk AutoArray ftab(_gh._ftabLen * 2, EBWT_CAT); AutoArray side(_gh._sideSz, EBWT_CAT); // Grab another 20 MB out of caution AutoArray extra(20*1024*1024, EBWT_CAT); // If we made it here without throwing bad_alloc, then we // passed the memory-usage stress test VMSG(" Passed! Constructing with these parameters: --bmax " << bmax << " --dcv " << dcv); if(isPacked()) { VMSG(" --packed"); } VMSG_NL(""); } VMSG_NL("Constructing suffix-array element generator"); KarkkainenBlockwiseSA bsa(s, bmax, _nthreads, dcv, seed, _sanity, _passMemExc, _verbose, outfile); assert(bsa.suffixItrIsReset()); assert_eq(bsa.size(), s.length()+1); VMSG_NL("Converting suffix-array elements to index image"); buildToDisk(bsa, s, out1, out2); } else { RefGraph* graph = new RefGraph( s, szs, _alts, _haplotypes, outfile, _nthreads, verbose); PathGraph* pg = new PathGraph( *graph, outfile, std::numeric_limits::max(), _nthreads, verbose); if(verbose) { cerr << "Generating edges... " << endl; } if(!pg->generateEdges(*graph)) { return; } // Re-initialize GFM parameters to reflect real number of edges (gbwt string) _gh.init( _gh.len(), pg->getNumEdges(), pg->getNumNodes(), _gh.lineRate(), _gh.offRate(), _gh.ftabChars(), 0, _gh.entireReverse()); buildToDisk(*pg, s, out1, out2); delete pg; pg = NULL; delete graph; graph = NULL; } out1.flush(); out2.flush(); if(out1.fail() || out2.fail()) { cerr << "An error occurred writing the index to disk. Please check if the disk is full." << endl; throw 1; } break; } catch(bad_alloc& e) { if(_passMemExc) { VMSG_NL(" Ran out of memory; automatically trying more memory-economical parameters."); } else { cerr << "Out of memory while constructing suffix array. Please try using a smaller" << endl << "number of blocks by specifying a smaller --bmax or a larger --bmaxdivn" << endl; throw 1; } } first = false; } assert(repOk()); // Now write reference sequence names on the end assert_eq(this->_refnames.size(), this->_nPat); for(index_t i = 0; i < this->_refnames.size(); i++) { out1 << this->_refnames[i].c_str() << endl; } out1 << '\0'; out1.flush(); out2.flush(); if(out1.fail() || out2.fail()) { cerr << "An error occurred writing the index to disk. Please check if the disk is full." << endl; throw 1; } } VMSG_NL("Returning from initFromVector"); } /** * Return the length that the joined string of the given string * list will have. Note that this is indifferent to how the text * fragments correspond to input sequences - it just cares about * the lengths of the fragments. */ index_t joinedLen(EList& szs) { index_t ret = 0; for(unsigned int i = 0; i < szs.size(); i++) { ret += (index_t)szs[i].len; } return ret; } /// Destruct an Ebwt ~GFM() { _fchr.reset(); _ftab.reset(); _eftab.reset(); _plen.reset(); _rstarts.reset(); _offs.reset(); _gfm.reset(); if(offs() != NULL && useShmem_) { FREE_SHARED(offs()); } if(gfm() != NULL && useShmem_) { FREE_SHARED(gfm()); } if (_in1 != NULL) fclose(_in1); if (_in2 != NULL) fclose(_in2); } /// Accessors inline const GFMParams& gh() const { return _gh; } index_t numZOffs() const { return _zOffs.size(); } index_t zOff(index_t i) const { assert_lt(i, _zOffs.size()); return _zOffs[i]; } index_t zGbwtByteOff(index_t i) const { assert_lt(i, _zGbwtByteOffs.size()); return _zGbwtByteOffs[i]; } int zGbwtBpOff(index_t i) const { assert_lt(i, _zGbwtBpOffs.size()); return _zGbwtBpOffs[i]; } index_t nPat() const { return _nPat; } index_t nFrag() const { return _nFrag; } inline index_t* fchr() { return _fchr.get(); } inline index_t* ftab() { return _ftab.get(); } inline index_t* eftab() { return _eftab.get(); } inline index_t* offs() { return _offs.get(); } inline index_t* plen() { return _plen.get(); } inline index_t* rstarts() { return _rstarts.get(); } inline uint8_t* gfm() { return _gfm.get(); } inline const index_t* fchr() const { return _fchr.get(); } inline const index_t* ftab() const { return _ftab.get(); } inline const index_t* eftab() const { return _eftab.get(); } inline const index_t* offs() const { return _offs.get(); } inline const index_t* plen() const { return _plen.get(); } inline const index_t* rstarts() const { return _rstarts.get(); } inline const uint8_t* gfm() const { return _gfm.get(); } inline const EList >& alts() const { return _alts; } inline const EList& altnames() const { return _altnames; } bool toBe() const { return _toBigEndian; } bool verbose() const { return _verbose; } bool sanityCheck() const { return _sanity; } EList& refnames() { return _refnames; } bool fw() const { return fw_; } bool repeat() const { return _repeat; } const EList& getRepeatIncluded() const { return _repeatIncluded; } #ifdef POPCNT_CAPABILITY bool _usePOPCNTinstruction; #endif /** * Returns true iff the index contains the given string (exactly). The * given string must contain only unambiguous characters. TODO: * support skipping of ambiguous characters. */ bool contains( const BTDnaString& str, index_t *top = NULL, index_t *bot = NULL) const; /** * Returns true iff the index contains the given string (exactly). The * given string must contain only unambiguous characters. TODO: * support skipping of ambiguous characters. */ bool contains( const char *str, index_t *top = NULL, index_t *bot = NULL) const { return contains(BTDnaString(str, true), top, bot); } /// Return true iff the Ebwt is currently in memory bool isInMemory() const { if(gfm() != NULL) { // Note: We might have skipped loading _offs, _ftab, // _eftab, and _rstarts depending on whether this is the // reverse index and what algorithm is being used. assert(_gh.repOk()); //assert(_ftab != NULL); //assert(_eftab != NULL); assert(fchr() != NULL); //assert(_offs != NULL); //assert(_rstarts != NULL); // assert_neq(_zGbwtByteOff, INDEX_MAX); // assert_neq(_zGbwtBpOff, -1); return true; } else { assert(ftab() == NULL); assert(eftab() == NULL); assert(fchr() == NULL); assert(offs() == NULL); assert(rstarts() == NULL); assert_eq(_zOffs.size(), 0); assert_eq(_zGbwtByteOffs.size(), 0); assert_eq(_zGbwtBpOffs.size(), 0); return false; } } /// Return true iff the Ebwt is currently stored on disk bool isEvicted() const { return !isInMemory(); } /** * Load this Ebwt into memory by reading it in from the _in1 and * _in2 streams. */ void loadIntoMemory( int needEntireReverse, bool loadSASamp, bool loadFtab, bool loadRstarts, bool loadNames, bool verbose) { readIntoMemory( needEntireReverse, // require reverse index to be concatenated reference reversed loadSASamp, // load the SA sample portion? loadFtab, // load the ftab (_ftab[] and _eftab[])? loadRstarts, // load the r-starts (_rstarts[])? false, // stop after loading the header portion? NULL, // params false, // mmSweep loadNames, // loadNames verbose); // startVerbose } /** * Frees memory associated with the Ebwt. */ void evictFromMemory() { assert(isInMemory()); _fchr.free(); _ftab.free(); _eftab.free(); _rstarts.free(); _offs.free(); // might not be under control of APtrWrap _gfm.free(); // might not be under control of APtrWrap // Keep plen; it's small and the client may want to seq it // even when the others are evicted. //_plen = NULL; _zOffs.clear(); _zGbwtByteOffs.clear(); _zGbwtBpOffs.clear(); } /** * Turn a substring of 'seq' starting at offset 'off' and having * length equal to the index's 'ftabChars' into an int that can be * used to index into the ftab array. */ index_t ftabSeqToInt( const BTDnaString& seq, index_t off, bool rev) const { int fc = _gh._ftabChars; index_t lo = off, hi = lo + fc; assert_leq(hi, seq.length()); index_t ftabOff = 0; for(int i = 0; i < fc; i++) { bool fwex = fw(); if(rev) fwex = !fwex; // We add characters to the ftabOff in the order they would // have been consumed in a normal search. For BWT, this // means right-to-left order; for BWT' it's left-to-right. int c = (fwex ? seq[lo + i] : seq[hi - i - 1]); if(c > 3) { return std::numeric_limits::max(); } assert_range(0, 3, c); ftabOff <<= 2; ftabOff |= c; } return ftabOff; } /** * Non-static facade for static function ftabHi. */ index_t ftabHi(index_t i) const { return GFM::ftabHi( ftab(), eftab(), _gh.linearFM() ? _gh._len : _gh._gbwtLen, _gh._ftabLen, _gh._eftabLen, i); } /** * Get "high interpretation" of ftab entry at index i. The high * interpretation of a regular ftab entry is just the entry * itself. The high interpretation of an extended entry is the * second correpsonding ui32 in the eftab. * * It's a static member because it's convenient to ask this * question before the Ebwt is fully initialized. */ static index_t ftabHi( const index_t *ftab, const index_t *eftab, index_t gbwtLen, index_t ftabLen, index_t eftabLen, index_t i) { assert_lt(i, ftabLen); if(ftab[i] <= gbwtLen) { return ftab[i]; } else { index_t efIdx = ftab[i] ^ (index_t)INDEX_MAX; assert_lt(efIdx*2+1, eftabLen); return eftab[efIdx*2+1]; } } /** * Non-static facade for static function ftabLo. */ index_t ftabLo(index_t i) const { return GFM::ftabLo( ftab(), eftab(), _gh.linearFM() ? _gh._len : _gh._gbwtLen, _gh._ftabLen, _gh._eftabLen, i); } /** * Get low bound of ftab range. */ index_t ftabLo(const BTDnaString& seq, index_t off) const { return ftabLo(ftabSeqToInt(seq, off, false)); } /** * Get high bound of ftab range. */ index_t ftabHi(const BTDnaString& seq, index_t off) const { return ftabHi(ftabSeqToInt(seq, off, false)); } /** * Extract characters from seq starting at offset 'off' and going either * forward or backward, depending on 'rev'. Order matters when compiling * the integer that gets looked up in the ftab. Each successive character * is ORed into the least significant bit-pair, and characters are * integrated in the direction of the search. */ bool ftabLoHi( const BTDnaString& seq, // sequence to extract from index_t off, // offset into seq to begin extracting bool rev, // reverse while extracting index_t& top, index_t& bot) const { index_t fi = ftabSeqToInt(seq, off, rev); if(fi == std::numeric_limits::max()) { return false; } top = ftabHi(fi); bot = ftabLo(fi+1); assert_geq(bot, top); return true; } /** * Get "low interpretation" of ftab entry at index i. The low * interpretation of a regular ftab entry is just the entry * itself. The low interpretation of an extended entry is the * first correpsonding ui32 in the eftab. * * It's a static member because it's convenient to ask this * question before the Ebwt is fully initialized. */ static index_t ftabLo( const index_t *ftab, const index_t *eftab, index_t gbwtLen, index_t ftabLen, index_t eftabLen, index_t i) { assert_lt(i, ftabLen); if(ftab[i] <= gbwtLen) { return ftab[i]; } else { index_t efIdx = ftab[i] ^ (index_t)INDEX_MAX; assert_lt(efIdx*2+1, eftabLen); return eftab[efIdx*2]; } } /** * Try to resolve the reference offset of the BW element 'elt'. If * it can be resolved immediately, return the reference offset. If * it cannot be resolved immediately, return 0xffffffff. */ index_t tryOffset(index_t elt, index_t node) const { assert(offs() != NULL); for(index_t i = 0; i < _zOffs.size(); i++) { if(elt == _zOffs[i]) return 0; } if((node & _gh._offMask) == node) { index_t nodeOff = node >> _gh._offRate; assert_lt(nodeOff, _gh._offsLen); index_t off = offs()[nodeOff]; return off; } else { // Try looking at zoff return (index_t)INDEX_MAX; } } /** * Try to resolve the reference offset of the BW element 'elt' such * that the offset returned is at the right-hand side of the * forward reference substring involved in the hit. */ index_t tryOffset( index_t elt, bool fw, index_t hitlen) const { index_t off = tryOffset(elt); if(off != (index_t)INDEX_MAX && !fw) { assert_lt(off, _gh._len); off = _gh._len - off - 1; assert_geq(off, hitlen-1); off -= (hitlen-1); assert_lt(off, _gh._len); } return off; } /** * Walk 'steps' steps to the left and return the row arrived at. */ index_t walkLeft(index_t row, index_t steps) const; /** * Resolve the reference offset of the BW element 'elt'. */ index_t getOffset(index_t row, index_t node = 0) const; /** * Resolve the reference offset of the BW element 'elt' such that * the offset returned is at the right-hand side of the forward * reference substring involved in the hit. */ index_t getOffset( index_t elt, bool fw, index_t hitlen) const; /** * When using read() to create an Ebwt, we have to set a couple of * additional fields in the Ebwt object that aren't part of the * parameter list and are not stored explicitly in the file. Right * now, this just involves initializing _zEbwtByteOff and * _zEbwtBpOff from _zOff. */ void postReadInit(const GFMParams& gh) { _zGbwtByteOffs.resizeExact(_zOffs.size()); _zGbwtBpOffs.resizeExact(_zOffs.size()); for(index_t i = 0; i < _zOffs.size(); i++) { index_t sideNum = _zOffs[i] / gh._sideGbwtLen; index_t sideCharOff = _zOffs[i] % gh._sideGbwtLen; index_t sideByteOff = sideNum * gh._sideSz; _zGbwtByteOffs[i] = sideCharOff >> 2; assert_lt(_zGbwtByteOffs[i], gh._sideGbwtSz); _zGbwtBpOffs[i] = sideCharOff & 3; assert_lt(_zGbwtBpOffs[i], 4); _zGbwtByteOffs[i] += sideByteOff; } assert(repOk(gh)); // Ebwt should be fully initialized now } /** * Given basename of an Ebwt index, read and return its flag. */ static int32_t readVersionFlags(const string& instr, int& major, int& minor, string& extra_version); static void readProgramVersion(int& major_version, int& minor_version, string& extra_version) { char extra[256] = {0,}; int second_version; sscanf(HISAT2_VERSION, "%d.%d.%d-%s", &second_version, &major_version, &minor_version, extra); extra_version = extra; } static void readIndexVersion(int index_version, int& major_version, int& minor_version, string& extra_version) { major_version = (index_version >> 16) & 0xff; minor_version = (index_version >> 8) & 0xff; if((index_version & 0xff) == 1) { extra_version = "alpha"; } else if((index_version & 0xff) == 2) { extra_version = "beta"; } else { extra_version = ""; } } static int getIndexVersion() { int major_version = 0, minor_version = 0; string extra_version; readProgramVersion(major_version, minor_version, extra_version); int version = 2; // HISAT2 version = (version << 8) | (major_version & 0xff); version = (version << 8) | (minor_version & 0xff); version = version << 8; if(extra_version == "alpha") { version |= 0x1; } else if(extra_version == "beta") { version |= 0x2; } return version; } /** * Pretty-print the Ebwt to the given output stream. */ void print(ostream& out) const { print(out, _gh); } /** * Pretty-print the Ebwt and given EbwtParams to the given output * stream. */ void print(ostream& out, const GFMParams& gh) const { gh.print(out); // print params return; out << "Ebwt (" << (isInMemory()? "memory" : "disk") << "):" << endl; for(index_t i = 0; i < _zOffs.size(); i++) { out << " " << (i+1) << " zOffs: " << _zOffs[i] << endl << " " << (i+1) << " zGbwtByteOff: " << _zGbwtByteOffs[i] << endl << " " << (i+1) << " zGbwtBpOff: " << _zGbwtBpOffs[i] << endl; } out << " nPat: " << _nPat << endl << " plen: "; if(plen() == NULL) { out << "NULL" << endl; } else { out << "non-NULL, [0] = " << plen()[0] << endl; } out << " rstarts: "; if(rstarts() == NULL) { out << "NULL" << endl; } else { out << "non-NULL, [0] = " << rstarts()[0] << endl; } out << " ebwt: "; if(gfm() == NULL) { out << "NULL" << endl; } else { out << "non-NULL, [0] = " << gfm()[0] << endl; } out << " fchr: "; if(fchr() == NULL) { out << "NULL" << endl; } else { out << "non-NULL, [0] = " << fchr()[0] << endl; } out << " ftab: "; if(ftab() == NULL) { out << "NULL" << endl; } else { out << "non-NULL, [0] = " << ftab()[0] << endl; } out << " eftab: "; if(eftab() == NULL) { out << "NULL" << endl; } else { out << "non-NULL, [0] = " << eftab()[0] << endl; } out << " offs: "; if(offs() == NULL) { out << "NULL" << endl; } else { out << "non-NULL, [0] = " << offs()[0] << endl; } } // Building template static TStr join(EList& l, uint32_t seed); template static void join(EList& l, EList& szs, index_t sztot, const RefReadInParams& refparams, uint32_t seed, TStr& s, bool include_rc = false, bool CGtoTG = false); template void joinToDisk(EList& l, EList& szs, index_t sztot, const RefReadInParams& refparams, TStr& ret, ostream& out1, ostream& out2); template void buildToDisk(PathGraph& gbwt, const TStr& s, ostream& out1, ostream& out2, streampos headerPos = -1); template void buildToDisk(InorderBlockwiseSA& sa, const TStr& s, ostream& out1, ostream& out2, streampos headerPos = -1); // I/O void readIntoMemory(int needEntireRev, bool loadSASamp, bool loadFtab, bool loadRstarts, bool justHeader, GFMParams *params, bool mmSweep, bool loadNames, bool startVerbose, bool subIndex = false); void writeFromMemory(bool justHeader, ostream& out1, ostream& out2) const; void writeFromMemory(bool justHeader, const string& out1, const string& out2) const; // Sanity checking void sanityCheckUpToSide(int upToSide) const; void sanityCheckAll(int reverse) const; void restore(SString& s) const; void checkOrigs(const EList >& os, bool mirror) const; // Searching and reporting bool joinedToTextOff(index_t qlen, index_t off, index_t& tidx, index_t& textoff, index_t& tlen, bool rejectStraddle, bool& straddled) const; bool textOffToJoined(index_t tid, index_t tlen, index_t& off) const; #define WITHIN_BWT_LEN(x) \ assert_leq(x[0], this->_gh._sideGbwtLen); \ assert_leq(x[1], this->_gh._sideGbwtLen); \ assert_leq(x[2], this->_gh._sideGbwtLen); \ assert_leq(x[3], this->_gh._sideGbwtLen) #define WITHIN_FCHR(x) \ assert_leq(x[0], this->fchr()[1]); \ assert_leq(x[1], this->fchr()[2]); \ assert_leq(x[2], this->fchr()[3]); \ assert_leq(x[3], this->fchr()[4]) #define WITHIN_FCHR_DOLLARA(x) \ assert_leq(x[0], this->fchr()[1]+1); \ assert_leq(x[1], this->fchr()[2]); \ assert_leq(x[2], this->fchr()[3]); \ assert_leq(x[3], this->fchr()[4]) /** * Count all occurrences of character c from the beginning of the * forward side to and add in the occ[] count up to the side * break just prior to the side. * * A Bowtie 2 side is shaped like: * * XXXXXXXXXXXXXXXX [A] [C] [G] [T] * --------48------ -4- -4- -4- -4- (numbers in bytes) */ inline index_t countBt2Side(const SideLocus& l, int c) const { assert_range(0, 3, c); assert_range(0, (int)this->_gh._sideGbwtSz-1, (int)l._by); assert_range(0, 3, (int)l._bp); const uint8_t *side = l.side(this->gfm()); index_t cCnt = countUpTo(l, c); assert_leq(cCnt, l.toBWRow(_gh)); assert_leq(cCnt, this->_gh._sideGbwtLen); assert_eq(_zGbwtByteOffs.size(), _zGbwtBpOffs.size()); for(index_t i = 0; i < _zGbwtByteOffs.size(); i++) { index_t zGbwtByteOff = _zGbwtByteOffs[i]; if(c == 0 && l._sideByteOff <= zGbwtByteOff && l._sideByteOff + l._by >= zGbwtByteOff) { // Adjust for the fact that we represented $ with an 'A', but // shouldn't count it as an 'A' here int zGbwtBpOff = _zGbwtBpOffs[i]; if((l._sideByteOff + l._by > zGbwtByteOff) || (l._sideByteOff + l._by == zGbwtByteOff && l._bp > zGbwtBpOff)) { cCnt--; // Adjust for '$' looking like an 'A' } } } index_t ret; // Now factor in the occ[] count at the side break const uint8_t *acgt8 = side + _gh._sideGbwtSz; if(!_gh._linearFM) { acgt8 += (sizeof(index_t) << 1); } const index_t *acgt = reinterpret_cast(acgt8); assert_leq(acgt[0], this->_gh._numSides * this->_gh._sideGbwtLen); // b/c it's used as padding assert_lt(acgt[1], this->_gh._gbwtLen); assert_lt(acgt[2], this->_gh._gbwtLen); assert_lt(acgt[3], this->_gh._gbwtLen); ret = acgt[c] + cCnt + this->fchr()[c]; #ifndef NDEBUG assert_leq(ret, this->fchr()[c+1]); // can't have jumpded into next char's section if(c == 0) { assert_leq(cCnt, this->_gh._sideGbwtLen); } else { assert_leq(ret, this->_gh._gbwtLen); } #endif return ret; } /** * Count all occurrences of all four nucleotides up to the starting * point (which must be in a forward side) given by 'l' storing the * result in 'cntsUpto', then count nucleotide occurrences within the * range of length 'num' storing the result in 'cntsIn'. Also, keep * track of the characters occurring within the range by setting * 'masks' accordingly (masks[1][10] == true -> 11th character is a * 'C', and masks[0][10] == masks[2][10] == masks[3][10] == false. */ inline void countBt2SideRange( const SideLocus& l, // top locus index_t num, // number of elts in range to tall index_t* cntsUpto, // A/C/G/T counts up to top index_t* cntsIn, // A/C/G/T counts within range EList *masks) const // masks indicating which range elts = A/C/G/T { assert_gt(num, 0); assert_range(0, (int)this->_gh._sideGbwtSz-1, (int)l._by); assert_range(0, 3, (int)l._bp); countUpToEx(l, cntsUpto); WITHIN_FCHR_DOLLARA(cntsUpto); WITHIN_BWT_LEN(cntsUpto); const uint8_t *side = l.side(this->gfm()); assert_eq(_zGbwtByteOffs.size(), _zGbwtBpOffs.size()); for(index_t i = 0; i < _zGbwtByteOffs.size(); i++) { index_t zGbwtByteOff = _zGbwtByteOffs[i]; if(l._sideByteOff <= zGbwtByteOff && l._sideByteOff + l._by >= zGbwtByteOff) { // Adjust for the fact that we represented $ with an 'A', but // shouldn't count it as an 'A' here int zGbwtBpOff = _zGbwtBpOffs[i]; if((l._sideByteOff + l._by > zGbwtByteOff) || (l._sideByteOff + l._by == zGbwtByteOff && l._bp > zGbwtBpOff)) { cntsUpto[0]--; // Adjust for '$' looking like an 'A' } } } // Now factor in the occ[] count at the side break const index_t *acgt = reinterpret_cast(side + _gh._sideGbwtSz); if(!this->_gh.linearFM()) acgt += 2; assert_leq(acgt[0], this->fchr()[1] + this->_gh.sideGbwtLen()); assert_leq(acgt[1], this->fchr()[2]-this->fchr()[1]); assert_leq(acgt[2], this->fchr()[3]-this->fchr()[2]); assert_leq(acgt[3], this->fchr()[4]-this->fchr()[3]); assert_leq(acgt[0], this->_gh._gbwtLen + this->_gh.sideGbwtLen()); assert_leq(acgt[1], this->_gh._gbwtLen); assert_leq(acgt[2], this->_gh._gbwtLen); assert_leq(acgt[3], this->_gh._gbwtLen); cntsUpto[0] += (acgt[0] + this->fchr()[0]); cntsUpto[1] += (acgt[1] + this->fchr()[1]); cntsUpto[2] += (acgt[2] + this->fchr()[2]); cntsUpto[3] += (acgt[3] + this->fchr()[3]); masks[0].resize(num); masks[1].resize(num); masks[2].resize(num); masks[3].resize(num); WITHIN_FCHR_DOLLARA(cntsUpto); WITHIN_FCHR_DOLLARA(cntsIn); // 'cntsUpto' is complete now. // Walk forward until we've tallied the entire 'In' range index_t nm = 0; // Rest of this side nm += countBt2SideRange2(l, true, num - nm, cntsIn, masks, nm); assert_eq(nm, cntsIn[0] + cntsIn[1] + cntsIn[2] + cntsIn[3]); assert_leq(nm, num); SideLocus lcopy = l; while(nm < num) { // Subsequent sides, if necessary lcopy.nextSide(this->_gh); nm += countBt2SideRange2(lcopy, false, num - nm, cntsIn, masks, nm); WITHIN_FCHR_DOLLARA(cntsIn); assert_leq(nm, num); assert_eq(nm, cntsIn[0] + cntsIn[1] + cntsIn[2] + cntsIn[3]); } assert_eq(num, cntsIn[0] + cntsIn[1] + cntsIn[2] + cntsIn[3]); WITHIN_FCHR_DOLLARA(cntsIn); } /** * Count all occurrences of character c from the beginning of the * forward side to and add in the occ[] count up to the side * break just prior to the side. * * A forward side is shaped like: * * [A] [C] XXXXXXXXXXXXXXXX * -4- -4- --------56------ (numbers in bytes) * ^ * Side ptr (result from SideLocus.side()) * * And following it is a reverse side shaped like: * * [G] [T] XXXXXXXXXXXXXXXX * -4- -4- --------56------ (numbers in bytes) * ^ * Side ptr (result from SideLocus.side()) * */ inline void countBt2SideEx(const SideLocus& l, index_t* arrs) const { assert_range(0, (int)this->_gh._sideGbwtSz-1, (int)l._by); assert_range(0, 3, (int)l._bp); countUpToEx(l, arrs); assert_eq(_zGbwtByteOffs.size(), _zGbwtBpOffs.size()); for(index_t i = 0; i < _zGbwtByteOffs.size(); i++) { index_t zGbwtByteOff = _zGbwtByteOffs[i]; if(l._sideByteOff <= zGbwtByteOff && l._sideByteOff + l._by >= zGbwtByteOff) { // Adjust for the fact that we represented $ with an 'A', but // shouldn't count it as an 'A' here int zGbwtBpOff = _zGbwtBpOffs[i]; if((l._sideByteOff + l._by > zGbwtByteOff) || (l._sideByteOff + l._by == zGbwtByteOff && l._bp > zGbwtBpOff)) { arrs[0]--; // Adjust for '$' looking like an 'A' } } } WITHIN_FCHR(arrs); WITHIN_BWT_LEN(arrs); // Now factor in the occ[] count at the side break const uint8_t *side = l.side(this->gfm()); const uint8_t *acgt16 = side + this->_gh._sideSz - sizeof(index_t) * 4; const index_t *acgt = reinterpret_cast(acgt16); assert_leq(acgt[0], this->fchr()[1] + this->_gh.sideGbwtLen()); assert_leq(acgt[1], this->fchr()[2]-this->fchr()[1]); assert_leq(acgt[2], this->fchr()[3]-this->fchr()[2]); assert_leq(acgt[3], this->fchr()[4]-this->fchr()[3]); assert_leq(acgt[0], this->_gh._len + this->_gh.sideGbwtLen()); assert_leq(acgt[1], this->_gh._len); assert_leq(acgt[2], this->_gh._len); assert_leq(acgt[3], this->_gh._len); arrs[0] += (acgt[0] + this->fchr()[0]); arrs[1] += (acgt[1] + this->fchr()[1]); arrs[2] += (acgt[2] + this->fchr()[2]); arrs[3] += (acgt[3] + this->fchr()[3]); WITHIN_FCHR(arrs); } /** * Count all occurrences of character 1 from the beginning of the * forward side to and add in the occ[] count up to the side * break just prior to the side. * */ inline index_t countMSide(const SideLocus& l) const { assert_range(0, (int)this->_gh._sideGbwtSz-1, (int)l._by); assert_range(0, 7, (int)l._bp); index_t cCnt = countUpTo_bits(l, false /* F? */); const uint8_t *side = l.side(this->gfm()); cCnt += *(index_t*)(side + _gh._sideGbwtSz + sizeof(index_t)); assert_leq(cCnt, l.toBWRow(_gh)); assert_leq(cCnt, this->_gh._numNodes); return cCnt; } /** * Counts the number of occurrences of character 'c' in the given Ebwt * side up to (but not including) the given byte/bitpair (by/bp). * * This is a performance-critical function. This is the top search- * related hit in the time profile. * * Function gets 11.09% in profile */ inline index_t countUpTo(const SideLocus& l, int c) const { // Count occurrences of c in each 64-bit (using bit trickery); // Someday countInU64() and pop() functions should be // vectorized/SSE-ized in case that helps. bool usePOPCNT = false; index_t cCnt = 0; const uint8_t *side = l.side(this->gfm()); int i = 0; #ifdef POPCNT_CAPABILITY if(_usePOPCNTinstruction) { usePOPCNT = true; int by = l._by + (l._bp > 0 ? 1 : 0); for(; i < by; i += 8) { if(i + 8 < by) { cCnt += countInU64(c, *(uint64_t*)&side[i]); } else { index_t by_shift = 8 - (by - i); index_t bp_shift = (l._bp > 0 ? 4 - l._bp : 0); index_t shift = (by_shift << 3) + (bp_shift << 1); uint64_t side_i = *(uint64_t*)&side[i]; side_i = (_toBigEndian ? side_i >> shift : side_i << shift); index_t cCnt_add = countInU64(c, side_i); if(c == 0) cCnt_add -= (shift >> 1); #ifndef NDEBUG index_t cCnt_temp = 0; for(int j = i; j < l._by; j++) { cCnt_temp += cCntLUT_4[0][c][side[j]]; } if(l._bp > 0) { cCnt_temp += cCntLUT_4[(int)l._bp][c][side[l._by]]; } assert_eq(cCnt_add, cCnt_temp); #endif cCnt += cCnt_add; break; } } } else { for(; i + 7 < l._by; i += 8) { cCnt += countInU64(c, *(uint64_t*)&side[i]); } } #else for(; i + 7 < l._by; i += 8) { cCnt += countInU64(c, *(uint64_t*)&side[i]); } #endif if(!usePOPCNT) { // Count occurences of c in the rest of the side (using LUT) for(; i < l._by; i++) { cCnt += cCntLUT_4[0][c][side[i]]; } // Count occurences of c in the rest of the byte if(l._bp > 0) { cCnt += cCntLUT_4[(int)l._bp][c][side[i]]; } } return cCnt; } /** * Counts the number of occurrences of character 'c' in the given Ebwt * side down to the given byte/bitpair (by/bp). * */ inline index_t countDownTo(const SideLocus& l, int c) const { // Count occurrences of c in each 64-bit (using bit trickery); // Someday countInU64() and pop() functions should be // vectorized/SSE-ized in case that helps. index_t cCnt = 0; const uint8_t *side = l.side(this->gfm()); int i = 64 - 4 * sizeof(index_t) - 1; #ifdef POPCNT_CAPABILITY if ( _usePOPCNTinstruction) { for(; i - 7 > l._by; i -= 8) { cCnt += countInU64(c, *(uint64_t*)&side[i-7]); } } else { for(; i + 7 > l._by; i -= 8) { cCnt += countInU64(c, *(uint64_t*)&side[i-7]); } } #else for(; i + 7 > l._by; i -= 8) { cCnt += countInU64(c, *(uint64_t*)&side[i-7]); } #endif // Count occurences of c in the rest of the side (using LUT) for(; i > l._by; i--) { cCnt += cCntLUT_4_rev[0][c][side[i]]; } // Count occurences of c in the rest of the byte if(l._bp > 0) { cCnt += cCntLUT_4_rev[4-(int)l._bp][c][side[i]]; } else { cCnt += cCntLUT_4_rev[0][c][side[i]]; } return cCnt; } /** * Tricky-bit-bashing bitpair counting for given two-bit value (0-3) * within a 64-bit argument. * * Function gets 2.32% in profile */ #ifdef POPCNT_CAPABILITY template #endif inline static void countInU64Ex(uint64_t dw, index_t* arrs) { uint64_t c0 = c_table[0]; uint64_t x0 = dw ^ c0; uint64_t x1 = (x0 >> 1); uint64_t x2 = x1 & (0x5555555555555555llu); uint64_t x3 = x0 & x2; #ifdef POPCNT_CAPABILITY uint64_t tmp = Operation().pop64(x3); #else uint64_t tmp = pop64(x3); #endif arrs[0] += (uint32_t) tmp; c0 = c_table[1]; x0 = dw ^ c0; x1 = (x0 >> 1); x2 = x1 & (0x5555555555555555llu); x3 = x0 & x2; #ifdef POPCNT_CAPABILITY tmp = Operation().pop64(x3); #else tmp = pop64(x3); #endif arrs[1] += (uint32_t) tmp; c0 = c_table[2]; x0 = dw ^ c0; x1 = (x0 >> 1); x2 = x1 & (0x5555555555555555llu); x3 = x0 & x2; #ifdef POPCNT_CAPABILITY tmp = Operation().pop64(x3); #else tmp = pop64(x3); #endif arrs[2] += (uint32_t) tmp; c0 = c_table[3]; x0 = dw ^ c0; x1 = (x0 >> 1); x2 = x1 & (0x5555555555555555llu); x3 = x0 & x2; #ifdef POPCNT_CAPABILITY tmp = Operation().pop64(x3); #else tmp = pop64(x3); #endif arrs[3] += (uint32_t) tmp; } /** * Counts the number of occurrences of all four nucleotides in the * given side up to (but not including) the given byte/bitpair (by/bp). * Count for 'a' goes in arrs[0], 'c' in arrs[1], etc. */ inline void countUpToEx(const SideLocus& l, index_t* arrs) const { int i = 0; // Count occurrences of each nucleotide in each 64-bit word using // bit trickery; note: this seems does not seem to lend a // significant boost to performance in practice. If you comment // out this whole loop (which won't affect correctness - it will // just cause the following loop to take up the slack) then runtime // does not change noticeably. Someday the countInU64() and pop() // functions should be vectorized/SSE-ized in case that helps. const uint8_t *side = l.side(this->gfm()); #ifdef POPCNT_CAPABILITY if (_usePOPCNTinstruction) { for(; i+7 < l._by; i += 8) { countInU64Ex(*(uint64_t*)&side[i], arrs); } } else { for(; i+7 < l._by; i += 8) { countInU64Ex(*(uint64_t*)&side[i], arrs); } } #else for(; i+7 < l._by; i += 8) { countInU64Ex(*(uint64_t*)&side[i], arrs); } #endif // Count occurences of nucleotides in the rest of the side (using LUT) // Many cache misses on following lines (~20K) for(; i < l._by; i++) { arrs[0] += cCntLUT_4[0][0][side[i]]; arrs[1] += cCntLUT_4[0][1][side[i]]; arrs[2] += cCntLUT_4[0][2][side[i]]; arrs[3] += cCntLUT_4[0][3][side[i]]; } // Count occurences of c in the rest of the byte if(l._bp > 0) { arrs[0] += cCntLUT_4[(int)l._bp][0][side[i]]; arrs[1] += cCntLUT_4[(int)l._bp][1][side[i]]; arrs[2] += cCntLUT_4[(int)l._bp][2][side[i]]; arrs[3] += cCntLUT_4[(int)l._bp][3][side[i]]; } } /** * Counts the number of occurrences of character 'c' in the given Ebwt * side up to (but not including) the given byte/bitpair (by/bp). * * This is a performance-critical function. This is the top search- * related hit in the time profile. */ inline index_t countUpTo_bits(const SideLocus& l, bool F) const { // Count occurrences of c in each 64-bit (using bit trickery); // Someday countInU64() and pop() functions should be // vectorized/SSE-ized in case that helps. bool usePOPCNT = false; index_t cCnt = 0; const uint8_t *side = l.side(this->gfm()); if(F) { side += (_gh._sideGbwtSz >> 1); } else { side += (_gh._sideGbwtSz - (_gh._sideGbwtSz >> 2)); } int i = 0; #ifdef POPCNT_CAPABILITY if(_usePOPCNTinstruction) { usePOPCNT = true; int by = l._by + (l._bp > 0 ? 1 : 0); for(; i < by; i += 8) { if(i + 8 < by) { cCnt += countInU64_bits(*(uint64_t*)&side[i]); } else { index_t by_shift = 8 - (by - i); index_t bp_shift = (l._bp > 0 ? 8 - l._bp : 0); index_t shift = (by_shift << 3) + bp_shift; uint64_t side_i = *(uint64_t*)&side[i]; side_i = (_toBigEndian ? side_i >> shift : side_i << shift); index_t cCnt_add = countInU64_bits(side_i); #ifndef NDEBUG index_t cCnt_temp = 0; for(int j = i; j < l._by; j++) { cCnt_temp += cCntBIT[0][side[j]]; } if(l._bp > 0) { cCnt_temp += cCntBIT[(int)l._bp][side[l._by]]; } assert_eq(cCnt_add, cCnt_temp); #endif cCnt += cCnt_add; break; } } } else { for(; i + 7 < l._by; i += 8) { cCnt += countInU64_bits(*(uint64_t*)&side[i]); } } #else for(; i + 7 < l._by; i += 8) { cCnt += countInU64_bits(*(uint64_t*)&side[i]); } #endif if(!usePOPCNT) { // Count occurences of c in the rest of the side (using LUT) for(; i < l._by; i++) { cCnt += cCntBIT[0][side[i]]; } // Count occurences of c in the rest of the byte if(l._bp > 0) { cCnt += cCntBIT[(int)l._bp][side[i]]; } } return cCnt; } #ifndef NDEBUG /** * Given top and bot loci, calculate counts of all four DNA chars up to * those loci. Used for more advanced backtracking-search. */ inline void mapLFEx( const SideLocus& l, index_t *arrs ASSERT_ONLY(, bool overrideSanity = false) ) const { assert_eq(0, arrs[0]); assert_eq(0, arrs[1]); assert_eq(0, arrs[2]); assert_eq(0, arrs[3]); countBt2SideEx(l, arrs); if(_sanity && !overrideSanity) { // Make sure results match up with individual calls to mapLF; // be sure to override sanity-checking in the callee, or we'll // have infinite recursion assert_eq(mapLF(l, 0, true), arrs[0]); assert_eq(mapLF(l, 1, true), arrs[1]); assert_eq(mapLF(l, 2, true), arrs[2]); assert_eq(mapLF(l, 3, true), arrs[3]); } } #endif /** * Given top and bot rows, calculate counts of all four DNA chars up to * those loci. */ inline void mapLFEx( index_t top, index_t bot, index_t *tops, index_t *bots ASSERT_ONLY(, bool overrideSanity = false) ) const { SideLocus ltop, lbot; SideLocus::initFromTopBot(top, bot, _gh, gfm(), ltop, lbot); mapLFEx(ltop, lbot, tops, bots ASSERT_ONLY(, overrideSanity)); } /** * Given top and bot loci, calculate counts of all four DNA chars up to * those loci. Used for more advanced backtracking-search. */ inline void mapLFEx( const SideLocus& ltop, const SideLocus& lbot, index_t *tops, index_t *bots ASSERT_ONLY(, bool overrideSanity = false) ) const { assert(ltop.repOk(this->gh())); assert(lbot.repOk(this->gh())); assert_eq(0, tops[0]); assert_eq(0, bots[0]); assert_eq(0, tops[1]); assert_eq(0, bots[1]); assert_eq(0, tops[2]); assert_eq(0, bots[2]); assert_eq(0, tops[3]); assert_eq(0, bots[3]); countBt2SideEx(ltop, tops); countBt2SideEx(lbot, bots); #ifndef NDEBUG if(_sanity && !overrideSanity) { // Make sure results match up with individual calls to mapLF; // be sure to override sanity-checking in the callee, or we'll // have infinite recursion assert_eq(mapLF(ltop, 0, true), tops[0]); assert_eq(mapLF(ltop, 1, true), tops[1]); assert_eq(mapLF(ltop, 2, true), tops[2]); assert_eq(mapLF(ltop, 3, true), tops[3]); assert_eq(mapLF(lbot, 0, true), bots[0]); assert_eq(mapLF(lbot, 1, true), bots[1]); assert_eq(mapLF(lbot, 2, true), bots[2]); assert_eq(mapLF(lbot, 3, true), bots[3]); } #endif } /** * Counts the number of occurrences of all four nucleotides in the * given side from the given byte/bitpair (l->_by/l->_bp) (or the * beginning of the side if l == 0). Count for 'a' goes in arrs[0], * 'c' in arrs[1], etc. * * Note: must account for $. * * Must fill in masks */ inline index_t countBt2SideRange2( const SideLocus& l, bool startAtLocus, index_t num, index_t* arrs, EList *masks, index_t maskOff) const { assert(!masks[0].empty()); assert_eq(masks[0].size(), masks[1].size()); assert_eq(masks[0].size(), masks[2].size()); assert_eq(masks[0].size(), masks[3].size()); ASSERT_ONLY(index_t myarrs[4] = {0, 0, 0, 0}); index_t nm = 0; // number of nucleotides tallied so far int iby = 0; // initial byte offset int ibp = 0; // initial base-pair offset if(startAtLocus) { iby = l._by; ibp = l._bp; } else { // Start at beginning } int by = iby, bp = ibp; assert_lt(bp, 4); index_t sideGbwtSz = this->_gh._sideGbwtSz >> (this->_gh.linearFM() ? 0 : 1); assert_lt(by, (int)sideGbwtSz); const uint8_t *side = l.side(this->gfm()); while(nm < num) { int c = (side[by] >> (bp * 2)) & 3; assert_lt(maskOff + nm, masks[c].size()); masks[0][maskOff + nm] = masks[1][maskOff + nm] = masks[2][maskOff + nm] = masks[3][maskOff + nm] = false; assert_range(0, 3, c); // Note: we tally $ just like an A arrs[c]++; // tally it ASSERT_ONLY(myarrs[c]++); masks[c][maskOff + nm] = true; // not dead nm++; if(++bp == 4) { bp = 0; by++; assert_leq(by, (int)sideGbwtSz); if(by == (int)sideGbwtSz) { // Fell off the end of the side break; } } } WITHIN_FCHR_DOLLARA(arrs); #ifndef NDEBUG if(_sanity) { // Make sure results match up with a call to mapLFEx. index_t tops[4] = {0, 0, 0, 0}; index_t bots[4] = {0, 0, 0, 0}; index_t top = l.toBWRow(gh()); index_t bot = top + nm; mapLFEx(top, bot, tops, bots, false); assert(myarrs[0] == (bots[0] - tops[0]) || myarrs[0] == (bots[0] - tops[0])+1); assert_eq(myarrs[1], bots[1] - tops[1]); assert_eq(myarrs[2], bots[2] - tops[2]); assert_eq(myarrs[3], bots[3] - tops[3]); } #endif return nm; } /** * Return the final character in row i (i.e. the i'th character in the * BWT transform). Note that the 'L' in the name of the function * stands for 'last', as in the literature. */ inline int rowL(const SideLocus& l) const { // Extract and return appropriate bit-pair return unpack_2b_from_8b(l.side(this->gfm())[l._by], l._bp); } /** * Return the final character in row i (i.e. the i'th character in the * BWT transform). Note that the 'L' in the name of the function * stands for 'last', as in the literature. */ inline int rowL(index_t i) const { // Extract and return appropriate bit-pair SideLocus l; l.initFromRow(i, _gh, gfm()); return rowL(l); } /** * Given top and bot loci, calculate counts of all four DNA chars up to * those loci. Used for more advanced backtracking-search. */ inline void mapLFRange( const SideLocus& ltop, const SideLocus& lbot, index_t num, // Number of elts index_t* cntsUpto, // A/C/G/T counts up to top index_t* cntsIn, // A/C/G/T counts within range EList *masks ASSERT_ONLY(, bool overrideSanity = false) ) const { assert(ltop.repOk(this->gh())); assert(lbot.repOk(this->gh())); assert_eq(num, lbot.toBWRow(this->gh()) - ltop.toBWRow(this->gh())); assert_eq(0, cntsUpto[0]); assert_eq(0, cntsIn[0]); assert_eq(0, cntsUpto[1]); assert_eq(0, cntsIn[1]); assert_eq(0, cntsUpto[2]); assert_eq(0, cntsIn[2]); assert_eq(0, cntsUpto[3]); assert_eq(0, cntsIn[3]); countBt2SideRange(ltop, num, cntsUpto, cntsIn, masks); assert_eq(num, cntsIn[0] + cntsIn[1] + cntsIn[2] + cntsIn[3]); #ifndef NDEBUG if(_sanity && !overrideSanity) { // Make sure results match up with individual calls to mapLF; // be sure to override sanity-checking in the callee, or we'll // have infinite recursion index_t tops[4] = {0, 0, 0, 0}; index_t bots[4] = {0, 0, 0, 0}; assert(ltop.repOk(this->gh())); assert(lbot.repOk(this->gh())); mapLFEx(ltop, lbot, tops, bots, false); for(int i = 0; i < 4; i++) { assert(cntsUpto[i] == tops[i] || tops[i] == bots[i]); if(i == 0) { assert(cntsIn[i] == bots[i]-tops[i] || cntsIn[i] == bots[i]-tops[i]+1); } else { assert_eq(cntsIn[i], bots[i]-tops[i]); } } } #endif } /** * Given row i, return the row that the LF mapping maps i to. */ inline index_t mapLF( const SideLocus& l ASSERT_ONLY(, bool overrideSanity = false) ) const { ASSERT_ONLY(index_t srcrow = l.toBWRow(_gh)); index_t ret; assert(l.side(this->gfm()) != NULL); int c = rowL(l); assert_lt(c, 4); assert_geq(c, 0); ret = countBt2Side(l, c); assert_lt(ret, this->_gh._gbwtLen); assert_neq(srcrow, ret); #ifndef NDEBUG if(_sanity && !overrideSanity) { // Make sure results match up with results from mapLFEx; // be sure to override sanity-checking in the callee, or we'll // have infinite recursion index_t arrs[] = { 0, 0, 0, 0 }; mapLFEx(l, arrs, true); assert_eq(arrs[c], ret); } #endif return ret; } /** * Given row i and character c, return the row that the LF mapping maps * i to on character c. */ inline index_t mapLF( const SideLocus& l, int c ASSERT_ONLY(, bool overrideSanity = false) ) const { index_t ret; assert_lt(c, 4); assert_geq(c, 0); ret = countBt2Side(l, c); assert_lt(ret, this->_gh._gbwtLen); #ifndef NDEBUG if(_sanity && !overrideSanity) { // Make sure results match up with results from mapLFEx; // be sure to override sanity-checking in the callee, or we'll // have infinite recursion index_t arrs[] = { 0, 0, 0, 0 }; mapLFEx(l, arrs, true); assert_eq(arrs[c], ret); } #endif return ret; } /** * Given row i and character c, return the row that the GLF mapping maps * i to on character c. */ inline pair mapLF( SideLocus& tloc, SideLocus& bloc, int c, pair* node_range = NULL ASSERT_ONLY(, bool overrideSanity = false) ) const { assert_lt(c, 4); assert_geq(c, 0); index_t top = mapLF(tloc, c); index_t bot = mapLF(bloc, c); if(node_range != NULL) { node_range->first = top; node_range->second = bot; } return pair(top, bot); } /** * Given row i and character c, return the row that the GLF mapping maps * i to on character c. */ inline pair mapGLF( SideLocus& tloc, SideLocus& bloc, int c, pair* node_range = NULL, EList >* node_iedges = NULL, index_t k = 5 ASSERT_ONLY(, bool overrideSanity = false) ) const { assert_lt(c, 4); assert_geq(c, 0); index_t top = mapLF(tloc, c); index_t bot = mapLF(bloc, c); if(gh().linearFM()) { if(node_range != NULL) { node_range->first = top; node_range->second = bot; } if(node_iedges != NULL) { node_iedges->clear(); } return pair(top, bot); } if(top + 1 >= gh()._gbwtLen || top >= bot) { assert_eq(top, bot); return pair(0, 0); } tloc.initFromRow_bit(top + 1, gh(), gfm()); index_t node_top = rank_M(tloc) - 1; index_t top_F_loc = 0, top_M_occ = 0; size_t iter = 0; while(true) { const uint8_t *side = tloc.side(gfm()) + gh()._sideGbwtSz - gh()._sideSz * iter; top_F_loc = *((index_t*)side); side += sizeof(index_t); top_M_occ = *((index_t*)side); assert_leq(top_M_occ, node_top + 1); if(top_M_occ <= node_top) break; iter++; } if(top_M_occ > 0) top_F_loc++; tloc.initFromRow_bit(top_F_loc, gh(), gfm()); if(node_top + 1 > top_M_occ) { top = select_F(tloc, node_top + 1 - top_M_occ); } else { top = top_F_loc; } bloc.initFromRow_bit(bot, gh(), gfm()); index_t node_bot = rank_M(bloc); const uint8_t *side = bloc.side(gfm()) + gh()._sideGbwtSz; index_t bot_F_loc = *((index_t*)side); side += sizeof(index_t); index_t bot_M_occ = *((index_t*)side); assert_leq(bot_M_occ, node_bot + 1); if(bot_M_occ > 0) bot_F_loc++; bloc.initFromRow_bit(bot_F_loc, gh(), gfm()); if(node_bot + 1 > bot_M_occ) { bot = select_F(bloc, node_bot + 1 - bot_M_occ); } else { bot = bot_F_loc; } if(node_range != NULL) { (*node_range).first = node_top; (*node_range).second = node_bot; } assert_leq(node_bot - node_top, bot - top); if(node_iedges != NULL && node_bot - node_top <= k && node_bot - node_top < bot - top) { getInEdgeCount(top, bot, *node_iedges); } return pair(top, bot); } /** * Given top and bot loci, calculate counts of all four DNA chars up to * those loci. Also, update a set of tops and bots for the reverse * index/direction using the idea from the bi-directional BWT paper. */ inline void mapBiLFEx( const SideLocus& ltop, const SideLocus& lbot, index_t *tops, index_t *bots, index_t *topsP, // topsP[0] = top index_t *botsP ASSERT_ONLY(, bool overrideSanity = false) ) const { #ifndef NDEBUG for(int i = 0; i < 4; i++) { assert_eq(0, tops[0]); assert_eq(0, bots[0]); } #endif countBt2SideEx(ltop, tops); countBt2SideEx(lbot, bots); #ifndef NDEBUG if(_sanity && !overrideSanity) { // Make sure results match up with individual calls to mapLF; // be sure to override sanity-checking in the callee, or we'll // have infinite recursion assert_eq(mapLF(ltop, 0, true), tops[0]); assert_eq(mapLF(ltop, 1, true), tops[1]); assert_eq(mapLF(ltop, 2, true), tops[2]); assert_eq(mapLF(ltop, 3, true), tops[3]); assert_eq(mapLF(lbot, 0, true), bots[0]); assert_eq(mapLF(lbot, 1, true), bots[1]); assert_eq(mapLF(lbot, 2, true), bots[2]); assert_eq(mapLF(lbot, 3, true), bots[3]); } #endif // bots[0..3] - tops[0..3] = # of ways to extend the suffix with an // A, C, G, T botsP[0] = topsP[0] + (bots[0] - tops[0]); topsP[1] = botsP[0]; botsP[1] = topsP[1] + (bots[1] - tops[1]); topsP[2] = botsP[1]; botsP[2] = topsP[2] + (bots[2] - tops[2]); topsP[3] = botsP[2]; botsP[3] = topsP[3] + (bots[3] - tops[3]); } /** * Given row and its locus information, proceed on the given character * and return the next row, or all-fs if we can't proceed on that * character. Returns 0xffffffff if this row ends in $. */ inline index_t mapLF1( index_t row, // starting row const SideLocus& l, // locus for starting row int c // character to proceed on ASSERT_ONLY(, bool overrideSanity = false) ) const { if(rowL(l) != c) return (index_t)INDEX_MAX; for(index_t i = 0; i < _zOffs.size(); i++) { if(row == _zOffs[i]) return (index_t)INDEX_MAX; } index_t ret; assert_lt(c, 4); assert_geq(c, 0); ret = countBt2Side(l, c); assert_lt(ret, this->_gh._gbwtLen); #ifndef NDEBUG if(_sanity && !overrideSanity) { // Make sure results match up with results from mapLFEx; // be sure to override sanity-checking in the callee, or we'll // have infinite recursion index_t arrs[] = { 0, 0, 0, 0 }; mapLFEx(l, arrs, true); assert_eq(arrs[c], ret); } #endif return ret; } /** * Given row and its locus information, set the row to LF(row) and * return the character that was in the final column. */ inline int mapLF1( index_t& row, // starting row const SideLocus& l // locus for starting row ASSERT_ONLY(, bool overrideSanity = false) ) const { for(index_t i = 0; i < _zOffs.size(); i++) { if(row == _zOffs[i]) return -1; } int c = rowL(l); assert_range(0, 3, c); row = countBt2Side(l, c); assert_lt(row, this->_gh._gbwtLen); #ifndef NDEBUG if(_sanity && !overrideSanity) { // Make sure results match up with results from mapLFEx; // be sure to override sanity-checking in the callee, or we'll // have infinite recursion index_t arrs[] = { 0, 0, 0, 0 }; mapLFEx(l, arrs, true); assert_eq(arrs[c], row); } #endif return c; } /** * Given row and its locus information, proceed on the given character * and return the next row, or all-fs if we can't proceed on that * character. Returns 0xffffffff if this row ends in $. */ inline pair mapGLF1( index_t row, // starting row SideLocus& l, // locus for starting row int c, // character to proceed pair* node_range = NULL ASSERT_ONLY(, bool overrideSanity = false) ) const { assert_lt(c, 4); assert_geq(c, 0); index_t top = mapLF1(row, l, c); if(top == (index_t)INDEX_MAX) return pair(0, 0); if(gh().linearFM()) { if(node_range != NULL) { node_range->first = top; node_range->second = top + 1; } return pair(top, top + 1); } index_t bot = top; l.initFromRow_bit(top + 1, gh(), gfm()); index_t node_top = rank_M(l) - 1; index_t F_loc = 0, M_occ = 0; size_t iter = 0; while(true) { const uint8_t *side = l.side(gfm()) + gh()._sideGbwtSz - gh()._sideSz * iter; F_loc = *((index_t*)side); side += sizeof(index_t); M_occ = *((index_t*)side); assert_leq(M_occ, node_top + 1); if(M_occ <= node_top) break; iter++; } if(M_occ > 0) F_loc++; l.initFromRow_bit(F_loc, gh(), gfm()); if(node_top + 1 > M_occ) { top = select_F(l, node_top + 1 - M_occ); } else { top = F_loc; } index_t node_bot = node_top + 1; if(node_bot + 1 > M_occ) { SideLocus l2; #if 0 l2.initFromRow_bit(top + 1, gh(), gfm()); bot = select_F(l2, 1); ASSERT_ONLY(index_t bot2 = select_F(l, node_bot + 1 - M_occ)); assert_eq(bot, bot2); #else bot = select_F(l, node_bot + 1 - M_occ); #endif } else { bot = F_loc; } if(node_range != NULL) { (*node_range).first = node_top; (*node_range).second = node_bot; } return pair(top, bot); } /** * Given row and its locus information, proceed on the given character * and return the next row, or all-fs if we can't proceed on that * character. Returns 0xffffffff if this row ends in $. */ inline pair mapGLF1( index_t row, // starting row SideLocus& l, // locus for starting row pair* node_range = NULL ASSERT_ONLY(, bool overrideSanity = false) ) const { for(index_t i = 0; i < _zOffs.size(); i++) { if(row == _zOffs[i]) return pair((index_t)INDEX_MAX, (index_t)INDEX_MAX); } mapLF1(row, l); index_t top = row; if(top == (index_t)INDEX_MAX) return pair(0, 0); if(gh().linearFM()) { if(node_range != NULL) { node_range->first = top; node_range->second = top + 1; } return pair(top, top + 1); } index_t bot = top; l.initFromRow_bit(top + 1, gh(), gfm()); index_t node_top = rank_M(l) - 1; index_t F_loc = 0, M_occ = 0; size_t iter = 0; while(true) { const uint8_t *side = l.side(gfm()) + gh()._sideGbwtSz - gh()._sideSz * iter; F_loc = *((index_t*)side); side += sizeof(index_t); M_occ = *((index_t*)side); assert_leq(M_occ, node_top + 1); if(M_occ <= node_top) break; iter++; } if(M_occ > 0) F_loc++; l.initFromRow_bit(F_loc, gh(), gfm()); if(node_top + 1 > M_occ) { top = select_F(l, node_top + 1 - M_occ); } else { top = F_loc; } index_t node_bot = node_top + 1; if(node_bot + 1 > M_occ) { #if 0 l2.initFromRow_bit(top + 1, gh(), gfm()); bot = select_F(l2, 1); ASSERT_ONLY(index_t bot2 = select_F(l, node_bot + 1 - M_occ)); assert_eq(bot, bot2); #else bot = select_F(l, node_bot + 1 - M_occ); #endif } else { bot = F_loc; } if(node_range != NULL) { (*node_range).first = node_top; (*node_range).second = node_bot; } return pair(top, bot); } /** * Given row i, return rank */ inline index_t rank_M( const SideLocus& l ASSERT_ONLY(, bool overrideSanity = false) ) const { index_t ret = countMSide(l); assert_leq(ret, this->_gh._numNodes); return ret; } /** * Given row i, return select */ inline index_t select_F( SideLocus l, index_t count ASSERT_ONLY(, bool overrideSanity = false) ) const { assert_gt(count, 0); const uint8_t *side = l.side(this->gfm()) + (_gh._sideGbwtSz >> 1); while(true) { index_t remainingBitsSide = (_gh._sideGbwtSz << 1) - l._charOff; assert_gt(remainingBitsSide, 0); index_t minSide = (count < remainingBitsSide ? count : remainingBitsSide); uint64_t bits = *(uint64_t*)&side[l._by]; uint8_t advance = 64; if(l._bp > 0) { bits >>= l._bp; advance -= l._bp; } if(minSide < advance) { advance = minSide; bits <<= (64 - minSide); } uint8_t tmp_count = 0; #ifdef POPCNT_CAPABILITY if(_usePOPCNTinstruction) { tmp_count = countInU64_bits(bits); } else { tmp_count = countInU64_bits(bits); } #else tmp_count = countInU64_bits(bits); #endif assert_leq(tmp_count, count); count -= tmp_count; if(count == 0) { assert_gt(advance, 0); l._charOff += (advance - 1); assert_lt(l._charOff, _gh._sideGbwtSz << 1); l._by = l._charOff >> 3; l._bp = l._charOff & 0x7; break; } assert_leq(l._charOff + advance, (_gh._sideGbwtSz << 1)); if(l._charOff + advance == (_gh._sideGbwtSz << 1)) { l.nextSide(_gh); side = l.side(this->gfm()) + (_gh._sideGbwtSz >> 1); } else { l._charOff += advance; l._by = l._charOff >> 3; l._bp = l._charOff & 0x7; } } return l.toBWRow(_gh); } /** * */ inline void getInEdgeCount( index_t top, index_t bot, EList >& node_iedges) const { assert_lt(top, bot); node_iedges.clear(); SideLocus l; l.initFromRow_bit(top, _gh, gfm()); const uint8_t *side = l.side(this->gfm()) + (_gh._sideGbwtSz >> 1); assert_lt(l._by, (_gh._sideGbwtSz >> 2)); assert_eq((side[l._by] >> l._bp) & 0x1, 0x1); bool first = true; index_t curr_node = 0; index_t num0s = 0; while(top < bot) { if(first) { first = false; } else { int bit = (side[l._by] >> l._bp) & 0x1; if(bit == 0x1) { curr_node++; num0s = 0; } else { num0s++; if(num0s == 1) { node_iedges.expand(); node_iedges.back().first = curr_node; } node_iedges.back().second = num0s; } } if(l._charOff + 1 == (_gh._sideGbwtSz << 1)) { l.nextSide(_gh); side = l.side(this->gfm()) + (_gh._sideGbwtSz >> 1); } else { l._charOff++; l._by = l._charOff >> 3; l._bp = l._charOff & 0x7; } top++; } } #ifndef NDEBUG /// Check that in-memory Ebwt is internally consistent with respect /// to given EbwtParams; assert if not bool inMemoryRepOk(const GFMParams& gh) const { assert_eq(_zOffs.size(), _zGbwtByteOffs.size()); assert_eq(_zOffs.size(), _zGbwtBpOffs.size()); for(index_t i = 0; i < _zOffs.size(); i++) { assert_geq(_zGbwtBpOffs[i], 0); assert_lt(_zGbwtBpOffs[i], 4); assert_lt(_zGbwtByteOffs[i], gh._gbwtTotSz); assert_lt(_zOffs[i], gh._gbwtLen); } assert_geq(_nFrag, _nPat); assert_eq(_alts.size(), _altnames.size()); return true; } /// Check that in-memory Ebwt is internally consistent; assert if /// not bool inMemoryRepOk() const { return repOk(_gh); } /// Check that Ebwt is internally consistent with respect to given /// EbwtParams; assert if not bool repOk(const GFMParams& gh) const { assert(_gh.repOk()); if(isInMemory()) { return inMemoryRepOk(gh); } return true; } /// Check that Ebwt is internally consistent; assert if not bool repOk() const { return repOk(_gh); } #endif bool _toBigEndian; int32_t _overrideOffRate; bool _verbose; bool _passMemExc; bool _sanity; bool fw_; // true iff this is a forward index FILE *_in1; // input fd for primary index file FILE *_in2; // input fd for secondary index file string _in1Str; // filename for primary index file string _in2Str; // filename for secondary index file EList _zOffs; EList _zGbwtByteOffs; EList _zGbwtBpOffs; index_t _nPat; /// number of reference texts index_t _nFrag; /// number of fragments APtrWrap _plen; APtrWrap _rstarts; // starting offset of fragments / text indexes // _fchr, _ftab and _eftab are expected to be relatively small // (usually < 1MB, perhaps a few MB if _fchr is particularly large // - like, say, 11). For this reason, we don't bother with writing // them to disk through separate output streams; we APtrWrap _fchr; APtrWrap _ftab; APtrWrap _eftab; // "extended" entries for _ftab // _offs may be extremely large. E.g. for DNA w/ offRate=4 (one // offset every 16 rows), the total size of _offs is the same as // the total size of the input sequence APtrWrap _offs; // _ebwt is the Extended Burrows-Wheeler Transform itself, and thus // is at least as large as the input sequence. APtrWrap _gfm; bool _useMm; /// use memory-mapped files to hold the index bool useShmem_; /// use shared memory to hold large parts of the index EList _refnames; /// names of the reference sequences EList _refnames_nospace; // names of the reference sequences (names stop at space) char *mmFile1_; char *mmFile2_; int _nthreads; GFMParams _gh; bool packed_; static const uint64_t default_bmax = INDEX_MAX; static const uint64_t default_bmaxMultSqrt = INDEX_MAX; static const uint64_t default_bmaxDivN = 4; static const int default_dcv = 1024; static const bool default_noDc = false; static const bool default_useBlockwise = true; static const uint32_t default_seed = 0; #ifdef BOWTIE_64BIT_INDEX static const int default_lineRate_gfm = 8; static const int default_lineRate_fm = 7; #else static const int default_lineRate_gfm = 7; static const int default_lineRate_fm = 6; #endif static const int default_offRate = 5; static const int default_offRatePlus = 0; static const int default_ftabChars = 10; static const bool default_bigEndian = false; // data used to build an index EList > _alts; EList _altnames; EList > _haplotypes; RepeatDB _repeatdb; EList _repeat_kmertables; bool _repeat; EList > _repeatLens; EList _repeatIncluded; protected: ostream& log() const { return cerr; // TODO: turn this into a parameter } /// Print a verbose message and flush (flushing is helpful for /// debugging) void verbose(const string& s) const { if(this->verbose()) { this->log() << s.c_str(); this->log().flush(); } } }; /** * Read reference names from an input stream 'in' for an Ebwt primary * file and store them in 'refnames'. */ template void readEbwtRefnames(istream& in, EList& refnames) { // _in1 must already be open with the get cursor at the // beginning and no error flags set. assert(in.good()); assert_eq((streamoff)in.tellg(), ios::beg); // Read endianness hints from both streams bool switchEndian = false; uint32_t one = readU32(in, switchEndian); // 1st word of primary stream if(one != 1) { assert_eq((1u<<24), one); switchEndian = true; } readU32(in, switchEndian); // version // Reads header entries one by one from primary stream index_t len = readIndex(in, switchEndian); index_t gbwtLen = readIndex(in, switchEndian); index_t numNodes = readIndex(in, switchEndian); int32_t lineRate = readI32(in, switchEndian); /*int32_t linesPerSide =*/ readI32(in, switchEndian); int32_t offRate = readI32(in, switchEndian); int32_t ftabChars = readI32(in, switchEndian); index_t eftabLen = readIndex(in, switchEndian); // BTL: chunkRate is now deprecated int32_t flags = readI32(in, switchEndian); bool entireReverse = false; if(flags < 0) { entireReverse = (((-flags) & GFM_ENTIRE_REV) != 0); } // Create a new EbwtParams from the entries read from primary stream GFMParams gh(len, gbwtLen, numNodes, lineRate, offRate, ftabChars, eftabLen, entireReverse); index_t nPat = readIndex(in, switchEndian); // nPat in.seekg(nPat*sizeof(index_t), ios_base::cur); // skip plen // Skip rstarts index_t nFrag = readIndex(in, switchEndian); in.seekg(nFrag*sizeof(index_t)*3, ios_base::cur); // Skip ebwt in.seekg(gh._gbwtTotLen, ios_base::cur); // Skip zOff from primary stream index_t numZOffs = readIndex(in, switchEndian); in.seekg(numZOffs * sizeof(index_t), ios_base::cur); // Skip fchr in.seekg(5 * sizeof(index_t), ios_base::cur); // Skip ftab in.seekg(gh._ftabLen*sizeof(index_t), ios_base::cur); // Skip eftab in.seekg(gh._eftabLen*sizeof(index_t), ios_base::cur); // Read reference sequence names from primary index file while(true) { char c = '\0'; in.read(&c, 1); if(in.eof()) break; if(c == '\0') break; else if(c == '\n') { refnames.push_back(""); } else { if(refnames.size() == 0) { refnames.push_back(""); } refnames.back().push_back(c); } } if(refnames.back().empty()) { refnames.pop_back(); } // Be kind in.clear(); in.seekg(0, ios::beg); assert(in.good()); } /** * Read reference names from the index with basename 'in' and store * them in 'refnames'. */ template void readEbwtRefnames(const string& instr, EList& refnames) { ifstream in; // Initialize our primary and secondary input-stream fields in.open((instr + ".1." + gfm_ext).c_str(), ios_base::in | ios::binary); if(!in.is_open()) { throw GFMFileOpenException("Cannot open file " + instr); } assert(in.is_open()); assert(in.good()); assert_eq((streamoff)in.tellg(), ios::beg); readEbwtRefnames(in, refnames); } /////////////////////////////////////////////////////////////////////// // // Functions for building Ebwts // /////////////////////////////////////////////////////////////////////// /** * Join several text strings together in a way that's compatible with * the text-chunking scheme dictated by chunkRate parameter. * * The non-static member Ebwt::join additionally builds auxilliary * arrays that maintain a mapping between chunks in the joined string * and the original text strings. */ template template TStr GFM::join(EList& l, uint32_t seed) { RandomSource rand; // reproducible given same seed rand.init(seed); TStr ret; index_t guessLen = 0; for(index_t i = 0; i < l.size(); i++) { guessLen += length(l[i]); } ret.resize(guessLen); index_t off = 0; for(size_t i = 0; i < l.size(); i++) { TStr& s = l[i]; assert_gt(s.length(), 0); for(size_t j = 0; j < s.size(); j++) { ret.set(s[j], off++); } } return ret; } /** * Join several text strings together in a way that's compatible with * the text-chunking scheme dictated by chunkRate parameter. * * The non-static member Ebwt::join additionally builds auxilliary * arrays that maintain a mapping between chunks in the joined string * and the original text strings. */ template template void GFM::join(EList& l, EList& szs, index_t sztot, const RefReadInParams& refparams, uint32_t seed, TStr& s, bool include_rc, bool CGtoTG) { RandomSource rand; // reproducible given same seed rand.init(seed); RefReadInParams rpcp = refparams; index_t guessLen = sztot; if(include_rc) { s.resize(guessLen << 1); } else { s.resize(guessLen); } ASSERT_ONLY(index_t szsi = 0); TIndexOffU dstoff = 0; for(index_t i = 0; i < l.size(); i++) { // For each sequence we can pull out of istream l[i]... assert(!l[i]->eof()); bool first = true; while(!l[i]->eof()) { RefRecord rec = fastaRefReadAppend(*l[i], first, s, dstoff, rpcp); first = false; index_t bases = (index_t)rec.len; assert_eq(rec.off, szs[szsi].off); assert_eq(rec.len, szs[szsi].len); assert_eq(rec.first, szs[szsi].first); ASSERT_ONLY(szsi++); if(bases == 0) continue; } } // Change 'C' in CG to 'T' so that CG becomes TG if(CGtoTG) { for(TIndexOffU i = 0; i + 1 < guessLen; i++) { int nt1 = s[i], nt2 = s[i+1]; if(nt1 == 1 && nt2 == 2) { s[i] = 3; } } } // Append reverse complement if(include_rc) { for(TIndexOffU i = 0; i < guessLen; i++) { int nt = s[guessLen - i - 1]; assert_range(0, 3, nt); s[guessLen + i] = dnacomp[nt]; } } } /** * Join several text strings together according to the text-chunking * scheme specified in the EbwtParams. Ebwt fields calculated in this * function are written directly to disk. * * It is assumed, but not required, that the header values have already * been written to 'out1' before this function is called. * * The static member Ebwt::join just returns a joined version of a * list of strings without building any of the auxilliary arrays. */ template template void GFM::joinToDisk( EList& l, EList& szs, index_t sztot, const RefReadInParams& refparams, TStr& ret, ostream& out1, ostream& out2) { RefReadInParams rpcp = refparams; assert_gt(szs.size(), 0); assert_gt(l.size(), 0); assert_gt(sztot, 0); // Not every fragment represents a distinct sequence - many // fragments may correspond to a single sequence. Count the // number of sequences here by counting the number of "first" // fragments. this->_nPat = 0; this->_nFrag = 0; for(index_t i = 0; i < szs.size(); i++) { if(szs[i].len > 0) this->_nFrag++; if(szs[i].first && szs[i].len > 0) this->_nPat++; } assert_gt(this->_nPat, 0); assert_geq(this->_nFrag, this->_nPat); _rstarts.reset(); writeIndex(out1, this->_nPat, this->toBe()); // Allocate plen[] try { this->_plen.init(new index_t[this->_nPat], this->_nPat); } catch(bad_alloc& e) { cerr << "Out of memory allocating plen[] in Ebwt::join()" << " at " << __FILE__ << ":" << __LINE__ << endl; throw e; } // For each pattern, set plen int npat = -1; for(index_t i = 0; i < szs.size(); i++) { if(szs[i].first && szs[i].len > 0) { if(npat >= 0) { writeIndex(out1, this->plen()[npat], this->toBe()); } npat++; this->plen()[npat] = (szs[i].len + szs[i].off); } else { this->plen()[npat] += (szs[i].len + szs[i].off); } } assert_eq((index_t)npat, this->_nPat-1); writeIndex(out1, this->plen()[npat], this->toBe()); // Write the number of fragments writeIndex(out1, this->_nFrag, this->toBe()); index_t seqsRead = 0; ASSERT_ONLY(index_t szsi = 0); ASSERT_ONLY(index_t entsWritten = 0); index_t dstoff = 0; // For each filebuf for(unsigned int i = 0; i < l.size(); i++) { assert(!l[i]->eof()); bool first = true; index_t patoff = 0; // For each *fragment* (not necessary an entire sequence) we // can pull out of istream l[i]... while(!l[i]->eof()) { string name; // Push a new name onto our vector _refnames.push_back(""); RefRecord rec = fastaRefReadAppend( *l[i], first, ret, dstoff, rpcp, &_refnames.back()); first = false; index_t bases = rec.len; if(rec.first && rec.len > 0) { if(_refnames.back().length() == 0) { // If name was empty, replace with an index ostringstream stm; stm << seqsRead; _refnames.back() = stm.str(); } } else { // This record didn't actually start a new sequence so // no need to add a name //assert_eq(0, _refnames.back().length()); _refnames.pop_back(); } // Increment seqsRead if this is the first fragment if(rec.first && rec.len > 0) seqsRead++; assert_lt(szsi, szs.size()); assert_eq(rec.off, szs[szsi].off); assert_eq(rec.len, szs[szsi].len); assert_eq(rec.first, szs[szsi].first); assert(rec.first || rec.off > 0); ASSERT_ONLY(szsi++); assert_leq(bases, this->plen()[seqsRead-1]); // Reset the patoff if this is the first fragment if(rec.first) patoff = 0; patoff += rec.off; // add fragment's offset from end of last frag. // Adjust rpcps //index_t seq = seqsRead-1; #ifndef NDEBUG if(bases > 0) { ASSERT_ONLY(entsWritten++); } #endif // This is where rstarts elements are written to the output stream //writeU32(out1, oldRetLen, this->toBe()); // offset from beginning of joined string //writeU32(out1, seq, this->toBe()); // sequence id //writeU32(out1, patoff, this->toBe()); // offset into sequence patoff += (index_t)bases; } assert_gt(szsi, 0); l[i]->reset(); assert(!l[i]->eof()); #ifndef NDEBUG int c = l[i]->get(); assert_eq('>', c); assert(!l[i]->eof()); l[i]->reset(); assert(!l[i]->eof()); #endif } assert_eq(entsWritten, this->_nFrag); } /** * Build an Ebwt from a string 's' and its suffix array 'sa' (which * might actually be a suffix array *builder* that builds blocks of the * array on demand). The bulk of the Ebwt, i.e. the ebwt and offs * arrays, is written directly to disk. This is by design: keeping * those arrays in memory needlessly increases the footprint of the * building process. Instead, we prefer to build the Ebwt directly * "to disk" and then read it back into memory later as necessary. * * It is assumed that the header values and join-related values (nPat, * plen) have already been written to 'out1' before this function * is called. When this function is finished, it will have * additionally written ebwt, zOff, fchr, ftab and eftab to the primary * file and offs to the secondary file. * * Assume DNA/RNA/any alphabet with 4 or fewer elements. * Assume occ array entries are 32 bits each. * * @param sa the suffix array to convert to a Ebwt * @param s the original string * @param out */ template template void GFM::buildToDisk( PathGraph& gbwt, const TStr& s, ostream& out1, ostream& out2, streampos headerPos) { const GFMParams& gh = this->_gh; assert(gh.repOk()); assert_lt(s.length(), gh.gbwtLen()); assert_eq(s.length(), gh._len); assert_gt(gh._lineRate, 3); index_t gbwtLen = gh._gbwtLen; streampos out1pos = out1.tellp(); if(headerPos < 0) { out1.seekp(8 + sizeof(index_t)); } else { out1.seekp(headerPos); } writeIndex(out1, gbwtLen, this->toBe()); writeIndex(out1, gh._numNodes, this->toBe()); out1.seekp(out1pos); index_t ftabLen = gh._ftabLen; index_t sideSz = gh._sideSz; index_t gbwtTotSz = gh._gbwtTotSz; index_t fchr[] = {0, 0, 0, 0, 0}; EList ftab(EBWT_CAT); EList zOffs; // Save # of occurrences of each character as we walk along the bwt index_t occ[4] = {0, 0, 0, 0}; index_t occSave[4] = {0, 0, 0, 0}; // # of occurrences of 1 in M arrays index_t M_occ = 0, M_occSave = 0; // Location in F that corresponds to 1 in M index_t F_loc = 0, F_locSave = 0; // Record rows that should "absorb" adjacent rows in the ftab. try { VMSG_NL("Allocating ftab, absorbFtab"); ftab.resize(ftabLen); ftab.fillZero(); } catch(bad_alloc &e) { cerr << "Out of memory allocating ftab[] " << "in GFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } // Allocate the side buffer; holds a single side as its being // constructed and then written to disk. Reused across all sides. #ifdef SIXTY4_FORMAT EList gfmSide(EBWT_CAT); #else EList gfmSide(EBWT_CAT); #endif try { // Used to calculate ftab and eftab, but having gfm costs a lot of memory _gfm.init(new uint8_t[gh._gbwtTotLen], gh._gbwtTotLen, true); #ifdef SIXTY4_FORMAT gfmSide.resize(sideSz >> 3); #else gfmSide.resize(sideSz); #endif } catch(bad_alloc &e) { cerr << "Out of memory allocating ebwtSide[] in " << "GFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } // Points to the base offset within ebwt for the side currently // being written index_t side = 0; // Whether we're assembling a forward or a reverse bucket bool fw = true; int sideCur = 0; index_t si = 0; // string offset (chars) ASSERT_ONLY(bool inSA = true); // true iff saI still points inside suffix // array (as opposed to the padding at the // end) // Iterate over packed bwt bytes VMSG_NL("Entering GFM loop"); ASSERT_ONLY(index_t beforeGbwtOff = (index_t)out1.tellp()); while(side < gbwtTotSz) { // Sanity-check our cursor into the side buffer assert_geq(sideCur, 0); assert_lt(sideCur, (int)gh._sideGbwtSz); assert_eq(0, side % sideSz); // 'side' must be on side boundary if(sideCur == 0) { memset(gfmSide.ptr(), 0, gh._sideGbwtSz); gfmSide[sideCur] = 0; // clear } assert_lt(side + sideCur, gbwtTotSz); // Iterate over bit-pairs in the si'th character of the BWT #ifdef SIXTY4_FORMAT for(int bpi = 0; bpi < 32; bpi++, si++) #else for(int bpi = 0; bpi < 4; bpi++, si++) #endif { int gbwtChar = 0; // one of A, C, G, T, and Z int F= 0, M = 0; // either 0 or 1 index_t pos = 0; // pos on joined string bool count = true; if(si < gbwtLen) { gbwt.nextRow(gbwtChar, F, M, pos); // (that might have triggered sa to calc next suf block) if(gbwtChar == 'Z') { // Don't add the 'Z' in the last column to the BWT // transform; we can't encode a $ (only A C T or G) // and counting it as, say, an A, will mess up the // LF mapping gbwtChar = 0; count = false; #ifndef NDEBUG if(zOffs.size() > 0) { assert_gt(si, zOffs.back()); } #endif zOffs.push_back(si); // remember GBWT row that corresponds to the 0th suffix } else { gbwtChar = asc2dna[gbwtChar]; assert_lt(gbwtChar, 4); // Update the fchr fchr[gbwtChar]++; } assert_lt(F, 2); assert_lt(M, 2); if(M == 1) { assert_neq(F_loc, numeric_limits::max()); F_loc = gbwt.nextFLocation(); #ifndef NDEBUG if(F_loc > 0) { assert_gt(F_loc, F_locSave); } #endif } // Suffix array offset boundary? - update offset array if(M == 1 && (M_occ & gh._offMask) == M_occ) { assert_lt((M_occ >> gh._offRate), gh._offsLen); // Write offsets directly to the secondary output // stream, thereby avoiding keeping them in memory writeIndex(out2, pos, this->toBe()); } } else { // Strayed off the end of the SA, now we're just // padding out a bucket #ifndef NDEBUG if(inSA) { // Assert that we wrote all the characters in the // string before now assert_eq(si, gbwtLen); inSA = false; } #endif // 'A' used for padding; important that padding be // counted in the occ[] array gbwtChar = 0; F = M = 0; } if(count) occ[gbwtChar]++; if(M) M_occ++; // Append BWT char to bwt section of current side if(fw) { // Forward bucket: fill from least to most #ifdef SIXTY4_FORMAT gfmSide[sideCur] |= ((uint64_t)gbwtChar << (bpi << 1)); if(gbwtChar > 0) assert_gt(gfmSide[sideCur], 0); // To be implemented ... assert(false); cerr << "Not implemented" << endl; exit(1); #else pack_2b_in_8b(gbwtChar, gfmSide[sideCur], bpi); assert_eq((gfmSide[sideCur] >> (bpi*2)) & 3, gbwtChar); int F_sideCur = (gh._sideGbwtSz + sideCur) >> 1; int F_bpi = bpi + ((sideCur & 0x1) << 2); // Can be used as M_bpi as well pack_1b_in_8b(F, gfmSide[F_sideCur], F_bpi); assert_eq((gfmSide[F_sideCur] >> F_bpi) & 1, F); int M_sideCur = F_sideCur + (gh._sideGbwtSz >> 2); pack_1b_in_8b(M, gfmSide[M_sideCur], F_bpi); assert_eq((gfmSide[M_sideCur] >> F_bpi) & 1, M); #endif } else { // Backward bucket: fill from most to least #ifdef SIXTY4_FORMAT gfmSide[sideCur] |= ((uint64_t)gbwtChar << ((31 - bpi) << 1)); if(gbwtChar > 0) assert_gt(gfmSide[sideCur], 0); // To be implemented ... assert(false); cerr << "Not implemented" << endl; exit(1); #else pack_2b_in_8b(gbwtChar, gfmSide[sideCur], 3-bpi); assert_eq((gfmSide[sideCur] >> ((3-bpi)*2)) & 3, gbwtChar); // To be implemented ... assert(false); cerr << "Not implemented" << endl; exit(1); #endif } } // end loop over bit-pairs assert_eq(0, (occ[0] + occ[1] + occ[2] + occ[3] + zOffs.size()) & 3); #ifdef SIXTY4_FORMAT assert_eq(0, si & 31); #else assert_eq(0, si & 3); #endif sideCur++; if((sideCur << 1) == (int)gh._sideGbwtSz) { sideCur = 0; index_t *uside = reinterpret_cast(gfmSide.ptr()); // Write 'A', 'C', 'G', 'T', and '1' in M tallies side += sideSz; assert_leq(side, gh._gbwtTotSz); uside[(sideSz / sizeof(index_t))-6] = endianizeIndex(F_locSave, this->toBe()); uside[(sideSz / sizeof(index_t))-5] = endianizeIndex(M_occSave, this->toBe()); uside[(sideSz / sizeof(index_t))-4] = endianizeIndex(occSave[0], this->toBe()); uside[(sideSz / sizeof(index_t))-3] = endianizeIndex(occSave[1], this->toBe()); uside[(sideSz / sizeof(index_t))-2] = endianizeIndex(occSave[2], this->toBe()); uside[(sideSz / sizeof(index_t))-1] = endianizeIndex(occSave[3], this->toBe()); F_locSave = F_loc; M_occSave = M_occ; occSave[0] = occ[0]; occSave[1] = occ[1]; occSave[2] = occ[2]; occSave[3] = occ[3]; // Write backward side to primary file out1.write((const char *)gfmSide.ptr(), sideSz); // memcpy(((char*)_gfm.get()) + side - sideSz, (const char *)gfmSide.ptr(), sideSz); } } VMSG_NL("Exited GFM loop"); // Assert that our loop counter got incremented right to the end assert_eq(side, gh._gbwtTotSz); // Assert that we wrote the expected amount to out1 assert_eq(((index_t)out1.tellp() - beforeGbwtOff), gh._gbwtTotSz); // assert that the last thing we did was write a forward bucket // // Write zOffs to primary stream // assert_gt(zOffs.size(), 0); writeIndex(out1, (index_t)zOffs.size(), this->toBe()); for(size_t i = 0; i < zOffs.size(); i++) { writeIndex(out1, zOffs[i], this->toBe()); } // // Finish building fchr // // Exclusive prefix sum on fchr for(int i = 1; i < 4; i++) { fchr[i] += fchr[i-1]; } assert_lt(fchr[3], gbwtLen); // Shift everybody up by one for(int i = 4; i >= 1; i--) { fchr[i] = fchr[i-1]; } fchr[0] = 0; if(_verbose) { for(int i = 0; i < 5; i++) cerr << "fchr[" << "ACGT$"[i] << "]: " << fchr[i] << endl; } // Write fchr to primary file for(int i = 0; i < 5; i++) { writeIndex(out1, fchr[i], this->toBe()); } _fchr.init(new index_t[5], 5, true); memcpy(_fchr.get(), fchr, sizeof(index_t) * 5); // Initialize _zGbwtByteOffs and _zGbwtBpOffs _zOffs = zOffs; postReadInit(gh); // Build ftab and eftab EList > tFtab; tFtab.resizeExact(ftabLen - 1); for(index_t i = 0; i + 1 < ftabLen; i++) { index_t q = i; pair range(0, gh._gbwtLen); SideLocus tloc, bloc; SideLocus::initFromTopBot(range.first, range.second, gh, gfm(), tloc, bloc); index_t j = 0; for(; j < (index_t)gh._ftabChars; j++) { int nt = q & 0x3; q >>= 2; if(bloc.valid()) { range = mapGLF(tloc, bloc, nt); } else { range = mapGLF1(range.first, tloc, nt); } if(range.first == (index_t)INDEX_MAX || range.first >= range.second) { break; } if(range.first + 1 == range.second) { tloc.initFromRow(range.first, gh, gfm()); bloc.invalidate(); } else { SideLocus::initFromTopBot(range.first, range.second, gh, gfm(), tloc, bloc); } } if(range.first >= range.second || j < (index_t)gh._ftabChars) { if(i == 0) { tFtab[i].first = tFtab[i].second = 0; } else { tFtab[i].first = tFtab[i].second = tFtab[i-1].second; } } else { tFtab[i].first = range.first; tFtab[i].second = range.second; } #ifndef NDEBUG if(gbwt.ftab.size() > i) { assert_eq(tFtab[i].first, gbwt.ftab[i].first); assert_eq(tFtab[i].second, gbwt.ftab[i].second); } #endif } // Clear memory _gfm.reset(); _fchr.reset(); _zOffs.clear(); _zGbwtByteOffs.clear(); _zGbwtBpOffs.clear(); // // Finish building ftab and build eftab // // Prefix sum on ftable index_t eftabLen = 0; for(index_t i = 1; i + 1 < ftabLen; i++) { if(tFtab[i-1].second != tFtab[i].first) { eftabLen += 2; } } if(gh._gbwtLen + (eftabLen >> 1) < gh._gbwtLen) { cerr << "Too many eftab entries: " << gh._gbwtLen << " + " << (eftabLen >> 1) << " > " << (index_t)INDEX_MAX << endl; throw 1; } EList eftab(EBWT_CAT); try { eftab.resize(eftabLen); eftab.fillZero(); } catch(bad_alloc &e) { cerr << "Out of memory allocating eftab[] " << "in GFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } index_t eftabCur = 0; ftab[0] = tFtab[0].first; ftab[1] = tFtab[0].second; for(index_t i = 1; i + 1 < ftabLen; i++) { if(ftab[i] != tFtab[i].first) { index_t lo = ftab[i]; index_t hi = tFtab[i].first; assert_lt(eftabCur*2+1, eftabLen); eftab[eftabCur*2] = lo; eftab[eftabCur*2+1] = hi; // one node can be shared, and one node can have at most four incoming edges assert_leq(lo, hi + 4); ftab[i] = (eftabCur++) ^ (index_t)INDEX_MAX; // insert pointer into eftab assert_eq(lo, GFM::ftabLo(ftab.ptr(), eftab.ptr(), gbwtLen, ftabLen, eftabLen, i)); assert_eq(hi, GFM::ftabHi(ftab.ptr(), eftab.ptr(), gbwtLen, ftabLen, eftabLen, i)); } ftab[i+1] = tFtab[i].second; } #ifndef NDEBUG for(index_t i = 0; i + 1 < ftabLen; i++ ){ assert_eq(tFtab[i].first, GFM::ftabHi(ftab.ptr(), eftab.ptr(), gbwtLen, ftabLen, eftabLen, i)); assert_eq(tFtab[i].second, GFM::ftabLo(ftab.ptr(), eftab.ptr(), gbwtLen, ftabLen, eftabLen, i+1)); } #endif // Write ftab to primary file for(index_t i = 0; i < ftabLen; i++) { writeIndex(out1, ftab[i], this->toBe()); } // Write eftab to primary file out1pos = out1.tellp(); if(headerPos < 0) { out1.seekp(24 + sizeof(index_t) * 3); } else { out1.seekp((int)headerPos + 16 + sizeof(index_t) * 2); } writeIndex(out1, eftabLen, this->toBe()); out1.seekp(out1pos); for(index_t i = 0; i < eftabLen; i++) { writeIndex(out1, eftab[i], this->toBe()); } // Note: if you'd like to sanity-check the Ebwt, you'll have to // read it back into memory first! assert(!isInMemory()); VMSG_NL("Exiting GFM::buildToDisk()"); } /** * Build an Ebwt from a string 's' and its suffix array 'sa' (which * might actually be a suffix array *builder* that builds blocks of the * array on demand). The bulk of the Ebwt, i.e. the ebwt and offs * arrays, is written directly to disk. This is by design: keeping * those arrays in memory needlessly increases the footprint of the * building process. Instead, we prefer to build the Ebwt directly * "to disk" and then read it back into memory later as necessary. * * It is assumed that the header values and join-related values (nPat, * plen) have already been written to 'out1' before this function * is called. When this function is finished, it will have * additionally written ebwt, zOff, fchr, ftab and eftab to the primary * file and offs to the secondary file. * * Assume DNA/RNA/any alphabet with 4 or fewer elements. * Assume occ array entries are 32 bits each. * * @param sa the suffix array to convert to a Ebwt * @param s the original string * @param out */ template template void GFM::buildToDisk( InorderBlockwiseSA& sa, const TStr& s, ostream& out1, ostream& out2, streampos headerPos) { const GFMParams& gh = this->_gh; assert(gh.repOk()); assert(gh.linearFM()); assert_lt(s.length(), gh.gbwtLen()); assert_eq(s.length(), gh._len); assert_gt(gh._lineRate, 3); index_t len = gh._len; index_t gbwtLen = gh._gbwtLen; assert_eq(len + 1, gbwtLen); streampos out1pos = out1.tellp(); if(headerPos < 0) { out1.seekp(8 + sizeof(index_t)); } else { out1.seekp(headerPos); } writeIndex(out1, gbwtLen, this->toBe()); writeIndex(out1, gh._numNodes, this->toBe()); out1.seekp(out1pos); index_t ftabLen = gh._ftabLen; index_t sideSz = gh._sideSz; index_t gbwtTotSz = gh._gbwtTotSz; index_t fchr[] = {0, 0, 0, 0, 0}; EList ftab(EBWT_CAT); EList zOffs; // Save # of occurrences of each character as we walk along the bwt index_t occ[4] = {0, 0, 0, 0}; index_t occSave[4] = {0, 0, 0, 0}; // Record rows that should "absorb" adjacent rows in the ftab. // The absorbed rows represent suffixes shorter than the ftabChars // cutoff. uint8_t absorbCnt = 0; EList absorbFtab(EBWT_CAT); try { VMSG_NL("Allocating ftab, absorbFtab"); ftab.resize(ftabLen); ftab.fillZero(); absorbFtab.resize(ftabLen); absorbFtab.fillZero(); } catch(bad_alloc &e) { cerr << "Out of memory allocating ftab[] or absorbFtab[] " << "in GFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } // Allocate the side buffer; holds a single side as its being // constructed and then written to disk. Reused across all sides. #ifdef SIXTY4_FORMAT EList gfmSide(EBWT_CAT); #else EList gfmSide(EBWT_CAT); #endif try { #ifdef SIXTY4_FORMAT gfmSide.resize(sideSz >> 3); #else gfmSide.resize(sideSz); #endif } catch(bad_alloc &e) { cerr << "Out of memory allocating gfmSide[] in " << "GFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } // Points to the base offset within ebwt for the side currently // being written index_t side = 0; // Whether we're assembling a forward or a reverse bucket bool fw = true; int sideCur = 0; // Have we skipped the '$' in the last column yet? ASSERT_ONLY(bool dollarSkipped = false); index_t si = 0; // string offset (chars) ASSERT_ONLY(index_t lastSufInt = 0); ASSERT_ONLY(bool inSA = true); // true iff saI still points inside suffix // array (as opposed to the padding at the // end) // Iterate over packed bwt bytes VMSG_NL("Entering GFM loop"); ASSERT_ONLY(index_t beforeGbwtOff = (index_t)out1.tellp()); while(side < gbwtTotSz) { // Sanity-check our cursor into the side buffer assert_geq(sideCur, 0); assert_lt(sideCur, (int)gh._sideGbwtSz); assert_eq(0, side % sideSz); // 'side' must be on side boundary gfmSide[sideCur] = 0; // clear assert_lt(side + sideCur, gbwtTotSz); // Iterate over bit-pairs in the si'th character of the BWT #ifdef SIXTY4_FORMAT for(int bpi = 0; bpi < 32; bpi++, si++) #else for(int bpi = 0; bpi < 4; bpi++, si++) #endif { int bwtChar; bool count = true; if(si <= len) { // Still in the SA; extract the bwtChar index_t saElt = sa.nextSuffix(); // (that might have triggered sa to calc next suf block) if(saElt == 0) { // Don't add the '$' in the last column to the BWT // transform; we can't encode a $ (only A C T or G) // and counting it as, say, an A, will mess up the // LR mapping bwtChar = 0; count = false; ASSERT_ONLY(dollarSkipped = true); zOffs.push_back(si); // remember the SA row that // corresponds to the 0th suffix } else { bwtChar = (int)(s[saElt-1]); assert_lt(bwtChar, 4); // Update the fchr fchr[bwtChar]++; } // Update ftab if((len-saElt) >= (index_t)gh._ftabChars) { // Turn the first ftabChars characters of the // suffix into an integer index into ftab. The // leftmost (lowest index) character of the suffix // goes in the most significant bit pair if the // integer. index_t sufInt = 0; for(int i = 0; i < gh._ftabChars; i++) { sufInt <<= 2; assert_lt((index_t)i, len-saElt); sufInt |= (unsigned char)(s[saElt+i]); } // Assert that this prefix-of-suffix is greater // than or equal to the last one (true b/c the // suffix array is sorted) #ifndef NDEBUG if(lastSufInt > 0) assert_geq(sufInt, lastSufInt); lastSufInt = sufInt; #endif // Update ftab assert_lt(sufInt+1, ftabLen); ftab[sufInt+1]++; if(absorbCnt > 0) { // Absorb all short suffixes since the last // transition into this transition absorbFtab[sufInt] = absorbCnt; absorbCnt = 0; } } else { // Otherwise if suffix is fewer than ftabChars // characters long, then add it to the 'absorbCnt'; // it will be absorbed into the next transition assert_lt(absorbCnt, 255); absorbCnt++; } // Suffix array offset boundary? - update offset array if((si & gh._offMask) == si) { assert_lt((si >> gh._offRate), gh._offsLen); // Write offsets directly to the secondary output // stream, thereby avoiding keeping them in memory writeIndex(out2, saElt, this->toBe()); } } else { // Strayed off the end of the SA, now we're just // padding out a bucket #ifndef NDEBUG if(inSA) { // Assert that we wrote all the characters in the // string before now assert_eq(si, len+1); inSA = false; } #endif // 'A' used for padding; important that padding be // counted in the occ[] array bwtChar = 0; } if(count) occ[bwtChar]++; // Append BWT char to bwt section of current side if(fw) { // Forward bucket: fill from least to most #ifdef SIXTY4_FORMAT ebwtSide[sideCur] |= ((uint64_t)bwtChar << (bpi << 1)); if(bwtChar > 0) assert_gt(ebwtSide[sideCur], 0); #else pack_2b_in_8b(bwtChar, gfmSide[sideCur], bpi); assert_eq((gfmSide[sideCur] >> (bpi*2)) & 3, bwtChar); #endif } else { // Backward bucket: fill from most to least #ifdef SIXTY4_FORMAT ebwtSide[sideCur] |= ((uint64_t)bwtChar << ((31 - bpi) << 1)); if(bwtChar > 0) assert_gt(ebwtSide[sideCur], 0); #else pack_2b_in_8b(bwtChar, gfmSide[sideCur], 3-bpi); assert_eq((gfmSide[sideCur] >> ((3-bpi)*2)) & 3, bwtChar); #endif } } // end loop over bit-pairs assert_eq(dollarSkipped ? 3 : 0, (occ[0] + occ[1] + occ[2] + occ[3]) & 3); #ifdef SIXTY4_FORMAT assert_eq(0, si & 31); #else assert_eq(0, si & 3); #endif sideCur++; if(sideCur == (int)gh._sideGbwtSz) { sideCur = 0; index_t *uside = reinterpret_cast(gfmSide.ptr()); // Write 'A', 'C', 'G', 'T', and '1' in M tallies side += sideSz; assert_leq(side, gh._gbwtTotSz); uside[(sideSz / sizeof(index_t))-4] = endianizeIndex(occSave[0], this->toBe()); uside[(sideSz / sizeof(index_t))-3] = endianizeIndex(occSave[1], this->toBe()); uside[(sideSz / sizeof(index_t))-2] = endianizeIndex(occSave[2], this->toBe()); uside[(sideSz / sizeof(index_t))-1] = endianizeIndex(occSave[3], this->toBe()); occSave[0] = occ[0]; occSave[1] = occ[1]; occSave[2] = occ[2]; occSave[3] = occ[3]; // Write backward side to primary file out1.write((const char *)gfmSide.ptr(), sideSz); } } VMSG_NL("Exited GFM loop"); if(absorbCnt > 0) { // Absorb any trailing, as-yet-unabsorbed short suffixes into // the last element of ftab absorbFtab[ftabLen-1] = absorbCnt; } // Assert that our loop counter got incremented right to the end assert_eq(side, gh._gbwtTotSz); // Assert that we wrote the expected amount to out1 assert_eq(((index_t)out1.tellp() - beforeGbwtOff), gh._gbwtTotSz); // assert that the last thing we did was write a forward bucket // // Write zOffs to primary stream // assert_eq(zOffs.size(), 1); writeIndex(out1, (index_t)zOffs.size(), this->toBe()); for(size_t i = 0; i < zOffs.size(); i++) { assert_neq(zOffs[i], (index_t)OFF_MASK); writeIndex(out1, zOffs[i], this->toBe()); } // // Finish building fchr // // Exclusive prefix sum on fchr for(int i = 1; i < 4; i++) { fchr[i] += fchr[i-1]; } assert_lt(fchr[3], gbwtLen); // Shift everybody up by one for(int i = 4; i >= 1; i--) { fchr[i] = fchr[i-1]; } fchr[0] = 0; if(_verbose) { for(int i = 0; i < 5; i++) cerr << "fchr[" << "ACGT$"[i] << "]: " << fchr[i] << endl; } // Write fchr to primary file for(int i = 0; i < 5; i++) { writeIndex(out1, fchr[i], this->toBe()); } // // Finish building ftab and build eftab // // Prefix sum on ftable index_t eftabLen = 0; assert_eq(0, absorbFtab[0]); for(index_t i = 1; i < ftabLen; i++) { if(absorbFtab[i] > 0) eftabLen += 2; } assert_leq(eftabLen, (index_t)gh._ftabChars*2); eftabLen = gh._ftabChars*2; EList eftab(EBWT_CAT); try { eftab.resize(eftabLen); eftab.fillZero(); } catch(bad_alloc &e) { cerr << "Out of memory allocating eftab[] " << "in GFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } index_t eftabCur = 0; for(index_t i = 1; i < ftabLen; i++) { index_t lo = ftab[i] + GFM::ftabHi(ftab.ptr(), eftab.ptr(), len, ftabLen, eftabLen, i-1); if(absorbFtab[i] > 0) { // Skip a number of short pattern indicated by absorbFtab[i] index_t hi = lo + absorbFtab[i]; assert_lt(eftabCur*2+1, eftabLen); eftab[eftabCur*2] = lo; eftab[eftabCur*2+1] = hi; ftab[i] = (eftabCur++) ^ (index_t)OFF_MASK; // insert pointer into eftab assert_eq(lo, GFM::ftabLo(ftab.ptr(), eftab.ptr(), len, ftabLen, eftabLen, i)); assert_eq(hi, GFM::ftabHi(ftab.ptr(), eftab.ptr(), len, ftabLen, eftabLen, i)); } else { ftab[i] = lo; } } assert_eq(GFM::ftabHi(ftab.ptr(), eftab.ptr(), len, ftabLen, eftabLen, ftabLen-1), len+1); // Write ftab to primary file for(index_t i = 0; i < ftabLen; i++) { writeIndex(out1, ftab[i], this->toBe()); } // Write eftab to primary file out1pos = out1.tellp(); if(headerPos < 0) { out1.seekp(24 + sizeof(index_t) * 3); } else { out1.seekp((int)headerPos + 16 + sizeof(index_t) * 2); } writeIndex(out1, eftabLen, this->toBe()); out1.seekp(out1pos); for(index_t i = 0; i < eftabLen; i++) { writeIndex(out1, eftab[i], this->toBe()); } // Note: if you'd like to sanity-check the Ebwt, you'll have to // read it back into memory first! assert(!isInMemory()); VMSG_NL("Exiting GFM::buildToDisk()"); } extern string gLastIOErrMsg; /* Checks whether a call to read() failed or not. */ inline bool is_read_err(int fdesc, ssize_t ret, size_t count) { if (ret < 0) { std::stringstream sstm; sstm << "ERRNO: " << errno << " ERR Msg:" << strerror(errno) << std::endl; gLastIOErrMsg = sstm.str(); return true; } return false; } /* Checks whether a call to fread() failed or not. */ inline bool is_fread_err(FILE* file_hd, size_t ret, size_t count) { if (ferror(file_hd)) { gLastIOErrMsg = "Error Reading File!"; return true; } return false; } /////////////////////////////////////////////////////////////////////// // // Functions for searching Ebwts // (But most of them are defined in the header) // /////////////////////////////////////////////////////////////////////// /** * Take an offset into the joined text and translate it into the * reference of the index it falls on, the offset into the reference, * and the length of the reference. Use a binary search through the * sorted list of reference fragment ranges t */ template bool GFM::joinedToTextOff( index_t qlen, index_t off, index_t& tidx, index_t& textoff, index_t& tlen, bool rejectStraddle, bool& straddled) const { assert(rstarts() != NULL); // must have loaded rstarts index_t top = 0; index_t bot = _nFrag; // 1 greater than largest addressable element index_t elt = (index_t)INDEX_MAX; // Begin binary search while(true) { index_t oldelt = elt; elt = top + ((bot - top) >> 1); if(oldelt == elt) { tidx = (index_t)INDEX_MAX; return false; } index_t lower = rstarts()[elt*3]; index_t upper; if(elt == _nFrag-1) { upper = _gh._len; } else { upper = rstarts()[((elt+1)*3)]; } assert_gt(upper, lower); index_t fraglen = upper - lower; if(lower <= off) { if(upper > off) { // not last element, but it's within // off is in this range; check if it falls off if(off + qlen > upper) { straddled = true; if(rejectStraddle) { // it falls off; signal no-go and return tidx = (index_t)INDEX_MAX; return false; } } // This is the correct text idx whether the index is // forward or reverse tidx = rstarts()[(elt*3)+1]; assert_lt(tidx, this->_nPat); assert_leq(fraglen, this->plen()[tidx]); // it doesn't fall off; now calculate textoff. // Initially it's the number of characters that precede // the alignment in the fragment index_t fragoff = off - rstarts()[(elt*3)]; if(!this->fw_) { fragoff = fraglen - fragoff - 1; fragoff -= (qlen-1); } // Add the alignment's offset into the fragment // ('fragoff') to the fragment's offset within the text textoff = fragoff + rstarts()[(elt*3)+2]; assert_lt(textoff, this->plen()[tidx]); break; // done with binary search } else { // 'off' belongs somewhere in the region between elt // and bot top = elt; } } else { // 'off' belongs somewhere in the region between top and // elt bot = elt; } // continue with binary search } tlen = this->plen()[tidx]; return true; } template bool GFM::textOffToJoined( index_t tid, index_t textoff, index_t& off) const { assert(rstarts() != NULL); // must have loaded rstarts index_t top = 0; index_t bot = _nFrag; // 1 greater than largest addressable element index_t elt = (index_t)INDEX_MAX; // Begin binary search while(true) { ASSERT_ONLY(index_t oldelt = elt); elt = top + ((bot - top) >> 1); assert_neq(oldelt, elt); // must have made progress index_t elt_tid = rstarts()[elt*3 + 1]; if(elt_tid == tid) { while(true) { if(tid != rstarts()[elt*3+1]) { return false; } if(rstarts()[elt*3 + 2] <= textoff) break; if(elt == 0) return false; elt--; } while(true) { assert_leq(rstarts()[elt*3+2], textoff); if(elt + 1 == _nFrag || tid + 1 == rstarts()[(elt+1)*3 + 1] || textoff < rstarts()[(elt+1)*3 + 2]) { off = rstarts()[elt*3] + (textoff - rstarts()[elt*3 + 2]); if(elt + 1 < _nFrag && tid == rstarts()[(elt+1)*3 + 1] && off >= rstarts()[(elt+1)*3]) { return false; } break; } elt++; } break; // done with binary search } else if(elt_tid < tid) { top = elt; } else { bot = elt; } // continue with binary search } return true; } /** * Walk 'steps' steps to the left and return the row arrived at. If we * walk through the dollar sign, return 0xffffffff. */ template index_t GFM::walkLeft(index_t row, index_t steps) const { assert(offs() != NULL); assert_neq((index_t)INDEX_MAX, row); SideLocus l; if(steps > 0) l.initFromRow(row, _gh, gfm()); while(steps > 0) { for(index_t i = 0; i < _zOffs.size(); i++) { if(row == _zOffs[i]) return (index_t)INDEX_MAX; } pair range = this->mapGLF1(row, l, (pair *)NULL ASSERT_ONLY(, false)); index_t newrow = range.first; assert_neq((index_t)INDEX_MAX, newrow); assert_neq(newrow, row); row = newrow; steps--; if(steps > 0) l.initFromRow(row, _gh, gfm()); } return row; } /** * Resolve the reference offset of the BW element 'elt'. */ template index_t GFM::getOffset(index_t row, index_t node) const { assert(offs() != NULL); assert_neq((index_t)INDEX_MAX, row); for(index_t i = 0; i < _zOffs.size(); i++) { if(row == _zOffs[i]) return 0; } if((node & _gh._offMask) == node) { index_t off = this->offs()[node >> _gh._offRate]; if(off != (index_t)INDEX_MAX) return off; } index_t jumps = 0; SideLocus l; l.initFromRow(row, _gh, gfm()); while(true) { pair node_range(0, 0); pair range = this->mapGLF1(row, l, &node_range ASSERT_ONLY(, false)); index_t newrow = range.first; jumps++; assert_neq((index_t)INDEX_MAX, newrow); assert_neq(newrow, row); row = newrow; for(index_t i = 0; i < _zOffs.size(); i++) { if(row == _zOffs[i]) return jumps; } if((node_range.first & _gh._offMask) == node_range.first) { index_t off = this->offs()[node_range.first >> _gh._offRate]; if(off != (index_t)INDEX_MAX) return jumps + off; } l.initFromRow(row, _gh, gfm()); } } /** * Resolve the reference offset of the BW element 'elt' such that * the offset returned is at the right-hand side of the forward * reference substring involved in the hit. */ template index_t GFM::getOffset( index_t elt, bool fw, index_t hitlen) const { index_t off = getOffset(elt); assert_neq((index_t)INDEX_MAX, off); if(!fw) { assert_lt(off, _gh._len); off = _gh._len - off - 1; assert_geq(off, hitlen-1); off -= (hitlen-1); assert_lt(off, _gh._len); } return off; } /** * Returns true iff the index contains the given string (exactly). The given * string must contain only unambiguous characters. TODO: support ambiguous * characters in 'str'. */ template bool GFM::contains( const BTDnaString& str, index_t *otop, index_t *obot) const { assert(isInMemory()); SideLocus tloc, bloc; if(str.empty()) { if(otop != NULL && obot != NULL) *otop = *obot = 0; return true; } int c = str[str.length()-1]; assert_range(0, 4, c); index_t top = 0, bot = 0; if(c < 4) { top = fchr()[c]; bot = fchr()[c+1]; } else { bool set = false; for(int i = 0; i < 4; i++) { if(fchr()[c] < fchr()[c+1]) { if(set) { return false; } else { set = true; top = fchr()[c]; bot = fchr()[c+1]; } } } } assert_geq(bot, top); tloc.initFromRow(top, gh(), gfm()); bloc.initFromRow(bot, gh(), gfm()); ASSERT_ONLY(index_t lastDiff = bot - top); for(int64_t i = (int64_t)str.length()-2; i >= 0; i--) { c = str[i]; assert_range(0, 4, c); if(c <= 3) { top = mapLF(tloc, c); bot = mapLF(bloc, c); } else { index_t sz = bot - top; int c1 = mapLF1(top, tloc ASSERT_ONLY(, false)); bot = mapLF(bloc, c1); assert_leq(bot - top, sz); if(bot - top < sz) { // Encountered an N and could not proceed through it because // there was more than one possible nucleotide we could replace // it with return false; } } assert_geq(bot, top); assert_leq(bot-top, lastDiff); ASSERT_ONLY(lastDiff = bot-top); if(i > 0) { tloc.initFromRow(top, gh(), gfm()); bloc.initFromRow(bot, gh(), gfm()); } } if(otop != NULL && obot != NULL) { *otop = top; *obot = bot; } return bot > top; } /////////////////////////////////////////////////////////////////////// // // Functions for reading and writing Ebwts // /////////////////////////////////////////////////////////////////////// /** * Read an Ebwt from file with given filename. */ template void GFM::readIntoMemory( int needEntireRev, bool loadSASamp, bool loadFtab, bool loadRstarts, bool justHeader, GFMParams *params, bool mmSweep, bool loadNames, bool startVerbose, bool subIndex) { bool switchEndian; // dummy; caller doesn't care #ifdef BOWTIE_MM char *mmFile[] = { NULL, NULL }; #endif if(_in1Str.length() > 0 && !subIndex) { if(_verbose || startVerbose) { cerr << " About to open input files: "; logTime(cerr); } // Initialize our primary and secondary input-stream fields if(_in1 != NULL) fclose(_in1); if(_verbose || startVerbose) cerr << "Opening \"" << _in1Str.c_str() << "\"" << endl; if((_in1 = fopen(_in1Str.c_str(), "rb")) == NULL) { cerr << "Could not open index file " << _in1Str.c_str() << endl; } if(loadSASamp) { if(_in2 != NULL) fclose(_in2); if(_verbose || startVerbose) cerr << "Opening \"" << _in2Str.c_str() << "\"" << endl; if((_in2 = fopen(_in2Str.c_str(), "rb")) == NULL) { cerr << "Could not open index file " << _in2Str.c_str() << endl; } } if(_verbose || startVerbose) { cerr << " Finished opening input files: "; logTime(cerr); } #ifdef BOWTIE_MM if(_useMm /*&& !justHeader*/) { const char *names[] = {_in1Str.c_str(), _in2Str.c_str()}; int fds[] = { fileno(_in1), fileno(_in2) }; for(int i = 0; i < (loadSASamp ? 2 : 1); i++) { if(_verbose || startVerbose) { cerr << " Memory-mapping input file " << (i+1) << ": "; logTime(cerr); } struct stat sbuf; if (stat(names[i], &sbuf) == -1) { perror("stat"); cerr << "Error: Could not stat index file " << names[i] << " prior to memory-mapping" << endl; throw 1; } mmFile[i] = (char*)mmap((void *)0, (size_t)sbuf.st_size, PROT_READ, MAP_SHARED, fds[(size_t)i], 0); if(mmFile[i] == (void *)(-1)) { perror("mmap"); cerr << "Error: Could not memory-map the index file " << names[i] << endl; throw 1; } if(mmSweep) { int sum = 0; for(off_t j = 0; j < sbuf.st_size; j += 1024) { sum += (int) mmFile[i][j]; } if(startVerbose) { cerr << " Swept the memory-mapped ebwt index file 1; checksum: " << sum << ": "; logTime(cerr); } } } mmFile1_ = mmFile[0]; mmFile2_ = loadSASamp ? mmFile[1] : NULL; } #endif } #ifdef BOWTIE_MM else if(_useMm && !justHeader) { mmFile[0] = mmFile1_; mmFile[1] = mmFile2_; } if(_useMm && !justHeader) { assert(mmFile[0] == mmFile1_); assert(mmFile[1] == mmFile2_); } #endif if(_verbose || startVerbose) { cerr << " Reading header: "; logTime(cerr); } // Read endianness hints from both streams size_t bytesRead = 0; if(!subIndex) { switchEndian = false; uint32_t one = readU32(_in1, switchEndian); // 1st word of primary stream bytesRead += 4; if(loadSASamp) { #ifndef NDEBUG assert_eq(one, readU32(_in2, switchEndian)); // should match! #else readU32(_in2, switchEndian); #endif } if(one != 1) { assert_eq((1u<<24), one); assert_eq(1, endianSwapU32(one)); switchEndian = true; } _toBigEndian = switchEndian; // Can't switch endianness and use memory-mapped files; in order to // support this, someone has to modify the file to switch // endiannesses appropriately, and we can't do this inside Bowtie // or we might be setting up a race condition with other processes. if(switchEndian && _useMm) { cerr << "Error: Can't use memory-mapped files when the index is the opposite endianness" << endl; throw 1; } // Reads header entries one by one from primary stream int index_version = (int)readU32(_in1, switchEndian); bytesRead += 4; int major_index_version, minor_index_version; string index_version_extra; readIndexVersion(index_version, major_index_version, minor_index_version, index_version_extra); int major_program_version, minor_program_version; string program_version_extra; readProgramVersion(major_program_version, minor_program_version, program_version_extra); if(major_program_version < major_index_version || (major_program_version == major_index_version && minor_program_version < minor_index_version)) { cerr << "Warning: the current version of HISAT2 (" << HISAT2_VERSION << ") is older than the version (2." << major_index_version << "." << minor_index_version; if(index_version_extra.length() > 0) { cerr << "-" << index_version_extra; } cerr << ") used to build the index." << endl; cerr << " Users are strongly recommended to update HISAT2 to the latest version." << endl; } } else { switchEndian = _toBigEndian; } index_t len = readIndex(_in1, switchEndian); bytesRead += sizeof(index_t); index_t gbwtLen = readIndex(_in1, switchEndian); bytesRead += sizeof(index_t); assert_lt(len, gbwtLen); index_t numNodes = readIndex(_in1, switchEndian); bytesRead += sizeof(index_t); int32_t lineRate = readI32(_in1, switchEndian); bytesRead += 4; /*int32_t linesPerSide =*/ readI32(_in1, switchEndian); bytesRead += 4; int32_t offRate = readI32(_in1, switchEndian); bytesRead += 4; // TODO: add isaRate to the actual file format (right now, the // user has to tell us whether there's an ISA sample and what the // sampling rate is. int32_t ftabChars = readI32(_in1, switchEndian); bytesRead += 4; index_t eftabLen = readIndex(_in1, switchEndian); bytesRead += sizeof(index_t); // chunkRate was deprecated in an earlier version of Bowtie; now // we use it to hold flags. int32_t flags = readI32(_in1, switchEndian); bool entireRev = false; if(flags < 0 && (((-flags) & GFM_ENTIRE_REV) == 0)) { if(needEntireRev != -1 && needEntireRev != 0) { cerr << "Error: This index is compatible with 0.* versions of Bowtie, but not with 2.*" << endl << "versions. Please build or download a version of the index that is compitble" << endl << "with Bowtie 2.* (i.e. built with bowtie-build 2.* or later)" << endl; throw 1; } } else entireRev = true; bytesRead += 4; // Create a new EbwtParams from the entries read from primary stream GFMParams *gh; bool deleteGh = false; if(params != NULL) { params->init(len, gbwtLen, numNodes, lineRate, offRate, ftabChars, eftabLen, entireRev); if(_verbose || startVerbose) params->print(cerr); gh = params; } else { gh = new GFMParams(len, gbwtLen, numNodes, lineRate, offRate, ftabChars, eftabLen, entireRev); deleteGh = true; } // Set up overridden suffix-array-sample parameters index_t offsLen = gh->_offsLen; index_t offRateDiff = 0; index_t offsLenSampled = offsLen; if(_overrideOffRate > offRate) { offRateDiff = _overrideOffRate - offRate; } if(offRateDiff > 0) { offsLenSampled >>= offRateDiff; if((offsLen & ~(((index_t)INDEX_MAX) << offRateDiff)) != 0) { offsLenSampled++; } } // Can't override the offrate or isarate and use memory-mapped // files; ultimately, all processes need to copy the sparser sample // into their own memory spaces. #if 0 if(_useMm && (offRateDiff)) { cerr << "Error: Can't use memory-mapped files when the offrate is overridden" << endl; throw 1; } #endif // Read nPat from primary stream this->_nPat = readIndex(_in1, switchEndian); bytesRead += sizeof(index_t); _plen.reset(); // Read plen from primary stream if(_useMm) { #ifdef BOWTIE_MM _plen.init((index_t*)(mmFile[0] + bytesRead), _nPat, false); bytesRead += _nPat*sizeof(index_t); fseek(_in1, _nPat*sizeof(index_t), SEEK_CUR); #endif } else { try { if(_verbose || startVerbose) { cerr << "Reading plen (" << this->_nPat << "): "; logTime(cerr); } _plen.init(new index_t[_nPat], _nPat, true); if(switchEndian) { for(index_t i = 0; i < this->_nPat; i++) { plen()[i] = readIndex(_in1, switchEndian); } } else { size_t r = MM_READ(_in1, (void*)(plen()), _nPat*sizeof(index_t)); if(r != (size_t)(_nPat*sizeof(index_t))) { cerr << "Error reading _plen[] array: " << r << ", " << _nPat*sizeof(index_t) << endl; throw 1; } } } catch(bad_alloc& e) { cerr << "Out of memory allocating plen[] in Ebwt::read()" << " at " << __FILE__ << ":" << __LINE__ << endl; throw e; } } // TODO: I'm not consistent on what "header" means. Here I'm using // "header" to mean everything that would exist in memory if we // started to build the Ebwt but stopped short of the build*() step // (i.e. everything up to and including join()). if(justHeader) { // Be kind if(deleteGh) delete gh; #ifdef BOWTIE_MM fseek(_in1, 0, SEEK_SET); if(loadSASamp) fseek(_in2, 0, SEEK_SET); #else rewind(_in1); if(loadSASamp) rewind(_in2); #endif return; } bool shmemLeader; this->_nFrag = readIndex(_in1, switchEndian); bytesRead += sizeof(index_t); if(_verbose || startVerbose) { cerr << "Reading rstarts (" << this->_nFrag*3 << "): "; logTime(cerr); } assert_geq(this->_nFrag, this->_nPat); _rstarts.reset(); if(loadRstarts) { if(_useMm) { #ifdef BOWTIE_MM _rstarts.init((index_t*)(mmFile[0] + bytesRead), _nFrag*3, false); bytesRead += this->_nFrag*sizeof(index_t)*3; fseek(_in1, this->_nFrag*sizeof(index_t)*3, SEEK_CUR); #endif } else { _rstarts.init(new index_t[_nFrag*3], _nFrag*3, true); if(switchEndian) { for(size_t i = 0; i < (size_t)(this->_nFrag*3); i += 3) { // fragment starting position in joined reference // string, text id, and fragment offset within text this->rstarts()[i] = readIndex(_in1, switchEndian); this->rstarts()[i+1] = readIndex(_in1, switchEndian); this->rstarts()[i+2] = readIndex(_in1, switchEndian); } } else { size_t r = MM_READ(_in1, (void *)rstarts(), this->_nFrag*sizeof(index_t)*3); if(r != (size_t)(this->_nFrag*sizeof(index_t)*3)) { cerr << "Error reading _rstarts[] array: " << r << ", " << (this->_nFrag*sizeof(index_t)*3) << endl; throw 1; } } } } else { // Skip em assert(rstarts() == NULL); bytesRead += this->_nFrag*sizeof(index_t)*3; fseek(_in1, this->_nFrag*sizeof(index_t)*3, SEEK_CUR); } _gfm.reset(); if(_useMm) { #ifdef BOWTIE_MM _gfm.init((uint8_t*)(mmFile[0] + bytesRead), gh->_gbwtTotLen, false); bytesRead += gh->_gbwtTotLen; fseek(_in1, gh->_gbwtTotLen, SEEK_CUR); #endif } else { // Allocate ebwt (big allocation) if(_verbose || startVerbose) { cerr << "Reading ebwt (" << gh->_gbwtTotLen << "): "; logTime(cerr); } bool shmemLeader = true; if(useShmem_) { uint8_t *tmp = NULL; shmemLeader = ALLOC_SHARED_U8( (_in1Str + "[ebwt]"), gh->_gbwtTotLen, &tmp, "gfm[]", (_verbose || startVerbose)); assert(tmp != NULL); _gfm.init(tmp, gh->_gbwtTotLen, false); if(_verbose || startVerbose) { cerr << " shared-mem " << (shmemLeader ? "leader" : "follower") << endl; } } else { try { _gfm.init(new uint8_t[gh->_gbwtTotLen], gh->_gbwtTotLen, true); } catch(bad_alloc& e) { cerr << "Out of memory allocating the gfm[] array for the Bowtie index. Please try" << endl << "again on a computer with more memory." << endl; throw 1; } } if(shmemLeader) { // Read ebwt from primary stream uint64_t bytesLeft = gh->_gbwtTotLen; char *pgbwt = (char*)this->gfm(); while (bytesLeft>0){ size_t r = MM_READ(this->_in1, (void *)pgbwt, bytesLeft); if(MM_IS_IO_ERR(this->_in1, r, bytesLeft)) { cerr << "Error reading _ebwt[] array: " << r << ", " << bytesLeft << endl; throw 1; } pgbwt += r; bytesLeft -= r; } if(switchEndian) { uint8_t *side = this->gfm(); for(size_t i = 0; i < gh->_numSides; i++) { index_t *cums = reinterpret_cast(side + gh->_sideSz - sizeof(index_t)*2); cums[0] = endianSwapIndex(cums[0]); cums[1] = endianSwapIndex(cums[1]); side += this->_gh._sideSz; } } #ifdef BOWTIE_SHARED_MEM if(useShmem_) NOTIFY_SHARED(gfm(), gh->_gbwtTotLen); #endif } else { // Seek past the data and wait until master is finished fseek(_in1, gh->_gbwtTotLen, SEEK_CUR); #ifdef BOWTIE_SHARED_MEM if(useShmem_) WAIT_SHARED(gfm(), gh->_gbwtTotLen); #endif } } // Read zOff from primary stream _zOffs.clear(); index_t num_zOffs = readIndex(_in1, switchEndian); bytesRead += sizeof(index_t); for(index_t i = 0; i < num_zOffs; i++) { index_t zOff = readIndex(_in1, switchEndian); bytesRead += sizeof(index_t); assert_lt(zOff, gbwtLen); _zOffs.push_back(zOff); } try { // Read fchr from primary stream if(_verbose || startVerbose) cerr << "Reading fchr (5)" << endl; _fchr.reset(); if(_useMm) { #ifdef BOWTIE_MM _fchr.init((index_t*)(mmFile[0] + bytesRead), 5, false); bytesRead += 5*sizeof(index_t); fseek(_in1, 5*sizeof(index_t), SEEK_CUR); #endif } else { _fchr.init(new index_t[5], 5, true); for(int i = 0; i < 5; i++) { this->fchr()[i] = readIndex(_in1, switchEndian); assert_leq(this->fchr()[i], gbwtLen); assert(i <= 0 || this->fchr()[i] >= this->fchr()[i-1]); } } assert_gt(this->fchr()[4], this->fchr()[0]); // Read ftab from primary stream if(_verbose || startVerbose) { if(loadFtab) { cerr << "Reading ftab (" << gh->_ftabLen << "): "; logTime(cerr); } else { cerr << "Skipping ftab (" << gh->_ftabLen << "): "; } } _ftab.reset(); if(loadFtab) { if(_useMm) { #ifdef BOWTIE_MM _ftab.init((index_t*)(mmFile[0] + bytesRead), gh->_ftabLen, false); bytesRead += gh->_ftabLen*sizeof(index_t); fseek(_in1, gh->_ftabLen*sizeof(index_t), SEEK_CUR); #endif } else { _ftab.init(new index_t[gh->_ftabLen], gh->_ftabLen, true); if(switchEndian) { for(size_t i = 0; i < gh->_ftabLen; i++) this->ftab()[i] = readIndex(_in1, switchEndian); } else { size_t r = MM_READ(_in1, (void *)ftab(), gh->_ftabLen*sizeof(index_t)); if(r != (size_t)(gh->_ftabLen*sizeof(index_t))) { cerr << "Error reading _ftab[] array: " << r << ", " << (gh->_ftabLen*sizeof(index_t)) << endl; throw 1; } } } // Read etab from primary stream if(_verbose || startVerbose) { if(loadFtab) { cerr << "Reading eftab (" << gh->_eftabLen << "): "; logTime(cerr); } else { cerr << "Skipping eftab (" << gh->_eftabLen << "): "; } } _eftab.reset(); if(_useMm) { #ifdef BOWTIE_MM _eftab.init((index_t*)(mmFile[0] + bytesRead), gh->_eftabLen, false); bytesRead += gh->_eftabLen*sizeof(index_t); fseek(_in1, gh->_eftabLen*sizeof(index_t), SEEK_CUR); #endif } else { _eftab.init(new index_t[gh->_eftabLen], gh->_eftabLen, true); if(switchEndian) { for(size_t i = 0; i < gh->_eftabLen; i++) this->eftab()[i] = readIndex(_in1, switchEndian); } else { size_t r = MM_READ(_in1, (void *)this->eftab(), gh->_eftabLen*sizeof(index_t)); if(r != (size_t)(gh->_eftabLen*sizeof(index_t))) { cerr << "Error reading _eftab[] array: " << r << ", " << (gh->_eftabLen*sizeof(index_t)) << endl; throw 1; } } } for(index_t i = 0; i < gh->_eftabLen; i++) { if(i > 0 && this->eftab()[i] > 0) { assert_geq(this->eftab()[i] + 4, this->eftab()[i-1]); } else if(i > 0 && this->eftab()[i-1] == 0) { assert_eq(0, this->eftab()[i]); } } } else { assert(ftab() == NULL); assert(eftab() == NULL); // Skip ftab bytesRead += gh->_ftabLen*sizeof(index_t); fseek(_in1, gh->_ftabLen*sizeof(index_t), SEEK_CUR); // Skip eftab bytesRead += sizeof(index_t); bytesRead += gh->_eftabLen*sizeof(index_t); fseek(_in1, gh->_eftabLen*sizeof(index_t), SEEK_CUR); } } catch(bad_alloc& e) { cerr << "Out of memory allocating fchr[], ftab[] or eftab[] arrays for the Bowtie index." << endl << "Please try again on a computer with more memory." << endl; throw 1; } // Read reference sequence names from primary index file (or not, // if --refidx is specified) if(loadNames) { while(true) { char c = '\0'; if(MM_READ(_in1, (void *)(&c), (size_t)1) != (size_t)1) break; bytesRead++; if(c == '\0') break; else if(c == '\n') { this->_refnames.push_back(""); } else { if(this->_refnames.size() == 0) { this->_refnames.push_back(""); } this->_refnames.back().push_back(c); } } } _offs.reset(); if(loadSASamp) { bytesRead = 4; // reset for secondary index file (already read 1-sentinel) shmemLeader = true; if(_verbose || startVerbose) { cerr << "Reading offs (" << offsLenSampled << " " << std::setw(2) << sizeof(index_t)*8 << "-bit words): "; logTime(cerr); } if(!_useMm) { if(!useShmem_) { // Allocate offs_ try { _offs.init(new index_t[offsLenSampled], offsLenSampled, true); } catch(bad_alloc& e) { cerr << "Out of memory allocating the offs[] array for the Bowtie index." << endl << "Please try again on a computer with more memory." << endl; throw 1; } } else { index_t *tmp = NULL; shmemLeader = ALLOC_SHARED_U32( (_in2Str + "[offs]"), offsLenSampled*sizeof(index_t), &tmp, "offs", (_verbose || startVerbose)); _offs.init((index_t*)tmp, offsLenSampled, false); } } if(_overrideOffRate < 32) { if(shmemLeader) { // Allocate offs (big allocation) if(switchEndian || offRateDiff > 0) { assert(!_useMm); const index_t blockMaxSz = (index_t)(2 * 1024 * 1024); // 2 MB block size const index_t blockMaxSzU = (blockMaxSz / sizeof(index_t)); // # U32s per block char *buf; try { buf = new char[blockMaxSz]; } catch(std::bad_alloc& e) { cerr << "Error: Out of memory allocating part of _offs array: '" << e.what() << "'" << endl; throw e; } for(index_t i = 0; i < offsLen; i += blockMaxSzU) { index_t block = min((index_t)blockMaxSzU, (index_t)(offsLen - i)); size_t r = MM_READ(_in2, (void *)buf, block * sizeof(index_t)); if(r != (size_t)(block * sizeof(index_t))) { cerr << "Error reading block of _offs[] array: " << r << ", " << (block * sizeof(index_t)) << endl; throw 1; } index_t idx = i >> offRateDiff; for(index_t j = 0; j < block; j += (1 << offRateDiff)) { assert_lt(idx, offsLenSampled); this->offs()[idx] = ((index_t*)buf)[j]; if(switchEndian) { this->offs()[idx] = endianSwapIndex(this->offs()[idx]); } idx++; } } delete[] buf; } else { if(_useMm) { #ifdef BOWTIE_MM _offs.init((index_t*)(mmFile[1] + bytesRead), offsLen, false); bytesRead += (offsLen * sizeof(index_t)); fseek(_in2, (offsLen * sizeof(index_t)), SEEK_CUR); #endif } else { // Workaround for small-index mode where MM_READ may // not be able to handle read amounts greater than 2^32 // bytes. uint64_t bytesLeft = (offsLen * sizeof(index_t)); char *offs = (char *)this->offs(); while(bytesLeft > 0) { size_t r = MM_READ(_in2, (void*)offs, bytesLeft); if(MM_IS_IO_ERR(_in2,r,bytesLeft)) { cerr << "Error reading block of _offs[] array: " << r << ", " << bytesLeft << gLastIOErrMsg << endl; throw 1; } offs += r; bytesLeft -= r; } } } #ifdef BOWTIE_SHARED_MEM if(useShmem_) NOTIFY_SHARED(offs(), offsLenSampled*sizeof(index_t)); #endif } else { // Not the shmem leader fseek(_in2, offsLenSampled*sizeof(index_t), SEEK_CUR); #ifdef BOWTIE_SHARED_MEM if(useShmem_) WAIT_SHARED(offs(), offsLenSampled*sizeof(index_t)); #endif } } } this->postReadInit(*gh); // Initialize fields of Ebwt not read from file if(_verbose || startVerbose) print(cerr, *gh); // The fact that _ebwt and friends actually point to something // (other than NULL) now signals to other member functions that the // Ebwt is loaded into memory. // Be kind if(deleteGh) delete gh; if(!subIndex) { #ifdef BOWTIE_MM fseek(_in1, 0, SEEK_SET); if(loadSASamp) fseek(_in2, 0, SEEK_SET); #else rewind(_in1); if(loadSASamp) rewind(_in2); #endif } } /** * Read reference names from an input stream 'in' for an Ebwt primary * file and store them in 'refnames'. */ template void readGFMRefnames(istream& in, EList& refnames) { // _in1 must already be open with the get cursor at the // beginning and no error flags set. assert(in.good()); assert_eq((streamoff)in.tellg(), ios::beg); // Read endianness hints from both streams bool switchEndian = false; uint32_t one = readU32(in, switchEndian); // 1st word of primary stream if(one != 1) { assert_eq((1u<<24), one); switchEndian = true; } // Reads header entries one by one from primary stream readU32(in, switchEndian); // version index_t len = readIndex(in, switchEndian); index_t gbwtLen = readIndex(in, switchEndian); index_t numNodes = readIndex(in, switchEndian); int32_t lineRate = readI32(in, switchEndian); /*int32_t linesPerSide =*/ readI32(in, switchEndian); int32_t offRate = readI32(in, switchEndian); int32_t ftabChars = readI32(in, switchEndian); index_t eftabLen = readIndex(in, switchEndian); // BTL: chunkRate is now deprecated int32_t flags = readI32(in, switchEndian); bool entireReverse = false; if(flags < 0) { entireReverse = (((-flags) & GFM_ENTIRE_REV) != 0); } // Create a new EbwtParams from the entries read from primary stream GFM gh(len, gbwtLen, numNodes, lineRate, offRate, ftabChars, eftabLen, entireReverse); index_t nPat = readIndex(in, switchEndian); // nPat in.seekg(nPat*sizeof(index_t), ios_base::cur); // skip plen // Skip rstarts index_t nFrag = readIndex(in, switchEndian); in.seekg(nFrag*sizeof(index_t)*3, ios_base::cur); // Skip ebwt in.seekg(gh._ebwtTotLen, ios_base::cur); // Skip zOff from primary stream index_t numZOffs = readIndex(in, switchEndian); in.seekg(numZOffs * sizeof(index_t), ios_base::cur); // Skip fchr in.seekg(5 * sizeof(index_t), ios_base::cur); // Skip ftab in.seekg(gh._ftabLen*sizeof(index_t), ios_base::cur); // Skip eftab in.seekg(gh._eftabLen*sizeof(index_t), ios_base::cur); // Read reference sequence names from primary index file while(true) { char c = '\0'; in.read(&c, 1); if(in.eof()) break; if(c == '\0') break; else if(c == '\n') { refnames.push_back(""); } else { if(refnames.size() == 0) { refnames.push_back(""); } refnames.back().push_back(c); } } if(refnames.back().empty()) { refnames.pop_back(); } // Be kind in.clear(); in.seekg(0, ios::beg); assert(in.good()); } /** * Read reference names from the index with basename 'in' and store * them in 'refnames'. */ template void readGFMRefnames(const string& instr, EList& refnames) { ifstream in; // Initialize our primary and secondary input-stream fields in.open((instr + ".1." + gfm_ext).c_str(), ios_base::in | ios::binary); if(!in.is_open()) { throw GFMFileOpenException("Cannot open file " + instr); } assert(in.is_open()); assert(in.good()); assert_eq((streamoff)in.tellg(), ios::beg); readGFMRefnames(in, refnames); } /** * Read just enough of the Ebwt's header to get its flags */ template int32_t GFM::readVersionFlags(const string& instr, int& major, int& minor, string& extra_version) { ifstream in; // Initialize our primary and secondary input-stream fields in.open((instr + ".1." + gfm_ext).c_str(), ios_base::in | ios::binary); if(!in.is_open()) { throw GFMFileOpenException("Cannot open file " + instr); } assert(in.is_open()); assert(in.good()); bool switchEndian = false; uint32_t one = readU32(in, switchEndian); // 1st word of primary stream if(one != 1) { assert_eq((1u<<24), one); assert_eq(1, endianSwapU32(one)); switchEndian = true; } index_t version = readU32(in, switchEndian); readIndexVersion(version, major, minor, extra_version); readIndex(in, switchEndian); readIndex(in, switchEndian); readIndex(in, switchEndian); readI32(in, switchEndian); readI32(in, switchEndian); readI32(in, switchEndian); readI32(in, switchEndian); readIndex(in, switchEndian); int32_t flags = readI32(in, switchEndian); return flags; } /** * Write an extended Burrows-Wheeler transform to a pair of output * streams. * * @param out1 output stream to primary file * @param out2 output stream to secondary file * @param be write in big endian? */ template void GFM::writeFromMemory(bool justHeader, ostream& out1, ostream& out2) const { const GFMParams& gh = this->_gh; assert(gh.repOk()); uint32_t be = this->toBe(); assert(out1.good()); assert(out2.good()); // When building an Ebwt, these header parameters are known // "up-front", i.e., they can be written to disk immediately, // before we join() or buildToDisk() writeI32(out1, 1, be); // endian hint for priamry stream writeI32(out2, 1, be); // endian hint for secondary stream int version = getIndexVersion(); writeI32(out1, version, be); // version writeIndex(out1, gh._len, be); // length of string (and bwt and suffix array) writeIndex(out1, 0, be); // dummy for gbwt len writeIndex(out1, 0, be); // dummy for number of nodes writeI32(out1, gh._lineRate, be); // 2^lineRate = size in bytes of 1 line writeI32(out1, 2, be); // not used writeI32(out1, gh._offRate, be); // every 2^offRate chars is "marked" writeI32(out1, gh._ftabChars, be); // number of 2-bit chars used to address ftab writeIndex(out1, 0, be); // eftab length int32_t flags = 1; if(gh._entireReverse) flags |= GFM_ENTIRE_REV; writeI32(out1, -flags, be); // BTL: chunkRate is now deprecated if(!justHeader) { assert(rstarts() != NULL); assert(offs() != NULL); assert(ftab() != NULL); assert(eftab() != NULL); assert(isInMemory()); // These Ebwt parameters are known after the inputs strings have // been joined() but before they have been built(). These can // written to the disk next and then discarded from memory. writeIndex(out1, this->_nPat, be); for(index_t i = 0; i < this->_nPat; i++) writeIndex(out1, this->plen()[i], be); assert_geq(this->_nFrag, this->_nPat); writeIndex(out1, this->_nFrag, be); for(size_t i = 0; i < this->_nFrag*3; i++) writeIndex(out1, this->rstarts()[i], be); // These Ebwt parameters are discovered only as the Ebwt is being // built (in buildToDisk()). Of these, only 'offs' and 'ebwt' are // terribly large. 'ebwt' is written to the primary file and then // discarded from memory as it is built; 'offs' is similarly // written to the secondary file and discarded. writeIndex(out1, gh._gbwtTotLen, be); out1.write((const char *)this->gfm(), gh._gbwtTotLen); writeIndex(out1, (index_t)_zOffs.size(), be); for(index_t i = 0; i < _zOffs.size(); i++) writeIndex(out1, _zOffs[i], be); index_t offsLen = gh._offsLen; for(index_t i = 0; i < offsLen; i++) writeIndex(out2, this->offs()[i], be); // 'fchr', 'ftab' and 'eftab' are not fully determined until the // loop is finished, so they are written to the primary file after // all of 'ebwt' has already been written and only then discarded // from memory. for(int i = 0; i < 5; i++) writeIndex(out1, this->fchr()[i], be); for(index_t i = 0; i < gh._ftabLen; i++) writeIndex(out1, this->ftab()[i], be); for(index_t i = 0; i < gh._eftabLen; i++) writeIndex(out1, this->eftab()[i], be); } } /** * Given a pair of strings representing output filenames, and assuming * this Ebwt object is currently in memory, write out this Ebwt to the * specified files. * * If sanity-checking is enabled, then once the streams have been * fully written and closed, we reopen them and read them into a * (hopefully) exact copy of this Ebwt. We then assert that the * current Ebwt and the copy match in all of their fields. */ template void GFM::writeFromMemory(bool justHeader, const string& out1, const string& out2) const { ASSERT_ONLY(const GFMParams& gh = this->_gh); assert(isInMemory()); assert(gh.repOk()); ofstream fout1(out1.c_str(), ios::binary); ofstream fout2(out2.c_str(), ios::binary); writeFromMemory(justHeader, fout1, fout2); fout1.close(); fout2.close(); // Read the file back in and assert that all components match if(_sanity) { #if 0 if(_verbose) cout << "Re-reading \"" << out1 << "\"/\"" << out2 << "\" for sanity check" << endl; Ebwt copy(out1, out2, _verbose, _sanity); assert(!isInMemory()); copy.loadIntoMemory(eh._color ? 1 : 0, true, false, false); assert(isInMemory()); assert_eq(eh._lineRate, copy.eh()._lineRate); assert_eq(eh._offRate, copy.eh()._offRate); assert_eq(eh._ftabChars, copy.eh()._ftabChars); assert_eq(eh._len, copy.eh()._len); assert_eq(_zOff, copy.zOff()); assert_eq(_zEbwtBpOff, copy.zEbwtBpOff()); assert_eq(_zEbwtByteOff, copy.zEbwtByteOff()); assert_eq(_nPat, copy.nPat()); for(index_t i = 0; i < _nPat; i++) assert_eq(this->_plen[i], copy.plen()[i]); assert_eq(this->_nFrag, copy.nFrag()); for(size_t i = 0; i < this->nFrag*3; i++) { assert_eq(this->_rstarts[i], copy.rstarts()[i]); } for(index_t i = 0; i < 5; i++) assert_eq(this->_fchr[i], copy.fchr()[i]); for(size_t i = 0; i < eh._ftabLen; i++) assert_eq(this->ftab()[i], copy.ftab()[i]); for(size_t i = 0; i < eh._eftabLen; i++) assert_eq(this->eftab()[i], copy.eftab()[i]); for(index_t i = 0; i < eh._offsLen; i++) assert_eq(this->_offs[i], copy.offs()[i]); for(index_t i = 0; i < eh._ebwtTotLen; i++) assert_eq(this->ebwt()[i], copy.ebwt()[i]); copy.sanityCheckAll(); if(_verbose) cout << "Read-in check passed for \"" << out1 << "\"/\"" << out2 << "\"" << endl; #endif } } /** * Write the rstarts array given the szs array for the reference. */ template void GFM::szsToDisk(const EList& szs, ostream& os, int reverse) { size_t seq = 0; index_t off = 0; index_t totlen = 0; for(size_t i = 0; i < szs.size(); i++) { if(szs[i].len == 0) continue; if(szs[i].first) off = 0; off += szs[i].off; if(szs[i].first && szs[i].len > 0) seq++; index_t seqm1 = (index_t)(seq-1); assert_lt(seqm1, _nPat); index_t fwoff = off; if(reverse == REF_READ_REVERSE) { // Invert pattern idxs seqm1 = _nPat - seqm1 - 1; // Invert pattern idxs assert_leq(off + szs[i].len, plen()[seqm1]); fwoff = plen()[seqm1] - (off + szs[i].len); } writeIndex(os, totlen, this->toBe()); // offset from beginning of joined string writeIndex(os, (index_t)seqm1, this->toBe()); // sequence id writeIndex(os, (index_t)fwoff, this->toBe()); // offset into sequence totlen += szs[i].len; off += szs[i].len; } } /////////////////////////////////////////////////////////////////////// // // Functions for printing and sanity-checking Ebwts // /////////////////////////////////////////////////////////////////////// /** * Check that the ebwt array is internally consistent up to (and not * including) the given side index by re-counting the chars and * comparing against the embedded occ[] arrays. */ template void GFM::sanityCheckUpToSide(int upToSide) const { assert(isInMemory()); index_t occ[] = {0, 0, 0, 0}; ASSERT_ONLY(index_t occ_save[] = {0, 0, 0, 0}); index_t cur = 0; // byte pointer const GFMParams& gh = this->_gh; bool fw = false; while(cur < (upToSide * gh._sideSz)) { assert_leq(cur + gh._sideSz, gh._gbwtTotLen); for(index_t i = 0; i < gh._sideGbwtSz; i++) { uint8_t by = this->gfm()[cur + (fw ? i : gh._sideGbwtSz-i-1)]; for(int j = 0; j < 4; j++) { // Unpack from lowest to highest bit pair int twoBit = unpack_2b_from_8b(by, fw ? j : 3-j); occ[twoBit]++; } assert_eq(0, (occ[0] + occ[1] + occ[2] + occ[3]) % 4); } assert_eq(0, (occ[0] + occ[1] + occ[2] + occ[3]) % gh._sideGbwtLen); // Finished forward bucket; check saved [A], [C], [G] and [T] // against the index_ts encoded here ASSERT_ONLY(const index_t *ugbwt = reinterpret_cast(&gfm()[cur + gh._sideGbwtSz])); ASSERT_ONLY(index_t as = ugbwt[0]); ASSERT_ONLY(index_t cs = ugbwt[1]); ASSERT_ONLY(index_t gs = ugbwt[2]); ASSERT_ONLY(index_t ts = ugbwt[3]); assert(as == occ_save[0] || as == occ_save[0]-1); assert_eq(cs, occ_save[1]); assert_eq(gs, occ_save[2]); assert_eq(ts, occ_save[3]); #ifndef NDEBUG occ_save[0] = occ[0]; occ_save[1] = occ[1]; occ_save[2] = occ[2]; occ_save[3] = occ[3]; #endif cur += gh._sideSz; } } /** * Sanity-check various pieces of the Ebwt */ template void GFM::sanityCheckAll(int reverse) const { const GFMParams& gh = this->_gh; assert(isInMemory()); // Check ftab for(index_t i = 1; i < gh._ftabLen; i++) { assert_geq(this->ftabHi(i), this->ftabLo(i-1)); assert_geq(this->ftabLo(i), this->ftabHi(i-1)); assert_leq(this->ftabHi(i), gh._gbwtLen); } assert_eq(this->ftabHi(gh._ftabLen-1), gh._gbwtLen); // Check offs int seenLen = (gh._gbwtLen + 31) >> ((index_t)5); uint32_t *seen; try { seen = new uint32_t[seenLen]; // bitvector marking seen offsets } catch(bad_alloc& e) { cerr << "Out of memory allocating seen[] at " << __FILE__ << ":" << __LINE__ << endl; throw e; } memset(seen, 0, 4 * seenLen); index_t offsLen = gh._offsLen; for(index_t i = 0; i < offsLen; i++) { assert_lt(this->offs()[i], gh._gbwtLen); int w = this->offs()[i] >> 5; int r = this->offs()[i] & 31; assert_eq(0, (seen[w] >> r) & 1); // shouldn't have been seen before seen[w] |= (1 << r); } delete[] seen; // Check nPat assert_gt(this->_nPat, 0); // Check plen, flen for(index_t i = 0; i < this->_nPat; i++) { assert_geq(this->plen()[i], 0); } // Check rstarts if(this->rstarts() != NULL) { for(index_t i = 0; i < this->_nFrag-1; i++) { assert_gt(this->rstarts()[(i+1)*3], this->rstarts()[i*3]); if(reverse == REF_READ_REVERSE) { assert(this->rstarts()[(i*3)+1] >= this->rstarts()[((i+1)*3)+1]); } else { assert(this->rstarts()[(i*3)+1] <= this->rstarts()[((i+1)*3)+1]); } } } // Check ebwt sanityCheckUpToSide(gh._numSides); VMSG_NL("Ebwt::sanityCheck passed"); } /** * Transform this Ebwt into the original string in linear time by using * the LF mapping to walk backwards starting at the row correpsonding * to the end of the string. The result is written to s. The Ebwt * must be in memory. */ template void GFM::restore(SString& s) const { assert(isInMemory()); s.resize(this->_gh._len); index_t jumps = 0; index_t i = this->_gh._len; // should point to final SA elt (starting with '$') SideLocus l(i, this->_gh, this->gfm()); while(true) { for(index_t j = 0; j < _zOffs.size(); j++) { if(i == _zOffs[j]) break; } assert_lt(jumps, this->_gh._len); //if(_verbose) cout << "restore: i: " << i << endl; // Not a marked row; go back a char in the original string index_t newi = mapLF(l ASSERT_ONLY(, false)); assert_neq(newi, i); s[this->_gh._len - jumps - 1] = rowL(l); i = newi; l.initFromRow(i, this->_gh, this->gfm()); jumps++; } assert_eq(jumps, this->_gh._len); } /** * Check that this Ebwt, when restored via restore(), matches up with * the given array of reference sequences. For sanity checking. */ template void GFM::checkOrigs( const EList >& os, bool mirror) const { SString rest; restore(rest); index_t restOff = 0; size_t i = 0, j = 0; if(mirror) { // TODO: FIXME return; } while(i < os.size()) { size_t olen = os[i].length(); int lastorig = -1; for(; j < olen; j++) { size_t joff = j; if(mirror) joff = olen - j - 1; if((int)os[i][joff] == 4) { // Skip over Ns lastorig = -1; if(!mirror) { while(j < olen && (int)os[i][j] == 4) j++; } else { while(j < olen && (int)os[i][olen-j-1] == 4) j++; } j--; continue; } assert_eq(os[i][joff], rest[restOff]); lastorig = (int)os[i][joff]; restOff++; } if(j == os[i].length()) { // Moved to next sequence i++; j = 0; } else { // Just jumped over a gap } } } /** * Try to find the Bowtie index specified by the user. First try the * exact path given by the user. Then try the user-provided string * appended onto the path of the "indexes" subdirectory below this * executable, then try the provided string appended onto * "$HISAT2_INDEXES/". */ string adjustEbwtBase(const string& cmdline, const string& ebwtFileBase, bool verbose = false); #endif /*GFM_H_*/ hisat2-2.2.1/gp.h000066400000000000000000000037201370663763200135040ustar00rootroot00000000000000/* * Copyright 2016, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ /* * gp.h * */ #ifndef GP_H_ #define GP_H_ #include #include /** * Encapsulates alignment policy for graph */ class GraphPolicy { public: GraphPolicy() { reset(); } GraphPolicy(size_t maxAltsTried, bool useHaplotype, bool haplotypeOnly, bool enableCODIS) { init(maxAltsTried, useHaplotype, haplotypeOnly, enableCODIS); } /** */ void reset() { init(0); } /** */ void init(size_t maxAltsTried, bool useHaplotype = false, bool haplotypeOnly = false, bool enableCODIS = false) { maxAltsTried_ = maxAltsTried; useHaplotype_ = useHaplotype; haplotypeOnly_ = haplotypeOnly; enableCODIS_ = enableCODIS; } size_t maxAltsTried() const { return maxAltsTried_; } bool useHaplotype() const { return useHaplotype_; } bool haplotypeOnly() const { return haplotypeOnly_; } bool enableCODIS() const { return enableCODIS_; } private: size_t maxAltsTried_; bool useHaplotype_; bool haplotypeOnly_; bool enableCODIS_; }; #endif /*ndef GP_H_*/ hisat2-2.2.1/group_walk.cpp000066400000000000000000000013671370663763200156100ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "group_walk.h" hisat2-2.2.1/group_walk.h000066400000000000000000001572251370663763200152620ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /* * group_walk.h * * Classes and routines for walking a set of BW ranges backwards from the edge * of a seed hit with the goal of resolving the offset of each row in each * range. Here "offset" means offset into the concatenated string of all * references. The main class is 'GroupWalk' and an important helper is * 'GWState'. * * For each combination of seed offset and orientation, there is an associated * QVal. Each QVal describes a (possibly empty) set of suffix array ranges. * Call these "seed range sets." Each range in the set is "backed" by a range * of the salist, represented as a PListSlice. Such a range is the origin of a * walk. * * When an offset is resolved, it is entered into the salist via the * PListSlice. Note that other routines in this same thread might also be * setting elements of the salist, so routines here should expect that elements * can go from unresolved to resolved at any time. * * What bookkeeping do we have to do as we walk? Before the first step, we * convert the initial QVal into a list of SATuples; the SATuples are our link * to the correpsonding ranges in the suffix array. The list of SATuples is * then converted to a list of GWState objects; these keep track of where we * are in our walk (e.g. what 'top' and 'bot' are, how many steps have we gone, * etc) as well as how the elements in the current range correspond to elements * from the original range. * * The user asks the GroupWalk to resolve another offset by calling advance(). * advance() can be called in various ways: * * (a) The user can request that the GroupWalk proceed until a * *particular* element is resolved, then return that resolved * element. Other elements may be resolved along the way, but * those results are buffered and may be dispensed in future calls * to advance(). * * (b) The user can request that the GroupWalk select an as-yet- * unreported element at random and and proceed until that element * is resolved and report it. Again, other elements may be * resolved along the way but they are buffered. * * (c) The user can request that the GroupWalk resolve elements in a * particular BW range (with a particular offset and orientation) * in an order of its choosing. The GroupWalk in this case * attempts to resolve as many offsets as possible as quickly as * possible, and returns them as soon as they're found. The res_ * buffer is used in this case. * * (d) Like (c) but resolving elements at a paritcular offset and * orientation instead of at a specific BW range. The res_ buffer * is used in this case, since there's a chance that the * * There are simple ways to heuristically reduce the problem size while * maintaining randomness. For instance, the user put a ceiling on the * number of elements that we walk from any given seed offset or range. * We can then trim away random subranges to reduce the size of the * problem. There is no need for the caller to do this for us. */ #ifndef GROUP_WALK_H_ #define GROUP_WALK_H_ #include #include #include "ds.h" #include "gfm.h" #include "read.h" #include "reference.h" #include "mem_ids.h" /** * Encapsulate an SA range and an associated list of slots where the resolved * offsets can be placed. */ template class SARangeWithOffs { public: SARangeWithOffs() { reset(); }; SARangeWithOffs( index_t tf, index_t bf, index_t ntf, index_t nbf, const EList >& n_iedge_count, size_t len, const T& o) { init(tf, bf, ntf, nbf, n_iedge_count, len, o); } void init( index_t tf, index_t bf, index_t ntf, index_t nbf, const EList >& n_iedge_count, size_t len_, const T& o) { topf = tf; botf = bf; assert_lt(topf, botf); node_top = ntf; node_bot = nbf; assert_leq(node_bot - node_top, botf - topf); node_iedge_count = n_iedge_count; len = len_, offs = o; } /** * Reset to uninitialized state. */ void reset() { topf = (index_t)INDEX_MAX; } /** * Return true if this is initialized. */ bool inited() const { return topf != (index_t)INDEX_MAX; } /** * Return the number of times this reference substring occurs in the * reference, which is also the size of the 'offs' TSlice. */ size_t size() const { return offs.size(); } index_t topf; // top in GBWT index index_t botf; index_t node_top; // top node index_t node_bot; EList > node_iedge_count; size_t len; // length of the reference sequence involved T offs; // offsets }; /** * A group of per-thread state that can be shared between all the GroupWalks * used in that thread. */ template struct GroupWalkState { GroupWalkState(int cat) : map(cat) { masks[0].setCat(cat); masks[1].setCat(cat); masks[2].setCat(cat); masks[3].setCat(cat); } EList masks[4]; // temporary list for masks; used in GWState EList map; // temporary list of GWState maps }; /** * Encapsulates counters that encode how much work the walk-left logic * has done. */ struct WalkMetrics { WalkMetrics() { reset(); } /** * Sum each across this object and 'm'. This is the only safe way * to update a WalkMetrics shared by many threads. */ void merge(const WalkMetrics& m, bool getLock = false) { ThreadSafe ts(&mutex_m, getLock); bwops += m.bwops; branches += m.branches; resolves += m.resolves; refresolves += m.refresolves; reports += m.reports; } /** * Set all to 0. */ void reset() { bwops = branches = resolves = refresolves = reports = 0; } uint64_t bwops; // Burrows-Wheeler operations uint64_t branches; // BW range branch-offs uint64_t resolves; // # offs resolved with BW walk-left uint64_t refresolves; // # resolutions caused by reference scanning uint64_t reports; // # offs reported (1 can be reported many times) MUTEX_T mutex_m; }; /** * Coordinates for a BW element that the GroupWalk might resolve. */ template struct GWElt { GWElt() { reset(); } /** * Reset GWElt to uninitialized state. */ void reset() { offidx = range = elt = len = (index_t)OFF_MASK; fw = false; } /** * Initialize this WalkResult. */ void init( index_t oi, bool f, index_t r, index_t e, index_t l) { offidx = oi; fw = f; range = r; elt = e; len = l; } /** * Return true iff this GWElt and the given GWElt refer to the same * element. */ bool operator==(const GWElt& o) const { return offidx == o.offidx && fw == o.fw && range == o.range && elt == o.elt && len == o.len; } /** * Return true iff this GWElt and the given GWElt refer to * different elements. */ bool operator!=(const GWElt& o) const { return !(*this == o); } index_t offidx; // seed offset index bool fw; // strand index_t range; // range index_t elt; // element index_t len; // length }; /** * A record encapsulating the result of looking up one BW element in * the Bowtie index. */ template struct WalkResult { WalkResult() { reset(); } /** * Reset GWElt to uninitialized state. */ void reset() { elt.reset(); bwrow = toff = (index_t)OFF_MASK; } /** * Initialize this WalkResult. */ void init( index_t oi, // seed offset index bool f, // strand index_t r, // range index_t e, // element index_t bwr, // BW row index_t len, // length index_t to) // text offset { elt.init(oi, f, r, e, len); bwrow = bwr; toff = to; } GWElt elt; // element resolved index_t bwrow; // SA row resolved index_t toff; // resolved offset from SA sample }; /** * A GW hit encapsulates an SATuple describing a reference substring * in the cache, along with a bool indicating whether each element of * the hit has been reported yet. */ template class GWHit { public: GWHit() : fmap(0, GW_CAT), offidx((index_t)OFF_MASK), fw(false), range((index_t)OFF_MASK), len((index_t)OFF_MASK), reported_(0, GW_CAT), nrep_(0) { assert(repOkBasic()); } /** * Initialize with a new SA range. Resolve the done vector so that * there's one bool per suffix array element. */ void init( SARangeWithOffs& sa, index_t oi, bool f, index_t r) { nrep_ = 0; offidx = oi; fw = f; range = r; len = (index_t)sa.len; reported_.resize(sa.offs.size()); reported_.fill(false); fmap.resize(sa.offs.size()); fmap.fill(make_pair((index_t)OFF_MASK, (index_t)OFF_MASK)); } /** * Clear contents of sat and done. */ void reset() { reported_.clear(); fmap.clear(); nrep_ = 0; offidx = (index_t)OFF_MASK; fw = false; range = (index_t)OFF_MASK; len = (index_t)OFF_MASK; } #ifndef NDEBUG /** * Check that GWHit is internally consistent. If a pointer to an * EList of GWStates is given, we assume that it is the EList * corresponding to this GWHit and check whether the forward and * reverse mappings match up for the as-yet-unresolved elements. */ bool repOk(const SARangeWithOffs& sa) const { assert_eq(reported_.size(), sa.offs.size()); assert_eq(fmap.size(), sa.offs.size()); // Shouldn't be any repeats among as-yet-unresolveds size_t nrep = 0; for(size_t i = 0; i < fmap.size(); i++) { if(reported_[i]) nrep++; if(sa.offs[i] != (index_t)OFF_MASK) { continue; } for(size_t j = i+1; j < fmap.size(); j++) { if(sa.offs[j] != (index_t)OFF_MASK) { continue; } assert(fmap[i] != fmap[j]); } } assert_eq(nrep_, nrep); return true; } /** * Return true iff this GWHit is not obviously corrupt. */ bool repOkBasic() { return true; } #endif /** * Set the ith element to be reported. */ void setReported(index_t i) { assert(!reported_[i]); assert_lt(i, reported_.size()); reported_[i] = true; nrep_++; } /** * Return true iff element i has been reported. */ bool reported(index_t i) const { assert_lt(i, reported_.size()); return reported_[i]; } /** * Return true iff all elements have been reported. */ bool done() const { assert_leq(nrep_, reported_.size()); return nrep_ == reported_.size(); } EList, 16> fmap; // forward map; to GWState & elt index_t offidx; // offset idx bool fw; // orientation index_t range; // original range index index_t len; // length of hit protected: EList reported_; // per-elt bool indicating whether it's been reported index_t nrep_; }; /** * Encapsulates the progress made along a particular path from the original * range. */ template class GWState { public: GWState() : map_(0, GW_CAT) { reset(); assert(repOkBasic()); } /** * Initialize this GWState with new gfm, top, bot, step, and sat. * * We assume map is already set up. * * Returns true iff at least one elt was resolved. */ template pair init( const GFM& gfm, // index to walk left in const BitPairReference& ref, // bitpair-encoded reference SARangeWithOffs& sa, // SA range with offsets EList& sts, // EList of GWStates for range being advanced GWHit& hit, // Corresponding hit structure index_t range, // which range is this? bool reportList, // if true, "report" resolved offsets immediately by adding them to 'res' list EList, 16>* res, // EList where resolved offsets should be appended index_t tp, // top of range at this step index_t bt, // bot of range at this step index_t n_tp, // node at top index_t n_bt, // node at bot const EList >& n_iedge_count, index_t st, // # steps taken to get to this step WalkMetrics& met) { assert_gt(bt, tp); assert_gt(n_bt, n_tp); assert_geq(bt - tp, n_bt - n_tp); assert_lt(range, sts.size()); top = tp; bot = bt; node_top = n_tp; node_bot = n_bt; node_iedge_count = n_iedge_count; step = st; assert(!inited_); ASSERT_ONLY(inited_ = true); ASSERT_ONLY(lastStep_ = step-1); return init(gfm, ref, sa, sts, hit, range, reportList, res, met); } /** * Initialize this GWState. * * We assume map is already set up, and that 'step' is equal to the * number of steps taken to get to the new top/bot pair *currently* * in the top and bot fields. * * Returns a pair of numbers, the first being the number of * resolved but unreported offsets found during this advance, the * second being the number of as-yet-unresolved offsets. */ template pair init( const GFM& gfm, // forward Bowtie index const BitPairReference& ref, // bitpair-encoded reference SARangeWithOffs& sa, // SA range with offsets EList& st, // EList of GWStates for advancing range GWHit& hit, // Corresponding hit structure index_t range, // range being inited bool reportList, // report resolutions, adding to 'res' list? EList, 16>* res, // EList to append resolutions WalkMetrics& met) // update these metrics { assert(inited_); assert_eq(step, lastStep_+1); ASSERT_ONLY(lastStep_++); assert_leq((index_t)step, gfm.gh().len()); assert_lt(range, st.size()); pair ret = make_pair(0, 0); index_t trimBegin = 0, trimEnd = 0; bool empty = true; // assume all resolved until proven otherwise // Commit new information, if any, to the PListSlide. Also, // trim and check if we're done. assert_eq(node_bot - node_top, map_.size()); ASSERT_ONLY(index_t num_orig_iedges = 0, orig_e = 0); index_t num_iedges = 0, e = 0; for(size_t i = mapi_; i < map_.size(); i++) { bool resolved = (off((index_t)i, sa) != (index_t)OFF_MASK); if(!resolved) { #ifndef NDEBUG while(orig_e < sa.node_iedge_count.size()) { if(map((index_t)i) <= sa.node_iedge_count[orig_e].first) { break; } num_orig_iedges += sa.node_iedge_count[orig_e].second; orig_e++; } #endif while(e < node_iedge_count.size()) { if(i <= node_iedge_count[e].first) { break; } num_iedges += node_iedge_count[e].second; e++; } // Elt not resolved yet; try to resolve it now index_t bwrow = (index_t)(top + i + num_iedges); index_t node = (index_t)(node_top + i); index_t toff = gfm.tryOffset(bwrow, node); ASSERT_ONLY(index_t origBwRow = sa.topf + map((index_t)i) + num_orig_iedges); ASSERT_ONLY(index_t origNode = sa.node_top + map((index_t)i)); assert_eq(bwrow, gfm.walkLeft(origBwRow, step)); if(toff != (index_t)OFF_MASK) { // Yes, toff was resolvable assert_eq(toff, gfm.getOffset(bwrow, node)); met.resolves++; toff += step; assert_eq(toff, gfm.getOffset(origBwRow, origNode)); setOff((index_t)i, toff, sa, met); if(!reportList) ret.first++; #if 0 // used to be #ifndef NDEBUG, but since we no longer require that the reference // string info be included, this is no longer relevant. // Sanity check that the reference characters under this // hit match the seed characters in hit.satup->key.seq. // This is NOT a check that we associated the exact right // text offset with the BW row. This is an important // distinction because when resolved offsets are filled in // via refernce scanning, they are not necessarily the // exact right text offsets to associate with the // respective BW rows but they WILL all be correct w/r/t // the reference sequence underneath, which is what really // matters here. index_t tidx = (index_t)OFF_MASK, tof, tlen; bool straddled = false; gfm.joinedToTextOff( hit.len, // length of seed toff, // offset in joined reference string tidx, // reference sequence id tof, // offset in reference coordinates tlen, // length of reference sequence true, // don't reject straddlers straddled); if(tidx != (index_t)OFF_MASK && hit.satup->key.seq != std::numeric_limits::max()) { // key: 2-bit characters packed into a 64-bit word with // the least significant bitpair corresponding to the // rightmost character on the Watson reference strand. uint64_t key = hit.satup->key.seq; for(int64_t j = tof + hit.len-1; j >= tof; j--) { // Get next reference base to the left int c = ref.getBase(tidx, j); assert_range(0, 3, c); // Must equal least significant bitpair of key if(c != (int)(key & 3)) { // Oops; when we jump to the piece of the // reference where the seed hit is, it doesn't // match the seed hit. Before dying, check // whether we have the right spot in the joined // reference string SString jref; gfm.restore(jref); uint64_t key2 = hit.satup->key.seq; for(int64_t k = toff + hit.len-1; k >= toff; k--) { int c = jref[k]; assert_range(0, 3, c); assert_eq(c, (int)(key2 & 3)); key2 >>= 2; } assert(false); } key >>= 2; } } #endif } } // Is the element resolved? We ask this regardless of how it was // resolved (whether this function did it just now, whether it did // it a while ago, or whether some other function outside GroupWalk // did it). if(off((index_t)i, sa) != (index_t)OFF_MASK) { if(reportList && !hit.reported(map((index_t)i))) { // Report it index_t toff = off((index_t)i, sa); assert(res != NULL); res->expand(); index_t origBwRow = sa.topf + map((index_t)i); res->back().init( hit.offidx, // offset idx hit.fw, // orientation hit.range, // original range index map((index_t)i), // original element offset origBwRow, // BW row resolved hit.len, // hit length toff); // text offset hit.setReported(map((index_t)i)); met.reports++; } // Offset resolved if(empty) { // Haven't seen a non-empty entry yet, so we // can trim this from the beginning. trimBegin++; } else { trimEnd++; } } else { // Offset not yet resolved ret.second++; trimEnd = 0; empty = false; // Set the forward map in the corresponding GWHit // object to point to the appropriate element of our // range assert_geq(i, mapi_); index_t bmap = map((index_t)i); hit.fmap[bmap].first = range; hit.fmap[bmap].second = (index_t)i; #ifndef NDEBUG for(size_t j = 0; j < bmap; j++) { if(sa.offs[j] == (index_t)OFF_MASK && hit.fmap[j].first == range) { assert_neq(i, hit.fmap[j].second); } } #endif } } // Trim from beginning assert_geq(trimBegin, 0); mapi_ += trimBegin; if(trimBegin > 0) { top += trimBegin; index_t e = 0; for(; e < node_iedge_count.size(); e++) { if(node_iedge_count[e].first >= trimBegin) break; assert_geq(top, node_iedge_count[e].second); top += node_iedge_count[e].second; } if(e > 0) node_iedge_count.erase(0, e); for(e = 0; e < node_iedge_count.size(); e++) { assert_geq(node_iedge_count[e].first, trimBegin); node_iedge_count[e].first -= trimBegin; } } node_top += trimBegin; if(trimEnd > 0) { // Trim from end map_.resize(map_.size() - trimEnd); bot -= trimEnd; index_t node_range = node_bot - node_top; while(node_iedge_count.size() > 0) { if(node_iedge_count.back().first < (node_range - trimEnd)) break; assert_geq(bot, node_iedge_count.back().second); bot -= node_iedge_count.back().second; node_iedge_count.pop_back(); } } node_bot -= trimEnd; #ifndef NDEBUG assert_leq(node_top, node_bot); index_t num_nodes = node_bot - node_top; index_t add = 0; for(index_t e = 0; e < node_iedge_count.size(); e++) { assert_lt(node_iedge_count[e].first, num_nodes); add += node_iedge_count[e].second; } assert_eq(bot - top, num_nodes + add); #endif if(empty) { assert(done()); #ifndef NDEBUG // If range is done, all elements from map should be // resolved for(size_t i = mapi_; i < map_.size(); i++) { assert_neq((index_t)OFF_MASK, off((index_t)i, sa)); } // If this range is done, then it should be the case that // all elements in the corresponding GWHit that point to // this range are resolved. for(size_t i = 0; i < hit.fmap.size(); i++) { if(sa.offs[i] == (index_t)OFF_MASK) { assert_neq(range, hit.fmap[i].first); } } #endif return ret; } else { assert(!done()); } // Is there a dollar sign in the middle of the range? tmp_zOffs.clear(); for(index_t i = 0; i < gfm._zOffs.size(); i++) { #ifndef NDEBUG if(i > 0) { assert_lt(gfm._zOffs[i-1], gfm._zOffs[i]); } #endif assert_neq(top, gfm._zOffs[i]); // assert_neq(bot-1, gfm._zOffs[i]); if(gfm._zOffs[i] > top && gfm._zOffs[i] < bot) { tmp_zOffs.push_back(gfm._zOffs[i]); } } // Yes, the dollar sign is in the middle of this range. We // must split it into the two ranges on either side of the // dollar. Let 'bot' and 'top' delimit the portion of the // range prior to the dollar. if(tmp_zOffs.size() > 0) { tmp_gbwt_to_node.clear(); index_t n = 0, e = 0; for(index_t r = 0; r < (bot - top); r++) { tmp_gbwt_to_node.push_back(n); if(e < node_iedge_count.size()) { assert_leq(n, node_iedge_count[e].first); if(n == node_iedge_count[e].first) { for(index_t a = 0; a < node_iedge_count[e].second; a++) { tmp_gbwt_to_node.push_back(n); r++; } e++; } } n++; } assert_eq(bot - top, tmp_gbwt_to_node.size()); for(index_t i = 0; i < tmp_zOffs.size(); i++) { // Note: might be able to do additional trimming off the end. // Create a new range for the portion after the dollar. index_t new_top = tmp_zOffs[i] + 1; if(i + 1 < tmp_zOffs.size() && new_top == tmp_zOffs[i+1]) { continue; } assert_leq(new_top - top, tmp_gbwt_to_node.size()); if(new_top - top == tmp_gbwt_to_node.size()) { break; } index_t new_node_top = tmp_gbwt_to_node[new_top - top] + node_top; assert_lt(new_node_top, node_bot); index_t new_bot; if(i + 1 < tmp_zOffs.size()) { new_bot = tmp_zOffs[i+1]; } else { new_bot = bot; } index_t new_node_bot = node_bot; if(new_bot - top < tmp_gbwt_to_node.size()) { new_node_bot = node_top + tmp_gbwt_to_node[new_bot - top]; if(new_bot - top > 0 && tmp_gbwt_to_node[new_bot - top] == tmp_gbwt_to_node[new_bot - top - 1]) { new_node_bot++; } } tmp_node_iedge_count.clear(); if(new_top >= new_bot) continue; for(index_t j = new_top - top; j + 1 < new_bot - top;) { index_t n = tmp_gbwt_to_node[j]; index_t j2 = j + 1; while(j2 < new_bot - top) { if(n != tmp_gbwt_to_node[j2]) { break; } j2++; } if(j + 1 < j2) { tmp_node_iedge_count.expand(); assert_lt(node_top, new_node_top); tmp_node_iedge_count.back().first = n - (new_node_top - node_top); tmp_node_iedge_count.back().second = j2 - j - 1; } j = j2; } st.expand(); st.back().reset(); st.back().initMap(new_node_bot - new_node_top); for(index_t j = new_node_top; j < new_node_bot; j++) { st.back().map_[j - new_node_top] = map(j - node_top + mapi_); } st.back().init( gfm, ref, sa, st, hit, (index_t)st.size()-1, reportList, res, new_top, new_bot, new_node_top, new_node_bot, tmp_node_iedge_count, step, met); } assert_eq((index_t)map_.size(), node_bot - node_top + mapi_); bot = tmp_zOffs[0]; assert_lt(bot - top, tmp_gbwt_to_node.size()); node_bot = tmp_gbwt_to_node[bot - top - 1] + node_top + 1; map_.resize(node_bot - node_top + mapi_); index_t width = node_bot - node_top; for(index_t e = 0; e < node_iedge_count.size(); e++) { if(node_iedge_count[e].first >= node_bot - node_top) { node_iedge_count.resize(e); break; } width += node_iedge_count[e].second; } if(width != bot - top) { assert_eq(width, bot - top + 1); assert_gt(node_iedge_count.size(), 0); assert_gt(node_iedge_count.back().second, 0); node_iedge_count.back().second -= 1; if(node_iedge_count.back().second == 0) { node_iedge_count.resize(node_iedge_count.size()- 1); } } } assert_gt(bot, top); // Prepare SideLocus's for next step if(bot-top > 1) { SideLocus::initFromTopBot(top, bot, gfm.gh(), gfm.gfm(), tloc, bloc); assert(tloc.valid()); assert(tloc.repOk(gfm.gh())); assert(bloc.valid()); assert(bloc.repOk(gfm.gh())); } else { tloc.initFromRow(top, gfm.gh(), gfm.gfm()); assert(tloc.valid()); assert(tloc.repOk(gfm.gh())); bloc.invalidate(); } return ret; } #ifndef NDEBUG /** * Check if this GWP is internally consistent. */ bool repOk( const GFM& gfm, GWHit& hit, index_t range) const { assert(done() || bot > top); assert(doneResolving(hit) || (tloc.valid() && tloc.repOk(gfm.gh()))); assert(doneResolving(hit) || bot == top+1 || (bloc.valid() && bloc.repOk(gfm.gh()))); assert_eq(map_.size()-mapi_, bot-top); // Make sure that 'done' is compatible with whether we have >= // 1 elements left to resolve. int left = 0; for(size_t i = mapi_; i < map_.size(); i++) { ASSERT_ONLY(index_t row = (index_t)(top + i - mapi_)); ASSERT_ONLY(index_t origRow = hit.satup->topf + map(i)); assert(step == 0 || row != origRow); assert_eq(row, gfm.walkLeft(origRow, step)); assert_lt(map_[i], hit.satup->offs.size()); if(off(i, hit) == (index_t)OFF_MASK) left++; } assert(repOkMapRepeats()); assert(repOkMapInclusive(hit, range)); return true; } /** * Return true iff this GWState is not obviously corrupt. */ bool repOkBasic() { assert_geq(bot, top); return true; } /** * Check that the fmap elements pointed to by our map_ include all * of the fmap elements that point to this range. */ bool repOkMapInclusive(GWHit& hit, index_t range) const { for(size_t i = 0; i < hit.fmap.size(); i++) { if(hit.satup->offs[i] == (index_t)OFF_MASK) { if(range == hit.fmap[i].first) { ASSERT_ONLY(bool found = false); for(size_t j = mapi_; j < map_.size(); j++) { if(map(j) == i) { ASSERT_ONLY(found = true); break; } } assert(found); } } } return true; } /** * Check that no two elements in map_ are the same. */ bool repOkMapRepeats() const { for(size_t i = mapi_; i < map_.size(); i++) { for(size_t j = i+1; j < map_.size(); j++) { assert_neq(map_[i], map_[j]); } } return true; } #endif /** * Return the offset currently assigned to the ith element. If it * has not yet been resolved, return 0xffffffff. */ index_t off( index_t i, const SARangeWithOffs& sa) { assert_geq(i, mapi_); assert_lt(i, map_.size()); assert_lt(map_[i], sa.offs.size()); return sa.offs.get(map_[i]); } /** * Return the offset of the element within the original range's * PListSlice that the ith element of this range corresponds to. */ index_t map(index_t i) const { assert_geq(i, mapi_); assert_lt(i, map_.size()); return map_[i]; } /** * Return the offset of the first untrimmed offset in the map. */ index_t mapi() const { return mapi_; } /** * Return number of active elements in the range being tracked by * this GWState. */ index_t size() const { return (index_t)(map_.size() - mapi_); } /** * Return true iff all elements in this leaf range have been * resolved. */ bool done() const { return size() == 0; } /** * Set the PListSlice element that corresponds to the ith element * of 'map' to the specified offset. */ void setOff( index_t i, index_t off, SARangeWithOffs& sa, WalkMetrics& met) { assert_lt(i + mapi_, map_.size()); assert_lt(map_[i + mapi_], sa.offs.size()); size_t saoff = map_[i + mapi_]; sa.offs[saoff] = off; assert_eq(off, sa.offs[saoff]); } /** * Advance this GWState by one step (i.e. one BW operation). In * the event of a "split", more elements are added to the EList * 'st', which must have room for at least 3 more elements without * needing another expansion. If an expansion of 'st' is * triggered, this GWState object becomes invalid. * * Returns a pair of numbers, the first being the number of * resolved but unreported offsets found during this advance, the * second being the number of as-yet-unresolved offsets. */ template pair advance( const GFM& gfm, // the forward Bowtie index, for stepping left const BitPairReference& ref, // bitpair-encoded reference SARangeWithOffs& sa, // SA range with offsets GWHit& hit, // the associated GWHit object index_t range, // which range is this? bool reportList, // if true, "report" resolved offsets immediately by adding them to 'res' list EList, 16>* res, // EList where resolved offsets should be appended EList& st, // EList of GWStates for range being advanced GroupWalkState& gws, // temporary storage for masks WalkMetrics& met, PerReadMetrics& prm) { ASSERT_ONLY(index_t origTop = top); ASSERT_ONLY(index_t origBot = bot); assert_geq(step, 0); assert_eq(step, lastStep_); // assert_geq(st.capacity(), st.size() + 4); assert(tloc.valid()); assert(tloc.repOk(gfm.gh())); assert_eq(node_bot-node_top, (index_t)(map_.size()-mapi_)); pair ret = make_pair(0, 0); assert_eq(top, tloc.toBWRow(gfm.gh())); if(bot - top > 1) { bool first = true; ASSERT_ONLY(index_t sum = 0); index_t newtop = 0, newbot = 0; index_t new_node_top = 0, new_node_bot = 0; gws.map.clear(); // Still multiple elements being tracked index_t curtop = top, curbot = bot; index_t cur_node_top = node_top, cur_node_bot = node_bot; for(index_t e = 0; e < node_iedge_count.size() + 1; e++) { if(e >= node_iedge_count.size()) { if(e > 0) { curtop = curbot + node_iedge_count[e-1].second; curbot = bot; if(curtop >= curbot) { assert_eq(curtop, curbot); break; } cur_node_top = cur_node_bot; cur_node_bot = node_bot; } } else { if(e > 0) { curtop = curbot + node_iedge_count[e-1].second; assert_lt(node_iedge_count[e-1].first, node_iedge_count[e].first); curbot = curtop + (node_iedge_count[e].first - node_iedge_count[e-1].first); cur_node_top = cur_node_bot; } else { curbot = curtop + node_iedge_count[e].first + 1; } cur_node_bot = node_top + node_iedge_count[e].first + 1; } assert_lt(curtop, curbot); index_t upto[4], in[4]; upto[0] = in[0] = upto[1] = in[1] = upto[2] = in[2] = upto[3] = in[3] = 0; // assert_eq(bot, bloc.toBWRow(gfm.gh())); met.bwops++; prm.nExFmops++; // Assert that there's not a dollar sign in the middle of // this range #ifndef NDEBUG for(index_t i = 0; i < gfm._zOffs.size(); i++) { assert(curbot <= gfm._zOffs[i] || curtop > gfm._zOffs[i]); } #endif SideLocus curtloc, curbloc; SideLocus::initFromTopBot(curtop, curbot, gfm.gh(), gfm.gfm(), curtloc, curbloc); gfm.mapLFRange(curtloc, curbloc, curbot-curtop, upto, in, gws.masks); #ifndef NDEBUG for(int i = 0; i < 4; i++) { assert_eq(curbot-curtop, (index_t)(gws.masks[i].size())); } #endif for(int i = 0; i < 4; i++) { if(in[i] > 0) { // Non-empty range resulted if(first) { // For the first one, first = false; pair range, node_range; backup_node_iedge_count.clear(); SideLocus::initFromTopBot(curtop, curbot, gfm.gh(), gfm.gfm(), curtloc, curbloc); range = gfm.mapGLF(curtloc, curbloc, i, &node_range, &backup_node_iedge_count, cur_node_bot - cur_node_top); newtop = range.first; newbot = range.second; new_node_top = node_range.first; new_node_bot = node_range.second; // Range narrowed so we have to look at the masks for(size_t j = 0; j < gws.masks[i].size(); j++) { assert_lt(j+mapi_+(cur_node_top - node_top), map_.size()); if(gws.masks[i][j]) { gws.map.push_back(map_[j+mapi_+(cur_node_top - node_top)]); assert(gws.map.size() <= 1 || gws.map.back() != gws.map[gws.map.size()-2]); #if 0 // If this element is not yet resolved, // then check that it really is the // expected number of steps to the left // of the corresponding element in the // root range assert_lt(gws.map.back(), sa.size()); if(sa.offs[gws.map.back()] == (index_t)OFF_MASK) { assert_eq(newtop + gws.map.size() - 1, gfm.walkLeft(sa.topf + gws.map.back(), step+1)); } #endif } } assert_lt(new_node_top, new_node_bot); if(new_node_bot - new_node_top < gws.map.size()) { assert_eq(curbot - curtop, cur_node_bot - cur_node_top); SideLocus tmptloc, tmpbloc; pair tmp_node_range; index_t j1 = 0, j2 = 0; for(index_t c = 0; c < gws.masks[i].size(); c++) { if(gws.masks[i][c]) { j1 = c; break; } } for(index_t j = 0; j + 1 < gws.map.size(); j++) { for(index_t c = j1 + 1; c < gws.masks[i].size(); c++) { if(gws.masks[i][c]) { j2 = c; break; } } assert_lt(j1, j2); SideLocus::initFromTopBot(curtop + j1, curtop + j2 + 1, gfm.gh(), gfm.gfm(), tmptloc, tmpbloc); gfm.mapGLF(tmptloc, tmpbloc, i, &tmp_node_range); assert_gt(tmp_node_range.second - tmp_node_range.first, 0); if(tmp_node_range.second - tmp_node_range.first == 1) { index_t jmap = gws.map[j]; assert_lt(jmap, sa.offs.size()); sa.offs[jmap] = gws.map[j]; gws.map[j] = (index_t)OFF_MASK; } j1 = j2; j2 = 0; } for(index_t j = 0; j < gws.map.size();) { if(gws.map[j] == (index_t)OFF_MASK) { gws.map.erase(j); } else j++; } #ifndef NDEBUG for(index_t j = 0; j < gws.map.size(); j++) { assert_neq(gws.map[j], (index_t)OFF_MASK); } #endif } assert_eq(new_node_bot - new_node_top, (index_t)(gws.map.size())); } else { // For each beyond the first, create a new // GWState and add it to the GWState list. // NOTE: this can cause the underlying list to // be expanded which in turn might leave 'st' // pointing to bad memory. st.expand(); st.back().reset(); tmp_node_iedge_count.clear(); pair range, node_range; SideLocus::initFromTopBot(curtop, curbot, gfm.gh(), gfm.gfm(), curtloc, curbloc); range = gfm.mapGLF(curtloc, curbloc, i, &node_range, &tmp_node_iedge_count, cur_node_bot - cur_node_top); assert_geq(range.second - range.first, node_range.second - node_range.first); index_t ntop = range.first; index_t nbot = range.second; st.back().mapi_ = 0; st.back().map_.clear(); met.branches++; // Range narrowed so we have to look at the masks for(size_t j = 0; j < gws.masks[i].size(); j++) { if(gws.masks[i][j]) st.back().map_.push_back(map_[j+mapi_+(cur_node_top - node_top)]); } assert_lt(node_range.first, node_range.second); if(node_range.second - node_range.first < st.back().map_.size()) { assert_eq(curbot - curtop, cur_node_bot - cur_node_top); SideLocus tmptloc, tmpbloc; pair tmp_node_range; index_t j1 = 0, j2 = 0; for(index_t c = 0; c < gws.masks[i].size(); c++) { if(gws.masks[i][c]) { j1 = c; break; } } for(index_t j = 0; j + 1 < st.back().map_.size(); j++) { for(index_t c = j1 + 1; c < gws.masks[i].size(); c++) { if(gws.masks[i][c]) { j2 = c; break; } } assert_lt(j1, j2); SideLocus::initFromTopBot(curtop + j1, curtop + j2 + 1, gfm.gh(), gfm.gfm(), tmptloc, tmpbloc); gfm.mapGLF(tmptloc, tmpbloc, i, &tmp_node_range); assert_gt(tmp_node_range.second - tmp_node_range.first, 0); if(tmp_node_range.second - tmp_node_range.first == 1) { index_t jmap = st.back().map_[j]; assert_lt(jmap, sa.offs.size()); sa.offs[jmap] = st.back().map_[j]; st.back().map_[j] = (index_t)OFF_MASK; } j1 = j2; j2 = 0; } for(index_t j = 0; j < st.back().map_.size();) { if(st.back().map_[j] == (index_t)OFF_MASK) { st.back().map_.erase(j); } else j++; } #ifndef NDEBUG for(index_t j = 0; j < st.back().map_.size(); j++) { assert_neq(st.back().map_[j], (index_t)OFF_MASK); } #endif } assert_eq(node_range.second - node_range.first, st.back().map_.size()); pair rret = st.back().init( gfm, // forward Bowtie index ref, // bitpair-encodede reference sa, // SA range with offsets st, // EList of all GWStates associated with original range hit, // associated GWHit object (index_t)st.size()-1, // range offset reportList, // if true, report hits to 'res' list res, // report hits here if reportList is true ntop, // BW top of new range nbot, // BW bot of new range node_range.first, node_range.second, tmp_node_iedge_count, step+1, // # steps taken to get to this new range met); // update these metrics ret.first += rret.first; ret.second += rret.second; } ASSERT_ONLY(sum += in[i]); } } } mapi_ = 0; // assert_eq(new_node_bot-new_node_top, sum); assert_gt(newbot, newtop); assert(top != newtop || bot != newbot); //assert(!(newtop < top && newbot > top)); top = newtop; bot = newbot; node_top = new_node_top; node_bot = new_node_bot; node_iedge_count = backup_node_iedge_count; backup_node_iedge_count.clear(); if(!gws.map.empty()) { map_ = gws.map; } //assert(repOkMapRepeats()); //assert(repOkMapInclusive(hit, range)); assert_eq(node_bot-node_top, (index_t)map_.size()); } else { // Down to one element assert_eq(bot, top+1); assert_eq(1, map_.size()-mapi_); // Sets top, returns char walked through (which we ignore) ASSERT_ONLY(index_t oldtop = top); met.bwops++; prm.nExFmops++; pair node_range(0, 0); pair range = gfm.mapGLF1(top, tloc, &node_range); top = range.first; assert_neq(top, oldtop); bot = top+1; node_top = node_range.first; node_bot = node_range.second; if(mapi_ > 0) { map_[0] = map_[mapi_]; mapi_ = 0; } map_.resize(1); } assert(top != origTop || bot != origBot); step++; assert_gt(step, 0); assert_leq((index_t)step, gfm.gh().len()); pair rret = init( gfm, // forward GFM index ref, // bitpair-encodede reference sa, // SA range with offsets st, // EList of all GWStates associated with original range hit, // associated GWHit object range, // range offset reportList, // if true, report hits to 'res' list res, // report hits here if reportList is true met); // update these metrics ret.first += rret.first; ret.second += rret.second; return ret; } /** * Clear all state in preparation for the next walk. */ void reset() { top = bot = node_top = node_bot = step = mapi_ = 0; ASSERT_ONLY(lastStep_ = -1); ASSERT_ONLY(inited_ = false); tloc.invalidate(); bloc.invalidate(); map_.clear(); node_iedge_count.clear(); backup_node_iedge_count.clear(); tmp_node_iedge_count.clear(); } /** * Resize the map_ field to the given size. */ void initMap(size_t newsz) { mapi_ = 0; map_.resize(newsz); for(size_t i = 0; i < newsz; i++) { map_[i] = (index_t)i; } } /** * Return true iff all rows corresponding to this GWState have been * resolved and reported. */ bool doneReporting(const GWHit& hit) const { for(size_t i = mapi_; i < map_.size(); i++) { if(!hit.reported(map(i))) return false; } return true; } /** * Return true iff all rows corresponding to this GWState have been * resolved (but not necessarily reported). */ bool doneResolving(const SARangeWithOffs& sa) const { for(size_t i = mapi_; i < map_.size(); i++) { if(sa.offs[map((index_t)i)] == (index_t)OFF_MASK) return false; } return true; } SideLocus tloc; // SideLocus for top SideLocus bloc; // SideLocus for bottom index_t top; // top elt of range in GBWT index_t bot; // bot elt of range in GBWT index_t node_top; index_t node_bot; EList > node_iedge_count; int step; // how many steps have we walked to the left so far // temporary EList > backup_node_iedge_count; EList > tmp_node_iedge_count; EList tmp_zOffs; EList tmp_gbwt_to_node; protected: ASSERT_ONLY(bool inited_); ASSERT_ONLY(int lastStep_); EList map_; // which elts in range 'range' we're tracking index_t mapi_; // first untrimmed element of map }; template class GroupWalk2S { public: typedef EList, S> TStateV; GroupWalk2S() : st_(8, GW_CAT) { reset(); } /** * Reset the GroupWalk in preparation for the next SeedResults. */ void reset() { elt_ = rep_ = 0; ASSERT_ONLY(inited_ = false); } /** * Initialize a new group walk w/r/t a QVal object. */ void init( const GFM& gfmFw, // forward Bowtie index for walking left const BitPairReference& ref, // bitpair-encoded reference SARangeWithOffs& sa, // SA range with offsets RandomSource& rnd, // pseudo-random generator for sampling rows WalkMetrics& met) // update metrics here { reset(); #ifndef NDEBUG inited_ = true; #endif // Init GWHit hit_.init(sa, 0, false, 0); // Init corresponding GWState st_.resize(1); st_.back().reset(); assert(st_.back().repOkBasic()); index_t top = sa.topf; index_t bot = sa.botf; index_t node_top = sa.node_top; index_t node_bot = (index_t)(node_top + sa.size()); st_.back().initMap(sa.size()); st_.ensure(4); st_.back().init( gfmFw, // Bowtie index ref, // bitpair-encoded reference sa, // SA range with offsets st_, // EList hit_, // GWHit 0, // range 0 false, // put resolved elements into res_? NULL, // put resolved elements here top, // GBW row at top bot, // GBW row at bot node_top, // node at top node_bot, // node at bot sa.node_iedge_count, 0, // # steps taken met); // update metrics here elt_ += sa.size(); assert(hit_.repOk(sa)); } // // ELEMENT-BASED // /** * Advance the GroupWalk until all elements have been resolved. */ void resolveAll(WalkMetrics& met, PerReadMetrics& prm) { WalkResult res; // ignore results for now for(size_t i = 0; i < elt_; i++) { advanceElement((index_t)i, res, met, prm); } } /** * Advance the GroupWalk until the specified element has been * resolved. */ bool advanceElement( index_t elt, // element within the range const GFM& gfmFw, // forward Bowtie index for walking left const BitPairReference& ref, // bitpair-encoded reference SARangeWithOffs& sa, // SA range with offsets GroupWalkState& gws, // GroupWalk state; scratch space WalkResult& res, // put the result here WalkMetrics& met, // metrics PerReadMetrics& prm) // per-read metrics { assert(inited_); assert(!done()); assert(hit_.repOk(sa)); assert_lt(elt, sa.size()); // elt must fall within range // Until we've resolved our element of interest... while(sa.offs[elt] == (index_t)OFF_MASK) { // Get the GWState that contains our element of interest size_t range = hit_.fmap[elt].first; assert_lt(range, st_.size()); st_.ensure(st_[range].node_bot - st_[range].node_top); // st_.ensure(4); GWState& st = st_[range]; assert(!st.doneResolving(sa)); // Returns a pair of numbers, the first being the number of // resolved but unreported offsets found during this advance, the // second being the number of as-yet-unresolved offsets. st.advance( gfmFw, ref, sa, hit_, (index_t)range, false, NULL, st_, gws, met, prm); assert(sa.offs[elt] != (index_t)OFF_MASK || !st_[hit_.fmap[elt].first].doneResolving(sa)); } assert_neq((index_t)OFF_MASK, sa.offs[elt]); // Report it! if(!hit_.reported(elt)) { hit_.setReported(elt); } met.reports++; res.init( 0, // seed offset false, // orientation 0, // range elt, // element sa.topf + elt, // bw row (index_t)sa.len, // length of hit sa.offs[elt]); // resolved text offset rep_++; return true; } /** * Return true iff all elements have been resolved and reported. */ bool done() const { return rep_ == elt_; } #ifndef NDEBUG /** * Check that GroupWalk is internally consistent. */ bool repOk(const SARangeWithOffs& sa) const { assert(hit_.repOk(sa)); assert_leq(rep_, elt_); // This is a lot of work size_t resolved = 0, reported = 0; // For each element const size_t sz = sa.size(); for(size_t m = 0; m < sz; m++) { // Is it resolved? if(sa.offs[m] != (index_t)OFF_MASK) { resolved++; } else { assert(!hit_.reported(m)); } // Is it reported? if(hit_.reported(m)) { reported++; } assert_geq(resolved, reported); } assert_geq(resolved, reported); assert_eq(rep_, reported); assert_eq(elt_, sz); return true; } #endif /** * Return the number of BW elements that we can resolve. */ index_t numElts() const { return elt_; } /** * Return the size occupied by this GroupWalk and all its constituent * objects. */ size_t totalSizeBytes() const { return 2 * sizeof(size_t) + st_.totalSizeBytes() + sizeof(GWHit); } /** * Return the capacity of this GroupWalk and all its constituent objects. */ size_t totalCapacityBytes() const { return 2 * sizeof(size_t) + st_.totalCapacityBytes() + sizeof(GWHit); } #ifndef NDEBUG bool initialized() const { return inited_; } #endif protected: ASSERT_ONLY(bool inited_); // initialized? index_t elt_; // # BW elements under the control of the GropuWalk index_t rep_; // # BW elements reported // For each orientation and seed offset, keep a GWState object that // holds the state of the walk so far. TStateV st_; // For each orientation and seed offset, keep an EList of GWHit. GWHit hit_; }; #endif /*GROUP_WALK_H_*/ hisat2-2.2.1/hgfm.h000066400000000000000000003142401370663763200140210ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef HGFM_H_ #define HGFM_H_ #include "hier_idx_common.h" #include "gfm.h" /** * Extended Burrows-Wheeler transform data. * LocalEbwt is a specialized Ebwt index that represents ~64K bps * and therefore uses two bytes as offsets within 64K bps. * This class has only two additional member variables to denote the genomic sequenuce it represents: * (1) the contig index and (2) the offset within the contig. * */ template class LocalGFM : public GFM { typedef GFM PARENT_CLASS; public: /// Construct an Ebwt from the given input file LocalGFM(const string& in, ALTDB* altdb, FILE *in5, FILE *in6, char *mmFile5, char *mmFile6, full_index_t& tidx, full_index_t& localOffset, full_index_t& joinedOffset, bool switchEndian, size_t& bytesRead, size_t& bytesRead2, int needEntireReverse, bool fw, int32_t overrideOffRate, // = -1, int32_t offRatePlus, // = -1, uint32_t lineRate, uint32_t offRate, uint32_t ftabChars, bool useMm, // = false, bool useShmem, // = false, bool mmSweep, // = false, bool loadNames, // = false, bool loadSASamp, // = true, bool loadFtab, // = true, bool loadRstarts, // = true, bool verbose, // = false, bool startVerbose, // = false, bool passMemExc, // = false, bool sanityCheck, // = false) bool useHaplotype) : // = false GFM(in, altdb, NULL, NULL, needEntireReverse, fw, overrideOffRate, offRatePlus, useMm, useShmem, mmSweep, loadNames, loadSASamp, loadFtab, loadRstarts, true, // load Splice Sites verbose, startVerbose, passMemExc, sanityCheck, useHaplotype, true) { this->_in1Str = in + ".5." + gfm_ext; this->_in2Str = in + ".5." + gfm_ext; readIntoMemory( in5, in6, mmFile5, mmFile6, tidx, localOffset, joinedOffset, switchEndian, bytesRead, bytesRead2, needEntireReverse, loadSASamp, loadFtab, loadRstarts, false, //justHeader lineRate, offRate, ftabChars, mmSweep, loadNames, startVerbose); _tidx = tidx; _localOffset = localOffset; _joinedOffset = joinedOffset; // If the offRate has been overridden, reflect that in the // _eh._offRate field if(offRatePlus > 0 && this->_overrideOffRate == -1) { this->_overrideOffRate = this->_gh._offRate + offRatePlus; } if(this->_overrideOffRate > this->_gh._offRate) { this->_gh.setOffRate(this->_overrideOffRate); assert_eq(this->_overrideOffRate, this->_gh._offRate); } assert(this->repOk()); } /// Construct an Ebwt from the given header parameters and string /// vector, optionally using a blockwise suffix sorter with the /// given 'bmax' and 'dcv' parameters. The string vector is /// ultimately joined and the joined string is passed to buildToDisk(). template LocalGFM( TStr& s, const EList& sa, PathGraph* pg, full_index_t tidx, full_index_t localOffset, full_index_t joinedOffset, EList >& alts, index_t local_size, bool packed, int needEntireReverse, int32_t lineRate, int32_t offRate, int32_t ftabChars, const string& file, // base filename for EBWT files bool fw, int dcv, EList& szs, index_t sztot, const RefReadInParams& refparams, uint32_t seed, ostream& out5, ostream& out6, int32_t overrideOffRate = -1, bool verbose = false, bool passMemExc = false, bool sanityCheck = false) : GFM(packed, needEntireReverse, lineRate, offRate, ftabChars, file, fw, dcv, szs, sztot, refparams, seed, overrideOffRate, verbose, passMemExc, sanityCheck) { const GFMParams& gh = this->_gh; assert(gh.repOk()); uint32_t be = this->toBe(); assert(out5.good()); assert(out6.good()); _tidx = tidx; _localOffset = localOffset; _joinedOffset = joinedOffset; writeIndex(out5, tidx, be); writeIndex(out5, localOffset, be); writeIndex(out5, joinedOffset, be); writeIndex(out5, gh._len, be); // length of string (and bwt and suffix array) streampos headerPos = out5.tellp(); writeIndex(out5, 0, be); // gbwtLen writeIndex(out5, 0, be); // num of nodes writeIndex(out5, 0, be); // eftabLen if(gh._len > 0) { assert_gt(szs.size(), 0); assert_gt(sztot, 0); // Not every fragment represents a distinct sequence - many // fragments may correspond to a single sequence. Count the // number of sequences here by counting the number of "first" // fragments. this->_nPat = 0; this->_nFrag = 0; for(size_t i = 0; i < szs.size(); i++) { if(szs[i].len > 0) this->_nFrag++; if(szs[i].first && szs[i].len > 0) this->_nPat++; } assert_eq(this->_nPat, 1); assert_geq(this->_nFrag, this->_nPat); this->_rstarts.reset(); writeIndex(out5, this->_nPat, be); assert_eq(this->_nPat, 1); this->_plen.init(new index_t[this->_nPat], this->_nPat); // For each pattern, set plen int npat = -1; for(size_t i = 0; i < szs.size(); i++) { if(szs[i].first && szs[i].len > 0) { if(npat >= 0) { writeIndex(out5, this->plen()[npat], be); } npat++; this->plen()[npat] = (szs[i].len + szs[i].off); } else { this->plen()[npat] += (szs[i].len + szs[i].off); } } assert_eq((index_t)npat, this->_nPat-1); writeIndex(out5, this->plen()[npat], be); // Write the number of fragments writeIndex(out5, this->_nFrag, be); if(refparams.reverse == REF_READ_REVERSE) { EList tmp(EBWT_CAT); reverseRefRecords(szs, tmp, false, verbose); this->szsToDisk(tmp, out5, refparams.reverse); } else { this->szsToDisk(szs, out5, refparams.reverse); } if(alts.empty()) { assert(pg == NULL); buildToDisk(sa, s, out5, out6, headerPos); } else { assert(pg != NULL); // Re-initialize GFM parameters to reflect real number of edges (gbwt string) this->_gh.init( this->_gh.len(), pg->getNumEdges(), pg->getNumNodes(), this->_gh.lineRate(), this->_gh.offRate(), this->_gh.ftabChars(), 0, this->_gh.entireReverse()); buildToDisk(*pg, s, out5, out6, headerPos); } } out5.flush(); out6.flush(); if(out5.fail() || out6.fail()) { cerr << "An error occurred writing the index to disk. Please check if the disk is full." << endl; throw 1; } } template void buildToDisk( PathGraph& gbwt, const TStr& s, ostream& out1, ostream& out2, streampos headerPos); template void buildToDisk( const EList& sa, const TStr& s, ostream& out1, ostream& out2, streampos headerPos); // I/O void readIntoMemory( FILE *in5, FILE *in6, char *mmFile5, char *mmFile6, full_index_t& tidx, full_index_t& localOffset, full_index_t& joinedOffset, bool switchEndian, size_t& bytesRead, size_t& bytesRead2, int needEntireRev, bool loadSASamp, bool loadFtab, bool loadRstarts, bool justHeader, int32_t lineRate, int32_t offRate, int32_t ftabChars, bool mmSweep, bool loadNames, bool startVerbose); /** * Sanity-check various pieces of the Ebwt */ void sanityCheckAll(int reverse) const { if(this->_gh._len > 0) { PARENT_CLASS::sanityCheckAll(reverse); } } bool empty() const { return this->_gh._len == 0; } public: full_index_t _tidx; full_index_t _localOffset; full_index_t _joinedOffset; }; /** * Build an Ebwt from a string 's' and its suffix array 'sa' (which * might actually be a suffix array *builder* that builds blocks of the * array on demand). The bulk of the Ebwt, i.e. the ebwt and offs * arrays, is written directly to disk. This is by design: keeping * those arrays in memory needlessly increases the footprint of the * building process. Instead, we prefer to build the Ebwt directly * "to disk" and then read it back into memory later as necessary. * * It is assumed that the header values and join-related values (nPat, * plen) have already been written to 'out1' before this function * is called. When this function is finished, it will have * additionally written ebwt, zOff, fchr, ftab and eftab to the primary * file and offs to the secondary file. * * Assume DNA/RNA/any alphabet with 4 or fewer elements. * Assume occ array entries are 32 bits each. * * @param sa the suffix array to convert to a Ebwt * @param s the original string * @param out */ template template void LocalGFM::buildToDisk( PathGraph& gbwt, const TStr& s, ostream& out5, ostream& out6, streampos headerPos) { assert_leq(s.length(), std::numeric_limits::max()); const GFMParams& gh = this->_gh; assert(gh.repOk()); assert_lt(s.length(), gh.gbwtLen()); assert_eq(s.length(), gh._len); assert_gt(gh._lineRate, 3); index_t gbwtLen = gh._gbwtLen; streampos out5pos = out5.tellp(); out5.seekp(headerPos); writeIndex(out5, gbwtLen, this->toBe()); writeIndex(out5, gh._numNodes, this->toBe()); headerPos = out5.tellp(); out5.seekp(out5pos); index_t ftabLen = gh._ftabLen; index_t sideSz = gh._sideSz; index_t gbwtTotSz = gh._gbwtTotSz; index_t fchr[] = {0, 0, 0, 0, 0}; EList ftab(EBWT_CAT); EList zOffs; // Save # of occurrences of each character as we walk along the bwt index_t occ[4] = {0, 0, 0, 0}; index_t occSave[4] = {0, 0, 0, 0}; // # of occurrences of 1 in M arrays index_t M_occ = 0, M_occSave = 0; // Location in F that corresponds to 1 in M index_t F_loc = 0, F_locSave = 0; try { VMSG_NL("Allocating ftab, absorbFtab"); ftab.resize(ftabLen); ftab.fillZero(); } catch(bad_alloc &e) { cerr << "Out of memory allocating ftab[] or absorbFtab[] " << "in LocalGFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } // Allocate the side buffer; holds a single side as its being // constructed and then written to disk. Reused across all sides. #ifdef SIXTY4_FORMAT EList gfmSide(EBWT_CAT); #else EList gfmSide(EBWT_CAT); #endif try { // Used to calculate ftab and eftab, but having gfm costs a lot of memory this->_gfm.init(new uint8_t[gh._gbwtTotLen], gh._gbwtTotLen, true); #ifdef SIXTY4_FORMAT gfmSide.resize(sideSz >> 3); #else gfmSide.resize(sideSz); #endif } catch(bad_alloc &e) { cerr << "Out of memory allocating ebwtSide[] in " << "LocalGFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } // Points to the base offset within ebwt for the side currently // being written index_t side = 0; // Whether we're assembling a forward or a reverse bucket bool fw = true; int sideCur = 0; index_t si = 0; // string offset (chars) ASSERT_ONLY(bool inSA = true); // true iff saI still points inside suffix // array (as opposed to the padding at the // end) // Iterate over packed bwt bytes VMSG_NL("Entering LocalGFM loop"); ASSERT_ONLY(uint32_t beforeGbwtOff = (uint32_t)out5.tellp()); while(side < gbwtTotSz) { // Sanity-check our cursor into the side buffer assert_geq(sideCur, 0); assert_lt(sideCur, (int)gh._sideGbwtSz); assert_eq(0, side % sideSz); // 'side' must be on side boundary gfmSide[sideCur] = 0; // clear if(sideCur == 0) { memset(gfmSide.ptr(), 0, gh._sideGbwtSz); gfmSide[sideCur] = 0; // clear } assert_lt(side + sideCur, gbwtTotSz); // Iterate over bit-pairs in the si'th character of the BWT #ifdef SIXTY4_FORMAT for(int bpi = 0; bpi < 32; bpi++, si++) { #else for(int bpi = 0; bpi < 4; bpi++, si++) { #endif int gbwtChar = 0; int F = 0, M = 0; full_index_t pos = 0; bool count = true; if(si < gbwtLen) { gbwt.nextRow(gbwtChar, F, M, pos); // (that might have triggered sa to calc next suf block) if(gbwtChar == 'Z') { // Don't add the '$' in the last column to the BWT // transform; we can't encode a $ (only A C T or G) // and counting it as, say, an A, will mess up the // LR mapping gbwtChar = 0; count = false; #ifndef NDEBUG if(zOffs.size() > 0) { assert_gt(si, zOffs.back()); } #endif zOffs.push_back(si); // remember GBWT row that corresponds to the 0th suffix } else { gbwtChar = asc2dna[gbwtChar]; assert_lt(gbwtChar, 4); // Update the fchr fchr[gbwtChar]++; } assert_lt(F, 2); assert_lt(M, 2); if(M == 1) { assert_neq(F_loc, numeric_limits::max()); F_loc = gbwt.nextFLocation(); #ifndef NDEBUG if(F_loc > 0) { assert_gt(F_loc, F_locSave); } #endif } // Suffix array offset boundary? - update offset array if(M == 1 && (M_occ & gh._offMask) == M_occ) { assert_lt((M_occ >> gh._offRate), gh._offsLen); // Write offsets directly to the secondary output // stream, thereby avoiding keeping them in memory writeIndex(out6, pos, this->toBe()); } } else { // Strayed off the end of the SA, now we're just // padding out a bucket #ifndef NDEBUG if(inSA) { // Assert that we wrote all the characters in the // string before now assert_eq(si, gbwtLen); inSA = false; } #endif // 'A' used for padding; important that padding be // counted in the occ[] array gbwtChar = 0; F = M = 0; } if(count) occ[gbwtChar]++; if(M) M_occ++; // Append BWT char to bwt section of current side if(fw) { // Forward bucket: fill from least to most #ifdef SIXTY4_FORMAT gfmSide[sideCur] |= ((uint64_t)gbwtChar << (bpi << 1)); if(gbwtChar > 0) assert_gt(gfmSide[sideCur], 0); assert(false); cerr << "Not implemented" << endl; exit(1); #else pack_2b_in_8b(gbwtChar, gfmSide[sideCur], bpi); assert_eq((gfmSide[sideCur] >> (bpi*2)) & 3, gbwtChar); int F_sideCur = (gh._sideGbwtSz + sideCur) >> 1; int F_bpi = bpi + ((sideCur & 0x1) << 2); // Can be used as M_bpi as well pack_1b_in_8b(F, gfmSide[F_sideCur], F_bpi); assert_eq((gfmSide[F_sideCur] >> F_bpi) & 1, F); int M_sideCur = F_sideCur + (gh._sideGbwtSz >> 2); pack_1b_in_8b(M, gfmSide[M_sideCur], F_bpi); assert_eq((gfmSide[M_sideCur] >> F_bpi) & 1, M); #endif } else { // Backward bucket: fill from most to least #ifdef SIXTY4_FORMAT gfmSide[sideCur] |= ((uint64_t)gbwtChar << ((31 - bpi) << 1)); if(gbwtChar > 0) assert_gt(gfmSide[sideCur], 0); // To be implemented ... assert(false); cerr << "Not implemented" << endl; exit(1); #else pack_2b_in_8b(gbwtChar, gfmSide[sideCur], 3-bpi); assert_eq((gfmSide[sideCur] >> ((3-bpi)*2)) & 3, gbwtChar); // To be implemented ... assert(false); cerr << "Not implemented" << endl; exit(1); #endif } } // end loop over bit-pairs assert_eq(0, (occ[0] + occ[1] + occ[2] + occ[3] + zOffs.size()) & 3); #ifdef SIXTY4_FORMAT assert_eq(0, si & 31); #else assert_eq(0, si & 3); #endif sideCur++; if((sideCur << 1) == (int)gh._sideGbwtSz) { sideCur = 0; index_t *uside = reinterpret_cast(gfmSide.ptr()); // Write 'A', 'C', 'G' and 'T' tallies side += sideSz; assert_leq(side, gh._gbwtTotSz); uside[(sideSz / sizeof(index_t))-6] = endianizeIndex(F_locSave, this->toBe()); uside[(sideSz / sizeof(index_t))-5] = endianizeIndex(M_occSave, this->toBe()); uside[(sideSz / sizeof(index_t))-4] = endianizeIndex(occSave[0], this->toBe()); uside[(sideSz / sizeof(index_t))-3] = endianizeIndex(occSave[1], this->toBe()); uside[(sideSz / sizeof(index_t))-2] = endianizeIndex(occSave[2], this->toBe()); uside[(sideSz / sizeof(index_t))-1] = endianizeIndex(occSave[3], this->toBe()); F_locSave = F_loc; M_occSave = M_occ; occSave[0] = occ[0]; occSave[1] = occ[1]; occSave[2] = occ[2]; occSave[3] = occ[3]; // Write backward side to primary file out5.write((const char *)gfmSide.ptr(), sideSz); // memcpy(((char*)this->_gfm.get()) + side - sideSz, (const char *)gfmSide.ptr(), sideSz); } } VMSG_NL("Exited LocalGFM loop"); // Assert that our loop counter got incremented right to the end assert_eq(side, gh._gbwtTotSz); // Assert that we wrote the expected amount to out5 assert_eq(((uint32_t)out5.tellp() - beforeGbwtOff), gh._gbwtTotSz); // assert that the last thing we did was write a forward bucket // // Write zOffs to primary stream // assert_gt(zOffs.size(), 0); writeIndex(out5, zOffs.size(), this->toBe()); for(size_t i = 0; i < zOffs.size(); i++) { writeIndex(out5, zOffs[i], this->toBe()); } // // Finish building fchr // // Exclusive prefix sum on fchr for(int i = 1; i < 4; i++) { fchr[i] += fchr[i-1]; } assert_lt(fchr[3], gbwtLen); // Shift everybody up by one for(int i = 4; i >= 1; i--) { fchr[i] = fchr[i-1]; } fchr[0] = 0; // Write fchr to primary file for(int i = 0; i < 5; i++) { writeIndex(out5, fchr[i], this->toBe()); } this->_fchr.init(new index_t[5], 5, true); memcpy(this->_fchr.get(), fchr, sizeof(index_t) * 5); // Initialize _zGbwtByteOffs and _zGbwtBpOffs this->_zOffs = zOffs; this->postReadInit(gh); // Build ftab and eftab EList > tFtab; tFtab.resizeExact(ftabLen - 1); for(index_t i = 0; i + 1 < ftabLen; i++) { index_t q = i; pair range(0, gh._gbwtLen); SideLocus tloc, bloc; SideLocus::initFromTopBot(range.first, range.second, gh, this->gfm(), tloc, bloc); index_t j = 0; for(; j < gh._ftabChars; j++) { int nt = q & 0x3; q >>= 2; if(bloc.valid()) { range = this->mapGLF(tloc, bloc, nt); } else { range = this->mapGLF1(range.first, tloc, nt); } if(range.first == (index_t)INDEX_MAX || range.first >= range.second) { break; } if(range.first + 1 == range.second) { tloc.initFromRow(range.first, gh, this->gfm()); bloc.invalidate(); } else { SideLocus::initFromTopBot(range.first, range.second, gh, this->gfm(), tloc, bloc); } } if(range.first >= range.second || j < gh._ftabChars) { if(i == 0) { tFtab[i].first = tFtab[i].second = 0; } else { tFtab[i].first = tFtab[i].second = tFtab[i-1].second; } } else { tFtab[i].first = range.first; tFtab[i].second = range.second; } #ifndef NDEBUG if(gbwt.ftab.size() > i) { assert_eq(tFtab[i].first, gbwt.ftab[i].first); assert_eq(tFtab[i].second, gbwt.ftab[i].second); } #endif } // Clear memory this->_gfm.reset(); this->_fchr.reset(); this->_zOffs.clear(); this->_zGbwtByteOffs.clear(); this->_zGbwtBpOffs.clear(); // // Finish building ftab and build eftab // // Prefix sum on ftable index_t eftabLen = 0; for(index_t i = 1; i + 1 < ftabLen; i++) { if(tFtab[i-1].second != tFtab[i].first) { eftabLen += 2; } } if(gh._gbwtLen + (eftabLen >> 1) < gh._gbwtLen) { cerr << "Too many eftab entries: " << gh._gbwtLen << " + " << (eftabLen >> 1) << " > " << (index_t)INDEX_MAX << endl; throw 1; } EList eftab(EBWT_CAT); try { eftab.resize(eftabLen); eftab.fillZero(); } catch(bad_alloc &e) { cerr << "Out of memory allocating eftab[] " << "in LocalGFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } index_t eftabCur = 0; ftab[0] = tFtab[0].first; ftab[1] = tFtab[0].second; for(index_t i = 1; i + 1 < ftabLen; i++) { if(ftab[i] != tFtab[i].first) { index_t lo = ftab[i]; index_t hi = tFtab[i].first; assert_lt(eftabCur*2+1, eftabLen); eftab[eftabCur*2] = lo; eftab[eftabCur*2+1] = hi; assert_leq(lo, hi + 4); ftab[i] = (eftabCur++) ^ (index_t)INDEX_MAX; // insert pointer into eftab assert_eq(lo, GFM::ftabLo(ftab.ptr(), eftab.ptr(), gbwtLen, ftabLen, eftabLen, i)); assert_eq(hi, GFM::ftabHi(ftab.ptr(), eftab.ptr(), gbwtLen, ftabLen, eftabLen, i)); } ftab[i+1] = tFtab[i].second; } #ifndef NDEBUG for(index_t i = 0; i + 1 < ftabLen; i++ ){ assert_eq(tFtab[i].first, GFM::ftabHi(ftab.ptr(), eftab.ptr(), gbwtLen, ftabLen, eftabLen, i)); assert_eq(tFtab[i].second, GFM::ftabLo(ftab.ptr(), eftab.ptr(), gbwtLen, ftabLen, eftabLen, i+1)); } #endif // Write ftab to primary file for(index_t i = 0; i < ftabLen; i++) { writeIndex(out5, ftab[i], this->toBe()); } // Write eftab to primary file out5pos = out5.tellp(); out5.seekp(headerPos); writeIndex(out5, eftabLen, this->toBe()); out5.seekp(out5pos); for(index_t i = 0; i < eftabLen; i++) { writeIndex(out5, eftab[i], this->toBe()); } // Note: if you'd like to sanity-check the Ebwt, you'll have to // read it back into memory first! assert(!this->isInMemory()); VMSG_NL("Exiting LocalGFM::buildToDisk()"); } /** * Build an Ebwt from a string 's' and its suffix array 'sa' (which * might actually be a suffix array *builder* that builds blocks of the * array on demand). The bulk of the Ebwt, i.e. the ebwt and offs * arrays, is written directly to disk. This is by design: keeping * those arrays in memory needlessly increases the footprint of the * building process. Instead, we prefer to build the Ebwt directly * "to disk" and then read it back into memory later as necessary. * * It is assumed that the header values and join-related values (nPat, * plen) have already been written to 'out1' before this function * is called. When this function is finished, it will have * additionally written ebwt, zOff, fchr, ftab and eftab to the primary * file and offs to the secondary file. * * Assume DNA/RNA/any alphabet with 4 or fewer elements. * Assume occ array entries are 32 bits each. * * @param sa the suffix array to convert to a Ebwt * @param s the original string * @param out */ template template void LocalGFM::buildToDisk( const EList& sa, const TStr& s, ostream& out5, ostream& out6, streampos headerPos) { assert_leq(s.length(), std::numeric_limits::max()); const GFMParams& gh = this->_gh; assert(gh.repOk()); assert(gh.linearFM()); assert_lt(s.length(), gh.gbwtLen()); assert_eq(s.length(), gh._len); assert_gt(gh._lineRate, 3); index_t len = gh._len; index_t gbwtLen = gh._gbwtLen; assert_eq(len + 1, gbwtLen); streampos out5pos = out5.tellp(); out5.seekp(headerPos); writeIndex(out5, gbwtLen, this->toBe()); writeIndex(out5, gh._numNodes, this->toBe()); headerPos = out5.tellp(); out5.seekp(out5pos); index_t ftabLen = gh._ftabLen; index_t sideSz = gh._sideSz; index_t gbwtTotSz = gh._gbwtTotSz; index_t fchr[] = {0, 0, 0, 0, 0}; EList ftab(EBWT_CAT); EList zOffs; // Save # of occurrences of each character as we walk along the bwt index_t occ[4] = {0, 0, 0, 0}; index_t occSave[4] = {0, 0, 0, 0}; // Record rows that should "absorb" adjacent rows in the ftab. // The absorbed rows represent suffixes shorter than the ftabChars // cutoff. uint8_t absorbCnt = 0; EList absorbFtab(EBWT_CAT); try { VMSG_NL("Allocating ftab, absorbFtab"); ftab.resize(ftabLen); ftab.fillZero(); absorbFtab.resize(ftabLen); absorbFtab.fillZero(); } catch(bad_alloc &e) { cerr << "Out of memory allocating ftab[] or absorbFtab[] " << "in LocalGFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } // Allocate the side buffer; holds a single side as its being // constructed and then written to disk. Reused across all sides. #ifdef SIXTY4_FORMAT EList gfmSide(EBWT_CAT); #else EList gfmSide(EBWT_CAT); #endif try { #ifdef SIXTY4_FORMAT gfmSide.resize(sideSz >> 3); #else gfmSide.resize(sideSz); #endif } catch(bad_alloc &e) { cerr << "Out of memory allocating gfmSide[] in " << "LocalGFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } // Points to the base offset within ebwt for the side currently // being written index_t side = 0; // Whether we're assembling a forward or a reverse bucket bool fw = true; int sideCur = 0; // Have we skipped the '$' in the last column yet? ASSERT_ONLY(bool dollarSkipped = false); index_t si = 0; // string offset (chars) ASSERT_ONLY(uint32_t lastSufInt = 0); ASSERT_ONLY(bool inSA = true); // true iff saI still points inside suffix // array (as opposed to the padding at the // end) // Iterate over packed bwt bytes VMSG_NL("Entering LocalGFM loop"); ASSERT_ONLY(uint32_t beforeGbwtOff = (uint32_t)out5.tellp()); while(side < gbwtTotSz) { // Sanity-check our cursor into the side buffer assert_geq(sideCur, 0); assert_lt(sideCur, (int)gh._sideGbwtSz); assert_eq(0, side % sideSz); // 'side' must be on side boundary gfmSide[sideCur] = 0; // clear assert_lt(side + sideCur, gbwtTotSz); // Iterate over bit-pairs in the si'th character of the BWT #ifdef SIXTY4_FORMAT for(int bpi = 0; bpi < 32; bpi++, si++) { #else for(int bpi = 0; bpi < 4; bpi++, si++) { #endif int bwtChar; bool count = true; if(si <= len) { // Still in the SA; extract the bwtChar index_t saElt = (index_t)sa[si]; // (that might have triggered sa to calc next suf block) if(saElt == 0) { // Don't add the '$' in the last column to the BWT // transform; we can't encode a $ (only A C T or G) // and counting it as, say, an A, will mess up the // LR mapping bwtChar = 0; count = false; ASSERT_ONLY(dollarSkipped = true); zOffs.push_back(si); // remember the SA row that // corresponds to the 0th suffix } else { bwtChar = (int)(s[saElt-1]); assert_lt(bwtChar, 4); // Update the fchr fchr[bwtChar]++; } // Update ftab if((len-saElt) >= (index_t)gh._ftabChars) { // Turn the first ftabChars characters of the // suffix into an integer index into ftab. The // leftmost (lowest index) character of the suffix // goes in the most significant bit pair if the // integer. uint32_t sufInt = 0; for(int i = 0; i < gh._ftabChars; i++) { sufInt <<= 2; assert_lt((index_t)i, len-saElt); sufInt |= (unsigned char)(s[saElt+i]); } // Assert that this prefix-of-suffix is greater // than or equal to the last one (true b/c the // suffix array is sorted) #ifndef NDEBUG if(lastSufInt > 0) assert_geq(sufInt, lastSufInt); lastSufInt = sufInt; #endif // Update ftab assert_lt(sufInt+1, ftabLen); ftab[sufInt+1]++; if(absorbCnt > 0) { // Absorb all short suffixes since the last // transition into this transition absorbFtab[sufInt] = absorbCnt; absorbCnt = 0; } } else { // Otherwise if suffix is fewer than ftabChars // characters long, then add it to the 'absorbCnt'; // it will be absorbed into the next transition assert_lt(absorbCnt, 255); absorbCnt++; } // Suffix array offset boundary? - update offset array if((si & gh._offMask) == si) { assert_lt((si >> gh._offRate), gh._offsLen); // Write offsets directly to the secondary output // stream, thereby avoiding keeping them in memory writeIndex(out6, saElt, this->toBe()); } } else { // Strayed off the end of the SA, now we're just // padding out a bucket #ifndef NDEBUG if(inSA) { // Assert that we wrote all the characters in the // string before now assert_eq(si, len+1); inSA = false; } #endif // 'A' used for padding; important that padding be // counted in the occ[] array bwtChar = 0; } if(count) occ[bwtChar]++; // Append BWT char to bwt section of current side if(fw) { // Forward bucket: fill from least to most #ifdef SIXTY4_FORMAT gfmSide[sideCur] |= ((uint64_t)bwtChar << (bpi << 1)); if(bwtChar > 0) assert_gt(gfmSide[sideCur], 0); #else pack_2b_in_8b(bwtChar, gfmSide[sideCur], bpi); assert_eq((gfmSide[sideCur] >> (bpi*2)) & 3, bwtChar); #endif } else { // Backward bucket: fill from most to least #ifdef SIXTY4_FORMAT gfmSide[sideCur] |= ((uint64_t)bwtChar << ((31 - bpi) << 1)); if(bwtChar > 0) assert_gt(gfmSide[sideCur], 0); #else pack_2b_in_8b(bwtChar, gfmSide[sideCur], 3-bpi); assert_eq((gfmSide[sideCur] >> ((3-bpi)*2)) & 3, bwtChar); #endif } } // end loop over bit-pairs assert_eq(dollarSkipped ? 3 : 0, (occ[0] + occ[1] + occ[2] + occ[3]) & 3); #ifdef SIXTY4_FORMAT assert_eq(0, si & 31); #else assert_eq(0, si & 3); #endif sideCur++; if(sideCur == (int)gh._sideGbwtSz) { sideCur = 0; index_t *uside = reinterpret_cast(gfmSide.ptr()); // Write 'A', 'C', 'G' and 'T' tallies side += sideSz; assert_leq(side, gh._gbwtTotSz); uside[(sideSz / sizeof(index_t))-4] = endianizeIndex(occSave[0], this->toBe()); uside[(sideSz / sizeof(index_t))-3] = endianizeIndex(occSave[1], this->toBe()); uside[(sideSz / sizeof(index_t))-2] = endianizeIndex(occSave[2], this->toBe()); uside[(sideSz / sizeof(index_t))-1] = endianizeIndex(occSave[3], this->toBe()); occSave[0] = occ[0]; occSave[1] = occ[1]; occSave[2] = occ[2]; occSave[3] = occ[3]; // Write backward side to primary file out5.write((const char *)gfmSide.ptr(), sideSz); } } VMSG_NL("Exited LocalGFM loop"); if(absorbCnt > 0) { // Absorb any trailing, as-yet-unabsorbed short suffixes into // the last element of ftab absorbFtab[ftabLen-1] = absorbCnt; } // Assert that our loop counter got incremented right to the end assert_eq(side, gh._gbwtTotSz); // Assert that we wrote the expected amount to out5 assert_eq(((uint32_t)out5.tellp() - beforeGbwtOff), gh._gbwtTotSz); // assert that the last thing we did was write a forward bucket // // Write zOffs to primary stream // assert_eq(zOffs.size(), 1); writeIndex(out5, zOffs.size(), this->toBe()); for(size_t i = 0; i < zOffs.size(); i++) { assert_neq(zOffs[i], (index_t)OFF_MASK); writeIndex(out5, zOffs[i], this->toBe()); } // // Finish building fchr // // Exclusive prefix sum on fchr for(int i = 1; i < 4; i++) { fchr[i] += fchr[i-1]; } assert_lt(fchr[3], gbwtLen); // Shift everybody up by one for(int i = 4; i >= 1; i--) { fchr[i] = fchr[i-1]; } fchr[0] = 0; // Write fchr to primary file for(int i = 0; i < 5; i++) { writeIndex(out5, fchr[i], this->toBe()); } // // Finish building ftab and build eftab // // Prefix sum on ftable index_t eftabLen = 0; assert_eq(0, absorbFtab[0]); for(index_t i = 1; i < ftabLen; i++) { if(absorbFtab[i] > 0) eftabLen += 2; } assert_leq(eftabLen, (index_t)gh._ftabChars*2); eftabLen = gh._ftabChars*2; EList eftab(EBWT_CAT); try { eftab.resize(eftabLen); eftab.fillZero(); } catch(bad_alloc &e) { cerr << "Out of memory allocating eftab[] " << "in LocalGFM::buildToDisk() at " << __FILE__ << ":" << __LINE__ << endl; throw e; } index_t eftabCur = 0; for(index_t i = 1; i < ftabLen; i++) { index_t lo = ftab[i] + GFM::ftabHi(ftab.ptr(), eftab.ptr(), len, ftabLen, eftabLen, i-1); if(absorbFtab[i] > 0) { // Skip a number of short pattern indicated by absorbFtab[i] index_t hi = lo + absorbFtab[i]; assert_lt(eftabCur*2+1, eftabLen); eftab[eftabCur*2] = lo; eftab[eftabCur*2+1] = hi; ftab[i] = (eftabCur++) ^ (index_t)OFF_MASK; // insert pointer into eftab assert_eq(lo, GFM::ftabLo(ftab.ptr(), eftab.ptr(), len, ftabLen, eftabLen, i)); assert_eq(hi, GFM::ftabHi(ftab.ptr(), eftab.ptr(), len, ftabLen, eftabLen, i)); } else { ftab[i] = lo; } } assert_eq(GFM::ftabHi(ftab.ptr(), eftab.ptr(), len, ftabLen, eftabLen, ftabLen-1), len+1); // Write ftab to primary file for(index_t i = 0; i < ftabLen; i++) { writeIndex(out5, ftab[i], this->toBe()); } // Write eftab to primary file out5pos = out5.tellp(); out5.seekp(headerPos); writeIndex(out5, eftabLen, this->toBe()); out5.seekp(out5pos); for(index_t i = 0; i < eftabLen; i++) { writeIndex(out5, eftab[i], this->toBe()); } // Note: if you'd like to sanity-check the Ebwt, you'll have to // read it back into memory first! assert(!this->isInMemory()); VMSG_NL("Exiting LocalGFM::buildToDisk()"); } /** * Read an Ebwt from file with given filename. */ template void LocalGFM::readIntoMemory( FILE *in5, FILE *in6, char *mmFile5, char *mmFile6, full_index_t& tidx, full_index_t& localOffset, full_index_t& joinedOffset, bool switchEndian, size_t& bytesRead, size_t& bytesRead2, int entireRev, bool loadSASamp, bool loadFtab, bool loadRstarts, bool justHeader, int32_t lineRate, int32_t offRate, int32_t ftabChars, bool mmSweep, bool loadNames, bool startVerbose) { #ifdef BOWTIE_MM char *mmFile[] = { mmFile5, mmFile6 }; #endif // Reads header entries one by one from primary stream tidx = readIndex(in5, switchEndian); bytesRead += sizeof(full_index_t); localOffset = readIndex(in5, switchEndian); bytesRead += sizeof(full_index_t); joinedOffset = readIndex(in5, switchEndian); bytesRead += sizeof(full_index_t); index_t len = readIndex(in5, switchEndian); bytesRead += sizeof(index_t); index_t gbwtLen = readIndex(in5, switchEndian); bytesRead += sizeof(index_t); index_t numNodes = readIndex(in5, switchEndian); bytesRead += sizeof(index_t); index_t eftabLen = readIndex(in5, switchEndian); bytesRead += sizeof(index_t); // Create a new EbwtParams from the entries read from primary stream this->_gh.init(len, gbwtLen, numNodes, lineRate, offRate, ftabChars, eftabLen, entireRev); if(len <= 0) { return; } // Set up overridden suffix-array-sample parameters uint32_t offsLen = this->_gh._offsLen; uint32_t offRateDiff = 0; uint32_t offsLenSampled = offsLen; if(this->_overrideOffRate > offRate) { offRateDiff = this->_overrideOffRate - offRate; } if(offRateDiff > 0) { offsLenSampled >>= offRateDiff; if((offsLen & ~((index_t)OFF_MASK << offRateDiff)) != 0) { offsLenSampled++; } } // Can't override the offrate or isarate and use memory-mapped // files; ultimately, all processes need to copy the sparser sample // into their own memory spaces. if(this->_useMm && (offRateDiff)) { cerr << "Error: Can't use memory-mapped files when the offrate is overridden" << endl; throw 1; } // Read nPat from primary stream this->_nPat = readIndex(in5, switchEndian); assert_eq(this->_nPat, 1); bytesRead += sizeof(index_t); this->_plen.reset(); // Read plen from primary stream if(this->_useMm) { #ifdef BOWTIE_MM this->_plen.init((index_t*)(mmFile[0] + bytesRead), this->_nPat, false); bytesRead += this->_nPat*sizeof(index_t); fseek(in5, this->_nPat*sizeof(index_t), SEEK_CUR); #endif } else { try { if(this->_verbose || startVerbose) { cerr << "Reading plen (" << this->_nPat << "): "; logTime(cerr); } this->_plen.init(new index_t[this->_nPat], this->_nPat, true); if(switchEndian) { for(index_t i = 0; i < this->_nPat; i++) { this->plen()[i] = readIndex(in5, switchEndian); } } else { size_t r = MM_READ(in5, (void*)(this->plen()), this->_nPat*sizeof(index_t)); if(r != (size_t)(this->_nPat*sizeof(index_t))) { cerr << "Error reading _plen[] array: " << r << ", " << this->_nPat*sizeof(index_t) << endl; throw 1; } } } catch(bad_alloc& e) { cerr << "Out of memory allocating plen[] in Ebwt::read()" << " at " << __FILE__ << ":" << __LINE__ << endl; throw e; } } bool shmemLeader; // TODO: I'm not consistent on what "header" means. Here I'm using // "header" to mean everything that would exist in memory if we // started to build the Ebwt but stopped short of the build*() step // (i.e. everything up to and including join()). if(justHeader) return; this->_nFrag = readIndex(in5, switchEndian); bytesRead += sizeof(index_t); if(this->_verbose || startVerbose) { cerr << "Reading rstarts (" << this->_nFrag*3 << "): "; logTime(cerr); } assert_geq(this->_nFrag, this->_nPat); this->_rstarts.reset(); if(loadRstarts) { if(this->_useMm) { #ifdef BOWTIE_MM this->_rstarts.init((index_t*)(mmFile[0] + bytesRead), this->_nFrag*3, false); bytesRead += this->_nFrag*sizeof(index_t)*3; fseek(in5, this->_nFrag*sizeof(index_t)*3, SEEK_CUR); #endif } else { this->_rstarts.init(new index_t[this->_nFrag*3], this->_nFrag*3, true); if(switchEndian) { for(index_t i = 0; i < this->_nFrag*3; i += 3) { // fragment starting position in joined reference // string, text id, and fragment offset within text this->rstarts()[i] = readIndex(in5, switchEndian); this->rstarts()[i+1] = readIndex(in5, switchEndian); this->rstarts()[i+2] = readIndex(in5, switchEndian); } } else { size_t r = MM_READ(in5, (void *)this->rstarts(), this->_nFrag*sizeof(index_t)*3); if(r != (size_t)(this->_nFrag*sizeof(index_t)*3)) { cerr << "Error reading _rstarts[] array: " << r << ", " << (this->_nFrag*sizeof(index_t)*3) << endl; throw 1; } } } } else { // Skip em assert(this->rstarts() == NULL); bytesRead += this->_nFrag*sizeof(index_t)*3; fseek(in5, this->_nFrag*sizeof(index_t)*3, SEEK_CUR); } this->_gfm.reset(); if(this->_useMm) { #ifdef BOWTIE_MM this->_gfm.init((uint8_t*)(mmFile[0] + bytesRead), this->_gh._gbwtTotLen, false); bytesRead += this->_gh._gbwtTotLen; fseek(in5, this->_gh._gbwtTotLen, SEEK_CUR); #endif } else { // Allocate ebwt (big allocation) if(this->_verbose || startVerbose) { cerr << "Reading ebwt (" << this->_gh._gbwtTotLen << "): "; logTime(cerr); } bool shmemLeader = true; if(this->useShmem_) { uint8_t *tmp = NULL; shmemLeader = ALLOC_SHARED_U8( (this->_in1Str + "[gfm]"), this->_gh._gbwtTotLen, &tmp, "gfm[]", (this->_verbose || startVerbose)); assert(tmp != NULL); this->_gfm.init(tmp, this->_gh._gbwtTotLen, false); if(this->_verbose || startVerbose) { cerr << " shared-mem " << (shmemLeader ? "leader" : "follower") << endl; } } else { try { this->_gfm.init(new uint8_t[this->_gh._gbwtTotLen], this->_gh._gbwtTotLen, true); } catch(bad_alloc& e) { cerr << "Out of memory allocating the ebwt[] array for the Bowtie index. Please try" << endl << "again on a computer with more memory." << endl; throw 1; } } if(shmemLeader) { // Read ebwt from primary stream uint64_t bytesLeft = this->_gh._gbwtTotLen; char *pgbwt = (char*)this->gfm(); while (bytesLeft>0){ size_t r = MM_READ(in5, (void *)pgbwt, bytesLeft); if(MM_IS_IO_ERR(in5, r, bytesLeft)) { cerr << "Error reading _ebwt[] array: " << r << ", " << bytesLeft << endl; throw 1; } pgbwt += r; bytesLeft -= r; } if(switchEndian) { uint8_t *side = this->gfm(); for(size_t i = 0; i < this->_gh._numSides; i++) { index_t *cums = reinterpret_cast(side + this->_gh._sideSz - sizeof(index_t)*2); cums[0] = endianSwapIndex(cums[0]); cums[1] = endianSwapIndex(cums[1]); side += this->_gh._sideSz; } } #ifdef BOWTIE_SHARED_MEM if(useShmem_) NOTIFY_SHARED(this->gfm(), this->_gh._gbwtTotLen); #endif } else { // Seek past the data and wait until master is finished fseek(in5, this->_gh._gbwtTotLen, SEEK_CUR); #ifdef BOWTIE_SHARED_MEM if(useShmem_) WAIT_SHARED(this->gfm(), this->_gh._gbwtTotLen); #endif } } // Read zOff from primary stream this->_zOffs.clear(); index_t num_zOffs = readIndex(in5, switchEndian); bytesRead += sizeof(index_t); for(index_t i = 0; i < num_zOffs; i++) { index_t zOff = readIndex(in5, switchEndian); bytesRead += sizeof(index_t); assert_lt(zOff, gbwtLen); this->_zOffs.push_back(zOff); } try { // Read fchr from primary stream if(this->_verbose || startVerbose) cerr << "Reading fchr (5)" << endl; this->_fchr.reset(); if(this->_useMm) { #ifdef BOWTIE_MM this->_fchr.init((index_t*)(mmFile[0] + bytesRead), 5, false); bytesRead += 5*sizeof(index_t); fseek(in5, 5*sizeof(index_t), SEEK_CUR); #endif } else { this->_fchr.init(new index_t[5], 5, true); for(index_t i = 0; i < 5; i++) { this->fchr()[i] = readIndex(in5, switchEndian); assert_leq(this->fchr()[i], gbwtLen); assert(i <= 0 || this->fchr()[i] >= this->fchr()[i-1]); } } assert_gt(this->fchr()[4], this->fchr()[0]); // Read ftab from primary stream if(this->_verbose || startVerbose) { if(loadFtab) { cerr << "Reading ftab (" << this->_gh._ftabLen << "): "; logTime(cerr); } else { cerr << "Skipping ftab (" << this->_gh._ftabLen << "): "; } } this->_ftab.reset(); if(loadFtab) { if(this->_useMm) { #ifdef BOWTIE_MM this->_ftab.init((index_t*)(mmFile[0] + bytesRead), this->_gh._ftabLen, false); bytesRead += this->_gh._ftabLen*sizeof(index_t); fseek(in5, this->_gh._ftabLen*sizeof(index_t), SEEK_CUR); #endif } else { this->_ftab.init(new index_t[this->_gh._ftabLen], this->_gh._ftabLen, true); if(switchEndian) { for(uint32_t i = 0; i < this->_gh._ftabLen; i++) this->ftab()[i] = readIndex(in5, switchEndian); } else { size_t r = MM_READ(in5, (void *)this->ftab(), this->_gh._ftabLen*sizeof(index_t)); if(r != (size_t)(this->_gh._ftabLen*sizeof(index_t))) { cerr << "Error reading _ftab[] array: " << r << ", " << (this->_gh._ftabLen*sizeof(index_t)) << endl; throw 1; } } } // Read etab from primary stream if(this->_verbose || startVerbose) { if(loadFtab) { cerr << "Reading eftab (" << this->_gh._eftabLen << "): "; logTime(cerr); } else { cerr << "Skipping eftab (" << this->_gh._eftabLen << "): "; } } this->_eftab.reset(); if(this->_useMm) { #ifdef BOWTIE_MM this->_eftab.init((index_t*)(mmFile[0] + bytesRead), this->_gh._eftabLen, false); bytesRead += this->_gh._eftabLen*sizeof(index_t); fseek(in5, this->_gh._eftabLen*sizeof(index_t), SEEK_CUR); #endif } else { this->_eftab.init(new index_t[this->_gh._eftabLen], this->_gh._eftabLen, true); if(switchEndian) { for(uint32_t i = 0; i < this->_gh._eftabLen; i++) this->eftab()[i] = readIndex(in5, switchEndian); } else { size_t r = MM_READ(in5, (void *)this->eftab(), this->_gh._eftabLen*sizeof(index_t)); if(r != (size_t)(this->_gh._eftabLen*sizeof(index_t))) { cerr << "Error reading _eftab[] array: " << r << ", " << (this->_gh._eftabLen*sizeof(index_t)) << endl; throw 1; } } } for(uint32_t i = 0; i < this->_gh._eftabLen; i++) { if(i > 0 && this->eftab()[i] > 0) { assert_geq(this->eftab()[i] + 4, this->eftab()[i-1]); } else if(i > 0 && this->eftab()[i-1] == 0) { assert_eq(0, this->eftab()[i]); } } } else { assert(this->ftab() == NULL); assert(this->eftab() == NULL); // Skip ftab bytesRead += this->_gh._ftabLen*sizeof(index_t); fseek(in5, this->_gh._ftabLen*sizeof(index_t), SEEK_CUR); // Skip eftab bytesRead += this->_gh._eftabLen*sizeof(index_t); fseek(in5, this->_gh._eftabLen*sizeof(index_t), SEEK_CUR); } } catch(bad_alloc& e) { cerr << "Out of memory allocating fchr[], ftab[] or eftab[] arrays for the Bowtie index." << endl << "Please try again on a computer with more memory." << endl; throw 1; } this->_offs.reset(); if(loadSASamp) { shmemLeader = true; if(this->_verbose || startVerbose) { cerr << "Reading offs (" << offsLenSampled << " " << std::setw(2) << sizeof(index_t)*8 << "-bit words): "; logTime(cerr); } if(!this->_useMm) { if(!this->useShmem_) { // Allocate offs_ try { this->_offs.init(new index_t[offsLenSampled], offsLenSampled, true); } catch(bad_alloc& e) { cerr << "Out of memory allocating the offs[] array for the Bowtie index." << endl << "Please try again on a computer with more memory." << endl; throw 1; } } else { index_t *tmp = NULL; shmemLeader = ALLOC_SHARED_U32( (this->_in2Str + "[offs]"), offsLenSampled*2, &tmp, "offs", (this->_verbose || startVerbose)); this->_offs.init((index_t*)tmp, offsLenSampled, false); } } if(this->_overrideOffRate < 32) { if(shmemLeader) { // Allocate offs (big allocation) if(switchEndian || offRateDiff > 0) { assert(!this->_useMm); const uint32_t blockMaxSz = (2 * 1024 * 1024); // 2 MB block size const uint32_t blockMaxSzUIndex = (blockMaxSz / sizeof(index_t)); // # UIndexs per block char *buf; try { buf = new char[blockMaxSz]; } catch(std::bad_alloc& e) { cerr << "Error: Out of memory allocating part of _offs array: '" << e.what() << "'" << endl; throw e; } for(index_t i = 0; i < offsLen; i += blockMaxSzUIndex) { index_t block = min((index_t)blockMaxSzUIndex, (index_t)(offsLen - i)); size_t r = MM_READ(in6, (void *)buf, block * sizeof(index_t)); if(r != (size_t)(block * sizeof(index_t))) { cerr << "Error reading block of _offs[] array: " << r << ", " << (block * sizeof(index_t)) << endl; throw 1; } index_t idx = i >> offRateDiff; for(index_t j = 0; j < block; j += (1 << offRateDiff)) { assert_lt(idx, offsLenSampled); this->offs()[idx] = ((index_t*)buf)[j]; if(switchEndian) { this->offs()[idx] = endianSwapIndex(this->offs()[idx]); } idx++; } } delete[] buf; } else { if(this->_useMm) { #ifdef BOWTIE_MM this->_offs.init((index_t*)(mmFile[1] + bytesRead2), offsLen, false); bytesRead2 += (offsLen * sizeof(index_t)); fseek(in6, (offsLen * sizeof(index_t)), SEEK_CUR); #endif } else { // If any of the high two bits are set if((offsLen & 0xc0000000) != 0) { if(sizeof(char *) <= 4) { cerr << "Sanity error: sizeof(char *) <= 4 but offsLen is " << hex << offsLen << endl; throw 1; } // offsLen << 2 overflows, so do it in four reads char *offs = (char *)this->offs(); for(size_t i = 0; i < sizeof(index_t); i++) { size_t r = MM_READ(in6, (void*)offs, offsLen); if(r != (size_t)(offsLen)) { cerr << "Error reading block of _offs[] array: " << r << ", " << offsLen << endl; throw 1; } offs += offsLen; } } else { // Do it all in one read size_t r = MM_READ(in6, (void*)this->offs(), offsLen * sizeof(index_t)); if(r != (size_t)(offsLen * sizeof(index_t))) { cerr << "Error reading _offs[] array: " << r << ", " << (offsLen * sizeof(index_t)) << endl; throw 1; } } } } #ifdef BOWTIE_SHARED_MEM if(this->useShmem_) NOTIFY_SHARED(this->offs(), offsLenSampled*sizeof(index_t)); #endif } else { // Not the shmem leader fseek(in6, offsLenSampled*sizeof(index_t), SEEK_CUR); #ifdef BOWTIE_SHARED_MEM if(this->useShmem_) WAIT_SHARED(this->offs(), offsLenSampled*sizeof(index_t)); #endif } } } this->postReadInit(this->_gh); // Initialize fields of Ebwt not read from file if(this->_verbose || startVerbose) this->print(cerr, this->_gh); } /** * Extended Burrows-Wheeler transform data. * HierEbwt is a specialized Ebwt index that represents one global index and a large set of local indexes. * */ template class HGFM : public GFM { typedef GFM PARENT_CLASS; public: /// Construct a GFM from the given input file HGFM(const string& in, ALTDB* altdb, RepeatDB* repeatdb, EList* readLens, int needEntireReverse, bool fw, int32_t overrideOffRate, // = -1, int32_t offRatePlus, // = -1, bool useMm, // = false, bool useShmem, // = false, bool mmSweep, // = false, bool loadNames, // = false, bool loadSASamp, // = true, bool loadFtab, // = true, bool loadRstarts, // = true, bool loadSpliceSites, // = true, bool verbose, // = false, bool startVerbose, // = false, bool passMemExc, // = false, bool sanityCheck, // = false bool useHaplotype, // = false bool skipLoading = false) : GFM(in, altdb, repeatdb, readLens, needEntireReverse, fw, overrideOffRate, offRatePlus, useMm, useShmem, mmSweep, loadNames, loadSASamp, loadFtab, loadRstarts, loadSpliceSites, verbose, startVerbose, passMemExc, sanityCheck, useHaplotype, skipLoading), _in5(NULL), _in6(NULL) { _in5Str = in + ".5." + gfm_ext; _in6Str = in + ".6." + gfm_ext; } /// Construct a HGFM from the given header parameters and string /// vector, optionally using a blockwise suffix sorter with the /// given 'bmax' and 'dcv' parameters. The string vector is /// ultimately joined and the joined string is passed to buildToDisk(). template HGFM( TStr& s, bool packed, int needEntireReverse, int32_t lineRate, int32_t offRate, int32_t ftabChars, int32_t localOffRate, int32_t localFtabChars, int nthreads, const string& snpfile, const string& htfile, const string& ssfile, const string& exonfile, const string& svfile, const string& repeatfile, const string& outfile, // base filename for GFM files bool fw, bool useBlockwise, TIndexOffU bmax, TIndexOffU bmaxSqrtMult, TIndexOffU bmaxDivN, int dcv, EList& is, EList& szs, index_t sztot, const RefReadInParams& refparams, bool localIndex, // create local indexes? EList* parent_szs, EList* parent_refnames, uint32_t seed, int32_t overrideOffRate = -1, bool verbose = false, bool passMemExc = false, bool sanityCheck = false); ~HGFM() { clearLocalGFMs(); } /** * Load this Ebwt into memory by reading it in from the _in1 and * _in2 streams. */ void loadIntoMemory( int needEntireReverse, bool loadSASamp, bool loadFtab, bool loadRstarts, bool loadNames, bool verbose) { readIntoMemory( needEntireReverse, // require reverse index to be concatenated reference reversed loadSASamp, // load the SA sample portion? loadFtab, // load the ftab (_ftab[] and _eftab[])? loadRstarts, // load the r-starts (_rstarts[])? false, // stop after loading the header portion? NULL, // params false, // mmSweep loadNames, // loadNames verbose); // startVerbose } // I/O void readIntoMemory( int needEntireRev, bool loadSASamp, bool loadFtab, bool loadRstarts, bool justHeader, GFMParams *params, bool mmSweep, bool loadNames, bool startVerbose); /** * Frees memory associated with the Ebwt. */ void evictFromMemory() { assert(PARENT_CLASS::isInMemory()); clearLocalGFMs(); PARENT_CLASS::evictFromMemory(); } /** * Sanity-check various pieces of the Ebwt */ void sanityCheckAll(int reverse) const { PARENT_CLASS::sanityCheckAll(reverse); for(size_t tidx = 0; tidx < _localGFMs.size(); tidx++) { for(size_t local_idx = 0; local_idx < _localGFMs[tidx].size(); local_idx++) { assert(_localGFMs[tidx][local_idx] != NULL); _localGFMs[tidx][local_idx]->sanityCheckAll(reverse); } } } const LocalGFM* getLocalGFM(index_t tidx, index_t offset) const { assert_lt(tidx, _localGFMs.size()); const EList*>& localGFMs = _localGFMs[tidx]; index_t offsetidx = offset / local_index_interval; if(offsetidx >= localGFMs.size()) { return NULL; } else { return localGFMs[offsetidx]; } } const LocalGFM* prevLocalGFM(const LocalGFM* currLocalGFM) const { assert(currLocalGFM != NULL); index_t tidx = currLocalGFM->_tidx; index_t offset = currLocalGFM->_localOffset; if(offset < local_index_interval) { return NULL; } else { return getLocalGFM(tidx, offset - local_index_interval); } } const LocalGFM* nextLocalGFM(const LocalGFM* currLocalGFM) const { assert(currLocalGFM != NULL); index_t tidx = currLocalGFM->_tidx; index_t offset = currLocalGFM->_localOffset; return getLocalGFM(tidx, offset + local_index_interval); } void clearLocalGFMs() { for(size_t tidx = 0; tidx < _localGFMs.size(); tidx++) { for(size_t local_idx = 0; local_idx < _localGFMs[tidx].size(); local_idx++) { assert(_localGFMs[tidx][local_idx] != NULL); delete _localGFMs[tidx][local_idx]; } _localGFMs[tidx].clear(); } _localGFMs.clear(); } public: index_t _nrefs; /// the number of reference sequences EList _refLens; /// approx lens of ref seqs (excludes trailing ambig chars) EList*> > _localGFMs; index_t _nlocalGFMs; //index_t _local_index_interval; FILE *_in5; // input fd for primary index file FILE *_in6; // input fd for secondary index file string _in5Str; string _in6Str; char *mmFile5_; char *mmFile6_; private: struct ThreadParam { // input SString s; EList > alts; EList > haplotypes; bool bigEndian; index_t local_offset; index_t curr_sztot; EList conv_local_szs; index_t local_sztot; index_t index_size; string file; EList sa; index_t dcv; index_t seed; // output RefGraph* rg; PathGraph* pg; // communication bool done; bool last; bool mainThread; }; static void gbwt_worker(void* vp); }; template void HGFM::gbwt_worker(void* vp) { ThreadParam& tParam = *(ThreadParam*)vp; while(!tParam.last) { if(tParam.mainThread) { assert(!tParam.done); if(tParam.s.length() <= 0) { tParam.done = true; return; } } else { while(tParam.done) { if(tParam.last) return; #if defined(_TTHREAD_WIN32_) Sleep(1); #elif defined(_TTHREAD_POSIX_) const static timespec ts = {0, 1000000}; // 1 millisecond nanosleep(&ts, NULL); #endif } if(tParam.s.length() <= 0) { tParam.done = true; continue; } } while(true) { if(tParam.alts.empty()) { KarkkainenBlockwiseSA > bsa( tParam.s, (index_t)(tParam.s.length()+1), 1, tParam.dcv, tParam.seed, false, /* this->_sanity */ false, /* this->_passMemExc */ false); /* this->_verbose */ assert(bsa.suffixItrIsReset()); assert_eq(bsa.size(), tParam.s.length()+1); tParam.sa.clear(); for(index_t i = 0; i < bsa.size(); i++) { tParam.sa.push_back(bsa.nextSuffix()); } } else { tParam.rg = NULL, tParam.pg = NULL; bool exploded = false; try { tParam.rg = new RefGraph( tParam.s, tParam.conv_local_szs, tParam.alts, tParam.haplotypes, tParam.file, 1, /* num threads */ false); /* verbose? */ tParam.pg = new PathGraph( *tParam.rg, tParam.file, local_max_gbwt, 1, /* num threads */ false); /* verbose? */ } catch (const NongraphException& err) { cerr << "Warning: no variants or splice sites in this graph (" << tParam.curr_sztot << ")" << endl; delete tParam.rg; delete tParam.pg; tParam.alts.clear(); continue; } catch (const ExplosionException& err) { exploded = true; } if(!exploded) { if(!tParam.pg->generateEdges(*tParam.rg)) { cerr << "An error occurred - generateEdges" << endl; throw 1; } exploded = tParam.pg->getNumEdges() > local_max_gbwt; } if(exploded) { cerr << "Warning: a local graph exploded (offset: " << tParam.curr_sztot << ", length: " << tParam.local_sztot << ")" << endl; delete tParam.pg; tParam.pg = NULL; delete tParam.rg; tParam.rg = NULL; if(tParam.alts.size() <= 1) { tParam.alts.clear(); } else { for(index_t s = 2; s < tParam.alts.size(); s += 2) { tParam.alts[s >> 1] = tParam.alts[s]; } tParam.alts.resize(tParam.alts.size() >> 1); tParam.haplotypes.clear(); for(index_t a = 0; a < tParam.alts.size(); a++) { const ALT& alt = tParam.alts[a]; if(!alt.snp()) continue; tParam.haplotypes.expand(); tParam.haplotypes.back().left = alt.pos; if(alt.deletion()) { tParam.haplotypes.back().right = alt.pos + alt.len - 1; } else { tParam.haplotypes.back().right = alt.pos; } tParam.haplotypes.back().alts.clear(); tParam.haplotypes.back().alts.push_back(a); } } continue; } } break; } tParam.done = true; if(tParam.mainThread) break; } } /// Construct a GFM from the given header parameters and string /// vector, optionally using a blockwise suffix sorter with the /// given 'bmax' and 'dcv' parameters. The string vector is /// ultimately joined and the joined string is passed to buildToDisk(). template template HGFM::HGFM( TStr& s, bool packed, int needEntireReverse, int32_t lineRate, int32_t offRate, int32_t ftabChars, int32_t localOffRate, int32_t localFtabChars, int nthreads, const string& snpfile, const string& htfile, const string& ssfile, const string& exonfile, const string& svfile, const string& repeatfile, const string& outfile, // base filename for EBWT files bool fw, bool useBlockwise, TIndexOffU bmax, TIndexOffU bmaxSqrtMult, TIndexOffU bmaxDivN, int dcv, EList& is, EList& szs, index_t sztot, const RefReadInParams& refparams, bool localIndex, EList* parent_szs, EList* parent_refnames, uint32_t seed, int32_t overrideOffRate, bool verbose, bool passMemExc, bool sanityCheck) : GFM(s, packed, needEntireReverse, lineRate, offRate, ftabChars, nthreads, snpfile, htfile, ssfile, exonfile, svfile, repeatfile, outfile, fw, useBlockwise, bmax, bmaxSqrtMult, bmaxDivN, dcv, is, szs, sztot, refparams, parent_szs, parent_refnames, seed, overrideOffRate, verbose, passMemExc, sanityCheck), _in5(NULL), _in6(NULL) { _in5Str = outfile + ".5." + gfm_ext; _in6Str = outfile + ".6." + gfm_ext; // const bool repeat_index = (parent_szs != NULL); int32_t local_lineRate; if(snpfile == "" && ssfile == "" && exonfile == "") { local_lineRate = local_lineRate_fm; } else { local_lineRate = local_lineRate_gfm; } // Open output files ofstream fout5(_in5Str.c_str(), ios::binary); if(!fout5.good()) { cerr << "Could not open index file for writing: \"" << _in5Str.c_str() << "\"" << endl << "Please make sure the directory exists and that permissions allow writing by" << endl << "HISAT2." << endl; throw 1; } ofstream fout6(_in6Str.c_str(), ios::binary); if(!fout6.good()) { cerr << "Could not open index file for writing: \"" << _in6Str.c_str() << "\"" << endl << "Please make sure the directory exists and that permissions allow writing by" << endl << "HISAT2." << endl; throw 1; } // Split the whole genome into a set of local indexes _nrefs = 0; _nlocalGFMs = 0; index_t cumlen = 0; typedef EList EList_RefRecord; ELList all_local_recs; if(localIndex) { // For each unambiguous stretch... for(index_t i = 0; i < szs.size(); i++) { const RefRecord& rec = szs[i]; if(rec.first) { if(_nrefs > 0) { // refLens_ links each reference sequence with the total number // of ambiguous and unambiguous characters in it. _refLens.push_back(cumlen); } cumlen = 0; _nrefs++; all_local_recs.expand(); assert_eq(_nrefs, all_local_recs.size()); } else if(i == 0) { cerr << "First record in reference index file was not marked as " << "'first'" << endl; throw 1; } assert_gt(_nrefs, 0); assert_eq(_nrefs, all_local_recs.size()); EList& ref_local_recs = all_local_recs[_nrefs-1]; index_t next_cumlen = cumlen + rec.off + rec.len; index_t local_off = (cumlen / local_index_interval) * local_index_interval; if(local_off >= local_index_interval) { local_off -= local_index_interval; } for(;local_off < next_cumlen; local_off += local_index_interval) { if(local_off + local_index_size <= cumlen) { continue; } index_t local_idx = local_off / local_index_interval; if(local_idx >= ref_local_recs.size()) { assert_eq(local_idx, ref_local_recs.size()); ref_local_recs.expand(); _nlocalGFMs++; } assert_lt(local_idx, ref_local_recs.size()); EList_RefRecord& local_recs = ref_local_recs[local_idx]; assert_gt(local_off + local_index_size, cumlen); local_recs.expand(); if(local_off + local_index_size < cumlen + rec.off) { local_recs.back().off = local_off + local_index_size - std::max(local_off, cumlen); local_recs.back().len = 0; } else { if(local_off < cumlen + rec.off) { local_recs.back().off = rec.off - (local_off > cumlen ? local_off - cumlen : 0); } else { local_recs.back().off = 0; } local_recs.back().len = std::min(next_cumlen, local_off + local_index_size) - std::max(local_off, cumlen + rec.off); } local_recs.back().first = (local_recs.size() == 1); } cumlen = next_cumlen; } // Store a cap entry for the end of the last reference seq _refLens.push_back(cumlen); #ifndef NDEBUG EList temp_szs; index_t temp_sztot = 0; index_t temp_nlocalGFMs = 0; for(size_t tidx = 0; tidx < all_local_recs.size(); tidx++) { assert_lt(tidx, _refLens.size()); EList& ref_local_recs = all_local_recs[tidx]; assert_eq((_refLens[tidx] + local_index_interval - 1) / local_index_interval, ref_local_recs.size()); temp_szs.expand(); temp_szs.back().off = 0; temp_szs.back().len = 0; temp_szs.back().first = true; index_t temp_ref_len = 0; index_t temp_ref_sztot = 0; temp_nlocalGFMs += ref_local_recs.size(); for(size_t i = 0; i < ref_local_recs.size(); i++) { EList_RefRecord& local_recs = ref_local_recs[i]; index_t local_len = 0; for(size_t j = 0; j < local_recs.size(); j++) { assert(local_recs[j].off != 0 || local_recs[j].len != 0); assert(j != 0 || local_recs[j].first); RefRecord local_rec = local_recs[j]; if(local_len < local_index_interval && local_recs[j].off > 0){ if(local_len + local_recs[j].off > local_index_interval) { temp_ref_len += (local_index_interval - local_len); local_rec.off = local_index_interval - local_len; } else { temp_ref_len += local_recs[j].off; } } else { local_rec.off = 0; } local_len += local_recs[j].off; if(local_len < local_index_interval && local_recs[j].len > 0) { if(local_len + local_recs[j].len > local_index_interval) { temp_ref_len += (local_index_interval - local_len); temp_ref_sztot += (local_index_interval - local_len); local_rec.len = local_index_interval - local_len; } else { temp_ref_len += local_recs[j].len; temp_ref_sztot += local_recs[j].len; } } else { local_rec.len = 0; } local_len += local_recs[j].len; if(local_rec.off > 0) { if(temp_szs.back().len > 0) { temp_szs.expand(); temp_szs.back().off = local_rec.off; temp_szs.back().len = local_rec.len; temp_szs.back().first = false; } else { temp_szs.back().off += local_rec.off; temp_szs.back().len = local_rec.len; } } else if(local_rec.len > 0) { temp_szs.back().len += local_rec.len; } } if(i + 2 < ref_local_recs.size()) { assert_eq(local_len, local_index_size); assert_eq(temp_ref_len % local_index_interval, 0); } else if (i + 1 < ref_local_recs.size()) { assert_leq(local_len, local_index_size); assert_geq(local_len, local_index_interval); } else { assert_eq(local_len % local_index_interval, _refLens[tidx] % local_index_interval); } } assert_eq(temp_ref_len, _refLens[tidx]); temp_sztot += temp_ref_sztot; } assert_eq(temp_sztot, sztot); for(size_t i = 0; i < temp_szs.size(); i++) { assert_lt(i, szs.size()); assert_eq(temp_szs[i].off, szs[i].off); assert_eq(temp_szs[i].len, szs[i].len); assert_eq(temp_szs[i].first, szs[i].first); } assert_eq(temp_szs.size(), szs.size()); assert_eq(_nlocalGFMs, temp_nlocalGFMs); #endif } uint32_t be = this->toBe(); assert(fout5.good()); assert(fout6.good()); // const local_index_t new_localFtabChars = (repeat_index ? 4 : localFtabChars); const local_index_t new_localFtabChars = localFtabChars; // When building an Ebwt, these header parameters are known // "up-front", i.e., they can be written to disk immediately, // before we join() or buildToDisk() writeI32(fout5, 1, be); // endian hint for priamry stream writeI32(fout6, 1, be); // endian hint for secondary stream writeIndex(fout5, _nlocalGFMs, be); // number of local Ebwts writeI32(fout5, local_lineRate, be); // 2^lineRate = size in bytes of 1 line writeI32(fout5, 2, be); // not used writeI32(fout5, (int32_t)localOffRate, be); // every 2^offRate chars is "marked" writeI32(fout5, (int32_t)new_localFtabChars, be); // number of 2-bit chars used to address ftab int32_t flags = 1; if(this->_gh._entireReverse) flags |= GFM_ENTIRE_REV; writeI32(fout5, -flags, be); // BTL: chunkRate is now deprecated if(localIndex) { assert_gt(this->_nthreads, 0); AutoArray threads(this->_nthreads - 1); EList tParams; tParams.reserveExact((size_t)this->_nthreads); for(index_t t = 0; t < (index_t)this->_nthreads; t++) { tParams.expand(); tParams.back().s.clear(); tParams.back().rg = NULL; tParams.back().pg = NULL; tParams.back().file = outfile; tParams.back().done = true; tParams.back().last = false; tParams.back().dcv = 1024; tParams.back().seed = seed; if(t + 1 < (index_t)this->_nthreads) { tParams.back().mainThread = false; threads[t] = new tthread::thread(gbwt_worker, (void*)&tParams.back()); } else { tParams.back().mainThread = true; } } // build local FM indexes index_t curr_sztot = 0; EList > alts; for(size_t tidx = 0; tidx < _refLens.size(); tidx++) { index_t refLen = _refLens[tidx]; index_t local_offset = 0; _localGFMs.expand(); assert_lt(tidx, _localGFMs.size()); while(local_offset < refLen) { index_t t = 0; while(local_offset < refLen && t < (index_t)this->_nthreads) { assert_lt(t, tParams.size()); ThreadParam& tParam = tParams[t]; tParam.index_size = std::min(refLen - local_offset, local_index_size); assert_lt(tidx, all_local_recs.size()); assert_lt(local_offset / local_index_interval, all_local_recs[tidx].size()); EList_RefRecord& local_szs = all_local_recs[tidx][local_offset / local_index_interval]; tParam.conv_local_szs.clear(); index_t local_len = 0, local_sztot = 0, local_sztot_interval = 0; for(size_t i = 0; i < local_szs.size(); i++) { assert(local_szs[i].off != 0 || local_szs[i].len != 0); assert(i != 0 || local_szs[i].first); tParam.conv_local_szs.push_back(local_szs[i]); local_len += local_szs[i].off; if(local_len < local_index_interval && local_szs[i].len > 0) { if(local_len + local_szs[i].len > local_index_interval) { local_sztot_interval += (local_index_interval - local_len); } else { local_sztot_interval += local_szs[i].len; } } local_sztot += local_szs[i].len; local_len += local_szs[i].len; } // Extract sequence corresponding to this local index tParam.s.resize(local_sztot); if(refparams.reverse == REF_READ_REVERSE) { tParam.s.install(s.buf() + s.length() - curr_sztot - local_sztot, local_sztot); } else { tParam.s.install(s.buf() + curr_sztot, local_sztot); } // Extract ALTs corresponding to this local index map alt_map; tParam.alts.clear(); ALT alt; alt.pos = curr_sztot; index_t alt_i = (index_t)this->_alts.bsearchLoBound(alt); for(; alt_i < this->_alts.size(); alt_i++) { const ALT& alt = this->_alts[alt_i]; if(alt.snp()) { if(alt.mismatch()) { if(curr_sztot + local_sztot <= alt.pos) break; } else if(alt.insertion()) { if(curr_sztot + local_sztot < alt.pos) break; } else { assert(alt.deletion()); if(curr_sztot + local_sztot < alt.pos + alt.len) break; } if(curr_sztot <= alt.pos) { alt_map[alt_i] = (index_t)tParam.alts.size(); tParam.alts.push_back(alt); tParam.alts.back().pos -= curr_sztot; } } else if(alt.splicesite()) { if(alt.excluded) continue; if(curr_sztot + local_sztot <= alt.right + 1) continue; if(curr_sztot <= alt.left) { tParam.alts.push_back(alt); tParam.alts.back().left -= curr_sztot; tParam.alts.back().right -= curr_sztot; } } else { assert(alt.exon()); } } // Extract haplotypes tParam.haplotypes.clear(); Haplotype haplotype; haplotype.left = curr_sztot; index_t haplotpye_i = (index_t)this->_haplotypes.bsearchLoBound(haplotype); for(; haplotpye_i < this->_haplotypes.size(); haplotpye_i++) { const Haplotype& haplotype = this->_haplotypes[haplotpye_i]; if(curr_sztot + local_sztot <= haplotype.right) continue; if(curr_sztot <= haplotype.left) { tParam.haplotypes.push_back(haplotype); tParam.haplotypes.back().left -= curr_sztot; tParam.haplotypes.back().right -= curr_sztot; for(index_t a = 0; a < tParam.haplotypes.back().alts.size(); a++) { index_t alt_i = tParam.haplotypes.back().alts[a]; if(alt_map.find(alt_i) == alt_map.end()) { assert(false); tParam.haplotypes.pop_back(); break; } tParam.haplotypes.back().alts[a] = alt_map[alt_i]; } } } tParam.local_offset = local_offset; tParam.curr_sztot = curr_sztot; tParam.local_sztot = local_sztot; assert(tParam.rg == NULL); assert(tParam.pg == NULL); tParam.done = false; curr_sztot += local_sztot_interval; local_offset += local_index_interval; t++; } if(!tParams.back().done) { gbwt_worker((void*)&tParams.back()); } for(index_t t2 = 0; t2 < t; t2++) { ThreadParam& tParam = tParams[t2]; while(!tParam.done) { #if defined(_TTHREAD_WIN32_) Sleep(1); #elif defined(_TTHREAD_POSIX_) const static timespec ts = {0, 1000000}; // 1 millisecond nanosleep(&ts, NULL); #endif } LocalGFM( tParam.s, tParam.sa, tParam.pg, (index_t)tidx, tParam.local_offset, tParam.curr_sztot, tParam.alts, tParam.index_size, packed, needEntireReverse, local_lineRate, localOffRate, // suffix-array sampling rate new_localFtabChars, // number of chars in initial arrow-pair calc outfile, // basename for .?.ebwt files fw, // fw dcv, // difference-cover period tParam.conv_local_szs, // list of reference sizes tParam.local_sztot, // total size of all unambiguous ref chars refparams, // reference read-in parameters seed, // pseudo-random number generator seed fout5, fout6, -1, // override offRate false, // be silent passMemExc, // pass exceptions up to the toplevel so that we can adjust memory settings automatically sanityCheck); // verify results and internal consistency tParam.s.clear(); if(tParam.rg != NULL) { assert(tParam.pg != NULL); delete tParam.rg; tParam.rg = NULL; delete tParam.pg; tParam.pg = NULL; } } } } assert_eq(curr_sztot, sztot); if(this->_nthreads > 1) { for(index_t i = 0; i + 1 < (index_t)this->_nthreads; i++) { tParams[i].last = true; threads[i]->join(); } } } fout5 << '\0'; fout5.flush(); fout6.flush(); if(fout5.fail() || fout6.fail()) { cerr << "An error occurred writing the index to disk. Please check if the disk is full." << endl; throw 1; } VMSG_NL("Returning from initFromVector"); // Close output files fout5.flush(); int64_t tellpSz5 = (int64_t)fout5.tellp(); VMSG_NL("Wrote " << fout5.tellp() << " bytes to primary GFM file: " << _in5Str.c_str()); fout5.close(); bool err = false; if(tellpSz5 > fileSize(_in5Str.c_str())) { err = true; cerr << "Index is corrupt: File size for " << _in5Str.c_str() << " should have been " << tellpSz5 << " but is actually " << fileSize(_in5Str.c_str()) << "." << endl; } fout6.flush(); int64_t tellpSz6 = (int64_t)fout6.tellp(); VMSG_NL("Wrote " << fout6.tellp() << " bytes to secondary GFM file: " << _in6Str.c_str()); fout6.close(); if(tellpSz6 > fileSize(_in6Str.c_str())) { err = true; cerr << "Index is corrupt: File size for " << _in6Str.c_str() << " should have been " << tellpSz6 << " but is actually " << fileSize(_in6Str.c_str()) << "." << endl; } if(err) { cerr << "Please check if there is a problem with the disk or if disk is full." << endl; throw 1; } // Reopen as input streams VMSG_NL("Re-opening _in5 and _in5 as input streams"); if(this->_sanity) { VMSG_NL("Sanity-checking ht2"); assert(!this->isInMemory()); readIntoMemory( fw ? -1 : needEntireReverse, // 1 -> need the reverse to be reverse-of-concat true, // load SA sample (_offs[])? true, // load ftab (_ftab[] & _eftab[])? true, // load r-starts (_rstarts[])? false, // just load header? NULL, // Params object to fill false, // mm sweep? true, // load names? false); // verbose startup? sanityCheckAll(refparams.reverse); evictFromMemory(); assert(!this->isInMemory()); } VMSG_NL("Returning from HGFM constructor"); } /** * Read an Ebwt from file with given filename. */ template void HGFM::readIntoMemory( int needEntireRev, bool loadSASamp, bool loadFtab, bool loadRstarts, bool justHeader, GFMParams *params, bool mmSweep, bool loadNames, bool startVerbose) { PARENT_CLASS::readIntoMemory(needEntireRev, loadSASamp, loadFtab, loadRstarts, justHeader || needEntireRev == 1, params, mmSweep, loadNames, startVerbose); bool switchEndian; // dummy; caller doesn't care #ifdef BOWTIE_MM char *mmFile[] = { NULL, NULL }; #endif if(_in5Str.length() > 0) { if(this->_verbose || startVerbose) { cerr << " About to open input files: "; logTime(cerr); } // Initialize our primary and secondary input-stream fields if(_in5 != NULL) fclose(_in5); if(this->_verbose || startVerbose) cerr << "Opening \"" << _in5Str.c_str() << "\"" << endl; if((_in5 = fopen(_in5Str.c_str(), "rb")) == NULL) { cerr << "Could not open index file " << _in5Str.c_str() << endl; } if(loadSASamp) { if(_in6 != NULL) fclose(_in6); if(this->_verbose || startVerbose) cerr << "Opening \"" << _in6Str.c_str() << "\"" << endl; if((_in6 = fopen(_in6Str.c_str(), "rb")) == NULL) { cerr << "Could not open index file " << _in6Str.c_str() << endl; } } if(this->_verbose || startVerbose) { cerr << " Finished opening input files: "; logTime(cerr); } #ifdef BOWTIE_MM if(this->_useMm /*&& !justHeader*/) { const char *names[] = {_in5Str.c_str(), _in6Str.c_str()}; int fds[] = { fileno(_in5), fileno(_in6) }; for(int i = 0; i < (loadSASamp ? 2 : 1); i++) { if(this->_verbose || startVerbose) { cerr << " ¯ " << (i+1) << ": "; logTime(cerr); } struct stat sbuf; if (stat(names[i], &sbuf) == -1) { perror("stat"); cerr << "Error: Could not stat index file " << names[i] << " prior to memory-mapping" << endl; throw 1; } mmFile[i] = (char*)mmap((void *)0, (size_t)sbuf.st_size, PROT_READ, MAP_SHARED, fds[(size_t)i], 0); if(mmFile[i] == (void *)(-1)) { perror("mmap"); cerr << "Error: Could not memory-map the index file " << names[i] << endl; throw 1; } if(mmSweep) { int sum = 0; for(off_t j = 0; j < sbuf.st_size; j += 1024) { sum += (int) mmFile[i][j]; } if(startVerbose) { cerr << " Swept the memory-mapped ebwt index file 1; checksum: " << sum << ": "; logTime(cerr); } } } mmFile5_ = mmFile[0]; mmFile6_ = loadSASamp ? mmFile[1] : NULL; } #endif } #ifdef BOWTIE_MM else if(this->_useMm && !justHeader) { mmFile[0] = mmFile5_; mmFile[1] = mmFile6_; } if(this->_useMm && !justHeader) { assert(mmFile[0] == mmFile5_); assert(mmFile[1] == mmFile6_); } #endif if(this->_verbose || startVerbose) { cerr << " Reading header: "; logTime(cerr); } // Read endianness hints from both streams size_t bytesRead = 0, bytesRead2 = 4; switchEndian = false; uint32_t one = readU32(_in5, switchEndian); // 1st word of primary stream bytesRead += 4; if(loadSASamp) { #ifndef NDEBUG assert_eq(one, readU32(_in6, switchEndian)); // should match! #else readU32(_in6, switchEndian); #endif } if(one != 1) { assert_eq((1u<<24), one); assert_eq(1, endianSwapU32(one)); switchEndian = true; } // Can't switch endianness and use memory-mapped files; in order to // support this, someone has to modify the file to switch // endiannesses appropriately, and we can't do this inside Bowtie // or we might be setting up a race condition with other processes. if(switchEndian && this->_useMm) { cerr << "Error: Can't use memory-mapped files when the index is the opposite endianness" << endl; throw 1; } _nlocalGFMs = readIndex(_in5, switchEndian); bytesRead += sizeof(index_t); int32_t lineRate = readI32(_in5, switchEndian); bytesRead += 4; readI32(_in5, switchEndian); bytesRead += 4; int32_t offRate = readI32(_in5, switchEndian); bytesRead += 4; // TODO: add isaRate to the actual file format (right now, the // user has to tell us whether there's an ISA sample and what the // sampling rate is. int32_t ftabChars = readI32(_in5, switchEndian); bytesRead += 4; /*int32_t flag =*/ readI32(_in5, switchEndian); bytesRead += 4; if(this->_verbose || startVerbose) { cerr << " number of local indexes: " << _nlocalGFMs << endl << " local offRate: " << offRate << endl << " local ftabLen: " << (1 << (2 * ftabChars)) << endl << " local ftabSz: " << (2 << (2 * ftabChars)) << endl ; } clearLocalGFMs(); index_t tidx = 0, localOffset = 0, joinedOffset = 0; string base = ""; for(size_t i = 0; i < _nlocalGFMs; i++) { LocalGFM *localGFM = new LocalGFM(base, NULL, _in5, _in6, mmFile5_, mmFile6_, tidx, localOffset, joinedOffset, switchEndian, bytesRead, bytesRead2, needEntireRev, this->fw_, -1, // overrideOffRate -1, // offRatePlus (uint32_t)lineRate, (uint32_t)offRate, (uint32_t)ftabChars, this->_useMm, this->useShmem_, mmSweep, loadNames, loadSASamp, loadFtab, loadRstarts, false, // _verbose false, this->_passMemExc, this->_sanity, false); // use haplotypes? if(tidx >= _localGFMs.size()) { assert_eq(tidx, _localGFMs.size()); _localGFMs.expand(); } assert_eq(tidx + 1, _localGFMs.size()); _localGFMs.back().push_back(localGFM); } #ifdef BOWTIE_MM fseek(_in5, 0, SEEK_SET); fseek(_in6, 0, SEEK_SET); #else rewind(_in5); rewind(_in6); #endif } #endif /*HGFM_H_*/ hisat2-2.2.1/hi_aligner.h000066400000000000000000011001711370663763200151760ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef HI_ALIGNER_H_ #define HI_ALIGNER_H_ #include #include #include #include "qual.h" #include "ds.h" #include "sstring.h" #include "alphabet.h" #include "edit.h" #include "read.h" // Threading is necessary to synchronize the classes that dump // intermediate alignment results to files. Otherwise, all data herein // is constant and shared, or per-thread. #include "threading.h" #include "aligner_result.h" #include "aligner_cache.h" #include "scoring.h" #include "mem_ids.h" #include "simple_func.h" #include "aligner_driver.h" #include "aligner_sw_driver.h" #include "group_walk.h" #include "tp.h" #include "gp.h" // Allow longer introns for long anchored reads involving canonical splice sites inline uint32_t MaxIntronLen(uint32_t anchor, uint32_t minAnchorLen) { uint32_t intronLen = 0; if(anchor >= minAnchorLen) { if(anchor < 2) anchor = 2; uint32_t shift = (anchor << 1) - 4; shift = min(max(shift, 13), 30); intronLen = 1 << shift; } return intronLen; } inline float intronLen_prob(uint32_t anchor, uint32_t intronLen, uint32_t maxIntronLen) { uint32_t expected_intron_len = maxIntronLen; if(anchor < 14) expected_intron_len = 1 << ((anchor << 1) + 4); if(expected_intron_len > maxIntronLen) expected_intron_len = maxIntronLen; assert_gt(expected_intron_len, 0); float result = ((float)intronLen) / ((float)expected_intron_len); if(result > 1.0f) result = 1.0f; return result; } // Allow longer introns for long anchored reads involving non-canonical splice sites inline uint32_t MaxIntronLen_noncan(uint32_t anchor, uint32_t minAnchorLen_noncan) { uint32_t intronLen = 0; if(anchor >= minAnchorLen_noncan) { if(anchor < 5) anchor = 5; uint32_t shift = (anchor << 1) - 10; shift = min(shift, 30); intronLen = 1 << shift; } return intronLen; } inline float intronLen_prob_noncan(uint32_t anchor, uint32_t intronLen, uint32_t maxIntronLen) { uint32_t expected_intron_len = maxIntronLen; if(anchor < 16) expected_intron_len = 1 << (anchor << 1); if(expected_intron_len > maxIntronLen) expected_intron_len = maxIntronLen; assert_gt(expected_intron_len, 0); float result = ((float)intronLen) / ((float)expected_intron_len); if(result > 1.0f) result = 1.0f; return result; } /** * Hit types for BWTHit class below * Three hit types to anchor a read on the genome * */ enum { CANDIDATE_HIT = 1, PSEUDOGENE_HIT, ANCHOR_HIT, }; /** * Simple struct for holding a partial alignment for the read * The alignment locations are represented by FM offsets [top, bot), * and later genomic offsets are calculated when necessary */ template struct BWTHit { BWTHit() { reset(); } void reset() { _top = _bot = 0; _node_top = _node_bot = 0; _node_iedge_count.clear(); _fw = true; _bwoff = (index_t)INDEX_MAX; _len = 0; _coords.clear(); _anchor_examined = false; _hit_type = CANDIDATE_HIT; } void init( index_t top, index_t bot, index_t node_top, index_t node_bot, const EList >& node_iedge_count, bool fw, uint32_t bwoff, uint32_t len, index_t hit_type = CANDIDATE_HIT) { assert_leq(node_bot - node_top, bot - top); #ifndef NDEBUG if(node_bot - node_top < bot - top) { assert_gt(node_iedge_count.size(), 0); } #endif _top = top; _bot = bot; _node_top = node_top; _node_bot = node_bot; _node_iedge_count = node_iedge_count; _fw = fw; _bwoff = bwoff; _len = len; _coords.clear(); _anchor_examined = false; _hit_type = hit_type; } bool hasGenomeCoords() const { return !_coords.empty(); } /** * Return true iff there is no hit. */ bool empty() const { return _bot <= _top; } /** * Higher score = higher priority. */ bool operator<(const BWTHit& o) const { return _len > o._len; } /** * Return the size of the alignments SA ranges. */ index_t size() const { assert_leq(_top, _bot); return _bot - _top; } index_t len() const { assert_gt(_len, 0); return _len; } #ifndef NDEBUG /** * Check that hit is sane w/r/t read. */ bool repOk(const Read& rd) const { assert_gt(_bot, _top); assert_neq(_bwoff, (index_t)INDEX_MAX); assert_gt(_len, 0); return true; } #endif index_t _top; // start of the range in the FM index index_t _bot; // end of the range in the FM index index_t _node_top; index_t _node_bot; EList > _node_iedge_count; bool _fw; // whether read is forward or reverse complemented index_t _bwoff; // current base of a read to search from the right end index_t _len; // read length EList _coords; // genomic offsets corresponding to [_top, _bot) bool _anchor_examined; // whether or not this hit is examined index_t _hit_type; // hit type (anchor hit, pseudogene hit, or candidate hit) }; /** * Simple struct for holding alignments for the read * The alignments are represented by chains of BWTHits */ template struct ReadBWTHit { ReadBWTHit() { reset(); } void reset() { _fw = true; _len = 0; _cur = 0; _done = false; _numPartialSearch = 0; _numUniqueSearch = 0; _repeat = false; _partialHits.clear(); } void init( bool fw, index_t len) { _fw = fw; assert_gt(len, 0); _len = len; _cur = 0; _done = false; _numPartialSearch = 0; _numUniqueSearch = 0; _repeat = false; _partialHits.clear(); } bool done() { #ifndef NDEBUG assert_gt(_len, 0); if(_cur >= _len) { assert(_done); } #endif return _done; } void done(bool done) { assert(!_done); assert(done); _done = done; } index_t len() const { return _len; } index_t cur() const { return _cur; } bool repeat() const { return _repeat; } index_t offsetSize() { return (index_t)_partialHits.size(); } size_t numPartialSearch() { return _numPartialSearch; } index_t numActualPartialSearch() { assert_leq(_numUniqueSearch, _numPartialSearch); return (index_t)(_numPartialSearch - _numUniqueSearch); } bool width(index_t offset_) { assert_lt(offset_, _partialHits.size()); return _partialHits[offset_].size(); } bool hasGenomeCoords(index_t offset_) { assert_lt(offset_, _partialHits.size()); index_t width_ = width(offset_); if(width_ == 0) { return true; } else { return _partialHits[offset_].hasGenomeCoords(); } } bool hasAllGenomeCoords() { if(_cur < _len) return false; if(_partialHits.size() <= 0) return false; for(size_t oi = 0; oi < _partialHits.size(); oi++) { if(!_partialHits[oi].hasGenomeCoords()) return false; } return true; } /** * */ index_t minWidth(index_t& offset) const { index_t minWidth_ = (index_t)INDEX_MAX; index_t minWidthLen_ = 0; for(size_t oi = 0; oi < _partialHits.size(); oi++) { const BWTHit& hit = _partialHits[oi]; if(hit.empty()) continue; // if(!hit.hasGenomeCoords()) continue; assert_gt(hit.size(), 0); if((minWidth_ > hit.size()) || (minWidth_ == hit.size() && minWidthLen_ < hit.len())) { minWidth_ = hit.size(); minWidthLen_ = hit.len(); offset = (index_t)oi; } } return minWidth_; } // add policy for calculating a search score int64_t searchScore(index_t minK) { int64_t score = 0; const int64_t penaltyPerOffset = minK * minK; for(size_t i = 0; i < _partialHits.size(); i++) { index_t len = _partialHits[i]._len; score += (len * len); } assert_geq(_numPartialSearch, _partialHits.size()); index_t actualPartialSearch = numActualPartialSearch(); score -= (actualPartialSearch * penaltyPerOffset); score -= (1 << (actualPartialSearch << 1)); return score; } BWTHit& getPartialHit(index_t offset_) { assert_lt(offset_, _partialHits.size()); return _partialHits[offset_]; } bool adjustOffset(index_t minK) { assert_gt(_partialHits.size(), 0); const BWTHit& hit = _partialHits.back(); if(hit.len() >= minK + 3) { return false; } assert_geq(_cur, hit.len()); index_t origCur = _cur - hit.len(); _cur = origCur + max(hit.len(), minK + 1) - minK; _partialHits.pop_back(); return true; } void setOffset(index_t offset) { assert_lt(offset, _len); _cur = offset; } #ifndef NDEBUG /** */ bool repOk() const { for(size_t i = 0; i < _partialHits.size(); i++) { if(i == 0) { assert_geq(_partialHits[i]._bwoff, 0); } if(i + 1 < _partialHits.size()) { assert_leq(_partialHits[i]._bwoff + _partialHits[i]._len, _partialHits[i+1]._bwoff); } else { assert_eq(i+1, _partialHits.size()); assert_eq(_partialHits[i]._bwoff + _partialHits[i]._len, _cur); } } return true; } #endif bool _fw; index_t _len; index_t _cur; bool _done; index_t _numPartialSearch; index_t _numUniqueSearch; index_t _cur_local; bool _repeat; EList > _partialHits; }; /** * this is per-thread data, which are shared by GenomeHit classes * the main purpose of this struct is to avoid extensive use of memory related functions * such as new and delete - those are really slow and lock based */ template struct SharedTempVars { SStringExpandable raw_refbuf; SStringExpandable raw_refbuf2; EList temp_scores; EList temp_scores2; // Align with alternatives EList > ssOffs; EList > offDiffs; EList > raw_refbufs; EList alt_edits; ELList candidate_edits; ELList > ht_llist; Haplotype cmp_ht; ASSERT_ONLY(SStringExpandable destU32); ASSERT_ONLY(BTDnaString editstr); ASSERT_ONLY(BTDnaString partialseq); ASSERT_ONLY(BTDnaString refstr); ASSERT_ONLY(EList reflens); ASSERT_ONLY(EList refoffs); LinkedEList > raw_edits; LinkedEList > > raw_ht_lists; }; /** * GenomeHit represents read alignment or alignment of a part of a read * Two GenomeHits that represents alignments of different parts of a read * can be combined together. Also, GenomeHit can be extended in both directions. */ template struct GenomeHit { GenomeHit() : _fw(false), _rdoff((index_t)INDEX_MAX), _len((index_t)INDEX_MAX), _trim5(0), _trim3(0), _tidx((index_t)INDEX_MAX), _toff((index_t)INDEX_MAX), _joinedOff((index_t)INDEX_MAX), _repeat(false), _edits(NULL), _ht_list(NULL), _score(MIN_I64), _localscore(MIN_I64), _hitcount(1), _edits_node(NULL), _ht_list_node(NULL), _sharedVars(NULL) { } GenomeHit(const GenomeHit& otherHit) : _edits(NULL), _ht_list(NULL), _hitcount(1), _edits_node(NULL), _ht_list_node(NULL), _sharedVars(NULL) { init(otherHit._fw, otherHit._rdoff, otherHit._len, otherHit._trim5, otherHit._trim3, otherHit._tidx, otherHit._toff, otherHit._joinedOff, *(otherHit._sharedVars), otherHit._repeat, otherHit._edits, otherHit._ht_list, otherHit._score, otherHit._localscore, otherHit._splicescore); } GenomeHit& operator=(const GenomeHit& otherHit) { if(this == &otherHit) return *this; init(otherHit._fw, otherHit._rdoff, otherHit._len, otherHit._trim5, otherHit._trim3, otherHit._tidx, otherHit._toff, otherHit._joinedOff, *(otherHit._sharedVars), otherHit._repeat, otherHit._edits, otherHit._ht_list, otherHit._score, otherHit._localscore, otherHit._splicescore); return *this; } ~GenomeHit() { if(_edits_node != NULL) { assert(_edits != NULL); assert(_sharedVars != NULL); _sharedVars->raw_edits.delete_node(_edits_node); _edits = NULL; _edits_node = NULL; } if(_ht_list_node != NULL) { assert(_ht_list != NULL); assert(_sharedVars != NULL); _sharedVars->raw_ht_lists.delete_node(_ht_list_node); _ht_list = NULL; _ht_list_node = NULL; } _sharedVars = NULL; } void init( bool fw, index_t rdoff, index_t len, index_t trim5, index_t trim3, index_t tidx, index_t toff, index_t joinedOff, SharedTempVars& sharedVars, bool repeat = false, EList* edits = NULL, EList >* ht_list = NULL, int64_t score = 0, int64_t localscore = 0, double splicescore = 0.0) { _fw = fw; _rdoff = rdoff; _len = len; _trim5 = trim5; _trim3 = trim3; _tidx = tidx; _toff = toff; _joinedOff = joinedOff; _repeat = repeat; _score = score; _localscore = localscore; _splicescore = splicescore; assert(_sharedVars == NULL || _sharedVars == &sharedVars); _sharedVars = &sharedVars; if(_edits == NULL) { assert(_edits_node == NULL); _edits_node = _sharedVars->raw_edits.new_node(); assert(_edits_node != NULL); _edits = &(_edits_node->payload); } assert(_edits != NULL); _edits->clear(); if(edits != NULL) *_edits = *edits; if(_ht_list == NULL) { assert(_ht_list_node == NULL); _ht_list_node = _sharedVars->raw_ht_lists.new_node(); assert(_ht_list_node != NULL); _ht_list = &(_ht_list_node->payload); } assert(_ht_list != NULL); _ht_list->clear(); if(ht_list != NULL) *_ht_list = *ht_list; _hitcount = 1; } bool inited() const { return _len >= 0 && _len < (index_t)INDEX_MAX; } /** * Check if it is compatible with another GenomeHit with respect to indels or introns */ bool compatibleWith( const GenomeHit& otherHit, index_t minIntronLen, index_t maxIntronLen, bool no_spliced_alignment = false) const; /** * Combine itself with another GenomeHit */ bool combineWith( const GenomeHit& otherHit, const Read& rd, const GFM& gfm, const BitPairReference& ref, const ALTDB& altdb, const RepeatDB& repeatdb, SpliceSiteDB& ssdb, SwAligner& swa, SwMetrics& swm, const Scoring& sc, TAlScore minsc, RandomSource& rnd, // pseudo-random source index_t minK_local, index_t minIntronLen, index_t maxIntronLen, index_t minAnchorLen, // minimum anchor length for canonical splice site index_t minAnchorLen_noncan, // minimum anchor length for non-canonical splice site const index_t maxAltsTried, const SpliceSite* spliceSite = NULL, // penalty for splice site bool no_spliced_alignment = false); /** * Extend the partial alignment (GenomeHit) bidirectionally */ bool extend( const Read& rd, const GFM& gfm, const BitPairReference& ref, const ALTDB& altdb, const RepeatDB& repeatdb, SpliceSiteDB& ssdb, SwAligner& swa, SwMetrics& swm, PerReadMetrics& prm, const Scoring& sc, TAlScore minsc, RandomSource& rnd, // pseudo-random source index_t minK_local, const TranscriptomePolicy& tpol, const GraphPolicy& gpol, index_t& leftext, index_t& rightext, index_t mm = 0); /** * Adjust alignment with respect to SNPs, usually updating Edits * */ static bool adjustWithALT( index_t rdoff, index_t len, const Coord& coord, SharedTempVars& sharedVars, EList >& genomeHits, const Read& rd, const GFM& gfm, const ALTDB& altdb, const BitPairReference& ref, const GraphPolicy& gpol); /** * Adjust alignment with respect to SNPs, usually updating Edits * */ bool adjustWithALT( const Read& rd, const GFM& gfm, const ALTDB& altdb, const BitPairReference& ref, const GraphPolicy& gpol); /* * */ static void findSSOffs( const GFM& gfm, const ALTDB& altdb, index_t start, index_t end, EList >& ssOffs); /* * Find offset differences due to deletions */ static index_t findOffDiffs( const GFM& gfm, const ALTDB& altdb, index_t start, index_t end, EList >& offDiffs); /* * */ static index_t alignWithALTs( const EList >& alts, const EList >& haplotypes, const EList& haplotype_maxrights, index_t joinedOff, const BTDnaString& rdseq, index_t base_rdoff, index_t rdoff, index_t rdlen, const BitPairReference& ref, SharedTempVars& sharedVar, index_t tidx, int rfoff, index_t rflen, bool left, const GraphPolicy& gpol, EList& edits, ELList >& ht_llist, EList >& ht_list, Haplotype& cmp_ht, ELList* candidate_edits = NULL, index_t mm = 0, index_t* numNs = NULL) { int best_rdoff = (int)rdoff; if(numNs != NULL) *numNs = 0; index_t numALTsTried = 0; EList& alt_edits = sharedVar.alt_edits; alt_edits = edits; index_t nedits = (index_t)edits.size(); if(candidate_edits != NULL) candidate_edits->clear(); ht_llist.clear(); // ht_llist.expand(); // ht_llist[0] = ht_list; alignWithALTs_recur( alts, haplotypes, haplotype_maxrights, joinedOff, rdseq, rdoff - base_rdoff, rdoff, rdlen, ref, sharedVar.raw_refbufs, ASSERT_ONLY(sharedVar.destU32,) alt_edits, best_rdoff, NULL, /* rfseq */ tidx, rfoff, rflen, left, edits, mm, ht_llist, cmp_ht, candidate_edits, 0, /* tmp_numNs */ numNs, 0, /* dep */ gpol, numALTsTried); index_t extlen = 0; if(left) { assert_geq(best_rdoff, -1); assert_leq(best_rdoff, (int)rdoff); extlen = rdoff - best_rdoff; } else { assert_leq(best_rdoff, (int)(rdoff + rdlen)); assert_geq(best_rdoff, (int)rdoff); extlen = best_rdoff - rdoff; } if(extlen > 0 && edits.size() > 0) { const Edit& f = edits.front(); if(f.pos + extlen == base_rdoff + 1) { if(f.type == EDIT_TYPE_READ_GAP || f.type == EDIT_TYPE_REF_GAP || f.type == EDIT_TYPE_SPL) { extlen = 0; } if(f.type == EDIT_TYPE_MM && f.chr == 'N') { extlen = 0; } } const Edit& b = edits.back(); if(extlen > 0 && b.pos == rdoff - base_rdoff + extlen - 1) { if(b.type == EDIT_TYPE_READ_GAP || b.type == EDIT_TYPE_REF_GAP) { extlen = 0; } } if(extlen == 0 && edits.size() > nedits) { if(left) { edits.erase(0, edits.size() - nedits); } else { edits.resize(nedits); } } } return extlen; } /* * */ static index_t alignWithALTs_recur( const EList >& alts, const EList >& haplotypes, const EList& haplotype_maxrights, index_t joinedOff, const BTDnaString& rdseq, index_t rdoff_add, index_t rdoff, index_t rdlen, const BitPairReference& ref, EList >& raw_refbufs, ASSERT_ONLY(SStringExpandable destU32,) EList& tmp_edits, int& best_rdoff, const char* rfseq, index_t tidx, int rfoff, index_t rflen, bool left, EList& edits, index_t mm, ELList >& ht_llist, Haplotype& cmp_ht, ELList* candidate_edits, index_t tmp_numNs, index_t* numNs, index_t dep, const GraphPolicy& gpol, index_t& numALTsTried, ALT_TYPE prev_alt_type = ALT_NONE); /** * For alignment involving indel, move the indels * to the left most possible position */ void leftAlign(const Read& rd); index_t rdoff() const { return _rdoff; } index_t len() const { return _len; } index_t trim5() const { return _trim5; } index_t trim3() const { return _trim3; } void trim5(index_t trim5, const Read& rd, SpliceSiteDB& ssdb, const Scoring& sc, index_t minK_local, index_t minIntronLen, index_t maxIntronLen, index_t minAnchorLen, index_t minAnchorLen_noncan, const BitPairReference& ref) { assert_eq(_rdoff, trim5); assert_eq(_trim5, 0); _trim5 = trim5; calculateScore(rd, ssdb, sc, minK_local, minIntronLen, maxIntronLen, minAnchorLen, minAnchorLen_noncan, ref); } void trim3(index_t trim3, const Read& rd, SpliceSiteDB& ssdb, const Scoring& sc, index_t minK_local, index_t minIntronLen, index_t maxIntronLen, index_t minAnchorLen, index_t minAnchorLen_noncan, const BitPairReference& ref) { _trim3 = trim3; calculateScore(rd, ssdb, sc, minK_local, minIntronLen, maxIntronLen, minAnchorLen, minAnchorLen_noncan, ref); } // for repeat alignments // reverse fw void reverse(const Read& rd) { _fw = !_fw; index_t end = _trim5 + _rdoff + _len; assert_leq(end, rd.length()); _rdoff = rd.length() - end; index_t tmp_trim = _trim5; _trim5 = _trim3; _trim3 = tmp_trim; Edit::invertPoss(*_edits, rd.length()); // complements Edit::complement(*_edits); } index_t ref() const { return _tidx; } index_t refoff() const { return _toff; } index_t fw() const { return _fw; } bool repeat() const { return _repeat; } void repeat(bool repeat) { _repeat = repeat;} index_t hitcount() const { return _hitcount; } /** * Leftmost coordinate */ Coord coord() const { return Coord(_tidx, _toff, _fw); } int64_t score() const { return _score; } int64_t localscore() const { return _localscore; } double splicescore() const { return _splicescore; } const EList& edits() const { return *_edits; } /** * Retrieve the partial alignment from the left until indel or intron */ void getLeft(index_t& rdoff, index_t& len, index_t& toff, int64_t* score = NULL, const Read* rd = NULL, const Scoring* sc = NULL) const { assert(inited()); toff = _toff, rdoff = _rdoff, len = _len; const BTString* qual = NULL; if(score != NULL) { assert(rd != NULL); assert(sc != NULL); *score = 0; qual = &(_fw ? rd->qual : rd->qualRev); } for(index_t i = 0; i < _edits->size(); i++) { const Edit& edit = (*_edits)[i]; if(edit.type == EDIT_TYPE_SPL || edit.type == EDIT_TYPE_READ_GAP || edit.type == EDIT_TYPE_REF_GAP || (edit.type == EDIT_TYPE_MM && edit.snpID != (index_t)INDEX_MAX)) { len = edit.pos; break; } if(score != NULL) { if(edit.type == EDIT_TYPE_MM) { assert(qual != NULL); if(edit.snpID == (index_t)INDEX_MAX) { *score += sc->score( dna2col[edit.qchr] - '0', asc2dnamask[edit.chr], (*qual)[this->_rdoff + edit.pos] - 33); } } } } assert_geq(len, 0); } /** * Retrieve the partial alignment from the right until indel or intron */ void getRight(index_t& rdoff, index_t& len, index_t& toff, int64_t* score = NULL, const Read* rd = NULL, const Scoring* sc = NULL) const { assert(inited()); toff = _toff, rdoff = _rdoff, len = _len; const BTString* qual = NULL; if(score != NULL) { assert(rd != NULL); assert(sc != NULL); *score = 0; qual = &(_fw ? rd->qual : rd->qualRev); } if(_edits->size() == 0) return; for(int i = (int)_edits->size() - 1; i >= 0; i--) { const Edit& edit = (*_edits)[i]; if(edit.type == EDIT_TYPE_SPL || edit.type == EDIT_TYPE_READ_GAP || edit.type == EDIT_TYPE_REF_GAP || (edit.type == EDIT_TYPE_MM && edit.snpID != (index_t)INDEX_MAX)) { rdoff = _rdoff + edit.pos; assert_lt(edit.pos, _len); len = _len - edit.pos; if(edit.type == EDIT_TYPE_REF_GAP) { assert_lt(edit.pos + 1, _len); assert_gt(len, 1); rdoff++; len--; } else if(edit.type == EDIT_TYPE_MM) { assert_leq(edit.pos + 1, _len); assert_geq(len, 1); rdoff++; len--; } toff = getRightOff() - len; break; } if(score != NULL) { if(edit.type == EDIT_TYPE_MM) { assert(qual != NULL); if(edit.snpID == (index_t)INDEX_MAX) { *score += sc->score( dna2col[edit.qchr] - '0', asc2dnamask[edit.chr], (*qual)[this->_rdoff + edit.pos] - 33); } } } } assert_geq(len, 0); } /** * Retrieve the genomic offset of the right end */ index_t getRightOff() const { assert(inited()); index_t toff = _toff + _len; for(index_t i = 0; i < _edits->size(); i++) { const Edit& ed = (*_edits)[i]; if(ed.type == EDIT_TYPE_SPL) { toff += ed.splLen; } else if(ed.type == EDIT_TYPE_READ_GAP) { toff++; } else if(ed.type == EDIT_TYPE_REF_GAP) { assert_gt(toff, 0); toff--; } } return toff; } /** * Retrieve left anchor length and number of edits in the anchor */ void getLeftAnchor(index_t& leftanchor, index_t& nedits) const { assert(inited()); leftanchor = _len; nedits = 0; for(index_t i = 0; i < _edits->size(); i++) { const Edit& edit = (*_edits)[i]; if(edit.type == EDIT_TYPE_SPL) { leftanchor = edit.pos; break; } else if(edit.type == EDIT_TYPE_MM || edit.type == EDIT_TYPE_READ_GAP || edit.type == EDIT_TYPE_REF_GAP) { nedits++; } } } /** * Retrieve right anchor length and number of edits in the anchor */ void getRightAnchor(index_t& rightanchor, index_t& nedits) const { rightanchor = _len; nedits = 0; if(_edits->size() == 0) return; for(int i = (int)_edits->size() - 1; i >= 0; i--) { const Edit& edit = (*_edits)[i]; if(edit.type == EDIT_TYPE_SPL) { rightanchor = _len - edit.pos - 1; break; } else if(edit.type == EDIT_TYPE_MM || edit.type == EDIT_TYPE_READ_GAP || edit.type == EDIT_TYPE_REF_GAP) { nedits++; } } } /** * Is it spliced alignment? * Return: first is spliced-alignment, second is spliced-alignment to known transcripts */ pair spliced() const { pair result(false, true); for(index_t i = 0; i < _edits->size(); i++) { const Edit& e = (*_edits)[i]; if(e.type == EDIT_TYPE_SPL) { result.first = true; result.second &= e.knownSpl; } } result.second &= result.first; return result; } /** * */ bool spliced_consistently() const { int splDir = SPL_UNKNOWN; for(index_t i = 0; i < _edits->size(); i++) { const Edit& edit = (*_edits)[i]; if(edit.type != EDIT_TYPE_SPL) continue; if(splDir != SPL_UNKNOWN) { if(edit.splDir != SPL_UNKNOWN) { if(splDir == SPL_FW || splDir == SPL_SEMI_FW) { if(edit.splDir != SPL_FW && edit.splDir != SPL_SEMI_FW) return false; } if(splDir == SPL_RC || splDir == SPL_SEMI_RC) { if(edit.splDir != SPL_RC && edit.splDir != SPL_SEMI_RC) return false; } } } else { splDir = edit.splDir; } } return true; } /** * return one of EDIT_SPL_FW, EDIT_SPL_RC, EDIT_SPL_UNKNOWN */ int splicing_dir() const { int splDir = SPL_UNKNOWN; for(index_t i = 0; i < _edits->size(); i++) { const Edit& edit = (*_edits)[i]; if(edit.type != EDIT_TYPE_SPL) continue; if(splDir != SPL_UNKNOWN) { if(edit.splDir != SPL_UNKNOWN) { if(splDir == SPL_FW || splDir == SPL_SEMI_FW) { if(edit.splDir != SPL_FW && edit.splDir != SPL_SEMI_FW) return SPL_UNKNOWN; } if(splDir == SPL_RC || splDir == SPL_SEMI_RC) { if(edit.splDir != SPL_RC && edit.splDir != SPL_SEMI_RC) return SPL_UNKNOWN; } } } else { splDir = edit.splDir; } } if(splDir == SPL_FW || splDir == SPL_SEMI_FW) return SPL_FW; else if(splDir == SPL_RC || splDir == SPL_SEMI_RC) return SPL_RC; else return SPL_UNKNOWN; } bool operator== (const GenomeHit& other) const { if(_fw != other._fw || _rdoff != other._rdoff || _len != other._len || _tidx != other._tidx || _toff != other._toff || _trim5 != other._trim5 || _trim3 != other._trim3) { return false; } if(_edits->size() != other._edits->size()) return false; for(index_t i = 0; i < _edits->size(); i++) { const Edit& e = (*_edits)[i]; const Edit& oe = (*other._edits)[i]; if(e.isReadGap()) { if(!oe.isReadGap()) return false; } else if(e.isRefGap()) { if(!oe.isRefGap()) return false; } else { if(!(e == oe)) { return false; } } } // daehwan - this may not be true when some splice sites are provided from outside // assert_eq(_score, other._score); return true; } bool contains(const GenomeHit& other) const { return (*this) == other; } /** * Return number of mismatches in the alignment. */ int mms() const { #if 0 if (_e2.inited()) return 2; else if(_e1.inited()) return 1; else return 0; #endif return 0; } /** * Return the number of Ns involved in the alignment. */ int ns() const { #if 0 int ns = 0; if(_e1.inited() && _e1.hasN()) { ns++; if(_e2.inited() && _e2.hasN()) { ns++; } } return ns; #endif return 0; } int ngaps() const { return 0; } #ifndef NDEBUG /** * Check that hit is sane w/r/t read. */ bool repOk(const Read& rd, const BitPairReference& ref); #endif void replace_edits_with_alts(const Read& rd, const EList >& alts, SpliceSiteDB& ssdb, const Scoring& sc, index_t minK_local, index_t minIntronLen, index_t maxIntronLen, index_t minAnchorLen, index_t minAnchorLen_noncan, const BitPairReference& ref) { assert(inited()); if(alts.size() <= 0) return; if(_edits->size() <= 0) return; index_t joinedOff = _joinedOff; int offset = 0; size_t i = 0, next_i; while(i < _edits->size()) { next_i = i + 1; Edit& ed = (*_edits)[i]; if(ed.type == EDIT_TYPE_SPL) { assert(false); } else if(ed.type == EDIT_TYPE_READ_GAP || ed.type == EDIT_TYPE_REF_GAP) { for(; next_i < _edits->size(); next_i++) { Edit& next_ed = (*_edits)[next_i]; if(ed.type != next_ed.type) break; } } if(ed.snpID == (index_t)INDEX_MAX) { ALT cmp_alt; cmp_alt.pos = joinedOff + ed.pos + offset; index_t alt_i = (index_t)alts.bsearchLoBound(cmp_alt); for(; alt_i < alts.size(); alt_i++) { const ALT& alt = alts[alt_i]; if(alt.left > cmp_alt.pos) break; if(ed.type == EDIT_TYPE_MM) { if(alt.type != ALT_SNP_SGL) continue; if("ACGT"[alt.seq] == ed.qchr) { ed.snpID = alt_i; break; } } else { size_t gap = next_i - i; if(ed.type == EDIT_TYPE_READ_GAP) { if(alt.type != ALT_SNP_DEL) continue; if(alt.len == gap) { for(size_t ii = i; ii < next_i; ii++) { Edit& ii_ed = (*_edits)[ii]; ii_ed.snpID = alt_i; } break; } } else { assert_eq(ed.type, EDIT_TYPE_REF_GAP); if(alt.type != ALT_SNP_INS) continue; if(alt.len == gap) { uint64_t seq = 0; for(size_t ii = i; ii < next_i; ii++) { Edit& ii_ed = (*_edits)[ii]; seq = (seq << 2) | asc2dna[ii_ed.qchr]; } if(alt.seq == seq) { for(size_t ii = i; ii < next_i; ii++) { Edit& ii_ed = (*_edits)[ii]; ii_ed.snpID = alt_i; } break; } } } } } } if(ed.type == EDIT_TYPE_SPL) { offset += ed.splLen; } else if(ed.type == EDIT_TYPE_READ_GAP || ed.type == EDIT_TYPE_REF_GAP) { size_t gap = next_i - i; if(ed.type == EDIT_TYPE_READ_GAP) { assert_gt(joinedOff, gap); offset += gap; } else { offset -= gap; } } i = next_i; } calculateScore(rd, ssdb, sc, minK_local, minIntronLen, maxIntronLen, minAnchorLen, minAnchorLen_noncan, ref); } private: /** * Calculate alignment score */ int64_t calculateScore( const Read& rd, SpliceSiteDB& ssdb, const Scoring& sc, index_t minK_local, index_t minIntronLen, index_t maxIntronLen, index_t minAnchorLen, index_t minAnchorLen_noncan, const BitPairReference& ref); public: bool _fw; index_t _rdoff; index_t _len; index_t _trim5; index_t _trim3; index_t _tidx; index_t _toff; index_t _joinedOff; bool _repeat; EList* _edits; EList >* _ht_list; int64_t _score; int64_t _localscore; double _splicescore; index_t _hitcount; // for selection purposes LinkedEListNode >* _edits_node; LinkedEListNode > >* _ht_list_node; SharedTempVars* _sharedVars; }; /** * Check if it is compatible with another GenomeHit with respect to indels or introns */ template bool GenomeHit::compatibleWith( const GenomeHit& otherHit, index_t minIntronLen, index_t maxIntronLen, bool no_spliced_alignment) const { if(this == &otherHit) return false; // check if they are on the same strand and on the same contig if(_fw != otherHit._fw || _tidx != otherHit._tidx) return false; // make sure itself is closer to the left end of read than otherHit if(_rdoff > otherHit._rdoff) return false; // do not consider a case itself (read portion) includes otherHit if(_rdoff + _len > otherHit._rdoff + otherHit._len) return false; // make sure itself comes before otherHit wrt. genomic positions if(_toff > otherHit._toff) return false; index_t this_rdoff, this_len, this_toff; this->getRight(this_rdoff, this_len, this_toff); assert_geq(this_len, 0); index_t other_rdoff, other_len, other_toff; otherHit.getLeft(other_rdoff, other_len, other_toff); assert_geq(other_len, 0); if(this_rdoff > other_rdoff) return false; if(this_rdoff + this_len > other_rdoff + other_len) return false; if(this_toff > other_toff) return false; index_t refdif = other_toff - this_toff; index_t rddif = other_rdoff - this_rdoff; // check if there is a deletion, an insertion, or a potential intron // between the two partial alignments if(!no_spliced_alignment) { if(refdif > rddif + maxIntronLen) { return false; } } return true; } /** * Combine itself with another GenomeHit * while allowing mismatches, an insertion, a deletion, or an intron */ template bool GenomeHit::combineWith( const GenomeHit& otherHit, const Read& rd, const GFM& gfm, const BitPairReference& ref, const ALTDB& altdb, const RepeatDB& repeatdb, SpliceSiteDB& ssdb, SwAligner& swa, SwMetrics& swm, const Scoring& sc, TAlScore minsc, RandomSource& rnd, // pseudo-random source index_t minK_local, index_t minIntronLen, index_t maxIntronLen, index_t minAnchorLen, // minimum anchor length for canonical splice site index_t minAnchorLen_noncan, // minimum anchor length for non-canonical splice site const index_t maxAltsTried, const SpliceSite* spliceSite, // penalty for splice site bool no_spliced_alignment) { if(this == &otherHit) return false; assert(compatibleWith(otherHit, minIntronLen, maxIntronLen, no_spliced_alignment)); assert_eq(this->_tidx, otherHit._tidx); assert_lt(this->_tidx, ref.numRefs()); // get the partial part of the alignment from the right // until an indel or splice sites index_t this_rdoff, this_len, this_toff; int64_t this_score; this->getRight(this_rdoff, this_len, this_toff, &this_score, &rd, &sc); assert_geq(this_len, 0); assert_leq(this_score, 0); assert_geq(this_score, this->_score); // get the partial part of the other alignment from the left // until an indel or splice sites index_t other_rdoff, other_len, other_toff; int64_t other_score; otherHit.getLeft(other_rdoff, other_len, other_toff, &other_score, &rd, &sc); assert_geq(other_len, 0); assert_leq(other_score, 0); assert_geq(other_score, otherHit._score); assert_leq(this_rdoff, other_rdoff); if(this_len != 0 && other_len != 0 && this_rdoff + this_len > other_rdoff + other_len) return false; assert_leq(this_rdoff + this_len, other_rdoff + other_len); index_t len = other_rdoff - this_rdoff + other_len; const index_t reflen = ref.approxLen(_tidx); if(this_toff + len > reflen) return false; assert_leq(this_toff + len, reflen); // check if an indel or an intron is necessary index_t refdif = other_toff - this_toff; index_t rddif = other_rdoff - this_rdoff; bool spliced = false, ins = false, del = false; if(refdif != rddif) { if(refdif > rddif) { if(!no_spliced_alignment && refdif - rddif >= minIntronLen) { assert_leq(refdif - rddif, maxIntronLen); spliced = true; } else { del = true; } } else { ins = true; } } #ifndef NDEBUG if(ins) { assert(!spliced && !del); } else { if(spliced) assert(!del); else assert(!spliced); } #endif if(no_spliced_alignment) { if(spliced) return false; } // if the combination of the two alignments does not involve an indel or an intron, // then simply combine them and return if(!spliced && !ins && !del && this_rdoff + this_len == other_rdoff) { index_t addoff = otherHit._rdoff - this->_rdoff; for(index_t i = 0; i < otherHit._edits->size(); i++) { _edits->push_back((*otherHit._edits)[i]); _edits->back().pos += addoff; } _len += otherHit._len; calculateScore( rd, ssdb, sc, minK_local, minIntronLen, maxIntronLen, minAnchorLen, minAnchorLen_noncan, ref); assert(repOk(rd, ref)); return true; } // calculate the maximum gap lengths based on the current score and the mimumimu alignment score to be reported const BTDnaString& seq = this->_fw ? rd.patFw : rd.patRc; const BTString& qual = this->_fw ? rd.qual : rd.qualRev; index_t rdlen = (index_t)seq.length(); int64_t remainsc = minsc - (_score - this_score) - (otherHit._score - other_score); if(remainsc > 0) remainsc = 0; int read_gaps = 0, ref_gaps = 0; if(!spliced) { read_gaps = sc.maxReadGaps(remainsc + sc.canSpl(), rdlen); ref_gaps = sc.maxRefGaps(remainsc + sc.canSpl(), rdlen); } if(ins) { if(refdif + ref_gaps < rddif) { return false; } } else if(del) { if(rddif + read_gaps < refdif) { return false; } } int this_ref_ext = read_gaps; if(spliced) this_ref_ext += (int)intronic_len; if(this_toff + len > reflen) return false; if(this_toff + len + this_ref_ext > reflen) this_ref_ext = reflen - (this_toff + len); assert(_sharedVars != NULL); SStringExpandable& raw_refbuf = _sharedVars->raw_refbuf; EList& temp_scores = _sharedVars->temp_scores; EList& temp_scores2 = _sharedVars->temp_scores2; ASSERT_ONLY(SStringExpandable& destU32 = _sharedVars->destU32); raw_refbuf.resize(len + this_ref_ext + 16); int off = ref.getStretch( reinterpret_cast(raw_refbuf.wbuf()), (size_t)this->_tidx, (size_t)this_toff, len + this_ref_ext ASSERT_ONLY(, destU32)); assert_lt(off, 16); char *refbuf = raw_refbuf.wbuf() + off, *refbuf2 = NULL; // discover a splice site, an insertion, or a deletion index_t maxscorei = (index_t)INDEX_MAX; int64_t maxscore = MIN_I64; uint32_t maxspldir = SPL_UNKNOWN; float maxsplscore = 0.0f; // allow an indel near a splice site index_t splice_gap_maxscorei = (index_t)INDEX_MAX; int64_t donor_seq = 0, acceptor_seq = 0; int splice_gap_off = 0; if(spliced || ins || del) { int other_ref_ext = min(read_gaps + (int)intronic_len, other_toff + other_len - len); SStringExpandable& raw_refbuf2 = _sharedVars->raw_refbuf2; raw_refbuf2.resize(len + other_ref_ext + 16); int off2 = ref.getStretch( reinterpret_cast(raw_refbuf2.wbuf()), (size_t)otherHit._tidx, (size_t)(other_toff + other_len - len - other_ref_ext), len + other_ref_ext ASSERT_ONLY(, destU32)); refbuf2 = raw_refbuf2.wbuf() + off2 + other_ref_ext; temp_scores.resize(len); temp_scores2.resize(len); if(spliced) { static const char GT = 0x23, AG = 0x02; static const char GTrc = 0x01, AGrc = 0x13; static const char GC = 0x21, GCrc = 0x21; static const char AT = 0x03, AC = 0x01; static const char ATrc = 0x03, ACrc = 0x20; int i; for(i = 0; i < (int)len; i++) { int rdc = seq[this_rdoff + i], rfc = refbuf[i]; if(i > 0) { temp_scores[i] = temp_scores[i-1]; } else { temp_scores[i] = 0; } if(rdc != rfc) { temp_scores[i] += sc.score(rdc, 1 << rfc, qual[this_rdoff + i] - 33); } if(temp_scores[i] < remainsc) { break; } } int i_limit = min(i, len); int i2; for(i2 = len - 1; i2 >= 0; i2--) { int rdc = seq[this_rdoff + i2], rfc = refbuf2[i2]; if((index_t)(i2 + 1) < len) { temp_scores2[i2] = temp_scores2[i2+1]; } else { temp_scores2[i2] = 0; } if(rdc != rfc) { temp_scores2[i2] += sc.score(rdc, 1 << rfc, qual[this_rdoff + i2] - 33); } if(temp_scores2[i2] < remainsc) { break; } } int i2_limit = max(i2, 0); if(spliceSite != NULL){ assert_leq(this_toff, (int)spliceSite->left()); if(i2_limit <= (int)(spliceSite->left() - this_toff)) { i2_limit = (int)(spliceSite->left() - this_toff); i_limit = i2_limit + 1; } else { i_limit = i2_limit; } } for(i = i2_limit, i2 = i2_limit + 1; i < i_limit && i2 < (int)len; i++, i2++) { int64_t tempscore = temp_scores[i] + temp_scores2[i2]; char donor = 0xff, acceptor = 0xff; if((index_t)(i + 2) < len + this_ref_ext) { donor = refbuf[i + 1]; donor = (donor << 4) | refbuf[i + 2]; } if(i2 - 2 >= -other_ref_ext) { acceptor = refbuf2[i2 - 2]; acceptor = (acceptor << 4) | refbuf2[i2 - 1]; } bool canonical = false, semi_canonical = false; uint32_t spldir = SPL_UNKNOWN; if((donor == GT && acceptor == AG) /* || (donor == AT && acceptor == AC) */) { spldir = SPL_FW; canonical = true; } else if((donor == AGrc && acceptor == GTrc) /* || (donor == ACrc && acceptor == ATrc) */) { spldir = SPL_RC; canonical = true; } else if((donor == GC && acceptor == AG) || (donor == AT && acceptor == AC)) { spldir = SPL_SEMI_FW; semi_canonical = true; } else if((donor == AGrc && acceptor == GCrc) || (donor == ACrc && acceptor == ATrc)) { spldir = SPL_SEMI_RC; semi_canonical = true; } tempscore -= (canonical ? sc.canSpl() : sc.noncanSpl()); int64_t temp_donor_seq = 0, temp_acceptor_seq = 0; float splscore = 0.0f; if(canonical) { // in case of canonical splice site, extract donor side sequence and acceptor side sequence // to calculate a score of the splicing event. if(spldir == SPL_FW) { if(i + 1 >= (int)donor_exonic_len && (int)(len + this_ref_ext) > i + (int)donor_intronic_len && i2 + (int)other_ref_ext >= (int)acceptor_intronic_len && (int)len > i2 + (int)acceptor_exonic_len - 1) { int from = i + 1 - (int)donor_exonic_len; int to = i + (int)donor_intronic_len; for(int j = from; j <= to; j++) { assert_geq(j, 0); assert_lt(j, (int)(len + this_ref_ext)); int base = refbuf[j]; if(base > 3) base = 0; temp_donor_seq = temp_donor_seq << 2 | base; } from = i2 - acceptor_intronic_len; to = i2 + acceptor_exonic_len - 1; for(int j = from; j <= to; j++) { assert_geq(j, -(int)other_ref_ext); assert_lt(j, (int)len); int base = refbuf2[j]; if(base > 3) base = 0; temp_acceptor_seq = temp_acceptor_seq << 2 | base; } } } else if(spldir == SPL_RC) { if(i + 1 >= (int)acceptor_exonic_len && (int)(len + this_ref_ext) > i + (int)acceptor_intronic_len && i2 + (int)other_ref_ext >= (int)donor_intronic_len && (int)len > i2 + (int)donor_exonic_len - 1) { int from = i + 1 - (int)acceptor_exonic_len; int to = i + (int)acceptor_intronic_len; for(int j = to; j >= from; j--) { assert_geq(j, 0); assert_lt(j, (int)(len + this_ref_ext)); int base = refbuf[j]; if(base > 3) base = 0; temp_acceptor_seq = temp_acceptor_seq << 2 | (base ^ 0x3); } from = i2 - donor_intronic_len; to = i2 + donor_exonic_len - 1; for(int j = to; j >= from; j--) { assert_geq(j, -(int)other_ref_ext); assert_lt(j, (int)len); int base = refbuf2[j]; if(base > 3) base = 0; temp_donor_seq = temp_donor_seq << 2 | (base ^ 0x3); } } } splscore = SpliceSiteDB::probscore(temp_donor_seq, temp_acceptor_seq); } // daehwan - for debugging purposes // choose a splice site with the better score if((maxspldir == SPL_UNKNOWN && spldir == SPL_UNKNOWN && maxscore < tempscore) || (maxspldir == SPL_UNKNOWN && spldir == SPL_UNKNOWN && maxscore == tempscore && semi_canonical) || (maxspldir != SPL_UNKNOWN && spldir != SPL_UNKNOWN && (maxscore < tempscore || (maxscore == tempscore && maxsplscore < splscore))) || (maxspldir == SPL_UNKNOWN && spldir != SPL_UNKNOWN)) { maxscore = tempscore; maxscorei = i; maxspldir = spldir; maxsplscore = splscore; if(maxspldir != SPL_UNKNOWN) { donor_seq = temp_donor_seq; acceptor_seq = temp_acceptor_seq; } else { donor_seq = 0; acceptor_seq = 0; } } } } else { // discover an insertion or a deletion assert(ins || del); int inslen = (ins ? rddif - refdif : 0); int dellen = (del ? refdif - rddif : 0); int64_t gap_penalty; if(ins) { gap_penalty = -(sc.refGapOpen() + sc.refGapExtend() * (inslen - 1)); } else { assert(del); gap_penalty = -(sc.readGapOpen() + sc.readGapExtend() * (dellen - 1)); } if(gap_penalty < remainsc) return false; int i; for(i = 0; i < (int)len; i++) { int rdc = seq[this_rdoff + i], rfc = refbuf[i]; if(i > 0) { temp_scores[i] = temp_scores[i-1]; } else { temp_scores[i] = 0; } if(rdc != rfc) { temp_scores[i] += sc.score(rdc, 1 << rfc, qual[this_rdoff + i] - 33); } if(temp_scores[i] + gap_penalty < remainsc) { break; } } int i_limit = min(i, len); int i2; for(i2 = len - 1; i2 >= 0; i2--) { int rdc = seq[this_rdoff + i2], rfc = refbuf2[i2]; if((index_t)(i2 + 1) < len) { temp_scores2[i2] = temp_scores2[i2+1]; } else { temp_scores2[i2] = 0; } if(rdc != rfc) { temp_scores2[i2] += sc.score(rdc, 1 << rfc, qual[this_rdoff + i2] - 33); } if(temp_scores2[i2] + gap_penalty < remainsc) { break; } } int i2_limit = (i2 < inslen ? 0 : i2 - inslen); for(i = i2_limit, i2 = i2_limit + 1 + inslen; i < i_limit && i2 < (int)len; i++, i2++) { int64_t tempscore = temp_scores[i] + temp_scores2[i2] + gap_penalty; if(maxscore < tempscore) { maxscore = tempscore; maxscorei = i; } } } if(maxscore == MIN_I64) return false; assert_lt(maxscorei, len); if(spliced && spliceSite == NULL) { uint32_t shorter_anchor_len = min(maxscorei + 1, len - maxscorei - 1); assert_leq(this_toff, other_toff); if(maxspldir == SPL_SEMI_FW || maxspldir == SPL_SEMI_RC || maxspldir == SPL_UNKNOWN) { if(shorter_anchor_len < minAnchorLen_noncan) { float intronLenProb = intronLen_prob_noncan(shorter_anchor_len, other_toff - this_toff, maxIntronLen); if(intronLenProb > 0.01f) return false; } } else { if(shorter_anchor_len < minAnchorLen) { float intronLenProb = intronLen_prob(shorter_anchor_len, other_toff - this_toff, maxIntronLen); if(intronLenProb > 0.01f) return false; } } } if(maxscore < remainsc) return false; } bool clear = true; for(int i = (int)_edits->size() - 1; i >= 0; i--) { const Edit& edit = (*_edits)[i]; if(edit.type == EDIT_TYPE_SPL || edit.type == EDIT_TYPE_READ_GAP || edit.type == EDIT_TYPE_REF_GAP || (edit.type == EDIT_TYPE_MM && edit.snpID != (index_t)INDEX_MAX)) { _edits->resize(i+1); clear = false; break; } } if(clear) this->_edits->clear(); // combine two alignments while updating edits if(spliced) { assert_geq(this_rdoff, this->_rdoff); index_t addoff = this_rdoff - this->_rdoff; int rd_gap_off = -min(splice_gap_off, 0); int ref_gap_off = max(splice_gap_off, 0); for(int i = 0; i < (int)len; i++) { assert_lt(this_rdoff + i, rdlen); int rdc = seq[this_rdoff + i]; assert_range(0, 4, rdc); int rfc; if(splice_gap_maxscorei <= maxscorei) { if(i <= (int)splice_gap_maxscorei) { rfc = refbuf[i]; } else if(i <= (int)maxscorei) { rfc = refbuf[i - ref_gap_off + rd_gap_off]; } else { rfc = refbuf2[i]; } } else { if(i <= (int)maxscorei) { rfc = refbuf[i]; } else if(i <= (int)splice_gap_maxscorei) { rfc = refbuf2[i + ref_gap_off - rd_gap_off]; } else { rfc = refbuf2[i]; } } assert_range(0, 4, rfc); if(rdc != rfc) { Edit e((uint32_t)(i + addoff), rfc, rdc, EDIT_TYPE_MM, false); _edits->push_back(e); } if(i == (int)maxscorei) { index_t left = this_toff + i + 1; if(splice_gap_maxscorei <= maxscorei) { left = left - ref_gap_off + rd_gap_off; } index_t right = other_toff + other_len - (len - i - 1); if(splice_gap_maxscorei > maxscorei) { right = right + ref_gap_off - rd_gap_off; } index_t skipLen = 0; assert_lt(left, right); skipLen = right - left; Edit e((uint32_t)(i + 1 + addoff), 0, 0, EDIT_TYPE_SPL, skipLen, maxspldir, spliceSite != NULL, false); e.donor_seq = donor_seq; e.acceptor_seq = acceptor_seq; _edits->push_back(e); } if(i == (int)splice_gap_maxscorei && splice_gap_off != 0) { if(rd_gap_off > 0) { assert_lt(left, right); for(index_t j = 0; j < (index_t)rd_gap_off; j++) { int temp_rfc_off = i + 1 + j; int temp_rfc; if(i < (int)maxscorei) { temp_rfc = refbuf[temp_rfc_off]; } else { temp_rfc = refbuf2[temp_rfc_off - rd_gap_off]; } assert_range(0, 4, temp_rfc); Edit e((uint32_t)(i + 1 + addoff), "ACGTN"[temp_rfc], '-', EDIT_TYPE_READ_GAP); _edits->push_back(e); } } else { assert_gt(ref_gap_off, 0); for(index_t j = 0; j < (index_t)ref_gap_off; j++) { assert_lt(this_rdoff + i + 1 + j, rdlen); int temp_rdc = seq[this_rdoff + i + 1 + j]; assert_range(0, 4, temp_rdc); Edit e((uint32_t)(i + 1 + j + addoff), '-', "ACGTN"[temp_rdc], EDIT_TYPE_REF_GAP); _edits->push_back(e); } i += ref_gap_off; } } } } else { index_t ins_len = 0; for(index_t i = 0; i < len; i++) { char rdc = seq[this_rdoff + i]; char rfc = (i <= maxscorei ? refbuf[i] : refbuf2[i]); assert_geq(this_rdoff, this->_rdoff); index_t addoff = this_rdoff - this->_rdoff; if(rdc != rfc) { ALT cmp_alt; assert_geq(this_toff, this->_toff); cmp_alt.pos = this->_joinedOff + i + (this_toff - this->_toff) - ins_len; index_t alt_i = (index_t)altdb.alts().bsearchLoBound(cmp_alt); index_t add_alt_i = std::numeric_limits::max(); for(; alt_i < altdb.alts().size(); alt_i++) { const ALT& alt = altdb.alts()[alt_i]; if(alt.left > cmp_alt.pos) break; if(alt.type != ALT_SNP_SGL) continue; if(alt.seq == rdc) { add_alt_i = alt_i; break; } } Edit e((uint32_t)(i + addoff), rfc, rdc, EDIT_TYPE_MM, false, add_alt_i); _edits->push_back(e); } if(i == maxscorei) { index_t left = this_toff + i + 1; if(other_toff + other_len < len - i - 1) return false; index_t right = other_toff + other_len - (len - i - 1); index_t skipLen = 0; if(del) { assert_lt(left, right); skipLen = right - left; for(index_t j = 0; j < skipLen; j++) { int temp_rfc; if(i + 1 + j < len) temp_rfc = refbuf[i + 1 + j]; else temp_rfc = ref.getBase(this->_tidx, this_toff + i + 1 + j); assert_range(0, 4, temp_rfc); Edit e((uint32_t)(i + 1 + addoff), "ACGTN"[temp_rfc], '-', EDIT_TYPE_READ_GAP); _edits->push_back(e); } } else { assert(ins); assert_lt(right, left); skipLen = left - right; for(index_t j = 0; j < skipLen; j++) { assert_lt(this_rdoff + i + 1 + j, seq.length()); int temp_rdc = seq[this_rdoff + i + 1 + j]; assert_range(0, 4, temp_rdc); Edit e((uint32_t)(i + 1 + j + addoff), '-', "ACGTN"[temp_rdc], EDIT_TYPE_REF_GAP); _edits->push_back(e); } i += skipLen; ins_len += skipLen; } } } } index_t fsi = (index_t)otherHit._edits->size(); for(index_t i = 0; i < otherHit._edits->size(); i++) { const Edit& edit = (*otherHit._edits)[i]; if(edit.type == EDIT_TYPE_SPL || edit.type == EDIT_TYPE_READ_GAP || edit.type == EDIT_TYPE_REF_GAP || (edit.type == EDIT_TYPE_MM && edit.snpID != (index_t)INDEX_MAX)) { fsi = i; break; } } assert_leq(this->_rdoff, otherHit._rdoff); index_t addoff = otherHit._rdoff - this->_rdoff; for(index_t i = fsi; i < otherHit._edits->size(); i++) { _edits->push_back((*otherHit._edits)[i]); _edits->back().pos += addoff; } // for alignment involving indel, left align so that // indels go to the left most of the combined alignment if(ins || del || (spliced && splice_gap_off != 0)) { leftAlign(rd); } // update alignment score, trims assert_leq(this->_rdoff + this->_len, otherHit._rdoff + otherHit._len); _len = otherHit._rdoff + otherHit._len - this->_rdoff; assert_eq(_trim3, 0); _trim3 += otherHit._trim3; calculateScore( rd, ssdb, sc, minK_local, minIntronLen, maxIntronLen, minAnchorLen, minAnchorLen_noncan, ref); #ifndef NDEBUG if(_joinedOff != (index_t)INDEX_MAX) { ASSERT_ONLY(bool straddled = false); ASSERT_ONLY(index_t tmp_tidx = 0, tmp_toff = 0, tmp_tlen = 0); gfm.joinedToTextOff( 0, _joinedOff, tmp_tidx, tmp_toff, tmp_tlen, true, // reject straddlers? straddled); // straddled? assert_eq(tmp_tidx, _tidx); assert_eq(tmp_toff, _toff); } #endif assert(repOk(rd, ref)); return true; } /** * Extend the partial alignment (GenomeHit) bidirectionally */ template bool GenomeHit::extend( const Read& rd, const GFM& gfm, const BitPairReference& ref, const ALTDB& altdb, const RepeatDB& repeatdb, SpliceSiteDB& ssdb, SwAligner& swa, SwMetrics& swm, PerReadMetrics& prm, const Scoring& sc, TAlScore minsc, RandomSource& rnd, // pseudo-random source index_t minK_local, const TranscriptomePolicy& tpol, const GraphPolicy& gpol, index_t& leftext, index_t& rightext, index_t mm) { assert_lt(this->_tidx, ref.numRefs()); index_t max_leftext = leftext, max_rightext = rightext; assert(max_leftext > 0 || max_rightext > 0); leftext = 0, rightext = 0; index_t rdlen = (index_t)rd.length(); bool doLeftAlign = false; assert(_sharedVars != NULL); const index_t minIntronLen = tpol.minIntronLen(); const index_t maxIntronLen = tpol.maxIntronLen(); const index_t minAnchorLen = tpol.minAnchorLen(); const index_t minAnchorLen_noncan = tpol.minAnchorLen_noncan(); // extend the alignment further in the left direction // with 'mm' mismatches allowed const BTDnaString& seq = _fw ? rd.patFw : rd.patRc; if(max_leftext > 0 && _rdoff > 0) { assert_gt(_rdoff, 0); index_t left_rdoff, left_len, left_toff; this->getLeft(left_rdoff, left_len, left_toff); assert_eq(left_rdoff, _rdoff); assert_eq(left_toff, _toff); if(_toff <= 0) return false; int rl = (int)_toff - (int)_rdoff; assert_geq(_score, minsc); index_t reflen = _rdoff + 10; rl -= (reflen - _rdoff); if(rl < 0) { reflen += rl; rl = 0; } index_t numNs = 0; index_t num_prev_edits = (index_t)_edits->size(); index_t best_ext = alignWithALTs( altdb.alts(), altdb.haplotypes(), altdb.haplotype_maxrights(), this->_joinedOff, seq, this->_rdoff - 1, this->_rdoff - 1, this->_rdoff, ref, *_sharedVars, _tidx, rl, reflen, true, /* left? */ gpol, *this->_edits, _sharedVars->ht_llist, *this->_ht_list, _sharedVars->cmp_ht, NULL, mm, &numNs); // Do not allow for any edits including known snps and splice sites when extending zero-length hit if(_len == 0 && mm == 0 && _edits->size() > 0) { _edits->clear(); return false; } if(best_ext > 0) { leftext = best_ext; assert_leq(num_prev_edits, _edits->size()); index_t added_edits = (index_t)_edits->size() - num_prev_edits; int ref_ext = (int)best_ext; for(index_t i = 0; i < added_edits; i++) { const Edit& edit = (*_edits)[i]; if(edit.type == EDIT_TYPE_REF_GAP) ref_ext--; else if(edit.type == EDIT_TYPE_READ_GAP) ref_ext++; else if(edit.type == EDIT_TYPE_SPL) ref_ext += edit.splLen; } assert_leq(best_ext, _rdoff); _rdoff -= best_ext; assert_leq(ref_ext, _toff); _toff -= ref_ext; _len += best_ext; assert_leq(_len, rdlen); assert_leq((int)numNs, ref_ext); assert_leq(ref_ext - (int)numNs, _joinedOff); _joinedOff -= (ref_ext - (int)numNs); for(index_t i = 0; i < _edits->size(); i++) { if(i < added_edits) { assert_geq((*_edits)[i].pos, _rdoff); (*_edits)[i].pos -= _rdoff; } else { (*_edits)[i].pos += best_ext; } } } } // extend the alignment further in the right direction // with 'mm' mismatches allowed if(max_rightext > 0 && _rdoff + _len < rdlen) { index_t right_rdoff, right_len, right_toff; this->getRight(right_rdoff, right_len, right_toff); index_t rl = right_toff + right_len; assert_eq(_rdoff + _len, right_rdoff + right_len); index_t rr = rdlen - (right_rdoff + right_len); index_t tlen = ref.approxLen(_tidx); if(rl < tlen) { index_t reflen = rr + 10; if(rl + reflen > tlen) { reflen = tlen - rl; } int ref_ext = (int)_len; for(index_t ei = 0; ei < _edits->size(); ei++) { const Edit& e = (*_edits)[ei]; if(e.type == EDIT_TYPE_REF_GAP) ref_ext--; else if(e.type == EDIT_TYPE_READ_GAP) ref_ext++; else if(e.type == EDIT_TYPE_SPL) ref_ext += e.splLen; else if(e.type == EDIT_TYPE_MM && e.chr == 'N') ref_ext--; } index_t best_ext = alignWithALTs( altdb.alts(), altdb.haplotypes(), altdb.haplotype_maxrights(), this->_joinedOff + ref_ext, seq, this->_rdoff, this->_rdoff + this->_len, rdlen - (this->_rdoff + this->_len), ref, *_sharedVars, _tidx, (int)rl, reflen, false, gpol, *this->_edits, _sharedVars->ht_llist, *this->_ht_list, _sharedVars->cmp_ht, NULL, mm); // Do not allow for any edits including known snps and splice sites when extending zero-length hit if(_len == 0 && mm == 0 && _edits->size() > 0) { _edits->clear(); return false; } if(best_ext > 0) { rightext = best_ext; _len += best_ext; } } } #ifndef NDEBUG if(_joinedOff != (index_t)INDEX_MAX && seq[_rdoff] < 4) { ASSERT_ONLY(bool straddled = false); ASSERT_ONLY(index_t tmp_tidx = 0, tmp_toff = 0, tmp_tlen = 0); gfm.joinedToTextOff( 0, _joinedOff, tmp_tidx, tmp_toff, tmp_tlen, true, // reject straddlers? straddled); // straddled? if(!gfm.repeat()) { assert_eq(tmp_tidx, _tidx); } assert_eq(tmp_toff, _toff); } #endif if(doLeftAlign) leftAlign(rd); assert_leq(_rdoff + _len, rdlen); calculateScore( rd, ssdb, sc, minK_local, minIntronLen, maxIntronLen, minAnchorLen, minAnchorLen_noncan, ref); assert(repOk(rd, ref)); return leftext > 0 || rightext > 0; } /** * Adjust alignment with respect to SNPs, usually updating Edits * */ template bool GenomeHit::adjustWithALT( index_t rdoff, index_t len, const Coord& coord, SharedTempVars& sharedVars, EList >& genomeHits, const Read& rd, const GFM& gfm, const ALTDB& altdb, const BitPairReference& ref, const GraphPolicy& gpol) { if(gfm.gh().linearFM()) { genomeHits.expand(); genomeHits.back().init( coord.orient(), rdoff, len, 0, // trim5 0, // trim3 (index_t)coord.ref(), (index_t)coord.off(), (index_t)coord.joinedOff(), sharedVars); return true; } index_t width = 1 << (gfm.gh()._offRate + 2); EList >& ssOffs = sharedVars.ssOffs; findSSOffs(gfm, altdb, (coord.joinedOff() >= width ? (index_t)(coord.joinedOff() - width) : 0), (index_t)(coord.joinedOff() + width), ssOffs); assert_gt(ssOffs.size(), 0); bool found = false; for(index_t s = 0; s < ssOffs.size(); s++) { index_t off = (index_t)coord.off(); index_t joinedOff = (index_t)coord.joinedOff(); pair& ssOff = ssOffs[s]; if(ssOff.first > 0) { assert_neq(ssOff.second, 0); if(ssOff.second > 0) { off += ssOff.first; joinedOff += ssOff.first; } else { off -= ssOff.first; joinedOff -= ssOff.first; } } size_t numGenomeHits = genomeHits.size(); genomeHits.expand(); genomeHits.back().init( coord.orient(), rdoff, len, 0, // trim5 0, // trim3 (index_t)coord.ref(), off, joinedOff, sharedVars); GenomeHit& genomeHit = genomeHits.back(); EList >& offDiffs = sharedVars.offDiffs; const index_t single_offDiffs_size = findOffDiffs(gfm, altdb, (genomeHit._joinedOff >= width ? genomeHit._joinedOff - width : 0), genomeHit._joinedOff + width, offDiffs); assert_leq(single_offDiffs_size, offDiffs.size()); const BTDnaString& seq = genomeHit._fw ? rd.patFw : rd.patRc; const EList >& alts = altdb.alts(); index_t orig_joinedOff = genomeHit._joinedOff; index_t orig_toff = genomeHit._toff; bool found2 = false; // maxAltsTried is not directly related to the size of offDiffs, // but let's make the size of offDiffs is determined by maxAltsTried const index_t max_offDiffs_size = max(4, gpol.maxAltsTried() / 4); if(offDiffs.size() - single_offDiffs_size > max_offDiffs_size) offDiffs.resize(single_offDiffs_size + max_offDiffs_size); for(index_t o = 0; o < offDiffs.size() && !found2; o++) { const pair& offDiff = offDiffs[o]; #ifndef NDEBUG if(o == 0) { assert_eq(offDiff.first, 0); assert_eq(offDiff.second, 0); } #endif if(offDiff.second >= 0) { genomeHit._joinedOff = orig_joinedOff + offDiff.first; genomeHit._toff = orig_toff + offDiff.first; } else { if(orig_toff < offDiff.first) continue; assert_geq(orig_joinedOff, offDiff.first); genomeHit._joinedOff = orig_joinedOff - offDiff.first; genomeHit._toff = orig_toff - offDiff.first; } genomeHit._edits->clear(); ELList& candidate_edits = sharedVars.candidate_edits; candidate_edits.clear(); index_t reflen = genomeHit._len + 10; index_t alignedLen = alignWithALTs( alts, altdb.haplotypes(), altdb.haplotype_maxrights(), genomeHit._joinedOff, seq, genomeHit._rdoff, genomeHit._rdoff, genomeHit._len, ref, sharedVars, genomeHit._tidx, (int)genomeHit._toff, reflen, false, /* left? */ gpol, *genomeHit._edits, sharedVars.ht_llist, *genomeHit._ht_list, sharedVars.cmp_ht, &candidate_edits); if(alignedLen == genomeHit._len) { found2 = true; assert(genomeHit.repOk(rd, ref)); for(index_t i = 0; i < genomeHits.size() - 1; i++) { if(genomeHits[i] == genomeHits.back()) { found2 = false; } } if(found2) { for(index_t e = 0; e < candidate_edits.size(); e++) { genomeHits.expand(); genomeHits.back() = genomeHits[genomeHits.size() - 2]; *(genomeHits.back()._edits) = candidate_edits[e]; assert(genomeHits.back().repOk(rd, ref)); for(size_t i = 0; i < genomeHits.size() - 1; i++) { if(genomeHits[i] == genomeHits.back()) { genomeHits.pop_back(); break; } } } } } else { genomeHit._edits->clear(); } } if(!found2) genomeHits.pop_back(); found = genomeHits.size() > numGenomeHits; } return found; } /** * Adjust alignment with respect to SNPs, usually updating Edits * */ template bool GenomeHit::adjustWithALT( const Read& rd, const GFM& gfm, const ALTDB& altdb, const BitPairReference& ref, const GraphPolicy& gpol) { if(gfm.gh().linearFM()) return true; assert_lt(this->_tidx, ref.numRefs()); assert(_sharedVars != NULL); EList >& offDiffs = _sharedVars->offDiffs; index_t width = 1 << (gfm.gh()._offRate + 2); const index_t single_offDiffs_size = findOffDiffs(gfm, altdb, (this->_joinedOff >= width ? this->_joinedOff - width : 0), this->_joinedOff + width, offDiffs); assert_leq(single_offDiffs_size, offDiffs.size()); const BTDnaString& seq = _fw ? rd.patFw : rd.patRc; const EList >& alts = altdb.alts(); index_t orig_joinedOff = this->_joinedOff; index_t orig_toff = this->_toff; bool found = false; // maxAltsTried is not directly related to the size of offDiffs, // but let's make the size of offDiffs is determined by maxAltsTried const index_t max_offDiffs_size = max(4, gpol.maxAltsTried() / 4); if(offDiffs.size() - single_offDiffs_size > max_offDiffs_size) offDiffs.resize(single_offDiffs_size + max_offDiffs_size); for(index_t o = 0; o < offDiffs.size() && !found; o++) { const pair& offDiff = offDiffs[o]; #ifndef NDEBUG if(o == 0) { assert_eq(offDiff.first, 0); assert_eq(offDiff.second, 0); } #endif if(offDiff.second >= 0) { this->_joinedOff = orig_joinedOff + offDiff.first; this->_toff = orig_toff + offDiff.first; } else { if(orig_toff < offDiff.first) continue; assert_geq(orig_joinedOff, offDiff.first); this->_joinedOff = orig_joinedOff - offDiff.first; this->_toff = orig_toff - offDiff.first; } index_t reflen = this->_len + 10; index_t alignedLen = alignWithALTs( alts, altdb.haplotypes(), altdb.haplotype_maxrights(), this->_joinedOff, seq, this->_rdoff, this->_rdoff, this->_len, ref, *_sharedVars, this->_tidx, (int)this->_toff, reflen, false, /* left? */ gpol, *this->_edits, _sharedVars->ht_llist, *this->_ht_list, _sharedVars->cmp_ht, &_sharedVars->candidate_edits); if(alignedLen == this->_len) { found = true; } else { this->_edits->clear(); } } #ifndef NDEBUG if(found) { assert(repOk(rd, ref)); } #endif return found; } /* * Find offset differences due to splice sites */ template void GenomeHit::findSSOffs( const GFM& gfm, const ALTDB& altdb, index_t start, index_t end, EList >& ssOffs) { ssOffs.clear(); ssOffs.expand(); ssOffs.back().first = ssOffs.back().second = 0; if(gfm.gh().linearFM() || !altdb.hasSpliceSites()) return; const EList >& alts = altdb.alts(); // Find splice sites included in this region ALT alt_search; alt_search.left = start; for(index_t i = (index_t)alts.bsearchLoBound(alt_search); i < alts.size(); i++) { const ALT& alt = alts[i]; if(alt.left >= end) break; if(!alt.splicesite()) continue; // if(alt.left < alt.right) { ssOffs.expand(); ssOffs.back().first = alt.right - alt.left + 1; ssOffs.back().second = 1; const index_t relax = 5; if(alt.right > relax) alt_search.left = alt.right - relax; else alt_search.left = 0; for(index_t j = (index_t)alts.bsearchLoBound(alt_search); j < alts.size(); j++) { const ALT& alt2 = alts[j]; if(!alt2.splicesite()) continue; if(alt2.left < alt2.right) continue; if(alt2.left + alt2.right == alt.left + alt.right) continue; if(alt2.left > alt.right + relax) break; ssOffs.expand(); if(alt2.right < alt.left) { ssOffs.back().first = alt.left - alt2.right; ssOffs.back().second = -1; } else { ssOffs.back().first = alt2.right - alt.left; ssOffs.back().second = 1; } } } else { ssOffs.expand(); ssOffs.back().first = alt.left - alt.right + 1; ssOffs.back().second = -1; } } if(ssOffs.size() > 1) { ssOffs.sort(); index_t new_size = (index_t)(unique(ssOffs.begin(), ssOffs.end()) - ssOffs.begin()); ssOffs.resize(new_size); } } /* * Find offset differences due to indels */ template index_t GenomeHit::findOffDiffs( const GFM& gfm, const ALTDB& altdb, index_t start, index_t end, EList >& offDiffs) { offDiffs.clear(); offDiffs.expand(); offDiffs.back().first = offDiffs.back().second = 0; if(gfm.gh().linearFM()) return offDiffs.size(); const EList >& alts = altdb.alts(); pair alt_range; // Find SNPs included in this region { ALT alt_search; alt_search.pos = start; alt_range.first = alt_range.second = (index_t)alts.bsearchLoBound(alt_search); for(alt_range.second = alt_range.first; alt_range.second < alts.size(); alt_range.second++) { const ALT& alt = alts[alt_range.second]; if(alt.splicesite() && alt.left > alt.right) continue; if(alt.deletion() && alt.reversed) continue; if(alt.pos >= end) break; } } if(alt_range.first >= alt_range.second) return offDiffs.size(); for(index_t second = alt_range.second; second > alt_range.first; second--) { assert_leq(second, alts.size()); const ALT& alt = alts[second - 1]; if(!alt.gap() || alt.splicesite() || (alt.deletion() && alt.reversed)) continue; int off = 0; if(alt.type == ALT_SNP_DEL) { off = alt.len; } else { assert_eq(alt.type, ALT_SNP_INS); off = -alt.len; } assert_neq(off, 0); offDiffs.expand(); offDiffs.back().first = abs(off); offDiffs.back().second = (off > 0 ? 1 : -1); } if(offDiffs.size() > 1) { offDiffs.sort(); index_t new_size = (index_t)(unique(offDiffs.begin(), offDiffs.end()) - offDiffs.begin()); offDiffs.resize(new_size); } const index_t single_offDiffs_size = offDiffs.size(); for(index_t second = alt_range.second; second > alt_range.first; second--) { assert_leq(alt_range.second, alts.size()); const ALT& alt = alts[second - 1]; if(!alt.gap() || alt.splicesite() || (alt.deletion() && alt.reversed)) continue; int off = 0; if(alt.type == ALT_SNP_DEL) { off = alt.len; } else { assert_eq(alt.type, ALT_SNP_INS); off = -alt.len; } for(index_t second2 = second - 1; second2 > alt_range.first; second2--) { const ALT& alt2 = alts[second2 - 1]; if(!alt2.gap() || alt2.splicesite() || (alt2.deletion() && alt2.reversed)) continue; if(alt2.type == ALT_SNP_DEL) { if(alt2.pos + alt2.len >= alt.pos) continue; off += alt2.len; } else { assert_eq(alt2.type, ALT_SNP_INS); if(alt2.pos >= alt.pos) continue; off -= alt2.len; } bool found = false; for(index_t i = 0; i < offDiffs.size(); i++) { int off_cmp = offDiffs[i].first * offDiffs[i].second; if(off == off_cmp) { found = true; break; } } if(!found) { offDiffs.expand(); offDiffs.back().first = abs(off); offDiffs.back().second = (off > 0 ? 1 : -1); } } } return single_offDiffs_size; } /* * */ template void add_haplotypes( const EList >& alts, const EList >& haplotypes, const EList& haplotype_maxrights, Haplotype& cmp_ht, EList >& ht_list, index_t rdlen, bool left_ext = true, bool initial = false) { pair ht_range; ht_range.first = ht_range.second = (int)haplotypes.bsearchLoBound(cmp_ht); if(ht_range.first >= haplotypes.size()) return; if(left_ext) { for(; ht_range.first >= 0; ht_range.first--) { const Haplotype& ht = haplotypes[ht_range.first]; if(!initial) { if(ht.right >= cmp_ht.left) continue; } index_t ht_maxright = haplotype_maxrights[ht_range.first]; assert_geq(ht_maxright, ht.right); if(ht_maxright + rdlen - 1 < cmp_ht.left) break; if(ht.alts.size() <= 0) continue; bool added = false; for(index_t h = 0; h < ht_list.size(); h++) { if(ht_list[h].first == ht_range.first) { added = true; break; } } if(added) continue; ht_list.expand(); ht_list.back().first = ht_range.first; assert_gt(ht.alts.size(), 0); if(ht.right < cmp_ht.left) { ht_list.back().second = ht.alts.size() - 1; } else { assert(initial); ht_list.back().second = ht.alts.size(); for(int a = (int)ht.alts.size() - 1; a >= 0; a--) { index_t alti = ht.alts[a]; assert_lt(alti, alts.size()); const ALT& alt = alts[alti]; assert(alt.snp()); ht_list.back().second = (index_t)a; if(cmp_ht.left > alt.pos) break; } if(ht_list.back().second == ht.alts.size()) { ht_list.pop_back(); } } } } else { if(initial) { for(; ht_range.first >= 0; ht_range.first--) { const Haplotype& ht = haplotypes[ht_range.first]; index_t ht_maxright = haplotype_maxrights[ht_range.first]; assert_geq(ht_maxright, ht.right); if(ht_maxright < cmp_ht.left) break; if(ht.right < cmp_ht.left || ht.left > cmp_ht.left) continue; if(ht.alts.size() <= 0) continue; bool added = false; for(index_t h = 0; h < ht_list.size(); h++) { if(ht_list[h].first == ht_range.first) { added = true; break; } } if(added) continue; ht_list.expand(); ht_list.back().first = ht_range.first; assert_gt(ht.alts.size(), 0); ht_list.back().second = ht.alts.size(); for(index_t a = 0; a < ht.alts.size(); a++) { index_t alti = ht.alts[a]; assert_lt(alti, alts.size()); const ALT& alt = alts[alti]; assert(alt.snp()); ht_list.back().second = a; if(cmp_ht.left <= alt.pos) break; } if(ht_list.back().second == ht.alts.size()) { ht_list.pop_back(); } } } for(; ht_range.second < haplotypes.size(); ht_range.second++) { const Haplotype& ht = haplotypes[ht_range.second]; if(ht.left < cmp_ht.right) continue; if(ht.left >= cmp_ht.right + rdlen) break; if(ht.alts.size() <= 0) continue; bool added = false; for(index_t h = 0; h < ht_list.size(); h++) { if(ht_list[h].first == ht_range.second) { added = true; break; } } if(added) continue; ht_list.expand(); ht_list.back().first = ht_range.second; assert_gt(ht.alts.size(), 0); ht_list.back().second = 0; } } } /* * */ template index_t GenomeHit::alignWithALTs_recur( const EList >& alts, const EList >& haplotypes, const EList& haplotype_maxrights, index_t joinedOff, const BTDnaString& rdseq, index_t rdoff_add, index_t rdoff, index_t rdlen, const BitPairReference& ref, EList >& raw_refbufs, ASSERT_ONLY(SStringExpandable destU32,) EList& tmp_edits, int& best_rdoff, const char* rfseq, index_t tidx, int rfoff, index_t rflen, bool left, EList& edits, index_t mm, ELList >& ht_llist, Haplotype& cmp_ht, ELList* candidate_edits, index_t tmp_numNs, index_t* numNs, index_t dep, const GraphPolicy& gpol, index_t& numALTsTried, ALT_TYPE prev_alt_type) { if(numALTsTried > gpol.maxAltsTried() + dep) return 0; assert_gt(rdlen, 0); assert_gt(rflen, 0); if(ht_llist.size() <= dep) ht_llist.expand(); if(raw_refbufs.size() <= dep) raw_refbufs.expand(); if(rfoff < -16) return 0; size_t contig_len = ref.approxLen(tidx); if(rfoff >= contig_len) return 0; if(rfoff >= 0 && rfoff + rflen > contig_len) { rflen = contig_len - rfoff; } else if(rfoff < 0 && rflen > contig_len) { rflen = contig_len; } if(rflen == 0) return 0; if(rfseq == NULL) { SStringExpandable& raw_refbuf = raw_refbufs[dep]; raw_refbuf.resize(rflen + 16 + 16); raw_refbuf.fill(0x4); int off = ref.getStretch( reinterpret_cast(raw_refbuf.wbuf() + 16), tidx, max(rfoff, 0), rfoff > 0 ? rflen : rflen + rfoff ASSERT_ONLY(, destU32)); assert_lt(off, 16); rfseq = raw_refbuf.wbuf() + 16 + off + min(rfoff, 0); } if(left) { index_t tmp_mm = 0; int min_rd_i = (int)rdoff; int mm_min_rd_i = (int)rdoff; index_t mm_tmp_numNs = 0; for(int rf_i = (int)rflen - 1; rf_i >= 0 && mm_min_rd_i >= 0; rf_i--, mm_min_rd_i--) { int rf_bp = rfseq[rf_i]; int rd_bp = rdseq[mm_min_rd_i]; if(rf_bp != rd_bp || rd_bp == 4) { if(tmp_mm == 0) { min_rd_i = mm_min_rd_i; } if(tmp_mm >= mm) break; tmp_mm++; Edit e( mm_min_rd_i, "ACGTN"[rf_bp], "ACGTN"[rd_bp], EDIT_TYPE_MM); tmp_edits.insert(e, 0); } if(rf_bp == 4) { if(tmp_mm == 0) tmp_numNs++; mm_tmp_numNs++; } } if(tmp_mm == 0) { min_rd_i = mm_min_rd_i; } if(mm_min_rd_i < best_rdoff) { best_rdoff = mm_min_rd_i; edits = tmp_edits; if(numNs != NULL) *numNs = mm_tmp_numNs; } if(mm_min_rd_i < 0) return rdlen; if(tmp_mm > 0) { tmp_edits.erase(0, tmp_mm); tmp_mm = 0; } // Find SNPs included in this region pair alt_range(0, 0); if(alts.size() > 0) { ALT cmp_alt; const index_t minK = 16; assert_leq(mm_min_rd_i, rdoff); index_t rd_diff = rdoff - mm_min_rd_i; rd_diff = (rd_diff > minK ? rd_diff - minK : 0); if(gpol.enableCODIS()) { rd_diff = 0; } if(rd_diff >= joinedOff) { cmp_alt.pos = joinedOff; } else { cmp_alt.pos = joinedOff - rd_diff; } alt_range.first = alt_range.second = (int)alts.bsearchLoBound(cmp_alt); if(alt_range.first >= alts.size()) { assert_gt(alts.size(), 0); alt_range.first = alt_range.second = alt_range.second - 1; } for(; alt_range.first >= 0; alt_range.first--) { const ALT& alt = alts[alt_range.first]; if(alt.snp()) { if(alt.deletion() && !alt.reversed) continue; if(alt.pos + rdlen < joinedOff) break; } else if(alt.splicesite()) { if(alt.left < alt.right) continue; if(alt.left + rdlen - 1 < joinedOff) break; } else { assert(alt.exon()); continue; } } } // Update and find Haplotypes EList >& ht_list = ht_llist[dep]; ht_list.clear(); if(gpol.useHaplotype() && haplotypes.size() > 0) { if(dep > 0) { EList >& ht_prev_list = ht_llist[dep-1]; for(index_t p = 0; p < ht_prev_list.size(); p++) { const pair& ht_ref = ht_prev_list[p]; const Haplotype& ht = haplotypes[ht_ref.first]; assert_lt(ht_ref.second, ht.alts.size()); index_t alt_id = ht.alts[ht_ref.second]; assert_gt(tmp_edits.size(), 0); const ALT& alt = alts[tmp_edits[0].snpID]; const ALT& ht_alt = alts[alt_id]; if(!alt.isSame(ht_alt)) continue; if(ht_ref.second == 0) { cmp_ht.left = cmp_ht.right = joinedOff; add_haplotypes(alts, haplotypes, haplotype_maxrights, cmp_ht, ht_list, rdlen); } else { ht_list.push_back(ht_ref); ht_list.back().second--; } } } if(ht_list.size() <= 0) { cmp_ht.left = cmp_ht.right = joinedOff; add_haplotypes(alts, haplotypes, haplotype_maxrights, cmp_ht, ht_list, rdlen, true, // left_ext? dep == 0); // initial? } } assert_geq(rdoff, 0); const index_t orig_nedits = (index_t)tmp_edits.size(); for(; alt_range.second > alt_range.first; alt_range.second--) { ALT alt = alts[alt_range.second]; if(alt.pos >= joinedOff) continue; if(alt.splicesite()) { if(alt.left < alt.right) continue; index_t tmp = alt.left; alt.left = alt.right; alt.right = tmp; } if(alt.deletion()) { if(!alt.reversed) continue; alt.pos = alt.pos - alt.len + 1; } if(alt.exon()) continue; bool alt_compatible = false; int rf_i = (int)rflen - 1, rd_i = (int)rdoff; int diff = 0; if(alt.type == ALT_SNP_SGL) { diff = joinedOff - alt.pos - 1; } else if(alt.type == ALT_SNP_DEL) { if(alt.pos + alt.len >= joinedOff) continue; diff = joinedOff - (alt.pos + alt.len); } else if(alt.type == ALT_SNP_INS) { diff = joinedOff - alt.pos; } else { assert(alt.splicesite()); diff = joinedOff - (alt.right + 1); } if(rf_i < diff || rd_i < diff) continue; rf_i -= diff; rd_i -= diff; int rd_bp = rdseq[rd_i]; if(rd_i < min_rd_i) { if(alt.type == ALT_SNP_INS) { if(rd_i + 1 >= min_rd_i) continue; } break; } // Check to see if there is a haplotype that supports this alt if(ht_list.size() > 0 && alt.snp()) { bool ht_found = false; for(index_t h = 0; h < ht_list.size(); h++) { const pair& ht_ref = ht_list[h]; const Haplotype& ht = haplotypes[ht_ref.first]; assert_lt(ht_ref.second, ht.alts.size()); index_t ht_alti = ht.alts[ht_ref.second]; const ALT& ht_alt = alts[ht_alti]; if(alts[alt_range.second].isSame(ht_alt)) { ht_found = true; break; } } if(!ht_found) continue; } if(alt.type == ALT_SNP_SGL) { if(rd_bp == (int)alt.seq) { int rf_bp = rfseq[rf_i]; Edit e( rd_i, "ACGTN"[rf_bp], "ACGTN"[rd_bp], EDIT_TYPE_MM, true, /* chars? */ alt_range.second); tmp_edits.insert(e, 0); rd_i--; rf_i--; alt_compatible = true; } } else if(alt.type == ALT_SNP_DEL) { if(rfoff + rf_i > (int)alt.len) { if(rf_i > (int)alt.len) { for(index_t i = 0; i < alt.len; i++) { int rf_bp = rfseq[rf_i - i]; Edit e( rd_i + 1, "ACGTN"[rf_bp], '-', EDIT_TYPE_READ_GAP, true, /* chars? */ alt_range.second); tmp_edits.insert(e, 0); } } else { // long deletions int new_rfoff = rfoff - alt.len; index_t new_rflen = rf_i + alt.len + 10; if(raw_refbufs.size() <= dep + 1) raw_refbufs.expand(); SStringExpandable& raw_refbuf = raw_refbufs[dep + 1]; raw_refbuf.resize(new_rflen + 16 + 16); raw_refbuf.fill(0x4); int off = ref.getStretch( reinterpret_cast(raw_refbuf.wbuf() + 16), tidx, max(new_rfoff, 0), new_rfoff > 0 ? new_rflen : new_rflen + new_rfoff ASSERT_ONLY(, destU32)); assert_lt(off, 16); const char* new_rfseq = raw_refbuf.wbuf() + 16 + off + min(new_rfoff, 0); for(int i = 0; i < alt.len; i++) { int rf_bp = new_rfseq[rf_i - i + alt.len]; Edit e( rd_i + 1, "ACGTN"[rf_bp], '-', EDIT_TYPE_READ_GAP, true, /* chars? */ alt_range.second); tmp_edits.insert(e, 0); } } rf_i -= (int)alt.len; alt_compatible = true; } } else if(alt.type == ALT_SNP_INS) { if(rd_i > (int)alt.len) { bool same_seq = true; for(index_t i = 0; i < alt.len; i++) { rd_bp = rdseq[rd_i - i]; int snp_bp = (alt.seq >> (i << 1)) & 0x3; if(rd_bp != snp_bp) { same_seq = false; break; } Edit e( rd_i - i, '-', "ACGTN"[rd_bp], EDIT_TYPE_REF_GAP, true, /* chars? */ alt_range.second); tmp_edits.insert(e, 0); } if(same_seq) { rd_i -= (int)alt.len; alt_compatible = true; } } } else if(alt.type == ALT_SPLICESITE) { bool add_splicesite = true; if(rd_i == rdoff && prev_alt_type == ALT_SPLICESITE) { add_splicesite = false; } if(add_splicesite) { assert_lt(rd_i, rflen); assert_lt(alt.left, alt.right); index_t intronLen = alt.right - alt.left + 1; Edit e(rd_i + 1, 0, 0, EDIT_TYPE_SPL, intronLen, alt.fw ? SPL_FW : SPL_RC, true, /* known splice site? */ false); /* chrs? */ tmp_edits.insert(e, 0); alt_compatible = true; } } if(alt_compatible) { numALTsTried++; assert_leq(rd_i, (int)rdoff); if(rd_i < 0) { best_rdoff = rd_i; edits = tmp_edits; return rdlen; } index_t next_joinedOff = alt.pos; int next_rfoff = rfoff, next_rdoff = rd_i; const char* next_rfseq = rfseq; int next_rflen = rf_i + 1, next_rdlen = rd_i + 1; if(alt.splicesite()) { assert_lt(alt.left, alt.right); next_joinedOff = alt.left; index_t intronLen = alt.right - alt.left + 1; assert_geq(next_rfoff, intronLen); next_rfoff -= intronLen; next_rfseq = NULL; } if(next_rflen < next_rdlen) { int add_len = next_rdlen + 10 - next_rflen; if(next_rfoff < add_len) add_len = next_rfoff; next_rfoff -= add_len; next_rflen += add_len; next_rfseq = NULL; } index_t alignedLen = alignWithALTs_recur( alts, haplotypes, haplotype_maxrights, next_joinedOff, rdseq, rdoff_add, next_rdoff, next_rdlen, ref, raw_refbufs, ASSERT_ONLY(destU32,) tmp_edits, best_rdoff, next_rfseq, tidx, next_rfoff, next_rflen, left, edits, mm, ht_llist, cmp_ht, candidate_edits, tmp_numNs, numNs, dep + 1, gpol, numALTsTried, alt.type); if(alignedLen == next_rdlen) return rdlen; } // Restore to the earlier state assert_leq(orig_nedits, tmp_edits.size()); if(orig_nedits < tmp_edits.size()) tmp_edits.erase(0, tmp_edits.size() - orig_nedits); } return 0; } else { index_t tmp_mm = 0; index_t max_rd_i = 0; index_t mm_max_rd_i = 0; index_t mm_tmp_numNs = 0; for(index_t rf_i = 0; rf_i < rflen && mm_max_rd_i < rdlen; rf_i++, mm_max_rd_i++) { int rf_bp = rfseq[rf_i]; int rd_bp = rdseq[rdoff + mm_max_rd_i]; if(rf_bp != rd_bp || rd_bp == 4) { if(tmp_mm == 0) { max_rd_i = mm_max_rd_i; } if(tmp_mm >= mm) break; tmp_mm++; Edit e( mm_max_rd_i + rdoff_add, "ACGTN"[rf_bp], "ACGTN"[rd_bp], EDIT_TYPE_MM); tmp_edits.push_back(e); } if(rf_bp == 4) { if(tmp_mm == 0) tmp_numNs++; mm_tmp_numNs++; } } if(tmp_mm == 0) { max_rd_i = mm_max_rd_i; } if(mm_max_rd_i + rdoff > best_rdoff) { best_rdoff = mm_max_rd_i + rdoff; edits = tmp_edits; if(numNs != NULL) *numNs = mm_tmp_numNs; if(candidate_edits != NULL) candidate_edits->clear(); } else if(mm_max_rd_i + rdoff == best_rdoff) { if(candidate_edits != NULL) { candidate_edits->expand(); candidate_edits->back() = tmp_edits; } } if(mm_max_rd_i == rflen) { return mm_max_rd_i; } // Find SNPs included in this region pair alt_range; { ALT cmp_alt; const index_t minK = 16; index_t rd_diff = (max_rd_i > minK ? max_rd_i - minK : 0); if(gpol.enableCODIS()) { rd_diff = 0; } cmp_alt.pos = joinedOff + rd_diff; alt_range.first = alt_range.second = (index_t)alts.bsearchLoBound(cmp_alt); if(alt_range.first >= alts.size()) return 0; for(; alt_range.second < alts.size(); alt_range.second++) { const ALT& alt = alts[alt_range.second]; if(alt.splicesite()) { if(alt.left > alt.right) continue; } if(alt.deletion()) { if(alt.reversed) continue; } if(alt.left > joinedOff + max_rd_i) break; } } if(mm_max_rd_i == rdlen) { bool further_search = false; for(index_t s = alt_range.first; s < alt_range.second; s++) { const ALT& alt = alts[s]; if(alt.splicesite() && alt.left < alt.right) { further_search = true; break; } } if(!further_search) return mm_max_rd_i; } if(tmp_mm > 0) { tmp_edits.resize(tmp_edits.size() - tmp_mm); tmp_mm = 0; } // Update and find Haplotypes EList >& ht_list = ht_llist[dep]; ht_list.clear(); if(gpol.useHaplotype() && haplotypes.size() > 0) { if(dep > 0) { EList >& ht_prev_list = ht_llist[dep-1]; for(index_t p = 0; p < ht_prev_list.size(); p++) { const pair& ht_ref = ht_prev_list[p]; const Haplotype& ht = haplotypes[ht_ref.first]; if(ht_ref.second < ht.alts.size()) { index_t alt_id = ht.alts[ht_ref.second]; assert_gt(tmp_edits.size(), 0); const ALT& alt = alts[tmp_edits.back().snpID]; const ALT& ht_alt = alts[alt_id]; if(!alt.isSame(ht_alt)) continue; } if(ht_ref.second + 1 >= ht.alts.size() && joinedOff > ht.right) { cmp_ht.left = cmp_ht.right = joinedOff; add_haplotypes(alts, haplotypes, haplotype_maxrights, cmp_ht, ht_list, rdlen, false); // left_ext? } else { ht_list.push_back(ht_ref); ht_list.back().second++; } } } if(ht_list.size() <= 0) { cmp_ht.left = cmp_ht.right = joinedOff; add_haplotypes(alts, haplotypes, haplotype_maxrights, cmp_ht, ht_list, rdlen, false, // left_ext? dep == 0 && rdoff_add == 0); // initial? } } const index_t orig_nedits = (index_t)tmp_edits.size(); for(; alt_range.first < alt_range.second; alt_range.first++) { const ALT& alt = alts[alt_range.first]; if(alt.splicesite()) { if(alt.left > alt.right) continue; } if(alt.exon()) continue; if(alt.deletion()) { if(alt.reversed) continue; } bool alt_compatible = false; assert_leq(joinedOff, alt.pos); index_t rf_i, rd_i; rf_i = rd_i = alt.pos - joinedOff; if(rd_i >= rdlen) continue; assert_leq(rd_i, max_rd_i); int rf_bp = rfseq[rf_i]; int rd_bp = rdseq[rdoff + rd_i]; // Check to see if there is a haplotype that supports this alt if(ht_list.size() > 0 && alt.snp()) { bool ht_found = false; for(index_t h = 0; h < ht_list.size(); h++) { const pair& ht_ref = ht_list[h]; const Haplotype& ht = haplotypes[ht_ref.first]; if(ht_ref.second >= ht.alts.size()) continue; index_t ht_alti = ht.alts[ht_ref.second]; const ALT& ht_alt = alts[ht_alti]; if(alts[alt_range.first].isSame(ht_alt)) { ht_found = true; break; } } if(!ht_found) continue; } if(alt.type == ALT_SNP_SGL) { if(rd_bp == (int)alt.seq) { Edit e( rd_i + rdoff_add, "ACGTN"[rf_bp], "ACGTN"[rd_bp], EDIT_TYPE_MM, true, /* chars? */ alt_range.first); tmp_edits.push_back(e); rd_i++; rf_i++; alt_compatible = true; } } else if(alt.type == ALT_SNP_DEL) { bool try_del = rd_i > 0; if(rd_i == 0 && dep > 0) { // Avoid consecutive deletions assert_gt(tmp_edits.size(), 0); const Edit& e = tmp_edits.back(); if(e.type != EDIT_TYPE_READ_GAP) { try_del = true; } } if(try_del) { if(rf_i + alt.len <= rflen) { for(index_t i = 0; i < alt.len; i++) { rf_bp = rfseq[rf_i + i]; Edit e( rd_i + rdoff_add, "ACGTN"[rf_bp], '-', EDIT_TYPE_READ_GAP, true, /* chars? */ alt_range.first); tmp_edits.push_back(e); } rf_i += alt.len; alt_compatible = true; } else { // long deletions index_t new_rflen = rf_i + alt.len + 10; if(raw_refbufs.size() <= dep + 1) raw_refbufs.expand(); SStringExpandable& raw_refbuf = raw_refbufs[dep + 1]; raw_refbuf.resize(new_rflen + 16 + 16); raw_refbuf.fill(0x4); int off = ref.getStretch( reinterpret_cast(raw_refbuf.wbuf() + 16), tidx, max(rfoff, 0), rfoff > 0 ? new_rflen : new_rflen + rfoff ASSERT_ONLY(, destU32)); assert_lt(off, 16); const char* new_rfseq = raw_refbuf.wbuf() + 16 + off + min(rfoff, 0); for(index_t i = 0; i < alt.len; i++) { rf_bp = new_rfseq[rf_i + i]; Edit e( rd_i + rdoff_add, "ACGTN"[rf_bp], '-', EDIT_TYPE_READ_GAP, true, /* chars? */ alt_range.first); tmp_edits.push_back(e); } rf_i += alt.len; alt_compatible = true; } } } else if(alt.type == ALT_SNP_INS) { if(rd_i + alt.len <= rdlen && rf_i > 0) { bool same_seq = true; for(index_t i = 0; i < alt.len; i++) { rd_bp = rdseq[rdoff + rd_i + i]; int snp_bp = (alt.seq >> ((alt.len - i - 1) << 1)) & 0x3; if(rd_bp != snp_bp) { same_seq = false; break; } Edit e( rd_i + i + rdoff_add, '-', "ACGTN"[rd_bp], EDIT_TYPE_REF_GAP, true, /* chars? */ alt_range.first); tmp_edits.push_back(e); } if(same_seq) { rd_i += alt.len; alt_compatible = true; } } } else if(alt.type == ALT_SPLICESITE) { bool try_splice = rd_i > 0; if(rd_i == 0 && dep > 0) { // Avoid consecutive introns assert_gt(tmp_edits.size(), 0); const Edit& e = tmp_edits.back(); if(e.type != EDIT_TYPE_SPL) { try_splice = true; } } if(try_splice) { assert_lt(rd_i, rflen); index_t intronLen = alt.right - alt.left + 1; Edit e(rd_i + rdoff_add, 0, 0, EDIT_TYPE_SPL, intronLen, alt.fw ? SPL_FW : SPL_RC, true, /* known splice site? */ false); /* chrs? */ tmp_edits.push_back(e); alt_compatible = true; } } if(alt_compatible) { numALTsTried++; if(rd_i == rdlen) { assert_leq(best_rdoff, rdoff + rd_i); if(best_rdoff < rdoff + rd_i) { if(candidate_edits != NULL) candidate_edits->clear(); } if(candidate_edits != NULL) { candidate_edits->expand(); candidate_edits->back() = tmp_edits; } best_rdoff = rdoff + rd_i; edits = tmp_edits; return rd_i; } index_t next_joinedOff = 0; int next_rfoff = rfoff + rf_i, next_rdoff = rdoff + rd_i; const char* next_rfseq = rfseq + rf_i; index_t next_rflen = rflen - rf_i, next_rdlen = rdlen - rd_i; if(alt.type == ALT_SNP_SGL) { next_joinedOff = alt.pos + 1; } else if(alt.type == ALT_SNP_DEL) { next_joinedOff = alt.pos + alt.len; if(rflen <= rf_i) { next_rflen = 0; // Will reset next_rfseq and next_rflen below } } else if(alt.type == ALT_SNP_INS) { next_joinedOff = alt.pos; } else if(alt.type == ALT_SPLICESITE) { next_joinedOff = alt.right + 1; index_t intronLen = alt.right - alt.left + 1; next_rfoff += intronLen; next_rfseq = NULL; } else { assert(false); } if(next_rflen < next_rdlen) { next_rflen = next_rdlen + 10; next_rfseq = NULL; } index_t alignedLen = alignWithALTs_recur( alts, haplotypes, haplotype_maxrights, next_joinedOff, rdseq, rdoff_add + rd_i, next_rdoff, next_rdlen, ref, raw_refbufs, ASSERT_ONLY(destU32,) tmp_edits, best_rdoff, next_rfseq, tidx, next_rfoff, next_rflen, left, edits, mm, ht_llist, cmp_ht, candidate_edits, tmp_numNs, numNs, dep + 1, gpol, numALTsTried, alt.type); if(alignedLen > 0) { assert_leq(rdoff + rd_i + alignedLen, best_rdoff); bool search_further = false; if(alt.splicesite()) { for(index_t sf = alt_range.first + 1; sf < alt_range.second; sf++) { const ALT& alt2 = alts[sf]; if(alt2.splicesite() && alt2.left < alt2.right) { search_further = true; break; } } } if(!search_further) { if(rd_i + alignedLen == rdlen) { return rd_i + alignedLen; } } } } // Restore to the earlier state assert_leq(orig_nedits, tmp_edits.size()); if(orig_nedits < tmp_edits.size()) tmp_edits.resize(orig_nedits); } return 0; } } /** * For alignment involving indel, move the indels * to the left most possible position */ template void GenomeHit::leftAlign(const Read& rd) { ASSERT_ONLY(const index_t rdlen = (index_t)rd.length()); const BTDnaString& seq = _fw ? rd.patFw : rd.patRc; for(index_t ei = 0; ei < _edits->size(); ei++) { Edit& edit = (*_edits)[ei]; if(edit.type != EDIT_TYPE_READ_GAP && edit.type != EDIT_TYPE_REF_GAP) continue; if(edit.snpID != (index_t)INDEX_MAX) continue; index_t ei2 = ei + 1; for(; ei2 < _edits->size(); ei2++) { const Edit& edit2 = (*_edits)[ei2]; if(edit2.type != edit.type) break; if(edit.type == EDIT_TYPE_READ_GAP) { if(edit.pos != edit2.pos) break; } else { assert_eq(edit.type, EDIT_TYPE_REF_GAP); if(edit.pos + ei2 - ei != edit2.pos) break; } } assert_gt(ei2, 0); ei2 -= 1; Edit& edit2 = (*_edits)[ei2]; int b = 0; if(ei > 0) { const Edit& prev_edit = (*_edits)[ei - 1]; b = prev_edit.pos; } int l = edit.pos - 1; while(l > b) { assert_lt(l, (int)rdlen); int rdc = seq[_rdoff + l]; assert_range(0, 4, rdc); char rfc = (edit.type == EDIT_TYPE_READ_GAP ? edit2.chr : edit2.qchr); if(rfc != "ACGTN"[rdc]) break; for(int ei3 = ei2; ei3 > (int)ei; ei3--) { if(edit.type == EDIT_TYPE_READ_GAP) { (*_edits)[ei3].chr = (*_edits)[ei3 - 1].chr; } else { (*_edits)[ei3].qchr = (*_edits)[ei3 - 1].qchr; } (*_edits)[ei3].pos -= 1; } rdc = seq[_rdoff + l]; assert_range(0, 4, rdc); if(edit.type == EDIT_TYPE_READ_GAP) { edit.chr = "ACGTN"[rdc]; } else { edit.qchr = "ACGTN"[rdc]; } edit.pos -= 1; l--; } ei = ei2; } } #ifndef NDEBUG /** * Check that hit is sane w/r/t read. */ template bool GenomeHit::repOk(const Read& rd, const BitPairReference& ref) { if(_len <= 0) return true; assert(_sharedVars != NULL); SStringExpandable& raw_refbuf = _sharedVars->raw_refbuf; SStringExpandable& destU32 = _sharedVars->destU32; BTDnaString& editstr = _sharedVars->editstr; BTDnaString& partialseq = _sharedVars->partialseq; BTDnaString& refstr = _sharedVars->refstr; EList& reflens = _sharedVars->reflens; EList& refoffs = _sharedVars->refoffs; editstr.clear(); partialseq.clear(); refstr.clear(); reflens.clear(); refoffs.clear(); const BTDnaString& seq = _fw ? rd.patFw : rd.patRc; partialseq.install(seq.buf() + this->_rdoff, (size_t)this->_len); Edit::toRef(partialseq, *_edits, editstr); index_t refallen = 0; int64_t reflen = 0; int64_t refoff = this->_toff; refoffs.push_back((index_t)refoff); size_t eidx = 0; for(size_t i = 0; i < _len; i++, reflen++, refoff++) { while(eidx < _edits->size() && (*_edits)[eidx].pos == i) { const Edit& edit = (*_edits)[eidx]; if(edit.isReadGap()) { reflen++; refoff++; } else if(edit.isRefGap()) { reflen--; refoff--; } if(edit.isSpliced()) { assert_gt(reflen, 0); refallen += reflen; reflens.push_back((index_t)reflen); reflen = 0; refoff += edit.splLen; assert_gt(refoff, 0); refoffs.push_back((index_t)refoff); } eidx++; } } assert_gt(reflen, 0); refallen += (index_t)reflen; reflens.push_back((index_t)reflen); assert_gt(reflens.size(), 0); assert_gt(refoffs.size(), 0); assert_eq(reflens.size(), refoffs.size()); refstr.clear(); for(index_t i = 0; i < reflens.size(); i++) { assert_gt(reflens[i], 0); if(i > 0) { assert_gt(refoffs[i], refoffs[i-1]); } raw_refbuf.resize(reflens[i] + 16); raw_refbuf.clear(); int off = ref.getStretch( reinterpret_cast(raw_refbuf.wbuf()), (size_t)this->_tidx, (size_t)max(refoffs[i], 0), reflens[i], destU32); assert_leq(off, 16); for(index_t j = 0; j < reflens[i]; j++) { char rfc = *(raw_refbuf.buf()+off+j); refstr.append(rfc); } } if(refstr != editstr) { cerr << "Decoded nucleotides and edits don't match reference:" << endl; //cerr << " score: " << score.score() //<< " (" << gaps << " gaps)" << endl; cerr << " edits: "; Edit::print(cerr, *_edits); cerr << endl; cerr << " decoded nucs: " << partialseq << endl; cerr << " edited nucs: " << editstr << endl; cerr << " reference nucs: " << refstr << endl; assert(0); } return true; } #endif /** * Calculate alignment score */ template int64_t GenomeHit::calculateScore( const Read& rd, SpliceSiteDB& ssdb, const Scoring& sc, index_t minK_local, index_t minIntronLen, index_t maxIntronLen, index_t minAnchorLen, index_t minAnchorLen_noncan, const BitPairReference& ref) { int64_t score = 0; double splicescore = 0; int64_t localscore = 0; index_t numsplices = 0; index_t mm = 0; const BTDnaString& seq = _fw ? rd.patFw : rd.patRc; const BTString& qual = _fw ? rd.qual : rd.qualRev; index_t rdlen = (index_t)seq.length(); int64_t toff_base = _toff; bool conflict_splicesites = false; uint8_t whichsense = SPL_UNKNOWN; for(index_t i = 0; i < _edits->size(); i++) { const Edit& edit = (*_edits)[i]; assert_lt(edit.pos, _len); if(edit.type == EDIT_TYPE_MM) { if(edit.snpID == std::numeric_limits::max()) { int pen = sc.score( dna2col[edit.qchr] - '0', asc2dnamask[edit.chr], qual[this->_rdoff + edit.pos] - 33); score += pen; mm++; } } else if(edit.type == EDIT_TYPE_SPL) { // int left = toff_base + edit.pos - 1; // assert_geq(left, 0); // int right = left + edit.splLen + 1; // assert_geq(right, 0); if(!edit.knownSpl) { int left_anchor_len = _rdoff + edit.pos; assert_gt(left_anchor_len, 0); assert_lt(left_anchor_len, (int)rdlen); int right_anchor_len = rdlen - left_anchor_len; index_t mm2 = 0; for(index_t j = i + 1; j < _edits->size(); j++) { const Edit& edit2 = (*_edits)[j]; if(edit2.type == EDIT_TYPE_MM || edit2.type == EDIT_TYPE_READ_GAP || edit2.type == EDIT_TYPE_REF_GAP) mm2++; } left_anchor_len -= (mm * 2); right_anchor_len -= (mm2 * 2); int shorter_anchor_len = min(left_anchor_len, right_anchor_len); if(shorter_anchor_len <= 0) shorter_anchor_len = 1; assert_gt(shorter_anchor_len, 0); uint32_t intronLen_thresh = ((edit.splDir == SPL_FW || edit.splDir == SPL_RC) ? MaxIntronLen(shorter_anchor_len, minAnchorLen) : MaxIntronLen_noncan(shorter_anchor_len, minAnchorLen_noncan)); if(intronLen_thresh < maxIntronLen) { if(edit.splLen > intronLen_thresh) { score += MIN_I32; } if(edit.splDir == SPL_FW || edit.splDir == SPL_RC) { float probscore = ssdb.probscore(edit.donor_seq, edit.acceptor_seq); float probscore_thresh = 0.8f; if(edit.splLen >> 16) probscore_thresh = 0.99f; else if(edit.splLen >> 15) probscore_thresh = 0.97f; else if(edit.splLen >> 14) probscore_thresh = 0.94f; else if(edit.splLen >> 13) probscore_thresh = 0.91f; else if(edit.splLen >> 12) probscore_thresh = 0.88f; if(probscore < probscore_thresh) score += MIN_I32; } if(shorter_anchor_len == left_anchor_len) { if(_trim5 > 0) score += MIN_I32; for(int j = (int)i - 1; j >= 0; j--) { if((*_edits)[j].type == EDIT_TYPE_MM || (*_edits)[j].type == EDIT_TYPE_READ_GAP || (*_edits)[j].type == EDIT_TYPE_REF_GAP) score += MIN_I32; } } else { if(_trim3 > 0) score += MIN_I32; for(index_t j = i + 1; j < _edits->size(); j++) { if((*_edits)[j].type == EDIT_TYPE_MM || (*_edits)[j].type == EDIT_TYPE_READ_GAP || (*_edits)[j].type == EDIT_TYPE_REF_GAP) score += MIN_I32; } } } if(edit.snpID == std::numeric_limits::max()) { if(edit.splDir == SPL_FW || edit.splDir == SPL_RC) { score -= sc.canSpl((int)edit.splLen); } else { score -= sc.noncanSpl((int)edit.splLen); } } // daehwan - for debugging purposes if(shorter_anchor_len <= 15) { numsplices += 1; splicescore += (double)edit.splLen; } } if(!conflict_splicesites) { if(whichsense == SPL_UNKNOWN) { whichsense = edit.splDir; } else if(edit.splDir != SPL_UNKNOWN) { assert_neq(whichsense, SPL_UNKNOWN); if(edit.splDir == SPL_FW || edit.splDir == SPL_SEMI_FW) { if(whichsense != SPL_FW && whichsense != SPL_SEMI_FW) { conflict_splicesites = true; } } if(edit.splDir == SPL_RC || edit.splDir == SPL_SEMI_RC) { if(whichsense != SPL_RC && whichsense != SPL_SEMI_RC) { conflict_splicesites = true; } } } } toff_base += edit.splLen; } else if(edit.type == EDIT_TYPE_READ_GAP) { bool open = true; if(i > 0 && (*_edits)[i-1].type == EDIT_TYPE_READ_GAP && (*_edits)[i-1].pos == edit.pos) { open = false; } if(edit.snpID == std::numeric_limits::max()) { if(open) score -= sc.readGapOpen(); else score -= sc.readGapExtend(); } toff_base++; } else if(edit.type == EDIT_TYPE_REF_GAP) { bool open = true; if(i > 0 && (*_edits)[i-1].type == EDIT_TYPE_REF_GAP && (*_edits)[i-1].pos + 1 == edit.pos) { open = false; } if(edit.snpID == std::numeric_limits::max()) { if(open) score -= sc.refGapOpen(); else score -= sc.refGapExtend(); } toff_base--; } #ifndef NDEBUG else { assert(false); } #endif } // Penalty for soft-clipping for(index_t i = 0; i < _trim5; i++) { score -= sc.sc(qual[i]); } for(index_t i = 0; i < _trim3; i++) { score -= sc.sc(qual[i]); } if(conflict_splicesites) { score -= sc.conflictSpl(); } if (numsplices > 1) splicescore /= (double)numsplices; score += (_len - mm) * sc.match(); _score = score; _splicescore = splicescore; _localscore = localscore; return score; } /** * Encapsulates counters that measure how much work has been done by * hierarchical indexing */ struct HIMetrics { HIMetrics() : mutex_m() { reset(); } void reset() { anchoratts = 0; localatts = 0; localindexatts = 0; localextatts = 0; localsearchrecur = 0; globalgenomecoords = 0; localgenomecoords = 0; } void init( uint64_t localatts_, uint64_t anchoratts_, uint64_t localindexatts_, uint64_t localextatts_, uint64_t localsearchrecur_, uint64_t globalgenomecoords_, uint64_t localgenomecoords_) { localatts = localatts_; anchoratts = anchoratts_; localindexatts = localindexatts_; localextatts = localextatts_; localsearchrecur = localsearchrecur_; globalgenomecoords = globalgenomecoords_; localgenomecoords = localgenomecoords_; } /** * Merge (add) the counters in the given HIMetrics object into this * object. This is the only safe way to update a HIMetrics shared * by multiple threads. */ void merge(const HIMetrics& r, bool getLock = false) { ThreadSafe ts(&mutex_m, getLock); localatts += r.localatts; anchoratts += r.anchoratts; localindexatts += r.localindexatts; localextatts += r.localextatts; localsearchrecur += r.localsearchrecur; globalgenomecoords += r.globalgenomecoords; localgenomecoords += r.localgenomecoords; } uint64_t localatts; // # attempts of local search uint64_t anchoratts; // # attempts of anchor search uint64_t localindexatts; // # attempts of local index search uint64_t localextatts; // # attempts of extension search uint64_t localsearchrecur; uint64_t globalgenomecoords; uint64_t localgenomecoords; MUTEX_T mutex_m; }; /** * With a hierarchical indexing, SplicedAligner provides several alignment strategies * , which enable effective alignment of RNA-seq reads */ template class HI_Aligner { public: /** * Initialize with index. */ HI_Aligner( const GFM& gfm, bool anchorStop = true, uint64_t threads_rids_mindist = 0) : _anchorStop(anchorStop), _gwstate(GW_CAT), _gwstate_local(GW_CAT), _thread_rids_mindist(threads_rids_mindist) { index_t genomeLen = gfm.gh().len(); _minK = 0; while(genomeLen > 0) { genomeLen >>= 2; _minK++; } _minK_local = 8; } HI_Aligner() { } /** */ void initRead(Read *rd, bool nofw, bool norc, TAlScore minsc, TAlScore maxpen, bool rightendonly = false) { assert(rd != NULL); _rds[0] = rd; _rds[1] = NULL; _paired = false; _rightendonly = rightendonly; _nofw[0] = nofw; _nofw[1] = true; _norc[0] = norc; _norc[1] = true; _minsc[0] = minsc; _minsc[1] = INDEX_MAX; _maxpen[0] = maxpen; _maxpen[1] = INDEX_MAX; for(size_t fwi = 0; fwi < 2; fwi++) { bool fw = (fwi == 0); _hits[0][fwi].init(fw, (index_t)_rds[0]->length()); } _genomeHits.clear(); _genomeHits_rep[0].clear(); _hits_searched[0].clear(); assert(!_paired); } /** */ void initReads(Read *rds[2], bool nofw[2], bool norc[2], TAlScore minsc[2], TAlScore maxpen[2]) { assert(rds[0] != NULL && rds[1] != NULL); _paired = true; _rightendonly = false; for(size_t rdi = 0; rdi < 2; rdi++) { _rds[rdi] = rds[rdi]; _nofw[rdi] = nofw[rdi]; _norc[rdi] = norc[rdi]; _minsc[rdi] = minsc[rdi]; _maxpen[rdi] = maxpen[rdi]; for(size_t fwi = 0; fwi < 2; fwi++) { bool fw = (fwi == 0); _hits[rdi][fwi].init(fw, (index_t)_rds[rdi]->length()); } _hits_searched[rdi].clear(); } _genomeHits.clear(); _genomeHits_rep[0].clear(); _genomeHits_rep[1].clear(); _concordantIdxInspected.first = _concordantIdxInspected.second = 0; assert(_paired); assert(!_rightendonly); } /** * Aligns a read or a pair * This funcion is called per read or pair */ virtual int go( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const GFM* rgfm, const ALTDB& altdb, const RepeatDB& repeatdb, const ALTDB& raltdb, const BitPairReference& ref, const BitPairReference* rref, SwAligner& swa, SpliceSiteDB& ssdb, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink) { const ReportingParams& rp = sink.reportingParams(); index_t rdi; bool fw; bool found[2][2] = {{true, true}, {this->_paired, this->_paired}}; // given read and its reverse complement // (and mate and the reverse complement of mate in case of pair alignment), // pick up one with best partial alignment while(nextBWT(sc, pepol, tpol, gpol, gfm, altdb, ref, rdi, fw, wlm, prm, him, rnd, sink)) { // given the partial alignment, try to extend it to full alignments index_t fwi = (fw == true ? 0 : 1); found[rdi][fwi] = align(sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, fw, wlm, prm, swm, him, rnd, sink); if(!found[0][0] && !found[0][1] && !found[1][0] && !found[1][1]) { break; } // try to combine this alignment with some of mate alignments // to produce pair alignment if(this->_paired) { pairReads(sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, wlm, prm, him, rnd, sink); // if(sink.bestPair() >= _minsc[0] + _minsc[1]) break; } } // if no concordant pair is found, try to use alignment of one-end // as an anchor to align the other-end if(this->_paired) { if(sink.numPair() == 0 && (sink.bestUnp1() >= _minsc[0] || sink.bestUnp2() >= _minsc[1])) { bool mate_found = false; const EList *rs[2] = {NULL, NULL}; sink.getUnp1(rs[0]); assert(rs[0] != NULL); sink.getUnp2(rs[1]); assert(rs[1] != NULL); index_t rs_size[2] = {(index_t)rs[0]->size(), (index_t)rs[1]->size()}; for(index_t i = 0; i < 2; i++) { for(index_t j = 0; j < rs_size[i]; j++) { const AlnRes& res = (*rs[i])[j]; bool fw = (res.orient() == 1); mate_found |= alignMate( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, i, fw, wlm, prm, swm, him, rnd, sink, (index_t)res.refid(), (index_t)res.refoff()); } } if(mate_found) { pairReads( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, wlm, prm, him, rnd, sink); } } } // Determine whether reads map to repetitive sequences bool repeat[2][2] = {{false, false}, {false, false}}; bool perform_repeat_alignment = false; index_t indexIdx[2] = {0, 0}; #if 1 if(rgfm != NULL && !((RFM*)rgfm)->empty()) { // use repeat index to decide whether a read or a pair is from repetitive sequences indexIdx[0] = ((RFM*)rgfm)->getLocalRFM_idx((*_rds)[0].length()); if(_paired) { indexIdx[1] = ((RFM*)rgfm)->getLocalRFM_idx((*_rds)[1].length()); } LocalRFM& rfm = ((RFM*)rgfm)->getLocalRFM(indexIdx[0]); bool skip_repeat[2][2] = {{false, false}, {false, false}}; if(_paired) { const EList *rs[2] = {NULL, NULL}; sink.getPair(rs[0], rs[1]); assert_eq(rs[0]->size(), rs[1]->size()); TAlScore bestScore[2][2] = {{_minsc[rdi], _minsc[rdi]}, {_minsc[rdi], _minsc[rdi]}}; for(size_t r = 0; r < rs[0]->size(); r++) { const AlnRes& rs1 = (*rs[0])[r]; const AlnRes& rs2 = (*rs[1])[r]; TAlScore score = rs1.score().score() + rs2.score().score(); int fwi[2] = {rs1.fw() ? 0 : 1, rs2.fw() ? 0 : 1}; if(score > bestScore[fwi[0]][fwi[1]]) { bestScore[fwi[0]][fwi[1]] = score; } } for(index_t fwi = 0; fwi < 2; fwi++) { for(index_t fwi2 = 0; fwi2 < 2; fwi2++) { if(bestScore[fwi][fwi2] < 0) continue; ReadBWTHit& hit = _hits[0][fwi]; bool unique = false; for(size_t hi = 0; hi < hit.offsetSize(); hi++) { BWTHit& partialHit = hit.getPartialHit(hi); if(partialHit.len() >= _minK + 8 && partialHit.size() == 1) { unique = true; break; } } if(!unique) continue; bool unique2 = false; ReadBWTHit& hit2 = _hits[1][fwi2]; for(size_t hi = 0; hi < hit2.offsetSize(); hi++) { BWTHit& partialHit = hit2.getPartialHit(hi); if(partialHit.len() >= _minK + 8 && partialHit.size() == 1) { unique2 = true; break; } } if(!unique2) continue; skip_repeat[0][fwi] = skip_repeat[1][fwi2] = true; } } } else { const EList *rs = NULL; if(rdi == 0) sink.getUnp1(rs); else sink.getUnp2(rs); TAlScore bestScore[2] = {_minsc[rdi], _minsc[rdi]}; for(index_t r = 0; r < rs->size(); r++) { TAlScore score = (*rs)[r].score().score(); if((*rs)[r].fw()) { if(score > bestScore[0]) { bestScore[0] = score; } } else { if(score > bestScore[1]) { bestScore[1] = score; } } } for(index_t fwi = 0; fwi < 2; fwi++) { if(bestScore[fwi] < 0) continue; ReadBWTHit& hit = _hits[rdi][fwi]; index_t offsetSize = hit.offsetSize(); for(size_t hi = 0; hi < offsetSize; hi++) { BWTHit& partialHit = hit.getPartialHit(hi); if(partialHit.len() >= _minK + 8 && partialHit.size() == 1) { skip_repeat[rdi][fwi] = true; break; } } if(skip_repeat[rdi][fwi]) break; } } for(size_t rdi = 0; rdi < (_paired ? 2 : 1); rdi++) { for(size_t fwi = 0; fwi < 2; fwi++) { if(skip_repeat[rdi][fwi]) continue; bool fw = (fwi == 0); _hits[rdi][fwi].init(fw, (index_t)_rds[rdi]->length()); } } while(nextBWT(sc, pepol, tpol, gpol, rfm, altdb, ref, rdi, fw, wlm, prm, him, rnd, sink)); for(size_t rdi = 0; rdi < (_paired ? 2 : 1); rdi++) { for(size_t fwi = 0; fwi < 2; fwi++) { if(skip_repeat[rdi][fwi]) continue; ReadBWTHit& hit = _hits[rdi][fwi]; index_t offsetSize = hit.offsetSize(); assert_gt(offsetSize, 0); for(size_t hi = 0; hi < offsetSize; hi++) { BWTHit& partialHit = hit.getPartialHit(hi); if(partialHit.len() >= (_minK << 1)) { repeat[rdi][fwi] = true; perform_repeat_alignment = true; break; } } } } } #else // use minimizer to decide whether a read or a pair is from repetitive sequences perform_repeat_alignment = false; for(size_t rdi = 0; rdi < (_paired ? 2 : 1); rdi++) { Read& read = *_rds[rdi]; for(size_t fwi = 0; fwi < 2; fwi++) { const BTDnaString& seq = (fwi == 0 ? read.patFw : read.patRc); repeat[rdi][fwi] = repeat_kmertable.isRepeat(seq, _tmp_minimizers); perform_repeat_alignment |= repeat[rdi][fwi]; } } #endif // Handle alignment to repetitive regions if(rgfm != NULL && perform_repeat_alignment) { LocalRFM& rfm = ((RFM*)rgfm)->getLocalRFM(indexIdx[0]); RB_KmerTable& repeatKmertable = ((RFM*)rgfm)->getKmertable(indexIdx[0]); _repeatConcordant.clear(); index_t prev_align_size[2] = {0, 0}; for(size_t rdi = 0; rdi < (_paired ? 2 : 1); rdi++) { const EList *rs = NULL; if(rdi == 0) sink.getUnp1(rs); else sink.getUnp2(rs); prev_align_size[rdi] = rs->size(); } for(size_t rdi = 0; rdi < (_paired ? 2 : 1); rdi++) { for(size_t fwi = 0; fwi < 2; fwi++) { if(!repeat[rdi][fwi]) continue; // choose candidate partial alignments for further alignment index_t maxsize = max(rp.khits, rp.kseeds); #if 0 ReadBWTHit& hit = _hits[rdi][fwi]; if(!hit.done()) continue; getAnchorHits(rfm, pepol, tpol, gpol, altdb, repeatdb, *rref, rnd, rdi, fwi == 0, // fw _genomeHits_rep[rdi], _genomeHits_rep[rdi].size() + maxsize, _sharedVars, wlm, prm, him, true); // repeat? #else getRepeatHits(rfm, pepol, tpol, gpol, raltdb, repeatdb, repeatKmertable, *rref, rnd, rdi, fwi == 0, // fw indexIdx[0], _genomeHits_rep[rdi], _genomeHits_rep[rdi].size() + maxsize, _sharedVars, sc, swa, ssdb, swm, wlm, prm, him, sink); #endif } } EList, RepeatCoord > >& positions = _positions; for(size_t rdi = 0; rdi < (_paired ? 2 : 1); rdi++) { for(size_t i = 0; i < _genomeHits_rep[rdi].size(); i++) { if(_genomeHits_rep[rdi][i].len() < (_minK << 1)) continue; // DK - debugging purposes #if 0 positions.clear(); repeatdb.getCoords(_genomeHits_rep[rdi][i]._tidx, _genomeHits_rep[rdi][i]._joinedOff, _genomeHits_rep[rdi][i]._joinedOff + _genomeHits_rep[rdi][i].len(), _snpIDs, raltdb, positions, rp.khits * 1000); #endif const EList *rs = NULL; if(rdi == 0) sink.getUnp2(rs); else sink.getUnp1(rs); assert(rs != NULL); bool candidate_found = false; for(size_t j = 0; j < prev_align_size[1-rdi]; j++) { const AlnRes& res = (*rs)[j]; if(res.repeat()) continue; TAlScore estScore = res.score().score() + _genomeHits_rep[rdi][i].score(); if(sink.bestPair() >= estScore && sink.numBestPair().first > rp.khits) break; positions.clear(); index_t joinedOff = 0; gfm.textOffToJoined(res.refid(), res.refoff(), joinedOff); repeatdb.findCoords(joinedOff, joinedOff + res.refExtent(), _genomeHits_rep[rdi][i]._tidx, _genomeHits_rep[rdi][i]._joinedOff, _genomeHits_rep[rdi][i]._joinedOff + _genomeHits_rep[rdi][i].len(), _snpIDs, raltdb, positions, rp.khits * 10); if(positions.size() <= 0) continue; for(size_t p = 0; p < positions.size(); p++) { if(positions[p].first.tid != res.refid()) continue; if(positions[p].first.toff + 1000 < res.refoff() || res.refoff() + 1000 < positions[p].first.toff) continue; if(sink.bestPair() >= estScore && sink.numBestPair().first > rp.khits) break; candidate_found = true; _genomeHits.clear(); _genomeHits.expand(); _genomeHits.back() = _genomeHits_rep[rdi][i]; _genomeHits.back()._tidx = positions[p].first.tid; _genomeHits.back()._toff = positions[p].first.toff; _genomeHits.back()._joinedOff = positions[p].first.joinedOff; if(!positions[p].first.fw) { _genomeHits.back().reverse(*_rds[rdi]); } // extend the partial alignments bidirectionally using // local search, extension, and (less often) global search hybridSearch(sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, _genomeHits.back()._fw, wlm, prm, swm, him, rnd, sink); } if(candidate_found) { pairReads( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, wlm, prm, him, rnd, sink); } } if(rdi == 0 && _paired) { for(size_t j = 0; j < _genomeHits_rep[1].size(); j++) { if(_genomeHits_rep[1][j].len() < (_minK << 1)) continue; TAlScore estScore = _genomeHits_rep[0][i].score() + _genomeHits_rep[1][j].score(); // if(sink.bestPair() >= estScore && sink.numBestPair().first > rp.khits) // break; positions.clear(); repeatdb.findCommonCoords(_genomeHits_rep[0][i]._tidx, _genomeHits_rep[0][i]._joinedOff, _genomeHits_rep[0][i]._joinedOff + _genomeHits_rep[0][i].len(), _snpIDs, _genomeHits_rep[1][j]._tidx, _genomeHits_rep[1][j]._joinedOff, _genomeHits_rep[1][j]._joinedOff + _genomeHits_rep[1][j].len(), _snpIDs2, raltdb, positions, rp.khits * 10); if(positions.size() <= 0) continue; _repeatConcordant.expand(); _repeatConcordant.back().first = _genomeHits_rep[0][i]._joinedOff; _repeatConcordant.back().second = _genomeHits_rep[1][j]._joinedOff; for(size_t p = 0; p < positions.size(); p++) { if(sink.bestPair() >= estScore && sink.numBestPair().first > rp.khits) break; _genomeHits.clear(); _genomeHits.expand(); _genomeHits.back() = _genomeHits_rep[0][i]; _genomeHits.back()._tidx = positions[p].first.tid; _genomeHits.back()._toff = positions[p].first.toff; _genomeHits.back()._joinedOff = positions[p].first.joinedOff; if(!positions[p].first.fw) { _genomeHits.back().reverse(*_rds[0]); } // extend the partial alignments bidirectionally using // local search, extension, and (less often) global search hybridSearch(sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, 0, _genomeHits.back()._fw, wlm, prm, swm, him, rnd, sink); _genomeHits.clear(); _genomeHits.expand(); _genomeHits.back() = _genomeHits_rep[1][j]; _genomeHits.back()._tidx = positions[p].second.tid; _genomeHits.back()._toff = positions[p].second.toff; _genomeHits.back()._joinedOff = positions[p].second.joinedOff; if(!positions[p].second.fw) { _genomeHits.back().reverse(*_rds[1]); } // extend the partial alignments bidirectionally using // local search, extension, and (less often) global search hybridSearch(sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, 1, _genomeHits.back()._fw, wlm, prm, swm, him, rnd, sink); } if(positions.size() > 0) { pairReads( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, wlm, prm, him, rnd, sink); } } } // if(rdi == 0) } // for(size_t i = 0; i < _genomeHits_rep[rdi].size() bool align2repeat = false; if(_paired) { index_t numBestPair = sink.numBestPair().first; align2repeat = (numBestPair == 0 || numBestPair > rp.khits); } else { const EList *rs = NULL; if(rdi == 0) sink.getUnp1(rs); else sink.getUnp2(rs); assert(rs != NULL); align2repeat = (rs->size() == 0 || sink.numBestUnp(rdi).first > rp.khits); } if(align2repeat) { for(size_t i = 0; i < _genomeHits_rep[rdi].size(); i++) { _genomeHits.clear(); _genomeHits.expand(); _genomeHits.back() = _genomeHits_rep[rdi][i]; _genomeHits.back()._repeat = true; hybridSearch(sc, pepol, tpol, gpol, rfm, altdb, repeatdb, *rref, swa, ssdb, rdi, _genomeHits.back()._fw, wlm, prm, swm, him, rnd, sink); } if(_paired && rdi == 1) { if(sink.numBestUnp(rdi).first > rp.khits) { pairReads( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, wlm, prm, him, rnd, sink); } } } } // for(size_t rdi = 0 } // repeat return EXTEND_POLICY_FULFILLED; } /** * Given a read or its reverse complement (or mate), * align the unmapped portion using the global FM index */ virtual bool nextBWT( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const BitPairReference& ref, index_t& rdi, bool& fw, WalkMetrics& wlm, PerReadMetrics& prm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink) { const ReportingParams& rp = sink.reportingParams(); // Pick up a candidate from a read or its reverse complement // (for pair, also consider mate and its reverse complement) while(pickNextReadToSearch(rdi, fw)) { size_t mineFw = 0, mineRc = 0; index_t fwi = (fw ? 0 : 1); ReadBWTHit& hit = _hits[rdi][fwi]; assert(!hit.done()); bool pseudogeneStop = gfm.gh().linearFM() && !tpol.no_spliced_alignment(); bool anchorStop = _anchorStop && !gfm.repeat(); if(!rp.secondary) { index_t numSearched = hit.numActualPartialSearch(); int64_t bestScore = 0; if(rdi == 0) { bestScore = sink.bestUnp1(); if(bestScore >= _minsc[rdi]) { // do not further align this candidate // unless it may be at least as good as the alignment of its reverse complement index_t maxmm = (index_t)((-bestScore + sc.mmpMax - 1) / sc.mmpMax); if(numSearched > maxmm + sink.bestSplicedUnp1() + 1) { hit.done(true); if(_paired) { if(sink.bestUnp2() >= _minsc[1-rdi] && sink.numPair() > 0) return false; else continue; } else { return false; } } } } else { assert(_paired); assert_eq(rdi, 1); bestScore = sink.bestUnp2(); if(bestScore >= _minsc[rdi]) { // Do not further extend this alignment // unless it may be at least as good as the previous alignemnt index_t maxmm = (index_t)((-bestScore + sc.mmpMax - 1) / sc.mmpMax); if(numSearched > maxmm + sink.bestSplicedUnp2() + 1) { hit.done(true); if(_paired) { if(sink.bestUnp1() >= _minsc[1-rdi] && sink.numPair() > 0) return false; else continue; } else { return false; } } } } ReadBWTHit& rchit = _hits[rdi][1-fwi]; if(rchit.done() && bestScore < _minsc[rdi]) { if(numSearched > rchit.numActualPartialSearch() + (anchorStop ? 1 : 0)) { hit.done(true); return false; } } } // Align this read beginning from previously stopped base // stops when it is uniquelly mapped with at least 28bp or // it may involve processed pseudogene partialSearch( gfm, *_rds[rdi], sc, sink.reportingParams(), fw, 0, mineFw, mineRc, hit, rnd, pseudogeneStop, anchorStop); assert(hit.repOk()); if(hit.done()) return true; // Advance hit._cur by 1 if(!pseudogeneStop) { if(hit._cur + 1 < hit._len) hit._cur++; } if(anchorStop) { hit.done(true); return true; } // hit.adjustOffset(_minK); } return false; } /** * Given partial alignments of a read, try to further extend * the alignment bidirectionally */ virtual bool align( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, bool fw, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink); /** * Given the alignment of its mate as an anchor, * align the read */ virtual bool alignMate( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, bool fw, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink, index_t tidx, index_t toff); /** * Given a partial alignment of a read, try to further extend * the alignment bidirectionally using a combination of * local search, extension, and global search */ virtual void hybridSearch( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, bool fw, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink) {} /** * Given a partial alignment of a read, try to further extend * the alignment bidirectionally using a combination of * local search, extension, and global search */ virtual int64_t hybridSearch_recur( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, const GenomeHit& hit, index_t hitoff, index_t hitlen, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink, bool alignMate = false, index_t dep = 0) { return numeric_limits::min(); } /** * Choose a candidate for alignment from a read or its reverse complement * (also from a mate or its reverse complement for pair) */ bool pickNextReadToSearch(index_t& rdi, bool& fw) { rdi = 0; fw = true; bool picked = false; int64_t maxScore = std::numeric_limits::min(); for(index_t rdi2 = 0; rdi2 < (_paired ? 2 : 1); rdi2++) { assert(_rds[rdi2] != NULL); for(index_t fwi = 0; fwi < 2; fwi++) { if (fwi == 0 && _nofw[rdi2]) continue; else if(fwi == 1 && _norc[rdi2]) continue; if(_hits[rdi2][fwi].done()) continue; int64_t curScore = _hits[rdi2][fwi].searchScore((index_t)_minK); if(_hits[rdi2][fwi].cur() == 0) { curScore = std::numeric_limits::max(); } assert_gt(curScore, std::numeric_limits::min()); if(curScore > maxScore) { maxScore = curScore; rdi = rdi2; fw = (fwi == 0); picked = true; } } } return picked; } /** * Align a part of a read without any edits */ index_t partialSearch( const GFM& gfm, // GFM index const Read& read, // read to align const Scoring& sc, // scoring scheme const ReportingParams& rp, bool fw, // don't align forward read size_t mineMax, // don't care about edit bounds > this size_t& mineFw, // minimum # edits for forward read size_t& mineRc, // minimum # edits for revcomp read ReadBWTHit& hit, // holds all the seed hits (and exact hit) RandomSource& rnd, bool& pseudogeneStop, // stop if mapped to multiple locations due to processed pseudogenes bool& anchorStop, index_t maxHitLen = (index_t)INDEX_MAX); /** * Global FM index search */ index_t globalGFMSearch( const GFM& gfm, // GFM index const Read& read, // read to align const Scoring& sc, // scoring scheme const ReportingParams& rp, bool fw, index_t hitoff, index_t& hitlen, index_t& top, index_t& bot, index_t& node_top, index_t& node_bot, EList >& node_iedge_count, RandomSource& rnd, bool& uniqueStop, index_t maxHitLen = (index_t)INDEX_MAX); /** * Local FM index search */ index_t localGFMSearch( const LocalGFM& gfm, // GFM index const Read& read, // read to align const Scoring& sc, // scoring scheme const ReportingParams& rp, bool fw, index_t rdoff, index_t& hitlen, local_index_t& top, local_index_t& bot, local_index_t& node_top, local_index_t& node_bot, EList >& local_node_iedge_count, RandomSource& rnd, bool& uniqueStop, local_index_t minUniqueLen, local_index_t maxHitLen = (local_index_t)INDEX_MAX, local_index_t maxHits = 0); /** * Convert FM offsets to the corresponding genomic offset (chromosome id, offset) **/ bool getGenomeCoords( const GFM& gfm, const ALTDB& altdb, const BitPairReference& ref, RandomSource& rnd, index_t top, index_t bot, index_t node_top, index_t node_bot, const EList >& node_iedge_count, bool fw, index_t maxelt, index_t rdoff, index_t rdlen, EList& coords, WalkMetrics& met, PerReadMetrics& prm, HIMetrics& him, bool rejectStraddle, bool& straddled); /** * Convert FM offsets to the corresponding genomic offset (chromosome id, offset) **/ bool getGenomeCoords_local( const GFM& gfm, const ALTDB& altdb, const BitPairReference& ref, RandomSource& rnd, local_index_t top, local_index_t bot, local_index_t node_top, local_index_t node_bot, const EList >& node_iedge_count, bool fw, index_t rdoff, index_t rdlen, EList& coords, WalkMetrics& met, PerReadMetrics& prm, HIMetrics& him, bool rejectStraddle, bool& straddled); /** * Given a set of partial alignments for a read, * choose some that are longer and mapped to fewer places */ index_t getAnchorHits( const GFM& gfm, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, RandomSource& rnd, index_t rdi, bool fw, EList >& genomeHits, index_t maxGenomeHitSize, SharedTempVars& sharedVars, WalkMetrics& wlm, PerReadMetrics& prm, HIMetrics& him, bool repeat = false) { index_t fwi = (fw ? 0 : 1); assert_lt(rdi, 2); assert(_rds[rdi] != NULL); ReadBWTHit& hit = _hits[rdi][fwi]; assert(hit.done()); index_t offsetSize = hit.offsetSize(); assert_gt(offsetSize, 0); for(size_t hi = 0; hi < offsetSize; hi++) { index_t hj = 0; for(; hj < offsetSize; hj++) { BWTHit& partialHit_j = hit.getPartialHit(hj); if(partialHit_j.empty() || partialHit_j.hasGenomeCoords() || partialHit_j.len() <= _minK + 2) continue; else break; } if(hj >= offsetSize) break; for(index_t hk = hj + 1; hk < offsetSize; hk++) { BWTHit& partialHit_j = hit.getPartialHit(hj); BWTHit& partialHit_k = hit.getPartialHit(hk); if(partialHit_k.empty() || partialHit_k.hasGenomeCoords() || partialHit_k.len() <= _minK + 2) continue; if(partialHit_j._hit_type == partialHit_k._hit_type) { if((partialHit_j.size() > partialHit_k.size()) || (partialHit_j.size() == partialHit_k.size() && partialHit_j.len() < partialHit_k.len())) { hj = hk; } } else { if(partialHit_k._hit_type > partialHit_j._hit_type) { hj = hk; } } } BWTHit& partialHit = hit.getPartialHit(hj); assert(!partialHit.hasGenomeCoords()); // Retrieve genomic coordinates // If there are too many genomic coordinates to get, // then we randomly choose and retrieve a small set of them assert_leq(genomeHits.size(), maxGenomeHitSize); index_t remainedGenomeHitSize = maxGenomeHitSize - genomeHits.size(); if(remainedGenomeHitSize <= 0) break; index_t expectedNumCoords = partialHit._node_bot - partialHit._node_top; bool straddled = false; if(expectedNumCoords <= remainedGenomeHitSize) { getGenomeCoords( gfm, altdb, ref, rnd, partialHit._top, partialHit._bot, partialHit._node_top, partialHit._node_bot, partialHit._node_iedge_count, fw, partialHit._bot - partialHit._top, hit._len - partialHit._bwoff - partialHit._len, partialHit._len, partialHit._coords, wlm, prm, him, false, // reject straddled straddled); } else { index_t edgeIdx = 0; index_t top = partialHit._top; index_t added = 0; for(index_t node = partialHit._node_top; node < partialHit._node_bot; node++, expectedNumCoords--) { index_t bot = top + 1; _tmp_node_iedge_count.clear(); if(edgeIdx < partialHit._node_iedge_count.size()) { assert_leq(node - partialHit._node_top, partialHit._node_iedge_count[edgeIdx].first); if(node - partialHit._node_top == partialHit._node_iedge_count[edgeIdx].first) { bot += partialHit._node_iedge_count[edgeIdx].second; _tmp_node_iedge_count.expand(); _tmp_node_iedge_count.back().first = 0; _tmp_node_iedge_count.back().second = partialHit._node_iedge_count[edgeIdx].second; edgeIdx++; } } assert_lt(added, remainedGenomeHitSize); uint32_t rndi = rnd.nextU32() % expectedNumCoords; if(rndi < remainedGenomeHitSize - added) { getGenomeCoords( gfm, altdb, ref, rnd, top, bot, node, node + 1, _tmp_node_iedge_count, fw, partialHit._bot - partialHit._top, hit._len - partialHit._bwoff - partialHit._len, partialHit._len, partialHit._coords, wlm, prm, him, false, // reject straddled straddled); added++; if(added >= remainedGenomeHitSize) break; } top = bot; } } if(!partialHit.hasGenomeCoords()) continue; EList& coords = partialHit._coords; assert_gt(coords.size(), 0); const index_t genomeHit_size = (index_t)genomeHits.size(); if(genomeHit_size + coords.size() > maxGenomeHitSize) { coords.shufflePortion(0, coords.size(), rnd); } for(index_t k = 0; k < coords.size(); k++) { const Coord& coord = coords[k]; if(coord.ref() == numeric_limits::max()) continue; index_t len = partialHit._len; index_t rdoff = hit._len - partialHit._bwoff - len; bool overlapped = false; for(index_t l = 0; l < genomeHit_size; l++) { GenomeHit& genomeHit = genomeHits[l]; if(genomeHit.ref() != (index_t)coord.ref() || genomeHit.fw() != coord.fw()) continue; assert_lt(genomeHit.rdoff(), hit._len); assert_lt(rdoff, hit._len); index_t hitoff = genomeHit.refoff() + hit._len - genomeHit.rdoff(); index_t hitoff2 = (index_t)coord.off() + hit._len - rdoff; int64_t hitoff_diff = (tpol.no_spliced_alignment() ? 0 : tpol.maxIntronLen()); if(abs((int64_t)hitoff - (int64_t)hitoff2) <= hitoff_diff) { overlapped = true; genomeHit._hitcount++; break; } } if(repeat) { if(!repeatdb.repeatExist(coord.ref(), coord.off(), coord.off() + len)) { continue; } } if(!overlapped) { GenomeHit::adjustWithALT( rdoff, len, coord, _sharedVars, genomeHits, *_rds[rdi], gfm, altdb, ref, gpol); } if(partialHit._hit_type == CANDIDATE_HIT && genomeHits.size() >= maxGenomeHitSize) break; } if(partialHit._hit_type == CANDIDATE_HIT && genomeHits.size() >= maxGenomeHitSize) break; } return (index_t)genomeHits.size(); } /** * */ index_t getRepeatHits( const GFM& gfm, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const ALTDB& altdb, const RepeatDB& repeatdb, const RB_KmerTable& repeat_kmertable, const BitPairReference& ref, RandomSource& rnd, index_t rdi, bool fw, index_t repID, EList >& genomeHits, index_t maxGenomeHitSize, SharedTempVars& sharedVars, const Scoring& sc, SwAligner& swa, SpliceSiteDB& ssdb, SwMetrics& swm, WalkMetrics& wlm, PerReadMetrics& prm, HIMetrics& him, AlnSinkWrap& sink) { assert_lt(rdi, 2); assert(_rds[rdi] != NULL); Read& rd = *_rds[rdi]; const BTDnaString& seq = fw ? rd.patFw : rd.patRc; repeat_kmertable.findAlignments(seq, _tmp_minimizers, _tmp_position2D, _tmp_alignments); const TAlScore cushion = sc.mmpMax; TAlScore bestScore = _minsc[rdi]; size_t prev_numHits = genomeHits.size(); for(index_t i = 0; i < _tmp_alignments.size(); i++) { const RB_Alignment& coord = _tmp_alignments[i]; index_t len = seq.length(); index_t rdoff = 0; if(!repeatdb.repeatExist(repID, coord.pos, coord.pos + len)) { continue; } genomeHits.expand(); GenomeHit& genomeHit = genomeHits.back(); genomeHit.init(fw, rdoff, 0, 0, // trim5 0, // trim3 repID, // ref, coord.pos, coord.pos, this->_sharedVars); index_t maxmm = (index_t)(-bestScore / sc.mmpMax); index_t leftext = 0, rightext = len; genomeHit.extend(rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext, maxmm); if(genomeHit.len() < len) { genomeHits.pop_back(); continue; } if(genomeHit.score() > bestScore) { bestScore = genomeHit.score(); size_t remove_count = 0; size_t k = prev_numHits; for(size_t j = prev_numHits; j < genomeHits.size(); j++) { if(genomeHits[j].score() >= max(_minsc[rdi], bestScore - cushion)) { assert_leq(k, j); if(k < j) { genomeHits[k] = genomeHits[j]; } k++; } else { remove_count++; } } assert_eq(k + remove_count, genomeHits.size()); assert_leq(prev_numHits + remove_count, genomeHits.size()); if(remove_count > 0) { genomeHits.resize(genomeHits.size() - remove_count); } } else if(genomeHit.score() < max(_minsc[rdi], bestScore - cushion)) { genomeHits.pop_back(); } } return (index_t)genomeHits.size(); } bool pairReads( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, WalkMetrics& wlm, PerReadMetrics& prm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink); /** * **/ bool reportHit( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, const SpliceSiteDB& ssdb, AlnSinkWrap& sink, index_t rdi, const GenomeHit& hit, bool alignMate = false, const GenomeHit* ohit = NULL); /** * check this alignment is already examined **/ bool redundant( AlnSinkWrap& sink, index_t rdi, index_t tidx, index_t toff); /** * check this alignment is already examined **/ bool redundant( AlnSinkWrap& sink, index_t rdi, const GenomeHit& hit); /** * **/ bool isSearched( const GenomeHit& hit, index_t rdi); /** * **/ void addSearched(const GenomeHit& hit, index_t rdi); protected: Read * _rds[2]; bool _paired; bool _rightendonly; bool _nofw[2]; bool _norc[2]; TAlScore _minsc[2]; TAlScore _maxpen[2]; bool _anchorStop; ReadBWTHit _hits[2][2]; EList _offs; SARangeWithOffs, index_t> _sas; GroupWalk2S, 16> _gws; GroupWalkState _gwstate; EList _offs_local; SARangeWithOffs, local_index_t> _sas_local; GroupWalk2S, 16> _gws_local; GroupWalkState _gwstate_local; // temporary and shared variables used for GenomeHit // this should be defined before _genomeHits and _hits_searched SharedTempVars _sharedVars; // temporary and shared variables for AlnRes LinkedEList > _rawEdits; // temporary EList > _genomeHits; EList > _genomeHits_rep[2]; EList _snpIDs; EList _snpIDs2; EList _genomeHits_done; ELList _coords; EList, RepeatCoord > > _positions; ELList _spliceSites; pair _concordantIdxInspected; EList > _repeatConcordant; size_t _minK; // log4 of the size of a genome size_t _minK_local; // log4 of the size of a local index (8) ELList > _local_genomeHits; EList _anchors_added; uint64_t max_localindexatts; uint64_t bwops_; // Burrows-Wheeler operations uint64_t bwedits_; // Burrows-Wheeler edits // EList > _hits_searched[2]; uint64_t _thread_rids_mindist; // EList > _node_iedge_count; EList > _tmp_node_iedge_count; EList > _local_node_iedge_count; EList > _tmp_local_node_iedge_count; EList > _tmp_minimizers; ELList _tmp_position2D; EList _tmp_alignments; // For AlnRes::matchesRef ASSERT_ONLY(EList raw_matches_); ASSERT_ONLY(BTDnaString tmp_rf_); ASSERT_ONLY(BTDnaString tmp_rdseq_); ASSERT_ONLY(BTString tmp_qseq_); }; #define HIER_INIT_LOCS(top, bot, tloc, bloc, e) { \ if(bot - top == 1) { \ tloc.initFromRow(top, (e).gh(), (e).gfm()); \ bloc.invalidate(); \ } else { \ SideLocus::initFromTopBot(top, bot, (e).gh(), (e).gfm(), tloc, bloc); \ assert(bloc.valid()); \ } \ } #define HIER_SANITY_CHECK_4TUP(t, b, tp, bp) { \ ASSERT_ONLY(cur_index_t tot = (b[0]-t[0])+(b[1]-t[1])+(b[2]-t[2])+(b[3]-t[3])); \ ASSERT_ONLY(cur_index_t totp = (bp[0]-tp[0])+(bp[1]-tp[1])+(bp[2]-tp[2])+(bp[3]-tp[3])); \ assert_eq(tot, totp); \ } #define LOCAL_INIT_LOCS(top, bot, tloc, bloc, e) { \ if(bot - top == 1) { \ tloc.initFromRow(top, (e).gh(), (e).gfm()); \ bloc.invalidate(); \ } else { \ SideLocus::initFromTopBot(top, bot, (e).gh(), (e).gfm(), tloc, bloc); \ assert(bloc.valid()); \ } \ } /** * Given partial alignments of a read, try to further extend * the alignment bidirectionally */ template bool HI_Aligner::align( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, bool fw, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink) { const ReportingParams& rp = sink.reportingParams(); index_t fwi = (fw ? 0 : 1); assert_lt(rdi, 2); assert(_rds[rdi] != NULL); ReadBWTHit& hit = _hits[rdi][fwi]; assert(hit.done()); index_t minOff = 0; if(hit.minWidth(minOff) == std::numeric_limits::max()) return false; // Don't try to align if the potential alignment for this read might be // worse than the best alignment of its reverse complement int64_t bestScore = (rdi == 0 ? sink.bestUnp1() : sink.bestUnp2()); index_t num_spliced = (rdi == 0 ? sink.bestSplicedUnp1() : sink.bestSplicedUnp2()); if(bestScore < _minsc[rdi]) bestScore = _minsc[rdi]; index_t maxmm = (index_t)((-bestScore + sc.mmpMax - 1) / sc.mmpMax); index_t numActualPartialSearch = hit.numActualPartialSearch(); if(!rp.secondary && numActualPartialSearch > maxmm + num_spliced + 1) return true; // choose candidate partial alignments for further alignment const index_t maxsize = max(rp.khits, rp.kseeds); _genomeHits.clear(); index_t numHits = getAnchorHits(gfm, pepol, tpol, gpol, altdb, repeatdb, ref, rnd, rdi, fw, _genomeHits, maxsize, _sharedVars, wlm, prm, him, gfm.repeat()); if(numHits <= 0) return false; // limit the number of local index searches used for alignment of the read uint64_t add = 0; if(rp.secondary) add = (-_minsc[rdi] / sc.mmpMax) * numHits * 2; else add = (-_minsc[rdi] / sc.mmpMax) * numHits; max_localindexatts = him.localindexatts + max(10, add); // extend the partial alignments bidirectionally using // local search, extension, and (less often) global search hybridSearch(sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, fw, wlm, prm, swm, him, rnd, sink); return true; } /** * Given the alignment of its mate as an anchor, * align the read */ template bool HI_Aligner::alignMate( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, bool fw, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink, index_t tidx, index_t toff) { const ReportingParams& rp = sink.reportingParams(); assert_lt(rdi, 2); index_t ordi = 1 - rdi; bool ofw = (fw == gMate2fw ? gMate1fw : gMate2fw); assert(_rds[ordi] != NULL); const Read& ord = *_rds[ordi]; index_t rdlen = (index_t)ord.length(); assert_gt(rdlen, 0); _genomeHits.clear(); if(_coords.size() == 0) { _coords.expand(); } EList& coords = _coords.front(); // local search to find anchors const HGFM* hGFM = (const HGFM*)(&gfm); const LocalGFM* lGFM = hGFM->getLocalGFM(tidx, toff); bool first = true; index_t count = 0; index_t max_hitlen = 0; while(count++ < 2) { if(first) { first = false; } else { if(_genomeHits.size() > 0) break; if(fw) { lGFM = hGFM->nextLocalGFM(lGFM); } else { lGFM = hGFM->prevLocalGFM(lGFM); } if(lGFM == NULL || lGFM->empty()) break; } index_t hitoff = rdlen - 1; while(hitoff >= _minK_local - 1) { index_t hitlen = 0; local_index_t top = (local_index_t)INDEX_MAX, bot = (local_index_t)INDEX_MAX; local_index_t node_top = (local_index_t)INDEX_MAX, node_bot = (local_index_t)INDEX_MAX; _local_node_iedge_count.clear(); bool uniqueStop = false; index_t nelt = localGFMSearch( *lGFM, // GFM index ord, // read to align sc, // scoring scheme sink.reportingParams(), ofw, hitoff, hitlen, top, bot, node_top, node_bot, _local_node_iedge_count, rnd, uniqueStop, _minK_local); assert_leq(top, bot); assert_eq(nelt, (index_t)(node_bot - node_top)); assert_leq(hitlen, hitoff + 1); if(nelt > 0 && nelt <= rp.kseeds && hitlen > max_hitlen) { coords.clear(); bool straddled = false; getGenomeCoords_local( *lGFM, altdb, ref, rnd, top, bot, node_top, node_bot, _local_node_iedge_count, ofw, hitoff - hitlen + 1, hitlen, coords, wlm, prm, him, true, // reject straddled? straddled); assert_leq(coords.size(), nelt); _genomeHits.clear(); for(index_t ri = 0; ri < coords.size(); ri++) { const Coord& coord = coords[ri]; if(tpol.no_spliced_alignment()) { if(coord.off() + pepol.maxFragLen() * 2 < toff || toff + pepol.maxFragLen() * 2 < coord.off()) continue; } GenomeHit::adjustWithALT( hitoff - hitlen + 1, hitlen, coord, _sharedVars, _genomeHits, *this->_rds[ordi], gfm, altdb, ref, gpol); } max_hitlen = hitlen; } assert_leq(hitlen, hitoff + 1); if(hitlen > 0) hitoff -= (hitlen - 1); if(hitoff > 0) hitoff -= 1; } // while(hitoff >= minHitLen - 1) } // while(count++ < 2) // randomly select const index_t maxsize = rp.kseeds; if(_genomeHits.size() > maxsize) { _genomeHits.shufflePortion(0, _genomeHits.size(), rnd); _genomeHits.resize(maxsize); } // local search using the anchor for(index_t hi = 0; hi < _genomeHits.size(); hi++) { him.anchoratts++; GenomeHit& genomeHit = _genomeHits[hi]; index_t leftext = (index_t)INDEX_MAX, rightext = (index_t)INDEX_MAX; genomeHit.extend( ord, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, _minsc[ordi], rnd, (index_t)_minK_local, tpol, gpol, leftext, rightext); hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, ordi, genomeHit, genomeHit.rdoff(), genomeHit.len(), wlm, prm, swm, him, rnd, sink, true); // alignMate? } return true; } /** * convert FM offsets to the corresponding genomic offset (chromosome id, offset) **/ template bool HI_Aligner::getGenomeCoords( const GFM& gfm, const ALTDB& altdb, const BitPairReference& ref, RandomSource& rnd, index_t top, index_t bot, index_t node_top, index_t node_bot, const EList >& node_iedge_count, bool fw, index_t maxelt, index_t rdoff, index_t rdlen, EList& coords, WalkMetrics& met, PerReadMetrics& prm, HIMetrics& him, bool rejectStraddle, bool& straddled) { straddled = false; assert_gt(bot, top); assert_leq(node_bot - node_top, bot - top); index_t nelt = node_bot - node_top; nelt = min(nelt, maxelt); him.globalgenomecoords += nelt; _offs.resize(nelt); _offs.fill((index_t)INDEX_MAX); _sas.init( top, bot, node_top, node_bot, node_iedge_count, rdlen, EListSlice(_offs, 0, nelt)); _gws.init(gfm, ref, _sas, rnd, met); for(index_t off = 0; off < nelt; off++) { WalkResult wr; index_t tidx = 0, toff = 0, tlen = 0; _gws.advanceElement( off, gfm, // forward Bowtie index for walking left ref, // bitpair-encoded reference _sas, // SA range with offsets _gwstate, // GroupWalk state; scratch space wr, // put the result here met, // metrics prm); // per-read metrics assert_neq(wr.toff, (index_t)INDEX_MAX); bool straddled2 = false; gfm.joinedToTextOff( wr.elt.len, wr.toff, tidx, toff, tlen, rejectStraddle, // reject straddlers? straddled2); // straddled? straddled |= straddled2; if(tidx == (index_t)INDEX_MAX) { // The seed hit straddled a reference boundary so the seed // hit isn't valid return false; } index_t global_toff = toff, global_tidx = tidx; // Coordinate of the seed hit w/r/t the pasted reference string coords.expand(); if(!straddled2) { coords.back().init(global_tidx, (int64_t)global_toff, fw, wr.toff); } else { coords.back().init(numeric_limits::max(), (int64_t)global_toff, fw, wr.toff); } } return true; } /** * convert FM offsets to the corresponding genomic offset (chromosome id, offset) **/ template bool HI_Aligner::getGenomeCoords_local( const GFM& gfm, const ALTDB& altdb, const BitPairReference& ref, RandomSource& rnd, local_index_t top, local_index_t bot, local_index_t node_top, local_index_t node_bot, const EList >& node_iedge_count, bool fw, index_t rdoff, index_t rdlen, EList& coords, WalkMetrics& met, PerReadMetrics& prm, HIMetrics& him, bool rejectStraddle, bool& straddled) { straddled = false; assert_gt(bot, top); assert_leq(node_bot - node_top, bot - top); index_t nelt = node_bot - node_top; him.localgenomecoords += nelt; _offs_local.resize(nelt); _offs_local.fill((local_index_t)INDEX_MAX); _sas_local.init( top, bot, node_top, node_bot, node_iedge_count, rdlen, EListSlice(_offs_local, 0, nelt)); _gws_local.init(gfm, ref, _sas_local, rnd, met); for(local_index_t off = 0; off < nelt; off++) { WalkResult wr; local_index_t tidx = 0, toff = 0, tlen = 0; _gws_local.advanceElement( off, gfm, // forward Bowtie index for walking left ref, // bitpair-encoded reference _sas_local, // SA range with offsets _gwstate_local, // GroupWalk state; scratch space wr, // put the result here met, // metrics prm); // per-read metrics assert_neq(wr.toff, (local_index_t)INDEX_MAX); bool straddled2 = false; bool result = gfm.joinedToTextOff( wr.elt.len, wr.toff, tidx, toff, tlen, rejectStraddle, // reject straddlers? straddled2); // straddled? if(!result) continue; straddled |= straddled2; if(tidx == (local_index_t)INDEX_MAX) { // The seed hit straddled a reference boundary so the seed // hit isn't valid return false; } LocalGFM* localGFM = (LocalGFM*)&gfm; index_t global_tidx = localGFM->_tidx; index_t global_toff = toff + localGFM->_localOffset; index_t joinedOff = wr.toff + localGFM->_joinedOffset; if(global_toff < rdoff) continue; // Coordinate of the seed hit w/r/t the pasted reference string coords.expand(); coords.back().init(global_tidx, (int64_t)global_toff, fw, joinedOff); } return true; } /** * examine alignments of left and right reads to produce concordant pair alignment **/ template bool HI_Aligner::pairReads( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, WalkMetrics& wlm, PerReadMetrics& prm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink) { const ReportingParams& rp = sink.reportingParams(); assert(_paired); const EList *rs1 = NULL, *rs2 = NULL; sink.getUnp1(rs1); assert(rs1 != NULL); sink.getUnp2(rs2); assert(rs2 != NULL); index_t start_i = _concordantIdxInspected.first, start_j = _concordantIdxInspected.second; _concordantIdxInspected.first = rs1->size(); _concordantIdxInspected.second = rs2->size(); for(index_t i = 0; i < rs1->size(); i++) { for(index_t j = (i >= start_i ? 0 : start_j); j < rs2->size(); j++) { if(sink.state().doneConcordant()) { return true; } const AlnRes& r1 = (*rs1)[i]; Coord left = r1.refcoord(), right = r1.refcoord_right(); assert_eq(left.ref(), right.ref()); const AlnRes& r2 = (*rs2)[j]; Coord left2 = r2.refcoord(), right2 = r2.refcoord_right(); assert_eq(left2.ref(), right2.ref()); if(r1.repeat() != r2.repeat()) continue; bool dna_frag_pass = true; if(r1.repeat() && r2.repeat()) { bool found = false; for(size_t r = 0; r < _repeatConcordant.size(); r++) { if(_repeatConcordant[r].first == left.off() && _repeatConcordant[r].second == left2.off()) { found = true; break; } } if(!found) { dna_frag_pass = false; } } else{ if(left.ref() != left2.ref()) continue; assert_eq(left.orient(), right.orient()); assert_eq(left2.orient(), right2.orient()); if(left.orient() == gMate1fw) { if(left2.orient() != gMate2fw) continue; } else { if(left2.orient() == gMate2fw) continue; Coord temp = left; left = left2; left2 = temp; temp = right; right = right2; right2 = temp; } if(left.off() > left2.off()) continue; if(right.off() > right2.off()) continue; if(right.off() + (int)tpol.maxIntronLen() < left2.off()) continue; assert_geq(r1.score().score(), _minsc[0]); assert_geq(r2.score().score(), _minsc[1]); if(tpol.no_spliced_alignment()){ int pairCl = PE_ALS_DISCORD; assert_eq(r1.refid(), r2.refid()); index_t off1, off2, len1, len2; bool fw1, fw2; if(r1.refoff() < r2.refoff()) { off1 = r1.refoff(); off2 = r2.refoff(); len1 = r1.refExtent(); len2 = r2.refExtent(); fw1 = r1.fw(); fw2 = r2.fw(); } else { off1 = r2.refoff(); off2 = r1.refoff(); len1 = r2.refExtent(); len2 = r1.refExtent(); fw1 = r2.fw(); fw2 = r1.fw(); } // Check that final mate alignments are consistent with // paired-end fragment constraints pairCl = pepol.peClassifyPair( off1, len1, fw1, off2, len2, fw2); dna_frag_pass = (pairCl != PE_ALS_DISCORD); } } if(!tpol.no_spliced_alignment() || dna_frag_pass) { TAlScore threshold = sink.bestPair(); if(sink.bestUnp1() >= _minsc[0] && sink.bestUnp2() >= _minsc[1]) { TAlScore tmp = sink.bestUnp1() + sink.bestUnp2() - (r1.readLength() + r2.readLength()) * 0.03 * sc.mm(255); if(tmp > threshold) { threshold = tmp; } } if(r1.score().score() + r2.score().score() >= threshold || rp.secondary) { sink.report(0, &r1, &r2); } } } } return true; } /** * report read (or pair) alignment **/ template bool HI_Aligner::reportHit( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, const SpliceSiteDB& ssdb, AlnSinkWrap& sink, index_t rdi, const GenomeHit& hit, bool alignMate, const GenomeHit* ohit) { assert_lt(rdi, 2); assert(_rds[rdi] != NULL); const Read& rd = *_rds[rdi]; index_t rdlen = (index_t)rd.length(); if(hit.rdoff() - hit.trim5() > 0 || hit.len() + hit.trim5() + hit.trim3() < rdlen) return false; if(hit.score() < _minsc[rdi]) return false; if(!sink.reportingParams().repeat && hit.repeat()) return false; // Edits are represented from 5' end of read to 3' end, not an alignment of read EList& edits = const_cast&>(hit.edits()); if(hit.trim5() > 0) { for(size_t i = 0; i < edits.size(); i++) { edits[i].pos += hit.trim5(); } } if(!hit.fw()) { Edit::invertPoss(edits, rdlen, false); } // in case of multiple exonic alignments, choose the ones near (known) splice sites // this helps eliminate cases of reads being mapped to pseudogenes pair spliced = hit.spliced(); // pair if(tpol.xs_only() && spliced.first) { if(hit.splicing_dir() == SPL_UNKNOWN) return false; } if(!tpol.no_spliced_alignment() && tpol.avoid_pseudogene()) { if(!spliced.first) { assert(!spliced.second); const index_t max_exon_size = 10000; index_t left = 0; if(hit.refoff() > max_exon_size) { left = hit.refoff() - max_exon_size; } index_t right = hit.refoff() + hit.len() + max_exon_size; spliced.first = ssdb.hasSpliceSites( hit.ref(), left, right, left, right, true); // include novel splice sites if(altdb.hasExons()) { spliced.second = ssdb.insideExon(hit.ref(), hit.refoff(), hit.refoff() + hit.len() - 1); } } } if(tpol.transcriptome_mapping_only() && !spliced.second) return false; AlnScore asc( hit.score(), // numeric score hit.ns(), // # Ns hit.ngaps(), // # gaps hit.repeat(), hit.splicescore(), // splice score spliced.second, // mapped to known transcripts? spliced.first, // spliced alignment or near splice sites (novel)? hit.trim5(), // left trim length hit.trim3()); // right trim length bool softTrim = hit.trim5() > 0 || hit.trim3() > 0; AlnRes rs; rs.init( rdlen, // # chars after hard trimming rd.rdid, // read ID asc, // alignment score &hit.edits(), // nucleotide edits array 0, // nucleotide edits first pos hit.edits().size(), // nucleotide edits last pos NULL, // ambig base array 0, // ambig base first pos 0, // ambig base last pos hit.coord(), // coord of leftmost aligned char in ref hit.repeat() ? gfm.plen()[0] : gfm.plen()[hit.ref()], // length of reference aligned to &_rawEdits, -1, // # seed mms allowed -1, // seed length -1, // seed interval 0, // minimum score for valid alignment (daehwan) -1, // nuc5p (for colorspace) -1, // nuc3p (for colorspace) false, // soft pre-trimming? 0, // 5p pre-trimming 0, // 3p pre-trimming softTrim, // soft trimming? hit.fw() ? hit.trim5() : hit.trim3(), // 5p trimming hit.fw() ? hit.trim3() : hit.trim5(), // 3p trimming hit.repeat()); // repeat? if(!hit.fw()) { Edit::invertPoss(edits, rdlen, false); } if(hit.trim5() > 0) { for(size_t i = 0; i < edits.size(); i++) { edits[i].pos -= hit.trim5(); } } //rs.setRefNs(nrefn); assert(rs.matchesRef( rd, ref, tmp_rf_, tmp_rdseq_, tmp_qseq_, _sharedVars.raw_refbuf, _sharedVars.destU32, raw_matches_, _sharedVars.raw_refbuf2, _sharedVars.reflens, _sharedVars.refoffs)); if(ohit == NULL) { bool done; if(rdi == 0 && !_rightendonly) { done = sink.report(0, &rs, NULL, alignMate); } else { done = sink.report(0, NULL, &rs, alignMate); } return done; } assert(ohit != NULL); const Read& ord = *_rds[1-rdi]; index_t ordlen = (index_t)ord.length(); if(ohit->rdoff() - ohit->trim5() > 0 || ohit->len() + ohit->trim5() + ohit->trim3() < ordlen) return false; if(ohit->score() < _minsc[1-rdi]) return false; EList& oedits = const_cast&>(ohit->edits()); if(ohit->trim5() > 0) { for(size_t i = 0; i < oedits.size(); i++) { oedits[i].pos += ohit->trim5(); } } if(!ohit->fw()) { Edit::invertPoss(oedits, ordlen, false); } AlnScore oasc( ohit->score(), // numeric score ohit->ns(), // # Ns ohit->ngaps(), // # gaps ohit->repeat()); // repeat? bool osoftTrim = ohit->trim5() > 0 || ohit->trim3() > 0; AlnRes ors; ors.init( ordlen, // # chars after hard trimming ord.rdid, // read ID oasc, // alignment score &ohit->edits(), // nucleotide edits array 0, // nucleotide edits first pos ohit->edits().size(), // nucleotide edits last pos NULL, // ambig base array 0, // ambig base first pos 0, // ambig base last pos ohit->coord(), // coord of leftmost aligned char in ref gfm.plen()[ohit->ref()], // length of reference aligned to &_rawEdits, -1, // # seed mms allowed -1, // seed length -1, // seed interval 0, // minimum score for valid alignment (daehwan) -1, // nuc5p (for colorspace) -1, // nuc3p (for colorspace) false, // soft pre-trimming? 0, // 5p pre-trimming 0, // 3p pre-trimming osoftTrim, // soft trimming? ohit->fw() ? ohit->trim5() : ohit->trim3(), // 5p trimming ohit->fw() ? ohit->trim3() : ohit->trim5(), // 3p trimming ohit->repeat()); // repeat? if(!ohit->fw()) { Edit::invertPoss(oedits, ordlen, false); } if(ohit->trim5() > 0) { for(size_t i = 0; i < oedits.size(); i++) { oedits[i].pos -= ohit->trim5(); } } //rs.setRefNs(nrefn); assert(ors.matchesRef( ord, ref, tmp_rf_, tmp_rdseq_, tmp_qseq_, _sharedVars.raw_refbuf, _sharedVars.destU32, raw_matches_, _sharedVars.raw_refbuf2, _sharedVars.reflens, _sharedVars.refoffs)); bool done; if(rdi == 0) { done = sink.report(0, &rs, &ors); } else { done = sink.report(0, &ors, &rs); } return done; } /** * check this alignment is already examined **/ template bool HI_Aligner::redundant( AlnSinkWrap& sink, index_t rdi, index_t tidx, index_t toff) { assert_lt(rdi, 2); const EList* rs = NULL; if(rdi == 0) sink.getUnp1(rs); else sink.getUnp2(rs); assert(rs != NULL); for(index_t i = 0; i < rs->size(); i++) { Coord coord_left = (*rs)[i].refcoord(), coord_right = (*rs)[i].refcoord_right(); assert_eq(coord_left.ref(), coord_right.ref()); assert_lt(coord_left.off(), coord_right.off()); assert_eq(coord_left.orient(), coord_right.orient()); if(tidx != coord_left.ref()) continue; if(toff >= coord_left.off() && toff <= coord_right.off()) return true; } return false; } /** * check this alignment is already examined **/ template bool HI_Aligner::redundant( AlnSinkWrap& sink, index_t rdi, const GenomeHit& hit) { assert_lt(rdi, 2); assert(_rds[rdi] != NULL); index_t rdlen = (index_t)_rds[rdi]->length(); const EList* rs = NULL; if(rdi == 0) sink.getUnp1(rs); else sink.getUnp2(rs); assert(rs != NULL); for(index_t i = 0; i < rs->size(); i++) { const AlnRes& rsi = (*rs)[i]; if(rsi.refcoord() == hit.coord()) { const EList& editsi = rsi.ned(); const EList& edits = hit.edits(); if(editsi.size() == edits.size()) { size_t eidx = 0; if(!hit.fw()) { Edit::invertPoss(const_cast&>(edits), rdlen, false); } // daehwan: this is a temporary solution to compare edits for(; eidx < editsi.size(); eidx++) { if(!(editsi[eidx] == edits[eidx])) { break; } } if(!hit.fw()) { Edit::invertPoss(const_cast&>(edits), rdlen, false); } if(eidx >= editsi.size()) { assert_eq(eidx, editsi.size()); return true; } } } } return false; } /** * Sweep right-to-left and left-to-right using exact matching. Remember all * the SA ranges encountered along the way. Report exact matches if there are * any. Calculate a lower bound on the number of edits in an end-to-end * alignment. */ template index_t HI_Aligner::partialSearch( const GFM& gfm, // BWT index const Read& read, // read to align const Scoring& sc, // scoring scheme const ReportingParams& rp, bool fw, size_t mineMax, // don't care about edit bounds > this size_t& mineFw, // minimum # edits for forward read size_t& mineRc, // minimum # edits for revcomp read ReadBWTHit& hit, // holds all the seed hits (and exact hit) RandomSource& rnd, // pseudo-random source bool& pseudogeneStop, bool& anchorStop, index_t maxHitLen) { bool pseudogeneStop_ = pseudogeneStop, anchorStop_ = anchorStop; pseudogeneStop = anchorStop = false; const index_t ftabLen = gfm.gh().ftabChars(); const bool linearFM = gfm.gh().linearFM(); SideLocus tloc, bloc; const index_t len = (index_t)read.length(); const BTDnaString& seq = fw ? read.patFw : read.patRc; assert(!seq.empty()); size_t nelt = 0; EList >& partialHits = hit._partialHits; index_t& cur = hit._cur; assert_lt(cur, hit._len); hit._numPartialSearch++; index_t offset = cur; index_t dep = offset; pair range(0, 0); pair rangeTemp(0, 0); pair node_range(0, 0); pair node_rangeTemp(0, 0); _node_iedge_count.clear(); _tmp_node_iedge_count.clear(); index_t left = len - dep; assert_gt(left, 0); if(left < ftabLen + 1) { cur = hit._len; partialHits.expand(); partialHits.back().init((index_t)INDEX_MAX, (index_t)INDEX_MAX, (index_t)INDEX_MAX, (index_t)INDEX_MAX, _node_iedge_count, fw, (index_t)offset, (index_t)(cur - offset)); hit.done(true); return 0; } // Does N interfere with use of Ftab? for(index_t i = 0; i < ftabLen; i++) { int c = seq[len-dep-1-i]; if(c > 3) { cur += (i+1); partialHits.expand(); partialHits.back().init((index_t)INDEX_MAX, (index_t)INDEX_MAX, (index_t)INDEX_MAX, (index_t)INDEX_MAX, _node_iedge_count, fw, (index_t)offset, (index_t)(cur - offset)); if(cur >= hit._len) { hit.done(true); } return 0; } } // Use ftab gfm.ftabLoHi(seq, len - dep - ftabLen, false, range.first, range.second); dep += ftabLen; if(range.first >= range.second) { cur = dep; partialHits.expand(); partialHits.back().init((index_t)INDEX_MAX, (index_t)INDEX_MAX, (index_t)INDEX_MAX, (index_t)INDEX_MAX, _node_iedge_count, fw, (index_t)offset, (index_t)(cur - offset)); if(cur >= hit._len) { hit.done(true); } return 0; } index_t same_range = 0, similar_range = 0; HIER_INIT_LOCS(range.first, range.second, tloc, bloc, gfm); // Keep going while(dep < len && dep - offset < maxHitLen) { int c = seq[len-dep-1]; if(c > 3) { rangeTemp.first = rangeTemp.second = 0; node_rangeTemp.first = node_rangeTemp.second = 0; _tmp_node_iedge_count.clear(); } else { if(bloc.valid()) { bwops_ += 2; if(linearFM) { rangeTemp = gfm.mapLF(tloc, bloc, c, &node_rangeTemp); } else { rangeTemp = gfm.mapGLF(tloc, bloc, c, &node_rangeTemp, &_tmp_node_iedge_count, (index_t)rp.kseeds); } } else { bwops_++; rangeTemp = gfm.mapGLF1(range.first, tloc, c, &node_rangeTemp); if(rangeTemp.first + 1 < rangeTemp.second) { assert_eq(node_rangeTemp.first + 1, node_rangeTemp.second); _tmp_node_iedge_count.clear(); _tmp_node_iedge_count.expand(); _tmp_node_iedge_count.back().first = 0; _tmp_node_iedge_count.back().second = rangeTemp.second - rangeTemp.first - 1; } } } if(rangeTemp.first >= rangeTemp.second) { break; } if(pseudogeneStop_) { if(node_rangeTemp.second - node_rangeTemp.first < node_range.second - node_range.first && node_range.second - node_range.first <= min(5, (index_t)rp.khits)) { static const index_t minLenForPseudogene = (index_t)_minK + 6; if(dep - offset >= minLenForPseudogene && similar_range >= 5) { hit._numUniqueSearch++; pseudogeneStop = true; break; } } if(node_rangeTemp.second - node_rangeTemp.first != 1) { if(node_rangeTemp.second - node_rangeTemp.first + 2 >= node_range.second - node_range.first) similar_range++; else if(node_rangeTemp.second - node_rangeTemp.first + 4 < node_range.second - node_range.first) similar_range = 0; } else { pseudogeneStop_ = false; } } if(anchorStop_) { if(node_rangeTemp.second - node_rangeTemp.first != 1 && node_range.second - node_range.first == node_rangeTemp.second - node_rangeTemp.first) { same_range++; if(same_range >= 5) { anchorStop_ = false; } } else { same_range = 0; } if(dep - offset >= _minK + 8 && node_rangeTemp.second - node_rangeTemp.first >= 4) { anchorStop_ = false; } } range = rangeTemp; node_range = node_rangeTemp; if(_tmp_node_iedge_count.size() > 0) { _node_iedge_count = _tmp_node_iedge_count; _tmp_node_iedge_count.clear(); } else { _node_iedge_count.clear(); } dep++; if(anchorStop_) { if(dep - offset >= _minK + 12 && range.second - range.first == 1) { hit._numUniqueSearch++; anchorStop = true; break; } } HIER_INIT_LOCS(range.first, range.second, tloc, bloc, gfm); } // Done if(range.first < range.second) { assert_leq(node_range.second - node_range.first, range.second - range.first); assert_gt(dep, offset); assert_leq(dep, len); partialHits.expand(); index_t hit_type = CANDIDATE_HIT; if(anchorStop) hit_type = ANCHOR_HIT; else if(pseudogeneStop) hit_type = PSEUDOGENE_HIT; bool report = node_range.first < node_range.second; if(node_range.second - node_range.first < range.second - range.first) { if(_node_iedge_count.size() == 0) report = false; } if(report) { #ifndef NDEBUG if(node_range.second - node_range.first < range.second - range.first) { ASSERT_ONLY(index_t add = 0); for(index_t e = 0; e < _node_iedge_count.size(); e++) { if(e > 0) { assert_lt(_node_iedge_count[e-1].first, _node_iedge_count[e].first); } assert_gt(_node_iedge_count[e].second, 0); add += _node_iedge_count[e].second; } assert_eq(node_range.second - node_range.first + add, range.second - range.first); } else { assert(_node_iedge_count.empty()); } #endif partialHits.back().init(range.first, range.second, node_range.first, node_range.second, _node_iedge_count, fw, (index_t)offset, (index_t)(dep - offset), hit_type); } else { _node_iedge_count.clear(); partialHits.back().init(INDEX_MAX, INDEX_MAX, INDEX_MAX, INDEX_MAX, _node_iedge_count, fw, (index_t)offset, (index_t)(dep - offset), hit_type); } nelt += (node_range.second - node_range.first); cur = dep; if(cur >= hit._len) { if(hit_type == CANDIDATE_HIT) hit._numUniqueSearch++; hit.done(true); } } return (index_t)nelt; } /** */ template index_t HI_Aligner::globalGFMSearch( const GFM& gfm, // BWT index const Read& read, // read to align const Scoring& sc, // scoring scheme const ReportingParams& rp, bool fw, index_t hitoff, index_t& hitlen, index_t& top, index_t& bot, index_t& node_top, index_t& node_bot, EList >& node_iedge_count, RandomSource& rnd, bool& uniqueStop, index_t maxHitLen) { bool uniqueStop_ = uniqueStop; uniqueStop = false; const index_t ftabLen = gfm.gh().ftabChars(); const bool linearFM = gfm.gh().linearFM(); SideLocus tloc, bloc; const index_t len = (index_t)read.length(); size_t nelt = 0; const BTDnaString& seq = fw ? read.patFw : read.patRc; assert(!seq.empty()); index_t offset = len - hitoff - 1; index_t dep = offset; pair range(0, 0); pair rangeTemp(0, 0); pair node_range(0, 0); pair node_rangeTemp(0, 0); node_iedge_count.clear(); _tmp_node_iedge_count.clear(); index_t left = len - dep; assert_gt(left, 0); if(left < ftabLen + 1) { hitlen = left; return 0; } // Does N interfere with use of Ftab? for(index_t i = 0; i < ftabLen; i++) { int c = seq[len-dep-1-i]; if(c > 3) { hitlen = (i+1); return 0; } } // Use ftab gfm.ftabLoHi(seq, len - dep - ftabLen, false, range.first, range.second); dep += ftabLen; if(range.first >= range.second) { hitlen = ftabLen; return 0; } HIER_INIT_LOCS(range.first, range.second, tloc, bloc, gfm); // Keep going while(dep < len) { int c = seq[len-dep-1]; if(c > 3) { rangeTemp.first = rangeTemp.second = 0; node_rangeTemp.first = node_rangeTemp.second = 0; _tmp_node_iedge_count.clear(); } else { if(bloc.valid()) { bwops_ += 2; if(linearFM) { rangeTemp = gfm.mapLF(tloc, bloc, c, &node_rangeTemp); } else { rangeTemp = gfm.mapGLF(tloc, bloc, c, &node_rangeTemp, &_tmp_node_iedge_count, (index_t)rp.kseeds); } } else { bwops_++; rangeTemp = gfm.mapGLF1(range.first, tloc, c, &node_rangeTemp); if(rangeTemp.first + 1 < rangeTemp.second) { assert_eq(node_rangeTemp.first + 1, node_rangeTemp.second); _tmp_node_iedge_count.clear(); _tmp_node_iedge_count.expand(); _tmp_node_iedge_count.back().first = 0; _tmp_node_iedge_count.back().second = rangeTemp.second - rangeTemp.first - 1; } } } if(rangeTemp.first >= rangeTemp.second) { break; } range = rangeTemp; node_range = node_rangeTemp; if(_tmp_node_iedge_count.size() > 0) { node_iedge_count = _tmp_node_iedge_count; _tmp_node_iedge_count.clear(); } else { node_iedge_count.clear(); } dep++; if(uniqueStop_) { if(range.second - range.first == 1 && dep - offset >= _minK) { uniqueStop = true; break; } } HIER_INIT_LOCS(range.first, range.second, tloc, bloc, gfm); } // Done if(node_range.first < node_range.second && node_range.second - node_range.first <= rp.kseeds) { assert_leq(node_range.second - node_range.first, range.second - range.first); #ifndef NDEBUG if(node_range.second - node_range.first < range.second - range.first) { ASSERT_ONLY(index_t add = 0); for(index_t e = 0; e < node_iedge_count.size(); e++) { if(e > 0) { assert_lt(node_iedge_count[e-1].first, node_iedge_count[e].first); } assert_gt(node_iedge_count[e].second, 0); add += node_iedge_count[e].second; } assert_eq(node_range.second - node_range.first + add, range.second - range.first); } else { assert(node_iedge_count.empty()); } #endif assert_gt(dep, offset); assert_leq(dep, len); top = range.first; bot = range.second; node_top = node_range.first; node_bot = node_range.second; nelt += (node_bot - node_top); hitlen = dep - offset; } return (index_t)nelt; } /** * **/ template index_t HI_Aligner::localGFMSearch( const LocalGFM& gfm, // GFM index const Read& read, // read to align const Scoring& sc, // scoring scheme const ReportingParams& rp, bool fw, index_t rdoff, index_t& hitlen, local_index_t& top, local_index_t& bot, local_index_t& node_top, local_index_t& node_bot, EList >& local_node_iedge_count, RandomSource& rnd, bool& uniqueStop, local_index_t minUniqueLen, local_index_t maxHitLen, local_index_t maxHits) { maxHits = max(maxHits, rp.kseeds); bool uniqueStop_ = uniqueStop; uniqueStop = false; const local_index_t ftabLen = (local_index_t)gfm.gh().ftabChars(); const bool linearFM = gfm.gh().linearFM(); SideLocus tloc, bloc; const local_index_t len = (local_index_t)read.length(); size_t nelt = 0; const BTDnaString& seq = fw ? read.patFw : read.patRc; assert(!seq.empty()); local_index_t offset = len - rdoff - 1; local_index_t dep = offset; pair range(0, 0); pair rangeTemp(0, 0); pair node_range(0, 0); pair node_rangeTemp(0, 0); top = bot = node_top = node_bot = 0; local_node_iedge_count.clear(); _tmp_local_node_iedge_count.clear(); local_index_t left = len - dep; assert_gt(left, 0); if(left < ftabLen + 1) { hitlen = left; return 0; } // Does N interfere with use of Ftab? for(local_index_t i = 0; i < ftabLen; i++) { int c = seq[len-dep-1-i]; if(c > 3) { hitlen = i + 1; return 0; } } gfm.ftabLoHi(seq, len - dep - ftabLen, false, range.first, range.second); dep += ftabLen; if(range.first >= range.second) { hitlen = ftabLen; return 0; } LOCAL_INIT_LOCS(range.first, range.second, tloc, bloc, gfm); // Keep going while(dep < len) { int c = seq[len-dep-1]; if(c > 3) { rangeTemp.first = rangeTemp.second = 0; node_rangeTemp.first = node_rangeTemp.second = 0; _tmp_local_node_iedge_count.clear(); } else { if(bloc.valid()) { bwops_ += 2; if(linearFM) { rangeTemp = gfm.mapLF(tloc, bloc, c, &node_rangeTemp); } else { rangeTemp = gfm.mapGLF(tloc, bloc, c, &node_rangeTemp, &_tmp_local_node_iedge_count, rp.kseeds); } } else { bwops_++; rangeTemp = gfm.mapGLF1(range.first, tloc, c, &node_rangeTemp); if(rangeTemp.first + 1 < rangeTemp.second) { assert_eq(node_rangeTemp.first + 1, node_rangeTemp.second); _tmp_local_node_iedge_count.clear(); _tmp_local_node_iedge_count.expand(); _tmp_local_node_iedge_count.back().first = 0; _tmp_local_node_iedge_count.back().second = rangeTemp.second - rangeTemp.first - 1; } } } if(rangeTemp.first >= rangeTemp.second) { break; } range = rangeTemp; node_range = node_rangeTemp; if(_tmp_local_node_iedge_count.size() > 0) { local_node_iedge_count = _tmp_local_node_iedge_count; _tmp_local_node_iedge_count.clear(); } else { local_node_iedge_count.clear(); } dep++; if(uniqueStop_) { if(range.second - range.first == 1 && dep - offset >= minUniqueLen) { uniqueStop = true; break; } } if(dep - offset >= maxHitLen) break; LOCAL_INIT_LOCS(range.first, range.second, tloc, bloc, gfm); } // Done if(node_range.first < node_range.second && node_range.second - node_range.first <= maxHits) { assert_leq(node_range.second - node_range.first, range.second - range.first); #ifndef NDEBUG if(node_range.second - node_range.first < range.second - range.first) { ASSERT_ONLY(index_t add = 0); for(index_t e = 0; e < local_node_iedge_count.size(); e++) { if(e > 0) { assert_lt(local_node_iedge_count[e-1].first, local_node_iedge_count[e].first); } assert_gt(local_node_iedge_count[e].second, 0); add += local_node_iedge_count[e].second; } assert_eq(node_range.second - node_range.first + add, range.second - range.first); } else { assert(local_node_iedge_count.empty()); } #endif assert_gt(dep, offset); assert_leq(dep, len); top = range.first; bot = range.second; node_top = node_range.first; node_bot = node_range.second; nelt += (node_bot - node_top); hitlen = dep - offset; } return (index_t)nelt; } /** * **/ template bool HI_Aligner::isSearched( const GenomeHit& hit, index_t rdi) { assert_lt(rdi, 2); EList >& searchedHits = _hits_searched[rdi]; for(index_t i = 0; i < searchedHits.size(); i++) { if(searchedHits[i].contains(hit)) return true; } return false; } /** * **/ template void HI_Aligner::addSearched( const GenomeHit& hit, index_t rdi) { assert_lt(rdi, 2); assert(!isSearched(hit, rdi)); EList >& searchedHits = _hits_searched[rdi]; searchedHits.push_back(hit); } #endif /*HI_ALIGNER_H_*/ hisat2-2.2.1/hier_idx_common.h000066400000000000000000000031751370663763200162450ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * Beast is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef HIERGBWT_COMMON_H_ #define HIERGBWT_COMMON_H_ // maximum size of a sequence represented by a local index static const uint32_t local_index_size = (1 << 16) - (1 << 13); // 1 << 5 is necessary for eftab index static const uint32_t local_max_gbwt = (1 << 16) - (1 << 11); // size of the overlapped sequence between the sequences represented by two consecutive local indexes static const uint32_t local_index_overlap = 1024; // interval between two consecutive local indexes static const uint32_t local_index_interval = local_index_size - local_index_overlap; // line rate in local indexes static const int32_t local_lineRate_fm = 6; static const int32_t local_lineRate_gfm = 7; // how many rows are marked in a local index, every 2^th row is marked static const int32_t local_offRate = 3; // the look table in a local index 4^ entries static const int32_t local_ftabChars = 6; #endif /*HIERGBWT_COMMON_H_*/ hisat2-2.2.1/hisat2000077500000000000000000000466611370663763200140600ustar00rootroot00000000000000#!/usr/bin/env perl # # Copyright 2015, Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # # hisat2: # # A wrapper script for hisat2. Provides various advantages over running # hisat directly, including: # # 1. Handling compressed inputs # 2. Redirecting output to various files # 3. Output directly to bam (not currently supported) use strict; use warnings; use Getopt::Long qw(GetOptions); use File::Spec; use POSIX; my ($vol,$script_path,$prog); $prog = File::Spec->rel2abs( __FILE__ ); while (-f $prog && -l $prog){ my (undef, $dir, undef) = File::Spec->splitpath($prog); $prog = File::Spec->rel2abs(readlink($prog), $dir); } ($vol,$script_path,$prog) = File::Spec->splitpath($prog); my $os_is_nix = ($^O eq "linux") || ($^O eq "darwin"); my $align_bin_s = $os_is_nix ? 'hisat2-align-s' : 'hisat2-align-s.exe'; my $build_bin = $os_is_nix ? 'hisat2-build' : 'hisat2-build.exe'; my $align_bin_l = $os_is_nix ? 'hisat2-align-l' : 'hisat2-align-l.exe'; my $align_prog_s= File::Spec->catpath($vol,$script_path,$align_bin_s); my $align_prog_l= File::Spec->catpath($vol,$script_path,$align_bin_l); my $align_prog = $align_prog_s; my $read_stat_prog = File::Spec->catpath($vol,$script_path,"hisat2_read_statistics.py"); my $idx_ext_l = 'ht2l'; my $idx_ext_s = 'ht2'; my $idx_ext = $idx_ext_s; my $seq_in_args = 0; my $skip_read_stat = 0; my %signo = (); my @signame = (); { # Get signal info use Config; my $i = 0; for my $name (split(' ', $Config{sig_name})) { $signo{$name} = $i; $signame[$i] = $name; $i++; } } (-x "$align_prog") || Fail("Expected hisat2 to be in same directory with hisat2-align-s and hisat2-align-l:\n$script_path\n"); (-x "$read_stat_prog") || ($skip_read_stat = 1); # Get description of arguments from HISAT so that we can distinguish HISAT # args from wrapper args sub getHt2Desc($) { my $d = shift; my $cmd = "'$align_prog' --wrapper basic-0 --arg-desc"; open(my $fh, "$cmd |") || Fail("Failed to run command '$cmd'\n"); while(readline $fh) { chomp; next if /^\s*$/; my @ts = split(/\t/); $d->{$ts[0]} = $ts[1]; } close($fh); $? == 0 || Fail("Description of arguments failed!\n"); } my %desc = (); my %wrapped = ("1" => 1, "2" => 1); getHt2Desc(\%desc); # Given an option like -1, determine whether it's wrapped (i.e. should be # handled by this script rather than being passed along to HISAT) sub isWrapped($) { return defined($wrapped{$_[0]}); } my @orig_argv = @ARGV; my @ht2w_args = (); # options for wrapper my @ht2_args = (); # options for HISAT my $saw_dd = 0; for(0..$#ARGV) { if($ARGV[$_] eq "--") { $saw_dd = 1; next; } push @ht2w_args, $ARGV[$_] if !$saw_dd; push @ht2_args, $ARGV[$_] if $saw_dd; } if(!$saw_dd) { @ht2_args = @ht2w_args; @ht2w_args= (); } my $debug = 0; my %read_fns = (); my %read_compress = (); my $cap_out = undef; # Filename for passthrough my $no_unal = 0; my $large_idx = 0; # Remove whitespace for my $i (0..$#ht2_args) { $ht2_args[$i]=~ s/^\s+//; $ht2_args[$i] =~ s/\s+$//; } # We've handled arguments that the user has explicitly directed either to the # wrapper or to hisat, now we capture some of the hisat arguments that # ought to be handled in the wrapper for(my $i = 0; $i < scalar(@ht2_args); $i++) { next unless defined($ht2_args[$i]); my $arg = $ht2_args[$i]; my @args = split(/=/, $arg); if(scalar(@args) > 2) { $args[1] = join("=", @args[1..$#args]); } $arg = $args[0]; if($arg eq "-U" || $arg eq "--unpaired") { $ht2_args[$i] = undef; $arg =~ s/^-U//; $arg =~ s/^--unpaired//; if($arg ne "") { # Argument was part of this token my @args = split(/,/, $arg); for my $a (@args) { push @ht2w_args, ("-U", $a); } } else { # Argument is in the next token $i < scalar(@ht2_args)-1 || Fail("Argument expected in next token!\n"); $i++; my @args = split(/,/, $ht2_args[$i]); for my $a (@args) { push @ht2w_args, ("-U", $a); } $ht2_args[$i] = undef; } } if($arg =~ /^--?([12])/ && $arg !~ /^--?12/) { my $mate = $1; $ht2_args[$i] = undef; $arg =~ s/^--?[12]//; if($arg ne "") { # Argument was part of this token my @args = split(/,/, $arg); for my $a (@args) { push @ht2w_args, ("-$mate", $a); } } else { # Argument is in the next token $i < scalar(@ht2_args)-1 || Fail("Argument expected in next token!\n"); $i++; my @args = split(/,/, $ht2_args[$i]); for my $a (@args) { push @ht2w_args, ("-$mate", $a); } $ht2_args[$i] = undef; } } if($arg eq "--debug") { $debug = 1; $ht2_args[$i] = undef; } if($arg eq "--no-unal") { $no_unal = 1; $ht2_args[$i] = undef; } if($arg eq "--large-index") { $large_idx = 1; $ht2_args[$i] = undef; } if($arg eq "--skip-read-lengths") { $skip_read_stat = 1; $ht2_args[$i] = undef } if($arg eq "-c") { $seq_in_args = 1; } for my $rarg ("un-conc", "al-conc", "al-conc-disc", "un", "al") { if($arg =~ /^--${rarg}$/ || $arg =~ /^--${rarg}-gz$/ || $arg =~ /^--${rarg}-bz2$/) { $ht2_args[$i] = undef; if(scalar(@args) > 1 && $args[1] ne "") { $read_fns{$rarg} = $args[1]; } else { $i < scalar(@ht2_args)-1 || Fail("--${rarg}* option takes an argument.\n"); $read_fns{$rarg} = $ht2_args[$i+1]; $ht2_args[$i+1] = undef; } $read_compress{$rarg} = ""; $read_compress{$rarg} = "gzip" if $arg eq "--${rarg}-gz"; $read_compress{$rarg} = "bzip2" if $arg eq "--${rarg}-bz2"; last; } } } # If the user asked us to redirect some reads to files, or to suppress # unaligned reads, then we need to capture the output from HISAT and pass it # through this wrapper. my $passthru = 0; if(scalar(keys %read_fns) > 0 || $no_unal) { $passthru = 1; push @ht2_args, "--passthrough"; $cap_out = "-"; for(my $i = 0; $i < scalar(@ht2_args); $i++) { next unless defined($ht2_args[$i]); my $arg = $ht2_args[$i]; if($arg eq "-S" || $arg eq "--output") { $i < scalar(@ht2_args)-1 || Fail("-S/--output takes an argument.\n"); $cap_out = $ht2_args[$i+1]; $ht2_args[$i] = undef; $ht2_args[$i+1] = undef; } } } my @tmp = (); for (@ht2_args) { push(@tmp, $_) if defined($_); } @ht2_args = @tmp; my @unps = (); my @mate1s = (); my @mate2s = (); my @to_delete = (); my @to_kills = (); my $temp_dir = "/tmp"; my $bam_out = 0; my $ref_str = undef; my $no_pipes = 0; my $keep = 0; my $verbose = 0; my $readpipe = undef; my $log_fName = undef; my $help = 0; my @ht2w_args_cp = (@ht2w_args>0) ? @ht2w_args : @ht2_args; Getopt::Long::Configure("pass_through","no_ignore_case"); my @old_ARGV = @ARGV; @ARGV = @ht2w_args_cp; GetOptions( "1=s" => \@mate1s, "2=s" => \@mate2s, "reads|U=s" => \@unps, "temp-directory=s" => \$temp_dir, "bam" => \$bam_out, "no-named-pipes" => \$no_pipes, "ref-string|reference-string=s" => \$ref_str, "keep" => \$keep, "verbose" => \$verbose, "log-file=s" => \$log_fName, "help|h" => \$help ); @ARGV = @old_ARGV; my $old_stderr; if ($log_fName) { open($old_stderr, ">&STDERR") or Fail("Cannot dup STDERR!\n"); open(STDERR, ">", $log_fName) or Fail("Cannot redirect to log file $log_fName.\n"); } Info("Before arg handling:\n"); Info(" Wrapper args:\n[ @ht2w_args ]\n"); Info(" Binary args:\n[ @ht2_args ]\n"); # check read lengths # if read_files have more than 1 files, use first one, my @read_files = (scalar(@unps) > 0) ? @unps : @mate1s; if ((scalar(@read_files) > 0) && ($seq_in_args == 0) && ($skip_read_stat == 0)) { Info("Check read length: $read_files[0]\n"); my $cmd = "'$read_stat_prog' $read_files[0]"; my $read_len_str = ""; open(my $fh, "$cmd |") || Fail("Failed to run command '$cmd'\n"); while(readline $fh) { chomp; next if /^\s*$/; my @ts = split(/ /); if (scalar(@ts) > 4) { $read_len_str = $ts[4]; } else { $read_len_str = ""; } } close($fh); if (($read_len_str ne "") && ($read_len_str ne "0")) { Info("Read Length String: $read_len_str\n"); push @ht2_args, ("--read-lengths", $read_len_str); } } sub check_file_exist($$$) { my ($unps, $mate1s, $mate2s) = @_; for my $fn (@$unps, @$mate1s, @$mate2s) { if (not -f $fn) { Fail("Read file '%s' doesn't exist\n", $fn); return 1; } } return 0; } sub cat_file($$) { my ($ifn, $ofh) = @_; my $ifh = undef; if($ifn =~ /\.gz$/) { open($ifh, "gzip -dc $ifn |") || Fail("Could not open gzipped read file: $ifn \n"); } elsif($ifn =~ /\.bz2/) { open($ifh, "bzip2 -dc $ifn |") || Fail("Could not open bzip2ed read file: $ifn \n"); } else { open($ifh, $ifn) || Fail("Could not open read file: $ifn \n"); } while(readline $ifh) { print {$ofh} $_; } close($ifh); } # Return non-zero if and only if the input should be wrapped (i.e. because # it's compressed). sub wrapInput($$$) { my ($unps, $mate1s, $mate2s) = @_; for my $fn (@$unps, @$mate1s, @$mate2s) { return 1 if $fn =~ /\.gz$/ || $fn =~ /\.bz2$/; } return 0; } sub Info { if ($verbose) { print STDERR "(INFO): " ,@_; } } sub Error { my @msg = @_; $msg[0] = "(ERR): ".$msg[0]; printf STDERR @msg; } sub Fail { Error(@_); die("Exiting now ...\n"); } sub Extract_IndexName_From { my $index_opt = $ref_str ? '--index' : '-x'; for (my $i=0; $i<@_; $i++) { if ($_[$i] eq $index_opt){ my $idx_basename = $_[$i+1]; my @idx_filenames = glob($idx_basename . "*.ht2{,l}"); unless(@idx_filenames) { if(exists $ENV{"HISAT2_INDEXES"}) { @idx_filenames = glob("$ENV{'HISAT2_INDEXES'}/$idx_basename" . "ht2{,l}"); } if(!@idx_filenames) { Fail("\"" . $idx_basename . "\" does not exist\n"); } $idx_basename = "$ENV{'HISAT2_INDEXES'}/$idx_basename" } return $idx_basename; } } Info("Cannot find any index option (--reference-string, --ref-string or -x) in the given command line.\n"); } if($seq_in_args == 0) { check_file_exist(\@unps, \@mate1s, \@mate2s); } if(wrapInput(\@unps, \@mate1s, \@mate2s)) { if(scalar(@mate2s) > 0) { # # Wrap paired-end inputs # # Put reads into temporary files or fork off processes to feed named pipes scalar(@mate2s) == scalar(@mate1s) || Fail("Different number of files specified with --reads/-1 as with -2\n"); # Make a named pipe for delivering mate #1s my $m1fn = "$temp_dir/$$.inpipe1"; push @to_delete, $m1fn; push @ht2_args, "-1 $m1fn"; # Create named pipe 1 for writing if(!$no_pipes) { mkfifo($m1fn, 0700) || Fail("mkfifo($m1fn) failed.\n"); } my $pid = 0; $pid = fork() unless $no_pipes; push @to_kills, $pid unless $no_pipes; if($pid == 0) { # Open named pipe 1 for writing open(my $ofh, ">$m1fn") || Fail("Can't open '$m1fn' for writing\n"); for my $ifn (@mate1s) { cat_file($ifn, $ofh); } close($ofh); exit 0 unless $no_pipes; } # Make a named pipe for delivering mate #2s my $m2fn = "$temp_dir/$$.inpipe2"; push @to_delete, $m2fn; push @ht2_args, "-2 $m2fn"; # Create named pipe 2 for writing if(!$no_pipes) { mkfifo($m2fn, 0700) || Fail("mkfifo($m2fn) failed.\n"); } $pid = 0; $pid = fork() unless $no_pipes; push @to_kills, $pid unless $no_pipes; if($pid == 0) { # Open named pipe 2 for writing open(my $ofh, ">$m2fn") || Fail("Can't open '$m2fn' for writing.\n"); for my $ifn (@mate2s) { cat_file($ifn, $ofh); } close($ofh); exit 0 unless $no_pipes; } } if(scalar(@unps) > 0) { # # Wrap unpaired inputs. # # Make a named pipe for delivering unpaired reads my $ufn = "$temp_dir/$$.unp"; push @to_delete, $ufn; push @ht2_args, "-U $ufn"; # Create named pipe 2 for writing if(!$no_pipes) { mkfifo($ufn, 0700) || Fail("mkfifo($ufn) failed.\n"); } my $pid = 0; $pid = fork() unless $no_pipes; if($pid == 0) { # Open named pipe 2 for writing open(my $ofh, ">$ufn") || Fail("Can't open '$ufn' for writing.\n"); for my $ifn (@unps) { cat_file($ifn, $ofh); } close($ofh); exit 0 unless $no_pipes; } } } else { if(scalar(@mate2s) > 0) { # Just pass all the mate arguments along to the binary push @ht2_args, ("-1", join(",", @mate1s)); push @ht2_args, ("-2", join(",", @mate2s)); } if(scalar(@unps) > 0) { push @ht2_args, ("-U", join(",", @unps)); } } if(defined($ref_str)) { my $ofn = "$temp_dir/$$.ref_str.fa"; open(my $ofh, ">$ofn") || Fail("could not open temporary fasta file '$ofn' for writing.\n"); print {$ofh} ">1\n$ref_str\n"; close($ofh); push @to_delete, $ofn; system("$build_bin $ofn $ofn") == 0 || Fail("hisat2-build returned non-0 exit level.\n"); push @ht2_args, ("--index", "$ofn"); push @to_delete, ("$ofn.1.".$idx_ext, "$ofn.2.".$idx_ext, "$ofn.3.".$idx_ext, "$ofn.4.".$idx_ext, "$ofn.5.".$idx_ext, "$ofn.6.".$idx_ext, "$ofn.7.".$idx_ext, "$ofn.8.".$idx_ext); } Info("After arg handling:\n"); Info(" Binary args:\n[ @ht2_args ]\n"); my $index_name = Extract_IndexName_From(@ht2_args); if ($large_idx) { Info("Using a large index enforced by user.\n"); $align_prog = $align_prog_l; $idx_ext = $idx_ext_l; if (not -f $index_name.".1.".$idx_ext_l) { Fail("Cannot find the large index ${index_name}.1.${idx_ext_l}\n"); } Info("Using large index (${index_name}.1.${idx_ext_l}).\n"); } else { if ((-f $index_name.".1.".$idx_ext_l) && (not -f $index_name.".1.".$idx_ext_s)) { Info("Cannot find a small index but a large one seems to be present.\n"); Info("Switching to using the large index (${index_name}.1.${idx_ext_l}).\n"); $align_prog = $align_prog_l; $idx_ext = $idx_ext_l; } else { Info("Using the small index (${index_name}.1.${idx_ext_s}).\n") } } my $debug_str = ($debug ? "-debug" : ""); # Construct command invoking hisat2-align my $cmd = "'$align_prog$debug_str' --wrapper basic-0 ".join(" ", @ht2_args); # Possibly add read input on an anonymous pipe $cmd = "$readpipe $cmd" if defined($readpipe); Info("$cmd\n"); my $ret; if(defined($cap_out)) { # Open HISAT2 pipe open(HT, "$cmd |") || Fail("Could not open HISAT2 pipe: '$cmd |'\n"); # Open output pipe my $ofh = *STDOUT; my @fhs_to_close = (); if($cap_out ne "-") { open($ofh, ">$cap_out") || Fail("Could not open output file '$cap_out' for writing.\n"); } my %read_fhs = (); for my $i ("al", "un", "al-conc", "al-conc-disc", "un-conc") { if(defined($read_fns{$i})) { my ($vol, $base_spec_dir, $base_fname) = File::Spec->splitpath($read_fns{$i}); if (-d $read_fns{$i}) { $base_spec_dir = $read_fns{$i}; $base_fname = undef; } if($i =~ /-conc$/ || $i =~ /-conc-disc$/) { # Open 2 output files, one for mate 1, one for mate 2 my ($fn1, $fn2); if ($base_fname) { ($fn1, $fn2) = ($base_fname,$base_fname); } else { ($fn1, $fn2) = ($i.'-mate',$i.'-mate'); } if($fn1 =~ /%/) { $fn1 =~ s/%/1/g; $fn2 =~ s/%/2/g; } elsif($fn1 =~ /\.[^.]*$/) { $fn1 =~ s/\.([^.]*)$/.1.$1/; $fn2 =~ s/\.([^.]*)$/.2.$1/; } else { $fn1 .= ".1"; $fn2 .= ".2"; } $fn1 = File::Spec->catpath($vol,$base_spec_dir,$fn1); $fn2 = File::Spec->catpath($vol,$base_spec_dir,$fn2); $fn1 ne $fn2 || Fail("$fn1\n$fn2\n"); my ($redir1, $redir2) = (">$fn1", ">$fn2"); $redir1 = "| gzip -c $redir1" if $read_compress{$i} eq "gzip"; $redir1 = "| bzip2 -c $redir1" if $read_compress{$i} eq "bzip2"; $redir2 = "| gzip -c $redir2" if $read_compress{$i} eq "gzip"; $redir2 = "| bzip2 -c $redir2" if $read_compress{$i} eq "bzip2"; open($read_fhs{$i}{1}, $redir1) || Fail("Could not open --$i mate-1 output file '$fn1'\n"); open($read_fhs{$i}{2}, $redir2) || Fail("Could not open --$i mate-2 output file '$fn2'\n"); push @fhs_to_close, $read_fhs{$i}{1}; push @fhs_to_close, $read_fhs{$i}{2}; } else { my $redir = ">".File::Spec->catpath($vol,$base_spec_dir,$i."-seqs"); if ($base_fname) { $redir = ">$read_fns{$i}"; } $redir = "| gzip -c $redir" if $read_compress{$i} eq "gzip"; $redir = "| bzip2 -c $redir" if $read_compress{$i} eq "bzip2"; open($read_fhs{$i}, $redir) || Fail("Could not open --$i output file '$read_fns{$i}'\n"); push @fhs_to_close, $read_fhs{$i}; } } } while() { chomp; my $filt = 0; unless(substr($_, 0, 1) eq "@") { # If we are supposed to output certain reads to files... my $tab1_i = index($_, "\t") + 1; my $tab2_i = index($_, "\t", $tab1_i); my $fl = substr($_, $tab1_i, $tab2_i - $tab1_i); my $unal = ($fl & 4) != 0; my $secondary = ($fl & 256) != 0; $filt = 1 if $no_unal && $unal; if($passthru) { if(scalar(keys %read_fhs) == 0) { # Next line is read with some whitespace escaped my $l = ; } else { my $mate1 = (($fl & 64) != 0); my $mate2 = (($fl & 128) != 0); my $unp = !$mate1 && !$mate2; my $pair = !$unp; # Next line is read with some whitespace escaped my $l = ; chomp($l); $l =~ s/%(..)/chr(hex($1))/eg; if((defined($read_fhs{un}) || defined($read_fhs{al})) && $unp && !$secondary) { if($unal) { # Failed to align print {$read_fhs{un}} $l if defined($read_fhs{un}); } else { # Aligned print {$read_fhs{al}} $l if defined($read_fhs{al}); } } if((defined($read_fhs{"un-conc"}) || defined($read_fhs{"al-conc"}) || defined($read_fhs{"al-conc-disc"})) && $pair && !$secondary) { my $conc = (($fl & 2) != 0); my $conc_disc = ($fl & 4) == 0 || ($fl & 8) == 0; if($conc && $mate1) { print {$read_fhs{"al-conc"}{1}} $l if defined($read_fhs{"al-conc"}); } elsif($conc && $mate2) { print {$read_fhs{"al-conc"}{2}} $l if defined($read_fhs{"al-conc"}); } elsif(!$conc && $mate1) { print {$read_fhs{"un-conc"}{1}} $l if defined($read_fhs{"un-conc"}); } elsif(!$conc && $mate2) { print {$read_fhs{"un-conc"}{2}} $l if defined($read_fhs{"un-conc"}); } if($conc_disc && $mate1) { print {$read_fhs{"al-conc-disc"}{1}} $l if defined($read_fhs{"al-conc-disc"}); } elsif($conc_disc && $mate2) { print {$read_fhs{"al-conc-disc"}{2}} $l if defined($read_fhs{"al-conc-disc"}); } } } } } print {$ofh} "$_\n" if !$filt; } for my $k (@fhs_to_close) { close($k); } close($ofh); close(HT); $ret = $?; } else { $ret = system($cmd); } kill 'TERM', @to_kills; if(!$keep) { for(@to_delete) { unlink($_); } } if ($ret == -1) { Error("Failed to execute hisat2-align: $!\n"); exit 1; } elsif ($ret & 127) { my $signm = "(unknown)"; $signm = $signame[$ret & 127] if defined($signame[$ret & 127]); my $ad = ""; $ad = "(core dumped)" if (($ret & 128) != 0); Error("hisat2-align died with signal %d (%s) $ad\n", ($ret & 127), $signm); exit 1; } elsif($ret != 0) { Error("hisat2-align exited with value %d\n", ($ret >> 8)); } exit ($ret >> 8); hisat2-2.2.1/hisat2-build000077500000000000000000000053671370663763200151530ustar00rootroot00000000000000#!/usr/bin/env python """ Copyright 2015, Daehwan Kim This file is part of HISAT 2. HISAT 2 is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. HISAT 2 is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. You should have received a copy of the GNU General Public License along with HISAT 2. If not, see . """ import os import sys import inspect import logging def build_args(): """ Parse the wrapper arguments. Returns the options, tuple. """ parsed_args = {} to_remove = [] argv = sys.argv[:] for i, arg in enumerate(argv): if arg == '--large-index': parsed_args[arg] = "" to_remove.append(i) elif arg == '--debug': parsed_args[arg] = "" to_remove.append(i) elif arg == '--verbose': parsed_args[arg] = "" to_remove.append(i) for i in reversed(to_remove): del argv[i] return parsed_args, argv def main(): logging.basicConfig(level=logging.ERROR, format='%(levelname)s: %(message)s' ) delta = 200 small_index_max_size= 4 * 1024**3 - delta build_bin_name = "hisat2-build" build_bin_s = "hisat2-build-s" build_bin_l = "hisat2-build-l" curr_script = os.path.realpath(inspect.getsourcefile(main)) ex_path = os.path.dirname(curr_script) build_bin_spec = os.path.join(ex_path,build_bin_s) options, argv = build_args() if '--verbose' in options: logging.getLogger().setLevel(logging.INFO) if '--debug' in options: build_bin_spec += '-debug' build_bin_l += '-debug' if '--large-index' in options: build_bin_spec = os.path.join(ex_path,build_bin_l) elif len(argv) >= 2: ref_fnames = argv[-2] tot_size = 0 for fn in ref_fnames.split(','): if os.path.exists(fn): statinfo = os.stat(fn) tot_size += statinfo.st_size if tot_size > small_index_max_size: build_bin_spec = os.path.join(ex_path,build_bin_l) argv[0] = build_bin_name argv.insert(1, 'basic-0') argv.insert(1, '--wrapper') logging.info('Command: %s %s' % (build_bin_spec, ' '.join(argv[1:]))) os.execv(build_bin_spec, argv) if __name__ == "__main__": main() hisat2-2.2.1/hisat2-build-new000077500000000000000000000057601370663763200157370ustar00rootroot00000000000000#!/usr/bin/env python """ Copyright 2018, Chanhee Park and Daehwan Kim This file is part of HISAT 2. HISAT 2 is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. HISAT 2 is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. You should have received a copy of the GNU General Public License along with HISAT 2. If not, see . """ import os import sys import inspect import logging def build_args(): """ Parse the wrapper arguments. Returns the options, tuple. """ parsed_args = {} to_remove = [] argv = sys.argv[:] for i, arg in enumerate(argv): if arg == '--large-index': parsed_args[arg] = "" to_remove.append(i) elif arg == '--debug': parsed_args[arg] = "" to_remove.append(i) elif arg == '--verbose': parsed_args[arg] = "" to_remove.append(i) for i in reversed(to_remove): del argv[i] return parsed_args, argv def main(): logging.basicConfig(level=logging.ERROR, format='%(levelname)s: %(message)s' ) delta = 200 small_index_max_size= 4 * 1024**3 - delta constuct_bin_name = "hisat2-construct-nonrepetitive-genome" constuct_bin_s = "hisat2-construct-nonrepetitive-genome-s" constuct_bin_l = "hisat2-construct-nonrepetitive-genome-l" build_bin_name = "hisat2-build" build_bin_s = "hisat2-build-s" build_bin_l = "hisat2-build-l" curr_script = os.path.realpath(inspect.getsourcefile(main)) ex_path = os.path.dirname(curr_script) build_bin_spec = os.path.join(ex_path,build_bin_s) options, argv = build_args() if '--verbose' in options: logging.getLogger().setLevel(logging.INFO) if '--debug' in options: build_bin_spec += '-debug' build_bin_l += '-debug' if '--large-index' in options: build_bin_spec = os.path.join(ex_path,build_bin_l) elif len(argv) >= 2: ref_fnames = argv[-2] tot_size = 0 for fn in ref_fnames.split(','): if os.path.exists(fn): statinfo = os.stat(fn) tot_size += statinfo.st_size if tot_size > small_index_max_size: build_bin_spec = os.path.join(ex_path,build_bin_l) argv[0] = build_bin_name argv.insert(1, 'basic-0') argv.insert(1, '--wrapper') logging.info('Command: %s %s' % (build_bin_spec, ' '.join(argv[1:]))) os.execv(build_bin_spec, argv) if __name__ == "__main__": main() hisat2-2.2.1/hisat2-inspect000077500000000000000000000052011370663763200155040ustar00rootroot00000000000000#!/usr/bin/env python """ Copyright 2015, Daehwan Kim This file is part of HISAT 2. HISAT 2 is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version. HISAT 2 is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. You should have received a copy of the GNU General Public License along with HISAT 2. If not, see . """ import os import imp import inspect import logging def main(): logging.basicConfig(level=logging.ERROR, format='%(levelname)s: %(message)s' ) inspect_bin_name = "hisat2-inspect" inspect_bin_s = "hisat2-inspect-s" inspect_bin_l = "hisat2-inspect-l" idx_ext_l = '.1.ht2l'; idx_ext_s = '.1.ht2'; curr_script = os.path.realpath(inspect.getsourcefile(main)) ex_path = os.path.dirname(curr_script) inspect_bin_spec = os.path.join(ex_path,inspect_bin_s) bld = imp.load_source('hisat2-build',os.path.join(ex_path,'hisat2-build')) options,arguments = bld.build_args() if '--verbose' in options: logging.getLogger().setLevel(logging.INFO) if '--debug' in options: inspect_bin_spec += '-debug' inspect_bin_l += '-debug' if '--large-index' in options: inspect_bin_spec = os.path.join(ex_path,inspect_bin_l) elif len(arguments) >= 1: idx_basename = arguments[-1] large_idx_exists = os.path.exists(idx_basename + idx_ext_l) small_idx_exists = os.path.exists(idx_basename + idx_ext_s) if not large_idx_exists and not small_idx_exists: env_path = os.getenv('HISAT2_INDEXES', '') large_idx_exists = os.path.exists(env_path + '/' + idx_basename + idx_ext_l) small_idx_exists = os.path.exists(env_path + '/' + idx_basename + idx_ext_s) if large_idx_exists and not small_idx_exists: inspect_bin_spec = os.path.join(ex_path,inspect_bin_l) arguments[0] = inspect_bin_name arguments.insert(1, 'basic-0') arguments.insert(1, '--wrapper') logging.info('Command: %s %s' % (inspect_bin_spec,' '.join(arguments[1:]))) os.execv(inspect_bin_spec, arguments) if __name__ == "__main__": main() hisat2-2.2.1/hisat2.cpp000066400000000000000000005377261370663763200146450ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include #include #include #include #include #include #include #include #include #include "alphabet.h" #include "assert_helpers.h" #include "endian_swap.h" #include "hgfm.h" #include "rfm.h" #include "formats.h" #include "sequence_io.h" #include "tokenize.h" #include "aln_sink.h" #include "pat.h" #include "threading.h" #include "ds.h" #include "aligner_metrics.h" #include "sam.h" #include "aligner_seed.h" #include "splice_site.h" #include "spliced_aligner.h" #include "aligner_seed_policy.h" #include "aligner_sw.h" #include "aligner_sw_driver.h" #include "aligner_cache.h" #include "util.h" #include "pe.h" #include "tp.h" #include "gp.h" #include "simple_func.h" #include "presets.h" #include "opts.h" #include "outq.h" #include "repeat_kmer.h" using namespace std; MemoryTally gMemTally; static EList mates1; // mated reads (first mate) static EList mates2; // mated reads (second mate) static EList mates12; // mated reads (1st/2nd interleaved in 1 file) static string adjIdxBase; bool gColor; // colorspace (not supported) int gVerbose; // be talkative static bool startVerbose; // be talkative at startup int gQuiet; // print nothing but the alignments static int sanityCheck; // enable expensive sanity checks static int format; // default read format is FASTQ static string origString; // reference text, or filename(s) static int seed; // srandom() seed static int timing; // whether to report basic timing data static int metricsIval; // interval between alignment metrics messages (0 = no messages) static string metricsFile;// output file to put alignment metrics in static bool metricsStderr;// output file to put alignment metrics in static bool metricsPerRead; // report a metrics tuple for every read static bool allHits; // for multihits, report just one static bool showVersion; // just print version and quit? static int ipause; // pause before maching? static uint32_t qUpto; // max # of queries to read int gTrim5; // amount to trim from 5' end int gTrim3; // amount to trim from 3' end static int offRate; // keep default offRate static bool solexaQuals; // quality strings are solexa quals, not phred, and subtract 64 (not 33) static bool phred64Quals; // quality chars are phred, but must subtract 64 (not 33) static bool integerQuals; // quality strings are space-separated strings of integers, not ASCII static int nthreads; // number of pthreads operating concurrently static int outType; // style of output static bool noRefNames; // true -> print reference indexes; not names static uint32_t khits; // number of hits per read; >1 is much slower static uint32_t mhits; // don't report any hits if there are > mhits static int partitionSz; // output a partitioning key in first field static bool useSpinlock; // false -> don't use of spinlocks even if they're #defines static bool fileParallel; // separate threads read separate input files in parallel static bool useShmem; // use shared memory to hold the index static bool useMm; // use memory-mapped files to hold the index static bool mmSweep; // sweep through memory-mapped files immediately after mapping int gMinInsert; // minimum insert size int gMaxInsert; // maximum insert size bool gMate1fw; // -1 mate aligns in fw orientation on fw strand bool gMate2fw; // -2 mate aligns in rc orientation on fw strand bool gFlippedMatesOK; // allow mates to be in wrong order bool gDovetailMatesOK; // allow one mate to extend off the end of the other bool gContainMatesOK; // allow one mate to contain the other in PE alignment bool gOlapMatesOK; // allow mates to overlap in PE alignment bool gExpandToFrag; // incr max frag length to =larger mate len if necessary bool gReportDiscordant; // find and report discordant paired-end alignments bool gReportMixed; // find and report unpaired alignments for paired reads static uint32_t cacheLimit; // ranges w/ size > limit will be cached static uint32_t cacheSize; // # words per range cache static uint32_t skipReads; // # reads/read pairs to skip bool gNofw; // don't align fw orientation of read bool gNorc; // don't align rc orientation of read static uint32_t fastaContLen; static uint32_t fastaContFreq; static bool hadoopOut; // print Hadoop status and summary messages static bool fuzzy; static bool fullRef; static bool samTruncQname; // whether to truncate QNAME to 255 chars static bool samOmitSecSeqQual; // omit SEQ/QUAL for 2ndary alignments? static bool samNoUnal; // don't print records for unaligned reads static bool samNoHead; // don't print any header lines in SAM output static bool samNoSQ; // don't print @SQ header lines static bool sam_print_as; static bool sam_print_xs; // XS:i static bool sam_print_xss; // Xs:i and Ys:i static bool sam_print_yn; // YN:i and Yn:i static bool sam_print_xn; static bool sam_print_cs; static bool sam_print_cq; static bool sam_print_x0; static bool sam_print_x1; static bool sam_print_xm; static bool sam_print_xo; static bool sam_print_xg; static bool sam_print_nm; static bool sam_print_md; static bool sam_print_yf; static bool sam_print_yi; static bool sam_print_ym; static bool sam_print_yp; static bool sam_print_yt; static bool sam_print_ys; static bool sam_print_zs; static bool sam_print_xr; static bool sam_print_xt; static bool sam_print_xd; static bool sam_print_xu; static bool sam_print_yl; static bool sam_print_ye; static bool sam_print_yu; static bool sam_print_xp; static bool sam_print_yr; static bool sam_print_zb; static bool sam_print_zr; static bool sam_print_zf; static bool sam_print_zm; static bool sam_print_zi; static bool sam_print_zp; static bool sam_print_zu; static bool sam_print_xs_a; static bool sam_print_nh; static bool bwaSwLike; static float bwaSwLikeC; static float bwaSwLikeT; static bool qcFilter; static bool sortByScore; // prioritize alignments to report by score? bool gReportOverhangs; // false -> filter out alignments that fall off the end of a reference sequence static string rgid; // ID: setting for @RG header line static string rgs; // SAM outputs for @RG header line static string rgs_optflag; // SAM optional flag to add corresponding to @RG ID static bool msample; // whether to report a random alignment when maxed-out via -m/-M int gGapBarrier; // # diags on top/bot only to be entered diagonally static EList qualities; static EList qualities1; static EList qualities2; static string polstr; // temporary holder for policy string static bool msNoCache; // true -> disable local cache static int bonusMatchType; // how to reward matches static int bonusMatch; // constant reward if bonusMatchType=constant static int penMmcType; // how to penalize mismatches static int penMmcMax; // max mm penalty static int penMmcMin; // min mm penalty static int penScMax; // max sc penalty static int penScMin; // min sc penalty static int penNType; // how to penalize Ns in the read static int penN; // constant if N pelanty is a constant static bool penNCatPair; // concatenate mates before N filtering? static bool localAlign; // do local alignment in DP steps static bool noisyHpolymer; // set to true if gap penalties should be reduced to be consistent with a sequencer that under- and overcalls homopolymers static int penRdGapConst; // constant cost of extending a gap in the read static int penRfGapConst; // constant cost of extending a gap in the reference static int penRdGapLinear; // coeff of linear term for cost of gap extension in read static int penRfGapLinear; // coeff of linear term for cost of gap extension in ref static SimpleFunc scoreMin; // minimum valid score as function of read len static SimpleFunc nCeil; // max # Ns allowed as function of read len static SimpleFunc msIval; // interval between seeds as function of read len static double descConsExp; // how to adjust score minimum as we descent further into index-assisted alignment static size_t descentLanding; // don't place a search root if it's within this many positions of end static SimpleFunc descentTotSz; // maximum space a DescentDriver can use in bytes static SimpleFunc descentTotFmops; // maximum # FM ops a DescentDriver can perform static int multiseedMms; // mismatches permitted in a multiseed seed static int multiseedLen; // length of multiseed seeds static size_t multiseedOff; // offset to begin extracting seeds static uint32_t seedCacheLocalMB; // # MB to use for non-shared seed alignment cacheing static uint32_t seedCacheCurrentMB; // # MB to use for current-read seed hit cacheing static uint32_t exactCacheCurrentMB; // # MB to use for current-read seed hit cacheing static size_t maxhalf; // max width on one side of DP table static bool seedSumm; // print summary information about seed hits, not alignments static bool doUngapped; // do ungapped alignment static size_t maxIters; // stop after this many extend loop iterations static size_t maxUg; // stop after this many ungap extends static size_t maxDp; // stop after this many DPs static size_t maxItersIncr; // amt to add to maxIters for each -k > 1 static size_t maxEeStreak; // stop after this many end-to-end fails in a row static size_t maxUgStreak; // stop after this many ungap fails in a row static size_t maxDpStreak; // stop after this many dp fails in a row static size_t maxStreakIncr; // amt to add to streak for each -k > 1 static size_t maxMateStreak; // stop seed range after this many mate-find fails static bool doExtend; // extend seed hits static bool enable8; // use 8-bit SSE where possible? static size_t cminlen; // longer reads use checkpointing static size_t cpow2; // checkpoint interval log2 static bool doTri; // do triangular mini-fills? static string defaultPreset; // default preset; applied immediately static bool ignoreQuals; // all mms incur same penalty, regardless of qual static string wrapper; // type of wrapper script, so we can print correct usage static EList queries; // list of query files static string outfile; // write SAM output to this file static int mapqv; // MAPQ calculation version static int tighten; // -M tighten mode (0=none, 1=best, 2=secbest+1) static bool doExactUpFront; // do exact search up front if seeds seem good enough static bool do1mmUpFront; // do 1mm search up front if seeds seem good enough static size_t do1mmMinLen; // length below which we disable 1mm e2e search static int seedBoostThresh; // if average non-zero position has more than this many elements static size_t maxSeeds; // maximum number of seeds allowed static size_t nSeedRounds; // # seed rounds static bool reorder; // true -> reorder SAM recs in -p mode static float sampleFrac; // only align random fraction of input reads static bool arbitraryRandom; // pseudo-randoms no longer a function of read properties static bool bowtie2p5; static bool useTempSpliceSite; static int penCanSplice; static int penNoncanSplice; static int penConflictSplice; static SimpleFunc penCanIntronLen; static SimpleFunc penNoncanIntronLen; static size_t minIntronLen; static size_t maxIntronLen; static string knownSpliceSiteInfile; // static string novelSpliceSiteInfile; // static string novelSpliceSiteOutfile; // static bool secondary; static bool no_spliced_alignment; static int rna_strandness; // static bool splicesite_db_only; // static bool anchorStop; static bool pseudogeneStop; static bool tranMapOnly; // transcriptome mapping only static bool tranAssm; // alignments selected for downstream transcript assembly such as StringTie and Cufflinks static string tranAssm_program; static bool avoid_pseudogene; #ifdef USE_SRA static EList sra_accs; #endif static string bt2index; // read Bowtie 2 index from files with this prefix static EList > extra_opts; static size_t extra_opts_cur; static EList thread_rids; static MUTEX_T thread_rids_mutex; static uint64_t thread_rids_mindist; static bool rmChrName; // remove "chr" from reference names (e.g., chr18 to 18) static bool addChrName; // add "chr" to reference names (e.g., 18 to chr18) static size_t max_alts_tried; static bool use_haplotype; static bool enable_codis; static bool templateLenAdjustment; static string alignSumFile; // write alignment summary stat. to this file static bool newAlignSummary; static int bowtie2_dp; // Bowtie2's dynamic programming alignment (0: no dynamic programming, 1: conditional dynamic programming, and 2: uncoditional dynamic programming) static bool fast; // --fast static bool sensitive; // --sensitive static bool very_sensitive; // --very-sensitive static bool repeat; static bool use_repeat_index; static EList readLens; #define DMAX std::numeric_limits::max() static void resetOptions() { mates1.clear(); mates2.clear(); mates12.clear(); adjIdxBase = ""; gColor = false; gVerbose = 0; startVerbose = 0; gQuiet = false; sanityCheck = 0; // enable expensive sanity checks format = FASTQ; // default read format is FASTQ origString = ""; // reference text, or filename(s) seed = 0; // srandom() seed timing = 0; // whether to report basic timing data metricsIval = 1; // interval between alignment metrics messages (0 = no messages) metricsFile = ""; // output file to put alignment metrics in metricsStderr = false; // print metrics to stderr (in addition to --metrics-file if it's specified metricsPerRead = false; // report a metrics tuple for every read? allHits = false; // for multihits, report just one showVersion = false; // just print version and quit? ipause = 0; // pause before maching? qUpto = 0xffffffff; // max # of queries to read gTrim5 = 0; // amount to trim from 5' end gTrim3 = 0; // amount to trim from 3' end offRate = -1; // keep default offRate solexaQuals = false; // quality strings are solexa quals, not phred, and subtract 64 (not 33) phred64Quals = false; // quality chars are phred, but must subtract 64 (not 33) integerQuals = false; // quality strings are space-separated strings of integers, not ASCII nthreads = 1; // number of pthreads operating concurrently outType = OUTPUT_SAM; // style of output noRefNames = false; // true -> print reference indexes; not names khits = 10; // number of hits per read; >1 is much slower mhits = 0; // stop after finding this many alignments+1 partitionSz = 0; // output a partitioning key in first field useSpinlock = true; // false -> don't use of spinlocks even if they're #defines fileParallel = false; // separate threads read separate input files in parallel useShmem = false; // use shared memory to hold the index useMm = false; // use memory-mapped files to hold the index mmSweep = false; // sweep through memory-mapped files immediately after mapping gMinInsert = 0; // minimum insert size gMaxInsert = 1000; // maximum insert size gMate1fw = true; // -1 mate aligns in fw orientation on fw strand gMate2fw = false; // -2 mate aligns in rc orientation on fw strand gFlippedMatesOK = false; // allow mates to be in wrong order gDovetailMatesOK = false; // allow one mate to extend off the end of the other gContainMatesOK = true; // allow one mate to contain the other in PE alignment gOlapMatesOK = true; // allow mates to overlap in PE alignment gExpandToFrag = true; // incr max frag length to =larger mate len if necessary gReportDiscordant = true; // find and report discordant paired-end alignments gReportMixed = true; // find and report unpaired alignments for paired reads cacheLimit = 5; // ranges w/ size > limit will be cached cacheSize = 0; // # words per range cache skipReads = 0; // # reads/read pairs to skip gNofw = false; // don't align fw orientation of read gNorc = false; // don't align rc orientation of read fastaContLen = 0; fastaContFreq = 0; hadoopOut = false; // print Hadoop status and summary messages fuzzy = false; // reads will have alternate basecalls w/ qualities fullRef = false; // print entire reference name instead of just up to 1st space samTruncQname = true; // whether to truncate QNAME to 255 chars samOmitSecSeqQual = false; // omit SEQ/QUAL for 2ndary alignments? samNoUnal = false; // omit SAM records for unaligned reads samNoHead = false; // don't print any header lines in SAM output samNoSQ = false; // don't print @SQ header lines sam_print_as = true; sam_print_xs = true; sam_print_xss = false; // Xs:i and Ys:i sam_print_yn = false; // YN:i and Yn:i sam_print_xn = true; sam_print_cs = false; sam_print_cq = false; sam_print_x0 = true; sam_print_x1 = true; sam_print_xm = true; sam_print_xo = true; sam_print_xg = true; sam_print_nm = true; sam_print_md = true; sam_print_yf = true; sam_print_yi = false; sam_print_ym = false; sam_print_yp = false; sam_print_yt = true; sam_print_ys = true; sam_print_zs = false; sam_print_xr = false; sam_print_xt = false; sam_print_xd = false; sam_print_xu = false; sam_print_yl = false; sam_print_ye = false; sam_print_yu = false; sam_print_xp = false; sam_print_yr = false; sam_print_zb = false; sam_print_zr = false; sam_print_zf = false; sam_print_zm = false; sam_print_zi = false; sam_print_zp = false; sam_print_zu = false; sam_print_xs_a = true; sam_print_nh = true; bwaSwLike = false; bwaSwLikeC = 5.5f; bwaSwLikeT = 20.0f; qcFilter = false; // don't believe upstream qc by default sortByScore = true; // prioritize alignments to report by score? rgid = ""; // SAM outputs for @RG header line rgs = ""; // SAM outputs for @RG header line rgs_optflag = ""; // SAM optional flag to add corresponding to @RG ID msample = true; gGapBarrier = 4; // disallow gaps within this many chars of either end of alignment qualities.clear(); qualities1.clear(); qualities2.clear(); polstr.clear(); msNoCache = true; // true -> disable local cache bonusMatchType = DEFAULT_MATCH_BONUS_TYPE; bonusMatch = DEFAULT_MATCH_BONUS; penMmcType = DEFAULT_MM_PENALTY_TYPE; penMmcMax = DEFAULT_MM_PENALTY_MAX; penMmcMin = DEFAULT_MM_PENALTY_MIN; penScMax = DEFAULT_SC_PENALTY_MAX; penScMin = DEFAULT_SC_PENALTY_MIN; penNType = DEFAULT_N_PENALTY_TYPE; penN = DEFAULT_N_PENALTY; penNCatPair = DEFAULT_N_CAT_PAIR; // concatenate mates before N filtering? localAlign = false; // do local alignment in DP steps noisyHpolymer = false; penRdGapConst = DEFAULT_READ_GAP_CONST; penRfGapConst = DEFAULT_REF_GAP_CONST; penRdGapLinear = DEFAULT_READ_GAP_LINEAR; penRfGapLinear = DEFAULT_REF_GAP_LINEAR; scoreMin.init (SIMPLE_FUNC_LINEAR, 0.0f, -0.2f); // scoreMin.init (SIMPLE_FUNC_CONST, -18, 0); nCeil.init (SIMPLE_FUNC_LINEAR, 0.0f, DMAX, 2.0f, 0.1f); msIval.init (SIMPLE_FUNC_LINEAR, 1.0f, DMAX, DEFAULT_IVAL_B, DEFAULT_IVAL_A); descConsExp = 2.0; descentLanding = 20; descentTotSz.init(SIMPLE_FUNC_LINEAR, 1024.0, DMAX, 0.0, 1024.0); descentTotFmops.init(SIMPLE_FUNC_LINEAR, 100.0, DMAX, 0.0, 10.0); multiseedMms = DEFAULT_SEEDMMS; multiseedLen = DEFAULT_SEEDLEN; multiseedOff = 0; seedCacheLocalMB = 32; // # MB to use for non-shared seed alignment cacheing seedCacheCurrentMB = 20; // # MB to use for current-read seed hit cacheing exactCacheCurrentMB = 20; // # MB to use for current-read seed hit cacheing maxhalf = 15; // max width on one side of DP table seedSumm = false; // print summary information about seed hits, not alignments doUngapped = true; // do ungapped alignment maxIters = 400; // max iterations of extend loop maxUg = 300; // stop after this many ungap extends maxDp = 300; // stop after this many dp extends maxItersIncr = 20; // amt to add to maxIters for each -k > 1 maxEeStreak = 15; // stop after this many end-to-end fails in a row maxUgStreak = 15; // stop after this many ungap fails in a row maxDpStreak = 15; // stop after this many dp fails in a row maxStreakIncr = 10; // amt to add to streak for each -k > 1 maxMateStreak = 10; // in PE: abort seed range after N mate-find fails doExtend = true; // do seed extensions enable8 = true; // use 8-bit SSE where possible? cminlen = 2000; // longer reads use checkpointing cpow2 = 4; // checkpoint interval log2 doTri = false; // do triangular mini-fills? defaultPreset = "sensitive%LOCAL%"; // default preset; applied immediately extra_opts.clear(); extra_opts_cur = 0; bt2index.clear(); // read Bowtie 2 index from files with this prefix ignoreQuals = false; // all mms incur same penalty, regardless of qual wrapper.clear(); // type of wrapper script, so we can print correct usage queries.clear(); // list of query files outfile.clear(); // write SAM output to this file mapqv = 2; // MAPQ calculation version tighten = 3; // -M tightening mode doExactUpFront = true; // do exact search up front if seeds seem good enough do1mmUpFront = true; // do 1mm search up front if seeds seem good enough seedBoostThresh = 300; // if average non-zero position has more than this many elements nSeedRounds = 2; // # rounds of seed searches to do for repetitive reads maxSeeds = 0; // maximum number of seeds allowed do1mmMinLen = 60; // length below which we disable 1mm search reorder = false; // reorder SAM records with -p > 1 sampleFrac = 1.1f; // align all reads arbitraryRandom = false; // let pseudo-random seeds be a function of read properties bowtie2p5 = false; useTempSpliceSite = true; penCanSplice = 0; penNoncanSplice = 12; penConflictSplice = 1000000; penCanIntronLen.init(SIMPLE_FUNC_LOG, -8, 1); penNoncanIntronLen.init(SIMPLE_FUNC_LOG, -8, 1); minIntronLen = 20; maxIntronLen = 500000; knownSpliceSiteInfile = ""; novelSpliceSiteInfile = ""; novelSpliceSiteOutfile = ""; secondary = false; // allow secondary alignments no_spliced_alignment = false; rna_strandness = RNA_STRANDNESS_UNKNOWN; splicesite_db_only = false; anchorStop = true; pseudogeneStop = true; tranMapOnly = false; tranAssm = false; tranAssm_program = ""; avoid_pseudogene = false; #ifdef USE_SRA sra_accs.clear(); #endif rmChrName = false; addChrName = false; max_alts_tried = 16; use_haplotype = false; enable_codis = false; templateLenAdjustment = true; alignSumFile = ""; newAlignSummary = false; bowtie2_dp = 0; // disable Bowtie2's dynamic programming alignment fast = false; sensitive = false; very_sensitive = false; repeat = false; // true iff alignments to repeat sequences are directly reported. use_repeat_index = true; readLens.clear(); } static const char *short_options = "fF:qbzhcu:rv:s:aP:t3:5:w:p:k:M:1:2:I:X:CQ:N:i:L:U:x:S:g:O:D:R:"; static struct option long_options[] = { {(char*)"verbose", no_argument, 0, ARG_VERBOSE}, {(char*)"startverbose", no_argument, 0, ARG_STARTVERBOSE}, {(char*)"quiet", no_argument, 0, ARG_QUIET}, {(char*)"sanity", no_argument, 0, ARG_SANITY}, {(char*)"pause", no_argument, &ipause, 1}, {(char*)"orig", required_argument, 0, ARG_ORIG}, {(char*)"all", no_argument, 0, 'a'}, {(char*)"solexa-quals", no_argument, 0, ARG_SOLEXA_QUALS}, {(char*)"integer-quals",no_argument, 0, ARG_INTEGER_QUALS}, {(char*)"int-quals", no_argument, 0, ARG_INTEGER_QUALS}, {(char*)"metrics", required_argument, 0, ARG_METRIC_IVAL}, {(char*)"metrics-file", required_argument, 0, ARG_METRIC_FILE}, {(char*)"metrics-stderr",no_argument, 0, ARG_METRIC_STDERR}, {(char*)"metrics-per-read", no_argument, 0, ARG_METRIC_PER_READ}, {(char*)"met-read", no_argument, 0, ARG_METRIC_PER_READ}, {(char*)"met", required_argument, 0, ARG_METRIC_IVAL}, {(char*)"met-file", required_argument, 0, ARG_METRIC_FILE}, {(char*)"met-stderr", no_argument, 0, ARG_METRIC_STDERR}, {(char*)"time", no_argument, 0, 't'}, {(char*)"trim3", required_argument, 0, '3'}, {(char*)"trim5", required_argument, 0, '5'}, {(char*)"seed", required_argument, 0, ARG_SEED}, {(char*)"qupto", required_argument, 0, 'u'}, {(char*)"upto", required_argument, 0, 'u'}, {(char*)"version", no_argument, 0, ARG_VERSION}, {(char*)"filepar", no_argument, 0, ARG_FILEPAR}, {(char*)"help", no_argument, 0, 'h'}, {(char*)"threads", required_argument, 0, 'p'}, {(char*)"khits", required_argument, 0, 'k'}, {(char*)"minins", required_argument, 0, 'I'}, {(char*)"maxins", required_argument, 0, 'X'}, {(char*)"quals", required_argument, 0, 'Q'}, {(char*)"Q1", required_argument, 0, ARG_QUALS1}, {(char*)"Q2", required_argument, 0, ARG_QUALS2}, {(char*)"refidx", no_argument, 0, ARG_REFIDX}, {(char*)"partition", required_argument, 0, ARG_PARTITION}, {(char*)"ff", no_argument, 0, ARG_FF}, {(char*)"fr", no_argument, 0, ARG_FR}, {(char*)"rf", no_argument, 0, ARG_RF}, {(char*)"cachelim", required_argument, 0, ARG_CACHE_LIM}, {(char*)"cachesz", required_argument, 0, ARG_CACHE_SZ}, {(char*)"nofw", no_argument, 0, ARG_NO_FW}, {(char*)"norc", no_argument, 0, ARG_NO_RC}, {(char*)"skip", required_argument, 0, 's'}, {(char*)"12", required_argument, 0, ARG_ONETWO}, {(char*)"tab5", required_argument, 0, ARG_TAB5}, {(char*)"tab6", required_argument, 0, ARG_TAB6}, {(char*)"phred33-quals", no_argument, 0, ARG_PHRED33}, {(char*)"phred64-quals", no_argument, 0, ARG_PHRED64}, {(char*)"phred33", no_argument, 0, ARG_PHRED33}, {(char*)"phred64", no_argument, 0, ARG_PHRED64}, {(char*)"solexa1.3-quals", no_argument, 0, ARG_PHRED64}, {(char*)"mm", no_argument, 0, ARG_MM}, {(char*)"shmem", no_argument, 0, ARG_SHMEM}, {(char*)"mmsweep", no_argument, 0, ARG_MMSWEEP}, {(char*)"hadoopout", no_argument, 0, ARG_HADOOPOUT}, {(char*)"fuzzy", no_argument, 0, ARG_FUZZY}, {(char*)"fullref", no_argument, 0, ARG_FULLREF}, {(char*)"usage", no_argument, 0, ARG_USAGE}, {(char*)"sam-no-qname-trunc", no_argument, 0, ARG_SAM_NO_QNAME_TRUNC}, {(char*)"sam-omit-sec-seq", no_argument, 0, ARG_SAM_OMIT_SEC_SEQ}, {(char*)"omit-sec-seq", no_argument, 0, ARG_SAM_OMIT_SEC_SEQ}, {(char*)"sam-no-head", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"sam-nohead", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"sam-noHD", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"sam-no-hd", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"sam-nosq", no_argument, 0, ARG_SAM_NOSQ}, {(char*)"sam-no-sq", no_argument, 0, ARG_SAM_NOSQ}, {(char*)"sam-noSQ", no_argument, 0, ARG_SAM_NOSQ}, {(char*)"no-head", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"no-hd", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"no-sq", no_argument, 0, ARG_SAM_NOSQ}, {(char*)"no-HD", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"no-SQ", no_argument, 0, ARG_SAM_NOSQ}, {(char*)"no-unal", no_argument, 0, ARG_SAM_NO_UNAL}, {(char*)"color", no_argument, 0, 'C'}, {(char*)"sam-RG", required_argument, 0, ARG_SAM_RG}, {(char*)"sam-rg", required_argument, 0, ARG_SAM_RG}, {(char*)"sam-rg-id", required_argument, 0, ARG_SAM_RGID}, {(char*)"RG", required_argument, 0, ARG_SAM_RG}, {(char*)"rg", required_argument, 0, ARG_SAM_RG}, {(char*)"rg-id", required_argument, 0, ARG_SAM_RGID}, {(char*)"snpphred", required_argument, 0, ARG_SNPPHRED}, {(char*)"snpfrac", required_argument, 0, ARG_SNPFRAC}, {(char*)"gbar", required_argument, 0, ARG_GAP_BAR}, {(char*)"qseq", no_argument, 0, ARG_QSEQ}, {(char*)"policy", required_argument, 0, ARG_ALIGN_POLICY}, {(char*)"preset", required_argument, 0, 'P'}, {(char*)"seed-summ", no_argument, 0, ARG_SEED_SUMM}, {(char*)"seed-summary", no_argument, 0, ARG_SEED_SUMM}, {(char*)"overhang", no_argument, 0, ARG_OVERHANG}, {(char*)"no-cache", no_argument, 0, ARG_NO_CACHE}, {(char*)"cache", no_argument, 0, ARG_USE_CACHE}, {(char*)"454", no_argument, 0, ARG_NOISY_HPOLY}, {(char*)"ion-torrent", no_argument, 0, ARG_NOISY_HPOLY}, {(char*)"no-mixed", no_argument, 0, ARG_NO_MIXED}, {(char*)"no-discordant",no_argument, 0, ARG_NO_DISCORDANT}, // {(char*)"local", no_argument, 0, ARG_LOCAL}, {(char*)"end-to-end", no_argument, 0, ARG_END_TO_END}, {(char*)"ungapped", no_argument, 0, ARG_UNGAPPED}, {(char*)"no-ungapped", no_argument, 0, ARG_UNGAPPED_NO}, {(char*)"sse8", no_argument, 0, ARG_SSE8}, {(char*)"no-sse8", no_argument, 0, ARG_SSE8_NO}, {(char*)"scan-narrowed",no_argument, 0, ARG_SCAN_NARROWED}, {(char*)"qc-filter", no_argument, 0, ARG_QC_FILTER}, {(char*)"bwa-sw-like", no_argument, 0, ARG_BWA_SW_LIKE}, {(char*)"multiseed", required_argument, 0, ARG_MULTISEED_IVAL}, {(char*)"ma", required_argument, 0, ARG_SCORE_MA}, {(char*)"mp", required_argument, 0, ARG_SCORE_MMP}, {(char*)"sp", required_argument, 0, ARG_SCORE_SCP}, {(char*)"no-softclip", no_argument, 0, ARG_NO_SOFTCLIP}, {(char*)"np", required_argument, 0, ARG_SCORE_NP}, {(char*)"rdg", required_argument, 0, ARG_SCORE_RDG}, {(char*)"rfg", required_argument, 0, ARG_SCORE_RFG}, {(char*)"score-min", required_argument, 0, ARG_SCORE_MIN}, {(char*)"min-score", required_argument, 0, ARG_SCORE_MIN}, {(char*)"n-ceil", required_argument, 0, ARG_N_CEIL}, {(char*)"dpad", required_argument, 0, ARG_DPAD}, {(char*)"mapq-print-inputs",no_argument, 0, ARG_SAM_PRINT_YI}, {(char*)"very-fast", no_argument, 0, ARG_PRESET_VERY_FAST}, {(char*)"fast", no_argument, 0, ARG_PRESET_FAST}, {(char*)"sensitive", no_argument, 0, ARG_PRESET_SENSITIVE}, {(char*)"very-sensitive", no_argument, 0, ARG_PRESET_VERY_SENSITIVE}, // {(char*)"very-fast-local", no_argument, 0, ARG_PRESET_VERY_FAST_LOCAL}, // {(char*)"fast-local", no_argument, 0, ARG_PRESET_FAST_LOCAL}, // {(char*)"sensitive-local", no_argument, 0, ARG_PRESET_SENSITIVE_LOCAL}, // {(char*)"very-sensitive-local", no_argument, 0, ARG_PRESET_VERY_SENSITIVE_LOCAL}, {(char*)"no-score-priority",no_argument, 0, ARG_NO_SCORE_PRIORITY}, {(char*)"seedlen", required_argument, 0, 'L'}, {(char*)"seedmms", required_argument, 0, 'N'}, {(char*)"seedival", required_argument, 0, 'i'}, {(char*)"ignore-quals", no_argument, 0, ARG_IGNORE_QUALS}, {(char*)"index", required_argument, 0, 'x'}, {(char*)"arg-desc", no_argument, 0, ARG_DESC}, {(char*)"wrapper", required_argument, 0, ARG_WRAPPER}, {(char*)"unpaired", required_argument, 0, 'U'}, {(char*)"output", required_argument, 0, 'S'}, {(char*)"mapq-v", required_argument, 0, ARG_MAPQ_V}, {(char*)"dovetail", no_argument, 0, ARG_DOVETAIL}, {(char*)"no-dovetail", no_argument, 0, ARG_NO_DOVETAIL}, {(char*)"contain", no_argument, 0, ARG_CONTAIN}, {(char*)"no-contain", no_argument, 0, ARG_NO_CONTAIN}, {(char*)"overlap", no_argument, 0, ARG_OVERLAP}, {(char*)"no-overlap", no_argument, 0, ARG_NO_OVERLAP}, {(char*)"tighten", required_argument, 0, ARG_TIGHTEN}, {(char*)"exact-upfront", no_argument, 0, ARG_EXACT_UPFRONT}, {(char*)"1mm-upfront", no_argument, 0, ARG_1MM_UPFRONT}, {(char*)"no-exact-upfront", no_argument, 0, ARG_EXACT_UPFRONT_NO}, {(char*)"no-1mm-upfront", no_argument, 0, ARG_1MM_UPFRONT_NO}, {(char*)"1mm-minlen", required_argument, 0, ARG_1MM_MINLEN}, {(char*)"seed-off", required_argument, 0, 'O'}, {(char*)"seed-boost", required_argument, 0, ARG_SEED_BOOST_THRESH}, {(char*)"max-seeds", required_argument, 0, ARG_MAX_SEEDS}, {(char*)"read-times", no_argument, 0, ARG_READ_TIMES}, {(char*)"show-rand-seed", no_argument, 0, ARG_SHOW_RAND_SEED}, {(char*)"dp-fail-streak", required_argument, 0, ARG_DP_FAIL_STREAK_THRESH}, {(char*)"ee-fail-streak", required_argument, 0, ARG_EE_FAIL_STREAK_THRESH}, {(char*)"ug-fail-streak", required_argument, 0, ARG_UG_FAIL_STREAK_THRESH}, {(char*)"fail-streak", required_argument, 0, 'D'}, {(char*)"dp-fails", required_argument, 0, ARG_DP_FAIL_THRESH}, {(char*)"ug-fails", required_argument, 0, ARG_UG_FAIL_THRESH}, {(char*)"extends", required_argument, 0, ARG_EXTEND_ITERS}, {(char*)"no-extend", no_argument, 0, ARG_NO_EXTEND}, {(char*)"mapq-extra", no_argument, 0, ARG_MAPQ_EX}, {(char*)"seed-rounds", required_argument, 0, 'R'}, {(char*)"reorder", no_argument, 0, ARG_REORDER}, {(char*)"passthrough", no_argument, 0, ARG_READ_PASSTHRU}, {(char*)"sample", required_argument, 0, ARG_SAMPLE}, {(char*)"cp-min", required_argument, 0, ARG_CP_MIN}, {(char*)"cp-ival", required_argument, 0, ARG_CP_IVAL}, {(char*)"tri", no_argument, 0, ARG_TRI}, {(char*)"nondeterministic", no_argument, 0, ARG_NON_DETERMINISTIC}, {(char*)"non-deterministic", no_argument, 0, ARG_NON_DETERMINISTIC}, // {(char*)"local-seed-cache-sz", required_argument, 0, ARG_LOCAL_SEED_CACHE_SZ}, {(char*)"seed-cache-sz", required_argument, 0, ARG_CURRENT_SEED_CACHE_SZ}, {(char*)"no-unal", no_argument, 0, ARG_SAM_NO_UNAL}, {(char*)"test-25", no_argument, 0, ARG_TEST_25}, // TODO: following should be a function of read length? {(char*)"desc-kb", required_argument, 0, ARG_DESC_KB}, {(char*)"desc-landing", required_argument, 0, ARG_DESC_LANDING}, {(char*)"desc-exp", required_argument, 0, ARG_DESC_EXP}, {(char*)"desc-fmops", required_argument, 0, ARG_DESC_FMOPS}, {(char*)"no-temp-splicesite", no_argument, 0, ARG_NO_TEMPSPLICESITE}, {(char*)"pen-cansplice", required_argument, 0, ARG_PEN_CANSPLICE}, {(char*)"pen-noncansplice", required_argument, 0, ARG_PEN_NONCANSPLICE}, {(char*)"pen-conflictsplice", required_argument, 0, ARG_PEN_CONFLICTSPLICE}, {(char*)"pen-intronlen", required_argument, 0, ARG_PEN_CANINTRONLEN}, {(char*)"pen-canintronlen", required_argument, 0, ARG_PEN_CANINTRONLEN}, {(char*)"pen-noncanintronlen", required_argument, 0, ARG_PEN_NONCANINTRONLEN}, {(char*)"min-intronlen", required_argument, 0, ARG_MIN_INTRONLEN}, {(char*)"max-intronlen", required_argument, 0, ARG_MAX_INTRONLEN}, {(char*)"known-splicesite-infile", required_argument, 0, ARG_KNOWN_SPLICESITE_INFILE}, {(char*)"novel-splicesite-infile", required_argument, 0, ARG_NOVEL_SPLICESITE_INFILE}, {(char*)"novel-splicesite-outfile", required_argument, 0, ARG_NOVEL_SPLICESITE_OUTFILE}, {(char*)"secondary", no_argument, 0, ARG_SECONDARY}, {(char*)"no-spliced-alignment", no_argument, 0, ARG_NO_SPLICED_ALIGNMENT}, {(char*)"rna-strandness", required_argument, 0, ARG_RNA_STRANDNESS}, {(char*)"splicesite-db-only", no_argument, 0, ARG_SPLICESITE_DB_ONLY}, {(char*)"no-anchorstop", no_argument, 0, ARG_NO_ANCHORSTOP}, {(char*)"transcriptome-mapping-only", no_argument, 0, ARG_TRANSCRIPTOME_MAPPING_ONLY}, {(char*)"tmo", no_argument, 0, ARG_TRANSCRIPTOME_MAPPING_ONLY}, {(char*)"downstream-transcriptome-assembly", no_argument, 0, ARG_TRANSCRIPTOME_ASSEMBLY}, {(char*)"dta", no_argument, 0, ARG_TRANSCRIPTOME_ASSEMBLY}, {(char*)"dta-cufflinks", no_argument, 0, ARG_TRANSCRIPTOME_ASSEMBLY_CUFFLINKS}, {(char*)"avoid-pseudogene",no_argument, 0, ARG_AVOID_PSEUDOGENE}, {(char*)"no-templatelen-adjustment", no_argument, 0, ARG_NO_TEMPLATELEN_ADJUSTMENT}, #ifdef USE_SRA {(char*)"sra-acc", required_argument, 0, ARG_SRA_ACC}, #endif {(char*)"remove-chrname", no_argument, 0, ARG_REMOVE_CHRNAME}, {(char*)"add-chrname", no_argument, 0, ARG_ADD_CHRNAME}, {(char*)"max-altstried", required_argument, 0, ARG_MAX_ALTSTRIED}, {(char*)"haplotype", no_argument, 0, ARG_HAPLOTYPE}, {(char*)"enable-codis", no_argument, 0, ARG_CODIS}, {(char*)"summary-file", required_argument, 0, ARG_SUMMARY_FILE}, {(char*)"new-summary", no_argument, 0, ARG_NEW_SUMMARY}, {(char*)"enable-dp", no_argument, 0, ARG_DP}, {(char*)"bowtie2-dp", required_argument, 0, ARG_DP}, {(char*)"repeat", no_argument, 0, ARG_REPEAT}, {(char*)"no-repeat-index", no_argument, 0, ARG_NO_REPEAT_INDEX}, {(char*)"read-lengths", required_argument, 0, ARG_READ_LENGTHS}, {(char*)0, 0, 0, 0} // terminator }; /** * Print out a concise description of what options are taken and whether they * take an argument. */ static void printArgDesc(ostream& out) { // struct option { // const char *name; // int has_arg; // int *flag; // int val; // }; size_t i = 0; while(long_options[i].name != 0) { out << long_options[i].name << "\t" << (long_options[i].has_arg == no_argument ? 0 : 1) << endl; i++; } size_t solen = strlen(short_options); for(i = 0; i < solen; i++) { // Has an option? Does if next char is : if(i == solen-1) { assert_neq(':', short_options[i]); cout << (char)short_options[i] << "\t" << 0 << endl; } else { if(short_options[i+1] == ':') { // Option with argument cout << (char)short_options[i] << "\t" << 1 << endl; i++; // skip the ':' } else { // Option with no argument cout << (char)short_options[i] << "\t" << 0 << endl; } } } } /** * Print a summary usage message to the provided output stream. */ static void printUsage(ostream& out) { out << "HISAT2 version " << string(HISAT2_VERSION).c_str() << " by Daehwan Kim (infphilo@gmail.com, www.ccb.jhu.edu/people/infphilo)" << endl; string tool_name = "hisat2-align"; if(wrapper == "basic-0") { tool_name = "hisat2"; } out << "Usage: " << endl #ifdef USE_SRA << " " << tool_name.c_str() << " [options]* -x {-1 -2 | -U | --sra-acc } [-S ]" << endl #else << " " << tool_name.c_str() << " [options]* -x {-1 -2 | -U } [-S ]" << endl #endif << endl << " Index filename prefix (minus trailing .X." << gfm_ext << ")." << endl << " Files with #1 mates, paired with files in ." << endl; if(wrapper == "basic-0") { out << " Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2)." << endl; } out << " Files with #2 mates, paired with files in ." << endl; if(wrapper == "basic-0") { out << " Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2)." << endl; } out << " Files with unpaired reads." << endl; if(wrapper == "basic-0") { out << " Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2)." << endl; } #ifdef USE_SRA out << " Comma-separated list of SRA accession numbers, e.g. --sra-acc SRR353653,SRR353654." << endl; #endif out << " File for SAM output (default: stdout)" << endl << endl << " , , can be comma-separated lists (no whitespace) and can be" << endl << " specified many times. E.g. '-U file1.fq,file2.fq -U file3.fq'." << endl // Wrapper script should write line next << endl << "Options (defaults in parentheses):" << endl << endl << " Input:" << endl << " -q query input files are FASTQ .fq/.fastq (default)" << endl << " --qseq query input files are in Illumina's qseq format" << endl << " -f query input files are (multi-)FASTA .fa/.mfa" << endl << " -r query input files are raw one-sequence-per-line" << endl << " -c , , are sequences themselves, not files" << endl << " -s/--skip skip the first reads/pairs in the input (none)" << endl << " -u/--upto stop after first reads/pairs (no limit)" << endl << " -5/--trim5 trim bases from 5'/left end of reads (0)" << endl << " -3/--trim3 trim bases from 3'/right end of reads (0)" << endl << " --phred33 qualities are Phred+33 (default)" << endl << " --phred64 qualities are Phred+64" << endl << " --int-quals qualities encoded as space-delimited integers" << endl #ifdef USE_SRA << " --sra-acc SRA accession ID" << endl #endif << endl << " Presets: Same as:" << endl // << " For --end-to-end:" << endl // << " --very-fast -D 5 -R 1 -N 0 -L 22 -i S,0,2.50" << endl // << " --fast -D 10 -R 2 -N 0 -L 22 -i S,0,2.50" << endl // << " --sensitive -D 15 -R 2 -N 0 -L 22 -i S,1,1.15 (default)" << endl // << " --very-sensitive -D 20 -R 3 -N 0 -L 20 -i S,1,0.50" << endl << " --fast --no-repeat-index" << endl << " --sensitive --bowtie2-dp 1 -k 30 --score-min L,0,-0.5" << endl << " --very-sensitive --bowtie2-dp 2 -k 50 --score-min L,0,-1" << endl << endl << " Alignment:" << endl //<< " -N max # mismatches in seed alignment; can be 0 or 1 (0)" << endl //<< " -L length of seed substrings; must be >3, <32 (22)" << endl //<< " -i interval between seed substrings w/r/t read len (S,1,1.15)" << endl << " --bowtie2-dp use Bowtie2's dynamic programming alignment algorithm (0) - 0: no dynamic programming, 1: conditional dynamic programming, and 2: unconditional dynamic programming (slowest)" << endl << " --n-ceil func for max # non-A/C/G/Ts permitted in aln (L,0,0.15)" << endl //<< " --dpad include extra ref chars on sides of DP table (15)" << endl //<< " --gbar disallow gaps within nucs of read extremes (4)" << endl << " --ignore-quals treat all quality values as 30 on Phred scale (off)" << endl << " --nofw do not align forward (original) version of read (off)" << endl << " --norc do not align reverse-complement version of read (off)" << endl << " --no-repeat-index do not use repeat index" << endl << endl << " Spliced Alignment:" << endl << " --pen-cansplice penalty for a canonical splice site (0)" << endl << " --pen-noncansplice penalty for a non-canonical splice site (12)" << endl // << " --pen-conflictsplice penalty for conflicting splice sites (1000000)" << endl << " --pen-canintronlen penalty for long introns (G,-8,1) with canonical splice sites" << endl << " --pen-noncanintronlen penalty for long introns (G,-8,1) with noncanonical splice sites" << endl << " --min-intronlen minimum intron length (20)" << endl << " --max-intronlen maximum intron length (500000)" << endl << " --known-splicesite-infile provide a list of known splice sites" << endl << " --novel-splicesite-outfile report a list of splice sites" << endl << " --novel-splicesite-infile provide a list of novel splice sites" << endl << " --no-temp-splicesite disable the use of splice sites found" << endl << " --no-spliced-alignment disable spliced alignment" << endl << " --rna-strandness specify strand-specific information (unstranded)" << endl << " --tmo reports only those alignments within known transcriptome" << endl << " --dta reports alignments tailored for transcript assemblers" << endl << " --dta-cufflinks reports alignments tailored specifically for cufflinks" << endl << " --avoid-pseudogene tries to avoid aligning reads to pseudogenes (experimental option)" << endl << " --no-templatelen-adjustment disables template length adjustment for RNA-seq reads" << endl << endl << " Scoring:" << endl //<< " --ma match bonus (0 for --end-to-end, 2 for --local) " << endl << " --mp , max and min penalties for mismatch; lower qual = lower penalty <6,2>" << endl << " --sp , max and min penalties for soft-clipping; lower qual = lower penalty <2,1>" << endl << " --no-softclip no soft-clipping" << endl << " --np penalty for non-A/C/G/Ts in read/ref (1)" << endl << " --rdg , read gap open, extend penalties (5,3)" << endl << " --rfg , reference gap open, extend penalties (5,3)" << endl << " --score-min min acceptable alignment score w/r/t read length" << endl << " (L,0.0,-0.2)" << endl << endl << " Reporting:" << endl << " -k It searches for at most distinct, primary alignments for each read. Primary alignments mean " << endl << " alignments whose alignment score is equal to or higher than any other alignments. The search terminates " << endl << " when it cannot find more distinct valid alignments, or when it finds , whichever happens first. " << endl << " The alignment score for a paired-end alignment equals the sum of the alignment scores of " << endl << " the individual mates. Each reported read or pair alignment beyond the first has the SAM ‘secondary’ bit " << endl << " (which equals 256) set in its FLAGS field. For reads that have more than distinct, " << endl << " valid alignments, hisat2 does not guarantee that the alignments reported are the best possible " << endl << " in terms of alignment score. Default: 5 (linear index) or 10 (graph index)." << endl << " Note: HISAT2 is not designed with large values for -k in mind, and when aligning reads to long, " << endl << " repetitive genomes, large -k could make alignment much slower." << endl << " --max-seeds HISAT2, like other aligners, uses seed-and-extend approaches. HISAT2 tries to extend seeds to " << endl << " full-length alignments. In HISAT2, --max-seeds is used to control the maximum number of seeds that " << endl << " will be extended. For DNA-read alignment (--no-spliced-alignment), HISAT2 extends up to these many seeds" << endl << " and skips the rest of the seeds. For RNA-read alignment, HISAT2 skips extending seeds and reports " << endl << " no alignments if the number of seeds is larger than the number specified with the option, " << endl << " to be compatible with previous versions of HISAT2. Large values for --max-seeds may improve alignment " << endl << " sensitivity, but HISAT2 is not designed with large values for --max-seeds in mind, and when aligning " << endl << " reads to long, repetitive genomes, large --max-seeds could make alignment much slower. " << endl << " The default value is the maximum of 5 and the value that comes with -k times 2." << endl << " -a/--all HISAT2 reports all alignments it can find. Using the option is equivalent to using both --max-seeds " << endl << " and -k with the maximum value that a 64-bit signed integer can represent (9,223,372,036,854,775,807)." << endl << " --repeat report alignments to repeat sequences directly" << endl << endl //<< " Effort:" << endl //<< " -D give up extending after failed extends in a row (15)" << endl //<< " -R for reads w/ repetitive seeds, try sets of seeds (2)" << endl //<< endl << " Paired-end:" << endl << " -I/--minins minimum fragment length (0), only valid with --no-spliced-alignment" << endl << " -X/--maxins maximum fragment length (500), only valid with --no-spliced-alignment" << endl << " --fr/--rf/--ff -1, -2 mates align fw/rev, rev/fw, fw/fw (--fr)" << endl << " --no-mixed suppress unpaired alignments for paired reads" << endl << " --no-discordant suppress discordant alignments for paired reads" << endl << endl << " Output:" << endl; //if(wrapper == "basic-0") { // out << " --bam output directly to BAM (by piping through 'samtools view')" << endl; //} out << " -t/--time print wall-clock time taken by search phases" << endl; if(wrapper == "basic-0") { out << " --un write unpaired reads that didn't align to " << endl << " --al write unpaired reads that aligned at least once to " << endl << " --un-conc write pairs that didn't align concordantly to " << endl << " --al-conc write pairs that aligned concordantly at least once to " << endl << " (Note: for --un, --al, --un-conc, or --al-conc, add '-gz' to the option name, e.g." << endl << " --un-gz , to gzip compress output, or add '-bz2' to bzip2 compress output.)" << endl; } out << " --summary-file print alignment summary to this file." << endl << " --new-summary print alignment summary in a new style, which is more machine-friendly." << endl << " --quiet print nothing to stderr except serious errors" << endl // << " --refidx refer to ref. seqs by 0-based index rather than name" << endl << " --met-file send metrics to file at (off)" << endl << " --met-stderr send metrics to stderr (off)" << endl << " --met report internal counters & metrics every secs (1)" << endl // Following is supported in the wrapper instead // << " --no-unal suppress SAM records for unaligned reads" << endl << " --no-head suppress header lines, i.e. lines starting with @" << endl << " --no-sq suppress @SQ header lines" << endl << " --rg-id set read group id, reflected in @RG line and RG:Z: opt field" << endl << " --rg add (\"lab:value\") to @RG line of SAM header." << endl << " Note: @RG line only printed when --rg-id is set." << endl << " --omit-sec-seq put '*' in SEQ and QUAL fields for secondary alignments." << endl << endl << " Performance:" << endl << " -o/--offrate override offrate of index; must be >= index's offrate" << endl << " -p/--threads number of alignment threads to launch (1)" << endl << " --reorder force SAM output order to match order of input reads" << endl #ifdef BOWTIE_MM << " --mm use memory-mapped I/O for index; many 'hisat2's can share" << endl #endif #ifdef BOWTIE_SHARED_MEM //<< " --shmem use shared mem for index; many 'hisat2's can share" << endl #endif << endl << " Other:" << endl << " --qc-filter filter out reads that are bad according to QSEQ filter" << endl << " --seed seed for random number generator (0)" << endl << " --non-deterministic seed rand. gen. arbitrarily instead of using read attributes" << endl << " --remove-chrname remove 'chr' from reference names in alignment" << endl << " --add-chrname add 'chr' to reference names in alignment " << endl // << " --verbose verbose output for debugging" << endl << " --version print version information and quit" << endl << " -h/--help print this usage message" << endl ; if(wrapper.empty()) { cerr << endl << "*** Warning ***" << endl << "'hisat2-align' was run directly. It is recommended that you run the wrapper script 'hisat2' instead." << endl << endl; } } /** * Parse an int out of optarg and enforce that it be at least 'lower'; * if it is less than 'lower', than output the given error message and * exit with an error and a usage message. */ static int parseInt(int lower, int upper, const char *errmsg, const char *arg) { long l; char *endPtr= NULL; l = strtol(arg, &endPtr, 10); if (endPtr != NULL) { if (l < lower || l > upper) { cerr << errmsg << endl; printUsage(cerr); throw 1; } return (int32_t)l; } cerr << errmsg << endl; printUsage(cerr); throw 1; return -1; } /** * Upper is maximum int by default. */ static int parseInt(int lower, const char *errmsg, const char *arg) { return parseInt(lower, std::numeric_limits::max(), errmsg, arg); } /** * Parse a T string 'str'. */ template T parse(const char *s) { T tmp; stringstream ss(s); ss >> tmp; return tmp; } /** * Parse a pair of Ts from a string, 'str', delimited with 'delim'. */ template pair parsePair(const char *str, char delim) { string s(str); EList ss; tokenize(s, delim, ss); pair ret; ret.first = parse(ss[0].c_str()); ret.second = parse(ss[1].c_str()); return ret; } /** * Parse a pair of Ts from a string, 'str', delimited with 'delim'. */ template void parseTuple(const char *str, char delim, EList& ret) { string s(str); EList ss; tokenize(s, delim, ss); for(size_t i = 0; i < ss.size(); i++) { ret.push_back(parse(ss[i].c_str())); } } static string applyPreset(const string& sorig, Presets& presets) { string s = sorig; size_t found = s.find("%LOCAL%"); if(found != string::npos) { s.replace(found, strlen("%LOCAL%"), localAlign ? "-local" : ""); } if(gVerbose) { cerr << "Applying preset: '" << s.c_str() << "' using preset menu '" << presets.name() << "'" << endl; } string pol; presets.apply(s, pol, extra_opts); return pol; } static bool saw_M; static bool saw_a; static bool saw_k; static EList presetList; /** * TODO: Argument parsing is very, very flawed. The biggest problem is that * there are two separate worlds of arguments, the ones set via polstr, and * the ones set directly in variables. This makes for nasty interactions, * e.g., with the -M option being resolved at an awkward time relative to * the -k and -a options. */ static void parseOption(int next_option, const char *arg) { switch (next_option) { case ARG_TEST_25: bowtie2p5 = true; break; case ARG_DESC_KB: descentTotSz = SimpleFunc::parse(arg, 0.0, 1024.0, 1024.0, DMAX); break; case ARG_DESC_FMOPS: descentTotFmops = SimpleFunc::parse(arg, 0.0, 10.0, 100.0, DMAX); break; case ARG_DESC_LANDING: descentLanding = parse(arg); break; case ARG_DESC_EXP: { descConsExp = parse(arg); if(descConsExp < 0.0) { cerr << "Error: --desc-exp must be greater than or equal to 0" << endl; throw 1; } break; } case '1': tokenize(arg, ",", mates1); break; case '2': tokenize(arg, ",", mates2); break; case ARG_ONETWO: tokenize(arg, ",", mates12); format = TAB_MATE5; break; case ARG_TAB5: tokenize(arg, ",", mates12); format = TAB_MATE5; break; case ARG_TAB6: tokenize(arg, ",", mates12); format = TAB_MATE6; break; case 'f': format = FASTA; break; case 'F': { format = FASTA_CONT; pair p = parsePair(arg, ','); fastaContLen = p.first; fastaContFreq = p.second; break; } case ARG_BWA_SW_LIKE: { bwaSwLikeC = 5.5f; bwaSwLikeT = 30; bwaSwLike = true; localAlign = true; // -a INT Score of a match [1] // -b INT Mismatch penalty [3] // -q INT Gap open penalty [5] // -r INT Gap extension penalty. The penalty for a contiguous // gap of size k is q+k*r. [2] polstr += ";MA=1;MMP=C3;RDG=5,2;RFG=5,2"; break; } case 'q': format = FASTQ; break; case 'r': format = RAW; break; case 'c': format = CMDLINE; break; case ARG_QSEQ: format = QSEQ; break; case 'C': { cerr << "Error: -C specified but Bowtie 2 does not support colorspace input." << endl; throw 1; break; } case 'I': gMinInsert = parseInt(0, "-I arg must be positive", arg); break; case 'X': gMaxInsert = parseInt(1, "-X arg must be at least 1", arg); break; case ARG_NO_DISCORDANT: gReportDiscordant = false; break; case ARG_NO_MIXED: gReportMixed = false; break; case 's': skipReads = (uint32_t)parseInt(0, "-s arg must be positive", arg); break; case ARG_FF: gMate1fw = true; gMate2fw = true; break; case ARG_RF: gMate1fw = false; gMate2fw = true; break; case ARG_FR: gMate1fw = true; gMate2fw = false; break; case ARG_SHMEM: useShmem = true; break; case ARG_SEED_SUMM: seedSumm = true; break; case ARG_MM: { #ifdef BOWTIE_MM useMm = true; break; #else cerr << "Memory-mapped I/O mode is disabled because bowtie was not compiled with" << endl << "BOWTIE_MM defined. Memory-mapped I/O is not supported under Windows. If you" << endl << "would like to use memory-mapped I/O on a platform that supports it, please" << endl << "refrain from specifying BOWTIE_MM=0 when compiling Bowtie." << endl; throw 1; #endif } case ARG_MMSWEEP: mmSweep = true; break; case ARG_HADOOPOUT: hadoopOut = true; break; case ARG_SOLEXA_QUALS: solexaQuals = true; break; case ARG_INTEGER_QUALS: integerQuals = true; break; case ARG_PHRED64: phred64Quals = true; break; case ARG_PHRED33: solexaQuals = false; phred64Quals = false; break; case ARG_OVERHANG: gReportOverhangs = true; break; case ARG_NO_CACHE: msNoCache = true; break; case ARG_USE_CACHE: msNoCache = false; break; case ARG_LOCAL_SEED_CACHE_SZ: seedCacheLocalMB = (uint32_t)parseInt(1, "--local-seed-cache-sz arg must be at least 1", arg); break; case ARG_CURRENT_SEED_CACHE_SZ: seedCacheCurrentMB = (uint32_t)parseInt(1, "--seed-cache-sz arg must be at least 1", arg); break; case ARG_REFIDX: noRefNames = true; break; case ARG_FUZZY: fuzzy = true; break; case ARG_FULLREF: fullRef = true; break; case ARG_GAP_BAR: gGapBarrier = parseInt(1, "--gbar must be no less than 1", arg); break; case ARG_SEED: seed = parseInt(0, "--seed arg must be at least 0", arg); break; case ARG_NON_DETERMINISTIC: arbitraryRandom = true; break; case 'u': qUpto = (uint32_t)parseInt(1, "-u/--qupto arg must be at least 1", arg); break; case 'Q': tokenize(arg, ",", qualities); integerQuals = true; break; case ARG_QUALS1: tokenize(arg, ",", qualities1); integerQuals = true; break; case ARG_QUALS2: tokenize(arg, ",", qualities2); integerQuals = true; break; case ARG_CACHE_LIM: cacheLimit = (uint32_t)parseInt(1, "--cachelim arg must be at least 1", arg); break; case ARG_CACHE_SZ: cacheSize = (uint32_t)parseInt(1, "--cachesz arg must be at least 1", arg); cacheSize *= (1024 * 1024); // convert from MB to B break; case ARG_WRAPPER: wrapper = arg; break; case 'p': nthreads = parseInt(1, "-p/--threads arg must be at least 1", arg); break; case ARG_FILEPAR: fileParallel = true; break; case '3': gTrim3 = parseInt(0, "-3/--trim3 arg must be at least 0", arg); break; case '5': gTrim5 = parseInt(0, "-5/--trim5 arg must be at least 0", arg); break; case 'h': printUsage(cout); throw 0; break; case ARG_USAGE: printUsage(cout); throw 0; break; // // NOTE that unlike in Bowtie 1, -M, -a and -k are mutually // exclusive here. // case 'M': { msample = true; mhits = parse(arg); if(saw_a || saw_k) { cerr << "Warning: -M, -k and -a are mutually exclusive. " << "-M will override" << endl; khits = 1; } assert_eq(1, khits); saw_M = true; cerr << "Warning: -M is deprecated. Use -D and -R to adjust " << "effort instead." << endl; break; } case ARG_EXTEND_ITERS: { maxIters = parse(arg); break; } case ARG_NO_EXTEND: { doExtend = false; break; } case 'R': { polstr += ";ROUNDS="; polstr += arg; break; } case 'D': { polstr += ";DPS="; polstr += arg; break; } case ARG_DP_MATE_STREAK_THRESH: { maxMateStreak = parse(arg); break; } case ARG_DP_FAIL_STREAK_THRESH: { maxDpStreak = parse(arg); break; } case ARG_EE_FAIL_STREAK_THRESH: { maxEeStreak = parse(arg); break; } case ARG_UG_FAIL_STREAK_THRESH: { maxUgStreak = parse(arg); break; } case ARG_DP_FAIL_THRESH: { maxDp = parse(arg); break; } case ARG_UG_FAIL_THRESH: { maxUg = parse(arg); break; } case ARG_MAX_SEEDS: { maxSeeds = parse(arg); break; } case ARG_SEED_BOOST_THRESH: { seedBoostThresh = parse(arg); break; } case 'a': { msample = false; allHits = true; mhits = 0; // disable -M if(saw_M || saw_k) { cerr << "Warning: -M, -k and -a are mutually exclusive. " << "-a will override" << endl; } saw_a = true; break; } case 'k': { msample = false; khits = (uint32_t)parseInt(1, "-k arg must be at least 1", arg); mhits = 0; // disable -M if(saw_M || saw_a) { cerr << "Warning: -M, -k and -a are mutually exclusive. " << "-k will override" << endl; } saw_k = true; break; } case ARG_VERBOSE: gVerbose = 1; break; case ARG_STARTVERBOSE: startVerbose = true; break; case ARG_QUIET: gQuiet = true; break; case ARG_SANITY: sanityCheck = true; break; case 't': timing = true; break; case ARG_METRIC_IVAL: { metricsIval = parseInt(1, "--metrics arg must be at least 1", arg); break; } case ARG_METRIC_FILE: metricsFile = arg; break; case ARG_METRIC_STDERR: metricsStderr = true; break; case ARG_METRIC_PER_READ: metricsPerRead = true; break; case ARG_NO_FW: gNofw = true; break; case ARG_NO_RC: gNorc = true; break; case ARG_SAM_NO_QNAME_TRUNC: samTruncQname = false; break; case ARG_SAM_OMIT_SEC_SEQ: samOmitSecSeqQual = true; break; case ARG_SAM_NO_UNAL: samNoUnal = true; break; case ARG_SAM_NOHEAD: samNoHead = true; break; case ARG_SAM_NOSQ: samNoSQ = true; break; case ARG_SAM_PRINT_YI: sam_print_yi = true; break; case ARG_REORDER: reorder = true; break; case ARG_MAPQ_EX: { sam_print_zp = true; sam_print_zu = true; sam_print_xp = true; sam_print_xss = true; sam_print_yn = true; break; } case ARG_SHOW_RAND_SEED: { sam_print_zs = true; break; } case ARG_SAMPLE: sampleFrac = parse(arg); break; case ARG_CP_MIN: cminlen = parse(arg); break; case ARG_CP_IVAL: cpow2 = parse(arg); break; case ARG_TRI: doTri = true; break; case ARG_READ_PASSTHRU: { sam_print_xr = true; break; } case ARG_READ_TIMES: { sam_print_xt = true; sam_print_xd = true; sam_print_xu = true; sam_print_yl = true; sam_print_ye = true; sam_print_yu = true; sam_print_yr = true; sam_print_zb = true; sam_print_zr = true; sam_print_zf = true; sam_print_zm = true; sam_print_zi = true; break; } case ARG_SAM_RG: { string argstr = arg; if(argstr.substr(0, 3) == "ID:") { rgid = "\t"; rgid += argstr; rgs_optflag = "RG:Z:" + argstr.substr(3); } else { rgs += '\t'; rgs += argstr; } break; } case ARG_SAM_RGID: { string argstr = arg; rgid = "\t"; rgid = "\tID:" + argstr; rgs_optflag = "RG:Z:" + argstr; break; } case ARG_PARTITION: partitionSz = parse(arg); break; case ARG_DPAD: maxhalf = parseInt(0, "--dpad must be no less than 0", arg); break; case ARG_ORIG: if(arg == NULL || strlen(arg) == 0) { cerr << "--orig arg must be followed by a string" << endl; printUsage(cerr); throw 1; } origString = arg; break; case ARG_LOCAL: localAlign = true; break; case ARG_END_TO_END: localAlign = false; break; case ARG_SSE8: enable8 = true; break; case ARG_SSE8_NO: enable8 = false; break; case ARG_UNGAPPED: doUngapped = true; break; case ARG_UNGAPPED_NO: doUngapped = false; break; // case ARG_NO_DOVETAIL: gDovetailMatesOK = false; break; // case ARG_NO_CONTAIN: gContainMatesOK = false; break; // case ARG_NO_OVERLAP: gOlapMatesOK = false; break; // case ARG_DOVETAIL: gDovetailMatesOK = true; break; // case ARG_CONTAIN: gContainMatesOK = true; break; // case ARG_OVERLAP: gOlapMatesOK = true; break; case ARG_QC_FILTER: qcFilter = true; break; case ARG_NO_SCORE_PRIORITY: sortByScore = false; break; case ARG_IGNORE_QUALS: ignoreQuals = true; break; case ARG_MAPQ_V: mapqv = parse(arg); break; case ARG_TIGHTEN: tighten = parse(arg); break; case ARG_EXACT_UPFRONT: doExactUpFront = true; break; case ARG_1MM_UPFRONT: do1mmUpFront = true; break; case ARG_EXACT_UPFRONT_NO: doExactUpFront = false; break; case ARG_1MM_UPFRONT_NO: do1mmUpFront = false; break; case ARG_1MM_MINLEN: do1mmMinLen = parse(arg); break; case ARG_NOISY_HPOLY: noisyHpolymer = true; break; case 'x': bt2index = arg; break; case ARG_PRESET_VERY_FAST_LOCAL: localAlign = true; case ARG_PRESET_VERY_FAST: { presetList.push_back("very-fast%LOCAL%"); break; } case ARG_PRESET_FAST_LOCAL: localAlign = true; case ARG_PRESET_FAST: { fast = true; presetList.push_back("fast%LOCAL%"); break; } case ARG_PRESET_SENSITIVE_LOCAL: localAlign = true; case ARG_PRESET_SENSITIVE: { sensitive = true; presetList.push_back("sensitive%LOCAL%"); break; } case ARG_PRESET_VERY_SENSITIVE_LOCAL: localAlign = true; case ARG_PRESET_VERY_SENSITIVE: { very_sensitive = true; presetList.push_back("very-sensitive%LOCAL%"); break; } case 'P': { presetList.push_back(arg); break; } case ARG_ALIGN_POLICY: { if(strlen(arg) > 0) { polstr += ";"; polstr += arg; } break; } case 'N': { polstr += ";SEED="; polstr += arg; break; } case 'L': { int64_t len = parse(arg); if(len < 0) { cerr << "Error: -L argument must be >= 0; was " << arg << endl; throw 1; } if(len > 32) { cerr << "Error: -L argument must be <= 32; was" << arg << endl; throw 1; } polstr += ";SEEDLEN="; polstr += arg; break; } case 'O': multiseedOff = parse(arg); break; case 'i': { EList args; tokenize(arg, ",", args); if(args.size() > 3 || args.size() == 0) { cerr << "Error: expected 3 or fewer comma-separated " << "arguments to -i option, got " << args.size() << endl; throw 1; } // Interval-settings arguments polstr += (";IVAL=" + args[0]); // Function type if(args.size() > 1) { polstr += ("," + args[1]); // Constant term } if(args.size() > 2) { polstr += ("," + args[2]); // Coefficient } break; } case ARG_MULTISEED_IVAL: { polstr += ";"; // Split argument by comma EList args; tokenize(arg, ",", args); if(args.size() > 5 || args.size() == 0) { cerr << "Error: expected 5 or fewer comma-separated " << "arguments to --multiseed option, got " << args.size() << endl; throw 1; } // Seed mm and length arguments polstr += "SEED="; polstr += (args[0]); // # mismatches if(args.size() > 1) polstr += ("," + args[ 1]); // length if(args.size() > 2) polstr += (";IVAL=" + args[2]); // Func type if(args.size() > 3) polstr += ("," + args[ 3]); // Constant term if(args.size() > 4) polstr += ("," + args[ 4]); // Coefficient break; } case ARG_N_CEIL: { // Split argument by comma EList args; tokenize(arg, ",", args); if(args.size() > 3) { cerr << "Error: expected 3 or fewer comma-separated " << "arguments to --n-ceil option, got " << args.size() << endl; throw 1; } if(args.size() == 0) { cerr << "Error: expected at least one argument to --n-ceil option" << endl; throw 1; } polstr += ";NCEIL="; if(args.size() == 3) { polstr += (args[0] + "," + args[1] + "," + args[2]); } else { if(args.size() == 1) { polstr += ("C," + args[0]); } else { polstr += (args[0] + "," + args[1]); } } break; } case ARG_SCORE_MA: polstr += ";MA="; polstr += arg; break; case ARG_SCORE_MMP: { EList args; tokenize(arg, ",", args); if(args.size() > 2 || args.size() == 0) { cerr << "Error: expected 1 or 2 comma-separated " << "arguments to --mp option, got " << args.size() << endl; throw 1; } if(args.size() >= 1) { polstr += ";MMP=Q,"; polstr += args[0]; if(args.size() >= 2) { polstr += ","; polstr += args[1]; } } break; } case ARG_SCORE_SCP: { EList args; tokenize(arg, ",", args); if(args.size() > 2 || args.size() == 0) { cerr << "Error: expected 1 or 2 comma-separated " << "arguments to --sp option, got " << args.size() << endl; throw 1; } if(args.size() >= 1) { polstr += ";SCP=Q,"; polstr += args[0]; if(args.size() >= 2) { polstr += ","; polstr += args[1]; } } break; } case ARG_NO_SOFTCLIP: { ostringstream convert; convert << std::numeric_limits::max(); polstr += ";SCP=Q,"; polstr += convert.str(); polstr += ","; polstr += convert.str(); break; } case ARG_SCORE_NP: polstr += ";NP=C"; polstr += arg; break; case ARG_SCORE_RDG: polstr += ";RDG="; polstr += arg; break; case ARG_SCORE_RFG: polstr += ";RFG="; polstr += arg; break; case ARG_SCORE_MIN: { polstr += ";"; EList args; tokenize(arg, ",", args); if(args.size() > 3 && args.size() == 0) { cerr << "Error: expected 3 or fewer comma-separated " << "arguments to --n-ceil option, got " << args.size() << endl; throw 1; } polstr += ("MIN=" + args[0]); if(args.size() > 1) { polstr += ("," + args[1]); } if(args.size() > 2) { polstr += ("," + args[2]); } break; } case ARG_DESC: printArgDesc(cout); throw 0; case 'S': outfile = arg; break; case 'U': { EList args; tokenize(arg, ",", args); for(size_t i = 0; i < args.size(); i++) { queries.push_back(args[i]); } break; } case ARG_VERSION: showVersion = 1; break; case ARG_NO_TEMPSPLICESITE: useTempSpliceSite = false; break; case ARG_PEN_CANSPLICE: { penCanSplice = parseInt(0, "--pen-cansplice arg must be at least 0", arg); break; } case ARG_PEN_NONCANSPLICE: { penNoncanSplice = parseInt(0, "--pen-noncansplice arg must be at least 0", arg); break; } case ARG_PEN_CONFLICTSPLICE: { penConflictSplice = parseInt(0, "--pen-conflictsplice arg must be at least 0", arg); break; } case ARG_PEN_CANINTRONLEN: { polstr += ";"; EList args; tokenize(arg, ",", args); if(args.size() > 3 && args.size() == 0) { cerr << "Error: expected 3 or fewer comma-separated " << "arguments to --n-ceil option, got " << args.size() << endl; throw 1; } polstr += ("CANINTRONLEN=" + args[0]); if(args.size() > 1) { polstr += ("," + args[1]); } if(args.size() > 2) { polstr += ("," + args[2]); } break; } case ARG_PEN_NONCANINTRONLEN: { polstr += ";"; EList args; tokenize(arg, ",", args); if(args.size() > 3 && args.size() == 0) { cerr << "Error: expected 3 or fewer comma-separated " << "arguments to --n-ceil option, got " << args.size() << endl; throw 1; } polstr += ("NONCANINTRONLEN=" + args[0]); if(args.size() > 1) { polstr += ("," + args[1]); } if(args.size() > 2) { polstr += ("," + args[2]); } break; } case ARG_MIN_INTRONLEN: { minIntronLen = parseInt(20, "--min-intronlen arg must be at least 20", arg); break; } case ARG_MAX_INTRONLEN: { maxIntronLen = parseInt(20, "--max-intronlen arg must be at least 20", arg); break; } case ARG_KNOWN_SPLICESITE_INFILE: knownSpliceSiteInfile = arg; break; case ARG_NOVEL_SPLICESITE_INFILE: novelSpliceSiteInfile = arg; break; case ARG_NOVEL_SPLICESITE_OUTFILE: novelSpliceSiteOutfile = arg; break; case ARG_SECONDARY: secondary = true; break; case ARG_NO_SPLICED_ALIGNMENT: no_spliced_alignment = true; break; case ARG_RNA_STRANDNESS: { string strandness = arg; if(strandness == "F") rna_strandness = RNA_STRANDNESS_F; else if(strandness == "R") rna_strandness = RNA_STRANDNESS_R; else if(strandness == "FR") rna_strandness = RNA_STRANDNESS_FR; else if(strandness == "RF") rna_strandness = RNA_STRANDNESS_RF; else { cerr << "Error: should be one of F, R, FR, or RF " << endl; throw 1; } break; } case ARG_SPLICESITE_DB_ONLY: { splicesite_db_only = true; break; } case ARG_NO_ANCHORSTOP: { anchorStop = false; break; } case ARG_TRANSCRIPTOME_MAPPING_ONLY: { tranMapOnly = true; break; } case ARG_TRANSCRIPTOME_ASSEMBLY: { tranAssm = true; break; } case ARG_TRANSCRIPTOME_ASSEMBLY_CUFFLINKS: { tranAssm = true; tranAssm_program = "cufflinks"; break; } case ARG_AVOID_PSEUDOGENE: { avoid_pseudogene = true; break; } #ifdef USE_SRA case ARG_SRA_ACC: { tokenize(arg, ",", sra_accs); format = SRA_FASTA; break; } #endif case ARG_REMOVE_CHRNAME: { rmChrName = true; break; } case ARG_ADD_CHRNAME: { addChrName = true; break; } case ARG_MAX_ALTSTRIED: { max_alts_tried = parseInt(8, "--max-altstried arg must be at least 8", arg); break; } case ARG_HAPLOTYPE: { use_haplotype = true; break; } case ARG_CODIS: { enable_codis = true; break; } case ARG_NO_TEMPLATELEN_ADJUSTMENT: { templateLenAdjustment = false; break; } case ARG_SUMMARY_FILE: { alignSumFile = arg; break; } case ARG_NEW_SUMMARY: { newAlignSummary = true; break; } case ARG_DP: { bowtie2_dp = parseInt(0, "--bowtie2-dp arg must be 0, 1, or 2", arg); break; } case ARG_REPEAT: { repeat = true; break; } case ARG_NO_REPEAT_INDEX: { use_repeat_index = false; break; } case ARG_READ_LENGTHS: { EList str_readLens; tokenize(arg, ",", str_readLens); for(size_t i = 0; i < str_readLens.size(); i++) { int readLen = parseInt(0, "--read-lengths arg must be at least 0", str_readLens[i].c_str()); readLens.push_back(readLen); } readLens.sort(); break; } default: printUsage(cerr); throw 1; } } /** * Read command-line arguments */ static void parseOptions(int argc, const char **argv) { int option_index = 0; int next_option; saw_M = false; saw_a = false; saw_k = false; presetList.clear(); if(startVerbose) { cerr << "Parsing options: "; logTime(cerr, true); } while(true) { next_option = getopt_long( argc, const_cast(argv), short_options, long_options, &option_index); const char * arg = optarg; if(next_option == EOF) { if(extra_opts_cur < extra_opts.size()) { next_option = extra_opts[extra_opts_cur].first; arg = extra_opts[extra_opts_cur].second.c_str(); extra_opts_cur++; } else { break; } } parseOption(next_option, arg); } // Now parse all the presets. Might want to pick which presets version to // use according to other parameters. auto_ptr presets(new PresetsV0()); // Apply default preset if(!defaultPreset.empty()) { polstr = applyPreset(defaultPreset, *presets.get()) + polstr; } // Apply specified presets for(size_t i = 0; i < presetList.size(); i++) { polstr += applyPreset(presetList[i], *presets.get()); } for(size_t i = 0; i < extra_opts.size(); i++) { next_option = extra_opts[extra_opts_cur].first; const char *arg = extra_opts[extra_opts_cur].second.c_str(); parseOption(next_option, arg); } // Remove initial semicolons while(!polstr.empty() && polstr[0] == ';') { polstr = polstr.substr(1); } if(gVerbose) { cerr << "Final policy string: '" << polstr.c_str() << "'" << endl; } size_t failStreakTmp = 0; SeedAlignmentPolicy::parseString( polstr, localAlign, noisyHpolymer, ignoreQuals, bonusMatchType, bonusMatch, penMmcType, penMmcMax, penMmcMin, penScMax, penScMin, penNType, penN, penRdGapConst, penRfGapConst, penRdGapLinear, penRfGapLinear, scoreMin, nCeil, penNCatPair, multiseedMms, multiseedLen, msIval, failStreakTmp, nSeedRounds, &penCanIntronLen, &penNoncanIntronLen); if(failStreakTmp > 0) { maxEeStreak = failStreakTmp; maxUgStreak = failStreakTmp; maxDpStreak = failStreakTmp; } if(saw_a || saw_k || true) { msample = false; mhits = 0; } else { assert_gt(mhits, 0); msample = true; } if(fast) { use_repeat_index = false; } else if(sensitive) { if(bowtie2_dp == 0) { bowtie2_dp = 1; } if(khits < 10) { khits = 10; saw_k = true; } scoreMin.init(SIMPLE_FUNC_LINEAR, 0.0f, -0.5f); } else if(very_sensitive) { bowtie2_dp = 2; if(khits < 30) { khits = 30; saw_k = true; } scoreMin.init(SIMPLE_FUNC_LINEAR, 0.0f, -1.0f); } if(mates1.size() != mates2.size()) { cerr << "Error: " << mates1.size() << " mate files/sequences were specified with -1, but " << mates2.size() << endl << "mate files/sequences were specified with -2. The same number of mate files/" << endl << "sequences must be specified with -1 and -2." << endl; throw 1; } if(qualities.size() && format != FASTA) { cerr << "Error: one or more quality files were specified with -Q but -f was not" << endl << "enabled. -Q works only in combination with -f and -C." << endl; throw 1; } if(qualities1.size() && format != FASTA) { cerr << "Error: one or more quality files were specified with --Q1 but -f was not" << endl << "enabled. --Q1 works only in combination with -f and -C." << endl; throw 1; } if(qualities2.size() && format != FASTA) { cerr << "Error: one or more quality files were specified with --Q2 but -f was not" << endl << "enabled. --Q2 works only in combination with -f and -C." << endl; throw 1; } if(qualities1.size() > 0 && mates1.size() != qualities1.size()) { cerr << "Error: " << mates1.size() << " mate files/sequences were specified with -1, but " << qualities1.size() << endl << "quality files were specified with --Q1. The same number of mate and quality" << endl << "files must sequences must be specified with -1 and --Q1." << endl; throw 1; } if(qualities2.size() > 0 && mates2.size() != qualities2.size()) { cerr << "Error: " << mates2.size() << " mate files/sequences were specified with -2, but " << qualities2.size() << endl << "quality files were specified with --Q2. The same number of mate and quality" << endl << "files must sequences must be specified with -2 and --Q2." << endl; throw 1; } if(!rgs.empty() && rgid.empty()) { cerr << "Warning: --rg was specified without --rg-id also " << "being specified. @RG line is not printed unless --rg-id " << "is specified." << endl; } // Check for duplicate mate input files if(format != CMDLINE) { for(size_t i = 0; i < mates1.size(); i++) { for(size_t j = 0; j < mates2.size(); j++) { if(mates1[i] == mates2[j] && !gQuiet) { cerr << "Warning: Same mate file \"" << mates1[i].c_str() << "\" appears as argument to both -1 and -2" << endl; } } } } // If both -s and -u are used, we need to adjust qUpto accordingly // since it uses rdid to know if we've reached the -u limit (and // rdids are all shifted up by skipReads characters) if(qUpto + skipReads > qUpto) { qUpto += skipReads; } if(useShmem && useMm && !gQuiet) { cerr << "Warning: --shmem overrides --mm..." << endl; useMm = false; } if(gGapBarrier < 1) { cerr << "Warning: --gbar was set less than 1 (=" << gGapBarrier << "); setting to 1 instead" << endl; gGapBarrier = 1; } if(multiseedMms >= multiseedLen) { assert_gt(multiseedLen, 0); cerr << "Warning: seed mismatches (" << multiseedMms << ") is less than seed length (" << multiseedLen << "); setting mismatches to " << (multiseedMms-1) << " instead" << endl; multiseedMms = multiseedLen-1; } sam_print_zm = sam_print_zm && bowtie2p5; #ifndef NDEBUG if(!gQuiet) { cerr << "Warning: Running in debug mode. Please use debug mode only " << "for diagnosing errors, and not for typical use of HISAT2." << endl; } #endif } static const char *argv0 = NULL; /// Create a PatternSourcePerThread for the current thread according /// to the global params and return a pointer to it static PatternSourcePerThreadFactory* createPatsrcFactory(PairedPatternSource& _patsrc, int tid) { PatternSourcePerThreadFactory *patsrcFact; patsrcFact = new WrappedPatternSourcePerThreadFactory(_patsrc); assert(patsrcFact != NULL); return patsrcFact; } #define PTHREAD_ATTRS (PTHREAD_CREATE_JOINABLE | PTHREAD_CREATE_DETACHED) typedef TIndexOffU index_t; typedef uint16_t local_index_t; static PairedPatternSource* multiseed_patsrc; static HGFM* multiseed_gfm; static RFM* multiseed_rgfm; static Scoring* multiseed_sc; static BitPairReference* multiseed_refs; static BitPairReference* multiseed_rrefs; static AlnSink* multiseed_msink; static OutFileBuf* multiseed_metricsOfb; static SpliceSiteDB* ssdb; static ALTDB* altdb; static RepeatDB* repeatdb; static ALTDB* raltdb; static TranscriptomePolicy* multiseed_tpol; static GraphPolicy* gpol; /** * Metrics for measuring the work done by the outer read alignment * loop. */ struct OuterLoopMetrics { OuterLoopMetrics() { reset(); } /** * Set all counters to 0. */ void reset() { reads = bases = srreads = srbases = freads = fbases = ureads = ubases = 0; } /** * Sum the counters in m in with the conters in this object. This * is the only safe way to update an OuterLoopMetrics that's shared * by multiple threads. */ void merge( const OuterLoopMetrics& m, bool getLock = false) { ThreadSafe ts(&mutex_m, getLock); reads += m.reads; bases += m.bases; srreads += m.srreads; srbases += m.srbases; freads += m.freads; fbases += m.fbases; ureads += m.ureads; ubases += m.ubases; } uint64_t reads; // total reads uint64_t bases; // total bases uint64_t srreads; // same-read reads uint64_t srbases; // same-read bases uint64_t freads; // filtered reads uint64_t fbases; // filtered bases uint64_t ureads; // unfiltered reads uint64_t ubases; // unfiltered bases MUTEX_T mutex_m; }; /** * Collection of all relevant performance metrics when aligning in * multiseed mode. */ struct PerfMetrics { PerfMetrics() : first(true) { reset(); } /** * Set all counters to 0. */ void reset() { olm.reset(); sdm.reset(); wlm.reset(); swmSeed.reset(); swmMate.reset(); rpm.reset(); dpSse8Seed.reset(); // 8-bit SSE seed extensions dpSse8Mate.reset(); // 8-bit SSE mate finds dpSse16Seed.reset(); // 16-bit SSE seed extensions dpSse16Mate.reset(); // 16-bit SSE mate finds nbtfiltst = 0; nbtfiltsc = 0; nbtfiltdo = 0; olmu.reset(); sdmu.reset(); wlmu.reset(); swmuSeed.reset(); swmuMate.reset(); rpmu.reset(); dpSse8uSeed.reset(); // 8-bit SSE seed extensions dpSse8uMate.reset(); // 8-bit SSE mate finds dpSse16uSeed.reset(); // 16-bit SSE seed extensions dpSse16uMate.reset(); // 16-bit SSE mate finds nbtfiltst_u = 0; nbtfiltsc_u = 0; nbtfiltdo_u = 0; him.reset(); } /** * Merge a set of specific metrics into this object. */ void merge( const OuterLoopMetrics *ol, const SeedSearchMetrics *sd, const WalkMetrics *wl, const SwMetrics *swSeed, const SwMetrics *swMate, const ReportingMetrics *rm, const SSEMetrics *dpSse8Ex, const SSEMetrics *dpSse8Ma, const SSEMetrics *dpSse16Ex, const SSEMetrics *dpSse16Ma, uint64_t nbtfiltst_, uint64_t nbtfiltsc_, uint64_t nbtfiltdo_, const HIMetrics *hi, bool getLock) { ThreadSafe ts(&mutex_m, getLock); if(ol != NULL) { olmu.merge(*ol, false); } if(sd != NULL) { sdmu.merge(*sd, false); } if(wl != NULL) { wlmu.merge(*wl, false); } if(swSeed != NULL) { swmuSeed.merge(*swSeed, false); } if(swMate != NULL) { swmuMate.merge(*swMate, false); } if(rm != NULL) { rpmu.merge(*rm, false); } if(dpSse8Ex != NULL) { dpSse8uSeed.merge(*dpSse8Ex, false); } if(dpSse8Ma != NULL) { dpSse8uMate.merge(*dpSse8Ma, false); } if(dpSse16Ex != NULL) { dpSse16uSeed.merge(*dpSse16Ex, false); } if(dpSse16Ma != NULL) { dpSse16uMate.merge(*dpSse16Ma, false); } nbtfiltst_u += nbtfiltst_; nbtfiltsc_u += nbtfiltsc_; nbtfiltdo_u += nbtfiltdo_; if(hi != NULL) { him.merge(*hi, false); } } /** * Reports a matrix of results, incl. column labels, to an OutFileBuf. * Optionally also sends results to stderr (unbuffered). Can optionally * print a per-read record with the read name at the beginning. */ void reportInterval( OutFileBuf* o, // file to send output to bool metricsStderr, // additionally output to stderr? bool total, // true -> report total, otherwise incremental bool sync, // synchronize output const BTString *name) // non-NULL name pointer if is per-read record { ThreadSafe ts(&mutex_m, sync); ostringstream stderrSs; time_t curtime = time(0); char buf[1024]; if(first) { const char *str = /* 1 */ "Time" "\t" /* 2 */ "Read" "\t" /* 3 */ "Base" "\t" /* 4 */ "SameRead" "\t" /* 5 */ "SameReadBase" "\t" /* 6 */ "UnfilteredRead" "\t" /* 7 */ "UnfilteredBase" "\t" /* 8 */ "Paired" "\t" /* 9 */ "Unpaired" "\t" /* 10 */ "AlConUni" "\t" /* 11 */ "AlConRep" "\t" /* 12 */ "AlConFail" "\t" /* 13 */ "AlDis" "\t" /* 14 */ "AlConFailUni" "\t" /* 15 */ "AlConFailRep" "\t" /* 16 */ "AlConFailFail" "\t" /* 17 */ "AlConRepUni" "\t" /* 18 */ "AlConRepRep" "\t" /* 19 */ "AlConRepFail" "\t" /* 20 */ "AlUnpUni" "\t" /* 21 */ "AlUnpRep" "\t" /* 22 */ "AlUnpFail" "\t" /* 23 */ "SeedSearch" "\t" /* 24 */ "IntraSCacheHit" "\t" /* 25 */ "InterSCacheHit" "\t" /* 26 */ "OutOfMemory" "\t" /* 27 */ "AlBWOp" "\t" /* 28 */ "AlBWBranch" "\t" /* 29 */ "ResBWOp" "\t" /* 30 */ "ResBWBranch" "\t" /* 31 */ "ResResolve" "\t" /* 34 */ "ResReport" "\t" /* 35 */ "RedundantSHit" "\t" /* 36 */ "BestMinEdit0" "\t" /* 37 */ "BestMinEdit1" "\t" /* 38 */ "BestMinEdit2" "\t" /* 39 */ "ExactAttempts" "\t" /* 40 */ "ExactSucc" "\t" /* 41 */ "ExactRanges" "\t" /* 42 */ "ExactRows" "\t" /* 43 */ "ExactOOMs" "\t" /* 44 */ "1mmAttempts" "\t" /* 45 */ "1mmSucc" "\t" /* 46 */ "1mmRanges" "\t" /* 47 */ "1mmRows" "\t" /* 48 */ "1mmOOMs" "\t" /* 49 */ "UngappedSucc" "\t" /* 50 */ "UngappedFail" "\t" /* 51 */ "UngappedNoDec" "\t" /* 52 */ "DPExLt10Gaps" "\t" /* 53 */ "DPExLt5Gaps" "\t" /* 54 */ "DPExLt3Gaps" "\t" /* 55 */ "DPMateLt10Gaps" "\t" /* 56 */ "DPMateLt5Gaps" "\t" /* 57 */ "DPMateLt3Gaps" "\t" /* 58 */ "DP16ExDps" "\t" /* 59 */ "DP16ExDpSat" "\t" /* 60 */ "DP16ExDpFail" "\t" /* 61 */ "DP16ExDpSucc" "\t" /* 62 */ "DP16ExCol" "\t" /* 63 */ "DP16ExCell" "\t" /* 64 */ "DP16ExInner" "\t" /* 65 */ "DP16ExFixup" "\t" /* 66 */ "DP16ExGathSol" "\t" /* 67 */ "DP16ExBt" "\t" /* 68 */ "DP16ExBtFail" "\t" /* 69 */ "DP16ExBtSucc" "\t" /* 70 */ "DP16ExBtCell" "\t" /* 71 */ "DP16ExCoreRej" "\t" /* 72 */ "DP16ExNRej" "\t" /* 73 */ "DP8ExDps" "\t" /* 74 */ "DP8ExDpSat" "\t" /* 75 */ "DP8ExDpFail" "\t" /* 76 */ "DP8ExDpSucc" "\t" /* 77 */ "DP8ExCol" "\t" /* 78 */ "DP8ExCell" "\t" /* 79 */ "DP8ExInner" "\t" /* 80 */ "DP8ExFixup" "\t" /* 81 */ "DP8ExGathSol" "\t" /* 82 */ "DP8ExBt" "\t" /* 83 */ "DP8ExBtFail" "\t" /* 84 */ "DP8ExBtSucc" "\t" /* 85 */ "DP8ExBtCell" "\t" /* 86 */ "DP8ExCoreRej" "\t" /* 87 */ "DP8ExNRej" "\t" /* 88 */ "DP16MateDps" "\t" /* 89 */ "DP16MateDpSat" "\t" /* 90 */ "DP16MateDpFail" "\t" /* 91 */ "DP16MateDpSucc" "\t" /* 92 */ "DP16MateCol" "\t" /* 93 */ "DP16MateCell" "\t" /* 94 */ "DP16MateInner" "\t" /* 95 */ "DP16MateFixup" "\t" /* 96 */ "DP16MateGathSol" "\t" /* 97 */ "DP16MateBt" "\t" /* 98 */ "DP16MateBtFail" "\t" /* 99 */ "DP16MateBtSucc" "\t" /* 100 */ "DP16MateBtCell" "\t" /* 101 */ "DP16MateCoreRej" "\t" /* 102 */ "DP16MateNRej" "\t" /* 103 */ "DP8MateDps" "\t" /* 104 */ "DP8MateDpSat" "\t" /* 105 */ "DP8MateDpFail" "\t" /* 106 */ "DP8MateDpSucc" "\t" /* 107 */ "DP8MateCol" "\t" /* 108 */ "DP8MateCell" "\t" /* 109 */ "DP8MateInner" "\t" /* 110 */ "DP8MateFixup" "\t" /* 111 */ "DP8MateGathSol" "\t" /* 112 */ "DP8MateBt" "\t" /* 113 */ "DP8MateBtFail" "\t" /* 114 */ "DP8MateBtSucc" "\t" /* 115 */ "DP8MateBtCell" "\t" /* 116 */ "DP8MateCoreRej" "\t" /* 117 */ "DP8MateNRej" "\t" /* 118 */ "DPBtFiltStart" "\t" /* 119 */ "DPBtFiltScore" "\t" /* 120 */ "DpBtFiltDom" "\t" /* 121 */ "MemPeak" "\t" /* 122 */ "UncatMemPeak" "\t" // 0 /* 123 */ "EbwtMemPeak" "\t" // EBWT_CAT /* 124 */ "CacheMemPeak" "\t" // CA_CAT /* 125 */ "ResolveMemPeak" "\t" // GW_CAT /* 126 */ "AlignMemPeak" "\t" // AL_CAT /* 127 */ "DPMemPeak" "\t" // DP_CAT /* 128 */ "MiscMemPeak" "\t" // MISC_CAT /* 129 */ "DebugMemPeak" "\t" // DEBUG_CAT /* 130 */ "LocalSearch" "\t" /* 131 */ "AnchorSearch" "\t" /* 132 */ "LocalIndexSearch" "\t" /* 133 */ "LocalExtSearch" "\t" /* 134 */ "LocalSearchRecur" "\t" /* 135 */ "GlobalGenomeCoords" "\t" /* 136 */ "LocalGenomeCoords" "\t" "\n"; if(name != NULL) { if(o != NULL) o->writeChars("Name\t"); if(metricsStderr) stderrSs << "Name\t"; } if(o != NULL) o->writeChars(str); if(metricsStderr) stderrSs << str; first = false; } if(total) mergeIncrementals(); // 0. Read name, if needed if(name != NULL) { if(o != NULL) { o->writeChars(name->toZBuf()); o->write('\t'); } if(metricsStderr) { stderrSs << (*name) << '\t'; } } // 1. Current time in secs itoa10(curtime, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const OuterLoopMetrics& ol = total ? olm : olmu; // 2. Reads itoa10(ol.reads, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 3. Bases itoa10(ol.bases, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 4. Same-read reads itoa10(ol.srreads, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 5. Same-read bases itoa10(ol.srbases, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 6. Unfiltered reads itoa10(ol.ureads, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 7. Unfiltered bases itoa10(ol.ubases, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const ReportingMetrics& rp = total ? rpm : rpmu; // 8. Paired reads itoa10(rp.npaired, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 9. Unpaired reads itoa10(rp.nunpaired, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 10. Pairs with unique concordant alignments itoa10(rp.nconcord_uni, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 11. Pairs with repetitive concordant alignments itoa10(rp.nconcord_rep, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 12. Pairs with 0 concordant alignments itoa10(rp.nconcord_0, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 13. Pairs with 1 discordant alignment itoa10(rp.ndiscord, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 14. Mates from unaligned pairs that align uniquely itoa10(rp.nunp_0_uni, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 15. Mates from unaligned pairs that align repetitively itoa10(rp.nunp_0_rep, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 16. Mates from unaligned pairs that fail to align itoa10(rp.nunp_0_0, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 17. Mates from repetitive pairs that align uniquely itoa10(rp.nunp_rep_uni, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 18. Mates from repetitive pairs that align repetitively itoa10(rp.nunp_rep_rep, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 19. Mates from repetitive pairs that fail to align itoa10(rp.nunp_rep_0, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 20. Unpaired reads that align uniquely itoa10(rp.nunp_uni, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 21. Unpaired reads that align repetitively itoa10(rp.nunp_rep, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 22. Unpaired reads that fail to align itoa10(rp.nunp_0, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const SeedSearchMetrics& sd = total ? sdm : sdmu; // 23. Seed searches itoa10(sd.seedsearch, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 24. Hits in 'current' cache itoa10(sd.intrahit, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 25. Hits in 'local' cache itoa10(sd.interhit, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 26. Out of memory itoa10(sd.ooms, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 27. Burrows-Wheeler ops in aligner itoa10(sd.bwops, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 28. Burrows-Wheeler branches (edits) in aligner itoa10(sd.bweds, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const WalkMetrics& wl = total ? wlm : wlmu; // 29. Burrows-Wheeler ops in resolver itoa10(wl.bwops, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 30. Burrows-Wheeler branches in resolver itoa10(wl.branches, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 31. Burrows-Wheeler offset resolutions itoa10(wl.resolves, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 34. Offset reports itoa10(wl.reports, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 35. Redundant seed hit itoa10(total ? swmSeed.rshit : swmuSeed.rshit, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 36. # times the best (out of fw/rc) minimum # edits was 0 itoa10(total ? sdm.bestmin0 : sdmu.bestmin0, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 37. # times the best (out of fw/rc) minimum # edits was 1 itoa10(total ? sdm.bestmin1 : sdmu.bestmin1, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 38. # times the best (out of fw/rc) minimum # edits was 2 itoa10(total ? sdm.bestmin2 : sdmu.bestmin2, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 39. Exact aligner attempts itoa10(total ? swmSeed.exatts : swmuSeed.exatts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 40. Exact aligner successes itoa10(total ? swmSeed.exsucc : swmuSeed.exsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 41. Exact aligner ranges itoa10(total ? swmSeed.exranges : swmuSeed.exranges, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 42. Exact aligner rows itoa10(total ? swmSeed.exrows : swmuSeed.exrows, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 43. Exact aligner OOMs itoa10(total ? swmSeed.exooms : swmuSeed.exooms, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 44. 1mm aligner attempts itoa10(total ? swmSeed.mm1atts : swmuSeed.mm1atts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 45. 1mm aligner successes itoa10(total ? swmSeed.mm1succ : swmuSeed.mm1succ, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 46. 1mm aligner ranges itoa10(total ? swmSeed.mm1ranges : swmuSeed.mm1ranges, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 47. 1mm aligner rows itoa10(total ? swmSeed.mm1rows : swmuSeed.mm1rows, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 48. 1mm aligner OOMs itoa10(total ? swmSeed.mm1ooms : swmuSeed.mm1ooms, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 49 Ungapped aligner success itoa10(total ? swmSeed.ungapsucc : swmuSeed.ungapsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 50. Ungapped aligner fail itoa10(total ? swmSeed.ungapfail : swmuSeed.ungapfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 51. Ungapped aligner no decision itoa10(total ? swmSeed.ungapnodec : swmuSeed.ungapnodec, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 52. # seed-extend DPs with < 10 gaps itoa10(total ? swmSeed.sws10 : swmuSeed.sws10, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 53. # seed-extend DPs with < 5 gaps itoa10(total ? swmSeed.sws5 : swmuSeed.sws5, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 54. # seed-extend DPs with < 3 gaps itoa10(total ? swmSeed.sws3 : swmuSeed.sws3, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 55. # seed-extend DPs with < 10 gaps itoa10(total ? swmMate.sws10 : swmuMate.sws10, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 56. # seed-extend DPs with < 5 gaps itoa10(total ? swmMate.sws5 : swmuMate.sws5, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 57. # seed-extend DPs with < 3 gaps itoa10(total ? swmMate.sws3 : swmuMate.sws3, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const SSEMetrics& dpSse16s = total ? dpSse16Seed : dpSse16uSeed; // 58. 16-bit SSE seed-extend DPs tried itoa10(dpSse16s.dp, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 59. 16-bit SSE seed-extend DPs saturated itoa10(dpSse16s.dpsat, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 60. 16-bit SSE seed-extend DPs failed itoa10(dpSse16s.dpfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 61. 16-bit SSE seed-extend DPs succeeded itoa10(dpSse16s.dpsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 62. 16-bit SSE seed-extend DP columns completed itoa10(dpSse16s.col, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 63. 16-bit SSE seed-extend DP cells completed itoa10(dpSse16s.cell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 64. 16-bit SSE seed-extend DP inner loop iters completed itoa10(dpSse16s.inner, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 65. 16-bit SSE seed-extend DP fixup loop iters completed itoa10(dpSse16s.fixup, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 66. 16-bit SSE seed-extend DP gather, cells with potential solutions itoa10(dpSse16s.gathsol, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 67. 16-bit SSE seed-extend DP backtrace attempts itoa10(dpSse16s.bt, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 68. 16-bit SSE seed-extend DP failed backtrace attempts itoa10(dpSse16s.btfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 69. 16-bit SSE seed-extend DP succesful backtrace attempts itoa10(dpSse16s.btsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 70. 16-bit SSE seed-extend DP backtrace cells itoa10(dpSse16s.btcell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 71. 16-bit SSE seed-extend DP core-diag rejections itoa10(dpSse16s.corerej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 72. 16-bit SSE seed-extend DP N rejections itoa10(dpSse16s.nrej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const SSEMetrics& dpSse8s = total ? dpSse8Seed : dpSse8uSeed; // 73. 8-bit SSE seed-extend DPs tried itoa10(dpSse8s.dp, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 74. 8-bit SSE seed-extend DPs saturated itoa10(dpSse8s.dpsat, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 75. 8-bit SSE seed-extend DPs failed itoa10(dpSse8s.dpfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 76. 8-bit SSE seed-extend DPs succeeded itoa10(dpSse8s.dpsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 77. 8-bit SSE seed-extend DP columns completed itoa10(dpSse8s.col, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 78. 8-bit SSE seed-extend DP cells completed itoa10(dpSse8s.cell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 79. 8-bit SSE seed-extend DP inner loop iters completed itoa10(dpSse8s.inner, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 80. 8-bit SSE seed-extend DP fixup loop iters completed itoa10(dpSse8s.fixup, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 81. 16-bit SSE seed-extend DP gather, cells with potential solutions itoa10(dpSse8s.gathsol, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 82. 16-bit SSE seed-extend DP backtrace attempts itoa10(dpSse8s.bt, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 83. 16-bit SSE seed-extend DP failed backtrace attempts itoa10(dpSse8s.btfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 84. 16-bit SSE seed-extend DP succesful backtrace attempts itoa10(dpSse8s.btsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 85. 16-bit SSE seed-extend DP backtrace cells itoa10(dpSse8s.btcell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 86. 16-bit SSE seed-extend DP core-diag rejections itoa10(dpSse8s.corerej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 87. 16-bit SSE seed-extend DP N rejections itoa10(dpSse8s.nrej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const SSEMetrics& dpSse16m = total ? dpSse16Mate : dpSse16uMate; // 88. 16-bit SSE mate-finding DPs tried itoa10(dpSse16m.dp, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 89. 16-bit SSE mate-finding DPs saturated itoa10(dpSse16m.dpsat, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 90. 16-bit SSE mate-finding DPs failed itoa10(dpSse16m.dpfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 91. 16-bit SSE mate-finding DPs succeeded itoa10(dpSse16m.dpsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 92. 16-bit SSE mate-finding DP columns completed itoa10(dpSse16m.col, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 93. 16-bit SSE mate-finding DP cells completed itoa10(dpSse16m.cell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 94. 16-bit SSE mate-finding DP inner loop iters completed itoa10(dpSse16m.inner, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 95. 16-bit SSE mate-finding DP fixup loop iters completed itoa10(dpSse16m.fixup, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 96. 16-bit SSE mate-finding DP gather, cells with potential solutions itoa10(dpSse16m.gathsol, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 97. 16-bit SSE mate-finding DP backtrace attempts itoa10(dpSse16m.bt, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 98. 16-bit SSE mate-finding DP failed backtrace attempts itoa10(dpSse16m.btfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 99. 16-bit SSE mate-finding DP succesful backtrace attempts itoa10(dpSse16m.btsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 100. 16-bit SSE mate-finding DP backtrace cells itoa10(dpSse16m.btcell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 101. 16-bit SSE mate-finding DP core-diag rejections itoa10(dpSse16m.corerej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 102. 16-bit SSE mate-finding DP N rejections itoa10(dpSse16m.nrej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const SSEMetrics& dpSse8m = total ? dpSse8Mate : dpSse8uMate; // 103. 8-bit SSE mate-finding DPs tried itoa10(dpSse8m.dp, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 104. 8-bit SSE mate-finding DPs saturated itoa10(dpSse8m.dpsat, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 105. 8-bit SSE mate-finding DPs failed itoa10(dpSse8m.dpfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 106. 8-bit SSE mate-finding DPs succeeded itoa10(dpSse8m.dpsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 107. 8-bit SSE mate-finding DP columns completed itoa10(dpSse8m.col, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 108. 8-bit SSE mate-finding DP cells completed itoa10(dpSse8m.cell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 109. 8-bit SSE mate-finding DP inner loop iters completed itoa10(dpSse8m.inner, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 110. 8-bit SSE mate-finding DP fixup loop iters completed itoa10(dpSse8m.fixup, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 111. 16-bit SSE mate-finding DP gather, cells with potential solutions itoa10(dpSse8m.gathsol, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 112. 16-bit SSE mate-finding DP backtrace attempts itoa10(dpSse8m.bt, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 113. 16-bit SSE mate-finding DP failed backtrace attempts itoa10(dpSse8m.btfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 114. 16-bit SSE mate-finding DP succesful backtrace attempts itoa10(dpSse8m.btsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 115. 16-bit SSE mate-finding DP backtrace cells itoa10(dpSse8m.btcell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 116. 16-bit SSE mate-finding DP core rejections itoa10(dpSse8m.corerej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 117. 16-bit SSE mate-finding N rejections itoa10(dpSse8m.nrej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 118. Backtrace candidates filtered due to starting cell itoa10(total ? nbtfiltst : nbtfiltst_u, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 119. Backtrace candidates filtered due to low score itoa10(total ? nbtfiltsc : nbtfiltsc_u, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 120. Backtrace candidates filtered due to domination itoa10(total ? nbtfiltdo : nbtfiltdo_u, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 121. Overall memory peak itoa10(gMemTally.peak() >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 122. Uncategorized memory peak itoa10(gMemTally.peak(0) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 123. Ebwt memory peak itoa10(gMemTally.peak(EBWT_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 124. Cache memory peak itoa10(gMemTally.peak(CA_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 125. Resolver memory peak itoa10(gMemTally.peak(GW_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 126. Seed aligner memory peak itoa10(gMemTally.peak(AL_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 127. Dynamic programming aligner memory peak itoa10(gMemTally.peak(DP_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 128. Miscellaneous memory peak itoa10(gMemTally.peak(MISC_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 129. Debug memory peak itoa10(gMemTally.peak(DEBUG_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 130 itoa10(him.localatts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 131 itoa10(him.anchoratts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 132 itoa10(him.localindexatts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 133 itoa10(him.localextatts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 134 itoa10(him.localsearchrecur, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 135 itoa10(him.globalgenomecoords, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 136 itoa10(him.localgenomecoords, buf); if(metricsStderr) stderrSs << buf; if(o != NULL) { o->writeChars(buf); } if(o != NULL) { o->write('\n'); } if(metricsStderr) cerr << stderrSs.str().c_str() << endl; if(!total) mergeIncrementals(); } void mergeIncrementals() { olm.merge(olmu, false); sdm.merge(sdmu, false); wlm.merge(wlmu, false); swmSeed.merge(swmuSeed, false); swmMate.merge(swmuMate, false); dpSse8Seed.merge(dpSse8uSeed, false); dpSse8Mate.merge(dpSse8uMate, false); dpSse16Seed.merge(dpSse16uSeed, false); dpSse16Mate.merge(dpSse16uMate, false); nbtfiltst_u += nbtfiltst; nbtfiltsc_u += nbtfiltsc; nbtfiltdo_u += nbtfiltdo; olmu.reset(); sdmu.reset(); wlmu.reset(); swmuSeed.reset(); swmuMate.reset(); rpmu.reset(); dpSse8uSeed.reset(); dpSse8uMate.reset(); dpSse16uSeed.reset(); dpSse16uMate.reset(); nbtfiltst_u = 0; nbtfiltsc_u = 0; nbtfiltdo_u = 0; } // Total over the whole job OuterLoopMetrics olm; // overall metrics SeedSearchMetrics sdm; // metrics related to seed alignment WalkMetrics wlm; // metrics related to walking left (i.e. resolving reference offsets) SwMetrics swmSeed; // metrics related to DP seed-extend alignment SwMetrics swmMate; // metrics related to DP mate-finding alignment ReportingMetrics rpm; // metrics related to reporting SSEMetrics dpSse8Seed; // 8-bit SSE seed extensions SSEMetrics dpSse8Mate; // 8-bit SSE mate finds SSEMetrics dpSse16Seed; // 16-bit SSE seed extensions SSEMetrics dpSse16Mate; // 16-bit SSE mate finds uint64_t nbtfiltst; uint64_t nbtfiltsc; uint64_t nbtfiltdo; // Just since the last update OuterLoopMetrics olmu; // overall metrics SeedSearchMetrics sdmu; // metrics related to seed alignment WalkMetrics wlmu; // metrics related to walking left (i.e. resolving reference offsets) SwMetrics swmuSeed; // metrics related to DP seed-extend alignment SwMetrics swmuMate; // metrics related to DP mate-finding alignment ReportingMetrics rpmu; // metrics related to reporting SSEMetrics dpSse8uSeed; // 8-bit SSE seed extensions SSEMetrics dpSse8uMate; // 8-bit SSE mate finds SSEMetrics dpSse16uSeed; // 16-bit SSE seed extensions SSEMetrics dpSse16uMate; // 16-bit SSE mate finds uint64_t nbtfiltst_u; uint64_t nbtfiltsc_u; uint64_t nbtfiltdo_u; // HIMetrics him; MUTEX_T mutex_m; // lock for when one ob bool first; // yet to print first line? time_t lastElapsed; // used in reportInterval to measure time since last call }; static PerfMetrics metrics; // Cyclic rotations #define ROTL(n, x) (((x) << (n)) | ((x) >> (32-n))) #define ROTR(n, x) (((x) >> (n)) | ((x) << (32-n))) static inline void printMmsSkipMsg( const PatternSourcePerThread& ps, bool paired, bool mate1, int seedmms) { ostringstream os; if(paired) { os << "Warning: skipping mate #" << (mate1 ? '1' : '2') << " of read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "' because length (" << (mate1 ? ps.bufa().patFw.length() : ps.bufb().patFw.length()) << ") <= # seed mismatches (" << seedmms << ")" << endl; } else { os << "Warning: skipping read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "' because length (" << (mate1 ? ps.bufa().patFw.length() : ps.bufb().patFw.length()) << ") <= # seed mismatches (" << seedmms << ")" << endl; } cerr << os.str().c_str(); } static inline void printLenSkipMsg( const PatternSourcePerThread& ps, bool paired, bool mate1) { ostringstream os; if(paired) { os << "Warning: skipping mate #" << (mate1 ? '1' : '2') << " of read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "' because it was < 2 characters long" << endl; } else { os << "Warning: skipping read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "' because it was < 2 characters long" << endl; } cerr << os.str().c_str(); } static inline void printLocalScoreMsg( const PatternSourcePerThread& ps, bool paired, bool mate1) { ostringstream os; if(paired) { os << "Warning: minimum score function gave negative number in " << "--local mode for mate #" << (mate1 ? '1' : '2') << " of read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "; setting to 0 instead" << endl; } else { os << "Warning: minimum score function gave negative number in " << "--local mode for read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "; setting to 0 instead" << endl; } cerr << os.str().c_str(); } static inline void printEEScoreMsg( const PatternSourcePerThread& ps, bool paired, bool mate1) { ostringstream os; if(paired) { os << "Warning: minimum score function gave positive number in " << "--end-to-end mode for mate #" << (mate1 ? '1' : '2') << " of read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "; setting to 0 instead" << endl; } else { os << "Warning: minimum score function gave positive number in " << "--end-to-end mode for read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "; setting to 0 instead" << endl; } cerr << os.str().c_str(); } #define MERGE_METRICS(met, sync) { \ msink.mergeMetrics(rpm); \ met.merge( \ &olm, \ &sdm, \ &wlm, \ &swmSeed, \ &swmMate, \ &rpm, \ &sseU8ExtendMet, \ &sseU8MateMet, \ &sseI16ExtendMet, \ &sseI16MateMet, \ nbtfiltst, \ nbtfiltsc, \ nbtfiltdo, \ &him, \ sync); \ olm.reset(); \ sdm.reset(); \ wlm.reset(); \ swmSeed.reset(); \ swmMate.reset(); \ rpm.reset(); \ sseU8ExtendMet.reset(); \ sseU8MateMet.reset(); \ sseI16ExtendMet.reset(); \ sseI16MateMet.reset(); \ him.reset(); \ } #define MERGE_SW(x) { \ x.merge( \ sseU8ExtendMet, \ sseU8MateMet, \ sseI16ExtendMet, \ sseI16MateMet, \ nbtfiltst, \ nbtfiltsc, \ nbtfiltdo); \ x.resetCounters(); \ } /** * Called once per thread. Sets up per-thread pointers to the shared global * data structures, creates per-thread structures, then enters the alignment * loop. The general flow of the alignment loop is: * * - If it's been a while and we're the master thread, report some alignment * metrics * - Get the next read/pair * - Check if this read/pair is identical to the previous * + If identical, check whether we can skip any or all alignment stages. If * we can skip all stages, report the result immediately and move to next * read/pair * + If not identical, continue * - */ static void multiseedSearchWorker_hisat2(void *vp) { int tid = *((int*)vp); assert(multiseed_gfm != NULL); assert(multiseedMms == 0); PairedPatternSource& patsrc = *multiseed_patsrc; const HGFM& gfm = *multiseed_gfm; const RFM* rgfm = multiseed_rgfm; const Scoring& sc = *multiseed_sc; const BitPairReference& ref = *multiseed_refs; const BitPairReference* rref = multiseed_rrefs; AlnSink& msink = *multiseed_msink; OutFileBuf* metricsOfb = multiseed_metricsOfb; // Sinks: these are so that we can print tables encoding counts for // events of interest on a per-read, per-seed, per-join, or per-SW // level. These in turn can be used to diagnose performance // problems, or generally characterize performance. //const BitPairReference& refs = *multiseed_refs; auto_ptr patsrcFact(createPatsrcFactory(patsrc, tid)); auto_ptr ps(patsrcFact->create()); // Instantiate an object for holding reporting-related parameters. if(maxSeeds == 0) { maxSeeds = max(5, khits * 2); } ReportingParams rp( (allHits ? std::numeric_limits::max() : khits), // -k (allHits ? std::numeric_limits::max() : maxSeeds), // --max-seeds mhits, // -m/-M 0, // penalty gap (not used now) msample, // true -> -M was specified, otherwise assume -m gReportDiscordant, // report discordang paired-end alignments? gReportMixed, // report unpaired alignments for paired reads? secondary, localAlign, bowtie2_dp, sensitive | very_sensitive, repeat); // Instantiate a mapping quality calculator auto_ptr bmapq(new_mapq(mapqv, scoreMin, sc)); // Make a per-thread wrapper for the global MHitSink object. AlnSinkWrap msinkwrap( msink, // global sink rp, // reporting parameters *bmapq.get(), // MAPQ calculator (size_t)tid, // thread id secondary, // secondary alignments no_spliced_alignment ? NULL : ssdb, thread_rids_mindist); SplicedAligner splicedAligner( gfm, anchorStop, thread_rids_mindist); SwAligner sw; OuterLoopMetrics olm; SeedSearchMetrics sdm; WalkMetrics wlm; SwMetrics swmSeed, swmMate; ReportingMetrics rpm; RandomSource rnd, rndArb; SSEMetrics sseU8ExtendMet; SSEMetrics sseU8MateMet; SSEMetrics sseI16ExtendMet; SSEMetrics sseI16MateMet; DescentMetrics descm; uint64_t nbtfiltst = 0; // TODO: find a new home for these uint64_t nbtfiltsc = 0; // TODO: find a new home for these uint64_t nbtfiltdo = 0; // TODO: find a new home for these HIMetrics him; ASSERT_ONLY(BTDnaString tmp); int pepolFlag; if(gMate1fw && gMate2fw) { pepolFlag = PE_POLICY_FF; } else if(gMate1fw && !gMate2fw) { pepolFlag = PE_POLICY_FR; } else if(!gMate1fw && gMate2fw) { pepolFlag = PE_POLICY_RF; } else { pepolFlag = PE_POLICY_RR; } assert_geq(gMaxInsert, gMinInsert); assert_geq(gMinInsert, 0); PairedEndPolicy pepol( pepolFlag, gMaxInsert, gMinInsert, localAlign, gFlippedMatesOK, gDovetailMatesOK, gContainMatesOK, gOlapMatesOK, gExpandToFrag); PerfMetrics metricsPt; // per-thread metrics object; for read-level metrics BTString nametmp; PerReadMetrics prm; // Used by thread with threadid == 1 to measure time elapsed time_t iTime = time(0); // Keep track of whether last search was exhaustive for mates 1 and 2 bool exhaustive[2] = { false, false }; // Keep track of whether mates 1/2 were filtered out last time through bool filt[2] = { true, true }; // Keep track of whether mates 1/2 were filtered out due Ns last time bool nfilt[2] = { true, true }; // Keep track of whether mates 1/2 were filtered out due to not having // enough characters to rise about the score threshold. bool scfilt[2] = { true, true }; // Keep track of whether mates 1/2 were filtered out due to not having // more characters than the number of mismatches permitted in a seed. bool lenfilt[2] = { true, true }; // Keep track of whether mates 1/2 were filtered out by upstream qc bool qcfilt[2] = { true, true }; rndArb.init((uint32_t)time(0)); int mergei = 0; int mergeival = 16; while(true) { bool success = false, done = false, paired = false; ps->nextReadPair(success, done, paired, outType != OUTPUT_SAM); if(!success && done) { break; } else if(!success) { continue; } TReadId rdid = ps->rdid(); if(nthreads > 1 && useTempSpliceSite) { assert_gt(tid, 0); assert_leq(tid, thread_rids.size()); assert(thread_rids[tid - 1] == 0 || rdid > thread_rids[tid - 1]); thread_rids[tid - 1] = (rdid > 0 ? rdid - 1 : 0); while(true) { uint64_t min_rdid = thread_rids[0]; { for(size_t i = 1; i < thread_rids.size(); i++) { if(thread_rids[i] < min_rdid) { min_rdid = thread_rids[i]; } } } if(min_rdid + thread_rids_mindist < rdid) { #if defined(_TTHREAD_WIN32_) Sleep(0); #elif defined(_TTHREAD_POSIX_) sched_yield(); #endif } else break; } } bool sample = true; if(arbitraryRandom) { ps->bufa().seed = rndArb.nextU32(); ps->bufb().seed = rndArb.nextU32(); } if(sampleFrac < 1.0f) { rnd.init(ROTL(ps->bufa().seed, 2)); sample = rnd.nextFloat() < sampleFrac; } if(rdid >= skipReads && rdid < qUpto && sample) { // Align this read/pair bool retry = true; // // Check if there is metrics reporting for us to do. // if(metricsIval > 0 && (metricsOfb != NULL || metricsStderr) && !metricsPerRead && ++mergei == mergeival) { // Do a periodic merge. Update global metrics, in a // synchronized manner if needed. MERGE_METRICS(metrics, nthreads > 1); mergei = 0; // Check if a progress message should be printed if(tid == 0) { // Only thread 1 prints progress messages time_t curTime = time(0); if(curTime - iTime >= metricsIval) { metrics.reportInterval(metricsOfb, metricsStderr, false, true, NULL); iTime = curTime; } } } prm.reset(); // per-read metrics prm.doFmString = false; if(sam_print_xt) { gettimeofday(&prm.tv_beg, &prm.tz_beg); } // Try to align this read while(retry) { retry = false; assert_eq(ps->bufa().color, false); olm.reads++; bool pair = paired; const size_t rdlen1 = ps->bufa().length(); const size_t rdlen2 = pair ? ps->bufb().length() : 0; olm.bases += (rdlen1 + rdlen2); msinkwrap.nextRead( &ps->bufa(), pair ? &ps->bufb() : NULL, rdid, sc.qualitiesMatter()); assert(msinkwrap.inited()); size_t rdlens[2] = { rdlen1, rdlen2 }; // Calculate the minimum valid score threshold for the read TAlScore minsc[2], maxpen[2]; maxpen[0] = maxpen[1] = 0; minsc[0] = minsc[1] = std::numeric_limits::max(); if(bwaSwLike) { // From BWA-SW manual: "Given an l-long query, the // threshold for a hit to be retained is // a*max{T,c*log(l)}." We try to recreate that here. float a = (float)sc.match(30); float T = bwaSwLikeT, c = bwaSwLikeC; minsc[0] = (TAlScore)max(a*T, a*c*log(rdlens[0])); if(paired) { minsc[1] = (TAlScore)max(a*T, a*c*log(rdlens[1])); } } else { minsc[0] = scoreMin.f(rdlens[0]); if(paired) minsc[1] = scoreMin.f(rdlens[1]); if(localAlign) { if(minsc[0] < 0) { if(!gQuiet) printLocalScoreMsg(*ps, paired, true); minsc[0] = 0; } if(paired && minsc[1] < 0) { if(!gQuiet) printLocalScoreMsg(*ps, paired, false); minsc[1] = 0; } } else { if(minsc[0] > 0) { if(!gQuiet) printEEScoreMsg(*ps, paired, true); minsc[0] = 0; } if(paired && minsc[1] > 0) { if(!gQuiet) printEEScoreMsg(*ps, paired, false); minsc[1] = 0; } } } // N filter; does the read have too many Ns? size_t readns[2] = {0, 0}; sc.nFilterPair( &ps->bufa().patFw, pair ? &ps->bufb().patFw : NULL, readns[0], readns[1], nfilt[0], nfilt[1]); // Score filter; does the read enough character to rise above // the score threshold? scfilt[0] = sc.scoreFilter(minsc[0], rdlens[0]); scfilt[1] = sc.scoreFilter(minsc[1], rdlens[1]); lenfilt[0] = lenfilt[1] = true; if(rdlens[0] <= (size_t)multiseedMms || rdlens[0] < 2) { if(!gQuiet) printMmsSkipMsg(*ps, paired, true, multiseedMms); lenfilt[0] = false; } if((rdlens[1] <= (size_t)multiseedMms || rdlens[1] < 2) && paired) { if(!gQuiet) printMmsSkipMsg(*ps, paired, false, multiseedMms); lenfilt[1] = false; } if(rdlens[0] < 2) { if(!gQuiet) printLenSkipMsg(*ps, paired, true); lenfilt[0] = false; } if(rdlens[1] < 2 && paired) { if(!gQuiet) printLenSkipMsg(*ps, paired, false); lenfilt[1] = false; } qcfilt[0] = qcfilt[1] = true; if(qcFilter) { qcfilt[0] = (ps->bufa().filter != '0'); qcfilt[1] = (ps->bufb().filter != '0'); } filt[0] = (nfilt[0] && scfilt[0] && lenfilt[0] && qcfilt[0]); filt[1] = (nfilt[1] && scfilt[1] && lenfilt[1] && qcfilt[1]); prm.nFilt += (filt[0] ? 0 : 1) + (filt[1] ? 0 : 1); Read* rds[2] = { &ps->bufa(), &ps->bufb() }; // For each mate... assert(msinkwrap.empty()); //size_t minedfw[2] = { 0, 0 }; //size_t minedrc[2] = { 0, 0 }; // Calcualte nofw / no rc bool nofw[2] = { false, false }; bool norc[2] = { false, false }; nofw[0] = paired ? (gMate1fw ? gNofw : gNorc) : gNofw; norc[0] = paired ? (gMate1fw ? gNorc : gNofw) : gNorc; nofw[1] = paired ? (gMate2fw ? gNofw : gNorc) : gNofw; norc[1] = paired ? (gMate2fw ? gNorc : gNofw) : gNorc; // Calculate nceil int nceil[2] = { 0, 0 }; nceil[0] = nCeil.f((double)rdlens[0]); nceil[0] = min(nceil[0], (int)rdlens[0]); if(paired) { nceil[1] = nCeil.f((double)rdlens[1]); nceil[1] = min(nceil[1], (int)rdlens[1]); } exhaustive[0] = exhaustive[1] = false; //size_t matemap[2] = { 0, 1 }; bool pairPostFilt = filt[0] && filt[1]; if(pairPostFilt) { rnd.init(ps->bufa().seed ^ ps->bufb().seed); } else { rnd.init(ps->bufa().seed); } // Calculate interval length for both mates int interval[2] = { 0, 0 }; for(size_t mate = 0; mate < (pair ? 2:1); mate++) { interval[mate] = msIval.f((double)rdlens[mate]); if(filt[0] && filt[1]) { // Boost interval length by 20% for paired-end reads interval[mate] = (int)(interval[mate] * 1.2 + 0.5); } interval[mate] = max(interval[mate], 1); } // Calculate streak length size_t streak[2] = { maxDpStreak, maxDpStreak }; size_t mtStreak[2] = { maxMateStreak, maxMateStreak }; size_t mxDp[2] = { maxDp, maxDp }; size_t mxUg[2] = { maxUg, maxUg }; size_t mxIter[2] = { maxIters, maxIters }; if(allHits) { streak[0] = streak[1] = std::numeric_limits::max(); mtStreak[0] = mtStreak[1] = std::numeric_limits::max(); mxDp[0] = mxDp[1] = std::numeric_limits::max(); mxUg[0] = mxUg[1] = std::numeric_limits::max(); mxIter[0] = mxIter[1] = std::numeric_limits::max(); } else if(khits > 1) { for(size_t mate = 0; mate < 2; mate++) { streak[mate] += (khits-1) * maxStreakIncr; mtStreak[mate] += (khits-1) * maxStreakIncr; mxDp[mate] += (khits-1) * maxItersIncr; mxUg[mate] += (khits-1) * maxItersIncr; mxIter[mate] += (khits-1) * maxItersIncr; } } if(filt[0] && filt[1]) { streak[0] = (size_t)ceil((double)streak[0] / 2.0); streak[1] = (size_t)ceil((double)streak[1] / 2.0); assert_gt(streak[1], 0); } assert_gt(streak[0], 0); // Calculate # seed rounds for each mate size_t nrounds[2] = { nSeedRounds, nSeedRounds }; if(filt[0] && filt[1]) { nrounds[0] = (size_t)ceil((double)nrounds[0] / 2.0); nrounds[1] = (size_t)ceil((double)nrounds[1] / 2.0); assert_gt(nrounds[1], 0); } assert_gt(nrounds[0], 0); // Increment counters according to what got filtered for(size_t mate = 0; mate < (pair ? 2:1); mate++) { if(!filt[mate]) { // Mate was rejected by N filter olm.freads++; // reads filtered out olm.fbases += rdlens[mate]; // bases filtered out } else { //shs[mate].clear(); //shs[mate].nextRead(mate == 0 ? ps->bufa() : ps->bufb()); //assert(shs[mate].empty()); olm.ureads++; // reads passing filter olm.ubases += rdlens[mate]; // bases passing filter } } //size_t eePeEeltLimit = std::numeric_limits::max(); // Whether we're done with mate1 / mate2 bool done[2] = { !filt[0], !filt[1] }; // size_t nelt[2] = {0, 0}; if(filt[0] && filt[1]) { splicedAligner.initReads(rds, nofw, norc, minsc, maxpen); } else if(filt[0]) { splicedAligner.initRead(rds[0], nofw[0], norc[0], minsc[0], maxpen[0], false); } else if(filt[1]) { splicedAligner.initRead(rds[1], nofw[1], norc[1], minsc[1], maxpen[1], true); } if(filt[0] || filt[1]) { int ret = splicedAligner.go( sc, pepol, *multiseed_tpol, *gpol, gfm, rgfm, *altdb, *repeatdb, *raltdb, ref, rref, sw, *ssdb, wlm, prm, swmSeed, him, rnd, msinkwrap); MERGE_SW(sw); // daehwan size_t mate = 0; assert_gt(ret, 0); // Clear out the exact hits so that we don't try to // extend them again later! if(ret == EXTEND_EXHAUSTED_CANDIDATES) { // Not done yet } else if(ret == EXTEND_POLICY_FULFILLED) { // Policy is satisfied for this mate at least if(msinkwrap.state().doneWithMate(mate == 0)) { done[mate] = true; } if(msinkwrap.state().doneWithMate(mate == 1)) { done[mate^1] = true; } } else if(ret == EXTEND_PERFECT_SCORE) { // We exhausted this mode at least done[mate] = true; } else if(ret == EXTEND_EXCEEDED_HARD_LIMIT) { // We exceeded a per-read limit done[mate] = true; } else if(ret == EXTEND_EXCEEDED_SOFT_LIMIT) { // Not done yet } else { // cerr << "Bad return value: " << ret << endl; throw 1; } if(!done[mate]) { TAlScore perfectScore = sc.perfectScore(rdlens[mate]); if(!done[mate] && minsc[mate] == perfectScore) { done[mate] = true; } } } for(size_t i = 0; i < 2; i++) { assert_leq(prm.nExIters, mxIter[i]); assert_leq(prm.nExDps, mxDp[i]); assert_leq(prm.nMateDps, mxDp[i]); assert_leq(prm.nExUgs, mxUg[i]); assert_leq(prm.nMateUgs, mxUg[i]); assert_leq(prm.nDpFail, streak[i]); assert_leq(prm.nUgFail, streak[i]); assert_leq(prm.nEeFail, streak[i]); } // Commit and report paired-end/unpaired alignments msinkwrap.finishRead( NULL, NULL, exhaustive[0], // exhausted seed hits for mate 1? exhaustive[1], // exhausted seed hits for mate 2? nfilt[0], nfilt[1], scfilt[0], scfilt[1], lenfilt[0], lenfilt[1], qcfilt[0], qcfilt[1], sortByScore, // prioritize by alignment score rnd, // pseudo-random generator rpm, // reporting metrics prm, // per-read metrics sc, // scoring scheme !seedSumm, // suppress seed summaries? seedSumm, // suppress alignments? templateLenAdjustment); assert(!retry || msinkwrap.empty()); } // while(retry) } // if(rdid >= skipReads && rdid < qUpto) else if(rdid >= qUpto) { break; } if(metricsPerRead) { MERGE_METRICS(metricsPt, nthreads > 1); nametmp = ps->bufa().name; metricsPt.reportInterval( metricsOfb, metricsStderr, true, true, &nametmp); metricsPt.reset(); } } // while(true) // One last metrics merge MERGE_METRICS(metrics, nthreads > 1); return; } /** * Called once per alignment job. Sets up global pointers to the * shared global data structures, creates per-thread structures, then * enters the search loop. */ static void multiseedSearch( Scoring& sc, TranscriptomePolicy& tpol, GraphPolicy& gp, PairedPatternSource& patsrc, // pattern source AlnSink& msink, // hit sink HGFM& gfm, // index of original text RFM* rgfm, // index of repeat sequences BitPairReference* refs, // base reference BitPairReference* rrefs, // repeat reference OutFileBuf *metricsOfb) { multiseed_patsrc = &patsrc; multiseed_msink = &msink; multiseed_gfm = &gfm; multiseed_rgfm = rgfm; multiseed_sc = ≻ multiseed_tpol = &tpol; gpol = &gp; multiseed_metricsOfb = metricsOfb; multiseed_refs = refs; multiseed_rrefs = rrefs; AutoArray threads(nthreads); AutoArray tids(nthreads); // Start the metrics thread { Timer _t(cerr, "Multiseed full-index search: ", timing); thread_rids.resize(nthreads); thread_rids.fill(0); thread_rids_mindist = (nthreads == 1 || !useTempSpliceSite ? 0 : 1000 * nthreads); for(int i = 0; i < nthreads; i++) { // Thread IDs start at 1 tids[i] = i+1; threads[i] = new tthread::thread(multiseedSearchWorker_hisat2, (void*)&tids[i]); } for (int i = 0; i < nthreads; i++) threads[i]->join(); } if(!metricsPerRead && (metricsOfb != NULL || metricsStderr)) { metrics.reportInterval(metricsOfb, metricsStderr, true, false, NULL); } } static string argstr; extern void initializeCntLut(); extern void initializeCntBit(); template static void driver( const char * type, const string& bt2indexBase, const string& outfile) { if(gVerbose || startVerbose) { cerr << "Entered driver(): "; logTime(cerr, true); } initializeCntLut(); initializeCntBit(); // Vector of the reference sequences; used for sanity-checking EList > names, os; EList nameLens, seqLens; // Read reference sequences from the command-line or from a FASTA file if(!origString.empty()) { // Read fasta file(s) EList origFiles; tokenize(origString, ",", origFiles); parseFastas(origFiles, names, nameLens, os, seqLens); } PatternParams pp( format, // file format fileParallel, // true -> wrap files with separate PairedPatternSources seed, // pseudo-random seed useSpinlock, // use spin locks instead of pthreads solexaQuals, // true -> qualities are on solexa64 scale phred64Quals, // true -> qualities are on phred64 scale integerQuals, // true -> qualities are space-separated numbers fuzzy, // true -> try to parse fuzzy fastq fastaContLen, // length of sampled reads for FastaContinuous... fastaContFreq, // frequency of sampled reads for FastaContinuous... skipReads // skip the first 'skip' patterns ); if(gVerbose || startVerbose) { cerr << "Creating PatternSource: "; logTime(cerr, true); } PairedPatternSource *patsrc = PairedPatternSource::setupPatternSources( queries, // singles, from argv mates1, // mate1's, from -1 arg mates2, // mate2's, from -2 arg mates12, // both mates on each line, from --12 arg #ifdef USE_SRA sra_accs, // SRA accessions #endif qualities, // qualities associated with singles qualities1, // qualities associated with m1 qualities2, // qualities associated with m2 pp, // read read-in parameters nthreads, gVerbose || startVerbose); // be talkative // Open hit output file if(gVerbose || startVerbose) { cerr << "Opening hit output file: "; logTime(cerr, true); } OutFileBuf *fout; if(!outfile.empty()) { fout = new OutFileBuf(outfile.c_str(), false); } else { fout = new OutFileBuf(); } // Initialize GFM object and read in header if(gVerbose || startVerbose) { cerr << "About to initialize fw GFM: "; logTime(cerr, true); } altdb = new ALTDB(); repeatdb = new RepeatDB(); raltdb = new ALTDB(); adjIdxBase = adjustEbwtBase(argv0, bt2indexBase, gVerbose); HGFM gfm( adjIdxBase, altdb, NULL, NULL, -1, // fw index true, // index is for the forward direction /* overriding: */ offRate, 0, // amount to add to index offrate or <= 0 to do nothing useMm, // whether to use memory-mapped files useShmem, // whether to use shared memory mmSweep, // sweep memory-mapped files !noRefNames, // load names? true, // load SA sample? true, // load ftab? true, // load rstarts? !no_spliced_alignment, // load splice sites? gVerbose, // whether to be talkative startVerbose, // talkative during initialization false /*passMemExc*/, sanityCheck, use_haplotype); //use haplotypes? if(sanityCheck && !os.empty()) { // Sanity check number of patterns and pattern lengths in GFM // against original strings assert_eq(os.size(), gfm.nPat()); for(size_t i = 0; i < os.size(); i++) { assert_eq(os[i].length(), gfm.plen()[i]); } } // Sanity-check the restored version of the GFM if(sanityCheck && !os.empty()) { gfm.loadIntoMemory( -1, // fw index true, // load SA sample true, // load ftab true, // load rstarts !noRefNames, startVerbose); gfm.checkOrigs(os, false); gfm.evictFromMemory(); } { // Load the other half of the index into memory assert(!gfm.isInMemory()); Timer _t(cerr, "Time loading forward index: ", timing); gfm.loadIntoMemory( -1, // not the reverse index true, // load SA samp? (yes, need forward index's SA samp) true, // load ftab (in forward index) true, // load rstarts (in forward index) !noRefNames, // load names? startVerbose); } RFM* rgfm = NULL; string rep_adjIdxBase = adjIdxBase + ".rep"; bool rep_index_exists = false; { std::ifstream infile((rep_adjIdxBase + ".1." + gfm_ext.c_str()).c_str()); rep_index_exists = infile.good(); } if(rep_index_exists && use_repeat_index) { rgfm = new RFM( rep_adjIdxBase, raltdb, repeatdb, &readLens, -1, // fw index true, // index is for the forward direction /* overriding: */ offRate, 0, // amount to add to index offrate or <= 0 to do nothing useMm, // whether to use memory-mapped files useShmem, // whether to use shared memory mmSweep, // sweep memory-mapped files !noRefNames, // load names? true, // load SA sample? true, // load ftab? true, // load rstarts? !no_spliced_alignment, // load splice sites? gVerbose, // whether to be talkative startVerbose, // talkative during initialization false /*passMemExc*/, sanityCheck, false); //use haplotypes? // CP to do #if 0 if(sanityCheck && !os.empty()) { // Sanity check number of patterns and pattern lengths in GFM // against original strings assert_eq(os.size(), gfm.nPat()); for(size_t i = 0; i < os.size(); i++) { assert_eq(os[i].length(), rgfm->plen()[i]); } } // Sanity-check the restored version of the GFM if(sanityCheck && !os.empty()) { rgfm->loadIntoMemory( -1, // fw index true, // load SA sample true, // load ftab true, // load rstarts !noRefNames, startVerbose); rgfm->checkOrigs(os, false); rgfm->evictFromMemory(); } #endif { // Load the other half of the index into memory assert(!rgfm->isInMemory()); Timer _t(cerr, "Time loading forward index: ", timing); rgfm->loadIntoMemory( -1, // not the reverse index true, // load SA samp? (yes, need forward index's SA samp) true, // load ftab (in forward index) true, // load rstarts (in forward index) !noRefNames, // load names? startVerbose); repeatdb->construct(gfm.rstarts(), gfm.nFrag()); } } if(!saw_k) { if(gfm.gh().linearFM()) khits = 5; else khits = 10; } OutputQueue oq( *fout, // out file buffer reorder && nthreads > 1, // whether to reorder when there's >1 thread nthreads, // # threads nthreads > 1, // whether to be thread-safe skipReads); // first read will have this rdid { Timer _t(cerr, "Time searching: ", timing); // Set up penalities if(bonusMatch > 0 && !localAlign) { cerr << "Warning: Match bonus always = 0 in --end-to-end mode; ignoring user setting" << endl; bonusMatch = 0; } if(tranAssm) { penNoncanIntronLen.init(SIMPLE_FUNC_LOG, -8, 2); } Scoring sc( bonusMatch, // constant reward for match penMmcType, // how to penalize mismatches penMmcMax, // max mm penalty penMmcMin, // min mm penalty penScMax, // max sc penalty penScMin, // min sc penalty scoreMin, // min score as function of read len nCeil, // max # Ns as function of read len penNType, // how to penalize Ns in the read penN, // constant if N pelanty is a constant penNCatPair, // whether to concat mates before N filtering penRdGapConst, // constant coeff for read gap cost penRfGapConst, // constant coeff for ref gap cost penRdGapLinear, // linear coeff for read gap cost penRfGapLinear, // linear coeff for ref gap cost gGapBarrier, // # rows at top/bot only entered diagonally penCanSplice, // canonical splicing penalty penNoncanSplice,// non-canonical splicing penalty penConflictSplice, // conflicting splice site penalty &penCanIntronLen, // penalty as to intron length &penNoncanIntronLen); // penalty as to intron length EList reflens; for(size_t i = 0; i < gfm.nPat(); i++) { reflens.push_back(gfm.plen()[i]); } EList refnames; readEbwtRefnames(adjIdxBase, refnames); EList replens; EList repnames; if(rep_index_exists && use_repeat_index) { rgfm->getReferenceNames(repnames); rgfm->getReferenceLens(replens); } if(rmChrName && addChrName) { cerr << "Error: --remove-chrname and --add-chrname cannot be used at the same time" << endl; throw 1; } if(rmChrName) { for(size_t i = 0; i < refnames.size(); i++) { string& refname = refnames[i]; if(refname.find("chr") == 0) { refname = refname.substr(3); } } } else if(addChrName) { for(size_t i = 0; i < refnames.size(); i++) { string& refname = refnames[i]; if(refname.find("chr") != 0) { refname = string("chr") + refname; } } } EList empty_replens; EList empty_repnames; SamConfig samc( refnames, // reference sequence names reflens, // reference sequence lengths repeat ? repnames : empty_repnames, // repeat sequence names repeat ? replens : empty_replens, // repeat sequence lengths samTruncQname, // whether to truncate QNAME to 255 chars samOmitSecSeqQual, // omit SEQ/QUAL for 2ndary alignments? samNoUnal, // omit unaligned-read records? string("hisat2"), // program id string("hisat2"), // program name string(HISAT2_VERSION), // program version argstr, // command-line rgs_optflag, // read-group string rna_strandness, sam_print_as, sam_print_xs, sam_print_xss, sam_print_yn, sam_print_xn, sam_print_cs, sam_print_cq, sam_print_x0, sam_print_x1, sam_print_xm, sam_print_xo, sam_print_xg, sam_print_nm, sam_print_md, sam_print_yf, sam_print_yi, sam_print_ym, sam_print_yp, sam_print_yt, sam_print_ys, sam_print_zs, sam_print_xr, sam_print_xt, sam_print_xd, sam_print_xu, sam_print_yl, sam_print_ye, sam_print_yu, sam_print_xp, sam_print_yr, sam_print_zb, sam_print_zr, sam_print_zf, sam_print_zm, sam_print_zi, sam_print_zp, sam_print_zu, sam_print_xs_a, sam_print_nh); // Set up hit sink; if sanityCheck && !os.empty() is true, // then instruct the sink to "retain" hits in a vector in // memory so that we can easily sanity check them later on AlnSink *mssink = NULL; Timer *_tRef = new Timer(cerr, "Time loading reference: ", timing); auto_ptr refs( new BitPairReference( adjIdxBase, NULL, false, sanityCheck, NULL, NULL, false, useMm, useShmem, mmSweep, gVerbose, startVerbose) ); delete _tRef; if(!refs->loaded()) throw 1; BitPairReference* rrefs = NULL; if(rep_index_exists && use_repeat_index) { const EList& included = rgfm->getRepeatIncluded(); rrefs = new BitPairReference( rep_adjIdxBase, &included, false, sanityCheck, NULL, NULL, false, useMm, useShmem, mmSweep, gVerbose, startVerbose); if(!rrefs->loaded()) throw 1; } bool xsOnly = (tranAssm_program == "cufflinks"); TranscriptomePolicy tpol(minIntronLen, maxIntronLen, tranAssm ? 15 : 7, tranAssm ? 20 : 14, no_spliced_alignment, tranMapOnly, tranAssm, xsOnly, avoid_pseudogene); GraphPolicy gpol(max_alts_tried, use_haplotype, altdb->haplotypes().size() > 0 && use_haplotype, enable_codis); init_junction_prob(); bool write = novelSpliceSiteOutfile != "" || useTempSpliceSite; bool read = knownSpliceSiteInfile != "" || novelSpliceSiteInfile != "" || useTempSpliceSite || altdb->hasSpliceSites(); ssdb = new SpliceSiteDB( *(refs.get()), refnames, nthreads > 1, // thread-safe write, // write? read); // read? ssdb->read(gfm, altdb->alts()); if(knownSpliceSiteInfile != "") { ifstream ssdb_file(knownSpliceSiteInfile.c_str(), ios::in); if(ssdb_file.is_open()) { ssdb->read(ssdb_file, true); // known splice sites ssdb_file.close(); } } if(novelSpliceSiteInfile != "") { ifstream ssdb_file(novelSpliceSiteInfile.c_str(), ios::in); if(ssdb_file.is_open()) { ssdb->read(ssdb_file, false); // novel splice sites ssdb_file.close(); } } switch(outType) { case OUTPUT_SAM: { mssink = new AlnSinkSam( oq, // output queue samc, // settings & routines for SAM output refnames, // reference names repnames, // repeat names gQuiet, // don't print alignment summary at end altdb, ssdb); if(!samNoHead) { bool printHd = true, printSq = true; BTString buf; samc.printHeader(buf, rgid, rgs, printHd, !samNoSQ, printSq); fout->writeString(buf); } break; } default: cerr << "Invalid output type: " << outType << endl; throw 1; } if(gVerbose || startVerbose) { cerr << "Dispatching to search driver: "; logTime(cerr, true); } // Set up global constraint OutFileBuf *metricsOfb = NULL; if(!metricsFile.empty() && metricsIval > 0) { metricsOfb = new OutFileBuf(metricsFile); } // Do the search for all input reads assert(patsrc != NULL); assert(mssink != NULL); multiseedSearch( sc, // scoring scheme tpol, gpol, *patsrc, // pattern source *mssink, // hit sink gfm, // BWT rgfm, refs.get(), rrefs, metricsOfb); // Evict any loaded indexes from memory if(gfm.isInMemory()) { gfm.evictFromMemory(); } if(!gQuiet && !seedSumm) { size_t repThresh = mhits; if(repThresh == 0) { repThresh = std::numeric_limits::max(); } mssink->finish(cerr, repThresh, gReportDiscordant, gReportMixed, newAlignSummary, hadoopOut); if(alignSumFile != "") { ofstream sumfile(alignSumFile.c_str(), ios::out); if(sumfile.is_open()) { mssink->finish(sumfile, repThresh, gReportDiscordant, gReportMixed, newAlignSummary, false); // hadoopOut sumfile.close(); } } } if(ssdb != NULL) { if(novelSpliceSiteOutfile != "") { ofstream ssdb_file(novelSpliceSiteOutfile.c_str(), ios::out); if(ssdb_file.is_open()) { ssdb->print(ssdb_file); ssdb_file.close(); } } } oq.flush(true); assert_eq(oq.numStarted(), oq.numFinished()); assert_eq(oq.numStarted(), oq.numFlushed()); delete patsrc; delete mssink; delete altdb; delete repeatdb; delete raltdb; delete ssdb; delete metricsOfb; delete rgfm; delete rrefs; if(fout != NULL) { delete fout; } } } // C++ name mangling is disabled for the bowtie() function to make it // easier to use Bowtie as a library. extern "C" { /** * Main bowtie entry function. Parses argc/argv style command-line * options, sets global configuration variables, and calls the driver() * function. */ int hisat2(int argc, const char **argv) { try { // Reset all global state, including getopt state opterr = optind = 1; resetOptions(); for(int i = 0; i < argc; i++) { argstr += argv[i]; if(i < argc-1) argstr += " "; } if(startVerbose) { cerr << "Entered main(): "; logTime(cerr, true); } parseOptions(argc, argv); argv0 = argv[0]; if(showVersion) { cout << argv0 << " version " << HISAT2_VERSION << endl; if(sizeof(void*) == 4) { cout << "32-bit" << endl; } else if(sizeof(void*) == 8) { cout << "64-bit" << endl; } else { cout << "Neither 32- nor 64-bit: sizeof(void*) = " << sizeof(void*) << endl; } cout << "Built on " << BUILD_HOST << endl; cout << BUILD_TIME << endl; cout << "Compiler: " << COMPILER_VERSION << endl; cout << "Options: " << COMPILER_OPTIONS << endl; cout << "Sizeof {int, long, long long, void*, size_t, off_t}: {" << sizeof(int) << ", " << sizeof(long) << ", " << sizeof(long long) << ", " << sizeof(void *) << ", " << sizeof(size_t) << ", " << sizeof(off_t) << "}" << endl; return 0; } { Timer _t(cerr, "Overall time: ", timing); if(startVerbose) { cerr << "Parsing index and read arguments: "; logTime(cerr, true); } // Get index basename (but only if it wasn't specified via --index) if(bt2index.empty()) { if(optind >= argc) { cerr << "No index, query, or output file specified!" << endl; printUsage(cerr); return 1; } bt2index = argv[optind++]; } // Get query filename bool got_reads = !queries.empty() || !mates1.empty() || !mates12.empty(); #ifdef USE_SRA got_reads = got_reads || !sra_accs.empty(); #endif if(minIntronLen > maxIntronLen) { cerr << "--min-intronlen(" << minIntronLen << ") should not be greater than --max-intronlen(" << maxIntronLen << ")" << endl; printUsage(cerr); return 1; } if(optind >= argc) { if(!got_reads) { printUsage(cerr); cerr << "***" << endl #ifdef USE_SRA << "Error: Must specify at least one read input with -U/-1/-2/--sra-acc" << endl; #else << "Error: Must specify at least one read input with -U/-1/-2" << endl; #endif return 1; } } else if(!got_reads) { // Tokenize the list of query files tokenize(argv[optind++], ",", queries); if(queries.empty()) { cerr << "Tokenized query file list was empty!" << endl; printUsage(cerr); return 1; } } // Get output filename if(optind < argc && outfile.empty()) { outfile = argv[optind++]; cerr << "Warning: Output file '" << outfile.c_str() << "' was specified without -S. This will not work in " << "future HISAT 2 versions. Please use -S instead." << endl; } // Extra parametesr? if(optind < argc) { cerr << "Extra parameter(s) specified: "; for(int i = optind; i < argc; i++) { cerr << "\"" << argv[i] << "\""; if(i < argc-1) cerr << ", "; } cerr << endl; if(mates1.size() > 0) { cerr << "Note that if files are specified using -1/-2, a file cannot" << endl << "also be specified. Please run HISAT2 separately for mates and singles." << endl; } throw 1; } // Optionally summarize if(gVerbose) { cout << "Input bt2 file: \"" << bt2index.c_str() << "\"" << endl; cout << "Query inputs (DNA, " << file_format_names[format].c_str() << "):" << endl; for(size_t i = 0; i < queries.size(); i++) { cout << " " << queries[i].c_str() << endl; } cout << "Quality inputs:" << endl; for(size_t i = 0; i < qualities.size(); i++) { cout << " " << qualities[i].c_str() << endl; } cout << "Output file: \"" << outfile.c_str() << "\"" << endl; cout << "Local endianness: " << (currentlyBigEndian()? "big":"little") << endl; cout << "Sanity checking: " << (sanityCheck? "enabled":"disabled") << endl; #ifdef NDEBUG cout << "Assertions: disabled" << endl; #else cout << "Assertions: enabled" << endl; #endif } if(ipause) { cout << "Press key to continue..." << endl; getchar(); } driver >("DNA", bt2index, outfile); } return 0; } catch(std::exception& e) { cerr << "Error: Encountered exception: '" << e.what() << "'" << endl; cerr << "Command: "; for(int i = 0; i < argc; i++) cerr << argv[i] << " "; cerr << endl; return 1; } catch(int e) { if(e != 0) { cerr << "Error: Encountered internal HISAT2 exception (#" << e << ")" << endl; cerr << "Command: "; for(int i = 0; i < argc; i++) cerr << argv[i] << " "; cerr << endl; } return e; } } // bowtie() } // extern "C" hisat2-2.2.1/hisat2.sln000066400000000000000000000125171370663763200146410ustar00rootroot00000000000000 Microsoft Visual Studio Solution File, Format Version 12.00 # Visual Studio 14 VisualStudioVersion = 14.0.25420.1 MinimumVisualStudioVersion = 10.0.40219.1 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*/; } hisat2-2.2.1/hisat2_build.cpp000066400000000000000000001005411370663763200160010ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include #include #include #include #include "assert_helpers.h" #include "endian_swap.h" #include "formats.h" #include "sequence_io.h" #include "tokenize.h" #include "timer.h" #include "ref_read.h" #include "filebuf.h" #include "reference.h" #include "ds.h" #include "gfm.h" #include "hgfm.h" #include "rfm.h" /** * \file Driver for the bowtie-build indexing tool. */ #include #include #include #include #include MemoryTally gMemTally; // Build parameters int verbose; static int sanityCheck; static int format; static TIndexOffU bmax; static TIndexOffU bmaxMultSqrt; static uint32_t bmaxDivN; static int dcv; static int noDc; static int entireSA; static int seed; static int showVersion; // GFM parameters static int32_t lineRate; static bool lineRate_provided; static int32_t linesPerSide; static int32_t offRate; static int32_t ftabChars; static int32_t localOffRate; static int32_t localFtabChars; static int bigEndian; static bool nsToAs; static bool autoMem; static bool packed; static bool writeRef; static bool justRef; static bool reverseEach; static int nthreads; // number of pthreads operating concurrently static string wrapper; static string snp_fname; static string ht_fname; static string ss_fname; static string exon_fname; static string sv_fname; static string repeat_ref_fname; static string repeat_info_fname; static string repeat_snp_fname; static string repeat_haplotype_fname; static void resetOptions() { verbose = true; // be talkative (default) sanityCheck = 0; // do slow sanity checks format = FASTA; // input sequence format bmax = OFF_MASK; // max blockwise SA bucket size bmaxMultSqrt = OFF_MASK; // same, as multplier of sqrt(n) bmaxDivN = 4; // same, as divisor of n dcv = 1024; // bwise SA difference-cover sample sz noDc = 0; // disable difference-cover sample entireSA = 0; // 1 = disable blockwise SA seed = 0; // srandom seed showVersion = 0; // just print version and quit? // GFM parameters lineRate = GFM::default_lineRate_gfm; lineRate_provided = false; linesPerSide = 1; // 1 64-byte line on a side offRate = 4; // sample 1 out of 16 SA elts ftabChars = 10; // 10 chars in initial lookup table localOffRate = 3; localFtabChars = 6; bigEndian = 0; // little endian nsToAs = false; // convert reference Ns to As prior to indexing autoMem = true; // automatically adjust memory usage parameters packed = false; // writeRef = true; // write compact reference to .3.ht2/.4.ht2 justRef = false; // *just* write compact reference, don't index reverseEach = false; nthreads = 1; wrapper.clear(); snp_fname = ""; ht_fname = ""; ss_fname = ""; exon_fname = ""; sv_fname = ""; repeat_ref_fname = ""; repeat_info_fname = ""; repeat_snp_fname = ""; repeat_haplotype_fname = ""; } // Argument constants for getopts enum { ARG_BMAX = 256, ARG_BMAX_MULT, ARG_BMAX_DIV, ARG_DCV, ARG_SEED, ARG_CUTOFF, ARG_PMAP, ARG_NTOA, ARG_USAGE, ARG_REVERSE_EACH, ARG_SA, ARG_WRAPPER, ARG_LOCAL_OFFRATE, ARG_LOCAL_FTABCHARS, ARG_SNP, ARG_HAPLOTYPE, ARG_SPLICESITE, ARG_EXON, ARG_SV, ARG_REPEAT_REF, ARG_REPEAT_INFO, ARG_REPEAT_SNP, ARG_REPEAT_HAPLOTYPE, }; /** * Print a detailed usage message to the provided output stream. */ static void printUsage(ostream& out) { out << "HISAT2 version " << string(HISAT2_VERSION).c_str() << " by Daehwan Kim (infphilo@gmail.com, http://www.ccb.jhu.edu/people/infphilo)" << endl; #ifdef BOWTIE_64BIT_INDEX string tool_name = "hisat2-build-l"; #else string tool_name = "hisat2-build-s"; #endif if(wrapper == "basic-0") { tool_name = "hisat2-build"; } out << "Usage: hisat2-build [options]* " << endl << " reference_in comma-separated list of files with ref sequences" << endl << " hisat2_index_base write " << gfm_ext << " data to files with this dir/basename" << endl << "Options:" << endl << " -c reference sequences given on cmd line (as" << endl << " )" << endl; if(wrapper == "basic-0") { out << " --large-index force generated index to be 'large', even if ref" << endl << " has fewer than 4 billion nucleotides" << endl; } out << " -a/--noauto disable automatic -p/--bmax/--dcv memory-fitting" << endl << " -p number of threads" << endl << " --bmax max bucket sz for blockwise suffix-array builder" << endl << " --bmaxdivn max bucket sz as divisor of ref len (default: 4)" << endl << " --dcv diff-cover period for blockwise (default: 1024)" << endl << " --nodc disable diff-cover (algorithm becomes quadratic)" << endl << " -r/--noref don't build .3/.4.ht2 (packed reference) portion" << endl << " -3/--justref just build .3/.4.ht2 (packed reference) portion" << endl << " -o/--offrate SA is sampled every 2^offRate BWT chars (default: 5)" << endl << " -t/--ftabchars # of chars consumed in initial lookup (default: 10)" << endl << " --localoffrate SA (local) is sampled every 2^offRate BWT chars (default: 3)" << endl << " --localftabchars # of chars consumed in initial lookup in a local index (default: 6)" << endl << " --snp SNP file name" << endl << " --haplotype haplotype file name" << endl << " --ss Splice site file name" << endl << " --exon Exon file name" << endl << " --repeat-ref Repeat reference file name" << endl << " --repeat-info Repeat information file name" << endl << " --repeat-snp Repeat snp file name" << endl << " --repeat-haplotype Repeat haplotype file name" << endl << " --seed seed for random number generator" << endl << " -q/--quiet disable verbose output (for debugging)" << endl << " -h/--help print detailed description of tool and its options" << endl << " --usage print this usage message" << endl << " --version print version information and quit" << endl ; if(wrapper.empty()) { cerr << endl << "*** Warning ***" << endl << "'" << tool_name << "' was run directly. It is recommended " << "that you run the wrapper script 'hisat2-build' instead." << endl << endl; } } static const char *short_options = "qrap:h?nscfl:i:o:t:h:3C"; static struct option long_options[] = { {(char*)"quiet", no_argument, 0, 'q'}, {(char*)"sanity", no_argument, 0, 's'}, {(char*)"threads", required_argument, 0, 'p'}, {(char*)"little", no_argument, &bigEndian, 0}, {(char*)"big", no_argument, &bigEndian, 1}, {(char*)"bmax", required_argument, 0, ARG_BMAX}, {(char*)"bmaxmultsqrt", required_argument, 0, ARG_BMAX_MULT}, {(char*)"bmaxdivn", required_argument, 0, ARG_BMAX_DIV}, {(char*)"dcv", required_argument, 0, ARG_DCV}, {(char*)"nodc", no_argument, &noDc, 1}, {(char*)"seed", required_argument, 0, ARG_SEED}, {(char*)"entiresa", no_argument, &entireSA, 1}, {(char*)"version", no_argument, &showVersion, 1}, {(char*)"noauto", no_argument, 0, 'a'}, {(char*)"noblocks", required_argument, 0, 'n'}, {(char*)"linerate", required_argument, 0, 'l'}, {(char*)"linesperside", required_argument, 0, 'i'}, {(char*)"offrate", required_argument, 0, 'o'}, {(char*)"ftabchars", required_argument, 0, 't'}, {(char*)"localoffrate", required_argument, 0, ARG_LOCAL_OFFRATE}, {(char*)"localftabchars", required_argument, 0, ARG_LOCAL_FTABCHARS}, {(char*)"snp", required_argument, 0, ARG_SNP}, {(char*)"haplotype", required_argument, 0, ARG_HAPLOTYPE}, {(char*)"ss", required_argument, 0, ARG_SPLICESITE}, {(char*)"exon", required_argument, 0, ARG_EXON}, {(char*)"sv", required_argument, 0, ARG_SV}, {(char*)"repeat-ref", required_argument, 0, ARG_REPEAT_REF}, {(char*)"repeat-info", required_argument, 0, ARG_REPEAT_INFO}, {(char*)"repeat-snp", required_argument, 0, ARG_REPEAT_SNP}, {(char*)"repeat-haplotype", required_argument, 0, ARG_REPEAT_HAPLOTYPE}, {(char*)"help", no_argument, 0, 'h'}, {(char*)"ntoa", no_argument, 0, ARG_NTOA}, {(char*)"justref", no_argument, 0, '3'}, {(char*)"noref", no_argument, 0, 'r'}, {(char*)"sa", no_argument, 0, ARG_SA}, {(char*)"reverse-each", no_argument, 0, ARG_REVERSE_EACH}, {(char*)"usage", no_argument, 0, ARG_USAGE}, {(char*)"wrapper", required_argument, 0, ARG_WRAPPER}, {(char*)0, 0, 0, 0} // terminator }; /** * Parse an int out of optarg and enforce that it be at least 'lower'; * if it is less than 'lower', then output the given error message and * exit with an error and a usage message. */ template static T parseNumber(T lower, const char *errmsg) { char *endPtr= NULL; T t = (T)strtoll(optarg, &endPtr, 10); if (endPtr != NULL) { if (t < lower) { cerr << errmsg << endl; printUsage(cerr); throw 1; } return t; } cerr << errmsg << endl; printUsage(cerr); throw 1; return -1; } /** * Read command-line arguments */ static void parseOptions(int argc, const char **argv) { int option_index = 0; int next_option; do { next_option = getopt_long( argc, const_cast(argv), short_options, long_options, &option_index); switch (next_option) { case ARG_WRAPPER: wrapper = optarg; break; case 'f': format = FASTA; break; case 'c': format = CMDLINE; break; //case 'p': packed = true; break; case 'C': cerr << "Error: -C specified but Bowtie 2 does not support colorspace input." << endl; throw 1; break; case 'l': lineRate = parseNumber(3, "-l/--lineRate arg must be at least 3"); lineRate_provided = true; break; case 'i': linesPerSide = parseNumber(1, "-i/--linesPerSide arg must be at least 1"); break; case 'o': offRate = parseNumber(0, "-o/--offRate arg must be at least 0"); break; case ARG_LOCAL_OFFRATE: localOffRate = parseNumber(0, "-o/--localoffrate arg must be at least 0"); break; case '3': justRef = true; break; case 't': ftabChars = parseNumber(1, "-t/--ftabChars arg must be at least 1"); break; case ARG_LOCAL_FTABCHARS: localFtabChars = parseNumber(1, "-t/--localftabchars arg must be at least 1"); break; case 'n': // all f-s is used to mean "not set", so put 'e' on end bmax = 0xfffffffe; break; case 'h': case ARG_USAGE: printUsage(cout); throw 0; break; case ARG_SNP: snp_fname = optarg; break; case ARG_HAPLOTYPE: ht_fname = optarg; break; case ARG_SPLICESITE: ss_fname = optarg; break; case ARG_EXON: exon_fname = optarg; break; case ARG_SV: sv_fname = optarg; break; case ARG_REPEAT_REF: repeat_ref_fname = optarg; break; case ARG_REPEAT_INFO: repeat_info_fname = optarg; break; case ARG_REPEAT_SNP: repeat_snp_fname = optarg; break; case ARG_REPEAT_HAPLOTYPE: repeat_haplotype_fname = optarg; break; case ARG_BMAX: bmax = parseNumber(1, "--bmax arg must be at least 1"); bmaxMultSqrt = OFF_MASK; // don't use multSqrt bmaxDivN = 0xffffffff; // don't use multSqrt break; case ARG_BMAX_MULT: bmaxMultSqrt = parseNumber(1, "--bmaxmultsqrt arg must be at least 1"); bmax = OFF_MASK; // don't use bmax bmaxDivN = 0xffffffff; // don't use multSqrt break; case ARG_BMAX_DIV: bmaxDivN = parseNumber(1, "--bmaxdivn arg must be at least 1"); bmax = OFF_MASK; // don't use bmax bmaxMultSqrt = OFF_MASK; // don't use multSqrt break; case ARG_DCV: dcv = parseNumber(3, "--dcv arg must be at least 3"); break; case ARG_SEED: seed = parseNumber(0, "--seed arg must be at least 0"); break; case ARG_REVERSE_EACH: reverseEach = true; break; case ARG_NTOA: nsToAs = true; break; case 'a': autoMem = false; break; case 'q': verbose = false; break; case 's': sanityCheck = true; break; case 'r': writeRef = false; break; case 'p': nthreads = parseNumber(1, "-p arg must be at least 1"); break; case -1: /* Done with options. */ break; case 0: if (long_options[option_index].flag != 0) break; default: printUsage(cerr); throw 1; } } while(next_option != -1); if(bmax < 40) { cerr << "Warning: specified bmax is very small (" << bmax << "). This can lead to" << endl << "extremely slow performance and memory exhaustion. Perhaps you meant to specify" << endl << "a small --bmaxdivn?" << endl; } } EList filesWritten; /** * Delete all the index files that we tried to create. For when we had to * abort the index-building process due to an error. */ static void deleteIdxFiles( const string& outfile, bool doRef, bool justRef) { for(size_t i = 0; i < filesWritten.size(); i++) { cerr << "Deleting \"" << filesWritten[i].c_str() << "\" file written during aborted indexing attempt." << endl; remove(filesWritten[i].c_str()); } } extern void initializeCntLut(); extern void initializeCntBit(); /** * Drive the index construction process and optionally sanity-check the * result. */ template static void driver( const string& infile, EList& infiles, const string& snpfile, const string& htfile, const string& ssfile, const string& exonfile, const string& svfile, const string& repeatfile, const string& outfile, bool packed, int reverse, bool localindex = true, EList* parent_szs = NULL, EList* parent_refnames = NULL, EList* output_szs = NULL, EList* output_refnames = NULL) { initializeCntLut(); initializeCntBit(); EList is(MISC_CAT); bool bisulfite = false; bool repeat = parent_szs != NULL; RefReadInParams refparams(false, reverse, nsToAs, bisulfite); assert_gt(infiles.size(), 0); if(format == CMDLINE) { // Adapt sequence strings to stringstreams open for input stringstream *ss = new stringstream(); for(size_t i = 0; i < infiles.size(); i++) { (*ss) << ">" << i << endl << infiles[i].c_str() << endl; } FileBuf *fb = new FileBuf(ss); assert(fb != NULL); assert(!fb->eof()); assert(fb->get() == '>'); ASSERT_ONLY(fb->reset()); assert(!fb->eof()); is.push_back(fb); } else { // Adapt sequence files to ifstreams for(size_t i = 0; i < infiles.size(); i++) { FILE *f = fopen(infiles[i].c_str(), "r"); if (f == NULL) { cerr << "Error: could not open "<< infiles[i].c_str() << endl; throw 1; } FileBuf *fb = new FileBuf(f); assert(fb != NULL); if(fb->peek() == -1 || fb->eof()) { cerr << "Warning: Empty fasta file: '" << infile.c_str() << "'" << endl; continue; } assert(!fb->eof()); assert(fb->get() == '>'); ASSERT_ONLY(fb->reset()); assert(!fb->eof()); is.push_back(fb); } } if(is.empty()) { cerr << "Warning: All fasta inputs were empty" << endl; throw 1; } filesWritten.push_back(outfile + ".1." + gfm_ext); filesWritten.push_back(outfile + ".2." + gfm_ext); // Vector for the ordered list of "records" comprising the input // sequences. A record represents a stretch of unambiguous // characters in one of the input sequences. EList szs(MISC_CAT); std::pair sztot; { if(verbose) cerr << "Reading reference sizes" << endl; Timer _t(cerr, " Time reading reference sizes: ", verbose); if(!reverse && (writeRef || justRef)) { filesWritten.push_back(outfile + ".3." + gfm_ext); filesWritten.push_back(outfile + ".4." + gfm_ext); sztot = BitPairReference::szsFromFasta(is, outfile, bigEndian, refparams, szs, sanityCheck); } else { assert(false); sztot = BitPairReference::szsFromFasta(is, string(), bigEndian, refparams, szs, sanityCheck); } } if(justRef) return; assert_gt(sztot.first, 0); assert_gt(sztot.second, 0); assert_gt(szs.size(), 0); // Construct index from input strings and parameters filesWritten.push_back(outfile + ".5." + gfm_ext); filesWritten.push_back(outfile + ".6." + gfm_ext); filesWritten.push_back(outfile + ".7." + gfm_ext); filesWritten.push_back(outfile + ".8." + gfm_ext); TStr s; GFM* gfm = NULL; if(!repeat) { // base index gfm = new HGFM( s, packed, 1, // TODO: maybe not? lineRate, offRate, // suffix-array sampling rate ftabChars, // number of chars in initial arrow-pair calc localOffRate, localFtabChars, nthreads, snpfile, htfile, ssfile, exonfile, svfile, repeatfile, outfile, // basename for .?.ht2 files reverse == 0, // fw !entireSA, // useBlockwise bmax, // block size for blockwise SA builder bmaxMultSqrt, // block size as multiplier of sqrt(len) bmaxDivN, // block size as divisor of len noDc? 0 : dcv,// difference-cover period is, // list of input streams szs, // list of reference sizes (TIndexOffU)sztot.first, // total size of all unambiguous ref chars refparams, // reference read-in parameters localindex, // create local indexes? parent_szs, // parent szs parent_refnames, // parent refence names seed, // pseudo-random number generator seed -1, // override offRate verbose, // be talkative autoMem, // pass exceptions up to the toplevel so that we can adjust memory settings automatically sanityCheck); // verify results and internal consistency } else { // repeat index gfm = new RFM( s, packed, 1, // TODO: maybe not? lineRate, offRate, // suffix-array sampling rate ftabChars, // number of chars in initial arrow-pair calc localOffRate, localFtabChars, nthreads, snpfile, htfile, ssfile, exonfile, svfile, repeatfile, outfile, // basename for .?.ht2 files reverse == 0, // fw !entireSA, // useBlockwise bmax, // block size for blockwise SA builder bmaxMultSqrt, // block size as multiplier of sqrt(len) bmaxDivN, // block size as divisor of len noDc? 0 : dcv,// difference-cover period is, // list of input streams szs, // list of reference sizes (TIndexOffU)sztot.first, // total size of all unambiguous ref chars refparams, // reference read-in parameters localindex, // create local indexes? parent_szs, // parent szs parent_refnames, // parent refence names seed, // pseudo-random number generator seed -1, // override offRate verbose, // be talkative autoMem, // pass exceptions up to the toplevel so that we can adjust memory settings automatically sanityCheck); // verify results and internal consistency } if(output_szs != NULL) { *output_szs = szs; } if(output_refnames != NULL) { *output_refnames = gfm->_refnames_nospace; } // Note that the Ebwt is *not* resident in memory at this time. To // load it into memory, call ebwt.loadIntoMemory() if(verbose) { // Print Ebwt's vital stats gfm->gh().print(cerr); } if(sanityCheck) { // Try restoring the original string (if there were // multiple texts, what we'll get back is the joined, // padded string, not a list) gfm->loadIntoMemory( reverse ? (refparams.reverse == REF_READ_REVERSE) : 0, true, // load SA sample? true, // load ftab? true, // load rstarts? false, false); SString s2; gfm->restore(s2); gfm->evictFromMemory(); { SString joinedss; GFM<>::join >( is, // list of input streams szs, // list of reference sizes (TIndexOffU)sztot.first, // total size of all unambiguous ref chars refparams, // reference read-in parameters seed, // pseudo-random number generator seed joinedss); if(refparams.reverse == REF_READ_REVERSE) { joinedss.reverse(); } assert_eq(joinedss.length(), s2.length()); assert(sstr_eq(joinedss, s2)); } if(verbose) { if(s2.length() < 1000) { cout << "Passed restore check: " << s2.toZBuf() << endl; } else { cout << "Passed restore check: (" << s2.length() << " chars)" << endl; } } } delete gfm; } static const char *argv0 = NULL; extern "C" { /** * main function. Parses command-line arguments. */ int hisat2_build(int argc, const char **argv) { string outfile; try { // Reset all global state, including getopt state opterr = optind = 1; resetOptions(); string infile; EList infiles(MISC_CAT); parseOptions(argc, argv); argv0 = argv[0]; if(showVersion) { cout << argv0 << " version " << string(HISAT2_VERSION).c_str() << endl; if(sizeof(void*) == 4) { cout << "32-bit" << endl; } else if(sizeof(void*) == 8) { cout << "64-bit" << endl; } else { cout << "Neither 32- nor 64-bit: sizeof(void*) = " << sizeof(void*) << endl; } cout << "Built on " << BUILD_HOST << endl; cout << BUILD_TIME << endl; cout << "Compiler: " << COMPILER_VERSION << endl; cout << "Options: " << COMPILER_OPTIONS << endl; cout << "Sizeof {int, long, long long, void*, size_t, off_t}: {" << sizeof(int) << ", " << sizeof(long) << ", " << sizeof(long long) << ", " << sizeof(void *) << ", " << sizeof(size_t) << ", " << sizeof(off_t) << "}" << endl; return 0; } // Get input filename if(optind >= argc) { cerr << "No input sequence or sequence file specified!" << endl; printUsage(cerr); return 1; } infile = argv[optind++]; // Get output filename if(optind >= argc) { cerr << "No output file specified!" << endl; printUsage(cerr); return 1; } outfile = argv[optind++]; tokenize(infile, ",", infiles); if(infiles.size() < 1) { cerr << "Tokenized input file list was empty!" << endl; printUsage(cerr); return 1; } if(!lineRate_provided) { if(snp_fname == "" && ss_fname == "" && exon_fname == "") { lineRate = GFM::default_lineRate_fm; } else { lineRate = GFM::default_lineRate_gfm; } } // Optionally summarize if(verbose) { cerr << "Settings:" << endl << " Output files: \"" << outfile.c_str() << ".*." << gfm_ext << "\"" << endl << " Line rate: " << lineRate << " (line is " << (1< parent_szs(MISC_CAT); EList parent_refnames; string dummy_fname = ""; driver >(infile, infiles, snp_fname, ht_fname, ss_fname, exon_fname, sv_fname, dummy_fname, outfile, false, REF_READ_FORWARD, true, // create local indexes NULL, // no parent szs NULL, // no parent refnames &parent_szs, // get parent szs &parent_refnames); // get parent refnames if(repeat_ref_fname.length() > 0) { EList repeat_infiles(MISC_CAT); tokenize(repeat_ref_fname, ",", repeat_infiles); driver >(repeat_ref_fname, repeat_infiles, repeat_snp_fname, repeat_haplotype_fname, dummy_fname, dummy_fname, dummy_fname, repeat_info_fname, outfile + ".rep", false, REF_READ_FORWARD, true, // create local index? &parent_szs, &parent_refnames); } } catch(bad_alloc& e) { if(autoMem) { cerr << "Switching to a packed string representation." << endl; packed = true; } else { throw e; } } } return 0; } catch(std::exception& e) { cerr << "Error: Encountered exception: '" << e.what() << "'" << endl; cerr << "Command: "; for(int i = 0; i < argc; i++) cerr << argv[i] << " "; cerr << endl; deleteIdxFiles(outfile, writeRef || justRef, justRef); return 1; } catch(int e) { if(e != 0) { cerr << "Error: Encountered internal HISAT2 exception (#" << e << ")" << endl; cerr << "Command: "; for(int i = 0; i < argc; i++) cerr << argv[i] << " "; cerr << endl; } deleteIdxFiles(outfile, writeRef || justRef, justRef); return e; } } } hisat2-2.2.1/hisat2_build_main.cpp000066400000000000000000000040441370663763200170060ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include #include #include #include "tokenize.h" #include "ds.h" #include "mem_ids.h" using namespace std; extern "C" { int hisat2_build(int argc, const char **argv); } /** * bowtie-build main function. It is placed in a separate source file * to make it slightly easier to compile as a library. * * If the user specifies -A as the first two arguments, main * will interpret that file as having one set of command-line arguments * per line, and will dispatch each batch of arguments one at a time to * bowtie-build. */ int main(int argc, const char **argv) { if(argc > 2 && strcmp(argv[1], "-A") == 0) { const char *file = argv[2]; ifstream in; in.open(file); char buf[4096]; int lastret = -1; while(in.getline(buf, 4095)) { EList args(MISC_CAT); args.push_back(string(argv[0])); tokenize(buf, " \t", args); const char **myargs = (const char**)malloc(sizeof(char*)*args.size()); for(size_t i = 0; i < args.size(); i++) { myargs[i] = args[i].c_str(); } if(args.size() == 1) continue; lastret = hisat2_build((int)args.size(), myargs); free(myargs); } if(lastret == -1) { cerr << "Warning: No arg strings parsed from " << file << endl; return 0; } return lastret; } else { return hisat2_build(argc, argv); } } hisat2-2.2.1/hisat2_extract_exons.py000077500000000000000000000126671370663763200174540ustar00rootroot00000000000000#!/usr/bin/env python3 # # Copyright 2015, Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # from sys import stderr, exit from collections import defaultdict as dd, Counter from argparse import ArgumentParser, FileType def extract_exons(gtf_file, verbose = False): genes = dd(list) trans = {} # Parse valid exon lines from the GTF file into a dict by transcript_id for line in gtf_file: line = line.strip() if not line or line.startswith('#'): continue if '#' in line: line = line.split('#')[0].strip() try: chrom, source, feature, left, right, score, \ strand, frame, values = line.split('\t') except ValueError: continue left, right = int(left), int(right) if feature != 'exon' or left >= right: continue values_dict = {} for attr in values.split(';')[:-1]: attr, _, val = attr.strip().partition(' ') values_dict[attr] = val.strip('"') if 'gene_id' not in values_dict or \ 'transcript_id' not in values_dict: continue transcript_id = values_dict['transcript_id'] if transcript_id not in trans: trans[transcript_id] = [chrom, strand, [[left, right]]] genes[values_dict['gene_id']].append(transcript_id) else: trans[transcript_id][2].append([left, right]) # Sort exons and merge where separating introns are <=5 bps for tran, [chrom, strand, exons] in trans.items(): exons.sort() tmp_exons = [exons[0]] for i in range(1, len(exons)): if exons[i][0] - tmp_exons[-1][1] <= 5: tmp_exons[-1][1] = exons[i][1] else: tmp_exons.append(exons[i]) trans[tran] = [chrom, strand, tmp_exons] # Calculate and print the unique junctions tmp_exons = set() for chrom, strand, texons in trans.values(): for i in range(len(texons)): tmp_exons.add((chrom, texons[i][0], texons[i][1], strand)) tmp_exons = sorted(tmp_exons) if len(tmp_exons) <= 0: return exons = [tmp_exons[0]] for exon in tmp_exons[1:]: prev_exon = exons[-1] if exon[0] != prev_exon[0]: exons.append(exon) continue assert prev_exon[1] <= exon[1] if prev_exon[2] < exon[1]: exons.append(exon) continue if prev_exon[2] < exon[2]: strand = prev_exon[3] if strand not in "+-": strand = exon[3] exons[-1] = (prev_exon[0], prev_exon[1], exon[2], strand) for chrom, left, right, strand in exons: # Zero-based offset print('{}\t{}\t{}\t{}'.format(chrom, left-1, right-1, strand)) # Print some stats if asked if verbose: None """ exon_lengths, intron_lengths, trans_lengths = \ Counter(), Counter(), Counter() for chrom, strand, exons in trans.values(): tran_len = 0 for i, exon in enumerate(exons): exon_len = exon[1]-exon[0]+1 exon_lengths[exon_len] += 1 tran_len += exon_len if i == 0: continue intron_lengths[exon[0] - exons[i-1][1]] += 1 trans_lengths[tran_len] += 1 print('genes: {}, genes with multiple isoforms: {}'.format( len(genes), sum(len(v) > 1 for v in genes.values())), file=stderr) print('transcripts: {}, transcript avg. length: {:d}'.format( len(trans), sum(trans_lengths.elements())/len(trans)), file=stderr) print('exons: {}, exon avg. length: {:d}'.format( sum(exon_lengths.values()), sum(exon_lengths.elements())/sum(exon_lengths.values())), file=stderr) print('introns: {}, intron avg. length: {:d}'.format( sum(intron_lengths.values()), sum(intron_lengths.elements())/sum(intron_lengths.values())), file=stderr) print('average number of exons per transcript: {:d}'.format( sum(exon_lengths.values())/len(trans)), file=stderr) """ if __name__ == '__main__': parser = ArgumentParser( description='Extract exons from a GTF file') parser.add_argument('gtf_file', nargs='?', type=FileType('r'), help='input GTF file (use "-" for stdin)') parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') args = parser.parse_args() if not args.gtf_file: parser.print_help() exit(1) extract_exons(args.gtf_file, args.verbose) hisat2-2.2.1/hisat2_extract_snps_haplotypes_UCSC.py000077500000000000000000000457011370663763200223630ustar00rootroot00000000000000#!/usr/bin/env python3 # # Copyright 2015, Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # import sys, subprocess import re from argparse import ArgumentParser, FileType from functools import cmp_to_key """ """ def reverse_complement(seq): result = "" for nt in seq: base = nt if nt == 'A': base = 'T' elif nt == 'a': base = 't' elif nt == 'C': base = 'G' elif nt == 'c': base = 'g' elif nt == 'G': base = 'C' elif nt == 'g': base = 'c' elif nt == 'T': base = 'A' elif nt == 't': base = 'a' result = base + result return result """ """ def read_genome(genome_file): chr_dic = {} chr_name, sequence = "", "" for line in genome_file: if line.startswith(">"): if chr_name and sequence: chr_dic[chr_name] = sequence chr_name = line.strip().split()[0][1:] sequence = "" else: sequence += line.strip() if chr_name and sequence: chr_dic[chr_name] = sequence return chr_dic """ Compare two variants [chr, pos, type, data, dic] """ def compare_vars(a, b): a_chr, a_pos, a_type, a_data = a[:4] b_chr, b_pos, b_type, b_data = b[:4] # daehwan - for debugging purposes if a_chr != b_chr: print(a) print(b) assert a_chr == b_chr if a_pos != b_pos: return a_pos - b_pos if a_type != b_type: if a_type == 'I': return -1 elif b_type == 'I': return 1 if a_type == 'S': return -1 else: return 1 if a_data < b_data: return -1 elif a_data > b_data: return 1 else: return 0 """ """ def compatible_vars(a, b): a_chr, a_pos, a_type, a_data = a[:4] b_chr, b_pos, b_type, b_data = b[:4] assert a_chr == b_chr assert a_pos <= b_pos if a_pos == b_pos: return False if a_type == 'D': if b_pos <= a_pos + a_data: return False return True """ """ def generate_haplotypes(snp_file, haplotype_file, vars, inter_gap, intra_gap, num_haplotypes): assert len(vars) > 0 # Sort variants and remove redundant variants vars = sorted(vars, key=cmp_to_key(compare_vars)) tmp_vars = [] v = 0 while v < len(vars): var = vars[v] for v2 in range(v + 1, len(vars)): var2 = vars[v2] if compare_vars(var, var2) == 0: v += 1 else: assert compare_vars(var, var2) < 0 break tmp_vars.append(var) v += 1 vars = tmp_vars # Create new variant ID for variants with the same ID # e.g. same two variant ID, rs60160543, are split into rs60160543.0 and rs60160543.1 vars_count = {} for var in vars: id = var[4]["id"] if id not in vars_count: vars_count[id] = 0 vars_count[id] += 1 vars_duplicate = set() for id, count in vars_count.items(): if count <= 1: continue vars_duplicate.add(id) vars_count = {} for var in vars: id = var[4]["id"] if id not in vars_count: vars_count[id] = 0 else: vars_count[id] += 1 if id not in vars_duplicate: var[4]["id2"] = id else: var[4]["id2"] = "%s.%d" % (id, vars_count[id]) # variant compatibility vars_cmpt = [-1 for i in range(len(vars))] for v in range(len(vars)): var_chr, var_pos, var_type, var_data = vars[v][:4] if var_type == 'D': var_pos += (var_data - 1) for v2 in range(v + 1, len(vars)): if vars_cmpt[v2] >= 0: continue var2_chr, var2_pos = vars[v2][:2] if var_chr != var2_chr: break if var_pos + inter_gap < var2_pos: break vars_cmpt[v2] = v # Assign genotypes for those missing genotypes genotypes_list = [] for v in range(len(vars)): var = vars[v] var_dic = var[4] freq = var_dic["freq"] used = [False for i in range(100)] if vars_cmpt[v] >= 0: v2 = v - 1 while v2 >= vars_cmpt[v]: var2 = vars[v2] if not compatible_vars(var2, var) or \ freq >= 0.1: var2_dic = var2[4] assert "genotype" in var2_dic genotype_num = var2_dic["genotype"] used[genotype_num] = True v2 -= 1 assert False in used for i in range(len(used)): if not used[i]: var_dic["genotype"] = i break genotypes_list.append(var_dic["genotype"]) # Write SNPs into a file (.snp) for var in vars: chr, pos, type, data, var_dic = var varID = var_dic["id2"] if type == 'S': type = "single" elif type == 'D': type = "deletion" else: assert type == 'I' type = "insertion" print("%s\t%s\t%s\t%s\t%s" % (varID, type, chr, pos, data), file=snp_file) # genotypes_list looks like # Var0: 0 # Var1: 0 # Var2: 1 # Var3: 2 # Get haplotypes from genotypes_list max_genotype_num = max(genotypes_list) haplotypes = ["" for i in range(max_genotype_num + 1)] for i in range(len(genotypes_list)): num = genotypes_list[i] if haplotypes[num] == "": haplotypes[num] = str(i) else: haplotypes[num] += ("#%d" % i) haplotypes = set(haplotypes) # haplotypes look like # '8#10#12#23', '8#12#23', '5#8#12#23#30' # Split some haplotypes that include large gaps inside def split_haplotypes(haplotypes): split_haplotypes = set() for haplotype in haplotypes: haplotype = haplotype.split('#') assert len(haplotype) > 0 if len(haplotype) == 1: split_haplotypes.add(haplotype[0]) continue prev_s, s = 0, 1 while s < len(haplotype): _, prev_locus, prev_type, prev_data, _ = vars[int(haplotype[s-1])] _, locus, type, data, _ = vars[int(haplotype[s])] prev_locus, locus = int(prev_locus), int(locus) if prev_type == 'D': prev_locus += (int(prev_data) - 1) if prev_locus + intra_gap < locus: split_haplotypes.add('#'.join(haplotype[prev_s:s])) prev_s = s s += 1 if s == len(haplotype): split_haplotypes.add('#'.join(haplotype[prev_s:s])) return split_haplotypes haplotypes2 = sorted(list(split_haplotypes(haplotypes))) def cmp_haplotype(a, b): a = a.split('#') _, a1_locus, _, _, _ = vars[int(a[0])] _, a2_locus, a2_type, a2_data, _ = vars[int(a[-1])] a_begin, a_end = int(a1_locus), int(a2_locus) if a2_type == 'D': a_end += (int(a2_data) - 1) b = b.split('#') _, b1_locus, _, _, _ = vars[int(b[0])] _, b2_locus, b2_type, b2_data, _ = vars[int(b[-1])] b_begin, b_end = int(b1_locus), int(b2_locus) if b2_type == 'D': b_end += (int(b2_data) - 1) if a_begin != b_begin: return a_begin - b_begin return a_end - b_end haplotypes = sorted(list(haplotypes2), key=cmp_to_key(cmp_haplotype)) # Write haplotypes for h_i in range(len(haplotypes)): h = haplotypes[h_i].split('#') chr, h1_locus, _, _, _ = vars[int(h[0])] _, h2_locus, h2_type, h2_data, _ = vars[int(h[-1])] h_begin, h_end = int(h1_locus), int(h2_locus) if h2_type == 'D': h_end += (int(h2_data) - 1) assert h_begin <= h_end h_new_begin = h_begin for h_j in reversed(range(0, h_i)): hc = haplotypes[h_j].split('#') _, hc_begin, hc_type, hc_data, _ = vars[int(hc[-1])] hc_begin = int(hc_begin) hc_end = hc_begin if hc_type == 'D': hc_end += (int(hc_data) - 1) if hc_end + inter_gap < h_begin: break if h_new_begin > hc_end: h_new_begin = hc_end assert h_new_begin <= h_begin h_add = [] for id in h: var_dic = vars[int(id)][4] h_add.append(var_dic["id2"]) print("ht%d\t%s\t%d\t%d\t%s" % (num_haplotypes, chr, h_new_begin, h_end, ','.join(h_add)), file=haplotype_file) num_haplotypes += 1 return num_haplotypes """ """ def main(genome_file, snp_fname, base_fname, inter_gap, intra_gap, verbose, testset): # load genomic sequences chr_dic = read_genome(genome_file) if testset: ref_testset_file = open(base_fname + ".ref.testset.fa", "w") alt_testset_file = open(base_fname + ".alt.testset.fa", "w") snp_out_file = open(base_fname + ".snp", 'w') haplotype_out_file = open(base_fname + ".haplotype", 'w') # load SNPs snp_list = [] prev_chr, curr_right = "", -1 num_haplotypes = 0 if snp_fname.endswith(".gz"): snp_cmd = ["gzip", "-cd", snp_fname] else: snp_cmd = ["cat", snp_fname] snp_proc = subprocess.Popen(snp_cmd, universal_newlines=True, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) ids_seen = set() for line in snp_proc.stdout: if not line or line.startswith('#'): continue line = line.strip() try: fields = line.split('\t') """ id, chr, start, end, rs_id, score, strand, refNCBI, refUCSC, observed, molType, classType, valid, \ avHet, avHetSE, func, locType, weight, exceptions, submitterCount, submitters, \ alleleFreqCount, alleles, alleleNs, alleleFreqs, bitfields = fields """ id, chr, start, end, rs_id, score, strand, refNCBI, refUCSC, observed, molType, classType = fields[:12] alleleFreqs = fields[-2].split(',')[:-1] if len(alleleFreqs) > 0: try: float(alleleFreqs[0]) except ValueError: alleleFreqs = [] except ValueError: continue start, end = int(start), int(end) score = int(score) if molType != "genomic": continue if classType not in ["single", "deletion", "insertion"]: continue if classType == "single": if start + 1 != end: continue elif classType == "deletion": assert start < end else: assert classType == "insertion" if start != end: continue if chr not in chr_dic: continue chr_seq = chr_dic[chr] chr_len = len(chr_seq) if start >= len(chr_seq): continue if rs_id in ids_seen: continue ids_seen.add(rs_id) if (prev_chr != chr or curr_right + inter_gap < start) and \ len(snp_list) > 0: num_haplotypes = generate_haplotypes(snp_out_file, haplotype_out_file, snp_list, inter_gap, intra_gap, num_haplotypes) snp_list = [] observed = observed.upper() allele_list = observed.split("/") if len(alleleFreqs) == 0: alleleFreqs = [0.0 for i in range(len(allele_list))] # Reverse complement alleles if strand is negative if strand == "-": tmp_allele_list = [] for allele in allele_list: tmp_allele_list.append(reverse_complement(allele)) allele_list = tmp_allele_list if classType == "single": allele_count = min(len(allele_list), len(alleleFreqs)) ref_base = chr_seq[start].upper() if ref_base not in allele_list: continue for a in range(allele_count): allele = allele_list[a] freq = float(alleleFreqs[a]) if allele not in "ACGT" or len(allele) != 1: continue if allele == ref_base: continue snp_list.append([chr, start, 'S', allele, {"id":rs_id, "freq":freq}]) if testset: ref_seq = chr_seq[start-50:start+50] alt_seq = chr_seq[start-50:start] + allele + chr_seq[start+1:start+50] print(">%s_single_%d" % (rs_id, start - 50), file=ref_testset_file) print(ref_seq, file=ref_testset_file) print(">%s_single_%d_%s" % (rs_id, start - 50, ref_seq), file=alt_testset_file) print(alt_seq, file=alt_testset_file) elif classType == "deletion": if start > 0: prev_base = chr_seq[start-1].upper() if prev_base not in "ACGT": continue if len(allele_list) != 2 or \ len(allele_list) != len(alleleFreqs): continue freq = 0.0 if allele_list[0] == "-": freq = float(alleleFreqs[1]) else: if allele_list[1] != "-": continue freq = float(alleleFreqs[0]) delLen = end - start snp_list.append([chr, start, 'D', delLen, {"id":rs_id, "freq":freq}]) if testset and delLen > 0 and delLen <= 10: ref_seq = chr_seq[start-50:start+50] alt_seq = chr_seq[start-50:start] + chr_seq[start+delLen:start+50+delLen] print(">%s_deletion_%d" % (rs_id, start - 50), file=ref_testset_file) print(ref_seq, file=ref_testset_file) print(">%s_deletion_%d_%s" % (rs_id, start - 50, ref_seq), file=alt_testset_file) print(alt_seq, file=alt_testset_file) else: assert classType == "insertion" if start > 0: prev_base = chr_seq[start-1].upper() if prev_base not in "ACGT": continue allele_count = min(len(allele_list), len(alleleFreqs)) for a in range(allele_count): allele = allele_list[a] freq = float(alleleFreqs[a]) if allele == "-" or len(allele) <= 0: continue if re.match('^[ACGT]+$', allele): snp_list.append([chr, start, 'I', allele, {"id":rs_id, "freq":freq}]) insLen = len(allele) if testset and insLen > 0 and insLen <= 10: ref_seq = chr_seq[start-50:start+50] alt_seq = chr_seq[start-50:start] + allele + chr_seq[start:start+50-insLen] print(">%s_insertion_%d" % (rs_id, start - 50), file=ref_testset_file) print(ref_seq, file=ref_testset_file) print(">%s_insertion_%d_%s" % (rs_id, start - 50, ref_seq), file=alt_testset_file) print(alt_seq, file=alt_testset_file) if curr_right < end: curr_right = end if prev_chr != chr: curr_right = end prev_chr = chr if testset: ref_testset_file.close() alt_testset_file.close() if len(snp_list) > 0: generate_haplotypes(snp_out_file, haplotype_out_file, snp_list, inter_gap, intra_gap, num_haplotypes) snp_list = [] snp_out_file.close() haplotype_out_file.close() if __name__ == '__main__': parser = ArgumentParser( description='Extract SNPs and haplotypes from a SNP file downloaded from UCSC (e.g. http://hgdownload.soe.ucsc.edu/goldenPath/hg38/database/snp144.txt.gz)') parser.add_argument('genome_file', nargs='?', type=FileType('r'), help='input genome file (e.g. genome.fa)') parser.add_argument('snp_fname', nargs='?', type=str, help='input snp file downloaded from UCSC (plain text or gzipped file is accepted: snp144Common.txt or snp144Common.txt.gz)') parser.add_argument("base_fname", nargs='?', type=str, help="base filename for SNPs and haplotypes") parser.add_argument("--inter-gap", dest="inter_gap", type=int, default=30, help="Maximum distance for variants to be in the same haplotype") parser.add_argument("--intra-gap", dest="intra_gap", type=int, default=50, help="Break a haplotype into several haplotypes") parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') parser.add_argument('--testset', dest='testset', action='store_true', help='print test reads') args = parser.parse_args() if not args.genome_file or \ not args.snp_fname or \ not args.base_fname: parser.print_help() exit(1) main(args.genome_file, args.snp_fname, args.base_fname, args.inter_gap, args.intra_gap, args.verbose, args.testset) hisat2-2.2.1/hisat2_extract_snps_haplotypes_VCF.py000077500000000000000000001046021370663763200222400ustar00rootroot00000000000000#!/usr/bin/env python3 # # Copyright 2016, Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # import sys, os, subprocess from argparse import ArgumentParser, FileType from functools import cmp_to_key digit2str = [str(i) for i in range(10)] """ """ def read_genome(genome_file): chr_dic = {} chr_name, sequence = "", "" for line in genome_file: if line.startswith(">"): if chr_name and sequence: chr_dic[chr_name] = sequence chr_name = line.strip().split()[0][1:] sequence = "" else: sequence += line.strip() if chr_name and sequence: chr_dic[chr_name] = sequence return chr_dic """ Compare two variants [chr, pos, type, data, dic] """ def compare_vars(a, b): a_chr, a_pos, a_type, a_data = a[:4] b_chr, b_pos, b_type, b_data = b[:4] assert a_chr == b_chr if a_pos != b_pos: return a_pos - b_pos if a_type != b_type: if a_type == 'I': return -1 elif b_type == 'I': return 1 if a_type == 'S': return -1 else: return 1 if a_data < b_data: return -1 elif a_data > b_data: return 1 else: return 0 """ """ def compatible_vars(a, b): a_chr, a_pos, a_type, a_data = a[:4] b_chr, b_pos, b_type, b_data = b[:4] assert a_chr == b_chr assert a_pos <= b_pos if a_pos == b_pos: return False if a_type == 'D': if b_pos <= a_pos + a_data: return False return True """ Given a VCF line, the function reports a list of variants [pos, type, data] type: 'S' for single nucleotide polymorphism, 'D' for deletion, and 'I' for insertion """ def extract_vars(chr_dic, chr, pos, ref_allele, alt_alleles, varID): chr_seq = chr_dic[chr] vars = [] assert ',' not in ref_allele alt_alleles = alt_alleles.split(',') for a in range(len(alt_alleles)): alt_allele = alt_allele2 = alt_alleles[a] if 'N' in alt_allele: continue ref_allele2, pos2 = ref_allele, pos if chr_seq[pos:pos+len(ref_allele)] != ref_allele: print("Error: the reference genome you provided seems to be incompatible with the VCF file at %d of chromosome %s where %s is in the reference genome while %s is in the VCF file" % (pos, chr, chr_seq[pos:pos+len(ref_allele)], ref_allele), file=sys.stderr) def warning_msg(): print("Warning) ref allele (%s) and alt allele (%s in %s) at chr%s:%d are excluded." % \ (ref_allele, alt_allele, ','.join(alt_alleles), chr, pos + 1), file=sys.stderr) min_len = min(len(ref_allele2), len(alt_allele2)) if min_len >= 2: if len(ref_allele2) != len(alt_allele2): if ref_allele2[:min_len-1] != alt_allele2[:min_len-1]: warning_msg() continue ref_allele2, alt_allele2 = ref_allele2[min_len-1:], alt_allele2[min_len-1:] pos2 += (min_len - 1) else: if ref_allele2[1:] != alt_allele2[1:]: warning_msg() continue ref_allele2, alt_allele2 = ref_allele2[0], alt_allele2[0] type, data = '', '' if len(ref_allele2) == 1 and len(alt_allele2) == 1: if ref_allele2 == alt_allele2: warning_msg() continue type = 'S' data = alt_allele2 elif len(ref_allele2) == 1: assert len(alt_allele2) > 1 if ref_allele2[0] != alt_allele2[0]: warning_msg() continue alt_allele2 = alt_allele2[1:] pos2 += 1 type = 'I' data = alt_allele2 if len(data) > 32: continue elif len(alt_allele2) == 1: assert len(ref_allele2) > 1 if ref_allele2[0] != alt_allele2[0]: warning_msg() continue ref_allele2 = ref_allele2[1:] pos2 += 1 type = 'D' data = len(ref_allele2) else: warning_msg() continue varID2 = varID if len(alt_alleles) > 1: varID2 = "%s.%d" % (varID, a) vars.append([chr, pos2, type, data, {"id":varID, "id2":varID2}]) return vars """ """ def generate_haplotypes(snp_file, haplotype_file, vars, inter_gap, intra_gap, num_genomes, num_haplotypes): assert len(vars) > 0 # Sort variants and remove redundant variants vars = sorted(vars, key=cmp_to_key(compare_vars)) tmp_vars = [] v = 0 while v < len(vars): var = vars[v] for v2 in range(v + 1, len(vars)): var2 = vars[v2] if compare_vars(var, var2) == 0: v += 1 if "CLNSIG" not in var[4]: if "CLNSIG" in var2[4]: var[4]["CLNSIG"] = var2[4]["CLNSIG"] if "genotype" not in var[4]: if "genotype" in var2[4]: var[4]["genotype"] = var2[4]["genotype"] else: assert compare_vars(var, var2) < 0 break tmp_vars.append(var) v += 1 vars = tmp_vars # Write SNPs into a file (.snp) for var in vars: chr, pos, type, data, var_dic = var varID = var_dic["id2"] if type == 'S': type = "single" elif type == 'D': type = "deletion" else: assert type == 'I' type = "insertion" print("%s\t%s\t%s\t%s\t%s" % \ (varID, type, chr, pos, data), file=snp_file) # variant compatibility vars_cmpt = [-1 for i in range(len(vars))] for v in range(len(vars)): var_chr, var_pos, var_type, var_data = vars[v][:4] if var_type == 'D': var_pos += (var_data - 1) for v2 in range(v + 1, len(vars)): if vars_cmpt[v2] >= 0: continue var2_chr, var2_pos, var2_type = vars[v2][:3] assert var_chr == var2_chr if var_type == 'D' and var2_type == 'D': if var_pos + 1 < var2_pos: break else: if var_pos < var2_pos: break vars_cmpt[v2] = v # Assign genotypes for those missing genotypes genotypes_list = [] if num_genomes > 0: max_genotype_num = 1 for v in range(len(vars)): var = vars[v] var_dic = var[4] if "genotype" not in var_dic: used = [True, True] + [False for i in range(8)] if vars_cmpt[v] >= 0: v2 = v - 1 while v2 >= vars_cmpt[v]: var2 = vars[v2] if not compatible_vars(var2, var): var2_dic = var2[4] assert "genotype" in var2_dic genotype_num = int(var2_dic["genotype"][0]) used[genotype_num] = True v2 -= 1 assert False in used for i in range(len(used)): if not used[i]: var_dic["genotype"] = ("%d" % i) * (num_genomes * 2) if i > max_genotype_num: max_genotype_num = i break genotypes_list.append(var_dic["genotype"]) num_chromosomes = len(genotypes_list[0]) # daehwan - for debugging purposes """ for v in range(len(vars)): var = vars[v] var_chr, var_pos, var_type, var_data, var_dic = var print v, var_chr, var_pos, var_type, var_data, var_dic["id"], var_dic["id2"], if "CLNSIG" in var_dic: print "CLNSIG:", var_dic["CLNSIG"], if "genotype" in var_dic: print var_dic["genotype"][:50], print """ # genotypes_list looks like # Var0: 000001000 # Var1: 010000000 # Var2: 001100000 # Var3: 222222222 # Get haplotypes from genotypes_list haplotypes = set() cnv_genotypes = ["" for i in range(num_chromosomes)] for genotypes in genotypes_list: for i in range(len(genotypes)): genotype = genotypes[i] cnv_genotypes[i] += genotype cnv_genotypes = set(cnv_genotypes) for raw_haplotype in cnv_genotypes: for num in range(1, max_genotype_num + 1): num_str = str(num) if num_str not in raw_haplotype: continue haplotype = "" for i in range(len(raw_haplotype)): if raw_haplotype[i] == num_str: if haplotype == "": haplotype = str(i) else: haplotype += ("#%d" % i) assert haplotype != "" haplotypes.add(haplotype) else: for v in range(len(vars)): var = vars[v] var_dic = var[4] used = [False for i in range(100)] if vars_cmpt[v] >= 0: v2 = v - 1 while v2 >= vars_cmpt[v]: var2 = vars[v2] if not compatible_vars(var2, var): var2_dic = var2[4] assert "genotype" in var2_dic genotype_num = var2_dic["genotype"] used[genotype_num] = True v2 -= 1 assert False in used for i in range(len(used)): if not used[i]: var_dic["genotype"] = i break genotypes_list.append(var_dic["genotype"]) # genotypes_list looks like # Var0: 0 # Var1: 0 # Var2: 1 # Var3: 2 # Get haplotypes from genotypes_list max_genotype_num = max(genotypes_list) haplotypes = ["" for i in range(max_genotype_num + 1)] for i in range(len(genotypes_list)): num = genotypes_list[i] if haplotypes[num] == "": haplotypes[num] = str(i) else: haplotypes[num] += ("#%d" % i) haplotypes = set(haplotypes) # haplotypes look like # '8#10#12#23', '8#12#23', '5#8#12#23#30' # Split some haplotypes that include large gaps inside def split_haplotypes(haplotypes): split_haplotypes = set() for haplotype in haplotypes: haplotype = haplotype.split('#') assert len(haplotype) > 0 if len(haplotype) == 1: split_haplotypes.add(haplotype[0]) continue prev_s, s = 0, 1 while s < len(haplotype): _, prev_locus, prev_type, prev_data, _ = vars[int(haplotype[s-1])] _, locus, type, data, _ = vars[int(haplotype[s])] prev_locus, locus = int(prev_locus), int(locus) if prev_type == 'D': prev_locus += (int(prev_data) - 1) if prev_locus + intra_gap < locus: split_haplotypes.add('#'.join(haplotype[prev_s:s])) prev_s = s s += 1 if s == len(haplotype): split_haplotypes.add('#'.join(haplotype[prev_s:s])) return split_haplotypes haplotypes2 = sorted(list(split_haplotypes(haplotypes))) def cmp_haplotype(a, b): a = a.split('#') _, a1_locus, _, _, _ = vars[int(a[0])] _, a2_locus, a2_type, a2_data, _ = vars[int(a[-1])] a_begin, a_end = int(a1_locus), int(a2_locus) if a2_type == 'D': a_end += (int(a2_data) - 1) b = b.split('#') _, b1_locus, _, _, _ = vars[int(b[0])] _, b2_locus, b2_type, b2_data, _ = vars[int(b[-1])] b_begin, b_end = int(b1_locus), int(b2_locus) if b2_type == 'D': b_end += (int(b2_data) - 1) if a_begin != b_begin: return a_begin - b_begin return a_end - b_end haplotypes = sorted(list(haplotypes2), key=cmp_to_key(cmp_haplotype)) # daehwan - for debugging purposes """ dis = prev_locus - locus print "\n[%d, %d]: %d haplotypes" % (i, j, len(haplotypes)), dis if len(cur_vars) in range(0, 1000): # print "vars:", sorted(list(cur_vars), cmp=cmp_varKey print "num:", len(haplotypes) for haplotype in haplotypes: print haplotype.split('#') print "\nnum:", len(haplotypes2) for haplotype in haplotypes2: print haplotype.split('#') """ # Write haplotypes for h_i in range(len(haplotypes)): h = haplotypes[h_i].split('#') chr, h1_locus, _, _, _ = vars[int(h[0])] _, h2_locus, h2_type, h2_data, _ = vars[int(h[-1])] h_begin, h_end = int(h1_locus), int(h2_locus) if h2_type == 'D': h_end += (int(h2_data) - 1) assert h_begin <= h_end h_new_begin = h_begin for h_j in reversed(range(0, h_i)): hc = haplotypes[h_j].split('#') _, hc_begin, hc_type, hc_data, _ = vars[int(hc[-1])] hc_begin = int(hc_begin) hc_end = hc_begin if hc_type == 'D': hc_end += (int(hc_data) - 1) if hc_end + inter_gap < h_begin: break if h_new_begin > hc_end: h_new_begin = hc_end assert h_new_begin <= h_begin h_add = [] for id in h: var_dic = vars[int(id)][4] h_add.append(var_dic["id2"]) print("ht%d\t%s\t%d\t%d\t%s" % \ (num_haplotypes, chr, h_new_begin, h_end, ','.join(h_add)), file=haplotype_file) num_haplotypes += 1 return num_haplotypes """ """ def main(genome_file, VCF_fnames, base_fname, inter_gap, intra_gap, only_rs, reference_type, genotype_vcf, genotype_gene_list, extra_files, verbose): # Load genomic sequences chr_dic = read_genome(genome_file) # GRCh38 - ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502/supporting/GRCh38_positions # ALL.chr22.phase3_shapeit2_mvncall_integrated_v3plus_nounphased.rsID.genotypes.GRCh38_dbSNP_no_SVs.vcf.gz # GRCh37 - ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20130502 # ALL.chr22.phase3_shapeit2_mvncall_integrated_v5a.20130502.genotypes.vcf.gz # List of variants (e.g. ClinVar database) genotype_var_list = {} # List of genomic regions to be processed genotype_ranges = {} if genotype_vcf != "": var_set = set() assert len(genotype_gene_list) > 0 if genotype_vcf.endswith(".gz"): vcf_cmd = ["gzip", "-cd", genotype_vcf] else: vcf_cmd = ["cat", genotype_vcf] vcf_proc = subprocess.Popen(vcf_cmd, universal_newlines=True, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) for line in vcf_proc.stdout: if line.startswith("#"): continue chr, pos, varID, ref_allele, alt_alleles, qual, filter, info = line.strip().split('\t') pos = int(pos) - 1 if chr not in chr_dic: continue gene = None for g in genotype_gene_list: if info.find(g) != -1: gene = g break if not gene: continue CLNSIG = -1 for item in info.split(';'): if not item.startswith("CLNSIG"): continue try: key, value = item.split('=') CLNSIG = int(value) except ValueError: continue if CLNSIG not in [4, 5]: continue if CLNSIG == 4: CLNSIG = "Likely pathogenic" else: CLNSIG = "Pathogenic" vars = extract_vars(chr_dic, chr, pos, ref_allele, alt_alleles, varID) if len(vars) == 0: continue if chr not in genotype_var_list: genotype_var_list[chr] = [] genotype_ranges[chr] = {} if gene not in genotype_ranges[chr]: genotype_ranges[chr][gene] = [len(chr_dic[chr]), -1] for var in vars: var_chr, var_pos, var_ref_allele, var_alt_allele = var[:4] var_str = "%s-%d-%s-%s" % (var_chr, var_pos, var_ref_allele, var_alt_allele) if var_str in var_set: continue var[4]["CLNSIG"] = CLNSIG var[4]["gene"] = gene genotype_var_list[chr].append(var) if var_pos < genotype_ranges[chr][gene][0]: genotype_ranges[chr][gene][0] = var_pos if var_pos > genotype_ranges[chr][gene][1]: genotype_ranges[chr][gene][1] = var_pos var_set.add(var_str) print("Number of variants in %s is:" % (genotype_vcf), file=sys.stderr) for chr, vars in genotype_var_list.items(): vars = sorted(vars, cmp=compare_vars) print("\tChromosome %s: %d variants" % (chr, len(vars)), file=sys.stderr) for chr, gene_ranges in genotype_ranges.items(): for gene, value in gene_ranges.items(): gene_ranges[gene] = [value[0] - 100, value[1] + 100] value = genotype_ranges[chr][gene] if verbose: print("%s\t%s\t%d-%d" % (chr, gene, value[0], value[1]), file=sys.stderr) if extra_files or True: clnsig_file = open("%s.clnsig" % base_fname, 'w') for chr, vars in genotype_var_list.items(): for var in vars: varID = var[4]["id2"] CLNSIG = var[4]["CLNSIG"] gene = var[4]["gene"] print("%s\t%s\t%s" % (varID, gene, CLNSIG), file=clnsig_file) clnsig_file.close() SNP_file = open("%s.snp" % base_fname, 'w') haplotype_file = open("%s.haplotype" % base_fname, 'w') # Write reference information and backbone sequences into files if extra_files: ref_file = open("%s.ref" % base_fname, 'w') for chr, gene_ranges in genotype_ranges.items(): for gene, value in gene_ranges.items(): left, right = value if reference_type == "gene": left, right = 0, right - left print("%s\t%s\t%d\t%d" % (gene, chr, left, right), file=ref_file) ref_file.close() if reference_type == "gene": backbone_file = open("%s_backbone.fa" % base_fname, 'w') for chr, gene_ranges in genotype_ranges.items(): for gene, value in gene_ranges.items(): left, right = value left, right = 0, right - left print(">%s" % (gene), file=backbone_file) backbone_seq = chr_dic[chr][value[0]:value[1]+1] for s in range(0, len(backbone_seq), 60): print(backbone_seq[s:s+60], file=backbone_file) backbone_file.close() elif reference_type == "chromosome": first = True for chr in genotype_ranges.keys(): if first: os.system("samtools faidx genome.fa %s > %s_backbone.fa" % (chr, base_fname)) first = False else: os.system("samtools faidx genome.fa %s >> %s_backbone.fa" % (chr, base_fname)) else: assert reference_type == "genome" os.system("cp genome.fa %s_backbone.fa" % base_fname) num_genomes = 0 num_haplotypes = 0 num_unassigned = 0 unnamed_var_count = 0 for VCF_fname in VCF_fnames: empty_VCF_file = False if VCF_fname == "/dev/null" or \ not os.path.exists(VCF_fname): empty_VCF_file = True if reference_type != "genome" and \ len(genotype_gene_list) > 0: continue if not empty_VCF_file: if VCF_fname.endswith(".gz"): vcf_cmd = ["gzip", "-cd", VCF_fname] else: vcf_cmd = ["cat", VCF_fname] vcf_proc = subprocess.Popen(vcf_cmd, universal_newlines=True, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) genomeIDs = [] vars, genotypes_list = [], [] prev_varID, prev_chr, prev_pos = "", "", -1 num_lines = 0 for line in vcf_proc.stdout: num_lines += 1 if line.startswith("##"): continue fields = line.strip().split('\t') chr, pos, varID, ref_allele, alt_alleles, qual, filter, info = fields[:8] if prev_chr != chr: curr_right = -1 if len(fields) >= 9: format = fields[8] genotypes = [] if len(fields) >= 10: genotypes = fields[9:] if line.startswith("#"): genomeIDs = genotypes num_genomes = len(genomeIDs) continue assert len(genotypes) == len(genomeIDs) if varID == ".": unnamed_var_count += 1 varID = "un%d" % unnamed_var_count if only_rs and not varID.startswith("rs"): continue if ';' in varID: continue if varID == prev_varID: continue if chr not in chr_dic: continue chr_seq = chr_dic[chr] chr_genotype_vars = [] chr_genotype_ranges = {} if len(genotype_gene_list) > 0: assert chr in genotype_var_list chr_genotype_vars = genotype_var_list[chr] assert chr in genotype_ranges chr_genotype_ranges = genotype_ranges[chr] pos = int(pos) - 1 offset = 0 gene = None if num_lines % 10000 == 1: print("\t%s:%d\r" % (chr, pos), file=sys.stderr) if chr_genotype_ranges: skip = True for gene_, range_ in chr_genotype_ranges.items(): if pos > range_[0] and pos < range_[1]: skip = False break if skip: continue if len(vars) == 0: for var in chr_genotype_vars: var_chr, var_pos, var_type, var_data, var_dic = var if var_pos < range_[0]: continue if var_pos > range_[1]: break if reference_type == "gene": var_pos -= range_[0] vars.append([gene_, var_pos, var_type, var_data, var_dic]) curr_right = range_[1] if reference_type == "gene": offset = range_[0] gene = gene_ if pos == prev_pos: continue if len(vars) > 0 and \ (curr_right + inter_gap < pos or prev_chr != chr): num_haplotypes = generate_haplotypes(SNP_file, haplotype_file, vars, inter_gap, intra_gap, num_genomes, num_haplotypes) vars = [] def add_vars(pos, offset, gene, varID, ref_allele, alt_alleles, vars, genotypes): tmp_vars = extract_vars(chr_dic, chr, pos, ref_allele, alt_alleles, varID) max_right = -1 for v in range(len(tmp_vars)): var = tmp_vars[v] _, pos2, type, data = var[:4] cnv_genotypes = [] for genotype in genotypes: P1, P2 = genotype[0], genotype[2] if P1 == digit2str[v + 1]: cnv_genotypes.append('1') else: cnv_genotypes.append('0') if P2 == digit2str[v + 1]: cnv_genotypes.append('1') else: cnv_genotypes.append('0') # Skip SNPs not present in a given population (e.g. 2,504 genomes in 1000 Genomes Project) if cnv_genotypes != [] and \ '1' not in cnv_genotypes: continue tmp_varID = var[4]["id2"] var_dic = {"id":varID, "id2":tmp_varID, "genotype":''.join(cnv_genotypes)} if reference_type == "gene": vars.append([gene, pos2 - offset, type, data, var_dic]) else: vars.append([chr, pos2, type, data, var_dic]) right = pos2 if type == 'D': right += (int(data) - 1) if max_right < right: max_right = right return max_right right = add_vars(pos, offset, gene, varID, ref_allele, alt_alleles, vars, genotypes) if curr_right < right: curr_right = right prev_varID = varID prev_chr = chr prev_pos = pos if len(vars) > 0: num_haplotypes = generate_haplotypes(SNP_file, haplotype_file, vars, inter_gap, intra_gap, num_genomes, num_haplotypes) vars = [] else: for chr in genotype_var_list.keys(): chr_seq = chr_dic[chr] chr_genotype_vars = genotype_var_list[chr] curr_right = -1 vars = [] for var in chr_genotype_vars: var_chr, var_pos, var_type, var_data, var_dic = var num_genomes = 0 if len(vars) > 0 and curr_right + inter_gap < var_pos: num_haplotypes = generate_haplotypes(SNP_file, haplotype_file, vars, inter_gap, intra_gap, num_genomes, num_haplotypes) vars = [] vars.append([var_chr, var_pos, var_type, var_data, var_dic]) curr_right = var_pos if var_type == 'D': curr_right += (var_data - 1) if len(vars) > 0: num_haplotypes = generate_haplotypes(SNP_file, haplotype_file, vars, inter_gap, intra_gap, num_genomes, num_haplotypes) vars = [] SNP_file.close() haplotype_file.close() if genotype_vcf != "": clnsig_file.close() if __name__ == '__main__': parser = ArgumentParser( description='Extract SNPs and haplotypes from VCF files') parser.add_argument('genome_file', nargs='?', type=FileType('r'), help='input genome file (e.g. genome.fa)') parser.add_argument('VCF_fnames', nargs='?', type=str, help='A comma-seperated VCF files (plain text or gzipped file is accepted: GRCh38_dbSNP_no_SVs.vcf or GRCh38_dbSNP_no_SVs.vcf.gz') parser.add_argument("base_fname", nargs='?', type=str, help="base filename for SNPs and haplotypes") parser.add_argument("--reference-type", dest="reference_type", type=str, default="genome", help="Reference type: gene, chromosome, and genome (default: genome)") parser.add_argument("--inter-gap", dest="inter_gap", type=int, default=30, help="Maximum distance for variants to be in the same haplotype (default: 30)") parser.add_argument("--intra-gap", dest="intra_gap", type=int, default=50, help="Break a haplotype into several haplotypes (default: 50)") parser.add_argument('--non-rs', dest='only_rs', action='store_false', help='Allow SNP IDs not beginning with rs') parser.add_argument('--genotype-vcf', dest='genotype_vcf', type=str, default="", help='VCF file name for genotyping (default: empty)') parser.add_argument('--genotype-gene-list', dest='genotype_gene_list', type=str, default="", help='A comma-separated list of genes to be genotyped (default: empty)') parser.add_argument('--extra-files', dest='extra_files', action='store_true', help='Output extra files such as _backbone.fa and .ref') parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') args = parser.parse_args() if not args.genome_file or \ not args.VCF_fnames or \ not args.base_fname: parser.print_help() exit(1) args.VCF_fnames = args.VCF_fnames.split(',') if args.genotype_vcf != "": if args.genotype_gene_list == "": genes = set() if args.genotype_vcf.endswith(".gz"): vcf_cmd = ["gzip", "-cd", args.genotype_vcf] else: vcf_cmd = ["cat", args.genotype_vcf] vcf_proc = subprocess.Popen(vcf_cmd, universal_newlines=True, stdout=subprocess.PIPE, stderr=open("/dev/null", 'w')) for line in vcf_proc.stdout: if line.startswith("#"): continue info = line.strip().split()[-1] if info.find("GENEINFO=") == -1: continue gene = info.split("GENEINFO=")[1] gene = gene.split(':')[0] genes.add(gene) args.genotype_gene_list = list(genes) else: args.genotype_gene_list = args.genotype_gene_list.split(',') if len(args.genotype_gene_list) == 0: print("Error: please specify --genotype-gene-list.", file=sys.stderr) sys.exit(1) else: args.genotype_gene_list = [] main(args.genome_file, args.VCF_fnames, args.base_fname, args.inter_gap, args.intra_gap, args.only_rs, args.reference_type, args.genotype_vcf, args.genotype_gene_list, args.extra_files, args.verbose) hisat2-2.2.1/hisat2_extract_splice_sites.py000077500000000000000000000116331370663763200207760ustar00rootroot00000000000000#!/usr/bin/env python3 # # Copyright 2015, Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # from sys import stderr, exit from collections import defaultdict as dd, Counter from argparse import ArgumentParser, FileType def extract_splice_sites(gtf_file, verbose=False): genes = dd(list) trans = {} # Parse valid exon lines from the GTF file into a dict by transcript_id for line in gtf_file: line = line.strip() if not line or line.startswith('#'): continue if '#' in line: line = line.split('#')[0].strip() try: chrom, source, feature, left, right, score, \ strand, frame, values = line.split('\t') except ValueError: continue left, right = int(left), int(right) if feature != 'exon' or left >= right: continue values_dict = {} for attr in values.split(';'): if attr: attr, _, val = attr.strip().partition(' ') values_dict[attr] = val.strip('"') if 'gene_id' not in values_dict or \ 'transcript_id' not in values_dict: continue transcript_id = values_dict['transcript_id'] if transcript_id not in trans: trans[transcript_id] = [chrom, strand, [[left, right]]] genes[values_dict['gene_id']].append(transcript_id) else: trans[transcript_id][2].append([left, right]) # Sort exons and merge where separating introns are <=5 bps for tran, [chrom, strand, exons] in trans.items(): exons.sort() tmp_exons = [exons[0]] for i in range(1, len(exons)): if exons[i][0] - tmp_exons[-1][1] <= 5: tmp_exons[-1][1] = exons[i][1] else: tmp_exons.append(exons[i]) trans[tran] = [chrom, strand, tmp_exons] # Calculate and print the unique junctions junctions = set() for chrom, strand, exons in trans.values(): for i in range(1, len(exons)): junctions.add((chrom, exons[i-1][1], exons[i][0], strand)) junctions = sorted(junctions) for chrom, left, right, strand in junctions: # Zero-based offset print('{}\t{}\t{}\t{}'.format(chrom, left-1, right-1, strand)) # Print some stats if asked if verbose: exon_lengths, intron_lengths, trans_lengths = \ Counter(), Counter(), Counter() for chrom, strand, exons in trans.values(): tran_len = 0 for i, exon in enumerate(exons): exon_len = exon[1]-exon[0]+1 exon_lengths[exon_len] += 1 tran_len += exon_len if i == 0: continue intron_lengths[exon[0] - exons[i-1][1]] += 1 trans_lengths[tran_len] += 1 print('genes: {}, genes with multiple isoforms: {}'.format( len(genes), sum(len(v) > 1 for v in genes.values())), file=stderr) print('transcripts: {}, transcript avg. length: {:.0f}'.format( len(trans), sum(trans_lengths.elements())//len(trans)), file=stderr) print('exons: {}, exon avg. length: {:.0f}'.format( sum(exon_lengths.values()), sum(exon_lengths.elements())//sum(exon_lengths.values())), file=stderr) print('introns: {}, intron avg. length: {:.0f}'.format( sum(intron_lengths.values()), sum(intron_lengths.elements())//sum(intron_lengths.values())), file=stderr) print('average number of exons per transcript: {:.0f}'.format( sum(exon_lengths.values())//len(trans)), file=stderr) if __name__ == '__main__': parser = ArgumentParser( description='Extract splice junctions from a GTF file') parser.add_argument('gtf_file', nargs='?', type=FileType('r'), help='input GTF file (use "-" for stdin)') parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') args = parser.parse_args() if not args.gtf_file: parser.print_help() exit(1) extract_splice_sites(args.gtf_file, args.verbose) hisat2-2.2.1/hisat2_inspect.cpp000066400000000000000000000677541370663763200163710ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include #include #include #include "assert_helpers.h" #include "endian_swap.h" #include "hgfm.h" #include "reference.h" #include "ds.h" #include "alt.h" using namespace std; MemoryTally gMemTally; static bool showVersion = false; // just print version and quit? int verbose = 0; // be talkative static int names_only = 0; // just print the sequence names in the index static int snp_only = 0; static int splicesite_only = 0; static int splicesite_all_only = 0; static int exon_only = 0; static int summarize_only = 0; // just print summary of index and quit static int across = 60; // number of characters across in FASTA output static bool refFromGFM = false; // true -> when printing reference, decode it from Gbwt instead of reading it from BitPairReference static string wrapper; static const char *short_options = "vhnsea:"; enum { ARG_VERSION = 256, ARG_WRAPPER, ARG_USAGE, ARG_SNP, ARG_SPLICESITE, ARG_SPLICESITE_ALL, ARG_EXON, }; static struct option long_options[] = { {(char*)"verbose", no_argument, 0, 'v'}, {(char*)"version", no_argument, 0, ARG_VERSION}, {(char*)"usage", no_argument, 0, ARG_USAGE}, {(char*)"names", no_argument, 0, 'n'}, {(char*)"snp", no_argument, 0, ARG_SNP}, {(char*)"ss", no_argument, 0, ARG_SPLICESITE}, {(char*)"ss-all", no_argument, 0, ARG_SPLICESITE_ALL}, {(char*)"exon", no_argument, 0, ARG_EXON}, {(char*)"summary", no_argument, 0, 's'}, {(char*)"help", no_argument, 0, 'h'}, {(char*)"across", required_argument, 0, 'a'}, {(char*)"gbwt-ref", no_argument, 0, 'g'}, {(char*)"wrapper", required_argument, 0, ARG_WRAPPER}, {(char*)0, 0, 0, 0} // terminator }; /** * Print a summary usage message to the provided output stream. */ static void printUsage(ostream& out) { out << "HISAT2 version " << string(HISAT2_VERSION).c_str() << " by Daehwan Kim (infphilo@gmail.com, http://www.ccb.jhu.edu/people/infphilo)" << endl; out << "Usage: hisat2-inspect [options]* " << endl << " ht2 filename minus trailing .1." << gfm_ext << "/.2." << gfm_ext << endl << endl << " By default, prints FASTA records of the indexed nucleotide sequences to" << endl << " standard out. With -n, just prints names. With -s, just prints a summary of" << endl << " the index parameters and sequences. With -e, preserves colors if applicable." << endl << endl << "Options:" << endl; if(wrapper == "basic-0") { out << " --large-index force inspection of the 'large' index, even if a" << endl << " 'small' one is present." << endl; } out << " -a/--across Number of characters across in FASTA output (default: 60)" << endl << " -s/--summary Print summary incl. ref names, lengths, index properties" << endl << " -n/--names Print reference sequence names only" << endl << " --snp Print SNPs" << endl << " --ss Print splice sites" << endl << " --ss-all Print splice sites including those not in the global index" << endl << " --exon Print exons" << endl << " -e/--ht2-ref Reconstruct reference from ." << gfm_ext << " (slow, preserves colors)" << endl << " -v/--verbose Verbose output (for debugging)" << endl << " -h/--help print detailed description of tool and its options" << endl << " --usage print this usage message" << endl ; if(wrapper.empty()) { cerr << endl << "*** Warning ***" << endl << "'hisat-inspect' was run directly. It is recommended " << "to use the wrapper script instead." << endl << endl; } } /** * Parse an int out of optarg and enforce that it be at least 'lower'; * if it is less than 'lower', than output the given error message and * exit with an error and a usage message. */ static int parseInt(int lower, const char *errmsg) { long l; char *endPtr= NULL; l = strtol(optarg, &endPtr, 10); if (endPtr != NULL) { if (l < lower) { cerr << errmsg << endl; printUsage(cerr); throw 1; } return (int32_t)l; } cerr << errmsg << endl; printUsage(cerr); throw 1; return -1; } /** * Read command-line arguments */ static void parseOptions(int argc, char **argv) { int option_index = 0; int next_option; do { next_option = getopt_long(argc, argv, short_options, long_options, &option_index); switch (next_option) { case ARG_WRAPPER: wrapper = optarg; break; case ARG_USAGE: case 'h': printUsage(cout); throw 0; break; case 'v': verbose = true; break; case ARG_VERSION: showVersion = true; break; case 'g': refFromGFM = true; break; case 'n': names_only = true; break; case ARG_SNP: snp_only = true; break; case ARG_SPLICESITE: splicesite_only = true; break; case ARG_SPLICESITE_ALL: splicesite_all_only = true; break; case ARG_EXON: exon_only = true; break; case 's': summarize_only = true; break; case 'a': across = parseInt(-1, "-a/--across arg must be at least 1"); break; case -1: break; /* Done with options. */ case 0: if (long_options[option_index].flag != 0) break; default: printUsage(cerr); throw 1; } } while(next_option != -1); } static void print_fasta_record( ostream& fout, const string& defline, const string& seq) { fout << ">"; fout << defline.c_str() << endl; if(across > 0) { size_t i = 0; while (i + across < seq.length()) { fout << seq.substr(i, across).c_str() << endl; i += across; } if (i < seq.length()) fout << seq.substr(i).c_str() << endl; } else { fout << seq.c_str() << endl; } } /** * Given output stream, BitPairReference, reference index, name and * length, print the whole nucleotide reference with the appropriate * number of columns. */ static void print_ref_sequence( ostream& fout, BitPairReference& ref, const string& name, size_t refi, size_t len) { bool newlines = across > 0; int myacross = across > 0 ? across : 60; size_t incr = myacross * 1000; uint32_t *buf = new uint32_t[(incr + 128)/4]; fout << ">" << name.c_str() << "\n"; ASSERT_ONLY(SStringExpandable destU32); for(size_t i = 0; i < len; i += incr) { size_t amt = min(incr, len-i); assert_leq(amt, incr); int off = ref.getStretch(buf, refi, i, amt ASSERT_ONLY(, destU32)); uint8_t *cb = ((uint8_t*)buf) + off; for(size_t j = 0; j < amt; j++) { if(newlines && j > 0 && (j % myacross) == 0) fout << "\n"; assert_range(0, 4, (int)cb[j]); fout << "ACGTN"[(int)cb[j]]; } fout << "\n"; } delete []buf; } /** * Create a BitPairReference encapsulating the reference portion of the * index at the given basename. Iterate through the reference * sequences, sending each one to print_ref_sequence to print. */ static void print_ref_sequences( ostream& fout, const EList& refnames, const TIndexOffU* plen, const string& adjustedGFMFileBase) { BitPairReference ref( adjustedGFMFileBase, // input basename NULL, false, // true -> expect colorspace reference false, // sanity-check reference NULL, // infiles NULL, // originals false, // infiles are sequences false, // memory-map false, // use shared memory false, // sweep mm-mapped ref verbose, // be talkative verbose); // be talkative at startup assert_eq(ref.numRefs(), refnames.size()); for(size_t i = 0; i < ref.numRefs(); i++) { print_ref_sequence( fout, ref, refnames[i], i, plen[i]); } } /** * Given an index, reconstruct the reference by LF mapping through the * entire thing. */ template static void print_index_sequences(ostream& fout, GFM& gfm) { EList* refnames = &(gfm.refnames()); TStr cat_ref; gfm.restore(cat_ref); TIndexOffU curr_ref = OFF_MASK; string curr_ref_seq = ""; TIndexOffU curr_ref_len = OFF_MASK; TIndexOffU last_text_off = 0; size_t orig_len = cat_ref.length(); TIndexOffU tlen = OFF_MASK; bool first = true; for(size_t i = 0; i < orig_len; i++) { TIndexOffU tidx = OFF_MASK; TIndexOffU textoff = OFF_MASK; tlen = OFF_MASK; bool straddled = false; gfm.joinedToTextOff(1 /* qlen */, (TIndexOffU)i, tidx, textoff, tlen, true, straddled); if (tidx != OFF_MASK && textoff < tlen) { if (curr_ref != tidx) { if (curr_ref != OFF_MASK) { // Add trailing gaps, if any exist if(curr_ref_seq.length() < curr_ref_len) { curr_ref_seq += string(curr_ref_len - curr_ref_seq.length(), 'N'); } print_fasta_record(fout, (*refnames)[curr_ref], curr_ref_seq); } curr_ref = tidx; curr_ref_seq = ""; curr_ref_len = tlen; last_text_off = 0; first = true; } TIndexOffU textoff_adj = textoff; if(first && textoff > 0) textoff_adj++; if (textoff_adj - last_text_off > 1) curr_ref_seq += string(textoff_adj - last_text_off - 1, 'N'); curr_ref_seq.push_back("ACGT"[int(cat_ref[i])]); last_text_off = textoff; first = false; } } if (curr_ref < refnames->size()) { // Add trailing gaps, if any exist if(curr_ref_seq.length() < curr_ref_len) { curr_ref_seq += string(curr_ref_len - curr_ref_seq.length(), 'N'); } print_fasta_record(fout, (*refnames)[curr_ref], curr_ref_seq); } } static char *argv0 = NULL; template static void print_index_sequence_names(const string& fname, ostream& fout) { EList p_refnames; readEbwtRefnames(fname, p_refnames); for(size_t i = 0; i < p_refnames.size(); i++) { cout << p_refnames[i].c_str() << endl; } } /** * Print a short summary of what's in the index and its flags. */ template static void print_snps( const string& fname, ostream& fout) { ALTDB altdb; GFM gfm( fname, &altdb, NULL, NULL, -1, // don't require entire reverse true, // index is for the forward direction -1, // offrate (-1 = index default) 0, // offrate-plus (0 = index default) false, // use memory-mapped IO false, // use shared memory false, // sweep memory-mapped memory true, // load names? false, // load SA sample? false, // load ftab? false, // load rstarts? true, // load splice sites? verbose, // be talkative? verbose, // be talkative at startup? false, // pass up memory exceptions? false, // sanity check? false); // use haplotypes? gfm.loadIntoMemory( -1, // need entire reverse true, // load SA sample true, // load ftab true, // load rstarts true, // load names verbose); // verbose EList p_refnames; readEbwtRefnames(fname, p_refnames); const EList >& alts = altdb.alts(); const EList& altnames = altdb.altnames(); assert_eq(alts.size(), altnames.size()); for(size_t i = 0; i < alts.size(); i++) { const ALT& alt = alts[i]; if(!alt.snp()) continue; if(alt.deletion() && alt.reversed) continue; string type = "single"; if(alt.type == ALT_SNP_DEL) { type = "deletion"; } else if(alt.type == ALT_SNP_INS) { type = "insertion"; } index_t tidx = 0, toff = 0, tlen = 0; bool straddled2 = false; gfm.joinedToTextOff( 1, alt.pos, tidx, toff, tlen, true, // reject straddlers? straddled2); // straddled? cout << altnames[i] << "\t" << type << "\t"; assert_lt(tidx, p_refnames.size()); cout << p_refnames[tidx] << "\t" << toff << "\t"; if(alt.type == ALT_SNP_SGL) { cout << "ACGT"[alt.seq & 0x3]; } else if(alt.type == ALT_SNP_DEL) { cout << alt.len; } else if(alt.type == ALT_SNP_INS) { for(index_t i = 0; i < alt.len; i++) { int nt = (alt.seq >> ((alt.len - i - 1) << 1)) & 0x3; cout << "ACGT"[nt]; } } cout << endl; } } /** * Print a short summary of what's in the index and its flags. */ template static void print_splicesites( const string& fname, ostream& fout) { ALTDB altdb; GFM gfm( fname, &altdb, NULL, NULL, -1, // don't require entire reverse true, // index is for the forward direction -1, // offrate (-1 = index default) 0, // offrate-plus (0 = index default) false, // use memory-mapped IO false, // use shared memory false, // sweep memory-mapped memory true, // load names? false, // load SA sample? false, // load ftab? false, // load rstarts? true, // load splice sites? verbose, // be talkative? verbose, // be talkative at startup? false, // pass up memory exceptions? false, // sanity check? false); // use haplotypes? gfm.loadIntoMemory( -1, // need entire reverse true, // load SA sample true, // load ftab true, // load rstarts true, // load names verbose); // verbose EList p_refnames; readEbwtRefnames(fname, p_refnames); const EList >& alts = altdb.alts(); for(size_t i = 0; i < alts.size(); i++) { const ALT& alt = alts[i]; if(!alt.splicesite()) continue; if(alt.left >= alt.right) continue; if(!splicesite_all_only && alt.excluded) continue; index_t tidx = 0, toff = 0, tlen = 0; bool straddled2 = false; gfm.joinedToTextOff( 1, alt.left, tidx, toff, tlen, true, // reject straddlers? straddled2); // straddled? index_t tidx2 = 0, toff2 = 0, tlen2 = 0; gfm.joinedToTextOff( 1, alt.right, tidx2, toff2, tlen2, true, // reject straddlers? straddled2); // straddled? assert_eq(tidx, tidx2); assert_lt(tidx, p_refnames.size()); cout << p_refnames[tidx] << "\t" << toff - 1 << "\t" << toff2 + 1 << "\t" << (alt.fw > 0 ? "+" : "-") << endl; } } /** * Print a short summary of what's in the index and its flags. */ template static void print_exons( const string& fname, ostream& fout) { ALTDB altdb; GFM gfm( fname, &altdb, NULL, NULL, -1, // don't require entire reverse true, // index is for the forward direction -1, // offrate (-1 = index default) 0, // offrate-plus (0 = index default) false, // use memory-mapped IO false, // use shared memory false, // sweep memory-mapped memory true, // load names? false, // load SA sample? false, // load ftab? false, // load rstarts? true, // load splice sites? verbose, // be talkative? verbose, // be talkative at startup? false, // pass up memory exceptions? false, // sanity check? false); // use haplotypes? gfm.loadIntoMemory( -1, // need entire reverse true, // load SA sample true, // load ftab true, // load rstarts true, // load names verbose); // verbose EList p_refnames; readEbwtRefnames(fname, p_refnames); const EList >& alts = altdb.alts(); for(size_t i = 0; i < alts.size(); i++) { const ALT& alt = alts[i]; if(!alt.exon()) continue; index_t tidx = 0, toff = 0, tlen = 0; bool straddled2 = false; gfm.joinedToTextOff( 1, alt.left, tidx, toff, tlen, true, // reject straddlers? straddled2); // straddled? index_t tidx2 = 0, toff2 = 0, tlen2 = 0; gfm.joinedToTextOff( 1, alt.right, tidx2, toff2, tlen2, true, // reject straddlers? straddled2); // straddled? assert_eq(tidx, tidx2); assert_lt(tidx, p_refnames.size()); cout << p_refnames[tidx] << "\t" << toff - 1 << "\t" << toff2 + 1 << "\t" << (alt.fw > 0 ? "+" : "-") << endl; } } /** * Print a short summary of what's in the index and its flags. */ template static void print_index_summary( const string& fname, ostream& fout) { int major, minor; string extra_version; int32_t flags = GFM::readVersionFlags(fname, major, minor, extra_version); bool entireReverse = false; ALTDB altdb; GFM gfm( fname, &altdb, NULL, NULL, -1, // don't require entire reverse true, // index is for the forward direction -1, // offrate (-1 = index default) 0, // offrate-plus (0 = index default) false, // use memory-mapped IO false, // use shared memory false, // sweep memory-mapped memory true, // load names? false, // load SA sample? false, // load ftab? false, // load rstarts? true, // load splice sites? verbose, // be talkative? verbose, // be talkative at startup? false, // pass up memory exceptions? false, // sanity check? false); // use haplotypes? EList p_refnames; readEbwtRefnames(fname, p_refnames); cout << "Index version" << "\t2." << major << '.' << minor; if(extra_version != "") { cout << "-" << extra_version; } cout << endl; cout << "Flags" << '\t' << (-flags) << endl; cout << "2.0-compatible" << '\t' << (entireReverse ? "1" : "0") << endl; cout << "SA-Sample" << "\t1 in " << (1 << gfm.gh().offRate()) << endl; cout << "FTab-Chars" << '\t' << gfm.gh().ftabChars() << endl; assert_eq(gfm.nPat(), p_refnames.size()); for(size_t i = 0; i < p_refnames.size(); i++) { cout << "Sequence-" << (i+1) << '\t' << p_refnames[i].c_str() << '\t' << gfm.plen()[i] << endl; } index_t numSnps = 0, numSpliceSites = 0, numExons = 0; const EList >& alts = altdb.alts(); for(size_t i = 0; i < alts.size(); i++) { const ALT& alt = alts[i]; if(alt.snp()) { numSnps++; } else if(alt.splicesite()) { if(alt.left < alt.right) { numSpliceSites++; } } else if(alt.exon()) { numExons++; } } cout << "Num. SNPs: " << numSnps << endl; cout << "Num. Splice Sites: " << numSpliceSites << endl; cout << "Num. Exons: " << numExons << endl; } extern void initializeCntLut(); extern void initializeCntBit(); static void driver( const string& ebwtFileBase, const string& query) { initializeCntLut(); initializeCntBit(); // Adjust string adjustedEbwtFileBase = adjustEbwtBase(argv0, ebwtFileBase, verbose); if (names_only) { print_index_sequence_names(adjustedEbwtFileBase, cout); } else if(summarize_only) { print_index_summary(adjustedEbwtFileBase, cout); } else if(snp_only) { print_snps(adjustedEbwtFileBase, cout); } else if(splicesite_only || splicesite_all_only) { print_splicesites(adjustedEbwtFileBase, cout); } else if(exon_only) { print_exons(adjustedEbwtFileBase, cout); } else { // Initialize Ebwt object ALTDB altdb; HGFM gfm( adjustedEbwtFileBase, &altdb, NULL, NULL, -1, // don't care about entire-reverse true, // index is for the forward direction -1, // offrate (-1 = index default) 0, // offrate-plus (0 = index default) false, // use memory-mapped IO false, // use shared memory false, // sweep memory-mapped memory true, // load names? true, // load SA sample? true, // load ftab? true, // load rstarts? true, // load splice sites? false, // be talkative? false, // be talkative at startup? false, // pass up memory exceptions? false, // sanity check? false); // use haplotypes? gfm.loadIntoMemory( -1, // need entire reverse true, // load SA sample true, // load ftab true, // load rstarts true, // load names verbose); // verbose // Load whole index into memory if(refFromGFM) { print_index_sequences >(cout, gfm); } else { EList refnames; readEbwtRefnames(adjustedEbwtFileBase, refnames); print_ref_sequences( cout, refnames, gfm.plen(), adjustedEbwtFileBase); } // Evict any loaded indexes from memory if(gfm.isInMemory()) { gfm.evictFromMemory(); } } } /** * main function. Parses command-line arguments. */ int main(int argc, char **argv) { try { string ebwtFile; // read serialized Ebwt from this file string query; // read query string(s) from this file EList queries; string outfile; // write query results to this file argv0 = argv[0]; parseOptions(argc, argv); if(showVersion) { cout << argv0 << " version " << HISAT2_VERSION << endl; if(sizeof(void*) == 4) { cout << "32-bit" << endl; } else if(sizeof(void*) == 8) { cout << "64-bit" << endl; } else { cout << "Neither 32- nor 64-bit: sizeof(void*) = " << sizeof(void*) << endl; } cout << "Built on " << BUILD_HOST << endl; cout << BUILD_TIME << endl; cout << "Compiler: " << COMPILER_VERSION << endl; cout << "Options: " << COMPILER_OPTIONS << endl; cout << "Sizeof {int, long, long long, void*, size_t, off_t}: {" << sizeof(int) << ", " << sizeof(long) << ", " << sizeof(long long) << ", " << sizeof(void *) << ", " << sizeof(size_t) << ", " << sizeof(off_t) << "}" << endl; return 0; } // Get input filename if(optind >= argc) { cerr << "No index name given!" << endl; printUsage(cerr); return 1; } ebwtFile = argv[optind++]; // Optionally summarize if(verbose) { cout << "Input ht2 file: \"" << ebwtFile.c_str() << "\"" << endl; cout << "Output file: \"" << outfile.c_str() << "\"" << endl; cout << "Local endianness: " << (currentlyBigEndian()? "big":"little") << endl; #ifdef NDEBUG cout << "Assertions: disabled" << endl; #else cout << "Assertions: enabled" << endl; #endif } driver(ebwtFile, query); return 0; } catch(std::exception& e) { cerr << "Error: Encountered exception: '" << e.what() << "'" << endl; cerr << "Command: "; for(int i = 0; i < argc; i++) cerr << argv[i] << " "; cerr << endl; return 1; } catch(int e) { if(e != 0) { cerr << "Error: Encountered internal HISAT2 exception (#" << e << ")" << endl; cerr << "Command: "; for(int i = 0; i < argc; i++) cerr << argv[i] << " "; cerr << endl; } return e; } } hisat2-2.2.1/hisat2_main.cpp000066400000000000000000000037521370663763200156340ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include #include #include #include "tokenize.h" #include "ds.h" using namespace std; extern "C" { int hisat2(int argc, const char **argv); } /** * Bowtie main function. It is placed in a separate source file to * make it slightly easier to compile Bowtie as a library. * * If the user specifies -A as the first two arguments, main * will interpret that file as having one set of command-line arguments * per line, and will dispatch each batch of arguments one at a time to * bowtie. */ int main(int argc, const char **argv) { if(argc > 2 && strcmp(argv[1], "-A") == 0) { const char *file = argv[2]; ifstream in; in.open(file); char buf[4096]; int lastret = -1; while(in.getline(buf, 4095)) { EList args; args.push_back(string(argv[0])); tokenize(buf, " \t", args); const char **myargs = (const char**)malloc(sizeof(char*)*args.size()); for(size_t i = 0; i < args.size(); i++) { myargs[i] = args[i].c_str(); } if(args.size() == 1) continue; lastret = hisat2((int)args.size(), myargs); free(myargs); } if(lastret == -1) { cerr << "Warning: No arg strings parsed from " << file << endl; return 0; } return lastret; } else { return hisat2(argc, argv); } } hisat2-2.2.1/hisat2_read_statistics.py000077500000000000000000000130551370663763200177430ustar00rootroot00000000000000#!/usr/bin/env python3 # # Copyright 2018, Chanhee Park and Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # import os, sys, math, gzip, bz2 from argparse import ArgumentParser, FileType """ """ COMPRESSION_NON = 0 COMPRESSION_GZIP = 1 COMPRESSION_BZIP2 = 2 SEQUENCE_UNKNOWN = -1 SEQUENCE_FASTA = 0 SEQUENCE_FASTQ = 1 FASTA_EXTENSIONS = ["fa", "fasta", "fna"] FASTQ_EXTENSIONS = ["fq", "fastq"] MAX_SKIP_LINES = 10000 """ """ def parser_FQ(fp): # skip empty line skip_line_count = 0 while skip_line_count < MAX_SKIP_LINES: line = fp.readline() if line == "": # end of file return if line[0] == '@': break skip_line_count += 1 if skip_line_count == MAX_SKIP_LINES: raise ValueError("Invalid file format") while True: id = line[1:].split()[0] seq = "" line = fp.readline() if line == "": return seq = line.strip() yield id, seq line = fp.readline() # '+' line = fp.readline() # quality line = fp.readline() # next ID if line == "": return """ """ def parser_FA(fp): # skip empty line skip_line_count = 0 while skip_line_count < MAX_SKIP_LINES: line = fp.readline() if line == "": # end of file return if line[0] == '>': break skip_line_count += 1 if skip_line_count == MAX_SKIP_LINES: raise ValueError("Invalid file format") while True: id = line[1:].split()[0] seq = "" while True: line = fp.readline() if line == "": break if line[0] == '>': break seq += line.strip() yield id, seq if line == "": return """ """ def parse_type(fname): compression_type = COMPRESSION_NON sequence_type = SEQUENCE_UNKNOWN ff = fname.split('.') ext = ff[-1] if ext.lower() == "gz": compression_type = COMPRESSION_GZIP ext = ff[-2] elif ext.lower() == "bz2": compression_type = COMPRESSION_BZIP2 ext = ff[-2] if ext.lower() in FASTA_EXTENSIONS: sequence_type = SEQUENCE_FASTA elif ext.lower() in FASTQ_EXTENSIONS: sequence_type = SEQUENCE_FASTQ return sequence_type, compression_type """ """ def generate_stats(length_map): mn = 0 # minimun read length mx = 0 # maximum read length cnt = 0 # number of reads avg = 0 # average read length sum = 0 if len(length_map) == 0: return cnt, mn, mx, avg # sort keys sorted_map = sorted(length_map) mn = sorted_map[0] mx = sorted_map[-1] for k, v in length_map.items(): sum += k * v cnt += v avg = sum // cnt return cnt, mn, mx, avg """ """ def reads_stat(read_file, read_count): length_map = {} try: sequence_type, compression_type = parse_type(read_file) if compression_type == COMPRESSION_GZIP: fp = gzip.open(read_file, 'rt') elif compression_type == COMPRESSION_BZIP2: fp = bz2.open(read_file, 'rt') else: assert (compression_type == COMPRESSION_NON) fp = open(read_file, 'r') if sequence_type == SEQUENCE_FASTA: fstream = parser_FA(fp) elif sequence_type == SEQUENCE_FASTQ: fstream = parser_FQ(fp) else: raise ValueError("Unsupported file format") cnt = 0 for id, seq in fstream: l = len(seq) if l in length_map: length_map[l] += 1 else: length_map[l] = 1 cnt += 1 if read_count > 0 and cnt >= read_count: break fp.close() except BaseException as e: print("Warning: {}".format(e), file=sys.stderr) cnt, mn, mx, avg = generate_stats(length_map) # sort by (read count, read length) length_map = sorted(length_map.items(), key=lambda t: (t[1], t[0]), reverse=True) if len(length_map) == 0: length_map.append((0, 0)) print(cnt, mn, mx, avg, ",".join([str(k) for (k,v) in length_map])) if __name__ == '__main__': parser = ArgumentParser( description='Compute statistics of reads. Show number of reads and minimum, maximum, average length of reads') parser.add_argument('read_file', nargs='?', type=str, help='reads file') parser.add_argument('-n', dest='read_count', action='store', type=int, default=10000, help='reads count (default: 10000)') args = parser.parse_args() if not args.read_file: parser.print_help() exit(1) reads_stat(args.read_file, args.read_count) hisat2-2.2.1/hisat2_repeat.cpp000066400000000000000000000760601370663763200161720ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include #include #include #include #include #include "assert_helpers.h" #include "endian_swap.h" #include "formats.h" #include "sequence_io.h" #include "tokenize.h" #include "timer.h" #include "ref_read.h" #include "filebuf.h" #include "reference.h" #include "ds.h" #include "gfm.h" #include "aligner_sw.h" #include "aligner_result.h" #include "search_globals.h" #include "scoring.h" #include "mask.h" #include "repeat_builder.h" /** * \file Driver for the bowtie-build indexing tool. */ #include #include #include #include #include MemoryTally gMemTally; // Build parameters int verbose; static int sanityCheck; static int format; static TIndexOffU bmax; static TIndexOffU bmaxMultSqrt; static uint32_t bmaxDivN; static int dcv; static int noDc; static int entireSA; static int seed; static int showVersion; // GFM parameters static int32_t lineRate; static bool lineRate_provided; static int32_t linesPerSide; static int32_t offRate; static int32_t ftabChars; static int32_t localOffRate; static int32_t localFtabChars; static int bigEndian; static bool autoMem; static int nthreads; // number of pthreads operating concurrently static string wrapper; static TIndexOffU seed_length; static TIndexOffU seed_count; static TIndexOffU repeat_count; static TIndexOffU min_repeat_length; static TIndexOffU max_repeat_length; static EList > repeat_length_pair; static TIndexOffU max_repeat_edit; static TIndexOffU max_repeat_matchlen; static bool symmetric_extend; static bool repeat_indel; static bool forward_only; static bool CGtoTG; static string repeat_str1; static string repeat_str2; TIndexOffU max_seed_mm; TIndexOffU max_seed_repeat; TIndexOffU max_seed_extlen; static bool save_sa; static bool load_sa; static void resetOptions() { verbose = true; // be talkative (default) sanityCheck = 0; // do slow sanity checks format = FASTA; // input sequence format bmax = OFF_MASK; // max blockwise SA bucket size bmaxMultSqrt = OFF_MASK; // same, as multplier of sqrt(n) bmaxDivN = 4; // same, as divisor of n dcv = 1024; // bwise SA difference-cover sample sz noDc = 0; // disable difference-cover sample entireSA = 0; // 1 = disable blockwise SA seed = 0; // srandom seed showVersion = 0; // just print version and quit? // GFM parameters lineRate = GFM::default_lineRate_gfm; lineRate_provided = false; linesPerSide = 1; // 1 64-byte line on a side offRate = 4; // sample 1 out of 16 SA elts ftabChars = 10; // 10 chars in initial lookup table localOffRate = 3; localFtabChars = 6; bigEndian = 0; // little endian autoMem = true; // automatically adjust memory usage parameters nthreads = 1; seed_length = 50; seed_count = 5; min_repeat_length = 100; max_repeat_length = numeric_limits::max(); repeat_length_pair.clear(); repeat_count = 5; max_repeat_edit = 10; max_repeat_matchlen = min_repeat_length / 2; // half of repeat_length repeat_indel = false; symmetric_extend = true; forward_only = false; CGtoTG = false; max_seed_mm = 5; max_seed_repeat = 5; max_seed_extlen = 25; save_sa = false; load_sa = false; wrapper.clear(); } // Argument constants for getopts enum { ARG_BMAX = 256, ARG_BMAX_MULT, ARG_BMAX_DIV, ARG_DCV, ARG_SEED, ARG_CUTOFF, ARG_PMAP, ARG_NTOA, ARG_USAGE, ARG_REVERSE_EACH, ARG_SA, ARG_SEED_LENGTH, ARG_SEED_COUNT, ARG_MIN_REPEAT_LENGTH, ARG_MAX_REPEAT_LENGTH, ARG_REPEAT_LENGTH, ARG_REPEAT_COUNT, ARG_REPEAT_EDIT, ARG_REPEAT_MATCHLEN, ARG_REPEAT_INDEL, ARG_WRAPPER, ARG_ASYMMETRIC_EXTEND, ARG_FORWARD_ONLY, ARG_CGTOTG, ARG_REPEAT_STR1, ARG_REPEAT_STR2, ARG_MAX_SEED_MM, ARG_MAX_SEED_REPEAT, ARG_MAX_SEED_EXTLEN, ARG_SAVE_SA, ARG_LOAD_SA, }; /** * Print a detailed usage message to the provided output stream. */ static void printUsage(ostream& out) { out << "HISAT2 version " << string(HISAT2_VERSION).c_str() << " by Chanhee Park and Daehwan Kim " << endl; string tool_name = "hisat2-repeat"; out << "Usage: " << tool_name << " [options]* " << endl << " reference_in comma-separated list of files with ref sequences" << endl << "Options:" << endl << " -c reference sequences given on cmd line (as" << endl << " )" << endl; if(wrapper == "basic-0") { out << " --large-index force generated index to be 'large', even if ref" << endl << " has fewer than 4 billion nucleotides" << endl; } out << " -a/--noauto disable automatic -p/--bmax/--dcv memory-fitting" << endl << " -p number of threads" << endl << " --bmax max bucket sz for blockwise suffix-array builder" << endl << " --bmaxdivn max bucket sz as divisor of ref len (default: 4)" << endl << " --dcv diff-cover period for blockwise (default: 1024)" << endl << " --nodc disable diff-cover (algorithm becomes quadratic)" << endl << " --seed-length seed length (default: 50)" << endl << " --seed-count seed count (default: 5)" << endl << " --min-repeat-length minimum repeat length (default: 100)" << endl << " --max-repeat-length maximum repeat length (default: 65535)" << endl << " --repeat-length -[,-] minimum-maximum repeat length pairs" << endl << " --repeat-count minimum repeat count (default: 5)" << endl << " --repeat-edit maximum repeat edit distance (default: 10)" << endl << " --repeat-matchlen " << endl << " --repeat-indel" << endl << " --repeat-str1" << endl << " --repeat-str2" << endl << " --asymmetric-extend extend seeds asymmetrically" << endl << " --forward-only use forward strand only" << endl << " --CGtoTG change CG to TG" << endl << " --max-seed-mm " << endl << " --max-seed-repeat " << endl << " --max-seed-extlen " << endl << " --save-sa" << endl << " --load-sa" << endl << " -q/--quiet disable verbose output (for debugging)" << endl << " -h/--help print detailed description of tool and its options" << endl << " --usage print this usage message" << endl << " --version print version information and quit" << endl ; } static const char *short_options = "qrap:h?nscfl:i:o:t:h:3C"; static struct option long_options[] = { {(char*)"quiet", no_argument, 0, 'q'}, {(char*)"sanity", no_argument, 0, 's'}, {(char*)"threads", required_argument, 0, 'p'}, {(char*)"little", no_argument, &bigEndian, 0}, {(char*)"big", no_argument, &bigEndian, 1}, {(char*)"bmax", required_argument, 0, ARG_BMAX}, {(char*)"bmaxmultsqrt", required_argument, 0, ARG_BMAX_MULT}, {(char*)"bmaxdivn", required_argument, 0, ARG_BMAX_DIV}, {(char*)"dcv", required_argument, 0, ARG_DCV}, {(char*)"nodc", no_argument, &noDc, 1}, {(char*)"seed", required_argument, 0, ARG_SEED}, {(char*)"entiresa", no_argument, &entireSA, 1}, {(char*)"version", no_argument, &showVersion, 1}, {(char*)"noauto", no_argument, 0, 'a'}, {(char*)"noblocks", required_argument, 0, 'n'}, {(char*)"linerate", required_argument, 0, 'l'}, {(char*)"linesperside", required_argument, 0, 'i'}, {(char*)"usage", no_argument, 0, ARG_USAGE}, {(char*)"seed-length", required_argument, 0, ARG_SEED_LENGTH}, {(char*)"seed-count", required_argument, 0, ARG_SEED_COUNT}, {(char*)"min-repeat-length", required_argument, 0, ARG_MIN_REPEAT_LENGTH}, {(char*)"max-repeat-length", required_argument, 0, ARG_MAX_REPEAT_LENGTH}, {(char*)"repeat-length", required_argument, 0, ARG_REPEAT_LENGTH}, {(char*)"repeat-count", required_argument, 0, ARG_REPEAT_COUNT}, {(char*)"repeat-edit", required_argument, 0, ARG_REPEAT_EDIT}, {(char*)"repeat-matchlen",required_argument, 0, ARG_REPEAT_MATCHLEN}, {(char*)"repeat-indel", no_argument, 0, ARG_REPEAT_INDEL}, {(char*)"wrapper", required_argument, 0, ARG_WRAPPER}, {(char*)"asymmetric-extend", no_argument, 0, ARG_ASYMMETRIC_EXTEND}, {(char*)"forward-only", no_argument, 0, ARG_FORWARD_ONLY}, {(char*)"CGtoTG", no_argument, 0, ARG_CGTOTG}, {(char*)"repeat-str1", required_argument, 0, ARG_REPEAT_STR1}, {(char*)"repeat-str2", required_argument, 0, ARG_REPEAT_STR2}, {(char*)"max-seed-mm", required_argument, 0, ARG_MAX_SEED_MM}, {(char*)"max-seed-repeat",required_argument, 0, ARG_MAX_SEED_REPEAT}, {(char*)"max-seed-extlen",required_argument, 0, ARG_MAX_SEED_EXTLEN}, {(char*)"save-sa", no_argument, 0, ARG_SAVE_SA}, {(char*)"load-sa", no_argument, 0, ARG_LOAD_SA}, {(char*)0, 0, 0, 0} // terminator }; /** * Parse an int out of optarg and enforce that it be at least 'lower'; * if it is less than 'lower', then output the given error message and * exit with an error and a usage message. */ template static T parseNumber(const char *str, T lower, const char *errmsg) { char *endPtr= NULL; T t = (T)strtoll(str, &endPtr, 10); if(endPtr != NULL) { if(t < lower) { cerr << errmsg << endl; printUsage(cerr); throw 1; } return t; } cerr << errmsg << endl; printUsage(cerr); throw 1; return -1; } template static T parseNumber(T lower, const char *errmsg) { return parseNumber(optarg, lower, errmsg); } static void parsePair(EList >& repeat_pair) { string tok; istringstream ss(optarg); while(getline(ss, tok, ',')) { if(tok.empty()) { continue; } TIndexOffU min_len, max_len; size_t pos = tok.find('-'); if(pos == string::npos) { // min min_len = parseNumber(tok.c_str(), 1, "min-repeat-length must be at least 1"); max_len = numeric_limits::max(); } else if(pos == tok.length() - 1) { // min- min_len = parseNumber(tok.substr(0, pos).c_str(), 1, "min-repeat-length must be at least 1"); max_len = numeric_limits::max(); } else if(pos == 0) { // -max // not support? min_len = 100; max_len = parseNumber(tok.substr(pos + 1).c_str(), 1, "max-repeat-length must be at least 1"); } else { min_len = parseNumber(tok.substr(0, pos).c_str(), 1, "min-repeat-length must be at least 1"); max_len = parseNumber(tok.substr(pos + 1).c_str(), 1, "max-repeat-length must be at least 1"); } if(min_len > max_len) { printUsage(cerr); throw 1; } if(max_len > numeric_limits::max()) { printUsage(cerr); throw 1; } repeat_pair.push_back(pair(min_len, max_len)); } } /** * Read command-line arguments */ static void parseOptions(int argc, const char **argv) { int option_index = 0; int next_option; bool saw_max_repeat_matchlen = false; do { next_option = getopt_long( argc, const_cast(argv), short_options, long_options, &option_index); switch (next_option) { case ARG_WRAPPER: wrapper = optarg; break; case 'f': format = FASTA; break; case 'c': format = CMDLINE; break; //case 'p': packed = true; break; case 'C': cerr << "Error: -C specified but Bowtie 2 does not support colorspace input." << endl; throw 1; break; case 'l': lineRate = parseNumber(3, "-l/--lineRate arg must be at least 3"); lineRate_provided = true; break; case 'i': linesPerSide = parseNumber(1, "-i/--linesPerSide arg must be at least 1"); break; case 'o': offRate = parseNumber(0, "-o/--offRate arg must be at least 0"); break; case 'n': // all f-s is used to mean "not set", so put 'e' on end bmax = 0xfffffffe; break; case 'h': case ARG_USAGE: printUsage(cout); throw 0; break; case ARG_BMAX: bmax = parseNumber(1, "--bmax arg must be at least 1"); bmaxMultSqrt = OFF_MASK; // don't use multSqrt bmaxDivN = 0xffffffff; // don't use multSqrt break; case ARG_BMAX_MULT: bmaxMultSqrt = parseNumber(1, "--bmaxmultsqrt arg must be at least 1"); bmax = OFF_MASK; // don't use bmax bmaxDivN = 0xffffffff; // don't use multSqrt break; case ARG_BMAX_DIV: bmaxDivN = parseNumber(1, "--bmaxdivn arg must be at least 1"); bmax = OFF_MASK; // don't use bmax bmaxMultSqrt = OFF_MASK; // don't use multSqrt break; case ARG_DCV: dcv = parseNumber(3, "--dcv arg must be at least 3"); break; case ARG_SEED: seed = parseNumber(0, "--seed arg must be at least 0"); break; case ARG_SEED_LENGTH: seed_length = parseNumber(1, "--seed-length arg must be at least 1"); break; case ARG_SEED_COUNT: seed_count = parseNumber(2, "--repeat-count arg must be at least 2"); break; case ARG_MIN_REPEAT_LENGTH: min_repeat_length = parseNumber(1, "--min-repeat-length arg must be at least 1"); break; case ARG_MAX_REPEAT_LENGTH: max_repeat_length = parseNumber(1, "--max-repeat-length arg must be at least 1"); break; case ARG_REPEAT_LENGTH: parsePair(repeat_length_pair); break; case ARG_REPEAT_COUNT: repeat_count = parseNumber(2, "--repeat-count arg must be at least 2"); break; case ARG_REPEAT_EDIT: max_repeat_edit = parseNumber(0, "--repeat-edit arg must be at least 0"); break; case ARG_REPEAT_MATCHLEN: max_repeat_matchlen = parseNumber(0, "--repeat-matchlen arg must be at least 0"); saw_max_repeat_matchlen = true; break; case ARG_REPEAT_INDEL: repeat_indel = true; break; case ARG_ASYMMETRIC_EXTEND: symmetric_extend = false; break; case ARG_FORWARD_ONLY: forward_only = true; break; case ARG_CGTOTG: CGtoTG = true; break; case ARG_REPEAT_STR1: repeat_str1 = optarg; break; case ARG_REPEAT_STR2: repeat_str2 = optarg; break; case ARG_MAX_SEED_MM: max_seed_mm = parseNumber(1, "--max_seed_mm arg must be at least 1"); break; case ARG_MAX_SEED_REPEAT: max_seed_repeat = parseNumber(5, "--max_seed_repeat arg must be at least 5"); break; case ARG_MAX_SEED_EXTLEN: max_seed_extlen = parseNumber(0, "--max_seed_extlen arg must be at least 0"); break; case ARG_SAVE_SA: save_sa = true; break; case ARG_LOAD_SA: load_sa = true; break; case 'a': autoMem = false; break; case 'q': verbose = false; break; case 's': sanityCheck = true; break; case 'p': nthreads = parseNumber(1, "-p arg must be at least 1"); break; case -1: /* Done with options. */ break; case 0: if (long_options[option_index].flag != 0) break; default: printUsage(cerr); throw 1; } } while(next_option != -1); if(bmax < 40) { cerr << "Warning: specified bmax is very small (" << bmax << "). This can lead to" << endl << "extremely slow performance and memory exhaustion. Perhaps you meant to specify" << endl << "a small --bmaxdivn?" << endl; } if(!saw_max_repeat_matchlen) { max_repeat_matchlen = min_repeat_length / 2; } } extern void initializeCntLut(); extern void initializeCntBit(); /** * Drive the index construction process and optionally sanity-check the * result. */ template static void driver( const string& infile, EList& infiles, const string& outfile, bool packed, bool forward_only, bool CGtoTG) { initializeCntLut(); initializeCntBit(); EList is(MISC_CAT); bool bisulfite = false; bool nsToAs = false; RefReadInParams refparams(false, false /* reverse */, nsToAs, bisulfite); assert_gt(infiles.size(), 0); if(format == CMDLINE) { // Adapt sequence strings to stringstreams open for input stringstream *ss = new stringstream(); for(size_t i = 0; i < infiles.size(); i++) { (*ss) << ">" << i << endl << infiles[i].c_str() << endl; } FileBuf *fb = new FileBuf(ss); assert(fb != NULL); assert(!fb->eof()); assert(fb->get() == '>'); ASSERT_ONLY(fb->reset()); assert(!fb->eof()); is.push_back(fb); } else { // Adapt sequence files to ifstreams for(size_t i = 0; i < infiles.size(); i++) { FILE *f = fopen(infiles[i].c_str(), "r"); if (f == NULL) { cerr << "Error: could not open "<< infiles[i].c_str() << endl; throw 1; } FileBuf *fb = new FileBuf(f); assert(fb != NULL); if(fb->peek() == -1 || fb->eof()) { cerr << "Warning: Empty fasta file: '" << infile.c_str() << "'" << endl; continue; } assert(!fb->eof()); assert(fb->get() == '>'); ASSERT_ONLY(fb->reset()); assert(!fb->eof()); is.push_back(fb); } } if(is.empty()) { cerr << "Warning: All fasta inputs were empty" << endl; throw 1; } // Vector for the ordered list of "records" comprising the input // sequences. A record represents a stretch of unambiguous // characters in one of the input sequences. EList szs; EList ref_names; std::pair sztot; { if(verbose) cerr << "Reading reference sizes" << endl; Timer _t(cerr, " Time reading reference sizes: ", verbose); sztot = BitPairReference::szsFromFasta(is, "", bigEndian, refparams, szs, sanityCheck, &ref_names); } assert_gt(sztot.first, 0); assert_gt(sztot.second, 0); assert_gt(szs.size(), 0); // Compose text strings into single string cerr << "Calculating joined length" << endl; TIndexOffU jlen = 0; for(unsigned int i = 0; i < szs.size(); i++) { jlen += (TIndexOffU)szs[i].len; } // assert_geq(jlen, sztot); cerr << " Joined length: " << jlen << endl; TStr s; { bool both_strand = forward_only ? false : true; cerr << "Reserving space for joined string" << endl; cerr << "Joining reference sequences" << endl; Timer timer(cerr, " Time to join reference sequences: ", verbose); GFM::join( is, szs, (TIndexOffU) sztot.first, refparams, seed, s, both_strand, // include reverse complemented sequence CGtoTG); //Change CG to TG } // Successfully obtained joined reference string #ifndef NDEBUG if(forward_only) { assert_geq(s.length(), jlen); } else { assert_geq(s.length(), jlen << 1); } #endif BitPackedArray suffix_array; bool sa_file_exist = false; string sa_fname = outfile + ".rep.sa"; if(load_sa) { ifstream fp(sa_fname, std::ifstream::binary); sa_file_exist = fp.is_open(); } if(load_sa && sa_file_exist) { cerr << "Load SA from " << sa_fname << endl; suffix_array.readFile(sa_fname); } else { suffix_array.init(s.length() + 1); if(bmax != (TIndexOffU) OFF_MASK) { // VMSG_NL("bmax according to bmax setting: " << bmax); } else if(bmaxDivN != (uint32_t) OFF_MASK) { bmax = max(jlen / (bmaxDivN * nthreads), 1); // VMSG_NL("bmax according to bmaxDivN setting: " << bmax); } else { bmax = (uint32_t) sqrt(s.length()); // VMSG_NL("bmax defaulted to: " << bmax); } int iter = 0; bool first = true; bool passMemExc = false, sanity = false; // Look for bmax/dcv parameters that work. while(true) { if(!first && bmax < 40 && passMemExc) { cerr << "Could not find appropriate bmax/dcv settings for building this index." << endl; cerr << "Please try indexing this reference on a computer with more memory." << endl; if (sizeof(void *) == 4) { cerr << "If this computer has more than 4 GB of memory, try using a 64-bit executable;" << endl << "this executable is 32-bit." << endl; } throw 1; } if(dcv > 4096) dcv = 4096; if((iter % 6) == 5 && dcv < 4096 && dcv != 0) { dcv <<= 1; // double difference-cover period } else { bmax -= (bmax >> 2); // reduce by 25% } iter++; suffix_array.reset(); try { cerr << "Using parameters --bmax " << bmax << endl; if(dcv == 0) { cerr << " and *no difference cover*" << endl; } else { cerr << " --dcv " << dcv << endl; } { cerr << " Doing ahead-of-time memory usage test" << endl; // Make a quick-and-dirty attempt to force a bad_alloc iff // we would have thrown one eventually as part of // constructing the DifferenceCoverSample dcv <<= 1; TIndexOffU sz = (TIndexOffU) DifferenceCoverSample::simulateAllocs(s, dcv >> 1); if(nthreads > 1) sz *= (nthreads + 1); AutoArray tmp(sz, EBWT_CAT); dcv >>= 1; // Likewise with the KarkkainenBlockwiseSA sz = (TIndexOffU) KarkkainenBlockwiseSA::simulateAllocs(s, bmax); AutoArray tmp2(sz, EBWT_CAT); // Grab another 20 MB out of caution AutoArray extra(20 * 1024 * 1024, EBWT_CAT); // If we made it here without throwing bad_alloc, then we // passed the memory-usage stress test cerr << " Passed! Constructing with these parameters: --bmax " << bmax << " --dcv " << dcv << endl; cerr << "" << endl; } cerr << "Constructing suffix-array element generator" << endl; KarkkainenBlockwiseSA bsa(s, bmax, nthreads, dcv, seed, sanity, passMemExc, false /* verbose */, outfile); assert(bsa.suffixItrIsReset()); assert_eq(bsa.size(), s.length() + 1); TIndexOffU count = 0; while(count < s.length() + 1) { TIndexOffU saElt = bsa.nextSuffix(); count++; if(count && (count % 10000000 == 0)) { cerr << "SA count " << count << endl; } if(saElt == s.length()) { assert_eq(count, s.length() + 1); break; } suffix_array.pushBack(saElt); } break; } catch (bad_alloc &e) { if(passMemExc) { cerr << " Ran out of memory; automatically trying more memory-economical parameters." << endl; } else { cerr << "Out of memory while constructing suffix array. Please try using a smaller" << endl << "number of blocks by specifying a smaller --bmax or a larger --bmaxdivn" << endl; throw 1; } } first = false; } if(save_sa) { suffix_array.writeFile(sa_fname); } } cerr << "suffix_array: " << endl; suffix_array.dump(); // Build Repeats { if(repeat_length_pair.empty()) { repeat_length_pair.push_back(pair(min_repeat_length, max_repeat_length)); } RepeatParameter rp; rp.seed_len = seed_length; rp.seed_count = seed_count; rp.seed_mm = max_seed_mm; rp.max_edit = max_repeat_edit; rp.symmetric_extend = symmetric_extend; rp.extend_unit_len = max_seed_extlen; for(size_t i = 0; i < repeat_length_pair.size(); i++) { rp.min_repeat_len = repeat_length_pair[i].first; rp.max_repeat_len = repeat_length_pair[i].second; rp.repeat_count = repeat_count; rp.append_result = (i != 0); RepeatBuilder repeatBuilder(s, szs, ref_names, forward_only, outfile); cerr << "RepeatBuilder: " << outfile << " " << rp.min_repeat_len << "-" << rp.max_repeat_len << endl; { Timer _t(cerr, " Time reading suffix array: ", verbose); repeatBuilder.readSA(rp, suffix_array); } repeatBuilder.build(rp); repeatBuilder.saveFile(rp); } } } static const char *argv0 = NULL; extern "C" { /** * main function. Parses command-line arguments. */ int hisat2_repeat(int argc, const char **argv) { string outfile; try { // Reset all global state, including getopt state opterr = optind = 1; resetOptions(); string infile; EList infiles(MISC_CAT); parseOptions(argc, argv); argv0 = argv[0]; if(showVersion) { cout << argv0 << " version " << string(HISAT2_VERSION).c_str() << endl; if(sizeof(void*) == 4) { cout << "32-bit" << endl; } else if(sizeof(void*) == 8) { cout << "64-bit" << endl; } else { cout << "Neither 32- nor 64-bit: sizeof(void*) = " << sizeof(void*) << endl; } cout << "Built on " << BUILD_HOST << endl; cout << BUILD_TIME << endl; cout << "Compiler: " << COMPILER_VERSION << endl; cout << "Options: " << COMPILER_OPTIONS << endl; cout << "Sizeof {int, long, long long, void*, size_t, off_t}: {" << sizeof(int) << ", " << sizeof(long) << ", " << sizeof(long long) << ", " << sizeof(void *) << ", " << sizeof(size_t) << ", " << sizeof(off_t) << "}" << endl; return 0; } // Get input filename if(optind >= argc) { cerr << "No input sequence or sequence file specified!" << endl; printUsage(cerr); return 1; } infile = argv[optind++]; if(optind >= argc) { cerr << "No output file specified!" << endl; printUsage(cerr); return 1; } outfile = argv[optind++]; tokenize(infile, ",", infiles); if(infiles.size() < 1) { cerr << "Tokenized input file list was empty!" << endl; printUsage(cerr); return 1; } // Optionally summarize if(verbose) { cerr << "Settings:" << endl; // << " Output files: \"" << outfile.c_str() << ".*." << gfm_ext << "\"" << endl; cerr << " Endianness: " << (bigEndian? "big":"little") << endl << " Actual local endianness: " << (currentlyBigEndian()? "big":"little") << endl << " Sanity checking: " << (sanityCheck? "enabled":"disabled") << endl; #ifdef NDEBUG cerr << " Assertions: disabled" << endl; #else cerr << " Assertions: enabled" << endl; #endif cerr << " Random seed: " << seed << endl; cerr << " Sizeofs: void*:" << sizeof(void*) << ", int:" << sizeof(int) << ", long:" << sizeof(long) << ", size_t:" << sizeof(size_t) << endl; cerr << "Input files DNA, " << file_format_names[format].c_str() << ":" << endl; for(size_t i = 0; i < infiles.size(); i++) { cerr << " " << infiles[i].c_str() << endl; } } // Seed random number generator srand(seed); { Timer timer(cerr, "Total time for call to driver() for forward index: ", verbose); try { driver >(infile, infiles, outfile, false, forward_only, CGtoTG); } catch(bad_alloc& e) { if(autoMem) { cerr << "Switching to a packed string representation." << endl; } else { throw e; } } } return 0; } catch(std::exception& e) { cerr << "Error: Encountered exception: '" << e.what() << "'" << endl; cerr << "Command: "; for(int i = 0; i < argc; i++) cerr << argv[i] << " "; cerr << endl; return 1; } catch(int e) { if(e != 0) { cerr << "Error: Encountered internal HISAT2 exception (#" << e << ")" << endl; cerr << "Command: "; for(int i = 0; i < argc; i++) cerr << argv[i] << " "; cerr << endl; } return e; } } } hisat2-2.2.1/hisat2_repeat_main.cpp000066400000000000000000000040471370663763200171720ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include #include #include #include "tokenize.h" #include "ds.h" #include "mem_ids.h" using namespace std; extern "C" { int hisat2_repeat(int argc, const char **argv); } /** * bowtie-build main function. It is placed in a separate source file * to make it slightly easier to compile as a library. * * If the user specifies -A as the first two arguments, main * will interpret that file as having one set of command-line arguments * per line, and will dispatch each batch of arguments one at a time to * bowtie-build. */ int main(int argc, const char **argv) { if(argc > 2 && strcmp(argv[1], "-A") == 0) { const char *file = argv[2]; ifstream in; in.open(file); char buf[4096]; int lastret = -1; while(in.getline(buf, 4095)) { EList args(MISC_CAT); args.push_back(string(argv[0])); tokenize(buf, " \t", args); const char **myargs = (const char**)malloc(sizeof(char*)*args.size()); for(size_t i = 0; i < args.size(); i++) { myargs[i] = args[i].c_str(); } if(args.size() == 1) continue; lastret = hisat2_repeat((int)args.size(), myargs); free(myargs); } if(lastret == -1) { cerr << "Warning: No arg strings parsed from " << file << endl; return 0; } return lastret; } else { return hisat2_repeat(argc, argv); } } hisat2-2.2.1/hisat2_simulate_reads.py000077500000000000000000001046171370663763200175640ustar00rootroot00000000000000#!/usr/bin/env python3 # # Copyright 2015, Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # import os, sys, math, random, re from collections import defaultdict, Counter from argparse import ArgumentParser, FileType """ """ def reverse_complement(seq): result = "" for nt in seq: base = nt if nt == 'A': base = 'T' elif nt == 'a': base = 't' elif nt == 'C': base = 'G' elif nt == 'c': base = 'g' elif nt == 'G': base = 'C' elif nt == 'g': base = 'c' elif nt == 'T': base = 'A' elif nt == 't': base = 'a' result = base + result return result """ python2 style randint """ def myrandint(m, x): s = x - m + 1 return m + int(random.random() * s) """ Random source for sequencing errors """ class ErrRandomSource: def __init__(self, prob = 0.0, size = 1 << 20): self.size = size self.rands = [] for i in range(self.size): if random.random() < prob: self.rands.append(1) else: self.rands.append(0) self.cur = 0 def getRand(self): assert self.cur < len(self.rands) rand = self.rands[self.cur] self.cur = (self.cur + 1) % len(self.rands) return rand """ """ def read_genome(genome_file): chr_dic = {} chr_name, sequence = "", "" for line in genome_file: if line[0] == ">": if chr_name and sequence: chr_dic[chr_name] = sequence chr_name = line.strip().split()[0][1:] sequence = "" else: sequence += line[:-1] if chr_name and sequence: chr_dic[chr_name] = sequence chr_filter = [str(x) for x in list(range(1, 23)) + ['X', 'Y']] #chr_filter = None if chr_filter: for chr_id, chr_seq in chr_dic.items(): if not chr_id in chr_filter: chr_dic.pop(chr_id, None) return chr_dic """ """ def read_transcript(genome_seq, gtf_file, frag_len): genes = defaultdict(list) transcripts = {} # Parse valid exon lines from the GTF file into a dict by transcript_id for line in gtf_file: line = line.strip() if not line or line.startswith('#'): continue if '#' in line: line = line.split('#')[0].strip() try: chrom, source, feature, left, right, score, \ strand, frame, values = line.split('\t') except ValueError: continue if chrom not in genome_seq: continue # Zero-based offset left, right = int(left) - 1, int(right) - 1 if feature != 'exon' or left >= right: continue values_dict = {} for attr in values.split(';')[:-1]: attr, _, val = attr.strip().partition(' ') values_dict[attr] = val.strip('"') if 'gene_id' not in values_dict or \ 'transcript_id' not in values_dict: continue transcript_id = values_dict['transcript_id'] if transcript_id not in transcripts: transcripts[transcript_id] = [chrom, strand, [[left, right]]] genes[values_dict['gene_id']].append(transcript_id) else: transcripts[transcript_id][2].append([left, right]) # Sort exons and merge where separating introns are <=5 bps for tran, [chr, strand, exons] in transcripts.items(): exons.sort() tmp_exons = [exons[0]] for i in range(1, len(exons)): if exons[i][0] - tmp_exons[-1][1] <= 5: tmp_exons[-1][1] = exons[i][1] else: tmp_exons.append(exons[i]) transcripts[tran] = [chr, strand, tmp_exons] tmp_transcripts = {} for tran, [chr, strand, exons] in transcripts.items(): exon_lens = [e[1] - e[0] + 1 for e in exons] transcript_len = sum(exon_lens) if transcript_len >= frag_len: tmp_transcripts[tran] = [chr, strand, transcript_len, exons] transcripts = tmp_transcripts return genes, transcripts """ """ def read_snp(snp_file): snps = defaultdict(list) for line in snp_file: line = line.strip() if not line or line.startswith('#'): continue try: snpID, type, chr, pos, data = line.split('\t') except ValueError: continue assert type in ["single", "deletion", "insertion"] if type == "deletion": data = int(data) snps[chr].append([snpID, type, int(pos), data]) return snps """ """ def sanity_check_input(genome_seq, genes, transcripts, snps, frag_len): num_canon_ss, num_ss = 0, 0 for transcript, [chr, strand, transcript_len, exons] in transcripts.items(): assert transcript_len >= frag_len if len(exons) <= 1: continue if chr not in genome_seq: continue chr_seq = genome_seq[chr] for i in range(len(exons) - 1): left1, right1 = exons[i] assert left1 < right1 left2, right2 = exons[i+1] assert left2 < right2 assert left1 < left2 and right1 < right2 donor = chr_seq[right1+1:right1+3] acceptor = chr_seq[left2-2:left2] if strand == "-": donor, acceptor = reverse_complement(acceptor), reverse_complement(donor) if donor == "GT" and acceptor == "AG": num_canon_ss += 1 num_ss += 1 if num_ss > 0: print("GT/AG splice sites: {}/{} ({:.2%})".format(num_canon_ss, num_ss, (float(num_canon_ss) / num_ss)), file=sys.stderr) num_alt_single, num_single = 0, 0 for chr, chr_snps in snps.items(): if chr not in genome_seq: continue chr_seq = genome_seq[chr] prev_snp = None for snp in chr_snps: snpID, type, pos, data = snp if prev_snp: assert prev_snp[2] <= pos prev_snp = snp if type != "single": continue assert pos < len(chr_seq) if chr_seq[pos] != data: num_alt_single += 1 num_single += 1 if num_single > 0: print("Alternative bases: {}/{} ({:.2%})".format(num_alt_single, num_single, (float(num_alt_single) / num_single)), file=sys.stderr) """ """ def generate_rna_expr_profile(expr_profile_type, num_transcripts = 10000): # Modelling and simulating generic RNA-Seq experiments with the flux simulator # http://nar.oxfordjournals.org/content/suppl/2012/06/29/gks666.DC1/nar-02667-n-2011-File002.pdf def calc_expr(x, a): x, a, b = float(x), 9500.0, 9500.0 k = -0.6 return (x**k) * math.exp(x/a * (x/b)**2) expr_profile = [0.0] * num_transcripts for i in range(len(expr_profile)): if expr_profile_type == "flux": expr_profile[i] = calc_expr(i + 1, num_transcripts) elif expr_profile_type == "constant": expr_profile[i] = 1.0 else: assert False expr_sum = sum(expr_profile) expr_profile = [expr_profile[i] / expr_sum for i in range(len(expr_profile))] assert abs(sum(expr_profile) - 1.0) < 0.001 return expr_profile """ """ def generate_dna_expr_profile(genome_seq): expr_profile = [] for chr_id, chr_seq in genome_seq.items(): expr_profile.append(len(chr_seq)) expr_sum = float(sum(expr_profile)) expr_profile = [expr_profile[i] / expr_sum for i in range(len(expr_profile))] assert abs(sum(expr_profile) - 1.0) < 0.001 return expr_profile """ """ def getSNPs(chr_snps, left, right): low, high = 0, len(chr_snps) while low < high: mid = (low + high) // 2 snpID, type, pos, data = chr_snps[mid] if pos < left: low = mid + 1 else: high = mid - 1 snps = [] for snp in chr_snps[low:]: snpID, type, pos, data = snp pos2 = pos if type == "deletion": pos2 += data if pos2 >= right: break if pos >= left: if len(snps) > 0: _, prev_type, prev_pos, prev_data = snps[-1] assert prev_pos <= pos prev_pos2 = prev_pos if prev_type == "deletion": prev_pos2 += prev_data if pos <= prev_pos2: continue snps.append(snp) return snps """ """ def getSamAlignment(rna, exons, chr_seq, trans_seq, frag_pos, read_len, chr_snps, snp_prob, err_rand_src, max_mismatch): # Find the genomic position for frag_pos and exon number tmp_frag_pos, tmp_read_len = frag_pos, read_len pos, cigars, cigar_descs = exons[0][0], [], [] e_pos = 0 prev_e = None for e_i in range(len(exons)): e = exons[e_i] if prev_e: i_len = e[0] - prev_e[1] - 1 pos += i_len e_len = e[1] - e[0] + 1 if e_len <= tmp_frag_pos: tmp_frag_pos -= e_len pos += e_len else: pos += tmp_frag_pos e_pos = tmp_frag_pos break prev_e = e # Define Cigar and its descriptions assert e_i < len(exons) e_len = exons[e_i][1] - exons[e_i][0] + 1 assert e_pos < e_len cur_pos = pos match_len = 0 prev_e = None mismatch, remain_trans_len = 0, len(trans_seq) - (frag_pos + read_len) assert remain_trans_len >= 0 for e_i in range(e_i, len(exons)): e = exons[e_i] if prev_e: i_len = e[0] - prev_e[1] - 1 cur_pos += i_len cigars.append(("{}N".format(i_len))) cigar_descs.append([]) tmp_e_left = e_left = e[0] + e_pos e_pos = 0 # Retreive SNPs if rna: snps = getSNPs(chr_snps, e_left, e[1]) else: snps = getSNPs(chr_snps, frag_pos, frag_pos + read_len) if snp_prob < 1.0 and len(snps) > 0: snps_ = [] for snp in snps: if random.random() <= snp_prob: snps_.append(snp) snps = snps_ # Simulate mismatches due to sequencing errors mms = [] for i in range(e_left, min(e[1], e_left + tmp_read_len - 1)): if err_rand_src.getRand() == 1: assert i < len(chr_seq) err_base = "A" #rand = random.randint(0, 2) rand = myrandint(0, 2) if chr_seq[i] == "A": err_base = "GCT"[rand] elif chr_seq[i] == "C": err_base = "AGT"[rand] elif chr_seq[i] == "G": err_base = "ACT"[rand] else: err_base = "ACG"[rand] mms.append(["", "single", i, err_base]) tmp_diffs = snps + mms # def diff_sort(a , b): # return a[2] - b[2] tmp_diffs = sorted(tmp_diffs, key=lambda t: t[2]) diffs = [] if len(tmp_diffs) > 0: diffs = tmp_diffs[:1] for diff in tmp_diffs[1:]: _, tmp_type, tmp_pos, tmp_data = diff _, prev_type, prev_pos, prev_data = diffs[-1] if prev_type == "deletion": prev_pos += prev_data if tmp_pos <= prev_pos: continue diffs.append(diff) cigar_descs.append([]) prev_diff = None for diff in diffs: diff_id, diff_type, diff_pos, diff_data = diff if prev_diff: prev_diff_id, prev_diff_type, prev_diff_pos, prev_diff_data = prev_diff if prev_diff_type == "deletion": prev_diff_pos += prev_diff_data assert prev_diff_pos < diff_pos diff_pos2 = diff_pos if diff_type == "deletion": diff_pos2 += diff_data if e_left + tmp_read_len - 1 < diff_pos2 or e[1] < diff_pos2: break if diff_type == "single": if mismatch + 1 > max_mismatch: continue cigar_descs[-1].append([diff_pos - tmp_e_left, diff_data, diff_id]) tmp_e_left = diff_pos + 1 mismatch += 1 elif diff_type == "deletion": del_len = diff_data if mismatch + del_len > max_mismatch: continue if len(cigars) <= 0 and diff_pos - e_left <= 0: continue if remain_trans_len < del_len: continue remain_trans_len -= del_len if diff_pos - e_left > 0: cigars.append("{}M".format(diff_pos - e_left)) cigar_descs[-1].append([diff_pos - tmp_e_left, "", ""]) cigar_descs.append([]) cigars.append("{}D".format(del_len)) cigar_descs[-1].append([0, del_len, diff_id]) cigar_descs.append([]) tmp_read_len -= (diff_pos - e_left) e_left = tmp_e_left = diff_pos + del_len elif diff_type == "insertion": ins_len = len(diff_data) if mismatch + ins_len > max_mismatch: continue if len(cigars) <= 0 and diff_pos - e_left <= 0: continue if e_left + tmp_read_len - 1 < diff_pos + ins_len: break if diff_pos - e_left > 0: cigars.append("{}M".format(diff_pos - e_left)) cigar_descs[-1].append([diff_pos - tmp_e_left, "", ""]) cigar_descs.append([]) cigars.append("{}I".format(ins_len)) cigar_descs[-1].append([0, diff_data, diff_id]) cigar_descs.append([]) tmp_read_len -= (diff_pos - e_left) tmp_read_len -= ins_len e_left = tmp_e_left = diff_pos else: assert False prev_diff = diff e_right = min(e[1], e_left + tmp_read_len - 1) e_len = e_right - e_left + 1 remain_e_len = e_right - tmp_e_left + 1 if remain_e_len > 0: cigar_descs[-1].append([remain_e_len, "", ""]) if e_len < tmp_read_len: tmp_read_len -= e_len cigars.append(("{}M".format(e_len))) else: assert e_len == tmp_read_len cigars.append(("{}M".format(tmp_read_len))) tmp_read_len = 0 break prev_e = e # Define MD, XM, NM, Zs, read_seq MD, XM, NM, Zs, read_seq = "", 0, 0, "", "" assert len(cigars) == len(cigar_descs) MD_match_len, Zs_match_len = 0, 0 cur_trans_pos = frag_pos for c in range(len(cigars)): cigar = cigars[c] cigar_len, cigar_op = int(cigar[:-1]), cigar[-1] cigar_desc = cigar_descs[c] if cigar_op == 'N': continue if cigar_op == 'M': for add_match_len, alt_base, snp_id in cigar_desc: MD_match_len += add_match_len Zs_match_len += add_match_len assert cur_trans_pos + add_match_len <= len(trans_seq) read_seq += trans_seq[cur_trans_pos:cur_trans_pos+add_match_len] cur_trans_pos += add_match_len if alt_base != "": if MD_match_len > 0: MD += ("{}".format(MD_match_len)) MD_match_len = 0 MD += trans_seq[cur_trans_pos] if snp_id != "": if Zs != "": Zs += "," Zs += ("{}|S|{}".format(Zs_match_len, snp_id)) Zs_match_len = 0 else: Zs_match_len += 1 if snp_id == "": XM += 1 NM += 1 read_seq += alt_base cur_trans_pos += 1 elif cigar_op == 'D': assert len(cigar_desc) == 1 add_match_len, del_len, snp_id = cigar_desc[0] MD_match_len += add_match_len Zs_match_len += add_match_len if MD_match_len > 0: MD += ("{}".format(MD_match_len)) MD_match_len = 0 MD += ("^{}".format(trans_seq[cur_trans_pos:cur_trans_pos+cigar_len])) read_seq += trans_seq[cur_trans_pos:cur_trans_pos+add_match_len] if Zs != "": Zs += "," Zs += ("{}|D|{}".format(Zs_match_len, cigar_desc[0][-1])) Zs_match_len = 0 cur_trans_pos += cigar_len elif cigar_op == 'I': assert len(cigar_desc) == 1 add_match_len, ins_seq, snp_id = cigar_desc[0] ins_len = len(ins_seq) MD_match_len += add_match_len Zs_match_len += add_match_len read_seq += trans_seq[cur_trans_pos:cur_trans_pos+add_match_len] read_seq += ins_seq if Zs != "": Zs += "," Zs += ("{}|I|{}".format(Zs_match_len, cigar_desc[0][-1])) Zs_match_len = 0 else: assert False if MD_match_len > 0: MD += ("{}".format(MD_match_len)) if len(read_seq) != read_len: print("read length differs:", len(read_seq), "vs.", read_len, file=sys.stderr) print(pos, "".join(cigars), cigar_descs, MD, XM, NM, Zs, file=sys.stderr) assert False return pos, cigars, cigar_descs, MD, XM, NM, Zs, read_seq """ """ cigar_re = re.compile('\d+\w') def samRepOk(genome_seq, read_seq, chr, pos, cigar, XM, NM, MD, Zs, max_mismatch): assert chr in genome_seq chr_seq = genome_seq[chr] assert pos < len(chr_seq) # Calculate XM and NM based on Cigar and Zs cigars = cigar_re.findall(cigar) cigars = [[int(cigars[i][:-1]), cigars[i][-1]] for i in range(len(cigars))] ref_pos, read_pos = pos, 0 ann_ref_seq, ann_ref_rel, ann_read_seq, ann_read_rel = [], [], [], [] for i in range(len(cigars)): cigar_len, cigar_op = cigars[i] if cigar_op == "M": partial_ref_seq = chr_seq[ref_pos:ref_pos+cigar_len] partial_read_seq = read_seq[read_pos:read_pos+cigar_len] assert len(partial_ref_seq) == len(partial_read_seq) ann_ref_seq += list(partial_ref_seq) ann_read_seq += list(partial_read_seq) for j in range(len(partial_ref_seq)): if partial_ref_seq[j] == partial_read_seq[j]: ann_ref_rel.append("=") ann_read_rel.append("=") else: ann_ref_rel.append("X") ann_read_rel.append("X") ref_pos += cigar_len read_pos += cigar_len elif cigar_op == "D": partial_ref_seq = chr_seq[ref_pos:ref_pos+cigar_len] ann_ref_rel += list(partial_ref_seq) ann_ref_seq += list(partial_ref_seq) ann_read_rel += (["-"] * cigar_len) ann_read_seq += (["-"] * cigar_len) ref_pos += cigar_len elif cigar_op == "I": partial_read_seq = read_seq[read_pos:read_pos+cigar_len] ann_ref_rel += (["-"] * cigar_len) ann_ref_seq += (["-"] * cigar_len) ann_read_rel += list(partial_read_seq) ann_read_seq += list(partial_read_seq) read_pos += cigar_len elif cigar_op == "N": ref_pos += cigar_len else: assert False assert len(ann_ref_seq) == len(ann_read_seq) assert len(ann_ref_seq) == len(ann_ref_rel) assert len(ann_ref_seq) == len(ann_read_rel) ann_Zs_seq = ["0" for i in range(len(ann_ref_seq))] Zss, Zs_i, snp_pos_add = [], 0, 0 if Zs != "": Zss = Zs.split(',') Zss = [zs.split('|') for zs in Zss] ann_read_pos = 0 for zs in Zss: zs_pos, zs_type, zs_id = zs zs_pos = int(zs_pos) for i in range(zs_pos): while ann_read_rel[ann_read_pos] == '-': ann_read_pos += 1 ann_read_pos += 1 if zs_type == "S": ann_Zs_seq[ann_read_pos] = "1" ann_read_pos += 1 elif zs_type == "D": while ann_read_rel[ann_read_pos] == '-': ann_Zs_seq[ann_read_pos] = "1" ann_read_pos += 1 elif zs_type == "I": while ann_ref_rel[ann_read_pos] == '-': ann_Zs_seq[ann_read_pos] = "1" ann_read_pos += 1 else: assert False tMD, tXM, tNM = "", 0, 0 match_len = 0 i = 0 while i < len(ann_ref_seq): if ann_ref_rel[i] == "=": assert ann_read_rel[i] == "=" match_len += 1 i += 1 continue assert ann_read_rel[i] != "=" if ann_ref_rel[i] == "X" and ann_read_rel[i] == "X": if match_len > 0: tMD += ("{}".format(match_len)) match_len = 0 tMD += ann_ref_seq[i] if ann_Zs_seq[i] == "0": tXM += 1 tNM += 1 i += 1 else: assert ann_ref_rel[i] == "-" or ann_read_rel[i] == "-" if ann_ref_rel[i] == '-': while ann_ref_rel[i] == '-': if ann_Zs_seq[i] == "0": tNM += 1 i += 1 else: assert ann_read_rel[i] == '-' del_seq = "" while ann_read_rel[i] == '-': del_seq += ann_ref_seq[i] if ann_Zs_seq[i] == "0": tNM += 1 i += 1 if match_len > 0: tMD += ("{}".format(match_len)) match_len = 0 tMD += ("^{}".format(del_seq)) if match_len > 0: tMD += ("{}".format(match_len)) if tMD != MD or tXM != XM or tNM != NM or XM > max_mismatch or XM != NM: print(chr, pos, cigar, MD, XM, NM, Zs, file=sys.stderr) print(tMD, tXM, tNM, file=sys.stderr) assert False """ """ def simulate_reads(genome_file, gtf_file, snp_file, base_fname, rna, paired_end, read_len, frag_len, num_frag, expr_profile_type, repeat_fname, error_rate, max_mismatch, random_seed, snp_prob, sanity_check, verbose): random.seed(random_seed, version=1) err_rand_src = ErrRandomSource(error_rate / 100.0) if read_len > frag_len: frag_len = read_len genome_seq = read_genome(genome_file) if rna: genes, transcripts = read_transcript(genome_seq, gtf_file, frag_len) else: genes, transcripts = {}, {} snps = read_snp(snp_file) if sanity_check: sanity_check_input(genome_seq, genes, transcripts, snps, frag_len) if rna: num_transcripts = min(len(transcripts), 10000) expr_profile = generate_rna_expr_profile(expr_profile_type, num_transcripts) else: expr_profile = generate_dna_expr_profile(genome_seq) expr_profile = [int(expr_profile[i] * num_frag) for i in range(len(expr_profile))] assert num_frag >= sum(expr_profile) while sum(expr_profile) < num_frag: for i in range(min(num_frag - sum(expr_profile), len(expr_profile))): expr_profile[i] += 1 assert num_frag == sum(expr_profile) repeat_loci = {} if repeat_fname != "" and os.path.exists(repeat_fname): for line in open(repeat_fname): if line.startswith('>'): continue coords = line.strip().split() for coord in coords: chr, pos, strand = coord.split(':') if chr not in repeat_loci: repeat_loci[chr] = [] repeat_loci[chr].append([int(pos), strand]) if rna: transcript_ids = sorted(list(transcripts.keys())) random.shuffle(transcript_ids, random=random.random) assert len(transcript_ids) >= len(expr_profile) else: chr_ids = list(genome_seq.keys()) sam_file = open(base_fname + ".sam", "w") # Write SAM header print("@HD\tVN:1.0\tSO:unsorted", file=sam_file) for chr in genome_seq.keys(): print("@SQ\tSN:%s\tLN:%d" % (chr, len(genome_seq[chr])), file=sam_file) read_file = open(base_fname + "_1.fa", "w") if paired_end: read2_file = open(base_fname + "_2.fa", "w") cur_read_id = 1 for t in range(len(expr_profile)): t_num_frags = expr_profile[t] if rna: transcript_id = transcript_ids[t] chr, strand, transcript_len, exons = transcripts[transcript_id] print(transcript_id, t_num_frags, file=sys.stderr) else: chr = chr_ids[t] print(chr, t_num_frags, file=sys.stderr) assert chr in genome_seq chr_seq = genome_seq[chr] chr_len = len(chr_seq) if chr in repeat_loci: chr_repeat_loci = repeat_loci[chr] else: chr_repeat_loci = [] if rna: t_seq = "" for e in exons: assert e[0] < e[1] t_seq += chr_seq[e[0]:e[1]+1] assert len(t_seq) == transcript_len else: t_seq = chr_seq exons = [[0, chr_len - 1]] if chr in snps: chr_snps = snps[chr] else: chr_snps = [] for f in range(t_num_frags): if rna: #frag_pos = random.randint(0, transcript_len - frag_len) frag_pos = myrandint(0, transcript_len - frag_len) else: while True: if len(chr_repeat_loci): #locus_id = random.randint(0, len(chr_repeat_loci) - 1) locus_id = myrandint(0, len(chr_repeat_loci) - 1) frag_pos = chr_repeat_loci[locus_id][0] else: #frag_pos = random.randint(0, chr_len - frag_len) frag_pos = myrandint(0, chr_len - frag_len) if 'N' not in chr_seq[frag_pos:frag_pos + frag_len]: break # SAM specification (v1.4) # http://samtools.sourceforge.net/ flag, flag2 = 99, 163 # 83, 147 pos, cigars, cigar_descs, MD, XM, NM, Zs, read_seq = getSamAlignment(rna, exons, chr_seq, t_seq, frag_pos, read_len, chr_snps, snp_prob, err_rand_src, max_mismatch) pos2, cigars2, cigar2_descs, MD2, XM2, NM2, Zs2, read2_seq = getSamAlignment(rna, exons, chr_seq, t_seq, frag_pos+frag_len-read_len, read_len, chr_snps, snp_prob, err_rand_src, max_mismatch) swapped = False if paired_end: #if random.randint(0, 1) == 1: if myrandint(0, 1) == 1: swapped = True if swapped: flag, flag2 = flag - 16, flag2 - 16 pos, pos2 = pos2, pos cigars, cigars2 = cigars2, cigars cigar_descs, cigar2_descs = cigar2_descs, cigar_descs read_seq, read2_seq = read2_seq, read_seq XM, XM2 = XM2, XM NM, NM2 = NM2, NM MD, MD2 = MD2, MD Zs, Zs2 = Zs2, Zs cigar_str, cigar2_str = "".join(cigars), "".join(cigars2) if sanity_check: samRepOk(genome_seq, read_seq, chr, pos, cigar_str, XM, NM, MD, Zs, max_mismatch) samRepOk(genome_seq, read2_seq, chr, pos2, cigar2_str, XM2, NM2, MD2, Zs2, max_mismatch) if Zs != "": Zs = ("\tZs:Z:{}".format(Zs)) if Zs2 != "": Zs2 = ("\tZs:Z:{}".format(Zs2)) if rna: XS = "\tXS:A:{}".format(strand) TI = "\tTI:Z:{}".format(transcript_id) else: XS, TI = "", "" print(">{}".format(cur_read_id), file=read_file) if swapped: print(reverse_complement(read_seq), file=read_file) else: print(read_seq, file=read_file) print("{}\t{}\t{}\t{}\t255\t{}\t{}\t{}\t0\t{}\t*\tXM:i:{}\tNM:i:{}\tMD:Z:{}{}{}{}".format(cur_read_id, flag, chr, pos + 1, cigar_str, chr, pos2 + 1, read_seq, XM, NM, MD, Zs, XS, TI), file=sam_file) if paired_end: print(">{}".format(cur_read_id), file=read2_file) if swapped: print(read2_seq, file=read2_file) else: print(reverse_complement(read2_seq), file=read2_file) print("{}\t{}\t{}\t{}\t255\t{}\t{}\t{}\t0\t{}\t*\tXM:i:{}\tNM:i:{}\tMD:Z:{}{}{}{}".format(cur_read_id, flag2, chr, pos2 + 1, cigar2_str, chr, pos + 1, read2_seq, XM2, NM2, MD2, Zs2, XS, TI), file=sam_file) cur_read_id += 1 sam_file.close() read_file.close() if paired_end: read2_file.close() if __name__ == '__main__': parser = ArgumentParser( description='Simulate reads from GENOME (fasta) and GTF files') parser.add_argument('genome_file', nargs='?', type=FileType('r'), help='input GENOME file') parser.add_argument('gtf_file', nargs='?', type=FileType('r'), help='input GTF file') parser.add_argument('snp_file', nargs='?', type=FileType('r'), help='input SNP file') parser.add_argument('base_fname', nargs='?', type=str, help='output base filename') parser.add_argument('-d', '--dna', dest='rna', action='store_false', default=True, help='DNA-seq reads (default: RNA-seq reads)') parser.add_argument('--single-end', dest='paired_end', action='store_false', default=True, help='single-end reads (default: paired-end reads)') parser.add_argument('-r', '--read-length', dest='read_len', action='store', type=int, default=100, help='read length (default: 100)') parser.add_argument('-f', '--fragment-length', dest='frag_len', action='store', type=int, default=250, help='fragment length (default: 250)') parser.add_argument('-n', '--num-fragment', dest='num_frag', action='store', type=int, default=1000000, help='number of fragments (default: 1000000)') parser.add_argument('-e', '--expr-profile', dest='expr_profile', action='store', type=str, default='flux', help='expression profile: flux or constant (default: flux)') parser.add_argument('--repeat-info', dest='repeat_fname', action='store', type=str, default='', help='repeat information filename') parser.add_argument('--error-rate', dest='error_rate', action='store', type=float, default=0.0, help='per-base sequencing error rate (%%) (default: 0.0)') parser.add_argument('--max-mismatch', dest='max_mismatch', action='store', type=int, default=3, help='max mismatches due to sequencing errors (default: 3)') parser.add_argument('--random-seed', dest='random_seed', action='store', type=int, default=0, help='random seeding value (default: 0)') parser.add_argument('--snp-prob', dest='snp_prob', action='store', type=float, default=1.0, help='probability of a read including a snp when the read spans the snp ranging from 0.0 to 1.0 (default: 1.0)') parser.add_argument('--sanity-check', dest='sanity_check', action='store_true', help='sanity check') parser.add_argument('-v', '--verbose', dest='verbose', action='store_true', help='also print some statistics to stderr') parser.add_argument('--version', action='version', version='%(prog)s 2.0.0-alpha') args = parser.parse_args() if not args.genome_file or not args.gtf_file or not args.snp_file: parser.print_help() exit(1) if not args.rna: args.expr_profile = "constant" simulate_reads(args.genome_file, args.gtf_file, args.snp_file, args.base_fname, args.rna, args.paired_end, args.read_len, args.frag_len, args.num_frag, args.expr_profile, args.repeat_fname, args.error_rate, args.max_mismatch, args.random_seed, args.snp_prob, args.sanity_check, args.verbose) hisat2-2.2.1/hisat2lib/000077500000000000000000000000001370663763200146045ustar00rootroot00000000000000hisat2-2.2.1/hisat2lib/ht2.h000066400000000000000000000071571370663763200154640ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef __HT2_H__ #define __HT2_H__ #ifdef __cplusplus extern "C" { #endif #include #include typedef int ht2_error_t; enum { HT2_OK = 0, HT2_ERR = -1, HT2_ERR_NOT_REPEAT = -2, }; #define HT2_RET_OK(x) ((x) == HT2_OK) typedef void* ht2_handle_t; struct ht2_options { int offRate; int useMm; int useShmem; int mmSweep; int noRefNames; int noSplicedAlignment; int gVerbose; int startVerbose; int sanityCheck; int useHaplotype; }; typedef struct ht2_options ht2_option_t; /************************************************************************** * * Initialize APIs * **************************************************************************/ ht2_handle_t ht2_init(const char *name, ht2_option_t *options); void ht2_close(ht2_handle_t); ht2_error_t ht2_init_options(ht2_option_t *options); /************************************************************************** * * Index APIs * **************************************************************************/ const char* ht2_index_getrefnamebyid(ht2_handle_t handle, uint32_t chr_id); struct ht2_index_getrefnames_result { int count; char* names[0]; }; /** * @brief * * @param handle * @param result_ptr pointer to result. Caller must relese memory by free(). * * @return */ ht2_error_t ht2_index_getrefnames(ht2_handle_t handle, struct ht2_index_getrefnames_result **result_ptr); /************************************************************************** * * Repeat APIs * **************************************************************************/ struct ht2_position { uint32_t chr_id; int direction; /* 0 - forward, 1 - reverse */ uint64_t pos; /* 0-based */ }; struct ht2_repeat_expand_result { int count; struct ht2_position positions[0]; }; /** * @brief * * @param handle * @param repeat_name * @param repeat_pos repeat position on repeat sequence(0-based) * @param repeat_len * @param result_ptr pointer to result. caller must release memory by free(). * ex) free(result_ptr); * * @return */ ht2_error_t ht2_repeat_expand(ht2_handle_t handle, const char *repeat_name, uint64_t repeat_pos, uint64_t repeat_len, struct ht2_repeat_expand_result **result_ptr); /************************************************************************** * * Alignment APIs * **************************************************************************/ /* TODO */ /************************************************************************** * * ETC APIs * **************************************************************************/ /* TODO */ void ht2_test_1(ht2_handle_t); void ht2_repeat_dump_repeatmap(ht2_handle_t handle); #ifdef __cplusplus } /* extern "C" */ #endif #endif /* __HT2_H__ */ hisat2-2.2.1/hisat2lib/ht2_alignment.cpp000066400000000000000000000014041370663763200200420ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ hisat2-2.2.1/hisat2lib/ht2_handle.h000066400000000000000000000024071370663763200167700ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef __HT2_HANDLE_H__ #define __HT2_HANDLE_H__ #if 1 #define EXPORT __attribute__((visibility("default"))) #else #define EXPORT #endif typedef TIndexOffU index_t; typedef uint16_t local_index_t; struct ht2_handle { ALTDB* altdb; ALTDB* raltdb; RepeatDB* repeatdb; HGFM* gfm; RFM *rgfm; string tmp_str; string ht2_idx_name; struct ht2_options options; }; #endif /* __HT2_HANDLE_H__ */ hisat2-2.2.1/hisat2lib/ht2_index.cpp000066400000000000000000000045131370663763200171770ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include "ds.h" #include "repeat.h" #include "rfm.h" #include "ht2.h" #include "ht2_handle.h" EXPORT const char* ht2_index_getrefnamebyid(ht2_handle_t handle, uint32_t chr_id) { struct ht2_handle *hp = (struct ht2_handle *)handle; size_t refname_size = hp->gfm->_refnames.size(); if(chr_id < (refname_size - 1)) { return hp->gfm->_refnames[chr_id].c_str(); } return NULL; } EXPORT ht2_error_t ht2_index_getrefnames(ht2_handle_t handle, struct ht2_index_getrefnames_result **result_ptr) { struct ht2_handle *hp = (struct ht2_handle *)handle; size_t refname_size = hp->gfm->_refnames.size(); if(refname_size == 0) { return HT2_ERR; } size_t result_hdr_size = sizeof(struct ht2_index_getrefnames_result) + sizeof(char *) * refname_size; size_t result_buf_size = 0; for(size_t i = 0; i < refname_size - 1; i++) { result_buf_size += strlen(hp->gfm->_refnames[i].c_str()) + 1; } void* ptr = malloc(result_hdr_size + result_buf_size); char* buf_ptr = (char *)ptr + result_hdr_size; memset(ptr, 0, result_hdr_size + result_buf_size); struct ht2_index_getrefnames_result* result = (struct ht2_index_getrefnames_result *)ptr; result->count = refname_size - 1; result->names[0] = buf_ptr; for(size_t i = 0; i < refname_size - 1; i++) { size_t rlen = strlen(hp->gfm->_refnames[i].c_str()); strcpy(result->names[i], hp->gfm->_refnames[i].c_str()); result->names[i + 1] = result->names[i] + rlen + 1; } (*result_ptr) = result; return HT2_OK; } hisat2-2.2.1/hisat2lib/ht2_init.cpp000066400000000000000000000112641370663763200170340ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include "ds.h" #include "repeat.h" #include "rfm.h" #include "ht2.h" #include "ht2_handle.h" using namespace std; MemoryTally gMemTally; static const struct ht2_options ht2_default_options = { .offRate = -1, .useMm = false, .useShmem = false, .mmSweep = false, .noRefNames = false, .noSplicedAlignment = false, .gVerbose = false, .startVerbose = false, .sanityCheck = 0, .useHaplotype = false, }; static void free_handle(struct ht2_handle *hp) { if(hp->altdb) { delete hp->altdb; } if(hp->raltdb) { delete hp->raltdb; } if(hp->repeatdb) { delete hp->repeatdb; } if(hp->gfm) { if(hp->gfm->isInMemory()) { hp->gfm->evictFromMemory(); } delete hp->gfm; } if(hp->rgfm) { if(hp->rgfm->isInMemory()) { hp->rgfm->evictFromMemory(); } delete hp->rgfm; } delete hp; } static void init_handle(struct ht2_handle *hp) { struct ht2_options *opt = &hp->options; hp->altdb = new ALTDB(); hp->repeatdb = new RepeatDB(); hp->raltdb = new ALTDB(); hp->gfm = new HGFM( hp->ht2_idx_name, hp->altdb, NULL, NULL, -1, true, opt->offRate, 0, opt->useMm, opt->useShmem, opt->mmSweep, !opt->noRefNames, true, true, true, !opt->noSplicedAlignment, opt->gVerbose, opt->startVerbose, false, opt->sanityCheck, opt->useHaplotype); // Load the other half of the index into memory assert(!hp->gfm->isInMemory()); hp->gfm->loadIntoMemory( -1, true, true, true, !opt->noRefNames, opt->startVerbose); hp->rgfm = new RFM( hp->ht2_idx_name + ".rep", hp->raltdb, hp->repeatdb, NULL, -1, true, opt->offRate, 0, opt->useMm, opt->useShmem, opt->mmSweep, !opt->noRefNames, true, true, true, !opt->noSplicedAlignment, opt->gVerbose, opt->startVerbose, false, opt->sanityCheck, false); assert(!hp->rgfm->isInMemory()); hp->rgfm->loadIntoMemory( -1, true, true, true, !opt->noRefNames, opt->startVerbose); hp->repeatdb->construct(hp->gfm->rstarts(), hp->gfm->nFrag()); } EXPORT ht2_handle_t ht2_init(const char *name, ht2_option_t *options) { struct ht2_handle *handle = new ht2_handle; handle->ht2_idx_name = name; if(options) { memcpy(&handle->options, options, sizeof(struct ht2_options)); } else { memcpy(&handle->options, &ht2_default_options, sizeof(struct ht2_options)); } // Init init_handle(handle); handle->tmp_str = name; return (ht2_handle_t)handle; } EXPORT void ht2_close(ht2_handle_t handle) { struct ht2_handle *hp = (struct ht2_handle *)handle; if(hp == NULL) { return; } free_handle(hp); } EXPORT ht2_error_t ht2_init_options(ht2_option_t *options) { if(options == NULL) { return HT2_ERR; } memcpy(options, &ht2_default_options, sizeof(ht2_default_options)); return HT2_OK; } EXPORT void ht2_test_1(ht2_handle_t handle) { struct ht2_handle *hp = (struct ht2_handle *)handle; size_t refname_size = hp->gfm->_refnames.size(); cerr << "ht2lib: " << "gfm refnames: " << refname_size << endl; for(size_t i = 0; i < refname_size; i++) { cerr << "ht2lib: " << " " << i << " -> " << hp->gfm->_refnames[i] << endl; } } hisat2-2.2.1/hisat2lib/ht2_repeat.cpp000066400000000000000000000055411370663763200173520ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include "ds.h" #include "repeat.h" #include "rfm.h" #include "ht2.h" #include "ht2_handle.h" EXPORT void ht2_repeat_dump_repeatmap(ht2_handle_t handle) { struct ht2_handle *hp = (struct ht2_handle *)handle; size_t localRFMSize = hp->rgfm->_localRFMs.size(); cerr << "ht2lib: " << "LocalRFM size: " << localRFMSize << endl; cerr << "ht2lib: " << "Dump repeatMap" << endl; // repID -> 0 const EList >& repeatMap = hp->repeatdb->repeatMap()[0]; cerr << repeatMap.size() << endl; for(size_t i = 0; i < repeatMap.size(); i++) { cerr << repeatMap[i].first << ", " << repeatMap[i].second << endl; } } EXPORT ht2_error_t ht2_repeat_expand(ht2_handle_t handle, const char *repeat_name, uint64_t repeat_pos, uint64_t repeat_len, struct ht2_repeat_expand_result **result_ptr) { struct ht2_handle *hp = (struct ht2_handle *)handle; index_t rep_id = hp->rgfm->getLocalRFM_idx(repeat_name); TIndexOffU left = repeat_pos; TIndexOffU right = left + repeat_len; bool ret = hp->repeatdb->repeatExist(rep_id, left, right); if(!ret) { return HT2_ERR_NOT_REPEAT; } /* get coord */ EList, RepeatCoord > > positions; EList snp_id_list; snp_id_list.clear(); hp->repeatdb->getCoords( rep_id, left, right, snp_id_list, *(hp->raltdb), positions ); /* build result */ size_t result_size = sizeof(struct ht2_repeat_expand_result) + positions.size() * sizeof(struct ht2_position); struct ht2_repeat_expand_result *result = (struct ht2_repeat_expand_result *)malloc(result_size); result->count = positions.size(); for(size_t i = 0; i < positions.size(); i++) { const RepeatCoord& coord = positions[i].first; result->positions[i].chr_id = coord.tid; result->positions[i].pos = coord.toff; result->positions[i].direction = coord.fw ? 0 : 1; } *(result_ptr) = result; return HT2_OK; } hisat2-2.2.1/hisat2lib/java_jni/000077500000000000000000000000001370663763200163655ustar00rootroot00000000000000hisat2-2.2.1/hisat2lib/java_jni/HT2Module.java000066400000000000000000000062031370663763200207740ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ import java.util.Map; import java.util.HashMap; import java.util.List; import java.util.ArrayList; public class HT2Module { static { System.loadLibrary("ht2jni"); } public static class HT2Position { int chr_id; int direction; long position; HT2Position(int id, int dir, int pos) { this.chr_id = id; this.direction = dir; this.position = pos; } @Override public String toString() { return getClass().getSimpleName() + "[chr_id=" + chr_id + ", direction=" + direction + ", position=" + position + "]"; } } private native long init(String indexName, Map options); private native void close(long handle); private native Map get_options(); private native String index_getrefnamebyid(long handle, int id); private native List index_getrefnames(long handle); private native List repeat_expand(long handle, String name, long rpos, long rlen); private long handle; HT2Module() { handle = 0; } public void initLibrary(String indexName) { initLibrary(indexName, null); } public void initLibrary(String indexName, Map options) { if(handle == 0) { handle = init(indexName, options); } } public Map initOption() { return get_options(); } public String getRefNameById(int chr_id) { if(handle == 0) { // TODO: Exception return ""; } // TODO: Exception(OutOfIndex) return index_getrefnamebyid(handle, chr_id); } public List getRefNames() { if(handle == 0) { // TODO: Exception return new ArrayList<>(); } return index_getrefnames(handle); } public List repeatExpand(String name, long position, long length) { if(handle == 0) { // TODO: Exception return new ArrayList<>(); } return repeat_expand(handle, name, position, length); } public void cleanup() { if(handle != 0) { close(handle); handle = 0; } } public void finalize() { cleanup(); } } hisat2-2.2.1/hisat2lib/java_jni/HT2ModuleExample.java000066400000000000000000000054751370663763200223220ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ import java.util.Map; import java.util.HashMap; import java.util.List; import java.util.ArrayList; public class HT2ModuleExample { public static void main(String[] args) { HT2Module module = null; Map ht2Options = null; String indexPath = "../../evaluation/indexes/HISAT2_22/22_rep"; //String indexPath = "../../evaluation/indexes/HISAT2/genome_rep"; try { module = new HT2Module(); // Get Default Options //ht2Options = module.InitOption(); // or ht2Options = new HashMap<>(); //ht2Options.put("gVerbose", 1); //ht2Options.put("startVerbose", 1); //ht2Options.put("sanityCheck", 1); System.out.println(ht2Options); module.initLibrary(indexPath, ht2Options); //module.initLibrary(indexPath); System.out.println("CHR_ID 0: " + module.getRefNameById(0)); // in 22_rep, OutOfIndex System.out.println("CHR_ID 1: " + module.getRefNameById(1)); List refnames = module.getRefNames(); System.out.println("Refnames size:" + refnames.size()); for(String name: refnames) { System.out.println(name); } // repeat for 22_rep List positions = module.repeatExpand("rep100-300", 8308, 100); // repeat for genome_rep //List positions = module.repeatExpand("rep100-300", 2446692, 100); System.out.println("Repeat expand size: " + positions.size()); for(HT2Module.HT2Position pos: positions) { String chrName = refnames.get(pos.chr_id).split(" ")[0]; String direction = pos.direction == 0 ? "+":"-"; System.out.println(chrName + ":" + pos.position + ":" + direction); } } finally { if(module != null) { module.cleanup(); } } } } hisat2-2.2.1/hisat2lib/java_jni/Makefile000066400000000000000000000033011370663763200200220ustar00rootroot00000000000000# # Copyright 2018, Chanhee Park and Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # JAVA_HOME := $(JAVA_HOME) HISAT2_DIR = ../.. CC = gcc MACOS = 0 ifneq (,$(findstring Darwin,$(shell uname))) MACOS = 1 endif INCLUDES = -I $(JAVA_HOME)/include INCLUDES += -I $(JAVA_HOME)/include/linux TARGET_LIB = libht2jni.so SHARED_FLAG = -shared ifeq (1,$(MACOS)) INCLUDES += -I $(JAVA_HOME)/include/darwin TARGET_LIB = libht2jni.jnilib SHARED_FLAG = -dynamiclib endif INCLUDES += -I $(HISAT2_DIR)/hisat2lib CFLAGS = -fPIC $(INCLUDES) #CFLAGS += -DDEBUG SRCS = ht2module.c OBJS = $(SRCS:.c=.o) all: lib lib: $(TARGET_LIB) $(TARGET_LIB): HT2Module.h $(OBJS) $(CC) $(SHARED_FLAG) -o $@ $(OBJS) $(HISAT2_DIR)/libhisat2lib.a -lstdc++ HT2Module.h HT2Module.class: HT2Module.java javac -h . HT2Module.java example: HT2ModuleExample.class HT2ModuleExample.class: HT2ModuleExample.java javac HT2ModuleExample.java clean: rm -f *.class HT2Module.h HT2Module_HT2Position.h *.so *.o $(TARGET_LIB) test: all example java -Djava.library.path=. HT2ModuleExample hisat2-2.2.1/hisat2lib/java_jni/ht2module.c000066400000000000000000000255201370663763200204400ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include #include "ht2.h" #include "HT2Module.h" #define CLASSPATH_INTEGER "java/lang/Integer" #define CLASSPATH_HASHMAP "java/util/HashMap" #define CLASSPATH_ARRAYLIST "java/util/ArrayList" #define CLASSPATH_HT2POSITION "HT2Module$HT2Position" #ifdef DEBUG #define DEBUGLOG(fmt, ...) do { fprintf(stderr, "%s:%d:%s(): " fmt, __FILE__, __LINE__, __func__, ##__VA_ARGS__); } while(0) #else #define DEBUGLOG(fmt, ...) #endif static jint JNI_VERSION = JNI_VERSION_1_2; static jclass classInteger; static jclass classHashMap; static jclass classArrayList; static jclass classHT2Position; static jmethodID mthIntegerInit; static jmethodID mthIntegerIntValue; static jmethodID mthHashMapInit; static jmethodID mthHashMapPut; static jmethodID mthHashMapGet; static jmethodID mthArrayListInit; static jmethodID mthArrayListEnsureCapacity; static jmethodID mthArrayListAdd; static jmethodID mthHT2PositionInit; static jobject NewHashMap(JNIEnv *env) { jobject obj = (*env)->NewObject(env, classHashMap, mthHashMapInit); return obj; } static jobject NewInteger(JNIEnv *env, int value) { jobject obj = (*env)->NewObject(env, classInteger, mthIntegerInit, value); return obj; } static jobject NewArrayList(JNIEnv *env) { jobject obj = (*env)->NewObject(env, classArrayList, mthArrayListInit); return obj; } static jobject NewHT2Position(JNIEnv *env, struct ht2_position *htpos) { jobject obj = (*env)->NewObject(env, classHT2Position, mthHT2PositionInit, htpos->chr_id, htpos->direction, htpos->pos); return obj; } static int GetInteger(JNIEnv *env, jobject jobjInt, int *val) { int value = (*env)->CallIntMethod(env, jobjInt, mthIntegerIntValue); *val = value; return 0; } static void hashmap_put(JNIEnv *env, jobject jobjMap, const char *key_str, const int val) { jstring keyObj; jobject valueObj; keyObj = (*env)->NewStringUTF(env, key_str); valueObj = NewInteger(env, val); (*env)->CallObjectMethod(env, jobjMap, mthHashMapPut, keyObj, valueObj); (*env)->DeleteLocalRef(env, keyObj); (*env)->DeleteLocalRef(env, valueObj); } static int hashmap_get(JNIEnv *env, jobject jobjMap, const char *key_str, int *val) { jstring keyObj; jobject valueObj; keyObj = (*env)->NewStringUTF(env, key_str); valueObj = (*env)->CallObjectMethod(env, jobjMap, mthHashMapGet, keyObj); if(valueObj == NULL) { DEBUGLOG("Can't find key: %s\n", key_str); (*env)->DeleteLocalRef(env, keyObj); return -1; } // Integer -> int int value = 0; if(GetInteger(env, valueObj, &value) < 0) { DEBUGLOG("Can't get Integer\n"); (*env)->DeleteLocalRef(env, keyObj); (*env)->DeleteLocalRef(env, valueObj); return -1; } (*env)->DeleteLocalRef(env, keyObj); (*env)->DeleteLocalRef(env, valueObj); *val = value; return 0; } static jobject conv_ht2option(JNIEnv *env, ht2_option_t *opts) { jobject hashMap = NewHashMap(env); #define HT2_OPT_BUILD(_name) \ hashmap_put(env, hashMap, #_name , opts->_name) HT2_OPT_BUILD(offRate); HT2_OPT_BUILD(useMm); HT2_OPT_BUILD(useShmem); HT2_OPT_BUILD(mmSweep); HT2_OPT_BUILD(noRefNames); HT2_OPT_BUILD(noSplicedAlignment); HT2_OPT_BUILD(gVerbose); HT2_OPT_BUILD(startVerbose); HT2_OPT_BUILD(sanityCheck); HT2_OPT_BUILD(useHaplotype); return hashMap; } static void update_ht2option(JNIEnv *env, ht2_option_t *opts, jobject jmapObject) { #define HT2_OPT_UPDATE(_name) \ do { \ int value = 0; \ if(hashmap_get(env, jmapObject, #_name, &value) == 0) { \ DEBUGLOG("Using %s, %d\n", #_name, value); \ opts->_name = value; \ } else { \ DEBUGLOG("Using default %s, %d\n", #_name, opts->_name); \ }\ } while(0) HT2_OPT_UPDATE(offRate); HT2_OPT_UPDATE(useMm); HT2_OPT_UPDATE(useShmem); HT2_OPT_UPDATE(mmSweep); HT2_OPT_UPDATE(noRefNames); HT2_OPT_UPDATE(noSplicedAlignment); HT2_OPT_UPDATE(gVerbose); HT2_OPT_UPDATE(startVerbose); HT2_OPT_UPDATE(sanityCheck); HT2_OPT_UPDATE(useHaplotype); } static void conv_refnames_result(JNIEnv *env, struct ht2_index_getrefnames_result *result, jobject jobjList) { size_t i; // Resize DEBUGLOG("count: %d\n", result->count); (*env)->CallVoidMethod(env, jobjList, mthArrayListEnsureCapacity, result->count); for(i = 0; i < result->count; i++) { DEBUGLOG("names[%d] %s\n", i, result->names[i]); jobject elem = (*env)->NewStringUTF(env, result->names[i]); (*env)->CallObjectMethod(env, jobjList, mthArrayListAdd, elem); (*env)->DeleteLocalRef(env, elem); } } static void conv_repeat_expand_result(JNIEnv *env, struct ht2_repeat_expand_result *result, jobject jobjList) { size_t i; // Resize DEBUGLOG("count: %d\n", result->count); (*env)->CallVoidMethod(env, jobjList, mthArrayListEnsureCapacity, result->count); // Add Items for(i = 0; i < result->count; i++) { struct ht2_position *htpos = &result->positions[i]; DEBUGLOG("position[%d]: %u, %d, %lu\n", i, htpos->chr_id, htpos->direction, htpos->pos); jobject elem = NewHT2Position(env, htpos); (*env)->CallObjectMethod(env, jobjList, mthArrayListAdd, elem); (*env)->DeleteLocalRef(env, elem); } } JNIEXPORT jlong JNICALL Java_HT2Module_init(JNIEnv *env, jobject thisObj, jstring indexNameObj, jobject optionMap) { DEBUGLOG("Init\n"); const char *index_path = (*env)->GetStringUTFChars(env, indexNameObj, NULL); if(index_path == NULL) { DEBUGLOG("Can't get string\n"); return 0; } DEBUGLOG("Index Path: %s\n", index_path); ht2_option_t ht2opt; ht2_init_options(&ht2opt); if(optionMap != NULL) { // update options update_ht2option(env, &ht2opt, optionMap); } ht2_handle_t handle = ht2_init(index_path, &ht2opt); DEBUGLOG("Initailzed %p\n", handle); (*env)->ReleaseStringUTFChars(env, indexNameObj, index_path); return (jlong)handle; } JNIEXPORT void JNICALL Java_HT2Module_close(JNIEnv *env, jobject thisObj, jlong handlePtr) { DEBUGLOG("Closing\n"); ht2_handle_t handle = (ht2_handle_t)handlePtr; DEBUGLOG("Received handle: %p\n", handle); ht2_close(handle); } JNIEXPORT jobject JNICALL Java_HT2Module_get_1options(JNIEnv *env, jobject thisObj) { DEBUGLOG("get_option\n"); // Get Default options ht2_option_t ht2opt; ht2_init_options(&ht2opt); // convert ht2opt to java hashmap jobject hashobj = conv_ht2option(env, &ht2opt); return hashobj; } JNIEXPORT jstring JNICALL Java_HT2Module_index_1getrefnamebyid(JNIEnv *env, jobject thisObj, jlong handlePtr, jint chr_id) { DEBUGLOG("index_getrefnamebyid\n"); ht2_handle_t handle = (ht2_handle_t)handlePtr; if(handle == NULL) { DEBUGLOG("Invalid handle\n"); return NULL; } const char *refname = ht2_index_getrefnamebyid(handle, chr_id); if(refname == NULL) { DEBUGLOG("Can't get refname(%d)\n", chr_id); return NULL; } return (*env)->NewStringUTF(env, refname); } JNIEXPORT jobject JNICALL Java_HT2Module_index_1getrefnames(JNIEnv *env, jobject thisObj, jlong handlePtr) { DEBUGLOG("index_getrefnames\n"); ht2_handle_t handle = (ht2_handle_t)handlePtr; if(handle == NULL) { DEBUGLOG("Invalid handle\n"); return NULL; } struct ht2_index_getrefnames_result *result = NULL; ht2_error_t ret = ht2_index_getrefnames(handle, &result); jobject refnames = NewArrayList(env); if(ret == HT2_OK) { DEBUGLOG("refnames size: %d\n", result->count); conv_refnames_result(env, result, refnames); free(result); } else { DEBUGLOG("Can't get refnames\n"); } return refnames; } JNIEXPORT jobject JNICALL Java_HT2Module_repeat_1expand(JNIEnv *env, jobject thisObj, jlong handlePtr, jstring nameObj, jlong rpos, jlong rlen) { DEBUGLOG("repeat_expand\n"); ht2_handle_t handle = (ht2_handle_t)handlePtr; if(handle == NULL) { DEBUGLOG("Invalid handle\n"); return NULL; } const char *name = (*env)->GetStringUTFChars(env, nameObj, NULL); if(name == NULL) { DEBUGLOG("Can't get string\n"); return NULL; } DEBUGLOG("Repeat Expand: %s, %lu, %lu\n", name, rpos, rlen); struct ht2_repeat_expand_result *result = NULL; ht2_error_t ret = ht2_repeat_expand(handle, name, rpos, rlen, &result); jobject positions = NewArrayList(env); if(ret == HT2_OK) { DEBUGLOG("expand position size: %d\n", result->count); conv_repeat_expand_result(env, result, positions); free(result); } else { DEBUGLOG("Can't expand repeat\n"); } (*env)->ReleaseStringUTFChars(env, nameObj, name); return positions; } jint JNI_OnLoad(JavaVM *vm, void *reserved) { DEBUGLOG("JNI Loaded\n"); JNIEnv *env; if((*vm)->GetEnv(vm, (void **)&env, JNI_VERSION) != JNI_OK) { return JNI_ERR; } // Load Class #define LOAD_CLASS(_globalRef, _clazz) \ do { \ jclass tmpClassRef = (*env)->FindClass(env, _clazz); \ if(tmpClassRef == NULL) { \ DEBUGLOG("Can't find class %s\n", _clazz); \ return JNI_ERR; \ } \ _globalRef = (jclass)(*env)->NewGlobalRef(env, tmpClassRef); \ (*env)->DeleteLocalRef(env, tmpClassRef); \ } while(0) LOAD_CLASS(classInteger, CLASSPATH_INTEGER); LOAD_CLASS(classHashMap, CLASSPATH_HASHMAP); LOAD_CLASS(classArrayList, CLASSPATH_ARRAYLIST); LOAD_CLASS(classHT2Position, CLASSPATH_HT2POSITION); // Load Method // Integer mthIntegerInit = (*env)->GetMethodID(env, classInteger, "", "(I)V"); mthIntegerIntValue = (*env)->GetMethodID(env, classInteger, "intValue", "()I"); // HashMap mthHashMapInit = (*env)->GetMethodID(env, classHashMap, "", "()V"); mthHashMapPut = (*env)->GetMethodID(env, classHashMap, "put", "(Ljava/lang/Object;Ljava/lang/Object;)Ljava/lang/Object;"); mthHashMapGet = (*env)->GetMethodID(env, classHashMap, "get", "(Ljava/lang/Object;)Ljava/lang/Object;"); // ArrayList mthArrayListInit = (*env)->GetMethodID(env, classArrayList, "", "()V"); mthArrayListEnsureCapacity = (*env)->GetMethodID(env, classArrayList, "ensureCapacity", "(I)V"); mthArrayListAdd = (*env)->GetMethodID(env, classArrayList, "add", "(Ljava/lang/Object;)Z"); // HT2Position mthHT2PositionInit = (*env)->GetMethodID(env, classHT2Position, "", "(III)V"); return JNI_VERSION; } void JNI_OnUnload(JavaVM *vm, void *reserved) { DEBUGLOG("JNI Unload\n"); JNIEnv *env; (*vm)->GetEnv(vm, (void **)&env, JNI_VERSION); (*env)->DeleteGlobalRef(env, classInteger); (*env)->DeleteGlobalRef(env, classHashMap); (*env)->DeleteGlobalRef(env, classArrayList); (*env)->DeleteGlobalRef(env, classHT2Position); } hisat2-2.2.1/hisat2lib/pymodule/000077500000000000000000000000001370663763200164425ustar00rootroot00000000000000hisat2-2.2.1/hisat2lib/pymodule/Makefile000066400000000000000000000016551370663763200201110ustar00rootroot00000000000000# # Copyright 2018, Chanhee Park and Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # all: lib lib: ARCHFLAGS="-arch x86_64" python ./setup.py build install: python ./setup.py install clean: python ./setup.py clean rm -rf build test test: lib python ./ht2example.py hisat2-2.2.1/hisat2lib/pymodule/ht2example.py000077500000000000000000000033221370663763200210700ustar00rootroot00000000000000#!/usr/bin/python # # Copyright 2018, Chanhee Park and Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . # # ./setup.py build # ./setup.py install # import ht2py # Path to index ht2_index = '../../evaluation/indexes/HISAT2_22/22_rep' # Get default options ht2_options = ht2py.get_options() print ht2_options ht2_options['gVerbose'] = 1 ht2_options['startVerbose'] = 1 # or ht2_options = {} handle = ht2py.init(ht2_index, ht2_options) print ht2py.index_getrefnamebyid(handle, 0) #print ht2py.index_getrefnamebyid(handle, 0, 1, 3, 5, 7, 9) # outofindex #print ht2py.index_getrefnamebyid(handle, 1) #print ht2py.index_getrefnamebyid(handle, -1) refnames = ht2py.index_getrefnames(handle) #for name in refnames: # print name # repeat expansion positions = ht2py.repeat_expand(handle, 'rep100-300', 8308, 100) for pos in positions: chr_id = pos[0] direction = pos[1] chr_pos = pos[2] chr_dir = '+' if direction == 1: chr_dir = '-' print refnames[chr_id].split()[0] + ":" + str(chr_pos) + ':' + chr_dir # close handle ht2py.close(handle) hisat2-2.2.1/hisat2lib/pymodule/ht2module.c000066400000000000000000000175701370663763200205230ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include #include "ht2.h" #define HT2_HANDLE_ID "handle" #ifdef DEBUG #define DEBUGLOG(fmt, ...) do { fprintf(stderr, "%s:%d:%s(): " fmt, __FILE__, __LINE__, __func__, ##__VA_ARGS__); } while(0) #else #define DEBUGLOG(fmt, ...) #endif static ht2_handle_t get_handle(PyObject *cap) { return PyCapsule_GetPointer(cap, HT2_HANDLE_ID); } static PyObject *conv_refnames_result(struct ht2_index_getrefnames_result *result) { PyObject *refnames = NULL; size_t i = 0; if(result == NULL) { return NULL; } refnames = PyList_New(result->count); for(i = 0; i < result->count; i++) { PyObject *str = PyString_FromString(result->names[i]); PyList_SetItem(refnames, i, str); } return refnames; } static PyObject *conv_repeat_expand_result(struct ht2_repeat_expand_result *result) { PyObject *positions = NULL; size_t i = 0; if(result == NULL) { return NULL; } positions = PyList_New(result->count); for(i = 0; i < result->count; i++) { struct ht2_position *htpos = &result->positions[i]; PyList_SetItem(positions, i, Py_BuildValue("(III)", htpos->chr_id, htpos->direction, htpos->pos) ); } return positions; } static PyObject *conv_ht2opt(ht2_option_t *opts) { PyObject *py_opt = NULL; py_opt = PyDict_New(); if(py_opt == NULL) { return NULL; } #define HT2_OPT_BUILD(_pobj, _popt, _name, _type) \ do {\ if(PyDict_SetItemString((_pobj), #_name, Py_BuildValue((_type), (_popt)->_name)) < 0) {\ DEBUGLOG("Can't set item %s\n", #_name);\ } \ } while(0) HT2_OPT_BUILD(py_opt, opts, offRate, "i"); HT2_OPT_BUILD(py_opt, opts, useMm, "i"); HT2_OPT_BUILD(py_opt, opts, useShmem, "i"); HT2_OPT_BUILD(py_opt, opts, mmSweep, "i"); HT2_OPT_BUILD(py_opt, opts, noRefNames, "i"); HT2_OPT_BUILD(py_opt, opts, noSplicedAlignment, "i"); HT2_OPT_BUILD(py_opt, opts, gVerbose, "i"); HT2_OPT_BUILD(py_opt, opts, startVerbose, "i"); HT2_OPT_BUILD(py_opt, opts, sanityCheck, "i"); HT2_OPT_BUILD(py_opt, opts, useHaplotype, "i"); return py_opt; } static void update_ht2_options(ht2_option_t *ht2opt, PyObject *py_opt) { #define HT2_OPT_UPDATE(_pobj, _ht2opt, _name) \ do {\ PyObject *p;\ if((p = PyDict_GetItemString((_pobj), #_name)) != NULL) { \ (_ht2opt)->_name = PyInt_AsLong(p); \ DEBUGLOG(#_name " %d\n", (ht2opt)->_name); \ if(PyErr_Occurred() != NULL) { \ DEBUGLOG("Error Occurred"); \ }\ }\ } while (0) HT2_OPT_UPDATE(py_opt, ht2opt, offRate); HT2_OPT_UPDATE(py_opt, ht2opt, useMm); HT2_OPT_UPDATE(py_opt, ht2opt, mmSweep); HT2_OPT_UPDATE(py_opt, ht2opt, noRefNames); HT2_OPT_UPDATE(py_opt, ht2opt, noSplicedAlignment); HT2_OPT_UPDATE(py_opt, ht2opt, gVerbose); HT2_OPT_UPDATE(py_opt, ht2opt, startVerbose); HT2_OPT_UPDATE(py_opt, ht2opt, sanityCheck); HT2_OPT_UPDATE(py_opt, ht2opt, useHaplotype); } static PyObject *ht2py_get_options(PyObject *self, PyObject *args) { ht2_option_t ht2opt; ht2_init_options(&ht2opt); /* convert ht2_option_t to PyObject(map) */ PyObject *pobj = conv_ht2opt(&ht2opt); return pobj; } static PyObject *ht2py_init(PyObject *self, PyObject *args) { ht2_handle_t handle; PyObject *popt = NULL; char *name = NULL; if(!PyArg_ParseTuple(args, "sO", &name, &popt)) { return NULL; } DEBUGLOG("name %s\n", name); DEBUGLOG("popt %p\n", popt); if(!PyDict_CheckExact(popt)) { // TODO // exception DEBUGLOG("Invalid data type\n"); return NULL; } ht2_option_t ht2opt; ht2_init_options(&ht2opt); update_ht2_options(&ht2opt, popt); handle = ht2_init(name, &ht2opt); DEBUGLOG("handle %p\n", handle); PyObject *cap = PyCapsule_New(handle, HT2_HANDLE_ID, NULL); return cap; } static PyObject *ht2py_close(PyObject *self, PyObject *args) { ht2_handle_t handle; PyObject *cap; // Parse Args // ht2py.close(handle) // if(!PyArg_ParseTuple(args, "O", &cap)) { DEBUGLOG("Can't parse args\n"); return NULL; } handle = get_handle(cap); if(handle == NULL) { DEBUGLOG("Can't get handle\n"); return NULL; } DEBUGLOG("handle %p\n", handle); ht2_close(handle); Py_RETURN_NONE; } static PyObject *ht2py_index_getrefnamebyid(PyObject *self, PyObject *args) { PyObject *cap; uint32_t chr_id; // ht2py.index_getrefnamebyid(handle, chr_id) if(!PyArg_ParseTuple(args, "Oi", &cap, &chr_id)) { DEBUGLOG("Can't parse args\n"); return NULL; } ht2_handle_t handle = get_handle(cap); if(handle == NULL) { DEBUGLOG("Can't get handle\n"); return NULL; } const char *refname = ht2_index_getrefnamebyid(handle, chr_id); if(refname == NULL) { DEBUGLOG("Can't get refname(%u)\n", chr_id); return Py_BuildValue("s", ""); } return Py_BuildValue("s", refname); } static PyObject *ht2py_index_getrefnames(PyObject *self, PyObject *args) { ht2_handle_t handle; PyObject *cap; // Parse Args // ht2py.index_getrefnames(handle) if(!PyArg_ParseTuple(args, "O", &cap)) { DEBUGLOG("Can't parse args\n"); return NULL; } handle = get_handle(cap); if(handle == NULL) { DEBUGLOG("Can't get handle\n"); return NULL; } struct ht2_index_getrefnames_result *result = NULL; ht2_error_t ret = ht2_index_getrefnames(handle, &result); PyObject *refnames = NULL; if(ret == HT2_OK) { /* Build List of names */ refnames = conv_refnames_result(result); free(result); } else { refnames = PyList_New(0); } return refnames; } static PyObject *ht2py_repeat_expand(PyObject *self, PyObject *args) { PyObject *cap; char *name = NULL; uint64_t rpos = 0; uint64_t rlen = 0; // Parse Args // ht2py.repeat_expand(handle, 'repeat_name', repeat_pos, repeat_len) if(!PyArg_ParseTuple(args, "OsLL", &cap, &name, &rpos, &rlen)) { DEBUGLOG("Can't parse args\n"); return NULL; } //fprintf(stderr, "%s, %lu, %lu\n", name, rpos, rlen); ht2_handle_t handle = get_handle(cap); if(handle == NULL) { DEBUGLOG("Can't get handle\n"); return NULL; } struct ht2_repeat_expand_result *result = NULL; ht2_error_t ret = ht2_repeat_expand(handle, name, rpos, rlen, &result); PyObject *positions = NULL; if(ret == HT2_OK) { /* Build list of position */ positions = conv_repeat_expand_result(result); free(result); } else { DEBUGLOG("error %d, %s, %lu, %lu\n", ret, name, rpos, rlen); positions = PyList_New(0); } return positions; } static PyMethodDef myMethods[] = { /* Initialize APIs */ {"get_options", ht2py_get_options, METH_NOARGS, "Get default options"}, {"init", ht2py_init, METH_VARARGS, "Initialize HT2Lib handle"}, {"close", ht2py_close, METH_VARARGS, "Release HT2Lib handle"}, /* Index APIs */ {"index_getrefnamebyid", ht2py_index_getrefnamebyid, METH_VARARGS, "Get reference name"}, {"index_getrefnames", ht2py_index_getrefnames, METH_VARARGS, "Get all reference names"}, /* Repeat APIs */ {"repeat_expand", ht2py_repeat_expand, METH_VARARGS, "Find reference positions"}, /* */ {NULL, NULL, 0, NULL} }; PyMODINIT_FUNC initht2py(void) { (void)Py_InitModule("ht2py", myMethods); } hisat2-2.2.1/hisat2lib/pymodule/setup.py000077500000000000000000000022251370663763200201600ustar00rootroot00000000000000#!/usr/bin/python # # Copyright 2018, Chanhee Park and Daehwan Kim # # This file is part of HISAT 2. # # HISAT 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # HISAT 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with HISAT 2. If not, see . from distutils.core import setup, Extension module1 = Extension('ht2py', # define_macros = [('DEBUG', '1')], include_dirs=['../'], libraries=['stdc++'], extra_objects = ['../../libhisat2lib.a'], sources = ['ht2module.c']) setup(name = 'ht2py', version = '1.0', ext_modules = [module1]) hisat2-2.2.1/hisat_bp.cpp000066400000000000000000004564421370663763200152370ustar00rootroot00000000000000/* * Copyright 2014, Daehwan Kim * * This file is part of HISAT. * * HISAT is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT. If not, see . */ #include #include #include #include #include #include #include #include #include #include #include "alphabet.h" #include "assert_helpers.h" #include "endian_swap.h" #include "bt2_idx.h" #include "bt2_io.h" #include "bt2_util.h" #include "hier_idx.h" #include "formats.h" #include "sequence_io.h" #include "tokenize.h" #include "aln_sink.h" #include "pat.h" #include "threading.h" #include "ds.h" #include "aligner_metrics.h" #include "sam.h" #include "aligner_seed.h" #include "splice_site.h" #include "bp_aligner.h" #include "aligner_seed_policy.h" #include "aligner_driver.h" #include "aligner_sw.h" #include "aligner_sw_driver.h" #include "aligner_cache.h" #include "util.h" #include "pe.h" #include "simple_func.h" #include "presets.h" #include "opts.h" #include "outq.h" #include "aligner_seed2.h" using namespace std; static EList mates1; // mated reads (first mate) static EList mates2; // mated reads (second mate) static EList mates12; // mated reads (1st/2nd interleaved in 1 file) static string adjIdxBase; bool gColor; // colorspace (not supported) int gVerbose; // be talkative static bool startVerbose; // be talkative at startup int gQuiet; // print nothing but the alignments static int sanityCheck; // enable expensive sanity checks static int format; // default read format is FASTQ static string origString; // reference text, or filename(s) static int seed; // srandom() seed static int timing; // whether to report basic timing data static int metricsIval; // interval between alignment metrics messages (0 = no messages) static string metricsFile;// output file to put alignment metrics in static bool metricsStderr;// output file to put alignment metrics in static bool metricsPerRead; // report a metrics tuple for every read static bool allHits; // for multihits, report just one static bool showVersion; // just print version and quit? static int ipause; // pause before maching? static uint32_t qUpto; // max # of queries to read int gTrim5; // amount to trim from 5' end int gTrim3; // amount to trim from 3' end static int offRate; // keep default offRate static bool solexaQuals; // quality strings are solexa quals, not phred, and subtract 64 (not 33) static bool phred64Quals; // quality chars are phred, but must subtract 64 (not 33) static bool integerQuals; // quality strings are space-separated strings of integers, not ASCII static int nthreads; // number of pthreads operating concurrently static int outType; // style of output static bool noRefNames; // true -> print reference indexes; not names static uint32_t khits; // number of hits per read; >1 is much slower static uint32_t mhits; // don't report any hits if there are > mhits static int partitionSz; // output a partitioning key in first field static bool useSpinlock; // false -> don't use of spinlocks even if they're #defines static bool fileParallel; // separate threads read separate input files in parallel static bool useShmem; // use shared memory to hold the index static bool useMm; // use memory-mapped files to hold the index static bool mmSweep; // sweep through memory-mapped files immediately after mapping int gMinInsert; // minimum insert size int gMaxInsert; // maximum insert size bool gMate1fw; // -1 mate aligns in fw orientation on fw strand bool gMate2fw; // -2 mate aligns in rc orientation on fw strand bool gFlippedMatesOK; // allow mates to be in wrong order bool gDovetailMatesOK; // allow one mate to extend off the end of the other bool gContainMatesOK; // allow one mate to contain the other in PE alignment bool gOlapMatesOK; // allow mates to overlap in PE alignment bool gExpandToFrag; // incr max frag length to =larger mate len if necessary bool gReportDiscordant; // find and report discordant paired-end alignments bool gReportMixed; // find and report unpaired alignments for paired reads static uint32_t cacheLimit; // ranges w/ size > limit will be cached static uint32_t cacheSize; // # words per range cache static uint32_t skipReads; // # reads/read pairs to skip bool gNofw; // don't align fw orientation of read bool gNorc; // don't align rc orientation of read static uint32_t fastaContLen; static uint32_t fastaContFreq; static bool hadoopOut; // print Hadoop status and summary messages static bool fuzzy; static bool fullRef; static bool samTruncQname; // whether to truncate QNAME to 255 chars static bool samOmitSecSeqQual; // omit SEQ/QUAL for 2ndary alignments? static bool samNoUnal; // don't print records for unaligned reads static bool samNoHead; // don't print any header lines in SAM output static bool samNoSQ; // don't print @SQ header lines static bool sam_print_as; static bool sam_print_xs; // XS:i static bool sam_print_xss; // Xs:i and Ys:i static bool sam_print_yn; // YN:i and Yn:i static bool sam_print_xn; static bool sam_print_cs; static bool sam_print_cq; static bool sam_print_x0; static bool sam_print_x1; static bool sam_print_xm; static bool sam_print_xo; static bool sam_print_xg; static bool sam_print_nm; static bool sam_print_md; static bool sam_print_yf; static bool sam_print_yi; static bool sam_print_ym; static bool sam_print_yp; static bool sam_print_yt; static bool sam_print_ys; static bool sam_print_zs; static bool sam_print_xr; static bool sam_print_xt; static bool sam_print_xd; static bool sam_print_xu; static bool sam_print_yl; static bool sam_print_ye; static bool sam_print_yu; static bool sam_print_xp; static bool sam_print_yr; static bool sam_print_zb; static bool sam_print_zr; static bool sam_print_zf; static bool sam_print_zm; static bool sam_print_zi; static bool sam_print_zp; static bool sam_print_zu; static bool sam_print_xs_a; static bool bwaSwLike; static float bwaSwLikeC; static float bwaSwLikeT; static bool qcFilter; static bool sortByScore; // prioritize alignments to report by score? bool gReportOverhangs; // false -> filter out alignments that fall off the end of a reference sequence static string rgid; // ID: setting for @RG header line static string rgs; // SAM outputs for @RG header line static string rgs_optflag; // SAM optional flag to add corresponding to @RG ID static bool msample; // whether to report a random alignment when maxed-out via -m/-M int gGapBarrier; // # diags on top/bot only to be entered diagonally static EList qualities; static EList qualities1; static EList qualities2; static string polstr; // temporary holder for policy string static bool msNoCache; // true -> disable local cache static int bonusMatchType; // how to reward matches static int bonusMatch; // constant reward if bonusMatchType=constant static int penMmcType; // how to penalize mismatches static int penMmcMax; // max mm penalty static int penMmcMin; // min mm penalty static int penNType; // how to penalize Ns in the read static int penN; // constant if N pelanty is a constant static bool penNCatPair; // concatenate mates before N filtering? static bool localAlign; // do local alignment in DP steps static bool noisyHpolymer; // set to true if gap penalties should be reduced to be consistent with a sequencer that under- and overcalls homopolymers static int penRdGapConst; // constant cost of extending a gap in the read static int penRfGapConst; // constant cost of extending a gap in the reference static int penRdGapLinear; // coeff of linear term for cost of gap extension in read static int penRfGapLinear; // coeff of linear term for cost of gap extension in ref static SimpleFunc scoreMin; // minimum valid score as function of read len static SimpleFunc nCeil; // max # Ns allowed as function of read len static SimpleFunc msIval; // interval between seeds as function of read len static double descConsExp; // how to adjust score minimum as we descent further into index-assisted alignment static size_t descentLanding; // don't place a search root if it's within this many positions of end static SimpleFunc descentTotSz; // maximum space a DescentDriver can use in bytes static SimpleFunc descentTotFmops; // maximum # FM ops a DescentDriver can perform static int multiseedMms; // mismatches permitted in a multiseed seed static int multiseedLen; // length of multiseed seeds static size_t multiseedOff; // offset to begin extracting seeds static uint32_t seedCacheLocalMB; // # MB to use for non-shared seed alignment cacheing static uint32_t seedCacheCurrentMB; // # MB to use for current-read seed hit cacheing static uint32_t exactCacheCurrentMB; // # MB to use for current-read seed hit cacheing static size_t maxhalf; // max width on one side of DP table static bool seedSumm; // print summary information about seed hits, not alignments static bool doUngapped; // do ungapped alignment static size_t maxIters; // stop after this many extend loop iterations static size_t maxUg; // stop after this many ungap extends static size_t maxDp; // stop after this many DPs static size_t maxItersIncr; // amt to add to maxIters for each -k > 1 static size_t maxEeStreak; // stop after this many end-to-end fails in a row static size_t maxUgStreak; // stop after this many ungap fails in a row static size_t maxDpStreak; // stop after this many dp fails in a row static size_t maxStreakIncr; // amt to add to streak for each -k > 1 static size_t maxMateStreak; // stop seed range after this many mate-find fails static bool doExtend; // extend seed hits static bool enable8; // use 8-bit SSE where possible? static size_t cminlen; // longer reads use checkpointing static size_t cpow2; // checkpoint interval log2 static bool doTri; // do triangular mini-fills? static string defaultPreset; // default preset; applied immediately static bool ignoreQuals; // all mms incur same penalty, regardless of qual static string wrapper; // type of wrapper script, so we can print correct usage static EList queries; // list of query files static string outfile; // write SAM output to this file static int mapqv; // MAPQ calculation version static int tighten; // -M tighten mode (0=none, 1=best, 2=secbest+1) static bool doExactUpFront; // do exact search up front if seeds seem good enough static bool do1mmUpFront; // do 1mm search up front if seeds seem good enough static size_t do1mmMinLen; // length below which we disable 1mm e2e search static int seedBoostThresh; // if average non-zero position has more than this many elements static size_t nSeedRounds; // # seed rounds static bool reorder; // true -> reorder SAM recs in -p mode static float sampleFrac; // only align random fraction of input reads static bool arbitraryRandom; // pseudo-randoms no longer a function of read properties static bool bowtie2p5; static bool useTempSpliceSite; static int penCanSplice; static int penNoncanSplice; static SimpleFunc penIntronLen; static string knownSpliceSiteInfile; // static string novelSpliceSiteInfile; // static string novelSpliceSiteOutfile; // static bool no_spliced_alignment; static int rna_strandness; // static string bt2index; // read Bowtie 2 index from files with this prefix static EList > extra_opts; static size_t extra_opts_cur; static EList thread_rids; static MUTEX_T thread_rids_mutex; static uint64_t thread_rids_mindist; #define DMAX std::numeric_limits::max() static void resetOptions() { mates1.clear(); mates2.clear(); mates12.clear(); adjIdxBase = ""; gColor = false; gVerbose = 0; startVerbose = 0; gQuiet = false; sanityCheck = 0; // enable expensive sanity checks format = FASTQ; // default read format is FASTQ origString = ""; // reference text, or filename(s) seed = 0; // srandom() seed timing = 0; // whether to report basic timing data metricsIval = 1; // interval between alignment metrics messages (0 = no messages) metricsFile = ""; // output file to put alignment metrics in metricsStderr = false; // print metrics to stderr (in addition to --metrics-file if it's specified metricsPerRead = false; // report a metrics tuple for every read? allHits = false; // for multihits, report just one showVersion = false; // just print version and quit? ipause = 0; // pause before maching? qUpto = 0xffffffff; // max # of queries to read gTrim5 = 0; // amount to trim from 5' end gTrim3 = 0; // amount to trim from 3' end offRate = -1; // keep default offRate solexaQuals = false; // quality strings are solexa quals, not phred, and subtract 64 (not 33) phred64Quals = false; // quality chars are phred, but must subtract 64 (not 33) integerQuals = false; // quality strings are space-separated strings of integers, not ASCII nthreads = 1; // number of pthreads operating concurrently outType = OUTPUT_SAM; // style of output noRefNames = false; // true -> print reference indexes; not names khits = 5; // number of hits per read; >1 is much slower mhits = 0; // stop after finding this many alignments+1 partitionSz = 0; // output a partitioning key in first field useSpinlock = true; // false -> don't use of spinlocks even if they're #defines fileParallel = false; // separate threads read separate input files in parallel useShmem = false; // use shared memory to hold the index useMm = false; // use memory-mapped files to hold the index mmSweep = false; // sweep through memory-mapped files immediately after mapping gMinInsert = 0; // minimum insert size gMaxInsert = 500; // maximum insert size gMate1fw = true; // -1 mate aligns in fw orientation on fw strand gMate2fw = false; // -2 mate aligns in rc orientation on fw strand gFlippedMatesOK = false; // allow mates to be in wrong order gDovetailMatesOK = false; // allow one mate to extend off the end of the other gContainMatesOK = true; // allow one mate to contain the other in PE alignment gOlapMatesOK = true; // allow mates to overlap in PE alignment gExpandToFrag = true; // incr max frag length to =larger mate len if necessary gReportDiscordant = true; // find and report discordant paired-end alignments gReportMixed = true; // find and report unpaired alignments for paired reads cacheLimit = 5; // ranges w/ size > limit will be cached cacheSize = 0; // # words per range cache skipReads = 0; // # reads/read pairs to skip gNofw = false; // don't align fw orientation of read gNorc = false; // don't align rc orientation of read fastaContLen = 0; fastaContFreq = 0; hadoopOut = false; // print Hadoop status and summary messages fuzzy = false; // reads will have alternate basecalls w/ qualities fullRef = false; // print entire reference name instead of just up to 1st space samTruncQname = true; // whether to truncate QNAME to 255 chars samOmitSecSeqQual = false; // omit SEQ/QUAL for 2ndary alignments? samNoUnal = false; // omit SAM records for unaligned reads samNoHead = false; // don't print any header lines in SAM output samNoSQ = false; // don't print @SQ header lines sam_print_as = true; sam_print_xs = true; sam_print_xss = false; // Xs:i and Ys:i sam_print_yn = false; // YN:i and Yn:i sam_print_xn = true; sam_print_cs = false; sam_print_cq = false; sam_print_x0 = true; sam_print_x1 = true; sam_print_xm = true; sam_print_xo = true; sam_print_xg = true; sam_print_nm = true; sam_print_md = true; sam_print_yf = true; sam_print_yi = false; sam_print_ym = false; sam_print_yp = false; sam_print_yt = true; sam_print_ys = true; sam_print_zs = false; sam_print_xr = false; sam_print_xt = false; sam_print_xd = false; sam_print_xu = false; sam_print_yl = false; sam_print_ye = false; sam_print_yu = false; sam_print_xp = false; sam_print_yr = false; sam_print_zb = false; sam_print_zr = false; sam_print_zf = false; sam_print_zm = false; sam_print_zi = false; sam_print_zp = false; sam_print_zu = false; sam_print_xs_a = true; bwaSwLike = false; bwaSwLikeC = 5.5f; bwaSwLikeT = 20.0f; qcFilter = false; // don't believe upstream qc by default sortByScore = true; // prioritize alignments to report by score? rgid = ""; // SAM outputs for @RG header line rgs = ""; // SAM outputs for @RG header line rgs_optflag = ""; // SAM optional flag to add corresponding to @RG ID msample = true; gGapBarrier = 4; // disallow gaps within this many chars of either end of alignment qualities.clear(); qualities1.clear(); qualities2.clear(); polstr.clear(); msNoCache = true; // true -> disable local cache bonusMatchType = DEFAULT_MATCH_BONUS_TYPE; bonusMatch = DEFAULT_MATCH_BONUS; penMmcType = DEFAULT_MM_PENALTY_TYPE; penMmcMax = DEFAULT_MM_PENALTY_MAX; penMmcMin = DEFAULT_MM_PENALTY_MIN; penNType = DEFAULT_N_PENALTY_TYPE; penN = DEFAULT_N_PENALTY; penNCatPair = DEFAULT_N_CAT_PAIR; // concatenate mates before N filtering? localAlign = false; // do local alignment in DP steps noisyHpolymer = false; penRdGapConst = DEFAULT_READ_GAP_CONST; penRfGapConst = DEFAULT_REF_GAP_CONST; penRdGapLinear = DEFAULT_READ_GAP_LINEAR; penRfGapLinear = DEFAULT_REF_GAP_LINEAR; // scoreMin.init (SIMPLE_FUNC_LINEAR, DEFAULT_MIN_CONST, DEFAULT_MIN_LINEAR); scoreMin.init (SIMPLE_FUNC_CONST, -18, 0); nCeil.init (SIMPLE_FUNC_LINEAR, 0.0f, DMAX, 2.0f, 0.1f); msIval.init (SIMPLE_FUNC_LINEAR, 1.0f, DMAX, DEFAULT_IVAL_B, DEFAULT_IVAL_A); descConsExp = 2.0; descentLanding = 20; descentTotSz.init(SIMPLE_FUNC_LINEAR, 1024.0, DMAX, 0.0, 1024.0); descentTotFmops.init(SIMPLE_FUNC_LINEAR, 100.0, DMAX, 0.0, 10.0); multiseedMms = DEFAULT_SEEDMMS; multiseedLen = DEFAULT_SEEDLEN; multiseedOff = 0; seedCacheLocalMB = 32; // # MB to use for non-shared seed alignment cacheing seedCacheCurrentMB = 20; // # MB to use for current-read seed hit cacheing exactCacheCurrentMB = 20; // # MB to use for current-read seed hit cacheing maxhalf = 15; // max width on one side of DP table seedSumm = false; // print summary information about seed hits, not alignments doUngapped = true; // do ungapped alignment maxIters = 400; // max iterations of extend loop maxUg = 300; // stop after this many ungap extends maxDp = 300; // stop after this many dp extends maxItersIncr = 20; // amt to add to maxIters for each -k > 1 maxEeStreak = 15; // stop after this many end-to-end fails in a row maxUgStreak = 15; // stop after this many ungap fails in a row maxDpStreak = 15; // stop after this many dp fails in a row maxStreakIncr = 10; // amt to add to streak for each -k > 1 maxMateStreak = 10; // in PE: abort seed range after N mate-find fails doExtend = true; // do seed extensions enable8 = true; // use 8-bit SSE where possible? cminlen = 2000; // longer reads use checkpointing cpow2 = 4; // checkpoint interval log2 doTri = false; // do triangular mini-fills? defaultPreset = "sensitive%LOCAL%"; // default preset; applied immediately extra_opts.clear(); extra_opts_cur = 0; bt2index.clear(); // read Bowtie 2 index from files with this prefix ignoreQuals = false; // all mms incur same penalty, regardless of qual wrapper.clear(); // type of wrapper script, so we can print correct usage queries.clear(); // list of query files outfile.clear(); // write SAM output to this file mapqv = 2; // MAPQ calculation version tighten = 3; // -M tightening mode doExactUpFront = true; // do exact search up front if seeds seem good enough do1mmUpFront = true; // do 1mm search up front if seeds seem good enough seedBoostThresh = 300; // if average non-zero position has more than this many elements nSeedRounds = 2; // # rounds of seed searches to do for repetitive reads do1mmMinLen = 60; // length below which we disable 1mm search reorder = false; // reorder SAM records with -p > 1 sampleFrac = 1.1f; // align all reads arbitraryRandom = false; // let pseudo-random seeds be a function of read properties bowtie2p5 = false; useTempSpliceSite = true; penCanSplice = 0; penNoncanSplice = 3; penIntronLen.init(SIMPLE_FUNC_LOG, -8, 1); knownSpliceSiteInfile = ""; novelSpliceSiteInfile = ""; novelSpliceSiteOutfile = ""; no_spliced_alignment = false; rna_strandness = RNA_STRANDNESS_UNKNOWN; } static const char *short_options = "fF:qbzhcu:rv:s:aP:t3:5:w:p:k:M:1:2:I:X:CQ:N:i:L:U:x:S:g:O:D:R:"; static struct option long_options[] = { {(char*)"verbose", no_argument, 0, ARG_VERBOSE}, {(char*)"startverbose", no_argument, 0, ARG_STARTVERBOSE}, {(char*)"quiet", no_argument, 0, ARG_QUIET}, {(char*)"sanity", no_argument, 0, ARG_SANITY}, {(char*)"pause", no_argument, &ipause, 1}, {(char*)"orig", required_argument, 0, ARG_ORIG}, {(char*)"all", no_argument, 0, 'a'}, {(char*)"solexa-quals", no_argument, 0, ARG_SOLEXA_QUALS}, {(char*)"integer-quals",no_argument, 0, ARG_INTEGER_QUALS}, {(char*)"int-quals", no_argument, 0, ARG_INTEGER_QUALS}, {(char*)"metrics", required_argument, 0, ARG_METRIC_IVAL}, {(char*)"metrics-file", required_argument, 0, ARG_METRIC_FILE}, {(char*)"metrics-stderr",no_argument, 0, ARG_METRIC_STDERR}, {(char*)"metrics-per-read", no_argument, 0, ARG_METRIC_PER_READ}, {(char*)"met-read", no_argument, 0, ARG_METRIC_PER_READ}, {(char*)"met", required_argument, 0, ARG_METRIC_IVAL}, {(char*)"met-file", required_argument, 0, ARG_METRIC_FILE}, {(char*)"met-stderr", no_argument, 0, ARG_METRIC_STDERR}, {(char*)"time", no_argument, 0, 't'}, {(char*)"trim3", required_argument, 0, '3'}, {(char*)"trim5", required_argument, 0, '5'}, {(char*)"seed", required_argument, 0, ARG_SEED}, {(char*)"qupto", required_argument, 0, 'u'}, {(char*)"upto", required_argument, 0, 'u'}, {(char*)"version", no_argument, 0, ARG_VERSION}, {(char*)"filepar", no_argument, 0, ARG_FILEPAR}, {(char*)"help", no_argument, 0, 'h'}, {(char*)"threads", required_argument, 0, 'p'}, {(char*)"khits", required_argument, 0, 'k'}, {(char*)"minins", required_argument, 0, 'I'}, {(char*)"maxins", required_argument, 0, 'X'}, {(char*)"quals", required_argument, 0, 'Q'}, {(char*)"Q1", required_argument, 0, ARG_QUALS1}, {(char*)"Q2", required_argument, 0, ARG_QUALS2}, {(char*)"refidx", no_argument, 0, ARG_REFIDX}, {(char*)"partition", required_argument, 0, ARG_PARTITION}, {(char*)"ff", no_argument, 0, ARG_FF}, {(char*)"fr", no_argument, 0, ARG_FR}, {(char*)"rf", no_argument, 0, ARG_RF}, {(char*)"cachelim", required_argument, 0, ARG_CACHE_LIM}, {(char*)"cachesz", required_argument, 0, ARG_CACHE_SZ}, {(char*)"nofw", no_argument, 0, ARG_NO_FW}, {(char*)"norc", no_argument, 0, ARG_NO_RC}, {(char*)"skip", required_argument, 0, 's'}, {(char*)"12", required_argument, 0, ARG_ONETWO}, {(char*)"tab5", required_argument, 0, ARG_TAB5}, {(char*)"tab6", required_argument, 0, ARG_TAB6}, {(char*)"phred33-quals", no_argument, 0, ARG_PHRED33}, {(char*)"phred64-quals", no_argument, 0, ARG_PHRED64}, {(char*)"phred33", no_argument, 0, ARG_PHRED33}, {(char*)"phred64", no_argument, 0, ARG_PHRED64}, {(char*)"solexa1.3-quals", no_argument, 0, ARG_PHRED64}, {(char*)"mm", no_argument, 0, ARG_MM}, {(char*)"shmem", no_argument, 0, ARG_SHMEM}, {(char*)"mmsweep", no_argument, 0, ARG_MMSWEEP}, {(char*)"hadoopout", no_argument, 0, ARG_HADOOPOUT}, {(char*)"fuzzy", no_argument, 0, ARG_FUZZY}, {(char*)"fullref", no_argument, 0, ARG_FULLREF}, {(char*)"usage", no_argument, 0, ARG_USAGE}, {(char*)"sam-no-qname-trunc", no_argument, 0, ARG_SAM_NO_QNAME_TRUNC}, {(char*)"sam-omit-sec-seq", no_argument, 0, ARG_SAM_OMIT_SEC_SEQ}, {(char*)"omit-sec-seq", no_argument, 0, ARG_SAM_OMIT_SEC_SEQ}, {(char*)"sam-no-head", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"sam-nohead", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"sam-noHD", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"sam-no-hd", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"sam-nosq", no_argument, 0, ARG_SAM_NOSQ}, {(char*)"sam-no-sq", no_argument, 0, ARG_SAM_NOSQ}, {(char*)"sam-noSQ", no_argument, 0, ARG_SAM_NOSQ}, {(char*)"no-head", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"no-hd", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"no-sq", no_argument, 0, ARG_SAM_NOSQ}, {(char*)"no-HD", no_argument, 0, ARG_SAM_NOHEAD}, {(char*)"no-SQ", no_argument, 0, ARG_SAM_NOSQ}, {(char*)"no-unal", no_argument, 0, ARG_SAM_NO_UNAL}, {(char*)"color", no_argument, 0, 'C'}, {(char*)"sam-RG", required_argument, 0, ARG_SAM_RG}, {(char*)"sam-rg", required_argument, 0, ARG_SAM_RG}, {(char*)"sam-rg-id", required_argument, 0, ARG_SAM_RGID}, {(char*)"RG", required_argument, 0, ARG_SAM_RG}, {(char*)"rg", required_argument, 0, ARG_SAM_RG}, {(char*)"rg-id", required_argument, 0, ARG_SAM_RGID}, {(char*)"snpphred", required_argument, 0, ARG_SNPPHRED}, {(char*)"snpfrac", required_argument, 0, ARG_SNPFRAC}, {(char*)"gbar", required_argument, 0, ARG_GAP_BAR}, {(char*)"qseq", no_argument, 0, ARG_QSEQ}, {(char*)"policy", required_argument, 0, ARG_ALIGN_POLICY}, {(char*)"preset", required_argument, 0, 'P'}, {(char*)"seed-summ", no_argument, 0, ARG_SEED_SUMM}, {(char*)"seed-summary", no_argument, 0, ARG_SEED_SUMM}, {(char*)"overhang", no_argument, 0, ARG_OVERHANG}, {(char*)"no-cache", no_argument, 0, ARG_NO_CACHE}, {(char*)"cache", no_argument, 0, ARG_USE_CACHE}, {(char*)"454", no_argument, 0, ARG_NOISY_HPOLY}, {(char*)"ion-torrent", no_argument, 0, ARG_NOISY_HPOLY}, {(char*)"no-mixed", no_argument, 0, ARG_NO_MIXED}, {(char*)"no-discordant",no_argument, 0, ARG_NO_DISCORDANT}, {(char*)"local", no_argument, 0, ARG_LOCAL}, {(char*)"end-to-end", no_argument, 0, ARG_END_TO_END}, {(char*)"ungapped", no_argument, 0, ARG_UNGAPPED}, {(char*)"no-ungapped", no_argument, 0, ARG_UNGAPPED_NO}, {(char*)"sse8", no_argument, 0, ARG_SSE8}, {(char*)"no-sse8", no_argument, 0, ARG_SSE8_NO}, {(char*)"scan-narrowed",no_argument, 0, ARG_SCAN_NARROWED}, {(char*)"qc-filter", no_argument, 0, ARG_QC_FILTER}, {(char*)"bwa-sw-like", no_argument, 0, ARG_BWA_SW_LIKE}, {(char*)"multiseed", required_argument, 0, ARG_MULTISEED_IVAL}, {(char*)"ma", required_argument, 0, ARG_SCORE_MA}, {(char*)"mp", required_argument, 0, ARG_SCORE_MMP}, {(char*)"np", required_argument, 0, ARG_SCORE_NP}, {(char*)"rdg", required_argument, 0, ARG_SCORE_RDG}, {(char*)"rfg", required_argument, 0, ARG_SCORE_RFG}, {(char*)"score-min", required_argument, 0, ARG_SCORE_MIN}, {(char*)"min-score", required_argument, 0, ARG_SCORE_MIN}, {(char*)"n-ceil", required_argument, 0, ARG_N_CEIL}, {(char*)"dpad", required_argument, 0, ARG_DPAD}, {(char*)"mapq-print-inputs",no_argument, 0, ARG_SAM_PRINT_YI}, {(char*)"very-fast", no_argument, 0, ARG_PRESET_VERY_FAST}, {(char*)"fast", no_argument, 0, ARG_PRESET_FAST}, {(char*)"sensitive", no_argument, 0, ARG_PRESET_SENSITIVE}, {(char*)"very-sensitive", no_argument, 0, ARG_PRESET_VERY_SENSITIVE}, {(char*)"very-fast-local", no_argument, 0, ARG_PRESET_VERY_FAST_LOCAL}, {(char*)"fast-local", no_argument, 0, ARG_PRESET_FAST_LOCAL}, {(char*)"sensitive-local", no_argument, 0, ARG_PRESET_SENSITIVE_LOCAL}, {(char*)"very-sensitive-local", no_argument, 0, ARG_PRESET_VERY_SENSITIVE_LOCAL}, {(char*)"no-score-priority",no_argument, 0, ARG_NO_SCORE_PRIORITY}, {(char*)"seedlen", required_argument, 0, 'L'}, {(char*)"seedmms", required_argument, 0, 'N'}, {(char*)"seedival", required_argument, 0, 'i'}, {(char*)"ignore-quals", no_argument, 0, ARG_IGNORE_QUALS}, {(char*)"index", required_argument, 0, 'x'}, {(char*)"arg-desc", no_argument, 0, ARG_DESC}, {(char*)"wrapper", required_argument, 0, ARG_WRAPPER}, {(char*)"unpaired", required_argument, 0, 'U'}, {(char*)"output", required_argument, 0, 'S'}, {(char*)"mapq-v", required_argument, 0, ARG_MAPQ_V}, {(char*)"dovetail", no_argument, 0, ARG_DOVETAIL}, {(char*)"no-dovetail", no_argument, 0, ARG_NO_DOVETAIL}, {(char*)"contain", no_argument, 0, ARG_CONTAIN}, {(char*)"no-contain", no_argument, 0, ARG_NO_CONTAIN}, {(char*)"overlap", no_argument, 0, ARG_OVERLAP}, {(char*)"no-overlap", no_argument, 0, ARG_NO_OVERLAP}, {(char*)"tighten", required_argument, 0, ARG_TIGHTEN}, {(char*)"exact-upfront", no_argument, 0, ARG_EXACT_UPFRONT}, {(char*)"1mm-upfront", no_argument, 0, ARG_1MM_UPFRONT}, {(char*)"no-exact-upfront", no_argument, 0, ARG_EXACT_UPFRONT_NO}, {(char*)"no-1mm-upfront", no_argument, 0, ARG_1MM_UPFRONT_NO}, {(char*)"1mm-minlen", required_argument, 0, ARG_1MM_MINLEN}, {(char*)"seed-off", required_argument, 0, 'O'}, {(char*)"seed-boost", required_argument, 0, ARG_SEED_BOOST_THRESH}, {(char*)"read-times", no_argument, 0, ARG_READ_TIMES}, {(char*)"show-rand-seed", no_argument, 0, ARG_SHOW_RAND_SEED}, {(char*)"dp-fail-streak", required_argument, 0, ARG_DP_FAIL_STREAK_THRESH}, {(char*)"ee-fail-streak", required_argument, 0, ARG_EE_FAIL_STREAK_THRESH}, {(char*)"ug-fail-streak", required_argument, 0, ARG_UG_FAIL_STREAK_THRESH}, {(char*)"fail-streak", required_argument, 0, 'D'}, {(char*)"dp-fails", required_argument, 0, ARG_DP_FAIL_THRESH}, {(char*)"ug-fails", required_argument, 0, ARG_UG_FAIL_THRESH}, {(char*)"extends", required_argument, 0, ARG_EXTEND_ITERS}, {(char*)"no-extend", no_argument, 0, ARG_NO_EXTEND}, {(char*)"mapq-extra", no_argument, 0, ARG_MAPQ_EX}, {(char*)"seed-rounds", required_argument, 0, 'R'}, {(char*)"reorder", no_argument, 0, ARG_REORDER}, {(char*)"passthrough", no_argument, 0, ARG_READ_PASSTHRU}, {(char*)"sample", required_argument, 0, ARG_SAMPLE}, {(char*)"cp-min", required_argument, 0, ARG_CP_MIN}, {(char*)"cp-ival", required_argument, 0, ARG_CP_IVAL}, {(char*)"tri", no_argument, 0, ARG_TRI}, {(char*)"nondeterministic", no_argument, 0, ARG_NON_DETERMINISTIC}, {(char*)"non-deterministic", no_argument, 0, ARG_NON_DETERMINISTIC}, {(char*)"local-seed-cache-sz", required_argument, 0, ARG_LOCAL_SEED_CACHE_SZ}, {(char*)"seed-cache-sz", required_argument, 0, ARG_CURRENT_SEED_CACHE_SZ}, {(char*)"no-unal", no_argument, 0, ARG_SAM_NO_UNAL}, {(char*)"test-25", no_argument, 0, ARG_TEST_25}, // TODO: following should be a function of read length? {(char*)"desc-kb", required_argument, 0, ARG_DESC_KB}, {(char*)"desc-landing", required_argument, 0, ARG_DESC_LANDING}, {(char*)"desc-exp", required_argument, 0, ARG_DESC_EXP}, {(char*)"desc-fmops", required_argument, 0, ARG_DESC_FMOPS}, {(char*)"no-temp-splicesite", no_argument, 0, ARG_NO_TEMPSPLICESITE}, {(char*)"pen-cansplice", required_argument, 0, ARG_PEN_CANSPLICE}, {(char*)"pen-noncansplice", required_argument, 0, ARG_PEN_NONCANSPLICE}, {(char*)"pen-intronlen", required_argument, 0, ARG_PEN_INTRONLEN}, {(char*)"known-splicesite-infile", required_argument, 0, ARG_KNOWN_SPLICESITE_INFILE}, {(char*)"novel-splicesite-infile", required_argument, 0, ARG_NOVEL_SPLICESITE_INFILE}, {(char*)"novel-splicesite-outfile", required_argument, 0, ARG_NOVEL_SPLICESITE_OUTFILE}, {(char*)"no-spliced-alignment", no_argument, 0, ARG_NO_SPLICED_ALIGNMENT}, {(char*)"rna-strandness", required_argument, 0, ARG_RNA_STRANDNESS}, {(char*)0, 0, 0, 0} // terminator }; /** * Print out a concise description of what options are taken and whether they * take an argument. */ static void printArgDesc(ostream& out) { // struct option { // const char *name; // int has_arg; // int *flag; // int val; // }; size_t i = 0; while(long_options[i].name != 0) { out << long_options[i].name << "\t" << (long_options[i].has_arg == no_argument ? 0 : 1) << endl; i++; } size_t solen = strlen(short_options); for(i = 0; i < solen; i++) { // Has an option? Does if next char is : if(i == solen-1) { assert_neq(':', short_options[i]); cout << (char)short_options[i] << "\t" << 0 << endl; } else { if(short_options[i+1] == ':') { // Option with argument cout << (char)short_options[i] << "\t" << 1 << endl; i++; // skip the ':' } else { // Option with no argument cout << (char)short_options[i] << "\t" << 0 << endl; } } } } /** * Print a summary usage message to the provided output stream. */ static void printUsage(ostream& out) { out << "HISAT version " << string(HISAT_VERSION).c_str() << " by Daehwan Kim (infphilo@gmail.com, www.ccb.jhu.edu/people/infphilo)" << endl; string tool_name = "hisat2-align"; if(wrapper == "basic-0") { tool_name = "hisat"; } out << "Usage: " << endl << " " << tool_name.c_str() << " [options]* -x {-1 -2 | -U } [-S ]" << endl << endl << " Index filename prefix (minus trailing .X." << gfm_ext << ")." << endl << " Files with #1 mates, paired with files in ." << endl; if(wrapper == "basic-0") { out << " Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2)." << endl; } out << " Files with #2 mates, paired with files in ." << endl; if(wrapper == "basic-0") { out << " Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2)." << endl; } out << " Files with unpaired reads." << endl; if(wrapper == "basic-0") { out << " Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2)." << endl; } out << " File for SAM output (default: stdout)" << endl << endl << " , , can be comma-separated lists (no whitespace) and can be" << endl << " specified many times. E.g. '-U file1.fq,file2.fq -U file3.fq'." << endl // Wrapper script should write line next << endl << "Options (defaults in parentheses):" << endl << endl << " Input:" << endl << " -q query input files are FASTQ .fq/.fastq (default)" << endl << " --qseq query input files are in Illumina's qseq format" << endl << " -f query input files are (multi-)FASTA .fa/.mfa" << endl << " -r query input files are raw one-sequence-per-line" << endl << " -c , , are sequences themselves, not files" << endl << " -s/--skip skip the first reads/pairs in the input (none)" << endl << " -u/--upto stop after first reads/pairs (no limit)" << endl << " -5/--trim5 trim bases from 5'/left end of reads (0)" << endl << " -3/--trim3 trim bases from 3'/right end of reads (0)" << endl << " --phred33 qualities are Phred+33 (default)" << endl << " --phred64 qualities are Phred+64" << endl << " --int-quals qualities encoded as space-delimited integers" << endl << endl << " Presets: Same as:" << endl << " For --end-to-end:" << endl << " --very-fast -D 5 -R 1 -N 0 -L 22 -i S,0,2.50" << endl << " --fast -D 10 -R 2 -N 0 -L 22 -i S,0,2.50" << endl << " --sensitive -D 15 -R 2 -N 0 -L 22 -i S,1,1.15 (default)" << endl << " --very-sensitive -D 20 -R 3 -N 0 -L 20 -i S,1,0.50" << endl << endl << " For --local:" << endl << " --very-fast-local -D 5 -R 1 -N 0 -L 25 -i S,1,2.00" << endl << " --fast-local -D 10 -R 2 -N 0 -L 22 -i S,1,1.75" << endl << " --sensitive-local -D 15 -R 2 -N 0 -L 20 -i S,1,0.75 (default)" << endl << " --very-sensitive-local -D 20 -R 3 -N 0 -L 20 -i S,1,0.50" << endl << endl << " Alignment:" << endl << " -N max # mismatches in seed alignment; can be 0 or 1 (0)" << endl << " -L length of seed substrings; must be >3, <32 (22)" << endl << " -i interval between seed substrings w/r/t read len (S,1,1.15)" << endl << " --n-ceil func for max # non-A/C/G/Ts permitted in aln (L,0,0.15)" << endl << " --dpad include extra ref chars on sides of DP table (15)" << endl << " --gbar disallow gaps within nucs of read extremes (4)" << endl << " --ignore-quals treat all quality values as 30 on Phred scale (off)" << endl << " --nofw do not align forward (original) version of read (off)" << endl << " --norc do not align reverse-complement version of read (off)" << endl << endl << " Spliced Alignment:" << endl << " --pen-cansplice penalty for a canonical splice site (0)" << endl << " --pen-noncansplice penalty for a non-canonical splice site (3)" << endl << " --pen-intronlen penalty for long introns (G,-8,1)" << endl << " --known-splicesite-infile provide a list of known splice sites" << endl << " --novel-splicesite-outfile report a list of splice sites" << endl << " --novel-splicesite-infile provide a list of novel splice sites" << endl << " --no-temp-splicesite disable the use of splice sites found" << endl << " --no-spliced-alignment disable spliced alignment" << endl << " --rna-strandness Specify strand-specific information (unstranded)" << endl << endl << " Scoring:" << endl << " --ma match bonus (0 for --end-to-end, 2 for --local) " << endl << " --mp max penalty for mismatch; lower qual = lower penalty (6)" << endl << " --np penalty for non-A/C/G/Ts in read/ref (1)" << endl << " --rdg , read gap open, extend penalties (5,3)" << endl << " --rfg , reference gap open, extend penalties (5,3)" << endl << " --score-min min acceptable alignment score w/r/t read length" << endl << " (G,20,8 for local, L,-0.6,-0.6 for end-to-end)" << endl << endl << " Reporting:" << endl << " (default) look for multiple alignments, report best, with MAPQ" << endl << " OR" << endl << " -k report up to alns per read; MAPQ not meaningful" << endl << " OR" << endl << " -a/--all report all alignments; very slow, MAPQ not meaningful" << endl << endl << " Effort:" << endl << " -D give up extending after failed extends in a row (15)" << endl << " -R for reads w/ repetitive seeds, try sets of seeds (2)" << endl << endl << " Paired-end:" << endl << " -I/--minins minimum fragment length (0)" << endl << " -X/--maxins maximum fragment length (500)" << endl << " --fr/--rf/--ff -1, -2 mates align fw/rev, rev/fw, fw/fw (--fr)" << endl << " --no-mixed suppress unpaired alignments for paired reads" << endl << " --no-discordant suppress discordant alignments for paired reads" << endl << " --no-dovetail not concordant when mates extend past each other" << endl << " --no-contain not concordant when one mate alignment contains other" << endl << " --no-overlap not concordant when mates overlap at all" << endl << endl << " Output:" << endl; //if(wrapper == "basic-0") { // out << " --bam output directly to BAM (by piping through 'samtools view')" << endl; //} out << " -t/--time print wall-clock time taken by search phases" << endl; if(wrapper == "basic-0") { out << " --un write unpaired reads that didn't align to " << endl << " --al write unpaired reads that aligned at least once to " << endl << " --un-conc write pairs that didn't align concordantly to " << endl << " --al-conc write pairs that aligned concordantly at least once to " << endl << " (Note: for --un, --al, --un-conc, or --al-conc, add '-gz' to the option name, e.g." << endl << " --un-gz , to gzip compress output, or add '-bz2' to bzip2 compress output.)" << endl; } out << " --quiet print nothing to stderr except serious errors" << endl // << " --refidx refer to ref. seqs by 0-based index rather than name" << endl << " --met-file send metrics to file at (off)" << endl << " --met-stderr send metrics to stderr (off)" << endl << " --met report internal counters & metrics every secs (1)" << endl // Following is supported in the wrapper instead // << " --no-unal supppress SAM records for unaligned reads" << endl << " --no-head supppress header lines, i.e. lines starting with @" << endl << " --no-sq supppress @SQ header lines" << endl << " --rg-id set read group id, reflected in @RG line and RG:Z: opt field" << endl << " --rg add (\"lab:value\") to @RG line of SAM header." << endl << " Note: @RG line only printed when --rg-id is set." << endl << " --omit-sec-seq put '*' in SEQ and QUAL fields for secondary alignments." << endl << endl << " Performance:" << endl << " -o/--offrate override offrate of index; must be >= index's offrate" << endl << " -p/--threads number of alignment threads to launch (1)" << endl << " --reorder force SAM output order to match order of input reads" << endl #ifdef BOWTIE_MM << " --mm use memory-mapped I/O for index; many 'hisat2's can share" << endl #endif #ifdef BOWTIE_SHARED_MEM //<< " --shmem use shared mem for index; many 'hisat2's can share" << endl #endif << endl << " Other:" << endl << " --qc-filter filter out reads that are bad according to QSEQ filter" << endl << " --seed seed for random number generator (0)" << endl << " --non-deterministic seed rand. gen. arbitrarily instead of using read attributes" << endl // << " --verbose verbose output for debugging" << endl << " --version print version information and quit" << endl << " -h/--help print this usage message" << endl ; if(wrapper.empty()) { cerr << endl << "*** Warning ***" << endl << "'hisat2-align' was run directly. It is recommended that you run the wrapper script 'hisat2' instead." << endl << endl; } } /** * Parse an int out of optarg and enforce that it be at least 'lower'; * if it is less than 'lower', than output the given error message and * exit with an error and a usage message. */ static int parseInt(int lower, int upper, const char *errmsg, const char *arg) { long l; char *endPtr= NULL; l = strtol(arg, &endPtr, 10); if (endPtr != NULL) { if (l < lower || l > upper) { cerr << errmsg << endl; printUsage(cerr); throw 1; } return (int32_t)l; } cerr << errmsg << endl; printUsage(cerr); throw 1; return -1; } /** * Upper is maximum int by default. */ static int parseInt(int lower, const char *errmsg, const char *arg) { return parseInt(lower, std::numeric_limits::max(), errmsg, arg); } /** * Parse a T string 'str'. */ template T parse(const char *s) { T tmp; stringstream ss(s); ss >> tmp; return tmp; } /** * Parse a pair of Ts from a string, 'str', delimited with 'delim'. */ template pair parsePair(const char *str, char delim) { string s(str); EList ss; tokenize(s, delim, ss); pair ret; ret.first = parse(ss[0].c_str()); ret.second = parse(ss[1].c_str()); return ret; } /** * Parse a pair of Ts from a string, 'str', delimited with 'delim'. */ template void parseTuple(const char *str, char delim, EList& ret) { string s(str); EList ss; tokenize(s, delim, ss); for(size_t i = 0; i < ss.size(); i++) { ret.push_back(parse(ss[i].c_str())); } } static string applyPreset(const string& sorig, Presets& presets) { string s = sorig; size_t found = s.find("%LOCAL%"); if(found != string::npos) { s.replace(found, strlen("%LOCAL%"), localAlign ? "-local" : ""); } if(gVerbose) { cerr << "Applying preset: '" << s.c_str() << "' using preset menu '" << presets.name() << "'" << endl; } string pol; presets.apply(s, pol, extra_opts); return pol; } static bool saw_M; static bool saw_a; static bool saw_k; static EList presetList; /** * TODO: Argument parsing is very, very flawed. The biggest problem is that * there are two separate worlds of arguments, the ones set via polstr, and * the ones set directly in variables. This makes for nasty interactions, * e.g., with the -M option being resolved at an awkward time relative to * the -k and -a options. */ static void parseOption(int next_option, const char *arg) { switch (next_option) { case ARG_TEST_25: bowtie2p5 = true; break; case ARG_DESC_KB: descentTotSz = SimpleFunc::parse(arg, 0.0, 1024.0, 1024.0, DMAX); break; case ARG_DESC_FMOPS: descentTotFmops = SimpleFunc::parse(arg, 0.0, 10.0, 100.0, DMAX); break; case ARG_DESC_LANDING: descentLanding = parse(arg); break; case ARG_DESC_EXP: { descConsExp = parse(arg); if(descConsExp < 0.0) { cerr << "Error: --desc-exp must be greater than or equal to 0" << endl; throw 1; } break; } case '1': tokenize(arg, ",", mates1); break; case '2': tokenize(arg, ",", mates2); break; case ARG_ONETWO: tokenize(arg, ",", mates12); format = TAB_MATE5; break; case ARG_TAB5: tokenize(arg, ",", mates12); format = TAB_MATE5; break; case ARG_TAB6: tokenize(arg, ",", mates12); format = TAB_MATE6; break; case 'f': format = FASTA; break; case 'F': { format = FASTA_CONT; pair p = parsePair(arg, ','); fastaContLen = p.first; fastaContFreq = p.second; break; } case ARG_BWA_SW_LIKE: { bwaSwLikeC = 5.5f; bwaSwLikeT = 30; bwaSwLike = true; localAlign = true; // -a INT Score of a match [1] // -b INT Mismatch penalty [3] // -q INT Gap open penalty [5] // -r INT Gap extension penalty. The penalty for a contiguous // gap of size k is q+k*r. [2] polstr += ";MA=1;MMP=C3;RDG=5,2;RFG=5,2"; break; } case 'q': format = FASTQ; break; case 'r': format = RAW; break; case 'c': format = CMDLINE; break; case ARG_QSEQ: format = QSEQ; break; case 'C': { cerr << "Error: -C specified but Bowtie 2 does not support colorspace input." << endl; throw 1; break; } case 'I': gMinInsert = parseInt(0, "-I arg must be positive", arg); break; case 'X': gMaxInsert = parseInt(1, "-X arg must be at least 1", arg); break; case ARG_NO_DISCORDANT: gReportDiscordant = false; break; case ARG_NO_MIXED: gReportMixed = false; break; case 's': skipReads = (uint32_t)parseInt(0, "-s arg must be positive", arg); break; case ARG_FF: gMate1fw = true; gMate2fw = true; break; case ARG_RF: gMate1fw = false; gMate2fw = true; break; case ARG_FR: gMate1fw = true; gMate2fw = false; break; case ARG_SHMEM: useShmem = true; break; case ARG_SEED_SUMM: seedSumm = true; break; case ARG_MM: { #ifdef BOWTIE_MM useMm = true; break; #else cerr << "Memory-mapped I/O mode is disabled because bowtie was not compiled with" << endl << "BOWTIE_MM defined. Memory-mapped I/O is not supported under Windows. If you" << endl << "would like to use memory-mapped I/O on a platform that supports it, please" << endl << "refrain from specifying BOWTIE_MM=0 when compiling Bowtie." << endl; throw 1; #endif } case ARG_MMSWEEP: mmSweep = true; break; case ARG_HADOOPOUT: hadoopOut = true; break; case ARG_SOLEXA_QUALS: solexaQuals = true; break; case ARG_INTEGER_QUALS: integerQuals = true; break; case ARG_PHRED64: phred64Quals = true; break; case ARG_PHRED33: solexaQuals = false; phred64Quals = false; break; case ARG_OVERHANG: gReportOverhangs = true; break; case ARG_NO_CACHE: msNoCache = true; break; case ARG_USE_CACHE: msNoCache = false; break; case ARG_LOCAL_SEED_CACHE_SZ: seedCacheLocalMB = (uint32_t)parseInt(1, "--local-seed-cache-sz arg must be at least 1", arg); break; case ARG_CURRENT_SEED_CACHE_SZ: seedCacheCurrentMB = (uint32_t)parseInt(1, "--seed-cache-sz arg must be at least 1", arg); break; case ARG_REFIDX: noRefNames = true; break; case ARG_FUZZY: fuzzy = true; break; case ARG_FULLREF: fullRef = true; break; case ARG_GAP_BAR: gGapBarrier = parseInt(1, "--gbar must be no less than 1", arg); break; case ARG_SEED: seed = parseInt(0, "--seed arg must be at least 0", arg); break; case ARG_NON_DETERMINISTIC: arbitraryRandom = true; break; case 'u': qUpto = (uint32_t)parseInt(1, "-u/--qupto arg must be at least 1", arg); break; case 'Q': tokenize(arg, ",", qualities); integerQuals = true; break; case ARG_QUALS1: tokenize(arg, ",", qualities1); integerQuals = true; break; case ARG_QUALS2: tokenize(arg, ",", qualities2); integerQuals = true; break; case ARG_CACHE_LIM: cacheLimit = (uint32_t)parseInt(1, "--cachelim arg must be at least 1", arg); break; case ARG_CACHE_SZ: cacheSize = (uint32_t)parseInt(1, "--cachesz arg must be at least 1", arg); cacheSize *= (1024 * 1024); // convert from MB to B break; case ARG_WRAPPER: wrapper = arg; break; case 'p': nthreads = parseInt(1, "-p/--threads arg must be at least 1", arg); break; case ARG_FILEPAR: fileParallel = true; break; case '3': gTrim3 = parseInt(0, "-3/--trim3 arg must be at least 0", arg); break; case '5': gTrim5 = parseInt(0, "-5/--trim5 arg must be at least 0", arg); break; case 'h': printUsage(cout); throw 0; break; case ARG_USAGE: printUsage(cout); throw 0; break; // // NOTE that unlike in Bowtie 1, -M, -a and -k are mutually // exclusive here. // case 'M': { msample = true; mhits = parse(arg); if(saw_a || saw_k) { cerr << "Warning: -M, -k and -a are mutually exclusive. " << "-M will override" << endl; khits = 1; } assert_eq(1, khits); saw_M = true; cerr << "Warning: -M is deprecated. Use -D and -R to adjust " << "effort instead." << endl; break; } case ARG_EXTEND_ITERS: { maxIters = parse(arg); break; } case ARG_NO_EXTEND: { doExtend = false; break; } case 'R': { polstr += ";ROUNDS="; polstr += arg; break; } case 'D': { polstr += ";DPS="; polstr += arg; break; } case ARG_DP_MATE_STREAK_THRESH: { maxMateStreak = parse(arg); break; } case ARG_DP_FAIL_STREAK_THRESH: { maxDpStreak = parse(arg); break; } case ARG_EE_FAIL_STREAK_THRESH: { maxEeStreak = parse(arg); break; } case ARG_UG_FAIL_STREAK_THRESH: { maxUgStreak = parse(arg); break; } case ARG_DP_FAIL_THRESH: { maxDp = parse(arg); break; } case ARG_UG_FAIL_THRESH: { maxUg = parse(arg); break; } case ARG_SEED_BOOST_THRESH: { seedBoostThresh = parse(arg); break; } case 'a': { msample = false; allHits = true; mhits = 0; // disable -M if(saw_M || saw_k) { cerr << "Warning: -M, -k and -a are mutually exclusive. " << "-a will override" << endl; } saw_a = true; break; } case 'k': { msample = false; khits = (uint32_t)parseInt(1, "-k arg must be at least 1", arg); mhits = 0; // disable -M if(saw_M || saw_a) { cerr << "Warning: -M, -k and -a are mutually exclusive. " << "-k will override" << endl; } saw_k = true; break; } case ARG_VERBOSE: gVerbose = 1; break; case ARG_STARTVERBOSE: startVerbose = true; break; case ARG_QUIET: gQuiet = true; break; case ARG_SANITY: sanityCheck = true; break; case 't': timing = true; break; case ARG_METRIC_IVAL: { metricsIval = parseInt(1, "--metrics arg must be at least 1", arg); break; } case ARG_METRIC_FILE: metricsFile = arg; break; case ARG_METRIC_STDERR: metricsStderr = true; break; case ARG_METRIC_PER_READ: metricsPerRead = true; break; case ARG_NO_FW: gNofw = true; break; case ARG_NO_RC: gNorc = true; break; case ARG_SAM_NO_QNAME_TRUNC: samTruncQname = false; break; case ARG_SAM_OMIT_SEC_SEQ: samOmitSecSeqQual = true; break; case ARG_SAM_NO_UNAL: samNoUnal = true; break; case ARG_SAM_NOHEAD: samNoHead = true; break; case ARG_SAM_NOSQ: samNoSQ = true; break; case ARG_SAM_PRINT_YI: sam_print_yi = true; break; case ARG_REORDER: reorder = true; break; case ARG_MAPQ_EX: { sam_print_zp = true; sam_print_zu = true; sam_print_xp = true; sam_print_xss = true; sam_print_yn = true; break; } case ARG_SHOW_RAND_SEED: { sam_print_zs = true; break; } case ARG_SAMPLE: sampleFrac = parse(arg); break; case ARG_CP_MIN: cminlen = parse(arg); break; case ARG_CP_IVAL: cpow2 = parse(arg); break; case ARG_TRI: doTri = true; break; case ARG_READ_PASSTHRU: { sam_print_xr = true; break; } case ARG_READ_TIMES: { sam_print_xt = true; sam_print_xd = true; sam_print_xu = true; sam_print_yl = true; sam_print_ye = true; sam_print_yu = true; sam_print_yr = true; sam_print_zb = true; sam_print_zr = true; sam_print_zf = true; sam_print_zm = true; sam_print_zi = true; break; } case ARG_SAM_RG: { string argstr = arg; if(argstr.substr(0, 3) == "ID:") { rgid = "\t"; rgid += argstr; rgs_optflag = "RG:Z:" + argstr.substr(3); } else { rgs += '\t'; rgs += argstr; } break; } case ARG_SAM_RGID: { string argstr = arg; rgid = "\t"; rgid = "\tID:" + argstr; rgs_optflag = "RG:Z:" + argstr; break; } case ARG_PARTITION: partitionSz = parse(arg); break; case ARG_DPAD: maxhalf = parseInt(0, "--dpad must be no less than 0", arg); break; case ARG_ORIG: if(arg == NULL || strlen(arg) == 0) { cerr << "--orig arg must be followed by a string" << endl; printUsage(cerr); throw 1; } origString = arg; break; case ARG_LOCAL: localAlign = true; break; case ARG_END_TO_END: localAlign = false; break; case ARG_SSE8: enable8 = true; break; case ARG_SSE8_NO: enable8 = false; break; case ARG_UNGAPPED: doUngapped = true; break; case ARG_UNGAPPED_NO: doUngapped = false; break; case ARG_NO_DOVETAIL: gDovetailMatesOK = false; break; case ARG_NO_CONTAIN: gContainMatesOK = false; break; case ARG_NO_OVERLAP: gOlapMatesOK = false; break; case ARG_DOVETAIL: gDovetailMatesOK = true; break; case ARG_CONTAIN: gContainMatesOK = true; break; case ARG_OVERLAP: gOlapMatesOK = true; break; case ARG_QC_FILTER: qcFilter = true; break; case ARG_NO_SCORE_PRIORITY: sortByScore = false; break; case ARG_IGNORE_QUALS: ignoreQuals = true; break; case ARG_MAPQ_V: mapqv = parse(arg); break; case ARG_TIGHTEN: tighten = parse(arg); break; case ARG_EXACT_UPFRONT: doExactUpFront = true; break; case ARG_1MM_UPFRONT: do1mmUpFront = true; break; case ARG_EXACT_UPFRONT_NO: doExactUpFront = false; break; case ARG_1MM_UPFRONT_NO: do1mmUpFront = false; break; case ARG_1MM_MINLEN: do1mmMinLen = parse(arg); break; case ARG_NOISY_HPOLY: noisyHpolymer = true; break; case 'x': bt2index = arg; break; case ARG_PRESET_VERY_FAST_LOCAL: localAlign = true; case ARG_PRESET_VERY_FAST: { presetList.push_back("very-fast%LOCAL%"); break; } case ARG_PRESET_FAST_LOCAL: localAlign = true; case ARG_PRESET_FAST: { presetList.push_back("fast%LOCAL%"); break; } case ARG_PRESET_SENSITIVE_LOCAL: localAlign = true; case ARG_PRESET_SENSITIVE: { presetList.push_back("sensitive%LOCAL%"); break; } case ARG_PRESET_VERY_SENSITIVE_LOCAL: localAlign = true; case ARG_PRESET_VERY_SENSITIVE: { presetList.push_back("very-sensitive%LOCAL%"); break; } case 'P': { presetList.push_back(arg); break; } case ARG_ALIGN_POLICY: { if(strlen(arg) > 0) { polstr += ";"; polstr += arg; } break; } case 'N': { polstr += ";SEED="; polstr += arg; break; } case 'L': { int64_t len = parse(arg); if(len < 0) { cerr << "Error: -L argument must be >= 0; was " << arg << endl; throw 1; } if(len > 32) { cerr << "Error: -L argument must be <= 32; was" << arg << endl; throw 1; } polstr += ";SEEDLEN="; polstr += arg; break; } case 'O': multiseedOff = parse(arg); break; case 'i': { EList args; tokenize(arg, ",", args); if(args.size() > 3 || args.size() == 0) { cerr << "Error: expected 3 or fewer comma-separated " << "arguments to -i option, got " << args.size() << endl; throw 1; } // Interval-settings arguments polstr += (";IVAL=" + args[0]); // Function type if(args.size() > 1) { polstr += ("," + args[1]); // Constant term } if(args.size() > 2) { polstr += ("," + args[2]); // Coefficient } break; } case ARG_MULTISEED_IVAL: { polstr += ";"; // Split argument by comma EList args; tokenize(arg, ",", args); if(args.size() > 5 || args.size() == 0) { cerr << "Error: expected 5 or fewer comma-separated " << "arguments to --multiseed option, got " << args.size() << endl; throw 1; } // Seed mm and length arguments polstr += "SEED="; polstr += (args[0]); // # mismatches if(args.size() > 1) polstr += ("," + args[ 1]); // length if(args.size() > 2) polstr += (";IVAL=" + args[2]); // Func type if(args.size() > 3) polstr += ("," + args[ 3]); // Constant term if(args.size() > 4) polstr += ("," + args[ 4]); // Coefficient break; } case ARG_N_CEIL: { // Split argument by comma EList args; tokenize(arg, ",", args); if(args.size() > 3) { cerr << "Error: expected 3 or fewer comma-separated " << "arguments to --n-ceil option, got " << args.size() << endl; throw 1; } if(args.size() == 0) { cerr << "Error: expected at least one argument to --n-ceil option" << endl; throw 1; } polstr += ";NCEIL="; if(args.size() == 3) { polstr += (args[0] + "," + args[1] + "," + args[2]); } else { if(args.size() == 1) { polstr += ("C," + args[0]); } else { polstr += (args[0] + "," + args[1]); } } break; } case ARG_SCORE_MA: polstr += ";MA="; polstr += arg; break; case ARG_SCORE_MMP: { EList args; tokenize(arg, ",", args); if(args.size() > 2 || args.size() == 0) { cerr << "Error: expected 1 or 2 comma-separated " << "arguments to --mmp option, got " << args.size() << endl; throw 1; } if(args.size() >= 1) { polstr += ";MMP=Q,"; polstr += args[0]; if(args.size() >= 2) { polstr += ","; polstr += args[1]; } } break; } case ARG_SCORE_NP: polstr += ";NP=C"; polstr += arg; break; case ARG_SCORE_RDG: polstr += ";RDG="; polstr += arg; break; case ARG_SCORE_RFG: polstr += ";RFG="; polstr += arg; break; case ARG_SCORE_MIN: { polstr += ";"; EList args; tokenize(arg, ",", args); if(args.size() > 3 && args.size() == 0) { cerr << "Error: expected 3 or fewer comma-separated " << "arguments to --n-ceil option, got " << args.size() << endl; throw 1; } polstr += ("MIN=" + args[0]); if(args.size() > 1) { polstr += ("," + args[1]); } if(args.size() > 2) { polstr += ("," + args[2]); } break; } case ARG_DESC: printArgDesc(cout); throw 0; case 'S': outfile = arg; break; case 'U': { EList args; tokenize(arg, ",", args); for(size_t i = 0; i < args.size(); i++) { queries.push_back(args[i]); } break; } case ARG_VERSION: showVersion = 1; break; case ARG_NO_TEMPSPLICESITE: useTempSpliceSite = false; break; case ARG_PEN_CANSPLICE: { penCanSplice = parseInt(0, "-k arg must be at least 0", arg); break; } case ARG_PEN_NONCANSPLICE: { penNoncanSplice = parseInt(0, "-k arg must be at least 0", arg); break; } case ARG_PEN_INTRONLEN: { polstr += ";"; EList args; tokenize(arg, ",", args); if(args.size() > 3 && args.size() == 0) { cerr << "Error: expected 3 or fewer comma-separated " << "arguments to --n-ceil option, got " << args.size() << endl; throw 1; } polstr += ("INTRONLEN=" + args[0]); if(args.size() > 1) { polstr += ("," + args[1]); } if(args.size() > 2) { polstr += ("," + args[2]); } break; } case ARG_KNOWN_SPLICESITE_INFILE: knownSpliceSiteInfile = arg; break; case ARG_NOVEL_SPLICESITE_INFILE: novelSpliceSiteInfile = arg; break; case ARG_NOVEL_SPLICESITE_OUTFILE: novelSpliceSiteOutfile = arg; break; case ARG_NO_SPLICED_ALIGNMENT: no_spliced_alignment = true; break; case ARG_RNA_STRANDNESS: { string strandness = arg; if(strandness == "F") rna_strandness = RNA_STRANDNESS_F; else if(strandness == "R") rna_strandness = RNA_STRANDNESS_R; else if(strandness == "FR") rna_strandness = RNA_STRANDNESS_FR; else if(strandness == "RF") rna_strandness = RNA_STRANDNESS_RF; else { // daehwan - throw exception with details cerr << "Error: should be one of F, R, FR, or RF " << endl; throw 1; } break; } default: printUsage(cerr); throw 1; } } /** * Read command-line arguments */ static void parseOptions(int argc, const char **argv) { int option_index = 0; int next_option; saw_M = false; saw_a = false; saw_k = true; presetList.clear(); if(startVerbose) { cerr << "Parsing options: "; logTime(cerr, true); } while(true) { next_option = getopt_long( argc, const_cast(argv), short_options, long_options, &option_index); const char * arg = optarg; if(next_option == EOF) { if(extra_opts_cur < extra_opts.size()) { next_option = extra_opts[extra_opts_cur].first; arg = extra_opts[extra_opts_cur].second.c_str(); extra_opts_cur++; } else { break; } } parseOption(next_option, arg); } // Now parse all the presets. Might want to pick which presets version to // use according to other parameters. auto_ptr presets(new PresetsV0()); // Apply default preset if(!defaultPreset.empty()) { polstr = applyPreset(defaultPreset, *presets.get()) + polstr; } // Apply specified presets for(size_t i = 0; i < presetList.size(); i++) { polstr += applyPreset(presetList[i], *presets.get()); } for(size_t i = 0; i < extra_opts.size(); i++) { next_option = extra_opts[extra_opts_cur].first; const char *arg = extra_opts[extra_opts_cur].second.c_str(); parseOption(next_option, arg); } // Remove initial semicolons while(!polstr.empty() && polstr[0] == ';') { polstr = polstr.substr(1); } if(gVerbose) { cerr << "Final policy string: '" << polstr.c_str() << "'" << endl; } size_t failStreakTmp = 0; SeedAlignmentPolicy::parseString( polstr, localAlign, noisyHpolymer, ignoreQuals, bonusMatchType, bonusMatch, penMmcType, penMmcMax, penMmcMin, penNType, penN, penRdGapConst, penRfGapConst, penRdGapLinear, penRfGapLinear, scoreMin, nCeil, penNCatPair, multiseedMms, multiseedLen, msIval, failStreakTmp, nSeedRounds, &penIntronLen); if(failStreakTmp > 0) { maxEeStreak = failStreakTmp; maxUgStreak = failStreakTmp; maxDpStreak = failStreakTmp; } if(saw_a || saw_k) { msample = false; mhits = 0; } else { assert_gt(mhits, 0); msample = true; } if(mates1.size() != mates2.size()) { cerr << "Error: " << mates1.size() << " mate files/sequences were specified with -1, but " << mates2.size() << endl << "mate files/sequences were specified with -2. The same number of mate files/" << endl << "sequences must be specified with -1 and -2." << endl; throw 1; } if(qualities.size() && format != FASTA) { cerr << "Error: one or more quality files were specified with -Q but -f was not" << endl << "enabled. -Q works only in combination with -f and -C." << endl; throw 1; } if(qualities1.size() && format != FASTA) { cerr << "Error: one or more quality files were specified with --Q1 but -f was not" << endl << "enabled. --Q1 works only in combination with -f and -C." << endl; throw 1; } if(qualities2.size() && format != FASTA) { cerr << "Error: one or more quality files were specified with --Q2 but -f was not" << endl << "enabled. --Q2 works only in combination with -f and -C." << endl; throw 1; } if(qualities1.size() > 0 && mates1.size() != qualities1.size()) { cerr << "Error: " << mates1.size() << " mate files/sequences were specified with -1, but " << qualities1.size() << endl << "quality files were specified with --Q1. The same number of mate and quality" << endl << "files must sequences must be specified with -1 and --Q1." << endl; throw 1; } if(qualities2.size() > 0 && mates2.size() != qualities2.size()) { cerr << "Error: " << mates2.size() << " mate files/sequences were specified with -2, but " << qualities2.size() << endl << "quality files were specified with --Q2. The same number of mate and quality" << endl << "files must sequences must be specified with -2 and --Q2." << endl; throw 1; } if(!rgs.empty() && rgid.empty()) { cerr << "Warning: --rg was specified without --rg-id also " << "being specified. @RG line is not printed unless --rg-id " << "is specified." << endl; } // Check for duplicate mate input files if(format != CMDLINE) { for(size_t i = 0; i < mates1.size(); i++) { for(size_t j = 0; j < mates2.size(); j++) { if(mates1[i] == mates2[j] && !gQuiet) { cerr << "Warning: Same mate file \"" << mates1[i].c_str() << "\" appears as argument to both -1 and -2" << endl; } } } } // If both -s and -u are used, we need to adjust qUpto accordingly // since it uses rdid to know if we've reached the -u limit (and // rdids are all shifted up by skipReads characters) if(qUpto + skipReads > qUpto) { qUpto += skipReads; } if(useShmem && useMm && !gQuiet) { cerr << "Warning: --shmem overrides --mm..." << endl; useMm = false; } if(gGapBarrier < 1) { cerr << "Warning: --gbar was set less than 1 (=" << gGapBarrier << "); setting to 1 instead" << endl; gGapBarrier = 1; } if(multiseedMms >= multiseedLen) { assert_gt(multiseedLen, 0); cerr << "Warning: seed mismatches (" << multiseedMms << ") is less than seed length (" << multiseedLen << "); setting mismatches to " << (multiseedMms-1) << " instead" << endl; multiseedMms = multiseedLen-1; } sam_print_zm = sam_print_zm && bowtie2p5; #ifndef NDEBUG if(!gQuiet) { cerr << "Warning: Running in debug mode. Please use debug mode only " << "for diagnosing errors, and not for typical use of HISAT." << endl; } #endif } static const char *argv0 = NULL; /// Create a PatternSourcePerThread for the current thread according /// to the global params and return a pointer to it static PatternSourcePerThreadFactory* createPatsrcFactory(PairedPatternSource& _patsrc, int tid) { PatternSourcePerThreadFactory *patsrcFact; patsrcFact = new WrappedPatternSourcePerThreadFactory(_patsrc); assert(patsrcFact != NULL); return patsrcFact; } #define PTHREAD_ATTRS (PTHREAD_CREATE_JOINABLE | PTHREAD_CREATE_DETACHED) typedef TIndexOffU index_t; typedef uint16_t local_index_t; static PairedPatternSource* multiseed_patsrc; static HierEbwt* multiseed_ebwtFw; static HierEbwt* multiseed_ebwtBw; static Scoring* multiseed_sc; static BitPairReference* multiseed_refs; static AlignmentCache* multiseed_ca; // seed cache static AlnSink* multiseed_msink; static EList multiseed_refnames; static OutFileBuf* multiseed_metricsOfb; static SpliceSiteDB* ssdb; static MUTEX_T multiseed_mutex; /** * Metrics for measuring the work done by the outer read alignment * loop. */ struct OuterLoopMetrics { OuterLoopMetrics() { reset(); } /** * Set all counters to 0. */ void reset() { reads = bases = srreads = srbases = freads = fbases = ureads = ubases = 0; } /** * Sum the counters in m in with the conters in this object. This * is the only safe way to update an OuterLoopMetrics that's shared * by multiple threads. */ void merge( const OuterLoopMetrics& m, bool getLock = false) { ThreadSafe ts(&mutex_m, getLock); reads += m.reads; bases += m.bases; srreads += m.srreads; srbases += m.srbases; freads += m.freads; fbases += m.fbases; ureads += m.ureads; ubases += m.ubases; } uint64_t reads; // total reads uint64_t bases; // total bases uint64_t srreads; // same-read reads uint64_t srbases; // same-read bases uint64_t freads; // filtered reads uint64_t fbases; // filtered bases uint64_t ureads; // unfiltered reads uint64_t ubases; // unfiltered bases MUTEX_T mutex_m; }; /** * Collection of all relevant performance metrics when aligning in * multiseed mode. */ struct PerfMetrics { PerfMetrics() : first(true) { reset(); } /** * Set all counters to 0. */ void reset() { olm.reset(); sdm.reset(); wlm.reset(); swmSeed.reset(); swmMate.reset(); rpm.reset(); dpSse8Seed.reset(); // 8-bit SSE seed extensions dpSse8Mate.reset(); // 8-bit SSE mate finds dpSse16Seed.reset(); // 16-bit SSE seed extensions dpSse16Mate.reset(); // 16-bit SSE mate finds nbtfiltst = 0; nbtfiltsc = 0; nbtfiltdo = 0; olmu.reset(); sdmu.reset(); wlmu.reset(); swmuSeed.reset(); swmuMate.reset(); rpmu.reset(); dpSse8uSeed.reset(); // 8-bit SSE seed extensions dpSse8uMate.reset(); // 8-bit SSE mate finds dpSse16uSeed.reset(); // 16-bit SSE seed extensions dpSse16uMate.reset(); // 16-bit SSE mate finds nbtfiltst_u = 0; nbtfiltsc_u = 0; nbtfiltdo_u = 0; him.reset(); } /** * Merge a set of specific metrics into this object. */ void merge( const OuterLoopMetrics *ol, const SeedSearchMetrics *sd, const WalkMetrics *wl, const SwMetrics *swSeed, const SwMetrics *swMate, const ReportingMetrics *rm, const SSEMetrics *dpSse8Ex, const SSEMetrics *dpSse8Ma, const SSEMetrics *dpSse16Ex, const SSEMetrics *dpSse16Ma, uint64_t nbtfiltst_, uint64_t nbtfiltsc_, uint64_t nbtfiltdo_, const HIMetrics *hi, bool getLock) { ThreadSafe ts(&mutex_m, getLock); if(ol != NULL) { olmu.merge(*ol, false); } if(sd != NULL) { sdmu.merge(*sd, false); } if(wl != NULL) { wlmu.merge(*wl, false); } if(swSeed != NULL) { swmuSeed.merge(*swSeed, false); } if(swMate != NULL) { swmuMate.merge(*swMate, false); } if(rm != NULL) { rpmu.merge(*rm, false); } if(dpSse8Ex != NULL) { dpSse8uSeed.merge(*dpSse8Ex, false); } if(dpSse8Ma != NULL) { dpSse8uMate.merge(*dpSse8Ma, false); } if(dpSse16Ex != NULL) { dpSse16uSeed.merge(*dpSse16Ex, false); } if(dpSse16Ma != NULL) { dpSse16uMate.merge(*dpSse16Ma, false); } nbtfiltst_u += nbtfiltst_; nbtfiltsc_u += nbtfiltsc_; nbtfiltdo_u += nbtfiltdo_; if(hi != NULL) { him.merge(*hi, false); } } /** * Reports a matrix of results, incl. column labels, to an OutFileBuf. * Optionally also sends results to stderr (unbuffered). Can optionally * print a per-read record with the read name at the beginning. */ void reportInterval( OutFileBuf* o, // file to send output to bool metricsStderr, // additionally output to stderr? bool total, // true -> report total, otherwise incremental bool sync, // synchronize output const BTString *name) // non-NULL name pointer if is per-read record { ThreadSafe ts(&mutex_m, sync); ostringstream stderrSs; time_t curtime = time(0); char buf[1024]; if(first) { const char *str = /* 1 */ "Time" "\t" /* 2 */ "Read" "\t" /* 3 */ "Base" "\t" /* 4 */ "SameRead" "\t" /* 5 */ "SameReadBase" "\t" /* 6 */ "UnfilteredRead" "\t" /* 7 */ "UnfilteredBase" "\t" /* 8 */ "Paired" "\t" /* 9 */ "Unpaired" "\t" /* 10 */ "AlConUni" "\t" /* 11 */ "AlConRep" "\t" /* 12 */ "AlConFail" "\t" /* 13 */ "AlDis" "\t" /* 14 */ "AlConFailUni" "\t" /* 15 */ "AlConFailRep" "\t" /* 16 */ "AlConFailFail" "\t" /* 17 */ "AlConRepUni" "\t" /* 18 */ "AlConRepRep" "\t" /* 19 */ "AlConRepFail" "\t" /* 20 */ "AlUnpUni" "\t" /* 21 */ "AlUnpRep" "\t" /* 22 */ "AlUnpFail" "\t" /* 23 */ "SeedSearch" "\t" /* 24 */ "IntraSCacheHit" "\t" /* 25 */ "InterSCacheHit" "\t" /* 26 */ "OutOfMemory" "\t" /* 27 */ "AlBWOp" "\t" /* 28 */ "AlBWBranch" "\t" /* 29 */ "ResBWOp" "\t" /* 30 */ "ResBWBranch" "\t" /* 31 */ "ResResolve" "\t" /* 34 */ "ResReport" "\t" /* 35 */ "RedundantSHit" "\t" /* 36 */ "BestMinEdit0" "\t" /* 37 */ "BestMinEdit1" "\t" /* 38 */ "BestMinEdit2" "\t" /* 39 */ "ExactAttempts" "\t" /* 40 */ "ExactSucc" "\t" /* 41 */ "ExactRanges" "\t" /* 42 */ "ExactRows" "\t" /* 43 */ "ExactOOMs" "\t" /* 44 */ "1mmAttempts" "\t" /* 45 */ "1mmSucc" "\t" /* 46 */ "1mmRanges" "\t" /* 47 */ "1mmRows" "\t" /* 48 */ "1mmOOMs" "\t" /* 49 */ "UngappedSucc" "\t" /* 50 */ "UngappedFail" "\t" /* 51 */ "UngappedNoDec" "\t" /* 52 */ "DPExLt10Gaps" "\t" /* 53 */ "DPExLt5Gaps" "\t" /* 54 */ "DPExLt3Gaps" "\t" /* 55 */ "DPMateLt10Gaps" "\t" /* 56 */ "DPMateLt5Gaps" "\t" /* 57 */ "DPMateLt3Gaps" "\t" /* 58 */ "DP16ExDps" "\t" /* 59 */ "DP16ExDpSat" "\t" /* 60 */ "DP16ExDpFail" "\t" /* 61 */ "DP16ExDpSucc" "\t" /* 62 */ "DP16ExCol" "\t" /* 63 */ "DP16ExCell" "\t" /* 64 */ "DP16ExInner" "\t" /* 65 */ "DP16ExFixup" "\t" /* 66 */ "DP16ExGathSol" "\t" /* 67 */ "DP16ExBt" "\t" /* 68 */ "DP16ExBtFail" "\t" /* 69 */ "DP16ExBtSucc" "\t" /* 70 */ "DP16ExBtCell" "\t" /* 71 */ "DP16ExCoreRej" "\t" /* 72 */ "DP16ExNRej" "\t" /* 73 */ "DP8ExDps" "\t" /* 74 */ "DP8ExDpSat" "\t" /* 75 */ "DP8ExDpFail" "\t" /* 76 */ "DP8ExDpSucc" "\t" /* 77 */ "DP8ExCol" "\t" /* 78 */ "DP8ExCell" "\t" /* 79 */ "DP8ExInner" "\t" /* 80 */ "DP8ExFixup" "\t" /* 81 */ "DP8ExGathSol" "\t" /* 82 */ "DP8ExBt" "\t" /* 83 */ "DP8ExBtFail" "\t" /* 84 */ "DP8ExBtSucc" "\t" /* 85 */ "DP8ExBtCell" "\t" /* 86 */ "DP8ExCoreRej" "\t" /* 87 */ "DP8ExNRej" "\t" /* 88 */ "DP16MateDps" "\t" /* 89 */ "DP16MateDpSat" "\t" /* 90 */ "DP16MateDpFail" "\t" /* 91 */ "DP16MateDpSucc" "\t" /* 92 */ "DP16MateCol" "\t" /* 93 */ "DP16MateCell" "\t" /* 94 */ "DP16MateInner" "\t" /* 95 */ "DP16MateFixup" "\t" /* 96 */ "DP16MateGathSol" "\t" /* 97 */ "DP16MateBt" "\t" /* 98 */ "DP16MateBtFail" "\t" /* 99 */ "DP16MateBtSucc" "\t" /* 100 */ "DP16MateBtCell" "\t" /* 101 */ "DP16MateCoreRej" "\t" /* 102 */ "DP16MateNRej" "\t" /* 103 */ "DP8MateDps" "\t" /* 104 */ "DP8MateDpSat" "\t" /* 105 */ "DP8MateDpFail" "\t" /* 106 */ "DP8MateDpSucc" "\t" /* 107 */ "DP8MateCol" "\t" /* 108 */ "DP8MateCell" "\t" /* 109 */ "DP8MateInner" "\t" /* 110 */ "DP8MateFixup" "\t" /* 111 */ "DP8MateGathSol" "\t" /* 112 */ "DP8MateBt" "\t" /* 113 */ "DP8MateBtFail" "\t" /* 114 */ "DP8MateBtSucc" "\t" /* 115 */ "DP8MateBtCell" "\t" /* 116 */ "DP8MateCoreRej" "\t" /* 117 */ "DP8MateNRej" "\t" /* 118 */ "DPBtFiltStart" "\t" /* 119 */ "DPBtFiltScore" "\t" /* 120 */ "DpBtFiltDom" "\t" /* 121 */ "MemPeak" "\t" /* 122 */ "UncatMemPeak" "\t" // 0 /* 123 */ "EbwtMemPeak" "\t" // EBWT_CAT /* 124 */ "CacheMemPeak" "\t" // CA_CAT /* 125 */ "ResolveMemPeak" "\t" // GW_CAT /* 126 */ "AlignMemPeak" "\t" // AL_CAT /* 127 */ "DPMemPeak" "\t" // DP_CAT /* 128 */ "MiscMemPeak" "\t" // MISC_CAT /* 129 */ "DebugMemPeak" "\t" // DEBUG_CAT /* 130 */ "LocalSearch" "\t" /* 131 */ "AnchorSearch" "\t" /* 132 */ "LocalIndexSearch" "\t" /* 133 */ "LocalExtSearch" "\t" /* 134 */ "LocalSearchRecur" "\t" /* 135 */ "GlobalGenomeCoords" "\t" /* 136 */ "LocalGenomeCoords" "\t" "\n"; if(name != NULL) { if(o != NULL) o->writeChars("Name\t"); if(metricsStderr) stderrSs << "Name\t"; } if(o != NULL) o->writeChars(str); if(metricsStderr) stderrSs << str; first = false; } if(total) mergeIncrementals(); // 0. Read name, if needed if(name != NULL) { if(o != NULL) { o->writeChars(name->toZBuf()); o->write('\t'); } if(metricsStderr) { stderrSs << (*name) << '\t'; } } // 1. Current time in secs itoa10(curtime, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const OuterLoopMetrics& ol = total ? olm : olmu; // 2. Reads itoa10(ol.reads, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 3. Bases itoa10(ol.bases, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 4. Same-read reads itoa10(ol.srreads, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 5. Same-read bases itoa10(ol.srbases, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 6. Unfiltered reads itoa10(ol.ureads, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 7. Unfiltered bases itoa10(ol.ubases, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const ReportingMetrics& rp = total ? rpm : rpmu; // 8. Paired reads itoa10(rp.npaired, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 9. Unpaired reads itoa10(rp.nunpaired, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 10. Pairs with unique concordant alignments itoa10(rp.nconcord_uni, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 11. Pairs with repetitive concordant alignments itoa10(rp.nconcord_rep, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 12. Pairs with 0 concordant alignments itoa10(rp.nconcord_0, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 13. Pairs with 1 discordant alignment itoa10(rp.ndiscord, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 14. Mates from unaligned pairs that align uniquely itoa10(rp.nunp_0_uni, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 15. Mates from unaligned pairs that align repetitively itoa10(rp.nunp_0_rep, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 16. Mates from unaligned pairs that fail to align itoa10(rp.nunp_0_0, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 17. Mates from repetitive pairs that align uniquely itoa10(rp.nunp_rep_uni, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 18. Mates from repetitive pairs that align repetitively itoa10(rp.nunp_rep_rep, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 19. Mates from repetitive pairs that fail to align itoa10(rp.nunp_rep_0, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 20. Unpaired reads that align uniquely itoa10(rp.nunp_uni, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 21. Unpaired reads that align repetitively itoa10(rp.nunp_rep, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 22. Unpaired reads that fail to align itoa10(rp.nunp_0, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const SeedSearchMetrics& sd = total ? sdm : sdmu; // 23. Seed searches itoa10(sd.seedsearch, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 24. Hits in 'current' cache itoa10(sd.intrahit, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 25. Hits in 'local' cache itoa10(sd.interhit, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 26. Out of memory itoa10(sd.ooms, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 27. Burrows-Wheeler ops in aligner itoa10(sd.bwops, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 28. Burrows-Wheeler branches (edits) in aligner itoa10(sd.bweds, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const WalkMetrics& wl = total ? wlm : wlmu; // 29. Burrows-Wheeler ops in resolver itoa10(wl.bwops, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 30. Burrows-Wheeler branches in resolver itoa10(wl.branches, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 31. Burrows-Wheeler offset resolutions itoa10(wl.resolves, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 34. Offset reports itoa10(wl.reports, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 35. Redundant seed hit itoa10(total ? swmSeed.rshit : swmuSeed.rshit, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 36. # times the best (out of fw/rc) minimum # edits was 0 itoa10(total ? sdm.bestmin0 : sdmu.bestmin0, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 37. # times the best (out of fw/rc) minimum # edits was 1 itoa10(total ? sdm.bestmin1 : sdmu.bestmin1, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 38. # times the best (out of fw/rc) minimum # edits was 2 itoa10(total ? sdm.bestmin2 : sdmu.bestmin2, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 39. Exact aligner attempts itoa10(total ? swmSeed.exatts : swmuSeed.exatts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 40. Exact aligner successes itoa10(total ? swmSeed.exsucc : swmuSeed.exsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 41. Exact aligner ranges itoa10(total ? swmSeed.exranges : swmuSeed.exranges, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 42. Exact aligner rows itoa10(total ? swmSeed.exrows : swmuSeed.exrows, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 43. Exact aligner OOMs itoa10(total ? swmSeed.exooms : swmuSeed.exooms, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 44. 1mm aligner attempts itoa10(total ? swmSeed.mm1atts : swmuSeed.mm1atts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 45. 1mm aligner successes itoa10(total ? swmSeed.mm1succ : swmuSeed.mm1succ, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 46. 1mm aligner ranges itoa10(total ? swmSeed.mm1ranges : swmuSeed.mm1ranges, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 47. 1mm aligner rows itoa10(total ? swmSeed.mm1rows : swmuSeed.mm1rows, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 48. 1mm aligner OOMs itoa10(total ? swmSeed.mm1ooms : swmuSeed.mm1ooms, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 49 Ungapped aligner success itoa10(total ? swmSeed.ungapsucc : swmuSeed.ungapsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 50. Ungapped aligner fail itoa10(total ? swmSeed.ungapfail : swmuSeed.ungapfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 51. Ungapped aligner no decision itoa10(total ? swmSeed.ungapnodec : swmuSeed.ungapnodec, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 52. # seed-extend DPs with < 10 gaps itoa10(total ? swmSeed.sws10 : swmuSeed.sws10, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 53. # seed-extend DPs with < 5 gaps itoa10(total ? swmSeed.sws5 : swmuSeed.sws5, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 54. # seed-extend DPs with < 3 gaps itoa10(total ? swmSeed.sws3 : swmuSeed.sws3, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 55. # seed-extend DPs with < 10 gaps itoa10(total ? swmMate.sws10 : swmuMate.sws10, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 56. # seed-extend DPs with < 5 gaps itoa10(total ? swmMate.sws5 : swmuMate.sws5, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 57. # seed-extend DPs with < 3 gaps itoa10(total ? swmMate.sws3 : swmuMate.sws3, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const SSEMetrics& dpSse16s = total ? dpSse16Seed : dpSse16uSeed; // 58. 16-bit SSE seed-extend DPs tried itoa10(dpSse16s.dp, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 59. 16-bit SSE seed-extend DPs saturated itoa10(dpSse16s.dpsat, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 60. 16-bit SSE seed-extend DPs failed itoa10(dpSse16s.dpfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 61. 16-bit SSE seed-extend DPs succeeded itoa10(dpSse16s.dpsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 62. 16-bit SSE seed-extend DP columns completed itoa10(dpSse16s.col, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 63. 16-bit SSE seed-extend DP cells completed itoa10(dpSse16s.cell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 64. 16-bit SSE seed-extend DP inner loop iters completed itoa10(dpSse16s.inner, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 65. 16-bit SSE seed-extend DP fixup loop iters completed itoa10(dpSse16s.fixup, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 66. 16-bit SSE seed-extend DP gather, cells with potential solutions itoa10(dpSse16s.gathsol, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 67. 16-bit SSE seed-extend DP backtrace attempts itoa10(dpSse16s.bt, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 68. 16-bit SSE seed-extend DP failed backtrace attempts itoa10(dpSse16s.btfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 69. 16-bit SSE seed-extend DP succesful backtrace attempts itoa10(dpSse16s.btsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 70. 16-bit SSE seed-extend DP backtrace cells itoa10(dpSse16s.btcell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 71. 16-bit SSE seed-extend DP core-diag rejections itoa10(dpSse16s.corerej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 72. 16-bit SSE seed-extend DP N rejections itoa10(dpSse16s.nrej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const SSEMetrics& dpSse8s = total ? dpSse8Seed : dpSse8uSeed; // 73. 8-bit SSE seed-extend DPs tried itoa10(dpSse8s.dp, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 74. 8-bit SSE seed-extend DPs saturated itoa10(dpSse8s.dpsat, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 75. 8-bit SSE seed-extend DPs failed itoa10(dpSse8s.dpfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 76. 8-bit SSE seed-extend DPs succeeded itoa10(dpSse8s.dpsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 77. 8-bit SSE seed-extend DP columns completed itoa10(dpSse8s.col, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 78. 8-bit SSE seed-extend DP cells completed itoa10(dpSse8s.cell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 79. 8-bit SSE seed-extend DP inner loop iters completed itoa10(dpSse8s.inner, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 80. 8-bit SSE seed-extend DP fixup loop iters completed itoa10(dpSse8s.fixup, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 81. 16-bit SSE seed-extend DP gather, cells with potential solutions itoa10(dpSse8s.gathsol, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 82. 16-bit SSE seed-extend DP backtrace attempts itoa10(dpSse8s.bt, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 83. 16-bit SSE seed-extend DP failed backtrace attempts itoa10(dpSse8s.btfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 84. 16-bit SSE seed-extend DP succesful backtrace attempts itoa10(dpSse8s.btsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 85. 16-bit SSE seed-extend DP backtrace cells itoa10(dpSse8s.btcell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 86. 16-bit SSE seed-extend DP core-diag rejections itoa10(dpSse8s.corerej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 87. 16-bit SSE seed-extend DP N rejections itoa10(dpSse8s.nrej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const SSEMetrics& dpSse16m = total ? dpSse16Mate : dpSse16uMate; // 88. 16-bit SSE mate-finding DPs tried itoa10(dpSse16m.dp, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 89. 16-bit SSE mate-finding DPs saturated itoa10(dpSse16m.dpsat, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 90. 16-bit SSE mate-finding DPs failed itoa10(dpSse16m.dpfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 91. 16-bit SSE mate-finding DPs succeeded itoa10(dpSse16m.dpsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 92. 16-bit SSE mate-finding DP columns completed itoa10(dpSse16m.col, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 93. 16-bit SSE mate-finding DP cells completed itoa10(dpSse16m.cell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 94. 16-bit SSE mate-finding DP inner loop iters completed itoa10(dpSse16m.inner, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 95. 16-bit SSE mate-finding DP fixup loop iters completed itoa10(dpSse16m.fixup, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 96. 16-bit SSE mate-finding DP gather, cells with potential solutions itoa10(dpSse16m.gathsol, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 97. 16-bit SSE mate-finding DP backtrace attempts itoa10(dpSse16m.bt, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 98. 16-bit SSE mate-finding DP failed backtrace attempts itoa10(dpSse16m.btfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 99. 16-bit SSE mate-finding DP succesful backtrace attempts itoa10(dpSse16m.btsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 100. 16-bit SSE mate-finding DP backtrace cells itoa10(dpSse16m.btcell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 101. 16-bit SSE mate-finding DP core-diag rejections itoa10(dpSse16m.corerej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 102. 16-bit SSE mate-finding DP N rejections itoa10(dpSse16m.nrej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } const SSEMetrics& dpSse8m = total ? dpSse8Mate : dpSse8uMate; // 103. 8-bit SSE mate-finding DPs tried itoa10(dpSse8m.dp, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 104. 8-bit SSE mate-finding DPs saturated itoa10(dpSse8m.dpsat, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 105. 8-bit SSE mate-finding DPs failed itoa10(dpSse8m.dpfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 106. 8-bit SSE mate-finding DPs succeeded itoa10(dpSse8m.dpsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 107. 8-bit SSE mate-finding DP columns completed itoa10(dpSse8m.col, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 108. 8-bit SSE mate-finding DP cells completed itoa10(dpSse8m.cell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 109. 8-bit SSE mate-finding DP inner loop iters completed itoa10(dpSse8m.inner, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 110. 8-bit SSE mate-finding DP fixup loop iters completed itoa10(dpSse8m.fixup, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 111. 16-bit SSE mate-finding DP gather, cells with potential solutions itoa10(dpSse8m.gathsol, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 112. 16-bit SSE mate-finding DP backtrace attempts itoa10(dpSse8m.bt, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 113. 16-bit SSE mate-finding DP failed backtrace attempts itoa10(dpSse8m.btfail, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 114. 16-bit SSE mate-finding DP succesful backtrace attempts itoa10(dpSse8m.btsucc, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 115. 16-bit SSE mate-finding DP backtrace cells itoa10(dpSse8m.btcell, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 116. 16-bit SSE mate-finding DP core rejections itoa10(dpSse8m.corerej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 117. 16-bit SSE mate-finding N rejections itoa10(dpSse8m.nrej, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 118. Backtrace candidates filtered due to starting cell itoa10(total ? nbtfiltst : nbtfiltst_u, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 119. Backtrace candidates filtered due to low score itoa10(total ? nbtfiltsc : nbtfiltsc_u, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 120. Backtrace candidates filtered due to domination itoa10(total ? nbtfiltdo : nbtfiltdo_u, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 121. Overall memory peak itoa10(gMemTally.peak() >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 122. Uncategorized memory peak itoa10(gMemTally.peak(0) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 123. Ebwt memory peak itoa10(gMemTally.peak(EBWT_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 124. Cache memory peak itoa10(gMemTally.peak(CA_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 125. Resolver memory peak itoa10(gMemTally.peak(GW_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 126. Seed aligner memory peak itoa10(gMemTally.peak(AL_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 127. Dynamic programming aligner memory peak itoa10(gMemTally.peak(DP_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 128. Miscellaneous memory peak itoa10(gMemTally.peak(MISC_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 129. Debug memory peak itoa10(gMemTally.peak(DEBUG_CAT) >> 20, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 130 itoa10(him.localatts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 131 itoa10(him.anchoratts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 132 itoa10(him.localindexatts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 133 itoa10(him.localextatts, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 134 itoa10(him.localsearchrecur, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 135 itoa10(him.globalgenomecoords, buf); if(metricsStderr) stderrSs << buf << '\t'; if(o != NULL) { o->writeChars(buf); o->write('\t'); } // 136 itoa10(him.localgenomecoords, buf); if(metricsStderr) stderrSs << buf; if(o != NULL) { o->writeChars(buf); } if(o != NULL) { o->write('\n'); } if(metricsStderr) cerr << stderrSs.str().c_str() << endl; if(!total) mergeIncrementals(); } void mergeIncrementals() { olm.merge(olmu, false); sdm.merge(sdmu, false); wlm.merge(wlmu, false); swmSeed.merge(swmuSeed, false); swmMate.merge(swmuMate, false); dpSse8Seed.merge(dpSse8uSeed, false); dpSse8Mate.merge(dpSse8uMate, false); dpSse16Seed.merge(dpSse16uSeed, false); dpSse16Mate.merge(dpSse16uMate, false); nbtfiltst_u += nbtfiltst; nbtfiltsc_u += nbtfiltsc; nbtfiltdo_u += nbtfiltdo; olmu.reset(); sdmu.reset(); wlmu.reset(); swmuSeed.reset(); swmuMate.reset(); rpmu.reset(); dpSse8uSeed.reset(); dpSse8uMate.reset(); dpSse16uSeed.reset(); dpSse16uMate.reset(); nbtfiltst_u = 0; nbtfiltsc_u = 0; nbtfiltdo_u = 0; } // Total over the whole job OuterLoopMetrics olm; // overall metrics SeedSearchMetrics sdm; // metrics related to seed alignment WalkMetrics wlm; // metrics related to walking left (i.e. resolving reference offsets) SwMetrics swmSeed; // metrics related to DP seed-extend alignment SwMetrics swmMate; // metrics related to DP mate-finding alignment ReportingMetrics rpm; // metrics related to reporting SSEMetrics dpSse8Seed; // 8-bit SSE seed extensions SSEMetrics dpSse8Mate; // 8-bit SSE mate finds SSEMetrics dpSse16Seed; // 16-bit SSE seed extensions SSEMetrics dpSse16Mate; // 16-bit SSE mate finds uint64_t nbtfiltst; uint64_t nbtfiltsc; uint64_t nbtfiltdo; // Just since the last update OuterLoopMetrics olmu; // overall metrics SeedSearchMetrics sdmu; // metrics related to seed alignment WalkMetrics wlmu; // metrics related to walking left (i.e. resolving reference offsets) SwMetrics swmuSeed; // metrics related to DP seed-extend alignment SwMetrics swmuMate; // metrics related to DP mate-finding alignment ReportingMetrics rpmu; // metrics related to reporting SSEMetrics dpSse8uSeed; // 8-bit SSE seed extensions SSEMetrics dpSse8uMate; // 8-bit SSE mate finds SSEMetrics dpSse16uSeed; // 16-bit SSE seed extensions SSEMetrics dpSse16uMate; // 16-bit SSE mate finds uint64_t nbtfiltst_u; uint64_t nbtfiltsc_u; uint64_t nbtfiltdo_u; // HIMetrics him; MUTEX_T mutex_m; // lock for when one ob bool first; // yet to print first line? time_t lastElapsed; // used in reportInterval to measure time since last call }; static PerfMetrics metrics; // Cyclic rotations #define ROTL(n, x) (((x) << (n)) | ((x) >> (32-n))) #define ROTR(n, x) (((x) >> (n)) | ((x) << (32-n))) static inline void printMmsSkipMsg( const PatternSourcePerThread& ps, bool paired, bool mate1, int seedmms) { ostringstream os; if(paired) { os << "Warning: skipping mate #" << (mate1 ? '1' : '2') << " of read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "' because length (" << (mate1 ? ps.bufa().patFw.length() : ps.bufb().patFw.length()) << ") <= # seed mismatches (" << seedmms << ")" << endl; } else { os << "Warning: skipping read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "' because length (" << (mate1 ? ps.bufa().patFw.length() : ps.bufb().patFw.length()) << ") <= # seed mismatches (" << seedmms << ")" << endl; } cerr << os.str().c_str(); } static inline void printLenSkipMsg( const PatternSourcePerThread& ps, bool paired, bool mate1) { ostringstream os; if(paired) { os << "Warning: skipping mate #" << (mate1 ? '1' : '2') << " of read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "' because it was < 2 characters long" << endl; } else { os << "Warning: skipping read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "' because it was < 2 characters long" << endl; } cerr << os.str().c_str(); } static inline void printLocalScoreMsg( const PatternSourcePerThread& ps, bool paired, bool mate1) { ostringstream os; if(paired) { os << "Warning: minimum score function gave negative number in " << "--local mode for mate #" << (mate1 ? '1' : '2') << " of read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "; setting to 0 instead" << endl; } else { os << "Warning: minimum score function gave negative number in " << "--local mode for read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "; setting to 0 instead" << endl; } cerr << os.str().c_str(); } static inline void printEEScoreMsg( const PatternSourcePerThread& ps, bool paired, bool mate1) { ostringstream os; if(paired) { os << "Warning: minimum score function gave positive number in " << "--end-to-end mode for mate #" << (mate1 ? '1' : '2') << " of read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "; setting to 0 instead" << endl; } else { os << "Warning: minimum score function gave positive number in " << "--end-to-end mode for read '" << (mate1 ? ps.bufa().name : ps.bufb().name) << "; setting to 0 instead" << endl; } cerr << os.str().c_str(); } #define MERGE_METRICS(met, sync) { \ msink.mergeMetrics(rpm); \ met.merge( \ &olm, \ &sdm, \ &wlm, \ &swmSeed, \ &swmMate, \ &rpm, \ &sseU8ExtendMet, \ &sseU8MateMet, \ &sseI16ExtendMet, \ &sseI16MateMet, \ nbtfiltst, \ nbtfiltsc, \ nbtfiltdo, \ &him, \ sync); \ olm.reset(); \ sdm.reset(); \ wlm.reset(); \ swmSeed.reset(); \ swmMate.reset(); \ rpm.reset(); \ sseU8ExtendMet.reset(); \ sseU8MateMet.reset(); \ sseI16ExtendMet.reset(); \ sseI16MateMet.reset(); \ him.reset(); \ } #define MERGE_SW(x) { \ x.merge( \ sseU8ExtendMet, \ sseU8MateMet, \ sseI16ExtendMet, \ sseI16MateMet, \ nbtfiltst, \ nbtfiltsc, \ nbtfiltdo); \ x.resetCounters(); \ } /** * Called once per thread. Sets up per-thread pointers to the shared global * data structures, creates per-thread structures, then enters the alignment * loop. The general flow of the alignment loop is: * * - If it's been a while and we're the master thread, report some alignment * metrics * - Get the next read/pair * - Check if this read/pair is identical to the previous * + If identical, check whether we can skip any or all alignment stages. If * we can skip all stages, report the result immediately and move to next * read/pair * + If not identical, continue * - */ static void multiseedSearchWorker_hisat_bp(void *vp) { int tid = *((int*)vp); assert(multiseed_ebwtFw != NULL); assert(multiseedMms == 0 || multiseed_ebwtBw != NULL); PairedPatternSource& patsrc = *multiseed_patsrc; const HierEbwt& ebwtFw = *multiseed_ebwtFw; const HierEbwt& ebwtBw = *multiseed_ebwtBw; const Scoring& sc = *multiseed_sc; const BitPairReference& ref = *multiseed_refs; AlignmentCache& scShared = *multiseed_ca; AlnSink& msink = *multiseed_msink; OutFileBuf* metricsOfb = multiseed_metricsOfb; // Sinks: these are so that we can print tables encoding counts for // events of interest on a per-read, per-seed, per-join, or per-SW // level. These in turn can be used to diagnose performance // problems, or generally characterize performance. //const BitPairReference& refs = *multiseed_refs; auto_ptr patsrcFact(createPatsrcFactory(patsrc, tid)); auto_ptr ps(patsrcFact->create()); // Thread-local cache for seed alignments PtrWrap > scLocal; if(!msNoCache) { scLocal.init(new AlignmentCache(seedCacheLocalMB * 1024 * 1024, false)); } AlignmentCache scCurrent(seedCacheCurrentMB * 1024 * 1024, false); // Thread-local cache for current seed alignments // Interfaces for alignment and seed caches AlignmentCacheIface ca( &scCurrent, scLocal.get(), msNoCache ? NULL : &scShared); // Instantiate an object for holding reporting-related parameters. ReportingParams rp( (allHits ? std::numeric_limits::max() : khits), // -k mhits, // -m/-M 0, // penalty gap (not used now) msample, // true -> -M was specified, otherwise assume -m gReportDiscordant, // report discordang paired-end alignments? gReportMixed); // report unpaired alignments for paired reads? // Instantiate a mapping quality calculator auto_ptr bmapq(new_mapq(mapqv, scoreMin, sc)); // Make a per-thread wrapper for the global MHitSink object. AlnSinkWrap msinkwrap( msink, // global sink rp, // reporting parameters *bmapq.get(), // MAPQ calculator (size_t)tid); // thread id BP_Aligner bp_Aligner( ebwtFw, multiseed_refnames, &multiseed_mutex, thread_rids_mindist, no_spliced_alignment); SwAligner sw; OuterLoopMetrics olm; SeedSearchMetrics sdm; WalkMetrics wlm; SwMetrics swmSeed, swmMate; ReportingMetrics rpm; RandomSource rnd, rndArb; SSEMetrics sseU8ExtendMet; SSEMetrics sseU8MateMet; SSEMetrics sseI16ExtendMet; SSEMetrics sseI16MateMet; DescentMetrics descm; uint64_t nbtfiltst = 0; // TODO: find a new home for these uint64_t nbtfiltsc = 0; // TODO: find a new home for these uint64_t nbtfiltdo = 0; // TODO: find a new home for these HIMetrics him; ASSERT_ONLY(BTDnaString tmp); int pepolFlag; if(gMate1fw && gMate2fw) { pepolFlag = PE_POLICY_FF; } else if(gMate1fw && !gMate2fw) { pepolFlag = PE_POLICY_FR; } else if(!gMate1fw && gMate2fw) { pepolFlag = PE_POLICY_RF; } else { pepolFlag = PE_POLICY_RR; } assert_geq(gMaxInsert, gMinInsert); assert_geq(gMinInsert, 0); PairedEndPolicy pepol( pepolFlag, gMaxInsert, gMinInsert, localAlign, gFlippedMatesOK, gDovetailMatesOK, gContainMatesOK, gOlapMatesOK, gExpandToFrag); PerfMetrics metricsPt; // per-thread metrics object; for read-level metrics BTString nametmp; PerReadMetrics prm; // Used by thread with threadid == 1 to measure time elapsed time_t iTime = time(0); // Keep track of whether last search was exhaustive for mates 1 and 2 bool exhaustive[2] = { false, false }; // Keep track of whether mates 1/2 were filtered out last time through bool filt[2] = { true, true }; // Keep track of whether mates 1/2 were filtered out due Ns last time bool nfilt[2] = { true, true }; // Keep track of whether mates 1/2 were filtered out due to not having // enough characters to rise about the score threshold. bool scfilt[2] = { true, true }; // Keep track of whether mates 1/2 were filtered out due to not having // more characters than the number of mismatches permitted in a seed. bool lenfilt[2] = { true, true }; // Keep track of whether mates 1/2 were filtered out by upstream qc bool qcfilt[2] = { true, true }; rndArb.init((uint32_t)time(0)); int mergei = 0; int mergeival = 16; while(true) { bool success = false, done = false, paired = false; ps->nextReadPair(success, done, paired, outType != OUTPUT_SAM); if(!success && done) { break; } else if(!success) { continue; } TReadId rdid = ps->rdid(); if(nthreads > 1 && useTempSpliceSite) { while(true) { uint64_t min_rdid = 0; { // ThreadSafe t(&thread_rids_mutex, nthreads > 1); assert_gt(thread_rids.size(), 0); min_rdid = thread_rids[0]; for(size_t i = 1; i < thread_rids.size(); i++) { if(thread_rids[i] < min_rdid) { min_rdid = thread_rids[i]; } } } if(min_rdid + thread_rids_mindist < rdid) { #if defined(_TTHREAD_WIN32_) Sleep(0); #elif defined(_TTHREAD_POSIX_) sched_yield(); #endif } else break; } } bool sample = true; if(arbitraryRandom) { ps->bufa().seed = rndArb.nextU32(); ps->bufb().seed = rndArb.nextU32(); } if(sampleFrac < 1.0f) { rnd.init(ROTL(ps->bufa().seed, 2)); sample = rnd.nextFloat() < sampleFrac; } if(rdid >= skipReads && rdid < qUpto && sample) { // Align this read/pair bool retry = true; // // Check if there is metrics reporting for us to do. // if(metricsIval > 0 && (metricsOfb != NULL || metricsStderr) && !metricsPerRead && ++mergei == mergeival) { // Do a periodic merge. Update global metrics, in a // synchronized manner if needed. MERGE_METRICS(metrics, nthreads > 1); mergei = 0; // Check if a progress message should be printed if(tid == 0) { // Only thread 1 prints progress messages time_t curTime = time(0); if(curTime - iTime >= metricsIval) { metrics.reportInterval(metricsOfb, metricsStderr, false, true, NULL); iTime = curTime; } } } prm.reset(); // per-read metrics prm.doFmString = false; if(sam_print_xt) { gettimeofday(&prm.tv_beg, &prm.tz_beg); } // Try to align this read while(retry) { retry = false; assert_eq(ps->bufa().color, false); ca.nextRead(); // clear the cache olm.reads++; assert(!ca.aligning()); bool pair = paired; const size_t rdlen1 = ps->bufa().length(); const size_t rdlen2 = pair ? ps->bufb().length() : 0; olm.bases += (rdlen1 + rdlen2); #if 0 msinkwrap.nextRead( &ps->bufa(), pair ? &ps->bufb() : NULL, rdid, sc.qualitiesMatter()); assert(msinkwrap.inited()); #endif size_t rdlens[2] = { rdlen1, rdlen2 }; // Calculate the minimum valid score threshold for the read TAlScore minsc[2], maxpen[2]; maxpen[0] = maxpen[1] = 0; minsc[0] = minsc[1] = std::numeric_limits::max(); if(bwaSwLike) { // From BWA-SW manual: "Given an l-long query, the // threshold for a hit to be retained is // a*max{T,c*log(l)}." We try to recreate that here. float a = (float)sc.match(30); float T = bwaSwLikeT, c = bwaSwLikeC; minsc[0] = (TAlScore)max(a*T, a*c*log(rdlens[0])); if(paired) { minsc[1] = (TAlScore)max(a*T, a*c*log(rdlens[1])); } } else { minsc[0] = scoreMin.f(rdlens[0]); if(paired) minsc[1] = scoreMin.f(rdlens[1]); if(localAlign) { if(minsc[0] < 0) { if(!gQuiet) printLocalScoreMsg(*ps, paired, true); minsc[0] = 0; } if(paired && minsc[1] < 0) { if(!gQuiet) printLocalScoreMsg(*ps, paired, false); minsc[1] = 0; } } else { if(minsc[0] > 0) { if(!gQuiet) printEEScoreMsg(*ps, paired, true); minsc[0] = 0; } if(paired && minsc[1] > 0) { if(!gQuiet) printEEScoreMsg(*ps, paired, false); minsc[1] = 0; } } } // N filter; does the read have too many Ns? size_t readns[2] = {0, 0}; sc.nFilterPair( &ps->bufa().patFw, pair ? &ps->bufb().patFw : NULL, readns[0], readns[1], nfilt[0], nfilt[1]); // Score filter; does the read enough character to rise above // the score threshold? scfilt[0] = sc.scoreFilter(minsc[0], rdlens[0]); scfilt[1] = sc.scoreFilter(minsc[1], rdlens[1]); lenfilt[0] = lenfilt[1] = true; if(rdlens[0] <= (size_t)multiseedMms || rdlens[0] < 2) { if(!gQuiet) printMmsSkipMsg(*ps, paired, true, multiseedMms); lenfilt[0] = false; } if((rdlens[1] <= (size_t)multiseedMms || rdlens[1] < 2) && paired) { if(!gQuiet) printMmsSkipMsg(*ps, paired, false, multiseedMms); lenfilt[1] = false; } if(rdlens[0] < 2) { if(!gQuiet) printLenSkipMsg(*ps, paired, true); lenfilt[0] = false; } if(rdlens[1] < 2 && paired) { if(!gQuiet) printLenSkipMsg(*ps, paired, false); lenfilt[1] = false; } qcfilt[0] = qcfilt[1] = true; if(qcFilter) { qcfilt[0] = (ps->bufa().filter != '0'); qcfilt[1] = (ps->bufb().filter != '0'); } filt[0] = (nfilt[0] && scfilt[0] && lenfilt[0] && qcfilt[0]); filt[1] = (nfilt[1] && scfilt[1] && lenfilt[1] && qcfilt[1]); prm.nFilt += (filt[0] ? 0 : 1) + (filt[1] ? 0 : 1); Read* rds[2] = { &ps->bufa(), &ps->bufb() }; // For each mate... assert(msinkwrap.empty()); //size_t minedfw[2] = { 0, 0 }; //size_t minedrc[2] = { 0, 0 }; // Calcualte nofw / no rc bool nofw[2] = { false, false }; bool norc[2] = { false, false }; nofw[0] = paired ? (gMate1fw ? gNofw : gNorc) : gNofw; norc[0] = paired ? (gMate1fw ? gNorc : gNofw) : gNorc; nofw[1] = paired ? (gMate2fw ? gNofw : gNorc) : gNofw; norc[1] = paired ? (gMate2fw ? gNorc : gNofw) : gNorc; // Calculate nceil int nceil[2] = { 0, 0 }; nceil[0] = nCeil.f((double)rdlens[0]); nceil[0] = min(nceil[0], (int)rdlens[0]); if(paired) { nceil[1] = nCeil.f((double)rdlens[1]); nceil[1] = min(nceil[1], (int)rdlens[1]); } exhaustive[0] = exhaustive[1] = false; //size_t matemap[2] = { 0, 1 }; bool pairPostFilt = filt[0] && filt[1]; if(pairPostFilt) { rnd.init(ps->bufa().seed ^ ps->bufb().seed); } else { rnd.init(ps->bufa().seed); } // Calculate interval length for both mates int interval[2] = { 0, 0 }; for(size_t mate = 0; mate < (pair ? 2:1); mate++) { interval[mate] = msIval.f((double)rdlens[mate]); if(filt[0] && filt[1]) { // Boost interval length by 20% for paired-end reads interval[mate] = (int)(interval[mate] * 1.2 + 0.5); } interval[mate] = max(interval[mate], 1); } // Calculate streak length size_t streak[2] = { maxDpStreak, maxDpStreak }; size_t mtStreak[2] = { maxMateStreak, maxMateStreak }; size_t mxDp[2] = { maxDp, maxDp }; size_t mxUg[2] = { maxUg, maxUg }; size_t mxIter[2] = { maxIters, maxIters }; if(allHits) { streak[0] = streak[1] = std::numeric_limits::max(); mtStreak[0] = mtStreak[1] = std::numeric_limits::max(); mxDp[0] = mxDp[1] = std::numeric_limits::max(); mxUg[0] = mxUg[1] = std::numeric_limits::max(); mxIter[0] = mxIter[1] = std::numeric_limits::max(); } else if(khits > 1) { for(size_t mate = 0; mate < 2; mate++) { streak[mate] += (khits-1) * maxStreakIncr; mtStreak[mate] += (khits-1) * maxStreakIncr; mxDp[mate] += (khits-1) * maxItersIncr; mxUg[mate] += (khits-1) * maxItersIncr; mxIter[mate] += (khits-1) * maxItersIncr; } } if(filt[0] && filt[1]) { streak[0] = (size_t)ceil((double)streak[0] / 2.0); streak[1] = (size_t)ceil((double)streak[1] / 2.0); assert_gt(streak[1], 0); } assert_gt(streak[0], 0); // Calculate # seed rounds for each mate size_t nrounds[2] = { nSeedRounds, nSeedRounds }; if(filt[0] && filt[1]) { nrounds[0] = (size_t)ceil((double)nrounds[0] / 2.0); nrounds[1] = (size_t)ceil((double)nrounds[1] / 2.0); assert_gt(nrounds[1], 0); } assert_gt(nrounds[0], 0); // Increment counters according to what got filtered for(size_t mate = 0; mate < (pair ? 2:1); mate++) { if(!filt[mate]) { // Mate was rejected by N filter olm.freads++; // reads filtered out olm.fbases += rdlens[mate]; // bases filtered out } else { //shs[mate].clear(); //shs[mate].nextRead(mate == 0 ? ps->bufa() : ps->bufb()); //assert(shs[mate].empty()); olm.ureads++; // reads passing filter olm.ubases += rdlens[mate]; // bases passing filter } } //size_t eePeEeltLimit = std::numeric_limits::max(); // Whether we're done with mate1 / mate2 bool done[2] = { !filt[0], !filt[1] }; // size_t nelt[2] = {0, 0}; if(filt[0] && filt[1]) { bp_Aligner.initReads(rds, nofw, norc, minsc, maxpen); } else if(filt[0] || filt[1]) { bp_Aligner.initRead(rds[0], nofw[0], norc[0], minsc[0], maxpen[0], filt[1]); } if(filt[0] || filt[1]) { int ret = bp_Aligner.go(sc, ebwtFw, ebwtBw, ref, sw, *ssdb, wlm, prm, swmSeed, him, rnd, msinkwrap); MERGE_SW(sw); // daehwan size_t mate = 0; assert_gt(ret, 0); // Clear out the exact hits so that we don't try to // extend them again later! if(ret == EXTEND_EXHAUSTED_CANDIDATES) { // Not done yet } else if(ret == EXTEND_POLICY_FULFILLED) { // Policy is satisfied for this mate at least if(msinkwrap.state().doneWithMate(mate == 0)) { done[mate] = true; } if(msinkwrap.state().doneWithMate(mate == 1)) { done[mate^1] = true; } } else if(ret == EXTEND_PERFECT_SCORE) { // We exhausted this mode at least done[mate] = true; } else if(ret == EXTEND_EXCEEDED_HARD_LIMIT) { // We exceeded a per-read limit done[mate] = true; } else if(ret == EXTEND_EXCEEDED_SOFT_LIMIT) { // Not done yet } else { // cerr << "Bad return value: " << ret << endl; throw 1; } if(!done[mate]) { TAlScore perfectScore = sc.perfectScore(rdlens[mate]); if(!done[mate] && minsc[mate] == perfectScore) { done[mate] = true; } } } for(size_t i = 0; i < 2; i++) { assert_leq(prm.nExIters, mxIter[i]); assert_leq(prm.nExDps, mxDp[i]); assert_leq(prm.nMateDps, mxDp[i]); assert_leq(prm.nExUgs, mxUg[i]); assert_leq(prm.nMateUgs, mxUg[i]); assert_leq(prm.nDpFail, streak[i]); assert_leq(prm.nUgFail, streak[i]); assert_leq(prm.nEeFail, streak[i]); } #if 0 // Commit and report paired-end/unpaired alignments msinkwrap.finishRead( NULL, NULL, exhaustive[0], // exhausted seed hits for mate 1? exhaustive[1], // exhausted seed hits for mate 2? nfilt[0], nfilt[1], scfilt[0], scfilt[1], lenfilt[0], lenfilt[1], qcfilt[0], qcfilt[1], sortByScore, // prioritize by alignment score rnd, // pseudo-random generator rpm, // reporting metrics prm, // per-read metrics sc, // scoring scheme !seedSumm, // suppress seed summaries? seedSumm); // suppress alignments? assert(!retry || msinkwrap.empty()); #endif if(nthreads > 1 && useTempSpliceSite) { // ThreadSafe t(&thread_rids_mutex, nthreads > 1); assert_gt(tid, 0); assert_leq(tid, thread_rids.size()); assert(thread_rids[tid - 1] == 0 || rdid > thread_rids[tid - 1]); thread_rids[tid - 1] = rdid; } } // while(retry) } // if(rdid >= skipReads && rdid < qUpto) else if(rdid >= qUpto) { break; } if(metricsPerRead) { MERGE_METRICS(metricsPt, nthreads > 1); nametmp = ps->bufa().name; metricsPt.reportInterval( metricsOfb, metricsStderr, true, true, &nametmp); metricsPt.reset(); } } // while(true) // One last metrics merge MERGE_METRICS(metrics, nthreads > 1); return; } /** * Called once per alignment job. Sets up global pointers to the * shared global data structures, creates per-thread structures, then * enters the search loop. */ static void multiseedSearch( Scoring& sc, PairedPatternSource& patsrc, // pattern source AlnSink& msink, // hit sink HierEbwt& ebwtFw, // index of original text HierEbwt& ebwtBw, // index of mirror text BitPairReference* refs, const EList& refnames, OutFileBuf *metricsOfb) { multiseed_patsrc = &patsrc; multiseed_msink = &msink; multiseed_ebwtFw = &ebwtFw; multiseed_ebwtBw = &ebwtBw; multiseed_sc = ≻ multiseed_refnames = refnames; multiseed_metricsOfb = metricsOfb; multiseed_refs = refs; AutoArray threads(nthreads); AutoArray tids(nthreads); { // Load the other half of the index into memory assert(!ebwtFw.isInMemory()); Timer _t(cerr, "Time loading forward index: ", timing); ebwtFw.loadIntoMemory( 0, // colorspace? -1, // not the reverse index true, // load SA samp? (yes, need forward index's SA samp) true, // load ftab (in forward index) true, // load rstarts (in forward index) !noRefNames, // load names? startVerbose); } #if 0 if(multiseedMms > 0 || do1mmUpFront) { // Load the other half of the index into memory assert(!ebwtBw.isInMemory()); Timer _t(cerr, "Time loading mirror index: ", timing); ebwtBw.loadIntoMemory( 0, // colorspace? // It's bidirectional search, so we need the reverse to be // constructed as the reverse of the concatenated strings. 1, true, // load SA samp in reverse index true, // yes, need ftab in reverse index true, // load rstarts in reverse index !noRefNames, // load names? startVerbose); } #endif // Start the metrics thread { Timer _t(cerr, "Multiseed full-index search: ", timing); thread_rids.resize(nthreads); thread_rids.fill(0); thread_rids_mindist = (nthreads == 1 || !useTempSpliceSite ? 0 : 1000 * nthreads); for(int i = 0; i < nthreads; i++) { // Thread IDs start at 1 tids[i] = i+1; threads[i] = new tthread::thread(multiseedSearchWorker_hisat_bp, (void*)&tids[i]); } for (int i = 0; i < nthreads; i++) threads[i]->join(); } if(!metricsPerRead && (metricsOfb != NULL || metricsStderr)) { metrics.reportInterval(metricsOfb, metricsStderr, true, false, NULL); } } static string argstr; extern void initializeCntLut(); extern void initializeCntBit(); template static void driver( const char * type, const string& bt2indexBase, const string& outfile) { if(gVerbose || startVerbose) { cerr << "Entered driver(): "; logTime(cerr, true); } initializeCntLut(); initializeCntBit(); // Vector of the reference sequences; used for sanity-checking EList > names, os; EList nameLens, seqLens; // Read reference sequences from the command-line or from a FASTA file if(!origString.empty()) { // Read fasta file(s) EList origFiles; tokenize(origString, ",", origFiles); parseFastas(origFiles, names, nameLens, os, seqLens); } PatternParams pp( format, // file format fileParallel, // true -> wrap files with separate PairedPatternSources seed, // pseudo-random seed useSpinlock, // use spin locks instead of pthreads solexaQuals, // true -> qualities are on solexa64 scale phred64Quals, // true -> qualities are on phred64 scale integerQuals, // true -> qualities are space-separated numbers fuzzy, // true -> try to parse fuzzy fastq fastaContLen, // length of sampled reads for FastaContinuous... fastaContFreq, // frequency of sampled reads for FastaContinuous... skipReads // skip the first 'skip' patterns ); if(gVerbose || startVerbose) { cerr << "Creating PatternSource: "; logTime(cerr, true); } PairedPatternSource *patsrc = PairedPatternSource::setupPatternSources( queries, // singles, from argv mates1, // mate1's, from -1 arg mates2, // mate2's, from -2 arg mates12, // both mates on each line, from --12 arg qualities, // qualities associated with singles qualities1, // qualities associated with m1 qualities2, // qualities associated with m2 pp, // read read-in parameters gVerbose || startVerbose); // be talkative // Open hit output file if(gVerbose || startVerbose) { cerr << "Opening hit output file: "; logTime(cerr, true); } OutFileBuf *fout; if(!outfile.empty()) { fout = new OutFileBuf(outfile.c_str(), false); } else { fout = new OutFileBuf(); } // Initialize Ebwt object and read in header if(gVerbose || startVerbose) { cerr << "About to initialize fw Ebwt: "; logTime(cerr, true); } adjIdxBase = adjustEbwtBase(argv0, bt2indexBase, gVerbose); HierEbwt ebwt( adjIdxBase, 0, // index is colorspace -1, // fw index true, // index is for the forward direction /* overriding: */ offRate, 0, // amount to add to index offrate or <= 0 to do nothing useMm, // whether to use memory-mapped files useShmem, // whether to use shared memory mmSweep, // sweep memory-mapped files !noRefNames, // load names? true, // load SA sample? true, // load ftab? true, // load rstarts? gVerbose, // whether to be talkative startVerbose, // talkative during initialization false /*passMemExc*/, sanityCheck); HierEbwt* ebwtBw = NULL; #if 0 // We need the mirror index if mismatches are allowed if(multiseedMms > 0 || do1mmUpFront) { if(gVerbose || startVerbose) { cerr << "About to initialize rev Ebwt: "; logTime(cerr, true); } ebwtBw = new HierEbwt( adjIdxBase + ".rev", 0, // index is colorspace 1, // TODO: maybe not false, // index is for the reverse direction /* overriding: */ offRate, 0, // amount to add to index offrate or <= 0 to do nothing useMm, // whether to use memory-mapped files useShmem, // whether to use shared memory mmSweep, // sweep memory-mapped files !noRefNames, // load names? true, // load SA sample? true, // load ftab? true, // load rstarts? gVerbose, // whether to be talkative startVerbose, // talkative during initialization false /*passMemExc*/, sanityCheck); } #endif if(sanityCheck && !os.empty()) { // Sanity check number of patterns and pattern lengths in Ebwt // against original strings assert_eq(os.size(), ebwt.nPat()); for(size_t i = 0; i < os.size(); i++) { assert_eq(os[i].length(), ebwt.plen()[i]); } } // Sanity-check the restored version of the Ebwt if(sanityCheck && !os.empty()) { ebwt.loadIntoMemory( 0, -1, // fw index true, // load SA sample true, // load ftab true, // load rstarts !noRefNames, startVerbose); ebwt.checkOrigs(os, false, false); ebwt.evictFromMemory(); } OutputQueue oq( *fout, // out file buffer reorder && nthreads > 1, // whether to reorder when there's >1 thread nthreads, // # threads nthreads > 1, // whether to be thread-safe skipReads); // first read will have this rdid { Timer _t(cerr, "Time searching: ", timing); // Set up penalities if(bonusMatch > 0 && !localAlign) { cerr << "Warning: Match bonus always = 0 in --end-to-end mode; ignoring user setting" << endl; bonusMatch = 0; } Scoring sc( bonusMatch, // constant reward for match penMmcType, // how to penalize mismatches penMmcMax, // max mm pelanty penMmcMin, // min mm pelanty scoreMin, // min score as function of read len nCeil, // max # Ns as function of read len penNType, // how to penalize Ns in the read penN, // constant if N pelanty is a constant penNCatPair, // whether to concat mates before N filtering penRdGapConst, // constant coeff for read gap cost penRfGapConst, // constant coeff for ref gap cost penRdGapLinear, // linear coeff for read gap cost penRfGapLinear, // linear coeff for ref gap cost gGapBarrier, // # rows at top/bot only entered diagonally penCanSplice, // canonical splicing penalty penNoncanSplice,// non-canonical splicing penalty &penIntronLen); // penalty as to intron length EList reflens; for(size_t i = 0; i < ebwt.nPat(); i++) { reflens.push_back(ebwt.plen()[i]); } EList refnames; readEbwtRefnames(adjIdxBase, refnames); SamConfig samc( refnames, // reference sequence names reflens, // reference sequence lengths samTruncQname, // whether to truncate QNAME to 255 chars samOmitSecSeqQual, // omit SEQ/QUAL for 2ndary alignments? samNoUnal, // omit unaligned-read records? string("hisat"), // program id string("hisat"), // program name string(HISAT_VERSION), // program version argstr, // command-line rgs_optflag, // read-group string rna_strandness, sam_print_as, sam_print_xs, sam_print_xss, sam_print_yn, sam_print_xn, sam_print_cs, sam_print_cq, sam_print_x0, sam_print_x1, sam_print_xm, sam_print_xo, sam_print_xg, sam_print_nm, sam_print_md, sam_print_yf, sam_print_yi, sam_print_ym, sam_print_yp, sam_print_yt, sam_print_ys, sam_print_zs, sam_print_xr, sam_print_xt, sam_print_xd, sam_print_xu, sam_print_yl, sam_print_ye, sam_print_yu, sam_print_xp, sam_print_yr, sam_print_zb, sam_print_zr, sam_print_zf, sam_print_zm, sam_print_zi, sam_print_zp, sam_print_zu, sam_print_xs_a); // Set up hit sink; if sanityCheck && !os.empty() is true, // then instruct the sink to "retain" hits in a vector in // memory so that we can easily sanity check them later on AlnSink *mssink = NULL; Timer *_tRef = new Timer(cerr, "Time loading reference: ", timing); auto_ptr refs( new BitPairReference( adjIdxBase, false, sanityCheck, NULL, NULL, false, useMm, useShmem, mmSweep, gVerbose, startVerbose) ); delete _tRef; if(!refs->loaded()) throw 1; init_junction_prob(); bool write = novelSpliceSiteOutfile != "" || useTempSpliceSite; bool read = knownSpliceSiteInfile != "" || novelSpliceSiteInfile != "" || useTempSpliceSite; ssdb = new SpliceSiteDB( *(refs.get()), refnames, nthreads > 1, // thread-safe write, // write? read); // read? if(ssdb != NULL) { if(knownSpliceSiteInfile != "") { ifstream ssdb_file(knownSpliceSiteInfile.c_str(), ios::in); if(ssdb_file.is_open()) { ssdb->read(ssdb_file, false); // known splice sites ssdb_file.close(); } } if(novelSpliceSiteInfile != "") { ifstream ssdb_file(novelSpliceSiteInfile.c_str(), ios::in); if(ssdb_file.is_open()) { ssdb->read(ssdb_file, true); // novel splice sites ssdb_file.close(); } } } switch(outType) { case OUTPUT_SAM: { mssink = new AlnSinkSam( oq, // output queue samc, // settings & routines for SAM output refnames, // reference names gQuiet, // don't print alignment summary at end ssdb); #if 0 if(!samNoHead) { bool printHd = true, printSq = true; BTString buf; samc.printHeader(buf, rgid, rgs, printHd, !samNoSQ, printSq); fout->writeString(buf); } #endif break; } default: cerr << "Invalid output type: " << outType << endl; throw 1; } if(gVerbose || startVerbose) { cerr << "Dispatching to search driver: "; logTime(cerr, true); } // Set up global constraint OutFileBuf *metricsOfb = NULL; if(!metricsFile.empty() && metricsIval > 0) { metricsOfb = new OutFileBuf(metricsFile); } // Do the search for all input reads assert(patsrc != NULL); assert(mssink != NULL); multiseedSearch( sc, // scoring scheme *patsrc, // pattern source *mssink, // hit sink ebwt, // BWT *ebwtBw, // BWT' refs.get(), refnames, metricsOfb); // Evict any loaded indexes from memory if(ebwt.isInMemory()) { ebwt.evictFromMemory(); } if(ebwtBw != NULL) { delete ebwtBw; } if(!gQuiet && !seedSumm) { size_t repThresh = mhits; if(repThresh == 0) { repThresh = std::numeric_limits::max(); } #if 0 mssink->finish( repThresh, gReportDiscordant, gReportMixed, hadoopOut); #endif } if(ssdb != NULL) { if(novelSpliceSiteOutfile != "") { ofstream ssdb_file(novelSpliceSiteOutfile.c_str(), ios::out); if(ssdb_file.is_open()) { ssdb->print(ssdb_file); ssdb_file.close(); } } } oq.flush(true); assert_eq(oq.numStarted(), oq.numFinished()); assert_eq(oq.numStarted(), oq.numFlushed()); delete patsrc; delete mssink; delete ssdb; delete metricsOfb; if(fout != NULL) { delete fout; } } } // C++ name mangling is disabled for the bowtie() function to make it // easier to use Bowtie as a library. extern "C" { /** * Main bowtie entry function. Parses argc/argv style command-line * options, sets global configuration variables, and calls the driver() * function. */ int hisat(int argc, const char **argv) { try { // Reset all global state, including getopt state opterr = optind = 1; resetOptions(); for(int i = 0; i < argc; i++) { argstr += argv[i]; if(i < argc-1) argstr += " "; } if(startVerbose) { cerr << "Entered main(): "; logTime(cerr, true); } parseOptions(argc, argv); argv0 = argv[0]; if(showVersion) { cout << argv0 << " version " << HISAT_VERSION << endl; if(sizeof(void*) == 4) { cout << "32-bit" << endl; } else if(sizeof(void*) == 8) { cout << "64-bit" << endl; } else { cout << "Neither 32- nor 64-bit: sizeof(void*) = " << sizeof(void*) << endl; } cout << "Built on " << BUILD_HOST << endl; cout << BUILD_TIME << endl; cout << "Compiler: " << COMPILER_VERSION << endl; cout << "Options: " << COMPILER_OPTIONS << endl; cout << "Sizeof {int, long, long long, void*, size_t, off_t}: {" << sizeof(int) << ", " << sizeof(long) << ", " << sizeof(long long) << ", " << sizeof(void *) << ", " << sizeof(size_t) << ", " << sizeof(off_t) << "}" << endl; return 0; } { Timer _t(cerr, "Overall time: ", timing); if(startVerbose) { cerr << "Parsing index and read arguments: "; logTime(cerr, true); } // Get index basename (but only if it wasn't specified via --index) if(bt2index.empty()) { if(optind >= argc) { cerr << "No index, query, or output file specified!" << endl; printUsage(cerr); return 1; } bt2index = argv[optind++]; } // Get query filename bool got_reads = !queries.empty() || !mates1.empty() || !mates12.empty(); if(optind >= argc) { if(!got_reads) { printUsage(cerr); cerr << "***" << endl << "Error: Must specify at least one read input with -U/-1/-2" << endl; return 1; } } else if(!got_reads) { // Tokenize the list of query files tokenize(argv[optind++], ",", queries); if(queries.empty()) { cerr << "Tokenized query file list was empty!" << endl; printUsage(cerr); return 1; } } // Get output filename if(optind < argc && outfile.empty()) { outfile = argv[optind++]; cerr << "Warning: Output file '" << outfile.c_str() << "' was specified without -S. This will not work in " << "future HISAT 2 versions. Please use -S instead." << endl; } // Extra parametesr? if(optind < argc) { cerr << "Extra parameter(s) specified: "; for(int i = optind; i < argc; i++) { cerr << "\"" << argv[i] << "\""; if(i < argc-1) cerr << ", "; } cerr << endl; if(mates1.size() > 0) { cerr << "Note that if files are specified using -1/-2, a file cannot" << endl << "also be specified. Please run HISAT2 separately for mates and singles." << endl; } throw 1; } // Optionally summarize if(gVerbose) { cout << "Input bt2 file: \"" << bt2index.c_str() << "\"" << endl; cout << "Query inputs (DNA, " << file_format_names[format].c_str() << "):" << endl; for(size_t i = 0; i < queries.size(); i++) { cout << " " << queries[i].c_str() << endl; } cout << "Quality inputs:" << endl; for(size_t i = 0; i < qualities.size(); i++) { cout << " " << qualities[i].c_str() << endl; } cout << "Output file: \"" << outfile.c_str() << "\"" << endl; cout << "Local endianness: " << (currentlyBigEndian()? "big":"little") << endl; cout << "Sanity checking: " << (sanityCheck? "enabled":"disabled") << endl; #ifdef NDEBUG cout << "Assertions: disabled" << endl; #else cout << "Assertions: enabled" << endl; #endif } if(ipause) { cout << "Press key to continue..." << endl; getchar(); } driver >("DNA", bt2index, outfile); } return 0; } catch(std::exception& e) { cerr << "Error: Encountered exception: '" << e.what() << "'" << endl; cerr << "Command: "; for(int i = 0; i < argc; i++) cerr << argv[i] << " "; cerr << endl; return 1; } catch(int e) { if(e != 0) { cerr << "Error: Encountered internal HISAT2 exception (#" << e << ")" << endl; cerr << "Command: "; for(int i = 0; i < argc; i++) cerr << argv[i] << " "; cerr << endl; } return e; } } // bowtie() } // extern "C" hisat2-2.2.1/ival_list.cpp000066400000000000000000000120661370663763200154220ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "ival_list.h" #ifdef MAIN_IVAL_DS #include #include "random_source.h" using namespace std; int main(void) { cerr << "Case 1 ... "; { EIvalMergeList list((size_t)5); list.add(Interval(0, 10, true, 10)); list.add(Interval(0, 30, true, 10)); list.add(Interval(0, 20, true, 10)); assert(!list.locusPresent(Coord(0, 5, true))); assert(!list.locusPresent(Coord(0, 9, true))); assert(list.locusPresent(Coord(0, 10, true))); assert(list.locusPresent(Coord(0, 11, true))); assert(list.locusPresent(Coord(0, 19, true))); assert(list.locusPresent(Coord(0, 20, true))); assert(list.locusPresent(Coord(0, 21, true))); assert(list.locusPresent(Coord(0, 29, true))); assert(list.locusPresent(Coord(0, 30, true))); assert(list.locusPresent(Coord(0, 31, true))); assert(list.locusPresent(Coord(0, 39, true))); assert(!list.locusPresent(Coord(0, 40, true))); assert(!list.locusPresent(Coord(0, 41, true))); } cerr << " PASSED" << endl; cerr << "Case 2 ... "; { EIvalMergeList list((size_t)5); list.add(Interval(0, 10, true, 10)); for(size_t i = 5; i < 45; i++) { assert(list.locusPresent(Coord(0, i, true)) == (i >= 10 && i < 20)); } list.clear(); list.add(Interval(0, 15, true, 10)); for(size_t i = 5; i < 45; i++) { assert(list.locusPresent(Coord(0, i, true)) == (i >= 15 && i < 25)); } } cerr << " PASSED" << endl; cerr << "Case 3 ... "; { EIvalMergeList list((size_t)5); for(size_t i = 0; i < 20; i++) { list.add(Interval(0, 10*i, true, 9)); } for(size_t i = 0; i < 200; i++) { assert(list.locusPresent(Coord(0, i, true)) == ((i % 10) != 9)); assert(!list.locusPresent(Coord(0, i, false))); assert(!list.locusPresent(Coord(1, i, true))); } } cerr << " PASSED" << endl; cerr << "Case 4 ... "; { EIvalMergeList list((size_t)5); for(int i = 19; i >= 0; i--) { list.add(Interval(0, 10*i, true, 9)); } for(size_t i = 0; i < 200; i++) { assert(list.locusPresent(Coord(0, i, true)) == ((i % 10) != 9)); assert(!list.locusPresent(Coord(0, i, false))); assert(!list.locusPresent(Coord(1, i, true))); } } cerr << " PASSED" << endl; cerr << "Random testing (1 ref) ... "; { RandomSource rnd(34523); for(size_t c = 0; c < 10; c++) { EIvalMergeList list1((size_t)16); EIvalMergeList list2((size_t)2000); size_t num_intervals = 20; uint32_t max_width = 100; for(size_t i = 0; i < num_intervals; i++) { uint32_t start = rnd.nextU32() % max_width/2; uint32_t end = (rnd.nextU32() % (max_width - start - 1) + start)+1; assert_lt(end, max_width); assert_gt(end, start); list1.add(Interval(0, start, false, end-start)); list2.add(Interval(0, start, false, end-start)); } assert_geq(num_intervals, list1.size()); assert_geq(num_intervals, list2.size()); assert(list1.repOk()); assert(list2.repOk()); for(uint32_t i = 0; i < max_width+1; i++) { assert(list1.repOk()); assert(list2.repOk()); ASSERT_ONLY(bool l1 = list1.locusPresent(Coord(0, i, true))); ASSERT_ONLY(bool l2 = list2.locusPresent(Coord(0, i, true))); assert_eq(l1, l2); } } } cerr << " PASSED" << endl; cerr << "Random testing (few refs) ... "; { RandomSource rnd(34523); for(size_t c = 0; c < 10; c++) { EIvalMergeList list1((size_t)16); EIvalMergeList list2((size_t)2000); size_t num_intervals = 20; uint32_t max_width = 100; for(size_t i = 0; i < num_intervals; i++) { uint32_t start = rnd.nextU32() % max_width/2; uint32_t end = (rnd.nextU32() % (max_width - start - 1) + start)+1; assert_lt(end, max_width); assert_gt(end, start); bool orient = (rnd.nextU2() == 0); TRefId ref = (TRefId)(rnd.nextU32() % 5); list1.add(Interval(ref, start, orient, end-start)); list2.add(Interval(ref, start, orient, end-start)); } assert_geq(num_intervals, list1.size()); assert_geq(num_intervals, list2.size()); assert(list1.repOk()); assert(list2.repOk()); for(uint32_t i = 0; i < max_width+1; i++) { assert(list1.repOk()); assert(list2.repOk()); for(int fwi = 0; fwi < 2; fwi++) { bool fw = (fwi == 0); for(TRefId refi = 0; refi < 5; refi++) { ASSERT_ONLY(bool l1 = list1.locusPresent(Coord(refi, i, fw))); ASSERT_ONLY(bool l2 = list2.locusPresent(Coord(refi, i, fw))); assert_eq(l1, l2); } } } } } cerr << " PASSED" << endl; } #endif /*def MAIN_IVAL_DS*/ hisat2-2.2.1/ival_list.h000066400000000000000000000150551370663763200150700ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef IVAL_LIST_H_ #define IVAL_LIST_H_ #include "ds.h" #include "ref_coord.h" #include /** * Encapsulates the "union" of a collection of intervals. Intervals are stored * in a sorted list. Intervals can be added but not removed. Supports just * one type of query for now: locusPresent(). */ class EIvalMergeList { public: static const size_t DEFAULT_UNSORTED_SZ = 16; explicit EIvalMergeList(int cat = 0) : sorted_(cat), sortedLhs_(cat), unsorted_(cat), unsortedSz_(DEFAULT_UNSORTED_SZ) { } explicit EIvalMergeList(size_t unsortedSz, int cat = 0) : sorted_(cat), sortedLhs_(cat), unsorted_(cat), unsortedSz_(unsortedSz) { } /** * Set the maximum size of the unsorted list. */ void setUnsortedSize(size_t usz) { unsortedSz_ = usz; } /** * Add a new interval to the list. */ void add(const Interval& i) { assert_leq(unsorted_.size(), unsortedSz_); if(unsorted_.size() < unsortedSz_) { unsorted_.push_back(i); } if(unsorted_.size() == unsortedSz_) { flush(); } } /** * Move all unsorted interval information into the sorted list and re-sort. * Merge overlapping intervals. */ void flush() { for(size_t i = 0; i < unsorted_.size(); i++) { sorted_.push_back(unsorted_[i]); } sorted_.sort(); merge(); sortedLhs_.clear(); for(size_t i = 0; i < sorted_.size(); i++) { sortedLhs_.push_back(sorted_[i].upstream()); } assert(sortedLhs_.sorted()); unsorted_.clear(); } #ifndef NDEBUG /** * Check that this interval list is internally consistent. */ bool repOk() const { assert_eq(sorted_.size(), sortedLhs_.size()); return true; } #endif /** * Remove all ranges from the list. */ void reset() { clear(); } /** * Remove all ranges from the list. */ void clear() { sorted_.clear(); sortedLhs_.clear(); unsorted_.clear(); } /** * Return true iff this locus is present in one of the intervals in the * list. */ bool locusPresent(const Coord& loc) const { return locusPresentUnsorted(loc) || locusPresentSorted(loc); } /** * Return the number of intervals added since the last call to reset() or * clear(). */ size_t size() const { return sorted_.size() + unsorted_.size(); } /** * Return true iff list is empty. */ bool empty() const { return sorted_.empty() && unsorted_.empty(); } protected: /** * Go through the sorted interval list and merge adjacent entries that * overlap. */ void merge() { size_t nmerged = 0; for(size_t i = 1; i < sorted_.size(); i++) { if(sorted_[i-1].downstream() >= sorted_[i].upstream()) { nmerged++; assert_leq(sorted_[i-1].upstream(), sorted_[i].upstream()); Coord up = std::min(sorted_[i-1].upstream(), sorted_[i].upstream()); Coord dn = std::max(sorted_[i-1].downstream(), sorted_[i].downstream()); sorted_[i].setUpstream(up); sorted_[i].setLength(dn.off() - up.off()); sorted_[i-1].reset(); } } sorted_.sort(); assert_lt(nmerged, sorted_.size()); sorted_.resize(sorted_.size()-nmerged); #ifndef NDEBUG for(size_t i = 0; i < sorted_.size(); i++) { assert(sorted_[i].inited()); } #endif } /** * Return true iff the given locus is present in one of the intervals in * the sorted list. */ bool locusPresentSorted(const Coord& loc) const { assert(repOk()); if(sorted_.empty()) { return false; } size_t beg = sortedLhs_.bsearchLoBound(loc); if(beg == sortedLhs_.size() || sortedLhs_[beg] > loc) { // Check element before if(beg == 0) { return false; } return sorted_[beg-1].contains(loc); } else { assert_eq(loc, sortedLhs_[beg]); return true; } } /** * Return true iff the given locus is present in one of the intervals in * the unsorted list. */ bool locusPresentUnsorted(const Coord& loc) const { for(size_t i = 0; i < unsorted_.size(); i++) { if(unsorted_[i].contains(loc)) { return true; } } return false; } EList sorted_; // LHS, RHS sorted EList sortedLhs_; // LHS, index into sorted_, sorted EList unsorted_; // unsorted size_t unsortedSz_; // max allowed size of unsorted_ }; /** * Binned version of the above. We bin using the low bits of the reference * sequence. */ class EIvalMergeListBinned { public: static const size_t NBIN = 7; explicit EIvalMergeListBinned(int cat = 0) : bins_(1 << NBIN, cat) { bins_.resize(1 << NBIN); } explicit EIvalMergeListBinned( size_t unsortedSz, int cat = 0) : bins_(1 << NBIN, cat) { bins_.resize(1 << NBIN); for(size_t i = 0; i < (1 << NBIN); i++) { bins_[i].setUnsortedSize(unsortedSz); } } /** * Add a new interval to the list. */ void add(const Interval& i) { size_t bin = i.ref() & ~(0xffffffff << NBIN); assert_lt(bin, bins_.size()); bins_[bin].add(i); } #ifndef NDEBUG /** * Check that this interval list is internally consistent. */ bool repOk() const { for(size_t i = 0; i < bins_.size(); i++) { assert(bins_[i].repOk()); } return true; } #endif /** * Remove all ranges from the list. */ void reset() { clear(); } /** * Remove all ranges from the list. */ void clear() { for(size_t i = 0; i < bins_.size(); i++) { bins_[i].clear(); } } /** * Return true iff this locus is present in one of the intervals in the * list. */ bool locusPresent(const Coord& loc) const { size_t bin = loc.ref() & ~(0xffffffff << NBIN); assert_lt(bin, bins_.size()); return bins_[bin].locusPresent(loc); } /** * Return the number of intervals added since the last call to reset() or * clear(). */ size_t size() const { // TODO: Keep track of size size_t sz = 0; for(size_t i = 0; i < bins_.size(); i++) { sz += bins_[i].size(); } return sz; } /** * Return true iff list is empty. */ bool empty() const { return size() == 0; } protected: EList bins_; }; #endif /*ndef IVAL_LIST_H_*/ hisat2-2.2.1/li_hla/000077500000000000000000000000001370663763200141535ustar00rootroot00000000000000hisat2-2.2.1/li_hla/Makefile000066400000000000000000000004331370663763200156130ustar00rootroot00000000000000CXX = g++ CXXFLAGS= -W LINKPATH= -I./samtools-0.1.19 -L./samtools-0.1.19 LINKFLAGS = -lbam -lz -lm -lpthread DEBUG= OBJECTS = all: hla hla: main.o $(CXX) -o $@ $(LINKPATH) $(CXXFLAGS) $(OBJECTS) main.o $(LINKFLAGS) main.o: main.cpp alignments.hpp clean: rm -f *.o *.gch hla hisat2-2.2.1/li_hla/alignments.hpp000066400000000000000000000113251370663763200170270ustar00rootroot00000000000000// The class handles reading the bam file #ifndef _LSONG_ALIGNMENT_HEADER #define _LSONG_ALIGNMENT_HEADER #include "samtools-0.1.19/sam.h" #include #include #include #include #include "defs.h" class Alignments { private: samfile_t *fpSam ; bam1_t *b ; char fileName[1024] ; bool opened ; std::map chrNameToId ; int onlyChromId ; // ignore other chromosomes void Open() { fpSam = samopen( fileName, "rb", 0 ) ; if ( !fpSam->header ) { fprintf( stderr, "Can not open %s.\n", fileName ) ; exit( 1 ) ; } // Collect the chromosome information for ( int i = 0 ; i < fpSam->header->n_targets ; ++i ) { std::string s( fpSam->header->target_name[i] ) ; chrNameToId[s] = i ; } opened = true ; } public: struct _pair segments[MAX_SEG_COUNT] ; unsigned int segCnt ; Alignments() { b = NULL ; opened = false ; onlyChromId = -1 ; } ~Alignments() {} void Open( char *file ) { strcpy( fileName, file ) ; Open() ; } void Rewind() { Close() ; Open() ; } void Close() { samclose( fpSam ) ; fpSam = NULL ; } bool IsOpened() { return opened ; } int Next() { int i ; int start = 0, len = 0 ; uint32_t *rawCigar ; while ( 1 ) { while ( 1 ) { if ( b ) bam_destroy1( b ) ; b = bam_init1() ; if ( samread( fpSam, b ) <= 0 ) return 0 ; if ( b->core.flag & 0xC ) continue ; if ( onlyChromId != -1 && onlyChromId != GetChromId() ) { continue ; } if ( ( b->core.flag & 0x900 ) == 0 ) break ; } // Compute the exons segments from the reads segCnt = 0 ; start = b->core.pos ; //+ 1 ; rawCigar = bam1_cigar( b ) ; for ( i = 0 ; i < b->core.n_cigar ; ++i ) { int op = rawCigar[i] & BAM_CIGAR_MASK ; int num = rawCigar[i] >> BAM_CIGAR_SHIFT ; switch ( op ) { case BAM_CMATCH: case BAM_CDEL: len += num ; break ; case BAM_CINS: case BAM_CSOFT_CLIP: case BAM_CHARD_CLIP: case BAM_CPAD: num = 0 ; break ; case BAM_CREF_SKIP: { segments[ segCnt ].a = start ; segments[ segCnt ].b = start + len - 1 ; ++segCnt ; start = start + len + num ; len = 0 ; } break ; default: len += num ; break ; } } segments[ segCnt ].a = start ; segments[ segCnt ].b = start + len - 1 ; ++segCnt ; /*for ( i = 0 ; i < segCnt ; ++i ) printf( "(%d %d) ", segments[i].a, segments[i].b ) ; printf( "\n" ) ;*/ // Check whether the mates are compatible int mChrId = b->core.mtid ; int64_t mPos = b->core.mpos ; if ( b->core.mtid == b->core.tid ) { for ( i = 0 ; i < segCnt - 1 ; ++i ) { if ( mPos >= segments[i].b && mPos <= segments[i + 1].a ) break ; } if ( i < segCnt - 1 ) continue ; } break ; } return 1 ; } int GetChromId() { return b->core.tid ; } char* GetChromName( int tid ) { return fpSam->header->target_name[ tid ] ; } int GetChromIdFromName( const char *s ) { std::string ss( s ) ; if ( chrNameToId.find( ss ) == chrNameToId.end() ) { printf( "Unknown genome name: %s\n", s ) ; exit( 1 ) ; } return chrNameToId[ss] ; } int GetChromLength( int tid ) { return fpSam->header->target_len[ tid ] ; } void GetMatePosition( int &chrId, int64_t &pos ) { chrId = b->core.mtid ; pos = b->core.mpos ; //+ 1 ; } int GetRepeatPosition( int &chrId, int64_t &pos ) { // Look at the CC field. if ( !bam_aux_get( b, "CC" ) || !bam_aux_get( b, "CP" ) ) { chrId = -1 ; pos = -1 ; return 0 ; } std::string s( bam_aux2Z( bam_aux_get(b, "CC" ) ) ) ; chrId = chrNameToId[ s ] ; pos = bam_aux2i( bam_aux_get( b, "CP" ) ) ;// Possible error for 64bit return 1 ; } bool IsReverse() { if ( b->core.flag & 0x10 ) return true ; return false ; } bool IsMateReverse() { if ( b->core.flag & 0x20 ) return true ; return false ; } char *GetReadId() { return bam1_qname( b ) ; } bool IsUnique() { if ( bam_aux_get( b, "NH" ) ) { if ( bam_aux2i( bam_aux_get( b, "NH" ) ) > 1 ) return false ; } return true ; } int GetFieldI( char *f ) { if ( bam_aux_get( b, f ) ) { return bam_aux2i( bam_aux_get( b, f ) ) ; } return -1 ; } char *GetFieldZ( char *f ) { if ( bam_aux_get( b, f ) ) { return bam_aux2Z( bam_aux_get( b, f ) ) ; } return NULL ; } // -1:minus, 0: unknown, 1:plus int GetStrand() { if ( segCnt == 1 ) return 0 ; if ( bam_aux_get( b, "XS" ) ) { if ( bam_aux2A( bam_aux_get( b, "XS" ) ) == '-' ) return -1 ; else return 1 ; } else return 0 ; } void OnlyChrom( const char *chr ) { onlyChromId = GetChromIdFromName( chr ) ; } } ; #endif hisat2-2.2.1/li_hla/defs.h000066400000000000000000000005131370663763200152440ustar00rootroot00000000000000#ifndef _LSONG_RSCAF_DEFS_HEADER #define _LSONG_RSCAF_DEFS_HEADER #include #define MAX_SEG_COUNT 127 struct _pair { int64_t a, b ; } ; char nucToNum[26] = { 0, -1, 1, -1, -1, -1, 2, -1, -1, -1, -1, -1, -1, -1, -1, -1, -1, -1, -1, 3, -1, -1, -1, -1, -1, -1 } ; char numToNuc[26] = {'A', 'C', 'G', 'T'} ; #endif hisat2-2.2.1/li_hla/main.cpp000066400000000000000000000300531370663763200156040ustar00rootroot00000000000000//usage: a.out prefix_of_allele_information alignment.bam [-b backbone_id] #include #include #include #include #include #include #include "alignments.hpp" struct _compatible { int weight ; double value ; } ; struct _snpInfo { char type ; // d, s, i int position ; char nucleotide ; int length ; } ; struct _mdComponent { char type ; int length ; int num ; } ; struct _result { int a, b ; double logLikelihood ; } ; std::map snpNameToId ; std::map alleleNameToId ; std::vector alleleIdToName ; std::vector snpInfo ; // When using this, we already convert the snp name into snp id std::vector< std::vector > snpLink ; // What are the allele ids associate with the snp id std::map > positionToSnp ; // map of the genomic coordinate to the snp id std::vector< std::vector > alleleSnpList ; // the list of snp ids associate with this allele std::vector alleleLength ; std::vector< struct _pair > alignmentCoords ; bool CompResult( struct _result a, struct _result b ) { return a.logLikelihood > b.logLikelihood ; } void Split( const char *s, char delimit, std::vector &fields ) { int i ; fields.clear() ; if ( s == NULL ) return ; std::string f ; for ( i = 0 ; s[i] ; ++i ) { if ( s[i] == delimit || s[i] == '\n' ) { fields.push_back( f ) ; f.clear() ; } else f.append( 1, s[i] ) ; } fields.push_back( f ) ; f.clear() ; } int main( int argc, char *argv[] ) { int i, j, k ; FILE *fp ; char buffer[10100] ; std::vector fields ; int binSize = 50 ; const char *backboneName = NULL ; // the compatilibity between the alignment and allele // The likelihood of this read from this allele struct _compatible **compatibility ; int **snpAllele ; // whether a snp showed up in the allele. Alignments alignments ; alignments.Open( argv[2] ) ; for ( i = 3 ; i < argc ; ++i ) { if ( !strcmp( argv[i], "-b" ) ) { backboneName = argv[i + 1] ; alignments.OnlyChrom( backboneName ) ; ++i ; } else { fprintf( stderr, "Unknown argument %s.\n", argv[i] ) ; exit( 1 ) ; } } // Parse the files associate with the snps // Firstly, read in the snp list have the information sprintf( buffer, "%s.snp", argv[1] ) ; fp = fopen( buffer, "r" ) ; k = 0 ; while ( fgets( buffer, sizeof( buffer ), fp ) ) { Split( buffer, '\t', fields ) ; if ( backboneName && strcmp( fields[2].c_str(), backboneName ) ) continue ; snpNameToId[ fields[0] ] = k ; struct _snpInfo info ; info.type = fields[1][0] ; if ( info.type == 'd' ) { info.position = atoi( fields[3].c_str() ) ; info.length = atoi( fields[4].c_str() ) ; } else if ( info.type == 'i' ) { info.position = atoi( fields[3].c_str() ) ; info.length = strlen( fields[4].c_str() ) ; } else { info.position = atoi( fields[3].c_str() ) ; // notice that the snp file is 0-based index. info.length = 1 ; info.nucleotide = fields[4][0] ; } snpInfo.push_back( info ) ; std::vector< int > tmpList ; snpLink.push_back( tmpList ) ; for ( int p = 0 ; p < info.length ; ++p ) { if ( info.type != 'i' || p == 0 ) { if ( positionToSnp.find( info.position + p ) == positionToSnp.end() ) { positionToSnp[ info.position + p] = tmpList ; } positionToSnp[ info.position + p].push_back( k ) ; } } ++k ; } fclose( fp ) ; // Read in the link file. Determine the id of alleles and the association // of alleles and snps. // TODO: obtain the length of each allele and take the length into account in the statistical model // Add the id for the backbound int backboneLength = 0 ; sprintf( buffer, "%s_backbone.fa", argv[1] ) ; fp = fopen( buffer, "r" ) ; /*for ( i = 1 ; buffer[i] && buffer[i] != ' ' && buffer[i] != '\n' ; ++i ) ; buffer[i] = '\0' ; std::string backboneName( buffer + 1 ) ; alleleNameToId[ backboneName ] = 0 ; alleleIdToName.push_back( backboneName ) ;*/ bool start = false ; while ( fgets( buffer, sizeof( buffer ), fp ) ) { if ( buffer[0] == '>' ) { for ( i = 1 ; buffer[i] && buffer[i] != ' ' && buffer[i] != '\n' ; ++i ) ; buffer[i] = '\0' ; if ( !strcmp( backboneName, buffer + 1 ) ) { start = true ; } else if ( start ) break ; } if ( start && buffer[0] != '>' ) { int len = strlen( buffer ) ; if ( buffer[len - 1 ] == '\n' ) backboneLength += len - 1 ; else backboneLength += len ; } } fclose( fp ) ; /*k = 0 ; if ( k == 0 ) { std::vector tmpList ; alleleSnpList.push_back( tmpList ) ; alleleLength.push_back( backboneLength ) ; }*/ // scanning the link file sprintf( buffer, "%s.link", argv[1] ) ; fp = fopen( buffer, "r" ) ; k = 0 ; while ( fgets( buffer, sizeof( buffer ), fp ) ) { std::vector tmpFields ; Split( buffer, '\t', tmpFields ) ; // skip the snps from other backbones if ( snpNameToId.find( tmpFields[0] ) == snpNameToId.end() ) continue ; int snpId = snpNameToId[ tmpFields[0] ] ; Split( tmpFields[1].c_str(), ' ', fields ) ; int size = fields.size() ; for ( i = 0 ; i < size ; ++i ) { if ( alleleNameToId.find( fields[i] ) == alleleNameToId.end() ) { //printf( "%s %d\n", fields[i].c_str(), k ) ; alleleNameToId[ fields[i] ] = k ; alleleIdToName.push_back( fields[i] ) ; std::vector tmpList ; alleleSnpList.push_back( tmpList ) ; alleleLength.push_back( backboneLength ) ; ++k ; } int alleleId = alleleNameToId[ fields[i] ] ; //if ( snpId == 118 ) // printf( "%s: %s %d\n", tmpFields[0].c_str(), fields[i].c_str(), alleleId ) ; snpLink[ snpId ].push_back( alleleId ) ; alleleSnpList[ alleleId ].push_back( snpId ) ; if ( snpInfo[ snpId ].type == 'd' ) { alleleLength[ alleleId ] -= snpInfo[ snpId ].length ; } else if ( snpInfo[ snpId ].type == 'i' ) { alleleLength[ alleleId ] += snpInfo[ snpId ].length ; } } } fclose( fp ) ; int numOfAllele = alleleIdToName.size() ; int numOfSnps = snpLink.size() ; snpAllele = new int* [numOfSnps] ; for ( i = 0 ; i < numOfSnps ; ++i ) { snpAllele[i] = new int[numOfAllele] ; memset( snpAllele[i], 0, sizeof( int ) * numOfAllele ) ; } for ( i = 0 ; i < numOfSnps ; ++i ) { int size = snpLink[i].size() ; for ( j = 0 ; j < size ; ++j ) { snpAllele[i][ snpLink[i][j] ] = 1 ; } } // Compute the compatbility score for each alignment and the allele // Get the number of alignment int numOfAlignments = 0 ; while ( alignments.Next() ) ++numOfAlignments ; alignments.Rewind() ; compatibility = new struct _compatible*[numOfAlignments] ; for ( i = 0 ; i < numOfAlignments ; ++i ) { compatibility[i] = new struct _compatible[ numOfAllele ] ; for ( j = 0 ; j < numOfAllele ; ++j ) { compatibility[i][j].value = 0 ;//-log( (double)alleleLength[j] ) / log( 10.0 ); } } i = 0 ; bool *snpHit = new bool[ numOfSnps ] ; while ( alignments.Next() ) { struct _pair coord = alignments.segments[0] ; alignmentCoords.push_back( coord ) ; memset( snpHit, 0, sizeof( bool ) * numOfSnps ) ; Split( alignments.GetFieldZ( "Zs" ), ',', fields ) ; int size = fields.size() ; for ( k = 0 ; k < size ; ++k ) { std::vector subfields ; Split( fields[k].c_str(), '|', subfields ) ; int snpId = snpNameToId[ subfields[2] ] ; snpHit[ snpId ] = true ; } for ( k = coord.a ; k <= coord.b ; ++k ) { int size = positionToSnp[k].size() ; for ( int l = 0 ; l < size ; ++l ) { // if this SNP is hit. Then other allele don't have this snp // will deduct its likelihood //TODO: the deduction can be based on the quality score of the read int tag = 0 ; int snpId = positionToSnp[k][l] ; if ( snpHit[ snpId ] ) { tag = 0 ; } else { // if this SNP is not hit, then every allele containing this snp // will deduct its likelihood tag = 1 ; } for ( j = 0 ; j < numOfAllele ; ++j ) { if ( snpAllele[ snpId ][j] == tag ) { int v = -2 ; //if ( snpInfo[ snpId ].type == 'd' || snpInfo[ snpId ].type == 'i' ) // v = -4 * snpInfo[ snpId ].length ; if ( snpInfo[ snpId ].type == 'd' && snpInfo[ snpId ].position < k && k != coord.a ) { // The penality has already been subtracted. v = 0 ; } compatibility[i][j].value += v ; /*if ( i == 8 && j == 78 ) { printf( "Bad snp %d: %d %d\n", tag, k, positionToSnp[k][l] ) ; }*/ } } } } ++i ; } //printf( "%d %d\n", numOfAlignments, numOfAllele ) ; // Now, let's consider every pair of alleles, and compute its log likelihood double **logLikelihood ; logLikelihood = new double *[ numOfAllele] ; for ( j = 0 ; j < numOfAllele ; ++j ) { logLikelihood[j] = new double[ numOfAllele ] ; //memset( logLikelihood[j], 0, sizeof( double ) * numOfAllele ) ; for ( k = 0 ; k < numOfAllele ; ++k ) logLikelihood[j][k] = 0 ; } int prevBin = -1 ; double assignJBin = 0 ; double assignKBin = 0 ; for ( j = 0 ; j < numOfAllele ; ++j ) { for ( k = j ; k < numOfAllele ; ++k ) { double binAdjust = 0 ; double averageRead = ( (double)numOfAlignments ) / (double)( alleleLength[j] + alleleLength[k] ) * binSize ; for ( i = 0 ; i < numOfAlignments ; ++i ) { double vj = compatibility[i][j].value ; double vk = compatibility[i][k].value ; double weightJ = 0, weightK = 0 ; if ( vj == vk ) { weightJ = weightK = 0.5 ; } else if ( vj == vk + 2 ) { if ( vj == 0 ) { weightJ = 1 ; } else { weightJ = 0.99 ; weightK = 0.01 ; } } else if ( vk == vj + 2 ) { if ( vk == 0 ) { weightK = 1 ; } else { weightJ = 0.01 ; weightK = 0.99 ; } } else { if ( vk > vj ) weightK = 1 ; else weightJ = 1 ; } double l = weightJ * compatibility[i][j].value + weightK * compatibility[i][k].value ; if ( alignmentCoords[i].a / binSize != prevBin ) { if ( prevBin != -1 && ( assignJBin > averageRead + 4 * sqrt( averageRead ) || assignKBin > averageRead + 4 * sqrt( averageRead ) ) ) { //if ( j == 8 && k == 78 ) // printf( "%lf: %lf %lf %d %d\n", averageRead, assignJBin, assignKBin, alleleLength[j], alleleLength[k] ) ; binAdjust -= 4 ; } prevBin = alignmentCoords[i].a / binSize ; assignJBin = 0 ; assignKBin = 0 ; } assignJBin += weightJ ; assignKBin += weightK ; /*if ( j == 8 && k == 78 && l < 0 ) { printf( "Bad alignment %d (%s %s). %lf %lf: %lf\n", i, alleleIdToName[j].c_str(), alleleIdToName[k].c_str(), compatibility[i][j].value, compatibility[i][k].value, l ) ; }*/ logLikelihood[j][k] += l ; } logLikelihood[j][k] += ( -log( (double)alleleLength[j] ) / log(10.0 ) - log( (double)alleleLength[k] ) / log(10.0) ) ; logLikelihood[j][k] += binAdjust ; } } // Find the result double max ; int maxj = -1 ; int maxk = -1 ; std::vector< struct _result > results ; for ( j = 0 ; j < numOfAllele ; ++j ) { for ( k = j ; k < numOfAllele ; ++k ) { if ( maxj == -1 || logLikelihood[j][k] > max ) { maxj = j ; maxk = k ; max = logLikelihood[j][k] ; } struct _result r ; r.a = j ; r.b = k ; r.logLikelihood = logLikelihood[j][k] ; results.push_back( r ) ; } } //printf( "%s %s %lf\n", alleleIdToName[ maxj ].c_str(), alleleIdToName[ maxk ].c_str(), max) ; //printf( "%lf\n", logLikelihood[124][128] ) ; if ( results.size() == 0 ) { printf( "-1 -1 -1\n" ) ; exit( 1 ) ; } std::sort( results.begin(), results.end(), CompResult ) ; i = 0 ; printf( "%s %s %lf\n", alleleIdToName[ results[i].a ].c_str(), alleleIdToName[ results[i].b ].c_str(), results[i].logLikelihood ) ; k = results.size() ; for ( i = 1 ; i < k ; ++i ) { if ( results[i].logLikelihood != results[0].logLikelihood ) break ; printf( "%s %s %lf\n", alleleIdToName[ results[i].a ].c_str(), alleleIdToName[ results[i].b ].c_str(), results[i].logLikelihood ) ; } return 0 ; } hisat2-2.2.1/limit.cpp000066400000000000000000000035461370663763200145550ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include "limit.h" uint8_t MIN_U8 = std::numeric_limits::min(); uint8_t MAX_U8 = std::numeric_limits::max(); uint16_t MIN_U16 = std::numeric_limits::min(); uint16_t MAX_U16 = std::numeric_limits::max(); uint32_t MIN_U32 = std::numeric_limits::min(); uint32_t MAX_U32 = std::numeric_limits::max(); uint64_t MIN_U64 = std::numeric_limits::min(); uint64_t MAX_U64 = std::numeric_limits::max(); size_t MIN_SIZE_T = std::numeric_limits::min(); size_t MAX_SIZE_T = std::numeric_limits::max(); int MIN_I = std::numeric_limits::min(); int MAX_I = std::numeric_limits::max(); int8_t MIN_I8 = std::numeric_limits::min(); int8_t MAX_I8 = std::numeric_limits::max(); int16_t MIN_I16 = std::numeric_limits::min(); int16_t MAX_I16 = std::numeric_limits::max(); int32_t MIN_I32 = std::numeric_limits::min(); int32_t MAX_I32 = std::numeric_limits::max(); int64_t MIN_I64 = std::numeric_limits::min(); int64_t MAX_I64 = std::numeric_limits::max(); hisat2-2.2.1/limit.h000066400000000000000000000024331370663763200142140ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef LIMIT_H_ #define LIMIT_H_ #include #include extern uint8_t MIN_U8; extern uint8_t MAX_U8; extern uint16_t MIN_U16; extern uint16_t MAX_U16; extern uint32_t MIN_U32; extern uint32_t MAX_U32; extern uint64_t MIN_U64; extern uint64_t MAX_U64; extern size_t MIN_SIZE_T; extern size_t MAX_SIZE_T; extern int MIN_I; extern int MAX_I; extern int8_t MIN_I8; extern int8_t MAX_I8; extern int16_t MIN_I16; extern int16_t MAX_I16; extern int32_t MIN_I32; extern int32_t MAX_I32; extern int64_t MIN_I64; extern int64_t MAX_I64; #endif hisat2-2.2.1/ls.cpp000066400000000000000000000070711370663763200140520ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifdef MAIN_LS #include #include #include "sstring.h" #include "ls.h" #include "ds.h" using namespace std; int main(void) { cerr << "Test LarssonSadakana for int..."; { typedef int T; const char *t = "banana"; EList sa; EList isa; for(size_t i = 0; i < strlen(t); i++) { isa.push_back(t[i]); } isa.push_back(0); // disregarded sa.resize(isa.size()); LarssonSadakane ls; ls.suffixsort(isa.ptr(), sa.ptr(), (T)sa.size()-1, 'z', 0); assert_eq((T)'a', t[sa[1]]); assert_eq(5, sa[1]); assert_eq((T)'a', t[sa[2]]); assert_eq(3, sa[2]); assert_eq((T)'a', t[sa[3]]); assert_eq(1, sa[3]); assert_eq((T)'b', t[sa[4]]); assert_eq(0, sa[4]); assert_eq((T)'n', t[sa[5]]); assert_eq(4, sa[5]); assert_eq((T)'n', t[sa[6]]); assert_eq(2, sa[6]); } cerr << "PASSED" << endl; cerr << "Test LarssonSadakana for uint32_t..."; { typedef uint32_t T; const char *t = "banana"; EList sa; EList isa; for(size_t i = 0; i < strlen(t); i++) { isa.push_back(t[i]); } isa.push_back(0); // disregarded sa.resize(isa.size()); LarssonSadakane ls; ls.suffixsort( (int*)isa.ptr(), (int*)sa.ptr(), (int)sa.size()-1, 'z', 0); assert_eq((T)'a', t[sa[1]]); assert_eq(5, sa[1]); assert_eq((T)'a', t[sa[2]]); assert_eq(3, sa[2]); assert_eq((T)'a', t[sa[3]]); assert_eq(1, sa[3]); assert_eq((T)'b', t[sa[4]]); assert_eq(0, sa[4]); assert_eq((T)'n', t[sa[5]]); assert_eq(4, sa[5]); assert_eq((T)'n', t[sa[6]]); assert_eq(2, sa[6]); } cerr << "PASSED" << endl; cerr << "Last elt is < or > others ..."; { { typedef int T; const char *t = "aaa"; EList sa; EList isa; for(size_t i = 0; i < strlen(t); i++) { isa.push_back(t[i]); } isa.push_back(0); // disregarded sa.resize(isa.size()); LarssonSadakane ls; ls.suffixsort(isa.ptr(), sa.ptr(), (T)sa.size()-1, 'z', 0); assert_eq(3, sa[0]); assert_eq(2, sa[1]); assert_eq(1, sa[2]); assert_eq(0, sa[3]); } { typedef int T; const char *t = "aaa"; EList sa; EList isa; for(size_t i = 0; i < strlen(t); i++) { isa.push_back(t[i]); } isa.push_back('y'); // doesn't matter if this is > others sa.resize(isa.size()); LarssonSadakane ls; ls.suffixsort(isa.ptr(), sa.ptr(), (T)sa.size()-1, 'z', 0); assert_eq(3, sa[0]); assert_eq(2, sa[1]); assert_eq(1, sa[2]); assert_eq(0, sa[3]); } { typedef int T; const char *t = "aaa"; EList sa; EList isa; for(size_t i = 0; i < strlen(t); i++) { isa.push_back(t[i]); } isa.push_back('y'); // breaks ties isa.push_back(0); // disregarded sa.resize(isa.size()); LarssonSadakane ls; ls.suffixsort(isa.ptr(), sa.ptr(), (T)sa.size()-1, 'z', 0); assert_eq(4, sa[0]); assert_eq(0, sa[1]); assert_eq(1, sa[2]); assert_eq(2, sa[3]); assert_eq(3, sa[4]); } } cerr << "PASSED" << endl; } #endif hisat2-2.2.1/ls.h000066400000000000000000000272621370663763200135230ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /* Code in this file is ultimately based on: qsufsort.c Copyright 1999, N. Jesper Larsson, all rights reserved. This file contains an implementation of the algorithm presented in "Faster Suffix Sorting" by N. Jesper Larsson (jesper@cs.lth.se) and Kunihiko Sadakane (sada@is.s.u-tokyo.ac.jp). This software may be used freely for any purpose. However, when distributed, the original source must be clearly stated, and, when the source code is distributed, the copyright notice must be retained and any alterations in the code must be clearly marked. No warranty is given regarding the quality of this software.*/ #ifndef LS_H_ #define LS_H_ #include #include #include template class LarssonSadakane { T *I, /* group array, ultimately suffix array.*/ *V, /* inverse array, ultimately inverse of I.*/ r, /* number of symbols aggregated by transform.*/ h; /* length of already-sorted prefixes.*/ #define LS_KEY(p) (V[*(p)+(h)]) #define LS_SWAP(p, q) (tmp=*(p), *(p)=*(q), *(q)=tmp) #define LS_SMED3(a, b, c) (LS_KEY(a)LS_KEY(c) ? (b) : LS_KEY(a)>LS_KEY(c) ? (c) : (a))) /* Subroutine for select_sort_split and sort_split. Sets group numbers for a group whose lowest position in I is pl and highest position is pm.*/ inline void update_group(T *pl, T *pm) { T g; g=(T)(pm-I); /* group number.*/ V[*pl]=g; /* update group number of first position.*/ if (pl==pm) *pl=-1; /* one element, sorted group.*/ else do /* more than one element, unsorted group.*/ V[*++pl]=g; /* update group numbers.*/ while (pl>1); /* small arrays, middle element.*/ if (n>7) { pl=p; pn=p+n-1; if (n>40) { /* big arrays, pseudomedian of 9.*/ s=n>>3; pl=LS_SMED3(pl, pl+s, pl+s+s); pm=LS_SMED3(pm-s, pm, pm+s); pn=LS_SMED3(pn-s-s, pn-s, pn); } pm=LS_SMED3(pl, pm, pn); /* midsize arrays, median of 3.*/ } return LS_KEY(pm); } /* Sorting routine called for each unsorted group. Sorts the array of integers (suffix numbers) of length n starting at p. The algorithm is a ternary-split quicksort taken from Bentley & McIlroy, "Engineering a Sort Function", Software -- Practice and Experience 23(11), 1249-1265 (November 1993). This function is based on Program 7.*/ inline void sort_split(T *p, T n) { T *pa, *pb, *pc, *pd, *pl, *pm, *pn; T f, v, s, t, tmp; if (n<7) { /* multi-selection sort smallest arrays.*/ select_sort_split(p, n); return; } v=choose_pivot(p, n); pa=pb=p; pc=pd=p+n-1; while (1) { /* split-end partition.*/ while (pb<=pc && (f=LS_KEY(pb))<=v) { if (f==v) { LS_SWAP(pa, pb); ++pa; } ++pb; } while (pc>=pb && (f=LS_KEY(pc))>=v) { if (f==v) { LS_SWAP(pc, pd); --pd; } --pc; } if (pb>pc) break; LS_SWAP(pb, pc); ++pb; --pc; } pn=p+n; if ((s=(T)(pa-p))>(t=(T)(pb-pa))) s=t; for (pl=p, pm=pb-s; s; --s, ++pl, ++pm) LS_SWAP(pl, pm); if ((s=(T)(pd-pc))>(t=(T)(pn-pd-1))) s=t; for (pl=pb, pm=pn-s; s; --s, ++pl, ++pm) LS_SWAP(pl, pm); s=(T)(pb-pa); t=(T)(pd-pc); if (s>0) sort_split(p, s); update_group(p+s, p+n-t-1); if (t>0) sort_split(p+n-t, t); } /* Bucketsort for first iteration. Input: x[0...n-1] holds integers in the range 1...k-1, all of which appear at least once. x[n] is 0. (This is the corresponding output of transform.) k must be at most n+1. p is array of size n+1 whose contents are disregarded. Output: x is V and p is I after the initial sorting stage of the refined suffix sorting algorithm.*/ inline void bucketsort(T *x, T *p, T n, T k) { T *pi, i, c, d, g; for (pi=p; pi=p; --pi) { d=x[c=*pi]; /* c is position, d is next in list.*/ x[c]=g=i; /* last position equals group number.*/ if (d == 0 || d > 0) { /* if more than one element in group.*/ p[i--]=c; /* p is permutation for the sorted x.*/ do { d=x[c=d]; /* next in linked list.*/ x[c]=g; /* group number in x.*/ p[i--]=c; /* permutation in p.*/ } while (d == 0 || d > 0); } else p[i--]=-1; /* one element, sorted group.*/ } } /* Transforms the alphabet of x by attempting to aggregate several symbols into one, while preserving the suffix order of x. The alphabet may also be compacted, so that x on output comprises all integers of the new alphabet with no skipped numbers. Input: x is an array of size n+1 whose first n elements are positive integers in the range l...k-1. p is array of size n+1, used for temporary storage. q controls aggregation and compaction by defining the maximum value for any symbol during transformation: q must be at least k-l; if q<=n, compaction is guaranteed; if k-l>n, compaction is never done; if q is INT_MAX, the maximum number of symbols are aggregated into one. Output: Returns an integer j in the range 1...q representing the size of the new alphabet. If j<=n+1, the alphabet is compacted. The global variable r is set to the number of old symbols grouped into one. Only x[n] is 0.*/ inline T transform(T *x, T *p, T n, T k, T l, T q) { T b, c, d, e, i, j, m, s; T *pi, *pj; for (s=0, i=k-l; i; i>>=1) ++s; /* s is number of bits in old symbol.*/ e=std::numeric_limits::max()>>s; /* e is for overflow checking.*/ for (b=d=r=0; r=k-l) { /* if bucketing possible,*/ j=transform(V, I, n, k, l, n); bucketsort(V, I, n, j); /* bucketsort on first r positions.*/ } else { transform(V, I, n, k, l, std::numeric_limits::max()); for (i=0; i<=n; ++i) I[i]=i; /* initialize I with suffix numbers.*/ h=0; sort_split(I, n+1); /* quicksort on first r positions.*/ } h=r; /* number of symbols aggregated by transform.*/ while (*I>=-n) { pi=I; /* pi is first position of group.*/ sl=0; /* sl is negated length of sorted groups.*/ do { if ((s=*pi) <= 0 && (s=*pi) != 0) { pi-=s; /* skip over sorted group.*/ sl+=s; /* add negated length to sl.*/ } else { if (sl) { *(pi+sl)=sl; /* combine sorted groups before pi.*/ sl=0; } pk=I+V[s]+1; /* pk-1 is last position of unsorted group.*/ sort_split(pi, (T)(pk-pi)); pi=pk; /* next group.*/ } } while (pi<=I+n); if (sl) /* if the array ends with a sorted group.*/ *(pi+sl)=sl; /* combine sorted groups at end of I.*/ h=2*h; /* double sorted-depth.*/ } for (i=0; i<=n; ++i) /* reconstruct suffix array from inverse.*/ I[V[i]]=i; } }; #endif /*def LS_H_*/ hisat2-2.2.1/mask.cpp000066400000000000000000000020331370663763200143600ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "mask.h" // 5-bit pop count int alts5[32] = { 0, 1, 1, 2, 1, 2, 2, 3, 1, 2, 2, 3, 2, 3, 3, 4, 1, 2, 2, 3, 2, 3, 3, 4, 2, 3, 3, 4, 3, 4, 4, 5 }; // Index of lowest set bit int firsts5[32] = { -1, 0, 1, 0, 2, 0, 1, 0, 3, 0, 1, 0, 2, 0, 1, 0, 4, 0, 1, 0, 2, 0, 1, 0, 3, 0, 1, 0, 2, 0, 1, 0 }; hisat2-2.2.1/mask.h000066400000000000000000000041721370663763200140330ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef MASK_H_ #define MASK_H_ #include #include "random_source.h" // 5-bit pop count extern int alts5[32]; // Index of lowest set bit extern int firsts5[32]; /** * Return 1 if a 2-bit-encoded base ('i') matches any bit in the mask ('j') and * the mask < 16. Returns -1 if either the reference or the read character was * ambiguous. Returns 0 if the characters unambiguously mismatch. */ static inline int matchesEx(int i, int j) { if(j >= 16 || i > 3) { // read and/or ref was ambiguous return -1; } return (((1 << i) & j) != 0) ? 1 : 0; } /** * Return 1 if a 2-bit-encoded base ('i') matches any bit in the mask ('j'). */ static inline bool matches(int i, int j) { return ((1 << i) & j) != 0; } /** * Given a mask with up to 5 bits, return an index corresponding to a * set bit in the mask, randomly chosen from among all set bits. */ static inline int randFromMask(RandomSource& rnd, int mask) { assert_gt(mask, 0); if(alts5[mask] == 1) { // only one to pick from, pick it via lookup table return firsts5[mask]; } assert_gt(mask, 0); assert_lt(mask, 32); int r = rnd.nextU32() % alts5[mask]; assert_geq(r, 0); assert_lt(r, alts5[mask]); // could do the following via lookup table too for(int i = 0; i < 5; i++) { if((mask & (1 << i)) != 0) { if(r == 0) return i; r--; } } std::cerr << "Shouldn't get here" << std::endl; throw 1; return -1; } #endif /*ndef MASK_H_*/ hisat2-2.2.1/mem_ids.h000066400000000000000000000023671370663763200145210ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ // For holding index data #define EBWT_CAT ((int) 1) // For holding index-building data #define EBWTB_CAT ((int) 2) // For holding cache data #define CA_CAT ((int) 3) // For holding group-walk-left bookkeeping data #define GW_CAT ((int) 4) // For holding alignment bookkeeping data #define AL_CAT ((int) 5) // For holding dynamic programming bookkeeping data #define DP_CAT ((int) 6) // For holding alignment results and other hit objects #define RES_CAT ((int) 7) #define MISC_CAT ((int) 9) #define DEBUG_CAT ((int)10) hisat2-2.2.1/mm.h000066400000000000000000000030151370663763200135040ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef MM_H_ #define MM_H_ /** * mm.h: * * Defines that make it easier to handle files in the two different MM * contexts: i.e. on Linux and Mac where MM is supported and POSIX I/O * functions work as expected, and on Windows where MM is not supported * and where there isn't POSIX I/O, */ #if 0 #ifdef BOWTIE_MM #define MM_FILE_CLOSE(x) if(x > 3) { close(x); } #define MM_READ_RET ssize_t // #define MM_READ read #define MM_SEEK lseek #define MM_FILE int #define MM_FILE_INIT -1 #else #define MM_FILE_CLOSE(x) if(x != NULL) { fclose(x); } #define MM_READ_RET size_t #define MM_SEEK fseek #define MM_FILE FILE* #define MM_FILE_INIT NULL #endif #endif #define MM_READ(file, dest, sz) fread(dest, 1, sz, file) #define MM_IS_IO_ERR(file_hd, ret, count) is_fread_err(file_hd, ret, count) #endif /* MM_H_ */ hisat2-2.2.1/msvcc/000077500000000000000000000000001370663763200140365ustar00rootroot00000000000000hisat2-2.2.1/msvcc/CodeStubs.vcxproj.filters000066400000000000000000000031011370663763200210100ustar00rootroot00000000000000 {4FC737F1-C7A5-4376-A066-2A32D752A2FF} cpp;c;cc;cxx;def;odl;idl;hpj;bat;asm;asmx {93995380-89BD-4b04-88EB-625FBE52EBFB} h;hpp;hxx;hm;inl;inc;xsd {67DA6AB6-F800-4c08-8B7A-83BB121AAD01} rc;ico;cur;bmp;dlg;rc2;rct;bin;rgs;gif;jpg;jpeg;jpe;resx;tiff;tif;png;wav;mfcribbon-ms Source Files Source Files Source Files Header Files Header Files Header Files Header Files hisat2-2.2.1/msvcc/CoreDefs.props000066400000000000000000000012141370663763200166130ustar00rootroot00000000000000 typeof=decltype;POPCNT_CAPABILITY;ssize_t=SSIZE_T;NOMINMAX;_CRT_SECURE_NO_WARNINGS;_LARGEFILE_SOURCE;_FILE_OFFSET_BITS=64;_GNU_SOURCE;_MBCS;%(PreprocessorDefinitions) codeStubs hisat2-2.2.1/msvcc/SetVersion.vbs000066400000000000000000000007101370663763200166510ustar00rootroot00000000000000 ' ask the scripting runtime environemt for access to files set FS = CreateObject("Scripting.FileSystemObject") set FileHandler = FS.GetFile("../VERSION") set inputTextStream = FileHandler.OpenAsTextStream(1) version = inputTextStream.ReadLine WScript.Echo "Version: " & version set outputTextStream = FS.CreateTextFile("../version.h",true) outputTextStream.WriteLine "#define HISAT2_VERSION """ & version & """" outputTextStream.close hisat2-2.2.1/msvcc/SysDefs.props000066400000000000000000000014041370663763200165020ustar00rootroot00000000000000 BUILD_TIME="$([System.DateTime]::Now)";BUILD_HOST="$(VisualStudioEdition) OS ver $(OSVersion)";COMPILER_VERSION="MSC $(MSBuildAssemblyVersion)";COMPILER_OPTIONS="$(NuGetRuntimeIdentifier) Runtime vers $(MSBuildRuntimeVersion)" "version.h" hisat2-2.2.1/msvcc/codeStubs.vcxproj000066400000000000000000000115541370663763200174140ustar00rootroot00000000000000 Debug x64 Release x64 {08C70FF5-9E0C-42DF-B7BF-C1AF1B0D2CFF} win32stubs 8.1 StaticLibrary true v140 MultiByte StaticLibrary false v140 true MultiByte $(Platform)\$(ProjectName)\$(Configuration)\ $(Platform)\$(ProjectName)\$(Configuration)\ Level3 Disabled true _CRT_SECURE_NO_WARNINGS true cscript.exe SetVersion.vbs ../version.h; ../VERSION; Setting version info Level3 MaxSpeed true true true _CRT_SECURE_NO_WARNINGS;NDEBUG MultiThreaded true true true cscript.exe SetVersion.vbs ../version.h; ../VERSION; Setting version info hisat2-2.2.1/msvcc/codeStubs/000077500000000000000000000000001370663763200157715ustar00rootroot00000000000000hisat2-2.2.1/msvcc/codeStubs/getopt.c000066400000000000000000000732121370663763200174440ustar00rootroot00000000000000/* Getopt for GNU. NOTE: getopt is now part of the C library, so if you don't know what "Keep this file name-space clean" means, talk to drepper@gnu.org before changing it! Copyright (C) 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 2005 Free Software Foundation, Inc. NOTE: This source is derived from an old version taken from the GNU C Library (glibc). This program is free software; you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2, or (at your option) any later version. This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. You should have received a copy of the GNU General Public License along with this program; if not, write to the Free Software Foundation, Inc., 51 Franklin Street - Fifth Floor, Boston, MA 02110-1301, USA. */ /* This tells Alpha OSF/1 not to define a getopt prototype in . Ditto for AIX 3.2 and . */ #ifndef _NO_PROTO # define _NO_PROTO #endif #ifdef HAVE_CONFIG_H #include #endif #if !defined __STDC__ || !__STDC__ /* This is a separate conditional since some stdc systems reject `defined (const)'. */ # ifndef const # define const # endif #endif #ifdef _WIN32 #include #define ATTRIBUTE_UNUSED #else #include "ansidecl.h" #endif #include /* Comment out all this code if we are using the GNU C Library, and are not actually compiling the library itself. This code is part of the GNU C Library, but also included in many other GNU distributions. Compiling and linking in this code is a waste when using the GNU C library (especially if it is a shared library). Rather than having every GNU program understand `configure --with-gnu-libc' and omit the object files, it is simpler to just do this in the source for each such file. */ #define GETOPT_INTERFACE_VERSION 2 #if !defined _LIBC && defined __GLIBC__ && __GLIBC__ >= 2 # include # if _GNU_GETOPT_INTERFACE_VERSION == GETOPT_INTERFACE_VERSION # define ELIDE_CODE # endif #endif #ifndef ELIDE_CODE /* This needs to come after some library #include to get __GNU_LIBRARY__ defined. */ #ifdef __GNU_LIBRARY__ /* Don't include stdlib.h for non-GNU C libraries because some of them contain conflicting prototypes for getopt. */ # include # include #endif /* GNU C library. */ #ifdef VMS # include # if HAVE_STRING_H - 0 # include # endif #endif #ifndef _ /* This is for other GNU distributions with internationalized messages. When compiling libc, the _ macro is predefined. */ # if (HAVE_LIBINTL_H && ENABLE_NLS) || defined _LIBC # include # define _(msgid) gettext (msgid) # else # define _(msgid) (msgid) # endif #endif /* This version of `getopt' appears to the caller like standard Unix `getopt' but it behaves differently for the user, since it allows the user to intersperse the options with the other arguments. As `getopt' works, it permutes the elements of ARGV so that, when it is done, all the options precede everything else. Thus all application programs are extended to handle flexible argument order. Setting the environment variable POSIXLY_CORRECT disables permutation. Then the behavior is completely standard. GNU application programs can use a third alternative mode in which they can distinguish the relative order of options and other arguments. */ #include "getopt.h" /* For communication from `getopt' to the caller. When `getopt' finds an option that takes an argument, the argument value is returned here. Also, when `ordering' is RETURN_IN_ORDER, each non-option ARGV-element is returned here. */ char *optarg = NULL; /* Index in ARGV of the next element to be scanned. This is used for communication to and from the caller and for communication between successive calls to `getopt'. On entry to `getopt', zero means this is the first call; initialize. When `getopt' returns -1, this is the index of the first of the non-option elements that the caller should itself scan. Otherwise, `optind' communicates from one call to the next how much of ARGV has been scanned so far. */ /* 1003.2 says this must be 1 before any call. */ int optind = 1; /* Formerly, initialization of getopt depended on optind==0, which causes problems with re-calling getopt as programs generally don't know that. */ int __getopt_initialized = 0; /* The next char to be scanned in the option-element in which the last option character we returned was found. This allows us to pick up the scan where we left off. If this is zero, or a null string, it means resume the scan by advancing to the next ARGV-element. */ static char *nextchar; /* Callers store zero here to inhibit the error message for unrecognized options. */ int opterr = 1; /* Set to an option character which was unrecognized. This must be initialized on some systems to avoid linking in the system's own getopt implementation. */ int optopt = '?'; /* Describe how to deal with options that follow non-option ARGV-elements. If the caller did not specify anything, the default is REQUIRE_ORDER if the environment variable POSIXLY_CORRECT is defined, PERMUTE otherwise. REQUIRE_ORDER means don't recognize them as options; stop option processing when the first non-option is seen. This is what Unix does. This mode of operation is selected by either setting the environment variable POSIXLY_CORRECT, or using `+' as the first character of the list of option characters. PERMUTE is the default. We permute the contents of ARGV as we scan, so that eventually all the non-options are at the end. This allows options to be given in any order, even with programs that were not written to expect this. RETURN_IN_ORDER is an option available to programs that were written to expect options and other ARGV-elements in any order and that care about the ordering of the two. We describe each non-option ARGV-element as if it were the argument of an option with character code 1. Using `-' as the first character of the list of option characters selects this mode of operation. The special argument `--' forces an end of option-scanning regardless of the value of `ordering'. In the case of RETURN_IN_ORDER, only `--' can cause `getopt' to return -1 with `optind' != ARGC. */ static enum { REQUIRE_ORDER, PERMUTE, RETURN_IN_ORDER } ordering; /* Value of POSIXLY_CORRECT environment variable. */ static char *posixly_correct; #ifdef __GNU_LIBRARY__ /* We want to avoid inclusion of string.h with non-GNU libraries because there are many ways it can cause trouble. On some systems, it contains special magic macros that don't work in GCC. */ # include # define my_index strchr #else # if HAVE_STRING_H # include # else # if HAVE_STRINGS_H # include # endif # endif /* Avoid depending on library functions or files whose names are inconsistent. */ #if HAVE_STDLIB_H && HAVE_DECL_GETENV # include #elif !defined(getenv) # ifdef __cplusplus extern "C" { # endif /* __cplusplus */ extern char *getenv (const char *); # ifdef __cplusplus } # endif /* __cplusplus */ #endif static char * my_index (const char *str, int chr) { while (*str) { if (*str == chr) return (char *) str; str++; } return 0; } /* If using GCC, we can safely declare strlen this way. If not using GCC, it is ok not to declare it. */ #ifdef __GNUC__ /* Note that Motorola Delta 68k R3V7 comes with GCC but not stddef.h. That was relevant to code that was here before. */ # if (!defined __STDC__ || !__STDC__) && !defined strlen /* gcc with -traditional declares the built-in strlen to return int, and has done so at least since version 2.4.5. -- rms. */ extern int strlen (const char *); # endif /* not __STDC__ */ #endif /* __GNUC__ */ #endif /* not __GNU_LIBRARY__ */ /* Handle permutation of arguments. */ /* Describe the part of ARGV that contains non-options that have been skipped. `first_nonopt' is the index in ARGV of the first of them; `last_nonopt' is the index after the last of them. */ static int first_nonopt; static int last_nonopt; #ifdef _LIBC /* Bash 2.0 gives us an environment variable containing flags indicating ARGV elements that should not be considered arguments. */ /* Defined in getopt_init.c */ extern char *__getopt_nonoption_flags; static int nonoption_flags_max_len; static int nonoption_flags_len; static int original_argc; static char *const *original_argv; /* Make sure the environment variable bash 2.0 puts in the environment is valid for the getopt call we must make sure that the ARGV passed to getopt is that one passed to the process. */ static void __attribute__ ((unused)) store_args_and_env (int argc, char *const *argv) { /* XXX This is no good solution. We should rather copy the args so that we can compare them later. But we must not use malloc(3). */ original_argc = argc; original_argv = argv; } # ifdef text_set_element text_set_element (__libc_subinit, store_args_and_env); # endif /* text_set_element */ # define SWAP_FLAGS(ch1, ch2) \ if (nonoption_flags_len > 0) \ { \ char __tmp = __getopt_nonoption_flags[ch1]; \ __getopt_nonoption_flags[ch1] = __getopt_nonoption_flags[ch2]; \ __getopt_nonoption_flags[ch2] = __tmp; \ } #else /* !_LIBC */ # define SWAP_FLAGS(ch1, ch2) #endif /* _LIBC */ /* Exchange two adjacent subsequences of ARGV. One subsequence is elements [first_nonopt,last_nonopt) which contains all the non-options that have been skipped so far. The other is elements [last_nonopt,optind), which contains all the options processed since those non-options were skipped. `first_nonopt' and `last_nonopt' are relocated so that they describe the new indices of the non-options in ARGV after they are moved. */ #if defined __STDC__ && __STDC__ static void exchange (char **); #endif static void exchange (char **argv) { int bottom = first_nonopt; int middle = last_nonopt; int top = optind; char *tem; /* Exchange the shorter segment with the far end of the longer segment. That puts the shorter segment into the right place. It leaves the longer segment in the right place overall, but it consists of two parts that need to be swapped next. */ #ifdef _LIBC /* First make sure the handling of the `__getopt_nonoption_flags' string can work normally. Our top argument must be in the range of the string. */ if (nonoption_flags_len > 0 && top >= nonoption_flags_max_len) { /* We must extend the array. The user plays games with us and presents new arguments. */ char *new_str = (char *) malloc (top + 1); if (new_str == NULL) nonoption_flags_len = nonoption_flags_max_len = 0; else { memset (mempcpy (new_str, __getopt_nonoption_flags, nonoption_flags_max_len), '\0', top + 1 - nonoption_flags_max_len); nonoption_flags_max_len = top + 1; __getopt_nonoption_flags = new_str; } } #endif while (top > middle && middle > bottom) { if (top - middle > middle - bottom) { /* Bottom segment is the short one. */ int len = middle - bottom; register int i; /* Swap it with the top part of the top segment. */ for (i = 0; i < len; i++) { tem = argv[bottom + i]; argv[bottom + i] = argv[top - (middle - bottom) + i]; argv[top - (middle - bottom) + i] = tem; SWAP_FLAGS (bottom + i, top - (middle - bottom) + i); } /* Exclude the moved bottom segment from further swapping. */ top -= len; } else { /* Top segment is the short one. */ int len = top - middle; register int i; /* Swap it with the bottom part of the bottom segment. */ for (i = 0; i < len; i++) { tem = argv[bottom + i]; argv[bottom + i] = argv[middle + i]; argv[middle + i] = tem; SWAP_FLAGS (bottom + i, middle + i); } /* Exclude the moved top segment from further swapping. */ bottom += len; } } /* Update records for the slots the non-options now occupy. */ first_nonopt += (optind - last_nonopt); last_nonopt = optind; } /* Initialize the internal data when the first call is made. */ #if defined __STDC__ && __STDC__ static const char *_getopt_initialize (int, char *const *, const char *); #endif static const char * _getopt_initialize (int argc ATTRIBUTE_UNUSED, char *const *argv ATTRIBUTE_UNUSED, const char *optstring) { /* Start processing options with ARGV-element 1 (since ARGV-element 0 is the program name); the sequence of previously skipped non-option ARGV-elements is empty. */ first_nonopt = last_nonopt = optind; nextchar = NULL; posixly_correct = getenv ("POSIXLY_CORRECT"); /* Determine how to handle the ordering of options and nonoptions. */ if (optstring[0] == '-') { ordering = RETURN_IN_ORDER; ++optstring; } else if (optstring[0] == '+') { ordering = REQUIRE_ORDER; ++optstring; } else if (posixly_correct != NULL) ordering = REQUIRE_ORDER; else ordering = PERMUTE; #ifdef _LIBC if (posixly_correct == NULL && argc == original_argc && argv == original_argv) { if (nonoption_flags_max_len == 0) { if (__getopt_nonoption_flags == NULL || __getopt_nonoption_flags[0] == '\0') nonoption_flags_max_len = -1; else { const char *orig_str = __getopt_nonoption_flags; int len = nonoption_flags_max_len = strlen (orig_str); if (nonoption_flags_max_len < argc) nonoption_flags_max_len = argc; __getopt_nonoption_flags = (char *) malloc (nonoption_flags_max_len); if (__getopt_nonoption_flags == NULL) nonoption_flags_max_len = -1; else memset (mempcpy (__getopt_nonoption_flags, orig_str, len), '\0', nonoption_flags_max_len - len); } } nonoption_flags_len = nonoption_flags_max_len; } else nonoption_flags_len = 0; #endif return optstring; } /* Scan elements of ARGV (whose length is ARGC) for option characters given in OPTSTRING. If an element of ARGV starts with '-', and is not exactly "-" or "--", then it is an option element. The characters of this element (aside from the initial '-') are option characters. If `getopt' is called repeatedly, it returns successively each of the option characters from each of the option elements. If `getopt' finds another option character, it returns that character, updating `optind' and `nextchar' so that the next call to `getopt' can resume the scan with the following option character or ARGV-element. If there are no more option characters, `getopt' returns -1. Then `optind' is the index in ARGV of the first ARGV-element that is not an option. (The ARGV-elements have been permuted so that those that are not options now come last.) OPTSTRING is a string containing the legitimate option characters. If an option character is seen that is not listed in OPTSTRING, return '?' after printing an error message. If you set `opterr' to zero, the error message is suppressed but we still return '?'. If a char in OPTSTRING is followed by a colon, that means it wants an arg, so the following text in the same ARGV-element, or the text of the following ARGV-element, is returned in `optarg'. Two colons mean an option that wants an optional arg; if there is text in the current ARGV-element, it is returned in `optarg', otherwise `optarg' is set to zero. If OPTSTRING starts with `-' or `+', it requests different methods of handling the non-option ARGV-elements. See the comments about RETURN_IN_ORDER and REQUIRE_ORDER, above. Long-named options begin with `--' instead of `-'. Their names may be abbreviated as long as the abbreviation is unique or is an exact match for some defined option. If they have an argument, it follows the option name in the same ARGV-element, separated from the option name by a `=', or else the in next ARGV-element. When `getopt' finds a long-named option, it returns 0 if that option's `flag' field is nonzero, the value of the option's `val' field if the `flag' field is zero. The elements of ARGV aren't really const, because we permute them. But we pretend they're const in the prototype to be compatible with other systems. LONGOPTS is a vector of `struct option' terminated by an element containing a name which is zero. LONGIND returns the index in LONGOPT of the long-named option found. It is only valid when a long-named option has been found by the most recent call. If LONG_ONLY is nonzero, '-' as well as '--' can introduce long-named options. */ int _getopt_internal (int argc, char *const *argv, const char *optstring, const struct option *longopts, int *longind, int long_only) { optarg = NULL; if (optind == 0 || !__getopt_initialized) { if (optind == 0) optind = 1; /* Don't scan ARGV[0], the program name. */ optstring = _getopt_initialize (argc, argv, optstring); __getopt_initialized = 1; } /* Test whether ARGV[optind] points to a non-option argument. Either it does not have option syntax, or there is an environment flag from the shell indicating it is not an option. The later information is only used when the used in the GNU libc. */ #ifdef _LIBC # define NONOPTION_P (argv[optind][0] != '-' || argv[optind][1] == '\0' \ || (optind < nonoption_flags_len \ && __getopt_nonoption_flags[optind] == '1')) #else # define NONOPTION_P (argv[optind][0] != '-' || argv[optind][1] == '\0') #endif if (nextchar == NULL || *nextchar == '\0') { /* Advance to the next ARGV-element. */ /* Give FIRST_NONOPT & LAST_NONOPT rational values if OPTIND has been moved back by the user (who may also have changed the arguments). */ if (last_nonopt > optind) last_nonopt = optind; if (first_nonopt > optind) first_nonopt = optind; if (ordering == PERMUTE) { /* If we have just processed some options following some non-options, exchange them so that the options come first. */ if (first_nonopt != last_nonopt && last_nonopt != optind) exchange ((char **) argv); else if (last_nonopt != optind) first_nonopt = optind; /* Skip any additional non-options and extend the range of non-options previously skipped. */ while (optind < argc && NONOPTION_P) optind++; last_nonopt = optind; } /* The special ARGV-element `--' means premature end of options. Skip it like a null option, then exchange with previous non-options as if it were an option, then skip everything else like a non-option. */ if (optind != argc && !strcmp (argv[optind], "--")) { optind++; if (first_nonopt != last_nonopt && last_nonopt != optind) exchange ((char **) argv); else if (first_nonopt == last_nonopt) first_nonopt = optind; last_nonopt = argc; optind = argc; } /* If we have done all the ARGV-elements, stop the scan and back over any non-options that we skipped and permuted. */ if (optind == argc) { /* Set the next-arg-index to point at the non-options that we previously skipped, so the caller will digest them. */ if (first_nonopt != last_nonopt) optind = first_nonopt; return -1; } /* If we have come to a non-option and did not permute it, either stop the scan or describe it to the caller and pass it by. */ if (NONOPTION_P) { if (ordering == REQUIRE_ORDER) return -1; optarg = argv[optind++]; return 1; } /* We have found another option-ARGV-element. Skip the initial punctuation. */ nextchar = (argv[optind] + 1 + (longopts != NULL && argv[optind][1] == '-')); } /* Decode the current option-ARGV-element. */ /* Check whether the ARGV-element is a long option. If long_only and the ARGV-element has the form "-f", where f is a valid short option, don't consider it an abbreviated form of a long option that starts with f. Otherwise there would be no way to give the -f short option. On the other hand, if there's a long option "fubar" and the ARGV-element is "-fu", do consider that an abbreviation of the long option, just like "--fu", and not "-f" with arg "u". This distinction seems to be the most useful approach. */ if (longopts != NULL && (argv[optind][1] == '-' || (long_only && (argv[optind][2] || !my_index (optstring, argv[optind][1]))))) { char *nameend; const struct option *p; const struct option *pfound = NULL; int exact = 0; int ambig = 0; int indfound = -1; int option_index; for (nameend = nextchar; *nameend && *nameend != '='; nameend++) /* Do nothing. */ ; /* Test all long options for either exact match or abbreviated matches. */ for (p = longopts, option_index = 0; p->name; p++, option_index++) if (!strncmp (p->name, nextchar, nameend - nextchar)) { if ((unsigned int) (nameend - nextchar) == (unsigned int) strlen (p->name)) { /* Exact match found. */ pfound = p; indfound = option_index; exact = 1; break; } else if (pfound == NULL) { /* First nonexact match found. */ pfound = p; indfound = option_index; } else /* Second or later nonexact match found. */ ambig = 1; } if (ambig && !exact) { if (opterr) fprintf (stderr, _("%s: option `%s' is ambiguous\n"), argv[0], argv[optind]); nextchar += strlen (nextchar); optind++; optopt = 0; return '?'; } if (pfound != NULL) { option_index = indfound; optind++; if (*nameend) { /* Don't test has_arg with >, because some C compilers don't allow it to be used on enums. */ if (pfound->has_arg) optarg = nameend + 1; else { if (opterr) { if (argv[optind - 1][1] == '-') /* --option */ fprintf (stderr, _("%s: option `--%s' doesn't allow an argument\n"), argv[0], pfound->name); else /* +option or -option */ fprintf (stderr, _("%s: option `%c%s' doesn't allow an argument\n"), argv[0], argv[optind - 1][0], pfound->name); nextchar += strlen (nextchar); optopt = pfound->val; return '?'; } } } else if (pfound->has_arg == 1) { if (optind < argc) optarg = argv[optind++]; else { if (opterr) fprintf (stderr, _("%s: option `%s' requires an argument\n"), argv[0], argv[optind - 1]); nextchar += strlen (nextchar); optopt = pfound->val; return optstring[0] == ':' ? ':' : '?'; } } nextchar += strlen (nextchar); if (longind != NULL) *longind = option_index; if (pfound->flag) { *(pfound->flag) = pfound->val; return 0; } return pfound->val; } /* Can't find it as a long option. If this is not getopt_long_only, or the option starts with '--' or is not a valid short option, then it's an error. Otherwise interpret it as a short option. */ if (!long_only || argv[optind][1] == '-' || my_index (optstring, *nextchar) == NULL) { if (opterr) { if (argv[optind][1] == '-') /* --option */ fprintf (stderr, _("%s: unrecognized option `--%s'\n"), argv[0], nextchar); else /* +option or -option */ fprintf (stderr, _("%s: unrecognized option `%c%s'\n"), argv[0], argv[optind][0], nextchar); } nextchar = (char *) ""; optind++; optopt = 0; return '?'; } } /* Look at and handle the next short option-character. */ { char c = *nextchar++; char *temp = my_index (optstring, c); /* Increment `optind' when we start to process its last character. */ if (*nextchar == '\0') ++optind; if (temp == NULL || c == ':') { if (opterr) { if (posixly_correct) /* 1003.2 specifies the format of this message. */ fprintf (stderr, _("%s: illegal option -- %c\n"), argv[0], c); else fprintf (stderr, _("%s: invalid option -- %c\n"), argv[0], c); } optopt = c; return '?'; } /* Convenience. Treat POSIX -W foo same as long option --foo */ if (temp[0] == 'W' && temp[1] == ';') { char *nameend; const struct option *p; const struct option *pfound = NULL; int exact = 0; int ambig = 0; int indfound = 0; int option_index; /* This is an option that requires an argument. */ if (*nextchar != '\0') { optarg = nextchar; /* If we end this ARGV-element by taking the rest as an arg, we must advance to the next element now. */ optind++; } else if (optind == argc) { if (opterr) { /* 1003.2 specifies the format of this message. */ fprintf (stderr, _("%s: option requires an argument -- %c\n"), argv[0], c); } optopt = c; if (optstring[0] == ':') c = ':'; else c = '?'; return c; } else /* We already incremented `optind' once; increment it again when taking next ARGV-elt as argument. */ optarg = argv[optind++]; /* optarg is now the argument, see if it's in the table of longopts. */ for (nextchar = nameend = optarg; *nameend && *nameend != '='; nameend++) /* Do nothing. */ ; /* Test all long options for either exact match or abbreviated matches. */ for (p = longopts, option_index = 0; p->name; p++, option_index++) if (!strncmp (p->name, nextchar, nameend - nextchar)) { if ((unsigned int) (nameend - nextchar) == strlen (p->name)) { /* Exact match found. */ pfound = p; indfound = option_index; exact = 1; break; } else if (pfound == NULL) { /* First nonexact match found. */ pfound = p; indfound = option_index; } else /* Second or later nonexact match found. */ ambig = 1; } if (ambig && !exact) { if (opterr) fprintf (stderr, _("%s: option `-W %s' is ambiguous\n"), argv[0], argv[optind]); nextchar += strlen (nextchar); optind++; return '?'; } if (pfound != NULL) { option_index = indfound; if (*nameend) { /* Don't test has_arg with >, because some C compilers don't allow it to be used on enums. */ if (pfound->has_arg) optarg = nameend + 1; else { if (opterr) fprintf (stderr, _("\ %s: option `-W %s' doesn't allow an argument\n"), argv[0], pfound->name); nextchar += strlen (nextchar); return '?'; } } else if (pfound->has_arg == 1) { if (optind < argc) optarg = argv[optind++]; else { if (opterr) fprintf (stderr, _("%s: option `%s' requires an argument\n"), argv[0], argv[optind - 1]); nextchar += strlen (nextchar); return optstring[0] == ':' ? ':' : '?'; } } nextchar += strlen (nextchar); if (longind != NULL) *longind = option_index; if (pfound->flag) { *(pfound->flag) = pfound->val; return 0; } return pfound->val; } nextchar = NULL; return 'W'; /* Let the application handle it. */ } if (temp[1] == ':') { if (temp[2] == ':') { /* This is an option that accepts an argument optionally. */ if (*nextchar != '\0') { optarg = nextchar; optind++; } else optarg = NULL; nextchar = NULL; } else { /* This is an option that requires an argument. */ if (*nextchar != '\0') { optarg = nextchar; /* If we end this ARGV-element by taking the rest as an arg, we must advance to the next element now. */ optind++; } else if (optind == argc) { if (opterr) { /* 1003.2 specifies the format of this message. */ fprintf (stderr, _("%s: option requires an argument -- %c\n"), argv[0], c); } optopt = c; if (optstring[0] == ':') c = ':'; else c = '?'; } else /* We already incremented `optind' once; increment it again when taking next ARGV-elt as argument. */ optarg = argv[optind++]; nextchar = NULL; } } return c; } } int getopt (int argc, char *const *argv, const char *optstring) { return _getopt_internal (argc, argv, optstring, (const struct option *) 0, (int *) 0, 0); } #endif /* Not ELIDE_CODE. */ #ifdef TEST /* Compile with -DTEST to make an executable for use in testing the above definition of `getopt'. */ int main (int argc, char **argv) { int c; int digit_optind = 0; while (1) { int this_option_optind = optind ? optind : 1; c = getopt (argc, argv, "abc:d:0123456789"); if (c == -1) break; switch (c) { case '0': case '1': case '2': case '3': case '4': case '5': case '6': case '7': case '8': case '9': if (digit_optind != 0 && digit_optind != this_option_optind) printf ("digits occur in two different argv-elements.\n"); digit_optind = this_option_optind; printf ("option %c\n", c); break; case 'a': printf ("option a\n"); break; case 'b': printf ("option b\n"); break; case 'c': printf ("option c with value `%s'\n", optarg); break; case '?': break; default: printf ("?? getopt returned character code 0%o ??\n", c); } } if (optind < argc) { printf ("non-option ARGV-elements: "); while (optind < argc) printf ("%s ", argv[optind++]); printf ("\n"); } exit (0); } #endif /* TEST */ hisat2-2.2.1/msvcc/codeStubs/getopt.h000066400000000000000000000120651370663763200174500ustar00rootroot00000000000000/* Declarations for getopt. Copyright 1989, 1990, 1991, 1992, 1993, 1994, 1996, 1997, 1998, 2000, 2002 Free Software Foundation, Inc. NOTE: The canonical source of this file is maintained with the GNU C Library. Bugs can be reported to bug-glibc@gnu.org. This program is free software; you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2, or (at your option) any later version. This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. You should have received a copy of the GNU General Public License along with this program; if not, write to the Free Software Foundation, Inc., 51 Franklin Street - Fifth Floor, Boston, MA 02110-1301, USA. */ #ifndef _GETOPT_H #define _GETOPT_H 1 #ifndef __STDC__ #define __STDC__ 1 #define __STDC__UNDEF__ #endif #ifdef __cplusplus extern "C" { #endif /* For communication from `getopt' to the caller. When `getopt' finds an option that takes an argument, the argument value is returned here. Also, when `ordering' is RETURN_IN_ORDER, each non-option ARGV-element is returned here. */ extern char *optarg; /* Index in ARGV of the next element to be scanned. This is used for communication to and from the caller and for communication between successive calls to `getopt'. On entry to `getopt', zero means this is the first call; initialize. When `getopt' returns -1, this is the index of the first of the non-option elements that the caller should itself scan. Otherwise, `optind' communicates from one call to the next how much of ARGV has been scanned so far. */ extern int optind; /* Callers store zero here to inhibit the error message `getopt' prints for unrecognized options. */ extern int opterr; /* Set to an option character which was unrecognized. */ extern int optopt; /* Describe the long-named options requested by the application. The LONG_OPTIONS argument to getopt_long or getopt_long_only is a vector of `struct option' terminated by an element containing a name which is zero. The field `has_arg' is: no_argument (or 0) if the option does not take an argument, required_argument (or 1) if the option requires an argument, optional_argument (or 2) if the option takes an optional argument. If the field `flag' is not NULL, it points to a variable that is set to the value given in the field `val' when the option is found, but left unchanged if the option is not found. To have a long-named option do something other than set an `int' to a compiled-in constant, such as set a value from `optarg', set the option's `flag' field to zero and its `val' field to a nonzero value (the equivalent single-letter option character, if there is one). For long options that have a zero `flag' field, `getopt' returns the contents of the `val' field. */ struct option { #if defined (__STDC__) && __STDC__ const char *name; #else char *name; #endif /* has_arg can't be an enum because some compilers complain about type mismatches in all the code that assumes it is an int. */ int has_arg; int *flag; int val; }; /* Names for the values of the `has_arg' field of `struct option'. */ #define no_argument 0 #define required_argument 1 #define optional_argument 2 #if defined (__STDC__) && __STDC__ /* HAVE_DECL_* is a three-state macro: undefined, 0 or 1. If it is undefined, we haven't run the autoconf check so provide the declaration without arguments. If it is 0, we checked and failed to find the declaration so provide a fully prototyped one. If it is 1, we found it so don't provide any declaration at all. */ #if !HAVE_DECL_GETOPT #if defined (__GNU_LIBRARY__) || defined (HAVE_DECL_GETOPT) /* Many other libraries have conflicting prototypes for getopt, with differences in the consts, in unistd.h. To avoid compilation errors, only prototype getopt for the GNU C library. */ extern int getopt (int argc, char *const *argv, const char *shortopts); #else #ifndef __cplusplus extern int getopt (); #endif /* __cplusplus */ #endif #endif /* !HAVE_DECL_GETOPT */ extern int getopt_long (int argc, char *const *argv, const char *shortopts, const struct option *longopts, int *longind); extern int getopt_long_only (int argc, char *const *argv, const char *shortopts, const struct option *longopts, int *longind); /* Internal only. Users should not call this directly. */ extern int _getopt_internal (int argc, char *const *argv, const char *shortopts, const struct option *longopts, int *longind, int long_only); #else /* not __STDC__ */ extern int getopt (); extern int getopt_long (); extern int getopt_long_only (); extern int _getopt_internal (); #endif /* __STDC__ */ #ifdef __cplusplus } #endif #ifdef __STDC__UNDEF__ #undef __STDC__ #endif #endif /* getopt.h */ hisat2-2.2.1/msvcc/codeStubs/getopt1.c000066400000000000000000000104571370663763200175270ustar00rootroot00000000000000/* getopt_long and getopt_long_only entry points for GNU getopt. Copyright (C) 1987,88,89,90,91,92,93,94,96,97,98,2005 Free Software Foundation, Inc. NOTE: This source is derived from an old version taken from the GNU C Library (glibc). This program is free software; you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2, or (at your option) any later version. This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. You should have received a copy of the GNU General Public License along with this program; if not, write to the Free Software Foundation, Inc., 51 Franklin Street - Fifth Floor, Boston, MA 02110-1301, USA. */ #ifdef HAVE_CONFIG_H #include #endif #if !defined __STDC__ || !__STDC__ /* This is a separate conditional since some stdc systems reject `defined (const)'. */ #ifndef const #define const #endif #endif #include #include "getopt.h" /* Comment out all this code if we are using the GNU C Library, and are not actually compiling the library itself. This code is part of the GNU C Library, but also included in many other GNU distributions. Compiling and linking in this code is a waste when using the GNU C library (especially if it is a shared library). Rather than having every GNU program understand `configure --with-gnu-libc' and omit the object files, it is simpler to just do this in the source for each such file. */ #define GETOPT_INTERFACE_VERSION 2 #if !defined _LIBC && defined __GLIBC__ && __GLIBC__ >= 2 #include #if _GNU_GETOPT_INTERFACE_VERSION == GETOPT_INTERFACE_VERSION #define ELIDE_CODE #endif #endif #ifndef ELIDE_CODE /* This needs to come after some library #include to get __GNU_LIBRARY__ defined. */ #ifdef __GNU_LIBRARY__ #include #endif #ifndef NULL #define NULL 0 #endif int getopt_long (int argc, char *const *argv, const char *options, const struct option *long_options, int *opt_index) { return _getopt_internal (argc, argv, options, long_options, opt_index, 0); } /* Like getopt_long, but '-' as well as '--' can indicate a long option. If an option that starts with '-' (not '--') doesn't match a long option, but does match a short option, it is parsed as a short option instead. */ int getopt_long_only (int argc, char *const *argv, const char *options, const struct option *long_options, int *opt_index) { return _getopt_internal (argc, argv, options, long_options, opt_index, 1); } #endif /* Not ELIDE_CODE. */ #ifdef TEST #include int main (int argc, char **argv) { int c; int digit_optind = 0; while (1) { int this_option_optind = optind ? optind : 1; int option_index = 0; static struct option long_options[] = { {"add", 1, 0, 0}, {"append", 0, 0, 0}, {"delete", 1, 0, 0}, {"verbose", 0, 0, 0}, {"create", 0, 0, 0}, {"file", 1, 0, 0}, {0, 0, 0, 0} }; c = getopt_long (argc, argv, "abc:d:0123456789", long_options, &option_index); if (c == -1) break; switch (c) { case 0: printf ("option %s", long_options[option_index].name); if (optarg) printf (" with arg %s", optarg); printf ("\n"); break; case '0': case '1': case '2': case '3': case '4': case '5': case '6': case '7': case '8': case '9': if (digit_optind != 0 && digit_optind != this_option_optind) printf ("digits occur in two different argv-elements.\n"); digit_optind = this_option_optind; printf ("option %c\n", c); break; case 'a': printf ("option a\n"); break; case 'b': printf ("option b\n"); break; case 'c': printf ("option c with value `%s'\n", optarg); break; case 'd': printf ("option d with value `%s'\n", optarg); break; case '?': break; default: printf ("?? getopt returned character code 0%o ??\n", c); } } if (optind < argc) { printf ("non-option ARGV-elements: "); while (optind < argc) printf ("%s ", argv[optind++]); printf ("\n"); } exit (0); } #endif /* TEST */ hisat2-2.2.1/msvcc/codeStubs/sys/000077500000000000000000000000001370663763200166075ustar00rootroot00000000000000hisat2-2.2.1/msvcc/codeStubs/sys/time.cpp000066400000000000000000000026531370663763200202570ustar00rootroot00000000000000#include #include /* * This file is a copy of the public domain code that can be found in * multiple locations and whose prevenance is unclear * * This is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #if defined(_MSC_VER) || defined(_MSC_EXTENSIONS) #define DELTA_EPOCH_IN_MICROSECS 11644473600000000Ui64 #else #define DELTA_EPOCH_IN_MICROSECS 11644473600000000ULL #endif int gettimeofday(struct timeval *tv, struct timezone *tz) { FILETIME ft; unsigned __int64 tmpres = 0; static int tzflag; if (NULL != tv) { GetSystemTimeAsFileTime(&ft); tmpres |= ft.dwHighDateTime; tmpres <<= 32; tmpres |= ft.dwLowDateTime; /*converting file time to unix epoch*/ tmpres -= DELTA_EPOCH_IN_MICROSECS; tmpres /= 10; /*convert into microseconds*/ tv->tv_sec = (long)(tmpres / 1000000UL); tv->tv_usec = (long)(tmpres % 1000000UL); } if (NULL != tz) { if (!tzflag) { _tzset(); tzflag++; } tz->tz_minuteswest = _timezone / 60; tz->tz_dsttime = _daylight; } return 0; } hisat2-2.2.1/msvcc/codeStubs/sys/time.h000066400000000000000000000003421370663763200177150ustar00rootroot00000000000000 #ifndef TIME_STUB_H #define TIME_STUB_H #include //For timeval defn struct timezone { int tz_minuteswest; int tz_dsttime; }; int gettimeofday(struct timeval *tv, struct timezone *tz); #endifhisat2-2.2.1/msvcc/codeStubs/unistd.h000066400000000000000000000000001370663763200174360ustar00rootroot00000000000000hisat2-2.2.1/msvcc/hisat2-align-l.vcxproj000066400000000000000000000124321370663763200201700ustar00rootroot00000000000000 Debug x64 Release x64 {08c70ff5-9e0c-42df-b7bf-c1af1b0d2cff} {d281e44b-92e9-4cea-a666-b05530f65000} {e1f391b2-4a3f-43ca-9192-14536abe783e} {55F86D23-4245-4050-BD2D-CC5D4FD0C36B} hsat2alignl 8.1 hisat2-align-l Application true v140 MultiByte Application false v140 true MultiByte $(Platform)\$(ProjectName)\$(Configuration)\ $(Platform)\$(ProjectName)\$(Configuration)\ Level3 Disabled true BOWTIE_64BIT_INDEX;%(PreprocessorDefinitions) %(AdditionalIncludeDirectories) "version.h" Level3 MaxSpeed true true true BOWTIE_64BIT_INDEX;NDEBUG;%(PreprocessorDefinitions) %(AdditionalIncludeDirectories) MultiThreaded "version.h" true true 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Level3 MaxSpeed true true true BOWTIE_64BIT_INDEX;NDEBUG;%(PreprocessorDefinitions) %(AdditionalIncludeDirectories) MultiThreaded true true true hisat2-2.2.1/msvcc/shared64.vcxproj.filters000066400000000000000000000063541370663763200205520ustar00rootroot00000000000000 {4FC737F1-C7A5-4376-A066-2A32D752A2FF} cpp;c;cc;cxx;def;odl;idl;hpj;bat;asm;asmx {93995380-89BD-4b04-88EB-625FBE52EBFB} h;hpp;hxx;hm;inl;inc;xsd {67DA6AB6-F800-4c08-8B7A-83BB121AAD01} rc;ico;cur;bmp;dlg;rc2;rct;bin;rgs;gif;jpg;jpeg;jpe;resx;tiff;tif;png;wav;mfcribbon-ms Source Files Source Files Source Files Source Files Source Files Source Files Source Files Source Files Source Files Source Files Source Files Source Files Header Files Header Files Header Files Header Files Header Files Header Files Header Files Header Files Header Files Header Files Header Files Header Files Header Files hisat2-2.2.1/multikey_qsort.cpp000066400000000000000000000013731370663763200165260ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "multikey_qsort.h" hisat2-2.2.1/multikey_qsort.h000066400000000000000000001140441370663763200161730ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef MULTIKEY_QSORT_H_ #define MULTIKEY_QSORT_H_ #include #include "sequence_io.h" #include "alphabet.h" #include "assert_helpers.h" #include "diff_sample.h" #include "sstring.h" #include "btypes.h" using namespace std; /** * Swap elements a and b in s */ template static inline void swap(TStr& s, size_t slen, TPos a, TPos b) { assert_lt(a, slen); assert_lt(b, slen); swap(s[a], s[b]); } /** * Swap elements a and b in array s */ template static inline void swap(TVal* s, size_t slen, TPos a, TPos b) { assert_lt(a, slen); assert_lt(b, slen); swap(s[a], s[b]); } /** * Helper macro for swapping elements a and b in s. Does some additional * sainty checking w/r/t begin and end (which are parameters to the sorting * routines below). */ #define SWAP(s, a, b) { \ assert_geq(a, begin); \ assert_geq(b, begin); \ assert_lt(a, end); \ assert_lt(b, end); \ swap(s, slen, a, b); \ } /** * Helper macro for swapping the same pair of elements a and b in two different * strings s and s2. This is a helpful variant if, for example, the caller * would like to see how their input was permuted by the sort routine (in that * case, the caller would let s2 be an array s2[] where s2 is the same length * as s and s2[i] = i). */ #define SWAP2(s, s2, a, b) { \ SWAP(s, a, b); \ swap(s2, slen, a, b); \ } #define SWAP1(s, s2, a, b) { \ SWAP(s, a, b); \ } /** * Helper macro that swaps a range of elements [i, i+n) with another * range [j, j+n) in s. */ #define VECSWAP(s, i, j, n) { \ if(n > 0) { vecswap(s, slen, i, j, n, begin, end); } \ } /** * Helper macro that swaps a range of elements [i, i+n) with another * range [j, j+n) both in s and s2. */ #define VECSWAP2(s, s2, i, j, n) { \ if(n > 0) { vecswap2(s, slen, s2, i, j, n, begin, end); } \ } /** * Helper function that swaps a range of elements [i, i+n) with another * range [j, j+n) in s. begin and end represent the current range under * consideration by the caller (one of the recursive multikey_quicksort * routines below). */ template static inline void vecswap(TStr& s, size_t slen, TPos i, TPos j, TPos n, TPos begin, TPos end) { assert_geq(i, begin); assert_geq(j, begin); assert_lt(i, end); assert_lt(j, end); while(n-- > 0) { assert_geq(n, 0); TPos a = i+n; TPos b = j+n; assert_geq(a, begin); assert_geq(b, begin); assert_lt(a, end); assert_lt(b, end); swap(s, slen, a, b); } } template static inline void vecswap(TVal *s, size_t slen, TPos i, TPos j, TPos n, TPos begin, TPos end) { assert_geq(i, begin); assert_geq(j, begin); assert_lt(i, end); assert_lt(j, end); while(n-- > 0) { assert_geq(n, 0); TPos a = i+n; TPos b = j+n; assert_geq(a, begin); assert_geq(b, begin); assert_lt(a, end); assert_lt(b, end); swap(s, slen, a, b); } } /** * Helper function that swaps a range of elements [i, i+n) with another range * [j, j+n) both in s and s2. begin and end represent the current range under * consideration by the caller (one of the recursive multikey_quicksort * routines below). */ template static inline void vecswap2( TStr& s, size_t slen, TStr& s2, TPos i, TPos j, TPos n, TPos begin, TPos end) { assert_geq(i, begin); assert_geq(j, begin); assert_lt(i, end); assert_lt(j, end); while(n-- > 0) { assert_geq(n, 0); TPos a = i+n; TPos b = j+n; assert_geq(a, begin); assert_geq(b, begin); assert_lt(a, end); assert_lt(b, end); swap(s, slen, a, b); swap(s2, slen, a, b); } } template static inline void vecswap2(TVal* s, size_t slen, TVal* s2, TPos i, TPos j, TPos n, TPos begin, TPos end) { assert_geq(i, begin); assert_geq(j, begin); assert_lt(i, end); assert_lt(j, end); while(n-- > 0) { assert_geq(n, 0); TPos a = i+n; TPos b = j+n; assert_geq(a, begin); assert_geq(b, begin); assert_lt(a, end); assert_lt(b, end); swap(s, slen, a, b); swap(s2, slen, a, b); } } /// Retrieve an int-ized version of the ath character of string s, or, /// if a goes off the end of s, return a (user-specified) int greater /// than any TAlphabet character - 'hi'. #define CHAR_AT(ss, aa) ((length(s[ss]) > aa) ? (int)(s[ss][aa]) : hi) /// Retrieve an int-ized version of the ath character of string s, or, /// if a goes off the end of s, return a (user-specified) int greater /// than any TAlphabet character - 'hi'. #define CHAR_AT_SUF(si, off) \ (((off + s[si]) < hlen) ? ((int)(host[off + s[si]])) : (hi)) /// Retrieve an int-ized version of the ath character of string s, or, /// if a goes off the end of s, return a (user-specified) int greater /// than any TAlphabet character - 'hi'. #define CHAR_AT_SUF_U8(si, off) char_at_suf_u8(host, hlen, s, si, off, hi) // Note that CHOOSE_AND_SWAP_RANDOM_PIVOT is unused #define CHOOSE_AND_SWAP_RANDOM_PIVOT(sw, ch) { \ /* Note: rand() didn't really cut it here; it seemed to run out of */ \ /* randomness and, after a time, returned the same thing over and */ \ /* over again */ \ a = (rand() % n) + begin; /* choose pivot between begin and end */ \ assert_lt(a, end); assert_geq(a, begin); \ sw(s, s2, begin, a); /* move pivot to beginning */ \ } /** * Ad-hoc DNA-centric way of choose a pretty good pivot without using * the pseudo-random number generator. We try to get a 1 or 2 if * possible, since they'll split things more evenly than a 0 or 4. We * also avoid swapping in the event that we choose the first element. */ #define CHOOSE_AND_SWAP_SMART_PIVOT(sw, ch) { \ a = begin; /* choose first elt */ \ /* now try to find a better elt */ \ if(n >= 5) { /* n is the difference between begin and end */ \ if (ch(begin+1, depth) == 1 || ch(begin+1, depth) == 2) a = begin+1; \ else if(ch(begin+2, depth) == 1 || ch(begin+2, depth) == 2) a = begin+2; \ else if(ch(begin+3, depth) == 1 || ch(begin+3, depth) == 2) a = begin+3; \ else if(ch(begin+4, depth) == 1 || ch(begin+4, depth) == 2) a = begin+4; \ if(a != begin) sw(s, s2, begin, a); /* move pivot to beginning */ \ } \ /* the element at [begin] now holds the pivot value */ \ } #define CHOOSE_AND_SWAP_PIVOT CHOOSE_AND_SWAP_SMART_PIVOT #ifndef NDEBUG /** * Assert that the range of chars at depth 'depth' in strings 'begin' * to 'end' in string-of-suffix-offsets s is parititioned properly * according to the ternary paritioning strategy of Bentley and McIlroy * (*prior to* swapping the = regions to the center) */ template bool assertPartitionedSuf( const THost& host, TIndexOffU *s, size_t slen, int hi, int pivot, size_t begin, size_t end, size_t depth) { size_t hlen = host.length(); int state = 0; // 0 -> 1st = section, 1 -> < section, 2 -> > section, 3 -> 2nd = section for(size_t i = begin; i < end; i++) { switch(state) { case 0: if (CHAR_AT_SUF(i, depth) < pivot) { state = 1; break; } else if (CHAR_AT_SUF(i, depth) > pivot) { state = 2; break; } assert_eq(CHAR_AT_SUF(i, depth), pivot); break; case 1: if (CHAR_AT_SUF(i, depth) > pivot) { state = 2; break; } else if (CHAR_AT_SUF(i, depth) == pivot) { state = 3; break; } assert_lt(CHAR_AT_SUF(i, depth), pivot); break; case 2: if (CHAR_AT_SUF(i, depth) == pivot) { state = 3; break; } assert_gt(CHAR_AT_SUF(i, depth), pivot); break; case 3: assert_eq(CHAR_AT_SUF(i, depth), pivot); break; } } return true; } /** * Assert that the range of chars at depth 'depth' in strings 'begin' * to 'end' in string-of-suffix-offsets s is parititioned properly * according to the ternary paritioning strategy of Bentley and McIlroy * (*after* swapping the = regions to the center) */ template bool assertPartitionedSuf2( const THost& host, TIndexOffU *s, size_t slen, int hi, int pivot, size_t begin, size_t end, size_t depth) { size_t hlen = host.length(); int state = 0; // 0 -> < section, 1 -> = section, 2 -> > section for(size_t i = begin; i < end; i++) { switch(state) { case 0: if (CHAR_AT_SUF(i, depth) == pivot) { state = 1; break; } else if (CHAR_AT_SUF(i, depth) > pivot) { state = 2; break; } assert_lt(CHAR_AT_SUF(i, depth), pivot); break; case 1: if (CHAR_AT_SUF(i, depth) > pivot) { state = 2; break; } assert_eq(CHAR_AT_SUF(i, depth), pivot); break; case 2: assert_gt(CHAR_AT_SUF(i, depth), pivot); break; } } return true; } #endif /** * Assert that string s of suffix offsets into string 'host' is a seemingly * legitimate suffix-offset list (at this time, we just check that it doesn't * list any suffix twice). */ static inline void sanityCheckInputSufs(TIndexOffU *s, size_t slen) { assert_gt(slen, 0); for(size_t i = 0; i < slen; i++) { // Actually, it's convenient to allow the caller to provide // suffix offsets thare are off the end of the host string. // See, e.g., build() in diff_sample.cpp. //assert_lt(s[i], length(host)); for(size_t j = i+1; j < slen; j++) { assert_neq(s[i], s[j]); } } } /** * Assert that the string s of suffix offsets into 'host' really are in * lexicographical order up to depth 'upto'. */ template void sanityCheckOrderedSufs( const T& host, size_t hlen, TIndexOffU *s, size_t slen, size_t upto, size_t lower = 0, size_t upper = OFF_MASK) { assert_lt(s[0], hlen); upper = min(upper, slen-1); for(size_t i = lower; i < upper; i++) { // Allow s[i+t] to point off the end of the string; this is // convenient for some callers if(s[i+1] >= hlen) continue; #ifndef NDEBUG if(upto == OFF_MASK) { assert(sstr_suf_lt(host, s[i], hlen, host, s[i+1], hlen, false)); } else { if(sstr_suf_upto_lt(host, s[i], host, s[i+1], upto, false)) { // operator > treats shorter strings as // lexicographically smaller, but we want to opposite //assert(isPrefix(suffix(host, s[i+1]), suffix(host, s[i]))); } } #endif } } /** * Main multikey quicksort function for suffixes. Based on Bentley & * Sedgewick's algorithm on p.5 of their paper "Fast Algorithms for * Sorting and Searching Strings". That algorithm has been extended in * three ways: * * 1. Deal with keys of different lengths by checking bounds and * considering off-the-end values to be 'hi' (b/c our goal is the * BWT transform, we're biased toward considring prefixes as * lexicographically *greater* than their extensions). * 2. The multikey_qsort_suffixes version takes a single host string * and a list of suffix offsets as input. This reduces memory * footprint compared to an approach that treats its input * generically as a set of strings (not necessarily suffixes), thus * requiring that we store at least two integers worth of * information for each string. * 3. Sorting functions take an extra "upto" parameter that upper- * bounds the depth to which the function sorts. * * TODO: Consult a tie-breaker (like a difference cover sample) if two * keys share a long prefix. */ template void mkeyQSortSuf( const T& host, size_t hlen, TIndexOffU *s, size_t slen, int hi, size_t begin, size_t end, size_t depth, size_t upto = OFF_MASK) { // Helper for making the recursive call; sanity-checks arguments to // make sure that the problem actually got smaller. #define MQS_RECURSE_SUF(nbegin, nend, ndepth) { \ assert(nbegin > begin || nend < end || ndepth > depth); \ if(ndepth < upto) { /* don't exceed depth of 'upto' */ \ mkeyQSortSuf(host, hlen, s, slen, hi, nbegin, nend, ndepth, upto); \ } \ } assert_leq(begin, slen); assert_leq(end, slen); size_t a, b, c, d, /*e,*/ r; size_t n = end - begin; if(n <= 1) return; // 1-element list already sorted CHOOSE_AND_SWAP_PIVOT(SWAP1, CHAR_AT_SUF); // pick pivot, swap it into [begin] int v = CHAR_AT_SUF(begin, depth); // v <- randomly-selected pivot value #ifndef NDEBUG { bool stillInBounds = false; for(size_t i = begin; i < end; i++) { if(depth < (hlen-s[i])) { stillInBounds = true; break; } else { /* already fell off this suffix */ } } assert(stillInBounds); // >=1 suffix must still be in bounds } #endif a = b = begin; c = d = end-1; while(true) { // Invariant: everything before a is = pivot, everything // between a and b is < int bc = 0; // shouldn't have to init but gcc on Mac complains while(b <= c && v >= (bc = CHAR_AT_SUF(b, depth))) { if(v == bc) { SWAP(s, a, b); a++; } b++; } // Invariant: everything after d is = pivot, everything // between c and d is > int cc = 0; // shouldn't have to init but gcc on Mac complains while(b <= c && v <= (cc = CHAR_AT_SUF(c, depth))) { if(v == cc) { SWAP(s, c, d); d--; } c--; } if(b > c) break; SWAP(s, b, c); b++; c--; } assert(a > begin || c < end-1); // there was at least one =s assert_lt(d-c, n); // they can't all have been > pivot assert_lt(b-a, n); // they can't all have been < pivot assert(assertPartitionedSuf(host, s, slen, hi, v, begin, end, depth)); // check pre-=-swap invariant r = min(a-begin, b-a); VECSWAP(s, begin, b-r, r); // swap left = to center r = min(d-c, end-d-1); VECSWAP(s, b, end-r, r); // swap right = to center assert(assertPartitionedSuf2(host, s, slen, hi, v, begin, end, depth)); // check post-=-swap invariant r = b-a; // r <- # of <'s if(r > 0) { MQS_RECURSE_SUF(begin, begin + r, depth); // recurse on <'s } // Do not recurse on ='s if the pivot was the off-the-end value; // they're already fully sorted if(v != hi) { MQS_RECURSE_SUF(begin + r, begin + r + (a-begin) + (end-d-1), depth+1); // recurse on ='s } r = d-c; // r <- # of >'s excluding those exhausted if(r > 0 && v < hi-1) { MQS_RECURSE_SUF(end-r, end, depth); // recurse on >'s } } /** * Toplevel function for multikey quicksort over suffixes. */ template void mkeyQSortSuf( const T& host, TIndexOffU *s, size_t slen, int hi, bool verbose = false, bool sanityCheck = false, size_t upto = OFF_MASK) { size_t hlen = host.length(); assert_gt(slen, 0); if(sanityCheck) sanityCheckInputSufs(s, slen); mkeyQSortSuf(host, hlen, s, slen, hi, (size_t)0, slen, (size_t)0, upto); if(sanityCheck) sanityCheckOrderedSufs(host, hlen, s, slen, upto); } /** * Just like mkeyQSortSuf but all swaps are applied to s2 as well as s. * This is a helpful variant if, for example, the caller would like to * see how their input was permuted by the sort routine (in that case, * the caller would let s2 be an array s2[] where s2 is the same length * as s and s2[i] = i). */ struct QSortRange { size_t begin; size_t end; size_t depth; }; template void mkeyQSortSuf2( const T& host, size_t hlen, TIndexOffU *s, size_t slen, TIndexOffU *s2, int hi, size_t _begin, size_t _end, size_t _depth, size_t upto = OFF_MASK, EList* boundaries = NULL) { ELList block_list; while(true) { size_t begin = 0, end = 0, depth = 0; if(block_list.size() == 0) { begin = _begin; end = _end; depth = _depth; } else { if(block_list.back().size() > 0) { begin = block_list.back()[0].begin; end = block_list.back()[0].end; depth = block_list.back()[0].depth; block_list.back().erase(0); } else { block_list.resize(block_list.size() - 1); if(block_list.size() == 0) { break; } } } if(depth == upto) { if(boundaries != NULL) { (*boundaries).push_back(end); } continue; } assert_leq(begin, slen); assert_leq(end, slen); size_t a, b, c, d, /*e,*/ r; size_t n = end - begin; if(n <= 1) { // 1-element list already sorted if(n == 1 && boundaries != NULL) { boundaries->push_back(end); } continue; } CHOOSE_AND_SWAP_PIVOT(SWAP2, CHAR_AT_SUF); // pick pivot, swap it into [begin] int v = CHAR_AT_SUF(begin, depth); // v <- randomly-selected pivot value #ifndef NDEBUG { bool stillInBounds = false; for(size_t i = begin; i < end; i++) { if(depth < (hlen-s[i])) { stillInBounds = true; break; } else { /* already fell off this suffix */ } } assert(stillInBounds); // >=1 suffix must still be in bounds } #endif a = b = begin; c = d = /*e =*/ end-1; while(true) { // Invariant: everything before a is = pivot, everything // between a and b is < int bc = 0; // shouldn't have to init but gcc on Mac complains while(b <= c && v >= (bc = CHAR_AT_SUF(b, depth))) { if(v == bc) { SWAP2(s, s2, a, b); a++; } b++; } // Invariant: everything after d is = pivot, everything // between c and d is > int cc = 0; // shouldn't have to init but gcc on Mac complains while(b <= c && v <= (cc = CHAR_AT_SUF(c, depth))) { if(v == cc) { SWAP2(s, s2, c, d); d--; /*e--;*/ } //else if(c == e && v == hi) e--; c--; } if(b > c) break; SWAP2(s, s2, b, c); b++; c--; } assert(a > begin || c < end-1); // there was at least one =s assert_lt(/*e*/d-c, n); // they can't all have been > pivot assert_lt(b-a, n); // they can't all have been < pivot assert(assertPartitionedSuf(host, s, slen, hi, v, begin, end, depth)); // check pre-=-swap invariant r = min(a-begin, b-a); VECSWAP2(s, s2, begin, b-r, r); // swap left = to center r = min(d-c, end-d-1); VECSWAP2(s, s2, b, end-r, r); // swap right = to center assert(assertPartitionedSuf2(host, s, slen, hi, v, begin, end, depth)); // check post-=-swap invariant r = b-a; // r <- # of <'s block_list.expand(); block_list.back().clear(); if(r > 0) { // recurse on <'s block_list.back().expand(); block_list.back().back().begin = begin; block_list.back().back().end = begin + r; block_list.back().back().depth = depth; } // Do not recurse on ='s if the pivot was the off-the-end value; // they're already fully sorted //if(v != hi) { // recurse on ='s block_list.back().expand(); block_list.back().back().begin = begin + r; block_list.back().back().end = begin + r + (a-begin) + (end-d-1); block_list.back().back().depth = depth + 1; //} r = d-c; // r <- # of >'s excluding those exhausted if(r > 0 /*&& v < hi-1*/) { // recurse on >'s block_list.back().expand(); block_list.back().back().begin = end - r; block_list.back().back().end = end; block_list.back().back().depth = depth; } } } /** * Toplevel function for multikey quicksort over suffixes with double * swapping. */ template void mkeyQSortSuf2( const T& host, TIndexOffU *s, size_t slen, TIndexOffU *s2, int hi, bool verbose = false, bool sanityCheck = false, size_t upto = OFF_MASK, EList* boundaries = NULL) { size_t hlen = host.length(); if(sanityCheck) sanityCheckInputSufs(s, slen); TIndexOffU *sOrig = NULL; if(sanityCheck) { sOrig = new TIndexOffU[slen]; memcpy(sOrig, s, OFF_SIZE * slen); } mkeyQSortSuf2(host, hlen, s, slen, s2, hi, (size_t)0, slen, (size_t)0, upto, boundaries); if(sanityCheck) { sanityCheckOrderedSufs(host, hlen, s, slen, upto); for(size_t i = 0; i < slen; i++) { assert_eq(s[i], sOrig[s2[i]]); } delete[] sOrig; } } // Ugly but necessary; otherwise the compiler chokes dramatically on // the DifferenceCoverSample<> template args to the next few functions template class DifferenceCoverSample; /** * Constant time */ template inline bool sufDcLt( const T1& host, const T2& s1, const T2& s2, const DifferenceCoverSample& dc, bool sanityCheck = false) { size_t diff = dc.tieBreakOff(s1, s2); ASSERT_ONLY(size_t hlen = host.length()); assert_lt(diff, dc.v()); assert_lt(diff, hlen-s1); assert_lt(diff, hlen-s2); if(sanityCheck) { for(size_t i = 0; i < diff; i++) { assert_eq(host[s1+i], host[s2+i]); } } bool ret = dc.breakTie(s1+diff, s2+diff) < 0; #ifndef NDEBUG if(sanityCheck && ret != sstr_suf_lt(host, s1, hlen, host, s2, hlen, false)) { assert(false); } #endif return ret; } /** * k log(k) */ template inline void qsortSufDc( const T& host, size_t hlen, TIndexOffU* s, size_t slen, const DifferenceCoverSample& dc, size_t begin, size_t end, bool sanityCheck = false) { assert_leq(end, slen); assert_lt(begin, slen); assert_gt(end, begin); size_t n = end - begin; if(n <= 1) return; // 1-element list already sorted // Note: rand() didn't really cut it here; it seemed to run out of // randomness and, after a time, returned the same thing over and // over again size_t a = (rand() % n) + begin; // choose pivot between begin and end assert_lt(a, end); assert_geq(a, begin); SWAP(s, end-1, a); // move pivot to end size_t cur = 0; for(size_t i = begin; i < end-1; i++) { if(sufDcLt(host, s[i], s[end-1], dc, sanityCheck)) { if(sanityCheck) assert(dollarLt(suffix(host, s[i]), suffix(host, s[end-1]))); assert_lt(begin + cur, end-1); SWAP(s, i, begin + cur); cur++; } } // Put pivot into place assert_lt(cur, end-begin); SWAP(s, end-1, begin+cur); if(begin+cur > begin) qsortSufDc(host, hlen, s, slen, dc, begin, begin+cur); if(end > begin+cur+1) qsortSufDc(host, hlen, s, slen, dc, begin+cur+1, end); } /** * Toplevel function for multikey quicksort over suffixes. */ template void mkeyQSortSufDcU8( const T1& host1, const T2& host, size_t hlen, TIndexOffU* s, size_t slen, const DifferenceCoverSample& dc, int hi, bool verbose = false, bool sanityCheck = false) { if(sanityCheck) sanityCheckInputSufs(s, slen); mkeyQSortSufDcU8(host1, host, hlen, s, slen, dc, hi, 0, slen, 0, sanityCheck); if(sanityCheck) sanityCheckOrderedSufs(host1, hlen, s, slen, OFF_MASK); } /** * Return a boolean indicating whether s1 < s2 using the difference * cover to break the tie. */ template inline bool sufDcLtU8( const T1& host1, const T2& host, size_t hlen, size_t s1, size_t s2, const DifferenceCoverSample& dc, bool sanityCheck = false) { hlen += 0; size_t diff = dc.tieBreakOff((TIndexOffU)s1, (TIndexOffU)s2); assert_lt(diff, dc.v()); assert_lt(diff, hlen-s1); assert_lt(diff, hlen-s2); if(sanityCheck) { for(size_t i = 0; i < diff; i++) { assert_eq(host[s1+i], host1[s2+i]); } } bool ret = dc.breakTie((TIndexOffU)(s1+diff), (TIndexOffU)(s2+diff)) < 0; // Sanity-check return value using dollarLt #ifndef NDEBUG bool ret2 = sstr_suf_lt(host1, s1, hlen, host, s2, hlen, false); assert(!sanityCheck || ret == ret2); #endif return ret; } /** * k log(k) */ template inline void qsortSufDcU8( const T1& host1, const T2& host, size_t hlen, TIndexOffU* s, size_t slen, const DifferenceCoverSample& dc, size_t begin, size_t end, bool sanityCheck = false) { assert_leq(end, slen); assert_lt(begin, slen); assert_gt(end, begin); size_t n = end - begin; if(n <= 1) return; // 1-element list already sorted // Note: rand() didn't really cut it here; it seemed to run out of // randomness and, after a time, returned the same thing over and // over again size_t a = (rand() % n) + begin; // choose pivot between begin and end assert_lt(a, end); assert_geq(a, begin); SWAP(s, end-1, a); // move pivot to end size_t cur = 0; for(size_t i = begin; i < end-1; i++) { if(sufDcLtU8(host1, host, hlen, s[i], s[end-1], dc, sanityCheck)) { #ifndef NDEBUG if(sanityCheck) { assert(sstr_suf_lt(host1, s[i], hlen, host1, s[end-1], hlen, false)); } assert_lt(begin + cur, end-1); #endif SWAP(s, i, begin + cur); cur++; } } // Put pivot into place assert_lt(cur, end-begin); SWAP(s, end-1, begin+cur); if(begin+cur > begin) qsortSufDcU8(host1, host, hlen, s, slen, dc, begin, begin+cur); if(end > begin+cur+1) qsortSufDcU8(host1, host, hlen, s, slen, dc, begin+cur+1, end); } #define BUCKET_SORT_CUTOFF (4 * 1024 * 1024) #define SELECTION_SORT_CUTOFF 6 /** * Straightforwardly obtain a uint8_t-ized version of t[off]. This * works fine as long as TStr is not packed. */ template inline uint8_t get_uint8(const TStr& t, size_t off) { return t[off]; } /** * For incomprehensible generic-programming reasons, getting a uint8_t * version of a character in a packed String<> requires casting first * to Dna then to uint8_t. */ template<> inline uint8_t get_uint8(const S2bDnaString& t, size_t off) { return (uint8_t)t[off]; } /** * Return character at offset 'off' from the 'si'th suffix in the array * 's' of suffixes. If the character is out-of-bounds, return hi. */ template static inline int char_at_suf_u8( const TStr& host, size_t hlen, TIndexOffU* s, size_t si, size_t off, uint8_t hi) { return ((off+s[si]) < hlen) ? get_uint8(host, off+s[si]) : (hi); } template static void selectionSortSufDcU8( const T1& host1, const T2& host, size_t hlen, TIndexOffU* s, size_t slen, const DifferenceCoverSample& dc, uint8_t hi, size_t begin, size_t end, size_t depth, bool sanityCheck = false) { #define ASSERT_SUF_LT(l, r) \ if(sanityCheck && \ !sstr_suf_lt(host1, s[l], hlen, host1, s[r], hlen, false)) { \ assert(false); \ } assert_gt(end, begin+1); assert_leq(end-begin, SELECTION_SORT_CUTOFF); assert_eq(hi, 4); size_t v = dc.v(); if(end == begin+2) { size_t off = dc.tieBreakOff(s[begin], s[begin+1]); if(off + s[begin] >= hlen || off + s[begin+1] >= hlen) { off = OFF_MASK; } if(off != OFF_MASK) { if(off < depth) { qsortSufDcU8(host1, host, hlen, s, slen, dc, begin, end, sanityCheck); // It's helpful for debugging if we call this here if(sanityCheck) { sanityCheckOrderedSufs(host1, hlen, s, slen, OFF_MASK, begin, end); } return; } v = off - depth + 1; } } assert_leq(v, dc.v()); size_t lim = v; assert_geq(lim, 0); for(size_t i = begin; i < end-1; i++) { size_t targ = i; size_t targoff = depth + s[i]; for(size_t j = i+1; j < end; j++) { assert_neq(j, targ); size_t joff = depth + s[j]; size_t k; for(k = 0; k <= lim; k++) { assert_neq(j, targ); uint8_t jc = (k + joff < hlen) ? get_uint8(host, k + joff) : hi; uint8_t tc = (k + targoff < hlen) ? get_uint8(host, k + targoff) : hi; assert(jc != hi || tc != hi); if(jc > tc) { // the jth suffix is greater than the current // smallest suffix ASSERT_SUF_LT(targ, j); break; } else if(jc < tc) { // the jth suffix is less than the current smallest // suffix, so update smallest to be j ASSERT_SUF_LT(j, targ); targ = j; targoff = joff; break; } else if(k == lim) { // Check whether either string ends immediately // after this character assert_leq(k + joff + 1, hlen); assert_leq(k + targoff + 1, hlen); if(k + joff + 1 == hlen) { // targ < j assert_neq(k + targoff + 1, hlen); ASSERT_SUF_LT(targ, j); break; } else if(k + targoff + 1 == hlen) { // j < targ ASSERT_SUF_LT(j, targ); targ = j; targoff = joff; break; } } else { // They're equal so far, keep going } } // The jth suffix was equal to the current smallest suffix // up to the difference-cover period, so disambiguate with // difference cover if(k == lim+1) { assert_neq(j, targ); if(sufDcLtU8(host1, host, hlen, s[j], s[targ], dc, sanityCheck)) { // j < targ assert(!sufDcLtU8(host1, host, hlen, s[targ], s[j], dc, sanityCheck)); ASSERT_SUF_LT(j, targ); targ = j; targoff = joff; } else { assert(sufDcLtU8(host1, host, hlen, s[targ], s[j], dc, sanityCheck)); ASSERT_SUF_LT(targ, j); // ! } } } if(i != targ) { ASSERT_SUF_LT(targ, i); // swap i and targ TIndexOffU tmp = s[i]; s[i] = s[targ]; s[targ] = tmp; } for(size_t j = i+1; j < end; j++) { ASSERT_SUF_LT(i, j); } } if(sanityCheck) { sanityCheckOrderedSufs(host1, hlen, s, slen, OFF_MASK, begin, end); } } template static void bucketSortSufDcU8( const T1& host1, const T2& host, size_t hlen, TIndexOffU* s, size_t slen, const DifferenceCoverSample& dc, uint8_t hi, size_t _begin, size_t _end, size_t _depth, bool sanityCheck = false) { // 5 64-element buckets for bucket-sorting A, C, G, T, $ TIndexOffU* bkts[4]; for(size_t i = 0; i < 4; i++) { bkts[i] = new TIndexOffU[4 * 1024 * 1024]; } ELList block_list; bool first = true; while(true) { size_t begin = 0, end = 0; if(first) { begin = _begin; end = _end; first = false; } else { if(block_list.size() == 0) { break; } if(block_list.back().size() > 1) { end = block_list.back().back(); block_list.back().pop_back(); begin = block_list.back().back(); } else { block_list.resize(block_list.size() - 1); if(block_list.size() == 0) { break; } } } size_t depth = block_list.size() + _depth; assert_leq(end-begin, BUCKET_SORT_CUTOFF); assert_eq(hi, 4); if(end <= begin + 1) { // 1-element list already sorted continue; } if(depth > dc.v()) { // Quicksort the remaining suffixes using difference cover // for constant-time comparisons; this is O(k*log(k)) where // k=(end-begin) qsortSufDcU8(host1, host, hlen, s, slen, dc, begin, end, sanityCheck); continue; } if(end-begin <= SELECTION_SORT_CUTOFF) { // Bucket sort remaining items selectionSortSufDcU8(host1, host, hlen, s, slen, dc, hi, begin, end, depth, sanityCheck); if(sanityCheck) { sanityCheckOrderedSufs(host1, hlen, s, slen, OFF_MASK, begin, end); } continue; } size_t cnts[] = { 0, 0, 0, 0, 0 }; for(size_t i = begin; i < end; i++) { size_t off = depth + s[i]; uint8_t c = (off < hlen) ? get_uint8(host, off) : hi; assert_leq(c, 4); if(c == 0) { s[begin + cnts[0]++] = s[i]; } else { bkts[c-1][cnts[c]++] = s[i]; } } assert_eq(cnts[0] + cnts[1] + cnts[2] + cnts[3] + cnts[4], end - begin); size_t cur = begin + cnts[0]; if(cnts[1] > 0) { memcpy(&s[cur], bkts[0], cnts[1] << (OFF_SIZE/4 + 1)); cur += cnts[1]; } if(cnts[2] > 0) { memcpy(&s[cur], bkts[1], cnts[2] << (OFF_SIZE/4 + 1)); cur += cnts[2]; } if(cnts[3] > 0) { memcpy(&s[cur], bkts[2], cnts[3] << (OFF_SIZE/4 + 1)); cur += cnts[3]; } if(cnts[4] > 0) { memcpy(&s[cur], bkts[3], cnts[4] << (OFF_SIZE/4 + 1)); } // This frame is now totally finished with bkts[][], so recursive // callees can safely clobber it; we're not done with cnts[], but // that's local to the stack frame. block_list.expand(); block_list.back().clear(); block_list.back().push_back(begin); for(size_t i = 0; i < 4; i++) { if(cnts[i] > 0) { block_list.back().push_back(block_list.back().back() + cnts[i]); } } } // Done for(size_t i = 0; i < 4; i++) { delete [] bkts[i]; } } /** * Main multikey quicksort function for suffixes. Based on Bentley & * Sedgewick's algorithm on p.5 of their paper "Fast Algorithms for * Sorting and Searching Strings". That algorithm has been extended in * three ways: * * 1. Deal with keys of different lengths by checking bounds and * considering off-the-end values to be 'hi' (b/c our goal is the * BWT transform, we're biased toward considring prefixes as * lexicographically *greater* than their extensions). * 2. The multikey_qsort_suffixes version takes a single host string * and a list of suffix offsets as input. This reduces memory * footprint compared to an approach that treats its input * generically as a set of strings (not necessarily suffixes), thus * requiring that we store at least two integers worth of * information for each string. * 3. Sorting functions take an extra "upto" parameter that upper- * bounds the depth to which the function sorts. */ template void mkeyQSortSufDcU8( const T1& host1, const T2& host, size_t hlen, TIndexOffU* s, size_t slen, const DifferenceCoverSample& dc, int hi, size_t begin, size_t end, size_t depth, bool sanityCheck = false) { // Helper for making the recursive call; sanity-checks arguments to // make sure that the problem actually got smaller. #define MQS_RECURSE_SUF_DC_U8(nbegin, nend, ndepth) { \ assert(nbegin > begin || nend < end || ndepth > depth); \ mkeyQSortSufDcU8(host1, host, hlen, s, slen, dc, hi, nbegin, nend, ndepth, sanityCheck); \ } assert_leq(begin, slen); assert_leq(end, slen); size_t n = end - begin; if(n <= 1) return; // 1-element list already sorted if(depth > dc.v()) { // Quicksort the remaining suffixes using difference cover // for constant-time comparisons; this is O(k*log(k)) where // k=(end-begin) qsortSufDcU8(host1, host, hlen, s, slen, dc, begin, end, sanityCheck); if(sanityCheck) { sanityCheckOrderedSufs(host1, hlen, s, slen, OFF_MASK, begin, end); } return; } if(n <= BUCKET_SORT_CUTOFF) { // Bucket sort remaining items bucketSortSufDcU8(host1, host, hlen, s, slen, dc, (uint8_t)hi, begin, end, depth, sanityCheck); if(sanityCheck) { sanityCheckOrderedSufs(host1, hlen, s, slen, OFF_MASK, begin, end); } return; } size_t a, b, c, d, r; CHOOSE_AND_SWAP_PIVOT(SWAP1, CHAR_AT_SUF_U8); // choose pivot, swap to begin int v = CHAR_AT_SUF_U8(begin, depth); // v <- pivot value #ifndef NDEBUG { bool stillInBounds = false; for(size_t i = begin; i < end; i++) { if(depth < (hlen-s[i])) { stillInBounds = true; break; } else { /* already fell off this suffix */ } } assert(stillInBounds); // >=1 suffix must still be in bounds } #endif a = b = begin; c = d = end-1; while(true) { // Invariant: everything before a is = pivot, everything // between a and b is < int bc = 0; // shouldn't have to init but gcc on Mac complains while(b <= c && v >= (bc = CHAR_AT_SUF_U8(b, depth))) { if(v == bc) { SWAP(s, a, b); a++; } b++; } // Invariant: everything after d is = pivot, everything // between c and d is > int cc = 0; // shouldn't have to init but gcc on Mac complains //bool hiLatch = true; while(b <= c && v <= (cc = CHAR_AT_SUF_U8(c, depth))) { if(v == cc) { SWAP(s, c, d); d--; } //else if(hiLatch && cc == hi) { } c--; } if(b > c) break; SWAP(s, b, c); b++; c--; } assert(a > begin || c < end-1); // there was at least one =s assert_lt(d-c, n); // they can't all have been > pivot assert_lt(b-a, n); // they can't all have been < pivot r = min(a-begin, b-a); VECSWAP(s, begin, b-r, r); // swap left = to center r = min(d-c, end-d-1); VECSWAP(s, b, end-r, r); // swap right = to center r = b-a; // r <- # of <'s if(r > 0) { MQS_RECURSE_SUF_DC_U8(begin, begin + r, depth); // recurse on <'s } // Do not recurse on ='s if the pivot was the off-the-end value; // they're already fully sorted if(v != hi) { MQS_RECURSE_SUF_DC_U8(begin + r, begin + r + (a-begin) + (end-d-1), depth+1); // recurse on ='s } r = d-c; // r <- # of >'s excluding those exhausted if(r > 0 && v < hi-1) { MQS_RECURSE_SUF_DC_U8(end-r, end, depth); // recurse on >'s } } #endif /*MULTIKEY_QSORT_H_*/ hisat2-2.2.1/opts.h000066400000000000000000000163251370663763200140700ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef OPTS_H_ #define OPTS_H_ enum { ARG_ORIG = 256, // --orig ARG_SEED, // --seed ARG_SOLEXA_QUALS, // --solexa-quals ARG_VERBOSE, // --verbose ARG_STARTVERBOSE, // --startverbose ARG_QUIET, // --quiet ARG_METRIC_IVAL, // --met ARG_METRIC_FILE, // --met-file ARG_METRIC_STDERR, // --met-stderr ARG_METRIC_PER_READ, // --met-per-read ARG_REFIDX, // --refidx ARG_SANITY, // --sanity ARG_PARTITION, // --partition ARG_INTEGER_QUALS, // --int-quals ARG_FILEPAR, // --filepar ARG_SHMEM, // --shmem ARG_MM, // --mm ARG_MMSWEEP, // --mmsweep ARG_FF, // --ff ARG_FR, // --fr ARG_RF, // --rf ARG_NO_MIXED, // --no-mixed ARG_NO_DISCORDANT, // --no-discordant ARG_CACHE_LIM, // -- ARG_CACHE_SZ, // -- ARG_NO_FW, // --nofw ARG_NO_RC, // --norc ARG_SKIP, // --skip ARG_ONETWO, // --12 ARG_PHRED64, // --phred64 ARG_PHRED33, // --phred33 ARG_HADOOPOUT, // --hadoopout ARG_FUZZY, // --fuzzy ARG_FULLREF, // --fullref ARG_USAGE, // --usage ARG_SNPPHRED, // --snpphred ARG_SNPFRAC, // --snpfrac ARG_SAM_NO_QNAME_TRUNC, // --sam-no-qname-trunc ARG_SAM_OMIT_SEC_SEQ, // --sam-omit-sec-seq ARG_SAM_NOHEAD, // --sam-noHD/--sam-nohead ARG_SAM_NOSQ, // --sam-nosq/--sam-noSQ ARG_SAM_RG, // --sam-rg ARG_SAM_RGID, // --sam-rg-id ARG_GAP_BAR, // --gbar ARG_QUALS1, // --Q1 ARG_QUALS2, // --Q2 ARG_QSEQ, // --qseq ARG_SEED_SUMM, // --seed-summary ARG_OVERHANG, // --overhang ARG_NO_CACHE, // --no-cache ARG_USE_CACHE, // --cache ARG_NOISY_HPOLY, // --454/--ion-torrent ARG_LOCAL, // --local ARG_END_TO_END, // --end-to-end ARG_SCAN_NARROWED, // --scan-narrowed ARG_QC_FILTER, // --qc-filter ARG_BWA_SW_LIKE, // --bwa-sw-like ARG_MULTISEED_IVAL, // --multiseed ARG_SCORE_MIN, // --score-min ARG_SCORE_MA, // --ma ARG_SCORE_MMP, // --mp ARG_SCORE_SCP, // --sp ARG_NO_SOFTCLIP, // --no-softclip ARG_SCORE_NP, // --nm ARG_SCORE_RDG, // --rdg ARG_SCORE_RFG, // --rfg ARG_N_CEIL, // --n-ceil ARG_DPAD, // --dpad ARG_SAM_PRINT_YI, // --mapq-print-inputs ARG_ALIGN_POLICY, // --policy ARG_PRESET_VERY_FAST, // --very-fast ARG_PRESET_FAST, // --fast ARG_PRESET_SENSITIVE, // --sensitive ARG_PRESET_VERY_SENSITIVE, // --very-sensitive ARG_PRESET_VERY_FAST_LOCAL, // --very-fast-local ARG_PRESET_FAST_LOCAL, // --fast-local ARG_PRESET_SENSITIVE_LOCAL, // --sensitive-local ARG_PRESET_VERY_SENSITIVE_LOCAL, // --very-sensitive-local ARG_NO_SCORE_PRIORITY, // --no-score-priority ARG_IGNORE_QUALS, // --ignore-quals ARG_DESC, // --arg-desc ARG_TAB5, // --tab5 ARG_TAB6, // --tab6 ARG_WRAPPER, // --wrapper ARG_DOVETAIL, // --dovetail ARG_NO_DOVETAIL, // --no-dovetail ARG_CONTAIN, // --contain ARG_NO_CONTAIN, // --no-contain ARG_OVERLAP, // --overlap ARG_NO_OVERLAP, // --no-overlap ARG_MAPQ_V, // --mapq-v ARG_SSE8, // --sse8 ARG_SSE8_NO, // --no-sse8 ARG_UNGAPPED, // --ungapped ARG_UNGAPPED_NO, // --no-ungapped ARG_TIGHTEN, // --tighten ARG_UNGAP_THRESH, // --ungap-thresh ARG_EXACT_UPFRONT, // --exact-upfront ARG_1MM_UPFRONT, // --1mm-upfront ARG_EXACT_UPFRONT_NO, // --no-exact-upfront ARG_1MM_UPFRONT_NO, // --no-1mm-upfront ARG_1MM_MINLEN, // --1mm-minlen ARG_VERSION, // --version ARG_SEED_OFF, // --seed-off ARG_SEED_BOOST_THRESH, // --seed-boost ARG_MAX_SEEDS, ARG_READ_TIMES, // --read-times ARG_EXTEND_ITERS, // --extends ARG_DP_MATE_STREAK_THRESH, // --db-mate-streak ARG_DP_FAIL_STREAK_THRESH, // --dp-fail-streak ARG_UG_FAIL_STREAK_THRESH, // --ug-fail-streak ARG_EE_FAIL_STREAK_THRESH, // --ee-fail-streak ARG_DP_FAIL_THRESH, // --dp-fails ARG_UG_FAIL_THRESH, // --ug-fails ARG_MAPQ_EX, // --mapq-extra ARG_NO_EXTEND, // --no-extend ARG_REORDER, // --reorder ARG_SHOW_RAND_SEED, // --show-rand-seed ARG_READ_PASSTHRU, // --passthrough ARG_SAMPLE, // --sample ARG_CP_MIN, // --cp-min ARG_CP_IVAL, // --cp-ival ARG_TRI, // --tri ARG_LOCAL_SEED_CACHE_SZ, // --local-seed-cache-sz ARG_CURRENT_SEED_CACHE_SZ, // --seed-cache-sz ARG_SAM_NO_UNAL, // --no-unal ARG_NON_DETERMINISTIC, // --non-deterministic ARG_TEST_25, // --test-25 ARG_DESC_KB, // --desc-kb ARG_DESC_LANDING, // --desc-landing ARG_DESC_EXP, // --desc-exp ARG_DESC_FMOPS, // --desc-fmops ARG_NO_TEMPSPLICESITE, ARG_PEN_CANSPLICE, ARG_PEN_NONCANSPLICE, ARG_PEN_CONFLICTSPLICE, ARG_PEN_CANINTRONLEN, ARG_PEN_NONCANINTRONLEN, ARG_MIN_INTRONLEN, ARG_MAX_INTRONLEN, ARG_KNOWN_SPLICESITE_INFILE, ARG_NOVEL_SPLICESITE_INFILE, ARG_NOVEL_SPLICESITE_OUTFILE, ARG_SECONDARY, ARG_NO_SPLICED_ALIGNMENT, ARG_RNA_STRANDNESS, ARG_SPLICESITE_DB_ONLY, ARG_NO_ANCHORSTOP, ARG_TRANSCRIPTOME_MAPPING_ONLY, ARG_TRANSCRIPTOME_ASSEMBLY, ARG_TRANSCRIPTOME_ASSEMBLY_CUFFLINKS, ARG_AVOID_PSEUDOGENE, #ifdef USE_SRA ARG_SRA_ACC, #endif ARG_REMOVE_CHRNAME, ARG_ADD_CHRNAME, ARG_MAX_ALTSTRIED, ARG_HAPLOTYPE, ARG_CODIS, ARG_NO_TEMPLATELEN_ADJUSTMENT, ARG_SUMMARY_FILE, ARG_NEW_SUMMARY, ARG_DP, ARG_REPEAT, ARG_NO_REPEAT_INDEX, ARG_READ_LENGTHS }; #endif hisat2-2.2.1/outq.cpp000066400000000000000000000125701370663763200144240ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "outq.h" /** * Caller is telling us that they're about to write output record(s) for * the read with the given id. */ void OutputQueue::beginRead(TReadId rdid, size_t threadId) { ThreadSafe t(&mutex_m, threadSafe_); nstarted_++; if(reorder_) { assert_geq(rdid, cur_); assert_eq(lines_.size(), finished_.size()); assert_eq(lines_.size(), started_.size()); if(rdid - cur_ >= lines_.size()) { // Make sure there's enough room in lines_, started_ and finished_ size_t oldsz = lines_.size(); lines_.resize(rdid - cur_ + 1); started_.resize(rdid - cur_ + 1); finished_.resize(rdid - cur_ + 1); for(size_t i = oldsz; i < lines_.size(); i++) { started_[i] = finished_[i] = false; } } started_[rdid - cur_] = true; finished_[rdid - cur_] = false; } } /** * Writer is finished writing to */ void OutputQueue::finishRead(const BTString& rec, TReadId rdid, size_t threadId) { ThreadSafe t(&mutex_m, threadSafe_); if(reorder_) { assert_geq(rdid, cur_); assert_eq(lines_.size(), finished_.size()); assert_eq(lines_.size(), started_.size()); assert_lt(rdid - cur_, lines_.size()); assert(started_[rdid - cur_]); assert(!finished_[rdid - cur_]); lines_[rdid - cur_] = rec; nfinished_++; finished_[rdid - cur_] = true; flush(false, false); // don't force; already have lock } else { // obuf_ is the OutFileBuf for the output file obuf_.writeString(rec); nfinished_++; nflushed_++; } } /** * Write already-finished lines starting from cur_. */ void OutputQueue::flush(bool force, bool getLock) { if(!reorder_) { return; } ThreadSafe t(&mutex_m, getLock && threadSafe_); size_t nflush = 0; while(nflush < finished_.size() && finished_[nflush]) { assert(started_[nflush]); nflush++; } // Waiting until we have several in a row to flush cuts down on copies // (but requires more buffering) if(force || nflush >= NFLUSH_THRESH) { for(size_t i = 0; i < nflush; i++) { assert(started_[i]); assert(finished_[i]); obuf_.writeString(lines_[i]); } lines_.erase(0, nflush); started_.erase(0, nflush); finished_.erase(0, nflush); cur_ += nflush; nflushed_ += nflush; } } #ifdef OUTQ_MAIN #include using namespace std; int main(void) { cerr << "Case 1 (one thread) ... "; { OutFileBuf ofb; OutputQueue oq(ofb, false); assert_eq(0, oq.numFlushed()); assert_eq(0, oq.numStarted()); assert_eq(0, oq.numFinished()); oq.beginRead(1); assert_eq(0, oq.numFlushed()); assert_eq(1, oq.numStarted()); assert_eq(0, oq.numFinished()); oq.beginRead(3); assert_eq(0, oq.numFlushed()); assert_eq(2, oq.numStarted()); assert_eq(0, oq.numFinished()); oq.beginRead(2); assert_eq(0, oq.numFlushed()); assert_eq(3, oq.numStarted()); assert_eq(0, oq.numFinished()); oq.flush(); assert_eq(0, oq.numFlushed()); assert_eq(3, oq.numStarted()); assert_eq(0, oq.numFinished()); oq.beginRead(0); assert_eq(0, oq.numFlushed()); assert_eq(4, oq.numStarted()); assert_eq(0, oq.numFinished()); oq.flush(); assert_eq(0, oq.numFlushed()); assert_eq(4, oq.numStarted()); assert_eq(0, oq.numFinished()); oq.finishRead(0); assert_eq(0, oq.numFlushed()); assert_eq(4, oq.numStarted()); assert_eq(1, oq.numFinished()); oq.flush(); assert_eq(0, oq.numFlushed()); assert_eq(4, oq.numStarted()); assert_eq(1, oq.numFinished()); oq.flush(true); assert_eq(1, oq.numFlushed()); assert_eq(4, oq.numStarted()); assert_eq(1, oq.numFinished()); oq.finishRead(2); assert_eq(1, oq.numFlushed()); assert_eq(4, oq.numStarted()); assert_eq(2, oq.numFinished()); oq.flush(true); assert_eq(1, oq.numFlushed()); assert_eq(4, oq.numStarted()); assert_eq(2, oq.numFinished()); oq.finishRead(1); assert_eq(1, oq.numFlushed()); assert_eq(4, oq.numStarted()); assert_eq(3, oq.numFinished()); oq.flush(true); assert_eq(3, oq.numFlushed()); assert_eq(4, oq.numStarted()); assert_eq(3, oq.numFinished()); } cerr << "PASSED" << endl; cerr << "Case 2 (one thread) ... "; { OutFileBuf ofb; OutputQueue oq(ofb, false); BTString& buf1 = oq.beginRead(0); BTString& buf2 = oq.beginRead(1); BTString& buf3 = oq.beginRead(2); BTString& buf4 = oq.beginRead(3); BTString& buf5 = oq.beginRead(4); assert_eq(5, oq.numStarted()); assert_eq(0, oq.numFinished()); buf1.install("A\n"); buf2.install("B\n"); buf3.install("C\n"); buf4.install("D\n"); buf5.install("E\n"); oq.finishRead(4); oq.finishRead(1); oq.finishRead(0); oq.finishRead(2); oq.finishRead(3); oq.flush(true); assert_eq(5, oq.numFlushed()); assert_eq(5, oq.numStarted()); assert_eq(5, oq.numFinished()); ofb.flush(); } cerr << "PASSED" << endl; return 0; } #endif /*def ALN_SINK_MAIN*/ hisat2-2.2.1/outq.h000066400000000000000000000063671370663763200141000ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef OUTQ_H_ #define OUTQ_H_ #include "assert_helpers.h" #include "ds.h" #include "sstring.h" #include "read.h" #include "threading.h" #include "mem_ids.h" /** * Encapsulates a list of lines of output. If the earliest as-yet-unreported * read has id N and Bowtie 2 wants to write a record for read with id N+1, we * resize the lines_ and committed_ lists to have at least 2 elements (1 for N, * 1 for N+1) and return the BTString * associated with the 2nd element. When * the user calls commit() for the read with id N, */ class OutputQueue { static const size_t NFLUSH_THRESH = 8; public: OutputQueue( OutFileBuf& obuf, bool reorder, size_t nthreads, bool threadSafe, TReadId rdid = 0) : obuf_(obuf), cur_(rdid), nstarted_(0), nfinished_(0), nflushed_(0), lines_(RES_CAT), started_(RES_CAT), finished_(RES_CAT), reorder_(reorder), threadSafe_(threadSafe), mutex_m() { assert(nthreads <= 1 || threadSafe); } /** * Caller is telling us that they're about to write output record(s) for * the read with the given id. */ void beginRead(TReadId rdid, size_t threadId); /** * Writer is finished writing to */ void finishRead(const BTString& rec, TReadId rdid, size_t threadId); /** * Return the number of records currently being buffered. */ size_t size() const { return lines_.size(); } /** * Return the number of records that have been flushed so far. */ TReadId numFlushed() const { return nflushed_; } /** * Return the number of records that have been started so far. */ TReadId numStarted() const { return nstarted_; } /** * Return the number of records that have been finished so far. */ TReadId numFinished() const { return nfinished_; } /** * Write already-committed lines starting from cur_. */ void flush(bool force = false, bool getLock = true); protected: OutFileBuf& obuf_; TReadId cur_; TReadId nstarted_; TReadId nfinished_; TReadId nflushed_; EList lines_; EList started_; EList finished_; bool reorder_; bool threadSafe_; MUTEX_T mutex_m; }; class OutputQueueMark { public: OutputQueueMark( OutputQueue& q, const BTString& rec, TReadId rdid, size_t threadId) : q_(q), rec_(rec), rdid_(rdid), threadId_(threadId) { q_.beginRead(rdid, threadId); } ~OutputQueueMark() { q_.finishRead(rec_, rdid_, threadId_); } protected: OutputQueue& q_; const BTString& rec_; TReadId rdid_; size_t threadId_; }; #endif hisat2-2.2.1/pat.cpp000066400000000000000000001345011370663763200142170ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include #include #include #include "sstring.h" #include "pat.h" #include "filebuf.h" #include "formats.h" #ifdef USE_SRA #include "tinythread.h" #include #include #include #include #include #endif using namespace std; /** * Return a new dynamically allocated PatternSource for the given * format, using the given list of strings as the filenames to read * from or as the sequences themselves (i.e. if -c was used). */ PatternSource* PatternSource::patsrcFromStrings( const PatternParams& p, const EList& qs, size_t nthreads) { switch(p.format) { case FASTA: return new FastaPatternSource(qs, p); case FASTA_CONT: return new FastaContinuousPatternSource(qs, p); case RAW: return new RawPatternSource(qs, p); case FASTQ: return new FastqPatternSource(qs, p); case TAB_MATE5: return new TabbedPatternSource(qs, p, false); case TAB_MATE6: return new TabbedPatternSource(qs, p, true); case CMDLINE: return new VectorPatternSource(qs, p); case QSEQ: return new QseqPatternSource(qs, p); #ifdef USE_SRA case SRA_FASTA: case SRA_FASTQ: return new SRAPatternSource(qs, p, nthreads); #endif default: { cerr << "Internal error; bad patsrc format: " << p.format << endl; throw 1; } } } /** * The main member function for dispensing patterns. * * Returns true iff a pair was parsed succesfully. */ bool PatternSource::nextReadPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired, bool fixName) { // nextPatternImpl does the reading from the ultimate source; // it is implemented in concrete subclasses success = done = paired = false; nextReadPairImpl(ra, rb, rdid, endid, success, done, paired); if(success) { // Construct reversed versions of fw and rc seqs/quals ra.finalize(); if(!rb.empty()) { rb.finalize(); } // Fill in the random-seed field using a combination of // information from the user-specified seed and the read // sequence, qualities, and name ra.seed = genRandSeed(ra.patFw, ra.qual, ra.name, seed_); if(!rb.empty()) { rb.seed = genRandSeed(rb.patFw, rb.qual, rb.name, seed_); } } return success; } /** * The main member function for dispensing patterns. */ bool PatternSource::nextRead( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { // nextPatternImpl does the reading from the ultimate source; // it is implemented in concrete subclasses nextReadImpl(r, rdid, endid, success, done); if(success) { // Construct the reversed versions of the fw and rc seqs // and quals r.finalize(); // Fill in the random-seed field using a combination of // information from the user-specified seed and the read // sequence, qualities, and name r.seed = genRandSeed(r.patFw, r.qual, r.name, seed_); } return success; } /** * Get the next paired or unpaired read from the wrapped * PairedPatternSource. */ bool WrappedPatternSourcePerThread::nextReadPair( bool& success, bool& done, bool& paired, bool fixName) { PatternSourcePerThread::nextReadPair(success, done, paired, fixName); ASSERT_ONLY(TReadId lastRdId = rdid_); buf1_.reset(); buf2_.reset(); patsrc_.nextReadPair(buf1_, buf2_, rdid_, endid_, success, done, paired, fixName); assert(!success || rdid_ != lastRdId); return success; } /** * The main member function for dispensing pairs of reads or * singleton reads. Returns true iff ra and rb contain a new * pair; returns false if ra contains a new unpaired read. */ bool PairedSoloPatternSource::nextReadPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired, bool fixName) { uint32_t cur = cur_; success = false; while(cur < src_->size()) { // Patterns from srca_[cur_] are unpaired do { (*src_)[cur]->nextReadPair( ra, rb, rdid, endid, success, done, paired, fixName); } while(!success && !done); if(!success) { assert(done); // If patFw is empty, that's our signal that the // input dried up lock(); if(cur + 1 > cur_) cur_++; cur = cur_; unlock(); continue; // on to next pair of PatternSources } assert(success); ra.seed = genRandSeed(ra.patFw, ra.qual, ra.name, seed_); if(!rb.empty()) { rb.seed = genRandSeed(rb.patFw, rb.qual, rb.name, seed_); if(fixName) { ra.fixMateName(1); rb.fixMateName(2); } } ra.rdid = rdid; ra.endid = endid; if(!rb.empty()) { rb.rdid = rdid; rb.endid = endid+1; } ra.mate = 1; rb.mate = 2; return true; // paired } assert_leq(cur, src_->size()); done = (cur == src_->size()); return false; } /** * The main member function for dispensing pairs of reads or * singleton reads. Returns true iff ra and rb contain a new * pair; returns false if ra contains a new unpaired read. */ bool PairedDualPatternSource::nextReadPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired, bool fixName) { // 'cur' indexes the current pair of PatternSources uint32_t cur; { lock(); cur = cur_; unlock(); } success = false; done = true; while(cur < srca_->size()) { if((*srcb_)[cur] == NULL) { paired = false; // Patterns from srca_ are unpaired do { (*srca_)[cur]->nextRead(ra, rdid, endid, success, done); } while(!success && !done); if(!success) { assert(done); lock(); if(cur + 1 > cur_) cur_++; cur = cur_; // Move on to next PatternSource unlock(); continue; // on to next pair of PatternSources } ra.rdid = rdid; ra.endid = endid; ra.mate = 0; return success; } else { paired = true; // Patterns from srca_[cur_] and srcb_[cur_] are paired TReadId rdid_a = 0, endid_a = 0; TReadId rdid_b = 0, endid_b = 0; bool success_a = false, done_a = false; bool success_b = false, done_b = false; // Lock to ensure that this thread gets parallel reads // in the two mate files lock(); do { (*srca_)[cur]->nextRead(ra, rdid_a, endid_a, success_a, done_a); } while(!success_a && !done_a); do { (*srcb_)[cur]->nextRead(rb, rdid_b, endid_b, success_b, done_b); } while(!success_b && !done_b); if(!success_a && success_b) { cerr << "Error, fewer reads in file specified with -1 than in file specified with -2" << endl; throw 1; } else if(!success_a) { assert(done_a && done_b); if(cur + 1 > cur_) cur_++; cur = cur_; // Move on to next PatternSource unlock(); continue; // on to next pair of PatternSources } else if(!success_b) { cerr << "Error, fewer reads in file specified with -2 than in file specified with -1" << endl; throw 1; } assert_eq(rdid_a, rdid_b); //assert_eq(endid_a+1, endid_b); assert_eq(success_a, success_b); unlock(); if(fixName) { ra.fixMateName(1); rb.fixMateName(2); } rdid = rdid_a; endid = endid_a; success = success_a; done = done_a; ra.rdid = rdid; ra.endid = endid; if(!rb.empty()) { rb.rdid = rdid; rb.endid = endid+1; } ra.mate = 1; rb.mate = 2; return success; } } return success; } /** * Return the number of reads attempted. */ pair PairedDualPatternSource::readCnt() const { uint64_t rets = 0llu, retp = 0llu; for(size_t i = 0; i < srca_->size(); i++) { if((*srcb_)[i] == NULL) { rets += (*srca_)[i]->readCnt(); } else { assert_eq((*srca_)[i]->readCnt(), (*srcb_)[i]->readCnt()); retp += (*srca_)[i]->readCnt(); } } return make_pair(rets, retp); } /** * Given the values for all of the various arguments used to specify * the read and quality input, create a list of pattern sources to * dispense them. */ PairedPatternSource* PairedPatternSource::setupPatternSources( const EList& si, // singles, from argv const EList& m1, // mate1's, from -1 arg const EList& m2, // mate2's, from -2 arg const EList& m12, // both mates on each line, from --12 arg #ifdef USE_SRA const EList& sra_accs, #endif const EList& q, // qualities associated with singles const EList& q1, // qualities associated with m1 const EList& q2, // qualities associated with m2 const PatternParams& p, // read-in parameters size_t nthreads, bool verbose) // be talkative? { EList* a = new EList(); EList* b = new EList(); EList* ab = new EList(); // Create list of pattern sources for paired reads appearing // interleaved in a single file for(size_t i = 0; i < m12.size(); i++) { const EList* qs = &m12; EList tmp; if(p.fileParallel) { // Feed query files one to each PatternSource qs = &tmp; tmp.push_back(m12[i]); assert_eq(1, tmp.size()); } ab->push_back(PatternSource::patsrcFromStrings(p, *qs, nthreads)); if(!p.fileParallel) { break; } } #ifdef USE_SRA for(size_t i = 0; i < sra_accs.size(); i++) { const EList* qs = &sra_accs; EList tmp; if(p.fileParallel) { // Feed query files one to each PatternSource qs = &tmp; tmp.push_back(sra_accs[i]); assert_eq(1, tmp.size()); } ab->push_back(PatternSource::patsrcFromStrings(p, *qs, nthreads)); if(!p.fileParallel) { break; } } #endif // Create list of pattern sources for paired reads for(size_t i = 0; i < m1.size(); i++) { const EList* qs = &m1; EList tmpSeq; EList tmpQual; if(p.fileParallel) { // Feed query files one to each PatternSource qs = &tmpSeq; tmpSeq.push_back(m1[i]); assert_eq(1, tmpSeq.size()); } a->push_back(PatternSource::patsrcFromStrings(p, *qs, nthreads)); if(!p.fileParallel) { break; } } // Create list of pattern sources for paired reads for(size_t i = 0; i < m2.size(); i++) { const EList* qs = &m2; EList tmpSeq; EList tmpQual; if(p.fileParallel) { // Feed query files one to each PatternSource qs = &tmpSeq; tmpSeq.push_back(m2[i]); assert_eq(1, tmpSeq.size()); } b->push_back(PatternSource::patsrcFromStrings(p, *qs, nthreads)); if(!p.fileParallel) { break; } } // All mates/mate files must be paired assert_eq(a->size(), b->size()); // Create list of pattern sources for the unpaired reads for(size_t i = 0; i < si.size(); i++) { const EList* qs = &si; PatternSource* patsrc = NULL; EList tmpSeq; EList tmpQual; if(p.fileParallel) { // Feed query files one to each PatternSource qs = &tmpSeq; tmpSeq.push_back(si[i]); assert_eq(1, tmpSeq.size()); } patsrc = PatternSource::patsrcFromStrings(p, *qs, nthreads); assert(patsrc != NULL); a->push_back(patsrc); b->push_back(NULL); if(!p.fileParallel) { break; } } PairedPatternSource *patsrc = NULL; #ifdef USE_SRA if(m12.size() > 0 || sra_accs.size() > 0) { #else if(m12.size() > 0) { #endif patsrc = new PairedSoloPatternSource(ab, p); for(size_t i = 0; i < a->size(); i++) delete (*a)[i]; for(size_t i = 0; i < b->size(); i++) delete (*b)[i]; delete a; delete b; } else { patsrc = new PairedDualPatternSource(a, b, p); for(size_t i = 0; i < ab->size(); i++) delete (*ab)[i]; delete ab; } return patsrc; } VectorPatternSource::VectorPatternSource( const EList& v, const PatternParams& p) : PatternSource(p), cur_(p.skip), skip_(p.skip), paired_(false), v_(), quals_() { for(size_t i = 0; i < v.size(); i++) { EList ss; tokenize(v[i], ":", ss, 2); assert_gt(ss.size(), 0); assert_leq(ss.size(), 2); // Initialize s string s = ss[0]; int mytrim5 = gTrim5; if(gColor && s.length() > 1) { // This may be a primer character. If so, keep it in the // 'primer' field of the read buf and parse the rest of the // read without it. int c = toupper(s[0]); if(asc2dnacat[c] > 0) { // First char is a DNA char int c2 = toupper(s[1]); // Second char is a color char if(asc2colcat[c2] > 0) { mytrim5 += 2; // trim primer and first color } } } if(gColor) { // Convert '0'-'3' to 'A'-'T' for(size_t i = 0; i < s.length(); i++) { if(s[i] >= '0' && s[i] <= '4') { s[i] = "ACGTN"[(int)s[i] - '0']; } if(s[i] == '.') s[i] = 'N'; } } if(s.length() <= (size_t)(gTrim3 + mytrim5)) { // Entire read is trimmed away s.clear(); } else { // Trim on 5' (high-quality) end if(mytrim5 > 0) { s.erase(0, mytrim5); } // Trim on 3' (low-quality) end if(gTrim3 > 0) { s.erase(s.length()-gTrim3); } } // Initialize vq string vq; if(ss.size() == 2) { vq = ss[1]; } // Trim qualities if(vq.length() > (size_t)(gTrim3 + mytrim5)) { // Trim on 5' (high-quality) end if(mytrim5 > 0) { vq.erase(0, mytrim5); } // Trim on 3' (low-quality) end if(gTrim3 > 0) { vq.erase(vq.length()-gTrim3); } } // Pad quals with Is if necessary; this shouldn't happen while(vq.length() < s.length()) { vq.push_back('I'); } // Truncate quals to match length of read if necessary; // this shouldn't happen if(vq.length() > s.length()) { vq.erase(s.length()); } assert_eq(vq.length(), s.length()); v_.expand(); v_.back().installChars(s); quals_.push_back(BTString(vq)); trimmed3_.push_back(gTrim3); trimmed5_.push_back(mytrim5); ostringstream os; os << (names_.size()); names_.push_back(BTString(os.str())); } assert_eq(v_.size(), quals_.size()); } bool VectorPatternSource::nextReadImpl( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { // Let Strings begin at the beginning of the respective bufs r.reset(); lock(); if(cur_ >= v_.size()) { unlock(); // Clear all the Strings, as a signal to the caller that // we're out of reads r.reset(); success = false; done = true; assert(r.empty()); return false; } // Copy v_*, quals_* strings into the respective Strings r.color = gColor; r.patFw = v_[cur_]; r.qual = quals_[cur_]; r.trimmed3 = trimmed3_[cur_]; r.trimmed5 = trimmed5_[cur_]; ostringstream os; os << cur_; r.name = os.str(); cur_++; done = cur_ == v_.size(); rdid = endid = readCnt_; readCnt_++; unlock(); success = true; return true; } /** * This is unused, but implementation is given for completeness. */ bool VectorPatternSource::nextReadPairImpl( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) { // Let Strings begin at the beginning of the respective bufs ra.reset(); rb.reset(); paired = true; if(!paired_) { paired_ = true; cur_ <<= 1; } lock(); if(cur_ >= v_.size()-1) { unlock(); // Clear all the Strings, as a signal to the caller that // we're out of reads ra.reset(); rb.reset(); assert(ra.empty()); assert(rb.empty()); success = false; done = true; return false; } // Copy v_*, quals_* strings into the respective Strings ra.patFw = v_[cur_]; ra.qual = quals_[cur_]; ra.trimmed3 = trimmed3_[cur_]; ra.trimmed5 = trimmed5_[cur_]; cur_++; rb.patFw = v_[cur_]; rb.qual = quals_[cur_]; rb.trimmed3 = trimmed3_[cur_]; rb.trimmed5 = trimmed5_[cur_]; ostringstream os; os << readCnt_; ra.name = os.str(); rb.name = os.str(); ra.color = rb.color = gColor; cur_++; done = cur_ >= v_.size()-1; rdid = endid = readCnt_; readCnt_++; unlock(); success = true; return true; } /** * Parse a single quality string from fb and store qualities in r. * Assume the next character obtained via fb.get() is the first * character of the quality string. When returning, the next * character returned by fb.peek() or fb.get() should be the first * character of the following line. */ int parseQuals( Read& r, FileBuf& fb, int firstc, int readLen, int trim3, int trim5, bool intQuals, bool phred64, bool solexa64) { int c = firstc; assert(c != '\n' && c != '\r'); r.qual.clear(); if (intQuals) { while (c != '\r' && c != '\n' && c != -1) { bool neg = false; int num = 0; while(!isspace(c) && !fb.eof()) { if(c == '-') { neg = true; assert_eq(num, 0); } else { if(!isdigit(c)) { char buf[2048]; cerr << "Warning: could not parse quality line:" << endl; fb.getPastNewline(); cerr << fb.copyLastN(buf); buf[2047] = '\0'; cerr << buf; throw 1; } assert(isdigit(c)); num *= 10; num += (c - '0'); } c = fb.get(); } if(neg) num = 0; // Phred-33 ASCII encode it and add it to the back of the // quality string r.qual.append('!' + num); // Skip over next stretch of whitespace while(c != '\r' && c != '\n' && isspace(c) && !fb.eof()) { c = fb.get(); } } } else { while (c != '\r' && c != '\n' && c != -1) { r.qual.append(charToPhred33(c, solexa64, phred64)); c = fb.get(); while(c != '\r' && c != '\n' && isspace(c) && !fb.eof()) { c = fb.get(); } } } if ((int)r.qual.length() < readLen-1 || ((int)r.qual.length() < readLen && !r.color)) { tooFewQualities(r.name); } r.qual.trimEnd(trim3); if(r.qual.length()-trim5 < r.patFw.length()) { assert(gColor && r.primer != -1); assert_gt(trim5, 0); trim5--; } r.qual.trimBegin(trim5); if(r.qual.length() <= 0) return 0; assert_eq(r.qual.length(), r.patFw.length()); while(fb.peek() == '\n' || fb.peek() == '\r') fb.get(); return (int)r.qual.length(); } /// Read another pattern from a FASTA input file bool FastaPatternSource::read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { int c, qc = 0; success = true; done = false; assert(fb_.isOpen()); r.reset(); r.color = gColor; // Pick off the first carat c = fb_.get(); if(c < 0) { bail(r); success = false; done = true; return success; } while(c == '#' || c == ';' || c == '\r' || c == '\n') { c = fb_.peekUptoNewline(); fb_.resetLastN(); c = fb_.get(); } assert_eq(1, fb_.lastNLen()); // Pick off the first carat if(first_) { if(c != '>') { cerr << "Error: reads file does not look like a FASTA file" << endl; throw 1; } first_ = false; } assert_eq('>', c); c = fb_.get(); // get next char after '>' // Read to the end of the id line, sticking everything after the '>' // into *name //bool warning = false; while(true) { if(c < 0 || qc < 0) { bail(r); success = false; done = true; return success; } if(c == '\n' || c == '\r') { // Break at end of line, after consuming all \r's, \n's while(c == '\n' || c == '\r') { if(fb_.peek() == '>') { // Empty sequence break; } c = fb_.get(); if(c < 0 || qc < 0) { bail(r); success = false; done = true; return success; } } break; } r.name.append(c); if(fb_.peek() == '>') { // Empty sequence break; } c = fb_.get(); } if(c == '>') { // Empty sequences! cerr << "Warning: skipping empty FASTA read with name '" << r.name << "'" << endl; fb_.resetLastN(); rdid = endid = readCnt_; readCnt_++; success = true; done = false; return success; } assert_neq('>', c); // _in now points just past the first character of a sequence // line, and c holds the first character int begin = 0; int mytrim5 = gTrim5; if(gColor) { // This is the primer character, keep it in the // 'primer' field of the read buf and keep parsing c = toupper(c); if(asc2dnacat[c] > 0) { // First char is a DNA char int c2 = toupper(fb_.peek()); if(asc2colcat[c2] > 0) { // Second char is a color char r.primer = c; r.trimc = c2; mytrim5 += 2; } } if(c < 0) { bail(r); success = false; done = true; return success; } } while(c != '>' && c >= 0) { if(gColor) { if(c >= '0' && c <= '4') c = "ACGTN"[(int)c - '0']; if(c == '.') c = 'N'; } if(asc2dnacat[c] > 0 && begin++ >= mytrim5) { r.patFw.append(asc2dna[c]); r.qual.append('I'); } if(fb_.peek() == '>') break; c = fb_.get(); } r.patFw.trimEnd(gTrim3); r.qual.trimEnd(gTrim3); r.trimmed3 = gTrim3; r.trimmed5 = mytrim5; // Set up a default name if one hasn't been set if(r.name.empty()) { char cbuf[20]; itoa10(readCnt_, cbuf); r.name.install(cbuf); } assert_gt(r.name.length(), 0); r.readOrigBuf.install(fb_.lastN(), fb_.lastNLen()); fb_.resetLastN(); rdid = endid = readCnt_; readCnt_++; return success; } /// Read another pattern from a FASTQ input file bool FastqPatternSource::read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { int c; int dstLen = 0; success = true; done = false; r.reset(); r.color = gColor; r.fuzzy = fuzzy_; // Pick off the first at if(first_) { c = fb_.get(); if(c != '@') { c = getOverNewline(fb_); if(c < 0) { bail(r); success = false; done = true; return success; } } if(c != '@') { cerr << "Error: reads file does not look like a FASTQ file" << endl; throw 1; } assert_eq('@', c); first_ = false; } // Read to the end of the id line, sticking everything after the '@' // into *name while(true) { c = fb_.get(); if(c < 0) { bail(r); success = false; done = true; return success; } if(c == '\n' || c == '\r') { // Break at end of line, after consuming all \r's, \n's while(c == '\n' || c == '\r') { c = fb_.get(); if(c < 0) { bail(r); success = false; done = true; return success; } } break; } r.name.append(c); } // fb_ now points just past the first character of a // sequence line, and c holds the first character int charsRead = 0; BTDnaString *sbuf = &r.patFw; int dstLens[] = {0, 0, 0, 0}; int *dstLenCur = &dstLens[0]; int mytrim5 = gTrim5; int altBufIdx = 0; if(gColor && c != '+') { // This may be a primer character. If so, keep it in the // 'primer' field of the read buf and parse the rest of the // read without it. c = toupper(c); if(asc2dnacat[c] > 0) { // First char is a DNA char int c2 = toupper(fb_.peek()); // Second char is a color char if(asc2colcat[c2] > 0) { r.primer = c; r.trimc = c2; mytrim5 += 2; // trim primer and first color } } if(c < 0) { bail(r); success = false; done = true; return success; } } int trim5 = 0; if(c != '+') { trim5 = mytrim5; while(c != '+') { // Convert color numbers to letters if necessary if(c == '.') c = 'N'; if(gColor) { if(c >= '0' && c <= '4') c = "ACGTN"[(int)c - '0']; } if(fuzzy_ && c == '-') c = 'A'; if(isalpha(c)) { // If it's past the 5'-end trim point if(charsRead >= trim5) { sbuf->append(asc2dna[c]); (*dstLenCur)++; } charsRead++; } else if(fuzzy_ && c == ' ') { trim5 = 0; // disable 5' trimming for now if(charsRead == 0) { c = fb_.get(); continue; } charsRead = 0; if(altBufIdx >= 3) { cerr << "At most 3 alternate sequence strings permitted; offending read: " << r.name << endl; throw 1; } // Move on to the next alternate-sequence buffer sbuf = &r.altPatFw[altBufIdx++]; dstLenCur = &dstLens[altBufIdx]; } c = fb_.get(); if(c < 0) { bail(r); success = false; done = true; return success; } } dstLen = dstLens[0]; charsRead = dstLen + mytrim5; } // Trim from 3' end if(gTrim3 > 0) { if((int)r.patFw.length() > gTrim3) { r.patFw.resize(r.patFw.length() - gTrim3); dstLen -= gTrim3; assert_eq((int)r.patFw.length(), dstLen); } else { // Trimmed the whole read; we won't be using this read, // but we proceed anyway so that fb_ is advanced // properly r.patFw.clear(); dstLen = 0; } } assert_eq('+', c); // Chew up the optional name on the '+' line ASSERT_ONLY(int pk =) peekToEndOfLine(fb_); if(charsRead == 0) { assert_eq('@', pk); fb_.get(); fb_.resetLastN(); rdid = endid = readCnt_; readCnt_++; return success; } // Now read the qualities if (intQuals_) { assert(!fuzzy_); int qualsRead = 0; char buf[4096]; if(gColor && r.primer != -1) { // In case the original quality string is one shorter mytrim5--; } qualToks_.clear(); tokenizeQualLine(fb_, buf, 4096, qualToks_); for(unsigned int j = 0; j < qualToks_.size(); ++j) { char c = intToPhred33(atoi(qualToks_[j].c_str()), solQuals_); assert_geq(c, 33); if (qualsRead >= mytrim5) { r.qual.append(c); } ++qualsRead; } // done reading integer quality lines if(gColor && r.primer != -1) mytrim5++; r.qual.trimEnd(gTrim3); if(r.qual.length() < r.patFw.length()) { tooFewQualities(r.name); } else if(r.qual.length() > r.patFw.length() + 1) { tooManyQualities(r.name); } if(r.qual.length() == r.patFw.length()+1 && gColor && r.primer != -1) { r.qual.remove(0); } // Trim qualities on 3' end if(r.qual.length() > r.patFw.length()) { r.qual.resize(r.patFw.length()); assert_eq((int)r.qual.length(), dstLen); } peekOverNewline(fb_); } else { // Non-integer qualities altBufIdx = 0; trim5 = mytrim5; int qualsRead[4] = {0, 0, 0, 0}; int *qualsReadCur = &qualsRead[0]; BTString *qbuf = &r.qual; if(gColor && r.primer != -1) { // In case the original quality string is one shorter trim5--; } while(true) { c = fb_.get(); if (!fuzzy_ && c == ' ') { wrongQualityFormat(r.name); } else if(c == ' ') { trim5 = 0; // disable 5' trimming for now if((*qualsReadCur) == 0) continue; if(altBufIdx >= 3) { cerr << "At most 3 alternate quality strings permitted; offending read: " << r.name << endl; throw 1; } qbuf = &r.altQual[altBufIdx++]; qualsReadCur = &qualsRead[altBufIdx]; continue; } if(c < 0) { break; // let the file end just at the end of a quality line //bail(r); success = false; done = true; return success; } if (c != '\r' && c != '\n') { if (*qualsReadCur >= trim5) { c = charToPhred33(c, solQuals_, phred64Quals_); assert_geq(c, 33); qbuf->append(c); } (*qualsReadCur)++; } else { break; } } qualsRead[0] -= gTrim3; r.qual.trimEnd(gTrim3); if(r.qual.length() < r.patFw.length()) { tooFewQualities(r.name); } else if(r.qual.length() > r.patFw.length()+1) { tooManyQualities(r.name); } if(r.qual.length() == r.patFw.length()+1 && gColor && r.primer != -1) { r.qual.remove(0); } if(fuzzy_) { // Trim from 3' end of alternate basecall and quality strings if(gTrim3 > 0) { for(int i = 0; i < 3; i++) { assert_eq(r.altQual[i].length(), r.altPatFw[i].length()); if((int)r.altQual[i].length() > gTrim3) { r.altPatFw[i].resize(gTrim3); r.altQual[i].resize(gTrim3); } else { r.altPatFw[i].clear(); r.altQual[i].clear(); } qualsRead[i+1] = dstLens[i+1] = max(0, dstLens[i+1] - gTrim3); } } // Shift to RHS, and install in Strings assert_eq(0, r.alts); for(int i = 1; i < 4; i++) { if(qualsRead[i] == 0) continue; if(qualsRead[i] > dstLen) { // Shift everybody up int shiftAmt = qualsRead[i] - dstLen; for(int j = 0; j < dstLen; j++) { r.altQual[i-1].set(r.altQual[i-1][j+shiftAmt], j); r.altPatFw[i-1].set(r.altPatFw[i-1][j+shiftAmt], j); } r.altQual[i-1].resize(dstLen); r.altPatFw[i-1].resize(dstLen); } else if (qualsRead[i] < dstLen) { r.altQual[i-1].resize(dstLen); r.altPatFw[i-1].resize(dstLen); // Shift everybody down int shiftAmt = dstLen - qualsRead[i]; for(int j = dstLen-1; j >= shiftAmt; j--) { r.altQual[i-1].set(r.altQual[i-1][j-shiftAmt], j); r.altPatFw[i-1].set(r.altPatFw[i-1][j-shiftAmt], j); } // Fill in unset positions for(int j = 0; j < shiftAmt; j++) { // '!' - indicates no alternate basecall at // this position r.altQual[i-1].set(33, j); } } r.alts++; } } if(c == '\r' || c == '\n') { c = peekOverNewline(fb_); } else { c = peekToEndOfLine(fb_); } } r.readOrigBuf.install(fb_.lastN(), fb_.lastNLen()); fb_.resetLastN(); c = fb_.get(); // Should either be at end of file or at beginning of next record assert(c == -1 || c == '@'); // Set up a default name if one hasn't been set if(r.name.empty()) { char cbuf[20]; itoa10(readCnt_, cbuf); r.name.install(cbuf); } r.trimmed3 = gTrim3; r.trimmed5 = mytrim5; rdid = endid = readCnt_; readCnt_++; return success; } /// Read another pattern from a FASTA input file bool TabbedPatternSource::read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { r.reset(); r.color = gColor; success = true; done = false; // fb_ is about to dish out the first character of the // name field if(parseName(r, NULL, '\t') == -1) { peekOverNewline(fb_); // skip rest of line r.reset(); success = false; done = true; return false; } assert_neq('\t', fb_.peek()); // fb_ is about to dish out the first character of the // sequence field int charsRead = 0; int mytrim5 = gTrim5; int dstLen = parseSeq(r, charsRead, mytrim5, '\t'); assert_neq('\t', fb_.peek()); if(dstLen < 0) { peekOverNewline(fb_); // skip rest of line r.reset(); success = false; done = true; return false; } // fb_ is about to dish out the first character of the // quality-string field char ct = 0; if(parseQuals(r, charsRead, dstLen, mytrim5, ct, '\n') < 0) { peekOverNewline(fb_); // skip rest of line r.reset(); success = false; done = true; return false; } r.trimmed3 = gTrim3; r.trimmed5 = mytrim5; assert_eq(ct, '\n'); assert_neq('\n', fb_.peek()); r.readOrigBuf.install(fb_.lastN(), fb_.lastNLen()); fb_.resetLastN(); rdid = endid = readCnt_; readCnt_++; return true; } /// Read another pair of patterns from a FASTA input file bool TabbedPatternSource::readPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) { success = true; done = false; // Skip over initial vertical whitespace if(fb_.peek() == '\r' || fb_.peek() == '\n') { fb_.peekUptoNewline(); fb_.resetLastN(); } // fb_ is about to dish out the first character of the // name field int mytrim5_1 = gTrim5; if(parseName(ra, &rb, '\t') == -1) { peekOverNewline(fb_); // skip rest of line ra.reset(); rb.reset(); fb_.resetLastN(); success = false; done = true; return false; } assert_neq('\t', fb_.peek()); // fb_ is about to dish out the first character of the // sequence field for the first mate int charsRead1 = 0; int dstLen1 = parseSeq(ra, charsRead1, mytrim5_1, '\t'); if(dstLen1 < 0) { peekOverNewline(fb_); // skip rest of line ra.reset(); rb.reset(); fb_.resetLastN(); success = false; done = true; return false; } assert_neq('\t', fb_.peek()); // fb_ is about to dish out the first character of the // quality-string field char ct = 0; if(parseQuals(ra, charsRead1, dstLen1, mytrim5_1, ct, '\t', '\n') < 0) { peekOverNewline(fb_); // skip rest of line ra.reset(); rb.reset(); fb_.resetLastN(); success = false; done = true; return false; } ra.trimmed3 = gTrim3; ra.trimmed5 = mytrim5_1; assert(ct == '\t' || ct == '\n' || ct == '\r' || ct == -1); if(ct == '\r' || ct == '\n' || ct == -1) { // Only had 3 fields prior to newline, so this must be an unpaired read rb.reset(); ra.readOrigBuf.install(fb_.lastN(), fb_.lastNLen()); fb_.resetLastN(); success = true; done = false; paired = false; rdid = endid = readCnt_; readCnt_++; return success; } paired = true; assert_neq('\t', fb_.peek()); // Saw another tab after the third field, so this must be a pair if(secondName_) { // The second mate has its own name if(parseName(rb, NULL, '\t') == -1) { peekOverNewline(fb_); // skip rest of line ra.reset(); rb.reset(); fb_.resetLastN(); success = false; done = true; return false; } assert_neq('\t', fb_.peek()); } // fb_ about to give the first character of the second mate's sequence int charsRead2 = 0; int mytrim5_2 = gTrim5; int dstLen2 = parseSeq(rb, charsRead2, mytrim5_2, '\t'); if(dstLen2 < 0) { peekOverNewline(fb_); // skip rest of line ra.reset(); rb.reset(); fb_.resetLastN(); success = false; done = true; return false; } assert_neq('\t', fb_.peek()); // fb_ is about to dish out the first character of the // quality-string field if(parseQuals(rb, charsRead2, dstLen2, mytrim5_2, ct, '\n') < 0) { peekOverNewline(fb_); // skip rest of line ra.reset(); rb.reset(); fb_.resetLastN(); success = false; done = true; return false; } ra.readOrigBuf.install(fb_.lastN(), fb_.lastNLen()); fb_.resetLastN(); rb.trimmed3 = gTrim3; rb.trimmed5 = mytrim5_2; rdid = endid = readCnt_; readCnt_++; return true; } /** * Parse a name from fb_ and store in r. Assume that the next * character obtained via fb_.get() is the first character of * the sequence and the string stops at the next char upto (could * be tab, newline, etc.). */ int TabbedPatternSource::parseName( Read& r, Read* r2, char upto /* = '\t' */) { // Read the name out of the first field int c = 0; if(r2 != NULL) r2->name.clear(); r.name.clear(); while(true) { if((c = fb_.get()) < 0) { return -1; } if(c == upto) { // Finished with first field break; } if(c == '\n' || c == '\r') { return -1; } if(r2 != NULL) r2->name.append(c); r.name.append(c); } // Set up a default name if one hasn't been set if(r.name.empty()) { char cbuf[20]; itoa10(readCnt_, cbuf); r.name.install(cbuf); if(r2 != NULL) r2->name.install(cbuf); } return (int)r.name.length(); } /** * Parse a single sequence from fb_ and store in r. Assume * that the next character obtained via fb_.get() is the first * character of the sequence and the sequence stops at the next * char upto (could be tab, newline, etc.). */ int TabbedPatternSource::parseSeq( Read& r, int& charsRead, int& trim5, char upto /*= '\t'*/) { int begin = 0; int c = fb_.get(); assert(c != upto); r.patFw.clear(); r.color = gColor; if(gColor) { // This may be a primer character. If so, keep it in the // 'primer' field of the read buf and parse the rest of the // read without it. c = toupper(c); if(asc2dnacat[c] > 0) { // First char is a DNA char int c2 = toupper(fb_.peek()); // Second char is a color char if(asc2colcat[c2] > 0) { r.primer = c; r.trimc = c2; trim5 += 2; // trim primer and first color } } if(c < 0) { return -1; } } while(c != upto) { if(gColor) { if(c >= '0' && c <= '4') c = "ACGTN"[(int)c - '0']; if(c == '.') c = 'N'; } if(isalpha(c)) { assert_in(toupper(c), "ACGTN"); if(begin++ >= trim5) { assert_neq(0, asc2dnacat[c]); r.patFw.append(asc2dna[c]); } charsRead++; } if((c = fb_.get()) < 0) { return -1; } } r.patFw.trimEnd(gTrim3); return (int)r.patFw.length(); } /** * Parse a single quality string from fb_ and store in r. * Assume that the next character obtained via fb_.get() is * the first character of the quality string and the string stops * at the next char upto (could be tab, newline, etc.). */ int TabbedPatternSource::parseQuals( Read& r, int charsRead, int dstLen, int trim5, char& c2, char upto /*= '\t'*/, char upto2 /*= -1*/) { int qualsRead = 0; int c = 0; if (intQuals_) { char buf[4096]; while (qualsRead < charsRead) { qualToks_.clear(); if(!tokenizeQualLine(fb_, buf, 4096, qualToks_)) break; for (unsigned int j = 0; j < qualToks_.size(); ++j) { char c = intToPhred33(atoi(qualToks_[j].c_str()), solQuals_); assert_geq(c, 33); if (qualsRead >= trim5) { r.qual.append(c); } ++qualsRead; } } // done reading integer quality lines if (charsRead > qualsRead) tooFewQualities(r.name); } else { // Non-integer qualities while((qualsRead < dstLen + trim5) && c >= 0) { c = fb_.get(); c2 = c; if (c == ' ') wrongQualityFormat(r.name); if(c < 0) { // EOF occurred in the middle of a read - abort return -1; } if(!isspace(c) && c != upto && (upto2 == -1 || c != upto2)) { if (qualsRead >= trim5) { c = charToPhred33(c, solQuals_, phred64Quals_); assert_geq(c, 33); r.qual.append(c); } qualsRead++; } else { break; } } if(qualsRead < dstLen + trim5) { tooFewQualities(r.name); } else if(qualsRead > dstLen + trim5) { tooManyQualities(r.name); } } r.qual.resize(dstLen); while(c != upto && (upto2 == -1 || c != upto2) && c != -1) { c = fb_.get(); c2 = c; } return qualsRead; } void wrongQualityFormat(const BTString& read_name) { cerr << "Error: Encountered one or more spaces while parsing the quality " << "string for read " << read_name << ". If this is a FASTQ file " << "with integer (non-ASCII-encoded) qualities, try re-running with " << "the --integer-quals option." << endl; throw 1; } void tooFewQualities(const BTString& read_name) { cerr << "Error: Read " << read_name << " has more read characters than " << "quality values." << endl; throw 1; } void tooManyQualities(const BTString& read_name) { cerr << "Error: Read " << read_name << " has more quality values than read " << "characters." << endl; throw 1; } #ifdef USE_SRA struct SRA_Read { SStringExpandable name; // read name SDnaStringExpandable<128, 2> patFw; // forward-strand sequence SStringExpandable qual; // quality values void reset() { name.clear(); patFw.clear(); qual.clear(); } }; static const uint64_t buffer_size_per_thread = 4096; struct SRA_Data { uint64_t read_pos; uint64_t write_pos; uint64_t buffer_size; bool done; EList > paired_reads; ngs::ReadIterator* sra_it; SRA_Data() { read_pos = 0; write_pos = 0; buffer_size = buffer_size_per_thread; done = false; sra_it = NULL; } bool isFull() { assert_leq(read_pos, write_pos); assert_geq(read_pos + buffer_size, write_pos); return read_pos + buffer_size <= write_pos; } bool isEmpty() { assert_leq(read_pos, write_pos); assert_geq(read_pos + buffer_size, write_pos); return read_pos == write_pos; } pair& getPairForRead() { assert(!isEmpty()); return paired_reads[read_pos % buffer_size]; } pair& getPairForWrite() { assert(!isFull()); return paired_reads[write_pos % buffer_size]; } void advanceReadPos() { assert(!isEmpty()); read_pos++; } void advanceWritePos() { assert(!isFull()); write_pos++; } }; static void SRA_IO_Worker(void *vp) { SRA_Data* sra_data = (SRA_Data*)vp; assert(sra_data != NULL); ngs::ReadIterator* sra_it = sra_data->sra_it; assert(sra_it != NULL); while(!sra_data->done) { while(sra_data->isFull()) { #if defined(_TTHREAD_WIN32_) Sleep(1); #elif defined(_TTHREAD_POSIX_) const static timespec ts = {0, 1000000}; // 1 millisecond nanosleep(&ts, NULL); #endif } pair& pair = sra_data->getPairForWrite(); SRA_Read& ra = pair.first; SRA_Read& rb = pair.second; bool exception_thrown = false; try { if(!sra_it->nextRead() || !sra_it->nextFragment()) { ra.reset(); rb.reset(); sra_data->done = true; return; } // Read the name out of the first field ngs::StringRef rname = sra_it->getReadId(); ra.name.install(rname.data(), rname.size()); assert(!ra.name.empty()); ngs::StringRef ra_seq = sra_it->getFragmentBases(); if(gTrim5 + gTrim3 < (int)ra_seq.size()) { ra.patFw.installChars(ra_seq.data() + gTrim5, ra_seq.size() - gTrim5 - gTrim3); } ngs::StringRef ra_qual = sra_it->getFragmentQualities(); if(ra_seq.size() == ra_qual.size() && gTrim5 + gTrim3 < (int)ra_qual.size()) { ra.qual.install(ra_qual.data() + gTrim5, ra_qual.size() - gTrim5 - gTrim3); } else { ra.qual.resize(ra.patFw.length()); ra.qual.fill('I'); } assert_eq(ra.patFw.length(), ra.qual.length()); if(!sra_it->nextFragment()) { rb.reset(); } else { // rb.name = ra.name; ngs::StringRef rb_seq = sra_it->getFragmentBases(); if(gTrim5 + gTrim3 < (int)rb_seq.size()) { rb.patFw.installChars(rb_seq.data() + gTrim5, rb_seq.size() - gTrim5 - gTrim3); } ngs::StringRef rb_qual = sra_it->getFragmentQualities(); if(rb_seq.size() == rb_qual.size() && gTrim5 + gTrim3 < (int)rb_qual.size()) { rb.qual.install(rb_qual.data() + gTrim5, rb_qual.size() - gTrim5 - gTrim3); } else { rb.qual.resize(rb.patFw.length()); rb.qual.fill('I'); } assert_eq(rb.patFw.length(), rb.qual.length()); } sra_data->advanceWritePos(); } catch(ngs::ErrorMsg & x) { cerr << x.toString () << endl; exception_thrown = true; } catch(exception & x) { cerr << x.what () << endl; exception_thrown = true; } catch(...) { cerr << "unknown exception\n"; exception_thrown = true; } if(exception_thrown) { ra.reset(); rb.reset(); sra_data->done = true; cerr << "An error happened while fetching SRA reads. Please rerun HISAT2. You may want to disable the SRA cache if you didn't (see the instructions at https://github.com/ncbi/sra-tools/wiki/Toolkit-Configuration).\n"; exit(1); } } } SRAPatternSource::~SRAPatternSource() { if(io_thread_) delete io_thread_; if(sra_data_) delete sra_data_; if(sra_it_) delete sra_it_; if(sra_run_) delete sra_run_; } /// Read another pair of patterns from a FASTA input file bool SRAPatternSource::readPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) { assert(sra_run_ != NULL && sra_it_ != NULL); success = true; done = false; while(sra_data_->isEmpty()) { if(sra_data_->done && sra_data_->isEmpty()) { ra.reset(); rb.reset(); success = false; done = true; return false; } #if defined(_TTHREAD_WIN32_) Sleep(1); #elif defined(_TTHREAD_POSIX_) const static timespec ts = {0, 1000000}; // 1 millisecond nanosleep(&ts, NULL); #endif } pair& pair = sra_data_->getPairForRead(); ra.name.install(pair.first.name.buf(), pair.first.name.length()); ra.patFw.install(pair.first.patFw.buf(), pair.first.patFw.length()); ra.qual.install(pair.first.qual.buf(), pair.first.qual.length()); ra.trimmed3 = gTrim3; ra.trimmed5 = gTrim5; if(pair.second.patFw.length() > 0) { rb.name.install(pair.first.name.buf(), pair.first.name.length()); rb.patFw.install(pair.second.patFw.buf(), pair.second.patFw.length()); rb.qual.install(pair.second.qual.buf(), pair.second.qual.length()); rb.trimmed3 = gTrim3; rb.trimmed5 = gTrim5; paired = true; } else { rb.reset(); } sra_data_->advanceReadPos(); rdid = endid = readCnt_; readCnt_++; return true; } void SRAPatternSource::open() { string version = "hisat2-"; version += HISAT2_VERSION; ncbi::NGS::setAppVersionString(version.c_str()); assert(!sra_accs_.empty()); while(sra_acc_cur_ < sra_accs_.size()) { // Open read if(sra_it_) { delete sra_it_; sra_it_ = NULL; } if(sra_run_) { delete sra_run_; sra_run_ = NULL; } try { // open requested accession using SRA implementation of the API sra_run_ = new ngs::ReadCollection(ncbi::NGS::openReadCollection(sra_accs_[sra_acc_cur_])); #if 0 string run_name = sra_run_->getName(); cerr << " ReadGroups for " << run_name << endl; ngs::ReadGroupIterator it = sra_run_->getReadGroups(); do { ngs::Statistics s = it.getStatistics(); cerr << "Statistics for group <" << it.getName() << ">" << endl; // for(string p = s.nextPath(""); p != ""; p = s.nextPath(p)){ // System.out.println("\t"+p+": "+s.getAsString(p)); } while(it.nextReadGroup()); exit(1); #endif // compute window to iterate through size_t MAX_ROW = sra_run_->getReadCount(); sra_it_ = new ngs::ReadIterator(sra_run_->getReadRange(1, MAX_ROW, ngs::Read::all)); // create a buffer for SRA data sra_data_ = new SRA_Data; sra_data_->sra_it = sra_it_; sra_data_->buffer_size = nthreads_ * buffer_size_per_thread; sra_data_->paired_reads.resize(sra_data_->buffer_size); // create a thread for handling SRA data access io_thread_ = new tthread::thread(SRA_IO_Worker, (void*)sra_data_); // io_thread_->join(); } catch(...) { if(!errs_[sra_acc_cur_]) { cerr << "Warning: Could not access \"" << sra_accs_[sra_acc_cur_].c_str() << "\" for reading; skipping..." << endl; errs_[sra_acc_cur_] = true; } sra_acc_cur_++; continue; } return; } cerr << "Error: No input SRA accessions were valid" << endl; exit(1); return; } #endif hisat2-2.2.1/pat.h000066400000000000000000001270731370663763200136720ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef PAT_H_ #define PAT_H_ #include #include #include #include #include #include #include #include #include "alphabet.h" #include "assert_helpers.h" #include "tokenize.h" #include "random_source.h" #include "threading.h" #include "filebuf.h" #include "qual.h" #include "search_globals.h" #include "sstring.h" #include "ds.h" #include "read.h" #include "util.h" /** * Classes and routines for reading reads from various input sources. */ using namespace std; /** * Calculate a per-read random seed based on a combination of * the read data (incl. sequence, name, quals) and the global * seed in '_randSeed'. */ static inline uint32_t genRandSeed(const BTDnaString& qry, const BTString& qual, const BTString& name, uint32_t seed) { // Calculate a per-read random seed based on a combination of // the read data (incl. sequence, name, quals) and the global // seed uint32_t rseed = (seed + 101) * 59 * 61 * 67 * 71 * 73 * 79 * 83; size_t qlen = qry.length(); // Throw all the characters of the read into the random seed for(size_t i = 0; i < qlen; i++) { int p = (int)qry[i]; assert_leq(p, 4); size_t off = ((i & 15) << 1); rseed ^= (p << off); } // Throw all the quality values for the read into the random // seed for(size_t i = 0; i < qlen; i++) { int p = (int)qual[i]; assert_leq(p, 255); size_t off = ((i & 3) << 3); rseed ^= (p << off); } // Throw all the characters in the read name into the random // seed size_t namelen = name.length(); for(size_t i = 0; i < namelen; i++) { int p = (int)name[i]; if(p == '/') break; assert_leq(p, 255); size_t off = ((i & 3) << 3); rseed ^= (p << off); } return rseed; } /** * Parameters affecting how reads and read in. */ struct PatternParams { PatternParams( int format_, bool fileParallel_, uint32_t seed_, bool useSpinlock_, bool solexa64_, bool phred64_, bool intQuals_, bool fuzzy_, int sampleLen_, int sampleFreq_, uint32_t skip_) : format(format_), fileParallel(fileParallel_), seed(seed_), useSpinlock(useSpinlock_), solexa64(solexa64_), phred64(phred64_), intQuals(intQuals_), fuzzy(fuzzy_), sampleLen(sampleLen_), sampleFreq(sampleFreq_), skip(skip_) { } int format; // file format bool fileParallel; // true -> wrap files with separate PairedPatternSources uint32_t seed; // pseudo-random seed bool useSpinlock; // use spin locks instead of pthreads bool solexa64; // true -> qualities are on solexa64 scale bool phred64; // true -> qualities are on phred64 scale bool intQuals; // true -> qualities are space-separated numbers bool fuzzy; // true -> try to parse fuzzy fastq int sampleLen; // length of sampled reads for FastaContinuous... int sampleFreq; // frequency of sampled reads for FastaContinuous... uint32_t skip; // skip the first 'skip' patterns }; /** * Encapsulates a synchronized source of patterns; usually a file. * Optionally reverses reads and quality strings before returning them, * though that is usually more efficiently done by the concrete * subclass. Concrete subclasses should delimit critical sections with * calls to lock() and unlock(). */ class PatternSource { public: PatternSource(const PatternParams& p) : seed_(p.seed), readCnt_(0), numWrappers_(0), doLocking_(true), useSpinlock_(p.useSpinlock), mutex() { } virtual ~PatternSource() { } /** * Call this whenever this PatternSource is wrapped by a new * WrappedPatternSourcePerThread. This helps us keep track of * whether locks will be contended. */ void addWrapper() { lock(); numWrappers_++; unlock(); } /** * The main member function for dispensing patterns. * * Returns true iff a pair was parsed succesfully. */ virtual bool nextReadPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired, bool fixName); /** * The main member function for dispensing patterns. */ virtual bool nextRead( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done); /** * Implementation to be provided by concrete subclasses. An * implementation for this member is only relevant for formats that * can read in a pair of reads in a single transaction with a * single input source. If paired-end input is given as a pair of * parallel files, this member should throw an error and exit. */ virtual bool nextReadPairImpl( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) = 0; /** * Implementation to be provided by concrete subclasses. An * implementation for this member is only relevant for formats * where individual input sources look like single-end-read * sources, e.g., formats where paired-end reads are specified in * parallel read files. */ virtual bool nextReadImpl( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) = 0; /// Reset state to start over again with the first read virtual void reset() { readCnt_ = 0; } /** * Concrete subclasses call lock() to enter a critical region. * What constitutes a critical region depends on the subclass. */ void lock() { if(!doLocking_) return; // no contention mutex.lock(); } /** * Concrete subclasses call unlock() to exit a critical region * What constitutes a critical region depends on the subclass. */ void unlock() { if(!doLocking_) return; // no contention mutex.unlock(); } /** * Return a new dynamically allocated PatternSource for the given * format, using the given list of strings as the filenames to read * from or as the sequences themselves (i.e. if -c was used). */ static PatternSource* patsrcFromStrings( const PatternParams& p, const EList& qs, size_t nthreads = 1); /** * Return the number of reads attempted. */ TReadId readCnt() const { return readCnt_ - 1; } protected: uint32_t seed_; /// The number of reads read by this PatternSource TReadId readCnt_; int numWrappers_; /// # threads that own a wrapper for this PatternSource bool doLocking_; /// override whether to lock (true = don't override) /// User can ask to use the normal pthreads-style lock even if /// spinlocks is enabled and compiled in. This is sometimes better /// if we expect bad I/O latency on some reads. bool useSpinlock_; MUTEX_T mutex; }; /** * Abstract parent class for synhconized sources of paired-end reads * (and possibly also single-end reads). */ class PairedPatternSource { public: PairedPatternSource(const PatternParams& p) : mutex_m(), seed_(p.seed) {} virtual ~PairedPatternSource() { } virtual void addWrapper() = 0; virtual void reset() = 0; virtual bool nextReadPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired, bool fixName) = 0; virtual pair readCnt() const = 0; /** * Lock this PairedPatternSource, usually because one of its shared * fields is being updated. */ void lock() { mutex_m.lock(); } /** * Unlock this PairedPatternSource. */ void unlock() { mutex_m.unlock(); } /** * Given the values for all of the various arguments used to specify * the read and quality input, create a list of pattern sources to * dispense them. */ static PairedPatternSource* setupPatternSources( const EList& si, // singles, from argv const EList& m1, // mate1's, from -1 arg const EList& m2, // mate2's, from -2 arg const EList& m12, // both mates on each line, from --12 arg #ifdef USE_SRA const EList& sra_accs, #endif const EList& q, // qualities associated with singles const EList& q1, // qualities associated with m1 const EList& q2, // qualities associated with m2 const PatternParams& p, // read-in params size_t nthreads, bool verbose); // be talkative? protected: MUTEX_T mutex_m; /// mutex for syncing over critical regions uint32_t seed_; }; /** * Encapsulates a synchronized source of both paired-end reads and * unpaired reads, where the paired-end must come from parallel files. */ class PairedSoloPatternSource : public PairedPatternSource { public: PairedSoloPatternSource( const EList* src, const PatternParams& p) : PairedPatternSource(p), cur_(0), src_(src) { assert(src_ != NULL); for(size_t i = 0; i < src_->size(); i++) { assert((*src_)[i] != NULL); } } virtual ~PairedSoloPatternSource() { delete src_; } /** * Call this whenever this PairedPatternSource is wrapped by a new * WrappedPatternSourcePerThread. This helps us keep track of * whether locks within PatternSources will be contended. */ virtual void addWrapper() { for(size_t i = 0; i < src_->size(); i++) { (*src_)[i]->addWrapper(); } } /** * Reset this object and all the PatternSources under it so that * the next call to nextReadPair gets the very first read pair. */ virtual void reset() { for(size_t i = 0; i < src_->size(); i++) { (*src_)[i]->reset(); } cur_ = 0; } /** * The main member function for dispensing pairs of reads or * singleton reads. Returns true iff ra and rb contain a new * pair; returns false if ra contains a new unpaired read. */ virtual bool nextReadPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired, bool fixName); /** * Return the number of reads attempted. */ virtual pair readCnt() const { uint64_t ret = 0llu; for(size_t i = 0; i < src_->size(); i++) ret += (*src_)[i]->readCnt(); return make_pair(ret, 0llu); } protected: volatile uint32_t cur_; // current element in parallel srca_, srcb_ vectors const EList* src_; /// PatternSources for paired-end reads }; /** * Encapsulates a synchronized source of both paired-end reads and * unpaired reads, where the paired-end must come from parallel files. */ class PairedDualPatternSource : public PairedPatternSource { public: PairedDualPatternSource( const EList* srca, const EList* srcb, const PatternParams& p) : PairedPatternSource(p), cur_(0), srca_(srca), srcb_(srcb) { assert(srca_ != NULL); assert(srcb_ != NULL); // srca_ and srcb_ must be parallel assert_eq(srca_->size(), srcb_->size()); for(size_t i = 0; i < srca_->size(); i++) { // Can't have NULL first-mate sources. Second-mate sources // can be NULL, in the case when the corresponding first- // mate source is unpaired. assert((*srca_)[i] != NULL); for(size_t j = 0; j < srcb_->size(); j++) { assert_neq((*srca_)[i], (*srcb_)[j]); } } } virtual ~PairedDualPatternSource() { delete srca_; delete srcb_; } /** * Call this whenever this PairedPatternSource is wrapped by a new * WrappedPatternSourcePerThread. This helps us keep track of * whether locks within PatternSources will be contended. */ virtual void addWrapper() { for(size_t i = 0; i < srca_->size(); i++) { (*srca_)[i]->addWrapper(); if((*srcb_)[i] != NULL) { (*srcb_)[i]->addWrapper(); } } } /** * Reset this object and all the PatternSources under it so that * the next call to nextReadPair gets the very first read pair. */ virtual void reset() { for(size_t i = 0; i < srca_->size(); i++) { (*srca_)[i]->reset(); if((*srcb_)[i] != NULL) { (*srcb_)[i]->reset(); } } cur_ = 0; } /** * The main member function for dispensing pairs of reads or * singleton reads. Returns true iff ra and rb contain a new * pair; returns false if ra contains a new unpaired read. */ virtual bool nextReadPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired, bool fixName); /** * Return the number of reads attempted. */ virtual pair readCnt() const; protected: volatile uint32_t cur_; // current element in parallel srca_, srcb_ vectors const EList* srca_; /// PatternSources for 1st mates and/or unpaired reads const EList* srcb_; /// PatternSources for 2nd mates }; /** * Encapsulates a single thread's interaction with the PatternSource. * Most notably, this class holds the buffers into which the * PatterSource will write sequences. This class is *not* threadsafe * - it doesn't need to be since there's one per thread. PatternSource * is thread-safe. */ class PatternSourcePerThread { public: PatternSourcePerThread() : buf1_(), buf2_(), rdid_(0xffffffff), endid_(0xffffffff) { } virtual ~PatternSourcePerThread() { } /** * Read the next read pair. */ virtual bool nextReadPair( bool& success, bool& done, bool& paired, bool fixName) { return success; } Read& bufa() { return buf1_; } Read& bufb() { return buf2_; } const Read& bufa() const { return buf1_; } const Read& bufb() const { return buf2_; } TReadId rdid() const { return rdid_; } TReadId endid() const { return endid_; } virtual void reset() { rdid_ = endid_ = 0xffffffff; } /** * Return the length of mate 1 or mate 2. */ size_t length(int mate) const { return (mate == 1) ? buf1_.length() : buf2_.length(); } protected: Read buf1_; // read buffer for mate a Read buf2_; // read buffer for mate b TReadId rdid_; // index of read just read TReadId endid_; // index of read just read }; /** * Abstract parent factory for PatternSourcePerThreads. */ class PatternSourcePerThreadFactory { public: virtual ~PatternSourcePerThreadFactory() { } virtual PatternSourcePerThread* create() const = 0; virtual EList* create(uint32_t n) const = 0; /// Free memory associated with a pattern source virtual void destroy(PatternSourcePerThread* patsrc) const { assert(patsrc != NULL); // Free the PatternSourcePerThread delete patsrc; } /// Free memory associated with a pattern source list virtual void destroy(EList* patsrcs) const { assert(patsrcs != NULL); // Free all of the PatternSourcePerThreads for(size_t i = 0; i < patsrcs->size(); i++) { if((*patsrcs)[i] != NULL) { delete (*patsrcs)[i]; (*patsrcs)[i] = NULL; } } // Free the vector delete patsrcs; } }; /** * A per-thread wrapper for a PairedPatternSource. */ class WrappedPatternSourcePerThread : public PatternSourcePerThread { public: WrappedPatternSourcePerThread(PairedPatternSource& __patsrc) : patsrc_(__patsrc) { patsrc_.addWrapper(); } /** * Get the next paired or unpaired read from the wrapped * PairedPatternSource. */ virtual bool nextReadPair( bool& success, bool& done, bool& paired, bool fixName); private: /// Container for obtaining paired reads from PatternSources PairedPatternSource& patsrc_; }; /** * Abstract parent factory for PatternSourcePerThreads. */ class WrappedPatternSourcePerThreadFactory : public PatternSourcePerThreadFactory { public: WrappedPatternSourcePerThreadFactory(PairedPatternSource& patsrc) : patsrc_(patsrc) { } /** * Create a new heap-allocated WrappedPatternSourcePerThreads. */ virtual PatternSourcePerThread* create() const { return new WrappedPatternSourcePerThread(patsrc_); } /** * Create a new heap-allocated vector of heap-allocated * WrappedPatternSourcePerThreads. */ virtual EList* create(uint32_t n) const { EList* v = new EList; for(size_t i = 0; i < n; i++) { v->push_back(new WrappedPatternSourcePerThread(patsrc_)); assert(v->back() != NULL); } return v; } private: /// Container for obtaining paired reads from PatternSources PairedPatternSource& patsrc_; }; /// Skip to the end of the current string of newline chars and return /// the first character after the newline chars, or -1 for EOF static inline int getOverNewline(FileBuf& in) { int c; while(isspace(c = in.get())); return c; } /// Skip to the end of the current string of newline chars such that /// the next call to get() returns the first character after the /// whitespace static inline int peekOverNewline(FileBuf& in) { while(true) { int c = in.peek(); if(c != '\r' && c != '\n') { return c; } in.get(); } } /// Skip to the end of the current line; return the first character /// of the next line or -1 for EOF static inline int getToEndOfLine(FileBuf& in) { while(true) { int c = in.get(); if(c < 0) return -1; if(c == '\n' || c == '\r') { while(c == '\n' || c == '\r') { c = in.get(); if(c < 0) return -1; } // c now holds first character of next line return c; } } } /// Skip to the end of the current line such that the next call to /// get() returns the first character on the next line static inline int peekToEndOfLine(FileBuf& in) { while(true) { int c = in.get(); if(c < 0) return c; if(c == '\n' || c == '\r') { c = in.peek(); while(c == '\n' || c == '\r') { in.get(); if(c < 0) return c; // consume \r or \n c = in.peek(); } // next get() gets first character of next line return c; } } } extern void wrongQualityFormat(const BTString& read_name); extern void tooFewQualities(const BTString& read_name); extern void tooManyQualities(const BTString& read_name); /** * Encapsulates a source of patterns which is an in-memory vector. */ class VectorPatternSource : public PatternSource { public: VectorPatternSource( const EList& v, const PatternParams& p); virtual ~VectorPatternSource() { } virtual bool nextReadImpl( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done); /** * This is unused, but implementation is given for completeness. */ virtual bool nextReadPairImpl( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired); virtual void reset() { PatternSource::reset(); cur_ = skip_; paired_ = false; } private: size_t cur_; uint32_t skip_; bool paired_; EList v_; // forward sequences EList quals_; // forward qualities EList names_; // names EList trimmed3_; // names EList trimmed5_; // names }; /** * */ class BufferedFilePatternSource : public PatternSource { public: BufferedFilePatternSource( const EList& infiles, const PatternParams& p) : PatternSource(p), infiles_(infiles), filecur_(0), fb_(), skip_(p.skip), first_(true) { assert_gt(infiles.size(), 0); errs_.resize(infiles_.size()); errs_.fill(0, infiles_.size(), false); assert(!fb_.isOpen()); open(); // open first file in the list filecur_++; } virtual ~BufferedFilePatternSource() { if(fb_.isOpen()) fb_.close(); } /** * Fill Read with the sequence, quality and name for the next * read in the list of read files. This function gets called by * all the search threads, so we must handle synchronization. */ virtual bool nextReadImpl( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { // We'll be manipulating our file handle/filecur_ state lock(); while(true) { do { read(r, rdid, endid, success, done); } while(!success && !done); if(!success && filecur_ < infiles_.size()) { assert(done); open(); resetForNextFile(); // reset state to handle a fresh file filecur_++; continue; } break; } assert(r.repOk()); // Leaving critical region unlock(); return success; } /** * */ virtual bool nextReadPairImpl( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) { // We'll be manipulating our file handle/filecur_ state lock(); while(true) { do { readPair(ra, rb, rdid, endid, success, done, paired); } while(!success && !done); if(!success && filecur_ < infiles_.size()) { assert(done); open(); resetForNextFile(); // reset state to handle a fresh file filecur_++; continue; } break; } assert(ra.repOk()); assert(rb.repOk()); // Leaving critical region unlock(); return success; } /** * Reset state so that we read start reading again from the * beginning of the first file. Should only be called by the * master thread. */ virtual void reset() { PatternSource::reset(); filecur_ = 0; open(); filecur_++; } protected: /// Read another pattern from the input file; this is overridden /// to deal with specific file formats virtual bool read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) = 0; /// Read another pattern pair from the input file; this is /// overridden to deal with specific file formats virtual bool readPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) = 0; /// Reset state to handle a fresh file virtual void resetForNextFile() { } void open() { if(fb_.isOpen()) fb_.close(); while(filecur_ < infiles_.size()) { // Open read FILE *in; if(infiles_[filecur_] == "-") { in = stdin; } else if((in = fopen(infiles_[filecur_].c_str(), "rb")) == NULL) { if(!errs_[filecur_]) { cerr << "Warning: Could not open read file \"" << infiles_[filecur_].c_str() << "\" for reading; skipping..." << endl; errs_[filecur_] = true; } filecur_++; continue; } fb_.newFile(in); return; } cerr << "Error: No input read files were valid" << endl; exit(1); return; } EList infiles_; // filenames for read files EList errs_; // whether we've already printed an error for each file size_t filecur_; // index into infiles_ of next file to read FileBuf fb_; // read file currently being read from TReadId skip_; // number of reads to skip bool first_; }; /** * Parse a single quality string from fb and store qualities in r. * Assume the next character obtained via fb.get() is the first * character of the quality string. When returning, the next * character returned by fb.peek() or fb.get() should be the first * character of the following line. */ int parseQuals( Read& r, FileBuf& fb, int firstc, int readLen, int trim3, int trim5, bool intQuals, bool phred64, bool solexa64); /** * Synchronized concrete pattern source for a list of FASTA or CSFASTA * (if color = true) files. */ class FastaPatternSource : public BufferedFilePatternSource { public: FastaPatternSource(const EList& infiles, const PatternParams& p) : BufferedFilePatternSource(infiles, p), first_(true), solexa64_(p.solexa64), phred64_(p.phred64), intQuals_(p.intQuals) { } virtual void reset() { first_ = true; BufferedFilePatternSource::reset(); } protected: /** * Scan to the next FASTA record (starting with >) and return the first * character of the record (which will always be >). */ static int skipToNextFastaRecord(FileBuf& in) { int c; while((c = in.get()) != '>') { if(in.eof()) return -1; } return c; } /// Called when we have to bail without having parsed a read. void bail(Read& r) { r.reset(); fb_.resetLastN(); } /// Read another pattern from a FASTA input file virtual bool read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done); /// Read another pair of patterns from a FASTA input file virtual bool readPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) { // (For now, we shouldn't ever be here) cerr << "In FastaPatternSource.readPair()" << endl; throw 1; return false; } virtual void resetForNextFile() { first_ = true; } private: bool first_; public: bool solexa64_; bool phred64_; bool intQuals_; }; /** * Tokenize a line of space-separated integer quality values. */ static inline bool tokenizeQualLine( FileBuf& filebuf, char *buf, size_t buflen, EList& toks) { size_t rd = filebuf.gets(buf, buflen); if(rd == 0) return false; assert(NULL == strrchr(buf, '\n')); tokenize(string(buf), " ", toks); return true; } /** * Synchronized concrete pattern source for a list of files with tab- * delimited name, seq, qual fields (or, for paired-end reads, * basename, seq1, qual1, seq2, qual2). */ class TabbedPatternSource : public BufferedFilePatternSource { public: TabbedPatternSource( const EList& infiles, const PatternParams& p, bool secondName) : BufferedFilePatternSource(infiles, p), solQuals_(p.solexa64), phred64Quals_(p.phred64), intQuals_(p.intQuals), secondName_(secondName) { } protected: /// Read another pattern from a FASTA input file virtual bool read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done); /// Read another pair of patterns from a FASTA input file virtual bool readPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired); private: /** * Parse a name from fb_ and store in r. Assume that the next * character obtained via fb_.get() is the first character of * the sequence and the string stops at the next char upto (could * be tab, newline, etc.). */ int parseName(Read& r, Read* r2, char upto = '\t'); /** * Parse a single sequence from fb_ and store in r. Assume * that the next character obtained via fb_.get() is the first * character of the sequence and the sequence stops at the next * char upto (could be tab, newline, etc.). */ int parseSeq(Read& r, int& charsRead, int& trim5, char upto = '\t'); /** * Parse a single quality string from fb_ and store in r. * Assume that the next character obtained via fb_.get() is * the first character of the quality string and the string stops * at the next char upto (could be tab, newline, etc.). */ int parseQuals(Read& r, int charsRead, int dstLen, int trim5, char& c2, char upto = '\t', char upto2 = -1); bool solQuals_; bool phred64Quals_; bool intQuals_; EList qualToks_; bool secondName_; }; /** * Synchronized concrete pattern source for Illumina Qseq files. In * Qseq files, each read appears on a separate line and the tab- * delimited fields are: * * 1. Machine name * 2. Run number * 3. Lane number * 4. Tile number * 5. X coordinate of spot * 6. Y coordinate of spot * 7. Index: "Index sequence or 0. For no indexing, or for a file that * has not been demultiplexed yet, this field should have a value of * 0." * 8. Read number: 1 for unpaired, 1 or 2 for paired * 9. Sequence * 10. Quality * 11. Filter: 1 = passed, 0 = didn't */ class QseqPatternSource : public BufferedFilePatternSource { public: QseqPatternSource( const EList& infiles, const PatternParams& p) : BufferedFilePatternSource(infiles, p), solQuals_(p.solexa64), phred64Quals_(p.phred64), intQuals_(p.intQuals) { } protected: #define BAIL_UNPAIRED() { \ peekOverNewline(fb_); \ r.reset(); \ success = false; \ done = true; \ return success; \ } /** * Parse a name from fb_ and store in r. Assume that the next * character obtained via fb_.get() is the first character of * the sequence and the string stops at the next char upto (could * be tab, newline, etc.). */ int parseName( Read& r, // buffer for mate 1 Read* r2, // buffer for mate 2 (NULL if mate2 is read separately) bool append, // true -> append characters, false -> skip them bool clearFirst, // clear the name buffer first bool warnEmpty, // emit a warning if nothing was added to the name bool useDefault, // if nothing is read, put readCnt_ as a default value int upto); // stop parsing when we first reach character 'upto' /** * Parse a single sequence from fb_ and store in r. Assume * that the next character obtained via fb_.get() is the first * character of the sequence and the sequence stops at the next * char upto (could be tab, newline, etc.). */ int parseSeq( Read& r, // buffer for read int& charsRead, int& trim5, char upto); /** * Parse a single quality string from fb_ and store in r. * Assume that the next character obtained via fb_.get() is * the first character of the quality string and the string stops * at the next char upto (could be tab, newline, etc.). */ int parseQuals( Read& r, // buffer for read int charsRead, int dstLen, int trim5, char& c2, char upto, char upto2); /** * Read another pattern from a Qseq input file. */ virtual bool read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done); /** * Read a pair of patterns from 1 Qseq file. Note: this is never used. */ virtual bool readPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) { // (For now, we shouldn't ever be here) cerr << "In QseqPatternSource.readPair()" << endl; throw 1; return false; } bool solQuals_; bool phred64Quals_; bool intQuals_; EList qualToks_; }; /** * Synchronized concrete pattern source for a list of FASTA files where * reads need to be extracted from long continuous sequences. */ class FastaContinuousPatternSource : public BufferedFilePatternSource { public: FastaContinuousPatternSource(const EList& infiles, const PatternParams& p) : BufferedFilePatternSource(infiles, p), length_(p.sampleLen), freq_(p.sampleFreq), eat_(length_-1), beginning_(true), bufCur_(0), subReadCnt_(0llu) { resetForNextFile(); } virtual void reset() { BufferedFilePatternSource::reset(); resetForNextFile(); } protected: /// Read another pattern from a FASTA input file virtual bool read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { success = true; done = false; r.reset(); while(true) { r.color = gColor; int c = fb_.get(); if(c < 0) { success = false; done = true; return success; } if(c == '>') { resetForNextFile(); c = fb_.peek(); bool sawSpace = false; while(c != '\n' && c != '\r') { if(!sawSpace) { sawSpace = isspace(c); } if(!sawSpace) { nameBuf_.append(c); } fb_.get(); c = fb_.peek(); } while(c == '\n' || c == '\r') { fb_.get(); c = fb_.peek(); } nameBuf_.append('_'); } else { int cat = asc2dnacat[c]; if(cat >= 2) c = 'N'; if(cat == 0) { // Encountered non-DNA, non-IUPAC char; skip it continue; } else { // DNA char buf_[bufCur_++] = c; if(bufCur_ == 1024) bufCur_ = 0; if(eat_ > 0) { eat_--; // Try to keep readCnt_ aligned with the offset // into the reference; that lets us see where // the sampling gaps are by looking at the read // name if(!beginning_) readCnt_++; continue; } for(size_t i = 0; i < length_; i++) { if(length_ - i <= bufCur_) { c = buf_[bufCur_ - (length_ - i)]; } else { // Rotate c = buf_[bufCur_ - (length_ - i) + 1024]; } r.patFw.append(asc2dna[c]); r.qual.append('I'); } // Set up a default name if one hasn't been set r.name = nameBuf_; char cbuf[20]; itoa10(readCnt_ - subReadCnt_, cbuf); r.name.append(cbuf); eat_ = freq_-1; readCnt_++; beginning_ = false; rdid = endid = readCnt_-1; break; } } } return true; } /// Shouldn't ever be here; it's not sensible to obtain read pairs // from a continuous input. virtual bool readPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) { cerr << "In FastaContinuousPatternSource.readPair()" << endl; throw 1; return false; } /** * Reset state to be read for the next file. */ virtual void resetForNextFile() { eat_ = length_-1; beginning_ = true; bufCur_ = 0; nameBuf_.clear(); subReadCnt_ = readCnt_; } private: size_t length_; /// length of reads to generate size_t freq_; /// frequency to sample reads size_t eat_; /// number of characters we need to skip before /// we have flushed all of the ambiguous or /// non-existent characters out of our read /// window bool beginning_; /// skipping over the first read length? char buf_[1024]; /// read buffer BTString nameBuf_; /// read buffer for name of fasta record being /// split into mers size_t bufCur_; /// buffer cursor; points to where we should /// insert the next character uint64_t subReadCnt_;/// number to subtract from readCnt_ to get /// the pat id to output (so it resets to 0 for /// each new sequence) }; /** * Read a FASTQ-format file. * See: http://maq.sourceforge.net/fastq.shtml */ class FastqPatternSource : public BufferedFilePatternSource { public: FastqPatternSource(const EList& infiles, const PatternParams& p) : BufferedFilePatternSource(infiles, p), first_(true), solQuals_(p.solexa64), phred64Quals_(p.phred64), intQuals_(p.intQuals), fuzzy_(p.fuzzy) { } virtual void reset() { first_ = true; fb_.resetLastN(); BufferedFilePatternSource::reset(); } protected: /** * Scan to the next FASTQ record (starting with @) and return the first * character of the record (which will always be @). Since the quality * line may start with @, we keep scanning until we've seen a line * beginning with @ where the line two lines back began with +. */ static int skipToNextFastqRecord(FileBuf& in, bool sawPlus) { int line = 0; int plusLine = -1; int c = in.get(); int firstc = c; while(true) { if(line > 20) { // If we couldn't find our desired '@' in the first 20 // lines, it's time to give up if(firstc == '>') { // That firstc is '>' may be a hint that this is // actually a FASTA file, so return it intact return '>'; } // Return an error return -1; } if(c == -1) return -1; if(c == '\n') { c = in.get(); if(c == '@' && sawPlus && plusLine == (line-2)) { return '@'; } else if(c == '+') { // Saw a '+' at the beginning of a line; remember where // we saw it sawPlus = true; plusLine = line; } else if(c == -1) { return -1; } line++; } c = in.get(); } } /// Read another pattern from a FASTQ input file virtual bool read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done); /// Read another read pair from a FASTQ input file virtual bool readPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) { // (For now, we shouldn't ever be here) cerr << "In FastqPatternSource.readPair()" << endl; throw 1; return false; } virtual void resetForNextFile() { first_ = true; } private: /** * Do things we need to do if we have to bail in the middle of a * read, usually because we reached the end of the input without * finishing. */ void bail(Read& r) { r.patFw.clear(); fb_.resetLastN(); } bool first_; bool solQuals_; bool phred64Quals_; bool intQuals_; bool fuzzy_; EList qualToks_; }; /** * Read a Raw-format file (one sequence per line). No quality strings * allowed. All qualities are assumed to be 'I' (40 on the Phred-33 * scale). */ class RawPatternSource : public BufferedFilePatternSource { public: RawPatternSource(const EList& infiles, const PatternParams& p) : BufferedFilePatternSource(infiles, p), first_(true) { } virtual void reset() { first_ = true; BufferedFilePatternSource::reset(); } protected: /// Read another pattern from a Raw input file virtual bool read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { int c; success = true; done = false; r.reset(); c = getOverNewline(this->fb_); if(c < 0) { bail(r); success = false; done = true; return success; } assert(!isspace(c)); r.color = gColor; int mytrim5 = gTrim5; if(first_) { // Check that the first character is sane for a raw file int cc = c; if(gColor) { if(cc >= '0' && cc <= '4') cc = "ACGTN"[(int)cc - '0']; if(cc == '.') cc = 'N'; } if(asc2dnacat[cc] == 0) { cerr << "Error: reads file does not look like a Raw file" << endl; if(c == '>') { cerr << "Reads file looks like a FASTA file; please use -f" << endl; } if(c == '@') { cerr << "Reads file looks like a FASTQ file; please use -q" << endl; } throw 1; } first_ = false; } if(gColor) { // This may be a primer character. If so, keep it in the // 'primer' field of the read buf and parse the rest of the // read without it. c = toupper(c); if(asc2dnacat[c] > 0) { // First char is a DNA char int c2 = toupper(fb_.peek()); // Second char is a color char if(asc2colcat[c2] > 0) { r.primer = c; r.trimc = c2; mytrim5 += 2; // trim primer and first color } } if(c < 0) { bail(r); success = false; done = true; return success; } } // _in now points just past the first character of a sequence // line, and c holds the first character int chs = 0; while(!isspace(c) && c >= 0) { if(gColor) { if(c >= '0' && c <= '4') c = "ACGTN"[(int)c - '0']; if(c == '.') c = 'N'; } // 5' trimming if(isalpha(c) && chs >= mytrim5) { //size_t len = chs - mytrim5; //if(len >= 1024) tooManyQualities(BTString("(no name)")); r.patFw.append(asc2dna[c]); r.qual.append('I'); } chs++; if(isspace(fb_.peek())) break; c = fb_.get(); } // 3' trimming r.patFw.trimEnd(gTrim3); r.qual.trimEnd(gTrim3); c = peekToEndOfLine(fb_); r.trimmed3 = gTrim3; r.trimmed5 = mytrim5; r.readOrigBuf.install(fb_.lastN(), fb_.lastNLen()); fb_.resetLastN(); // Set up name char cbuf[20]; itoa10(readCnt_, cbuf); r.name.install(cbuf); readCnt_++; rdid = endid = readCnt_-1; return success; } /// Read another read pair from a FASTQ input file virtual bool readPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) { // (For now, we shouldn't ever be here) cerr << "In RawPatternSource.readPair()" << endl; throw 1; return false; } virtual void resetForNextFile() { first_ = true; } private: /** * Do things we need to do if we have to bail in the middle of a * read, usually because we reached the end of the input without * finishing. */ void bail(Read& r) { r.patFw.clear(); fb_.resetLastN(); } bool first_; }; #ifdef USE_SRA namespace ngs { class ReadCollection; class ReadIterator; } namespace tthread { class thread; }; struct SRA_Data; /** * */ class SRAPatternSource : public PatternSource { public: SRAPatternSource( const EList& sra_accs, const PatternParams& p, const size_t nthreads = 1) : PatternSource(p), sra_accs_(sra_accs), sra_acc_cur_(0), skip_(p.skip), first_(true), nthreads_(nthreads), sra_run_(NULL), sra_it_(NULL), sra_data_(NULL), io_thread_(NULL) { assert_gt(sra_accs_.size(), 0); errs_.resize(sra_accs_.size()); errs_.fill(0, sra_accs_.size(), false); open(); // open first file in the list sra_acc_cur_++; } virtual ~SRAPatternSource(); /** * Fill Read with the sequence, quality and name for the next * read in the list of read files. This function gets called by * all the search threads, so we must handle synchronization. */ virtual bool nextReadImpl( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { // We'll be manipulating our file handle/filecur_ state lock(); while(true) { do { read(r, rdid, endid, success, done); } while(!success && !done); if(!success && sra_acc_cur_ < sra_accs_.size()) { assert(done); open(); resetForNextFile(); // reset state to handle a fresh file sra_acc_cur_++; continue; } break; } assert(r.repOk()); // Leaving critical region unlock(); return success; } /** * */ virtual bool nextReadPairImpl( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired) { // We'll be manipulating our file handle/filecur_ state lock(); while(true) { do { readPair(ra, rb, rdid, endid, success, done, paired); } while(!success && !done); if(!success && sra_acc_cur_ < sra_accs_.size()) { assert(done); open(); resetForNextFile(); // reset state to handle a fresh file sra_acc_cur_++; continue; } break; } assert(ra.repOk()); assert(rb.repOk()); // Leaving critical region unlock(); return success; } /** * Reset state so that we read start reading again from the * beginning of the first file. Should only be called by the * master thread. */ virtual void reset() { PatternSource::reset(); sra_acc_cur_ = 0, open(); sra_acc_cur_++; } /// Read another pattern from the input file; this is overridden /// to deal with specific file formats virtual bool read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { return true; } /// Read another pattern pair from the input file; this is /// overridden to deal with specific file formats virtual bool readPair( Read& ra, Read& rb, TReadId& rdid, TReadId& endid, bool& success, bool& done, bool& paired); protected: /// Reset state to handle a fresh file virtual void resetForNextFile() { } void open(); EList sra_accs_; // filenames for read files EList errs_; // whether we've already printed an error for each file size_t sra_acc_cur_; // index into infiles_ of next file to read TReadId skip_; // number of reads to skip bool first_; size_t nthreads_; ngs::ReadCollection* sra_run_; ngs::ReadIterator* sra_it_; SRA_Data* sra_data_; tthread::thread* io_thread_; }; #endif #endif /*PAT_H_*/ hisat2-2.2.1/pe.cpp000066400000000000000000000755471370663763200140550ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include "assert_helpers.h" #include "pe.h" using namespace std; /** * Return a PE_TYPE flag indicating, given a PE_POLICY and coordinates * for a paired-end alignment, what type of alignment it is, i.e., * whether it's: * * 1. Straightforwardly concordant * 2. Mates dovetail (one extends beyond the end of the other) * 3. One mate contains the other but they don't dovetail * 4. One mate overlaps the other but neither contains the other and * they don't dovetail * 5. Discordant */ int PairedEndPolicy::peClassifyPair( int64_t off1, // offset of mate 1 size_t len1, // length of mate 1 bool fw1, // whether mate 1 aligned to Watson int64_t off2, // offset of mate 2 size_t len2, // length of mate 2 bool fw2) // whether mate 2 aligned to Watson const { assert_gt(len1, 0); assert_gt(len2, 0); // Expand the maximum fragment length if necessary to accomodate // the longer mate size_t maxfrag = maxfrag_; if(len1 > maxfrag && expandToFit_) maxfrag = len1; if(len2 > maxfrag && expandToFit_) maxfrag = len2; size_t minfrag = minfrag_; if(minfrag < 1) { minfrag = 1; } bool oneLeft = false; if(pol_ == PE_POLICY_FF) { if(fw1 != fw2) { // Bad combination of orientations return PE_ALS_DISCORD; } oneLeft = fw1; } else if(pol_ == PE_POLICY_RR) { if(fw1 != fw2) { // Bad combination of orientations return PE_ALS_DISCORD; } oneLeft = !fw1; } else if(pol_ == PE_POLICY_FR) { if(fw1 == fw2) { // Bad combination of orientations return PE_ALS_DISCORD; } oneLeft = fw1; } else if(pol_ == PE_POLICY_RF) { if(fw1 == fw2) { // Bad combination of orientations return PE_ALS_DISCORD; } oneLeft = !fw1; } // Calc implied fragment size int64_t fraglo = min(off1, off2); int64_t fraghi = max(off1+len1, off2+len2); assert_gt(fraghi, fraglo); size_t frag = (size_t)(fraghi - fraglo); if(frag > maxfrag || frag < minfrag) { // Pair is discordant by virtue of the extents return PE_ALS_DISCORD; } int64_t lo1 = off1; int64_t hi1 = off1 + len1 - 1; int64_t lo2 = off2; int64_t hi2 = off2 + len2 - 1; bool containment = false; // Check whether one mate entirely contains the other if((lo1 >= lo2 && hi1 <= hi2) || (lo2 >= lo1 && hi2 <= hi1)) { containment = true; } int type = PE_ALS_NORMAL; // Check whether one mate overlaps the other bool olap = false; if((lo1 <= lo2 && hi1 >= lo2) || (lo1 <= hi2 && hi1 >= hi2) || containment) { // The mates overlap olap = true; if(!olapOk_) return PE_ALS_DISCORD; type = PE_ALS_OVERLAP; } // Check if the mates are in the wrong relative orientation, // without any overlap if(!olap) { if((oneLeft && lo2 < lo1) || (!oneLeft && lo1 < lo2)) { return PE_ALS_DISCORD; } } // If one mate contained the other, report that if(containment) { if(!containOk_) return PE_ALS_DISCORD; type = PE_ALS_CONTAIN; } // Check whether there's dovetailing; i.e. does the left mate // extend past the right end of the right mate, or vice versa if(( oneLeft && (hi1 > hi2 || lo2 < lo1)) || (!oneLeft && (hi2 > hi1 || lo1 < lo2))) { if(!dovetailOk_) return PE_ALS_DISCORD; type = PE_ALS_DOVETAIL; } return type; } /** * Given details about how one mate aligns, and some details about the * reference sequence it aligned to, calculate a window and orientation s.t. * a paired-end alignment is concordant iff the opposite mate aligns in the * calculated window with the calculated orientation. The "window" is really a * cosntraint on which positions the extreme end of the opposite mate can fall. * This is a different type of constraint from the one placed on seed-extend * dynamic programming problems. That constraints requires that alignments at * one point pass through one of a set of "core" columns. * * When the opposite mate is to the left, we're constraining where its * left-hand extreme can fall, i.e., which cells in the top row of the matrix * it can end in. When the opposite mate is to the right, we're cosntraining * where its right-hand extreme can fall, i.e., which cells in the bottom row * of the matrix it can end in. However, in practice we can only constrain * where we start the backtrace, i.e. where the RHS of the alignment falls. * See frameFindMateRect for details. * * This calculaton does not consider gaps - the dynamic programming framer will * take gaps into account. * * Returns false if no concordant alignments are possible, true otherwise. */ bool PairedEndPolicy::otherMate( bool is1, // true -> mate 1 aligned and we're looking // for 2, false -> vice versa bool fw, // orientation of aligned mate int64_t off, // offset into the reference sequence int64_t maxalcols, // max # columns spanned by alignment size_t reflen, // length of reference sequence aligned to size_t len1, // length of mate 1 size_t len2, // length of mate 2 bool& oleft, // out: true iff opp mate must be to right of anchor int64_t& oll, // out: leftmost Watson off for LHS of opp alignment int64_t& olr, // out: rightmost Watson off for LHS of opp alignment int64_t& orl, // out: leftmost Watson off for RHS of opp alignment int64_t& orr, // out: rightmost Watson off for RHS of opp alignment bool& ofw) // out: true iff opp mate must be on Watson strand const { assert_gt(len1, 0); assert_gt(len2, 0); assert_gt(maxfrag_, 0); assert_geq(minfrag_, 0); assert_geq(maxfrag_, minfrag_); assert(maxalcols == -1 || maxalcols > 0); // Calculate whether opposite mate should align to left or to right // of given mate, and what strand it should align to pePolicyMateDir(pol_, is1, fw, oleft, ofw); size_t alen = is1 ? len1 : len2; // length of opposite mate // Expand the maximum fragment length if necessary to accomodate // the longer mate size_t maxfrag = maxfrag_; size_t minfrag = minfrag_; if(minfrag < 1) { minfrag = 1; } if(len1 > maxfrag && expandToFit_) maxfrag = len1; if(len2 > maxfrag && expandToFit_) maxfrag = len2; if(!expandToFit_ && (len1 > maxfrag || len2 > maxfrag)) { // Not possible to find a concordant alignment; one of the // mates is too long return false; } // Now calculate bounds within which a dynamic programming // algorithm should search for an alignment for the opposite mate if(oleft) { // -----------FRAG MAX---------------- // -------FRAG MIN------- // |-alen-| // Anchor mate // ^off // |------| // Not concordant: LHS not outside min // |------| // Concordant // |------| // Concordant // |------| // Not concordant: LHS outside max // -----------FRAG MAX---------------- // -------FRAG MIN------- // |-alen-| // Anchor mate // ^off // |------------| // LHS can't be outside this range // -----------FRAG MAX---------------- // |------------------------------------------------------------| // LHS can't be outside this range, assuming no restrictions on // flipping, dovetailing, containment, overlap, etc. // |-------| // maxalcols // |-----------------------------------------| // LHS can't be outside this range, assuming no flipping // |---------------------------------| // LHS can't be outside this range, assuming no dovetailing // |-------------------------| // LHS can't be outside this range, assuming no overlap oll = off + alen - maxfrag; olr = off + alen - minfrag; assert_geq(olr, oll); orl = oll; orr = off + maxfrag - 1; assert_geq(olr, oll); // What if overlapping alignments are not allowed? if(!olapOk_) { // RHS can't be flush with or to the right of off orr = min(orr, off-1); if(orr < olr) olr = orr; assert_leq(oll, olr); assert_leq(orl, orr); assert_geq(orr, olr); } // What if dovetail alignments are not allowed? else if(!dovetailOk_) { // RHS can't be past off+alen-1 orr = min(orr, off + alen - 1); assert_leq(oll, olr); assert_leq(orl, orr); } // What if flipped alignments are not allowed? else if(!flippingOk_ && maxalcols != -1) { // RHS can't be right of ??? orr = min(orr, off + alen - 1 + (maxalcols-1)); assert_leq(oll, olr); assert_leq(orl, orr); } assert_geq(olr, oll); assert_geq(orr, orl); assert_geq(orr, olr); assert_geq(orl, oll); } else { // -----------FRAG MAX---------------- // -------FRAG MIN------- // -----------FRAG MAX---------------- // |-alen-| // Anchor mate // ^off // |------| // Not concordant: RHS not outside min // |------| // Concordant // |------| // Concordant // |------| // Not concordant: RHS outside max // // -----------FRAG MAX---------------- // -------FRAG MIN------- // -----------FRAG MAX---------------- // |-alen-| // Anchor mate // ^off // |------------| // RHS can't be outside this range // |------------------------------------------------------------| // LHS can't be outside this range, assuming no restrictions on // dovetailing, containment, overlap, etc. // |-------| // maxalcols // |-----------------------------------------| // LHS can't be outside this range, assuming no flipping // |---------------------------------| // LHS can't be outside this range, assuming no dovetailing // |-------------------------| // LHS can't be outside this range, assuming no overlap orr = off + (maxfrag - 1); orl = off + (minfrag - 1); assert_geq(orr, orl); oll = off + alen - maxfrag; olr = orr; assert_geq(olr, oll); // What if overlapping alignments are not allowed? if(!olapOk_) { // LHS can't be left of off+alen oll = max(oll, off+alen); if(oll > orl) orl = oll; assert_leq(oll, olr); assert_leq(orl, orr); assert_geq(orl, oll); } // What if dovetail alignments are not allowed? else if(!dovetailOk_) { // LHS can't be left of off oll = max(oll, off); assert_leq(oll, olr); assert_leq(orl, orr); } // What if flipped alignments are not allowed? else if(!flippingOk_ && maxalcols != -1) { // LHS can't be left of off - maxalcols + 1 oll = max(oll, off - maxalcols + 1); assert_leq(oll, olr); assert_leq(orl, orr); } assert_geq(olr, oll); assert_geq(orr, orl); assert_geq(orr, olr); assert_geq(orl, oll); } // Boundaries and orientation determined return true; } #ifdef MAIN_PE #include #include void testCaseClassify( const string& name, int pol, size_t maxfrag, size_t minfrag, bool local, bool flip, bool dove, bool cont, bool olap, bool expand, int64_t off1, size_t len1, bool fw1, int64_t off2, size_t len2, bool fw2, int expect_class) { PairedEndPolicy pepol( pol, maxfrag, minfrag, local, flip, dove, cont, olap, expand); int ret = pepol.peClassifyPair( off1, // offset of mate 1 len1, // length of mate 1 fw1, // whether mate 1 aligned to Watson off2, // offset of mate 2 len2, // length of mate 2 fw2); // whether mate 2 aligned to Watson assert_eq(expect_class, ret); cout << "peClassifyPair: " << name << "...PASSED" << endl; } void testCaseOtherMate( const string& name, int pol, size_t maxfrag, size_t minfrag, bool local, bool flip, bool dove, bool cont, bool olap, bool expand, bool is1, bool fw, int64_t off, int64_t maxalcols, size_t reflen, size_t len1, size_t len2, bool expect_ret, bool expect_oleft, int64_t expect_oll, int64_t expect_olr, int64_t expect_orl, int64_t expect_orr, bool expect_ofw) { PairedEndPolicy pepol( pol, maxfrag, minfrag, local, flip, dove, cont, olap, expand); int64_t oll = 0, olr = 0; int64_t orl = 0, orr = 0; bool oleft = false, ofw = false; bool ret = pepol.otherMate( is1, fw, off, maxalcols, reflen, len1, len2, oleft, oll, olr, orl, orr, ofw); assert(ret == expect_ret); if(ret) { assert_eq(expect_oleft, oleft); assert_eq(expect_oll, oll); assert_eq(expect_olr, olr); assert_eq(expect_orl, orl); assert_eq(expect_orr, orr); assert_eq(expect_ofw, ofw); } cout << "otherMate: " << name << "...PASSED" << endl; } int main(int argc, char **argv) { // Set of 8 cases where we look for the opposite mate to the right // of the anchor mate, with various combinations of policies and // anchor-mate orientations. // |--------| // |--------| // ^110 ^119 // |------------------| // min frag // |--------| // ^120 ^129 // |----------------------------| // max frag // ^ // 100 { int policies[] = { PE_POLICY_FF, PE_POLICY_RR, PE_POLICY_FR, PE_POLICY_RF, PE_POLICY_FF, PE_POLICY_RR, PE_POLICY_FR, PE_POLICY_RF }; bool is1[] = { true, true, true, true, false, false, false, false }; bool fw[] = { true, false, true, false, false, true, true, false }; bool oleft[] = { false, false, false, false, false, false, false, false }; bool ofw[] = { true, false, false, true, false, true, false, true }; for(int i = 0; i < 8; i++) { ostringstream oss; oss << "Simple"; oss << i; testCaseOtherMate( oss.str(), policies[i], // policy 30, // maxfrag 20, // minfrag false, // local true, // flipping OK true, // dovetail OK true, // containment OK true, // overlap OK true, // expand-to-fit is1[i], // mate 1 is anchor fw[i], // anchor aligned to Watson 100, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate oleft[i], // wheter to look for opposite to left 80, // expected leftmost pos for opp mate LHS 129, // expected rightmost pos for opp mate LHS 119, // expected leftmost pos for opp mate RHS 129, // expected rightmost pos for opp mate RHS ofw[i]); // expected orientation in which opposite mate must align } } // Set of 8 cases where we look for the opposite mate to the left // of the anchor mate, with various combinations of policies and // anchor-mate orientations. // |--------| // ^100 ^109 // |--------| // ^110 ^119 // |------------------| // min frag // |-Anchor-| // ^120 ^129 // |----------------------------| // max frag // ^ // 100 { int policies[] = { PE_POLICY_FF, PE_POLICY_RR, PE_POLICY_FR, PE_POLICY_RF, PE_POLICY_FF, PE_POLICY_RR, PE_POLICY_FR, PE_POLICY_RF }; bool is1[] = { false, false, false, false, true, true, true, true }; bool fw[] = { true, false, false, true, false, true, false, true }; bool oleft[] = { true, true, true, true, true, true, true, true }; bool ofw[] = { true, false, true, false, false, true, true, false }; for(int i = 0; i < 8; i++) { ostringstream oss; oss << "Simple"; oss << (i+8); testCaseOtherMate( oss.str(), policies[i], // policy 30, // maxfrag 20, // minfrag false, // local true, // flipping OK true, // dovetail OK true, // containment OK true, // overlap OK true, // expand-to-fit is1[i], // mate 1 is anchor fw[i], // anchor aligned to Watson 120, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate oleft[i], // wheter to look for opposite to left 100, // expected leftmost pos for opp mate LHS 110, // expected rightmost pos for opp mate LHS 100, // expected leftmost pos for opp mate RHS 149, // expected rightmost pos for opp mate RHS ofw[i]); // expected orientation in which opposite mate must align } } // Case where min frag == max frag and opposite is to the right // |----------------------------| // min frag // |--------| // ^120 ^129 // |----------------------------| // max frag // ^ // 100 testCaseOtherMate( "MinFragEqMax1", PE_POLICY_FR, // policy 30, // maxfrag 30, // minfrag false, // local true, // flipping OK true, // dovetail OK true, // containment OK true, // overlap OK true, // expand-to-fit false, // mate 1 is anchor false, // anchor aligned to Watson 120, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate true, // wheter to look for opposite to left 100, // expected leftmost pos for opp mate LHS 100, // expected rightmost pos for opp mate LHS 100, // expected leftmost pos for opp mate RHS 149, // expected rightmost pos for opp mate RHS true); // expected orientation in which opposite mate must align // Case where min frag == max frag and opposite is to the right // |----------------------------| // min frag ^129 // |--------| // ^100 ^109 // |----------------------------| // max frag testCaseOtherMate( "MinFragEqMax2", PE_POLICY_FR, // policy 30, // maxfrag 30, // minfrag false, // local true, // flipping OK true, // dovetail OK true, // containment OK true, // overlap OK true, // expand-to-fit true, // mate 1 is anchor true, // anchor aligned to Watson 100, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate false, // wheter to look for opposite to left 80, // expected leftmost pos for opp mate LHS 129, // expected rightmost pos for opp mate LHS 129, // expected leftmost pos for opp mate RHS 129, // expected rightmost pos for opp mate RHS false); // expected orientation in which opposite mate must align testCaseOtherMate( "MinFragEqMax4NoDove1", PE_POLICY_FR, // policy 30, // maxfrag 25, // minfrag false, // local true, // flipping OK false, // dovetail OK true, // containment OK true, // overlap OK true, // expand-to-fit true, // mate 1 is anchor true, // anchor aligned to Watson 100, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate false, // wheter to look for opposite to left 100, // expected leftmost pos for opp mate LHS 129, // expected rightmost pos for opp mate LHS 124, // expected leftmost pos for opp mate RHS 129, // expected rightmost pos for opp mate RHS false); // expected orientation in which opposite mate must align testCaseOtherMate( "MinFragEqMax4NoCont1", PE_POLICY_FR, // policy 30, // maxfrag 25, // minfrag false, // local true, // flipping OK false, // dovetail OK false, // containment OK true, // overlap OK true, // expand-to-fit true, // mate 1 is anchor true, // anchor aligned to Watson 100, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate false, // wheter to look for opposite to left 100, // expected leftmost pos for opp mate LHS 129, // expected rightmost pos for opp mate LHS 124, // expected leftmost pos for opp mate RHS 129, // expected rightmost pos for opp mate RHS false); // expected orientation in which opposite mate must align testCaseOtherMate( "MinFragEqMax4NoOlap1", PE_POLICY_FR, // policy 30, // maxfrag 25, // minfrag false, // local true, // flipping OK false, // dovetail OK false, // containment OK false, // overlap OK true, // expand-to-fit true, // mate 1 is anchor true, // anchor aligned to Watson 100, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate false, // wheter to look for opposite to left 110, // expected leftmost pos for opp mate LHS 129, // expected rightmost pos for opp mate LHS 124, // expected leftmost pos for opp mate RHS 129, // expected rightmost pos for opp mate RHS false); // expected orientation in which opposite mate must align testCaseOtherMate( "MinFragEqMax4NoDove2", PE_POLICY_FR, // policy 30, // maxfrag 25, // minfrag false, // local true, // flipping OK false, // dovetail OK true, // containment OK true, // overlap OK true, // expand-to-fit false, // mate 1 is anchor false, // anchor aligned to Watson 120, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate true, // whether to look for opposite to left 100, // expected leftmost pos for opp mate LHS 105, // expected rightmost pos for opp mate LHS 100, // expected leftmost pos for opp mate RHS 129, // expected rightmost pos for opp mate RHS true); // expected orientation in which opposite mate must align testCaseOtherMate( "MinFragEqMax4NoOlap2", PE_POLICY_FR, // policy 30, // maxfrag 25, // minfrag false, // local true, // flipping OK false, // dovetail OK false, // containment OK false, // overlap OK true, // expand-to-fit false, // mate 1 is anchor false, // anchor aligned to Watson 120, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate true, // whether to look for opposite to left 100, // expected leftmost pos for opp mate LHS 105, // expected rightmost pos for opp mate LHS 100, // expected leftmost pos for opp mate RHS 119, // expected rightmost pos for opp mate RHS true); // expected orientation in which opposite mate must align { int olls[] = { 110 }; int olrs[] = { 299 }; int orls[] = { 149 }; int orrs[] = { 299 }; for(int i = 0; i < 1; i++) { ostringstream oss; oss << "Overhang1_"; oss << (i+1); testCaseOtherMate( oss.str(), PE_POLICY_FR, // policy 200, // maxfrag 50, // minfrag false, // local true, // flipping OK true, // dovetail OK true, // containment OK false, // overlap OK true, // expand-to-fit true, // mate 1 is anchor true, // anchor aligned to Watson 100, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate false, // whether to look for opposite to left olls[i], // expected leftmost pos for opp mate LHS olrs[i], // expected rightmost pos for opp mate LHS orls[i], // expected leftmost pos for opp mate RHS orrs[i], // expected rightmost pos for opp mate RHS false); // expected orientation in which opposite mate must align } } { int olls[] = { -100 }; int olrs[] = { 50 }; int orls[] = { -100 }; int orrs[] = { 89 }; for(int i = 0; i < 1; i++) { ostringstream oss; oss << "Overhang2_"; oss << (i+1); testCaseOtherMate( oss.str(), PE_POLICY_FR, // policy 200, // maxfrag 50, // minfrag false, // local true, // flipping OK true, // dovetail OK true, // containment OK false, // overlap OK true, // expand-to-fit true, // mate 1 is anchor false, // anchor aligned to Watson 90, // anchor's offset into ref -1, // max # alignment cols 200, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate true, // whether to look for opposite to left olls[i], // expected leftmost pos for opp mate LHS olrs[i], // expected rightmost pos for opp mate LHS orls[i], // expected leftmost pos for opp mate RHS orrs[i], // expected rightmost pos for opp mate RHS true); // expected orientation in which opposite mate must align } } { int mate2offs[] = { 150, 149, 149, 100, 99, 299, 1, 250, 250 }; int mate2lens[] = { 50, 50, 51, 100, 101, 1, 50, 50, 51 }; int peExpects[] = { PE_ALS_NORMAL, PE_ALS_DISCORD, PE_ALS_OVERLAP, PE_ALS_CONTAIN, PE_ALS_DOVETAIL, PE_ALS_NORMAL, PE_ALS_DISCORD, PE_ALS_NORMAL, PE_ALS_DISCORD }; for(int i = 0; i < 9; i++) { ostringstream oss; oss << "Simple1_"; oss << (i); testCaseClassify( oss.str(), PE_POLICY_FR, // policy 200, // maxfrag 100, // minfrag false, // local true, // flipping OK true, // dovetail OK true, // containment OK true, // overlap OK true, // expand-to-fit 100, // offset of mate 1 50, // length of mate 1 true, // whether mate 1 aligned to Watson mate2offs[i], // offset of mate 2 mate2lens[i], // length of mate 2 false, // whether mate 2 aligned to Watson peExpects[i]);// expectation for PE_ALS flag returned } } { int mate1offs[] = { 200, 201, 200, 200, 200, 100, 400, 100, 99 }; int mate1lens[] = { 50, 49, 51, 100, 101, 1, 50, 50, 51 }; int peExpects[] = { PE_ALS_NORMAL, PE_ALS_DISCORD, PE_ALS_OVERLAP, PE_ALS_CONTAIN, PE_ALS_DOVETAIL, PE_ALS_NORMAL, PE_ALS_DISCORD, PE_ALS_NORMAL, PE_ALS_DISCORD }; for(int i = 0; i < 9; i++) { ostringstream oss; oss << "Simple2_"; oss << (i); testCaseClassify( oss.str(), PE_POLICY_FR, // policy 200, // maxfrag 100, // minfrag false, // local true, // flipping OK true, // dovetail OK true, // containment OK true, // overlap OK true, // expand-to-fit mate1offs[i], // offset of mate 1 mate1lens[i], // length of mate 1 true, // whether mate 1 aligned to Watson 250, // offset of mate 2 50, // length of mate 2 false, // whether mate 2 aligned to Watson peExpects[i]);// expectation for PE_ALS flag returned } } testCaseOtherMate( "Regression1", PE_POLICY_FF, // policy 50, // maxfrag 0, // minfrag false, // local true, // flipping OK true, // dovetail OK true, // containment OK true, // overlap OK true, // expand-to-fit true, // mate 1 is anchor false, // anchor aligned to Watson 3, // anchor's offset into ref -1, // max # alignment cols 53, // ref length 10, // mate 1 length 10, // mate 2 length true, // expected return val from otherMate true, // whether to look for opposite to left -37, // expected leftmost pos for opp mate LHS 13, // expected rightmost pos for opp mate LHS -37, // expected leftmost pos for opp mate RHS 52, // expected rightmost pos for opp mate RHS false); // expected orientation in which opposite mate must align } #endif /*def MAIN_PE*/ hisat2-2.2.1/pe.h000066400000000000000000000230621370663763200135030ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /* * pe.h * * A class encapsulating a paired-end policy and routines for * identifying intervals according to the policy. For instance, * contains a routine that, given a policy and details about a match * for one mate, returns details about where to search for the other * mate. */ #ifndef PE_H_ #define PE_H_ #include #include // In description below "To the left" = "Upstream of w/r/t the Watson strand" // The 4 possible policies describing how mates 1 and 2 should be // oriented with respect to the reference genome and each other enum { // (fw) Both mates from Watson with 1 to the left, or // (rc) Both mates from Crick with 2 to the left PE_POLICY_FF = 1, // (fw) Both mates from Crick with 1 to the left, or // (rc) Both mates from Watson with 2 to the left PE_POLICY_RR, // (fw) Mate 1 from Watson and mate 2 from Crick with 1 to the left, or // (rc) Mate 2 from Watson and mate 1 from Crick with 2 to the left PE_POLICY_FR, // (fw) Mate 1 from Crick and mate 2 from Watson with 1 to the left, or // (rc) Mate 2 from Crick and mate 1 from Watson with 2 to the left PE_POLICY_RF }; // Various distinct ways that the mates might align with respect to // each other in a concordant alignment. We distinguish between them // because in some cases a user may want to consider some of these // categories to be discordant, even if the alignment otherwise // conforms to the paired-end policy. enum { // Describes a paired-end alignment where the mates // straightforwardly conform to the paired-end policy without any // overlap between the mates PE_ALS_NORMAL = 1, // Describes a paired-end alignment where the mate overlap, but // neither contains the other and they do not dovetail, but the // alignment conforms to the paired-end policy PE_ALS_OVERLAP, // Describes a paired-end alignment where the mates conform to the // paired-end policy, but one mate strictly contains the other but // they don't dovetail. We distinguish this from a "normal" // concordant alignment because some users may wish to categorize // such an alignment as discordant. PE_ALS_CONTAIN, // Describes a paired-end alignment where the mates conform to the // paired-end policy, but mates "fall off" each other. E.g. if the // policy is FR and any of these happen: // 1: >>>>> >>>>> // 2: <<<<<< <<<<<< // And the overall extent is consistent with the minimum fragment // length, this is a dovetail alignment. We distinguish this from // a "normal" concordant alignment because some users may wish to // categorize such an alignment as discordant. PE_ALS_DOVETAIL, // The mates are clearly discordant, owing to their orientations // and/or implied fragment length PE_ALS_DISCORD }; /** * Return true iff the orientations and relative positions of mates 1 * and 2 are compatible with the given PE_POLICY. */ static inline bool pePolicyCompat( int policy, // PE_POLICY bool oneLeft, // true iff mate 1 is to the left of mate 2 bool oneWat, // true iff mate 1 aligned to Watson strand bool twoWat) // true iff mate 2 aligned to Watson strand { switch(policy) { case PE_POLICY_FF: return oneWat == twoWat && oneWat == oneLeft; case PE_POLICY_RR: return oneWat == twoWat && oneWat != oneLeft; case PE_POLICY_FR: return oneWat != twoWat && oneWat == oneLeft; case PE_POLICY_RF: return oneWat != twoWat && oneWat != oneLeft; default: { std::cerr << "Bad PE_POLICY: " << policy << std::endl; throw 1; } } throw 1; } /** * Given that the given mate aligns in the given orientation, return * true iff the other mate must appear "to the right" of the given mate * in order for the alignment to be concordant. */ static inline void pePolicyMateDir( int policy,// in: PE_POLICY bool is1, // in: true iff mate 1 is the one that already aligned bool fw, // in: true iff already-aligned mate aligned to Watson bool& left, // out: set =true iff other mate must be to the left bool& mfw) // out: set =true iff other mate must align to watson { switch(policy) { case PE_POLICY_FF: { left = (is1 != fw); mfw = fw; break; } case PE_POLICY_RR: { left = (is1 == fw); mfw = fw; break; } case PE_POLICY_FR: { left = !fw; mfw = !fw; break; } case PE_POLICY_RF: { left = fw; mfw = !fw; break; } default: { std::cerr << "Error: No such PE_POLICY: " << policy << std::endl; throw 1; } } return; } /** * Encapsulates paired-end alignment parameters. */ class PairedEndPolicy { public: PairedEndPolicy() { reset(); } PairedEndPolicy( int pol, size_t maxfrag, size_t minfrag, bool local, bool flippingOk, bool dovetailOk, bool containOk, bool olapOk, bool expandToFit) { init( pol, maxfrag, minfrag, local, flippingOk, dovetailOk, containOk, olapOk, expandToFit); } /** * Initialize with nonsense values. */ void reset() { init(-1, 0xffffffff, 0xffffffff, false, false, false, false, false, false); } /** * Initialize given policy, maximum & minimum fragment lengths. */ void init( int pol, size_t maxfrag, size_t minfrag, bool local, bool flippingOk, bool dovetailOk, bool containOk, bool olapOk, bool expandToFit) { pol_ = pol; maxfrag_ = maxfrag; minfrag_ = minfrag; local_ = local; flippingOk_ = flippingOk; dovetailOk_ = dovetailOk; containOk_ = containOk; olapOk_ = olapOk; expandToFit_ = expandToFit; } /** * Given details about how one mate aligns, and some details about the * reference sequence it aligned to, calculate a window and orientation s.t. * a paired-end alignment is concordant iff the opposite mate aligns in the * calculated window with the calculated orientation. The calculaton does not * consider gaps. The dynamic programming framer will take gaps into account. * * Returns false if no concordant alignments are possible, true otherwise. */ bool otherMate( bool is1, // true -> mate 1 aligned and we're looking // for 2, false -> vice versa bool fw, // orientation of aligned mate int64_t off, // offset into the reference sequence int64_t maxalcols, // max # columns spanned by alignment size_t reflen, // length of reference sequence aligned to size_t len1, // length of mate 1 size_t len2, // length of mate 2 bool& oleft, // out: true iff opp mate must be to right of anchor int64_t& oll, // out: leftmost Watson off for LHS of opp alignment int64_t& olr, // out: rightmost Watson off for LHS of opp alignment int64_t& orl, // out: leftmost Watson off for RHS of opp alignment int64_t& orr, // out: rightmost Watson off for RHS of opp alignment bool& ofw) // out: true iff opp mate must be on Watson strand const; /** * Return a PE_TYPE flag indicating, given a PE_POLICY and coordinates * for a paired-end alignment, qwhat type of alignment it is, i.e., * whether it's: * * 1. Straightforwardly concordant * 2. Mates dovetail (one extends beyond the end of the other) * 3. One mate contains the other but they don't dovetail * 4. One mate overlaps the other but neither contains the other and * they don't dovetail * 5. Discordant */ int peClassifyPair( int64_t off1, // offset of mate 1 size_t len1, // length of mate 1 bool fw1, // whether mate 1 aligned to Watson int64_t off2, // offset of mate 2 size_t len2, // length of mate 2 bool fw2) // whether mate 2 aligned to Watson const; int policy() const { return pol_; } size_t maxFragLen() const { return maxfrag_; } size_t minFragLen() const { return minfrag_; } protected: // Use local alignment to search for the opposite mate, rather than // a type of alignment that requires the read to align end-to-end bool local_; // Policy governing how mates should be oriented with respect to // each other and the reference genome int pol_; // true iff settings are such that mates that violate the expected relative // orientation but are still consistent with maximum fragment length are OK bool flippingOk_; // true iff settings are such that dovetailed mates should be // considered concordant. bool dovetailOk_; // true iff paired-end alignments where one mate's alignment is // strictly contained within the other's should be considered // concordant bool containOk_; // true iff paired-end alignments where one mate's alignment // overlaps the other's should be considered concordant bool olapOk_; // What to do when a mate length is > maxfrag_? If expandToFit_ is // true, we temporarily increase maxfrag_ to equal the mate length. // Otherwise we say that any paired-end alignment involving the // long mate is discordant. bool expandToFit_; // Maximum fragment size to consider size_t maxfrag_; // Minimum fragment size to consider size_t minfrag_; }; #endif /*ndef PE_H_*/ hisat2-2.2.1/presets.cpp000066400000000000000000000052301370663763200151140ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include "presets.h" #include "opts.h" using namespace std; void PresetsV0::apply( const std::string& preset, std::string& policy, EList >& opts) { // Presets: Same as: // For --end-to-end: // --very-fast -M 5 -R 1 -N 0 -L 22 -i S,1,2.50 // --fast -M 10 -R 2 -N 0 -L 22 -i S,1,2.50 // --sensitive -M 15 -R 2 -N 0 -L 22 -i S,1,1.15 // --very-sensitive -M 25 -R 3 -N 0 -L 19 -i S,1,0.50 if(preset == "very-fast") { policy += ";SEED=0,22"; policy += ";DPS=5"; policy += ";ROUNDS=1"; policy += ";IVAL=S,0,2.50"; } else if(preset == "fast") { policy += ";SEED=0,22"; policy += ";DPS=10"; policy += ";ROUNDS=2"; policy += ";IVAL=S,0,2.50"; } else if(preset == "sensitive") { policy += ";SEED=0,22"; policy += ";DPS=15"; policy += ";ROUNDS=2"; policy += ";IVAL=S,1,1.15"; } else if(preset == "very-sensitive") { policy += ";SEED=0,20"; policy += ";DPS=20"; policy += ";ROUNDS=3"; policy += ";IVAL=S,1,0.50"; } // For --local: // --very-fast-local -M 1 -N 0 -L 25 -i S,1,2.00 // --fast-local -M 2 -N 0 -L 22 -i S,1,1.75 // --sensitive-local -M 2 -N 0 -L 20 -i S,1,0.75 (default) // --very-sensitive-local -M 3 -N 0 -L 20 -i S,1,0.50 else if(preset == "very-fast-local") { policy += ";SEED=0,25"; policy += ";DPS=5"; policy += ";ROUNDS=1"; policy += ";IVAL=S,1,2.00"; } else if(preset == "fast-local") { policy += ";SEED=0,22"; policy += ";DPS=10"; policy += ";ROUNDS=2"; policy += ";IVAL=S,1,1.75"; } else if(preset == "sensitive-local") { policy += ";SEED=0,20"; policy += ";DPS=15"; policy += ";ROUNDS=2"; policy += ";IVAL=S,1,0.75"; } else if(preset == "very-sensitive-local") { policy += ";SEED=0,20"; policy += ";DPS=20"; policy += ";ROUNDS=3"; policy += ";IVAL=S,1,0.50"; } else { cerr << "Unknown preset: " << preset.c_str() << endl; } } hisat2-2.2.1/presets.h000066400000000000000000000030431370663763200145610ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /** * presets.h * * Maps simple command-line options to more complicated combinations of * options for ease-of-use. */ #ifndef PRESETS_H_ #define PRESETS_H_ #include #include #include "ds.h" class Presets { public: Presets() { } virtual ~Presets() { } virtual void apply( const std::string& preset, std::string& policy, EList >& opts) = 0; virtual const char * name() = 0; }; /** * Initial collection of presets: 8/14/2011 prior to first Bowtie 2 release. */ class PresetsV0 : public Presets { public: PresetsV0() : Presets() { } virtual ~PresetsV0() { } virtual void apply( const std::string& preset, std::string& policy, EList >& opts); virtual const char * name() { return "V0"; } }; #endif /*ndef PRESETS_H_*/ hisat2-2.2.1/processor_support.h000066400000000000000000000045321370663763200167130ustar00rootroot00000000000000#ifndef PROCESSOR_SUPPORT_H_ #define PROCESSOR_SUPPORT_H_ // Utility class ProcessorSupport provides POPCNTenabled() to determine // processor support for POPCNT instruction. It uses CPUID to // retrieve the processor capabilities. // for Intel ICC compiler __cpuid() is an intrinsic // for Microsoft compiler __cpuid() is provided by #include // for GCC compiler __get_cpuid() is provided by #include // Intel compiler defines __GNUC__, so this is needed to disambiguate #if defined(__INTEL_COMPILER) # define USING_INTEL_COMPILER #elif defined(__GNUC__) # define USING_GCC_COMPILER # include #elif defined(_MSC_VER) // __MSC_VER defined by Microsoft compiler #define USING_MSC_COMPILER #endif struct regs_t {unsigned int EAX, EBX, ECX, EDX;}; #define BIT(n) ((1< * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /// An array that transforms Phred qualities into their maq-like /// equivalents by dividing by ten and rounding to the nearest 10, /// but saturating at 3. unsigned char qualRounds[] = { 0, 0, 0, 0, 0, // 0 - 4 10, 10, 10, 10, 10, 10, 10, 10, 10, 10, // 5 - 14 20, 20, 20, 20, 20, 20, 20, 20, 20, 20, // 15 - 24 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 25 - 34 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 35 - 44 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 45 - 54 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 55 - 64 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 65 - 74 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 75 - 84 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 85 - 94 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 95 - 104 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 105 - 114 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 115 - 124 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 125 - 134 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 135 - 144 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 145 - 154 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 155 - 164 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 165 - 174 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 175 - 184 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 185 - 194 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 195 - 204 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 205 - 214 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 215 - 224 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 225 - 234 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 235 - 244 30, 30, 30, 30, 30, 30, 30, 30, 30, 30, // 245 - 254 30 // 255 }; /** * Lookup table for converting from Solexa-scaled (log-odds) quality * values to Phred-scaled quality values. */ unsigned char solToPhred[] = { /* -10 */ 0, 1, 1, 1, 1, 1, 1, 2, 2, 3, /* 0 */ 3, 4, 4, 5, 5, 6, 7, 8, 9, 10, /* 10 */ 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, /* 20 */ 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, /* 30 */ 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, /* 40 */ 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, /* 50 */ 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, /* 60 */ 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, /* 70 */ 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, /* 80 */ 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, /* 90 */ 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, /* 100 */ 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, /* 110 */ 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, /* 120 */ 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, /* 130 */ 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, /* 140 */ 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, /* 150 */ 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, /* 160 */ 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, /* 170 */ 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, /* 180 */ 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, /* 190 */ 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, /* 200 */ 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, /* 210 */ 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, /* 220 */ 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, /* 230 */ 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, /* 240 */ 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, /* 250 */ 250, 251, 252, 253, 254, 255 }; hisat2-2.2.1/qual.h000066400000000000000000000147221370663763200140440ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef QUAL_H_ #define QUAL_H_ #include #include #include "search_globals.h" #include "sstring.h" extern unsigned char qualRounds[]; extern unsigned char solToPhred[]; /// Translate a Phred-encoded ASCII character into a Phred quality static inline uint8_t phredcToPhredq(char c) { return ((uint8_t)c >= 33 ? ((uint8_t)c - 33) : 0); } /** * Convert a Solexa-scaled quality value into a Phred-scale quality * value. * * p = probability that base is miscalled * Qphred = -10 * log10 (p) * Qsolexa = -10 * log10 (p / (1 - p)) * See: http://en.wikipedia.org/wiki/FASTQ_format * */ static inline uint8_t solexaToPhred(int sol) { assert_lt(sol, 256); if(sol < -10) return 0; return solToPhred[sol+10]; } class SimplePhredPenalty { public: static uint8_t mmPenalty (uint8_t qual) { return qual; } static uint8_t delPenalty(uint8_t qual) { return qual; } static uint8_t insPenalty(uint8_t qual_left, uint8_t qual_right) { return std::max(qual_left, qual_right); } }; class MaqPhredPenalty { public: static uint8_t mmPenalty (uint8_t qual) { return qualRounds[qual]; } static uint8_t delPenalty(uint8_t qual) { return qualRounds[qual]; } static uint8_t insPenalty(uint8_t qual_left, uint8_t qual_right) { return qualRounds[std::max(qual_left, qual_right)]; } }; static inline uint8_t mmPenalty(bool maq, uint8_t qual) { if(maq) { return MaqPhredPenalty::mmPenalty(qual); } else { return SimplePhredPenalty::mmPenalty(qual); } } static inline uint8_t delPenalty(bool maq, uint8_t qual) { if(maq) { return MaqPhredPenalty::delPenalty(qual); } else { return SimplePhredPenalty::delPenalty(qual); } } static inline uint8_t insPenalty(bool maq, uint8_t qual_left, uint8_t qual_right) { if(maq) { return MaqPhredPenalty::insPenalty(qual_left, qual_right); } else { return SimplePhredPenalty::insPenalty(qual_left, qual_right); } } /** * Take an ASCII-encoded quality value and convert it to a Phred33 * ASCII char. */ inline static char charToPhred33(char c, bool solQuals, bool phred64Quals) { using namespace std; if(c == ' ') { std::cerr << "Saw a space but expected an ASCII-encoded quality value." << endl << "Are quality values formatted as integers? If so, try --integer-quals." << endl; throw 1; } if (solQuals) { // Convert solexa-scaled chars to phred // http://maq.sourceforge.net/fastq.shtml char cc = solexaToPhred((int)c - 64) + 33; if (cc < 33) { std::cerr << "Saw ASCII character " << ((int)c) << " but expected 64-based Solexa qual (converts to " << (int)cc << ")." << endl << "Try not specifying --solexa-quals." << endl; throw 1; } c = cc; } else if(phred64Quals) { if (c < 64) { cerr << "Saw ASCII character " << ((int)c) << " but expected 64-based Phred qual." << endl << "Try not specifying --solexa1.3-quals/--phred64-quals." << endl; throw 1; } // Convert to 33-based phred c -= (64-33); } else { // Keep the phred quality if (c < 33) { cerr << "Saw ASCII character " << ((int)c) << " but expected 33-based Phred qual." << endl; throw 1; } } return c; } /** * Take an integer quality value and convert it to a Phred33 ASCII * char. */ inline static char intToPhred33(int iQ, bool solQuals) { using namespace std; int pQ; if (solQuals) { // Convert from solexa quality to phred // quality and translate to ASCII // http://maq.sourceforge.net/qual.shtml pQ = solexaToPhred((int)iQ) + 33; } else { // Keep the phred quality and translate // to ASCII pQ = (iQ <= 93 ? iQ : 93) + 33; } if (pQ < 33) { cerr << "Saw negative Phred quality " << ((int)pQ-33) << "." << endl; throw 1; } assert_geq(pQ, 0); return (int)pQ; } inline static uint8_t roundPenalty(uint8_t p) { if(gNoMaqRound) return p; return qualRounds[p]; } /** * Fill the q[] array with the penalties that are determined by * subtracting the quality values of the alternate basecalls from * the quality of the primary basecall. */ inline static uint8_t penaltiesAt(size_t off, uint8_t *q, int alts, const BTString& qual, const BTDnaString *altQry, const BTString *altQual) { uint8_t primQ = qual[off]; // qual of primary call uint8_t bestPenalty = roundPenalty(phredcToPhredq(primQ)); // By default, any mismatch incurs a penalty equal to the quality // of the called base q[0] = q[1] = q[2] = q[3] = bestPenalty; for(int i = 0; i < alts; i++) { uint8_t altQ = altQual[i][off]; // qual of alt call if(altQ == 33) break; // no alt call assert_leq(altQ, primQ); uint8_t pen = roundPenalty(primQ - altQ); if(pen < bestPenalty) { bestPenalty = pen; } // Get the base int altC = (int)altQry[i][off]; assert_lt(altC, 4); q[altC] = pen; } // Return the best penalty so that the caller can evaluate whether // any of the penalties are within-budget return bestPenalty; } /** * Fill the q[] array with the penalties that are determined by * subtracting the quality values of the alternate basecalls from * the quality of the primary basecall. */ inline static uint8_t loPenaltyAt(size_t off, int alts, const BTString& qual, const BTString *altQual) { uint8_t primQ = qual[off]; // qual of primary call uint8_t bestPenalty = roundPenalty(phredcToPhredq(primQ)); for(int i = 0; i < alts; i++) { uint8_t altQ = altQual[i][off]; // qual of alt call if(altQ == 33) break; // no more alt calls at this position assert_leq(altQ, primQ); uint8_t pen = roundPenalty(primQ - altQ); if(pen < bestPenalty) { bestPenalty = pen; } } return bestPenalty; } #endif /*QUAL_H_*/ hisat2-2.2.1/radix_sort.h000066400000000000000000000250041370663763200152530ustar00rootroot00000000000000#ifndef RADIX_SORT_H_ #define RADIX_SORT_H_ #include // in place radix sort using a single thread, should not be called directly // used for leaves of both in and out of place radix sorts template static void _radix_sort(T* begin, T* end, index_t (*hash)(T&), int log_size) { const int SHIFT = 8; const int BLOCKS = (1 << (SHIFT + 1)); const int BLOCKS_MASK = BLOCKS - 1; // compute maximum of log_size - 7 and 0 int right_shift = (log_size - SHIFT) * (log_size > SHIFT); // count number in each bin index_t count[BLOCKS] = {0}; for(T* curr = begin; curr != end; curr++) { count[(hash(*curr) >> right_shift) & BLOCKS_MASK]++; } // sum numbers to create an index T* index[BLOCKS + 1]; T* place[BLOCKS]; index[0] = place[0] = begin; for(int i = 1; i < BLOCKS; i++) { index[i] = place[i] = index[i - 1] + count[i - 1]; } index[BLOCKS] = end; //put objects in proper place for(int bin = 0; bin < BLOCKS; bin++) { while(place[bin] != index[bin + 1]) { T curr = *place[bin]; int x = (hash(curr) >> right_shift) & BLOCKS_MASK; while(x != bin) { T temp = *place[x]; *place[x]++ = curr; curr = temp; x = (hash(curr) >> right_shift) & BLOCKS_MASK; } *place[bin]++ = curr; } } //sort partitions for(int bin = 0; bin < BLOCKS; bin++) { if(index[bin + 1] - index[bin] > 64 && right_shift) { _radix_sort(index[bin], index[bin + 1], hash, right_shift); } else if (index[bin + 1] - index[bin] > 1) { sort(index[bin], index[bin + 1], CMP()); } } } template struct RecurseParams { index_t (*hash)(T&); T** begin; int log_size; int num; }; //basically used to wrap together calls to bin_sort template static void _radix_sort_worker(void* vp) { RecurseParams* params = (RecurseParams*)vp; index_t (*hash)(T&) = params->hash; T** begin = params->begin; int log_size = params->log_size; int num = params->num; for(int i = 0; i < num; i++) { if(begin[i + 1] - begin[i] > 1) _radix_sort(begin[i], begin[i + 1], hash, log_size); } } template void radix_sort_in_place(T* begin, T* end, index_t (*hash)(T&), index_t maxv, int nthreads = 1) { const int SHIFT = 8; const int BLOCKS = (1 << (SHIFT + 1)); int log_size = sizeof(maxv) * 8; while(!((1 << log_size) & maxv)) log_size--; int right_shift = log_size - SHIFT; // {(maxv >> right_shift) + 1 <= BLOCKS}, int occupied = (maxv >> right_shift) + 1; time_t start = time(0); // count number in each bin index_t count[BLOCKS] = {0}; for(T* curr = begin; curr != end; curr++) { count[hash(*curr) >> right_shift]++; } // sum numbers to create an index T* index[BLOCKS + 1]; T* place[BLOCKS]; index[0] = place[0] = begin; for(int i = 1; i < occupied; i++) { index[i] = place[i] = index[i - 1] + count[i - 1]; } index[occupied] = end; if(nthreads != 1) cerr << "COUNT NUMBER IN EACH BIN: " << time(0) - start << endl; start = time(0); //put objects in proper place for(int bin = 0; bin < occupied; bin++) { while(place[bin] != index[bin + 1]) { T curr = *place[bin]; int x = hash(curr) >> right_shift; while(x != bin) { // switched inner loop here, removed branch statement T temp = *place[x]; *place[x]++ = curr; curr = temp; x = hash(curr) >> right_shift; } *place[bin]++ = curr; } } if(nthreads != 1) cerr << "PLACE IN CORRECT BIN: " << time(0) - start << endl; start = time(0); //sort partitions if(nthreads == 1) { for(int bin = 0; bin < occupied; bin++) { if(index[bin + 1] - index[bin] > 1) _radix_sort(index[bin], index[bin + 1], hash, right_shift); } } else { AutoArray threads(nthreads); EList > params; params.resizeExact(nthreads); int st = 0; for(int i = 0; i < nthreads; i++) { params[i].hash = hash; params[i].begin = index + st; params[i].log_size = right_shift; params[i].num = occupied / nthreads; threads[i] = new tthread::thread(&_radix_sort_worker, (void*)¶ms[i]); st += params[i].num; } //do any remaining bins using main thread for(int bin = st; bin < occupied; bin++) { if(index[bin + 1] - index[bin] > 1) _radix_sort(index[bin], index[bin + 1], hash, right_shift); } for(int i = 0; i < nthreads; i++) { threads[i]->join(); } } if(nthreads != 1) cerr << "FINISHED RECURSIVE SORTS: " << time(0) - start << endl; } template struct CountParams { T* begin; T* end; T* o; index_t* count; index_t (*hash)(T&); int occupied; int right_shift; }; template static void _count_worker(void* vp) { CountParams* params = (CountParams*)vp; T* begin = params->begin; T* end = params->end; index_t (*hash)(T&) = params->hash; int occupied = params->occupied; int right_shift = params->right_shift; params->count = new index_t[occupied + 1](); for(T* curr = begin; curr != end; curr++) { params->count[hash(*curr) >> right_shift]++; } } template static void _write_worker(void* vp) { CountParams* params = (CountParams*)vp; T* begin = params->begin; T* end = params->end; T* o = params->o; index_t* count = params->count; index_t (*hash)(T&) = params->hash; int right_shift = params->right_shift; for(T* curr = begin; curr != end; curr++) { o[count[hash(*curr) >> right_shift]++] = *curr; } } template void radix_sort_copy(T* begin, T* end, T* o, index_t (*hash)(T&), index_t maxv, int nthreads = 1) { //set parameters const int SHIFT = 8; const int BLOCKS = (1 << (SHIFT + 1)); int log_size = sizeof(maxv) * 8; while(!((1 << log_size) & maxv)) log_size--; int right_shift = log_size - SHIFT; int occupied = (maxv >> right_shift) + 1; //count nodes time_t start = time(0); EList > cparams; cparams.resizeExact(nthreads); AutoArray threads1(nthreads); T* st = begin; T* en = st + (end - begin) / nthreads; for(int i = 0; i < nthreads; i++) { cparams[i].begin = st; cparams[i].end = en; cparams[i].hash = hash; cparams[i].o = o; cparams[i].occupied = occupied; cparams[i].right_shift= right_shift; if(nthreads == 1) { _count_worker((void*)&cparams[i]); } else { threads1[i] = new tthread::thread(&_count_worker, (void*)&cparams[i]); } st = en; if(i + 2 == nthreads) { en = end; } else { en = st + (end - begin) / nthreads; } } if(nthreads > 1) { for(int i = 0; i < nthreads; i++) { threads1[i]->join(); delete threads1[i]; } } if(nthreads != 1) cerr << "COUNT NUMBER IN EACH BIN: " << time(0) - start << endl; start = time(0); //transform counts into index index_t tot = cparams[0].count[0]; cparams[0].count[0] = 0; for(int i = 1; i < nthreads; i++) { tot += cparams[i].count[0]; cparams[i].count[0] = tot - cparams[i].count[0]; } for(int j = 1; j < occupied + 1; j++) { for(int i = 0; i < nthreads; i++) { tot += cparams[i].count[j]; cparams[i].count[j] = tot - cparams[i].count[j]; } } T* index[BLOCKS + 1]; for(int i = 0; i < occupied + 1; i++) { index[i] = o + cparams[0].count[i]; } //write T's to correct bin if(nthreads == 1) { _write_worker((void*)&cparams[0]); } else { for(int i = 0; i < nthreads; i++) threads1[i] = new tthread::thread(&_write_worker, (void*)&cparams[i]); for(int i = 0; i < nthreads; i++) { threads1[i]->join(); } } for(int i = 0; i < nthreads; i++) { delete[] cparams[i].count; delete threads1[i]; } if(nthreads != 1) cerr << "FINISHED FIRST ROUND: " << time(0) - start << endl; start = time(0); //sort partitions if(nthreads == 1) { for(int bin = 0; bin < occupied; bin++) if(index[bin + 1] - index[bin] > 1) _radix_sort(index[bin], index[bin + 1], hash, right_shift); } else { AutoArray threads(nthreads); EList > params; params.resizeExact(nthreads); int st = 0; for(int i = 0; i < nthreads; i++) { params[i].hash = hash; params[i].begin = index + st; params[i].log_size = right_shift; params[i].num = 0; index_t remaining_elements = (index_t)(index[occupied] - index[st]); while(params[i].num + st < occupied && (index_t)(index[params[i].num + st] - index[st]) < remaining_elements / (nthreads - i)) params[i].num++; cerr << params[i].num << " " << (index_t)(index[params[i].num + st] - index[st]) << endl; threads[i] = new tthread::thread(&_radix_sort_worker, (void*)¶ms[i]); st += params[i].num; } //do any remaining bins using main thread for(int bin = st; bin < occupied; bin++) { if(index[bin + 1] - index[bin] > 1) _radix_sort(index[bin], index[bin + 1], hash, right_shift); } for(int i = 0; i < nthreads; i++) { threads[i]->join(); delete threads[i]; } } if(nthreads != 1) cerr << "FINISHED RECURSIVE SORTS: " << time(0) - start << endl; } #endif //RADIX_SORT_H_ hisat2-2.2.1/random_source.cpp000066400000000000000000000063201370663763200162700ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "random_source.h" #include "random_util.h" #ifdef MERSENNE_TWISTER void RandomSource::gen_state() { for(int i = 0; i < (n - m); ++i) { state_[i] = state_[i + m] ^ twiddle(state_[i], state_[i + 1]); } for(int i = n - m; i < (n - 1); ++i) { state_[i] = state_[i + m - n] ^ twiddle(state_[i], state_[i + 1]); } state_[n - 1] = state_[m - 1] ^ twiddle(state_[n - 1], state_[0]); p_ = 0; // reset position } void RandomSource::init(uint32_t s) { // init by 32 bit seed reset(); state_[0] = s; for(int i = 1; i < n; ++i) { state_[i] = 1812433253UL * (state_[i - 1] ^ (state_[i - 1] >> 30)) + i; } p_ = n; // force gen_state() to be called for next random number inited_ = true; } void RandomSource::init(const uint32_t* array, int size) { // init by array init(19650218UL); int i = 1, j = 0; for(int k = ((n > size) ? n : size); k; --k) { state_[i] = (state_[i] ^ ((state_[i - 1] ^ (state_[i - 1] >> 30)) * 1664525UL)) + array[j] + j; // non linear ++j; j %= size; if((++i) == n) { state_[0] = state_[n - 1]; i = 1; } } for(int k = n - 1; k; --k) { state_[i] = (state_[i] ^ ((state_[i - 1] ^ (state_[i - 1] >> 30)) * 1566083941UL)) - i; if((++i) == n) { state_[0] = state_[n - 1]; i = 1; } } state_[0] = 0x80000000UL; // MSB is 1; assuring non-zero initial array p_ = n; // force gen_state() to be called for next random number inited_ = true; } #endif #ifdef MAIN_RANDOM_SOURCE using namespace std; int main(void) { cerr << "Test 1" << endl; { RandomSource rnd; int cnts[32]; for(size_t i = 0; i < 32; i++) { cnts[i] = 0; } for(uint32_t j = 0; j < 10; j++) { rnd.init(j); for(size_t i = 0; i < 10000; i++) { uint32_t rndi = rnd.nextU32(); for(size_t i = 0; i < 32; i++) { if((rndi & 1) != 0) { cnts[i]++; } rndi >>= 1; } } for(size_t i = 0; i < 32; i++) { cerr << i << ": " << cnts[i] << endl; } } } cerr << "Test 2" << endl; { int cnts[4][4]; for(size_t i = 0; i < 4; i++) { for(size_t j = 0; j < 4; j++) { cnts[i][j] = 0; } } RandomSource rnd; Random1toN rn1n; for(size_t i = 0; i < 100; i++) { rnd.init((uint32_t)i); rn1n.init(4, true); uint32_t ri = rn1n.next(rnd); cnts[ri][0]++; ri = rn1n.next(rnd); cnts[ri][1]++; ri = rn1n.next(rnd); cnts[ri][2]++; ri = rn1n.next(rnd); cnts[ri][3]++; } for(size_t i = 0; i < 4; i++) { for(size_t j = 0; j < 4; j++) { cerr << cnts[i][j]; if(j < 3) { cerr << ", "; } } cerr << endl; } } } #endif hisat2-2.2.1/random_source.h000066400000000000000000000123461370663763200157420ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef RANDOM_GEN_H_ #define RANDOM_GEN_H_ #include #include "assert_helpers.h" //#define MERSENNE_TWISTER #ifndef MERSENNE_TWISTER /** * Simple pseudo-random linear congruential generator, a la Numerical * Recipes. */ class RandomSource { public: static const uint32_t DEFUALT_A = 1664525; static const uint32_t DEFUALT_C = 1013904223; RandomSource() : a(DEFUALT_A), c(DEFUALT_C), inited_(false) { } RandomSource(uint32_t _last) : a(DEFUALT_A), c(DEFUALT_C), last(_last), inited_(true) { } RandomSource(uint32_t _a, uint32_t _c) : a(_a), c(_c), inited_(false) { } void init(uint32_t seed = 0) { last = seed; inited_ = true; lastOff = 30; } uint32_t nextU32() { assert(inited_); uint32_t ret; last = a * last + c; ret = last >> 16; last = a * last + c; ret ^= last; lastOff = 0; return ret; } uint64_t nextU64() { assert(inited_); uint64_t first = nextU32(); first = first << 32; uint64_t second = nextU32(); return first | second; } /** * Return a pseudo-random unsigned 32-bit integer sampled uniformly * from [lo, hi]. */ uint32_t nextU32Range(uint32_t lo, uint32_t hi) { uint32_t ret = lo; if(hi > lo) { ret += (nextU32() % (hi-lo+1)); } return ret; } /** * Get next 2-bit unsigned integer. */ uint32_t nextU2() { assert(inited_); if(lastOff > 30) { nextU32(); } uint32_t ret = (last >> lastOff) & 3; lastOff += 2; return ret; } /** * Get next boolean. */ bool nextBool() { assert(inited_); if(lastOff > 31) { nextU32(); } uint32_t ret = (last >> lastOff) & 1; lastOff++; return ret; } /** * Return an unsigned int chosen by picking randomly from among * options weighted by probabilies supplied as the elements of the * 'weights' array of length 'numWeights'. The weights should add * to 1. */ uint32_t nextFromProbs( const float* weights, size_t numWeights) { float f = nextFloat(); float tot = 0.0f; // total weight seen so far for(uint32_t i = 0; i < numWeights; i++) { tot += weights[i]; if(f < tot) return i; } return (uint32_t)(numWeights-1); } float nextFloat() { assert(inited_); return (float)nextU32() / (float)0xffffffff; } static uint32_t nextU32(uint32_t last, uint32_t a = DEFUALT_A, uint32_t c = DEFUALT_C) { return (a * last) + c; } uint32_t currentA() const { return a; } uint32_t currentC() const { return c; } uint32_t currentLast() const { return last; } private: uint32_t a; uint32_t c; uint32_t last; uint32_t lastOff; bool inited_; }; #else class RandomSource { // Mersenne Twister random number generator public: // default constructor: uses default seed only if this is the first instance RandomSource() { reset(); } // constructor with 32 bit int as seed RandomSource(uint32_t s) { init(s); } // constructor with array of size 32 bit ints as seed RandomSource(const uint32_t* array, int size) { init(array, size); } void reset() { state_[0] = 0; p_ = 0; inited_ = false; } virtual ~RandomSource() { } // the two seed functions void init(uint32_t); // seed with 32 bit integer void init(const uint32_t*, int size); // seed with array /** * Return next 1-bit unsigned integer. */ bool nextBool() { return (nextU32() & 1) == 0; } /** * Get next unsigned 32-bit integer. */ inline uint32_t nextU32() { assert(inited_); if(p_ == n) { gen_state(); // new state vector needed } // gen_state() is split off to be non-inline, because it is only called once // in every 624 calls and otherwise irand() would become too big to get inlined uint32_t x = state_[p_++]; x ^= (x >> 11); x ^= (x << 7) & 0x9D2C5680UL; x ^= (x << 15) & 0xEFC60000UL; x ^= (x >> 18); return x; } /** * Return next float between 0 and 1. */ float nextFloat() { assert(inited_); return (float)nextU32() / (float)0xffffffff; } protected: // used by derived classes, otherwise not accessible; use the ()-operator static const int n = 624, m = 397; // compile time constants // the variables below are static (no duplicates can exist) uint32_t state_[n]; // state vector array int p_; // position in state array bool inited_; // true if init function has been called // private functions used to generate the pseudo random numbers uint32_t twiddle(uint32_t u, uint32_t v) { return (((u & 0x80000000UL) | (v & 0x7FFFFFFFUL)) >> 1) ^ ((v & 1UL) ? 0x9908B0DFUL : 0x0UL); } void gen_state(); // generate new state }; #endif #endif /*RANDOM_GEN_H_*/ hisat2-2.2.1/random_util.cpp000066400000000000000000000016131370663763200157450ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "random_util.h" const size_t Random1toN::SWAPLIST_THRESH = 128; const size_t Random1toN::CONVERSION_THRESH = 16; const float Random1toN::CONVERSION_FRAC = 0.10f; hisat2-2.2.1/random_util.h000066400000000000000000000135001370663763200154100ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef RANDOM_UTIL_H_ #define RANDOM_UTIL_H_ #include #include "random_source.h" #include "ds.h" /** * Return a random integer in [1, N]. Each time it's called it samples again * without replacement. done() indicates when all elements have been given * out. */ class Random1toN { typedef uint32_t T; public: // A set with fewer than this many elements should kick us into swap-list // mode immediately. Otherwise we start in seen-list mode and then // possibly proceed to swap-list mode later. static const size_t SWAPLIST_THRESH; // Convert seen-list to swap-list after this many entries in the seen-list. static const size_t CONVERSION_THRESH; // Convert seen-list to swap-list after this (this times n_) many entries // in the seen-list. static const float CONVERSION_FRAC; Random1toN(int cat = 0) : sz_(0), n_(0), cur_(0), list_(SWAPLIST_THRESH, cat), seen_(CONVERSION_THRESH, cat), thresh_(0) {} Random1toN(size_t n, int cat = 0) : sz_(0), n_(n), cur_(0), list_(SWAPLIST_THRESH, cat), seen_(CONVERSION_THRESH, cat), thresh_(0) {} /** * Initialize the set of pseudo-randoms to be given out without replacement. */ void init(size_t n, bool withoutReplacement) { sz_ = n_ = n; converted_ = false; swaplist_ = n < SWAPLIST_THRESH || withoutReplacement; cur_ = 0; list_.clear(); seen_.clear(); thresh_ = std::max(CONVERSION_THRESH, (size_t)(CONVERSION_FRAC * n)); } /** * Reset in preparation for giving out a fresh collection of pseudo-randoms * without replacement. */ void reset() { sz_ = n_ = cur_ = 0; swaplist_ = converted_ = false; list_.clear(); seen_.clear(); thresh_ = 0; } /** * Get next pseudo-random element without replacement. */ T next(RandomSource& rnd) { assert(!done()); if(cur_ == 0 && !converted_) { // This is the first call to next() if(n_ == 1) { // Trivial case: set of 1 cur_ = 1; return 0; } if(swaplist_) { // The set is small, so we go immediately to the random // swapping list list_.resize(n_); for(size_t i = 0; i < n_; i++) { list_[i] = (T)i; } } } if(swaplist_) { // Get next pseudo-random using the swap-list size_t r = cur_ + (rnd.nextU32() % (n_ - cur_)); if(r != cur_) { std::swap(list_[cur_], list_[r]); } return list_[cur_++]; } else { assert(!converted_); // Get next pseudo-random but reject it if it's in the seen-list bool again = true; T rn = 0; size_t seenSz = seen_.size(); while(again) { rn = rnd.nextU32() % (T)n_; again = false; for(size_t i = 0; i < seenSz; i++) { if(seen_[i] == rn) { again = true; break; } } } // Add it to the seen-list seen_.push_back(rn); cur_++; assert_leq(cur_, n_); // Move on to using the swap-list? assert_gt(thresh_, 0); if(seen_.size() >= thresh_ && cur_ < n_) { // Add all elements not already in the seen list to the // swap-list assert(!seen_.empty()); seen_.sort(); list_.resize(n_ - cur_); size_t prev = 0; size_t cur = 0; for(size_t i = 0; i <= seenSz; i++) { // Add all the elements between the previous element and // this one for(size_t j = prev; j < seen_[i]; j++) { list_[cur++] = (T)j; } prev = seen_[i]+1; } for(size_t j = prev; j < n_; j++) { list_[cur++] = (T)j; } assert_eq(cur, n_ - cur_); seen_.clear(); cur_ = 0; n_ = list_.size(); converted_ = true; swaplist_ = true; } return rn; } } /** * Return true iff the generator was initialized. */ bool inited() const { return n_ > 0; } /** * Set so that there are no pseudo-randoms remaining. */ void setDone() { assert(inited()); cur_ = n_; } /** * Return true iff all pseudo-randoms have already been given out. */ bool done() const { return inited() && cur_ >= n_; } /** * Return the total number of pseudo-randoms we are initialized to give * out, including ones already given out. */ size_t size() const { return n_; } /** * Return the number of pseudo-randoms left to give out. */ size_t left() const { return n_ - cur_; } /** * Return the total size occupued by the Descent driver and all its * constituent parts. */ size_t totalSizeBytes() const { return list_.totalSizeBytes() + seen_.totalSizeBytes(); } /** * Return the total capacity of the Descent driver and all its constituent * parts. */ size_t totalCapacityBytes() const { return list_.totalCapacityBytes() + seen_.totalCapacityBytes(); } protected: size_t sz_; // domain to pick elts from size_t n_; // number of elements in active list bool swaplist_; // if small, use swapping bool converted_; // true iff seen-list was converted to swap-list size_t cur_; // # times next() was called EList list_; // pseudo-random swapping list EList seen_; // prior to swaplist_ mode, list of // pseudo-randoms given out size_t thresh_; // conversion threshold for this instantiation, which // depends both on CONVERSION_THRESH and on n_ }; #endif hisat2-2.2.1/read.h000066400000000000000000000337311370663763200140160ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef READ_H_ #define READ_H_ #include #include #include "ds.h" #include "sstring.h" #include "filebuf.h" #include "util.h" enum rna_strandness_format { RNA_STRANDNESS_UNKNOWN = 0, RNA_STRANDNESS_F, RNA_STRANDNESS_R, RNA_STRANDNESS_FR, RNA_STRANDNESS_RF }; typedef uint64_t TReadId; typedef size_t TReadOff; typedef int64_t TAlScore; class HitSet; /** * A buffer for keeping all relevant information about a single read. */ struct Read { Read() { reset(); } Read(const char *nm, const char *seq, const char *ql) { init(nm, seq, ql); } void reset() { rdid = 0; endid = 0; alts = 0; trimmed5 = trimmed3 = 0; readOrigBuf.clear(); patFw.clear(); patRc.clear(); qual.clear(); patFwRev.clear(); patRcRev.clear(); qualRev.clear(); name.clear(); for(int j = 0; j < 3; j++) { altPatFw[j].clear(); altPatFwRev[j].clear(); altPatRc[j].clear(); altPatRcRev[j].clear(); altQual[j].clear(); altQualRev[j].clear(); } color = fuzzy = false; primer = '?'; trimc = '?'; filter = '?'; seed = 0; ns_ = 0; } /** * Finish initializing a new read. */ void finalize() { for(size_t i = 0; i < patFw.length(); i++) { if((int)patFw[i] > 3) { ns_++; } } constructRevComps(); constructReverses(); } /** * Simple init function, used for testing. */ void init( const char *nm, const char *seq, const char *ql) { reset(); patFw.installChars(seq); qual.install(ql); for(size_t i = 0; i < patFw.length(); i++) { if((int)patFw[i] > 3) { ns_++; } } constructRevComps(); constructReverses(); if(nm != NULL) name.install(nm); } /// Return true iff the read (pair) is empty bool empty() const { return patFw.empty(); } /// Return length of the read in the buffer size_t length() const { return patFw.length(); } /** * Return the number of Ns in the read. */ size_t ns() const { return ns_; } /** * Construct reverse complement of the pattern and the fuzzy * alternative patters. If read is in colorspace, just reverse * them. */ void constructRevComps() { if(color) { patRc.installReverse(patFw); for(int j = 0; j < alts; j++) { altPatRc[j].installReverse(altPatFw[j]); } } else { patRc.installReverseComp(patFw); for(int j = 0; j < alts; j++) { altPatRc[j].installReverseComp(altPatFw[j]); } } } /** * Given patFw, patRc, and qual, construct the *Rev versions in * place. Assumes constructRevComps() was called previously. */ void constructReverses() { patFwRev.installReverse(patFw); patRcRev.installReverse(patRc); qualRev.installReverse(qual); for(int j = 0; j < alts; j++) { altPatFwRev[j].installReverse(altPatFw[j]); altPatRcRev[j].installReverse(altPatRc[j]); altQualRev[j].installReverse(altQual[j]); } } /** * Append a "/1" or "/2" string onto the end of the name buf if * it's not already there. */ void fixMateName(int i) { assert(i == 1 || i == 2); size_t namelen = name.length(); bool append = false; if(namelen < 2) { // Name is too short to possibly have /1 or /2 on the end append = true; } else { if(i == 1) { // append = true iff mate name does not already end in /1 append = name[namelen-2] != '/' || name[namelen-1] != '1'; } else { // append = true iff mate name does not already end in /2 append = name[namelen-2] != '/' || name[namelen-1] != '2'; } } if(append) { name.append('/'); name.append("012"[i]); } } /** * Dump basic information about this read to the given ostream. */ void dump(std::ostream& os) const { using namespace std; os << name << ' '; if(color) { os << patFw.toZBufXForm("0123."); } else { os << patFw; } os << ' '; // Print out the fuzzy alternative sequences for(int j = 0; j < 3; j++) { bool started = false; if(!altQual[j].empty()) { for(size_t i = 0; i < length(); i++) { if(altQual[j][i] != '!') { started = true; } if(started) { if(altQual[j][i] == '!') { os << '-'; } else { if(color) { os << "0123."[(int)altPatFw[j][i]]; } else { os << altPatFw[j][i]; } } } } } cout << " "; } os << qual.toZBuf() << " "; // Print out the fuzzy alternative quality strings for(int j = 0; j < 3; j++) { bool started = false; if(!altQual[j].empty()) { for(size_t i = 0; i < length(); i++) { if(altQual[j][i] != '!') { started = true; } if(started) { os << altQual[j][i]; } } } if(j == 2) { os << endl; } else { os << " "; } } } /** * Check whether two reads are the same in the sense that they will * lead to us finding the same set of alignments. */ static bool same( const BTDnaString& seq1, const BTString& qual1, const BTDnaString& seq2, const BTString& qual2, bool qualitiesMatter) { if(seq1.length() != seq2.length()) { return false; } for(size_t i = 0; i < seq1.length(); i++) { if(seq1[i] != seq2[i]) return false; } if(qualitiesMatter) { if(qual1.length() != qual2.length()) { return false; } for(size_t i = 0; i < qual1.length(); i++) { if(qual1[i] != qual2[i]) return false; } } return true; } /** * Get the nucleotide and quality value at the given offset from 5' end. * If 'fw' is false, get the reverse complement. */ std::pair get(TReadOff off5p, bool fw) const { assert_lt(off5p, length()); int c = (int)patFw[off5p]; int q = qual[off5p]; assert_geq(q, 33); return make_pair((!fw && c < 4) ? (c ^ 3) : c, q - 33); } /** * Get the nucleotide at the given offset from 5' end. * If 'fw' is false, get the reverse complement. */ int getc(TReadOff off5p, bool fw) const { assert_lt(off5p, length()); int c = (int)patFw[off5p]; return (!fw && c < 4) ? (c ^ 3) : c; } /** * Get the quality value at the given offset from 5' end. */ int getq(TReadOff off5p) const { assert_lt(off5p, length()); int q = qual[off5p]; assert_geq(q, 33); return q-33; } #ifndef NDEBUG /** * Check that read info is internally consistent. */ bool repOk() const { if(patFw.empty()) return true; assert_eq(qual.length(), patFw.length()); return true; } #endif BTDnaString patFw; // forward-strand sequence BTDnaString patRc; // reverse-complement sequence BTString qual; // quality values BTDnaString altPatFw[3]; BTDnaString altPatRc[3]; BTString altQual[3]; BTDnaString patFwRev; BTDnaString patRcRev; BTString qualRev; BTDnaString altPatFwRev[3]; BTDnaString altPatRcRev[3]; BTString altQualRev[3]; // For remembering the exact input text used to define a read SStringExpandable readOrigBuf; BTString name; // read name TReadId rdid; // 0-based id based on pair's offset in read file(s) TReadId endid; // 0-based id based on pair's offset in read file(s) // and which mate ("end") this is int mate; // 0 = single-end, 1 = mate1, 2 = mate2 uint32_t seed; // random seed size_t ns_; // # Ns int alts; // number of alternatives bool fuzzy; // whether to employ fuzziness bool color; // whether read is in color space char primer; // primer base, for csfasta files char trimc; // trimmed color, for csfasta files char filter; // if read format permits filter char, set it here int trimmed5; // amount actually trimmed off 5' end int trimmed3; // amount actually trimmed off 3' end HitSet *hitset; // holds previously-found hits; for chaining }; /** * A string of FmStringOps represent a string of tasks performed by the * best-first alignment search. We model the search as a series of FM ops * interspersed with reported alignments. */ struct FmStringOp { bool alignment; // true -> found an alignment TAlScore pen; // penalty of the FM op or alignment size_t n; // number of FM ops (only relevant for non-alignment) }; /** * A string that summarizes the progress of an FM-index-assistet best-first * search. Useful for trying to figure out what the aligner is spending its * time doing for a given read. */ struct FmString { /** * Add one or more FM index ops to the op string */ void add(bool alignment, TAlScore pen, size_t nops) { if(ops.empty() || ops.back().pen != pen) { ops.expand(); ops.back().alignment = alignment; ops.back().pen = pen; ops.back().n = 0; } ops.back().n++; } /** * Reset FmString to uninitialized state. */ void reset() { pen = std::numeric_limits::max(); ops.clear(); } /** * Print a :Z optional field where certain characters (whitespace, colon * and percent) are escaped using % escapes. */ void print(BTString& o, char *buf) const { for(size_t i = 0; i < ops.size(); i++) { if(i > 0) { o.append(';'); } if(ops[i].alignment) { o.append("A,"); itoa10(ops[i].pen, buf); o.append(buf); } else { o.append("F,"); itoa10(ops[i].pen, buf); o.append(buf); o.append(','); itoa10(ops[i].n, buf); o.append(buf); } } } TAlScore pen; // current penalty EList ops; // op string }; /** * Key per-read metrics. These are used for thresholds, allowing us to bail * for unproductive reads. They also the basis of what's printed when the user * specifies --read-times. */ struct PerReadMetrics { PerReadMetrics() { reset(); } void reset() { nExIters = nExDps = nExDpSuccs = nExDpFails = nMateDps = nMateDpSuccs = nMateDpFails = nExUgs = nExUgSuccs = nExUgFails = nMateUgs = nMateUgSuccs = nMateUgFails = nExEes = nExEeSuccs = nExEeFails = nRedundants = nEeFmops = nSdFmops = nExFmops = nDpFail = nDpFailStreak = nDpLastSucc = nUgFail = nUgFailStreak = nUgLastSucc = nEeFail = nEeFailStreak = nEeLastSucc = nFilt = 0; nFtabs = 0; nRedSkip = 0; nRedFail = 0; nRedIns = 0; doFmString = false; nSeedRanges = nSeedElts = 0; nSeedRangesFw = nSeedEltsFw = 0; nSeedRangesRc = nSeedEltsRc = 0; seedMedian = seedMean = 0; bestLtMinscMate1 = bestLtMinscMate2 = std::numeric_limits::min(); fmString.reset(); } struct timeval tv_beg; // timer start to measure how long alignment takes struct timezone tz_beg; // timer start to measure how long alignment takes uint64_t nExIters; // iterations of seed hit extend loop uint64_t nExDps; // # extend DPs run on this read uint64_t nExDpSuccs; // # extend DPs run on this read uint64_t nExDpFails; // # extend DPs run on this read uint64_t nExUgs; // # extend ungapped alignments run on this read uint64_t nExUgSuccs; // # extend ungapped alignments run on this read uint64_t nExUgFails; // # extend ungapped alignments run on this read uint64_t nExEes; // # extend ungapped alignments run on this read uint64_t nExEeSuccs; // # extend ungapped alignments run on this read uint64_t nExEeFails; // # extend ungapped alignments run on this read uint64_t nMateDps; // # mate DPs run on this read uint64_t nMateDpSuccs; // # mate DPs run on this read uint64_t nMateDpFails; // # mate DPs run on this read uint64_t nMateUgs; // # mate ungapped alignments run on this read uint64_t nMateUgSuccs; // # mate ungapped alignments run on this read uint64_t nMateUgFails; // # mate ungapped alignments run on this read uint64_t nRedundants; // # redundant seed hits uint64_t nSeedRanges; // # BW ranges found for seeds uint64_t nSeedElts; // # BW elements found for seeds uint64_t nSeedRangesFw; // # BW ranges found for seeds from fw read uint64_t nSeedEltsFw; // # BW elements found for seeds from fw read uint64_t nSeedRangesRc; // # BW ranges found for seeds from fw read uint64_t nSeedEltsRc; // # BW elements found for seeds from fw read uint64_t seedMedian; // median seed hit count uint64_t seedMean; // rounded mean seed hit count uint64_t nEeFmops; // FM Index ops for end-to-end alignment uint64_t nSdFmops; // FM Index ops used to align seeds uint64_t nExFmops; // FM Index ops used to resolve offsets uint64_t nFtabs; // # ftab lookups uint64_t nRedSkip; // # times redundant path was detected and aborted uint64_t nRedFail; // # times a path was deemed non-redundant uint64_t nRedIns; // # times a path was added to redundancy list uint64_t nDpFail; // number of dp failures in a row up until now uint64_t nDpFailStreak; // longest streak of dp failures uint64_t nDpLastSucc; // index of last dp attempt that succeeded uint64_t nUgFail; // number of ungap failures in a row up until now uint64_t nUgFailStreak; // longest streak of ungap failures uint64_t nUgLastSucc; // index of last ungap attempt that succeeded uint64_t nEeFail; // number of ungap failures in a row up until now uint64_t nEeFailStreak; // longest streak of ungap failures uint64_t nEeLastSucc; // index of last ungap attempt that succeeded uint64_t nFilt; // # mates filtered TAlScore bestLtMinscMate1; // best invalid score observed for mate 1 TAlScore bestLtMinscMate2; // best invalid score observed for mate 2 // For collecting information to go into an FM string bool doFmString; FmString fmString; }; #endif /*READ_H_*/ hisat2-2.2.1/read_qseq.cpp000066400000000000000000000203521370663763200153750ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "pat.h" /** * Parse a name from fb_ and store in r. Assume that the next * character obtained via fb_.get() is the first character of * the sequence and the string stops at the next char upto (could * be tab, newline, etc.). */ int QseqPatternSource::parseName( Read& r, // buffer for mate 1 Read* r2, // buffer for mate 2 (NULL if mate2 is read separately) bool append, // true -> append characters, false -> skip them bool clearFirst, // clear the name buffer first bool warnEmpty, // emit a warning if nothing was added to the name bool useDefault, // if nothing is read, put readCnt_ as a default value int upto) // stop parsing when we first reach character 'upto' { if(clearFirst) { if(r2 != NULL) r2->name.clear(); r.name.clear(); } while(true) { int c; if((c = fb_.get()) < 0) { // EOF reached in the middle of the name return -1; } if(c == '\n' || c == '\r') { // EOL reached in the middle of the name return -1; } if(c == upto) { // Finished with field break; } if(append) { if(r2 != NULL) r2->name.append(c); r.name.append(c); } } // Set up a default name if one hasn't been set if(r.name.empty() && useDefault && append) { char cbuf[20]; itoa10(readCnt_, cbuf); r.name.append(cbuf); if(r2 != NULL) r2->name.append(cbuf); } if(r.name.empty() && warnEmpty) { cerr << "Warning: read had an empty name field" << endl; } return (int)r.name.length(); } /** * Parse a single sequence from fb_ and store in r. Assume * that the next character obtained via fb_.get() is the first * character of the sequence and the sequence stops at the next * char upto (could be tab, newline, etc.). */ int QseqPatternSource::parseSeq( Read& r, int& charsRead, int& trim5, char upto) { int begin = 0; int c = fb_.get(); assert(c != upto); r.patFw.clear(); r.color = gColor; if(gColor) { // NOTE: clearly this is not relevant for Illumina output, but // I'm keeping it here in case there's some reason to put SOLiD // data in this format in the future. // This may be a primer character. If so, keep it in the // 'primer' field of the read buf and parse the rest of the // read without it. c = toupper(c); if(asc2dnacat[c] > 0) { // First char is a DNA char int c2 = toupper(fb_.peek()); // Second char is a color char if(asc2colcat[c2] > 0) { r.primer = c; r.trimc = c2; trim5 += 2; // trim primer and first color } } if(c < 0) { return -1; } } while(c != upto) { if(c == '.') c = 'N'; if(gColor) { if(c >= '0' && c <= '4') c = "ACGTN"[(int)c - '0']; } if(isalpha(c)) { assert_in(toupper(c), "ACGTN"); if(begin++ >= trim5) { assert_neq(0, asc2dnacat[c]); r.patFw.append(asc2dna[c]); } charsRead++; } if((c = fb_.get()) < 0) { return -1; } } r.patFw.trimEnd(gTrim3); return (int)r.patFw.length(); } /** * Parse a single quality string from fb_ and store in r. * Assume that the next character obtained via fb_.get() is * the first character of the quality string and the string stops * at the next char upto (could be tab, newline, etc.). */ int QseqPatternSource::parseQuals( Read& r, int charsRead, int dstLen, int trim5, char& c2, char upto = '\t', char upto2 = -1) { int qualsRead = 0; int c = 0; if (intQuals_) { // Probably not relevant char buf[4096]; while (qualsRead < charsRead) { qualToks_.clear(); if(!tokenizeQualLine(fb_, buf, 4096, qualToks_)) break; for (unsigned int j = 0; j < qualToks_.size(); ++j) { char c = intToPhred33(atoi(qualToks_[j].c_str()), solQuals_); assert_geq(c, 33); if (qualsRead >= trim5) { r.qual.append(c); } ++qualsRead; } } // done reading integer quality lines if (charsRead > qualsRead) tooFewQualities(r.name); } else { // Non-integer qualities while((qualsRead < dstLen + trim5) && c >= 0) { c = fb_.get(); c2 = c; if (c == ' ') wrongQualityFormat(r.name); if(c < 0) { // EOF occurred in the middle of a read - abort return -1; } if(!isspace(c) && c != upto && (upto2 == -1 || c != upto2)) { if (qualsRead >= trim5) { c = charToPhred33(c, solQuals_, phred64Quals_); assert_geq(c, 33); r.qual.append(c); } qualsRead++; } else { break; } } } if(r.qual.length() < (size_t)dstLen) { tooFewQualities(r.name); } // TODO: How to detect too many qualities?? r.qual.resize(dstLen); while(c != -1 && c != upto && (upto2 == -1 || c != upto2)) { c = fb_.get(); c2 = c; } return qualsRead; } /** * Read another pattern from a Qseq input file. */ bool QseqPatternSource::read( Read& r, TReadId& rdid, TReadId& endid, bool& success, bool& done) { r.reset(); r.color = gColor; success = true; done = false; readCnt_++; rdid = endid = readCnt_-1; peekOverNewline(fb_); fb_.resetLastN(); // 1. Machine name if(parseName(r, NULL, true, true, true, false, '\t') == -1) BAIL_UNPAIRED(); assert_neq('\t', fb_.peek()); r.name.append('_'); // 2. Run number if(parseName(r, NULL, true, false, true, false, '\t') == -1) BAIL_UNPAIRED(); assert_neq('\t', fb_.peek()); r.name.append('_'); // 3. Lane number if(parseName(r, NULL, true, false, true, false, '\t') == -1) BAIL_UNPAIRED(); assert_neq('\t', fb_.peek()); r.name.append('_'); // 4. Tile number if(parseName(r, NULL, true, false, true, false, '\t') == -1) BAIL_UNPAIRED(); assert_neq('\t', fb_.peek()); r.name.append('_'); // 5. X coordinate of spot if(parseName(r, NULL, true, false, true, false, '\t') == -1) BAIL_UNPAIRED(); assert_neq('\t', fb_.peek()); r.name.append('_'); // 6. Y coordinate of spot if(parseName(r, NULL, true, false, true, false, '\t') == -1) BAIL_UNPAIRED(); assert_neq('\t', fb_.peek()); r.name.append('_'); // 7. Index if(parseName(r, NULL, true, false, true, false, '\t') == -1) BAIL_UNPAIRED(); assert_neq('\t', fb_.peek()); r.name.append('/'); // 8. Mate number if(parseName(r, NULL, true, false, true, false, '\t') == -1) BAIL_UNPAIRED(); // Empty sequence?? if(fb_.peek() == '\t') { // Get tab that separates seq from qual ASSERT_ONLY(int c =) fb_.get(); assert_eq('\t', c); assert_eq('\t', fb_.peek()); // Get tab that separates qual from filter ASSERT_ONLY(c =) fb_.get(); assert_eq('\t', c); // Next char is first char of filter flag assert_neq('\t', fb_.peek()); fb_.resetLastN(); cerr << "Warning: skipping empty QSEQ read with name '" << r.name << "'" << endl; } else { assert_neq('\t', fb_.peek()); int charsRead = 0; int mytrim5 = gTrim5; // 9. Sequence int dstLen = parseSeq(r, charsRead, mytrim5, '\t'); assert_neq('\t', fb_.peek()); if(dstLen < 0) BAIL_UNPAIRED(); char ct = 0; // 10. Qualities if(parseQuals(r, charsRead, dstLen, mytrim5, ct, '\t', -1) < 0) BAIL_UNPAIRED(); r.trimmed3 = gTrim3; r.trimmed5 = mytrim5; if(ct != '\t') { cerr << "Error: QSEQ with name " << r.name << " did not have tab after qualities" << endl; throw 1; } assert_eq(ct, '\t'); } // 11. Filter flag int filt = fb_.get(); if(filt == -1) BAIL_UNPAIRED(); r.filter = filt; if(filt != '0' && filt != '1') { // Bad value for filt } if(fb_.peek() != -1 && fb_.peek() != '\n') { // Bad value right after the filt field } fb_.get(); r.readOrigBuf.install(fb_.lastN(), fb_.lastNLen()); fb_.resetLastN(); if(r.qual.length() < r.patFw.length()) { tooFewQualities(r.name); } else if(r.qual.length() > r.patFw.length()) { tooManyQualities(r.name); } #ifndef NDEBUG assert_eq(r.patFw.length(), r.qual.length()); for(size_t i = 0; i < r.qual.length(); i++) { assert_geq((int)r.qual[i], 33); } #endif return true; } hisat2-2.2.1/ref_coord.cpp000066400000000000000000000017661370663763200154030ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "ref_coord.h" #include using namespace std; ostream& operator<<(ostream& out, const Interval& c) { out << c.upstream() << "+" << c.len(); return out; } ostream& operator<<(ostream& out, const Coord& c) { out << c.ref() << ":" << c.off(); return out; } hisat2-2.2.1/ref_coord.h000066400000000000000000000242421370663763200150420ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef REF_COORD_H_ #define REF_COORD_H_ #include #include #include #include "assert_helpers.h" typedef int64_t TRefId; typedef int64_t TRefOff; /** * Encapsulates a reference coordinate; i.e. identifiers for (a) a * reference sequence, and (b) a 0-based offset into that sequence. */ class Coord { public: Coord() { reset(); } Coord(const Coord& c) { init(c); } Coord(TRefId rf, TRefOff of, bool fw, TRefOff jof = 0) { init(rf, of, fw, jof); } /** * Copy given fields into this Coord. */ void init(TRefId rf, TRefOff of, bool fw, TRefOff jof = 0) { ref_ = rf; off_ = of; orient_ = (fw ? 1 : 0); joinedOff_ = jof; } /** * Copy contents of given Coord into this one. */ void init(const Coord& c) { ref_ = c.ref_; off_ = c.off_; orient_ = c.orient_; joinedOff_ = c.joinedOff_; } /** * Return true iff this Coord is identical to the given Coord. */ bool operator==(const Coord& o) const { assert(inited()); assert(o.inited()); return ref_ == o.ref_ && off_ == o.off_ && fw() == o.fw(); } /** * Return true iff this Coord is less than the given Coord. One Coord is * less than another if (a) its reference id is less, (b) its orientation is * less, or (c) its offset is less. */ bool operator<(const Coord& o) const { if(ref_ < o.ref_) return true; if(ref_ > o.ref_) return false; if(orient_ < o.orient_) return true; if(orient_ > o.orient_) return false; if(off_ < o.off_) return true; if(off_ > o.off_) return false; return false; } /** * Return the opposite result from operator<. */ bool operator>=(const Coord& o) const { return !((*this) < o); } /** * Return true iff this Coord is greater than the given Coord. One Coord * is greater than another if (a) its reference id is greater, (b) its * orientation is greater, or (c) its offset is greater. */ bool operator>(const Coord& o) const { if(ref_ > o.ref_) return true; if(ref_ < o.ref_) return false; if(orient_ > o.orient_) return true; if(orient_ < o.orient_) return false; if(off_ > o.off_) return true; if(off_ < o.off_) return false; return false; } /** * Return the opposite result from operator>. */ bool operator<=(const Coord& o) const { return !((*this) > o); } /** * Reset this coord to uninitialized state. */ void reset() { ref_ = std::numeric_limits::max(); off_ = std::numeric_limits::max(); orient_ = -1; joinedOff_ = std::numeric_limits::max(); } /** * Return true iff this Coord is initialized (i.e. ref and off have both * been set since the last call to reset()). */ bool inited() const { if(ref_ != std::numeric_limits::max() && off_ != std::numeric_limits::max()) { assert(orient_ == 0 || orient_ == 1); return true; } return false; } /** * Get orientation of the Coord. */ bool fw() const { assert(inited()); assert(orient_ == 0 || orient_ == 1); return orient_ == 1; } #ifndef NDEBUG /** * Check that coord is internally consistent. */ bool repOk() const { if(ref_ != std::numeric_limits::max() && off_ != std::numeric_limits::max()) { assert(orient_ == 0 || orient_ == 1); } return true; } #endif /** * Check whether an interval defined by this coord and having * length 'len' is contained within an interval defined by * 'inbegin' and 'inend'. */ bool within(int64_t len, int64_t inbegin, int64_t inend) const { return off_ >= inbegin && off_ + len <= inend; } inline TRefId ref() const { return ref_; } inline TRefOff off() const { return off_; } inline int orient() const { return orient_; } inline TRefOff joinedOff() const { return joinedOff_; } inline void setRef(TRefId id) { ref_ = id; } inline void setOff(TRefOff off) { off_ = off; } inline void setJoinedOff(TRefOff joinedOff) { joinedOff_ = joinedOff; } inline void adjustOff(TRefOff off) { off_ += off; } protected: TRefId ref_; // which reference? TRefOff off_; // 0-based offset into reference int orient_; // true -> Watson strand TRefOff joinedOff_; // offset in a joined ref. sequence }; std::ostream& operator<<(std::ostream& out, const Coord& c); /** * Encapsulates a reference interval, which consists of a Coord and a length. */ class Interval { public: Interval() { reset(); } explicit Interval(const Coord& upstream, TRefOff len) { init(upstream, len); } explicit Interval(TRefId rf, TRefOff of, bool fw, TRefOff len) { init(rf, of, fw, len); } void init(const Coord& upstream, TRefOff len) { upstream_ = upstream; len_ = len; } void init(TRefId rf, TRefOff of, bool fw, TRefOff len) { upstream_.init(rf, of, fw); len_ = len; } /** * Set offset. */ void setOff(TRefOff of) { upstream_.setOff(of); } /** * Set length. */ void setLen(TRefOff len) { len_ = len; } /** * Reset this interval to uninitialized state. */ void reset() { upstream_.reset(); len_ = 0; } /** * Return true iff this Interval is initialized. */ bool inited() const { if(upstream_.inited()) { assert_gt(len_, 0); return true; } else { return false; } } /** * Return true iff this Interval is equal to the given Interval, * i.e. if they cover the same set of positions. */ bool operator==(const Interval& o) const { return upstream_ == o.upstream_ && len_ == o.len_; } /** * Return true iff this Interval is less than the given Interval. * One interval is less than another if its upstream location is * prior to the other's or, if their upstream locations are equal, * if its length is less than the other's. */ bool operator<(const Interval& o) const { if(upstream_ < o.upstream_) return true; if(upstream_ > o.upstream_) return false; if(len_ < o.len_) return true; return false; } /** * Return opposite result from operator<. */ bool operator>=(const Interval& o) const { return !((*this) < o); } /** * Return true iff this Interval is greater than than the given * Interval. One interval is greater than another if its upstream * location is after the other's or, if their upstream locations * are equal, if its length is greater than the other's. */ bool operator>(const Interval& o) const { if(upstream_ > o.upstream_) return true; if(upstream_ < o.upstream_) return false; if(len_ > o.len_) return true; return false; } /** * Return opposite result from operator>. */ bool operator<=(const Interval& o) const { return !((*this) > o); } /** * Set upstream Coord. */ void setUpstream(const Coord& c) { upstream_ = c; } /** * Set length. */ void setLength(TRefOff l) { len_ = l; } inline TRefId ref() const { return upstream_.ref(); } inline TRefOff off() const { return upstream_.off(); } inline TRefOff dnoff() const { return upstream_.off() + len_; } inline int orient() const { return upstream_.orient(); } /** * Return a Coord encoding the coordinate just past the downstream edge of * the interval. */ inline Coord downstream() const { return Coord( upstream_.ref(), upstream_.off() + len_, upstream_.orient()); } /** * Return true iff the given Coord is inside this Interval. */ inline bool contains(const Coord& c) const { return c.ref() == ref() && c.orient() == orient() && c.off() >= off() && c.off() < dnoff(); } /** * Return true iff the given Coord is inside this Interval, without * requiring orientations to match. */ inline bool containsIgnoreOrient(const Coord& c) const { return c.ref() == ref() && c.off() >= off() && c.off() < dnoff(); } /** * Return true iff the given Interval is inside this Interval. */ inline bool contains(const Interval& c) const { return c.ref() == ref() && c.orient() == orient() && c.off() >= off() && c.dnoff() <= dnoff(); } /** * Return true iff the given Interval is inside this Interval, without * requiring orientations to match. */ inline bool containsIgnoreOrient(const Interval& c) const { return c.ref() == ref() && c.off() >= off() && c.dnoff() <= dnoff(); } /** * Return true iff the given Interval overlaps this Interval. */ inline bool overlaps(const Interval& c) const { return c.ref() == upstream_.ref() && c.orient() == upstream_.orient() && ((off() <= c.off() && dnoff() > c.off()) || (off() <= c.dnoff() && dnoff() > c.dnoff()) || (c.off() <= off() && c.dnoff() > off()) || (c.off() <= dnoff() && c.dnoff() > dnoff())); } /** * Return true iff the given Interval overlaps this Interval, without * requiring orientations to match. */ inline bool overlapsIgnoreOrient(const Interval& c) const { return c.ref() == upstream_.ref() && ((off() <= c.off() && dnoff() > c.off()) || (off() <= c.dnoff() && dnoff() > c.dnoff()) || (c.off() <= off() && c.dnoff() > off()) || (c.off() <= dnoff() && c.dnoff() > dnoff())); } inline const Coord& upstream() const { return upstream_; } inline TRefOff len() const { return len_; } #ifndef NDEBUG /** * Check that the Interval is internally consistent. */ bool repOk() const { assert(upstream_.repOk()); assert_geq(len_, 0); return true; } #endif inline void adjustOff(TRefOff off) { upstream_.adjustOff(off); } protected: Coord upstream_; TRefOff len_; }; std::ostream& operator<<(std::ostream& out, const Interval& c); #endif /*ndef REF_COORD_H_*/ hisat2-2.2.1/ref_read.cpp000066400000000000000000000301151370663763200151760ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "ref_read.h" /** * Reads past the next ambiguous or unambiguous stretch of sequence * from the given FASTA file and returns its length. Does not do * anything with the sequence characters themselves; this is purely for * measuring lengths. */ RefRecord fastaRefReadSize( FileBuf& in, const RefReadInParams& rparms, bool first, BitpairOutFileBuf* bpout, string* name // put parsed FASTA name here ) { int c; static int lastc = '>'; // last character seen // RefRecord params TIndexOffU len = 0; // 'len' counts toward total length // 'off' counts number of ambiguous characters before first // unambiguous character size_t off = 0; // Pick off the first carat and any preceding whitespace if(first) { assert(!in.eof()); lastc = '>'; c = in.getPastWhitespace(); if(in.eof()) { // Got eof right away; emit warning cerr << "Warning: Empty input file" << endl; lastc = -1; return RefRecord(0, 0, true); } assert(c == '>'); } first = true; // Skip to the end of the id line; if the next line is either // another id line or a comment line, keep skipping if(lastc == '>') { #if 0 // Skip to the end of the name line do { if((c = in.getPastNewline()) == -1) { // No more input cerr << "Warning: Encountered empty reference sequence" << endl; lastc = -1; return RefRecord(0, 0, true); } if(c == '>') { cerr << "Warning: Encountered empty reference sequence" << endl; } // continue until a non-name, non-comment line } while (c == '>'); #else c = lastc; // Skip to the end of the name line do { // get name while (true) { c = in.get(); if (c == -1) { // No more input cerr << "Warning: Encountered empty reference sequence" << endl; lastc = -1; return RefRecord(0, 0, true); } if(c == '\n' || c == '\r') { while(c == '\r' || c == '\n') c = in.get(); if (c == -1) { // No more input cerr << "Warning: Encountered empty reference sequence" << endl; lastc = -1; return RefRecord(0, 0, true); } break; } if (name) name->push_back(c); } if (c == '>') { cerr << "Warning: Encountered empty reference sequence" << endl; // If there's another name line immediately after this one, // discard the previous name and start fresh with the new one if (name) name->clear(); } } while (c == '>'); #endif } else { first = false; // not the first in a sequence off = 1; // The gap has already been consumed, so count it if((c = in.get()) == -1) { // Don't emit a warning, since this might legitimately be // a gap on the end of the final sequence in the file lastc = -1; return RefRecord((TIndexOffU)off, (TIndexOffU)len, first); } } // Now skip to the first DNA character, counting gap characters // as we go int lc = -1; // last-DNA char variable for color conversion while(true) { int cat = asc2dnacat[c]; if(rparms.nsToAs && cat >= 2) c = 'A'; if(cat == 1) { // This is a DNA character if(rparms.color) { if(lc != -1) { // Got two consecutive unambiguous DNAs break; // to read-in loop } // Keep going; we need two consecutive unambiguous DNAs lc = asc2dna[(int)c]; // The 'if(off > 0)' takes care of the case where // the reference is entirely unambiguous and we don't // want to incorrectly increment off. if(off > 0) off++; } else { break; // to read-in loop } } else if(cat >= 2) { if(lc != -1 && off == 0) off++; lc = -1; off++; // skip over gap character and increment } else if(c == '>') { if(off > 0 && lastc == '>') { cerr << "Warning: Encountered reference sequence with only gaps" << endl; } else if(lastc == '>') { cerr << "Warning: Encountered empty reference sequence" << endl; } lastc = '>'; //return RefRecord(off, 0, false); return RefRecord((TIndexOffU)off, 0, first); } c = in.get(); if(c == -1) { // End-of-file if(off > 0 && lastc == '>') { cerr << "Warning: Encountered reference sequence with only gaps" << endl; } else if(lastc == '>') { cerr << "Warning: Encountered empty reference sequence" << endl; } lastc = -1; //return RefRecord(off, 0, false); return RefRecord((TIndexOffU)off, 0, first); } } assert(!rparms.color || (lc != -1)); assert_eq(1, asc2dnacat[c]); // C must be unambiguous base if(off > 0 && rparms.color && first) { // Handle the case where the first record has ambiguous // characters but we're in color space; one of those counts is // spurious off--; } // in now points just past the first character of a sequence // line, and c holds the first character while(c != -1 && c != '>') { if(rparms.nsToAs && asc2dnacat[c] >= 2) c = 'A'; uint8_t cat = asc2dnacat[c]; int cc = toupper(c); if(rparms.bisulfite && cc == 'C') c = cc = 'T'; if(cat == 1) { // It's a DNA character assert(cc == 'A' || cc == 'C' || cc == 'G' || cc == 'T'); // Check for overflow if((TIndexOffU)(len + 1) < len) { throw RefTooLongException(); } // Consume it len++; // Output it if(bpout != NULL) { if(rparms.color) { // output color bpout->write(dinuc2color[asc2dna[(int)c]][lc]); } else if(!rparms.color) { // output nucleotide bpout->write(asc2dna[c]); } } lc = asc2dna[(int)c]; } else if(cat >= 2) { // It's an N or a gap lastc = c; assert(cc != 'A' && cc != 'C' && cc != 'G' && cc != 'T'); return RefRecord((TIndexOffU)off, (TIndexOffU)len, first); } else { // Not DNA and not a gap, ignore it #ifndef NDEBUG if(!isspace(c)) { cerr << "Unexpected character in sequence: "; if(isprint(c)) { cerr << ((char)c) << endl; } else { cerr << "(" << c << ")" << endl; } } #endif } c = in.get(); } lastc = c; return RefRecord((TIndexOffU)off, (TIndexOffU)len, first); } #if 0 static void printRecords(ostream& os, const EList& l) { for(size_t i = 0; i < l.size(); i++) { os << l[i].first << ", " << l[i].off << ", " << l[i].len << endl; } } #endif /** * Reverse the 'src' list of RefRecords into the 'dst' list. Don't * modify 'src'. */ void reverseRefRecords( const EList& src, EList& dst, bool recursive, bool verbose) { dst.clear(); { EList cur; for(int i = (int)src.size()-1; i >= 0; i--) { bool first = (i == (int)src.size()-1 || src[i+1].first); // Clause after the || on next line is to deal with empty FASTA // records at the end of the 'src' list, which would be wrongly // omitted otherwise. if(src[i].len || (first && src[i].off == 0)) { cur.push_back(RefRecord(0, src[i].len, first)); first = false; } if(src[i].off) cur.push_back(RefRecord(src[i].off, 0, first)); } for(int i = 0; i < (int)cur.size(); i++) { assert(cur[i].off == 0 || cur[i].len == 0); if(i < (int)cur.size()-1 && cur[i].off != 0 && !cur[i+1].first) { dst.push_back(RefRecord(cur[i].off, cur[i+1].len, cur[i].first)); i++; } else { dst.push_back(cur[i]); } } } //if(verbose) { // cout << "Source: " << endl; // printRecords(cout, src); // cout << "Dest: " << endl; // printRecords(cout, dst); //} #ifndef NDEBUG size_t srcnfirst = 0, dstnfirst = 0; for(size_t i = 0; i < src.size(); i++) { if(src[i].first) { srcnfirst++; } } for(size_t i = 0; i < dst.size(); i++) { if(dst[i].first) { dstnfirst++; } } assert_eq(srcnfirst, dstnfirst); if(!recursive) { EList tmp; reverseRefRecords(dst, tmp, true); assert_eq(tmp.size(), src.size()); for(size_t i = 0; i < src.size(); i++) { assert_eq(src[i].len, tmp[i].len); assert_eq(src[i].off, tmp[i].off); assert_eq(src[i].first, tmp[i].first); } } #endif } /** * Calculate a vector containing the sizes of all of the patterns in * all of the given input files, in order. Returns the total size of * all references combined. Rewinds each istream before returning. */ std::pair fastaRefReadSizes( EList& in, EList& recs, const RefReadInParams& rparms, BitpairOutFileBuf* bpout, TIndexOff& numSeqs) { TIndexOffU unambigTot = 0; size_t bothTot = 0; assert_gt(in.size(), 0); // For each input istream for(size_t i = 0; i < in.size(); i++) { bool first = true; assert(!in[i]->eof()); // For each pattern in this istream while(!in[i]->eof()) { RefRecord rec; try { rec = fastaRefReadSize(*in[i], rparms, first, bpout); if((unambigTot + rec.len) < unambigTot) { throw RefTooLongException(); } } catch(RefTooLongException& e) { cerr << e.what() << endl; throw 1; } // Add the length of this record. if(rec.first) numSeqs++; unambigTot += rec.len; bothTot += rec.len; bothTot += rec.off; first = false; if(rec.len == 0 && rec.off == 0 && !rec.first) continue; recs.push_back(rec); } // Reset the input stream in[i]->reset(); assert(!in[i]->eof()); #ifndef NDEBUG // Check that it's really reset int c = in[i]->get(); assert_eq('>', c); in[i]->reset(); assert(!in[i]->eof()); #endif } // Remove empty reference sequences for(int64_t i = 0; (size_t)i < recs.size(); i++) { const RefRecord& rec = recs[i]; if(rec.first && rec.len == 0) { if(i + 1 >= recs.size() || recs[i+1].first) { bothTot -= rec.len; bothTot -= rec.off; recs.erase(i); i -= 1; } } } assert_geq(bothTot, 0); assert_geq(unambigTot, 0); return make_pair( unambigTot, // total number of unambiguous DNA characters read bothTot); // total number of DNA characters read, incl. ambiguous ones } std::pair fastaRefReadFragsNames( EList& in, EList& recs, const RefReadInParams& rparms, BitpairOutFileBuf* bpout, TIndexOff& numSeqs, EList& names) { TIndexOffU unambigTot = 0; size_t bothTot = 0; assert_gt(in.size(), 0); // For each input istream for(size_t i = 0; i < in.size(); i++) { bool first = true; assert(!in[i]->eof()); // For each pattern in this istream while(!in[i]->eof()) { RefRecord rec; string name; try { rec = fastaRefReadSize(*in[i], rparms, first, bpout, &name); if((unambigTot + rec.len) < unambigTot) { throw RefTooLongException(); } } catch(RefTooLongException& e) { cerr << e.what() << endl; throw 1; } // Add the length of this record. if(rec.first) numSeqs++; if(rec.first) names.push_back(name); unambigTot += rec.len; bothTot += rec.len; bothTot += rec.off; first = false; if(rec.len == 0 && rec.off == 0 && !rec.first) continue; recs.push_back(rec); } // Reset the input stream in[i]->reset(); assert(!in[i]->eof()); #ifndef NDEBUG // Check that it's really reset int c = in[i]->get(); assert_eq('>', c); in[i]->reset(); assert(!in[i]->eof()); #endif } // Remove empty reference sequences for(int64_t i = 0; (size_t)i < recs.size(); i++) { const RefRecord& rec = recs[i]; if(rec.first && rec.len == 0) { if(i + 1 >= recs.size() || recs[i+1].first) { bothTot -= rec.len; bothTot -= rec.off; recs.erase(i); i -= 1; } } } assert_geq(bothTot, 0); assert_geq(unambigTot, 0); return make_pair( unambigTot, // total number of unambiguous DNA characters read bothTot); // total number of DNA characters read, incl. ambiguous ones } hisat2-2.2.1/ref_read.h000066400000000000000000000206661370663763200146550ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef REF_READ_H_ #define REF_READ_H_ #include #include #include #include #include #include #include "alphabet.h" #include "assert_helpers.h" #include "filebuf.h" #include "word_io.h" #include "ds.h" #include "endian_swap.h" using namespace std; class RefTooLongException : public exception { public: RefTooLongException() { #ifdef BOWTIE_64BIT_INDEX // This should never happen! msg = "Error: Reference sequence has more than 2^64-1 characters! " "Please divide the reference into smaller chunks and index each " "independently."; #else msg = "Error: Reference sequence has more than 2^32-1 characters! " "Please build a large index by passing the --large-index option " "to bowtie2-build"; #endif } ~RefTooLongException() throw() {} const char* what() const throw() { return msg.c_str(); } protected: string msg; }; /** * Encapsulates a stretch of the reference containing only unambiguous * characters. From an ordered list of RefRecords, one can (almost) * deduce the "shape" of the reference sequences (almost because we * lose information about stretches of ambiguous characters at the end * of reference sequences). */ struct RefRecord { RefRecord() : off(), len(), first() { } RefRecord(TIndexOffU _off, TIndexOffU _len, bool _first) : off(_off), len(_len), first(_first) { } RefRecord(FILE *in, bool swap) { assert(in != NULL); if(!fread(&off, OFF_SIZE, 1, in)) { cerr << "Error reading RefRecord offset from FILE" << endl; throw 1; } if(swap) off = endianSwapIndex(off); if(!fread(&len, OFF_SIZE, 1, in)) { cerr << "Error reading RefRecord offset from FILE" << endl; throw 1; } if(swap) len = endianSwapIndex(len); first = fgetc(in) ? true : false; } void write(std::ostream& out, bool be) { writeIndex(out, off, be); writeIndex(out, len, be); out.put(first ? 1 : 0); } TIndexOffU off; /// Offset of the first character in the record TIndexOffU len; /// Length of the record bool first; /// Whether this record is the first for a reference sequence }; enum { REF_READ_FORWARD = 0, // don't reverse reference sequence REF_READ_REVERSE, // reverse entire reference sequence REF_READ_REVERSE_EACH // reverse each unambiguous stretch of reference }; /** * Parameters governing treatment of references as they're read in. */ struct RefReadInParams { RefReadInParams(bool col, int r, bool nsToA, bool bisulf) : color(col), reverse(r), nsToAs(nsToA), bisulfite(bisulf) { } // extract colors from reference bool color; // reverse each reference sequence before passing it along int reverse; // convert ambiguous characters to As bool nsToAs; // bisulfite-convert the reference bool bisulfite; }; extern RefRecord fastaRefReadSize( FileBuf& in, const RefReadInParams& rparms, bool first, BitpairOutFileBuf* bpout, string *name = NULL); extern std::pair fastaRefReadSizes( EList& in, EList& recs, const RefReadInParams& rparms, BitpairOutFileBuf* bpout, TIndexOff& numSeqs); extern std::pair fastaRefReadFragsNames( EList& in, EList& recs, const RefReadInParams& rparms, BitpairOutFileBuf* bpout, TIndexOff& numSeqs, EList& names); extern void reverseRefRecords( const EList& src, EList& dst, bool recursive = false, bool verbose = false); /** * Reads the next sequence from the given FASTA file and appends it to * the end of dst, optionally reversing it. */ template static RefRecord fastaRefReadAppend( FileBuf& in, // input file bool first, // true iff this is the first record in the file TStr& dst, // destination buf for parsed characters TIndexOffU& dstoff, // index of next character in dst to assign RefReadInParams& rparms, // string* name = NULL) // put parsed FASTA name here { int c; static int lastc = '>'; if(first) { c = in.getPastWhitespace(); if(c != '>') { cerr << "Reference file does not seem to be a FASTA file" << endl; throw 1; } lastc = c; } assert_neq(-1, lastc); // RefRecord params size_t len = 0; size_t off = 0; first = true; size_t ilen = dstoff; // Chew up the id line; if the next line is either // another id line or a comment line, keep chewing int lc = -1; // last-DNA char variable for color conversion c = lastc; if(c == '>' || c == '#') { do { while (c == '#') { if((c = in.getPastNewline()) == -1) { lastc = -1; goto bail; } } assert_eq('>', c); while(true) { c = in.get(); if(c == -1) { lastc = -1; goto bail; } if(c == '\n' || c == '\r') { while(c == '\r' || c == '\n') c = in.get(); if(c == -1) { lastc = -1; goto bail; } break; } if (name) name->push_back(c); } // c holds the first character on the line after the name // line if(c == '>') { // If there's another name line immediately after this one, // discard the previous name and start fresh with the new one if (name) name->clear(); } } while (c == '>' || c == '#'); } else { ASSERT_ONLY(int cc = toupper(c)); assert(cc != 'A' && cc != 'C' && cc != 'G' && cc != 'T'); first = false; } // Skip over an initial stretch of gaps or ambiguous characters. // For colorspace we skip until we see two consecutive unambiguous // characters (i.e. the first unambiguous color). while(true) { int cat = asc2dnacat[c]; if(rparms.nsToAs && cat >= 2) { c = 'A'; } int cc = toupper(c); if(rparms.bisulfite && cc == 'C') c = cc = 'T'; if(cat == 1) { // This is a DNA character if(rparms.color) { if(lc != -1) { // Got two consecutive unambiguous DNAs break; // to read-in loop } // Keep going; we need two consecutive unambiguous DNAs lc = asc2dna[(int)c]; // The 'if(off > 0)' takes care of the case where // the reference is entirely unambiguous and we don't // want to incorrectly increment off. if(off > 0) off++; } else { break; // to read-in loop } } else if(cat >= 2) { if(lc != -1 && off == 0) { off++; } lc = -1; off++; // skip it } else if(c == '>') { lastc = '>'; goto bail; } c = in.get(); if(c == -1) { lastc = -1; goto bail; } } if(first && rparms.color && off > 0) { // Handle the case where the first record has ambiguous // characters but we're in color space; one of those counts is // spurious off--; } assert(!rparms.color || lc != -1); assert_eq(1, asc2dnacat[c]); // in now points just past the first character of a sequence // line, and c holds the first character while(true) { // Note: can't have a comment in the middle of a sequence, // though a comment can end a sequence int cat = asc2dnacat[c]; assert_neq(2, cat); if(cat == 1) { // Consume it if(!rparms.color || lc != -1) len++; // Add it to reference buffer if(rparms.color) { dst.set((char)dinuc2color[asc2dna[(int)c]][lc], dstoff++); } else if(!rparms.color) { dst.set(asc2dna[c], dstoff++); } assert_lt((int)dst[dstoff-1], 4); lc = asc2dna[(int)c]; } c = in.get(); if(rparms.nsToAs && asc2dnacat[c] >= 2) c = 'A'; if (c == -1 || c == '>' || c == '#' || asc2dnacat[c] >= 2) { lastc = c; break; } if(rparms.bisulfite && toupper(c) == 'C') c = 'T'; } bail: // Optionally reverse the portion that we just appended. // ilen = length of buffer before this last sequence was appended. if(rparms.reverse == REF_READ_REVERSE_EACH) { // Find limits of the portion we just appended size_t nlen = dstoff; dst.reverseWindow(ilen, nlen); } return RefRecord((TIndexOffU)off, (TIndexOffU)len, first); } #endif /*ndef REF_READ_H_*/ hisat2-2.2.1/reference.cpp000066400000000000000000000551031370663763200153710ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include #include "reference.h" #include "mem_ids.h" using namespace std; /** * Load from .3.gfm_ext/.4.gfm_ext HISAT2 index files. */ BitPairReference::BitPairReference( const string& in, const EList* included, bool color, bool sanity, EList* infiles, EList >* origs, bool infilesSeq, bool useMm, bool useShmem, bool mmSweep, bool verbose, bool startVerbose) : buf_(NULL), sanityBuf_(NULL), loaded_(true), sanity_(sanity), useMm_(useMm), useShmem_(useShmem), verbose_(verbose) { string s3 = in + ".3." + gfm_ext; string s4 = in + ".4." + gfm_ext; FILE *f3, *f4; if((f3 = fopen(s3.c_str(), "rb")) == NULL) { cerr << "Could not open reference-string index file " << s3 << " for reading." << endl; cerr << "This is most likely because your index was built with an older version" << endl << "(<= 0.9.8.1) of bowtie-build. Please re-run bowtie-build to generate a new" << endl << "index (or download one from the Bowtie website) and try again." << endl; loaded_ = false; return; } if((f4 = fopen(s4.c_str(), "rb")) == NULL) { cerr << "Could not open reference-string index file " << s4 << " for reading." << endl; loaded_ = false; return; } #ifdef BOWTIE_MM char *mmFile = NULL; if(useMm_) { if(verbose_ || startVerbose) { cerr << " Memory-mapping reference index file " << s4.c_str() << ": "; logTime(cerr); } struct stat sbuf; if (stat(s4.c_str(), &sbuf) == -1) { perror("stat"); cerr << "Error: Could not stat index file " << s4.c_str() << " prior to memory-mapping" << endl; throw 1; } mmFile = (char*)mmap((void *)0, (size_t)sbuf.st_size, PROT_READ, MAP_SHARED, fileno(f4), 0); if(mmFile == (void *)(-1) || mmFile == NULL) { perror("mmap"); cerr << "Error: Could not memory-map the index file " << s4.c_str() << endl; throw 1; } if(mmSweep) { TIndexOff sum = 0; for(off_t i = 0; i < sbuf.st_size; i += 1024) { sum += (TIndexOff) mmFile[i]; } if(startVerbose) { cerr << " Swept the memory-mapped ref index file; checksum: " << sum << ": "; logTime(cerr); } } } #endif // Read endianness sentinel, set 'swap' uint32_t one; bool swap = false; one = readIndex(f3, swap); if(one != 1) { if(useMm_) { cerr << "Error: Can't use memory-mapped files when the index is the opposite endianness" << endl; throw 1; } assert_eq(0x1000000, one); swap = true; // have to endian swap U32s } // Read # records TIndexOffU sz; sz = readIndex(f3, swap); if(sz == 0) { cerr << "Error: number of reference records is 0 in " << s3.c_str() << endl; throw 1; } // Read records nrefs_ = 0; // Cumulative count of all unambiguous characters on a per- // stretch 8-bit alignment (i.e. count of bytes we need to // allocate in buf_) TIndexOffU cumsz = 0; TIndexOffU cumlen = 0; EList seq_poss; TIndexOffU seq_cumpos = 0; TIndexOffU skips = 0; // For each unambiguous stretch... for(TIndexOffU i = 0; i < sz; i++) { recs_.push_back(RefRecord(f3, swap)); if(included != NULL && !(*included)[i]) { seq_cumpos += recs_.back().len; recs_.pop_back(); skips++; continue; } seq_poss.push_back(seq_cumpos); if(recs_.back().first) { // This is the first record for this reference sequence (and the // last record for the one before) refRecOffs_.push_back((TIndexOffU)recs_.size()-1); // refOffs_ links each reference sequence with the total number of // unambiguous characters preceding it in the pasted reference refOffs_.push_back(cumsz); if(nrefs_ > 0) { // refLens_ links each reference sequence with the total number // of ambiguous and unambiguous characters in it. refLens_.push_back(cumlen); } cumlen = 0; nrefs_++; } else if(i == 0) { cerr << "First record in reference index file was not marked as " << "'first'" << endl; throw 1; } cumUnambig_.push_back(cumsz); cumRefOff_.push_back(cumlen); cumsz += recs_.back().len; cumlen += recs_.back().off; cumlen += recs_.back().len; seq_cumpos += recs_.back().len; } if(verbose_ || startVerbose) { cerr << "Read " << nrefs_ << " reference strings from " << sz << " records: "; logTime(cerr); } // Store a cap entry for the end of the last reference seq refRecOffs_.push_back((TIndexOffU)recs_.size()); refOffs_.push_back(cumsz); refLens_.push_back(cumlen); cumUnambig_.push_back(cumsz); cumRefOff_.push_back(cumlen); bufSz_ = cumsz; assert_eq(nrefs_, refLens_.size()); assert_eq(sz, recs_.size() + skips); if (f3 != NULL) fclose(f3); // done with .3.gfm_ext file // Round cumsz up to nearest byte boundary if((cumsz & 3) != 0) { cumsz += (4 - (cumsz & 3)); } bufAllocSz_ = cumsz >> 2; assert_eq(0, cumsz & 3); // should be rounded up to nearest 4 if(useMm_) { #ifdef BOWTIE_MM buf_ = (uint8_t*)mmFile; if(sanity_) { FILE *ftmp = fopen(s4.c_str(), "rb"); sanityBuf_ = new uint8_t[cumsz >> 2]; size_t ret = fread(sanityBuf_, 1, cumsz >> 2, ftmp); if(ret != (cumsz >> 2)) { cerr << "Only read " << ret << " bytes (out of " << (cumsz >> 2) << ") from reference index file " << s4.c_str() << endl; throw 1; } fclose(ftmp); for(size_t i = 0; i < (cumsz >> 2); i++) { assert_eq(sanityBuf_[i], buf_[i]); } } #else cerr << "Shouldn't be at " << __FILE__ << ":" << __LINE__ << " without BOWTIE_MM defined" << endl; throw 1; #endif } else { bool shmemLeader = true; if(!useShmem_) { // Allocate a buffer to hold the reference string try { buf_ = new uint8_t[cumsz >> 2]; if(buf_ == NULL) throw std::bad_alloc(); } catch(std::bad_alloc& e) { cerr << "Error: Ran out of memory allocating space for the bitpacked reference. Please" << endl << "re-run on a computer with more memory." << endl; throw 1; } } else { shmemLeader = ALLOC_SHARED_U8( (s4 + "[ref]"), (cumsz >> 2), &buf_, "ref", (verbose_ || startVerbose)); } if(shmemLeader) { // Open the bitpair-encoded reference file FILE *f4 = fopen(s4.c_str(), "rb"); if(f4 == NULL) { cerr << "Could not open reference-string index file " << s4.c_str() << " for reading." << endl; cerr << "This is most likely because your index was built with an older version" << endl << "(<= 0.9.8.1) of bowtie-build. Please re-run bowtie-build to generate a new" << endl << "index (or download one from the Bowtie website) and try again." << endl; loaded_ = false; return; } if(included == NULL) { // Read the whole thing in size_t ret = fread(buf_, 1, cumsz >> 2, f4); // Didn't read all of it? if(ret != (cumsz >> 2)) { cerr << "Only read " << ret << " bytes (out of " << (cumsz >> 2) << ") from reference index file " << s4.c_str() << endl; throw 1; } // Make sure there's no more char c; ret = fread(&c, 1, 1, f4); assert_eq(0, ret); // should have failed } else { TIndexOffU buf_pos = 0; uint8_t four_buf = 0, four_buf2 = 0; for(size_t i = 0; i < seq_poss.size(); i++) { TIndexOffU seq_pos = seq_poss[i]; TIndexOffU cur_len = refLens_[i]; TIndexOffU seq_pos2 = seq_pos + cur_len; TIndexOffU left_pad = seq_pos & 3; assert_eq((seq_pos - left_pad) & 3, 0); TIndexOffU right_pad = 4 - (seq_pos2 & 3); if(right_pad == 4) right_pad = 0; assert_eq((seq_pos2 + right_pad) & 3, 0); TIndexOffU cur_len2 = left_pad + cur_len + right_pad; assert_eq(cur_len2 & 3, 0); uint8_t *buf2_ = new uint8_t[cur_len2 >> 2]; // Read sequences selectively fseek(f4, (seq_pos - left_pad) >> 2, SEEK_SET); size_t ret = fread(buf2_, 1, cur_len2 >> 2, f4); // Didn't read all of it? if(ret != (cur_len2 >> 2)) { cerr << "Only read " << ret << " bytes (out of " << (cur_len2 >> 2) << ") from reference index file " << s4.c_str() << endl; throw 1; } four_buf2 = buf2_[0] >> (left_pad << 1); for(TIndexOffU j = seq_pos; j < seq_pos2; j++, buf_pos++) { if((j & 3) == 0) { four_buf2 = buf2_[(j - (seq_pos - left_pad)) >> 2]; } uint8_t nt = four_buf2 & 3; four_buf2 >>= 2; four_buf |= (nt << ((buf_pos & 3) << 1)); if((buf_pos & 3) == 3) { buf_[buf_pos >> 2] = four_buf; four_buf = 0; } } delete [] buf2_; seq_pos += cur_len; } #ifndef NDEBUG TIndexOffU cumsz2 = 0; for(size_t i = 0; i < refLens_.size(); i++) { cumsz2 += refLens_[i]; } assert_eq(buf_pos, cumsz2); #endif if((buf_pos & 3) != 0) { buf_[buf_pos >> 2] = four_buf; } assert_eq(nrefs_, refLens_.size()); } fclose(f4); #ifdef BOWTIE_SHARED_MEM if(useShmem_) NOTIFY_SHARED(buf_, (cumsz >> 2)); #endif } else { #ifdef BOWTIE_SHARED_MEM if(useShmem_) WAIT_SHARED(buf_, (cumsz >> 2)); #endif } } // Populate byteToU32_ bool big = currentlyBigEndian(); for(int i = 0; i < 256; i++) { uint32_t word = 0; if(big) { word |= ((i >> 0) & 3) << 24; word |= ((i >> 2) & 3) << 16; word |= ((i >> 4) & 3) << 8; word |= ((i >> 6) & 3) << 0; } else { word |= ((i >> 0) & 3) << 0; word |= ((i >> 2) & 3) << 8; word |= ((i >> 4) & 3) << 16; word |= ((i >> 6) & 3) << 24; } byteToU32_[i] = word; } #ifndef NDEBUG if(sanity_) { // Compare the sequence we just read from the compact index // file to the true reference sequence. EList > *os; // for holding references EList > osv(DEBUG_CAT); // for holding ref seqs EList > osn(DEBUG_CAT); // for holding ref names EList osvLen(DEBUG_CAT); // for holding ref seq lens EList osnLen(DEBUG_CAT); // for holding ref name lens SStringExpandable tmp_destU32_; if(infiles != NULL) { if(infilesSeq) { for(size_t i = 0; i < infiles->size(); i++) { // Remove initial backslash; that's almost // certainly being used to protect the first // character of the sequence from getopts (e.g., // when the first char is -) if((*infiles)[i].at(0) == '\\') { (*infiles)[i].erase(0, 1); } osv.push_back(SString((*infiles)[i])); } } else { parseFastas(*infiles, osn, osnLen, osv, osvLen); } os = &osv; } else { assert(origs != NULL); os = origs; } // Go through the loaded reference files base-by-base and // sanity check against what we get by calling getBase and // getStretch for(size_t i = 0; i < os->size(); i++) { size_t olen = ((*os)[i]).length(); size_t olenU32 = (olen + 12) / 4; uint32_t *buf = new uint32_t[olenU32]; uint8_t *bufadj = (uint8_t*)buf; bufadj += getStretch(buf, i, 0, olen, tmp_destU32_); for(size_t j = 0; j < olen; j++) { assert_eq((int)(*os)[i][j], (int)bufadj[j]); assert_eq((int)(*os)[i][j], (int)getBase(i, j)); } delete[] buf; } } #endif } BitPairReference::~BitPairReference() { if(buf_ != NULL && !useMm_ && !useShmem_) delete[] buf_; if(sanityBuf_ != NULL) delete[] sanityBuf_; } /** * Return a single base of the reference. Calling this repeatedly * is not an efficient way to retrieve bases from the reference; * use loadStretch() instead. * * This implementation scans linearly through the records for the * unambiguous stretches of the target reference sequence. When * there are many records, binary search would be more appropriate. */ int BitPairReference::getBase(size_t tidx, size_t toff) const { uint64_t reci = refRecOffs_[tidx]; // first record for target reference sequence uint64_t recf = refRecOffs_[tidx+1]; // last record (exclusive) for target seq assert_gt(recf, reci); uint64_t bufOff = refOffs_[tidx]; uint64_t off = 0; // For all records pertaining to the target reference sequence... for(uint64_t i = reci; i < recf; i++) { assert_geq(toff, off); off += recs_[i].off; if(toff < off) { return 4; } assert_geq(toff, off); uint64_t recOff = off + recs_[i].len; if(toff < recOff) { toff -= off; bufOff += (uint64_t)toff; assert_lt(bufOff, bufSz_); const uint64_t bufElt = (bufOff) >> 2; const uint64_t shift = (bufOff & 3) << 1; return ((buf_[bufElt] >> shift) & 3); } bufOff += recs_[i].len; off = recOff; assert_geq(toff, off); } // end for loop over records return 4; } /** * Load a stretch of the reference string into memory at 'dest'. * * This implementation scans linearly through the records for the * unambiguous stretches of the target reference sequence. When * there are many records, binary search would be more appropriate. */ int BitPairReference::getStretchNaive( uint32_t *destU32, size_t tidx, size_t toff, size_t count) const { uint8_t *dest = (uint8_t*)destU32; uint64_t reci = refRecOffs_[tidx]; // first record for target reference sequence uint64_t recf = refRecOffs_[tidx+1]; // last record (exclusive) for target seq assert_gt(recf, reci); uint64_t cur = 0; uint64_t bufOff = refOffs_[tidx]; uint64_t off = 0; // For all records pertaining to the target reference sequence... for(uint64_t i = reci; i < recf; i++) { assert_geq(toff, off); off += recs_[i].off; for(; toff < off && count > 0; toff++) { dest[cur++] = 4; count--; } if(count == 0) break; assert_geq(toff, off); if(toff < off + recs_[i].len) { bufOff += (TIndexOffU)(toff - off); // move bufOff pointer forward } else { bufOff += recs_[i].len; } off += recs_[i].len; for(; toff < off && count > 0; toff++) { assert_lt(bufOff, bufSz_); const uint64_t bufElt = (bufOff) >> 2; const uint64_t shift = (bufOff & 3) << 1; dest[cur++] = (buf_[bufElt] >> shift) & 3; bufOff++; count--; } if(count == 0) break; assert_geq(toff, off); } // end for loop over records // In any chars are left after scanning all the records, // they must be ambiguous while(count > 0) { count--; dest[cur++] = 4; } assert_eq(0, count); return 0; } /** * Load a stretch of the reference string into memory at 'dest'. */ int BitPairReference::getStretch( uint32_t *destU32, size_t tidx, size_t toff, size_t count ASSERT_ONLY(, SStringExpandable& destU32_2)) const { ASSERT_ONLY(size_t origCount = count); ASSERT_ONLY(size_t origToff = toff); if(count == 0) return 0; uint8_t *dest = (uint8_t*)destU32; #ifndef NDEBUG destU32_2.clear(); uint8_t *dest_2 = NULL; int off2; if((rand() % 10) == 0) { destU32_2.resize((origCount >> 2) + 2); off2 = getStretchNaive(destU32_2.wbuf(), tidx, origToff, origCount); dest_2 = ((uint8_t*)destU32_2.wbuf()) + off2; } #endif destU32[0] = 0x04040404; // Add Ns, which we might end up using later uint64_t reci = refRecOffs_[tidx]; // first record for target reference sequence uint64_t recf = refRecOffs_[tidx+1]; // last record (exclusive) for target seq assert_gt(recf, reci); uint64_t cur = 4; // keep a cushion of 4 bases at the beginning uint64_t bufOff = refOffs_[tidx]; uint64_t off = 0; int64_t offset = 4; bool firstStretch = true; bool binarySearched = false; uint64_t left = reci; uint64_t right = recf; uint64_t mid = 0; // For all records pertaining to the target reference sequence... for(uint64_t i = reci; i < recf; i++) { uint64_t origBufOff = bufOff; assert_geq(toff, off); if (firstStretch && recf > reci + 16){ // binary search finds smallest i s.t. toff >= cumRefOff_[i] while (left < right-1) { mid = left + ((right - left) >> 1); if (cumRefOff_[mid] <= toff) left = mid; else right = mid; } off = cumRefOff_[left]; bufOff = cumUnambig_[left]; origBufOff = bufOff; i = left; assert(cumRefOff_[i+1] == 0 || cumRefOff_[i+1] > toff); binarySearched = true; } off += recs_[i].off; // skip Ns at beginning of stretch assert_gt(count, 0); if(toff < off) { size_t cpycnt = min((size_t)(off - toff), count); memset(&dest[cur], 4, cpycnt); count -= cpycnt; toff += cpycnt; cur += cpycnt; if(count == 0) break; } assert_geq(toff, off); if(toff < off + recs_[i].len) { bufOff += toff - off; // move bufOff pointer forward } else { bufOff += recs_[i].len; } off += recs_[i].len; assert(off == cumRefOff_[i+1] || cumRefOff_[i+1] == 0); assert(!binarySearched || toff < off); if(toff < off) { if(firstStretch) { if(toff + 8 < off && count > 8) { // We already added some Ns, so we have to do // a fixup at the beginning of the buffer so // that we can start clobbering at cur >> 2 if(cur & 3) { offset -= (cur & 3); } uint64_t curU32 = cur >> 2; // Do the initial few bases if(bufOff & 3) { const uint64_t bufElt = (bufOff) >> 2; const int64_t low2 = bufOff & 3; // Lots of cache misses on the following line destU32[curU32] = byteToU32_[buf_[bufElt]]; for(int j = 0; j < low2; j++) { ((char *)(&destU32[curU32]))[j] = 4; } curU32++; offset += low2; const int64_t chars = 4 - low2; count -= chars; bufOff += chars; toff += chars; } assert_eq(0, bufOff & 3); uint64_t bufOffU32 = bufOff >> 2; uint64_t countLim = count >> 2; uint64_t offLim = ((off - (toff + 4)) >> 2); uint64_t lim = min(countLim, offLim); // Do the fast thing for as far as possible for(uint64_t j = 0; j < lim; j++) { // Lots of cache misses on the following line destU32[curU32] = byteToU32_[buf_[bufOffU32++]]; #ifndef NDEBUG if(dest_2 != NULL) { assert_eq(dest[(curU32 << 2) + 0], dest_2[(curU32 << 2) - offset + 0]); assert_eq(dest[(curU32 << 2) + 1], dest_2[(curU32 << 2) - offset + 1]); assert_eq(dest[(curU32 << 2) + 2], dest_2[(curU32 << 2) - offset + 2]); assert_eq(dest[(curU32 << 2) + 3], dest_2[(curU32 << 2) - offset + 3]); } #endif curU32++; } toff += (lim << 2); assert_leq(toff, off); assert_leq((lim << 2), count); count -= (lim << 2); bufOff = bufOffU32 << 2; cur = curU32 << 2; } // Do the slow thing for the rest for(; toff < off && count > 0; toff++) { assert_lt(bufOff, bufSz_); const uint64_t bufElt = (bufOff) >> 2; const uint64_t shift = (bufOff & 3) << 1; dest[cur++] = (buf_[bufElt] >> shift) & 3; bufOff++; count--; } firstStretch = false; } else { // Do the slow thing for(; toff < off && count > 0; toff++) { assert_lt(bufOff, bufSz_); const uint64_t bufElt = (bufOff) >> 2; const uint64_t shift = (bufOff & 3) << 1; dest[cur++] = (buf_[bufElt] >> shift) & 3; bufOff++; count--; } } } if(count == 0) break; assert_eq(recs_[i].len, bufOff - origBufOff); assert_geq(toff, off); } // end for loop over records // In any chars are left after scanning all the records, // they must be ambiguous while(count > 0) { count--; dest[cur++] = 4; } assert_eq(0, count); return (int)offset; } /** * Parse the input fasta files, populating the szs list and writing the * .3.gfm_ext and .4.gfm_ext portions of the index as we go. */ pair BitPairReference::szsFromFasta( EList& is, const string& outfile, bool bigEndian, const RefReadInParams& refparams, EList& szs, bool sanity, EList *names) { RefReadInParams parms = refparams; std::pair sztot; if(!outfile.empty()) { string file3 = outfile + ".3." + gfm_ext; string file4 = outfile + ".4." + gfm_ext; // Open output stream for the '.3.gfm_ext' file which will // hold the size records. ofstream fout3(file3.c_str(), ios::binary); if(!fout3.good()) { cerr << "Could not open index file for writing: \"" << file3.c_str() << "\"" << endl << "Please make sure the directory exists and that permissions allow writing by" << endl << "HISAT2." << endl; throw 1; } BitpairOutFileBuf bpout(file4.c_str()); // Read in the sizes of all the unambiguous stretches of the genome // into a vector of RefRecords. The input streams are reset once // it's done. writeIndex(fout3, 1, bigEndian); // endianness sentinel TIndexOff numSeqs = 0; sztot = fastaRefReadSizes(is, szs, parms, &bpout, numSeqs); writeIndex(fout3, (TIndexOffU)szs.size(), bigEndian); // write # records for(size_t i = 0; i < szs.size(); i++) szs[i].write(fout3, bigEndian); if(sztot.first == 0) { cerr << "Error: No unambiguous stretches of characters in the input. Aborting..." << endl; throw 1; } assert_gt(sztot.first, 0); assert_gt(sztot.second, 0); bpout.close(); fout3.close(); } else { // Read in the sizes of all the unambiguous stretches of the // genome into a vector of RefRecords TIndexOff numSeqs = 0; //sztot = fastaRefReadSizes(is, szs, parms, NULL, numSeqs); sztot = fastaRefReadFragsNames(is, szs, parms, NULL, numSeqs, *names); #ifndef NDEBUG if(parms.color) { parms.color = false; EList szs2(EBWTB_CAT); TIndexOff numSeqs2 = 0; ASSERT_ONLY(std::pair sztot2 =) fastaRefReadSizes(is, szs2, parms, NULL, numSeqs2); assert_eq(numSeqs, numSeqs2); // One less color than base assert_geq(sztot2.second, sztot.second + numSeqs); parms.color = true; } #endif } return sztot; } hisat2-2.2.1/reference.h000066400000000000000000000135001370663763200150310ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef REFERENCE_H_ #define REFERENCE_H_ #include #include #include #include #ifdef BOWTIE_MM #include #include #endif #include "endian_swap.h" #include "ref_read.h" #include "sequence_io.h" #include "mm.h" #include "shmem.h" #include "timer.h" #include "sstring.h" #include "btypes.h" /** * Concrete reference representation that bulk-loads the reference from * the bit-pair-compacted binary file and stores it in memory also in * bit-pair-compacted format. The user may request reference * characters either on a per-character bases or by "stretch" using * getBase(...) and getStretch(...) respectively. * * Most of the complexity in this class is due to the fact that we want * to represent references with ambiguous (non-A/C/G/T) characters but * we don't want to use more than two bits per base. This means we * need a way to encode the ambiguous stretches of the reference in a * way that is external to the bitpair sequence. To accomplish this, * we use the RefRecords vector, which is stored in the .3.ebwt index * file. The bitpairs themselves are stored in the .4.ebwt index file. * * Once it has been loaded, a BitPairReference is read-only, and is * safe for many threads to access at once. */ class BitPairReference { public: /** * Load from .3.ebwt/.4.ebwt Bowtie index files. */ BitPairReference( const string& in, const EList* included, bool color, bool sanity = false, EList* infiles = NULL, EList >* origs = NULL, bool infilesSeq = false, bool useMm = false, bool useShmem = false, bool mmSweep = false, bool verbose = false, bool startVerbose = false); ~BitPairReference(); /** * Return a single base of the reference. Calling this repeatedly * is not an efficient way to retrieve bases from the reference; * use loadStretch() instead. * * This implementation scans linearly through the records for the * unambiguous stretches of the target reference sequence. When * there are many records, binary search would be more appropriate. */ int getBase(size_t tidx, size_t toff) const; /** * Load a stretch of the reference string into memory at 'dest'. * * This implementation scans linearly through the records for the * unambiguous stretches of the target reference sequence. When * there are many records, binary search would be more appropriate. */ int getStretchNaive( uint32_t *destU32, size_t tidx, size_t toff, size_t count) const; /** * Load a stretch of the reference string into memory at 'dest'. * * This implementation scans linearly through the records for the * unambiguous stretches of the target reference sequence. When * there are many records, binary search would be more appropriate. */ int getStretch( uint32_t *destU32, size_t tidx, size_t toff, size_t count ASSERT_ONLY(, SStringExpandable& destU32_2)) const; /** * Return the number of reference sequences. */ TIndexOffU numRefs() const { return nrefs_; } /** * Return the approximate length of a reference sequence (it might leave * off some Ns on the end). * * TODO: Is it still true that it might leave off Ns? */ TIndexOffU approxLen(TIndexOffU elt) const { assert_lt(elt, nrefs_); return refLens_[elt]; } /** * Return true iff buf_ and all the vectors are populated. */ bool loaded() const { return loaded_; } /** * Given a reference sequence id, return its offset into the pasted * reference string; i.e., return the number of unambiguous nucleotides * preceding it. */ TIndexOffU pastedOffset(TIndexOffU idx) const { return refOffs_[idx]; } /** * Parse the input fasta files, populating the szs list and writing the * .3.ebwt and .4.ebwt portions of the index as we go. */ static std::pair szsFromFasta( EList& is, const string& outfile, bool bigEndian, const RefReadInParams& refparams, EList& szs, bool sanity, EList *names = NULL); protected: uint32_t byteToU32_[256]; EList recs_; /// records describing unambiguous stretches // following two lists are purely for the binary search in getStretch EList cumUnambig_; // # unambig ref chars up to each record EList cumRefOff_; // # ref chars up to each record EList refLens_; /// approx lens of ref seqs (excludes trailing ambig chars) EList refOffs_; /// buf_ begin offsets per ref seq EList refRecOffs_; /// record begin/end offsets per ref seq uint8_t *buf_; /// the whole reference as a big bitpacked byte array uint8_t *sanityBuf_;/// for sanity-checking buf_ TIndexOffU bufSz_; /// size of buf_ TIndexOffU bufAllocSz_; TIndexOffU nrefs_; /// the number of reference sequences bool loaded_; /// whether it's loaded bool sanity_; /// do sanity checking bool useMm_; /// load the reference as a memory-mapped file bool useShmem_; /// load the reference into shared memory bool verbose_; ASSERT_ONLY(SStringExpandable tmp_destU32_); }; #endif hisat2-2.2.1/repeat.h000066400000000000000000000607341370663763200143660ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef REPEAT_H_ #define REPEAT_H_ #include #include #include #include #include "assert_helpers.h" #include "word_io.h" #include "mem_ids.h" #include "ref_coord.h" #include "alt.h" using namespace std; template class RepeatCoord { public: bool operator< (const RepeatCoord& o) const { if(joinedOff != o.joinedOff) return joinedOff < o.joinedOff; if(fw != o.fw) return fw; if(alleleID != o.alleleID) return alleleID < o.alleleID; return false; } public: RepeatCoord() {}; RepeatCoord(index_t l_tid, index_t l_toff, index_t l_joinedOff, bool l_fw, index_t l_alleleID) : tid(l_tid), toff(l_toff), joinedOff(l_joinedOff), fw(l_fw) {}; index_t tid; index_t toff; index_t joinedOff; bool fw; index_t alleleID; }; template class RepeatAllele { public: RepeatAllele() { reset(); } void init(index_t allelePos_, index_t alleleLen_) { allelePos = allelePos_; alleleLen = alleleLen_; } void reset() { allelePos = 0; alleleLen = 0; } bool operator< (const RepeatAllele& o) const { if(allelePos != o.allelePos) return allelePos < o.allelePos; return alleleLen < o.alleleLen; } #ifndef NDEBUG bool repOk() const { return true; } #endif bool write(ofstream& f_out, bool bigEndian) const { writeU16(f_out, allelePos, bigEndian); writeU16(f_out, alleleLen, bigEndian); return true; } bool read(ifstream& f_in, bool bigEndian) { allelePos = readU16(f_in, bigEndian); alleleLen = readU16(f_in, bigEndian); return true; } bool compatible(index_t left, index_t right) const { if(left < allelePos || right > allelePos + alleleLen) return false; return true; } public: uint16_t allelePos; uint16_t alleleLen; }; // sorting functions template struct sort_pair_loci { bool operator()(const pair, index_t>& a, const pair, index_t>& b) { return a.first.joinedOff < b.first.joinedOff; } }; template struct sort_pair_loci_by_index { bool operator()(const pair, index_t>& a, const pair, index_t>& b) { return a.second < b.second; } }; template class Repeat { public: void init(const string& repName_, index_t repID_, index_t repPos_, index_t repLen_) { repName = repName_; repID = repID_; repPos = repPos_; repLen = repLen_; } bool write(ofstream& f_out, bool bigEndian) const { writeIndex(f_out, repID, bigEndian); writeIndex(f_out, repPos, bigEndian); writeIndex(f_out, repLen, bigEndian); writeIndex(f_out, alleles.size(), bigEndian); for(index_t i = 0; i < alleles.size(); i++) { alleles[i].write(f_out, bigEndian); } writeIndex(f_out, positions.size(), bigEndian); for(index_t i = 0; i < positions.size(); i++) { writeIndex(f_out, positions[i].joinedOff, bigEndian); writeU8(f_out, positions[i].fw); writeIndex(f_out, positions[i].alleleID, bigEndian); } return true; } bool read(ifstream& f_in, bool bigEndian) { repID = readIndex(f_in, bigEndian); repPos = readIndex(f_in, bigEndian); repLen = readIndex(f_in, bigEndian); index_t numAlleles = readIndex(f_in, bigEndian); alleles.resizeExact(numAlleles); for(index_t i = 0; i < numAlleles; i++) { alleles[i].read(f_in, bigEndian); } index_t numPositions = readIndex(f_in, bigEndian); positions.resizeExact(numPositions); for(index_t i = 0; i < numPositions; i++) { positions[i].tid = 0; positions[i].toff = 0; positions[i].joinedOff = readIndex(f_in, bigEndian); positions[i].fw = readU8(f_in); positions[i].alleleID = readIndex(f_in, bigEndian); assert_lt(positions[i].alleleID, alleles.size()); } return true; } public: string repName; index_t repID; index_t repPos; index_t repLen; EList > alleles; EList > positions; }; template class RepeatDB { public: RepeatDB() {} virtual ~RepeatDB() {} bool empty() const { return _repeats.size() == 0; } EList >& repeats() { return _repeats; } const EList >& repeats() const { return _repeats; } const ELList >& repeatMap() const { return _repeatMap; } void write(ofstream& f_out, bool bigEndian) const { if(_repeats.size() <= 0) { writeIndex(f_out, 0, bigEndian); return; } EList repeatGroup; for(index_t i = 0; i < _repeats.size(); i++) { #ifndef NDEBUG if(i + 1 < _repeats.size()) { assert_leq(_repeats[i].repID, _repeats[i+1].repID); } #endif if(_repeats[i].repID > repeatGroup.size()) { repeatGroup.push_back(i); assert_eq(_repeats[i].repID, repeatGroup.size()); } } repeatGroup.push_back(_repeats.size()); assert_eq(_repeats.back().repID + 1, repeatGroup.size()); writeIndex(f_out, repeatGroup.size(), bigEndian); streampos filepos = f_out.tellp(); EList repeatFilePos; for(index_t i = 0; i < repeatGroup.size(); i++) { writeIndex(f_out, 0, bigEndian); } for(index_t i = 0; i < repeatGroup.size(); i++) { index_t begin = (i == 0 ? 0 : repeatGroup[i-1]); index_t end = repeatGroup[i]; writeIndex(f_out, end - begin, bigEndian); for(index_t j = begin; j < end; j++) { _repeats[j].write(f_out, bigEndian); } repeatFilePos.push_back(f_out.tellp()); } assert_eq(repeatFilePos.size(), repeatGroup.size()); streampos origpos = f_out.tellp(); f_out.seekp(filepos); for(index_t i = 0; i < repeatFilePos.size(); i++) { writeIndex(f_out, repeatFilePos[i], bigEndian); } f_out.seekp(origpos); } void read(ifstream& f_in, bool bigEndian, const EList& includeRepeat) { index_t numRepeatGroup = readIndex(f_in, bigEndian); EList filePos; filePos.resizeExact(numRepeatGroup); for(index_t i = 0; i < numRepeatGroup; i++) { filePos[i] = readIndex(f_in, bigEndian); } assert_eq(numRepeatGroup, includeRepeat.size()); for(index_t i = 0, repID = 0; i < numRepeatGroup; i++) { if(!includeRepeat[i]) continue; if(i > 0) { f_in.seekg(filePos[i-1]); } index_t numRepeats = readIndex(f_in, bigEndian); index_t repeat_size = _repeats.size(); _repeats.resizeExact(repeat_size + numRepeats); for(index_t j = 0; j < numRepeats; j++) { _repeats[repeat_size+j].read(f_in, bigEndian); _repeats[repeat_size+j].repID = repID; } repID++; } f_in.seekg(filePos.back()); } // Build an internal table to enable rapid search of repeats // and converts joined offsets to chromosome IDs (tid) and loci (toff) void construct(const index_t* rstarts, index_t rlen) { _repeatMap.clear(); if(_repeats.empty()) return; for(index_t r = 0; r < _repeats.size(); r++) { if(_repeats[r].repID >= _repeatMap.size()) { _repeatMap.expand(); _repeatMap.back().clear(); } EList >& repeatMap = _repeatMap.back(); repeatMap.expand(); if(repeatMap.size() == 1) { repeatMap.back().first = _repeats[r].repLen; } else { repeatMap.back().first = repeatMap[repeatMap.size() - 2].first + _repeats[r].repLen; } repeatMap.back().second = r; } EList, index_t> > joinedOffList; for(index_t r = 0; r < _repeats.size(); r++) { Repeat& repeat = _repeats[r]; EList >& positions = repeat.positions; for(index_t p = 0; p < positions.size(); p++) { joinedOffList.expand(); joinedOffList.back().first.joinedOff = positions[p].joinedOff; joinedOffList.back().first.tid = 0; joinedOffList.back().first.toff = 0; joinedOffList.back().first.fw = positions[p].fw; joinedOffList.back().first.alleleID = positions[p].alleleID; joinedOffList.back().second = joinedOffList.size() - 1; } } sort(joinedOffList.begin(), joinedOffList.end(), sort_pair_loci()); index_t j = 0, r = 0; while(j < joinedOffList.size() && r < rlen) { index_t off = joinedOffList[j].first.joinedOff; index_t lower = rstarts[r*3]; index_t upper; if(r == rlen - 1) { upper = numeric_limits::max(); } else { upper = rstarts[(r+1)*3]; } assert_gt(upper, lower); if(off >= upper) { r++; continue; } assert_geq(off, lower); joinedOffList[j].first.tid = rstarts[(r*3)+1]; joinedOffList[j].first.toff = off - lower + rstarts[(r*3)+2]; j++; } sort(joinedOffList.begin(), joinedOffList.end(), sort_pair_loci_by_index()); index_t count = 0; for(index_t r = 0; r < _repeats.size(); r++) { Repeat& repeat = _repeats[r]; EList >& positions = _repeats[r].positions; for(index_t p = 0; p < positions.size(); p++) { assert_lt(count, joinedOffList.size()); assert_eq(positions[p].joinedOff, joinedOffList[count].first.joinedOff); positions[p] = joinedOffList[count].first; RepeatAllele& allele = repeat.alleles[positions[p].alleleID]; if(positions[p].fw) { positions[p].joinedOff -= allele.allelePos; positions[p].toff -= allele.allelePos; } else { assert_leq(allele.allelePos + allele.alleleLen, repeat.repLen); index_t subLen = repeat.repLen - allele.allelePos - allele.alleleLen; positions[p].joinedOff -= subLen; positions[p].toff -= subLen; } count++; } } } bool repeatExist(index_t repID, index_t left, index_t right) const { if(repID >= _repeatMap.size()) return false; // Find a repeat corresponding to a given location (left, right) const EList >& repeatMap = _repeatMap[repID]; pair repeat(left, numeric_limits::max()); index_t repeatIdx = repeatMap.bsearchLoBound(repeat); assert_lt(repeatIdx, repeatMap.size()); if(right > repeatMap[repeatIdx].first) return false; return true; } bool getCoords(index_t repID, index_t left, // left offset in the repeat sequence index_t right, // right offset const EList& snpIDs, // SNP IDs const ALTDB& altdb, EList, RepeatCoord > >& near_positions, index_t max_positions = numeric_limits::max()) const { near_positions.clear(); if(repID >= _repeatMap.size()) return false; // Find a repeat corresponding to a given location (left, right) const EList >& repeatMap = _repeatMap[repID]; pair repeat(left, numeric_limits::max()); index_t repeatIdx = repeatMap.bsearchLoBound(repeat); assert_lt(repeatIdx, repeatMap.size()); if(right > repeatMap[repeatIdx].first) return false; index_t repeatIdx_ = repeatMap[repeatIdx].second; assert_lt(repeatIdx_, _repeats.size()); const EList >& alleles = _repeats[repeatIdx_].alleles; index_t adjLeft = left, adjRight = right; if(repeatIdx > 0) { adjLeft -= repeatMap[repeatIdx-1].first; adjRight -= repeatMap[repeatIdx-1].first; } const EList >& positions = _repeats[repeatIdx_].positions; for(index_t p = 0; p < positions.size(); p++) { const RepeatCoord& position = positions[p]; assert_lt(position.alleleID, alleles.size()); const RepeatAllele& allele = alleles[position.alleleID]; if(!allele.compatible(adjLeft, adjRight)) continue; near_positions.expand(); near_positions.back().first = position; if(positions[p].fw) { near_positions.back().first.joinedOff += adjLeft; near_positions.back().first.toff += adjLeft; } else { const index_t len = right - left; assert_leq(adjLeft + len, _repeats[repeatIdx_].repLen); index_t rc_adjLeft = _repeats[repeatIdx_].repLen - adjLeft - len; near_positions.back().first.joinedOff += rc_adjLeft; near_positions.back().first.toff += rc_adjLeft; } if(near_positions.size() >= max_positions) break; } return near_positions.size() > 0; } bool findCoords(index_t anchor_left, index_t anchor_right, index_t repID, index_t left, // left offset in the repeat sequence index_t right, // right offset const EList& snpIDs, // SNP IDs const ALTDB& altdb, EList, RepeatCoord > >& near_positions, index_t max_positions = numeric_limits::max(), index_t dist = 1000) const { near_positions.clear(); if(repID >= _repeatMap.size()) return false; // Find a repeat corresponding to a given location (left, right) const EList >& repeatMap = _repeatMap[repID]; pair repeat(left, numeric_limits::max()); index_t repeatIdx = repeatMap.bsearchLoBound(repeat); assert_lt(repeatIdx, repeatMap.size()); if(right > repeatMap[repeatIdx].first) return false; index_t repeatIdx_ = repeatMap[repeatIdx].second; assert_lt(repeatIdx_, _repeats.size()); const EList >& alleles = _repeats[repeatIdx_].alleles; index_t adjLeft = left, adjRight = right; if(repeatIdx > 0) { adjLeft -= repeatMap[repeatIdx-1].first; adjRight -= repeatMap[repeatIdx-1].first; } const EList >& positions = _repeats[repeatIdx_].positions; RepeatCoord cmp; cmp.joinedOff = (anchor_left >= dist ? anchor_left - dist : 0); index_t p = positions.bsearchLoBound(cmp); for(; p < positions.size(); p++) { const RepeatCoord& position = positions[p]; index_t pos = positions[p].joinedOff + adjLeft; if(pos + dist < anchor_left) continue; if(anchor_right + dist < pos) break; assert_lt(position.alleleID, alleles.size()); const RepeatAllele& allele = alleles[position.alleleID]; if(!allele.compatible(adjLeft, adjRight)) continue; near_positions.expand(); near_positions.back().first = position; if(positions[p].fw) { near_positions.back().first.joinedOff += adjLeft; near_positions.back().first.toff += adjLeft; } else { const index_t len = right - left; assert_leq(adjLeft + len, _repeats[repeatIdx_].repLen); index_t rc_adjLeft = _repeats[repeatIdx_].repLen - adjLeft - len; near_positions.back().first.joinedOff += rc_adjLeft; near_positions.back().first.toff += rc_adjLeft; } if(near_positions.size() >= max_positions) break; } return near_positions.size() > 0; } bool findCommonCoords(index_t repID, index_t left, // left offset in the repeat sequence index_t right, // right offset const EList& snpIDs, // SNP IDs index_t repID2, index_t left2, // left offset 2 in the repeat sequence index_t right2, // right offset 2 const EList& snpIDs2, // SNP IDs const ALTDB& altdb, EList, RepeatCoord > >& common_positions, index_t max_positions = numeric_limits::max(), index_t dist = 1000) const { common_positions.clear(); if(repID >= _repeatMap.size() || repID2 >= _repeatMap.size()) return false; // Find a repeat corresponding to a given location (left, right) const EList >& repeatMap = _repeatMap[repID]; assert_lt(left, right); pair repeat(left, numeric_limits::max()); index_t repeatIdx = repeatMap.bsearchLoBound(repeat); assert_lt(repeatIdx, repeatMap.size()); if(right > repeatMap[repeatIdx].first) return false; index_t repeatIdx_ = repeatMap[repeatIdx].second; assert_lt(repeatIdx_, _repeats.size()); const EList >& alleles = _repeats[repeatIdx_].alleles; index_t adjLeft = left, adjRight = right; if(repeatIdx > 0) { adjLeft -= repeatMap[repeatIdx-1].first; adjRight -= repeatMap[repeatIdx-1].first; } // Find a repeat cooresponding to a given location (left2, right2) const EList >& repeatMap2 = _repeatMap[repID2]; assert_lt(left2, right2); pair repeat2(left2, numeric_limits::max()); index_t repeatIdx2 = repeatMap2.bsearchLoBound(repeat2); assert_lt(repeatIdx2, repeatMap2.size()); if(right2 > repeatMap2[repeatIdx2].first) return false; index_t repeatIdx2_ = repeatMap2[repeatIdx2].second; assert_lt(repeatIdx2_, _repeats.size()); const EList >& alleles2 = _repeats[repeatIdx2_].alleles; index_t adjLeft2 = left2, adjRight2 = right2; if(repeatIdx2 > 0) { adjLeft2 -= repeatMap2[repeatIdx2-1].first; adjRight2 -= repeatMap2[repeatIdx2-1].first; } const EList >& positions = _repeats[repeatIdx_].positions; const EList >& positions2 = _repeats[repeatIdx2_].positions; index_t jsave = 0; for(index_t i = 0; i < positions.size(); i++) { const RepeatAllele& allele = alleles[positions[i].alleleID]; if(!allele.compatible(adjLeft, adjRight)) continue; index_t i_pos = positions[i].joinedOff + adjLeft; for(index_t j = jsave; j < positions2.size(); j++) { index_t j_pos = positions2[j].joinedOff + adjLeft2; if(j_pos + dist < i_pos) { jsave = j + 1; continue; } if(i_pos + dist < j_pos) break; const RepeatAllele& allele2 = alleles2[positions2[j].alleleID]; if(!allele2.compatible(adjLeft2, adjRight2)) continue; common_positions.expand(); common_positions.back().first = positions[i]; if(positions[i].fw) { common_positions.back().first.joinedOff += adjLeft; common_positions.back().first.toff += adjLeft; } else { const index_t len = right - left; assert_leq(adjLeft + len, _repeats[repeatIdx_].repLen); index_t rc_adjLeft = _repeats[repeatIdx_].repLen - adjLeft - len; common_positions.back().first.joinedOff += rc_adjLeft; common_positions.back().first.toff += rc_adjLeft; } common_positions.back().second = positions2[j]; if(positions2[j].fw) { common_positions.back().second.toff += adjLeft2; common_positions.back().second.joinedOff += adjLeft2; } else { const index_t len = right2 - left2; assert_leq(adjLeft2 + len, _repeats[repeatIdx2_].repLen); index_t rc_adjLeft2 = _repeats[repeatIdx2_].repLen - adjLeft2 - len; common_positions.back().second.joinedOff += rc_adjLeft2; common_positions.back().second.toff += rc_adjLeft2; } if(common_positions.size() >= max_positions) break; } if(common_positions.size() >= max_positions) break; } return common_positions.size() > 0; } private: pair get_alt_range(const ALTDB& altdb, index_t left, index_t right) const { pair alt_range; ALT cmp_alt; cmp_alt.pos = left; alt_range.first = alt_range.second = (index_t)altdb.alts().bsearchLoBound(cmp_alt); for(; alt_range.second < altdb.alts().size(); alt_range.second++) { const ALT& alt = altdb.alts()[alt_range.second]; if(alt.left > right) break; } return alt_range; } private: EList > _repeats; ELList > _repeatMap; // pos to repeat id }; #endif /*ifndef REPEAT_H_*/ hisat2-2.2.1/repeat_builder.cpp000066400000000000000000005000611370663763200164170ustar00rootroot00000000000000 /* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #include #include #include #include #include "timer.h" #include "aligner_sw.h" #include "aligner_result.h" #include "scoring.h" #include "sstring.h" #include "repeat_builder.h" #include "repeat_kmer.h" #include "bit_packed_array.h" unsigned int levenshtein_distance(const std::string& s1, const std::string& s2) { const std::size_t len1 = s1.size(), len2 = s2.size(); std::vector col(len2+1), prevCol(len2+1); for (unsigned int i = 0; i < prevCol.size(); i++) prevCol[i] = i; for (unsigned int i = 0; i < len1; i++) { col[0] = i+1; for (unsigned int j = 0; j < len2; j++) // note that std::min({arg1, arg2, arg3}) works only in C++11, // for C++98 use std::min(std::min(arg1, arg2), arg3) col[j+1] = std::min( std::min(prevCol[1 + j] + 1, col[j] + 1), prevCol[j] + (s1[i]==s2[j] ? 0 : 1) ); col.swap(prevCol); } return prevCol[len2]; } unsigned int hamming_distance(const string& s1, const string& s2) { assert_eq(s1.length(), s2.length()); uint32_t cnt = 0; for(size_t i = 0; i < s1.length(); i++) { if(s1[i] != s2[i]) { cnt++; } } return cnt; } string reverse(const string& str) { string rev = str; size_t str_len = str.length(); for(size_t i = 0; i < str_len; i++) { rev[i] = str[str_len - i - 1]; } return rev; } string reverse_complement(const string& str) { string rev = str; size_t str_len = str.length(); for(size_t i = 0; i < str_len; i++) { char nt = str[str_len - i - 1]; rev[i] = asc2dnacomp[nt]; } return rev; } template TIndexOffU getLCP(const TStr& s, CoordHelper& coordHelper, TIndexOffU a, TIndexOffU b, TIndexOffU max_len = 1000) { TIndexOffU a_end = coordHelper.getEnd(a); TIndexOffU b_end = coordHelper.getEnd(b); assert_leq(a_end, s.length()); assert_leq(b_end, s.length()); TIndexOffU k = 0; while((a + k) < a_end && (b + k) < b_end && k < max_len) { if(s[a + k] != s[b + k]) { break; } k++; } return k; } template bool isSameSequenceUpto(const TStr& s, CoordHelper& coordHelper, TIndexOffU a, TIndexOffU b, TIndexOffU upto) { TIndexOffU a_end = coordHelper.getEnd(a); TIndexOffU b_end = coordHelper.getEnd(b); assert_leq(a_end, s.length()); assert_leq(b_end, s.length()); if(a + upto > a_end || b + upto > b_end) return false; for(int k = upto - 1; k >= 0; k--) { if(s[a + k] != s[b + k]) { return false; } } return true; } bool isSenseDominant(CoordHelper& coordHelper, const EList& positions, size_t seed_len) { // count the number of seeds on the sense strand size_t sense_mer_count = 0; for(size_t i = 0; i < positions.size(); i++) { if(positions[i] < coordHelper.forward_length()) sense_mer_count++; } // there exists two sets of seeds that are essentially the same // due to sense and antisense strands except palindromic sequences // choose one and skip the other one assert_leq(sense_mer_count, positions.size()); size_t antisense_mer_count = positions.size() - sense_mer_count; if(sense_mer_count < antisense_mer_count) { return false; } else if(sense_mer_count == antisense_mer_count) { assert_geq(positions.back(), coordHelper.forward_length()); TIndexOffU sense_pos = coordHelper.length() - positions.back() - seed_len; if(positions[0] > sense_pos) return false; } return true; } static const Range EMPTY_RANGE = Range(1, 0); struct RepeatRange { RepeatRange() { forward = true; }; RepeatRange(Range r, int id) : range(r), rg_id(id), forward(true) {}; RepeatRange(Range r, int id, bool fw) : range(r), rg_id(id), forward(fw) {}; Range range; int rg_id; bool forward; }; Range reverseRange(const Range& r, TIndexOffU size) { size_t len = r.second - r.first; Range rc; rc.first = size - r.second; rc.second = rc.first + len; return rc; } string reverseComplement(const string& str) { string rc; for(TIndexOffU si = str.length(); si > 0; si--) { rc.push_back(asc2dnacomp[str[si - 1]]); } return rc; } string toMDZ(const EList& edits, const string& read) { StackedAln stk; BTDnaString btread; BTString buf; btread.install(read.c_str(), true); stk.init(btread, edits, 0, 0, 0, 0); stk.buildMdz(); stk.writeMdz(&buf, NULL); return string(buf.toZBuf()); } string applyEdits(const string& ref, size_t read_len, const EList& edits, const Coord& coord) { string read; size_t ref_pos = coord.off(); size_t read_pos = 0; for(size_t i = 0; i < edits.size(); i++) { for(; read_pos < edits[i].pos; read_pos++, ref_pos++) { read.push_back(ref[ref_pos]); } if(edits[i].type == EDIT_TYPE_READ_GAP) { // delete on read ref_pos++; } else if(edits[i].type == EDIT_TYPE_REF_GAP) { // insert on read read.push_back(edits[i].qchr); read_pos++; } else if(edits[i].type == EDIT_TYPE_MM) { assert_eq(ref[ref_pos], edits[i].chr); read.push_back(edits[i].qchr); read_pos++; ref_pos++; } else { assert(false); } } for(; read_pos < read_len; read_pos++, ref_pos++) { read.push_back(ref[ref_pos]); } return read; } template static bool compareRepeatCoordByJoinedOff(const RepeatCoord& a, const RepeatCoord& b) { return a.joinedOff < b.joinedOff; } template string getString(const TStr& ref, TIndexOffU start, size_t len) { ASSERT_ONLY(const size_t ref_len = ref.length()); assert_leq(start + len, ref_len); string s; for(size_t i = 0; i < len; i++) { char nt = "ACGT"[ref[start + i]]; s.push_back(nt); } return s; } template void getString(const TStr& ref, TIndexOffU start, size_t len, string& s) { s.clear(); ASSERT_ONLY(const size_t ref_len = ref.length()); assert_leq(start + len, ref_len); for(size_t i = 0; i < len; i++) { char nt = "ACGT"[ref[start + i]]; s.push_back(nt); } } template inline uint8_t getSequenceBase(const TStr& ref, TIndexOffU pos) { assert_lt(pos, ref.length()); return (uint8_t)ref[pos]; } template void dump_tstr(const TStr& s) { static int print_width = 60; size_t s_len = s.length(); for(size_t i = 0; i < s_len; i += print_width) { string buf; for(size_t j = 0; (j < print_width) && (i + j < s_len); j++) { buf.push_back("ACGTN"[s[i + j]]); } cerr << buf << endl; } cerr << endl; } size_t getMaxMatchLen(const EList& edits, const size_t read_len) { size_t max_length = 0; uint32_t last_edit_pos = 0; uint32_t len = 0; if (edits.size() == 0) { // no edits --> exact match return read_len; } for(size_t i = 0; i < edits.size(); i++) { if (last_edit_pos > edits[i].pos) { continue; } len = edits[i].pos - last_edit_pos; if(len > max_length) { max_length = len; } last_edit_pos = edits[i].pos + 1; } if (last_edit_pos < read_len) { len = read_len - last_edit_pos; if(len > max_length) { max_length = len; } } return max_length; } int max_index(size_t array[4]) { int max_idx = 0; for(size_t i = 1; i < 4; i++) { if(array[max_idx] < array[i]) { max_idx = i; } } return max_idx; } CoordHelper::CoordHelper(TIndexOffU length, TIndexOffU forward_length, const EList& szs, const EList& ref_names) : length_(length), forward_length_(forward_length), szs_(szs), ref_namelines_(ref_names) { // build ref_names_ from ref_namelines_ buildNames(); buildJoinedFragment(); } CoordHelper::~CoordHelper() { } void CoordHelper::buildNames() { ref_names_.resize(ref_namelines_.size()); for(size_t i = 0; i < ref_namelines_.size(); i++) { string& nameline = ref_namelines_[i]; for(size_t j = 0; j < nameline.length(); j++) { char n = nameline[j]; if(n == ' ') { break; } ref_names_[i].push_back(n); } } } int CoordHelper::mapJoinedOffToSeq(TIndexOffU joinedOff) { /* search from cached_list */ if(num_cached_ > 0) { for(size_t i = 0; i < num_cached_; i++) { Fragments *frag = &cached_[i]; if(frag->contain(joinedOff)) { return frag->frag_id; } } /* fall through */ } /* search list */ int top = 0; int bot = fraglist_.size() - 1; int pos = 0; Fragments *frag = &fraglist_[pos]; while((bot - top) > 1) { pos = top + ((bot - top) >> 1); frag = &fraglist_[pos]; if (joinedOff < frag->joinedOff) { bot = pos; } else { top = pos; } } frag = &fraglist_[top]; if (frag->contain(joinedOff)) { // update cache if (num_cached_ < CACHE_SIZE_JOINEDFRG) { cached_[num_cached_] = *frag; num_cached_++; } else { cached_[victim_] = *frag; victim_++; // round-robin victim_ %= CACHE_SIZE_JOINEDFRG; } return top; } return -1; } int CoordHelper::getGenomeCoord(TIndexOffU joinedOff, string& chr_name, TIndexOffU& pos_in_chr) { int seq_id = mapJoinedOffToSeq(joinedOff); if (seq_id < 0) { return -1; } Fragments *frag = &fraglist_[seq_id]; TIndexOffU offset = joinedOff - frag->joinedOff; pos_in_chr = frag->seqOff + offset; chr_name = ref_names_[frag->seq_id]; return 0; } void CoordHelper::buildJoinedFragment() { TIndexOffU acc_joinedOff = 0; TIndexOffU acc_seqOff = 0; TIndexOffU seq_id = 0; TIndexOffU frag_id = 0; for(size_t i = 0; i < szs_.size(); i++) { const RefRecord& rec = szs_[i]; if(rec.len == 0) { continue; } if (rec.first) { acc_seqOff = 0; seq_id++; } fraglist_.expand(); fraglist_.back().joinedOff = acc_joinedOff; fraglist_.back().length = rec.len; fraglist_.back().frag_id = frag_id++; fraglist_.back().seq_id = seq_id - 1; fraglist_.back().first = rec.first; acc_seqOff += rec.off; fraglist_.back().seqOff = acc_seqOff; acc_joinedOff += rec.len; acc_seqOff += rec.len; } // Add Last Fragment(empty) fraglist_.expand(); fraglist_.back().joinedOff = acc_joinedOff; fraglist_.back().length = 0; fraglist_.back().seqOff = acc_seqOff; fraglist_.back().first = false; fraglist_.back().frag_id = frag_id; fraglist_.back().seq_id = seq_id; } TIndexOffU CoordHelper::getEnd(TIndexOffU e) { assert_lt(e, length_) TIndexOffU end = 0; if(e < forward_length_) { int frag_id = mapJoinedOffToSeq(e); assert_geq(frag_id, 0); end = fraglist_[frag_id].joinedOff + fraglist_[frag_id].length; } else { // ReverseComplement // a, b are positions w.r.t reverse complement string. // fragment map is based on forward string int frag_id = mapJoinedOffToSeq(length_ - e - 1); assert_geq(frag_id, 0); end = length_ - fraglist_[frag_id].joinedOff; } assert_leq(end, length_); return end; } TIndexOffU CoordHelper::getStart(TIndexOffU e) { assert_lt(e, length_) TIndexOffU start = 0; if(e < forward_length_) { int frag_id = mapJoinedOffToSeq(e); assert_geq(frag_id, 0); start = fraglist_[frag_id].joinedOff; } else { // ReverseComplement // a, b are positions w.r.t reverse complement string. // fragment map is based on forward string int frag_id = mapJoinedOffToSeq(length_ - e - 1); assert_geq(frag_id, 0); start = length_ - (fraglist_[frag_id].joinedOff + fraglist_[frag_id].length); } assert_leq(start, length_); return start; } template void SeedExt::getExtendedSeedSequence(const TStr& s, string& seq) const { seq.clear(); TIndexOffU prev_end = orig_pos.first; for(size_t j = 0; j < left_gaps.size(); j++) { TIndexOffU curr_end = orig_pos.first - left_gaps[j].first; assert_leq(curr_end, prev_end); if(curr_end < prev_end) { seq = getString(s, curr_end, prev_end - curr_end) + seq; } int gap_len = left_gaps[j].second; assert_neq(gap_len, 0); if(gap_len > 0) { // deletion string gap_str(gap_len, '-'); seq = gap_str + seq; } else { curr_end += gap_len; } prev_end = curr_end; } assert_leq(pos.first, prev_end); if(pos.first < prev_end) { seq = getString(s, pos.first, prev_end - pos.first) + seq; } if(orig_pos.second - orig_pos.first > 0) seq += getString(s, orig_pos.first, orig_pos.second - orig_pos.first); TIndexOffU prev_begin = orig_pos.second; for(size_t j = 0; j < right_gaps.size(); j++) { TIndexOffU curr_begin = orig_pos.second + right_gaps[j].first; assert_leq(prev_begin, curr_begin); if(prev_begin < curr_begin) { seq += getString(s, prev_begin, curr_begin - prev_begin); } int gap_len = right_gaps[j].second; assert_neq(gap_len, 0); if(gap_len > 0) { // deletion string gap_str(gap_len, '-'); seq += gap_str; } else { curr_begin -= gap_len; } prev_begin = curr_begin; } assert_leq(prev_begin, pos.second); if(prev_begin < pos.second) { seq += getString(s, prev_begin, pos.second - prev_begin); } } SeedSNP *lookup_add_SNP(EList& repeat_snps, SeedSNP& snp) { for(size_t i = 0; i < repeat_snps.size(); i++) { if(*repeat_snps[i] == snp) { return repeat_snps[i]; } } repeat_snps.expand(); repeat_snps.back() = new SeedSNP(); *(repeat_snps.back()) = snp; return repeat_snps.back(); } template void SeedExt::generateSNPs(const TStr &s, const string& consensus, EList& repeat_snps) { EList > gaps; // Merge Gaps { TIndexOffU left_ext_len = getLeftExtLength(); for(size_t g = 0; g < left_gaps.size(); g++) { gaps.expand(); gaps.back().first = left_ext_len - left_gaps[g].first + left_gaps[g].second; gaps.back().second = left_gaps[g].second; } TIndexOffU right_base = orig_pos.second - pos.first; for(size_t g = 0; g < right_gaps.size(); g++) { gaps.expand(); gaps.back().first = right_base + right_gaps[g].first; gaps.back().second = right_gaps[g].second; } gaps.sort(); } // string con_ref; string seq_read; TIndexOffU prev_con_pos = consensus_pos.first; TIndexOffU prev_seq_pos = pos.first; for(size_t g = 0; g < gaps.size(); g++) { TIndexOffU curr_seq_pos = pos.first + gaps[g].first; TIndexOffU curr_con_pos = prev_con_pos + (curr_seq_pos - prev_seq_pos); con_ref = consensus.substr(prev_con_pos, curr_con_pos - prev_con_pos); seq_read = getString(s, prev_seq_pos, curr_seq_pos - prev_seq_pos); for(size_t l = 0; l < con_ref.length(); l++) { if(seq_read[l] != con_ref[l]) { // Single SeedSNP snp; snp.init(EDIT_TYPE_MM, prev_con_pos + l, 1, seq_read[l]); // lookup repeat_snp snps.expand(); snps.back() = lookup_add_SNP(repeat_snps, snp); } } int gap_len = gaps[g].second; assert_neq(gap_len, 0); if(gap_len > 0) { // Deletion (gap on read) string gap_str(gap_len, '-'); SeedSNP snp; snp.init(EDIT_TYPE_READ_GAP, curr_con_pos, gap_len, consensus.substr(curr_con_pos, gap_len)); snps.expand(); snps.back() = lookup_add_SNP(repeat_snps, snp); curr_con_pos += gap_len; } else { // Insertion (gap on reference) string gap_str(abs(gap_len), '-'); SeedSNP snp; snp.init(EDIT_TYPE_REF_GAP, curr_con_pos, abs(gap_len), getString(s, curr_seq_pos, abs(gap_len))); snps.expand(); snps.back() = lookup_add_SNP(repeat_snps, snp); curr_seq_pos += abs(gap_len); } prev_con_pos = curr_con_pos; prev_seq_pos = curr_seq_pos; } assert_eq(consensus_pos.second - prev_con_pos, pos.second - prev_seq_pos); con_ref = consensus.substr(prev_con_pos, consensus_pos.second - prev_con_pos); seq_read = getString(s, prev_seq_pos, pos.second - prev_seq_pos); for(size_t l = 0; l < con_ref.length(); l++) { if(seq_read[l] != con_ref[l]) { // Single SeedSNP snp; snp.init(EDIT_TYPE_MM, prev_con_pos + l, 1, seq_read[l]); snps.expand(); snps.back() = lookup_add_SNP(repeat_snps, snp); } } } bool seedCmp(const SeedExt& s, const SeedExt& s2) { if(s.getLength() != s2.getLength()) return s.getLength() > s2.getLength(); if(s.pos.first != s2.pos.first) return s.pos.first < s2.pos.first; if(s.pos.second != s2.pos.second) return s.pos.second < s2.pos.second; return false; } template void RB_Repeat::init(const RepeatParameter& rp, const TStr& s, CoordHelper& coordHelper, const RB_SubSA& subSA, const RB_RepeatBase& repeatBase) { consensus_ = repeatBase.seq; assert_geq(consensus_.length(), rp.min_repeat_len); assert_eq(consensus_.length(), rp.min_repeat_len + repeatBase.nodes.size() - 1); EList positions, next_positions; const EList& repeat_index = subSA.getRepeatIndex(); seeds_.clear(); for(size_t n = 0; n < repeatBase.nodes.size(); n++) { TIndexOffU idx = repeatBase.nodes[n]; TIndexOffU saBegin = repeat_index[idx]; TIndexOffU saEnd = (idx + 1 < repeat_index.size() ? repeat_index[idx+1] : subSA.size()); next_positions.clear(); for(size_t sa = saBegin; sa < saEnd; sa++) { TIndexOffU left = subSA.get(sa); TIndexOffU right = left + subSA.seed_len(); next_positions.push_back(left); if(left > 0) { size_t idx = positions.bsearchLoBound(left - 1); if(idx < positions.size() && positions[idx] == left - 1) continue; } seeds_.expand(); SeedExt& seed = seeds_.back(); seed.reset(); seed.orig_pos = pair(left, right); seed.pos = seed.orig_pos; seed.consensus_pos.first = n; seed.consensus_pos.second = n + rp.min_repeat_len; seed.bound = pair(coordHelper.getStart(left), coordHelper.getEnd(left)); #ifndef NDEBUG string tmp_str = getString(s, seed.pos.first, seed.pos.second - seed.pos.first); assert_eq(tmp_str, consensus_.substr(n, seed.pos.second - seed.pos.first)); #endif for(size_t p = seed.consensus_pos.second; p < consensus_.length(); p++) { size_t pos = seed.pos.second; if(pos >= seed.bound.second) break; int nt = getSequenceBase(s, pos); if("ACGT"[nt] != consensus_[p]) break; seed.pos.second++; seed.consensus_pos.second++; } } positions = next_positions; } internal_update(); } template void RB_Repeat::extendConsensus(const RepeatParameter& rp, const TStr& s) { size_t remain = seeds_.size(); EList left_consensuses, right_consensuses, empty_consensuses; EList ed_seed_nums; const TIndexOffU default_max_ext_len = (rp.max_repeat_len - rp.seed_len) / 2; const TIndexOffU seed_mm = 1; string left_ext_consensus = "", right_ext_consensus = ""; empty_consensuses.resize(seed_mm + 1); empty_consensuses.fill(""); while(remain >= rp.repeat_count) { for(size_t i = 0; i < remain; i++) { seeds_[i].done = false; seeds_[i].curr_ext_len = 0; } // extend seeds in left or right direction TIndexOffU max_ext_len = min(default_max_ext_len, (TIndexOffU)(rp.max_repeat_len - consensus_.length())); get_consensus_seq(s, seeds_, 0, // seed begin remain, // seed end max_ext_len, max_ext_len, seed_mm, rp, ed_seed_nums, &left_consensuses, &right_consensuses); size_t allowed_seed_mm = 0; left_ext_consensus.clear(); right_ext_consensus.clear(); for(int i = 0 /* (int)seed_mm */; i >= 0; i--) { size_t extlen = (ed_seed_nums[i] < rp.repeat_count ? 0 : (left_consensuses[i].length() + right_consensuses[i].length())); // if(extlen <= 0 || extlen < max_ext_len * i / seed_mm) //if(extlen < max_ext_len * 2) // continue; if(extlen <= 0) continue; left_ext_consensus = left_consensuses[i]; right_ext_consensus = right_consensuses[i]; allowed_seed_mm = (size_t)i; assert_gt(left_ext_consensus.length(), 0); assert_gt(right_ext_consensus.length(), 0); break; } size_t num_passed_seeds = 0; if(left_ext_consensus.length() > 0 && right_ext_consensus.length()) { consensus_ = reverse(left_ext_consensus) + consensus_; consensus_ += right_ext_consensus; #if 0 calc_edit_dist(s, seeds_, 0, remain, left ? consensuses : empty_consensuses, left ? empty_consensuses : consensuses, allowed_seed_mm); #endif // update seeds for(size_t i = 0; i < seeds_.size(); i++) { SeedExt& seed = seeds_[i]; if(i < remain) { if(seed.ed <= allowed_seed_mm) { num_passed_seeds++; seed.done = true; seed.total_ed += seed.ed; seed.pos.first -= left_ext_consensus.length(); seed.pos.second += right_ext_consensus.length(); seed.consensus_pos.first = 0; seed.consensus_pos.second = consensus_.length(); if(seed.left_gaps.size() > 0 && seed.left_gaps.back().first >= seed.orig_pos.first - seed.pos.first) { int gap_len = seed.left_gaps.back().second; seed.pos.first += gap_len; } if(seed.right_gaps.size() > 0 && seed.right_gaps.back().first >= seed.pos.second - seed.orig_pos.second) { int gap_len = seed.right_gaps.back().second; seed.pos.second -= gap_len; } } } } // move up "done" seeds size_t j = 0; for(size_t i = 0; i < remain; i++) { if(!seeds_[i].done) continue; assert_geq(i, j); if(i > j) { SeedExt temp = seeds_[j]; seeds_[j] = seeds_[i]; seeds_[i] = temp; // Find next "undone" seed j++; while(j < i && seeds_[j].done) { j++; } assert(j < remain && !seeds_[j].done); } else { j = i + 1; } } } remain = num_passed_seeds; break; } // while(remain >= rp.repeat_count) #ifndef NDEBUG // make sure seed positions are unique EList > seed_poses; for(size_t i = 0; i < seeds_.size(); i++) { seed_poses.expand(); seed_poses.back() = seeds_[i].orig_pos; } seed_poses.sort(); for(size_t i = 0; i + 1 < seed_poses.size(); i++) { if(seed_poses[i].first == seed_poses[i+1].first) { assert_lt(seed_poses[i].second, seed_poses[i+1].second); } else { assert_lt(seed_poses[i].first, seed_poses[i+1].first); } } for(size_t i = 0; i < seeds_.size(); i++) { assert(seeds_[i].valid()); } #endif RB_Repeat::internal_update(); // check repeats within a repeat sequence self_repeat_ = false; for(size_t i = 0; i + 1 < seed_ranges_.size(); i++) { const RB_AlleleCoord& range = seed_ranges_[i]; for(size_t j = i + 1; j < seed_ranges_.size(); j++) { RB_AlleleCoord& range2 = seed_ranges_[j]; if(range.right <= range2.left) break; self_repeat_ = true; } } } template void RB_Repeat::getNextRepeat(const RepeatParameter& rp, const TStr& s, RB_Repeat& o) { o.reset(); size_t i = 0; for(i = 0; i < seeds_.size() && seeds_[i].done; i++); if(i >= seeds_.size()) return; for(size_t j = i; j < seeds_.size(); j++) { assert(!seeds_[j].done); o.seeds_.expand(); o.seeds_.back() = seeds_[j]; } seeds_.resizeExact(i); internal_update(); assert_gt(o.seeds_.size(), 0); o.consensus_ = getString(s, o.seeds_[0].orig_pos.first, o.seeds_[0].orig_pos.second - o.seeds_[0].orig_pos.first); for(size_t j = 0; j < o.seeds_.size(); j++) { o.seeds_[j].pos = o.seeds_[j].orig_pos; o.seeds_[j].left_gaps.clear(); o.seeds_[j].right_gaps.clear(); o.seeds_[j].ed = o.seeds_[j].total_ed = 0; } } void RB_Repeat::internal_update() { sort(seeds_.begin(), seeds_.end(), seedCmp); seed_ranges_.resizeExact(seeds_.size()); for(size_t i = 0; i < seeds_.size(); i++) { seed_ranges_[i].left = seeds_[i].pos.first; seed_ranges_[i].right = seeds_[i].pos.second; seed_ranges_[i].idx = i; } seed_ranges_.sort(); // check repeats within a repeat sequence size_t remove_count = 0; for(size_t i = 0; i + 1 < seed_ranges_.size(); i++) { const RB_AlleleCoord& range = seed_ranges_[i]; if(range.left == numeric_limits::max()) continue; for(size_t j = i + 1; j < seed_ranges_.size(); j++) { RB_AlleleCoord& range2 = seed_ranges_[j]; if(range2.left == numeric_limits::max()) continue; if(range.right <= range2.left) break; if(range.right >= range2.right) { range2.left = numeric_limits::max(); remove_count++; } } } if(remove_count <= 0) return; for(size_t i = 0; i < seed_ranges_.size(); i++) { if(seed_ranges_[i].left == numeric_limits::max()) seeds_[seed_ranges_[i].idx].reset(); } sort(seeds_.begin(), seeds_.end(), seedCmp); #ifndef NDEBUG for(size_t i = 0; i < seeds_.size(); i++) { if(i < seeds_.size() - remove_count) { assert_lt(seeds_[i].pos.first, seeds_[i].pos.second); } else { assert_eq(seeds_[i].pos.first, seeds_[i].pos.second); } } #endif seeds_.resize(seeds_.size() - remove_count); seed_ranges_.resize(seeds_.size()); for(size_t i = 0; i < seeds_.size(); i++) { seed_ranges_[i].left = seeds_[i].pos.first; seed_ranges_[i].right = seeds_[i].pos.second; seed_ranges_[i].idx = i; } seed_ranges_.sort(); } bool RB_Repeat::contain(TIndexOffU left, TIndexOffU right) const { size_t l = 0, r = seed_ranges_.size(); while(l < r) { size_t m = (l + r) / 2; const RB_AlleleCoord& coord = seed_ranges_[m]; if(right <= coord.left) { r = m; } else if(left >= coord.right) { l = m + 1; } else { return coord.left <= left && right <= coord.right; } } return false; } template void RB_Repeat::saveSeedExtension(const RepeatParameter& rp, const TStr& s, CoordHelper& coordHelper, TIndexOffU grp_id, ostream& fp, size_t& total_repeat_seq_len, size_t& total_allele_seq_len) const { // apply color, which is compatible with linux commands such as cat and less -r #if 1 const string red = "\033[31m", reset = "\033[0m", resetline = "\033[4m", redline = "\033[4;31m"; #else const string red = "", reset = "", resetline = "", redline = ""; #endif bool show_exterior_seq = true; const size_t max_show_seq_len = 700; size_t total_count = 0; for(size_t i = 0; i < seeds_.size(); i++) { const SeedExt& seed = seeds_[i]; size_t ext_len = seed.getLength(); if(ext_len < rp.min_repeat_len) continue; total_allele_seq_len += ext_len; total_count++; bool sense_strand = seed.pos.first < coordHelper.forward_length(); fp << setw(6) << repeat_id_ << " " << setw(5) << seeds_.size(); fp << " " << setw(4) << i; fp << " " << setw(4) << ext_len; fp << " " << (sense_strand ? '+' : '-'); fp << " " << setw(10) << seed.pos.first << " " << setw(10) << seed.pos.second; fp << " " << setw(10) << seed.orig_pos.first << " " << setw(10) << seed.orig_pos.second; string chr_name; TIndexOffU pos_in_chr; if(sense_strand) { coordHelper.getGenomeCoord(seed.pos.first, chr_name, pos_in_chr); } else { coordHelper.getGenomeCoord(s.length() - seed.pos.first - (seed.pos.second - seed.pos.first), chr_name, pos_in_chr); } fp << " " << setw(5) << chr_name << ":" << setw(10) << std::left << pos_in_chr << std::right; if(sense_strand) { coordHelper.getGenomeCoord(seed.pos.second, chr_name, pos_in_chr); } else { coordHelper.getGenomeCoord(s.length() - seed.pos.second - (seed.pos.second - seed.pos.first), chr_name, pos_in_chr); } fp << " " << setw(5) << chr_name << ":" << setw(10) << std::left << pos_in_chr << std::right; if(!seed.aligned) { fp << endl; continue; } string deststr = ""; seed.getExtendedSeedSequence(s, deststr); // add exterior sequences if(seed.consensus_pos.first > 0) { TIndexOffU seq_pos, seq_len; if(seed.pos.first >= seed.consensus_pos.first) { seq_pos = seed.pos.first - seed.consensus_pos.first; seq_len = seed.consensus_pos.first; } else { seq_pos = 0; seq_len = seed.pos.first; } deststr = getString(s, seq_pos, seq_len) + deststr; if(seq_len < seed.consensus_pos.first) { deststr = string(seed.consensus_pos.first - seq_len, 'N') + deststr; } } if(seed.consensus_pos.second < consensus_.length()) { deststr += getString(s, seed.pos.second, consensus_.length() - seed.consensus_pos.second); } assert_eq(consensus_.length(), deststr.length()); fp << " "; // print sequence w.r.t. the current group for(size_t j = 0; j < min(consensus_.length(), max_show_seq_len); j++) { bool outside = j < seed.consensus_pos.first || j >= seed.consensus_pos.second; bool different = (consensus_[j] != deststr[j]); if(outside) { if(show_exterior_seq) { if(different) { fp << redline; fp << deststr[j]; fp << reset; } else { fp << resetline; fp << deststr[j]; fp << reset; } } else { if(j < seed.consensus_pos.first) fp << " "; } } else { if(different) fp << red; fp << deststr[j]; if(different) fp << reset; } } #if 0 fp << "\t"; for(size_t ei = 0; ei < seed.edits.size(); ei++) { const Edit& edit = seed.edits[ei]; if(ei > 0) fp << ","; fp << edit; if (edit.snpID != std::numeric_limits::max()) { fp << "@" << edit.snpID; } } #endif fp << endl; } if(total_count > 0) fp << setw(5) << total_count << endl << endl; total_repeat_seq_len += consensus_.length(); } template size_t calc_edit_dist(const TStr& s, const SeedExt& seed, const SeedExt& seed2, size_t left_ext, size_t right_ext, size_t max_ed) { if(seed.bound.first + left_ext > seed.pos.first || seed.pos.second + right_ext > seed.bound.second || seed2.bound.first + left_ext > seed2.pos.first || seed2.pos.second + right_ext > seed2.bound.second) return max_ed + 1; size_t ed = 0; for(size_t i = 0; i < left_ext; i++) { int ch = getSequenceBase(s, seed.pos.first - i - 1); assert_range(0, 3, ch); int ch2 = getSequenceBase(s, seed2.pos.first - i - 1); assert_range(0, 3, ch2); if(ch != ch2) ed++; if(ed > max_ed) return ed; } for(size_t i = 0; i < right_ext; i++) { int ch = getSequenceBase(s, seed.pos.second + i); assert_range(0, 3, ch); int ch2 = getSequenceBase(s, seed2.pos.second + i); assert_range(0, 3, ch2); if(ch != ch2) ed++; if(ed > max_ed) return ed; } return ed; } size_t extract_kmer(const string& seq, size_t offset, size_t k = 5) { assert_leq(offset + k, seq.length()); size_t kmer = 0; for(size_t i = 0; i < k; i++) { kmer = (kmer << 2) | asc2dna[seq[offset + i]]; } return kmer; } size_t next_kmer(size_t kmer, char base, size_t k = 5) { kmer &= ((1 << ((k-1)*2))) - 1; kmer = (kmer << 2) | asc2dna[base]; return kmer; } void build_kmer_table(const string& consensus, EList >& kmer_table, size_t k = 5) { kmer_table.clear(); if(consensus.length() < k) return; size_t kmer = 0; for(size_t i = 0; i + k <= consensus.length(); i++) { if(i == 0) { kmer = extract_kmer(consensus, i, k); } else { kmer = next_kmer(kmer, consensus[i+k-1], k); } kmer_table.expand(); kmer_table.back().first = kmer; kmer_table.back().second = i; } kmer_table.sort(); } void find_gap_pos(const string& s, const string& s2, EList& ed, EList& ed2, bool del, size_t gap_len, size_t max_mm, size_t& gap_pos, size_t& mm, bool debug = false) { assert_eq(s.length(), s2.length()); size_t seq_len = s.length(); ed.resizeExact(seq_len); ed.fill(max_mm + 1); ed2.resizeExact(seq_len); ed2.fill(max_mm + 1); // from left to right for(size_t i = 0; i < seq_len; i++) { size_t add = (s[i] == s2[i] ? 0 : 1); if(i == 0) ed[i] = add; else ed[i] = ed[i-1] + add; if(ed[i] >= max_mm + 1) break; } // from right to left size_t s_sub = del ? 0 : gap_len; size_t s2_sub = del ? gap_len : 0; for(int i = seq_len - 1; i >= gap_len; i--) { size_t add = (s[i - s_sub] == s2[i - s2_sub] ? 0 : 1); if(i == seq_len - 1) ed2[i] = add; else ed2[i] = ed2[i+1] + add; if(ed2[i] > max_mm) break; } if(debug) { cout << s << endl << s2 << endl; for(size_t i = 0; i < ed.size(); i++) { cout << (ed[i] % 10); } cout << endl; for(size_t i = 0; i < ed2.size(); i++) { cout << (ed2[i] % 10); } cout << endl; } size_t min_mm = ed2[gap_len]; int min_mm_i = -1; assert_eq(ed.size(), ed2.size()); for(size_t i = 0; i + gap_len + 1 < ed.size(); i++) { if(ed[i] > max_mm) break; size_t cur_mm = ed[i] + ed2[i + gap_len + 1]; if(cur_mm < min_mm) { min_mm = cur_mm; min_mm_i = i; } } mm = min_mm; if(mm > max_mm) return; gap_pos = (size_t)(min_mm_i + 1); } void align_with_one_gap(const string& s, const EList >& s_kmer_table, const string& s2, EList& counts, size_t max_gap, size_t max_mm, size_t& mm, size_t& gap_pos, int& gap_len, size_t k = 5) { mm = max_mm + 1; assert_eq(s.length(), s2.length()); counts.resizeExact(max_gap * 2 + 1); counts.fillZero(); size_t max_count = 0, max_count_i = 0; for(size_t i = 0; i + k <= s2.length(); i += 1) { pair kmer(0, 0); kmer.first = extract_kmer(s2, i, k); size_t lb = s_kmer_table.bsearchLoBound(kmer); while(lb < s_kmer_table.size() && kmer.first == s_kmer_table[lb].first) { int gap = (int)s_kmer_table[lb].second - (int)i; if(gap != 0 && abs(gap) < max_gap) { size_t gap_i = (size_t)(gap + max_gap); counts[gap_i]++; if(counts[gap_i] > max_count) { max_count = counts[gap_i]; max_count_i = gap_i; } } lb++; } } if(max_count <= 0) return; assert_lt(max_count_i, counts.size()); int gap = (int)max_count_i - (int)max_gap; assert_neq(gap, 0); size_t abs_gap = abs(gap); bool del = gap > 0; EList ed, ed2; find_gap_pos(s, s2, ed, ed2, del, abs_gap, max_mm, gap_pos, mm); if(mm > max_mm) return; gap_len = del ? abs_gap : -abs_gap; #ifndef NDEBUG assert_leq(mm, max_mm); string ds = s, ds2 = s2; size_t debug_mm = 0; if(del) ds.erase(gap_pos, abs_gap); else ds2.erase(gap_pos, abs_gap); for(size_t i = 0; i < min(ds.length(), ds2.length()); i++) { if(ds[i] != ds2[i]) debug_mm++; } assert_eq(debug_mm, mm); #endif } template void calc_edit_dist(const TStr& s, EList& seeds, size_t sb, size_t se, const EList& left_consensuses, const EList& right_consensuses, uint32_t max_ed) { string left_consensus = left_consensuses[max_ed]; string right_consensus = right_consensuses[max_ed]; EList > left_kmer_table, right_kmer_table; build_kmer_table(left_consensus, left_kmer_table); build_kmer_table(right_consensus, right_kmer_table); string left_seq, right_seq; const size_t max_gap = 10; EList counts; size_t left_ext = left_consensus.length(); size_t right_ext = right_consensus.length(); for(size_t i = sb; i < se; i++) { SeedExt& seed = seeds[i]; if(seed.bound.first + left_ext > seed.pos.first || seed.pos.second + right_ext > seed.bound.second) { seed.ed = max_ed + 1; continue; } size_t left_ed = 0; if(left_ext > 0) { getString(s, seed.pos.first - left_ext, left_ext, left_seq); reverse(left_seq.begin(), left_seq.end()); for(size_t j = 0; j < left_ext; j++) { if(left_seq[j] != left_consensus[j]) left_ed++; if(left_ed <= max_ed && j < left_consensuses[left_ed].length()) { seed.curr_ext_len = j + 1; } } if(left_ed > max_ed) { size_t gap_pos = 0; int gap_len = 0; align_with_one_gap(left_consensus, left_kmer_table, left_seq, counts, min(left_ed - max_ed, max_gap), max_ed, left_ed, gap_pos, gap_len); if(left_ed <= max_ed) { seed.left_gaps.expand(); seed.left_gaps.back().first = seed.getLeftExtLength() + gap_pos; seed.left_gaps.back().second = gap_len; } } } else { left_seq.clear(); } size_t right_ed = 0; if(right_ext > 0) { getString(s, seed.pos.second, right_ext, right_seq); for(size_t j = 0; j < right_ext; j++) { if(right_seq[j] != right_consensus[j]) right_ed++; if(right_ed <= max_ed && j < right_consensuses[right_ed].length()) { seed.curr_ext_len = j + 1; } } if(right_ed > max_ed) { size_t gap_pos = 0; int gap_len = 0; align_with_one_gap(right_consensus, right_kmer_table, right_seq, counts, min(right_ed - max_ed, max_gap), max_ed, right_ed, gap_pos, gap_len); if(right_ed <= max_ed) { seed.right_gaps.expand(); seed.right_gaps.back().first = seed.getRightExtLength() + gap_pos; seed.right_gaps.back().second = gap_len; } } } else { right_seq.clear(); } seed.ed = left_ed + right_ed; } } template void RB_Repeat::get_consensus_seq(const TStr& s, EList& seeds, size_t sb, size_t se, size_t min_left_ext, size_t min_right_ext, size_t max_ed, const RepeatParameter& rp, EList& ed_seed_nums, EList* left_consensuses, EList* right_consensuses) const { assert_lt(sb, se); assert_geq(se - sb, rp.seed_count); assert_leq(se, seeds.size()); if(left_consensuses != NULL) { left_consensuses->clear(); left_consensuses->resize(max_ed + 1); for(size_t i = 0; i < max_ed + 1; i++) { (*left_consensuses)[i].clear(); } } if(right_consensuses != NULL) { right_consensuses->clear(); right_consensuses->resize(max_ed + 1); for(size_t i = 0; i < max_ed + 1; i++) { (*right_consensuses)[i].clear(); } } assert_eq(min_left_ext, min_right_ext); EList seqs; seqs.reserveExact(seeds.size()); size_t max_i = 0, max_count = 0; while(min_left_ext > 0) { seqs.clear(); max_i = 0; max_count = 0; for(size_t i = sb; i < se; i++) { const SeedExt& seed = seeds[i]; if(seeds[i].bound.first + min_left_ext > seed.pos.first || seed.pos.second + min_right_ext > seed.bound.second) continue; seqs.expand(); if(min_left_ext > 0) { getString(s, seed.pos.first - min_left_ext, min_left_ext, seqs.back()); } if(min_right_ext > 0) { seqs.back() += getString(s, seed.pos.second, min_right_ext); } } seqs.sort(); for(size_t i = 0; i + max_count < seqs.size();) { size_t count = 1; for(size_t j = i + 1; j < seqs.size(); j++) { if(seqs[i] == seqs[j]) count++; else break; } if(count >= max_count) { max_count = count; max_i = i; } i = i + count; } if(max_count >= rp.seed_count) break; min_left_ext--; min_right_ext--; } // update ed // extend consensus string ed_seed_nums.resize(max_ed + 1); ed_seed_nums.fillZero(); EList next_ed_seed_nums; next_ed_seed_nums.resize(max_ed + 1); if(max_count < rp.seed_count) return; for(size_t i = sb; i < se; i++) seeds[i].ed = 0; size_t seed_ext_len = 0; while(seed_ext_len < max(min_left_ext, min_right_ext)) { // select base to be used for extension uint8_t left_ext_base = 0; if(seed_ext_len < min_left_ext) left_ext_base = seqs[max_i][min_left_ext - seed_ext_len - 1]; uint8_t right_ext_base = 0; if(seed_ext_len < min_right_ext) right_ext_base = seqs[max_i][min_left_ext + seed_ext_len]; // estimate extended ed next_ed_seed_nums.fillZero(); for(size_t i = sb; i < se; i++) { uint32_t est_ed = seeds[i].ed; if(seed_ext_len < min_left_ext) { if(seeds[i].bound.first + seed_ext_len + 1 <= seeds[i].pos.first) { TIndexOffU left_pos = seeds[i].pos.first - seed_ext_len - 1; int ch = getSequenceBase(s, left_pos); assert_range(0, 3, ch); if ("ACGT"[ch] != left_ext_base) { est_ed++; } } else { est_ed = max_ed + 1; } } if(seed_ext_len < min_right_ext) { TIndexOffU right_pos = seeds[i].pos.second + seed_ext_len; if(right_pos >= seeds[i].bound.second) { est_ed = max_ed + 1; } else { int ch = getSequenceBase(s, right_pos); assert_range(0, 3, ch); if ("ACGT"[ch] != right_ext_base) { est_ed++; } } } if(est_ed <= max_ed) { next_ed_seed_nums[est_ed]++; } } for(size_t i = 1; i < next_ed_seed_nums.size(); i++) next_ed_seed_nums[i] += next_ed_seed_nums[i-1]; if(next_ed_seed_nums[max_ed] < rp.repeat_count) { break; } for(size_t i = sb; i < se; i++) { if(seed_ext_len < min_left_ext) { if(seeds[i].bound.first + seed_ext_len + 1 <= seeds[i].pos.first) { TIndexOffU left_pos = seeds[i].pos.first - seed_ext_len - 1; int ch = getSequenceBase(s, left_pos); assert_range(0, 3, ch); if ("ACGT"[ch] != left_ext_base) { seeds[i].ed++; } } else { seeds[i].ed = max_ed + 1; } } if(seed_ext_len < min_right_ext) { TIndexOffU right_pos = seeds[i].pos.second + seed_ext_len; if(right_pos >= seeds[i].bound.second) { seeds[i].ed = max_ed + 1; } else { int ch = getSequenceBase(s, right_pos); assert_range(0, 3, ch); if ("ACGT"[ch] != right_ext_base) { seeds[i].ed++; } } } } for(size_t i = 0; i < next_ed_seed_nums.size(); i++) { if(next_ed_seed_nums[i] < rp.repeat_count) continue; ed_seed_nums[i] = next_ed_seed_nums[i]; if(seed_ext_len < min_left_ext) { assert(left_consensuses != NULL); (*left_consensuses)[i] += left_ext_base; } if(seed_ext_len < min_right_ext) { assert(right_consensuses != NULL); (*right_consensuses)[i] += right_ext_base; } } seed_ext_len++; } } template void RB_RepeatExt::get_consensus_seq(const TStr& s, EList& seeds, size_t sb, size_t se, size_t min_left_ext, size_t min_right_ext, size_t max_ed, const RepeatParameter& rp, EList& ed_seed_nums, EList* left_consensuses, EList* right_consensuses) const { assert_lt(sb, se); assert_leq(se, seeds.size()); if(left_consensuses != NULL) { left_consensuses->clear(); left_consensuses->resize(max_ed + 1); for(size_t i = 0; i < max_ed + 1; i++) { (*left_consensuses)[i].clear(); } } if(right_consensuses != NULL) { right_consensuses->clear(); right_consensuses->resize(max_ed + 1); for(size_t i = 0; i < max_ed + 1; i++) { (*right_consensuses)[i].clear(); } } // cluster sequences EList belongto; belongto.resizeExact(se - sb); for(size_t i = 0; i < belongto.size(); i++) belongto[i] = i; for(size_t i = 0; i + 1 < belongto.size(); i++) { for(size_t j = i + 1; j < belongto.size(); j++) { if(belongto[j] != j) continue; size_t ed = calc_edit_dist(s, seeds[sb + i], seeds[sb + j], min_left_ext, min_right_ext, max_ed + 1); if(ed <= max_ed + 1) { belongto[j] = belongto[i]; } } } // find the maximum group that has the most sequences EList vote; vote.resizeExact(seeds.size()); vote.fillZero(); size_t max_group = 0; for(size_t i = 0; i < belongto.size(); i++) { size_t cur_group = belongto[i]; assert_lt(cur_group, vote.size()); vote[cur_group]++; if(cur_group != max_group && vote[cur_group] > vote[max_group]) { max_group = cur_group; } } // reuse vote to store seeds EList& consensus_group = vote; consensus_group.clear(); for(size_t i = 0; i < belongto.size(); i++) { if(belongto[i] == max_group) consensus_group.push_back(i); } // update ed // extend consensus string ed_seed_nums.resize(max_ed + 1); ed_seed_nums.fillZero(); EList next_ed_seed_nums; next_ed_seed_nums.resize(max_ed + 1); for(size_t i = sb; i < se; i++) seeds[i].ed = 0; size_t seed_ext_len = 0; while(seed_ext_len < max(min_left_ext, min_right_ext)) { // count base size_t l_count[4] = {0, }; size_t r_count[4] = {0, }; for(size_t i = 0; i < consensus_group.size(); i++) { size_t si = sb + consensus_group[i]; int ch; if(seed_ext_len < min_left_ext) { if(seeds[i].bound.first + seed_ext_len + 1 <= seeds[i].pos.first) { ch = getSequenceBase(s, seeds[si].pos.first - seed_ext_len - 1); assert_range(0, 3, ch); l_count[ch]++; } } if(seed_ext_len < min_right_ext) { if(seeds[i].bound.second + seed_ext_len) { ch = getSequenceBase(s, seeds[si].pos.second + seed_ext_len); assert_range(0, 3, ch); r_count[ch]++; } } } // select base to be used for extension uint8_t left_ext_base = 0; if(seed_ext_len < min_left_ext) left_ext_base = max_index(l_count); uint8_t right_ext_base = 0; if(seed_ext_len < min_right_ext) right_ext_base = max_index(r_count); // estimate extended ed next_ed_seed_nums.fillZero(); for(size_t i = sb; i < se; i++) { uint32_t est_ed = seeds[i].ed; if(seed_ext_len < min_left_ext) { if(seeds[i].bound.first + seed_ext_len + 1 <= seeds[i].pos.first) { TIndexOffU left_pos = seeds[i].pos.first - seed_ext_len - 1; int ch = getSequenceBase(s, left_pos); assert_range(0, 3, ch); if (ch != left_ext_base) { est_ed++; } } else { est_ed = max_ed + 1; } } if(seed_ext_len < min_right_ext) { TIndexOffU right_pos = seeds[i].pos.second + seed_ext_len; if(right_pos >= seeds[i].bound.second) { est_ed = max_ed + 1; } else { int ch = getSequenceBase(s, right_pos); assert_range(0, 3, ch); if (ch != right_ext_base) { est_ed++; } } } if(est_ed <= max_ed) { next_ed_seed_nums[est_ed]++; } } for(size_t i = 1; i < next_ed_seed_nums.size(); i++) next_ed_seed_nums[i] += next_ed_seed_nums[i-1]; if(next_ed_seed_nums[max_ed] < rp.repeat_count) { break; } for(size_t i = sb; i < se; i++) { if(seed_ext_len < min_left_ext) { if(seeds[i].bound.first + seed_ext_len + 1 <= seeds[i].pos.first) { TIndexOffU left_pos = seeds[i].pos.first - seed_ext_len - 1; int ch = getSequenceBase(s, left_pos); assert_range(0, 3, ch); if (ch != left_ext_base) { seeds[i].ed++; } } else { seeds[i].ed = max_ed + 1; } } if(seed_ext_len < min_right_ext) { TIndexOffU right_pos = seeds[i].pos.second + seed_ext_len; if(right_pos >= seeds[i].bound.second) { seeds[i].ed = max_ed + 1; } else { int ch = getSequenceBase(s, right_pos); assert_range(0, 3, ch); if (ch != right_ext_base) { seeds[i].ed++; } } } } for(size_t i = 0; i < next_ed_seed_nums.size(); i++) { if(next_ed_seed_nums[i] < rp.repeat_count) continue; ed_seed_nums[i] = next_ed_seed_nums[i]; if(seed_ext_len < min_left_ext) { assert(left_consensuses != NULL); (*left_consensuses)[i] += (char)("ACGT"[left_ext_base]); } if(seed_ext_len < min_right_ext) { assert(right_consensuses != NULL); (*right_consensuses)[i] += (char)("ACGT"[right_ext_base]); } } seed_ext_len++; } } EList RB_Repeat::ca_ed_; EList RB_Repeat::ca_ed2_; string RB_Repeat::ca_s_; string RB_Repeat::ca_s2_; size_t RB_Repeat::seed_merge_tried = 0; size_t RB_Repeat::seed_merged = 0; template void RB_RepeatExt::extendConsensus(const RepeatParameter& rp, const TStr& s) { size_t remain = seeds_.size(); EList consensuses, empty_consensuses; EList ed_seed_nums; bool left = true; const TIndexOffU default_max_ext_len = 100; const TIndexOffU seed_mm = 4; string ext_consensus = ""; empty_consensuses.resize(seed_mm + 1); empty_consensuses.fill(""); while(remain >= rp.repeat_count) { for(size_t i = 0; i < remain; i++) { seeds_[i].done = false; seeds_[i].curr_ext_len = 0; } // extend seeds in left or right direction TIndexOffU max_ext_len = min(default_max_ext_len, (TIndexOffU)(rp.max_repeat_len - consensus_.length())); get_consensus_seq(s, seeds_, 0, // seed begin remain, // seed end left ? max_ext_len : 0, left ? 0 : max_ext_len, seed_mm, rp, ed_seed_nums, left ? &consensuses : NULL, left ? NULL : &consensuses); size_t allowed_seed_mm = 0; ext_consensus.clear(); for(int i = (int)seed_mm; i >= 0; i--) { size_t extlen = (ed_seed_nums[i] < rp.repeat_count ? 0 : consensuses[i].length()); if(extlen <= 0 || extlen < max_ext_len * i / seed_mm) continue; if(i > 0 && consensuses[i].length() <= consensuses[i-1].length() + 5) continue; ext_consensus = consensuses[i]; allowed_seed_mm = (size_t)i; assert(ext_consensus.length() > 0); break; } size_t num_passed_seeds = 0; if(ext_consensus.length() > 0) { if(left) consensus_ = reverse(ext_consensus) + consensus_; else consensus_ += ext_consensus; calc_edit_dist(s, seeds_, 0, remain, left ? consensuses : empty_consensuses, left ? empty_consensuses : consensuses, allowed_seed_mm); // update seeds for(size_t i = 0; i < seeds_.size(); i++) { SeedExt& seed = seeds_[i]; if(i < remain) { if(seed.ed <= allowed_seed_mm) { num_passed_seeds++; seed.done = true; seed.total_ed += seed.ed; if(left) { if(seed.left_gaps.size() > 0 && seed.left_gaps.back().first >= seed.orig_pos.first - seed.pos.first) { int gap_len = seed.left_gaps.back().second; seed.pos.first += gap_len; } seed.pos.first -= ext_consensus.length(); seed.consensus_pos.first = 0; seed.consensus_pos.second = consensus_.length(); } else { if(seed.right_gaps.size() > 0 && seed.right_gaps.back().first >= seed.pos.second - seed.orig_pos.second) { int gap_len = seed.right_gaps.back().second; seed.pos.second -= gap_len; } seed.pos.second += ext_consensus.length(); seed.consensus_pos.second = consensus_.length(); } } else { if(left) { assert_leq(seed.curr_ext_len, ext_consensus.length()); TIndexOffU adjust = ext_consensus.length() - seed.curr_ext_len; seed.consensus_pos.first += adjust; seed.consensus_pos.second += ext_consensus.length(); assert_leq(seed.curr_ext_len, seed.pos.first); seed.pos.first -= seed.curr_ext_len; } else { assert_leq(seed.curr_ext_len, ext_consensus.length()); seed.consensus_pos.second += seed.curr_ext_len; seed.pos.second += seed.curr_ext_len; } } } else { if(left) { seed.consensus_pos.first += ext_consensus.length(); seed.consensus_pos.second += ext_consensus.length(); } } } // move up "done" seeds size_t j = 0; for(size_t i = 0; i < remain; i++) { if(!seeds_[i].done) continue; assert_geq(i, j); if(i > j) { SeedExt temp = seeds_[j]; seeds_[j] = seeds_[i]; seeds_[i] = temp; // Find next "undone" seed j++; while(j < i && seeds_[j].done) { j++; } assert(j < remain && !seeds_[j].done); } else { j = i + 1; } } } remain = num_passed_seeds; if(remain < rp.repeat_count) { if(left) { left = false; remain = seeds_.size(); } } } // while(remain >= rp.repeat_count) #ifndef NDEBUG // make sure seed positions are unique EList > seed_poses; for(size_t i = 0; i < seeds_.size(); i++) { seed_poses.expand(); seed_poses.back() = seeds_[i].orig_pos; } seed_poses.sort(); for(size_t i = 0; i + 1 < seed_poses.size(); i++) { if(seed_poses[i].first == seed_poses[i+1].first) { assert_lt(seed_poses[i].second, seed_poses[i+1].second); } else { assert_lt(seed_poses[i].first, seed_poses[i+1].first); } } for(size_t i = 0; i < seeds_.size(); i++) { assert(seeds_[i].valid()); } #endif internal_update(); // check repeats within a repeat sequence self_repeat_ = false; for(size_t i = 0; i + 1 < seed_ranges_.size(); i++) { const RB_AlleleCoord& range = seed_ranges_[i]; for(size_t j = i + 1; j < seed_ranges_.size(); j++) { RB_AlleleCoord& range2 = seed_ranges_[j]; if(range.right <= range2.left) break; self_repeat_ = true; } } } bool RB_Repeat::overlap(const RB_Repeat& o, bool& contain, bool& left, size_t& seed_i, size_t& seed_j, bool debug) const { contain = left = false; seed_i = seed_j = 0; size_t p = 0, p2 = 0; while(p < seed_ranges_.size() && p2 < o.seed_ranges_.size()) { const RB_AlleleCoord& range = seed_ranges_[p]; const SeedExt& seed = seeds_[range.idx]; Range range_ = seed.getExtendedRange(consensus_.length()); RB_AlleleCoord ex_range(range_.first, range_.second, p); bool representative = (float)range.len() >= consensus_.length() * 0.95f; const RB_AlleleCoord& range2 = o.seed_ranges_[p2]; const SeedExt& seed2 = o.seeds_[range2.idx]; Range range2_ = seed2.getExtendedRange(o.consensus().length()); RB_AlleleCoord ex_range2(range2_.first, range2_.second, p2); bool representative2 = (float)range2.len() >= o.consensus_.length() * 0.95f; seed_i = range.idx; seed_j = range2.idx; const size_t relax = 5; if(representative && representative2) { if(ex_range.overlap_len(ex_range2) > 0) { if(ex_range.contain(ex_range2, relax)) { contain = true; left = true; } else if(ex_range2.contain(ex_range, relax)) { contain = true; left = false; } else { left = ex_range.left <= ex_range2.left; } return true; } } else if(representative) { if(range2.contain(ex_range, relax)) { contain = true; left = false; return true; } } else if(representative2) { if(range.contain(ex_range2, relax)) { contain = true; left = true; return true; } } if(range.right <= range2.right) p++; if(range2.right <= range.right) p2++; } return false; } void RB_Repeat::showInfo(const RepeatParameter& rp, CoordHelper& coordHelper) const { cerr << "\trepeat id: " << repeat_id_ << endl; cerr << "\tnumber of alleles: " << seeds_.size() << endl; cerr << "\tconsensus length: " << consensus_.length() << endl; cerr << consensus_ << endl; for(size_t i = 0; i < seeds_.size(); i++) { const SeedExt& seed = seeds_[i]; size_t ext_len = seed.getLength(); if(ext_len < rp.min_repeat_len) continue; bool sense_strand = seed.pos.first < coordHelper.forward_length(); cerr << "\t\t" << setw(4) << i << " " << setw(4) << ext_len; cerr << " " << (sense_strand ? '+' : '-'); cerr << " " << setw(10) << seed.pos.first << " " << setw(10) << seed.pos.second; cerr << " " << setw(4) << seed.consensus_pos.first << " " << setw(4) << seed.consensus_pos.second; cerr << " " << (seed.aligned ? "aligned" : "unaligned"); cerr << endl; } } template void RB_Repeat::generateSNPs(const RepeatParameter& rp, const TStr& s, TIndexOffU grp_id) { EList& seeds = this->seeds(); for(size_t i = 0; i < seeds.size(); i++) { SeedExt& seed = seeds[i]; if(!seed.aligned) { continue; } if(seed.getLength() < rp.min_repeat_len) { continue; } assert_eq(seed.getLength(), seed.pos.second - seed.pos.first); seed.generateSNPs(s, consensus(), snps()); } if(snps().size() > 0) { sort(snps_.begin(), snps().end(), SeedSNP::cmpSeedSNPByPos); } } void RB_Repeat::saveSNPs(ofstream &fp, TIndexOffU grp_id, TIndexOffU& snp_id_base) { string chr_name = "rep" + to_string(repeat_id_); for(size_t j = 0; j < snps_.size(); j++) { SeedSNP& snp = *snps_[j]; // assign SNPid snp.id = (snp_id_base++); fp << "rps" << snp.id; fp << "\t"; if(snp.type == EDIT_TYPE_MM) { fp << "single"; } else if(snp.type == EDIT_TYPE_READ_GAP) { fp << "deletion"; } else if(snp.type == EDIT_TYPE_REF_GAP) { fp << "insertion"; } else { assert(false); } fp << "\t"; fp << chr_name; fp << "\t"; fp << snp.pos; fp << "\t"; if(snp.type == EDIT_TYPE_MM || snp.type == EDIT_TYPE_REF_GAP) { fp << snp.base; } else if(snp.type == EDIT_TYPE_READ_GAP) { fp << snp.len; } else { assert(false); } fp << endl; } } float RB_RepeatExt::mergeable(const RB_Repeat& o) const { const EList& ranges = seed_ranges(); const EList& ranges2 = o.seed_ranges(); size_t num_overlap_bp = 0; size_t p = 0, p2 = 0; while(p < ranges.size() && p2 < ranges2.size()) { const RB_AlleleCoord& range = ranges[p]; const RB_AlleleCoord& range2 = ranges2[p2]; TIndexOffU overlap = range.overlap_len(range2); num_overlap_bp += overlap; if(range.right <= range2.right) p++; else p2++; } size_t num_total_bp = 0, num_total_bp2 = 0; for(size_t r = 0; r < ranges.size(); r++) num_total_bp += (ranges[r].right - ranges[r].left); for(size_t r = 0; r < ranges2.size(); r++) num_total_bp2 += (ranges2[r].right - ranges2[r].left); float portion = float(num_overlap_bp) / float(min(num_total_bp, num_total_bp2)); return portion; } inline void get_next_range(const EList& offsets, size_t i, Range& r, float& avg) { r.first = i; for(; r.first < offsets.size() && offsets[r.first] < 0; r.first++); r.second = r.first + 1; avg = 0.0f; if(r.first < offsets.size()) { float diff = (float)offsets[r.first] - (float)r.first; avg += diff; } for(; r.second < offsets.size() && offsets[r.second] >= 0 && (r.second == 0 || offsets[r.second] >= offsets[r.second - 1]); r.second++) { float diff = (float)offsets[r.second] - (float)r.second; avg += diff; } avg /= (float)(r.second - r.first); return; } template bool RB_RepeatExt::align(const RepeatParameter& rp, const TStr& ref, const string& s, const EList >& s_kmer_table, const string& s2, EList& offsets, size_t k, SeedExt& seed, int consensus_approx_left, int consensus_approx_right, size_t left, size_t right, bool debug) { RB_Repeat::seed_merge_tried++; seed.reset(); seed.pos.first = left; seed.pos.second = right; seed.orig_pos.first = seed.pos.first; seed.orig_pos.second = seed.orig_pos.first; seed.consensus_pos.first = 0; seed.consensus_pos.second = consensus_.length(); seed.aligned = false; { const int query_len = right - left; const int consensus_len = consensus_approx_right - consensus_approx_left; const int abs_gap_len = max(abs(consensus_len - query_len), 5); offsets.resize(s2.length()); offsets.fill(-1); pair kmer(0, 0); for(size_t i = 0; i + k <= s2.length(); i++) { if(i == 0) { kmer.first = extract_kmer(s2, i, k); } else { kmer.first = next_kmer(kmer.first, s2[i+k-1], k); } size_t lb = s_kmer_table.bsearchLoBound(kmer); while(lb < s_kmer_table.size() && kmer.first == s_kmer_table[lb].first) { int expected = (int)i + consensus_approx_left; int real = (int)s_kmer_table[lb].second; int abs_diff = abs(expected - real); if(abs_diff <= abs_gap_len * 2 || debug) { if(offsets[i] == -1) { offsets[i] = (int)s_kmer_table[lb].second; } else if(offsets[i] >= 0) { offsets[i] = -2; } else { assert_lt(offsets[i], -1); offsets[i] -= 1; } } lb++; } if(offsets[i] > 0 && i + k == s2.length()) { for(size_t j = i + 1; j < s2.length(); j++) { offsets[j] = offsets[j-1] + 1; } } } } if(debug) { cerr << "initial offsets" << endl; for(size_t j = 0; j < offsets.size(); j++) { cout << j << ": " << offsets[j] << " " << s2[j] << ": " << (offsets[j] < 0 ? ' ' : s[offsets[j]]) << endl; } } // remove inconsistent positions Range range; float range_avg; get_next_range(offsets, 0, range, range_avg); while(range.second < offsets.size()) { Range range2; float range_avg2; get_next_range(offsets, range.second, range2, range_avg2); if(range2.first >= offsets.size()) break; assert_leq(range.second, range2.first); float abs_diff = abs(range_avg - range_avg2); if(offsets[range.second - 1] > offsets[range2.first] || (abs_diff > 10.0f && (range.second - range.first < 5 || range2.second - range2.first < 5)) || abs_diff > 20.0f) { if(range.second - range.first < range2.second - range2.first) { for(size_t i = range.first; i < range.second; i++) { offsets[i] = -1; } range = range2; range_avg = range_avg2; } else { for(size_t i = range2.first; i < range2.second; i++) { offsets[i] = -1; } } } else { range = range2; range_avg = range_avg2; } } bool weighted_avg_inited = false; float weighted_avg = -1.0f; for(size_t i = 0; i < offsets.size(); i++) { if(offsets[i] < 0) continue; float diff = (float)offsets[i] - (float)i; if(weighted_avg_inited) { if(abs(diff - weighted_avg) > 20.0f) { offsets[i] = -1; continue; } } if(weighted_avg < 0.0f) { weighted_avg = diff; weighted_avg_inited = true; } else { weighted_avg = 0.8f * weighted_avg + 0.2f * diff; } } if(debug) { cerr << "after filtering" << endl; for(size_t j = 0; j < offsets.size(); j++) { cout << j << ": " << offsets[j] << " " << s2[j] << ": " << (offsets[j] < 0 ? ' ' : s[offsets[j]]) << endl; } } int i = 0; while(i < offsets.size()) { // skip non-negative offsets for(; i < offsets.size() && offsets[i] >= 0; i++); if(i >= offsets.size()) break; int j = i; for(; j < offsets.size(); j++) { if(offsets[j] >= 0) break; } assert(i >= offsets.size() || offsets[i] < 0); assert(j >= offsets.size() || offsets[j] >= 0); if(i > 0 && j < offsets.size()) { i -= 1; int left = offsets[i], right = offsets[j]; assert_geq(left, 0); if(left > right) return false; assert_leq(left, right); int ref_len = right - left + 1; int query_len = j - i + 1; if(query_len == ref_len) { // match for(size_t i2 = i + 1; i2 < j; i2++) offsets[i2] = offsets[i2-1] + 1; } else { // deletion or insertion bool del = query_len < ref_len; size_t gap_len = del ? ref_len - query_len : query_len - ref_len; size_t max_len = max(ref_len, query_len); const size_t max_mm = max_len / 25 + 1; const size_t very_max_mm = max(max_len / 2, max_mm); ca_s_ = s.substr(left, max_len); ca_s2_ = s2.substr(i, max_len); size_t gap_pos = 0, mm = very_max_mm + 1; find_gap_pos(ca_s_, ca_s2_, ca_ed_, ca_ed2_, del, gap_len, very_max_mm, gap_pos, mm, debug); if(mm > very_max_mm) { return false; } assert_lt(gap_pos, query_len); if(del) { for(size_t i2 = i + 1; i2 < j; i2++) { if(i2 - i == gap_pos) { offsets[i2] = offsets[i2-1] + gap_len; } else { offsets[i2] = offsets[i2-1] + 1; } } } else { for(size_t i2 = i + 1; i2 < j; i2++) { if(i2 - i >= gap_pos && i2 - i < gap_pos + gap_len) { offsets[i2] = offsets[i2-1]; } else { offsets[i2] = offsets[i2-1] + 1; } } } } } i = j; } if(debug) { cerr << "final offsets" << endl; for(size_t j = 0; j < offsets.size(); j++) { cout << j << ": " << offsets[j] << " " << s2[j] << ": " << (offsets[j] < 0 ? ' ' : s[offsets[j]]) << endl; } } #ifndef NDEBUG for(size_t i = 1; i < offsets.size(); i++) { if(offsets[i-1] < 0 || offsets[i] < 0) continue; assert_leq(offsets[i-1], offsets[i]); } #endif size_t b, e; for(b = 0; b < offsets.size() && offsets[b] < 0; b++); if(b >= offsets.size()) return false; assert_lt((size_t)b, offsets.size()); for(e = offsets.size() - 1; e > b && offsets[e] < 0; e--); if(b == e) return false; for(size_t p = b; p <= e; p++) { if(offsets[p] < 0) return false; } // try to fill the ends if(b > 0) { int mm = 0, pb = (int)b; for(int i = pb - 1; i >= 0; i--) { assert_geq(offsets[i+1], 0); if(offsets[i+1] == 0) break; if(s2[i] != s[offsets[i+1] - 1]) mm++; if(pb - i < 25 * (mm - 1)) break; offsets[i] = offsets[i+1] - 1; b = (size_t)i; } } if(e + 1 < offsets.size()) { int mm = 0, prev_end = (int)e; for(int i = prev_end + 1; i < offsets.size(); i++) { assert_geq(offsets[i-1], 0); if(offsets[i-1] + 1 >= s.length()) break; if(s2[i] != s[offsets[i-1] + 1]) mm++; if(i - prev_end < 25 * (mm - 1)) break; offsets[i] = offsets[i-1] + 1; e = (size_t)i; } } assert_geq(seed.pos.first, (TIndexOffU)b); seed.pos.first += (TIndexOffU)b; seed.pos.second = seed.pos.first + e - b + 1; seed.orig_pos.first = seed.pos.first; seed.orig_pos.second = seed.pos.first; seed.consensus_pos.first = offsets[b]; seed.consensus_pos.second = offsets[e] + 1; seed.aligned = true; for(int p = b; p < e; p++) { assert_geq(offsets[p], 0); assert_leq(offsets[p], offsets[p+1]); if(offsets[p] + 1 == offsets[p+1]) { // match continue; } else if(offsets[p] + 1 < offsets[p+1]) { // deletion seed.right_gaps.expand(); seed.right_gaps.back().first = p + 1 - b; seed.right_gaps.back().second = offsets[p+1] - offsets[p] - 1; } else { assert_eq(offsets[p], offsets[p+1]); int p2 = p + 1; for(; offsets[p2] == offsets[p2+1]; p2++); seed.right_gaps.expand(); seed.right_gaps.back().first = p + 1 - b; seed.right_gaps.back().second = (int)p - (int)p2; p = p2; } } if(debug) { string consensus_str = consensus_.substr(seed.consensus_pos.first, seed.consensus_pos.second - seed.consensus_pos.first); string seed_str; seed.getExtendedSeedSequence(ref, seed_str); assert_eq(consensus_str.length(), seed_str.length()); cerr << "consensus: " << consensus_str << endl; cerr << "seed : " << seed_str << endl; } RB_Repeat::seed_merged++; return true; } bool RB_RepeatExt::isSelfRepeat(const RepeatParameter& rp, const string& s, const EList >& s_kmer_table, EList& offsets, size_t k, bool debug) { offsets.resize(s.length()); offsets.fill(-1); pair kmer(0, 0); for(size_t i = 0; i + k <= s.length(); i++) { if(i == 0) { kmer.first = extract_kmer(s, i, k); } else { kmer.first = next_kmer(kmer.first, s[i+k-1], k); } size_t lb = s_kmer_table.bsearchLoBound(kmer); while(lb < s_kmer_table.size() && kmer.first == s_kmer_table[lb].first) { if(offsets[i] == -1) { offsets[i] = (int)s_kmer_table[lb].second; } else if(offsets[i] >= 0) { offsets[i] = -2; } else { assert_lt(offsets[i], -1); offsets[i] -= 1; } lb++; } if(offsets[i] > 0 && i + k == s.length()) { for(size_t j = i + 1; j < s.length(); j++) { offsets[j] = offsets[j-1] + 1; } } } if(debug) { cerr << "offsets" << endl; for(size_t j = 0; j < offsets.size(); j++) { cout << j << ": " << offsets[j] << " " << s[j] << ": " << (offsets[j] < 0 ? ' ' : s[offsets[j]]) << endl; } } const size_t min_repeat_size = 100; size_t repeat_count = 0; for(size_t i = 0; i + min_repeat_size < offsets.size(); i++) { if(offsets[i] >= -1) continue; size_t j = i + 1; for(; j < offsets.size() && offsets[j] <= -2; j++); if(j - i >= min_repeat_size) repeat_count++; i = j; } return repeat_count >= 2; } template bool RB_RepeatExt::merge(const RepeatParameter& rp, const TStr& s, RB_SWAligner& swalginer, const RB_RepeatExt& o, bool contain, size_t seed_i, size_t seed_j, bool debug) { // construct a new consensus sequence string prev_consensus = consensus_; size_t consensus_add_len = 0; { assert_lt(seed_i, seeds_.size()); assert_lt(seed_j, o.seeds_.size()); const SeedExt& seed = seeds_[seed_i]; const SeedExt& oseed = o.seeds_[seed_j]; Range range = seed.getExtendedRange(consensus_.length()); Range orange = oseed.getExtendedRange(o.consensus_.length()); assert_leq(range.first, orange.first + 10); consensus_add_len = (int)orange.first - (int)range.first; if(!contain) { if(range.second <= orange.first) { cerr << "something wrong: " << range.first << "-" << range.second << " "; cerr << orange.first << "-" << orange.second << " " << o.consensus_.length() << endl; assert(false); } if(range.second > orange.first && range.second - orange.first < o.consensus_.length()) { consensus_ += o.consensus_.substr(range.second - orange.first); } } } EList > merge_list; merge_list.reserveExact(o.seed_ranges_.size()); size_t p = 0, p2 = 0; const size_t relax = 5; while(p < seed_ranges_.size() && p2 < o.seed_ranges_.size()) { const RB_AlleleCoord& range = seed_ranges_[p]; assert_lt(range.idx, seeds_.size()); const RB_AlleleCoord& range2 = o.seed_ranges_[p2]; assert_lt(range2.idx, o.seeds_.size()); if(range.contain(range2, relax)) { merge_list.expand(); merge_list.back().first = p; merge_list.back().second = p2; if(debug) { cerr << p << ":" << range.left << "-" << range.right << " > "; cerr << p2 << ":" << range2.left << "-" << range2.right << endl; } } else if(range2.contain(range, relax)) { merge_list.expand(); merge_list.back().first = p; merge_list.back().second = p2; if(debug) { cerr << p << ":" << range.left << "-" << range.right << " < "; cerr << p2 << ":" << range2.left << "-" << range2.right << endl; } } else { TIndexOffU overlap_len = range.overlap_len(range2); bool stored = !merge_list.empty() && merge_list.back().first == p; bool stored2 = !merge_list.empty() && merge_list.back().second == p2; if(overlap_len > 0) { if(!stored && !stored2) { merge_list.expand(); merge_list.back().first = p; merge_list.back().second = p2; if(debug) { cerr << p << ":" << range.left << "-" << range.right << " ol "; cerr << p2 << ":" << range2.left << "-" << range2.right << endl; } } } else { if(range2.right <= range.left) { if(!stored2) { merge_list.expand(); merge_list.back().first = numeric_limits::max(); merge_list.back().second = p2; if(debug) { cerr << p << ":" << range.left << "-" << range.right << " <> "; cerr << p2 << ":" << range2.left << "-" << range2.right << endl; } } } else { assert_leq(range.right, range2.left); if(!stored) { merge_list.expand(); merge_list.back().first = p; merge_list.back().second = numeric_limits::max(); if(debug) { cerr << p << ":" << range.left << "-" << range.right << " <> "; cerr << p2 << ":" << range2.left << "-" << range2.right << endl; } } } } } if(range.right <= range2.right) p++; if(range2.right <= range.right) p2++; } assert(p == seeds_.size() || p2 == o.seeds_.size()); while(p < seed_ranges_.size()) { bool stored = !merge_list.empty() && merge_list.back().first == p; if(!stored) { const RB_AlleleCoord& range = seed_ranges_[p]; merge_list.expand(); merge_list.back().first = p; merge_list.back().second = numeric_limits::max(); if(debug) { cerr << p << ":" << range.left << "-" << range.right << " : <> " << endl; } } p++; } while(p2 < o.seed_ranges_.size()) { bool stored2 = !merge_list.empty() && merge_list.back().second == p2; if(!stored2) { const RB_AlleleCoord& range2 = o.seed_ranges_[p2]; merge_list.expand(); merge_list.back().first = numeric_limits::max(); merge_list.back().second = p2; if(debug) { cerr << ": <> " << p2 << ":" << range2.left << "-" << range2.right << endl; } } p2++; } assert(!merge_list.empty()); if(debug) { for(size_t i = 0; i < merge_list.size(); i++) { cerr << "merge list:" << endl; cerr << "\t" << (merge_list[i].first < seed_ranges_.size() ? (int)merge_list[i].first : -1); cerr << " " << (merge_list[i].second < o.seed_ranges_.size() ? (int)merge_list[i].second : -1) << endl; } } const size_t kmer_len = 12; EList > kmer_table; build_kmer_table(consensus_, kmer_table, kmer_len); EList offsets; bool self_repeat = isSelfRepeat(rp, consensus_, kmer_table, offsets, kmer_len, debug); if(self_repeat) { consensus_ = prev_consensus; return false; } string seq; for(size_t i = 0; i < merge_list.size(); i++) { size_t seed_id = (merge_list[i].first < seed_ranges_.size() ? seed_ranges_[merge_list[i].first].idx : merge_list[i].first); size_t oseed_id = (merge_list[i].second < o.seed_ranges_.size() ? o.seed_ranges_[merge_list[i].second].idx : merge_list[i].second); if(seed_id < seeds_.size()) { if(oseed_id >= o.seeds_.size()) continue; else if(seed_ranges_[merge_list[i].first].contain(o.seed_ranges_[merge_list[i].second])) continue; } const SeedExt* seed = (seed_id < seeds_.size() ? &seeds_[seed_id] : NULL); const SeedExt* oseed = (oseed_id < o.seeds_.size() ? &o.seeds_[oseed_id] : NULL); assert(seed != NULL || oseed != NULL); size_t left = (seed != NULL ? seed->pos.first : numeric_limits::max()); size_t right = (seed != NULL ? seed->pos.second : 0); int consensus_approx_left = (seed != NULL ? seed->consensus_pos.first : numeric_limits::max()); int consensus_approx_right = (seed != NULL ? seed->consensus_pos.second : 0); if(oseed != NULL) { if(oseed->pos.first < left) { left = oseed->pos.first; } if(oseed->pos.second > right) { right = oseed->pos.second; } int oconsensus_approx_left = oseed->consensus_pos.first + consensus_add_len; if(oconsensus_approx_left < consensus_approx_left) { consensus_approx_left = oconsensus_approx_left; } int oconsensus_approx_right = oseed->consensus_pos.second + consensus_add_len; if(oconsensus_approx_right > consensus_approx_right) { consensus_approx_right = oconsensus_approx_right; } } getString(s, left, right - left, seq); if(seed_id >= seeds_.size()) { seed_id = seeds_.size(); seeds_.expand(); } /* bool succ = */ align(rp, s, consensus_, kmer_table, seq, offsets, kmer_len, seeds_[seed_id], consensus_approx_left, consensus_approx_right, left, right, debug && right - left == 1080); } while(true) { internal_update(); size_t remove_count = 0; for(size_t i = 0; i + 1 < seed_ranges_.size(); i++) { RB_AlleleCoord& range = seed_ranges_[i]; if(range.left == numeric_limits::max()) break; for(size_t j = i + 1; j < seed_ranges_.size(); j++) { RB_AlleleCoord& range2 = seed_ranges_[j]; if(range2.left == numeric_limits::max()) break; if(range.right <= range2.left) break; getString(s, range.left, range2.right - range.left, seq); /* bool succ = */ align(rp, s, consensus_, kmer_table, seq, offsets, kmer_len, seeds_[range.idx], seeds_[range.idx].consensus_pos.first, seeds_[range2.idx].consensus_pos.second, range.left, range2.right, debug && range.left == 692422); range.left = seeds_[range.idx].pos.first; range.right = seeds_[range.idx].pos.second; seeds_[range2.idx].reset(); range2.left = numeric_limits::max(); remove_count++; } } if(remove_count <= 0) break; sort(seeds_.begin(), seeds_.end(), seedCmp); seeds_.resize(seeds_.size() - remove_count); } return true; } #define DMAX std::numeric_limits::max() RB_SWAligner::RB_SWAligner() { rnd_.init(0); } RB_SWAligner::~RB_SWAligner() { if(sc_) { delete sc_; } } void RB_SWAligner::init_dyn(const RepeatParameter& rp) { const int MM_PEN = 3; // const int MM_PEN = 6; const int GAP_PEN_LIN = 2; // const int GAP_PEN_LIN = (((MM_PEN) * rpt_edit_ + 1) * 1.0); const int GAP_PEN_CON = 4; // const int GAP_PEN_CON = (((MM_PEN) * rpt_edit_ + 1) * 1.0); const int MAX_PEN = MAX_I16; scoreMin_.init(SIMPLE_FUNC_LINEAR, rp.max_edit * MM_PEN * -1.0, 0.0); nCeil_.init(SIMPLE_FUNC_LINEAR, 0.0, 0.0); penCanIntronLen_.init(SIMPLE_FUNC_LOG, -8, 1); penNoncanIntronLen_.init(SIMPLE_FUNC_LOG, -8, 1); sc_ = new Scoring( DEFAULT_MATCH_BONUS, // constant reward for match DEFAULT_MM_PENALTY_TYPE, // how to penalize mismatches MM_PEN, // max mm penalty MM_PEN, // min mm penalty MAX_PEN, // max sc penalty MAX_PEN, // min sc penalty scoreMin_, // min score as function of read len nCeil_, // max # Ns as function of read len DEFAULT_N_PENALTY_TYPE, // how to penalize Ns in the read DEFAULT_N_PENALTY, // constant if N pelanty is a constant DEFAULT_N_CAT_PAIR, // whether to concat mates before N filtering GAP_PEN_CON, // constant coeff for read gap cost GAP_PEN_CON, // constant coeff for ref gap cost GAP_PEN_LIN, // linear coeff for read gap cost GAP_PEN_LIN, // linear coeff for ref gap cost 1 /* gGapBarrier */ // # rows at top/bot only entered diagonally ); } void RB_SWAligner::makePadString(const string& ref, const string& read, string& pad, size_t len) { pad.resize(len); for(size_t i = 0; i < len; i++) { // shift A->C, C->G, G->T, T->A pad[i] = "CGTA"[asc2dna[ref[i]]]; if(read[i] == pad[i]) { // shift pad[i] = "CGTA"[asc2dna[pad[i]]]; } } int head_len = len / 2; size_t pad_start = len - head_len; for(size_t i = 0; i < head_len; i++) { if(read[i] == pad[pad_start + i]) { // shift pad[pad_start + i] = "CGTA"[asc2dna[pad[pad_start + i]]]; } } } int RB_SWAligner::alignStrings(const string &ref, const string &read, EList& edits, Coord& coord) { // Prepare Strings // Read -> BTDnaString // Ref -> bit-encoded string //SwAligner swa; BTDnaString btread; BTString btqual; BTString btref; BTString btref2; BTDnaString btreadrc; BTString btqualrc; string qual = ""; for(size_t i = 0; i < read.length(); i++) { qual.push_back('I'); } #if 0 cerr << "REF : " << ref << endl; cerr << "READ: " << read << endl; cerr << "QUAL: " << qual << endl; #endif btread.install(read.c_str(), true); btreadrc = btread; btreadrc.reverseComp(); btqual.install(qual.c_str()); btqualrc = btqual; btref.install(ref.c_str()); TAlScore min_score = sc_->scoreMin.f((double)btread.length()); btref2 = btref; size_t nceil = 0; // size_t nrow = btread.length(); // Convert reference string to mask for(size_t i = 0; i < btref2.length(); i++) { if(toupper(btref2[i]) == 'N') { btref2.set(16, i); } else { int num = 0; int alts[] = {4, 4, 4, 4}; decodeNuc(toupper(btref2[i]), num, alts); assert_leq(num, 4); assert_gt(num, 0); btref2.set(0, i); for(int j = 0; j < num; j++) { btref2.set(btref2[i] | (1 << alts[j]), i); } } } bool fw = true; uint32_t refidx = 0; swa.initRead( btread, // read sequence btreadrc, btqual, // read qualities btqualrc, 0, // offset of first character within 'read' to consider btread.length(), // offset of last char (exclusive) in 'read' to consider *sc_); // local-alignment score floor DynProgFramer dpframe(false); size_t readgaps = 0; size_t refgaps = 0; size_t maxhalf = 0; DPRect rect; dpframe.frameSeedExtensionRect( 0, // ref offset implied by seed hit assuming no gaps btread.length(), // length of read sequence used in DP table btref2.length(), // length of reference readgaps, // max # of read gaps permitted in opp mate alignment refgaps, // max # of ref gaps permitted in opp mate alignment (size_t)nceil, // # Ns permitted maxhalf, // max width in either direction rect); // DP Rectangle assert(rect.repOk()); size_t cminlen = 2000, cpow2 = 4; swa.initRef( fw, // whether to align forward or revcomp read refidx, // reference ID rect, // DP rectangle btref2.wbuf(), // reference strings 0, // offset of first reference char to align to btref2.length(), // offset of last reference char to align to btref2.length(), // length of reference sequence *sc_, // scoring scheme min_score, // minimum score true, // use 8-bit SSE if positions cminlen, // minimum length for using checkpointing scheme cpow2, // interval b/t checkpointed diags; 1 << this false, // triangular mini-fills? false // is this a seed extension? ); TAlScore best = std::numeric_limits::min(); bool found = swa.align(rnd_, best); #ifdef DEBUGLOG cerr << "found: " << found << "\t" << best << "\t" << "minsc: " << min_score << endl; #endif if (found) { #ifdef DEBUGLOG //cerr << "CP " << "found: " << found << "\t" << best << "\t" << "minsc: " << min_score << endl; cerr << "REF : " << ref << endl; cerr << "READ: " << read << endl; #endif SwResult res; int max_match_len = 0; res.reset(); res.alres.init_raw_edits(&rawEdits_); found = swa.nextAlignment(res, best, rnd_); if (found) { edits = res.alres.ned(); //const TRefOff ref_off = res.alres.refoff(); //const Coord& coord = res.alres.refcoord(); coord = res.alres.refcoord(); //assert_geq(genomeHit._joinedOff + coord.off(), genomeHit.refoff()); #ifdef DEBUGLOG cerr << "num edits: " << edits.size() << endl; cerr << "coord: " << coord.off(); cerr << ", " << coord.ref(); cerr << ", " << coord.orient(); cerr << ", " << coord.joinedOff(); cerr << endl; Edit::print(cerr, edits); cerr << endl; Edit::printQAlign(cerr, btread, edits); #endif max_match_len = getMaxMatchLen(edits, btread.length()); #ifdef DEBUGLOG cerr << "max match length: " << max_match_len << endl; #endif } #ifdef DEBUGLOG cerr << "nextAlignment: " << found << endl; cerr << "-------------------------" << endl; #endif } return 0; } void RB_SWAligner::doTest(const RepeatParameter& rp, const string& refstr, const string& readstr) { init_dyn(rp); doTestCase1(refstr, readstr, rp.max_edit); } void RB_SWAligner::doTestCase1(const string& refstr, const string& readstr, TIndexOffU rpt_edit) { cerr << "doTestCase1----------------" << endl; EList edits; Coord coord; if (refstr.length() == 0 || readstr.length() == 0) { return; } EList ed; string pad; makePadString(refstr, readstr, pad, 5); string ref2 = pad + refstr + pad; string read2 = pad + readstr + pad; alignStrings(refstr, readstr, edits, coord); size_t left = pad.length(); size_t right = left + readstr.length(); edits.reserveExact(ed.size()); for(size_t i = 0; i < ed.size(); i++) { if(ed[i].pos >= left && ed[i].pos <= right) { edits.push_back(ed[i]); edits.back().pos -= left; } } #if 0 RepeatGroup rg; rg.edits = edits; rg.coord = coord; rg.seq = readstr; rg.base_offset = 0; string chr_name = "rep"; cerr << "REF : " << refstr << endl; cerr << "READ: " << readstr << endl; size_t snpids = 0; rg.buildSNPs(snpids); rg.writeSNPs(cerr, chr_name); cerr << endl; #endif } template RepeatBuilder::RepeatBuilder(TStr& s, const EList& szs, const EList& ref_names, bool forward_only, const string& filename) : s_(s), coordHelper_(s.length(), forward_only ? s.length() : s.length() / 2, szs, ref_names), forward_only_(forward_only), filename_(filename), forward_length_(forward_only ? s.length() : s.length() / 2) { cerr << "RepeatBuilder: " << filename_ << endl; } template RepeatBuilder::~RepeatBuilder() { for(map::iterator it = repeat_map_.begin(); it != repeat_map_.end(); it++) { delete it->second; } repeat_map_.clear(); } template void RepeatBuilder::readSA(const RepeatParameter& rp, BlockwiseSA& sa) { TIndexOffU count = 0; subSA_.init(s_.length() + 1, rp.min_repeat_len, rp.repeat_count); while(count < s_.length() + 1) { TIndexOffU saElt = sa.nextSuffix(); count++; if(count && (count % 10000000 == 0)) { cerr << "SA count " << count << endl; } if(saElt == s_.length()) { assert_eq(count, s_.length() + 1); break; } subSA_.push_back(s_, coordHelper_, saElt, count == s_.length()); } cerr << "subSA size is " << subSA_.size() << endl; subSA_.dump(); #if 0 for(size_t i = 0; i < subSA_.size(); i++) { TIndexOffU joinedOff = subSA_[i]; fp << setw(10) << joinedOff << " " << getString(s_, joinedOff, rp.seed_len) << endl; } #endif cerr << "subSA mem Usage: " << subSA_.getMemUsage() << endl << endl; } template void RepeatBuilder::readSA(const RepeatParameter &rp, const BitPackedArray &sa) { TIndexOffU count = 0; subSA_.init(s_.length() + 1, rp.min_repeat_len, rp.repeat_count); for(size_t i = 0; i < sa.size(); i++) { TIndexOffU saElt = sa[i]; count++; if(count && (count % 10000000 == 0)) { cerr << "RB count " << count << endl; } if(saElt == s_.length()) { assert_eq(count, s_.length() + 1); break; } subSA_.push_back(s_, coordHelper_, saElt, count == s_.length()); } cerr << "subSA size: " << endl; subSA_.dump(); #if 0 for(size_t i = 0; i < subSA_.size(); i++) { TIndexOffU joinedOff = subSA_[i]; fp << setw(10) << joinedOff << " " << getString(s_, joinedOff, rp.seed_len) << endl; } #endif cerr << "subSA mem Usage: " << subSA_.getMemUsage() << endl << endl; } template void RepeatBuilder::build(const RepeatParameter& rp) { string rpt_len_str; rpt_len_str = to_string(rp.min_repeat_len) + "-" + to_string(rp.max_repeat_len); string seed_filename = filename_ + ".rep." + rpt_len_str + ".seed"; ofstream fp(seed_filename.c_str()); swaligner_.init_dyn(rp); RB_RepeatManager* repeat_manager = new RB_RepeatManager; EList repeatBases; subSA_.buildRepeatBase(s_, coordHelper_, rp.max_repeat_len, repeatBases); size_t repeat_id = 0; for(size_t i = 0; i < repeatBases.size(); i++) { addRepeatGroup(rp, repeat_id, *repeat_manager, repeatBases[i], fp); } { // Build and test minimizer-based k-mer table const size_t window = RB_Minimizer::default_w; const size_t k = RB_Minimizer::default_k; RB_KmerTable kmer_table; EList seqs; seqs.reserveExact(repeat_map_.size()); for(map::const_iterator it = repeat_map_.begin(); it != repeat_map_.end(); it++) { const RB_Repeat& repeat = *(it->second); assert(repeat.satisfy(rp)); seqs.expand(); seqs.back() = repeat.consensus(); } kmer_table.build(seqs, window, k); kmer_table.dump(cerr); cerr << endl; string query, rc_query; EList > minimizers; size_t total = 0, num_repeat = 0, correct = 0, false_positive = 0, false_negative = 0; for(size_t i = 0; i + rp.min_repeat_len <= forward_length_; i += 1000) { if(coordHelper_.getEnd(i) != coordHelper_.getEnd(i + rp.min_repeat_len)) continue; query = getString(s_, i, rp.min_repeat_len); rc_query = reverseComplement(query); TIndexOffU idx = subSA_.find_repeat_idx(s_, query); const EList& test_repeat_index = subSA_.getRepeatIndex(); bool repeat = (idx < test_repeat_index.size()); bool est_repeat = kmer_table.isRepeat(query, rc_query, minimizers); total++; if(repeat) num_repeat++; if(repeat == est_repeat) { correct++; } else { if(est_repeat) { false_positive++; } else { false_negative++; assert(false); } } } cerr << "total: " << total << endl; cerr << "repeat: " << num_repeat << endl; cerr << "correct: " << correct << endl; cerr << "false positive: " << false_positive << endl; cerr << "false negative: " << false_negative << endl; cerr << endl; ELList position2D; EList alignments; size_t repeat_total = 0, repeat_aligned = 0; const EList& test_repeat_index = subSA_.getRepeatIndex(); size_t interval = 1; if(test_repeat_index.size() >= 1000) { interval = test_repeat_index.size() / 1000; } size_t total_alignments = 0, max_alignments = 0; string query2, rc_query2; for(size_t i = 0; i < test_repeat_index.size(); i += interval) { TIndexOffU saElt_idx = test_repeat_index[i]; TIndexOffU saElt_idx_end = (i + 1 < test_repeat_index.size() ? test_repeat_index[i+1] : subSA_.size()); TIndexOffU saElt_size = saElt_idx_end - saElt_idx; TIndexOffU saElt = subSA_[saElt_idx]; query = getString(s_, saElt, rp.min_repeat_len); query2 = query; // introduce three mismatches into a query when the query is at lest 100-bp if(rp.min_repeat_len >= 100) { const size_t mid_pos1 = (size_t)(rp.min_repeat_len * 0.1); if(query2[mid_pos1] == 'A') { query2[mid_pos1] = 'C'; } else { query2[mid_pos1] = 'A'; } const size_t mid_pos2 = (size_t)(rp.min_repeat_len * 0.5); if(query2[mid_pos2] == 'C') { query2[mid_pos2] = 'G'; } else { query2[mid_pos2] = 'C'; } const size_t mid_pos3 = (size_t)(rp.min_repeat_len * 0.9); if(query2[mid_pos3] == 'G') { query2[mid_pos3] = 'T'; } else { query2[mid_pos3] = 'G'; } } repeat_total += saElt_size; size_t found = 0; size_t cur_alignments = 0; if(kmer_table.isRepeat(query2, minimizers)) { kmer_table.findAlignments(query2, minimizers, position2D, alignments); total_alignments += (alignments.size() * saElt_size); cur_alignments = alignments.size(); TIndexOffU baseoff = 0; for(size_t s = 0; s < seqs.size(); s++) { int spos = seqs[s].find(query); if(spos != string::npos) { for(size_t a = 0; a < alignments.size(); a++) { if(alignments[a].pos == baseoff + spos) { found++; } } } baseoff += seqs[s].length(); } assert_leq(found, 1); } rc_query = reverseComplement(query); rc_query2 = reverseComplement(query2); size_t rc_found = 0; if(kmer_table.isRepeat(rc_query2, minimizers)) { kmer_table.findAlignments(rc_query2, minimizers, position2D, alignments); total_alignments += (alignments.size() * saElt_size); cur_alignments += alignments.size(); if(cur_alignments > max_alignments) { max_alignments = cur_alignments; } TIndexOffU baseoff = 0; for(size_t s = 0; s < seqs.size(); s++) { int spos = seqs[s].find(rc_query); if(spos != string::npos) { for(size_t a = 0; a < alignments.size(); a++) { if(alignments[a].pos == baseoff + spos) { rc_found++; } } } baseoff += seqs[s].length(); } assert_leq(rc_found, 1); } if(found + rc_found == 1) { repeat_aligned += saElt_size; } } cerr << "num repeats: " << repeat_total << endl; cerr << "repeat aligned using minimizers: " << repeat_aligned << endl; cerr << "average number of alignments: " << (float)total_alignments / repeat_total << endl; cerr << "max alignment: " << max_alignments << endl; cerr << endl; } cerr << "number of repeats is " << repeat_map_.size() << endl; size_t total_rep_seq_len = 0, total_allele_seq_len = 0; size_t i = 0; for(map::iterator it = repeat_map_.begin(); it != repeat_map_.end(); it++, i++) { RB_Repeat& repeat = *(it->second); if(!repeat.satisfy(rp)) continue; repeat.saveSeedExtension(rp, s_, coordHelper_, i, fp, total_rep_seq_len, total_allele_seq_len); } size_t total_qual_seeds = 0; for(map::iterator it = repeat_map_.begin(); it != repeat_map_.end(); it++, i++) { RB_Repeat& repeat = *(it->second); EList& seeds = repeat.seeds(); for(size_t i = 0; i < seeds.size(); i++) { if(seeds[i].getLength() < rp.min_repeat_len) continue; total_qual_seeds++; } } cerr << "total repeat sequence length: " << total_rep_seq_len << endl; cerr << "total allele sequence length: " << total_allele_seq_len << endl; cerr << "total number of seeds including those that are position-wise different, but sequence-wise identical: " << total_qual_seeds << endl; cerr << endl; fp << "total repeat sequence length: " << total_rep_seq_len << endl; fp << "total allele sequence length: " << total_allele_seq_len << endl; fp << "total number of seeds including those that are position-wise different, but sequence-wise identical: " << total_qual_seeds << endl; fp.close(); delete repeat_manager; repeat_manager = NULL; // remove non-qualifying repeats // and update repeat IDs for those remaining { map temp_repeat_map; size_t i = 0; for(map::iterator it = repeat_map_.begin(); it != repeat_map_.end(); it++) { RB_Repeat* repeat = it->second; if(!repeat->satisfy(rp)) { delete repeat; continue; } repeat->repeat_id(i); temp_repeat_map[repeat->repeat_id()] = repeat; i++; } repeat_map_ = temp_repeat_map; } const bool sanity_check = true; if(sanity_check) { EList > kmer_table; string query; size_t total = 0, match = 0; EList positions; const EList& test_repeat_index = subSA_.getRepeatIndex(); size_t interval = 1; if(test_repeat_index.size() >= 10000) { interval = test_repeat_index.size() / 10000; } for(size_t i = 0; i < test_repeat_index.size(); i += interval) { TIndexOffU saElt_idx = test_repeat_index[i]; TIndexOffU saElt_idx_end = (i + 1 < test_repeat_index.size() ? test_repeat_index[i+1] : subSA_.size()); positions.clear(); for(size_t j = saElt_idx; j < saElt_idx_end; j++) { positions.push_back(subSA_[j]); #ifndef NDEBUG if(j > saElt_idx) { TIndexOffU lcp_len = getLCP(s_, coordHelper_, positions[0], positions.back(), rp.min_repeat_len); assert_eq(lcp_len, rp.min_repeat_len); } TIndexOffU saElt = subSA_[j]; TIndexOffU start = coordHelper_.getStart(saElt); TIndexOffU start2 = coordHelper_.getStart(saElt + rp.min_repeat_len - 1); assert_eq(start, start2); #endif } TIndexOffU saElt = subSA_[saElt_idx]; size_t true_count = saElt_idx_end - saElt_idx; getString(s_, saElt, rp.min_repeat_len, query); total++; size_t count = 0, rc_count = 0; string rc_query = reverse_complement(query); for(map::iterator it = repeat_map_.begin(); it != repeat_map_.end(); it++) { RB_Repeat& repeat = *(it->second); int pos = repeat.consensus().find(query); if(pos != string::npos) { for(size_t s = 0; s < repeat.seeds().size(); s++) { SeedExt& seed = repeat.seeds()[s]; string seq; seed.getExtendedSeedSequence(s_, seq); if(seq.find(query) != string::npos) count++; } } pos = repeat.consensus().find(rc_query); if(pos != string::npos) { for(size_t s = 0; s < repeat.seeds().size(); s++) { SeedExt& seed = repeat.seeds()[s]; string seq; seed.getExtendedSeedSequence(s_, seq); if(seq.find(rc_query) != string::npos) rc_count++; } } } if(count == true_count || rc_count == true_count) { match++; } else if(total - match <= 10) { cerr << " query: " << query << endl; cerr << "rc_query: " << rc_query << endl; cerr << "true count: " << true_count << endl; cerr << "found count: " << count << endl; cerr << "rc found count: " << rc_count << endl; cerr << endl; } } cerr << "RepeatBuilder: sanity check: " << match << " passed (out of " << total << ")" << endl << endl; } } template void RepeatBuilder::writeHaploType(const EList& haplo_lists, const EList& seeds, TIndexOffU& hapl_id_base, const string& seq_name, ostream &fp) { if(haplo_lists.size() == 0) { return; } for(size_t i = 0; i < haplo_lists.size(); i++) { const SeedHP &haploType = haplo_lists[i]; fp << "rpht" << hapl_id_base++; fp << "\t" << seq_name; fp << "\t" << haploType.range.first; fp << "\t" << haploType.range.second; fp << "\t"; assert_gt(haploType.snpIDs.size(), 0); for (size_t j = 0; j < haploType.snpIDs.size(); j++) { if(j) { fp << ","; } fp << haploType.snpIDs[j]; } fp << endl; } } template void RepeatBuilder::writeAllele(TIndexOffU grp_id, TIndexOffU allele_id, Range range, const string& seq_name, TIndexOffU baseoff, const EList& seeds, ostream &fp) { // >rpt_name*0\trep\trep_pos\trep_len\tpos_count\t0 // chr_name:pos:direction chr_name:pos:direction // // >rep1*0 rep 0 100 470 0 // 22:112123123:+ 22:1232131113:+ // size_t snp_size = seeds[range.first].snps.size(); size_t pos_size = range.second - range.first; fp << ">"; fp << "rpt_" << grp_id << "*" << allele_id; fp << "\t" << seq_name; fp << "\t" << baseoff + seeds[range.first].consensus_pos.first; fp << "\t" << seeds[range.first].consensus_pos.second - seeds[range.first].consensus_pos.first; fp << "\t" << pos_size; fp << "\t" << snp_size; fp << "\t"; for(size_t i = 0; i < snp_size; i++) { if(i > 0) {fp << ",";} fp << "rps" << seeds[range.first].snps[i]->id; } fp << endl; // print positions for(size_t i = 0; i < pos_size; i++) { if(i > 0 && (i % 10 == 0)) { fp << endl; } if(i % 10 != 0) { fp << " "; } string chr_name; TIndexOffU pos_in_chr; TIndexOffU joinedOff = seeds[range.first + i].pos.first; bool fw = true; if(joinedOff < forward_length_) { fw = true; } else { fw = false; joinedOff = s_.length() - joinedOff - seeds[range.first].getLength(); } coordHelper_.getGenomeCoord(joinedOff, chr_name, pos_in_chr); char direction = fw ? '+' : '-'; fp << chr_name << ":" << pos_in_chr << ":" << direction; } fp << endl; } template void RepeatBuilder::writeSNPs(ostream& fp, const string& rep_chr_name, const ESet& editset) { for(size_t i = 0; i < editset.size(); i++) { const Edit& ed = editset[i]; if(ed.isMismatch()) { fp << "rps" << ed.snpID; fp << "\t" << "single"; fp << "\t" << rep_chr_name; fp << "\t" << ed.pos; fp << "\t" << ed.qchr; fp << endl; } else { assert(false); } } } template void RepeatBuilder::saveFile(const RepeatParameter& rp) { saveRepeats(rp); } bool RB_RepeatManager::checkRedundant(const RepeatParameter& rp, const map& repeat_map, const EList& positions, EList& to_remove) const { to_remove.clear(); bool replace = false; for(size_t i = 0; i < positions.size(); i++) { TIndexOffU seed_pos = positions[i]; Range seed_range(seed_pos + rp.seed_len, 0); map >::const_iterator it = range_to_repeats_.upper_bound(seed_range); if(it == range_to_repeats_.end()) continue; for(map >::const_reverse_iterator rit(it); rit != range_to_repeats_.rend(); rit++) { Range repeat_range = rit->first; if(repeat_range.first + rp.max_repeat_len <= seed_pos) break; const EList& repeat_ids = rit->second; assert_gt(repeat_ids.size(), 0); for(size_t r = 0; r < repeat_ids.size(); r++) { size_t repeat_id = repeat_ids[r]; size_t idx = to_remove.bsearchLoBound(repeat_id); if(idx < to_remove.size() && to_remove[idx] == repeat_id) continue; bool overlap = seed_pos < repeat_range.second && seed_pos + rp.seed_len > repeat_range.first; if(!overlap) continue; map::const_iterator it2 = repeat_map.find(repeat_id); assert(it2 != repeat_map.end()); const RB_Repeat& repeat = *(it2->second); const EList& allele_ranges = repeat.seed_ranges(); size_t num_contain = 0, num_overlap = 0, num_close = 0, num_overlap_bp = 0; size_t p = 0, p2 = 0; while(p < positions.size() && p2 < allele_ranges.size()) { RB_AlleleCoord range; range.left = positions[p]; range.right = positions[p] + rp.seed_len; RB_AlleleCoord range2 = allele_ranges[p2]; if(range2.contain(range)) { num_contain++; num_overlap_bp += rp.seed_len; } else { TIndexOffU overlap = range2.overlap_len(range); if(overlap > 0) { num_overlap++; num_overlap_bp += (range2.right - range.left); } else if(range.right + 10 > range2.left && range2.right + 10 > range.left) { num_close++; } } if(range.right <= range2.right) p++; else p2++; } // if the number of matches is >= 90% of positions in the smaller group if((num_contain + num_overlap) * 10 + num_close * 8 >= min(positions.size(), allele_ranges.size()) * 9) { if(positions.size() <= allele_ranges.size()) { return true; } else { replace = true; to_remove.push_back(repeat_id); to_remove.sort(); } } } } // DK - check this out if(replace) break; } return false; } void RB_RepeatManager::addRepeat(const RB_Repeat* repeat) { const EList& allele_ranges = repeat->seed_ranges(); for(size_t i = 0; i < allele_ranges.size(); i++) { Range allele_range(allele_ranges[i].left, allele_ranges[i].right); addRepeat(allele_range, repeat->repeat_id()); } } void RB_RepeatManager::addRepeat(Range range, size_t repeat_id) { if(range_to_repeats_.find(range) == range_to_repeats_.end()) { range_to_repeats_[range] = EList(); } EList& repeat_ids = range_to_repeats_[range]; size_t idx = repeat_ids.bsearchLoBound(repeat_id); if(idx < repeat_ids.size() && repeat_ids[idx] == repeat_id) return; repeat_ids.push_back(repeat_id); repeat_ids.sort(); } void RB_RepeatManager::removeRepeat(const RB_Repeat* repeat) { const EList& allele_ranges = repeat->seed_ranges(); for(size_t p = 0; p < allele_ranges.size(); p++) { Range range(allele_ranges[p].left, allele_ranges[p].right); removeRepeat(range, repeat->repeat_id()); } } void RB_RepeatManager::removeRepeat(Range range, size_t repeat_id) { EList& repeat_ids = range_to_repeats_[range]; TIndexOffU idx = repeat_ids.bsearchLoBound(repeat_id); if(idx < repeat_ids.size() && repeat_id == repeat_ids[idx]) { repeat_ids.erase(idx); if(repeat_ids.empty()) range_to_repeats_.erase(range); } } void RB_RepeatManager::showInfo(const RepeatParameter& rp, CoordHelper& coordHelper, const map& repeat_map, size_t num_case) const { size_t count = 0; for(map >::const_iterator it = range_to_repeats_.begin(); it != range_to_repeats_.end(); it++) { const Range& range = it->first; if(range.second - range.first < rp.min_repeat_len) continue; map >::const_iterator jt = it; jt++; for(; jt != range_to_repeats_.end(); jt++) { const Range& range2 = jt->first; if(range2.second - range2.first < rp.min_repeat_len) continue; if(range.second <= range2.first) break; if(count < num_case) { cerr << "range (" << range.first << ", " << range.second << ") vs. range2 ("; cerr << range2.first << ", " << range2.second << ")" << endl; for(size_t i = 0; i < it->second.size(); i++) { cerr << "\t1 " << it->second[i] << endl; const RB_Repeat* repeat = repeat_map.find(it->second[i])->second; repeat->showInfo(rp, coordHelper); } for(size_t i = 0; i < jt->second.size(); i++) { cerr << "\t2 " << jt->second[i] << endl; const RB_Repeat* repeat = repeat_map.find(jt->second[i])->second; repeat->showInfo(rp, coordHelper); } cerr << endl << endl; } count++; } } cerr << "ShowInfo - count: " << count << endl; } template void RepeatBuilder::reassignSeeds(const RepeatParameter& rp, size_t repeat_bid, // repeat begin id size_t repeat_eid, // repeat end id EList& seeds) { assert_lt(repeat_bid, repeat_eid); EList updated; updated.resizeExact(repeat_eid - repeat_bid); updated.fillZero(); string seq; for(size_t s = 0; s < seeds.size(); s++) { SeedExt& seed = seeds[s]; size_t max_repeat_id = repeat_bid; size_t max_ext_len = 0; for(size_t i = repeat_bid; i < repeat_eid; i++) { map::iterator it = repeat_map_.find(i); assert(it != repeat_map_.end()); const RB_Repeat& repeat = *(it->second); const string& consensus = repeat.consensus(); const size_t ext_len = (consensus.length() - rp.seed_len) / 2; if(seed.bound.first + ext_len > seed.pos.first) continue; if(seed.pos.second + ext_len > seed.bound.second) continue; getString(s_, seed.pos.first - ext_len, rp.seed_len + ext_len * 2, seq); assert_eq(consensus.length(), seq.length()); size_t tmp_ext_len = 0; for(size_t j = 0; j < ext_len; j++, tmp_ext_len++) { if(seq[ext_len - j - 1] != consensus[ext_len - j - 1] || seq[ext_len + rp.seed_len + j] != consensus[ext_len + rp.seed_len + j]) { break; } } if(tmp_ext_len > max_ext_len) { max_repeat_id = i; max_ext_len = tmp_ext_len; } } if(rp.seed_len + max_ext_len * 2 >= rp.min_repeat_len) { seed.pos.first -= max_ext_len; seed.pos.second += max_ext_len; map::iterator it = repeat_map_.find(max_repeat_id); assert(it != repeat_map_.end()); RB_Repeat& repeat = *(it->second); const string& consensus = repeat.consensus(); const size_t ext_len = (consensus.length() - rp.seed_len) / 2; assert_leq(max_ext_len, ext_len); seed.consensus_pos.first = ext_len - max_ext_len; seed.consensus_pos.second = consensus.length() - (ext_len - max_ext_len); assert_leq(seed.consensus_pos.second - seed.consensus_pos.first, consensus.length()); repeat.addSeed(seed); updated[max_repeat_id - repeat_bid] = true; } } for(size_t i = repeat_bid; i < repeat_eid; i++) { if(!updated[i - repeat_bid]) continue; map::iterator it = repeat_map_.find(i); assert(it != repeat_map_.end()); RB_Repeat& repeat = *(it->second); repeat.update(); } } /** * TODO * @brief * * @param rpt_seq * @param rpt_range */ template void RepeatBuilder::addRepeatGroup(const RepeatParameter& rp, size_t& repeat_id, RB_RepeatManager& repeat_manager, const RB_RepeatBase& repeatBase, ostream& fp) { RB_Repeat* repeat = new RB_Repeat; repeat->repeat_id(repeat_id); repeat->init(rp, s_, coordHelper_, subSA_, repeatBase); assert(repeat_map_.find(repeat->repeat_id()) == repeat_map_.end()); repeat_map_[repeat->repeat_id()] = repeat; repeat_id++; } template bool RepeatBuilder::checkSequenceMergeable(const string& ref, const string& read, EList& edits, Coord& coord, TIndexOffU rpt_len, TIndexOffU max_edit) { size_t max_matchlen = 0; EList ed; string pad; swaligner_.makePadString(ref, read, pad, 5); string ref2 = pad + ref; string read2 = pad + read; swaligner_.alignStrings(ref2, read2, ed, coord); // match should start from pad string if(coord.off() != 0) { return false; } // no edits on pad string if(ed.size() > 0 && ed[0].pos < pad.length()) { return false; } size_t left = pad.length(); size_t right = left + read.length(); edits.clear(); edits.reserveExact(ed.size()); for(size_t i = 0; i < ed.size(); i++) { if(ed[i].pos >= left && ed[i].pos <= right) { edits.push_back(ed[i]); edits.back().pos -= left; } } max_matchlen = getMaxMatchLen(edits, read.length()); #ifdef DEBUGLOG { cerr << "After pad removed" << endl; BTDnaString btread; btread.install(read.c_str(), true); Edit::print(cerr, edits); cerr << endl; Edit::printQAlign(cerr, btread, edits); } #endif return (max_matchlen >= rpt_len); } template void RepeatBuilder::saveRepeats(const RepeatParameter &rp) { ios_base::openmode mode = ios_base::out; if(rp.append_result) { mode |= ios_base::app; } else { mode |= ios_base::trunc; } // Generate SNPs size_t i = 0; for(map::iterator it = repeat_map_.begin(); it != repeat_map_.end(); it++, i++) { RB_Repeat& repeat = *(it->second); if(!repeat.satisfy(rp)) continue; // for each repeats repeat.generateSNPs(rp, s_, i); } // save snp, consensus sequenuce, info string snp_fname = filename_ + ".rep.snp"; string info_fname = filename_ + ".rep.info"; string hapl_fname = filename_ + ".rep.haplotype"; ofstream snp_fp(snp_fname.c_str(), mode); ofstream info_fp(info_fname.c_str(), mode); ofstream hapl_fp(hapl_fname.c_str(), mode); const string repName = "rep" + to_string(rp.min_repeat_len) + "-" + to_string(rp.max_repeat_len); i = 0; TIndexOffU consensus_baseoff = 0; TIndexOffU snp_id_base = 0; TIndexOffU hapl_id_base = 0; for(map::iterator it = repeat_map_.begin(); it != repeat_map_.end(); it++, i++) { RB_Repeat& repeat = *(it->second); if(!repeat.satisfy(rp)) continue; // for each repeats repeat.saveSNPs(snp_fp, i, snp_id_base); saveAlleles(rp, repName, repeat, info_fp, hapl_fp, i, hapl_id_base, consensus_baseoff); consensus_baseoff += repeat.consensus().length(); } snp_fp.close(); info_fp.close(); hapl_fp.close(); // save all consensus sequence saveConsensus(rp, repName); } template void RepeatBuilder::saveConsensus(const RepeatParameter &rp, const string& repName) { ios_base::openmode mode = ios_base::out; if(rp.append_result) { mode |= ios_base::app; } else { mode |= ios_base::trunc; } string fa_fname = filename_ + ".rep.fa"; ofstream fa_fp(fa_fname.c_str(), mode); fa_fp << ">" << repName << endl; size_t oskip = 0; for(map::iterator it = repeat_map_.begin(); it != repeat_map_.end(); it++) { RB_Repeat& repeat = *(it->second); if(!repeat.satisfy(rp)) continue; // for each repeats const string& constr = repeat.consensus(); size_t si = 0; size_t constr_len = constr.length(); while(si < constr_len) { size_t out_len = std::min((size_t)(output_width - oskip), (size_t)(constr_len - si)); fa_fp << constr.substr(si, out_len); if((oskip + out_len) == output_width) { fa_fp << endl; oskip = 0; } else { // last line oskip = oskip + out_len; } si += out_len; } } if(oskip) { fa_fp << endl; } fa_fp.close(); } template void RepeatBuilder::saveAlleles( const RepeatParameter& rp, const string& repName, RB_Repeat& repeat, ofstream& fp, ofstream& hapl_fp, TIndexOffU grp_id, TIndexOffU& hapl_id_base, TIndexOffU baseoff) { const EList& seeds = repeat.seeds(); EList haplo_lists; Range range(0, seeds.size()); int allele_id = 0; for(size_t sb = range.first; sb < range.second;) { size_t se = sb + 1; for(; se < range.second; se++) { if(!(SeedExt::isSameConsensus(seeds[sb], seeds[se]) && SeedExt::isSameSNPs(seeds[sb], seeds[se]) && seeds[sb].aligned == seeds[se].aligned)) { break; } } if(!seeds[sb].aligned) { sb = se; continue; } if(seeds[sb].getLength() < rp.min_repeat_len) { sb = se; continue; } // [sb, se) are same alleles writeAllele(grp_id, allele_id, Range(sb, se), repName, baseoff, seeds, fp); generateHaploType(Range(sb, se), seeds, haplo_lists); allele_id++; sb = se; } // sort HaploType List by pos haplo_lists.sort(); writeHaploType(haplo_lists, seeds, hapl_id_base, repName, hapl_fp); } template void RepeatBuilder::generateHaploType(Range range, const EList &seeds, EList &haplo_list) { const EList& snps = seeds[range.first].snps; if(snps.size() == 0) return; // right-most position TIndexOffU max_right_pos = seeds[range.first].consensus_pos.second - 1; #ifndef NDEBUG for(size_t i = 0; i < snps.size(); i++) { assert_leq(snps[i]->pos, max_right_pos); } #endif // create haplotypes of at least 16 bp long to prevent combinations of SNPs // break a list of SNPs into several haplotypes if a SNP is far from the next SNP in the list const TIndexOffU min_ht_len = 16; size_t eb = 0, ee = 1; while(ee < snps.size() + 1) { if(ee == snps.size() || snps[eb]->pos + (min_ht_len << 1) < snps[ee]->pos) { TIndexOffU left_pos = snps[eb]->pos; TIndexOffU right_pos = snps[ee-1]->pos; if(snps[ee-1]->type == EDIT_TYPE_READ_GAP) { right_pos += snps[ee-1]->len; } if(left_pos + min_ht_len - 1 > right_pos) { right_pos = left_pos + min_ht_len - 1; } right_pos = min(max_right_pos, right_pos); assert_leq(left_pos, right_pos); SeedHP seedHP; seedHP.range.first = left_pos; seedHP.range.second = right_pos; for(size_t i = eb; i < ee; i++) { string snp_ids = "rps" + to_string(snps[i]->id); seedHP.snpIDs.push_back(snp_ids); } // Add to haplo_list bool found = false; for(size_t i = 0; i < haplo_list.size(); i++) { if(haplo_list[i] == seedHP) { found = true; break; } } if(!found) { // Add haplo_list.push_back(seedHP); } eb = ee; } ee++; } } void RB_SubSA::init(TIndexOffU sa_size, TIndexOffU seed_len, TIndexOffU seed_count) { assert_gt(sa_size, 1); sa_size_ = sa_size; assert_gt(seed_len, 0); seed_len_ = seed_len; assert_gt(seed_count, 1); seed_count_ = seed_count; temp_suffixes_.clear(); repeat_list_.clear(); repeat_index_.clear(); } template void RB_SubSA::push_back(const TStr& s, CoordHelper& coordHelper, TIndexOffU saElt, bool lastInput) { if(saElt + seed_len() <= coordHelper.getEnd(saElt)) { if(seed_count_ == 1) { repeat_list_.push_back(saElt); } else { assert_gt(seed_count_, 1); if(temp_suffixes_.empty()) { temp_suffixes_.push_back(saElt); return; } TIndexOffU prev_saElt = temp_suffixes_.back(); // calculate common prefix length between two text. // text1 is started from prev_saElt and text2 is started from saElt bool same = isSameSequenceUpto(s, coordHelper, prev_saElt, saElt, seed_len_); if(same) { temp_suffixes_.push_back(saElt); } if(!same || lastInput) { if(temp_suffixes_.size() >= seed_count_) { repeat_index_.push_back(repeat_list_.size()); temp_suffixes_.sort(); for(size_t pi = 0; pi < temp_suffixes_.size(); pi++) { repeat_list_.push_back(temp_suffixes_[pi]); } } temp_suffixes_.clear(); if(!lastInput) { temp_suffixes_.push_back(saElt); } else { temp_suffixes_.nullify(); } } } } if(lastInput) { size_t bit = sizeof(uint32_t) * 8; size_t num = (repeat_list_.size() + bit - 1) / bit; done_.resizeExact(num); done_.fillZero(); #ifndef NDEBUG string prev_seq = ""; for(size_t i = 0; i < repeat_index_.size(); i++) { TIndexOffU saBegin = repeat_index_[i]; TIndexOffU saEnd = (i + 1 < repeat_index_.size() ? repeat_index_[i+1] : repeat_list_.size()); string seq = getString(s, repeat_list_[saBegin], seed_len()); for(size_t j = saBegin + 1; j < saEnd; j++) { string tmp_seq = getString(s, repeat_list_[j], seed_len()); assert_eq(seq, tmp_seq); } if(prev_seq != "" ) { assert_lt(prev_seq, seq); } prev_seq = seq; } #endif } } template Range RB_SubSA::find(const TStr& s, const string& seq) const { TIndexOffU i = find_repeat_idx(s, seq); if(i >= repeat_index_.size()) return Range(0, 0); Range range(repeat_index_[i], i + 1 < repeat_index_.size() ? repeat_index_[i+1] : repeat_list_.size()); return range; } template TIndexOffU RB_SubSA::find_repeat_idx(const TStr& s, const string& seq) const { assert_eq(seq.length(), seed_len_); string temp; size_t l = 0, r = repeat_index_.size(); while(l < r) { size_t m = (r + l) >> 1; TIndexOffU saElt = repeat_list_[repeat_index_[m]]; getString(s, saElt, seed_len_, temp); if(seq == temp) { return m; } else if(seq < temp) { r = m; } else { assert(seq > temp); l = m + 1; } } return repeat_index_.size(); } void RB_SubSA::setDone(TIndexOffU off, TIndexOffU len) { assert_leq(off + len, repeat_list_.size()); const TIndexOffU bit = sizeof(uint32_t) * 8; for(TIndexOffU i = off; i < off + len; i++) { TIndexOffU quotient = i / bit; TIndexOffU remainder = i % bit; assert_lt(quotient, done_.size()); uint32_t num = done_[quotient]; num = num | (1 << remainder); done_[quotient] = num; } } bool RB_SubSA::isDone(TIndexOffU off, TIndexOffU len) const { assert_leq(off + len, repeat_list_.size()); const TIndexOffU bit = sizeof(uint32_t) * 8; for(TIndexOffU i = off; i < off + len; i++) { TIndexOffU quotient = i / bit; TIndexOffU remainder = i % bit; assert_lt(quotient, done_.size()); uint32_t num = done_[quotient]; num = num & (1 << remainder); if(num == 0) return false; } return true; } template void RB_SubSA::buildRepeatBase(const TStr& s, CoordHelper& coordHelper, const size_t max_len, EList& repeatBases) { if(repeat_index_.empty()) return; done_.fillZero(); EList senseDominant; senseDominant.resizeExact(repeat_index_.size()); senseDominant.fillZero(); EList repeatStack; EList > size_table; size_table.reserveExact(repeat_index_.size() / 2 + 1); for(size_t i = 0; i < repeat_index_.size(); i++) { TIndexOffU begin = repeat_index_[i]; TIndexOffU end = (i + 1 < repeat_index_.size() ? repeat_index_[i+1] : repeat_list_.size()); assert_lt(begin, end); EList positions; positions.reserveExact(end - begin); for(size_t j = begin; j < end; j++) positions.push_back(repeat_list_[j]); if(!isSenseDominant(coordHelper, positions, seed_len_)) continue; senseDominant[i] = 1; size_table.expand(); size_table.back().first = end - begin; size_table.back().second = i; } size_table.sort(); size_t bundle_count = 0; string tmp_str; EList tmp_ranges; tmp_ranges.resizeExact(4); EList > tmp_sort_ranges; tmp_sort_ranges.resizeExact(4); for(int64_t i = (int64_t)size_table.size() - 1; i >= 0; i--) { TIndexOffU idx = size_table[i].second; ASSERT_ONLY(TIndexOffU num = size_table[i].first); assert_lt(idx, repeat_index_.size()); #ifndef NDEBUG if(idx + 1 < repeat_index_.size()) { assert_eq(repeat_index_[idx] + num, repeat_index_[idx+1]); } else { assert_eq(repeat_index_[idx] + num, repeat_list_.size()); } #endif TIndexOffU saBegin = repeat_index_[idx]; if(isDone(saBegin)) { assert(isDone(saBegin, num)); continue; } ASSERT_ONLY(size_t rb_done = 0); assert_lt(saBegin, repeat_list_.size()); repeatStack.push_back(idx); while(!repeatStack.empty()) { TIndexOffU idx = repeatStack.back(); assert(senseDominant[idx]); repeatStack.pop_back(); assert_lt(idx, repeat_index_.size()); TIndexOffU saBegin = repeat_index_[idx]; TIndexOffU saEnd = (idx + 1 < repeat_index_.size() ? repeat_index_[idx + 1] : repeat_list_.size()); if(isDone(saBegin)) { assert(isDone(saBegin, saEnd - saBegin)); continue; } TIndexOffU saElt = repeat_list_[saBegin]; size_t ri = repeatBases.size(); repeatBases.expand(); repeatBases.back().seq = getString(s, saElt, seed_len_); repeatBases.back().nodes.clear(); repeatBases.back().nodes.push_back(idx); ASSERT_ONLY(rb_done++); setDone(saBegin, saEnd - saBegin); bool left = true; while(repeatBases[ri].seq.length() <= max_len) { if(left) { tmp_str = "N"; tmp_str += repeatBases[ri].seq.substr(0, seed_len_ - 1); } else { tmp_str = repeatBases[ri].seq.substr(repeatBases[ri].seq.length() - seed_len_ + 1, seed_len_ - 1); tmp_str.push_back('N'); } assert_eq(tmp_str.length(), seed_len_); for(size_t c = 0; c < 4; c++) { if(left) tmp_str[0] = "ACGT"[c]; else tmp_str.back() = "ACGT"[c]; assert_eq(tmp_str.length(), seed_len_); TIndexOffU idx = find_repeat_idx(s, tmp_str); size_t num = 0; if(idx < repeat_index_.size()) { if(idx + 1 < repeat_index_.size()) { num = repeat_index_[idx+1] - repeat_index_[idx]; } else { num = repeat_list_.size() - repeat_index_[idx]; } } tmp_ranges[c].first = idx; tmp_ranges[c].second = num; assert(num == 0 || num >= seed_count_); tmp_sort_ranges[c].first = num; tmp_sort_ranges[c].second = c; if(idx == repeat_index_.size() || isDone(repeat_index_[idx]) || !senseDominant[idx]) { #ifndef NDEBUG if(idx < repeat_index_.size()) { assert(isDone(repeat_index_[idx], num) || !senseDominant[idx]); } #endif tmp_sort_ranges[c].first = 0; tmp_ranges[c].second = 0; } } tmp_sort_ranges.sort(); if(tmp_sort_ranges[3].first < seed_count_) { if(left) { left = false; continue; } else { break; } } for(size_t cc = 0; cc < 3; cc++) { assert_leq(tmp_sort_ranges[cc].first, tmp_sort_ranges[cc+1].first); if(tmp_sort_ranges[cc].first < seed_count_) continue; size_t c = tmp_sort_ranges[cc].second; repeatStack.push_back(tmp_ranges[c].first); } size_t c = tmp_sort_ranges[3].second; if(repeatBases[ri].seq.length() >= max_len) { assert_eq(repeatBases[ri].seq.length(), max_len); TIndexOffU idx = tmp_ranges[c].first; assert(!isDone(repeat_index_[idx])); repeatStack.push_back(idx); if(left) { left = false; continue; } else { break; } } else { TIndexOffU idx = tmp_ranges[c].first; TIndexOffU num = tmp_ranges[c].second; setDone(repeat_index_[idx], num); if(left) { repeatBases[ri].seq.insert(0, 1, "ACGT"[c]); repeatBases[ri].nodes.insert(idx, 0); } else { repeatBases[ri].seq.push_back("ACGT"[c]); repeatBases[ri].nodes.push_back(idx); } } } } assert_gt(rb_done, 0); bundle_count++; } cerr << "Bundle count: " << bundle_count << endl; #ifndef NDEBUG { set idx_set; for(size_t i = 0; i < repeatBases.size(); i++) { const RB_RepeatBase& repeatBase = repeatBases[i]; for(size_t j = 0; j < repeatBase.nodes.size(); j++) { assert(idx_set.find(repeatBase.nodes[j]) == idx_set.end()); idx_set.insert(repeatBase.nodes[j]); } } } #endif #if 0 { EList > kmer_table; string query; size_t total = 0, match = 0; size_t interval = 1; if(repeat_index_.size() >= 10000) { interval = repeat_index_.size() / 10000; } EList positions; for(size_t i = 0; i < repeat_index_.size(); i += interval) { TIndexOffU saElt_idx = repeat_index_[i]; TIndexOffU saElt_idx_end = (i + 1 < repeat_index_.size() ? repeat_index_[i+1] : repeat_list_.size()); positions.clear(); for(size_t j = saElt_idx; j < saElt_idx_end; j++) { positions.push_back(repeat_list_[j]); #ifndef NDEBUG if(j > saElt_idx) { TIndexOffU lcp_len = getLCP(s, coordHelper, positions[0], positions.back(), seed_len_); assert_geq(lcp_len, seed_len_); } TIndexOffU saElt = repeat_list_[j]; TIndexOffU start = coordHelper.getStart(saElt); TIndexOffU start2 = coordHelper.getStart(saElt + seed_len_ - 1); assert_eq(start, start2); #endif } TIndexOffU saElt = repeat_list_[saElt_idx]; size_t true_count = saElt_idx_end - saElt_idx; getString(s, saElt, seed_len_, query); total++; size_t count = 0; for(size_t r = 0; r < repeatBases.size(); r++) { const RB_RepeatBase& repeat = repeatBases[r]; int pos = repeat.seq.find(query); if(pos != string::npos) { for(size_t j = 0; j < repeat.nodes.size(); j++) { TIndexOffU _node = repeat.nodes[j]; TIndexOffU _saElt_idx = repeat_index_[_node]; TIndexOffU _saElt_idx_end = (_node + 1 < repeat_index_.size() ? repeat_index_[_node+1] : repeat_list_.size()); TIndexOffU _saElt = repeat_list_[_saElt_idx]; string seq = getString(s, _saElt, seed_len()); if(query == seq) { count += (_saElt_idx_end - _saElt_idx); } } } } size_t rc_count = 0; string rc_query = reverse_complement(query); for(size_t r = 0; r < repeatBases.size(); r++) { const RB_RepeatBase& repeat = repeatBases[r]; int pos = repeat.seq.find(rc_query); if(pos != string::npos) { for(size_t j = 0; j < repeat.nodes.size(); j++) { TIndexOffU _node = repeat.nodes[j]; TIndexOffU _saElt_idx = repeat_index_[_node]; TIndexOffU _saElt_idx_end = (_node + 1 < repeat_index_.size() ? repeat_index_[_node+1] : repeat_list_.size()); TIndexOffU _saElt = repeat_list_[_saElt_idx]; string seq = getString(s, _saElt, seed_len()); if(rc_query == seq) { rc_count += (_saElt_idx_end - _saElt_idx); } } } } if(count == true_count || rc_count == true_count) { match++; } else if(total - match <= 10) { cerr << "query: " << query << endl; cerr << "true count: " << true_count << endl; cerr << "found count: " << count << endl; cerr << "rc found count: " << rc_count << endl; cerr << endl; } } cerr << "RB_SubSA: sanity check: " << match << " passed (out of " << total << ")" << endl << endl; } #endif } #define write_fp(x) fp.write((const char *)&(x), sizeof((x))) void RB_SubSA::writeFile(ofstream& fp) { write_fp(sa_size_); write_fp(seed_len_); write_fp(seed_count_); size_t sz = temp_suffixes_.size(); write_fp(sz); for(size_t i = 0; i < sz; i++) { write_fp(temp_suffixes_[i]); } sz = repeat_index_.size(); write_fp(sz); for(size_t i = 0; i < sz; i++) { write_fp(repeat_index_[i]); } sz = done_.size(); write_fp(sz); for(size_t i = 0; i < sz; i++) { write_fp(done_[i]); } } #define read_fp(x) fp.read((char *)&(x), sizeof((x))) void RB_SubSA::readFile(ifstream& fp) { TIndexOffU val; read_fp(val); rt_assert_eq(val, sa_size_); read_fp(val); rt_assert_eq(val, seed_len_); read_fp(val); rt_assert_eq(val, seed_count_); size_t sz; read_fp(sz); temp_suffixes_.resizeExact(sz); for(size_t i = 0; i < sz; i++) { read_fp(temp_suffixes_[i]); } size_t val_sz; // repeat_index_ read_fp(val_sz); repeat_index_.resizeExact(val_sz); for(size_t i = 0; i < val_sz; i++) { read_fp(repeat_index_[i]); } // done read_fp(val_sz); done_.resizeExact(val_sz); for(size_t i = 0; i < val_sz; i++) { read_fp(done_[i]); } } /****************************/ template class RepeatBuilder >; template void dump_tstr(const SString& ); template bool compareRepeatCoordByJoinedOff(const RepeatCoord& , const RepeatCoord&); hisat2-2.2.1/repeat_builder.h000066400000000000000000000654661370663763200161030ustar00rootroot00000000000000 /* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef __REPEAT_BUILDER_H__ #define __REPEAT_BUILDER_H__ #include #include #include #include #include "assert_helpers.h" #include "word_io.h" #include "mem_ids.h" #include "ref_coord.h" #include "ref_read.h" #include "edit.h" #include "ds.h" #include "repeat.h" #include "blockwise_sa.h" #include "simple_func.h" #include "scoring.h" #include "aligner_sw.h" #include "bit_packed_array.h" //#define DEBUGLOG using namespace std; /** * Encapsulates repeat parameters. */ class RepeatParameter { public: TIndexOffU seed_len; // seed length TIndexOffU seed_count; // seed count TIndexOffU seed_mm; // maximum edit distance allowed during initial seed extension TIndexOffU repeat_count; // repeat count TIndexOffU min_repeat_len; // minimum repeat length TIndexOffU max_repeat_len; // maximum repeat length TIndexOffU max_edit; // maximum edit distance allowed bool symmetric_extend; // extend symmetrically TIndexOffU extend_unit_len; // extend seeds no longer than this length at a time bool append_result; }; typedef pair Range; struct Fragments { bool contain(TIndexOffU pos) { if (pos >= joinedOff && pos < (joinedOff + length)) { return true; } return false; } TIndexOffU joinedOff; // index within joined text TIndexOffU length; int frag_id; int seq_id; TIndexOffU seqOff; // index within sequence bool first; }; class CoordHelper { public: CoordHelper(TIndexOffU length, TIndexOffU forward_length, const EList& szs, const EList& ref_names); ~CoordHelper(); int mapJoinedOffToSeq(TIndexOffU joined_pos); int getGenomeCoord(TIndexOffU joined_pos, string& chr_name, TIndexOffU& pos_in_chr); TIndexOffU getEnd(TIndexOffU e); TIndexOffU getStart(TIndexOffU e); TIndexOffU forward_length() const { return forward_length_; } TIndexOffU length() const { return length_; } private: void buildNames(); void buildJoinedFragment(); private: TIndexOffU length_; TIndexOffU forward_length_; EList szs_; EList ref_names_; EList ref_namelines_; // mapping info from joined string to genome EList fraglist_; // Fragments Cache #define CACHE_SIZE_JOINEDFRG 10 Fragments cached_[CACHE_SIZE_JOINEDFRG]; int num_cached_ = 0; int victim_ = 0; /* round-robin */ }; struct SeedHP { bool operator==(const SeedHP &rhs) const { return range == rhs.range && snpIDs == rhs.snpIDs; } bool operator!=(const SeedHP &rhs) const { return !(rhs == *this); } bool operator<(const SeedHP &rhs) const { return range < rhs.range; } bool operator>(const SeedHP &rhs) const { return rhs < *this; } bool operator<=(const SeedHP &rhs) const { return !(rhs < *this); } bool operator>=(const SeedHP &rhs) const { return !(*this < rhs); } Range range; EList snpIDs; }; struct SeedSNP { int type; // EDIT_TYPE_MM, EDIT_TYPE_READ_GAP, EDIT_TYPE_REF_GAP TIndexOffU pos; size_t len; string base; // valid when ty = MM, REF_GAP TIndexOffU id; SeedSNP() {} void init(int ty, TIndexOffU po, size_t ln, string bs) { type = ty; pos = po; len = ln; base = bs; } void init(int ty, TIndexOffU po, size_t ln, char bs) { type = ty; pos = po; len = ln; base.assign(1, bs); } friend ostream &operator<<(ostream &os, const SeedSNP &snp) { if(snp.type == EDIT_TYPE_MM) { os << "single"; } else if(snp.type == EDIT_TYPE_REF_GAP) { os << "insertion"; } else if(snp.type == EDIT_TYPE_READ_GAP) { os << "deletion"; } os << " "; os << snp.pos << " "; if(snp.type == EDIT_TYPE_MM || snp.type == EDIT_TYPE_REF_GAP) { os << snp.base; } else if(snp.type == EDIT_TYPE_READ_GAP) { os << snp.len; } return os; } bool operator==(const SeedSNP &rhs) const { return type == rhs.type && pos == rhs.pos && len == rhs.len && base == rhs.base; } bool operator!=(const SeedSNP &rhs) const { return !(rhs == *this); } static bool cmpSeedSNPByPos( SeedSNP * const& a, SeedSNP * const& b) { return a->pos < b->pos; } }; class SeedExt { public: SeedExt() { reset(); }; void reset() { done = false; curr_ext_len = 0; ed = total_ed = 0; orig_pos.first = 0; orig_pos.second = 0; pos.first = 0; pos.second = 0; bound.first = 0; bound.second = 0; consensus_pos.first = 0; consensus_pos.second = 0; left_gaps.clear(); right_gaps.clear(); aligned = true; }; TIndexOffU getLeftExtLength() const { assert_leq(pos.first, orig_pos.first); TIndexOffU len = orig_pos.first - pos.first; return len; } TIndexOffU getRightExtLength() const { assert_geq(pos.second, orig_pos.second); return pos.second - orig_pos.second; } TIndexOffU getLength() const { TIndexOffU len = orig_pos.second - orig_pos.first; len += getLeftExtLength(); len += getRightExtLength(); return len; } template void getLeftExtString(const TStr& s, string& seq) { seq.clear(); seq = getString(s, pos.first, orig_pos.first - pos.first); } template void getRightExtString(const TStr& s, string& seq) { seq.clear(); seq = getString(s, orig_pos.second, pos.second - orig_pos.second); } template void getExtendedSeedSequence(const TStr& s, string& seq) const; template void generateSNPs(const TStr& s, const string& consensus, EList& repeat_snps); static bool isSameConsensus(const SeedExt& a, const SeedExt& b) { return (a.consensus_pos == b.consensus_pos); //&& (a.getLength() == b.getLength()); } static bool isSameSNPs(const SeedExt& a, const SeedExt& b) { const EList& a_edit = a.snps; const EList& b_edit = b.snps; if(a_edit.size() != b_edit.size()) { return false; } for(size_t i = 0; i < a_edit.size(); i++) { if(!(*a_edit[i] == *b_edit[i])) { return false; } } return true; } static bool isSameAllele(const SeedExt& a, const SeedExt& b) { const EList& a_edit = a.edits; const EList& b_edit = b.edits; if(a_edit.size() != b_edit.size()) { return false; } for(size_t i = 0; i < a_edit.size(); i++) { if(!(a_edit[i] == b_edit[i])) { return false; } } return true; } static bool sort_by_edits(const SeedExt& a, const SeedExt& b) { const EList& a_edit = a.edits; const EList& b_edit = b.edits; size_t i = 0; while((i < a_edit.size()) && (i < b_edit.size())) { if(!(a_edit[i] == b_edit[i])) { if(a_edit[i] < b_edit[i]) { return true; } else { return false; } } i++; } if(a_edit.size() < b_edit.size()) { return true; } if((a_edit.size() == b_edit.size()) && (a.pos.first < b.pos.first)) { return true; } return false; } void generateEdits(const string& consensus_merged, const string& seed_ext) { size_t ed_done = 0; size_t ext_len = getLength(); assert_eq(ext_len, seed_ext.length()); edits.clear(); for(size_t i = 0; i < ext_len && ed_done < total_ed; i++) { char con_base = consensus_merged[consensus_pos.first + i]; char seed_base = seed_ext[i]; if (con_base != seed_base) { edits.expand(); edits.back().init(consensus_pos.first + i, con_base, seed_base, EDIT_TYPE_MM); ed_done++; } } } Range getExtendedRange(size_t consensus_len) const { assert_leq(consensus_pos.second, consensus_len); Range range; range.first = (pos.first < consensus_pos.first ? 0 : pos.first - consensus_pos.first); range.second = pos.second + (consensus_len - consensus_pos.second); return range; } #ifndef NDEBUG bool valid() const { assert_leq(consensus_pos.first, consensus_pos.second); TIndexOffU constr_len = consensus_pos.second - consensus_pos.first; TIndexOffU allele_len = getLength(); TIndexOffU cur_off = 0; for(size_t i = 0; i < left_gaps.size(); i++) { assert_geq(left_gaps[i].first, cur_off); cur_off = left_gaps[i].first; int gap_len = left_gaps[i].second; assert_neq(gap_len, 0); if(gap_len > 0) { // deletion allele_len += gap_len; } else { allele_len += gap_len; cur_off += (-gap_len); } } cur_off = 0; for(size_t i = 0; i < right_gaps.size(); i++) { assert_geq(right_gaps[i].first, cur_off); cur_off = right_gaps[i].first; int gap_len = right_gaps[i].second; assert_neq(gap_len, 0); if(gap_len > 0) { // deletion allele_len += gap_len; } else { allele_len += gap_len; cur_off += (-gap_len); } } assert_eq(constr_len, allele_len); return true; } #endif public: // seed extended position [first, second) pair orig_pos; pair pos; // extension bound. the seed must be placed on same fragment // [first, second) pair bound; // positions relative to consensus sequence pair consensus_pos; // a list of gaps (deletions and insertions) in both directions // offsets from seed's left and right ("pos" above) // positive and negative values indicate deletions and insertions, resp. EList > left_gaps; EList > right_gaps; uint32_t ed; // edit distance uint32_t total_ed; // total edit distance bool done; // done flag uint32_t curr_ext_len; // bool aligned; EList edits; // edits w.r.t. consensus_merged EList snps; }; class RB_AlleleCoord { public: RB_AlleleCoord() : left(0), right(0), idx(0) {} RB_AlleleCoord(TIndexOffU l, TIndexOffU r, size_t i) : left(l), right(r), idx(i) {} TIndexOffU len() const { return right - left; } bool operator<(const RB_AlleleCoord& o) const { if(left != o.left) return left < o.left; if(right != o.right) return right > o.right; return false; } bool contain(const RB_AlleleCoord& o, size_t relax = 0) const { if(o.left + relax >= left && o.right <= right + relax) return true; else return false; } bool contained(const RB_AlleleCoord& o) const { return o.contain(*this); } TIndexOffU overlap_len(const RB_AlleleCoord& o) const { if(contain(o)) return o.len(); else if(o.contain(*this)) return len(); else if(left < o.right && right > o.left) { if(left <= o.left) { return right - o.left; } else { return o.right - left; } } return 0; } public: TIndexOffU left; TIndexOffU right; size_t idx; }; class RB_RepeatManager; class RB_SWAligner; class RB_SubSA; class RB_RepeatBase { public: string seq; EList nodes; }; class RB_Repeat { public: RB_Repeat() {} ~RB_Repeat() { if(snps_.size() > 0) { for(size_t i = 0; i < snps_.size(); i++) { delete snps_[i]; } } } void repeat_id(size_t repeat_id) { repeat_id_ = repeat_id; } size_t repeat_id() const { return repeat_id_; } void parent_id(size_t parent_id) { parent_id_ = parent_id; } size_t parent_id() const { return parent_id_; } string& consensus() { return consensus_; } const string& consensus() const { return consensus_; } EList& seeds() { return seeds_; } const EList& seeds() const { return seeds_; } EList& seed_ranges() { return seed_ranges_; } const EList& seed_ranges() const { return seed_ranges_; } EList& snps() { return snps_; } const EList& snps() const { return snps_; } template void init(const RepeatParameter& rp, const TStr& s, CoordHelper& coordHelper, const RB_SubSA& subSA, const RB_RepeatBase& repeatBase); template void extendConsensus(const RepeatParameter& rp, const TStr& s); template void getNextRepeat(const RepeatParameter& rp, const TStr& s, RB_Repeat& o); template void saveSeedExtension(const RepeatParameter& rp, const TStr& s, CoordHelper& coordHelper, TIndexOffU seed_grp_id, ostream& fp, size_t& total_repeat_seq_len, size_t& total_allele_seq_len) const; void saveSNPs(ofstream& fp, TIndexOffU grp_id, TIndexOffU& snp_id_base); void saveConsensus(ofstream& fp, TIndexOffU grp_id); bool overlap(const RB_Repeat& o, bool& contain, bool& left, size_t& seed_i, size_t& seed_j, bool debug = false) const; bool satisfy(const RepeatParameter& rp) const { if(consensus_.length() < rp.min_repeat_len) return false; if(seeds_.size() < rp.repeat_count) return false; if(seeds_[rp.repeat_count - 1].getLength() < rp.min_repeat_len) return false; return true; } void reset() { consensus_.clear(); for(size_t i = 0; i < seeds_.size(); i++) seeds_[i].reset(); seeds_.clear(); seed_ranges_.clear(); } void showInfo(const RepeatParameter& rp, CoordHelper& coordHelper) const; template void generateSNPs(const RepeatParameter&, const TStr& s, TIndexOffU grp_id); bool self_repeat() const { return self_repeat_; } void update() { internal_update(); } void addSeed(const SeedExt& seed) { seeds_.expand(); seeds_.back() = seed; } bool contain(TIndexOffU left, TIndexOffU right) const; protected: template void get_consensus_seq(const TStr& s, EList& seeds, size_t sb, size_t se, size_t min_left_ext, size_t min_right_ext, size_t max_ed, const RepeatParameter& rp, EList& ed_seed_nums, EList* left_consensuses, EList* right_consensuses) const; void internal_update(); protected: size_t repeat_id_; size_t parent_id_; string consensus_; EList seeds_; EList seed_ranges_; EList snps_; bool self_repeat_; static EList ca_ed_; static EList ca_ed2_; static string ca_s_; static string ca_s2_; public: static size_t seed_merge_tried; static size_t seed_merged; }; class RB_RepeatExt : public RB_Repeat { public: RB_RepeatExt() {} ~RB_RepeatExt() {} template void extendConsensus(const RepeatParameter& rp, const TStr& s); float mergeable(const RB_Repeat& o) const; template bool merge(const RepeatParameter& rp, const TStr& s, RB_SWAligner& swalginer, const RB_RepeatExt& o, bool contain, size_t seed_i, size_t seed_j, bool debug = false); protected: template void get_consensus_seq(const TStr& s, EList& seeds, size_t sb, size_t se, size_t min_left_ext, size_t min_right_ext, size_t max_ed, const RepeatParameter& rp, EList& ed_seed_nums, EList* left_consensuses, EList* right_consensuses) const; template bool align(const RepeatParameter& rp, const TStr& ref, const string& s, const EList >& s_kmer_table, const string& s2, EList& offsets, size_t k, SeedExt& seed, int consensus_approx_left, int consensus_approx_right, size_t left, size_t right, bool debug); bool isSelfRepeat(const RepeatParameter& rp, const string& s, const EList >& s_kmer_table, EList& offsets, size_t k, bool debug); }; // check if a set of seeds are already processed class RB_RepeatManager { public: size_t numCoords() const { return range_to_repeats_.size(); } bool checkRedundant(const RepeatParameter& rp, const map& repeat_map, const EList& positions, EList& to_remove) const; void addRepeat(const RB_Repeat* repeat); void addRepeat(Range range, size_t repeat_id); void removeRepeat(const RB_Repeat* repeat); void removeRepeat(Range range, size_t repeat_id); public: void showInfo(const RepeatParameter& rp, CoordHelper& coordHelper, const map& repeat_map, size_t num_case = 5) const; private: map > range_to_repeats_; }; class RB_SWAligner { public: RB_SWAligner(); ~RB_SWAligner(); void init_dyn(const RepeatParameter& rp); int alignStrings(const string &ref, const string &read, EList& edits, Coord& coord); void makePadString(const string& ref, const string& read, string& pad, size_t len); void doTest(const RepeatParameter& rp, const string& refstr, const string& readstr); void doTestCase1(const string& refstr, const string& readstr, TIndexOffU rpt_edit); private: // SimpleFunc scoreMin_; SimpleFunc nCeil_; SimpleFunc penCanIntronLen_; SimpleFunc penNoncanIntronLen_; Scoring *sc_; SwAligner swa; LinkedEList > rawEdits_; RandomSource rnd_; }; // SA Subset class RB_SubSA { public: RB_SubSA() {} ~RB_SubSA() {} void writeFile(ofstream& fp); void readFile(ifstream &fp); void init(TIndexOffU sa_size, TIndexOffU seed_len, TIndexOffU seed_count); template void push_back(const TStr& s, CoordHelper& coordHelper, TIndexOffU saElt, bool lastInput = false); inline TIndexOffU seed_len() const { return seed_len_; } inline TIndexOffU seed_count() const { return seed_count_; } inline size_t size() const { return repeat_list_.size(); } TIndexOffU get(size_t idx) const { return repeat_list_[idx]; } inline TIndexOffU operator[](size_t i) const { return get(i); } const EList& getRepeatIndex() const { return repeat_index_; } template Range find(const TStr& s, const string& seq) const; template TIndexOffU find_repeat_idx(const TStr& s, const string& seq) const; void setDone(TIndexOffU off, TIndexOffU len = 1); bool isDone(TIndexOffU off, TIndexOffU len = 1) const; void dump() const { cerr << "seed length: " << seed_len_ << endl; cerr << "minimum seed count: " << seed_count_ << endl; cerr << "number of seed groups: " << repeat_index_.size() << endl; cerr << "number of seeds: " << repeat_list_.size() << endl; } size_t getMemUsage() const { size_t tot = 0; tot += repeat_list_.totalCapacityBytes(); tot += repeat_index_.totalCapacityBytes(); tot += done_.totalCapacityBytes(); tot += temp_suffixes_.totalCapacityBytes(); return tot; } template void buildRepeatBase(const TStr& s, CoordHelper& coordHelper, const size_t max_len, EList& repeatBases); private: TIndexOffU sa_size_; TIndexOffU seed_len_; TIndexOffU seed_count_; EList temp_suffixes_; // repeat index EList repeat_list_; EList repeat_index_; EList done_; }; // find and write repeats template class RepeatBuilder { public: RepeatBuilder(TStr& s, const EList& szs, const EList& ref_names, bool forward_only, const string& filename); ~RepeatBuilder(); public: void readSA(const RepeatParameter& rp, BlockwiseSA& sa); void readSA(const RepeatParameter& rp, const string& filename); void readSA(const RepeatParameter& rp, const BitPackedArray& sa); void writeSA(const RepeatParameter& rp, const string& filename); void build(const RepeatParameter& rp); void sortRepeatGroup(); void saveRepeats(const RepeatParameter& rp); void saveConsensus(const RepeatParameter& rp, const string& repName); void saveFile(const RepeatParameter& rp); void addRepeatGroup(const RepeatParameter& rp, size_t& repeat_id, RB_RepeatManager& repeat_manger, const RB_RepeatBase& repeatBase, ostream& fp); void reassignSeeds(const RepeatParameter& rp, size_t repeat_bid, size_t repeat_eid, EList& seeds); bool checkSequenceMergeable(const string& ref, const string& read, EList& edits, Coord& coord, TIndexOffU rpt_len, TIndexOffU max_edit = 10); void doTest(const RepeatParameter& rp, const string& refstr, const string& readstr) { swaligner_.doTest(rp, refstr, readstr); } private: void saveAlleles(const RepeatParameter& rp, const string& repName, RB_Repeat& repeat, ofstream&, ofstream&, TIndexOffU grp_id, TIndexOffU&, TIndexOffU baseoff); void writeAllele(TIndexOffU grp_id, TIndexOffU allele_id, Range range, const string& seq_name, TIndexOffU baseoff, const EList& seeds, ostream &fp); void writeSNPs(ostream& fp, const string& rep_chr_name, const ESet& editset); void writeHaploType(const EList& haplo_list, const EList& seeds, TIndexOffU& hapl_id_base, const string& seq_name, ostream& fp); void generateHaploType(Range range, const EList& seeds, EList& haplo_list); private: const int output_width = 60; TStr& s_; bool forward_only_; string filename_; RB_SubSA subSA_; TIndexOffU forward_length_; CoordHelper coordHelper_; // RB_SWAligner swaligner_; // Seeds EList consensus_all_; ELList seeds_; map repeat_map_; }; int strcmpPos(const string&, const string&, TIndexOffU&); template void dump_tstr(TStr& s); #endif /* __REPEAT_BUILDER_H__ */ hisat2-2.2.1/repeat_kmer.h000066400000000000000000000504711370663763200154010ustar00rootroot00000000000000 /* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef __REPEAT_KMER_H__ #define __REPEAT_KMER_H__ #include #include #include #include #include #include "assert_helpers.h" #include "word_io.h" #include "mem_ids.h" #include "ds.h" template class RB_Minimizer { public: static const size_t default_w = 5; static const size_t default_k = 31; public: static pair get_minimizer(const TStr& seq, size_t off, size_t window, size_t k) { assert_leq(k, 32); assert_leq(off + window + k - 1, seq.length()); pair minimizer(get_kmer(seq, off, k), off); uint64_t kmer = minimizer.first; for(size_t i = off + 1; i < off + window; i++) { uint64_t next_kmer = get_next_kmer(kmer, seq[i+k-1], k); if(minimizer_leq(next_kmer, minimizer.first)) { minimizer.first = next_kmer; minimizer.second = i; } kmer = next_kmer; } return minimizer; } static void get_minimizer(const TStr& seq, size_t window, size_t k, EList >& minimizers) { assert_leq(k, 32); assert_leq(window + k - 1, seq.length()); minimizers.clear(); pair minimizer = get_minimizer(seq, 0, window, k); minimizers.push_back(minimizer); uint64_t kmer = get_kmer(seq, window - 1, k); for(size_t i = 1; i + window + k - 1 <= seq.length(); i++) { uint64_t next_kmer = get_next_kmer(kmer, seq[i+window+k-2], k); if(minimizer.second < i) { minimizer = get_minimizer(seq, i, window, k); } else if(minimizer_leq(next_kmer, minimizer.first)) { minimizer.first = next_kmer; minimizer.second = i + window - 1; } minimizers.push_back(minimizer); kmer = next_kmer; } #ifndef NDEBUG assert_eq(minimizers.size() + window + k - 2, seq.length()); for(size_t i = 0; i + window + k - 1 <= seq.length(); i++) { pair minimizer = get_minimizer(seq, i, window, k); assert(minimizer == minimizers[i]); } #endif } protected: static bool minimizer_leq(uint64_t kmer, uint64_t kmer2) { #if 1 kmer = convert_minimizer(kmer); kmer2 = convert_minimizer(kmer2); #endif return kmer <= kmer2; } // Heng Li's minimap and minaisam paper, 2016 static uint64_t convert_minimizer(uint64_t x) { x = (~x) + (x << 21); x = x ^ (x >> 24); x = x + (x << 3) + (x << 8); x = x ^ (x >> 14); x = x + (x << 2) + (x << 4); x = x ^ (x >> 28); x = x + (x << 31); return x; } static uint64_t get_kmer(const TStr& seq, size_t offset, size_t k) { assert_leq(offset + k, seq.length()); uint64_t kmer = 0; for(size_t i = 0; i < k; i++) { size_t c = seq[offset + i]; if(c > 3) c = asc2dna[c]; kmer = (kmer << 2 | c); } return kmer; } static uint64_t get_next_kmer(uint64_t kmer, size_t base, size_t k) { kmer &= (((uint64_t)1 << ((k-1)*2))) - 1; if(base > 3) base = asc2dna[base]; kmer = (kmer << 2) | base; return kmer; } static TStr get_string(uint64_t kmer, size_t k) { TStr seq = ""; for(size_t i = 0; i < k; i++) { size_t nt = kmer & 0x3; seq.push_back("ACGT"[nt]); kmer >>= 2; } reverse(seq.begin(), seq.end()); return seq; } }; struct RB_Alignment { TIndexOffU pos; TIndexOffU off; TIndexOffU len; bool operator<(const RB_Alignment& o) const { if(pos != o.pos) return pos < o.pos; return len > o.len; } }; struct RB_Alignment_CMPbyLen { bool operator()(const RB_Alignment& a, const RB_Alignment& b) { if(a.len != b.len) return a.len > b.len; return a.pos < b.pos; } }; class RB_KmerTable { public: RB_KmerTable() { w_ = k_ = 0; } ~RB_KmerTable() {} public: bool isIn(uint64_t kmer) const { #if 1 pair key(kmer, 0); size_t idx = kmer_table_.bsearchLoBound(key); return idx < kmer_table_.size() && kmer_table_[idx].first == kmer; #else return kmers_.find(kmer) != kmers_.end(); #endif } template bool isRepeat(const TStr& query, const TStr& rc_query, EList >& minimizers) const { return isRepeat(query, minimizers) || isRepeat(rc_query, minimizers); } template bool isRepeat(const TStr& query, EList >& minimizers) const { RB_Minimizer::get_minimizer(query, w_, k_, minimizers); size_t est_count = 0; uint64_t prev_minimizer = 0; bool prev_in = false; for(size_t j = 0; j < minimizers.size(); j++) { bool curr_in = false; if(minimizers[j].first == prev_minimizer) { if(prev_in) est_count++; curr_in = prev_in; } else if(isIn(minimizers[j].first)) { curr_in = true; est_count++; #if 1 return true; #endif } prev_minimizer = minimizers[j].first; prev_in = curr_in; } #if 1 return false; #else bool est_repeat = est_count * 10 >= minimizers.size(); return est_repeat; #endif } template void findRepeats(const TStr& query, EList >& minimizers, EList& repeats) const { repeats.clear(); RB_Minimizer::get_minimizer(query, w_, k_, minimizers); for(size_t i = 0; i < minimizers.size(); i++) { if(i > 0 && minimizers[i].first == minimizers[i-1].first) continue; pair minimizer(minimizers[i].first, 0); size_t j = kmer_table_.bsearchLoBound(minimizer); for(; j < kmer_table_.size() && minimizer.first == kmer_table_[j].first; j++) { repeats.push_back(kmer_table_[j].second); } } if(repeats.empty()) return; size_t remove_count = 0; repeats.sort(); for(size_t i = 0; i + 1 < repeats.size();) { size_t j = i + 1; for(; j < repeats.size(); j++) { if(repeats[i] == repeats[j]) { repeats[j] = std::numeric_limits::max(); remove_count++; } else break; } i = j; } repeats.sort(); assert_lt(remove_count, repeats.size()); repeats.resize(repeats.size() - remove_count); } template void findAlignments(const TStr& query, EList >& minimizers, ELList& position2D, EList& alignments, TIndexOffU max_num_alignment = 1000) const { minimizers.clear(); RB_Minimizer::get_minimizer(query, w_, k_, minimizers); position2D.clear(); for(size_t i = 0; i < minimizers.size(); i++) { if(i > 0 && minimizers[i].first == minimizers[i-1].first) continue; pair minimizer(minimizers[i].first, 0); size_t idx = kmer_table_.bsearchLoBound(minimizer); if(idx < kmer_table_.size() && kmer_table_[idx].first == minimizer.first) { TIndexOffU begin = kmer_table_[idx].second; TIndexOffU end = (idx + 1 < kmer_table_.size() ? kmer_table_[idx+1].second : pos_list_.size()); position2D.expand(); EList& positions = position2D.back(); positions.clear(); positions.expand(); positions.back().pos = begin; // suffix begin positions.back().off = i; // minimizer index positions.expand(); positions.back().pos = end; // suffix end positions.back().off = i; // minimizer index } } alignments.clear(); if(position2D.empty()) return; for(size_t i = 0; i < position2D.size(); i++) { size_t num_i = 0, num_pos = numeric_limits::max(); for(size_t j = 0; j < position2D.size(); j++) { EList& positions = position2D[j]; if(positions.empty()) continue; assert_eq(positions.size(), 2); TIndexOffU cur_num_pos = positions[1].pos - positions[0].pos; if(cur_num_pos == 0) continue; if(cur_num_pos < num_pos) { num_i = j; num_pos = cur_num_pos; } } if(num_pos > max_num_alignment && alignments.size() > 0) break; if(num_pos > max_num_alignment) break; EList& positions = position2D[num_i]; TIndexOffU begin = positions[0].pos; TIndexOffU end = positions[1].pos; TIndexOffU min_i = positions[0].off; assert_eq(num_pos, end - begin); positions.clear(); for(TIndexOffU j = begin; j < end; j++) { if(pos_list_[j] < minimizers[min_i].second) continue; positions.expand(); positions.back().pos = pos_list_[j]; positions.back().off = minimizers[min_i].second; positions.back().len = k_; } if(i == 0) { for(size_t j = 0; j < positions.size(); j++) { alignments.expand(); alignments.back() = positions[j]; } } else { size_t a = 0, p = 0; size_t num_alignment = alignments.size(); while(a < num_alignment && p < positions.size()) { RB_Alignment& alignment = alignments[a]; RB_Alignment& position = positions[p]; if(alignment.pos < position.pos) { TIndexOffU offDiff = position.off - alignment.off; if(position.pos - alignment.pos == offDiff) { alignment.len = min(offDiff, alignment.len) + position.len; a++; p++; } else { a++; } } else if(alignment.pos > position.pos) { TIndexOffU offDiff = alignment.off - position.off; if(alignment.pos - position.pos == offDiff) { alignment.pos = position.pos; alignment.off = position.off; alignment.len = min(offDiff, position.len) + alignment.len; assert_geq(alignment.pos, alignment.off); a++; p++; } else { alignments.expand(); alignments.back() = position; p++; } } else { assert_eq(alignment.pos, position.pos); a++; p++; } } while(p < positions.size()) { RB_Alignment& position = positions[p]; alignments.expand(); alignments.back() = position; p++; } if(i + 1 < position2D.size()) { alignments.sort(); } } positions.clear(); if(alignments.size() >= max_num_alignment) { break; } } if(alignments.empty()) return; for(size_t i = 0; i < alignments.size(); i++) { alignments[i].pos -= alignments[i].off; } alignments.sort(); // remove duplicates size_t remove_count = 0; for(size_t i = 0; i + 1 + remove_count < alignments.size(); i++) { size_t j = i + 1 + remove_count; for(; j < alignments.size(); j++) { if(alignments[i].pos != alignments[j].pos) { assert_geq(j, i + 1); if(j > i + 1) { alignments[i+1] = alignments[j]; } break; } else { remove_count++; } } } assert_lt(remove_count, alignments.size()); if(remove_count > 0) { alignments.resize(alignments.size() - remove_count); } if(alignments.size() > 1) { sort(alignments.begin(), alignments.end(), RB_Alignment_CMPbyLen()); } } bool write(ofstream& f_out, bool bigEndian) const { writeIndex(f_out, w_, bigEndian); writeIndex(f_out, k_, bigEndian); writeIndex(f_out, kmer_table_.size(), bigEndian); for(size_t i = 0; i < kmer_table_.size(); i++) { writeIndex(f_out, kmer_table_[i].first, bigEndian); if(sizeof(TIndexOffU) == 4) { writeU32(f_out, kmer_table_[i].second, bigEndian); } else { assert_eq(sizeof(TIndexOffU), 8); writeIndex(f_out, kmer_table_[i].second, bigEndian); } } writeIndex(f_out, pos_list_.size(), bigEndian); for(size_t i = 0; i < pos_list_.size(); i++) { if(sizeof(TIndexOffU) == 4) { writeU32(f_out, pos_list_[i], bigEndian); } else { assert_eq(sizeof(TIndexOffU), 8); writeIndex(f_out, pos_list_[i], bigEndian); } } return true; } bool read(ifstream& f_in, bool bigEndian) { w_ = readIndex(f_in, bigEndian); k_ = readIndex(f_in, bigEndian); size_t kmer_size = readIndex(f_in, bigEndian); kmer_table_.reserveExact(kmer_size); while(kmer_table_.size() < kmer_size) { kmer_table_.expand(); kmer_table_.back().first = readIndex(f_in, bigEndian); if(sizeof(TIndexOffU) == 4) { kmer_table_.back().second = readU32(f_in, bigEndian); } else { assert_eq(sizeof(TIndexOffU), 8); kmer_table_.back().second = readIndex(f_in, bigEndian); } #if 0 kmers_.insert(kmer_table_.back().first); #endif } size_t pos_size = readIndex(f_in, bigEndian); pos_list_.reserveExact(pos_size); while(pos_list_.size() < pos_size) { pos_list_.expand(); if(sizeof(TIndexOffU) == 4) { pos_list_.back() = readU32(f_in, bigEndian); } else { assert_eq(sizeof(TIndexOffU), 8); pos_list_.back() = readIndex(f_in, bigEndian); } } return true; } public: template void build(const EList& seqs, size_t w, size_t k) { w_ = w; k_ = k; kmer_table_.clear(); pos_list_.clear(); EList > tmp_table; set tmp_kmers; TIndexOffU baseoff = 0; EList > minimizers; for(size_t s = 0; s < seqs.size(); s++) { const TStr& seq = seqs[s]; RB_Minimizer::get_minimizer(seq, w_, k_, minimizers); for(size_t i = 0; i < minimizers.size(); i++) { if(!tmp_table.empty() && tmp_table.back().first == minimizers[i].first && tmp_table.back().second == baseoff + minimizers[i].second) continue; tmp_table.expand(); tmp_table.back().first = minimizers[i].first; tmp_table.back().second = baseoff + minimizers[i].second; tmp_kmers.insert(minimizers[i].first); #if 0 kmers_.insert(minimizers[i].first); #endif } baseoff += seq.length(); } tmp_table.sort(); kmer_table_.reserveExact(tmp_kmers.size()); pos_list_.reserveExact(tmp_table.size()); for(size_t i = 0; i < tmp_table.size(); i++) { #ifndef NDEBUG if(!pos_list_.empty()) { assert_neq(pos_list_.back(), tmp_table[i].second); } #endif if(kmer_table_.empty() || kmer_table_.back().first != tmp_table[i].first) { kmer_table_.expand(); kmer_table_.back().first = tmp_table[i].first; kmer_table_.back().second = pos_list_.size(); } pos_list_.push_back(tmp_table[i].second); } assert_eq(kmer_table_.size(), tmp_kmers.size()); assert_eq(pos_list_.size(), tmp_table.size()); } void dump(ostream& o) const { o << "window : " << w_ << endl; o << "k length : " << k_ << endl; o << "number of kmer : " << kmer_table_.size() << endl; o << "number of pos : " << pos_list_.size() << endl; EList counts, counts_10; counts.resizeExact(10); counts_10.resizeExact(10); counts.fillZero(); counts_10.fillZero(); for(size_t i = 0; i < kmer_table_.size(); i++) { size_t count = 0; if(i + 1 < kmer_table_.size()) { count = kmer_table_[i+1].second - kmer_table_[i].second; } else { count = pos_list_.size() - kmer_table_[i].second; } assert_gt(count, 0); count -= 1; if(count < counts.size()) { counts[count]++; } size_t count_10 = count / 10; if(count_10 < counts_10.size()) { counts_10[count_10]++; } else { counts_10.back()++; } } for(size_t i = 0; i < counts.size(); i++) { o << "\t" << i + 1 << ": " << counts[i] << endl; } for(size_t i = 1; i < counts_10.size(); i++) { o << "\t" << i * 10 + 1; if(i + 1 < counts_10.size()) { o << " to " << (i+1) * 10 << ": "; } else { o << " or more: "; } o << counts_10[i] << endl; } } private: size_t w_; size_t k_; EList > kmer_table_; EList pos_list_; unordered_set kmers_; }; #endif /* __REPEAT_KMER_H__ */ hisat2-2.2.1/rfm.h000066400000000000000000001220571370663763200136670ustar00rootroot00000000000000/* * Copyright 2018, Chanhee Park and Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef RFM_H_ #define RFM_H_ #include "hgfm.h" /** * Extended Burrows-Wheeler transform data. * LocalEbwt is a specialized Ebwt index that represents ~64K bps * and therefore uses two bytes as offsets within 64K bps. * This class has only two additional member variables to denote the genomic sequenuce it represents: * (1) the contig index and (2) the offset within the contig. * */ template class LocalRFM : public GFM { typedef GFM PARENT_CLASS; public: /// Construct an Ebwt from the given input file LocalRFM(const string& in, ALTDB* altdb, FILE *in1, FILE *in2, char *mmFile1, char *mmFile2, bool switchEndian, size_t& bytesRead, size_t& bytesRead2, int needEntireReverse, bool fw, int32_t overrideOffRate, // = -1, int32_t offRatePlus, // = -1, bool useMm, // = false, bool useShmem, // = false, bool mmSweep, // = false, bool loadNames, // = false, bool loadSASamp, // = true, bool loadFtab, // = true, bool loadRstarts, // = true, bool verbose, // = false, bool startVerbose, // = false, bool passMemExc, // = false, bool sanityCheck, // = false) bool useHaplotype) : // = false GFM(in, altdb, NULL, NULL, needEntireReverse, fw, overrideOffRate, offRatePlus, useMm, useShmem, mmSweep, loadNames, loadSASamp, loadFtab, loadRstarts, true, // load Splice Sites verbose, startVerbose, passMemExc, sanityCheck, useHaplotype, true) { this->_in1 = in1; this->_in2 = in2; this->_repeat = true; bool subIndex = true; PARENT_CLASS::readIntoMemory( needEntireReverse, loadSASamp, loadFtab, loadRstarts, false, //justHeader &(this->_gh), mmSweep, loadNames, startVerbose, subIndex); // If the offRate has been overridden, reflect that in the // _eh._offRate field if(offRatePlus > 0 && this->_overrideOffRate == -1) { this->_overrideOffRate = this->_gh._offRate + offRatePlus; } if(this->_overrideOffRate > this->_gh._offRate) { this->_gh.setOffRate(this->_overrideOffRate); assert_eq(this->_overrideOffRate, this->_gh._offRate); } assert(this->repOk()); } /// Construct an Ebwt from the given header parameters and string /// vector, optionally using a blockwise suffix sorter with the /// given 'bmax' and 'dcv' parameters. The string vector is /// ultimately joined and the joined string is passed to buildToDisk(). template LocalRFM( TStr& s, InorderBlockwiseSA* sa, PathGraph* pg, EList >& alts, index_t local_size, const EList& refnames, bool packed, int needEntireReverse, int32_t lineRate, int32_t offRate, int32_t ftabChars, const string& file, // base filename for EBWT files bool fw, int dcv, EList& szs, index_t sztot, const RefReadInParams& refparams, uint32_t seed, ostream& out1, ostream& out2, int32_t overrideOffRate = -1, bool verbose = false, bool passMemExc = false, bool sanityCheck = false) : GFM(packed, needEntireReverse, lineRate, offRate, ftabChars, file, fw, dcv, szs, sztot, refparams, seed, overrideOffRate, verbose, passMemExc, sanityCheck) { const GFMParams& gh = this->_gh; assert(gh.repOk()); uint32_t be = this->toBe(); assert(out1.good()); assert(out2.good()); assert_eq(refnames.size(), 1); this->_refnames.push_back(refnames[0]); writeIndex(out1, gh._len, be); // length of string (and bwt and suffix array) streampos headerPos = out1.tellp(); writeIndex(out1, 0, be); // gbwtLen writeIndex(out1, 0, be); // num of nodes writeI32(out1, lineRate, be); writeI32(out1, 0, be); writeI32(out1, offRate, be); writeI32(out1, ftabChars, be); writeIndex(out1, 0, be); // eftabLen writeI32(out1, 0, be); // flag if(gh._len > 0) { assert_gt(szs.size(), 0); assert_gt(sztot, 0); // Not every fragment represents a distinct sequence - many // fragments may correspond to a single sequence. Count the // number of sequences here by counting the number of "first" // fragments. this->_nPat = 0; this->_nFrag = 0; for(size_t i = 0; i < szs.size(); i++) { if(szs[i].len > 0) this->_nFrag++; if(szs[i].first && szs[i].len > 0) this->_nPat++; } assert_eq(this->_nPat, 1); assert_geq(this->_nFrag, this->_nPat); this->_rstarts.reset(); writeIndex(out1, this->_nPat, be); assert_eq(this->_nPat, 1); this->_plen.init(new index_t[this->_nPat], this->_nPat); // For each pattern, set plen int npat = -1; for(size_t i = 0; i < szs.size(); i++) { if(szs[i].first && szs[i].len > 0) { if(npat >= 0) { writeIndex(out1, this->plen()[npat], be); } npat++; this->plen()[npat] = (szs[i].len + szs[i].off); } else { this->plen()[npat] += (szs[i].len + szs[i].off); } } assert_eq((index_t)npat, this->_nPat-1); writeIndex(out1, this->plen()[npat], be); // Write the number of fragments writeIndex(out1, this->_nFrag, be); if(refparams.reverse == REF_READ_REVERSE) { EList tmp(EBWT_CAT); reverseRefRecords(szs, tmp, false, verbose); this->szsToDisk(tmp, out1, refparams.reverse); } else { this->szsToDisk(szs, out1, refparams.reverse); } if(alts.empty()) { assert(pg == NULL); PARENT_CLASS::buildToDisk(*sa, s, out1, out2, headerPos); } else { assert(pg != NULL); // Re-initialize GFM parameters to reflect real number of edges (gbwt string) this->_gh.init( this->_gh.len(), pg->getNumEdges(), pg->getNumNodes(), this->_gh.lineRate(), this->_gh.offRate(), this->_gh.ftabChars(), 0, this->_gh.entireReverse()); PARENT_CLASS::buildToDisk(*pg, s, out1, out2, headerPos); } } // Now write reference sequence names on the end assert_eq(this->_refnames.size(), this->_nPat); for(index_t i = 0; i < this->_refnames.size(); i++) { out1 << this->_refnames[i].c_str(); if(i + 1 < this->_refnames.size()) cout << endl; else out1 << '\0'; } out1.flush(); out2.flush(); if(out1.fail() || out2.fail()) { cerr << "An error occurred writing the index to disk. Please check if the disk is full." << endl; throw 1; } } /** * Sanity-check various pieces of the Ebwt */ void sanityCheckAll(int reverse) const { if(this->_gh._len > 0) { PARENT_CLASS::sanityCheckAll(reverse); } } bool empty() const { return this->_gh._len == 0; } }; /** * Extended Burrows-Wheeler transform data. * RFM is a specialized Ebwt index that represents one global index and a large set of local indexes. * */ template class RFM : public GFM { typedef GFM PARENT_CLASS; public: /// Construct a GFM from the given input file RFM(const string& in, ALTDB* altdb, RepeatDB* repeatdb, EList* readLens, int needEntireReverse, bool fw, int32_t overrideOffRate, // = -1, int32_t offRatePlus, // = -1, bool useMm, // = false, bool useShmem, // = false, bool mmSweep, // = false, bool loadNames, // = false, bool loadSASamp, // = true, bool loadFtab, // = true, bool loadRstarts, // = true, bool loadSpliceSites, // = true, bool verbose, // = false, bool startVerbose, // = false, bool passMemExc, // = false, bool sanityCheck, // = false bool useHaplotype, // = false bool skipLoading = false) : GFM(in, altdb, repeatdb, readLens, needEntireReverse, fw, overrideOffRate, offRatePlus, useMm, useShmem, mmSweep, loadNames, loadSASamp, loadFtab, loadRstarts, loadSpliceSites, verbose, startVerbose, passMemExc, sanityCheck, useHaplotype, skipLoading), _in1(NULL), _in2(NULL) { _in1Str = in + ".1." + gfm_ext; _in2Str = in + ".2." + gfm_ext; } /// Construct a HGFM from the given header parameters and string /// vector, optionally using a blockwise suffix sorter with the /// given 'bmax' and 'dcv' parameters. The string vector is /// ultimately joined and the joined string is passed to buildToDisk(). template RFM( TStr& s, bool packed, int needEntireReverse, int32_t lineRate, int32_t offRate, int32_t ftabChars, int32_t localOffRate, int32_t localFtabChars, int nthreads, const string& snpfile, const string& htfile, const string& ssfile, const string& exonfile, const string& svfile, const string& repeatfile, const string& outfile, // base filename for GFM files bool fw, bool useBlockwise, TIndexOffU bmax, TIndexOffU bmaxSqrtMult, TIndexOffU bmaxDivN, int dcv, EList& is, EList& szs, index_t sztot, const RefReadInParams& refparams, bool localIndex, // create local indexes? EList* parent_szs, EList* parent_refnames, uint32_t seed, int32_t overrideOffRate = -1, bool verbose = false, bool passMemExc = false, bool sanityCheck = false); RFM() { clearLocalRFMs(); } /** * Load this Ebwt into memory by reading it in from the _in1 and * _in2 streams. */ void loadIntoMemory( int needEntireReverse, bool loadSASamp, bool loadFtab, bool loadRstarts, bool loadNames, bool verbose) { readIntoMemory( needEntireReverse, // require reverse index to be concatenated reference reversed loadSASamp, // load the SA sample portion? loadFtab, // load the ftab (_ftab[] and _eftab[])? loadRstarts, // load the r-starts (_rstarts[])? false, // stop after loading the header portion? NULL, // params false, // mmSweep loadNames, // loadNames verbose); // startVerbose } // I/O void readIntoMemory( int needEntireRev, bool loadSASamp, bool loadFtab, bool loadRstarts, bool justHeader, GFMParams *params, bool mmSweep, bool loadNames, bool startVerbose); /** * Frees memory associated with the Ebwt. */ void evictFromMemory() { assert(PARENT_CLASS::isInMemory()); clearLocalRFMs(); PARENT_CLASS::evictFromMemory(); } /** * Sanity-check various pieces of the Ebwt */ void sanityCheckAll(int reverse) const { PARENT_CLASS::sanityCheckAll(reverse); for(size_t i = 0; i < _localRFMs.size(); i++) { assert(_localRFMs[i] != NULL); _localRFMs[i]->sanityCheckAll(reverse); } } void getReferenceNames(EList& refnames) { for(size_t i = 0; i < _localRFMs.size(); i++) { assert_eq(_localRFMs[i]->refnames().size(), 1); refnames.push_back(_localRFMs[i]->refnames()[0]); } } void getReferenceLens(EList& reflens) { for(size_t i = 0; i < _localRFMs.size(); i++) { reflens.push_back(_localRFMs[i]->plen()[0]); } } index_t getLocalRFM_idx(index_t readLen) { for(size_t i = 0; i < this->_repeatLens.size(); i++) { if(this->_repeatLens[i].first >= readLen) { return i; } } return this->_repeatLens.size() - 1; } index_t getLocalRFM_idx(const char *refname) { for(size_t i = 0; i < this->_repeatLens.size(); i++) { assert_eq(_localRFMs[i]->refnames().size(), 1); string& ref = _localRFMs[i]->refnames()[0]; if(ref.compare(refname) == 0) { return i; } } return this->_repeatLens.size() - 1; } bool empty() const { return _localRFMs.empty(); } LocalRFM& getLocalRFM(index_t idx) { assert_lt(idx, this->_repeatLens.size()); return *_localRFMs[idx]; } RB_KmerTable& getKmertable(index_t idx) { assert_lt(idx, this->_repeat_kmertables.size()); return this->_repeat_kmertables[idx]; } void clearLocalRFMs() { for(size_t i = 0; i < _localRFMs.size(); i++) { assert(_localRFMs[i] != NULL); delete _localRFMs[i]; } _localRFMs.clear(); } public: EList _refLens; /// approx lens of ref seqs (excludes trailing ambig chars) EList*> _localRFMs; EList > _localRFMFilePos; FILE *_in1; // input fd for primary index file FILE *_in2; // input fd for secondary index file string _in1Str; string _in2Str; char *mmFile1_; char *mmFile2_; private: struct WorkerParam { // input SString s; EList > alts; EList > haplotypes; bool bigEndian; index_t local_offset; index_t curr_sztot; EList conv_local_szs; index_t local_sztot; index_t index_size; string file; InorderBlockwiseSA >* sa; index_t dcv; index_t seed; EList refnames; // output RefGraph* rg; PathGraph* pg; int threads; }; static void build_worker(void* vp); }; template void RFM::build_worker(void* vp) { WorkerParam& tParam = *(WorkerParam*)vp; if(tParam.alts.empty()) { tParam.sa = NULL; tParam.sa = new KarkkainenBlockwiseSA >( tParam.s, (index_t)(tParam.s.length()+1), tParam.threads, tParam.dcv, tParam.seed, false, /* this->_sanity */ false, /* this->_passMemExc */ false); /* this->_verbose */ assert(tParam.sa->suffixItrIsReset()); assert_eq(tParam.sa->size(), tParam.s.length()+1); } else { while(true) { tParam.rg = NULL, tParam.pg = NULL; bool exploded = false; try { tParam.rg = new RefGraph( tParam.s, tParam.conv_local_szs, tParam.alts, tParam.haplotypes, tParam.file, tParam.threads, /* num threads */ false); /* verbose? */ tParam.pg = new PathGraph( *tParam.rg, tParam.file, local_max_gbwt, 1, /* num threads */ false); /* verbose? */ } catch (const NongraphException& err) { cerr << "Warning: no variants or splice sites in this graph (" << tParam.curr_sztot << ")" << endl; delete tParam.rg; delete tParam.pg; tParam.alts.clear(); continue; } catch (const ExplosionException& err) { exploded = true; } if(!exploded) { if(!tParam.pg->generateEdges(*tParam.rg)) { cerr << "An error occurred - generateEdges" << endl; throw 1; } exploded = tParam.pg->getNumEdges() > local_max_gbwt; } if(exploded) { cerr << "Warning: a local graph exploded (offset: " << tParam.curr_sztot << ", length: " << tParam.local_sztot << ")" << endl; delete tParam.pg; tParam.pg = NULL; delete tParam.rg; tParam.rg = NULL; if(tParam.alts.size() <= 1) { tParam.alts.clear(); } else { for(index_t s = 2; s < tParam.alts.size(); s += 2) { tParam.alts[s >> 1] = tParam.alts[s]; } tParam.alts.resize(tParam.alts.size() >> 1); tParam.haplotypes.clear(); for(index_t a = 0; a < tParam.alts.size(); a++) { const ALT& alt = tParam.alts[a]; if(!alt.snp()) continue; tParam.haplotypes.expand(); tParam.haplotypes.back().left = alt.pos; if(alt.deletion()) { tParam.haplotypes.back().right = alt.pos + alt.len - 1; } else { tParam.haplotypes.back().right = alt.pos; } tParam.haplotypes.back().alts.clear(); tParam.haplotypes.back().alts.push_back(a); } } continue; } break; } } } /// Construct a GFM from the given header parameters and string /// vector, optionally using a blockwise suffix sorter with the /// given 'bmax' and 'dcv' parameters. The string vector is /// ultimately joined and the joined string is passed to buildToDisk(). template template RFM::RFM( TStr& s, bool packed, int needEntireReverse, int32_t lineRate, int32_t offRate, int32_t ftabChars, int32_t localOffRate, int32_t localFtabChars, int nthreads, const string& snpfile, const string& htfile, const string& ssfile, const string& exonfile, const string& svfile, const string& repeatfile, const string& outfile, // base filename for EBWT files bool fw, bool useBlockwise, TIndexOffU bmax, TIndexOffU bmaxSqrtMult, TIndexOffU bmaxDivN, int dcv, EList& is, EList& szs, index_t sztot, const RefReadInParams& refparams, bool localIndex, EList* parent_szs, EList* parent_refnames, uint32_t seed, int32_t overrideOffRate, bool verbose, bool passMemExc, bool sanityCheck) : GFM(s, packed, needEntireReverse, lineRate, offRate, ftabChars, nthreads, snpfile, htfile, ssfile, exonfile, svfile, repeatfile, outfile, fw, useBlockwise, bmax, bmaxSqrtMult, bmaxDivN, dcv, is, szs, sztot, refparams, parent_szs, parent_refnames, seed, overrideOffRate, verbose, passMemExc, sanityCheck), _in1(NULL), _in2(NULL) { _in1Str = outfile + ".1." + gfm_ext; _in2Str = outfile + ".2." + gfm_ext; // Open output files ofstream fout1(_in1Str.c_str(), ios::binary); if(!fout1.good()) { cerr << "Could not open index file for writing: \"" << _in1Str.c_str() << "\"" << endl << "Please make sure the directory exists and that permissions allow writing by" << endl << "HISAT2." << endl; throw 1; } ofstream fout2(_in2Str.c_str(), ios::binary); if(!fout2.good()) { cerr << "Could not open index file for writing: \"" << _in2Str.c_str() << "\"" << endl << "Please make sure the directory exists and that permissions allow writing by" << endl << "HISAT2." << endl; throw 1; } // Split the whole genome into a set of local indexes uint32_t be = this->toBe(); assert(fout1.good()); assert(fout2.good()); // When building an Ebwt, these header parameters are known // "up-front", i.e., they can be written to disk immediately, // before we join() or buildToDisk() writeI32(fout1, 1, be); // endian hint for priamry stream writeI32(fout2, 1, be); // endian hint for secondary stream int version = GFM::getIndexVersion(); writeI32(fout1, version, be); // version index_t nLocalRFMs = this->_repeatLens.size(); writeIndex(fout1, nLocalRFMs, be); // number of local Ebwts for(size_t i = 0; i < this->_repeatLens.size(); i++) { writeIndex(fout1, this->_repeatLens[i].first, be); writeIndex(fout1, this->_repeatLens[i].second, be); } streampos filepos = fout1.tellp(); _localRFMFilePos.resizeExact(szs.size()); for(size_t i = 0; i < _localRFMFilePos.size(); i++) { writeIndex(fout1, 0, be); writeIndex(fout1, 0, be); } assert_gt(this->_nthreads, 0); WorkerParam tParam; tParam.rg = NULL; tParam.pg = NULL; tParam.sa = NULL; tParam.file = outfile; tParam.dcv = 1024; tParam.seed = seed; tParam.threads = this->_nthreads; // build local FM indexes assert_eq(szs.size(), this->_refnames.size()); index_t curr_sztot = 0; EList > alts; for(size_t tidx = 0; tidx < szs.size(); tidx++) { assert(szs[tidx].first); index_t refLen = szs[tidx].len; _localRFMs.expand(); assert_lt(tidx, _localRFMs.size()); tParam.index_size = refLen; tParam.conv_local_szs.clear(); tParam.conv_local_szs.push_back(szs[tidx]); tParam.refnames.clear(); tParam.refnames.push_back(this->_refnames[tidx]); // Extract sequence corresponding to this local index tParam.s.resize(refLen); if(refparams.reverse == REF_READ_REVERSE) { tParam.s.install(s.buf() + s.length() - curr_sztot - refLen, refLen); } else { tParam.s.install(s.buf() + curr_sztot, refLen); } #if 0 // Extract ALTs corresponding to this local index map alt_map; tParam.alts.clear(); ALT alt; alt.pos = curr_sztot; index_t alt_i = (index_t)this->_alts.bsearchLoBound(alt); for(; alt_i < this->_alts.size(); alt_i++) { const ALT& alt = this->_alts[alt_i]; if(alt.snp()) { if(alt.mismatch()) { if(curr_sztot + local_sztot <= alt.pos) break; } else if(alt.insertion()) { if(curr_sztot + local_sztot < alt.pos) break; } else { assert(alt.deletion()); if(curr_sztot + local_sztot < alt.pos + alt.len) break; } if(curr_sztot <= alt.pos) { alt_map[alt_i] = (index_t)tParam.alts.size(); tParam.alts.push_back(alt); tParam.alts.back().pos -= curr_sztot; } } else if(alt.splicesite()) { if(alt.excluded) continue; if(curr_sztot + local_sztot <= alt.right + 1) continue; if(curr_sztot <= alt.left) { tParam.alts.push_back(alt); tParam.alts.back().left -= curr_sztot; tParam.alts.back().right -= curr_sztot; } } else { assert(alt.exon()); } } // Extract haplotypes tParam.haplotypes.clear(); Haplotype haplotype; haplotype.left = curr_sztot; index_t haplotpye_i = (index_t)this->_haplotypes.bsearchLoBound(haplotype); for(; haplotpye_i < this->_haplotypes.size(); haplotpye_i++) { const Haplotype& haplotype = this->_haplotypes[haplotpye_i]; if(curr_sztot + local_sztot <= haplotype.right) continue; if(curr_sztot <= haplotype.left) { tParam.haplotypes.push_back(haplotype); tParam.haplotypes.back().left -= curr_sztot; tParam.haplotypes.back().right -= curr_sztot; for(index_t a = 0; a < tParam.haplotypes.back().alts.size(); a++) { index_t alt_i = tParam.haplotypes.back().alts[a]; if(alt_map.find(alt_i) == alt_map.end()) { assert(false); tParam.haplotypes.pop_back(); break; } tParam.haplotypes.back().alts[a] = alt_map[alt_i]; } } } #endif tParam.local_offset = 0; tParam.curr_sztot = curr_sztot; tParam.local_sztot = refLen; assert(tParam.rg == NULL); assert(tParam.pg == NULL); assert(tParam.sa == NULL); curr_sztot += refLen; build_worker(&tParam); LocalRFM( tParam.s, tParam.sa, tParam.pg, tParam.alts, tParam.index_size, tParam.refnames, packed, needEntireReverse, lineRate, offRate, // suffix-array sampling rate ftabChars, // number of chars in initial arrow-pair calc outfile, // basename for .?.ebwt files fw, // fw dcv, // difference-cover period tParam.conv_local_szs, // list of reference sizes tParam.local_sztot, // total size of all unambiguous ref chars refparams, // reference read-in parameters seed, // pseudo-random number generator seed fout1, fout2, -1, // override offRate false, // be silent passMemExc, // pass exceptions up to the toplevel so that we can adjust memory settings automatically sanityCheck); // verify results and internal consistency if(tParam.rg != NULL) { assert(tParam.pg != NULL); delete tParam.rg; tParam.rg = NULL; delete tParam.pg; tParam.pg = NULL; } if(tParam.sa != NULL) { delete tParam.sa; tParam.sa = NULL; } _localRFMFilePos[tidx].first = fout1.tellp(); _localRFMFilePos[tidx].second = fout2.tellp(); } assert_eq(curr_sztot, sztot); streampos origpos = fout1.tellp(); fout1.seekp(filepos); for(size_t i = 0; i < _localRFMFilePos.size(); i++) { writeIndex(fout1, _localRFMFilePos[i].first, be); writeIndex(fout1, _localRFMFilePos[i].second, be); } fout1.seekp(origpos); fout1 << '\0'; fout1.flush(); fout2.flush(); if(fout1.fail() || fout2.fail()) { cerr << "An error occurred writing the index to disk. Please check if the disk is full." << endl; throw 1; } VMSG_NL("Returning from initFromVector"); // Close output files fout1.flush(); int64_t tellpSz1 = (int64_t)fout1.tellp(); VMSG_NL("Wrote " << fout1.tellp() << " bytes to primary GFM file: " << _in1Str.c_str()); fout1.close(); bool err = false; if(tellpSz1 > fileSize(_in1Str.c_str())) { err = true; cerr << "Index is corrupt: File size for " << _in1Str.c_str() << " should have been " << tellpSz1 << " but is actually " << fileSize(_in1Str.c_str()) << "." << endl; } fout2.flush(); int64_t tellpSz2 = (int64_t)fout2.tellp(); VMSG_NL("Wrote " << fout2.tellp() << " bytes to secondary GFM file: " << _in2Str.c_str()); fout2.close(); if(tellpSz2 > fileSize(_in2Str.c_str())) { err = true; cerr << "Index is corrupt: File size for " << _in2Str.c_str() << " should have been " << tellpSz2 << " but is actually " << fileSize(_in2Str.c_str()) << "." << endl; } if(err) { cerr << "Please check if there is a problem with the disk or if disk is full." << endl; throw 1; } // Reopen as input streams VMSG_NL("Re-opening _in1 and _in2 as input streams"); if(this->_sanity) { VMSG_NL("Sanity-checking ht2"); assert(!this->isInMemory()); readIntoMemory( fw ? -1 : needEntireReverse, // 1 -> need the reverse to be reverse-of-concat true, // load SA sample (_offs[])? true, // load ftab (_ftab[] & _eftab[])? true, // load r-starts (_rstarts[])? false, // just load header? NULL, // Params object to fill false, // mm sweep? true, // load names? false); // verbose startup? sanityCheckAll(refparams.reverse); evictFromMemory(); assert(!this->isInMemory()); } VMSG_NL("Returning from HGFM constructor"); } /** * Read an Ebwt from file with given filename. */ template void RFM::readIntoMemory( int needEntireRev, bool loadSASamp, bool loadFtab, bool loadRstarts, bool justHeader, GFMParams *params, bool mmSweep, bool loadNames, bool startVerbose) { bool switchEndian; // dummy; caller doesn't care #ifdef BOWTIE_MM char *mmFile[] = { NULL, NULL }; #endif if(_in1Str.length() > 0) { if(this->_verbose || startVerbose) { cerr << " About to open input files: "; logTime(cerr); } // Initialize our primary and secondary input-stream fields if(_in1 != NULL) fclose(_in1); if(this->_verbose || startVerbose) cerr << "Opening \"" << _in1Str.c_str() << "\"" << endl; if((_in1 = fopen(_in1Str.c_str(), "rb")) == NULL) { cerr << "Could not open index file " << _in1Str.c_str() << endl; } if(loadSASamp) { if(_in2 != NULL) fclose(_in2); if(this->_verbose || startVerbose) cerr << "Opening \"" << _in2Str.c_str() << "\"" << endl; if((_in2 = fopen(_in2Str.c_str(), "rb")) == NULL) { cerr << "Could not open index file " << _in2Str.c_str() << endl; } } if(this->_verbose || startVerbose) { cerr << " Finished opening input files: "; logTime(cerr); } #ifdef BOWTIE_MM if(this->_useMm /*&& !justHeader*/) { const char *names[] = {_in1Str.c_str(), _in2Str.c_str()}; int fds[] = { fileno(_in1), fileno(_in2) }; for(int i = 0; i < (loadSASamp ? 2 : 1); i++) { if(this->_verbose || startVerbose) { cerr << " ¯ " << (i+1) << ": "; logTime(cerr); } struct stat sbuf; if (stat(names[i], &sbuf) == -1) { perror("stat"); cerr << "Error: Could not stat index file " << names[i] << " prior to memory-mapping" << endl; throw 1; } mmFile[i] = (char*)mmap((void *)0, (size_t)sbuf.st_size, PROT_READ, MAP_SHARED, fds[(size_t)i], 0); if(mmFile[i] == (void *)(-1)) { perror("mmap"); cerr << "Error: Could not memory-map the index file " << names[i] << endl; throw 1; } if(mmSweep) { int sum = 0; for(off_t j = 0; j < sbuf.st_size; j += 1024) { sum += (int) mmFile[i][j]; } if(startVerbose) { cerr << " Swept the memory-mapped ebwt index file 1; checksum: " << sum << ": "; logTime(cerr); } } } mmFile1_ = mmFile[0]; mmFile2_ = loadSASamp ? mmFile[1] : NULL; } #endif } #ifdef BOWTIE_MM else if(this->_useMm && !justHeader) { mmFile[0] = mmFile1_; mmFile[1] = mmFile2_; } if(this->_useMm && !justHeader) { assert(mmFile[0] == mmFile1_); assert(mmFile[1] == mmFile2_); } #endif if(this->_verbose || startVerbose) { cerr << " Reading header: "; logTime(cerr); } // Read endianness hints from both streams size_t bytesRead = 0, bytesRead2 = 4; switchEndian = false; uint32_t one = readU32(_in1, switchEndian); // 1st word of primary stream bytesRead += 4; if(loadSASamp) { #ifndef NDEBUG assert_eq(one, readU32(_in2, switchEndian)); // should match! #else readU32(_in2, switchEndian); #endif } if(one != 1) { assert_eq((1u<<24), one); assert_eq(1, endianSwapU32(one)); switchEndian = true; } // Can't switch endianness and use memory-mapped files; in order to // support this, someone has to modify the file to switch // endiannesses appropriately, and we can't do this inside Bowtie // or we might be setting up a race condition with other processes. if(switchEndian && this->_useMm) { cerr << "Error: Can't use memory-mapped files when the index is the opposite endianness" << endl; throw 1; } readI32(_in1, switchEndian); bytesRead += 4; index_t nlocalRFMs = readIndex(_in1, switchEndian); bytesRead += sizeof(index_t); EList > localRFMLens; localRFMLens.resizeExact(nlocalRFMs); for(size_t i = 0; i < localRFMLens.size(); i++) { localRFMLens[i].first = readIndex(_in1, switchEndian); localRFMLens[i].second = readIndex(_in1, switchEndian); } _localRFMFilePos.resizeExact(nlocalRFMs); for(size_t i = 0; i < _localRFMFilePos.size(); i++) { _localRFMFilePos[i].first = readIndex(_in1, switchEndian); _localRFMFilePos[i].second = readIndex(_in1, switchEndian); } clearLocalRFMs(); assert_eq(this->_repeatIncluded.size(), nlocalRFMs); string base = ""; for(size_t i = 0; i < nlocalRFMs; i++) { if(!this->_repeatIncluded[i]) continue; if(i > 0) { fseek(_in1, _localRFMFilePos[i-1].first, SEEK_SET); fseek(_in2, _localRFMFilePos[i-1].second, SEEK_SET); } LocalRFM *localRFM = new LocalRFM(base, NULL, _in1, _in2, mmFile1_, mmFile2_, switchEndian, bytesRead, bytesRead2, needEntireRev, this->fw_, -1, // overrideOffRate -1, // offRatePlus this->_useMm, this->useShmem_, mmSweep, loadNames, loadSASamp, loadFtab, loadRstarts, false, // _verbose false, this->_passMemExc, this->_sanity, false); // use haplotypes? _localRFMs.push_back(localRFM); } fseek(_in1, _localRFMFilePos.back().first, SEEK_SET); fseek(_in2, _localRFMFilePos.back().second, SEEK_SET); #ifdef BOWTIE_MM fseek(_in1, 0, SEEK_SET); fseek(_in2, 0, SEEK_SET); #else rewind(_in1); rewind(_in2); #endif } #endif /*RFM_H_*/ hisat2-2.2.1/sam.h000066400000000000000000001156221370663763200136630ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef SAM_H_ #define SAM_H_ #include #include #include "ds.h" #include "read.h" #include "util.h" #include "aligner_result.h" #include "scoring.h" #include "alt.h" #include "filebuf.h" enum { // Comments use language from v1.4-r962 spec SAM_FLAG_PAIRED = 1, // templ. having mult. frag.s in sequencing SAM_FLAG_MAPPED_PAIRED = 2, // each frag properly aligned SAM_FLAG_UNMAPPED = 4, // fragment unmapped SAM_FLAG_MATE_UNMAPPED = 8, // next fragment in template unmapped SAM_FLAG_QUERY_STRAND = 16, // SEQ is reverse comp'ed from original SAM_FLAG_MATE_STRAND = 32, // next fragment SEQ reverse comp'ed SAM_FLAG_FIRST_IN_PAIR = 64, // first fragment in template SAM_FLAG_SECOND_IN_PAIR = 128, // last fragment in template SAM_FLAG_NOT_PRIMARY = 256, // secondary alignment SAM_FLAG_FAILS_CHECKS = 512, // not passing quality controls SAM_FLAG_DUPLICATE = 1024 // PCR or optical duplicate }; class AlnRes; class AlnFlags; class AlnSetSumm; /** * Encapsulates all the various ways that a user may wish to customize SAM * output. */ template class SamConfig { typedef EList StrList; typedef EList LenList; public: SamConfig( const StrList& refnames, // reference sequence names const LenList& reflens, // reference sequence lengths const StrList& repnames, // repeat sequence names const LenList& replens, // repeat sequence lengths bool truncQname, // truncate read name to 255? bool omitsec, // omit secondary SEQ/QUAL bool noUnal, // omit unaligned reads const std::string& pg_id, // id const std::string& pg_pn, // name const std::string& pg_vn, // version const std::string& pg_cl, // command-line const std::string& rgs, // read groups string int rna_strandness, bool print_as, bool print_xs, bool print_xss, bool print_yn, bool print_xn, bool print_cs, bool print_cq, bool print_x0, bool print_x1, bool print_xm, bool print_xo, bool print_xg, bool print_nm, bool print_md, bool print_yf, bool print_yi, bool print_ym, bool print_yp, bool print_yt, bool print_ys, bool print_zs, bool print_xr, bool print_xt, bool print_xd, bool print_xu, bool print_ye, // streak of failed DPs at end bool print_yl, // longest streak of failed DPs bool print_yu, // index of last succeeded DP bool print_xp, // print seed hit information bool print_yr, // # redundant seed hits bool print_zb, // # Ftab lookups bool print_zr, // # redundant path checks bool print_zf, // # FM Index ops bool print_zm, // FM Index op string for best-first search bool print_zi, // # seed extend loop iters bool print_zp, bool print_zu, bool print_xs_a, bool print_nh) : truncQname_(truncQname), omitsec_(omitsec), noUnal_(noUnal), pg_id_(pg_id), pg_pn_(pg_pn), pg_vn_(pg_vn), pg_cl_(pg_cl), rgs_(rgs), refnames_(refnames), reflens_(reflens), repnames_(repnames), replens_(replens), rna_strandness_(rna_strandness), print_as_(print_as), // alignment score of best alignment print_xs_(print_xs), // alignment score of second-best alignment print_xss_(print_xss), print_yn_(print_yn), // minimum valid score and perfect score print_xn_(print_xn), print_cs_(print_cs), print_cq_(print_cq), print_x0_(print_x0), print_x1_(print_x1), print_xm_(print_xm), print_xo_(print_xo), print_xg_(print_xg), print_nm_(print_nm), print_md_(print_md), print_yf_(print_yf), print_yi_(print_yi), print_ym_(print_ym), print_yp_(print_yp), print_yt_(print_yt), print_ys_(print_ys), print_zs_(print_zs), print_xr_(print_xr), print_xt_(print_xt), // time elapsed in microseconds print_xd_(print_xd), // DP extend attempts print_xu_(print_xu), // ungapped extend attempts print_ye_(print_ye), // streak of failed DPs at end print_yl_(print_yl), // longest streak of failed DPs print_yu_(print_yu), // index of last succeeded DP print_xp_(print_xp), // print seed hit information print_yr_(print_yr), // index of last succeeded DP print_zb_(print_zb), // # Ftab lookups print_zr_(print_zr), // # redundant path checks print_zf_(print_zf), // # FM Index ops print_zm_(print_zm), // FM Index op string for best-first search print_zi_(print_zi), // # seed extend loop iters print_zp_(print_zp), // # seed extend loop iters print_zu_(print_zu), // # seed extend loop iters print_xs_a_(print_xs_a), print_nh_(print_nh) { assert_eq(refnames_.size(), reflens_.size()); } /** * Print a reference name in a way that doesn't violate SAM's character * constraints. \*|[!-()+-<>-~][!-~]* */ void printRefName( BTString& o, const std::string& name) const; /** * Print a :Z optional field where certain characters (whitespace, colon * and percent) are escaped using % escapes. */ template void printOptFieldEscapedZ(BTString& o, const T& s) const { size_t len = s.length(); for(size_t i = 0; i < len; i++) { if(s[i] < 33 || s[i] > 126 || s[i] == ':' || s[i] == '%') { // percent-encode it o.append('%'); int ms = s[i] >> 4; int ls = s[i] & 15; assert_range(0, 15, ms); assert_range(0, 15, ls); o.append("0123456789ABCDEF"[ms]); o.append("0123456789ABCDEF"[ls]); } else { o.append(s[i]); } } } /** * Print a :Z optional field where newline characters are escaped using % * escapes. */ template void printOptFieldNewlineEscapedZ(BTString& o, const T& s) const { size_t len = s.length(); for(size_t i = 0; i < len; i++) { if(s[i] == 10 || s[i] == 13 || s[i] == '%') { // percent-encode it o.append('%'); int ms = s[i] >> 4; int ls = s[i] & 15; assert_range(0, 15, ms); assert_range(0, 15, ls); o.append("0123456789ABCDEF"[ms]); o.append("0123456789ABCDEF"[ls]); } else { o.append(s[i]); } } } /** * Print a read name in a way that doesn't violate SAM's character * constraints. [!-?A-~]{1,255} (i.e. [33, 63], [65, 126]) */ template void printReadName( BTString& o, const TStr& name, bool omitSlashMate) const { size_t namelen = name.length(); if(omitSlashMate && namelen >= 2 && name[namelen-2] == '/' && (name[namelen-1] == '1' || name[namelen-1] == '2' || name[namelen-1] == '3')) { namelen -= 2; } if(truncQname_ && namelen > 255) { namelen = 255; } for(size_t i = 0; i < namelen; i++) { if(truncQname_ && isspace(name[i])) { return; } o.append(name[i]); } } /** * Print a reference name given a reference index. */ void printRefNameFromIndex( BTString& o, size_t i, bool repeat = false) const; /** * Print SAM header to given output buffer. */ void printHeader( BTString& o, const std::string& rgid, const std::string& rgs, bool printHd, bool printSq, bool printPg) const; /** * Print the @HD header line to the given string. */ void printHdLine(BTString& o, const char *samver) const; /** * Print the @SQ header lines to the given string. */ void printSqLines(BTString& o) const; /** * Print the @PG header line to the given string. */ void printPgLine(BTString& o) const; /** * Print the optional flags to the given string. */ void printAlignedOptFlags( BTString& o, // output buffer bool first, // first opt flag printed is first overall? const Read& rd, // the read AlnRes& res, // individual alignment result StackedAln& staln, // stacked alignment const AlnFlags& flags, // alignment flags const AlnSetSumm& summ, // summary of alignments for this read const SeedAlSumm& ssm, // seed alignment summary const PerReadMetrics& prm, // per-read metics const Scoring& sc, // scoring scheme const char *mapqInp, // inputs to MAPQ calculation const ALTDB* altdb) const; /** * Print the optional flags to the given string. */ void printEmptyOptFlags( BTString& o, // output buffer bool first, // first opt flag printed is first overall? const Read& rd, // the read const AlnFlags& flags, // alignment flags const AlnSetSumm& summ, // summary of alignments for this read const SeedAlSumm& ssm, // seed alignment summary const PerReadMetrics& prm, // per-read metrics const Scoring& sc) // scoring scheme const; /** * Return true iff we should try to obey the SAM spec's recommendations * that: * * SEQ and QUAL of secondary alignments should be set to ‘*’ to reduce the * file size. */ bool omitSecondarySeqQual() const { return omitsec_; } bool omitUnalignedReads() const { return noUnal_; } protected: bool truncQname_; // truncate QNAME to 255 chars? bool omitsec_; // omit secondary bool noUnal_; // omit unaligned reads std::string pg_id_; // @PG ID: Program record identifier std::string pg_pn_; // @PG PN: Program name std::string pg_vn_; // @PG VN: Program version std::string pg_cl_; // @PG CL: Program command-line std::string rgs_; // Read-group string to add to all records const StrList& refnames_; // reference sequence names const LenList& reflens_; // reference sequence lengths const StrList& repnames_; // repeat sequence names const LenList& replens_; // repeat sequence lengths int rna_strandness_; // Which alignment flags to print? // Following are printed by BWA-SW bool print_as_; // AS:i: Alignment score generated by aligner bool print_xs_; // XS:i: Suboptimal alignment score bool print_xss_;// Xs:i: Best invalid alignment score found bool print_yn_; // YN:i:, Yn:i: minimum valid score and perfect score bool print_xn_; // XN:i: Number of ambiguous bases in the referenece // Other optional flags bool print_cs_; // CS:Z: Color read sequence on the original strand bool print_cq_; // CQ:Z: Color read quality on the original strand // Following are printed by BWA bool print_x0_; // X0:i: Number of best hits bool print_x1_; // X1:i: Number of sub-optimal best hits bool print_xm_; // XM:i: Number of mismatches in the alignment bool print_xo_; // XO:i: Number of gap opens bool print_xg_; // XG:i: Number of gap extensions (incl. opens) bool print_nm_; // NM:i: Edit dist. to the ref, Ns count, clipping doesn't bool print_md_; // MD:Z: String for mms. [0-9]+(([A-Z]|\^[A-Z]+)[0-9]+)*2 // Following are Bowtie2-specific bool print_yf_; // YF:i: Read was filtered out? bool print_yi_; // YI:Z: Summary of inputs to MAPQ calculation bool print_ym_; // YM:i: Read was repetitive when aligned unpaired? bool print_yp_; // YP:i: Read was repetitive when aligned paired? bool print_yt_; // YT:Z: String representing alignment type bool print_ys_; // YS:i: Score of other mate bool print_zs_; // ZS:i: Pseudo-random seed bool print_xr_; // XR:Z: Original read string bool print_xt_; // XT:i: Time taken to align bool print_xd_; // XD:i: DP problems bool print_xu_; // XU:i: ungapped alignment bool print_ye_; // YE:i: streak of failed DPs at end bool print_yl_; // YL:i: longest streak of failed DPs bool print_yu_; // YU:i: index of last succeeded DP bool print_xp_; // XP:BI: seed hit information bool print_yr_; // YR:i: # redundant seed hits bool print_zb_; // ZB:i: # Ftab lookups bool print_zr_; // ZR:i: # redundant path checks bool print_zf_; // ZF:i: # FM Index ops bool print_zm_; // ZM:i: FM ops string for best-first search bool print_zi_; // ZI:i: # extend loop iters bool print_zp_; // ZP:i: Score of best/second-best paired-end alignment bool print_zu_; // ZU:i: Score of best/second-best unpaired alignment bool print_xs_a_; // XS:A:[+=] Sense/anti-sense strand splice sites correspond to bool print_nh_; // NH:i: # alignments }; /** * Print a reference name in a way that doesn't violate SAM's character * constraints. \*|[!-()+-<>-~][!-~]* (i.e. [33, 63], [65, 126]) */ template void SamConfig::printRefName( BTString& o, const std::string& name) const { size_t namelen = name.length(); for(size_t i = 0; i < namelen; i++) { if(isspace(name[i])) { return; } o.append(name[i]); } } /** * Print a reference name given a reference index. */ template void SamConfig::printRefNameFromIndex(BTString& o, size_t i, bool repeat) const { if(repeat) { printRefName(o, repnames_[i]); } else { printRefName(o, refnames_[i]); } } /** * Print SAM header to given output buffer. */ template void SamConfig::printHeader( BTString& o, const string& rgid, const string& rgs, bool printHd, bool printSq, bool printPg) const { if(printHd) printHdLine(o, "1.0"); if(printSq) printSqLines(o); if(!rgid.empty()) { o.append("@RG"); o.append(rgid.c_str()); o.append(rgs.c_str()); o.append('\n'); } if(printPg) printPgLine(o); } /** * Print the @HD header line to the given string. */ template void SamConfig::printHdLine(BTString& o, const char *samver) const { o.append("@HD\tVN:"); o.append(samver); o.append("\tSO:unsorted\n"); } /** * Print the @SQ header lines to the given string. */ template void SamConfig::printSqLines(BTString& o) const { char buf[1024]; for(size_t i = 0; i < refnames_.size(); i++) { o.append("@SQ\tSN:"); printRefName(o, refnames_[i]); o.append("\tLN:"); itoa10(reflens_[i], buf); o.append(buf); o.append('\n'); } for(size_t i = 0; i < repnames_.size(); i++) { o.append("@SQ\tSN:"); printRefName(o, repnames_[i]); o.append("\tLN:"); itoa10(replens_[i], buf); o.append(buf); o.append('\n'); } } /** * Print the @PG header line to the given string. */ template void SamConfig::printPgLine(BTString& o) const { o.append("@PG\tID:"); o.append(pg_id_.c_str()); o.append("\tPN:"); o.append(pg_pn_.c_str()); o.append("\tVN:"); o.append(pg_vn_.c_str()); o.append("\tCL:\""); o.append(pg_cl_.c_str()); o.append('"'); o.append('\n'); } #define WRITE_SEP() { \ if(!first) o.append('\t'); \ first = false; \ } /** * Print the optional flags to the given string. */ template void SamConfig::printAlignedOptFlags( BTString& o, // output buffer bool first, // first opt flag printed is first overall? const Read& rd, // the read AlnRes& res, // individual alignment result StackedAln& staln, // stacked alignment buffer const AlnFlags& flags, // alignment flags const AlnSetSumm& summ, // summary of alignments for this read const SeedAlSumm& ssm, // seed alignment summary const PerReadMetrics& prm, // per-read metrics const Scoring& sc, // scoring scheme const char *mapqInp, // inputs to MAPQ calculation const ALTDB* altdb) const { char buf[1024]; if(print_as_) { // AS:i: Alignment score generated by aligner itoa10(res.score().score(), buf); WRITE_SEP(); o.append("AS:i:"); o.append(buf); } // Do not output suboptimal alignment score, which conflicts with Cufflinks and StringTie if(print_xs_) { // XS:i: Suboptimal alignment score // Use ZS:i: to avoid conflict with XS:A: AlnScore sco = summ.secbestMate(rd.mate < 2); if(sco.valid()) { itoa10(sco.score(), buf); WRITE_SEP(); o.append("ZS:i:"); o.append(buf); } } if(print_xn_) { // XN:i: Number of ambiguous bases in the referenece itoa10(res.refNs(), buf); WRITE_SEP(); o.append("XN:i:"); o.append(buf); } if(print_x0_) { // X0:i: Number of best hits } if(print_x1_) { // X1:i: Number of sub-optimal best hits } size_t num_mm = 0; size_t num_go = 0; size_t num_gx = 0; for(size_t i = 0; i < res.ned().size(); i++) { if(res.ned()[i].isMismatch()) { if(res.ned()[i].snpID >= altdb->alts().size()) { num_mm++; } } else if(res.ned()[i].isReadGap()) { if(res.ned()[i].snpID >= altdb->alts().size()) { num_go++; num_gx++; } while(i < res.ned().size()-1 && res.ned()[i+1].pos == res.ned()[i].pos && res.ned()[i+1].isReadGap()) { i++; if(res.ned()[i].snpID >= altdb->alts().size()) { num_gx++; } } } else if(res.ned()[i].isRefGap()) { if(res.ned()[i].snpID >= altdb->alts().size()) { num_go++; num_gx++; } while(i < res.ned().size()-1 && res.ned()[i+1].pos == res.ned()[i].pos+1 && res.ned()[i+1].isRefGap()) { i++; if(res.ned()[i].snpID >= altdb->alts().size()) { num_gx++; } } } } if(print_xm_) { // XM:i: Number of mismatches in the alignment itoa10(num_mm, buf); WRITE_SEP(); o.append("XM:i:"); o.append(buf); } if(print_xo_) { // XO:i: Number of gap opens itoa10(num_go, buf); WRITE_SEP(); o.append("XO:i:"); o.append(buf); } if(print_xg_) { // XG:i: Number of gap extensions (incl. opens) itoa10(num_gx, buf); WRITE_SEP(); o.append("XG:i:"); o.append(buf); } if(print_nm_) { // NM:i: Edit dist. to the ref, Ns count, clipping doesn't size_t NM = 0; for(size_t i = 0; i < res.ned().size(); i++) { if(res.ned()[i].type != EDIT_TYPE_SPL) { if(res.ned()[i].snpID >= altdb->alts().size()) { NM++; } } } itoa10(NM, buf); WRITE_SEP(); o.append("NM:i:"); o.append(buf); } if(print_md_) { // MD:Z: String for mms. [0-9]+(([A-Z]|\^[A-Z]+)[0-9]+)*2 WRITE_SEP(); o.append("MD:Z:"); staln.buildMdz(); staln.writeMdz( &o, // output buffer NULL); // no char buffer } if(print_ys_ && summ.paired()) { // YS:i: Alignment score of opposite mate assert(res.oscore().valid()); itoa10(res.oscore().score(), buf); WRITE_SEP(); o.append("YS:i:"); o.append(buf); } if(print_yn_) { // YN:i: Minimum valid score for this mate TAlScore mn = sc.scoreMin.f(rd.length()); itoa10(mn, buf); WRITE_SEP(); o.append("YN:i:"); o.append(buf); // Yn:i: Perfect score for this mate TAlScore pe = sc.perfectScore(rd.length()); itoa10(pe, buf); WRITE_SEP(); o.append("Yn:i:"); o.append(buf); } if(print_xss_) { // Xs:i: Best invalid alignment score of this mate bool one = true; if(flags.partOfPair() && !flags.readMate1()) { one = false; } TAlScore bst = one ? prm.bestLtMinscMate1 : prm.bestLtMinscMate2; if(bst > std::numeric_limits::min()) { itoa10(bst, buf); WRITE_SEP(); o.append("Xs:i:"); o.append(buf); } if(flags.partOfPair()) { // Ys:i: Best invalid alignment score of opposite mate bst = one ? prm.bestLtMinscMate2 : prm.bestLtMinscMate1; if(bst > std::numeric_limits::min()) { itoa10(bst, buf); WRITE_SEP(); o.append("Ys:i:"); o.append(buf); } } } if(print_zs_) { // ZS:i: Pseudo-random seed for read itoa10(rd.seed, buf); WRITE_SEP(); o.append("ZS:i:"); o.append(buf); } if(print_yt_) { // YT:Z: String representing alignment type WRITE_SEP(); flags.printYT(o); } if(print_yp_ && flags.partOfPair() && flags.canMax()) { // YP:i: Read was repetitive when aligned paired? WRITE_SEP(); flags.printYP(o); } if(print_ym_ && flags.canMax() && (flags.isMixedMode() || !flags.partOfPair())) { // YM:i: Read was repetitive when aligned unpaired? WRITE_SEP(); flags.printYM(o); } if(print_yf_ && flags.filtered()) { // YF:i: Read was filtered? first = flags.printYF(o, first) && first; } if(print_yi_) { // Print MAPQ calibration info if(mapqInp[0] != '\0') { // YI:i: Suboptimal alignment score WRITE_SEP(); o.append("YI:Z:"); o.append(mapqInp); } } if(flags.partOfPair() && print_zp_) { // ZP:i: Score of best concordant paired-end alignment WRITE_SEP(); o.append("ZP:Z:"); if(summ.bestPaired().valid()) { itoa10(summ.bestPaired().score(), buf); o.append(buf); } else { o.append("NA"); } // Zp:i: Second-best concordant paired-end alignment score WRITE_SEP(); o.append("Zp:Z:"); if(summ.secbestPaired().valid()) { itoa10(summ.secbestPaired().score(), buf); o.append(buf); } else { o.append("NA"); } } if(print_zu_) { // ZU:i: Score of best unpaired alignment AlnScore best = (rd.mate <= 1 ? summ.best1() : summ.best2()); AlnScore secbest = (rd.mate <= 1 ? summ.secbest1() : summ.secbest2()); WRITE_SEP(); o.append("ZU:i:"); if(best.valid()) { itoa10(best.score(), buf); o.append(buf); } else { o.append("NA"); } // Zu:i: Score of second-best unpaired alignment WRITE_SEP(); o.append("Zu:i:"); if(secbest.valid()) { itoa10(secbest.score(), buf); o.append(buf); } else { o.append("NA"); } } if(!rgs_.empty()) { WRITE_SEP(); o.append(rgs_.c_str()); } if(print_xt_) { // XT:i: Timing WRITE_SEP(); struct timeval tv_end; struct timezone tz_end; gettimeofday(&tv_end, &tz_end); size_t total_usecs = (tv_end.tv_sec - prm.tv_beg.tv_sec) * 1000000 + (tv_end.tv_usec - prm.tv_beg.tv_usec); itoa10(total_usecs, buf); o.append("XT:i:"); o.append(buf); } if(print_xd_) { // XD:i: Extend DPs WRITE_SEP(); itoa10(prm.nExDps, buf); o.append("XD:i:"); o.append(buf); // Xd:i: Mate DPs WRITE_SEP(); itoa10(prm.nMateDps, buf); o.append("Xd:i:"); o.append(buf); } if(print_xu_) { // XU:i: Extend ungapped tries WRITE_SEP(); itoa10(prm.nExUgs, buf); o.append("XU:i:"); o.append(buf); // Xu:i: Mate ungapped tries WRITE_SEP(); itoa10(prm.nMateUgs, buf); o.append("Xu:i:"); o.append(buf); } if(print_ye_) { // YE:i: Streak of failed DPs at end WRITE_SEP(); itoa10(prm.nDpFail, buf); o.append("YE:i:"); o.append(buf); // Ye:i: Streak of failed ungaps at end WRITE_SEP(); itoa10(prm.nUgFail, buf); o.append("Ye:i:"); o.append(buf); } if(print_yl_) { // YL:i: Longest streak of failed DPs WRITE_SEP(); itoa10(prm.nDpFailStreak, buf); o.append("YL:i:"); o.append(buf); // Yl:i: Longest streak of failed ungaps WRITE_SEP(); itoa10(prm.nUgFailStreak, buf); o.append("Yl:i:"); o.append(buf); } if(print_yu_) { // YU:i: Index of last succesful DP WRITE_SEP(); itoa10(prm.nDpLastSucc, buf); o.append("YU:i:"); o.append(buf); // Yu:i: Index of last succesful DP WRITE_SEP(); itoa10(prm.nUgLastSucc, buf); o.append("Yu:i:"); o.append(buf); } if(print_xp_) { // XP:Z: String describing seed hits WRITE_SEP(); o.append("XP:B:I,"); itoa10(prm.nSeedElts, buf); o.append(buf); o.append(','); itoa10(prm.nSeedEltsFw, buf); o.append(buf); o.append(','); itoa10(prm.nSeedEltsRc, buf); o.append(buf); o.append(','); itoa10(prm.seedMean, buf); o.append(buf); o.append(','); itoa10(prm.seedMedian, buf); o.append(buf); } if(print_yr_) { // YR:i: Redundant seed hits WRITE_SEP(); itoa10(prm.nRedundants, buf); o.append("YR:i:"); o.append(buf); } if(print_zb_) { // ZB:i: Ftab ops for seed alignment WRITE_SEP(); itoa10(prm.nFtabs, buf); o.append("ZB:i:"); o.append(buf); } if(print_zr_) { // ZR:Z: Redundant path skips in seed alignment WRITE_SEP(); o.append("ZR:Z:"); itoa10(prm.nRedSkip, buf); o.append(buf); o.append(','); itoa10(prm.nRedFail, buf); o.append(buf); o.append(','); itoa10(prm.nRedIns, buf); o.append(buf); } if(print_zf_) { // ZF:i: FM Index ops for seed alignment WRITE_SEP(); itoa10(prm.nSdFmops, buf); o.append("ZF:i:"); o.append(buf); // Zf:i: FM Index ops for offset resolution WRITE_SEP(); itoa10(prm.nExFmops, buf); o.append("Zf:i:"); o.append(buf); } if(print_zm_) { // ZM:Z: Print FM index op string for best-first search WRITE_SEP(); o.append("ZM:Z:"); prm.fmString.print(o, buf); } if(print_zi_) { // ZI:i: Seed extend loop iterations WRITE_SEP(); itoa10(prm.nExIters, buf); o.append("ZI:i:"); o.append(buf); } if(print_xs_a_) { if(rna_strandness_ == RNA_STRANDNESS_UNKNOWN) { uint8_t whichsense = res.spliced_whichsense_transcript(); if(whichsense != SPL_UNKNOWN) { WRITE_SEP(); o.append("XS:A:"); if(whichsense == SPL_FW || whichsense == SPL_SEMI_FW) { o.append('+'); } else { assert(whichsense == SPL_RC || whichsense == SPL_SEMI_RC); o.append('-'); } } } else { WRITE_SEP(); o.append("XS:A:"); char strandness = '+'; if(res.readMate1()) { if(res.orient()) { if(rna_strandness_ == RNA_STRANDNESS_R || rna_strandness_ == RNA_STRANDNESS_RF) { strandness = '-'; } } else { if(rna_strandness_ == RNA_STRANDNESS_F || rna_strandness_ == RNA_STRANDNESS_FR) { strandness = '-'; } } } else { assert(res.readMate2()); assert(rna_strandness_ == RNA_STRANDNESS_FR || rna_strandness_ == RNA_STRANDNESS_RF); if(res.orient()) { if(rna_strandness_ == RNA_STRANDNESS_FR) { strandness = '-'; } } else { if(rna_strandness_ == RNA_STRANDNESS_RF) { strandness = '-'; } } } o.append(strandness); } } if(print_nh_) { if(flags.alignedPaired()) { WRITE_SEP(); itoa10(summ.numAlnsPaired(), buf); o.append("NH:i:"); o.append(buf); } else if(flags.alignedUnpaired() || flags.alignedUnpairedMate()) { WRITE_SEP(); itoa10((flags.alignedUnpaired() || flags.readMate1()) ? summ.numAlns1() : summ.numAlns2(), buf); o.append("NH:i:"); o.append(buf); } } bool snp_first = true; index_t prev_snp_idx = INDEX_MAX; size_t len_trimmed = rd.length() - res.trimmed5p(true) - res.trimmed3p(true); if(!res.fw()) { Edit::invertPoss(const_cast&>(res.ned()), len_trimmed, false); } for(size_t i = 0; i < res.ned().size(); i++) { if(res.ned()[i].snpID >= altdb->alts().size()) continue; index_t snp_idx = res.ned()[i].snpID; assert_lt(snp_idx, altdb->alts().size()); const ALT& snp = altdb->alts()[snp_idx]; const string& snpID = altdb->altnames()[snp_idx]; if(snp_idx == prev_snp_idx) continue; if(snp_first) { WRITE_SEP(); o.append("Zs:Z:"); } if(!snp_first) o.append(","); uint64_t pos = res.ned()[i].pos; size_t j = i; while(j > 0) { if(res.ned()[j-1].snpID < altdb->alts().size()) { const ALT& snp2 = altdb->alts()[res.ned()[j-1].snpID]; if(snp2.type == ALT_SNP_SGL) { pos -= (res.ned()[j-1].pos + 1); } else if(snp2.type == ALT_SNP_DEL) { pos -= res.ned()[j-1].pos; } else if(snp2.type == ALT_SNP_INS) { pos -= (res.ned()[j-1].pos + snp.len); } break; } j--; } itoa10(pos, buf); o.append(buf); o.append("|"); if(snp.type == ALT_SNP_SGL) { o.append("S"); } else if(snp.type == ALT_SNP_DEL) { o.append("D"); } else { assert_eq(snp.type, ALT_SNP_INS); o.append("I"); } o.append("|"); o.append(snpID.c_str()); if(snp_first) snp_first = false; prev_snp_idx = snp_idx; } if(!res.fw()) { Edit::invertPoss(const_cast&>(res.ned()), len_trimmed, false); } if(print_xr_) { // Original read string o.append("\n"); printOptFieldNewlineEscapedZ(o, rd.readOrigBuf); } } /** * Print the optional flags to the given string. */ template void SamConfig::printEmptyOptFlags( BTString& o, // output buffer bool first, // first opt flag printed is first overall? const Read& rd, // read const AlnFlags& flags, // alignment flags const AlnSetSumm& summ, // summary of alignments for this read const SeedAlSumm& ssm, // seed alignment summary const PerReadMetrics& prm, // per-read metrics const Scoring& sc) // scoring scheme const { char buf[1024]; if(print_yn_) { // YN:i: Minimum valid score for this mate TAlScore mn = sc.scoreMin.f(rd.length()); itoa10(mn, buf); WRITE_SEP(); o.append("YN:i:"); o.append(buf); // Yn:i: Perfect score for this mate TAlScore pe = sc.perfectScore(rd.length()); itoa10(pe, buf); WRITE_SEP(); o.append("Yn:i:"); o.append(buf); } if(print_zs_) { // ZS:i: Pseudo-random seed for read itoa10(rd.seed, buf); WRITE_SEP(); o.append("ZS:i:"); o.append(buf); } if(print_yt_) { // YT:Z: String representing alignment type WRITE_SEP(); flags.printYT(o); } if(print_yp_ && flags.partOfPair() && flags.canMax()) { // YP:i: Read was repetitive when aligned paired? WRITE_SEP(); flags.printYP(o); } if(print_ym_ && flags.canMax() && (flags.isMixedMode() || !flags.partOfPair())) { // YM:i: Read was repetitive when aligned unpaired? WRITE_SEP(); flags.printYM(o); } if(print_yf_ && flags.filtered()) { // YM:i: Read was repetitive when aligned unpaired? first = flags.printYF(o, first) && first; } if(!rgs_.empty()) { WRITE_SEP(); o.append(rgs_.c_str()); } if(print_xt_) { // XT:i: Timing WRITE_SEP(); struct timeval tv_end; struct timezone tz_end; gettimeofday(&tv_end, &tz_end); size_t total_usecs = (tv_end.tv_sec - prm.tv_beg.tv_sec) * 1000000 + (tv_end.tv_usec - prm.tv_beg.tv_usec); itoa10(total_usecs, buf); o.append("XT:i:"); o.append(buf); } if(print_xd_) { // XD:i: Extend DPs WRITE_SEP(); itoa10(prm.nExDps, buf); o.append("XD:i:"); o.append(buf); // Xd:i: Mate DPs WRITE_SEP(); itoa10(prm.nMateDps, buf); o.append("Xd:i:"); o.append(buf); } if(print_xu_) { // XU:i: Extend ungapped tries WRITE_SEP(); itoa10(prm.nExUgs, buf); o.append("XU:i:"); o.append(buf); // Xu:i: Mate ungapped tries WRITE_SEP(); itoa10(prm.nMateUgs, buf); o.append("Xu:i:"); o.append(buf); } if(print_ye_) { // YE:i: Streak of failed DPs at end WRITE_SEP(); itoa10(prm.nDpFail, buf); o.append("YE:i:"); o.append(buf); // Ye:i: Streak of failed ungaps at end WRITE_SEP(); itoa10(prm.nUgFail, buf); o.append("Ye:i:"); o.append(buf); } if(print_yl_) { // YL:i: Longest streak of failed DPs WRITE_SEP(); itoa10(prm.nDpFailStreak, buf); o.append("YL:i:"); o.append(buf); // Yl:i: Longest streak of failed ungaps WRITE_SEP(); itoa10(prm.nUgFailStreak, buf); o.append("Yl:i:"); o.append(buf); } if(print_yu_) { // YU:i: Index of last succesful DP WRITE_SEP(); itoa10(prm.nDpLastSucc, buf); o.append("YU:i:"); o.append(buf); // Yu:i: Index of last succesful DP WRITE_SEP(); itoa10(prm.nUgLastSucc, buf); o.append("Yu:i:"); o.append(buf); } if(print_xp_) { // XP:Z: String describing seed hits WRITE_SEP(); o.append("XP:B:I,"); itoa10(prm.nSeedElts, buf); o.append(buf); o.append(','); itoa10(prm.nSeedEltsFw, buf); o.append(buf); o.append(','); itoa10(prm.nSeedEltsRc, buf); o.append(buf); o.append(','); itoa10(prm.seedMean, buf); o.append(buf); o.append(','); itoa10(prm.seedMedian, buf); o.append(buf); } if(print_yr_) { // YR:i: Redundant seed hits WRITE_SEP(); itoa10(prm.nRedundants, buf); o.append("YR:i:"); o.append(buf); } if(print_zb_) { // ZB:i: Ftab ops for seed alignment WRITE_SEP(); itoa10(prm.nFtabs, buf); o.append("ZB:i:"); o.append(buf); } if(print_zr_) { // ZR:Z: Redundant path skips in seed alignment WRITE_SEP(); o.append("ZR:Z:"); itoa10(prm.nRedSkip, buf); o.append(buf); o.append(','); itoa10(prm.nRedFail, buf); o.append(buf); o.append(','); itoa10(prm.nRedIns, buf); o.append(buf); } if(print_zf_) { // ZF:i: FM Index ops for seed alignment WRITE_SEP(); itoa10(prm.nSdFmops, buf); o.append("ZF:i:"); o.append(buf); // Zf:i: FM Index ops for offset resolution WRITE_SEP(); itoa10(prm.nExFmops, buf); o.append("Zf:i:"); o.append(buf); } if(print_zm_) { // ZM:Z: Print FM index op string for best-first search WRITE_SEP(); o.append("ZM:Z:"); prm.fmString.print(o, buf); } if(print_zi_) { // ZI:i: Seed extend loop iterations WRITE_SEP(); itoa10(prm.nExIters, buf); o.append("ZI:i:"); o.append(buf); } if(print_xr_) { // Original read string o.append("\n"); printOptFieldNewlineEscapedZ(o, rd.readOrigBuf); } } #endif /* SAM_H_ */ hisat2-2.2.1/scoring.cpp000066400000000000000000000224271370663763200151020ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include "scoring.h" using namespace std; /** * Return true iff a read of length 'rdlen' passes the score filter, i.e., * has enough characters to rise above the minimum score threshold. */ bool Scoring::scoreFilter( int64_t minsc, size_t rdlen) const { int64_t sc = (int64_t)(rdlen * match(30)); return sc >= minsc; } /** * Given the score floor for valid alignments and the length of the read, * calculate the maximum possible number of read gaps that could occur in a * valid alignment. */ int Scoring::maxReadGaps( int64_t minsc, size_t rdlen) const { // Score if all characters match. TODO: remove assumption that match bonus // is independent of quality value. int64_t sc = (int64_t)(rdlen * match(30)); assert_geq(sc, minsc); // Now convert matches to read gaps until sc calls below minsc bool first = true; int num = 0; while(sc >= minsc) { if(first) { first = false; // Subtract both penalties sc -= readGapOpen(); } else { // Subtract just the extension penalty sc -= readGapExtend(); } num++; } assert_gt(num, 0); return num-1; } /** * Given the score floor for valid alignments and the length of the read, * calculate the maximum possible number of reference gaps that could occur * in a valid alignment. */ int Scoring::maxRefGaps( int64_t minsc, size_t rdlen) const { // Score if all characters match. TODO: remove assumption that match bonus // is independent of quality value. int64_t sc = (int64_t)(rdlen * match(30)); assert_geq(sc, minsc); // Now convert matches to read gaps until sc calls below minsc bool first = true; int num = 0; while(sc >= minsc) { sc -= match(30); if(first) { first = false; // Subtract both penalties sc -= refGapOpen(); } else { // Subtract just the extension penalty sc -= refGapExtend(); } num++; } assert_gt(num, 0); return num-1; } /** * Given a read sequence, return true iff the read passes the N filter. * The N filter rejects reads with more than the number of Ns. */ bool Scoring::nFilter(const BTDnaString& rd, size_t& ns) const { size_t rdlen = rd.length(); size_t maxns = nCeil.f((double)rdlen); assert_geq(rd.length(), 0); for(size_t i = 0; i < rdlen; i++) { if(rd[i] == 4) { ns++; if(ns > maxns) { return false; // doesn't pass } } } return true; // passes } /** * Given a read sequence, return true iff the read passes the N filter. * The N filter rejects reads with more than the number of Ns. * * For paired-end reads, there is a question of how to apply the filter. * The filter could be applied to both mates separately, which might then * prevent paired-end alignment. Or the filter could be applied to the * reads as though they're concatenated together. The latter approach has * pros and cons. The pro is that we can use paired-end information to * recover alignments for mates that would not have passed the N filter on * their own. The con is that we might not want to do that, since the * non-N portion of the bad mate might contain particularly unreliable * information. */ void Scoring::nFilterPair( const BTDnaString* rd1, // mate 1 const BTDnaString* rd2, // mate 2 size_t& ns1, // # Ns in mate 1 size_t& ns2, // # Ns in mate 2 bool& filt1, // true -> mate 1 rejected by filter bool& filt2) // true -> mate 2 rejected by filter const { // Both fail to pass by default filt1 = filt2 = false; if(rd1 != NULL && rd2 != NULL && ncatpair) { size_t rdlen1 = rd1->length(); size_t rdlen2 = rd2->length(); size_t maxns = nCeil.f((double)(rdlen1 + rdlen2)); for(size_t i = 0; i < rdlen1; i++) { if((*rd1)[i] == 4) ns1++; if(ns1 > maxns) { // doesn't pass return; } } for(size_t i = 0; i < rdlen2; i++) { if((*rd2)[i] == 4) ns2++; if(ns2 > maxns) { // doesn't pass return; } } // Both pass filt1 = filt2 = true; } else { if(rd1 != NULL) filt1 = nFilter(*rd1, ns1); if(rd2 != NULL) filt2 = nFilter(*rd2, ns2); } } #ifdef SCORING_MAIN int main() { { cout << "Case 1: Simple 1 ... "; Scoring sc = Scoring::base1(); assert_eq(COST_MODEL_CONSTANT, sc.matchType); assert_eq(0, sc.maxRefGaps(0, 10)); // 10 - 1 - 15 = -6 assert_eq(0, sc.maxRefGaps(0, 11)); // 11 - 1 - 15 = -5 assert_eq(0, sc.maxRefGaps(0, 12)); // 12 - 1 - 15 = -4 assert_eq(0, sc.maxRefGaps(0, 13)); // 13 - 1 - 15 = -3 assert_eq(0, sc.maxRefGaps(0, 14)); // 14 - 1 - 15 = -2 assert_eq(0, sc.maxRefGaps(0, 15)); // 15 - 1 - 15 = -1 assert_eq(1, sc.maxRefGaps(0, 16)); // 16 - 1 - 15 = 0 assert_eq(1, sc.maxRefGaps(0, 17)); // 17 - 2 - 19 = -4 assert_eq(1, sc.maxRefGaps(0, 18)); // 18 - 2 - 19 = -3 assert_eq(1, sc.maxRefGaps(0, 19)); // 19 - 2 - 19 = -2 assert_eq(1, sc.maxRefGaps(0, 20)); // 20 - 2 - 19 = -1 assert_eq(2, sc.maxRefGaps(0, 21)); // 21 - 2 - 19 = 0 assert_eq(0, sc.maxReadGaps(0, 10)); // 10 - 0 - 15 = -5 assert_eq(0, sc.maxReadGaps(0, 11)); // 11 - 0 - 15 = -4 assert_eq(0, sc.maxReadGaps(0, 12)); // 12 - 0 - 15 = -3 assert_eq(0, sc.maxReadGaps(0, 13)); // 13 - 0 - 15 = -2 assert_eq(0, sc.maxReadGaps(0, 14)); // 14 - 0 - 15 = -1 assert_eq(1, sc.maxReadGaps(0, 15)); // 15 - 0 - 15 = 0 assert_eq(1, sc.maxReadGaps(0, 16)); // 16 - 0 - 19 = -3 assert_eq(1, sc.maxReadGaps(0, 17)); // 17 - 0 - 19 = -2 assert_eq(1, sc.maxReadGaps(0, 18)); // 18 - 0 - 19 = -1 assert_eq(2, sc.maxReadGaps(0, 19)); // 19 - 0 - 19 = 0 assert_eq(2, sc.maxReadGaps(0, 20)); // 20 - 0 - 23 = -3 assert_eq(2, sc.maxReadGaps(0, 21)); // 21 - 0 - 23 = -2 // N ceiling: const=2, linear=0.1 assert_eq(1, sc.nCeil(1)); assert_eq(2, sc.nCeil(3)); assert_eq(2, sc.nCeil(5)); assert_eq(2, sc.nCeil(7)); assert_eq(2, sc.nCeil(9)); assert_eq(3, sc.nCeil(10)); for(int i = 0; i < 30; i++) { assert_eq(3, sc.n(i)); assert_eq(3, sc.mm(i)); } assert_eq(5, sc.gapbar); cout << "PASSED" << endl; } { cout << "Case 2: Simple 2 ... "; Scoring sc( 4, // reward for a match COST_MODEL_QUAL, // how to penalize mismatches 0, // constant if mm pelanty is a constant 30, // penalty for nuc mm in decoded colorspace als -3.0f, // constant coeff for minimum score -3.0f, // linear coeff for minimum score DEFAULT_FLOOR_CONST, // constant coeff for score floor DEFAULT_FLOOR_LINEAR, // linear coeff for score floor 3.0f, // max # ref Ns allowed in alignment; const coeff 0.4f, // max # ref Ns allowed in alignment; linear coeff COST_MODEL_QUAL, // how to penalize Ns in the read 0, // constant if N pelanty is a constant true, // whether to concatenate mates before N filtering 25, // constant coeff for cost of gap in the read 25, // constant coeff for cost of gap in the ref 10, // coeff of linear term for cost of gap in read 10, // coeff of linear term for cost of gap in ref 5, // 5 rows @ top/bot diagonal-entrance-only -1, // no restriction on row false // score prioritized over row ); assert_eq(COST_MODEL_CONSTANT, sc.matchType); assert_eq(4, sc.matchConst); assert_eq(COST_MODEL_QUAL, sc.mmcostType); assert_eq(COST_MODEL_QUAL, sc.npenType); assert_eq(0, sc.maxRefGaps(0, 8)); // 32 - 4 - 35 = -7 assert_eq(0, sc.maxRefGaps(0, 9)); // 36 - 4 - 35 = -3 assert_eq(1, sc.maxRefGaps(0, 10)); // 40 - 4 - 35 = 1 assert_eq(1, sc.maxRefGaps(0, 11)); // 44 - 8 - 45 = -9 assert_eq(1, sc.maxRefGaps(0, 12)); // 48 - 8 - 45 = -5 assert_eq(1, sc.maxRefGaps(0, 13)); // 52 - 8 - 45 = -1 assert_eq(2, sc.maxRefGaps(0, 14)); // 56 - 8 - 45 = 3 assert_eq(0, sc.maxReadGaps(0, 8)); // 32 - 0 - 35 = -3 assert_eq(1, sc.maxReadGaps(0, 9)); // 36 - 0 - 35 = 1 assert_eq(1, sc.maxReadGaps(0, 10)); // 40 - 0 - 45 = -5 assert_eq(1, sc.maxReadGaps(0, 11)); // 44 - 0 - 45 = -1 assert_eq(2, sc.maxReadGaps(0, 12)); // 48 - 0 - 45 = 3 assert_eq(2, sc.maxReadGaps(0, 13)); // 52 - 0 - 55 = -3 assert_eq(3, sc.maxReadGaps(0, 14)); // 56 - 0 - 55 = 1 // N ceiling: const=3, linear=0.4 assert_eq(1, sc.nCeil(1)); assert_eq(2, sc.nCeil(2)); assert_eq(3, sc.nCeil(3)); assert_eq(4, sc.nCeil(4)); assert_eq(5, sc.nCeil(5)); assert_eq(5, sc.nCeil(6)); assert_eq(5, sc.nCeil(7)); assert_eq(6, sc.nCeil(8)); assert_eq(6, sc.nCeil(9)); for(int i = 0; i < 256; i++) { assert_eq(i, sc.n(i)); assert_eq(i, sc.mm(i)); } assert_eq(5, sc.gapbar); cout << "PASSED" << endl; } } #endif /*def SCORING_MAIN*/ hisat2-2.2.1/scoring.h000066400000000000000000000432231370663763200145440ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef SCORING_H_ #define SCORING_H_ #include #include "qual.h" #include "simple_func.h" #include "limit.h" // Default type of bonus to added for matches #define DEFAULT_MATCH_BONUS_TYPE COST_MODEL_CONSTANT // When match bonus type is constant, use this constant #define DEFAULT_MATCH_BONUS 0 // Same settings but different defaults for --local mode #define DEFAULT_MATCH_BONUS_TYPE_LOCAL COST_MODEL_CONSTANT #define DEFAULT_MATCH_BONUS_LOCAL 2 // Default type of penalty to assess against mismatches #define DEFAULT_MM_PENALTY_TYPE COST_MODEL_QUAL // Default type of penalty to assess against mismatches #define DEFAULT_MM_PENALTY_TYPE_IGNORE_QUALS COST_MODEL_CONSTANT // When mismatch penalty type is constant, use this constant #define DEFAULT_MM_PENALTY_MAX 6 #define DEFAULT_MM_PENALTY_MIN 2 // When softclip penalty type is constant, use this constant #define DEFAULT_SC_PENALTY_MAX 2 #define DEFAULT_SC_PENALTY_MIN 1 // Default type of penalty to assess against mismatches #define DEFAULT_N_PENALTY_TYPE COST_MODEL_CONSTANT // When mismatch penalty type is constant, use this constant #define DEFAULT_N_PENALTY 1 // Constant coefficient b in linear function f(x) = ax + b determining // minimum valid score f when read length is x #define DEFAULT_MIN_CONST (-0.6f) // Linear coefficient a #define DEFAULT_MIN_LINEAR (-0.6f) // Different defaults for --local mode #define DEFAULT_MIN_CONST_LOCAL (0.0f) #define DEFAULT_MIN_LINEAR_LOCAL (10.0f) // Constant coefficient b in linear function f(x) = ax + b determining // maximum permitted number of Ns f in a read before it is filtered & // the maximum number of Ns in an alignment before it is considered // invalid. #define DEFAULT_N_CEIL_CONST 0.0f // Linear coefficient a #define DEFAULT_N_CEIL_LINEAR 0.15f // Default for whether to concatenate mates before the N filter (as opposed to // filting each mate separately) #define DEFAULT_N_CAT_PAIR false // Default read gap penalties for when homopolymer calling is reliable #define DEFAULT_READ_GAP_CONST 5 #define DEFAULT_READ_GAP_LINEAR 3 // Default read gap penalties for when homopolymer calling is not reliable #define DEFAULT_READ_GAP_CONST_BADHPOLY 3 #define DEFAULT_READ_GAP_LINEAR_BADHPOLY 1 // Default reference gap penalties for when homopolymer calling is reliable #define DEFAULT_REF_GAP_CONST 5 #define DEFAULT_REF_GAP_LINEAR 3 // Default reference gap penalties for when homopolymer calling is not reliable #define DEFAULT_REF_GAP_CONST_BADHPOLY 3 #define DEFAULT_REF_GAP_LINEAR_BADHPOLY 1 enum { COST_MODEL_ROUNDED_QUAL = 1, COST_MODEL_QUAL, COST_MODEL_CONSTANT }; /** * How to penalize various types of sequence dissimilarity, and other settings * that govern how dynamic programming tables should be filled in and how to * backtrace to find solutions. */ class Scoring { /** * Init an array that maps quality to penalty or bonus according to 'type' * and 'cons' */ template void initPens( T *pens, // array to fill int type, // penalty type; qual | rounded qual | constant int consMin, // constant for when penalty type is constant int consMax) // constant for when penalty type is constant { if(type == COST_MODEL_ROUNDED_QUAL) { for(int i = 0; i < 256; i++) { pens[i] = (T)qualRounds[i]; } } else if(type == COST_MODEL_QUAL) { assert_neq(consMin, 0); assert_neq(consMax, 0); for(int i = 0; i < 256; i++) { int ii = min(i, 40); // TODO: Bit hacky, this float frac = (float)ii / 40.0f; pens[i] = consMin + (T)(frac * (consMax-consMin)); assert_gt(pens[i], 0); //if(pens[i] == 0) { // pens[i] = ((consMax > 0) ? (T)1 : (T)-1); //} } } else if(type == COST_MODEL_CONSTANT) { for(int i = 0; i < 256; i++) { pens[i] = (T)consMax; } } else { throw 1; } } public: Scoring( int mat, // reward for a match int mmcType, // how to penalize mismatches int mmpMax_, // maximum mismatch penalty int mmpMin_, // minimum mismatch penalty int scpMax_, // maximum softclip penalty int scpMin_, // minimum softclip penalty const SimpleFunc& scoreMin_, // minimum score for valid alignment; const coeff const SimpleFunc& nCeil_, // max # ref Ns allowed in alignment; const coeff int nType, // how to penalize Ns in the read int n, // constant if N pelanty is a constant bool ncat, // whether to concatenate mates before N filtering int rdGpConst, // constant coeff for cost of gap in the read int rfGpConst, // constant coeff for cost of gap in the ref int rdGpLinear, // coeff of linear term for cost of gap in read int rfGpLinear, // coeff of linear term for cost of gap in ref int gapbar_, // # rows at top/bot can only be entered diagonally int cp_ = 0, // canonical splicing penalty int ncp_ = 12, // non-canonical splicing penalty int csp_ = 24, // conflicting splice site penalty const SimpleFunc* icp_ = NULL, // penalty as to intron length const SimpleFunc* incp_ = NULL) // penalty as to intron length { matchType = COST_MODEL_CONSTANT; matchConst = mat; mmcostType = mmcType; mmpMax = mmpMax_; mmpMin = mmpMin_; scpMax = scpMax_; scpMin = scpMin_; scoreMin = scoreMin_; nCeil = nCeil_; npenType = nType; npen = n; ncatpair = ncat; rdGapConst = rdGpConst; rfGapConst = rfGpConst; rdGapLinear = rdGpLinear; rfGapLinear = rfGpLinear; qualsMatter_ = mmcostType != COST_MODEL_CONSTANT; gapbar = gapbar_; monotone = matchType == COST_MODEL_CONSTANT && matchConst == 0; initPens(mmpens, mmcostType, mmpMin_, mmpMax_); initPens(npens, npenType, npen, npen); initPens(matchBonuses, matchType, matchConst, matchConst); cp = cp_; ncp = ncp_; csp = csp_; if(icp_ != NULL) icp = *icp_; if(incp_ != NULL) incp = *incp_; assert(repOk()); } /** * Set a constant match bonus. */ void setMatchBonus(int bonus) { matchType = COST_MODEL_CONSTANT; matchConst = bonus; initPens(matchBonuses, matchType, matchConst, matchConst); assert(repOk()); } /** * Set the mismatch penalty. */ void setMmPen(int mmType_, int mmpMax_, int mmpMin_) { mmcostType = mmType_; mmpMax = mmpMax_; mmpMin = mmpMin_; initPens(mmpens, mmcostType, mmpMin, mmpMax); } /** * Set the N penalty. */ void setNPen(int nType, int n) { npenType = nType; npen = n; initPens(npens, npenType, npen, npen); } #ifndef NDEBUG /** * Check that scoring scheme is internally consistent. */ bool repOk() const { assert_geq(matchConst, 0); assert_gt(rdGapConst, 0); assert_gt(rdGapLinear, 0); assert_gt(rfGapConst, 0); assert_gt(rfGapLinear, 0); return true; } #endif /** * Return a linear function of x where 'cnst' is the constant coefficiant * and 'lin' is the linear coefficient. */ static float linearFunc(int64_t x, float cnst, float lin) { return (float)((double)cnst + ((double)lin * x)); } /** * Return the penalty incurred by a mismatch at an alignment column * with read character 'rdc' reference mask 'refm' and quality 'q'. * * qs should be clamped to 63 on the high end before this query. */ inline int mm(int rdc, int refm, int q) const { assert_range(0, 255, q); return (rdc > 3 || refm > 15) ? npens[q] : mmpens[q]; } /** * Return the score of the given read character with the given quality * aligning to the given reference mask. Take Ns into account. */ inline int score(int rdc, int refm, int q) const { assert_range(0, 255, q); if(rdc > 3 || refm > 15) { return -npens[q]; } if((refm & (1 << rdc)) != 0) { return (int)matchBonuses[q]; } else { return -mmpens[q]; } } /** * Return the score of the given read character with the given quality * aligning to the given reference mask. Take Ns into account. Increment * a counter if it's an N. */ inline int score(int rdc, int refm, int q, int& ns) const { assert_range(0, 255, q); if(rdc > 3 || refm > 15) { ns++; return -npens[q]; } if((refm & (1 << rdc)) != 0) { return (int)matchBonuses[q]; } else { return -mmpens[q]; } } /** * Return the penalty incurred by a mismatch at an alignment column * with read character 'rdc' and quality 'q'. We assume the * reference character is non-N. */ inline int mm(int rdc, int q) const { assert_range(0, 255, q); return (rdc > 3) ? npens[q] : mmpens[q]; } /** * Return the marginal penalty incurred by a mismatch at a read * position with quality 'q'. */ inline int mm(int q) const { assert_geq(q, 0); return q < 255 ? mmpens[q] : mmpens[255]; } /** * Return the marginal penalty incurred by a mismatch at a read * position with quality 'q'. */ inline int sc(int q) const { assert_geq(q, 0); if(q <= 33) return scpMin; q -= 33; if(q > 40) q = 40; return (int)((q / 40.0f) * (scpMax - scpMin) + scpMin); } /** * Return the marginal penalty incurred by a mismatch at a read * position with quality 30. */ inline int64_t match() const { return match(30); } /** * Return the marginal penalty incurred by a mismatch at a read * position with quality 'q'. */ inline int64_t match(int q) const { assert_geq(q, 0); return (int64_t)((q < 255 ? matchBonuses[q] : matchBonuses[255]) + 0.5f); } /** * Return the best score achievable by a read of length 'rdlen'. */ inline int64_t perfectScore(size_t rdlen) const { if(monotone) { return 0; } else { return rdlen * match(30); } } /** * Return true iff the penalities are such that two reads with the * same sequence but different qualities might yield different * alignments. */ inline bool qualitiesMatter() const { return qualsMatter_; } /** * Return the marginal penalty incurred by an N mismatch at a read * position with quality 'q'. */ inline int n(int q) const { assert_geq(q, 0); return q < 255 ? npens[q] : npens[255]; } /** * Return the marginal penalty incurred by a gap in the read, * given that this is the 'ext'th extension of the gap (0 = open, * 1 = first, etc). */ inline int ins(int ext) const { assert_geq(ext, 0); if(ext == 0) return readGapOpen(); return readGapExtend(); } /** * Return the marginal penalty incurred by a gap in the reference, * given that this is the 'ext'th extension of the gap (0 = open, * 1 = first, etc). */ inline int del(int ext) const { assert_geq(ext, 0); if(ext == 0) return refGapOpen(); return refGapExtend(); } /** * Return true iff a read of length 'rdlen' passes the score filter, i.e., * has enough characters to rise above the minimum score threshold. */ bool scoreFilter( int64_t minsc, size_t rdlen) const; /** * Given the score floor for valid alignments and the length of the read, * calculate the maximum possible number of read gaps that could occur in a * valid alignment. */ int maxReadGaps( int64_t minsc, size_t rdlen) const; /** * Given the score floor for valid alignments and the length of the read, * calculate the maximum possible number of reference gaps that could occur * in a valid alignment. */ int maxRefGaps( int64_t minsc, size_t rdlen) const; /** * Given a read sequence, return true iff the read passes the N filter. * The N filter rejects reads with more than the number of Ns calculated by * taking nCeilConst + nCeilLinear * read length. */ bool nFilter(const BTDnaString& rd, size_t& ns) const; /** * Given a read sequence, return true iff the read passes the N filter. * The N filter rejects reads with more than the number of Ns calculated by * taking nCeilConst + nCeilLinear * read length. * * For paired-end reads, there is a question of how to apply the filter. * The filter could be applied to both mates separately, which might then * prevent paired-end alignment. Or the filter could be applied to the * reads as though they're concatenated together. The latter approach has * pros and cons. The pro is that we can use paired-end information to * recover alignments for mates that would not have passed the N filter on * their own. The con is that we might not want to do that, since the * non-N portion of the bad mate might contain particularly unreliable * information. */ void nFilterPair( const BTDnaString* rd1, // mate 1 const BTDnaString* rd2, // mate 2 size_t& ns1, // # Ns in mate 1 size_t& ns2, // # Ns in mate 2 bool& filt1, // true -> mate 1 rejected by filter bool& filt2) // true -> mate 2 rejected by filter const; /** * The penalty associated with opening a new read gap. */ inline int readGapOpen() const { return rdGapConst + rdGapLinear; } /** * The penalty associated with opening a new ref gap. */ inline int refGapOpen() const { return rfGapConst + rfGapLinear; } /** * The penalty associated with extending a read gap by one character. */ inline int readGapExtend() const { return rdGapLinear; } /** * The penalty associated with extending a ref gap by one character. */ inline int refGapExtend() const { return rfGapLinear; } // avg. known score: -22.96, avg. random score: -33.70 inline int64_t canSpl(int intronlen = 0, int minanchor = 100, float probscore = 0.0f) const { int penintron = (intronlen > 0 ? icp.f((double)intronlen) : 0); if(penintron < 0) penintron = 0; if(minanchor < 10 && probscore < -24.0f + (10 - minanchor)) { return MAX_I32; } return penintron + cp; } inline int64_t noncanSpl(int intronlen = 0, int minanchor = 100, float probscore = 0.0f) const { if(minanchor < 14) return MAX_I32; int penintron = (intronlen > 0 ? incp.f((double)intronlen) : 0); if(penintron < 0) penintron = 0; return penintron + ncp; } inline int conflictSpl() const { return (int)csp; } int matchType; // how to reward matches int matchConst; // reward for a match int mmcostType; // based on qual? rounded? just a constant? int mmpMax; // maximum mismatch penalty int mmpMin; // minimum mismatch penalty int scpMax; // maximum softclip penalty int scpMin; // minimum softclip penalty SimpleFunc scoreMin; // minimum score for valid alignment, constant coeff SimpleFunc nCeil; // max # Ns involved in alignment, constant coeff int npenType; // N: based on qual? rounded? just a constant? int npen; // N: if mmcosttype=constant, this is the const bool ncatpair; // true -> do N filtering on concated pair int rdGapConst; // constant term coeffecient in extend cost int rfGapConst; // constant term coeffecient in extend cost int rdGapLinear; // linear term coeffecient in extend cost int rfGapLinear; // linear term coeffecient in extend cost int gapbar; // # rows at top/bot can only be entered diagonally bool monotone; // scores can only go down? float matchBonuses[256]; // map from qualities to match bonus int mmpens[256]; // map from qualities to mm penalty int npens[256]; // map from N qualities to penalty int64_t cp; // canonical splicing penalty int64_t ncp; // non-canonical splicing penalty int64_t csp; // conflicting splice site penalty SimpleFunc icp; // intron length penalty SimpleFunc incp; // intron length penalty static Scoring base1() { const double DMAX = std::numeric_limits::max(); SimpleFunc scoreMin(SIMPLE_FUNC_LINEAR, 0.0f, DMAX, 37.0f, 0.3f); SimpleFunc nCeil(SIMPLE_FUNC_LINEAR, 0.0f, DMAX, 2.0f, 0.1f); return Scoring( 1, // reward for a match COST_MODEL_CONSTANT, // how to penalize mismatches 3, // max mismatch penalty 3, // min mismatch penalty 2, // max softclip penalty 2, // min softclip penalty scoreMin, // score min: 37 + 0.3x nCeil, // n ceiling: 2 + 0.1x COST_MODEL_CONSTANT, // how to penalize Ns in the read 3, // constant if N pelanty is a constant false, // concatenate mates before N filtering? 11, // constant coeff for gap in read 11, // constant coeff for gap in ref 4, // linear coeff for gap in read 4, // linear coeff for gap in ref 5); // 5 rows @ top/bot diagonal-entrance-only } protected: bool qualsMatter_; }; #endif /*SCORING_H_*/ hisat2-2.2.1/scripts/000077500000000000000000000000001370663763200144125ustar00rootroot00000000000000hisat2-2.2.1/scripts/convert_quals.pl000077500000000000000000000064621370663763200176470ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # # # convert_quals.pl # # Modify scale/encoding of quality values in a FASTQ file. # # Author: Ben Langmead # Date: 5/5/2009 # # p = probability that base is miscalled # Qphred = -10 * log10 (p) # Qsolexa = -10 * log10 (p / (1 - p)) # See: http://en.wikipedia.org/wiki/FASTQ_format # use strict; use warnings; use Getopt::Long; my $inphred = 33; my $insolexa = 0; my $outphred = 0; my $outsolexa = 64; # Default: convert 33-based Phred quals into 64-based Solexa qualss my $result = GetOptions ("inphred=i" => \$inphred, "insolexa=i" => \$insolexa, "outphred=i" => \$outphred, "outsolexa=i" => \$outsolexa); $result == 1 || die "One or more errors parsing script arguments"; if($inphred > 0) { $inphred >= 33 || die "Input base must be >= 33, was $inphred"; } else { $insolexa >= 33 || die "Input base must be >= 33, was $insolexa"; } sub log10($) { return log(shift) / log(10.0); } sub round { my($number) = shift; return int($number + .5 * ($number <=> 0)); } # Convert from phred qual to probability of miscall sub phredToP($) { my $phred = shift; my $p = (10.0 ** (($phred) / -10.0)); ($p >= 0.0 && $p <= 1.0) || die "Bad prob: $p, from sol $phred"; return $p; } # Convert from solexa qual to probability of miscall sub solToP($) { my $sol = shift; my $x = (10.0 ** (($sol) / -10.0)); my $p = $x / (1.0 + $x); ($p >= 0.0 && $p <= 1.0) || die "Bad prob: $p, from x $x, phred $sol"; return $p; } # Convert from probability of miscall to phred qual sub pToPhred($) { my $p = shift; ($p >= 0.0 && $p <= 1.0) || die "Bad prob: $p"; return round(-10.0 * log10($p)); } # Convert from probability of miscall to solexa qual sub pToSol($) { my $p = shift; ($p >= 0.0 && $p <= 1.0) || die "Bad prob: $p"; return 0.0 if $p == 1.0; return round(-10.0 * log10($p / (1.0 - $p))); } while(<>) { my $name = $_; print $name; my $seq = <>; print $seq; my $name2 = <>; print $name2; my $quals = <>; chomp($quals); my @qual = split(//, $quals); for(my $i = 0; $i <= $#qual; $i++) { my $co = ord($qual[$i]); my $p; # Convert input qual to p if($inphred > 0) { $co -= $inphred; $co >= 0 || die "Bad Phred input quality: $co"; $p = phredToP($co); } else { $co -= $insolexa; $p = solToP($co); } # Convert p to output qual if($outphred > 0) { $co = pToPhred($p); $co >= 0 || die "Bad Phred output quality: $co"; $co += $outphred; } else { $co = pToSol($p); $co += $outsolexa; } $co >= 33 || die "Error: Output qual " . $co . " char is less than 33. Try a larger output base."; print chr($co); } print "\n"; } hisat2-2.2.1/scripts/gen_2b_occ_lookup.pl000077500000000000000000000057361370663763200203360ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # # # Generate lookup table that, given two packed DNA bytes (eight bases) # and a character (A, C, G or T), returns how many times that character # occurs in that packed pair of bytes. Useful for quickly counting # character occurrences in long strings. The LUT is indexed first by # character (0 - 3) then by byte (0 - 2^16-1). # # See ebwt.h. # my @as = (); my @cs = (); my @gs = (); my @ts = (); # Compile character arrays my $i; for($i = 0; $i < (256*256); $i++) { my $b01 = ($i >> 0) & 3; my $b23 = ($i >> 2) & 3; my $b45 = ($i >> 4) & 3; my $b67 = ($i >> 6) & 3; my $b89 = ($i >> 8) & 3; my $b1011 = ($i >> 10) & 3; my $b1213 = ($i >> 12) & 3; my $b1415 = ($i >> 14) & 3; my $a = ($b01 == 0) + ($b23 == 0) + ($b45 == 0) + ($b67 == 0) + ($b89 == 0) + ($b1011 == 0) + ($b1213 == 0) + ($b1415 == 0); my $c = ($b01 == 1) + ($b23 == 1) + ($b45 == 1) + ($b67 == 1) + ($b89 == 1) + ($b1011 == 1) + ($b1213 == 1) + ($b1415 == 1); my $g = ($b01 == 2) + ($b23 == 2) + ($b45 == 2) + ($b67 == 2) + ($b89 == 2) + ($b1011 == 2) + ($b1213 == 2) + ($b1415 == 2); my $t = ($b01 == 3) + ($b23 == 3) + ($b45 == 3) + ($b67 == 3) + ($b89 == 3) + ($b1011 == 3) + ($b1213 == 3) + ($b1415 == 3); push @as, $a; push @cs, $c; push @gs, $g; push @ts, $t; } my $entsPerLine = 16; # Count occurrences in all 4 bit pairs print "uint8_t cCntLUT_16b_4[4][256*256] = {\n"; # Print As array print "\t/* As */ {\n"; for($i = 0; $i < (256*256); $i++) { print "\t\t" if(($i % $entsPerLine) == 0); print "$as[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t},\n"; # Print Cs array print "\t/* Cs */ {\n"; for($i = 0; $i < (256*256); $i++) { print "\t\t" if(($i % $entsPerLine) == 0); print "$cs[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t},\n"; # Print Gs array print "\t/* Gs */ {\n"; for($i = 0; $i < (256*256); $i++) { print "\t\t" if(($i % $entsPerLine) == 0); print "$gs[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t},\n"; # Print Ts array print "\t/* Ts */ {\n"; for($i = 0; $i < (256*256); $i++) { print "\t\t" if(($i % $entsPerLine) == 0); print "$ts[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t}\n"; print "};\n"; hisat2-2.2.1/scripts/gen_occ_lookup.pl000077500000000000000000000150201370663763200177360ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # # # Generate lookup table that, given a packed DNA byte (four bases) and # a character (A, C, G or T), returns how many times that character # occurs in that packed byte. Useful for quickly counting character # occurrences in long strings. The LUT is indexed first by character # (0-3) then by byte (0-255). # # Larger lookup tables are also possible, though they seem # counterproductive. E.g., looking up eight bases at a time yields a # 256K LUT, which doesn't fit in L1. A four-base LUT is 1KB, easily # fitting in L1. # # See ebwt.h. # my @as4 = (), @as3 = (), @as2 = (), @as1 = (); my @cs4 = (), @cs3 = (), @cs2 = (), @cs1 = (); my @gs4 = (), @gs3 = (), @gs2 = (), @gs1 = (); my @ts4 = (), @ts3 = (), @ts2 = (), @ts1 = (); # Compile character arrays my $i; for($i = 0; $i < 256; $i++) { my $b01 = ($i >> 0) & 3; my $b23 = ($i >> 2) & 3; my $b45 = ($i >> 4) & 3; my $b67 = ($i >> 6) & 3; my $a4 = ($b01 == 0) + ($b23 == 0) + ($b45 == 0) + ($b67 == 0); my $c4 = ($b01 == 1) + ($b23 == 1) + ($b45 == 1) + ($b67 == 1); my $g4 = ($b01 == 2) + ($b23 == 2) + ($b45 == 2) + ($b67 == 2); my $t4 = ($b01 == 3) + ($b23 == 3) + ($b45 == 3) + ($b67 == 3); push @as4, $a4; push @cs4, $c4; push @gs4, $g4; push @ts4, $t4; my $a3 = ($b01 == 0) + ($b23 == 0) + ($b45 == 0); my $c3 = ($b01 == 1) + ($b23 == 1) + ($b45 == 1); my $g3 = ($b01 == 2) + ($b23 == 2) + ($b45 == 2); my $t3 = ($b01 == 3) + ($b23 == 3) + ($b45 == 3); push @as3, $a3; push @cs3, $c3; push @gs3, $g3; push @ts3, $t3; my $a2 = ($b01 == 0) + ($b23 == 0); my $c2 = ($b01 == 1) + ($b23 == 1); my $g2 = ($b01 == 2) + ($b23 == 2); my $t2 = ($b01 == 3) + ($b23 == 3); push @as2, $a2; push @cs2, $c2; push @gs2, $g2; push @ts2, $t2; my $a1 = ($b01 == 0) + 0; my $c1 = ($b01 == 1) + 0; my $g1 = ($b01 == 2) + 0; my $t1 = ($b01 == 3) + 0; push @as1, $a1; push @cs1, $c1; push @gs1, $g1; push @ts1, $t1; } my $entsPerLine = 16; print "#include \n\n"; print "/* Generated by gen_lookup_tables.pl */\n\n"; # Count occurrences in all 4 bit pairs print "uint8_t cCntLUT_4[4][4][256] = {\n"; print "\t/* All 4 bit pairs */ {\n"; # Print As array print "\t\t/* As */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$as4[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Cs array print "\t\t/* Cs */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$cs4[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Gs array print "\t\t/* Gs */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$gs4[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Ts array print "\t\t/* Ts */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$ts4[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t}\n\t},\n"; # Count occurrences in low 1 bit pair print "\t/* Least significant 1 bit pair */ {\n"; # Print As array print "\t\t/* As */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$as1[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Cs array print "\t\t/* Cs */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$cs1[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Gs array print "\t\t/* Gs */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$gs1[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Ts array print "\t\t/* Ts */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$ts1[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t}\n\t},\n"; # Count occurrences in low 2 bit pairs print "\t/* Least significant 2 bit pairs */ {\n"; # Print As array print "\t\t/* As */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$as2[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Cs array print "\t\t/* Cs */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$cs2[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Gs array print "\t\t/* Gs */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$gs2[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Ts array print "\t\t/* Ts */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$ts2[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t}\n\t},\n"; # Count occurrences in low 3 bit pairs print "\t/* Least significant 3 bit pairs */ {\n"; # Print As array print "\t\t/* As */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$as3[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Cs array print "\t\t/* Cs */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$cs3[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Gs array print "\t\t/* Gs */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$gs3[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t},\n"; # Print Ts array print "\t\t/* Ts */ {\n"; for($i = 0; $i < 256; $i++) { print "\t\t\t" if(($i % $entsPerLine) == 0); print "$ts3[$i], "; print "\n" if(($i % $entsPerLine) == ($entsPerLine-1)); } print "\t\t}\n\t}\n"; print "};\n"; hisat2-2.2.1/scripts/gen_solqual_lookup.pl000077500000000000000000000040241370663763200206540ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # use warnings; use strict; sub log10($) { return log(shift) / log(10.0); } sub round { my($number) = shift; return int($number + .5 * ($number <=> 0)); } # Convert from solexa qual to probability of miscall sub phredToP($) { my $sol = shift; my $p = (10.0 ** (($sol) / -10.0)); ($p >= 0.0 && $p <= 1.0) || die "Bad prob: $p, from sol $sol"; return $p; } # Convert from phred qual to probability of miscall sub solToP($) { my $phred = shift; my $x = (10.0 ** (($phred) / -10.0)); my $p = $x / (1.0 + $x); ($p >= 0.0 && $p <= 1.0) || die "Bad prob: $p, from x $x, phred $phred"; return $p; } # Convert from probability of miscall to phred qual sub pToPhred($) { my $p = shift; ($p >= 0.0 && $p <= 1.0) || die "Bad prob: $p"; return round(-10.0 * log10($p)); } # Convert from probability of miscall to solexa qual sub pToSol($) { my $p = shift; ($p >= 0.0 && $p <= 1.0) || die "Bad prob: $p"; return 0 if($p == 1.0); return round(-10.0 * log10($p / (1.0 - $p))); } # Print conversion table from Phred to Solexa print "uint8_t solToPhred[] = {"; my $cols = 10; my $cnt = 0; for(my $i = -10; $i < 256; $i++) { # Solexa qual = $i my $p = solToP($i); my $ph = pToPhred($p); print "\n\t/* $i */ " if($cnt == 0); $cnt++; $cnt = 0 if($cnt == 10); print "$ph"; print ", " if($i < 255); } print "\n};\n"; hisat2-2.2.1/scripts/infer_fraglen.pl000077500000000000000000000100131370663763200175460ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # ## # infer_fraglen.pl # # Infer fragment length by looking for unique alignments for mates # (separately), then piecing those together and building a distribution. # use strict; use warnings; use Getopt::Long; use FindBin qw($Bin); my $m1 = ""; my $m2 = ""; my $index = ""; my $bowtie_args = ""; my $bowtie2 = "$Bin/../bowtie2"; my $debug = 0; my $binsz = 10; my $mapq_cutoff = 30; my $upto = undef; sub dieusage { my $msg = shift; my $exitlevel = shift; $exitlevel = $exitlevel || 1; print STDERR "$msg\n"; exit $exitlevel; } ## # Given a basename, return true iff all index files exist. # sub checkIndex($) { my $idx = shift; my $ext = "bt2"; return -f "$idx.1.$ext" && -f "$idx.2.$ext" && -f "$idx.3.$ext" && -f "$idx.4.$ext" && -f "$idx.rev.1.$ext" && -f "$idx.rev.2.$ext"; } GetOptions ( "bowtie2=s" => \$bowtie2, "index=s" => \$index, "m1=s" => \$m1, "m2=s" => \$m2, "upto=i" => \$upto, "mapq_cutoff=i" => \$mapq_cutoff, "debug" => \$debug, "bowtie-args=s" => \$bowtie_args) || dieusage("Bad option", 1); die "Must specify --m1" if $m1 eq ""; die "Must specify --m2" if $m2 eq ""; die "Must specify --index" if $index eq ""; $m1 =~ s/^~/$ENV{HOME}/; $m2 =~ s/^~/$ENV{HOME}/; $index =~ s/^~/$ENV{HOME}/; die "Bad bowtie path: $bowtie2" if system("$bowtie2 --version >/dev/null 2>/dev/null") != 0; die "Bad index: $index" if !checkIndex($index); # Hash holding all the observed fragment orientations and lengths my %fragments = (); my $m1cmd = ($m1 =~ /\.gz$/) ? "gzip -dc $m1" : "cat $m1"; my $m2cmd = ($m2 =~ /\.gz$/) ? "gzip -dc $m2" : "cat $m2"; my $cmd1 = "$m1cmd | $bowtie2 $bowtie_args --sam-nohead -x $index - > .infer_fraglen.tmp"; my $cmd2 = "$m2cmd | $bowtie2 $bowtie_args --sam-nohead -x $index - |"; my $tot = 0; system($cmd1) == 0 || die "Error running '$cmd1'"; open (M1, ".infer_fraglen.tmp") || die "Could not open '.infer_fraglen.tmp'"; open (M2, $cmd2) || die "Could not open '$cmd2'"; while() { my $lm1 = $_; my $lm2 = ; chomp($lm1); chomp($lm2); my @lms1 = split(/\t/, $lm1); my @lms2 = split(/\t/, $lm2); my ($name1, $flags1, $chr1, $off1, $mapq1, $slen1) = ($lms1[0], $lms1[1], $lms1[2], $lms1[3], $lms1[4], length($lms1[9])); my ($name2, $flags2, $chr2, $off2, $mapq2, $slen2) = ($lms2[0], $lms2[1], $lms2[2], $lms2[3], $lms2[4], length($lms2[9])); # One or both mates didn't align uniquely? next if $chr1 eq "*" || $chr2 eq "*"; # Mates aligned to different chromosomes? next if $chr1 ne $chr2; # MAPQs too low? next if $mapq1 < $mapq_cutoff || $mapq2 < $mapq_cutoff; # This pairing can be used as an observation of fragment orientation and length my $fw1 = (($flags1 & 16) == 0) ? "F" : "R"; my $fw2 = (($flags2 & 16) == 0) ? "F" : "R"; my $frag = $off2 - $off1; # This can overestimate if one mate is entirely subsumed in the other if($frag > 0) { $frag += $slen2; } else { $frag -= $slen1; } # Install into bin $frag = int(($frag + ($binsz/2))/$binsz); # Round to nearest bin $fragments{"$fw1$fw2"}{$frag}++; $tot++; } close(M1); close(M2); unlink(".infer_fraglen.tmp"); # ditch temporary file # Print out the bins for my $k (keys %fragments) { for my $k2 (sort {$a <=> $b} keys %{$fragments{$k}}) { print "$k, ".($k2*$binsz).", ".$fragments{$k}{$k2}."\n"; } } print STDERR "DONE\n"; hisat2-2.2.1/scripts/make_a_thaliana_tair.sh000077500000000000000000000022501370663763200210450ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for A. thaliana from TAIR v10 and build Bowtie 2 index. # GENOMES_MIRROR=ftp://ftp.arabidopsis.org/home/tair get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } BOWTIE_BUILD_EXE=./bowtie2-build if [ ! -x "$BOWTIE_BUILD_EXE" ] ; then if ! which bowtie2-build ; then echo "Could not find bowtie2-build in current directory or in PATH" exit 1 else BOWTIE_BUILD_EXE=`which bowtie2-build` fi fi FC= for c in 1 2 3 4 5 C M ; do if [ ! -f TAIR10_chr$c.fas ] ; then FN=TAIR10_chr$c.fas F=${GENOMES_MIRROR}/Sequences/whole_chromosomes/${FN} [ -n "$FC" ] && FC="$FC,$FN" [ -z "$FC" ] && FC=$FN get $F || (echo "Error getting $F" && exit 1) fi if [ ! -f TAIR10_chr$c.fas ] ; then echo "Could not find chr$c.fas file!" exit 2 fi done CMD="${BOWTIE_BUILD_EXE} $* $FC a_thaliana" echo $CMD if $CMD ; then echo "a_thaliana index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_b_taurus_UMD3.sh000077500000000000000000000026721370663763200203710ustar00rootroot00000000000000#!/bin/sh # # Builds an index from UMD Freeze 3.0 of the Bos Taurus (cow) genome. # BASE_CHRS="\ Chr1 \ Chr2 \ Chr3 \ Chr4 \ Chr5 \ Chr6 \ Chr7 \ Chr8 \ Chr9 \ Chr10 \ Chr11 \ Chr12 \ Chr13 \ Chr14 \ Chr15 \ Chr16 \ Chr17 \ Chr18 \ Chr19 \ Chr20 \ Chr21 \ Chr22 \ Chr23 \ Chr24 \ Chr25 \ Chr26 \ Chr27 \ Chr28 \ Chr29 \ ChrX \ ChrU \ ChrY-contigs \ ChrY-contigs.SHOTGUN_ONLY" CHRS_TO_INDEX=$BASE_CHRS get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } BOWTIE_BUILD_EXE=./bowtie2-build if [ ! -x "$BOWTIE_BUILD_EXE" ] ; then if ! which bowtie2-build ; then echo "Could not find bowtie2-build in current directory or in PATH" exit 1 else BOWTIE_BUILD_EXE=`which bowtie2-build` fi fi FTP_BASE=ftp://ftp.cbcb.umd.edu/pub/data/Bos_taurus/Bos_taurus_UMD_3.0 OUTPUT=b_taurus INPUTS= for c in $CHRS_TO_INDEX ; do if [ ! -f ${c}.fa ] ; then F=${c}.fa.gz get ${FTP_BASE}/$F || (echo "Error getting $F" && exit 1) gunzip $F || (echo "Error unzipping $F" && exit 1) fi [ -n "$INPUTS" ] && INPUTS=$INPUTS,${c}.fa [ -z "$INPUTS" ] && INPUTS=${c}.fa done CMD="$BOWTIE_BUILD_EXE $* $INPUTS $OUTPUT" echo $CMD if $CMD ; then echo "$OUTPUT index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_bdgp6.sh000077500000000000000000000027421370663763200167550ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the BDGP6 release 84 version of drosophila melanogaster (fly) from # Ensembl. # # Note that Ensembl's build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/drosophila_melanogaster/dna get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa F=Drosophila_melanogaster.BDGP6.dna.toplevel.fa if [ ! -f $F ] ; then get ${ENSEMBL_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_bdgp6_tran.sh000077500000000000000000000050611370663763200177760ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the BDGP6 release 84 version of drosophila melanogaster (fly) from # Ensembl. # # Note that Ensembl's build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/drosophila_melanogaster/dna ENSEMBL_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/drosophila_melanogaster GTF_FILE=Drosophila_melanogaster.BDGP6.${ENSEMBL_RELEASE}.gtf get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Drosophila_melanogaster.BDGP6.dna.toplevel.fa if [ ! -f $F ] ; then get ${ENSEMBL_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --ss genome.ss --exon genome.exon genome_tran" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_canFam2.sh000077500000000000000000000024521370663763200172200ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the canFam2 version of C. familiaris (dog) # from UCSC. # i=2 BASE_CHRS=chr1 while [ $i -lt 39 ] ; do BASE_CHRS="$BASE_CHRS chr$i" i=`expr $i + 1` done BASE_CHRS="$BASE_CHRS chrX chrM chrUn" CHRS_TO_INDEX=$BASE_CHRS CANFAM2_BASE=ftp://hgdownload.cse.ucsc.edu/goldenPath/canFam2/chromosomes get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } BOWTIE_BUILD_EXE=./bowtie2-build if [ ! -x "$BOWTIE_BUILD_EXE" ] ; then if ! which bowtie2-build ; then echo "Could not find bowtie2-build in current directory or in PATH" exit 1 else BOWTIE_BUILD_EXE=`which bowtie2-build` fi fi INPUTS= for c in $CHRS_TO_INDEX ; do if [ ! -f ${c}.fa ] ; then F=${c}.fa.gz get ${CANFAM2_BASE}/$F || (echo "Error getting $F" && exit 1) gunzip $F || (echo "Error unzipping $F" && exit 1) fi [ -n "$INPUTS" ] && INPUTS=$INPUTS,${c}.fa [ -z "$INPUTS" ] && INPUTS=${c}.fa done CMD="${BOWTIE_BUILD_EXE} $* ${INPUTS} canFam2" echo Running $CMD if $CMD ; then echo "canFam2 index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_ce10.sh000077500000000000000000000016721370663763200165040ustar00rootroot00000000000000#!/bin/sh CE10_BASE=ftp://hgdownload.cse.ucsc.edu/goldenPath/ce10/bigZips F=chromFa.tar.gz get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget -O `basename $1` $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa get ${CE10_BASE}/$F || (echo "Error getting $F" && exit 1) tar xvzfO $F > genome.fa || (echo "Error unzipping $F" && exit 1) rm $F CMD="${HISAT2_BUILD_EXE} genome.fa genome" echo "Running $CMD" if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_dm6.sh000077500000000000000000000022531370663763200164360ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for a D. melanogaster from flybase. Currently set # to download 5.22, but F, REL, and IDX_NAME can be edited to reflect a # different version number. (But note that you will usually also have # to change the date in REL.) # DM6_BASE=ftp://hgdownload.cse.ucsc.edu/goldenPath/dm6/bigZips F=dm6.fa.gz get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget -O `basename $1` $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa get ${DM6_BASE}/$F || (echo "Error getting $F" && exit 1) gzip -cd $F > genome.fa || (echo "Error unzipping $F" && exit 1) rm $F CMD="${HISAT2_BUILD_EXE} genome.fa genome" echo "Running $CMD" if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_e_coli.sh000077500000000000000000000021131370663763200171750ustar00rootroot00000000000000#!/bin/sh # # Downloads the sequence for a strain of e. coli from NCBI and builds a # Bowtie index for it # GENOMES_MIRROR=ftp://ftp.ncbi.nlm.nih.gov/genomes BOWTIE_BUILD_EXE=./bowtie2-build if [ ! -x "$BOWTIE_BUILD_EXE" ] ; then if ! which bowtie2-build ; then echo "Could not find bowtie2-build in current directory or in PATH" exit 1 else BOWTIE_BUILD_EXE=`which bowtie2-build` fi fi if [ ! -f NC_008253.fna ] ; then if ! which wget > /dev/null ; then echo wget not found, looking for curl... if ! which curl > /dev/null ; then echo curl not found either, aborting... else # Use curl curl ${GENOMES_MIRROR}/Bacteria/Escherichia_coli_536_uid58531/NC_008253.fna -o NC_008253.fna fi else # Use wget wget ${GENOMES_MIRROR}/Bacteria/Escherichia_coli_536_uid58531/NC_008253.fna fi fi if [ ! -f NC_008253.fna ] ; then echo "Could not find NC_008253.fna file!" exit 2 fi CMD="${BOWTIE_BUILD_EXE} $* -t 8 NC_008253.fna e_coli" echo $CMD if $CMD ; then echo "e_coli index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch37.sh000077500000000000000000000027561370663763200170550ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh37 release 75 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh37 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=75 ENSEMBL_GRCh37_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa F=Homo_sapiens.GRCh37.${ENSEMBL_RELEASE}.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh37_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi CMD="${HISAT2_BUILD_EXE} genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch37_snp.sh000077500000000000000000000046641370663763200177350ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh37 release 75 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh37 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=75 ENSEMBL_GRCh37_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna DBSNP_RELEASE=144 SNP_FILE=snp${DBSNP_RELEASE}Common.txt UCSC_COMMON_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/${SNP_FILE} get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi rm -f genome.fa F=Homo_sapiens.GRCh37.${ENSEMBL_RELEASE}.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh37_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $SNP_FILE ] ; then get ${UCSC_COMMON_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) gunzip ${SNP_FILE}.gz || (echo "Error unzipping ${SNP_FILE}" && exit 1) awk 'BEGIN{OFS="\t"} {if($2 ~ /^chr/) {$2 = substr($2, 4)}; if($2 == "M") {$2 = "MT"} print}' ${SNP_FILE} > ${SNP_FILE}.tmp mv ${SNP_FILE}.tmp ${SNP_FILE} ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE} genome fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype genome_snp" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch37_snp_tran.sh000077500000000000000000000067741370663763200207650ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh37 release 75 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh37 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=75 ENSEMBL_GRCh37_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna ENSEMBL_GRCh37_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/homo_sapiens GTF_FILE=Homo_sapiens.GRCh37.${ENSEMBL_RELEASE}.gtf DBSNP_RELEASE=144 SNP_FILE=snp${DBSNP_RELEASE}Common.txt UCSC_COMMON_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/${SNP_FILE} get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Homo_sapiens.GRCh37.${ENSEMBL_RELEASE}.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh37_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $SNP_FILE ] ; then get ${UCSC_COMMON_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) gunzip ${SNP_FILE}.gz || (echo "Error unzipping ${SNP_FILE}" && exit 1) awk 'BEGIN{OFS="\t"} {if($2 ~ /^chr/) {$2 = substr($2, 4)}; if($2 == "M") {$2 = "MT"} print}' ${SNP_FILE} > ${SNP_FILE}.tmp mv ${SNP_FILE}.tmp ${SNP_FILE} ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE} genome fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GRCh37_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype --ss genome.ss --exon genome.exon genome_snp_tran" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch37_snp_tran_ercc.sh000077500000000000000000000074111370663763200217460ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh37 release 75 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh37 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=75 ENSEMBL_GRCh37_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna ENSEMBL_GRCh37_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/homo_sapiens GTF_FILE=Homo_sapiens.GRCh37.${ENSEMBL_RELEASE}.gtf DBSNP_RELEASE=144 SNP_FILE=snp${DBSNP_RELEASE}Common.txt UCSC_COMMON_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/${SNP_FILE} ERCC_FILE=ERCC92 ERCC_FTP=https://tools.thermofisher.com/content/sfs/manuals/${ERCC_FILE}.zip get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Homo_sapiens.GRCh37.${ENSEMBL_RELEASE}.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh37_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa get ${ERCC_FTP} || (echo "Error getting ${ERCC_FILE}.zip" && exit 1) unzip ${ERCC_FILE}.zip cat ${ERCC_FILE}.fa >> genome.fa fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GRCh37_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) cat ${ERCC_FILE}.gtf >> ${GTF_FILE} ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi if [ ! -f $SNP_FILE ] ; then get ${UCSC_COMMON_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) gunzip ${SNP_FILE}.gz || (echo "Error unzipping ${SNP_FILE}" && exit 1) awk 'BEGIN{OFS="\t"} {if($2 ~ /^chr/) {$2 = substr($2, 4)}; if($2 == "M") {$2 = "MT"} print}' ${SNP_FILE} > ${SNP_FILE}.tmp mv ${SNP_FILE}.tmp ${SNP_FILE} ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE} genome fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype --ss genome.ss --exon genome.exon genome_snp_tran_ercc" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch37_tran.sh000077500000000000000000000050731370663763200200740ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh37 release 75 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh37 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=75 ENSEMBL_GRCh37_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna ENSEMBL_GRCh37_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/homo_sapiens GTF_FILE=Homo_sapiens.GRCh37.${ENSEMBL_RELEASE}.gtf get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Homo_sapiens.GRCh37.${ENSEMBL_RELEASE}.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh37_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GRCh37_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --ss genome.ss --exon genome.exon genome_tran" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch38.sh000077500000000000000000000027401370663763200170470ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh38 release 84 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh38 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCh38_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa F=Homo_sapiens.GRCh38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh38_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch38_rep.sh000077500000000000000000000040151370663763200177120ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh38 release 84 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh38 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCh38_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_REPEAT_EXE=./hisat2-repeat if [ ! -x "$HISAT2_REPEAT_EXE" ]; then if ! which hisat2-repeat ; then echo "Could not find hisat2-repeat in current directory or in PATH" exit 1 else HISAT2_REPEAT_EXE=`which hisat2-repeat` fi fi rm -f genome.fa F=Homo_sapiens.GRCh38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh38_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi # Build repeat CMD="${HISAT2_REPEAT_EXE} -p 4 --repeat-count 5 --repeat-length 51-300,76-300,100-300,101-300,151-300 genome.fa genome_rep" echo Running $CMD $CMD [[ "$?" -eq 0 ]] || { echo "Index building failed; see error message"; exit 1; }; CMD="${HISAT2_BUILD_EXE} -p 4 --repeat-ref genome_rep.rep.fa --repeat-info genome_rep.rep.info genome.fa genome_rep" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch38_snp.sh000077500000000000000000000046411370663763200177310ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh38 release 84 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh38 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCh38_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna DBSNP_RELEASE=144 SNP_FILE=snp${DBSNP_RELEASE}Common.txt UCSC_COMMON_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/hg38/database/${SNP_FILE} get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi rm -f genome.fa F=Homo_sapiens.GRCh38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh38_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $SNP_FILE ] ; then get ${UCSC_COMMON_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) gunzip ${SNP_FILE}.gz || (echo "Error unzipping ${SNP_FILE}" && exit 1) awk 'BEGIN{OFS="\t"} {if($2 ~ /^chr/) {$2 = substr($2, 4)}; if($2 == "M") {$2 = "MT"} print}' ${SNP_FILE} > ${SNP_FILE}.tmp mv ${SNP_FILE}.tmp ${SNP_FILE} ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE} genome fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype genome_snp" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch38_snp_rep.sh000077500000000000000000000057161370663763200206030ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh38 release 84 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh38 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCh38_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna DBSNP_RELEASE=144 SNP_FILE=snp${DBSNP_RELEASE}Common.txt UCSC_COMMON_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/hg38/database/${SNP_FILE} get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi HISAT2_REPEAT_EXE=./hisat2-repeat if [ ! -x "$HISAT2_REPEAT_EXE" ]; then if ! which hisat2-repeat ; then echo "Could not find hisat2-repeat in current directory or in PATH" exit 1 else HISAT2_REPEAT_EXE=`which hisat2-repeat` fi fi rm -f genome.fa F=Homo_sapiens.GRCh38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh38_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $SNP_FILE ] ; then get ${UCSC_COMMON_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) gunzip ${SNP_FILE}.gz || (echo "Error unzipping ${SNP_FILE}" && exit 1) awk 'BEGIN{OFS="\t"} {if($2 ~ /^chr/) {$2 = substr($2, 4)}; if($2 == "M") {$2 = "MT"} print}' ${SNP_FILE} > ${SNP_FILE}.tmp mv ${SNP_FILE}.tmp ${SNP_FILE} ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE} genome fi # Build repeat CMD="${HISAT2_REPEAT_EXE} -p 4 --repeat-count 5 --repeat-length 51-300,76-300,100-300,101-300,151-300 genome.fa genome_rep" echo Running $CMD $CMD [[ "$?" -eq 0 ]] || { echo "Index building failed; see error message"; exit 1; }; CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype --repeat-ref genome_rep.rep.fa --repeat-info genome_rep.rep.info genome_snp_rep" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch38_snp_tran.sh000077500000000000000000000067511370663763200207610ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh38 release 84 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh38 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCh38_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna ENSEMBL_GRCh38_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/homo_sapiens GTF_FILE=Homo_sapiens.GRCh38.${ENSEMBL_RELEASE}.gtf DBSNP_RELEASE=144 SNP_FILE=snp${DBSNP_RELEASE}Common.txt UCSC_COMMON_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/hg38/database/${SNP_FILE} get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Homo_sapiens.GRCh38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh38_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $SNP_FILE ] ; then get ${UCSC_COMMON_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) gunzip ${SNP_FILE}.gz || (echo "Error unzipping ${SNP_FILE}" && exit 1) awk 'BEGIN{OFS="\t"} {if($2 ~ /^chr/) {$2 = substr($2, 4)}; if($2 == "M") {$2 = "MT"} print}' ${SNP_FILE} > ${SNP_FILE}.tmp mv ${SNP_FILE}.tmp ${SNP_FILE} ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE} genome fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GRCh38_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype --ss genome.ss --exon genome.exon genome_snp_tran" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch38_snp_tran_ercc.sh000077500000000000000000000073661370663763200217600ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh38 release 84 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh38 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCh37_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna ENSEMBL_GRCh37_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/homo_sapiens GTF_FILE=Homo_sapiens.GRCh38.${ENSEMBL_RELEASE}.gtf DBSNP_RELEASE=144 SNP_FILE=snp${DBSNP_RELEASE}Common.txt UCSC_COMMON_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/${SNP_FILE} ERCC_FILE=ERCC92 ERCC_FTP=https://tools.thermofisher.com/content/sfs/manuals/${ERCC_FILE}.zip get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Homo_sapiens.GRCh38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh37_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa get ${ERCC_FTP} || (echo "Error getting ${ERCC_FILE}.zip" && exit 1) unzip ${ERCC_FILE}.zip cat ${ERCC_FILE}.fa >> genome.fa fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GRCh37_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) cat ${ERCC_FILE}.gtf >> ${GTF_FILE} ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi if [ ! -f $SNP_FILE ] ; then get ${UCSC_COMMON_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) gunzip ${SNP_FILE}.gz || (echo "Error unzipping ${SNP_FILE}" && exit 1) awk 'BEGIN{OFS="\t"} {if($2 ~ /^chr/) {$2 = substr($2, 4)}; if($2 == "M") {$2 = "MT"} print}' ${SNP_FILE} > ${SNP_FILE}.tmp mv ${SNP_FILE}.tmp ${SNP_FILE} ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE} genome fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype --ss genome.ss --exon genome.exon genome_snp_tran_ercc" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grch38_tran.sh000077500000000000000000000050501370663763200200700ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCh38 release 84 version of H. sapiens (human) from # Ensembl. # # Note that Ensembl's GRCh38 build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCh38_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/homo_sapiens/dna ENSEMBL_GRCh38_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/homo_sapiens GTF_FILE=Homo_sapiens.GRCh38.${ENSEMBL_RELEASE}.gtf get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Homo_sapiens.GRCh38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCh38_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GRCh38_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --ss genome.ss --exon genome.exon genome_tran" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grcm38.sh000077500000000000000000000025421370663763200170540ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCm38 release 81 version of M. Musculus (mouse) from # Ensembl. # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCm38_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/mus_musculus/dna get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa F=Mus_musculus.GRCm38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCm38_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi CMD="${HISAT2_BUILD_EXE} genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grcm38_snp.sh000077500000000000000000000044501370663763200177340ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCm37 release 81 version of M. Musculus (mouse) from # Ensembl. # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCm38_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/mus_musculus/dna DBSNP_RELEASE=142 SNP_FILE=snp${DBSNP_RELEASE}Common.txt UCSC_COMMON_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/mm10/database/${SNP_FILE} get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi rm -f genome.fa F=Mus_musculus.GRCm38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCm38_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $SNP_FILE ] ; then get ${UCSC_COMMON_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) gunzip ${SNP_FILE}.gz || (echo "Error unzipping ${SNP_FILE}" && exit 1) awk 'BEGIN{OFS="\t"} {if($2 ~ /^chr/) {$2 = substr($2, 4)}; if($2 == "M") {$2 = "MT"} print}' ${SNP_FILE} > ${SNP_FILE}.tmp mv ${SNP_FILE}.tmp ${SNP_FILE} ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE} genome fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype genome_snp" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grcm38_snp_tran.sh000077500000000000000000000065601370663763200207640ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCm38 release 81 version of M. Musculus (mouse) from # Ensembl. # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCm38_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/mus_musculus/dna ENSEMBL_GRCm38_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/mus_musculus GTF_FILE=Mus_musculus.GRCm38.${ENSEMBL_RELEASE}.gtf DBSNP_RELEASE=142 SNP_FILE=snp${DBSNP_RELEASE}Common.txt UCSC_COMMON_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/mm10/database/${SNP_FILE} get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Mus_musculus.GRCm38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCm38_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $SNP_FILE ] ; then get ${UCSC_COMMON_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) gunzip ${SNP_FILE}.gz || (echo "Error unzipping ${SNP_FILE}" && exit 1) awk 'BEGIN{OFS="\t"} {if($2 ~ /^chr/) {$2 = substr($2, 4)}; if($2 == "M") {$2 = "MT"} print}' ${SNP_FILE} > ${SNP_FILE}.tmp mv ${SNP_FILE}.tmp ${SNP_FILE} ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE} genome fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GRCm38_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype --ss genome.ss --exon genome.exon genome_snp_tran" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_grcm38_tran.sh000077500000000000000000000046601370663763200201030ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the GRCm38 release 81 version of M. musculus (mouse) from # Ensembl. # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_GRCm38_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/mus_musculus/dna ENSEMBL_GRCm38_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/mus_musculus GTF_FILE=Mus_musculus.GRCm38.${ENSEMBL_RELEASE}.gtf get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Mus_musculus.GRCm38.dna.primary_assembly.fa if [ ! -f $F ] ; then get ${ENSEMBL_GRCm38_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GRCm38_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --ss genome.ss --exon genome.exon genome_tran" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_hg19.sh000077500000000000000000000024101370663763200165130ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the HG19 version of H. sapiens (human) from # UCSC. # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # UCSC_HG19_BASE=http://hgdownload.cse.ucsc.edu/goldenPath/hg19/bigZips F=chromFa.tar.gz get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa get ${UCSC_HG19_BASE}/$F || (echo "Error getting $F" && exit 1) tar xvzfO $F > genome.fa || (echo "Error unzipping $F" && exit 1) rm $F CMD="${HISAT2_BUILD_EXE} genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_hg38.sh000077500000000000000000000024151370663763200165210ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the HG38 version of H. sapiens (human) from # UCSC. # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # UCSC_HG38_BASE=http://hgdownload.cse.ucsc.edu/goldenPath/hg38/bigZips F=hg38.chromFa.tar.gz get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa get ${UCSC_HG38_BASE}/$F || (echo "Error getting $F" && exit 1) tar xvzfO $F > genome.fa || (echo "Error unzipping $F" && exit 1) rm $F CMD="${HISAT2_BUILD_EXE} genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_hg38_allsnp.sh000077500000000000000000000040461370663763200200740ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the HG38 version of H. spiens (human) from # UCSC. # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # UCSC_HG38_BASE=http://hgdownload.cse.ucsc.edu/goldenPath/hg38/bigZips F=hg38.chromFa.tar.gz DBSNP_RELEASE=144 SNP_FILE=snp${DBSNP_RELEASE}.txt UCSC_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/hg38/database/${SNP_FILE} get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi rm -f genome.fa get ${UCSC_HG38_BASE}/$F || (echo "Error getting $F" && exit 1) tar xvzf $F || (echo "Error unzipping $F" && exit 1) for i in {1..22}; do cat chroms/chr$i.fa >> genome.fa; done cat chroms/chr[XYM].fa >> genome.fa rm $F if [ ! -f $SNP_FILE ] ; then get ${UCSC_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE}.gz genome fi CMD="${HISAT2_BUILD_EXE} -p 4 --large-index genome.fa --snp genome.snp --haplotype genome.haplotype genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_hg38_snp.sh000077500000000000000000000040541370663763200174020ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the HG38 version of H. spiens (human) from # UCSC. # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # UCSC_HG38_BASE=http://hgdownload.cse.ucsc.edu/goldenPath/hg38/bigZips F=hg38.chromFa.tar.gz DBSNP_RELEASE=144 SNP_FILE=snp${DBSNP_RELEASE}Common.txt UCSC_COMMON_SNP=http://hgdownload.cse.ucsc.edu/goldenPath/hg38/database/${SNP_FILE} get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SNP_SCRIPT=./hisat2_extract_snps_haplotypes_UCSC.py if [ ! -x "$HISAT2_SNP_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_UCSC.py ; then echo "Could not find hisat2_extract_snps_haplotypes_UCSC.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_UCSC.py` fi fi rm -f genome.fa get ${UCSC_HG38_BASE}/$F || (echo "Error getting $F" && exit 1) tar xvzf $F || (echo "Error unzipping $F" && exit 1) for i in {1..22}; do cat chroms/chr$i.fa >> genome.fa; done cat chroms/chr[XYM].fa >> genome.fa rm $F if [ ! -f $SNP_FILE ] ; then get ${UCSC_COMMON_SNP}.gz || (echo "Error getting ${UCSC_COMMON_SNP}" && exit 1) ${HISAT2_SNP_SCRIPT} genome.fa ${SNP_FILE}.gz genome fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_hg38_snp_tran.sh000077500000000000000000000024141370663763200204240ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the HG38 version of H. spiens (human) from # UCSC. # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # UCSC_HG38_BASE=http://hgdownload.cse.ucsc.edu/goldenPath/hg38/bigZips F=hg38.chromFa.tar.gz get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa get ${UCSC_HG38_BASE}/$F || (echo "Error getting $F" && exit 1) tar xvzfO $F > genome.fa || (echo "Error unzipping $F" && exit 1) rm $F CMD="${HISAT2_BUILD_EXE} genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_mm10.sh000077500000000000000000000024111370663763200165160ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the MM10 version of M. musculus (mouse) from # UCSC. # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # UCSC_MM10_BASE=http://hgdownload.cse.ucsc.edu/goldenPath/mm10/bigZips F=chromFa.tar.gz get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa get ${UCSC_MM10_BASE}/$F || (echo "Error getting $F" && exit 1) tar xvzfO $F > genome.fa || (echo "Error unzipping $F" && exit 1) rm $F CMD="${HISAT2_BUILD_EXE} genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_mm9.sh000077500000000000000000000035471370663763200164610ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the mm9 version of M. musculus (mouse) from # UCSC. # # Note that UCSC's mm9 build has two categories of compressed fasta # files: # # 1. The base files, named chr??.fa.gz # 2. The unplaced-sequence files, named chr??_random.fa.gz # # By default, this script indexes all these files. To change which # categories are built by this script, edit the CHRS_TO_INDEX # variable below. # BASE_CHRS="\ chr1 \ chr2 \ chr3 \ chr4 \ chr5 \ chr6 \ chr7 \ chr8 \ chr9 \ chr10 \ chr11 \ chr12 \ chr13 \ chr14 \ chr15 \ chr16 \ chr17 \ chr18 \ chr19 \ chrX \ chrY \ chrM" RANDOM_CHRS="\ chr1_random \ chr3_random \ chr4_random \ chr5_random \ chr7_random \ chr8_random \ chr9_random \ chr13_random \ chr16_random \ chr17_random \ chrX_random \ chrY_random \ chrUn_random" CHRS_TO_INDEX="$BASE_CHRS $RANDOM_CHRS" UCSC_MM9_BASE=ftp://hgdownload.cse.ucsc.edu/goldenPath/mm9/chromosomes get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } BOWTIE_BUILD_EXE=./bowtie2-build if [ ! -x "$BOWTIE_BUILD_EXE" ] ; then if ! which bowtie2-build ; then echo "Could not find bowtie2-build in current directory or in PATH" exit 1 else BOWTIE_BUILD_EXE=`which bowtie2-build` fi fi INPUTS= for c in $CHRS_TO_INDEX ; do if [ ! -f ${c}.fa ] ; then F=${c}.fa.gz get ${UCSC_MM9_BASE}/$F || (echo "Error getting $F" && exit 1) gunzip $F || (echo "Error unzipping $F" && exit 1) fi [ -n "$INPUTS" ] && INPUTS=$INPUTS,${c}.fa [ -z "$INPUTS" ] && INPUTS=${c}.fa done CMD="${BOWTIE_BUILD_EXE} $* ${INPUTS} mm9" echo Running $CMD if $CMD ; then echo "mm9 index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_r64.sh000077500000000000000000000027531370663763200163700ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the R64-1-1 release 84 version of saccharomyces cerevisiae (yeast) from # Ensembl. # # Note that Ensembl's build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/saccharomyces_cerevisiae/dna get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa F=Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa if [ ! -f $F ] ; then get ${ENSEMBL_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_r64_tran.sh000077500000000000000000000050761370663763200174150ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the R64-1-1 release 84 version of saccharomyces cerevisiae (yeast) from # Ensembl. # # Note that Ensembl's build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/saccharomyces_cerevisiae/dna ENSEMBL_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/saccharomyces_cerevisiae GTF_FILE=Saccharomyces_cerevisiae.R64-1-1.${ENSEMBL_RELEASE}.gtf get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Saccharomyces_cerevisiae.R64-1-1.dna.toplevel.fa if [ ! -f $F ] ; then get ${ENSEMBL_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --ss genome.ss --exon genome.exon genome_tran" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_rn4.sh000077500000000000000000000036721370663763200164610ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the rn4 version of R. norvegicus (rat) from # UCSC. # # Note that UCSC's rn4 build has two categories of compressed fasta # files: # # 1. The base files, named chr??.fa.gz # 2. The unplaced-sequence files, named chr??_random.fa.gz # # By default, this script indexes all these files. To change which # categories are built by this script, edit the CHRS_TO_INDEX # variable below. # i=2 BASE_CHRS=chr1 while [ $i -lt 21 ] ; do BASE_CHRS="$BASE_CHRS chr$i" i=`expr $i + 1` done BASE_CHRS="$BASE_CHRS chrX chrM chrUn" RANDOM_CHRS="\ chr1_random \ chr2_random \ chr3_random \ chr4_random \ chr5_random \ chr6_random \ chr7_random \ chr8_random \ chr9_random \ chr10_random \ chr11_random \ chr12_random \ chr13_random \ chr14_random \ chr15_random \ chr16_random \ chr17_random \ chr18_random \ chr19_random \ chr20_random \ chrX_random \ chrUn_random" CHRS_TO_INDEX="$BASE_CHRS $RANDOM_CHRS" RN4_BASE=ftp://hgdownload.cse.ucsc.edu/goldenPath/rn4/chromosomes get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } BOWTIE_BUILD_EXE=./bowtie2-build if [ ! -x "$BOWTIE_BUILD_EXE" ] ; then if ! which bowtie2-build ; then echo "Could not find bowtie2-build in current directory or in PATH" exit 1 else BOWTIE_BUILD_EXE=`which bowtie2-build` fi fi INPUTS= for c in $CHRS_TO_INDEX ; do if [ ! -f ${c}.fa ] ; then F=${c}.fa.gz get ${RN4_BASE}/$F || (echo "Error getting $F" && exit 1) gunzip $F || (echo "Error unzipping $F" && exit 1) fi [ -n "$INPUTS" ] && INPUTS=$INPUTS,${c}.fa [ -z "$INPUTS" ] && INPUTS=${c}.fa done CMD="${BOWTIE_BUILD_EXE} $* ${INPUTS} rn4" echo Running $CMD if $CMD ; then echo "rn4 index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_rn6.sh000077500000000000000000000024751370663763200164630ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the rn6 version of R. norvegicus (rat) from # UCSC. # # Note that UCSC's rn6 build has two categories of compressed fasta # files: # # 1. The base files, named chr??.fa.gz # 2. The unplaced-sequence files, named chr??_random.fa.gz # # By default, this script indexes all these files. To change which # categories are built by this script, edit the CHRS_TO_INDEX # variable below. # RN6_BASE=ftp://hgdownload.cse.ucsc.edu/goldenPath/rn6/bigZips F=rn6.fa.gz get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa get ${RN6_BASE}/$F || (echo "Error getting $F" && exit 1) gzip -cd $F > genome.fa || (echo "Error unzipping $F" && exit 1) rm $F CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_rnor6.sh000077500000000000000000000027421370663763200170210ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the RNor_6.0 release 84 version of rattus_norvegicus (rat) from # Ensembl. # # Note that Ensembl's build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_RNOR6_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/rattus_norvegicus/dna get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa F=Rattus_norvegicus.Rnor_6.0.dna.toplevel.fa if [ ! -f $F ] ; then get ${ENSEMBL_RNOR6_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_rnor6_tran.sh000077500000000000000000000050641370663763200200450ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the RNor_6.0 release 84 version of rattus_norvegicus (rat) from # Ensembl. # # Note that Ensembl's build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_RNOR6_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/rattus_norvegicus/dna ENSEMBL_RNOR6_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/rattus_norvegicus GTF_FILE=Rattus_norvegicus.Rnor_6.0.${ENSEMBL_RELEASE}.gtf get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Rattus_norvegicus.Rnor_6.0.dna.toplevel.fa if [ ! -f $F ] ; then get ${ENSEMBL_RNOR6_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_RNOR6_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --ss genome.ss --exon genome.exon genome_tran" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_sc3.sh000077500000000000000000000016541370663763200164440ustar00rootroot00000000000000#!/bin/sh SC3_BASE=ftp://hgdownload.cse.ucsc.edu/goldenPath/sacCer3/bigZips F=chromFa.tar.gz get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa get ${SC3_BASE}/$F || (echo "Error getting $F" && exit 1) tar xvzfO $F > genome.fa || (echo "Error unzipping $F" && exit 1) rm $F CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_wbcel235.sh000077500000000000000000000027461370663763200173050ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the WBcel235 release 84 version of caenorhabditis elegans (worm) from # Ensembl. # # Note that Ensembl's build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/caenorhabditis_elegans/dna get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi rm -f genome.fa F=Caenorhabditis_elegans.WBcel235.dna.toplevel.fa if [ ! -f $F ] ; then get ${ENSEMBL_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa genome" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_wbcel235_tran.sh000077500000000000000000000050661370663763200203270ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for the WBcel235 release 84 version of caenorhabditis elegans (worm) from # Ensembl. # # Note that Ensembl's build has three categories of compressed fasta # files: # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=84 ENSEMBL_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/fasta/caenorhabditis_elegans/dna ENSEMBL_GTF_BASE=ftp://ftp.ensembl.org/pub/release-${ENSEMBL_RELEASE}/gtf/caenorhabditis_elegans GTF_FILE=Caenorhabditis_elegans.WBcel235.${ENSEMBL_RELEASE}.gtf get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Caenorhabditis_elegans.WBcel235.dna.toplevel.fa if [ ! -f $F ] ; then get ${ENSEMBL_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --ss genome.ss --exon genome.exon genome_tran" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/make_zm3_snp_tran_ercc.sh000077500000000000000000000065601370663763200213660ustar00rootroot00000000000000#!/bin/sh # # Downloads sequence for Zea mays AGPv3.31 from # Ensembl. # # The base files, named ??.fa.gz # # By default, this script builds and index for just the base files, # since alignments to those sequences are the most useful. To change # which categories are built by this script, edit the CHRS_TO_INDEX # variable below. # ENSEMBL_RELEASE=31 ENSEMBL_DNA_BASE=ftp://ftp.ensemblgenomes.org/pub/plants/release-${ENSEMBL_RELEASE}/fasta/zea_mays/dna/ ENSEMBL_GTF_BASE=ftp://ftp.ensemblgenomes.org/pub/plants/release-${ENSEMBL_RELEASE}/gtf/zea_mays/ GTF_FILE=Zea_mays.AGPv3.31.gtf ENSEMBL_VCF_BASE=ftp://ftp.ensemblgenomes.org/pub/plants/release-${ENSEMBL_RELEASE}/vcf/zea_mays/ VCF_FILE=zea_mays.vcf.gz ERCC_FILE=ERCC92 ERCC_FTP=https://tools.thermofisher.com/content/sfs/manuals/${ERCC_FILE}.zip get() { file=$1 if ! wget --version >/dev/null 2>/dev/null ; then if ! curl --version >/dev/null 2>/dev/null ; then echo "Please install wget or curl somewhere in your PATH" exit 1 fi curl -o `basename $1` $1 return $? else wget $1 return $? fi } HISAT2_BUILD_EXE=./hisat2-build if [ ! -x "$HISAT2_BUILD_EXE" ] ; then if ! which hisat2-build ; then echo "Could not find hisat2-build in current directory or in PATH" exit 1 else HISAT2_BUILD_EXE=`which hisat2-build` fi fi HISAT2_VCF_SCRIPT=./hisat2_extract_snps_haplotypes_VCF.py if [ ! -x "$HISAT2_VCF_SCRIPT" ] ; then if ! which hisat2_extract_snps_haplotypes_VCF.py ; then echo "Could not find hisat2_extract_snps_haplotypes_VCF.py in current directory or in PATH" exit 1 else HISAT2_SNP_SCRIPT=`which hisat2_extract_snps_haplotypes_VCF.py` fi fi HISAT2_SS_SCRIPT=./hisat2_extract_splice_sites.py if [ ! -x "$HISAT2_SS_SCRIPT" ] ; then if ! which hisat2_extract_splice_sites.py ; then echo "Could not find hisat2_extract_splice_sites.py in current directory or in PATH" exit 1 else HISAT2_SS_SCRIPT=`which hisat2_extract_splice_sites.py` fi fi HISAT2_EXON_SCRIPT=./hisat2_extract_exons.py if [ ! -x "$HISAT2_EXON_SCRIPT" ] ; then if ! which hisat2_extract_exons.py ; then echo "Could not find hisat2_extract_exons.py in current directory or in PATH" exit 1 else HISAT2_EXON_SCRIPT=`which hisat2_extract_exons.py` fi fi rm -f genome.fa F=Zea_mays.AGPv3.31.dna.genome.fa if [ ! -f $F ] ; then get ${ENSEMBL_DNA_BASE}/$F.gz || (echo "Error getting $F" && exit 1) gunzip $F.gz || (echo "Error unzipping $F" && exit 1) mv $F genome.fa get ${ERCC_FTP} || (echo "Error getting ${ERCC_FILE}.zip" && exit 1) unzip ${ERCC_FILE}.zip cat ${ERCC_FILE}.fa >> genome.fa fi if [ ! -f $GTF_FILE ] ; then get ${ENSEMBL_GTF_BASE}/${GTF_FILE}.gz || (echo "Error getting ${GTF_FILE}" && exit 1) gunzip ${GTF_FILE}.gz || (echo "Error unzipping ${GTF_FILE}" && exit 1) cat ${ERCC_FILE}.gtf >> ${GTF_FILE} ${HISAT2_SS_SCRIPT} ${GTF_FILE} > genome.ss ${HISAT2_EXON_SCRIPT} ${GTF_FILE} > genome.exon fi if [ ! -f $VCF_FILE ] ; then get ${ENSEMBL_VCF_BASE}/${VCF_FILE} || (echo "Error getting ${ENSEMBL_VCF_BASE}/${VCF_FILE}" && exit 1) ${HISAT2_VCF_SCRIPT} --non-rs genome.fa ${VCF_FILE} genome fi CMD="${HISAT2_BUILD_EXE} -p 4 genome.fa --snp genome.snp --haplotype genome.haplotype --ss genome.ss --exon genome.exon genome_snp_tran_ercc" echo Running $CMD if $CMD ; then echo "genome index built; you may remove fasta files" else echo "Index building failed; see error message" fi hisat2-2.2.1/scripts/sa.py000066400000000000000000000041541370663763200153730ustar00rootroot00000000000000#!/usr/bin/env python """ sa.py Parse and possibly sanity-check a .sa file output by bowtie2-build in --sa mode. These files have a very simple format: first is a uint32_t containing the length of the suffix array, the rest is an array of that many uint32_ts containing the suffix array. """ import sys import struct def loadBowtieSa(fh): """ Load a .sa file from handle into an array of ints """ nsa = struct.unpack('I', fh.read(4))[0] return [ struct.unpack('I', fh.read(4))[0] for i in xrange(0, nsa) ] def loadBowtieSaFilename(fn): """ Load a .sa file from filename into an array of ints """ with open(fn, 'rb') as fh: return loadBowtieSa(fh) def loadFasta(fns): """ Load the concatenation of all the A/C/G/T characters """ falist = [] dna = set(['A', 'C', 'G', 'T', 'a', 'c', 'g', 't']) for fn in fns: with open(fn, 'r') as fh: for line in fh: if line[0] == '>': continue for c in line: if c in dna: falist.append(c) return ''.join(falist) if __name__ == "__main__": import argparse parser = argparse.ArgumentParser(\ description='Parse suffix array built from bowtie2-build') parser.add_argument(\ '--sa', metavar='string', required=True, type=str, help='Suffix array file') parser.add_argument(\ '--fa', metavar='string', type=str, nargs='+', help='FASTA file') args = parser.parse_args() def go(): ref = None if args.fa is not None: ref = loadFasta(args.fa) sas = loadBowtieSaFilename(args.sa) # Suffix array is in sas; note that $ is considered greater than all # other characters if ref is not None: for i in xrange(1, len(sas)): sa1, sa2 = sas[i-1], sas[i] assert sa1 != sa2 # Sanity check that suffixes are really in order while sa1 < len(ref) and sa2 < len(ref): if ref[sa1] < ref[sa2]: break assert ref[sa1] == ref[sa2] sa1 += 1 sa2 += 1 else: # Note: Bowtie treats $ as greater than all other # characters; so if these strings are tied up to the end of # one or the other, the longer string is prior assert sa1 < sa2, "%d, %d" % (sas[i-1], sas[i]) assert sas[-1] == len(ref) go() hisat2-2.2.1/scripts/sim/000077500000000000000000000000001370663763200152025ustar00rootroot00000000000000hisat2-2.2.1/scripts/sim/AlignmentCheck.pm000066400000000000000000000604311370663763200204200ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # ## # AlignmentCheck.pm # # Read in fasta files containing reference sequences that might be # aligned to, then read in alignment files, checking each alignment to # be sure it's sane and consistent with the reference sequence it # aligns to. # package AlignmentCheck; use strict; use warnings; use FindBin qw($Bin); use lib $Bin; use DNA; use Data::Dumper; ## # Parse a fasta file into the %ref hash # sub parseFasta($$) { my ($fa, $ref) = @_; print STDERR "Parsing FASTA file $fa...\n"; my $fapipe = "$fa"; $fapipe = "gzip -dc $fa |" if $fa =~ /\.gz$/; open(FA, $fapipe) || die "Could not open '$fapipe' for reading"; my $name = ""; my $bases = 0; while() { chomp; if(/^>/) { $name = substr($_, 1); $name =~ s/\s.*//; print STDERR " parsing sequence $name...\n"; $ref->{std}{$name} = ""; } else { $name ne "" || die "sequence before name"; $bases += length($_); $ref->{std}{$name} .= $_; } } print STDERR "Parsed $bases reference bases\n"; close(FA); } ## # Create a new alignment checker # sub new { my ( $class, $name, # name of checker $fas, # list of fasta files containing reference sequences # or hash of the references themselves (former if it's # an array ref latter if it's a hash ref) $format, # alignment format $bisC, # whether alignment was w/r/t ref w/ all Cs converted to Ys $bisCpG, # whether alignment was w/r/t ref w/ all CpGs converted to YpGs ) = @_; (defined($fas)) || die "Must specify non-empty list of fasta files"; # Set defaults for format, bisC, bisCpG, name $format = "bowtie" unless defined($format); $bisC = 0 unless defined($bisC); $bisCpG = 0 unless defined($bisCpG); $name = "noname" unless defined($name); # Parse all the fasta files into the ref hash my %ref = (); if(ref($fas) eq "HASH") { for (keys %$fas) { $ref{std}{$_} = $fas->{$_} } } else { ref($fas) eq "ARRAY" || die; foreach (@$fas) { parseFasta($_, \%ref); } } return bless { _name => $name, _fas => $fas, _format => $format, _bisC => $bisC, _bisCpG => $bisCpG, _refs => \%ref, _nals => 0, _nedits => 0 }, $class; } sub name { return $_[0]->{_name} } sub fas { return $_[0]->{_fas} } sub format { return $_[0]->{_format} } sub bisC { return $_[0]->{_bisC} } sub bisCpG { return $_[0]->{_bisCpG} } sub refs { return $_[0]->{_refs} } sub nals { return $_[0]->{_nals} } sub nedits { return $_[0]->{_nedits} } sub nrefs { return scalar(keys %{$_[0]->{_refs}}); } ## # Given a sequence that represents the read oriented s.t. the Watson- # upstream end is on the left, and given a set of edits to apply to the # read, also oriented assuming that Watson-upstream is on the left, # return the string corresponding to the read mutated to match the # reference. # my $nedits = 0; sub applyEdits($$$) { my ($seq, $edits, $line) = @_; my $rfseq = $seq; my $lpos = length($seq)+1; $nedits += scalar(@$edits); foreach (reverse @$edits) { next unless defined($_); #print STDERR "Applying edit at $_->{pos}\n"; # Apply the edit $_->{pos} <= $lpos || die "Edit position $_->{pos} was greater than previous $lpos"; if($_->{qchr} eq "-") { # Insert $_->{pos} <= length($rfseq) || die "Read gap pos $_->{pos} was not <= string len ".length($rfseq)."\n$line"; substr($rfseq, $_->{pos}, 0) = $_->{chr}; } elsif($_->{chr} eq "-") { # Deletion $_->{pos} < length($rfseq) || die "Ref gap pos $_->{pos} was not < string len ".length($rfseq)."\n$line"; my $dc = substr($rfseq, $_->{pos}, 1); $dc eq $_->{qchr} || die "Edit: $_->{pos}:$_->{chr}>$_->{qchr} but ref char was $dc". "\n$rfseq\n$line"; substr($rfseq, $_->{pos}, 1) = ""; } else { # Mismatch $_->{pos} < length($rfseq) || die "Mismatch pos $_->{pos} was not < string len ".length($rfseq)."\n$line"; substr($rfseq, $_->{pos}, 1) eq $_->{qchr} || die "Edit: $_->{pos}:$_->{chr}>$_->{qchr}\n$rfseq\n$line"; substr($rfseq, $_->{pos}, 1) = $_->{chr}; } } return $rfseq; } ## # Given a list of Bowtie edits, invert them by reversing the list and # changing the poss to be with respect to the other end of the read. # sub invertEdits($$) { my ($edits, $len) = @_; @$edits = reverse @$edits; for (@$edits) { next unless defined($_); defined($_->{qchr}) || die; if($_->{qchr} eq "-") { $_->{pos} = $len - $_->{pos}; length($_->{chr}) >= 1 || die; $_->{chr} = reverse $_->{chr}; } else { $_->{pos} = $len - $_->{pos} - 1; length($_->{chr}) == 1 || die; } } } ## # Given an edit string, parses it into a list of hashes and returns # the list. # sub parseEdits($) { my $editstr = shift; return undef if (!defined($editstr) || $editstr eq "-" || $editstr eq ""); my @edits = (); # For each edit for (split(/,/, $editstr)) { # Parse pos my ($pos, $ed) = split(/:/); defined($ed) || die; # Parse chr, qchr my ($chr, $qchr) = split(/>/, $ed); push @edits, { pos => $pos, chr => $chr, qchr => $qchr }; } return \@edits; } ## # Given an edit string, possibly with 4 semicolon-delimited fields, # parse it into a set of 4 lists of hashes and return the set as an # array ref. # sub parseAllEdits($) { my $editstr = shift; return [ undef, undef, undef, undef ] if ($editstr eq "-" || $editstr eq ""); my @editls = split(/;/, $editstr, -1); if(scalar(@editls) > 1) { scalar(@editls) == 4 || die; return [ parseEdits($editls[0]), parseEdits($editls[1]), parseEdits($editls[2]), parseEdits($editls[3]) ]; } else { scalar(@editls) == 1 || die; return [ parseEdits($editls[0]), undef, undef, undef ]; } } ## # Given array refs for two lists of edits, one corresponding to the # nucleotide edit list and the other corresponding to the resolved # ambiguous base list, eliminate any edits that appear in both lists. # Really this shouldn't happen, but I observe that merman does report # an edit in both categories if the reference base is being resolved to # an incompatible nucleotide, e.g. R>C. # sub removeDups($$) { my ($ned, $aed) = @_; return unless (defined($ned) && defined($aed)); for my $i (0..scalar(@$ned)-1) { next unless defined($ned->[$i]); for my $j (0..scalar(@$aed)-1) { next unless defined($aed->[$j]); if($ned->[$i]->{qchr} eq $aed->[$j]->{qchr} && $ned->[$i]->{chr} eq $aed->[$j]->{chr} && $ned->[$i]->{pos} eq $aed->[$j]->{pos}) { #print STDERR " Eliminated a duplicate edit\n"; $aed->[$j] = undef; } } } } ## # Take all the references in %ref and make both Watson and Crick # versions where the sequence is in-silico bisulfite treated. # sub bisulfiteC($) { my $ref = shift; for(keys %{$ref->{std}}) { $ref->{bisc_fw}{$_} = $ref->{std}{$_}; $ref->{bisc_fw}{$_} = s/C/Y/g; $ref->{bisc_rc}{$_} = $ref->{std}{$_}; $ref->{bisc_rc}{$_} = s/G/R/g; } } ## # Take all the references in %ref and make both Watson and Crick # versions where the sequence is in-silico bisulfite treated. # sub bisulfiteCpG($) { my $ref = shift; for(keys %{$ref->{std}}) { $ref->{biscpg_fw}{$_} = $ref->{std}{$_}; $ref->{biscpg_fw}{$_} =~ s/CG/YG/g; $ref->{biscpg_fw}{$_} =~ s/C/T/g; $ref->{biscpg_rc}{$_} = $ref->{std}{$_}; $ref->{biscpg_rc}{$_} =~ s/CG/CR/g; $ref->{biscpg_rc}{$_} =~ s/G/A/g; } } ## # Given a Bowtie orientation string, return true iff the 5' end of the # read is at the left of the printed sequence. # sub fivePrimeLeft($) { return ($_[0] eq "+" || $_[0] eq "W" || $_[0] eq "CR"); } ## # Given the orientation of the read and the state of the global # bisulfite variables, determine which version of the reference to # compare against. # sub calcRefType { my ($self, $orient) = @_; if($self->bisC || $self->bisCpG) { if($orient eq "W" || $orient eq "WR" || $orient eq "+") { return $self->bisC ? "bisc_fw" : "biscpg_fw"; } else { $orient eq "C" || $orient eq "CR" || $orient eq "-" || die; return $self->bisC ? "bisc_rc" : "biscpg_rc"; } } else { return "std"; } } ## # Parse a CIGAR string into parallel arrays of CIGAR operations (M, I, D) # sub cigarParse($$$) { my ($cigar, $ops, $runs) = @_; my $i = 0; while($i < length($cigar)) { substr($cigar, $i) =~ /^([0-9]+)/; defined($1) || die "Could not parse number at pos $i: '$cigar'"; $i += length($1); $i < length($cigar) || die; push @$runs, $1; my $op = substr($cigar, $i, 1); defined($op) || die "Could not parse operation at pos $i: '$cigar'"; push @$ops, $op; $i++; } } ## # Trim a read sequence according to the soft clipping in the CIGAR string. # sub cigarTrim($$) { my ($seq, $cigar) = @_; my @ops = (); my @runs = (); cigarParse($cigar, \@ops, \@runs); my ($trimup, $trimdn) = (0, 0); if($ops[0] eq 'S') { $runs[0] < length($seq) || die "Soft clipped entire alignment!"; $seq = substr($seq, $runs[0]); $trimup = $runs[0]; } if(scalar(@ops) > 1 && $ops[-1] eq 'S') { $runs[-1] < length($seq) || die "Soft clipped entire alignment!"; $seq = substr($seq, 0, -$runs[-1]); $trimdn = $runs[-1]; } return ($seq, $trimup, $trimdn); } ## # Parse a CIGAR string into a string of operators. Operators are expanded into # runs where appropriate. = and X are collapsed into M. # sub parse_cigar($) { my ($cigar) = @_; my $ret = ""; my $i = 0; my ($rdlen, $rflen) = (0, 0); while($i < length($cigar)) { substr($cigar, $i) =~ /^([0-9]+)/; defined($1) || die "Could not parse number at pos $i: '$cigar'"; my $runlen = $1; $i += length($1); $i < length($cigar) || die; my $op = substr($cigar, $i, 1); defined($op) || die "Could not parse operation at pos $i: '$cigar'"; if($op eq "X" || $op eq "=") { $op = "M"; } $rdlen += $runlen if $op ne "D"; $rflen += $runlen if $op ne "I"; $ret .= ($op x $runlen); $i++; } return ($ret, $rdlen, $rflen); } ## # Parse an MD:Z string into a string with length equal to query length. Each # position contains either a space, if the read matches the reference at that # position, or a character, if the reference contains a character that doesn't # match its opposite in the alignment. In the latter case, the character in # the string is the reference character. # sub parse_md($) { my ($md) = @_; my $i = 0; my $ret = ""; while($i < length($md)) { # Starts with a number? my $ch = substr($md, $i, 1); if($ch =~ /[0-9]/) { # Parse the number off the beginning substr($md, $i) =~ /^([0-9]+)/; defined($1) || die "Could not parse number at pos $i: '$md'"; my $runlen = $1; $ret .= (" " x $runlen) if $runlen > 0; $i += length($runlen); } elsif($ch eq "^") { # Read gap $i++; substr($md, $i) =~ /^([A-Za-z]+)/; defined($1) || die "Could not parse read gap at pos $i: '$md'"; my $chrs = $1; $i += length($chrs); $ret .= $chrs; } else { # DNA character $ch =~ /[ACGTN.]/i || die "Bad char '$ch' at pos $i: '$md'"; $ret .= $ch; $i++; } } return $ret; } ## # Given a read sequence (with characters in upstream-to-downstream order with # respect to the reference - NOT necessarily 5'-to-3') and a CIGAR string and # an MD:Z string, build the alignment strings. The alignment strings will only # contain the portion of the read that aligned. Any portions that were either # hard-trimmed or soft-trimmed are trimmed from this function's result. # # For now, I'm assuming that the MD:Z string only describes aligned characters, # i.e. *after* trimming. # sub _read_md_cigar_to_al($$$) { my ($seq, $md, $cigar) = @_; my $alread = ""; my $alref = ""; $cigar ne "*" || die "CIGAR string was star!"; $seq ne "" || die "Empty sequence given to _read_md_cigar_to_al"; my $parsed_md = parse_md($md); my ($parsed_cig, $cigar_rdlen, $cigar_rflen) = parse_cigar($cigar); my ($rdoff, $mdoff) = (0, 0); my ($htriml, $striml, $htrimr, $strimr) = (0, 0, 0, 0); my $nonsh = 0; # have I seen a non-S, non-H CIGAR op? my $nonh = 0; # have I seen a non-H CIGAR op? for(my $i = 0; $i < length($parsed_cig); $i++) { my $cigop = substr($parsed_cig, $i, 1); $nonh++ if $cigop ne "H"; $nonsh++ if ($cigop ne "H" && $cigop ne "S"); if($cigop eq "S") { if($nonsh) { $strimr++; } else { $striml++; } $rdoff++; next; } if($cigop eq "H") { if($nonh) { $htrimr++; } else { $htriml++; } next; } $cigop = "M" if $cigop eq "=" || $cigop eq "X"; if($cigop eq "P") { # Padding $alread .= "-"; $alref .= "-"; } elsif($cigop eq "M") { my $rdc = substr($seq, $rdoff, 1); $mdoff < length($parsed_md) || die "Bad mdoff ($mdoff)\nlength(parsed_md)=".length($parsed_md)."\nseq:\n$seq\ncigar:\n$cigar\nmd:\n$md\nparsed md:\n$parsed_md"; my $rfc = substr($parsed_md, $mdoff, 1); $rfc = $rdc if $rfc eq " "; $alread .= $rdc; $alref .= $rfc; $rdoff++; $mdoff++; } elsif($cigop eq "D") { # Read gap # Read: AAA-AAA # Ref: AAAAAAA my $rfc = substr($parsed_md, $mdoff, 1); $rfc ne " " || die "Must have a ref character opposite a gap in the read:\n". "cig: $parsed_cig ($i)\nmd: $parsed_md ($mdoff)\n"; $alread .= "-"; $alref .= $rfc; $mdoff++; } else { # Reference gap # Read: AAAAAAA # Ref: AAA-AAA $cigop eq "I" || die "Unsupported cigop: $cigop in cigar $cigar"; my $rdc = substr($seq, $rdoff, 1); $alread .= $rdc; $alref .= "-"; $rdoff++; # $mdoff SHOULD NOT be incremented here } $rdoff <= length($seq) || die "Bad rdoff:$rdoff for seq '$seq' cigop=$cigop\nseq: $seq\ncigar=$cigar\nmd=$md"; } return ($alread, $alref, $htriml, $striml, $htrimr, $strimr); } ## # Parse a line from a Bowtie alignment file and check that the # alignment is sane and consistent with the reference. # sub parseBowtieLines { my ($self, $lines) = @_; for my $line (@$lines) { chomp($line); my ($rdname, $orient, $refname, $off, $seq, $qual, $oms, $editstr, $flags) = split(/\t/, $line, -1); next if $refname eq "*"; $flags =~ /XC:([^,\s]+)/; my $cigar = $1; defined($cigar) || die "Could not parse CIGAR string from flags: '$flags'"; defined($editstr) || die "Failed to get 8 tokens from line:\n$_"; $off == int($off) || die "Offset field (col 4) must be an integer:\n$_"; $oms == int($oms) || die "OMS field (col 7) must be an integer:\n$_"; my $reftype = $self->calcRefType($orient); defined($self->refs->{$reftype}{$refname}) || die "No such refname as $refname for reftype $reftype:\n". Dumper($self->refs->{$reftype}); my $edits4 = parseAllEdits($editstr); my ($ned, $aed) = ($edits4->[0], $edits4->[1]); removeDups($ned, $aed); my $fpl = fivePrimeLeft($orient); # Trim seq according to CIGAR string my $rfseq = $seq; my ($trimup, $trimdn); ($rfseq, $trimup, $trimdn) = cigarTrim($rfseq, $cigar); invertEdits($ned, length($rfseq)) unless ($fpl || !defined($ned)); invertEdits($aed, length($rfseq)) unless ($fpl || !defined($aed)); $rfseq = applyEdits($rfseq, $ned, $line) if defined($ned); $rfseq = applyEdits($rfseq, $aed, $line) if defined($aed); # Check if our alignment falls off the end of the reference, in # which case we need to pad the reference string with Ns my $exoff = $off; my $padleft = ""; my $exlen = length($rfseq); my $tlen = length($self->refs->{$reftype}{$refname}); if($exoff < 0) { # Alignment hangs off LHS; pad it my $npad = -$exoff; $padleft = "N" x $npad; $exlen += $exoff; $exlen >= 0 || die "Read was entirely off the LHS of the reference\n". "Referemce len=$tlen\n". "Alignment referemce len=$tlen\n". "$line\n"; $exoff = 0; } my $padright = ""; my $roverhang = $off + length($rfseq) - $tlen; if($roverhang > 0) { $padright = "N" x $roverhang; $exlen -= $roverhang; $exlen >= 0 || die "Read was entirely off the RHS of the reference\n". "Referemce len=$tlen\n". "Alignment referemce len=$tlen\n". "$line\n"; } my $refsub = substr($self->refs->{$reftype}{$refname}, $exoff, $exlen); length($refsub) == $exlen || die "Tried to extract ref substring of length $exlen, got ". "\"$refsub\" from \"".$self->refs->{$reftype}{$refname}."\"". "\n$line\n". "\noff=$off, rfseq=$rfseq\n"; $refsub = DNA::iupacSubN($refsub); my $trueRfseq = $padleft . $refsub . $padright; length($trueRfseq) == length($rfseq) || die "Different lengths for edited read and ref:\n". " Read: $seq\n". "Edited read: $rfseq\n". " Ref: $trueRfseq\n"; $rfseq eq $trueRfseq || die "Did not match:\n". " Read: $seq\n". "Edited read: $rfseq\n". " Ref: $trueRfseq\n"; $self->{_nals}++; } } ## # Parse a line from a SAM alignment file and check that the # alignment is sane and consistent with the reference. # sub parseSamLines { my ($self, $lines) = @_; my ($lastseq, $lastqual) = ("", ""); my $idx = 0; for my $line (@$lines) { $idx++; print STDERR "Processing line...\n"; chomp($line); next if $line =~ /^\@/; my @toks = split(/\t/, $line, -1); my ( $qname, #1 $flag, #2 $rname, #3 $pos, #4 $mapq, #5 $cigar, #6 $rnext, #7 $pnext, #8 $tlen, #9 $seq, #10 $qual) = @toks; defined($qual) || die "Not enough SAM tokens:\n$line\n"; my @opt_flags_list = @toks[11..$#toks]; my %opt_flags = (); next if $cigar eq "*"; # Failed to align # Get the read sequence & qualities from previous record if necessary if($seq eq "*") { $lastseq ne "" || die "Line #$idx:\n$line"; $seq = $lastseq; $qual = $lastqual; } else { $lastseq = $seq; $lastqual = $qual; } $seq ne "*" || die; my ($parsed_cigar, $rdlen_cigar, $rflen_cigar) = parse_cigar($cigar); length($seq) == $rdlen_cigar || die "Sequence length and parsed cigar string length ($rdlen_cigar) mismatch:\n". "$seq\n$parsed_cigar\nLine:\n$line"; # Stick optional flags into a hash for my $fl (@opt_flags_list) { my @fs = split(/:/, $fl, -1); scalar(@fs) > 2 || die "Bad optional flag: $fl\n$line\n"; $opt_flags{$fs[0]}{type} = $fs[1]; $opt_flags{$fs[0]}{value} = join(":", @fs[2..$#fs]); } defined($opt_flags{"MD"}) || die "No MD:Z flag:\n$line\n"; $opt_flags{"MD"}{type} eq "Z" || die "Bad type for MD:Z flag\n$line\n"; my $md = $opt_flags{"MD"}{value}; $pos == int($pos) || die "POS field (col 4) must be an int:\n$line\n"; $pnext == int($pnext) || die "PNEXT field (col 8) must be an int:\n$line\n"; $tlen == int($tlen) || die "TLEN field (col 9) must be an int:\n$line\n"; $mapq == int($mapq) || die "MAPQ field (col 5) must be an int:\n$line\n"; # TODO: deal with bisulfite strands?? my $fw = (($flag & 0x10) == 0); my $orient = $fw ? "+" : "-"; my $reftype = $self->calcRefType($orient); defined($self->refs->{$reftype}{$rname}) || die "No such refname as $rname for reftype $reftype:\n$line\n". Dumper($self->refs->{$reftype}); my $exoff = $pos-1; # expected 0-based reference offset my ($alread, $alref, $htriml, $striml, $htrimr, $strimr) = _read_md_cigar_to_al($seq, $md, $cigar); print STDERR "$alread\n$alref\n"; my $rfseq = $alref; $rfseq =~ s/[ -]//g; # remove spaces & gaps my $exlen = length($rfseq); my $refsub = substr($self->refs->{$reftype}{$rname}, $exoff, $exlen); length($refsub) == $exlen || die "Tried to extract ref substring of length $exlen from:\n". $self->refs->{$reftype}{$rname}. "\ngot string of length ".length($refsub).":\n". $refsub. "\nfrom:\n". $line. "\nexlen is the length of:\n$rfseq\npos=$pos, rfseq=$rfseq\n"; $refsub = DNA::iupacSubN($refsub); my $trueRfseq = $refsub; length($trueRfseq) == length($rfseq) || die "Different lengths for edited read and ref:\n". " Read: $seq\n". "Edited read: $rfseq\n". " Ref: $trueRfseq\n"; $rfseq eq $trueRfseq || die "Did not match:\n". " Read: $seq\n". "Edited read: $rfseq\n". " Ref: $trueRfseq\n". "$line"; $self->{_nals}++; } } ## # Parse lines from a Bowtie alignment file and check that the # alignments are sane and consistent with the reference. # sub parseBowtie { my ($self, $fh) = @_; while(<$fh>) { $self->parseBowtieLines([$_]); } } ## # Parse lines from a SAM alignment file and check that alignments are # sane and consistent with the reference. # sub parseSam { my ($self, $fh) = @_; my @lines = (); while(<$fh>) { push @lines, $_; } $self->parseSamLines(\@lines); } ## # Parse lines from an alignment file of the type given by self->format # sub parseLines { my ($self, $lines) = @_; if($self->format eq "bowtie") { $self->parseBowtieLines($lines); } else { $self->format eq "sam" || die; $self->parseSamLines($lines); } } ## # Parse lines from an alignment file of the type given by self->format # sub parse { my ($self, $fh) = @_; if($self->format eq "bowtie") { $self->parseBowtie($fh); } else { $self->format eq "sam" || die; $self->parseSam($fh); } } ## # Print summary of how many alignments and edits were checked. # sub printSummary { my $self = shift; print STDERR "--- Summary ---\n"; print STDERR "Read ".scalar(keys %{$self->refs})." reference strings\n"; print STDERR "Checked $self->{_nals} alignments, $self->{_nedits} edits\n"; print STDERR "---------------\n"; print STDERR "PASSED\n"; } ## # Check the given batch of alignments. We check that they're # internally consistent in some basic ways, and we check that the # sequence and edits are consistent with the reference. # # The $als argument is either a list of (possibly compressed) filenames # of files containing alignments, or a list of alignment strings. If # the former, $filenames is non-zero. # sub checkAlignments { my ($self, $als, $filenames) = @_; if($self->bisC) { print STDERR "Generating all-C bisulfite-treated references\n"; bisulfiteC($self->refs); } if($self->bisCpG) { print STDERR "Generating all-CpG bisulfite-treated references\n"; bisulfiteCpG($self->refs); } if($filenames) { foreach (@$als) { my $alnpipe = $_; print STDERR "Processing alignment file '$_'\n"; $alnpipe = "gzip -dc $_ |" if ($_ =~ /\.gz$/); my $alnfh = undef; open($alnfh, $alnpipe) || die "Could not open '$alnpipe' for reading"; $self->parse($alnfh); close($alnfh); } } else { $self->parseLines($als); } } ## # Check simple alignments # sub test1 { my $ac = AlignmentCheck->new( "AlignmentCheck.pm test1 checker", { "r1" => "TTGTTCGT" }, "bowtie", 0, 0 ); $ac->checkAlignments([ "0\t+\tr1\t1\tTGTTCGT\tIIIIIII\t40\t-", "1\t+\tr1\t0\tTTGTTCG\tIIIIIII\t40\t-", "2\t+\tr1\t2\tGTTCGTA\tIIIIIII\t40\t6:N>A", "3\t+\tr1\t-1\tATTGTTC\tIIIIIII\t40\t0:N>A"], 0); return 1; } ## # Check simple alignments from files # sub test2 { open(TMP, ">/tmp/.AlignmentCheck.pm.fa") || die; print TMP ">r1\n"; print TMP "TTGTTCGT\n"; close(TMP); my $ac = AlignmentCheck->new( "AlignmentCheck.pm test1 checker", [ "/tmp/.AlignmentCheck.pm.fa" ], "bowtie", 0, 0 ); $ac->checkAlignments([ "0\t+\tr1\t1\tTGTTCGT\tIIIIIII\t40\t-", "1\t+\tr1\t0\tTTGTTCG\tIIIIIII\t40\t-", "2\t+\tr1\t2\tGTTCGTA\tIIIIIII\t40\t6:N>A", "3\t+\tr1\t-1\tATTGTTC\tIIIIIII\t40\t0:N>A"], 0); return 1; } if($0 =~ /[^0-9a-zA-Z_]?AlignmentCheck\.pm$/) { my @fas = (); my @als = (); my $format = "sam"; my $bisC = 0; my $bisCpG = 0; my $test = 0; use Getopt::Long; GetOptions ( "test" => \$test, "fasta|ref=s" => \@fas, "als|aln=s" => \@als, "format=s" => \$format, "bis-C|bisulfite-C" => \$bisC, "bis-CpG|bisulfite-CpG" => \$bisCpG, "bowtie" => sub {$format = "bowtie"}, "sam" => sub {$format = "sam"}) || die; if($test) { use Test; print "Running unit tests\n"; # Run unit tests Test::shouldSucceed("test1", \&test1); Test::shouldSucceed("test2", \&test2); exit 0; } my $ac = AlignmentCheck->new( "AlignmentCheck.pm checker", \@fas, $format, $bisC, $bisCpG); $ac->checkAlignments(\@als, 1); } 1; hisat2-2.2.1/scripts/sim/DNA.pm000066400000000000000000000114731370663763200161500ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # package DNA; use strict; use warnings; use Carp; use FindBin qw($Bin); use lib $Bin; use Test; ## # Set up uppercase IUPAC characters minus N # my %iupac_u_nn = ( "R" => 1, "Y" => 1, "M" => 1, "K" => 1, "S" => 1, "W" => 1, "B" => 1, "V" => 1, "H" => 1, "D" => 1 ); ## # Return true iff arg is an IUPAC code and not ACGT or N. # sub isIUPAC($) { return defined($iupac_u_nn{$_[0]}); } ## # Replace IUPAC characters with Ns. # sub iupacSubN($) { $_[0] =~ tr/RYMKSWBVHDrymkswbvhd/NNNNNNNNNNnnnnnnnnnn/; return $_[0]; } my %compat = ( "A" => "A", "T" => "T", "C" => "C", "G" => "G", "R" => "AG", "Y" => "CT", "M" => "AC", "K" => "GT", "S" => "CG", "W" => "AT", "B" => "CGT", "V" => "ACG", "H" => "ACT", "D" => "AGT", "N" => "ACGT" ); ## # Pick a random character that's compatible with input character. # sub pickCompat($) { my $c = uc $_[0]; defined($compat{$c}) || die "Bad input $c"; my $cc = $compat{$c}; if(length($cc) == 1) { return $cc; } else { length($cc) > 1 || die; return substr($cc, int(rand(length($cc))), 1); } } my %incompat = ( "A" => "CGT", "T" => "ACG", "C" => "AGT", "G" => "ACT", "R" => "CT", "Y" => "AG", "M" => "GT", "K" => "AC", "S" => "AT", "W" => "CG", "B" => "A", "V" => "T", "H" => "G", "D" => "C", "N" => "ACGT" ); ## # Pick a random character that's compatible with input character. # sub pickIncompat($) { my $c = uc $_[0]; defined($incompat{$c}) || die "Bad input $c"; my $cc = $incompat{$c}; if(length($cc) == 1) { return $cc; } else { length($cc) > 1 || die; return substr($cc, int(rand(length($cc))), 1); } } ## # Set up lowercase IUPAC characters minus N # my %iupac_l_nn = ( "r" => 1, "y" => 1, "m" => 1, "k" => 1, "s" => 1, "w" => 1, "b" => 1, "v" => 1, "h" => 1, "d" => 1 ); my %revcompMap = ( "A" => "T", "T" => "A", "C" => "G", "G" => "C", "R" => "Y", "Y" => "R", "M" => "K", "K" => "M", "S" => "S", "W" => "W", "B" => "V", "V" => "B", "H" => "D", "D" => "H", "N" => "N", "a" => "t", "t" => "a", "c" => "g", "g" => "c", "r" => "y", "y" => "r", "m" => "k", "k" => "m", "s" => "s", "w" => "w", "b" => "v", "v" => "b", "h" => "d", "d" => "h", "n" => "n" ); my %unambigSet = ( "A" => 1, "a" => 1, "C" => 1, "c" => 1, "G" => 1, "g" => 1, "T" => 1, "t" => 1 ); ## # Return the complement, incl. if it's IUPAC. # sub comp($) { my $s = uc shift; return $s =~ tr/ACGTRYMKSWBVHDN/TGCAYRKMSWVBDHN/; } ## # Return the reverse complement of a string. # sub revcomp($) { my $s = uc shift; $s = reverse $s; $s =~ tr/ACGTRYMKSWBVHDN/TGCAYRKMSWVBDHN/; return $s; } ## # Return true iff it's unambiguous. # sub unambig($) { return $unambigSet{$_[0]}; } ## # Manipulate DNA in an integer-indexed fashion. # sub plus($$) { my ($c, $amt) = @_; my %ctoi = ("A" => 0, "C" => 1, "G" => 2, "T" => 3); my %itoc = (0 => "A", 1 => "C", 2 => "G", 3 => "T"); $c = uc $c; defined($ctoi{$c}) || die "Not an unambiguous nucleotide: $c"; return $itoc{($ctoi{$c}+$amt) % 4}; } my %dinucToColorMap = ( "AA" => "0", "AC" => "1", "AG" => "2", "AT" => "3", "CC" => "0", "CG" => "3", "CT" => "2", "GG" => "0", "GT" => "1", "TT" => "0", "AN" => ".", "CN" => ".", "GN" => ".", "NT" => ".", "NN" => ".", ); sub dinucToColor($$) { my ($n1, $n2) = @_; if(ord($n2) < ord($n1)) { my $tmp = $n1; $n1 = $n2; $n2 = $tmp; } ord($n1) <= ord($n2) || die; defined($dinucToColorMap{"$n1$n2"}) || die "Bad nucleotide dinuc: '$n1$n2'"; return $dinucToColorMap{"$n1$n2"}; } sub test1 { plus("A", 1) eq "C" || die; plus("C", 1) eq "G" || die; plus("G", 1) eq "T" || die; plus("T", 1) eq "A" || die; return 1; } sub test2 { plus("A", 2) eq "G" || die; plus("C", 2) eq "T" || die; plus("G", 2) eq "A" || die; plus("T", 2) eq "C" || die; return 1; } sub test3 { revcomp("ACGT") eq "ACGT" || die; return 1; } sub test4 { revcomp("ACGTYR") eq "YRACGT" || die; return 1; } if($0 =~ /[^0-9a-zA-Z_]?DNA\.pm$/) { print "Running unit tests\n"; # Run unit tests Test::shouldSucceed("test1", \&test1); Test::shouldSucceed("test2", \&test2); Test::shouldSucceed("test3", \&test3); Test::shouldSucceed("test4", \&test4); } 1; hisat2-2.2.1/scripts/sim/Mutate.pm000066400000000000000000000211571370663763200170050ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # package Mutate; use strict; use Carp; use FindBin qw($Bin); use lib $Bin; use DNA; use Test; use List::Util qw(max min); use Math::Random; ## # Default SNP rate generator. Doesn't generate the SNP per se, just # the rate. # sub defaultSNPGen() { return Math::Random::random_uniform(1, 0, 0.05); } ## # Default read gap rate generator. Doesn't generate the gaps or # lengths, just the rate. # sub defaultRdGapGen() { return Math::Random::random_uniform(1, 0, 0.005); } ## # Default reference gap rate generator. Doesn't generate the gaps or # lengths, just the rate. # sub defaultRfGapGen() { return Math::Random::random_uniform(1, 0, 0.005); } ## # Default rearrangement rate generator. # sub defaultRearrGen() { return Math::Random::random_uniform(1, 0, 0.005); } ## # Default function for generating gap lengths when introducing a gap. # sub defaultGapLenGen() { return int(Math::Random::random_exponential(1, 3))+1; } ## # Default function for generating random sequence to insert into a gap. # sub defaultSeqGen($) { my $len = shift; ($len == int($len) && $len > 0) || die "Bad length for sequence generator: $len"; my $ret = ""; for (1..$len) { $ret .= substr("ACGT", int(rand(4)), 1); } return $ret; } ## # Create a new DNA mutator # sub new { my ( $class, $name, # name $snp, # SNP rate $rdgap, # read gap rate $rfgap, # ref gap rate $rearr, # rearrangement rate $gaplen, # gap length $seqgen, # DNA generator ) = @_; $name = "noname" unless defined($name); $snp = \&defaultSNPGen unless defined($snp); $rdgap = \&defaultRdGapGen unless defined($rdgap); $rfgap = \&defaultRfGapGen unless defined($rfgap); $rearr = \&defaultRearrGen unless defined($rearr); $gaplen = \&defaultGapLenGen unless defined($gaplen); $seqgen = \&defaultSeqGen unless defined($seqgen); return bless { _name => $name, _snp => $snp, _rdgap => $rdgap, _rfgap => $rfgap, _rearr => $rearr, _gaplen => $gaplen, _seqgen => $seqgen, }, $class; } sub snp { return $_[0]->{_snp} } sub rdgap { return $_[0]->{_rdgap} } sub rfgap { return $_[0]->{_rfgap} } sub rearr { return $_[0]->{_rearr} } sub gaplen { return $_[0]->{_gaplen} } sub seqgen { return $_[0]->{_seqgen} } ## # Given a sequence (i.e. a key $srcchr into the reference hash), # mutate that string. Note that rearrangement mutations can affect # more than one sequence at a time. # # Returns a list containing counts for: # # 1: number of SNPs added # 2: number of read gaps added # 3: number of ref gaps added # 4: number of rearrangements added # sub mutateSeq { my ($self, $srcchr, $ref) = @_; my ($nsnp, $nrfgap, $nrdgap, $nrearr) = (0, 0, 0, 0); my $mutseq = $ref->{$srcchr}; # Calculate # SNPs to add my $len = length($mutseq); my $snpRate = $self->snp->(); my $rfgapRate = $self->rfgap->(); my $rdgapRate = $self->rdgap->(); my $rearrRate = $self->rearr->(); $nsnp = Math::Random::random_binomial(1, $len, $snpRate); $nrfgap = Math::Random::random_binomial(1, $len, $rfgapRate); $nrdgap = Math::Random::random_binomial(1, $len, $rdgapRate); $nrearr = Math::Random::random_binomial(1, $len, $rearrRate); print STDERR " Introducing $nsnp SNPs, $nrfgap/$nrdgap ref/read gaps, and $nrearr rearrangements\n"; $nsnp = min($nsnp, $len); # Add the SNPs for (1..$nsnp) { my $off = int(rand($len)); # where to mutate my $add = int(rand(3))+1; # how to mutate my $c = substr($mutseq, $off, 1); $c eq "A" || $c eq "C" || $c eq "G" || $c eq "T" || $c eq "N" || die "Bad char '$c' in:\n$ref->{$srcchr}"; substr($mutseq, $off, 1) = DNA::plus(substr($mutseq, $off, 1), $add); } print STDERR " Finished SNPs\n"; # Calculate # ref gaps to add for (1..$nrfgap) { my $off = int(rand($len)); # where to mutate my $gaplen = $self->gaplen->(); # how many gap positions in ref # Insert characters into the subject genome my $insseq = $self->seqgen->($gaplen); substr($mutseq, $off, 0) = $insseq; $len = length($mutseq); } print STDERR " Finished ref gaps\n"; # Calculate # read gaps to add for (1..$nrdgap) { my $off = int(rand($len)); # where to mutate my $gaplen = $self->gaplen->(); # how many gap positions in ref # Delete characters from subject genome substr($mutseq, $off, $gaplen) = ""; $len = length($mutseq); } print STDERR " Finished read gaps\n"; $ref->{$srcchr} = $mutseq; return ($nsnp, $nrfgap, $nrdgap, $nrearr); my $totlen = 0; for (keys %$ref) { $totlen += length($ref->{$_}); } # Calculate # rearrangements to add for (1..$nrearr) { # Pick two loci, at least one on this reference sequence and # then cross them over somehow my $off = int(rand($len)); my @refkeys = keys %$ref; my $ochr = $refkeys[int(rand(scalar(@refkeys)))]; my $oseq = $ref->{$ochr}; my $ooff = int(rand(length($oseq))); my $srcleft = int(rand(2)); my $dstleft = int(rand(2)); my $srcrc = int(rand(2)); my $dstrc = int(rand(2)); # Check that the source and dest don't overlap next if $srcchr eq $ochr; # Get the sequence to move my $presrclen = length($mutseq); my $predstlen = length($oseq); my $srcseq; if($srcleft) { $srcseq = substr($mutseq, 0, $off); } else { $srcseq = substr($mutseq, $off); } my $dstseq; if($dstleft) { $dstseq = substr($oseq, 0, $ooff); } else { $dstseq = substr($oseq, $ooff); } # Delete the sequence from the source length($srcseq) <= length($mutseq) || die; length($dstseq) <= length($oseq) || die; if($srcleft) { substr($mutseq, 0, length($srcseq)) = ""; } else { substr($mutseq, -length($srcseq)) = ""; } if($dstleft) { substr($oseq, 0, length($dstseq)) = ""; } else { substr($oseq, -length($dstseq)) = ""; } # Possibly reverse the pieces we broke off my $len1 = length($srcseq); my $len2 = length($dstseq); $srcseq = DNA::revcomp($srcseq) if $srcrc; $dstseq = DNA::revcomp($dstseq) if $dstrc; length($srcseq) == $len1 || die "$srcseq"; length($dstseq) == $len2 || die; # Mutate the current chromosome if($srcleft) { $mutseq = $dstseq . $mutseq; } else { $mutseq = $mutseq . $dstseq; } # Mutate the other chromosome if($dstleft) { $oseq = $srcseq . $oseq; } else { $oseq = $oseq . $srcseq; } my $postsrclen = length($mutseq); my $postdstlen = length($oseq); ($presrclen + $presrclen) == ($postsrclen + $postsrclen) || die "from $srcchr to $ochr: $presrclen + $presrclen != $postsrclen + $postsrclen"; $ref->{$srcchr} = $mutseq; $ref->{$ochr} = $oseq; my $ntotlen = 0; for (keys %$ref) { $ntotlen += length($ref->{$_}); } $totlen == $ntotlen || die "Total length changed after rearrangements from $srcchr to $ochr ($totlen -> $ntotlen)"; } print STDERR " Finished rearrangements\n"; $ref->{$srcchr} = $mutseq; return ($nsnp, $nrfgap, $nrdgap, $nrearr); } sub test1 { my $mut = Mutate->new("UnitTest mutator"); my %refs = ( "r1" => "TATGACGGTCGAAACCAGGCGA", "r2" => "TATATTTAGTCTCGTCTGGCTGTCTCGGCTGCGCGCGAGTAAAGACCGGCCTGATC"); $mut->mutateSeq("r1", \%refs); $mut->mutateSeq("r2", \%refs); return 1; } sub test2 { my $mut = Mutate->new( "UnitTest mutator", \&defaultSNPGen, \&defaultRdGapGen, \&defaultRfGapGen, sub { return 0.1 }, \&defaultGapLenGen, \&defaultSeqGen); my %refs = ( "r1" => "TATGACGGTCGAAACCAGGCGA", "r2" => "TATATTTAGTCTCGTCTGGCTGTCTCGGCTGCGCGCGAGTAAAGACCGGCCTGATC", "r3" => "TATATTTAGTCTCGTCTGGCTGTCTCGGCTGCGCGCGAGTAAAGACCGGCCTGATC". "ATTGGTGTCGCGGCGCGCGTATATATATATATATATAGCCTGCTACGTCAGCTAGC", "r4" => "TATATTTAGTCTCGTCTGGCTGTCTCGGCTGCGCGCGAGTAAAGACCGGCCTGATC". "ATTGGTGTCGCGGCGCGCGTATATATATATATATATAGCCTGCTACGTCAGCTAGC". "ATATAACAAAAAAACCCCACACGACGCGGACTCTAGCACTATCGGACTATCATCGG"); $mut->mutateSeq("r1", \%refs); $mut->mutateSeq("r2", \%refs); $mut->mutateSeq("r3", \%refs); $mut->mutateSeq("r4", \%refs); return 1; } if($0 =~ /[^0-9a-zA-Z_]?Mutate\.pm$/) { print "Running unit tests\n"; # Run unit tests Test::shouldSucceed("test1", \&test1); Test::shouldSucceed("test2", \&test2); } 1; hisat2-2.2.1/scripts/sim/RandDNA.pm000066400000000000000000000107551370663763200167570ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # package RandDNA; use strict; use Carp; use FindBin qw($Bin); use lib $Bin; use DNA; use Test; use Math::Random; ## # Create a new random DNA generator # sub new { my ( $class, $name, # name of generator $n, # N fraction $iupac, # Non-A/C/G/T/N IUPAC fraction (after N fraction removed) $at, # AT fraction (after N/IUPAC fractions removed) $a, # A fraction (of AT) $c, # C fraction (of CG) ) = @_; $name = "noname" unless defined($name); return bless { _name => $name, _n => $n || croak("No N frac"), _iupac => $iupac || croak("No IUPAC frac"), _at => $at || rand(), _a => $a || rand(), _c => $c || rand() }, $class; } sub name { return $_[0]->{_name} } sub nFrac { return $_[0]->{_n} } sub iupacFrac { return $_[0]->{_iupac} } sub atFrac { return $_[0]->{_at} } sub aFrac { return $_[0]->{_a} } sub cFrac { return $_[0]->{_c} } ## # Return a random IUPAC character. # sub randIUPAC() { my @iu = ( "R", "Y", "M", "K", "S", "W", "B", "V", "H", "D" ); my $iui = int(rand(scalar(@iu))); defined($iu[$iui]) || die "Bad index $iui"; return $iu[$iui]; } ## # Given parameters controlling character frequencies, build a palette # of short sequences that can be composed to make longer sequences. # sub genBuildingBlocks { my ($self, $arr, $num, $bbrnd) = @_; defined($arr) || die; $num = 30 unless defined($num); # Random generator for length $bbrnd = sub { return int(rand(100))+1 } unless defined($bbrnd); for my $i (1..$num) { my $seq = ""; # Generate length my $len = $bbrnd->(); $len > 0 || die "Bad length: $len"; # Generate characters for my $j (1..$len) { my $c = ""; if(rand() < $self->nFrac()) { $c = "N"; } elsif(rand() < $self->iupacFrac()) { $c = randIUPAC(); defined($c) || die; } else { $c = (rand() < $self->atFrac()) ? "AT" : "CG"; if($c eq "AT") { $c = (rand() < $self->aFrac()) ? "A" : "T"; } else { $c = (rand() < $self->cFrac()) ? "C" : "G"; } } $seq .= $c; } # Add to return list push @$arr, $seq; } } ## # Use this generator to generate another random sequence. # sub nextSeq { my ($self, $len, $bbsr, $runrnd) = @_; my $seq = ""; # Generate building blocks my @bbs = (); if(defined($bbsr)) { @bbs = @$bbsr; } else { $self->genBuildingBlocks(\@bbs); } scalar(@bbs) > 0 || die; # Random generator for run length my $defaultRnd = sub { # Mean of exp is 1/lambda return int(Math::Random::random_exponential(1, 2))+1; }; $runrnd = $defaultRnd unless defined($runrnd); # Build the sequence by repeatedly inserting runs of building blocks while(length($seq) < $len * 5) { # Choose building block my $bbi = int(rand(scalar(@bbs))); # Choose how many times to add it my $runlen = $runrnd->(); # Choose insert point my $insat = int(rand(length($seq))); # Repeatedly insert building lock for my $i (1..$runlen) { substr($seq, $insat, 0) = $bbs[$bbi]; } } # Return chopped out piece return substr($seq, $len * 2, $len); } sub test1 { my $rd = RandDNA->new( "randtest1", # name 0.02, # n frac 0.01, # IUPAC frac 0.4, # AT frac 0.45, # A/AT frac 0.35); # C/CG frac my $seq = $rd->nextSeq(200, undef); length($seq) == 200 || die; return 1; } sub test2 { my @bb = ("AAA", "CCCC"); my $rd = RandDNA->new( "randtest2", # name 0.02, # n frac 0.01, # IUPAC frac 0.4, # AT frac 0.45, # A/AT frac 0.35); # C/CG frac my $seq = $rd->nextSeq(300, \@bb); length($seq) == 300 || die; return 1; } if($0 =~ /[^0-9a-zA-Z_]?RandDNA\.pm$/) { print "Running unit tests\n"; # Run unit tests Test::shouldSucceed("test1", \&test1); Test::shouldSucceed("test2", \&test2); } 1; hisat2-2.2.1/scripts/sim/SampleRead.pm000066400000000000000000000154471370663763200175700ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # package SampleRead; use strict; use Carp; use FindBin qw($Bin); use lib $Bin; use DNA; use Test; use List::Util qw(max min); use Math::Random; ## # Default sequencing miscall rate generator. # sub defaultSeqMmGen() { return Math::Random::random_uniform(1, 0, 0.1); } ## # Default random generator for read length. # sub defaultFragLenGen() { return int(Math::Random::random_normal(1, 200, 40))+1; } ## # Default random generator for read length. # sub defaultReadLenGen() { my $r = int(rand(3)); if($r == 0) { return int(Math::Random::random_exponential(1, 60))+1; } elsif($r == 1) { return int(Math::Random::random_exponential(1, 20))+1; } else { return int(Math::Random::random_exponential(1, 150))+1; } } ## # Create a new read sampler # sub new { my ( $class, $name, # name of simulator $fraglengen, # paired-end fragment length generator $m1lengen, # random mate1 length generator $m2lengen, # random mate2 length generator ) = @_; $fraglengen = \&defaultFragLenGen unless defined($fraglengen); $m1lengen = \&defaultReadLenGen unless defined($m1lengen); $m2lengen = \&defaultReadLenGen unless defined($m2lengen); $name = "noname" unless defined($name); return bless { _name => $name, _fraglengen => $fraglengen, _m1lengen => $m1lengen, _m2lengen => $m2lengen, }, $class; } sub name { return $_[0]->{_name} } sub fraglengen { return $_[0]->{_fraglengen} } sub m1lengen { return $_[0]->{_m1lengen} } sub m2lengen { return $_[0]->{_m2lengen} } ## # Generate a set of reads from a subject genome encoded in a hash ref. # sub genReads { my ( $self, $num, # number of reads/fragments to generate $color, # colorize? $refs, # hash ref holding reference sequences $seqs, # put generated read sequences here $quals, # put generated quality sequences here $lengen) = @_; # length generator ref($refs) eq "HASH" || die "Reference input must be hash ref, is ".ref($refs); ref($seqs) eq "ARRAY" || die "seqs input must be array ref, is ".ref($seqs); ref($quals) eq "ARRAY" || die "quals input must be array ref, is".ref($quals); $lengen = $self->m1lengen() unless defined($lengen); my $totreflen = 0; my @keys = keys %$refs; for (@keys) { $totreflen += length($refs->{$_}); } for(1..$num) { if(rand() < 0.05 && scalar(@$seqs) > 0) { # Clone a previous read my $ci = int(rand(scalar(@$seqs))); push @$seqs, $seqs->[$ci]; push @$quals, $quals->[$ci]; } else { while(1) { my $ro = int(rand($totreflen)); my $len = $lengen->(); $len = 1 if $len < 1; my $key = undef; my $rflen = 0; for (@keys) { $rflen = length($refs->{$_}); if($ro < $rflen) { $key = $_; last; } $ro -= $rflen; } defined($key) || die; $rflen > 0 || die; # If we are overhanging the end, discard and try again next if $ro + $len > $rflen; my $rfseq = substr($refs->{$key}, $ro, $len); length($rfseq) == $len || die; my $rc = int(rand(2)); # Possibly reverse-complement it $rfseq = DNA::revcomp($rfseq) if $rc == 1; # Possible colorize if($color) { my $cseq = ""; for(0..$len-2) { my ($c1, $c2) = (substr($rfseq, $_, 1), substr($rfseq, $_+1, 1)); my $col = DNA::dinucToColor($c1, $c2); $cseq .= $col; } $rfseq = $cseq; $len = length($rfseq); } push @$seqs, $rfseq; # TODO: generate interesting qualities push @$quals, "I" x $len; last; } } # Simulate next read } } ## # Generate a set of read pairs from a subject genome encoded in a hash # ref. First we extract unpaired fragments, then take sequences from # either end to make the mates. # sub genReadPairs { my ( $self, $num, # number of reads/fragments to generate $color, # colorize? $refs, # hash ref holding reference sequences $m1fw, # orientation of mate 1 when fragment comes from Watson strand $m2fw, # orientation of mate 2 when fragment comes from Watson strand $seq1s, # put generated mate1 sequences here $seq2s, # put generated mate2 sequences here $qual1s, # put generated mate1 quality sequences here $qual2s) = @_; # put generated mate2 quality sequences here # First simulate fragments ref($refs) eq "HASH" || die "Reference input must be hash ref"; ref($seq1s) eq "ARRAY" || die "seq1s input must be array ref"; ref($seq2s) eq "ARRAY" || die "seq2s input must be array ref"; ref($qual1s) eq "ARRAY" || die "qual1s input must be array ref"; ref($qual2s) eq "ARRAY" || die "qual2s input must be array ref"; my @fragseqs = (); my @fragquals = (); $self->genReads( $num, $color, $refs, \@fragseqs, \@fragquals, $self->fraglengen); scalar(@fragseqs) == scalar(@fragquals) || die; # For each fragment for (1..scalar(@fragseqs)) { # Take mates from either end my $m1len = $self->m1lengen->(); my $m2len = $self->m2lengen->(); $m1len = min($m1len, length($fragseqs[$_-1])); $m2len = min($m2len, length($fragseqs[$_-1])); my $m1seq = substr($fragseqs [$_-1], 0, $m1len); my $m2seq = substr($fragseqs [$_-1], -$m2len); my $m1qual = substr($fragquals[$_-1], 0, $m1len); my $m2qual = substr($fragquals[$_-1], -$m2len); if(!$m1fw) { $m1seq = DNA::revcomp($m1seq); $m1qual = reverse $m1qual; } if(!$m2fw) { $m2seq = DNA::revcomp($m2seq); $m2qual = reverse $m2qual; } # Commit new pair to the list push @$seq1s, $m1seq; push @$seq2s, $m2seq; push @$qual1s, $m1qual; push @$qual2s, $m2qual; # Simulate next pair } } sub test1 { my $samp = SampleRead->new("UnitTest read sampler"); my %refs = ( "r1" => "TATGACGGTCGAAACCAGGCGA", "r2" => "TATATTTAGTCTCGTCTGGCTGTCTCGGCTGCGCGCGAGTAAAGACCGGCCTGATC"); my @seqs = (); my @quals = (); $samp->genReads(10, 0, \%refs, \@seqs, \@quals, \&defaultReadLenGen); scalar(@seqs) == 10 || die; scalar(@quals) == 10 || die; return 1; } sub test2 { return 1; } if($0 =~ /[^0-9a-zA-Z_]?SampleRead\.pm$/) { print "Running unit tests\n"; # Run unit tests Test::shouldSucceed("test1", \&test1); Test::shouldSucceed("test2", \&test2); } 1; hisat2-2.2.1/scripts/sim/Sim.pm000066400000000000000000000745561370663763200163110ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # package Sim; use strict; use Carp; use FindBin qw($Bin); use lib $Bin; use DNA; use Test; use RandDNA; use SampleRead; use Mutate; use AlignmentCheck; use Math::Random; use List::Util qw(max min); use POSIX; ## # Replacement for "die" that additionally writes error message to file so that # run.pl can read it later. # sub mydie($) { my $fn = ".run.pl.child.$$"; open(EO, ">$fn") || die "Could not open $fn for writing"; print EO "$_[0]\n"; close(EO); confess $_[0]; } # Generates random printable strings of a given length sub randStr($) { my $len = shift; my @chars = ('a'..'z', 'A'..'Z', '0'..'9', '_'); my $str = ""; foreach (1..$len) { $str .= $chars[int(rand(scalar(@chars)))]; } return $str; } ## # Default random generator for number of reference per test case. # sub defaultRefNumGen() { return int(Math::Random::random_exponential(1, 8))+1; } ## # Default random generator for reference length. # sub defaultRefLenGen() { return int(Math::Random::random_exponential(1, 50000))+1; } ## # Default random generator for number of reference per test case. # sub defaultReadNumGen() { return int(Math::Random::random_exponential(1, 10000))+1; } ## # Default random generator for read length. # sub defaultFragLenGen() { return int(Math::Random::random_normal(1, 200, 40))+1; } ## # Default random generator for reference length. # sub defaultReadLenGen() { my $r = int(rand(3)); if($r == 0) { return int(Math::Random::random_exponential(1, 60))+1; } elsif($r == 1) { return int(Math::Random::random_exponential(1, 20))+1; } else { return int(Math::Random::random_exponential(1, 150))+1; } } ## # Default random generator for fraction of reference characters = N. # sub defaultNGen() { return Math::Random::random_uniform(1, 0, 0.05); } ## # Default random generator for fraction of reference characters = an # ambiguous IUPAC code. # sub defaultIupacGen() { return Math::Random::random_uniform(1, 0, 0.01); } ## # Default random generator for AT/ACGT fraction. # sub defaultAtGen() { return min(max(Math::Random::random_normal(1, 0.5, 0.18), 0), 1); } ## # Default random generator for A/AT fraction. # sub defaultAGen() { return min(max(Math::Random::random_normal(1, 0.5, 0.18), 0), 1); } ## # Default random generator for C/CG fraction. # sub defaultCGen() { return min(max(Math::Random::random_normal(1, 0.5, 0.18), 0), 1); } ## # Default SNP rate generator. Doesn't generate the SNP per se, just # the rate. # sub defaultSNPGen() { return Math::Random::random_uniform(1, 0, 0.05); } ## # Default read gap rate generator. Doesn't generate the gaps or # lengths, just the rate. # sub defaultRdGapGen() { return Math::Random::random_uniform(1, 0, 0.005); } ## # Default reference gap rate generator. Doesn't generate the gaps or # lengths, just the rate. # sub defaultRfGapGen() { return Math::Random::random_uniform(1, 0, 0.005); } ## # Default rearrangement rate generator. # sub defaultRearrGen() { return Math::Random::random_uniform(1, 0, 0.005); } ## # Default function for generating gap lengths when introducing a gap. # sub defaultGapLenGen($) { return int(Math::Random::random_exponential(1, 3))+1; } ## # Default function for generating random sequence to insert into a gap. # sub defaultSeqGen($) { my $len = shift; ($len == int($len) && $len > 0) || mydie("Bad length for sequence generator: $len"); my $ret = ""; for (1..$len) { $ret .= substr("ACGT", int(rand(4)), 1); } return $ret; } ## # Default sequencing miscall rate generator. # sub defaultSeqMmGen() { return Math::Random::random_uniform(1, 0, 0.1); } ## # Create a new test case simulator # sub new { my ( $class, $name, # name of simulator $rfnumgen, # number of reference sequences $rflengen, # reference length $rdnumgen, # number of read sequences per run $rdlengen, # read length generator $fraglengen, # fragment length generator $ngen, # N fraction $iupacgen, # Non-A/C/G/T/N IUPAC fraction (after N fraction removed) $atgen, # AT fraction (after N/IUPAC fractions removed) $agen, # A fraction (of AT) $cgen, # C fraction (of CG) $snpgen, # SNP rate gen $rdgapgen, # read gap generator $rfgapgen, # ref gap generator $rearrgen, # rearrangement generator $gaplengen, # gap length generator $seqgen, # gap filler sequence generator $seqmm, # sequencing error generator ) = @_; $rfnumgen = \&defaultRefNumGen unless defined($rfnumgen); $rflengen = \&defaultRefLenGen unless defined($rflengen); $rdnumgen = \&defaultReadNumGen unless defined($rdnumgen); $rdlengen = \&defaultReadLenGen unless defined($rdlengen); $fraglengen = \&defaultFragLenGen unless defined($fraglengen); $ngen = \&defaultNGen unless defined($ngen); $iupacgen = \&defaultIupacGen unless defined($iupacgen); $atgen = \&defaultAtGen unless defined($atgen); $agen = \&defaultAGen unless defined($agen); $cgen = \&defaultCGen unless defined($cgen); $snpgen = \&defaultSNPGen unless defined($snpgen); $rdgapgen = \&defaultRdGapGen unless defined($rdgapgen); $rfgapgen = \&defaultRfGapGen unless defined($rfgapgen); $rearrgen = \&defaultRearrGen unless defined($rearrgen); $gaplengen = \&defaultGapLenGen unless defined($gaplengen); $seqgen = \&defaultSeqGen unless defined($seqgen); $seqmm = \&defaultSeqMmGen unless defined($seqmm); $name = "noname" unless defined($name); return bless { _name => $name, _rfnumgen => $rfnumgen, _rflengen => $rflengen, _rdnumgen => $rdnumgen, _rdlengen => $rdlengen, _fraglengen => $fraglengen, _ngen => $ngen, _iupacgen => $iupacgen, _atgen => $atgen, _agen => $agen, _cgen => $cgen, _snpgen => $snpgen, _rdgapgen => $rdgapgen, _rfgapgen => $rfgapgen, _rearrgen => $rearrgen, _gaplengen => $gaplengen, _seqgen => $seqgen, _seqmm => $seqmm, }, $class; } sub rfnumgen { return $_[0]->{_rfnumgen} } sub rflengen { return $_[0]->{_rflengen} } sub rdnumgen { return $_[0]->{_rdnumgen} } sub rdlengen { return $_[0]->{_rdlengen} } sub fraglengen { return $_[0]->{_fraglengen} } sub ngen { return $_[0]->{_ngen} } sub iupacgen { return $_[0]->{_iupacgen} } sub atgen { return $_[0]->{_atgen} } sub agen { return $_[0]->{_agen} } sub cgen { return $_[0]->{_cgen} } sub snpgen { return $_[0]->{_snpgen} } sub rdgapgen { return $_[0]->{_rdgapgen} } sub rfgapgen { return $_[0]->{_rfgapgen} } sub rearrgen { return $_[0]->{_rearrgen} } sub gaplengen { return $_[0]->{_gaplengen} } sub seqgen { return $_[0]->{_seqgen} } sub seqmm { return $_[0]->{_seqmm} } ## # Generate DNA generator. # sub genDNAgen { my $self = shift; my $nfrac = $self->ngen->(); my $iupacfrac = $self->iupacgen->(); my $atfrac = $self->atgen->(); my $afrac = $self->agen->(); my $cfrac = $self->cgen->(); my $refdnagen = RandDNA->new( "Sim.pm gen", $nfrac, $iupacfrac, $atfrac, $afrac, $cfrac); printf STDERR "Created DNA generator\n"; printf STDERR " N frac: %0.3f\n", $nfrac; printf STDERR " IUPAC frac: %0.3f\n", $iupacfrac; printf STDERR " AT/ACGT frac: %0.3f\n", $atfrac; printf STDERR " A/AT frac: %0.3f\n", $afrac; printf STDERR " C/CG frac: %0.3f\n", $cfrac; return $refdnagen; } ## # Generate and print reference sequences to file of given name. Also, # install reference sequences into hash ref $ref. To allow for # "overhang" (alignment that hang off the end of the reference), we # actually write out a little bit less than the full reference sequence # for each sequence. # sub genRef { my ($self, $ref, $refdnagen, $conf, $tmpfn) = @_; # Get a generator for reference length my $reflen = $self->rflengen; # Generate the number of references my $refnum = $self->rfnumgen->(); $refnum = sqrt($refnum) if $conf->{small}; $refnum = 1 if $refnum <= 0; $refnum = sqrt($refnum) if $conf->{small}; $refnum = 1 if $refnum <= 0; $refnum = ceil($refnum); $refnum = $conf->{numrefs} if defined($conf->{numrefs}); # Open output file open (FA, ">$tmpfn") || mydie("Could not open temporary fasta file '$tmpfn' for writing"); my %ccnt = (); print STDERR "Generating $refnum references\n"; for (1..$refnum) { # Randomly generate length my $len = $reflen->(); $len = sqrt($len) if $conf->{small}; $len = 1 if $len <= 0; $len = ceil($len); my $seq = $refdnagen->nextSeq($len); length($seq) >= $len || die; my $name = "Sim.pm.$_"; $ref->{$name} = $seq; # Select amount to trim from LHS my $trimleft = int(Math::Random::random_exponential(1, 200)); # Select amount to trim from RHS my $trimright = int(Math::Random::random_exponential(1, 200)); # Make sure we're leaving some sequence after trimming while($trimleft + $trimright > $len) { if(int(rand(2))) { $trimleft = int($trimleft*0.5); } else { $trimright = int($trimright*0.5); } } # Trim the sequence substr($seq, 0, $trimleft) = ""; $seq = substr($seq, 0, length($seq)-$trimright); my $trimlen = length($seq); $trimlen == $len - $trimleft - $trimright || mydie("Unexpected trim combo: $len, $trimleft, $trimright, $trimlen"); print STDERR " Generated reference '$name' of untrimmed length $len, trimmed length $trimlen\n"; print FA ">$name\n"; my $buf = ""; length($seq) >= $trimlen || die; for my $i (1..$trimlen) { my $c = substr($seq, $i-1, 1); defined($c) || die; $ccnt{$c}++; $buf .= $c; $ref->{$name} .= $c; if($i % 60 == 0) { print FA "$buf\n"; $buf = ""; } } print FA "$buf\n" if $buf ne ""; } close(FA); print STDERR "Wrote references to $tmpfn\n"; for my $k (sort keys %ccnt) { print STDERR " $k: $ccnt{$k}\n"; } } ## # Generate a hash of key/value arguments to pass to bowtie2. # sub genBuildArgs { my ($self) = @_; my %args = (); my $r1 = int(rand(3)); if($r1 == 0) { $args{"--bmaxdivn"} = int(Math::Random::random_exponential(1, 4))+1; } elsif($r1 == 1) { $args{"--bmax"} = int(Math::Random::random_exponential(1, 10000))+100; } my $r2 = int(rand(2)); if($r2 == 0) { $args{"--dcv"} = 2 ** (int(rand(10))+4); } my $r3 = int(rand(5)); if($r3 == 0) { $args{"--packed"} = ""; } my $r4 = int(rand(3)); if($r4 == 0) { $args{"--offrate"} = int(rand(8))+1; } return \%args; } ## # Given a fasta filename, an index basename, and a path to the # bowtie2-build executable, build nucleotide-space and colorpace # indexes for the sequences in the fasta file. # sub build { my ($self, $fa, $idx, $conf, $args) = @_; my $argstr = ""; for (keys %$args) { $argstr .= " $_"; if($args->{$_} ne "") { $argstr .= " ".$args->{$_}; } } $argstr .= " --sanity"; # Build nucleotide index my $cmd = "$conf->{bowtie2_build_debug} $argstr $fa $idx"; print STDERR "$cmd\n"; system($cmd); $? == 0 || mydie("Error running '$cmd'; exitlevel=$?"); print STDERR "Built nucleotide index '$idx'\n"; # Build colorspace index unless($conf->{no_color}) { $cmd = "$conf->{bowtie2_build_debug} $argstr -C $fa ${idx}.c"; print STDERR "$cmd\n"; system($cmd); $? == 0 || mydie("Error running '$cmd'; exitlevel=$?"); print STDERR "Built colorspace index '$idx'\n"; } } ## # Given a hash of sequences, flatten all IUPAC codes into unambiguous # nucleotides. # sub flattenIUPAC() { my ($self, $h) = @_; for my $c (keys %$h) { my $len = length($h->{$c}); for my $i (0..$len-1) { my $ch = uc substr($h->{$c}, $i, 1); my $nc = $ch; if(DNA::isIUPAC($ch) || $ch eq "N") { if(rand() < $self->snpgen->()) { $nc = DNA::pickIncompat($ch); defined($nc) || mydie("Couldn't find incompatible base for $ch"); } else { $nc = DNA::pickCompat($ch); defined($nc) || mydie("Couldn't find compatible base for $ch"); } } if($ch ne $nc) { substr($h->{$c}, $i, 1) = $nc; } } } } ## # Mutate reference genome into a subject genome. # sub mutate() { my ($self, $refs) = @_; my %subj = %$refs; $self->flattenIUPAC(\%subj); print STDERR "Flattened IUPAC characters\n"; my $mutator = Mutate->new( "Sim.pm mutator", $self->snpgen, $self->rdgapgen, $self->rfgapgen, $self->rearrgen, $self->gaplengen, $self->seqgen); my ($nsnp, $nrfgap, $nrdgap, $nrearr) = (0, 0, 0, 0); for(keys %subj) { print STDERR " Mutating sequence $_\n"; my ($nsnp_, $nrfgap_, $nrdgap_, $nrearr_) = $mutator->mutateSeq($_, \%subj); $nsnp += $nsnp_; $nrfgap += $nrfgap_; $nrdgap += $nrdgap_; $nrearr += $nrearr_; } print STDERR "Mutated reference genome to subject genome\n"; print STDERR " SNPs introduced: $nsnp\n"; print STDERR " Reference gaps introduced: $nrfgap\n"; print STDERR " Read gaps introduced: $nrdgap\n"; print STDERR " Rearrangements introduced: $nrearr\n"; return \%subj; } sub dumpFastq { my ($self, $input, $fh1, $fh2) = @_; for (1..scalar(@{$input->{seq1s}})) { my $seq1 = $input->{seq1s}->[$_-1]; my $qual1 = $input->{qual1s}->[$_-1]; print {$fh1} "\@$_\n"; print {$fh1} "$seq1\n"; print {$fh1} "+$_\n"; print {$fh1} "$qual1\n"; if($input->{paired}) { my $seq2 = $input->{seq2s}->[$_-1]; my $qual2 = $input->{qual2s}->[$_-1]; print {$fh2} "\@$_\n"; print {$fh2} "$seq2\n"; print {$fh2} "+$_\n"; print {$fh2} "$qual2\n"; } } } sub dumpQseq { my ($self, $input, $fh1, $fh2) = @_; for (1..scalar(@{$input->{seq1s}})) { my $seq1 = $input->{seq1s}->[$_-1]; my $qual1 = $input->{qual1s}->[$_-1]; print {$fh1} "R\t1\t1\t1\t$_\t$_\t1\t1\t$seq1\t$qual1\t1\n"; if($input->{paired}) { my $seq2 = $input->{seq2s}->[$_-1]; my $qual2 = $input->{qual2s}->[$_-1]; print {$fh2} "R\t1\t1\t1\t$_\t$_\t1\t1\t$seq2\t$qual2\t1\n"; } } } sub dumpFasta { my ($self, $input, $fh1, $fh2) = @_; for (1..scalar(@{$input->{seq1s}})) { my $seq1 = $input->{seq1s}->[$_-1]; print {$fh1} ">$_\n"; print {$fh1} "$seq1\n"; if($input->{paired}) { my $seq2 = $input->{seq2s}->[$_-1]; print {$fh2} ">$_\n"; print {$fh2} "$seq2\n"; } } } sub dumpRaw { my ($self, $input, $fh1, $fh2) = @_; for (1..scalar(@{$input->{seq1s}})) { my $seq1 = $input->{seq1s}->[$_-1]; print {$fh1} "$seq1\n"; if($input->{paired}) { my $seq2 = $input->{seq2s}->[$_-1]; print {$fh2} "$seq2\n"; } } } ## # Generate the input (reads plus paired/fragment information) # sub genInput { my ($self, $refs, $conf) = @_; # Select whether we're doing colorspace my $color = int(rand(2)); $color = 0 if $conf->{no_color}; # Select whether we're doing unpaired or paired-end. my $paired = int(rand(2)); $paired = 0 if $conf->{no_paired}; # Select format for read file my @formats = ("fastq", "qseq", "fasta", "raw"); my @format_arg = ( "-q", "--qseq", "-f", "-r"); my $formati = int(rand(scalar(@formats))); my $format = $formats[$formati]; my $format_arg = $format_arg[$formati]; my $tmprdfn1 = "$conf->{tempdir}/Sim.pm.$conf->{randstr}_1.$format"; my $tmprdfn2 = "$conf->{tempdir}/Sim.pm.$conf->{randstr}_2.$format"; # Generate reads from the subject genome; no sequencing error yet my %input = ( seq1s => [], seq2s => [], qual1s => [], qual2s => [], mate1fw => 1, mate2fw => 0, paired => $paired, color => $color, format => $format, format_arg => $format_arg, file1 => $tmprdfn1, file2 => $tmprdfn2 ); my $read_sampler = SampleRead->new( "Sim.pm read sampler", $self->fraglengen, $self->rdlengen, $self->rdlengen); print STDERR "Created read sampler\n"; my $numreads = $self->rdnumgen->(); $numreads = ceil(sqrt($numreads)) if $conf->{small}; $numreads == int($numreads) || mydie("numreads $numreads not a number"); my $tmp = int(rand(3)); if($tmp == 0) { $input{mate2fw} = 1; } elsif($tmp == 1) { $input{mate1fw} = 0; $input{mate2fw} = 1; } print STDERR "Sampling $numreads reads\n"; ref($refs) eq "HASH" || mydie("Reference input must be hash ref"); if($paired) { $read_sampler->genReadPairs( $numreads, # number of reads/fragments to generate $input{color}, # colorize? $refs, # hash ref holding reference sequences $input{mate1fw}, # orientation of mate 1 when fragment comes from Watson strand $input{mate2fw}, # orientation of mate 2 when fragment comes from Watson strand $input{seq1s}, # put generated mate1 sequences here $input{seq2s}, # put generated mate2 sequences here $input{qual1s}, # put generated mate1 quality sequences here $input{qual2s}); # put generated mate2 quality sequences here } else { $read_sampler->genReads( $numreads, # number of reads/fragments to generate $input{color}, # colorize? $refs, # hash ref holding reference sequences $input{seq1s}, # put generated sequences here $input{qual1s}); # put generated quality sequences here } # TODO: with some probability, sort the reads print STDERR "Dumping reads to temporary files $tmprdfn1 & $tmprdfn2\n"; # Dump reads to output file my ($fh1, $fh2); open($fh1, ">$tmprdfn1") || mydie("Could not open '$tmprdfn1' for writing"); open($fh2, ">$tmprdfn2") || mydie("Could not open '$tmprdfn2' for writing"); if($format eq "fastq") { $self->dumpFastq(\%input, $fh1, $fh2); } elsif($format eq "qseq") { $self->dumpQseq(\%input, $fh1, $fh2); } elsif($format eq "fasta") { $self->dumpFasta(\%input, $fh1, $fh2); } elsif($format eq "raw") { $self->dumpRaw(\%input, $fh1, $fh2); } close($fh1); close($fh2); return \%input; } ## # Mutate reads according to sequencing error model. # sub mutateSeq { my ($self, $input) = @_; return $input; } ## # Generate a setting for MA (match bonus). # sub genPolicyMA($) { my $local = shift; return "" if ($local || int(rand(2)) == 0); return "MA=".Math::Random::random_uniform(1, 1, 40).";"; } ## # Generate a setting for MMP (mismatch penalty). # sub genPolicyMMP() { return "" if int(rand(2)) == 0; my $op = substr("CQR", int(rand(3)), 1); if($op eq "C") { $op .= Math::Random::random_uniform(1, 1, 40); } return "MMP=$op;"; } ## # Generate a setting for NP (penalty for a mismatch involving an N). # sub genPolicyNP() { return "" if int(rand(2)) == 0; my $op = substr("CQR", int(rand(3)), 1); if($op eq "C") { $op .= int(Math::Random::random_exponential(1, 3))+1; } return "NP=$op;"; } ## # Generate a setting for RDG (read gap open and extend penalties). # sub genPolicyRDG() { return undef if int(rand(2)) == 0; my $op = Math::Random::random_uniform(1, 1, 50); if(int(rand(2)) == 0) { $op .= ","; $op .= Math::Random::random_uniform(1, 1, 20); } return "$op"; } ## # Generate a setting for RFG (ref gap open and extend penalties). # sub genPolicyRFG() { return undef if int(rand(2)) == 0; my $op = Math::Random::random_uniform(1, 1, 50); if(int(rand(2)) == 0) { $op .= ","; $op .= Math::Random::random_uniform(1, 1, 20); } return "$op"; } ## # Generate a setting for MIN (function determining minimum acceptable score). # sub genPolicyMIN($) { my $local = shift; return undef if ($local || int(rand(2)) == 0); my $xx = Math::Random::random_uniform(1, 1, 10); my $yy = Math::Random::random_uniform(1, 1, 10); if(!$local) { $xx = -$xx if int(rand(2)) == 0; $yy = -$yy; } return "L,$xx,$yy"; } ## # Generate a setting for NCEIL (function determining maximum number of Ns # allowed). # sub genPolicyNCEIL() { return undef if int(rand(2)) == 0; my $xx = Math::Random::random_uniform(1, 0, 1.5); my $yy = Math::Random::random_uniform(1, 0, 1.5); return "$xx,$yy"; } ## # Generate a setting for SEED (# mismatches, length, interval). # sub genPolicySEED() { return undef if int(rand(2)) == 0; # Pick a number of mismatches my $sd = substr("012", int(rand(2)), 1); if(rand() < 0.9) { # Length $sd .= ",".int(Math::Random::random_uniform(1, 12, 32)); } return $sd; } ## # Generate a setting for -D (# DP fails in a row). # sub genPolicyFailStreak() { return undef if int(rand(2)) == 0; return int(Math::Random::random_uniform(1, 2, 50)); } ## # Generate a setting for -R (# seeding rounds). # sub genPolicySeedRounds() { return undef if int(rand(2)) == 0; return int(Math::Random::random_uniform(1, 1, 5)); } ## # Generate a setting for IVAL. Interval between seeds is a function of the # read length OR sqaure root of read length OR cube root of read length. # sub genPolicyIVAL() { return "" if int(rand(2)) == 0; # Pick a number of mismatches my $iv = substr("LSC", int(rand(3)), 1); if($iv eq "L") { if(rand() < 0.9) { # Multiplier $iv .= ",".Math::Random::random_uniform(1, 0.0, 0.5); } if(rand() < 0.3) { # Offset $iv .= ",".Math::Random::random_uniform(1, 0.0, 4.0); } } elsif($iv eq "S") { if(rand() < 0.9) { # Multiplier $iv .= ",".Math::Random::random_uniform(1, 0.0, 3.0); } if(rand() < 0.3) { # Offset $iv .= ",".Math::Random::random_uniform(1, 0.0, 7.0); } } elsif($iv eq "C") { if(rand() < 0.9) { # Multiplier $iv .= ",".Math::Random::random_uniform(1, 0.0, 5.0); } if(rand() < 0.3) { # Offset $iv .= ",".Math::Random::random_uniform(1, 0.0, 14.0); } } return "IVAL=$iv;"; } ## # Generate a random but meaningful string of policy arguments to specify using # the -P option. # sub genPolicyArg($) { my $local = shift; my $args = ""; $args .= genPolicyMA($local); $args .= genPolicyMMP(); $args .= genPolicyNP(); $args .= genPolicyIVAL(); if($args ne "") { return substr($args, 0, -1); } else { return ""; } } ## # Generate a hash of key/value arguments to pass to bowtie2. # sub genAlignArgs { my ($self, $input, $color, $conf) = @_; my %args = (); my $local = int(rand(2)) == 0; $args{"-u"} = $conf->{maxreads} if defined($conf->{maxreads}); $args{"--mm"} = "" if int(rand(2)) == 0; #$args{"--overhang"} = "" if int(rand(2)) == 0; $args{"--trim3"} = int(rand(10)) if int(rand(2)) == 0; $args{"--trim5"} = int(rand(10)) if int(rand(2)) == 0; $args{"--nofw"} = "" if int(rand(4)) == 0; $args{"--norc"} = "" if int(rand(4)) == 0; $args{"--col-keepends"} = "" if ($color && int(rand(3)) == 0); $args{"--gbar"} = int(Math::Random::random_exponential(1, 3))+1 if int(rand(4)) == 0; $args{"--local"} = "" if $local; my $rep = int(rand(5)); if($rep == 0) { $args{"-a"} = ""; } elsif($rep == 1) { $args{"-k"} = int(Math::Random::random_exponential(1, 3))+2; } elsif($rep == 2) { $args{"-M"} = int(Math::Random::random_exponential(1, 3))+2; } $args{"--rdg"} = genPolicyRDG(); $args{"--rfg"} = genPolicyRFG(); $args{"--score-min"} = genPolicyMIN($local); $args{"--n-ceil"} = genPolicyNCEIL(); $args{"-N"} = genPolicySEED(); $args{"-D"} = genPolicyFailStreak(); $args{"-R"} = genPolicySeedRounds(); $args{"--policy"} = ("\"".genPolicyArg($local)."\"") if rand() < 0.9; $args{"--cp-min"} = int(Math::Random::random_exponential(1, 3)) + 2; $args{"--cp-ival"} = int(Math::Random::random_exponential(1, 1)) + 1; return \%args; } ## # Align the given input set against the given index using the given # bowtie2 binary and arguments. Sanity-check the SAM output. # sub align { my ($self, $fa, $idx, $input, $conf, $args) = @_; my $argstr = ""; for (keys %$args) { if(defined($args->{$_})) { $argstr .= " $_"; if($args->{$_} ne "") { $argstr .= " ".$args->{$_}; } } } $argstr .= " -C" if $input->{color}; $argstr .= " ".$input->{format_arg}; $idx .= ".c" if $input->{color}; my $inputfn; if($input->{paired}) { $inputfn = "-1 $input->{file1} -2 $input->{file2}"; } else { $inputfn = $input->{file1}; } # Create object that will help us sanity-check alignments my $ac = AlignmentCheck->new( "Sim.pm alignment checker", # name [ $fa ], # fasta "sam", # SAM-formatted alignments 0, # no bis-C 0 # no bis-CpG ); $ac->nrefs() > 0 || mydie("No references"); # Run normal (non-debug) Bowtie defined($conf->{tempdir}) || mydie("No tmp dir"); my $als = "$conf->{tempdir}/Sim.pm.$conf->{randstr}.als"; my $als_debug = "$conf->{tempdir}/Sim.pm.$conf->{randstr}.debug.als"; my $als_px = "$conf->{tempdir}/Sim.pm.$conf->{randstr}.px.als"; my $als_px_reord = "$conf->{tempdir}/Sim.pm.$conf->{randstr}.px.reord.als"; my $cmd = "$conf->{bowtie2_debug} $argstr $idx $inputfn"; print "$cmd\n"; open(ALSDEB, ">$als_debug") || mydie("Could not open '$als_debug' for writing"); open(ALSDEBCMD, "$cmd |") || mydie("Could not open pipe '$cmd |'"); my $ival = 50; my $nals = 0; my @lines = (); while() { # Remove @PG line because CL: tag can legitimately differ print ALSDEB $_ unless /^\@PG/; push @lines, $_; $nals++; print STDERR " Read $nals alignments...\n" if ($nals % $ival) == 0; } close(ALSDEBCMD); $ac->checkAlignments(\@lines, 0); $? == 0 || mydie("bowtie2-align-debug exited with exitlevel $?:\n$cmd\n"); close(ALSDEB); $ac->printSummary(); # With some probability, also run debug Bowtie and check that # results are identical if(int(rand(3)) == 0) { print STDERR "ALSO checking that bowtie2 and bowtie2-align-debug match up\n"; # Remove @PG line because CL: tag can legitimately differ $cmd = "$conf->{bowtie2} $argstr $idx $inputfn | grep -v '^\@PG' > $als"; print "$cmd\n"; system($cmd); $? == 0 || mydie("Command '$cmd' failed with exitlevel $?"); $cmd = "diff -uw $als $als_debug"; print "$cmd\n"; system($cmd); $? == 0 || mydie("diff found a difference between bowtie2 and bowtie2-align-debug ". "output for same input (above)\n"); } # With some probability, also run debug Bowtie in -p X mode with X > 1 and # without the --reorder argument and check that results are identical if(int(rand(3)) == 0) { print STDERR "ALSO checking that bowtie2 and bowtie2 -p X w/ X > 1 match up\n"; my $p = int(rand(3))+2; $cmd = "$conf->{bowtie2} $argstr -p $p $idx $inputfn | grep -v '^\@PG' > $als_px"; print "$cmd\n"; system($cmd); $? == 0 || mydie("Command '$cmd' failed with exitlevel $?"); # Sort the $als_px and $als_debug files to guarantee that reads and # alignments for a given read appear in the same order in both $cmd = "sort -k 1,1 -n -k 2,2 -k 3,3 -k 4,4 < $als_px | grep -v '^\@PG' > $als_px.sorted"; print "$cmd\n"; system($cmd); $? == 0 || mydie("Failed to sort alignment file $als_px\n"); # Sort the $als_px and $als_debug files to guarantee that reads and # alignments for a given read appear in the same order in both $cmd = "sort -k 1,1 -n -k 2,2 -k 3,3 -k 4,4 < $als_debug | grep -v '^\@PG' > $als_debug.sorted"; print "$cmd\n"; system($cmd); $? == 0 || mydie("Failed to sort alignment file $als_debug\n"); $cmd = "diff -uw $als_debug.sorted $als_px.sorted"; print "$cmd\n"; system($cmd); $? == 0 || mydie("diff found a difference between bowtie2-align-debug and bowtie2 ". "-p output for same input (above)\n"); } # With some probability, also run debug Bowtie in -p X mode with X > 1 and # with the --reorder argument and check that results are identical if(int(rand(3)) == 0) { print STDERR "ALSO checking that bowtie2 and bowtie2 -p X --reorder w/ X > 1 match up\n"; my $p = int(rand(3))+2; $cmd = "$conf->{bowtie2} $argstr -p $p $idx --reorder $inputfn | grep -v '^\@PG' > $als_px_reord"; print "$cmd\n"; system($cmd); $? == 0 || mydie("Command '$cmd' failed with exitlevel $?"); $cmd = "diff -uw $als_debug $als_px_reord"; print "$cmd\n"; system($cmd); $? == 0 || mydie("diff found a difference between bowtie2-align-debug and bowtie2 ". "-p --reorder output for same input (above)\n"); } } ## # Generate a new test case # # Possible key/value pairs in $conf hash: # # 1. bowtie2_build: path to bowtie2-build binary # 2. bowtie2: path to bowtie2 binary # 3. bowtie2_build_debug: path to bowtie2-build-debug binary # 4. bowtie2_debug: path to bowtie2-debug binary # 5. tempdir: temporary directory for reference/reads/index # 6. no_paired: defined & non-0 -> don't generate paired-end datasets # 7. no_color: defined & non-0 -> don't generate colorspace datasets # 8. single_thread: defined & non-0 -> don't use -p X where X > 1 # sub nextCase { my ($self, $conf) = @_; $conf->{bowtie2_build} = "bowtie2-build" unless defined($conf->{bowtie2_build}); $conf->{bowtie2} = "bowtie2-align" unless defined($conf->{bowtie2}); $conf->{bowtie2_build_debug} = "bowtie2-build-debug" unless defined($conf->{bowtie2_build_debug}); $conf->{bowtie2_debug} = "bowtie2-align-debug" unless defined($conf->{bowtie2_debug}); $conf->{tempdir} = "/tmp" unless defined($conf->{tempdir}); srand(time ^ $$); $conf->{randstr} = randStr(8); print "*** TEST CASE ***\n"; # Generate the references my $refdnagen = $self->genDNAgen(); # Generate references and write them to a temporary fasta file my $tmpfn = "$conf->{tempdir}/Sim.pm.$conf->{randstr}.fa"; my %refs = (); $self->genRef(\%refs, $refdnagen, $conf, $tmpfn); # Run bowtie2-build my $tmpidxfn = "$conf->{tempdir}/Sim.pm.$conf->{randstr}"; my $buildArgs = $self->genBuildArgs(); $self->build($tmpfn, $tmpidxfn, $conf, $buildArgs); my $numruns = 10; $numruns *= 10 if $conf->{small}; # Lots of short runs # For each batch of reads / bowtie options for(1..$numruns) { print "*** Run $_ of $numruns\n"; # Generate mutated version of the reference as our subject genome my $subj = $self->mutate(\%refs); # Generate all the input, including reads, pairedness, # fragment information, whether it's colorspace, etc my $input = $self->genInput($subj, $conf); # Mutate the input my $mutinput = $self->mutateSeq($input); # Select Bowtie arguments my $args = $self->genAlignArgs($mutinput, $input->{color}, $conf); $self->align($tmpfn, $tmpidxfn, $mutinput, $conf, $args); # Sanity check output. Possible sanity checks are: # 1. Check alignments & edits against reference # 2. Compare bowtie2 and bowtie2-debug # 3. Compare -p X>1 and -p 1 } } if($0 =~ /Sim\.pm$/) { print "Running unit tests\n"; # Run unit tests } 1; hisat2-2.2.1/scripts/sim/Test.pm000066400000000000000000000021131370663763200164540ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # package Test; use strict; use Carp; sub test($$) { my ($name, $f) = @_; print "Test \"$name\"..."; $f->(); print "PASSED\n"; } sub shouldSucceed($$) { my ($name, $f) = @_; print "Test \"$name\"..."; $f->() || die ""; print "PASSED\n"; } sub shouldFail($$) { my ($name, $f) = @_; print "Test \"$name\"..."; $f->() && die ""; print "PASSED\n"; } 1; hisat2-2.2.1/scripts/sim/contrib/000077500000000000000000000000001370663763200166425ustar00rootroot00000000000000hisat2-2.2.1/scripts/sim/contrib/ForkManager.pm000066400000000000000000000261751370663763200214070ustar00rootroot00000000000000=head1 NAME Parallel::ForkManager - A simple parallel processing fork manager =head1 SYNOPSIS use Parallel::ForkManager; $pm = new Parallel::ForkManager($MAX_PROCESSES); foreach $data (@all_data) { # Forks and returns the pid for the child: my $pid = $pm->start and next; ... do some work with $data in the child process ... $pm->finish; # Terminates the child process } =head1 DESCRIPTION This module is intended for use in operations that can be done in parallel where the number of processes to be forked off should be limited. Typical use is a downloader which will be retrieving hundreds/thousands of files. The code for a downloader would look something like this: use LWP::Simple; use Parallel::ForkManager; ... @links=( ["http://www.foo.bar/rulez.data","rulez_data.txt"], ["http://new.host/more_data.doc","more_data.doc"], ... ); ... # Max 30 processes for parallel download my $pm = new Parallel::ForkManager(30); foreach my $linkarray (@links) { $pm->start and next; # do the fork my ($link,$fn) = @$linkarray; warn "Cannot get $fn from $link" if getstore($link,$fn) != RC_OK; $pm->finish; # do the exit in the child process } $pm->wait_all_children; First you need to instantiate the ForkManager with the "new" constructor. You must specify the maximum number of processes to be created. If you specify 0, then NO fork will be done; this is good for debugging purposes. Next, use $pm->start to do the fork. $pm returns 0 for the child process, and child pid for the parent process (see also L). The "and next" skips the internal loop in the parent process. NOTE: $pm->start dies if the fork fails. $pm->finish terminates the child process (assuming a fork was done in the "start"). NOTE: You cannot use $pm->start if you are already in the child process. If you want to manage another set of subprocesses in the child process, you must instantiate another Parallel::ForkManager object! =head1 METHODS =over 5 =item new $processes Instantiate a new Parallel::ForkManager object. You must specify the maximum number of children to fork off. If you specify 0 (zero), then no children will be forked. This is intended for debugging purposes. =item start [ $process_identifier ] This method does the fork. It returns the pid of the child process for the parent, and 0 for the child process. If the $processes parameter for the constructor is 0 then, assuming you're in the child process, $pm->start simply returns 0. An optional $process_identifier can be provided to this method... It is used by the "run_on_finish" callback (see CALLBACKS) for identifying the finished process. =item finish [ $exit_code ] Closes the child process by exiting and accepts an optional exit code (default exit code is 0) which can be retrieved in the parent via callback. If you use the program in debug mode ($processes == 0), this method doesn't do anything. =item set_max_procs $processes Allows you to set a new maximum number of children to maintain. Returns the previous setting. =item wait_all_children You can call this method to wait for all the processes which have been forked. This is a blocking wait. =back =head1 CALLBACKS You can define callbacks in the code, which are called on events like starting a process or upon finish. The callbacks can be defined with the following methods: =over 4 =item run_on_finish $code [, $pid ] You can define a subroutine which is called when a child is terminated. It is called in the parent process. The paremeters of the $code are the following: - pid of the process, which is terminated - exit code of the program - identification of the process (if provided in the "start" method) - exit signal (0-127: signal name) - core dump (1 if there was core dump at exit) =item run_on_start $code You can define a subroutine which is called when a child is started. It called after the successful startup of a child in the parent process. The parameters of the $code are the following: - pid of the process which has been started - identification of the process (if provided in the "start" method) =item run_on_wait $code, [$period] You can define a subroutine which is called when the child process needs to wait for the startup. If $period is not defined, then one call is done per child. If $period is defined, then $code is called periodically and the module waits for $period seconds betwen the two calls. Note, $period can be fractional number also. The exact "$period seconds" is not guarranteed, signals can shorten and the process scheduler can make it longer (on busy systems). The $code called in the "start" and the "wait_all_children" method also. No parameters are passed to the $code on the call. =back =head1 EXAMPLE =head2 Parallel get This small example can be used to get URLs in parallel. use Parallel::ForkManager; use LWP::Simple; my $pm=new Parallel::ForkManager(10); for my $link (@ARGV) { $pm->start and next; my ($fn)= $link =~ /^.*\/(.*?)$/; if (!$fn) { warn "Cannot determine filename from $fn\n"; } else { $0.=" ".$fn; print "Getting $fn from $link\n"; my $rc=getstore($link,$fn); print "$link downloaded. response code: $rc\n"; }; $pm->finish; }; =head2 Callbacks Example of a program using callbacks to get child exit codes: use strict; use Parallel::ForkManager; my $max_procs = 5; my @names = qw( Fred Jim Lily Steve Jessica Bob Dave Christine Rico Sara ); # hash to resolve PID's back to child specific information my $pm = new Parallel::ForkManager($max_procs); # Setup a callback for when a child finishes up so we can # get it's exit code $pm->run_on_finish( sub { my ($pid, $exit_code, $ident) = @_; print "** $ident just got out of the pool ". "with PID $pid and exit code: $exit_code\n"; } ); $pm->run_on_start( sub { my ($pid,$ident)=@_; print "** $ident started, pid: $pid\n"; } ); $pm->run_on_wait( sub { print "** Have to wait for one children ...\n" }, 0.5 ); foreach my $child ( 0 .. $#names ) { my $pid = $pm->start($names[$child]) and next; # This code is the child process print "This is $names[$child], Child number $child\n"; sleep ( 2 * $child ); print "$names[$child], Child $child is about to get out...\n"; sleep 1; $pm->finish($child); # pass an exit code to finish } print "Waiting for Children...\n"; $pm->wait_all_children; print "Everybody is out of the pool!\n"; =head1 BUGS AND LIMITATIONS Do not use Parallel::ForkManager in an environment, where other child processes can affect the run of the main program, so using this module is not recommended in an environment where fork() / wait() is already used. If you want to use more than one copies of the Parallel::ForkManager, then you have to make sure that all children processes are terminated, before you use the second object in the main program. You are free to use a new copy of Parallel::ForkManager in the child processes, although I don't think it makes sense. =head1 COPYRIGHT Copyright (c) 2000 Szab, Balzs (dLux) All right reserved. This program is free software; you can redistribute it and/or modify it under the same terms as Perl itself. =head1 AUTHOR dLux (Szab, Balzs) =head1 CREDITS Noah Robin (documentation tweaks) Chuck Hirstius (callback exit status, example) Grant Hopwood (win32 port) Mark Southern (bugfix) =cut package Parallel::ForkManager; use POSIX ":sys_wait_h"; use strict; use vars qw($VERSION); $VERSION='0.7.5'; sub new { my ($c,$processes)=@_; my $h={ max_proc => $processes, processes => {}, in_child => 0, }; return bless($h,ref($c)||$c); }; sub start { my ($s,$identification)=@_; die "Cannot start another process while you are in the child process" if $s->{in_child}; while ($s->{max_proc} && ( keys %{ $s->{processes} } ) >= $s->{max_proc}) { $s->on_wait; $s->wait_one_child(defined $s->{on_wait_period} ? &WNOHANG : undef); }; $s->wait_children; if ($s->{max_proc}) { my $pid=fork(); die "Cannot fork: $!" if !defined $pid; if ($pid) { $s->{processes}->{$pid}=$identification; $s->on_start($pid,$identification); } else { $s->{in_child}=1 if !$pid; } return $pid; } else { $s->{processes}->{$$}=$identification; $s->on_start($$,$identification); return 0; # Simulating the child which returns 0 } } sub finish { my ($s, $x)=@_; if ( $s->{in_child} ) { exit ($x || 0); } if ($s->{max_proc} == 0) { # max_proc == 0 $s->on_finish($$, $x ,$s->{processes}->{$$}, 0, 0); delete $s->{processes}->{$$}; } return 0; } sub wait_children { my ($s)=@_; return if !keys %{$s->{processes}}; my $kid; do { $kid = $s->wait_one_child(&WNOHANG); } while $kid > 0 || $kid < -1; # AS 5.6/Win32 returns negative PIDs }; sub wait_one_child { my ($s,$par)=@_; my $kid; while (1) { $kid = $s->_waitpid(-1,$par||=0); last if $kid == 0 || $kid == -1; # AS 5.6/Win32 returns negative PIDs redo if !exists $s->{processes}->{$kid}; my $id = delete $s->{processes}->{$kid}; $s->on_finish( $kid, $? >> 8 , $id, $? & 0x7f, $? & 0x80 ? 1 : 0); last; } $kid; }; sub wait_all_children { my ($s)=@_; while (keys %{ $s->{processes} }) { $s->on_wait; $s->wait_one_child(defined $s->{on_wait_period} ? &WNOHANG : undef); }; } sub run_on_finish { my ($s,$code,$pid)=@_; $s->{on_finish}->{$pid || 0}=$code; } sub on_finish { my ($s,$pid,@par)=@_; my $code=$s->{on_finish}->{$pid} || $s->{on_finish}->{0} or return 0; $code->($pid,@par); }; sub run_on_wait { my ($s,$code, $period)=@_; $s->{on_wait}=$code; $s->{on_wait_period} = $period; } sub on_wait { my ($s)=@_; if(ref($s->{on_wait}) eq 'CODE') { $s->{on_wait}->(); if (defined $s->{on_wait_period}) { local $SIG{CHLD} = sub { } if ! defined $SIG{CHLD}; select undef, undef, undef, $s->{on_wait_period} }; }; }; sub run_on_start { my ($s,$code)=@_; $s->{on_start}=$code; } sub on_start { my ($s,@par)=@_; $s->{on_start}->(@par) if ref($s->{on_start}) eq 'CODE'; }; sub set_max_procs { my ($s, $mp)=@_; $s->{max_proc} = $mp; } # OS dependant code follows... sub _waitpid { # Call waitpid() in the standard Unix fashion. return waitpid($_[1],$_[2]); } # On ActiveState Perl 5.6/Win32 build 625, waitpid(-1, &WNOHANG) always # blocks unless an actual PID other than -1 is given. sub _NT_waitpid { my ($s, $pid, $par) = @_; if ($par == &WNOHANG) { # Need to nonblock on each of our PIDs in the pool. my @pids = keys %{ $s->{processes} }; # Simulate -1 (no processes awaiting cleanup.) return -1 unless scalar(@pids); # Check each PID in the pool. my $kid; foreach $pid (@pids) { $kid = waitpid($pid, $par); return $kid if $kid != 0; # AS 5.6/Win32 returns negative PIDs. } return $kid; } else { # Normal waitpid() call. return waitpid($pid, $par); } } { local $^W = 0; if ($^O eq 'NT' or $^O eq 'MSWin32') { *_waitpid = \&_NT_waitpid; } } 1; hisat2-2.2.1/scripts/sim/run.pl000066400000000000000000000075701370663763200163540ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # use strict; use warnings; use Getopt::Long; use FindBin qw($Bin); use lib "$Bin"; use lib "$Bin/contrib"; use Sim; use ForkManager; # Simulator configuration my %conf = ( bowtie2_build => "bowtie2-build", bowtie2 => "bowtie2", bowtie2_build_debug => "bowtie2-build-debug", bowtie2_debug => "bowtie2-debug", tempdir => "/tmp", no_color => 1, small => 1 ); # Number of parallel processes to use my $cpus = 1; my $usage = qq! run.pl [options*] Options: --bowtie2 Path to bowtie2 release binary --bowtie2-debug Path to bowtie2 debug binary --bowtie2-build Path to bowtie2-build release binary --bowtie2-build-debug Path to bowtie2-build debug binary --tempdir Put temporary files here --cases Each thread runs around cases (def: 5) --cpus / -p Run test cases in threads at once --maxreads Handle at most reads per case --numrefs Generate refs per case --die-with-child Kill parent as soon as 1 child dies --no-die-with-child Don\'t kill parent as soon as 1 child dies --small Make small test cases --help Print this usage message !; my $help = 0; my $ncases = 5; my $dieWithChild = 1; GetOptions( "bowtie2=s" => \$conf{bowtie2}, "bowtie2-debug=s" => \$conf{bowtie2_debug}, "bowtie2-build=s" => \$conf{bowtie2_build}, "bowtie2-build-debug=s" => \$conf{bowtie2_build_debug}, "tempdir|tmpdir=s" => \$conf{tempdir}, "cases-per-thread=i" => \$ncases, "small" => \$conf{small}, "large" => sub { $conf{small} = 0 }, "no-paired" => \$conf{no_paired}, "color" => sub { $conf{no_color} = 0 }, "no-color" => \$conf{no_color}, "help" => \$help, "die-with-child" => \$dieWithChild, "no-die-with-child" => sub { $dieWithChild = 0 }, "p|cpus=i" => \$cpus, "u|qupto|maxreads=i" => \$conf{maxreads}, "numrefs|num-refs=i" => \$conf{numrefs}, ) || die "Bad options;"; if($help) { print $usage; exit 0; } my $sim = Sim->new(); my $pm = new Parallel::ForkManager($cpus); # Callback for when a child finishes so we can get its exit code my @childFailed = (); my @childFailedPid = (); $pm->run_on_finish(sub { my ($pid, $exit_code, $ident) = @_; if($exit_code != 0) { push @childFailed, $exit_code; push @childFailedPid, $pid; !$dieWithChild || die "Dying with child with PID $pid"; } }); my $totcases = $ncases * $cpus; for(1..$totcases) { my $childPid = $pm->start; if($childPid != 0) { next; # spawn the next child } $sim->nextCase(\%conf); $pm->finish; } $pm->wait_all_children; for(@childFailedPid) { print STDERR "Error message from child with pid $_:\n"; my $fn = ".run.pl.child.$_"; if(open(ER, $fn)) { print STDERR "---------\n"; while() { print STDERR $_; } print STDERR "---------\n"; close(ER); } else { print STDERR "(could not open $fn)\n"; } } print STDERR "PASSED\n"; hisat2-2.2.1/scripts/sim/run.sh000066400000000000000000000020021370663763200163340ustar00rootroot00000000000000#!/bin/sh # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # CPUS=$1 shift make -j $CPUS bowtie2-align bowtie2-align-debug bowtie2-build bowtie2-build-debug && \ perl scripts/sim/run.pl \ --bowtie2=./bowtie2-align \ --bowtie2-debug=./bowtie2-align-debug \ --bowtie2-build=./bowtie2-build \ --bowtie2-build-debug=./bowtie2-build-debug \ --cpus=$CPUS \ $* hisat2-2.2.1/scripts/sim/unit.sh000066400000000000000000000014411370663763200165150ustar00rootroot00000000000000#!/bin/sh # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # d=`dirname $0` for i in `ls $d/*.pm` ; do echo $i ; perl $i --test ; done hisat2-2.2.1/scripts/test/000077500000000000000000000000001370663763200153715ustar00rootroot00000000000000hisat2-2.2.1/scripts/test/DNA.pm000066400000000000000000000047301370663763200163350ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # package DNA; use strict; use warnings; use Carp; my %revcompMap = ( "A" => "T", "a" => "t", "T" => "A", "t" => "a", "C" => "G", "c" => "g", "G" => "C", "g" => "c", "R" => "Y", "r" => "y", "Y" => "R", "y" => "r", "M" => "K", "m" => "k", "K" => "M", "k" => "m", "S" => "S", "s" => "s", "W" => "W", "w" => "w", "B" => "V", "b" => "v", "V" => "B", "v" => "b", "H" => "D", "h" => "d", "D" => "H", "d" => "h", "N" => "N", "n" => "n" ); my %compat = ( "A" => "A", "T" => "T", "C" => "C", "G" => "G", "R" => "AG", "Y" => "CT", "M" => "AC", "K" => "GT", "S" => "CG", "W" => "AT", "B" => "CGT", "V" => "ACG", "H" => "ACT", "D" => "AGT", "N" => "N" ); my %incompat = ( "A" => "CGT", "T" => "ACG", "C" => "AGT", "G" => "ACT", "R" => "CT", "Y" => "AG", "M" => "GT", "K" => "AC", "S" => "AT", "W" => "CG", "B" => "A", "V" => "T", "H" => "G", "D" => "C", "N" => "N" ); my %unambigSet = ( "A" => 1, "a" => 1, "C" => 1, "c" => 1, "G" => 1, "g" => 1, "T" => 1, "t" => 1 ); ## # Return the complement, incl. if it's IUPAC. # sub comp($) { my $ret = $revcompMap{$_[0]} || die "Can't reverse-complement '$_[0]'"; return $ret; } ## # Return the complement, incl. if it's IUPAC. # sub revcomp { my ($ret, $color) = @_; $ret = reverse $ret; unless($color) { for(my $i = 0; $i < length($ret); $i++) { substr($ret, $i, 1) = comp(substr($ret, $i, 1)); } } return $ret; } ## # Return true iff it's unambiguous. # sub unambig($) { return $unambigSet{$_[0]}; } ## # Manipulate DNA in an integer-indexed fashion. # sub plus($$) { my ($c, $amt) = @_; my %ctoi = ("A" => 0, "C" => 1, "G" => 2, "T" => 3); my %itoc = (0 => "A", 1 => "C", 2 => "G", 3 => "T"); $c = uc $c; defined($ctoi{$c}) || die; return $itoc{($ctoi{$c}+$amt) % 4}; } 1; hisat2-2.2.1/scripts/test/Read.pm000066400000000000000000000116131370663763200166040ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # package Read; use strict; use Carp; use FindBin qw($Bin); use lib $Bin; use DNA; use Test; sub new { my ($class, $name, $seq, $qual, $color, $fw, $orig) = @_; $name = "noname" unless defined($name); return bless { _name => $name, _seq => $seq || croak("No sequence"), _qual => $qual || croak("No qualities"), _color => $color || 0, _fw => $fw || croak("No orientation"), _orig => $orig || croak("No original read string") }, $class; } sub name { return $_[0]->{_name} } sub seq { return $_[0]->{_seq} } sub qual { return $_[0]->{_qual} } sub color { return $_[0]->{_color} } sub fw { return $_[0]->{_fw} } sub orig { return $_[0]->{_orig} } sub len { return length($_[0]->seq()) } ## # Obtain a character from the read. # sub at { my ($self, $off, $ori) = @_; my ($c, $q) = ""; if($ori eq "RtL") { $c = uc substr($self->seq(), -$off-1, 1); $q = substr($self->qual(), -$off-1, 1); } else { $c = uc substr($self->seq(), $off, 1); $q = substr($self->qual(), $off, 1); } length($c) == 1 || die; return ($c, $q); } ## # Load a set of FASTQ reads into the given reads array. # sub fromFastq { my ($fh, $color, $reads) = @_; $reads = [] unless defined($reads); while(<$fh>) { my $l1 = $_; my $l2 = <$fh>; defined($l2) || croak("Name line followed by EOF"); my $l3 = <$fh>; defined($l3) || croak("Sequence line followed by EOF"); my $l4 = <$fh>; defined($l4) || croak("Name2 line followed by EOF"); my $orig = "$l1$l2$l3$l4"; chomp($l1); chomp($l2); chomp($l3); chomp($l4); push @{$reads}, Read->new(substr($l1, 1), $l2, $l4, $color, "FW", $orig); } return $reads; } ## # Load a set of FASTQ reads into the given reads array. # sub fromFastqs { my ($fqs, $color, $reads) = @_; $reads = [] unless defined($reads); for my $f (@$fqs) { my $fqfh; open($fqfh, $f =~ /\.gz$/ ? "gzip -dc $f |" : "$f") || croak("Could not open $f for reading"); fromFastq($fqfh, $color, $reads); close($fqfh); } return $reads; } ## # Load a set of FASTA reads into the given reads array. # sub fromFasta { my ($fh, $color, $reads) = @_; $reads = [] unless defined($reads); while(<$fh>) { my $l1 = $_; my $l2 = <$fh>; defined($l2) || croak("Name line followed by EOF"); my $orig = "$l1$l2"; chomp($l1); chomp($l2); my $qual = "I" x length($l2); push @{$reads}, Read->new(substr($l1, 1), $l2, $qual, $color, "FW", $orig); } return $reads; } ## # Load a set of FASTA reads into the given reads array. # sub fromFastas { my ($fas, $color, $reads) = @_; $reads = [] unless defined($reads); for my $f (@$fas) { my $fafh; open($fafh, $f =~ /\.gz$/ ? "gzip -dc $f |" : "$f") || croak("Could not open $f for reading"); fromFasta($fafh, $color, $reads); close($fafh); } return $reads; } ## # Load a set of FASTA reads into the given reads array. # sub fromStrings { my ($strs, $color, $reads) = @_; $reads = [] unless defined($reads); my $idx = 0; for my $str (@$strs) { my $qual = "I" x length($str); push @{$reads}, new Read($idx, $str, $qual, $color, "FW", $str); $idx++; } return $reads; } sub test1 { my $r = new Read("blah", "TTACGAACCACAACGTATCG", "I"x20, 0, "FW", "?"); my ($c, $q) = $r->at(0, "LtR"); ($c eq "T" && $q eq "I") || croak("Expected (T, I), got ($c, $q)\n"); ($c, $q) = $r->at(0, "RtL"); ($c eq "G" && $q eq "I") || croak("Expected (G, I), got ($c, $q)\n"); ($c, $q) = $r->at(1, "LtR"); ($c eq "T" && $q eq "I") || croak("Expected (T, I), got ($c, $q)\n"); ($c, $q) = $r->at(1, "RtL"); ($c eq "C" && $q eq "I") || croak("Expected (C, I), got ($c, $q)\n"); return 1; } sub test2 { my $rs = fromStrings(["ACGATGCTACG", "TGACGATGCTAG"], 0); $rs->[0]->seq() eq "ACGATGCTACG" || croak($rs->[0]->seq()); $rs->[0]->qual() eq "IIIIIIIIIII" || croak($rs->[0]->qual()); $rs->[0]->name() eq "0" || croak($rs->[0]->name()); $rs->[1]->seq() eq "TGACGATGCTAG" || croak($rs->[1]->seq()); $rs->[1]->qual() eq "IIIIIIIIIIII" || croak($rs->[1]->qual()); $rs->[1]->name() eq "1" || croak($rs->[1]->name()); return 1; } if($0 =~ /Read\.pm$/) { print "Running unit tests\n"; # Run unit tests print "Test \"test1\"..."; test1(); print "PASSED\n"; print "Test \"test2\"..."; test2(); print "PASSED\n"; } 1; hisat2-2.2.1/scripts/test/args.pl000066400000000000000000000075531370663763200166740ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # ## # args.pl # # Basic tests to ensure that bad combinations of arguments are rejected # and good ones are accepted. # my $bowtie2 = "./bowtie2"; my $bowtie2_d = "./bowtie2-debug"; if(system("$bowtie2 --version") != 0) { print STDERR "Could not execute ./bowtie2; looking in PATH...\n"; $bowtie2 = `which bowtie2`; chomp($bowtie2); if(system("$bowtie2 --version") != 0) { die "Could not find bowtie2 in current directory or in PATH\n"; } } if(system("$bowtie2_d --version") != 0) { print STDERR "Could not execute ./bowtie2-debug; looking in PATH...\n"; $bowtie2_d = `which bowtie2-debug`; chomp($bowtie2_d); if(system("$bowtie2_d --version") != 0) { die "Could not find bowtie2-debug in current directory or in PATH\n"; } } if(! -f "e_coli_c.1.ebwt") { print STDERR "Making colorspace e_coli index\n"; my $bowtie2_build = "./bowtie2-build"; if(system("$bowtie2_build --version") != 0) { print STDERR "Could not execute ./bowtie2-build; looking in PATH...\n"; $bowtie2_build = `which $bowtie2_build`; chomp($bowtie2_build); if(system("$bowtie2_build --version") != 0) { die "Could not find bowtie2-build in current directory or in PATH\n"; } } system("$bowtie2_build -C genomes/NC_008253.fna e_coli_c") && die; } else { print STDERR "Colorspace e_coli index already present...\n"; } open TMP, ">.args.pl.1.fa" || die; print TMP ">\nT0120012002012030303023\n"; close(TMP); open TMP, ">.args.pl.1.qv" || die; print TMP ">\n10 11 12 10 10 11 12 10 10 12 10 22 33 23 13 10 12 23 24 25 26 27\n"; close(TMP); open TMP, ">.args.pl.2.qv" || die; print TMP ">\n9 10 11 12 10 10 11 12 10 10 12 10 22 33 23 13 10 12 23 24 25 26 27\n"; close(TMP); my @bad = ( "-N 6", "-N 5", "-N 4", "-N 3" ); my @badEx = ( "e_coli -f .args.pl.1.fa -Q .args.pl.1.qv", "e_coli_c -f .args.pl.1.fa -Q .args.pl.1.qv" ); my @good = ( "-N 0", "-N 1", "-N 2" ); my @goodEx = ( "-C e_coli_c -f .args.pl.1.fa -Q .args.pl.1.qv", "-C e_coli_c -f .args.pl.1.fa -Q .args.pl.2.qv" ); sub run($) { my $cmd = shift; print "$cmd\n"; return system($cmd); } print "Bad:\n"; for my $a (@bad) { run("$bowtie2 $a e_coli reads/e_coli_1000.fq /dev/null") != 0 || die "bowtie2 should have rejected: \"$a\"\n"; run("$bowtie2_d $a e_coli reads/e_coli_1000.fq /dev/null") != 0 || die "bowtie2-debug should have rejected: \"$a\"\n"; print "PASSED: bad args \"$a\"\n"; } print "\nBadEx:\n"; for my $a (@badEx) { run("$bowtie2 $a /dev/null") != 0 || die "bowtie2 should have rejected: \"$a\"\n"; run("$bowtie2_d $a /dev/null") != 0 || die "bowtie2-debug should have rejected: \"$a\"\n"; print "PASSED: bad args \"$a\"\n"; } print "\nGood:\n"; for my $a (@good) { run("$bowtie2 $a e_coli reads/e_coli_1000.fq /dev/null") == 0 || die "bowtie2 should have accepted: \"$a\"\n"; run("$bowtie2_d $a e_coli reads/e_coli_1000.fq /dev/null") == 0 || die "bowtie2-debug should have accepted: \"$a\"\n"; print "PASSED: good args \"$a\"\n"; } print "\nGoodEx:\n"; for my $a (@goodEx) { run("$bowtie2 $a /dev/null") == 0 || die "bowtie2 should have accepted: \"$a\"\n"; run("$bowtie2_d $a /dev/null") == 0 || die "bowtie2-debug should have accepted: \"$a\"\n"; print "PASSED: good args \"$a\"\n"; } hisat2-2.2.1/scripts/test/simple_tests.pl000066400000000000000000010540331370663763200204470ustar00rootroot00000000000000#!/usr/bin/perl -w # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # ## # Give simple tests with known results to bowtie2. # use strict; use warnings; use Getopt::Long; use FindBin qw($Bin); use lib $Bin; use List::Util qw(max min); use Data::Dumper; use DNA; use Clone qw(clone); use Test::Deep; my $bowtie2 = ""; my $bowtie2_build = ""; my $skipColor = 1; GetOptions( "bowtie2=s" => \$bowtie2, "bowtie2-build=s" => \$bowtie2_build, "skip-color" => \$skipColor) || die "Bad options"; if(! -x $bowtie2 || ! -x $bowtie2_build) { my $bowtie2_dir = `dirname $bowtie2`; my $bowtie2_exe = `basename $bowtie2`; my $bowtie2_build_exe = `basename $bowtie2_build`; chomp($bowtie2_dir); chomp($bowtie2_exe); chomp($bowtie2_build_exe); system("make -C $bowtie2_dir $bowtie2_exe $bowtie2_build_exe") && die; } (-x $bowtie2) || die "Cannot run '$bowtie2'"; (-x $bowtie2_build) || die "Cannot run '$bowtie2_build'"; my @cases = ( { name => "Left-align insertion", ref => [ "GCGATATCTACGACTGCTACGTACAAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGATCTGACAGC" ], norc => 1, reads => [ "ACAAAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGA" ], # ref: AC-AAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGA # read: ACAAAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGA # 0123456789012345678901234567890123456789 cigar => [ "2M1I40M" ], samoptflags => [ { "MD:Z:42" => 1, "YT:Z:UU" => 1, "NM:i:1" => 1, "XG:i:1" => 1, "XO:i:1" => 1, "AS:i:-8" => 1 } ], report => "", args => "" }, { name => "Left-align deletion", ref => [ "GCGATATCTACGACTGCTACGTACAAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGATCTGACAGC" ], norc => 1, reads => [ "ACGTACAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGA" ], # ref: ACGTACAAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGA # read: ACGTAC-AAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGA # 012345678901234567890123456789012345678 cigar => [ "6M1D39M" ], samoptflags => [ { "MD:Z:6^A39" => 1, "YT:Z:UU" => 1, "NM:i:1" => 1, "XG:i:1" => 1, "XO:i:1" => 1, "AS:i:-8" => 1 } ], report => "", args => "" }, { name => "Left-align insertion with mismatch at LHS", ref => [ "GCGATATCTACGACTGCTACGCCCAAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGATCTGACAGC" ], norc => 1, reads => [ "TATCTACGACTGCTACGCCCTAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGATCTGAC" ], # ref: GCGATATCTACGACTGCTACGCCCAAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGATCTGACAGC # read: TATCTACGACTGCTACGCCC-TAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGATCTGAC # 01234567890123456789-012345678901234567890123456789012345678901234 # 0 1 2 3 4 5 6 cigar => [ "20M1D45M" ], samoptflags => [ { "MD:Z:20^A0A44" => 1, "YT:Z:UU" => 1, "NM:i:2" => 1, "XG:i:1" => 1, "XO:i:1" => 1, "XM:i:1" => 1, "AS:i:-14" => 1 } ], report => "", args => "" }, # This won't necessarily pass because the original location of the deletion # might #{ name => "Left-align deletion with mismatch at LHS", # ref => [ "GCGATATCTACGACTGCTACGCCCAAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGATCTGACAGC" ], # norc => 1, # reads => [ "TATCTACGACTGCTACGCCAAAAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGATCTGAC" ], # # ref: GCGATATCTACGACTGCTACGCCC-AAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGATCTGACAGC # # read: TATCTACGACTGCTACGCCAAAAAAAAAAAAAAAAGTGTTTACGTTGCTAGACTCGATCGATCTGAC # # 01234567890123456789-012345678901234567890123456789012345678901234 # # 0 1 0 1 2 3 4 # cigar => [ "20M1I45M" ], # samoptflags => [ { # "MD:Z:19C45" => 1, # "YT:Z:UU" => 1, # "NM:i:2" => 1, # "XG:i:1" => 1, # "XO:i:1" => 1, # "XM:i:1" => 1, # "AS:i:-14" => 1 } ], # report => "", # args => "" #}, { name => "Flags for when mates align non-concordantly, with many alignments for one", # 012345678 ref => [ "CAGCGGCTAGCTATCGATCGTCCGGCAGCTATCATTATGATNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGGATAGATCGCTCGCCTGACCTATATCGCTCGCGATTACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGATCGAGGATAGATCGCTCGCCTGACCTATATCGCTCGCGATTACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGATCGAGGATAGATCGCTCGCCTGACCTATATCGCTCGCGATTACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGATCG" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 # 0 1 2 3 4 5 6 7 8 9 0 1 # 0 1 norc => 1, mate1s => [ "GCGGCTAGCTATCGATCGTCCGGCAGCTATCATTATGA" ], mate2s => [ "ACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGA" ], # 981 1064 1147 # ACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGA ACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGA ACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGA samflags_map => [{ 981 => (1 | 128), 1064 => (1 | 128), 1147 => (1 | 128), 2 => (1 | 64) }], report => "", args => "" }, { name => "Flags for when mates align non-concordantly, with many alignments for one", # 012345678 ref => [ "CAGCGGCTAGCTATCGATCGTCCGGCAGCTATCATTATGATNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGGATAGATCGCTCGCCTGACCTATATCGCTCGCGATTACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGATCGAG" ], # 01234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 # 0 1 2 3 4 5 6 7 8 9 0 1 # 0 1 norc => 1, mate1s => [ "GCGGCTAGCTATCGATCGTCCGGCAGCTATCATTATGA" ], mate2s => [ "ACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGA" ], tlen_map => [{ 2 => 1021, 981 => -1021 }], samflags_map => [{ 981 => (1 | 128), 2 => (1 | 64) }], report => "", args => "" }, { name => "Flags for when mates align non-concordantly, with many alignments for one", # 012345678 ref => [ "CAGCGGCTAGCTATCGATCGTCCGGCAGCTATCATTATGATAGGATAGATCGCTCGCCTGACCTATATCGCTCGCGATTACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGATCGAGGATAGATCGCTCGCCTGACCTATATCGCTCGCGATTACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGATCGAGGATAGATCGCTCGCCTGACCTATATCGCTCGCGATTACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGATCG" ], # 01234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 # 0 * 1 * 2 * # 0 1 norc => 1, mate1s => [ "GCGGCTAGCTATCGATCGTCCGGCAGCTATCATTATGA" ], mate2s => [ "TCGTCGTGATGCGTCAGCTCGGATAGCCAGTACGTAGCTCGT" ], # 981 1064 1147 # ACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGA ACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGA ACGAGCTACGTACTGGCTATCCGAGCTGACGCATCACGACGA samflags_map => [{ 79 => (1 | 2 | 16 | 128), 162 => (1 | 2 | 16 | 128), 245 => (1 | 2 | 16 | 128), 2 => (1 | 2 | 32 | 64) }], report => "", args => "" }, # Checking MD:Z strings for alignment { name => "MD:Z 1", ref => [ "CACGATCGACTTGA"."C"."TCATCGACGCTATCATTAATATATATAAGCCCGCATCTA" ], reads => [ "CACGATCGACTTGG". "TCATCGACGCTATCATTAATATATATAAGCCCGCATCTA" ], hits => [ { 0 => 1 } ], samoptflags => [ { "AS:i:-14" => 1, # alignment score "XM:i:1" => 1, # num mismatches "XO:i:1" => 1, # num gap opens "XG:i:1" => 1, # num gap extensions "NM:i:2" => 1, # num edits "MD:Z:13^A0C39" => 1, # mismatching positions/bases "YT:Z:UU" => 1, # type of alignment (concordant/discordant/etc) } ] }, { name => "MD:Z 2", ref => [ "CACGATCGACTTGA"."A"."TCATCGACGCTATCATTAATATATATAAGCCCGCATCTA" ], reads => [ "CACGATCGACTTGG". "TCATCGACGCTATCATTAATATATATAAGCCCGCATCTA" ], # 0123456789012 012345678901234567890123456789012345678 hits => [ { 0 => 1 } ], samoptflags => [ { "AS:i:-14" => 1, # alignment score "XM:i:1" => 1, # num mismatches "XO:i:1" => 1, # num gap opens "XG:i:1" => 1, # num gap extensions "NM:i:2" => 1, # num edits "MD:Z:13^A0A39" => 1, # mismatching positions/bases "YT:Z:UU" => 1, # type of alignment (concordant/discordant/etc) } ] }, { name => "MD:Z 3", ref => [ "CACGATCGACTTGT"."AA"."TCATCGACGCTATCATTAATATATATAAGCCCGCATCTA" ], reads => [ "CACGATCGACTTGC". "TCATCGACGCTATCATTAATATATATAAGCCCGCATCTA" ], # 0123456789012 012345678901234567890123456789012345678 hits => [ { 0 => 1 } ], samoptflags => [ { "AS:i:-17" => 1, # alignment score "XM:i:1" => 1, # num mismatches "XO:i:1" => 1, # num gap opens "XG:i:2" => 1, # num gap extensions "NM:i:3" => 1, # num edits "MD:Z:13^TA0A39" => 1, # mismatching positions/bases "YT:Z:UU" => 1, # type of alignment (concordant/discordant/etc) } ] }, { name => "MD:Z 4", ref => [ "CACGATCGACTTGN"."NN"."TCATCGACGCTATCATTAATATATATAAGCCCGCATCTA" ], reads => [ "CACGATCGACTTGC". "TCATCGACGCTATCATTAATATATATAAGCCCGCATCTA" ], # 0123456789012 012345678901234567890123456789012345678 hits => [ { 0 => 1 } ], samoptflags => [ { "AS:i:-12" => 1, # alignment score "XN:i:3" => 1, # num ambiguous ref bases "XM:i:1" => 1, # num mismatches "XO:i:1" => 1, # num gap opens "XG:i:2" => 1, # num gap extensions "NM:i:3" => 1, # num edits "MD:Z:13^NN0N39" => 1, # mismatching positions/bases "YT:Z:UU" => 1, # type of alignment (concordant/discordant/etc) } ] }, # # Local alignment # # Local alignment for a short perfect hit where hit spans the read { name => "Local alignment 1", ref => [ "TTGT" ], reads => [ "TTGT" ], args => "--local --policy \"MIN=L,0.0,0.75\"", hits => [ { 0 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:4=" ], cigar => [ "4M" ], samoptflags => [ { "AS:i:8" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:4" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YP:i:0" => 1, # read aligned repetitively in paired fashion "YT:Z:UU" => 1, # type of alignment (concordant/discordant/etc) } ] }, # T T G A T T G A # T x T x # T x T # G x G # T T # Local alignment for a short hit where hit is trimmed at one end { name => "Local alignment 2", ref => [ "TTGA" ], reads => [ "TTGT" ], args => "--local --policy \"MIN=L,0.0,0.75\\;SEED=0,3\\;IVAL=C,1,0\"", report => "-a", hits => [ { 0 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:3=1S" ], cigar => [ "3M1S" ], samoptflags => [ { "AS:i:6" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:3" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YP:i:0" => 1, # read aligned repetitively in paired fashion "YT:Z:UU" => 1, # type of alignment (concordant/discordant/etc) } ] }, # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 # T T G T T C G T T T G T T C G T # 0 T x # 1 T x # 2 G x # 3 T x # 4 T x # 5 C x # 6 G x # 7 T x # 8 T x # 9 T x # 0 G x # 1 T x # 2 T x # # Score=130 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 # T T G T T C G T T T G T T C G T # 0 T x # 1 T x # 2 G x # 3 T x # 4 T x # 5 C x # 6 G x # 7 T x # 8 T # 9 T # 0 G # 1 T # 2 T # # Score=80 # Local alignment for a perfect hit { name => "Local alignment 3", # TTGTTCGT # TTGTTCGT ref => [ "TTGTTCGTTTGTTCGT" ], # 0123456789012345 # TTGTTCGTTTGTT # TTGTTCGT----- reads => [ "TTGTTCGTTTGTT" ], args => "--local -L 8 -i C,1,0 --score-min=C,12", report => "-a", hits => [ { 0 => 1, 8 => 1 } ], flags_map => [{ 0 => "XM:0,XP:0,XT:UU,XC:13=", 8 => "XM:0,XP:0,XT:UU,XC:8=" }], cigar_map => [{ 0 => "13M", 8 => "8M5S" }], samoptflags_map => [{ 0 => { "AS:i:26" => 1, "XS:i:16" => 1, "YT:Z:UU" => 1, "MD:Z:13" => 1 }, 8 => { "AS:i:16" => 1, "XS:i:16" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1 } }] }, # 1 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 # T T G T T C G T T T G T T C G T # 0 T x # 1 T x # 2 G x # 3 T x # 4 T x # 5 C x # 6 G x # 7 T x # 8 T # 9 T # 10 G # 1 T # Local alignment for a hit that should be trimmed from the right end { name => "Local alignment 4", ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCGTTTGT" ], args => "--local --policy \"SEED=0,3\\;IVAL=C,1,0\" --score-min=C,12", report => "-a", hits => [ { 0 => 1, 8 => 1 } ], flags_map => [{ 0 => "XM:0,XP:0,XT:UU,XC:12=", 8 => "XM:0,XP:0,XT:UU,XC:8=4S" }], cigar_map => [{ 0 => "12M", 8 => "8M4S" }], samoptflags_map => [{ 0 => { "AS:i:24" => 1, "XS:i:16" => 1, "YT:Z:UU" => 1, "MD:Z:12" => 1 }, 8 => { "AS:i:16" => 1, "XS:i:16" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1 } }] }, # # Test some common featuers for the manual. E.g. when more than one # alignment is reported in -k mode, what order are they reported in? They # should be in order by alignment score. # { name => "Alignment order -k", # 012345678 ref => [ "GCGCATGCACATATCANNNNNGCGCATGCACATATCTNNNNNNNNGCGCATGCACATATTTNNNNNNNNNGCGCATGGTGTTATCA" ], reads => [ "GCGCATGCACATATCA" ], quals => [ "GOAIYEFGFIWDSFIU" ], args => "--min-score C,-24,0 -L 4", report => "-k 4" }, { name => "Alignment order -a", # 012345678 ref => [ "GCGCATGCACATATCANNNNNGCGCATGCACATATCTNNNNNNNNGCGCATGCACATATTTNNNNNNNNNGCGCATGGTGTTATCA" ], reads => [ "GCGCATGCACATATCA" ], quals => [ "GOAIYEFGFIWDSFIU" ], args => "--min-score C,-24,0 -L 4", report => "-a" }, # # What order are mates reported in? Should be reporting in mate1/mate2 # order. # { name => "Mate reporting order, -a", # 012345678 ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAAATAGACGACTCGATCGCGGATTAGGGGTAGACCCCCCCCCGACTNNNNNNNNNNAGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAAATAGACGACTCGATCGCGGATTAGGGGTAGACCCCCCCCCGACTNNNNNNNNNNAGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAAATAGACGACTCGATCGCGGATTAGGGGTAGACCCCCCCCCGACTNNNNNNNNCGGTAATACGGCCATCGCGGCGGCATTACTCGGCGACTGCACGAGCAGATATTGGGGGTCTAATATAACGTCTCATTAAAACGCTCTAGTCAGCTCATTGGCTCTA" ], mate1s => [ "CTATCATCACGCGGATATT", "GGGGGGGGTCTACCCCTAA", "ATACGGCCATCGCGGCGGCATTACTCGGCG" ], mate2s => [ "GGGGGGGGTCTACCCCTAA", "CTATCATCACGCGGATATT", "AGCCAATGAGCTGACTAGAGCGTTTT" ], quals => [ "GOAIYEFGFIWDSFIU" ], args => "", report => "-a" }, { name => "Mate reporting order, -M 1", # 012345678 ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAAATAGACGACTCGATCGCGGATTAGGGGTAGACCCCCCCCCGACTNNNNNNNNNNAGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAAATAGACGACTCGATCGCGGATTAGGGGTAGACCCCCCCCCGACTNNNNNNNNNNAGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAAATAGACGACTCGATCGCGGATTAGGGGTAGACCCCCCCCCGACTNNNNNNNNCGGTAATACGGCCATCGCGGCGGCATTACTCGGCGACTGCACGAGCAGATATTGGGGGTCTAATATAACGTCTCATTAAAACGCTCTAGTCAGCTCATTGGCTCTA" ], mate1s => [ "CTATCATCACGCGGATATT", "GGGGGGGGTCTACCCCTAA", "ATACGGCCATCGCGGCGGCATTACTCGGCG" ], mate2s => [ "GGGGGGGGTCTACCCCTAA", "CTATCATCACGCGGATATT", "AGCCAATGAGCTGACTAGAGCGTTTT" ], quals => [ "GOAIYEFGFIWDSFIU" ], args => "", report => "-M 1" }, # # Test dovetailing, containment, and overlapping # { name => "Non-overlapping; no args", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATA" ], mate2s => [ "CGCATCGACATTAATATCC" ], pairhits => [{ "1,23" => 1 }], mate1fw => 1, mate2fw => 1, report => "-M 1" }, { name => "Non-overlapping; --no-discordant", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATA" ], mate2s => [ "CGCATCGACATTAATATCC" ], pairhits => [{ "1,23" => 1 }], mate1fw => 1, mate2fw => 1, report => "-M 1 --no-discordant" }, { name => "Non-overlapping; --no-discordant --no-mixed", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATA" ], mate2s => [ "CGCATCGACATTAATATCC" ], pairhits => [{ "1,23" => 1 }], mate1fw => 1, mate2fw => 1, report => "-M 1 --no-discordant --no-mixed" }, { name => "Non-overlapping; --no-discordant --no-mixed", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATA" ], mate2s => [ "CGCATCGACATTAATATCC" ], pairhits => [{ "1,23" => 1 }], mate1fw => 1, mate2fw => 1, report => "-M 1 --no-discordant --no-mixed" }, { name => "Non-overlapping; --no-dovetail", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATA" ], mate2s => [ "CGCATCGACATTAATATCC" ], pairhits => [{ "1,23" => 1 }], mate1fw => 1, mate2fw => 1, args => "--no-dovetail", report => "-M 1" }, { name => "Non-overlapping; --un-conc=.tmp.simple_tests.pl", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATA" ], mate2s => [ "CGCATCGACATTAATATCC" ], pairhits => [{ "1,23" => 1 }], mate1fw => 1, mate2fw => 1, args => "--un-conc=.tmp.simple_tests.pl", report => "-M 1" }, { name => "Non-overlapping; --no-overlap", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATA" ], mate2s => [ "CGCATCGACATTAATATCC" ], pairhits => [{ "1,23" => 1 }], mate1fw => 1, mate2fw => 1, args => "--no-overlap", report => "-M 1" }, { name => "Overlapping; no args", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATATTA" ], mate2s => [ "TTAGCGCATCGACATTAATATCC" ], pairhits => [{ "1,19" => 1 }], mate1fw => 1, mate2fw => 1, args => "", report => "-M 1" }, { name => "Overlapping; --no-dovetail", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATATTA" ], mate2s => [ "TTAGCGCATCGACATTAATATCC" ], pairhits => [{ "1,19" => 1 }], mate1fw => 1, mate2fw => 1, args => "--no-dovetail", report => "-M 1" }, { name => "Overlapping; --no-contain", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATATTA" ], mate2s => [ "TTAGCGCATCGACATTAATATCC" ], pairhits => [{ "1,19" => 1 }], mate1fw => 1, mate2fw => 1, args => "--no-contain", report => "-M 1" }, { name => "Overlapping; --no-overlap", ref => [ "AGCTATCATCACGCGGATATTAGCGCATCGACATTAATATCCCCAAA" ], # 01234567890123456789012345678901234567890123456 mate1s => [ "GCTATCATCACGCGGATATTA" ], mate2s => [ "TTAGCGCATCGACATTAATATCC" ], pairhits => [], mate1fw => 1, mate2fw => 1, args => "--no-overlap", report => "-M 1" }, # # Test XS:i with quality scaling # { name => "Scoring params 1", # 012345678 ref => [ "ACTATTGCGCGCATGCACATATCAATTAAGCCGTCTCTCTAAAGAGACCCCAATCTCGCGCGCTAGACGTCAGTAGTTTAATTTTATAAACACCTCGCTGCGGGG" ], reads => [ "GCGCATGCACATATCAATTAAGCCGTCTCTCTAAAGAGACCCCAATCTCGCGCGCTAGACGTCAGTAGTTTAATTTTATAAACACCTC" ], quals => [ "GOAIYEFGFIWDSFIUYWEHRIWQWLFNSLDKkjdfglduhiuevhsiuqkAUHFIUEHGIUDJFHSKseuweyriwfskdgbiuuhh" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567 # 0 1 2 3 4 5 6 7 8 args => "", report => "-M 1", hits => [ { 8 => 1 } ], cigar => [ "88M" ], samoptflags => [ { "AS:i:0" => 1, "YT:Z:UU" => 1, "MD:Z:88" => 1 } ], }, { name => "Scoring params 2", # 012345678 ref => [ "ACTATTGCGCGCATGCACATATCAATTAAGCCGTCTCTCTAAAGAGACCCCAATCTCGCGCGCTAGACGTCAGTAGTTT"."TTTATAAACACCTCGCTGCGGGG" ], reads => [ "NCGCATGCACATtTCAATTAAGCCGTCTCTCTAAAGA". "CCAATCTCGCGCGCTAGACGTCAGTAGTTTAAATTTATAAACACCTC" ], # * -1 * -6 **** -5 -3 -3 -3 -3 *** -5 -3 -3 -3 quals => [ "GOAIYEFGFIWDSFIUYWEHRIWQWLFNSLDKkjdfg". "iuevhsiuqkAUHFIUEHGIUDJFHSKseuweyriwfskdgbiuuhh" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567 # 0 1 2 3 4 5 6 7 8 args => "--ignore-quals --score-min C,-40,0 -N 1 -L 20", report => "-M 1", hits => [ { 8 => 1 } ], cigar => [ "37M4D30M3I14M" ], # 37M4D30M13I4M samoptflags => [ { "AS:i:-38" => 1, "YT:Z:UU" => 1, "MD:Z:0G11A24^GACC44" => 1, "NM:i:9" => 1, "XM:i:2" => 1, "XG:i:7" => 1, "XO:i:2" => 1 } ], }, { name => "Scoring params 3", # 012345678 ref => [ "ACTATTGCGCGCATGCACATATCAATTAAGCCGTCTCTCTAAAGAGACCCCAATCTCGCGCGCTAGACGTCAGTAGTTT"."TTTATAAACACCTCGCTGCGGGG" ], reads => [ "NCGCATGCACATtTCAATTAAGCCGTCTCTCTAAAGA". "CCAATCTCGCGCGCTAGACGTCAGTAGTTTAAATTTATAAACACCTC" ], # * -1 * -6 **** -5 -3 -3 -3 -3 *** -1 -2 -2 -2 quals => [ "GOAIYEFGFIWDSFIUYWEHRIWQWLFNSLDKkjdfg". "iuevhsiuqkAUHFIUEHGIUDJFHSKseuweyriwfskdgbiuuhh" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567 # 0 1 2 3 4 5 6 7 8 args => "--ignore-quals --rfg 1,2 --score-min C,-40,0 -N 1 -L 20", report => "-M 1", hits => [ { 8 => 1 } ], cigar => [ "37M4D30M3I14M" ], samoptflags => [ { "AS:i:-31" => 1, "YT:Z:UU" => 1, "MD:Z:0G11A24^GACC44" => 1, "NM:i:9" => 1, "XM:i:2" => 1, "XG:i:7" => 1, "XO:i:2" => 1 } ], }, { name => "Scoring params 4", # 012345678 ref => [ "ACTATTGCGCGCATGCACATATCAATTAAGCCGTCTCTCTAAAGAGACCCCAATCTCGCGCGCTAGACGTCAGTAGTTT"."TTTATAAACACCTCGCTGCGGGG" ], reads => [ "NCGCATGCACATtTCAATTAAGCCGTCTCTCTAAAGA". "CCAATCTCGCGCGCTAGACGTCAGTAGTTTAAATTTATAAACACCTC" ], # * -1 * -6 **** -1 -2 -2 -2 -2 *** -5 -3 -3 -3 quals => [ "GOAIYEFGFIWDSFIUYWEHRIWQWLFNSLDKkjdfg". "iuevhsiuqkAUHFIUEHGIUDJFHSKseuweyriwfskdgbiuuhh" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567 # 0 1 2 3 4 5 6 7 8 args => "--ignore-quals --rdg 1,2 --score-min C,-40,0 -N 1 -L 20", report => "-M 1", hits => [ { 8 => 1 } ], cigar => [ "37M4D30M3I14M" ], samoptflags => [ { "AS:i:-30" => 1, "YT:Z:UU" => 1, "MD:Z:0G11A24^GACC44" => 1, "NM:i:9" => 1, "XM:i:2" => 1, "XG:i:7" => 1, "XO:i:2" => 1 } ], }, { name => "Scoring params 5", # 012345678 ref => [ "ACTATTGCGCGCATGCACATATCAATTAAGCCGTCTCTCTAAAGAGACCCCAATCTCGCGCGCTAGACGTCAGTAGTTT"."TTTATAAACACCTCGCTGCGGGG" ], reads => [ "NCGCATGCACATtTCAATTAAGCCGTCTCTCTAAAGA". "CCAATCTCGCGCGCTAGACGTCAGTAGTTTAAATTTATAAACACCTC" ], # * -1 * -8 **** -5 -3 -3 -3 -3 *** -5 -3 -3 -3 quals => [ "GOAIYEFGFIWDSFIUYWEHRIWQWLFNSLDKkjdfg". "iuevhsiuqkAUHFIUEHGIUDJFHSKseuweyriwfskdgbiuuhh" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567 # 0 1 2 3 4 5 6 7 8 args => "--ignore-quals --mp 8 --score-min C,-40,0 -N 1 -L 20", report => "-M 1", hits => [ { 8 => 1 } ], cigar => [ "37M4D30M3I14M" ], samoptflags => [ { "AS:i:-40" => 1, "YT:Z:UU" => 1, "MD:Z:0G11A24^GACC44" => 1, "NM:i:9" => 1, "XM:i:2" => 1, "XG:i:7" => 1, "XO:i:2" => 1 } ], }, { name => "Scoring params 6", # 012345678 ref => [ "ACTATTGCGCGCATGCACATATCAATTAAGCCGTCTCTCTAAAGAGACCCCAATCTCGCGCGCTAGACGTCAGTAGTTT"."TTTATAAACACCTCGCTGCGGGG" ], reads => [ "NCGCATGCACATtTCAATTAAGCCGTCTCTCTAAAGA". "CCAATCTCGCGCGCTAGACGTCAGTAGTTTAAATTTATAAACACCTC" ], # * -4 * -6 **** -5 -3 -3 -3 -3 *** -5 -3 -3 -3 quals => [ "GOAIYEFGFIWDSFIUYWEHRIWQWLFNSLDKkjdfg". "iuevhsiuqkAUHFIUEHGIUDJFHSKseuweyriwfskdgbiuuhh" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567 # 0 1 2 3 4 5 6 7 8 args => "--ignore-quals --np 4 --score-min C,-41,0 -N 1 -L 20", report => "-M 1", hits => [ { 8 => 1 } ], cigar => [ "37M4D30M3I14M" ], samoptflags => [ { "AS:i:-41" => 1, "YT:Z:UU" => 1, "MD:Z:0G11A24^GACC44" => 1, "NM:i:9" => 1, "XM:i:2" => 1, "XG:i:7" => 1, "XO:i:2" => 1 } ], }, # # Test XS:i with quality scaling # { name => "Q XS:i 1a", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], quals => [ "IIIIIIIA" ], args => "--multiseed=0,7,C,1 --score-min=C,-6", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-5" => 1, "XS:i:-5" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, { name => "Q XS:i 1a ! --mp 3,3", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], quals => [ "IIIIIII!" ], args => "-L 6 --mp 3,3 --score-min=C,-6", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-3" => 1, "XS:i:-3" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, { name => "Q XS:i 1a ! --mp 3,6", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], quals => [ "IIIIIII!" ], args => "-L 6 --mp 6,3 --score-min=C,-6", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-3" => 1, "XS:i:-3" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, { name => "Q XS:i 1a I --mp 3,3", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], quals => [ "IIIIIIII" ], args => "-L 6 --mp 3,3 --score-min=C,-6", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-3" => 1, "XS:i:-3" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, { name => "Q XS:i 1a I --mp 3,6", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], quals => [ "IIIIIIII" ], args => "-L 6 --mp 6,3 --score-min=C,-6", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-6" => 1, "XS:i:-6" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, { name => "Q XS:i 1a --ignore-quals", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], quals => [ "IIIIIIIA" ], args => "--multiseed=0,7,C,1 --score-min=C,-6 --ignore-quals", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-6" => 1, "XS:i:-6" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, { name => "Q XS:i 1b", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], quals => [ "IIIIIII5" ], args => "--multiseed=0,7,C,1 --score-min=C,-6", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-4" => 1, "XS:i:-4" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, { name => "Q XS:i 1b --ignore-quals", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], quals => [ "IIIIIII5" ], args => "--multiseed=0,7,C,1 --score-min=C,-6 --ignore-quals", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-6" => 1, "XS:i:-6" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, { name => "Q XS:i 1c", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], quals => [ "IIIIIII4" ], args => "--multiseed=0,7,C,1 --score-min=C,-6", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-3" => 1, "XS:i:-3" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, { name => "Q XS:i 1c --ignore-quals", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], quals => [ "IIIIIII4" ], args => "--multiseed=0,7,C,1 --score-min=C,-6 --ignore-quals", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-6" => 1, "XS:i:-6" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, # One mate aligns. Ensuring that the unmapped mate gets reference # information filled in from the other mate. { ref => [ "CATCGACTGAGACTCGTACGACAATTACGCGCATTATTCGCATCACCAGCGCGGCGCGCGCCCCCTAT" ], # 01234567890123456789012345678901234567890123456789012345678901234567 # 0 1 2 3 4 5 6 # ATCACCAGCGTTTCGCGCGAAACCTA mate1s => [ "ATCGACTGAGACTCGTACGACAATTAC" ], mate2s => [ "TAGGTTTCGCGCGAAACGCTGGTGAT" ], pairhits_orig => [{ "1,1" => 1}] }, { ref => [ "TTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCGT" ], args => "--multiseed=0,4,C,1,0", report => "-M 1" }, # Testing that DEFAULT is -M 1 { ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCGT" ], report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1, "YM:i:1" => 1 } ], }, { ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCGT" ], report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1, "YM:i:1" => 1 } ], }, # # Test XS:i # { name => "XS:i 1", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], args => "--multiseed=0,7,C,1 --score-min=C,-6", report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-6" => 1, "XS:i:-6" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:7A0" => 1, "NM:i:1" => 1, "XM:i:1" => 1 } ], }, { name => "XS:i 2", ref => [ "TTGTTCGATTGTTCGA" ], reads => [ "TTGTTCGT" ], args => "--multiseed=0,7,C,1 --score-min=C,-5", report => "", cigar => [ "*" ], samoptflags => [{ "YT:Z:UU" => 1, "YM:i:0" => 1 }], }, { name => "XS:i 3a", ref => [ "TTGTTCGATTGTTCGT" ], # TTGTTCGT reads => [ "TTGTTCGT" ], args => "--multiseed=0,7,C,1 --score-min=C,-6", report => "-M 1", hits => [ { 8 => 1 } ], cigar => [ "8M" ], samoptflags => [ { "AS:i:0" => 1, "XS:i:-6" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1, "NM:i:0" => 1, "XM:i:0" => 1 } ], }, { name => "XS:i 3b", ref => [ "TTGTTCGATTGTTCGT" ], # TTGTTCGT reads => [ "TTGTTCGT" ], args => "--multiseed=0,7,C,1 --score-min=C,-6 --seed=52", report => "-M 1", hits => [ { 8 => 1 } ], cigar => [ "8M" ], samoptflags => [ { "AS:i:0" => 1, "XS:i:-6" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1, "NM:i:0" => 1, "XM:i:0" => 1 } ], }, { name => "XS:i 3c", ref => [ "TTGTTCGATTGTTCGT" ], # TTGTTCGT reads => [ "TTGTTCGT" ], args => "--multiseed=0,7,C,1 --score-min=C,-6 --seed=53", report => "-M 2", hits => [ { 8 => 1 } ], cigar => [ "8M" ], samoptflags => [ { "AS:i:0" => 1, "XS:i:-6" => 1, "YM:i:0" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1, "NM:i:0" => 1, "XM:i:0" => 1 } ], }, { name => "XS:i 4a", ref => [ "TTGTTCAATTGTTCGATTGTTCGT" ], # |||||| ||||||| |||||||| # TTGTTCGT||||||| |||||||| # TTGTTCGT|||||||| # TTGTTCGT reads => [ "TTGTTCGT" ], args => "--multiseed=0,6,C,1 --score-min=C,-12 --seed=53", report => "-M 2", hits => [ { 16 => 1 } ], cigar => [ "8M" ], samoptflags => [ { "AS:i:0" => 1, "XS:i:-6" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1, "NM:i:0" => 1, "XM:i:0" => 1 } ], }, { name => "XS:i 4b", ref => [ "TTGTTCAATTGTTCGATTGTTCGT" ], # |||||| ||||||| |||||||| # TTGTTCGT||||||| |||||||| # TTGTTCGT|||||||| # TTGTTCGT reads => [ "TTGTTCGT" ], args => "--multiseed=0,6,C,1 --score-min=C,-12 --seed=54", report => "-M 3", hits => [ { 16 => 1 } ], cigar => [ "8M" ], samoptflags => [ { "AS:i:0" => 1, "XS:i:-6" => 1, "YM:i:0" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1, "NM:i:0" => 1, "XM:i:0" => 1 } ], }, { name => "XS:i 5a", ref => [ "TTGTTCAATTGTTCGATTGTTCGTTTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAA" ], # |||||| ||||||| |||||||||||||| |||||| |||||| |||||| |||||| |||||| |||||| |||||| |||||| |||||| |||||| |||||| # TTGTTCGT||||||| ||||||||TTGTTCGT|||||| TTGTTCGT|||||| TTGTTCGT|||||| TTGTTCGT|||||| TTGTTCGT|||||| TTGTTCGT|||||| # TTGTTCGT|||||||| TTGTTCGT TTGTTCGT TTGTTCGT TTGTTCGT TTGTTCGT TTGTTCGT # TTGTTCGT reads => [ "TTGTTCGT" ], args => "--multiseed=0,6,C,1,1 --score-min=C,-12 --seed=54", report => "-M 1", hits => [ { 16 => 1 } ], cigar => [ "8M" ], samoptflags => [ { "AS:i:0" => 1, "XS:i:-6" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1, "NM:i:0" => 1, "XM:i:0" => 1 } ], }, { name => "XS:i 5b", ref => [ "TTGTTCAATTGTTCGATTGTTCGTTTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAATTGTTCAA" ], # |||||| ||||||| |||||||||||||| |||||| |||||| |||||| |||||| |||||| |||||| |||||| |||||| |||||| |||||| |||||| # TTGTTCGT||||||| ||||||||TTGTTCGT|||||| TTGTTCGT|||||| TTGTTCGT|||||| TTGTTCGT|||||| TTGTTCGT|||||| TTGTTCGT|||||| # TTGTTCGT|||||||| TTGTTCGT TTGTTCGT TTGTTCGT TTGTTCGT TTGTTCGT TTGTTCGT # TTGTTCGT reads => [ "TTGTTCGT" ], args => "--multiseed=0,5,C,1,1 --score-min=C,-12 --seed=55", report => "-M 1", hits => [ { 16 => 1 } ], cigar => [ "8M" ], samoptflags => [ { "AS:i:0" => 1, "XS:i:-6" => 1, "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1, "NM:i:0" => 1, "XM:i:0" => 1 } ], }, # Testing BWA-SW-like scoring # # a*max{T,c*log(l)} = 1 * max(30, 5.5 * log(56)) = 1 * max(30, 22.139) = 30 # { name => "BWA-SW-like 1", ref => [ "GTTTAGATTCCACTACGCTAACCATCGAGAACTCGTCTCAGAGTTTCGATAGGAAAATCTGCGA" ], # |||||||||||||||||||||||||||||||||||||||||||||||||||||||| reads => [ "TAGATTCCACTACGCTAACCATCGAGAACTCGTCTCAGAGTTTCGATAGGAAAATC" ], # 01234567890123456789012345678901234567890123456789012345 # 1 2 3 4 5 args => "--bwa-sw-like", hits => [{ 3 => 1 }], samoptflags => [{ "AS:i:56" => 1, "NM:i:0" => 1, "MD:Z:56" => 1, "YT:Z:UU" => 1 }] }, { name => "BWA-SW-like 2", # 0123 ref => [ "GTTTAGATTCCACTACGCTAACCATCGAGAACTCGTCTCAGAGTTTCGATAGGAAAATCTGCGA" ], # |||||||||||||||||||||||||| |||||||||||||||||||||||||||| reads => [ "TAGATTCCACTACGCTAACCATCGAGTTCTCGTCTCAGAGTTTCGATAGGAAAATC" ], # 01234567890123456789012345678901234567890123456789012345 # 1 2 3 4 5 args => "--bwa-sw-like -L 18", hits => [{ 3 => 1 }], # Tot matches = 54 # Tot penalties = 6 samoptflags => [{ "AS:i:48" => 1, "NM:i:2" => 1, "XM:i:2" => 1, "MD:Z:26A0A28" => 1, "YT:Z:UU" => 1 }] }, { name => "BWA-SW-like 3", # 0123 ref => [ "GTTTAGATTCCACTACGCTAACCATCGAGAACTCGTCTCAGAGTTTCGATAGGAAAATCTGCGA" ], # |||||||||||||||||||||||||| ||||||||||||||||||||||||||| reads => [ "TAGATTCCACTACGCTAACCATCGAG"."TCGTCTCAGAGTTTCGATAGGAAAATC" ], # 01234567890123456789012345678901234567890123456789012345 # 1 2 3 4 5 args => "--bwa-sw-like -i C,1,0", hits => [{ 3 => 1 }], # Tot matches = 53 # Tot penalties = 11 samoptflags => [{ "AS:i:42" => 1, "NM:i:3" => 1, "XM:i:0" => 1, "XO:i:1" => 1, "XG:i:3" => 1, "MD:Z:26^AAC27" => 1, "YT:Z:UU" => 1 }] }, # Some tricky SAM FLAGS field tests { name => "SAM paired-end where both mates align 1", ref => [ "GCACTATCTACGCTTCGGCGTCGGCGAAAAAACGCACGACCGGGTGTGTGACAATCATATATAGCGCGC" ], # 012345678901234567890123456789012345678901234567890123456789012345678 # 0 1 2 3 4 5 6 mate1s => [ "CTATCTACGCTTCGGCGTCGGTGA" ], mate2s => [ "GATTGTCACACACCCGGTCGT" ], # ----------------------------------------------------- # 01234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 # 0x1 template having multiple fragments in sequencing # 0x2 each fragment properly aligned according to the aligner # 0x4 fragment unmapped # 0x8 next fragment in the template unmapped # 0x10 SEQ being reverse complemented # 0x20 SEQ of the next fragment in the template being reversed # 0x40 the first fragment in the template # 0x80 the last fragment in the template pairhits => [{ "3,35" => 1 }], norc => 1, samflags_map => [{ 3 => (1 | 2 | 32 | 64), 35 => (1 | 2 | 16 | 128) }], tlen_map => [{ 3 => 53, 35 => -53 }] }, { name => "SAM paired-end where both mates align 2", ref => [ "GCACTATCTACGCTTCGGCGTCGGCGAAAAAACGCACGACCGGGTGTGTGACAATCATATATAGCGCGC" ], # 012345678901234567890123456789012345678901234567890123456789012345678 # 0 1 2 3 4 5 6 mate1s => [ "TCACCGACGCCGAAGCGTAGATAG" ], mate2s => [ "ACGACCGGGTGTGTGACAATC" ], # ----------------------------------------------------- # 01234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 # 0x1 template having multiple fragments in sequencing # 0x2 each fragment properly aligned according to the aligner # 0x4 fragment unmapped # 0x8 next fragment in the template unmapped # 0x10 SEQ being reverse complemented # 0x20 SEQ of the next fragment in the template being reversed # 0x40 the first fragment in the template # 0x80 the last fragment in the template mate1fw => 0, mate2fw => 1, pairhits => [{ "3,35" => 1 }], norc => 1, samflags_map => [{ 3 => (1 | 2 | 16 | 64), 35 => (1 | 2 | 32 | 128) }], tlen_map => [{ 3 => 53, 35 => -53 }] }, { name => "SAM paired-end where both mates align 3", ref => [ "GCACTATCTACGCTTCGGCGTCGGCGAAAAAACGCACGACCGGGTGTGTGACAATCATATATAGCGCGC" ], # 012345678901234567890123456789012345678901234567890123456789012345678 # 0 1 2 3 4 5 6 mate1s => [ "CTATCTACGCTTCGGCGTCGGTGA" ], mate2s => [ "ACGACCGGGTGTGTGACAATC" ], # ----------------------------------------------------- # 01234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 # 0x1 template having multiple fragments in sequencing # 0x2 each fragment properly aligned according to the aligner # 0x4 fragment unmapped # 0x8 next fragment in the template unmapped # 0x10 SEQ being reverse complemented # 0x20 SEQ of the next fragment in the template being reversed # 0x40 the first fragment in the template # 0x80 the last fragment in the template mate1fw => 1, mate2fw => 1, pairhits => [{ "3,35" => 1 }], norc => 1, samflags_map => [{ 3 => (1 | 2 | 64), 35 => (1 | 2 | 128) }], tlen_map => [{ 3 => 53, 35 => -53 }] }, { name => "SAM paired-end where mate #1 aligns but mate #2 doesn't", ref => [ "GCACTATCTACGCTTCGGCGTCGGCGAAAAAACGCACGACCGGGTGTGTGACAATCATATATAGCGCGC" ], # 012345678901234567890123456789012345678901234567890123456789012345678 # 0 1 2 3 4 5 6 mate1s => [ "CTATCTACGCTTCGGCGTCGGCGA" ], mate2s => [ "GATTGTCTTTTCCCGGAAAAATCGT" ], # 0x1 template having multiple fragments in sequencing # 0x2 each fragment properly aligned according to the aligner # 0x4 fragment unmapped # 0x8 next fragment in the template unmapped # 0x10 SEQ being reverse complemented # 0x20 SEQ of the next fragment in the template being reversed # 0x40 the first fragment in the template # 0x80 the last fragment in the template pairhits => [{ "*,3" => 1 }], norc => 1, samflags_map => [{ 3 => (1 | 8 | 64), "*" => (1 | 4 | 128) }] }, { name => "SAM paired-end where neither mate aligns", ref => [ "GCACTATCTACGCTTCGGCGTCGGCGAAAAAACGCACGACCGGGTGTGTGACAATCATATATAGCGCGC" ], # 012345678901234567890123456789012345678901234567890123456789012345678 # 0 1 2 3 4 5 6 mate1s => [ "CTATATACGAAAAAGCGTCGGCGA" ], mate2s => [ "GATTGTCTTTTCCCGGAAAAATCGT" ], # 0x1 template having multiple fragments in sequencing # 0x2 each fragment properly aligned according to the aligner # 0x4 fragment unmapped # 0x8 next fragment in the template unmapped # 0x10 SEQ being reverse complemented # 0x20 SEQ of the next fragment in the template being reversed # 0x40 the first fragment in the template # 0x80 the last fragment in the template pairhits => [{ "*,*" => 1 }], norc => 1, samoptflags_flagmap => [{ (1 | 4 | 8 | 64) => { "YT:Z:UP" => 1 }, (1 | 4 | 8 | 128) => { "YT:Z:UP" => 1 } }] }, { name => "SAM paired-end where both mates align, but discordantly", ref => [ "GCACTATCTACGCTTCGGCGTCGGCGAAAAAACGCACGACCGGGTGTGTGACAATCATATATAGCGCGC" ], # 012345678901234567890123456789012345678901234567890123456789012345678 # 0 1 2 3 4 5 6 mate1s => [ "CTATCTACGCTTCGGCGTCGGCGA" ], mate2s => [ "ACGACCGGGTGTGTGACAATC" ], # ----------------------------------------------------- # 01234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 # 0x1 template having multiple fragments in sequencing # 0x2 each fragment properly aligned according to the aligner # 0x4 fragment unmapped # 0x8 next fragment in the template unmapped # 0x10 SEQ being reverse complemented # 0x20 SEQ of the next fragment in the template being reversed # 0x40 the first fragment in the template # 0x80 the last fragment in the template pairhits => [{ "3,35" => 1 }], norc => 1, samflags_map => [{ 3 => (1 | 64), 35 => (1 | 128) }], # Which TLEN is right? Depends on criteria for when to infer TLEN. If # criterion is mates are concordant, then it should be 0 here. If the # criterion is that both mates align to the same chromosome, should be # +-53 #tlen_map => [{ 3 => 0, 35 => 0 }] }, tlen_map => [{ 3 => 53, 35 => -53 }] }, { name => "matchesRef regression 4", ref => [ "CCGGGTCGTCACGCCCCGCTTGCGTCANGCCCCTCACCCTCCCTTTGTCGGCTCCCACCCCTCCCCATCCGTTGTCCCCGCCCCCGCCCGCCGGGTCGTCACGCCCCGCTTGCGTCANGC", "GCTCGGAATTCGTGCTCCGNCCCGTACGGTT" ], # # NNNNNGA------A-------------------G-NTTT # |||||||||||||||||||||||||||||||||| # CCAAT-ATTTTTAATTTCCTCTATTTTTCTCTCGTCTTG args => "--policy \"NP=Q\\;RDG=46.3220993654702\\;RFG=41.3796024365659\\;MIN=L,5.57015383125426,-3.28597145122829\\;NCEIL=L,0.263054599454459,0.130843661549367\\;SEED=1,29\\;IVAL=L,0.0169183264663712,3.75762168662522\" --overhang --trim5 6", reads => [ "CTTTGCACCCCTCCCTTGTCGGCTCCCACCCATCCCCATCCGTTGTCCCCGCCCCCGCCCGCCGGTCGTCACTCCCCGTTTGCGTCATGCCCCTCACCCTCCCTTTGTCGGCTCGCACCCCTCCCCATCCGTTGTCCCCGCCCCCGCTCTCGGGGTCTTCACGCCCCGCTTGCTTCATGCCCCTCACTCGCACCCCG" ], }, { name => "matchesRef regression 3", ref => [ "GAAGNTTTTCCAATATTTTTAATTTCCTCTATTTTTCTCTCGTCTTGNTCTAC" ], # # NNNNNGA------A-------------------G-NTTT # |||||||||||||||||||||||||||||||||| # CCAAT-ATTTTTAATTTCCTCTATTTTTCTCTCGTCTTG args => "--policy \"MMP=R\\;MIN=L,8.8,-8.1\" --overhang", reads => [ "CAAGACGAGAGAAAAATAGAGGAAATTAAAAATATTGG" ], }, { name => "matchesRef regression 2", ref => ["GTTGTCGGCAGCTCTGGATATGTGNTCTCGGGTTTATNTCGTTGTCG", "CCTTGTTNTTAATGCTGCCTGGTTTNG"], args => "--policy \"RDG=2.02030755427021,2.81949533273331\\;MIN=L,-6.52134769703939,-3.39889659588514\\;IVAL=L,0.127835912101927\" --overhang --trim5 5", mate1s => ["TCTGGCGGTTGCGAAGGCCCCTGGCGGTTGCTATGTCCTCTGGCGGTTGCGTTGTCGGCAGCTCG"], mate2s => ["AGAACACATATCCAGAGCTGCCGACAACGAAATGAACCCGAGAGCACAAATCCAGAG"] }, # Regression test for an issue observed once { name => "matchesRef regression 1", # 0 1 2 3 4 5 6 7 # 01234567890123456789012345678901234567890123456789012345678901234567890 ref => [ "AGGTCGACCGAAAGGCCTAGAGGTCGACCGACAATCTGACCATGGGGCGAGGAGCGAGTAC" ], # |||||||||||||||||||||||||||||||||||||||||||||||||| reads => [ "AAGGCCTAGAGGTCGACCGACAATCTGACCATGGGGCGAGGAGCGAGTACTGGTCTGGGG" ], # 012345678901234567890123456789012345678901234567890123456789 # 0 1 2 3 4 5 args => "--overhang" }, # 1 discordant alignment and one concordant alignment. Discordant because # the fragment is too long. { name => "Discordant with different chromosomes", ref => [ "TTTATAAAAATATTTCCCCCCCC", "CCCCCCTGTCGCTACCGCCCCCCCCCCC" ], # ATAAAAATAT GTCGCTACCG # ATAAAAATAT TGTCGCTACC # 01234567890123456789012 # 0 1 2 # 0123456789012345678901234567 # 0 1 2 mate1s => [ "ATAAAAATAT", "ATAAAAATAT" ], mate2s => [ "GTCGCTACCG", "TGTCGCTACC" ], mate1fw => 1, mate2fw => 1, args => "-I 0 -X 35", # Not really any way to flag an alignment as discordant pairhits => [ { "3,7" => 1 }, { "3,6" => 1 } ], rnext_map => [ { 3 => 1, 7 => 0 }, { 3 => 1, 6 => 0 } ], pnext_map => [ { 3 => 7, 7 => 3 }, { 3 => 6, 6 => 3 } ] }, { name => "Fastq 1", ref => [ "AGCATCGATCAGTATCTGA" ], fastq => "\@r0\nCATCGATCAGTATCTG\n+\nIIIIIIIIIIIIIIII", hits => [{ 2 => 1 }] }, { name => "Tabbed 1", ref => [ "AGCATCGATCAGTATCTGA" ], tabbed => "r0\tCATCGATCAGTATCTG\tIIIIIIIIIIIIIIII", hits => [{ 2 => 1 }] }, { name => "Fasta 1", ref => [ "AGCATCGATCAGTATCTGA" ], fasta => ">r0\nCATCGATCAGTATCTG", hits => [{ 2 => 1 }] }, { name => "Qseq 1", ref => [ "AGCATCGATCAGTATCTGA" ], qseq => join("\t", "MachName", "RunNum", "Lane", "Tile", "X", "Y", "Index", "0", # Mate "CATCGATCAGTATCTG", "IIIIIIIIIIIIIIII", "1"), hits => [{ 2 => 1 }] }, { name => "Raw 1", ref => [ "AGCATCGATCAGTATCTGA" ], raw => "CATCGATCAGTATCTG", hits => [{ 2 => 1 }] }, # Like Fastq 1 but with extra newline { name => "Fastq 2", ref => [ "AGCATCGATCAGTATCTGA" ], fastq => "\@r0\nCATCGATCAGTATCTG\n+\nIIIIIIIIIIIIIIII\n", hits => [{ 2 => 1 }] }, { name => "Tabbed 1", ref => [ "AGCATCGATCAGTATCTGA" ], tabbed => "r0\tCATCGATCAGTATCTG\tIIIIIIIIIIIIIIII\n", hits => [{ 2 => 1 }] }, { name => "Fasta 2", ref => [ "AGCATCGATCAGTATCTGA" ], fasta => ">r0\nCATCGATCAGTATCTG\n", hits => [{ 2 => 1 }] }, { name => "Qseq 2", ref => [ "AGCATCGATCAGTATCTGA" ], qseq => join("\t", "MachName", "RunNum", "Lane", "Tile", "X", "Y", "Index", "0", # Mate "CATCGATCAGTATCTG", "IIIIIIIIIIIIIIII", "1")."\n", hits => [{ 2 => 1 }] }, { name => "Raw 2", ref => [ "AGCATCGATCAGTATCTGA" ], raw => "CATCGATCAGTATCTG\n", hits => [{ 2 => 1 }] }, # Like Fastq 1 but with many extra newlines { name => "Fastq 3", ref => [ "AGCATCGATCAGTATCTGA" ], fastq => "\n\n\r\n\@r0\nCATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIIII\n\n", hits => [{ 2 => 1 }] }, { name => "Tabbed 3", ref => [ "AGCATCGATCAGTATCTGA" ], tabbed => "\n\n\r\nr0\tCATCGATCAGTATCTG\tIIIIIIIIIIIIIIII\n\n", hits => [{ 2 => 1 }] }, { name => "Fasta 3", ref => [ "AGCATCGATCAGTATCTGA" ], fasta => "\n\n\r\n>r0\nCATCGATCAGTATCTG\r\n\n", hits => [{ 2 => 1 }] }, { name => "Qseq 3", ref => [ "AGCATCGATCAGTATCTGA" ], qseq => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "X", "Y", "Index", "0", # Mate "CATCGATCAGTATCTG", "IIIIIIIIIIIIIIII", "1")."\n\n", hits => [{ 2 => 1 }] }, { name => "Raw 3", ref => [ "AGCATCGATCAGTATCTGA" ], raw => "\n\n\nCATCGATCAGTATCTG\n\n", hits => [{ 2 => 1 }] }, # Quality string length doesn't match (too short by 1) { name => "Fastq 4", ref => [ "AGCATCGATCAGTATCTGA" ], fastq => "\n\n\r\n\@r0\nCATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIII\n\n", should_abort => 1}, { name => "Tabbed 4", ref => [ "AGCATCGATCAGTATCTGA" ], tabbed => "\n\n\r\nr0\tCATCGATCAGTATCTG\tIIIIIIIIIIIIIII\n\n", should_abort => 1}, { name => "Qseq 4", ref => [ "AGCATCGATCAGTATCTGA" ], qseq => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "X", "Y", "Index", "0", # Mate "CATCGATCAGTATCTG", "IIIIIIIIIIIIIII", "1")."\n\n", should_abort => 1}, # Name line doesn't start with @ { name => "Fastq 5", ref => [ "AGCATCGATCAGTATCTGA" ], fastq => "\n\n\r\nr0\nCATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIII\n\n", should_abort => 1, hits => [{ }] }, # Name line doesn't start with > { name => "Fasta 5", ref => [ "AGCATCGATCAGTATCTGA" ], fasta => "\n\n\r\nr0\nCATCGATCAGTATCTG\r", should_abort => 1, hits => [{ }] }, # Name line doesn't start with @ (2) { name => "Fastq 6", ref => [ "AGCATCGATCAGTATCTGA" ], fastq => "r0\nCATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIII\n\n", should_abort => 1, hits => [{ }] }, # Name line doesn't start with > (2) { name => "Fasta 6", ref => [ "AGCATCGATCAGTATCTGA" ], fasta => "r0\nCATCGATCAGTATCTG\r", should_abort => 1, hits => [{ }] }, # Part of sequence is trimmed { name => "Fastq 7", ref => [ "AGCATCGATCAGTATCTGA" ], fastq => "\n\n\r\n\@r0\nCATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIIII\n\n", args => "--trim3 4", norc => 1, hits => [{ 2 => 1 }] }, { name => "Tabbed 7", ref => [ "AGCATCGATCAGTATCTGA" ], tabbed => "\n\n\r\nr0\tCATCGATCAGTATCTG\tIIIIIIIIIIIIIIII\n\n", args => "--trim3 4", norc => 1, hits => [{ 2 => 1 }] }, { name => "Fasta 7", ref => [ "AGCATCGATCAGTATCTGA" ], fasta => "\n\n\r\n\>r0\nCATCGATCAGTATCTG\r\n", args => "--trim3 4", norc => 1, hits => [{ 2 => 1 }] }, { name => "Qseq 7", ref => [ "AGCATCGATCAGTATCTGA" ], qseq => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "X", "Y", "Index", "0", # Mate "CATCGATCAGTATCTG", "IIIIIIIIIIIIIIII", "1")."\n\n", args => "--trim3 4", norc => 1, hits => [{ 2 => 1 }] }, { name => "Raw 7", ref => [ "AGCATCGATCAGTATCTGA" ], raw => "\n\n\r\nCATCGATCAGTATCTG\r\n", args => "--trim3 4", norc => 1, hits => [{ 2 => 1 }] }, # Whole sequence is trimmed { name => "Fastq 8", ref => [ "AGCATCGATCAGTATCTGA" ], fastq => "\n\n\r\n\@r0\nCATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIIII\n\n", args => "--trim5 16", hits => [{ "*" => 1 }] }, { name => "Tabbed 8", ref => [ "AGCATCGATCAGTATCTGA" ], tabbed => "\n\n\r\nr0\tCATCGATCAGTATCTG\tIIIIIIIIIIIIIIII\n\n", args => "--trim5 16", hits => [{ "*" => 1 }] }, { name => "Fasta 8", ref => [ "AGCATCGATCAGTATCTGA" ], fasta => "\n\n\r\n\>r0\nCATCGATCAGTATCTG\r\n", args => "--trim3 16", hits => [{ "*" => 1 }] }, { name => "Qseq 8", ref => [ "AGCATCGATCAGTATCTGA" ], qseq => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "X", "Y", "Index", "0", # Mate "CATCGATCAGTATCTG", "IIIIIIIIIIIIIIII", "1")."\n\n", args => "--trim3 16", hits => [{ "*" => 1 }] }, { name => "Raw 8", ref => [ "AGCATCGATCAGTATCTGA" ], raw => "\n\n\r\nCATCGATCAGTATCTG\r\n", args => "--trim3 16", hits => [{ "*" => 1 }] }, # Sequence is skipped { name => "Fastq 9", ref => [ "AGCATCGATCAGTATCTGA" ], fastq => "\n\n\r\n\@r0\nCATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIIII\n\n", args => "-s 1", hits => [{ }] }, { name => "Tabbed 9", ref => [ "AGCATCGATCAGTATCTGA" ], tabbed => "\n\n\r\nr0\tCATCGATCAGTATCTG\tIIIIIIIIIIIIIIII\n\n", args => "-s 1", hits => [{ }] }, { name => "Fasta 9", ref => [ "AGCATCGATCAGTATCTGA" ], fasta => "\n\n\r\n>r0\nCATCGATCAGTATCTG\r\n", args => "-s 1", hits => [{ }] }, { name => "Qseq 9", ref => [ "AGCATCGATCAGTATCTGA" ], qseq => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "X", "Y", "Index", "0", # Mate "CATCGATCAGTATCTG", "IIIIIIIIIIIIIIII", "1")."\n\n", args => "-s 1", hits => [{ }] }, { name => "Raw 9", ref => [ "AGCATCGATCAGTATCTGA" ], raw => "CATCGATCAGTATCTG\n", args => "-s 1", hits => [{ }] }, # Like Fastq 1 but with many extra newlines { name => "Fastq multiread 1", ref => [ "AGCATCGATCAGTATCTGA" ], fastq => "\n\n\r\n\@r0\nCATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIIII\n\n". "\n\n\r\n\@r1\nATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIII\n\n", hits => [{ 2 => 1 }, { 3 => 1 }] }, { name => "Tabbed multiread 1", ref => [ "AGCATCGATCAGTATCTGA" ], tabbed => "\n\n\r\nr0\tCATCGATCAGTATCTG\tIIIIIIIIIIIIIIII\n\n". "\n\n\r\nr1\tATCGATCAGTATCTG\tIIIIIIIIIIIIIII\n\n", hits => [{ 2 => 1 }, { 3 => 1 }] }, { name => "Fasta multiread 1", ref => [ "AGCATCGATCAGTATCTGA" ], fasta => "\n\n\r\n>r0\nCATCGATCAGTATCTG\n\n". "\n\n\r\n>r1\nATCGATCAGTATCTG\n\n", hits => [{ 2 => 1 }, { 3 => 1 }] }, { name => "Qseq multiread 1", ref => [ "AGCATCGATCAGTATCTGA" ], qseq => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "10", "10", "Index", "0", # Mate "CATCGATCAGTATCTG", "IIIIIIIIIIIIIIII", "1")."\n\n". join("\t", "MachName", "RunNum", "Lane", "Tile", "12", "15", "Index", "0", # Mate "ATCGATCAGTATCTG", "IIIIIIIIIIIIIII", "1")."\n\n", idx_map => { "MachName_RunNum_Lane_Tile_10_10_Index" => 0, "MachName_RunNum_Lane_Tile_12_15_Index" => 1 }, hits => [{ 2 => 1 }, { 3 => 1 }] }, { name => "Raw multiread 1", ref => [ "AGCATCGATCAGTATCTGA" ], raw => "\n\n\r\nCATCGATCAGTATCTG\n\n". "\n\n\r\nATCGATCAGTATCTG\n\n", hits => [{ 2 => 1 }, { 3 => 1 }] }, # Like Fastq multiread 1 but with -u 1 { name => "Fastq multiread 2", ref => [ "AGCATCGATCAGTATCTGA" ], args => "-u 1", fastq => "\n\n\r\n\@r0\nCATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIIII\n\n". "\n\n\r\n\@r1\nATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIII\n\n", hits => [{ 2 => 1 }] }, { name => "Tabbed multiread 2", ref => [ "AGCATCGATCAGTATCTGA" ], args => "-u 1", tabbed => "\n\n\r\nr0\tCATCGATCAGTATCTG\tIIIIIIIIIIIIIIII\n\n". "\n\n\r\nr1\tATCGATCAGTATCTG\tIIIIIIIIIIIIIII\n\n", hits => [{ 2 => 1 }] }, { name => "Fasta multiread 2", ref => [ "AGCATCGATCAGTATCTGA" ], args => "-u 1", fasta => "\n\n\r\n>r0\nCATCGATCAGTATCTG\r\n". "\n\n\r\n>r1\nATCGATCAGTATCTG\r\n", hits => [{ 2 => 1 }] }, { name => "Qseq multiread 2", ref => [ "AGCATCGATCAGTATCTGA" ], args => "-u 1", qseq => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "10", "10", "Index", "0", # Mate "CATCGATCAGTATCTG", "IIIIIIIIIIIIIIII", "1")."\n\n". join("\t", "MachName", "RunNum", "Lane", "Tile", "12", "15", "Index", "0", # Mate "ATCGATCAGTATCTG", "IIIIIIIIIIIIIII", "1")."\n\n", idx_map => { "MachName_RunNum_Lane_Tile_10_10_Index" => 0, "MachName_RunNum_Lane_Tile_12_15_Index" => 1 }, hits => [{ 2 => 1 }] }, { name => "Raw multiread 2", ref => [ "AGCATCGATCAGTATCTGA" ], args => "-u 1", raw => "\n\n\r\nCATCGATCAGTATCTG\r\n". "\n\n\r\nATCGATCAGTATCTG\r\n", hits => [{ 2 => 1 }] }, # Like Fastq multiread 1 but with -u 2 { name => "Fastq multiread 3", ref => [ "AGCATCGATCAGTATCTGA" ], args => "-u 2", fastq => "\n\n\r\n\@r0\nCATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIIII\n\n". "\n\n\r\n\@r1\nATCGATCAGTATCTG\r\n+\n\nIIIIIIIIIIIIIII\n\n", hits => [{ 2 => 1 }, { 3 => 1 }] }, { name => "Tabbed multiread 3", ref => [ "AGCATCGATCAGTATCTGA" ], args => "-u 2", tabbed => "\n\n\r\nr0\tCATCGATCAGTATCTG\tIIIIIIIIIIIIIIII\n\n". "\n\n\r\nr1\tATCGATCAGTATCTG\tIIIIIIIIIIIIIII\n\n", hits => [{ 2 => 1 }, { 3 => 1 }] }, { name => "Fasta multiread 3", ref => [ "AGCATCGATCAGTATCTGA" ], args => "-u 2", fasta => "\n\n\r\n>r0\nCATCGATCAGTATCTG\r\n". "\n\n\r\n>r1\nATCGATCAGTATCTG\r\n", hits => [{ 2 => 1 }, { 3 => 1 }] }, { name => "Qseq multiread 3", ref => [ "AGCATCGATCAGTATCTGA" ], args => "-u 2", qseq => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "10", "10", "Index", "0", # Mate "CATCGATCAGTATCTG", "IIIIIIIIIIIIIIII", "1")."\n\n". join("\t", "MachName", "RunNum", "Lane", "Tile", "12", "15", "Index", "0", # Mate "ATCGATCAGTATCTG", "IIIIIIIIIIIIIII", "1")."\n\n", idx_map => { "MachName_RunNum_Lane_Tile_10_10_Index" => 0, "MachName_RunNum_Lane_Tile_12_15_Index" => 1 }, hits => [{ 2 => 1 }, { 3 => 1 }] }, { name => "Raw multiread 3", ref => [ "AGCATCGATCAGTATCTGA" ], args => "-u 2", raw => "\n\n\r\nCATCGATCAGTATCTG\r\n". "\n\n\r\nATCGATCAGTATCTG\r\n", hits => [{ 2 => 1 }, { 3 => 1 }] }, # Paired-end reads that should align { name => "Fastq paired 1", ref => [ "AGCATCGATCAAAAACTGA" ], # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 fastq1 => "\n\n\r\n\@r0\nAGCATCGATC\r\n+\n\nIIIIIIIIII\n\n". "\n\n\@r1\nTCAGTTTTTGA\r\n+\n\nIIIIIIIIIII\n\n", fastq2 => "\n\n\r\n\@r0\nTCAGTTTTTGA\n+\n\nIIIIIIIIIII\n\n". "\n\n\r\n\@r1\nAGCATCGATC\r\n+\n\nIIIIIIIIII", pairhits => [ { "0,8" => 1 }, { "0,8" => 1 } ] }, { name => "Tabbed paired 1", ref => [ "AGCATCGATCAAAAACTGA" ], # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 tabbed => "\n\n\r\nr0\tAGCATCGATC\tIIIIIIIIII\tTCAGTTTTTGA\tIIIIIIIIIII\n\n". "\n\nr1\tTCAGTTTTTGA\tIIIIIIIIIII\tAGCATCGATC\tIIIIIIIIII\n\n", paired => 1, pairhits => [ { "0,8" => 1 }, { "0,8" => 1 } ] }, { name => "Fasta paired 1", ref => [ "AGCATCGATCAAAAACTGA" ], # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 fasta1 => "\n\n\r\n>r0\nAGCATCGATC\r\n". "\n\n>r1\nTCAGTTTTTGA\r\n", fasta2 => "\n\n\r\n>r0\nTCAGTTTTTGA\n". "\n\n\r\n>r1\nAGCATCGATC", pairhits => [ { "0,8" => 1 }, { "0,8" => 1 } ] }, { name => "Qseq paired 1", ref => [ "AGCATCGATCAAAAACTGA" ], # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 qseq1 => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "10", "10", "Index", "1", # Mate "AGCATCGATC", "ABCBGACBCB", "1")."\n\n". join("\t", "MachName", "RunNum", "Lane", "Tile", "12", "15", "Index", "1", # Mate "TCAGTTTTTGA", "95849456875", "1")."\n\n", qseq2 => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "10", "10", "Index", "2", # Mate "TCAGTTTTTGA", "ABCBGACBCBA", "1")."\n\n". join("\t", "MachName", "RunNum", "Lane", "Tile", "12", "15", "Index", "2", # Mate "AGCATCGATC", "AGGCBBGCBG", "1")."\n\n", idx_map => { "MachName_RunNum_Lane_Tile_10_10_Index" => 0, "MachName_RunNum_Lane_Tile_12_15_Index" => 1 }, pairhits => [ { "0,8" => 1 }, { "0,8" => 1 } ] }, { name => "Raw paired 1", ref => [ "AGCATCGATCAAAAACTGA" ], # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 raw1 => "\n\n\r\nAGCATCGATC\r\n". "\n\nTCAGTTTTTGA\r\n", raw2 => "\n\n\r\nTCAGTTTTTGA\n". "\n\n\r\nAGCATCGATC", pairhits => [ { "0,8" => 1 }, { "0,8" => 1 } ] }, # Paired-end reads that should align { name => "Fastq paired 2", ref => [ "AGCATCGATCAAAAACTGA" ], args => "-s 1", # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 fastq1 => "\@r0\nAGCATCGATC\r\n+\n\nIIIIIIIIII\n\n". "\n\n\@r1\nTCAGTTTTTGA\n+\n\nIIIIIIIIIII\n\n", fastq2 => "\n\n\r\n\@r0\nTCAGTTTTTGA\n+\n\nIIIIIIIIIII\n\n". "\n\n\r\n\@r1\nAGCATCGATC\r\n+\n\nIIIIIIIIII", pairhits => [ { }, { "0,8" => 1 } ] }, { name => "Tabbed paired 2", ref => [ "AGCATCGATCAAAAACTGA" ], args => "-s 1", # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 tabbed => "r0\tAGCATCGATC\tIIIIIIIIII\tTCAGTTTTTGA\tIIIIIIIIIII\n\n". "\nr1\tTCAGTTTTTGA\tIIIIIIIIIII\tAGCATCGATC\tIIIIIIIIII", paired => 1, pairhits => [ { }, { "0,8" => 1 } ] }, { name => "Fasta paired 2", ref => [ "AGCATCGATCAAAAACTGA" ], args => "-s 1", # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 fasta1 => ">r0\nAGCATCGATC\r\n". "\n\n>r1\nTCAGTTTTTGA\n", fasta2 => "\n\n\r\n>r0\nTCAGTTTTTGA\n". "\n\n\r\n>r1\nAGCATCGATC", pairhits => [ { }, { "0,8" => 1 } ] }, { name => "Qseq paired 1", ref => [ "AGCATCGATCAAAAACTGA" ], # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 args => "-s 1", qseq1 => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "10", "10", "Index", "1", # Mate "AGCATCGATC", "ABCBGACBCB", "1")."\n\n". join("\t", "MachName", "RunNum", "Lane", "Tile", "12", "15", "Index", "1", # Mate "TCAGTTTTTGA", "95849456875", "1")."\n\n", qseq2 => "\n\n\n".join("\t", "MachName", "RunNum", "Lane", "Tile", "10", "10", "Index", "2", # Mate "TCAGTTTTTGA", "ABCBGACBCBA", "1")."\n\n". join("\t", "MachName", "RunNum", "Lane", "Tile", "12", "15", "Index", "2", # Mate "AGCATCGATC", "AGGCBBGCBG", "1")."\n\n", idx_map => { "MachName_RunNum_Lane_Tile_10_10_Index" => 0, "MachName_RunNum_Lane_Tile_12_15_Index" => 1 }, pairhits => [ { }, { "0,8" => 1 } ] }, { name => "Raw paired 2", ref => [ "AGCATCGATCAAAAACTGA" ], args => "-s 1", # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 raw1 => "AGCATCGATC\r\n". "\n\nTCAGTTTTTGA\n", raw2 => "\n\n\r\nTCAGTTTTTGA\n". "\n\n\r\nAGCATCGATC", pairhits => [ { }, { "0,8" => 1 } ] }, # Paired-end reads that should align { name => "Fastq paired 3", ref => [ "AGCATCGATCAAAAACTGA" ], args => "-u 1", # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 fastq1 => "\n\n\r\n\@r0\nAGCATCGATC\r\n+\n\nIIIIIIIIII\n\n". "\n\n\@r1\nTCAGTTTTTGA\r\n+\n\nIIIIIIIIIII\n\n", fastq2 => "\n\n\r\n\@r0\nTCAGTTTTTGA\n+\n\nIIIIIIIIIII\n\n". "\n\n\r\n\@r1\nAGCATCGATC\r\n+\nIIIIIIIIII", pairhits => [ { "0,8" => 1 }, { } ] }, { name => "Tabbed paired 3", ref => [ "AGCATCGATCAAAAACTGA" ], args => "-u 1", # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 tabbed => "\n\n\r\nr0\tAGCATCGATC\tIIIIIIIIII\tTCAGTTTTTGA\tIIIIIIIIIII\n\n". "\n\nr1\tTCAGTTTTTGA\tIIIIIIIIIII\tAGCATCGATC\tIIIIIIIIII", paired => 1, pairhits => [ { "0,8" => 1 }, { } ] }, { name => "Fasta paired 3", ref => [ "AGCATCGATCAAAAACTGA" ], args => "-u 1", # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 fasta1 => "\n\n\r\n>r0\nAGCATCGATC\r\n". "\n\n>r1\nTCAGTTTTTGA\r\n", fasta2 => "\n\n\r\n>r0\nTCAGTTTTTGA\n". "\n\n\r\n>r1\nAGCATCGATC", pairhits => [ { "0,8" => 1 }, { } ] }, { name => "Raw paired 3", ref => [ "AGCATCGATCAAAAACTGA" ], args => "-u 1", # AGCATCGATC # TCAAAAACTGA # 0123456789012345678 raw1 => "\n\n\r\nAGCATCGATC\r\n". "\n\nTCAGTTTTTGA\r\n", raw2 => "\n\n\r\nTCAGTTTTTGA\n". "\n\n\r\nAGCATCGATC", pairhits => [ { "0,8" => 1 }, { } ] }, # Paired-end reads that should align #{ name => "Fastq paired 4", # ref => [ "AGCATCGATCAAAAACTGA" ], # args => "-s 1 -L 4 -i C,1,0", # # AGCATCGATC # # TCAAAAACTGA # # 0123456789012345678 # fastq1 => "\n\n\r\n\@r0\nAGCATCGATC\r\n+\n\nIIIIIIIIII\n\n". # #"\n\n\@r1\nTC\r\n+\n\nII\n\n". # "\n\n\@r2\nTCAGTTTTTGA\r\n+\n\nIIIIIIIIIII\n\n", # fastq2 => "\n\n\r\n\@r0\nTCAGTTTTTGA\n+\n\nIIIIIIIIIII\n\n". # #"\n\n\r\n\@r1\nAG\r\n+\nII". # "\n\@r2\nAGCATCGATC\r\n+\nIIIIIIIIII", # paired => 1, # pairhits => [ { }, { "*,*" => 1 }, { "0,8" => 1 } ], # pairhits => [ { "0,8" => 1 } ], # samoptflags_map => [ # { }, # { "*" => { "YT:Z:UP" => 1, "YF:Z:LN" => 1 } }, ## { 0 => { "MD:Z:10" => 1, "YT:Z:CP" => 1 }, # 8 => { "MD:Z:11" => 1, "YT:Z:CP" => 1 } }] #}, #{ name => "Tabbed paired 4", # ref => [ "AGCATCGATCAAAAACTGA" ], # args => "-s 1 -L 4 -i C,1,0", # # AGCATCGATC # # TCAAAAACTGA # # 0123456789012345678 # tabbed => "\n\n\r\nr0\tAGCATCGATC\tIIIIIIIIII\tTCAGTTTTTGA\tIIIIIIIIIII\n\n". # "\n\nr1\tTC\tII\tAG\tII". # "\n\nr2\tTCAGTTTTTGA\tIIIIIIIIIII\tAGCATCGATC\tIIIIIIIIII\n\n", # paired => 1, # #pairhits => [ { }, { "*,*" => 1 }, { "0,8" => 1 } ], # pairhits => [ { }, { "0,8" => 1 } ], # samoptflags_map => [ # { }, # #{ "*" => { "YT:Z:UP" => 1, "YF:Z:LN" => 1 } }, # { 0 => { "MD:Z:10" => 1, "YT:Z:CP" => 1 }, # 8 => { "MD:Z:11" => 1, "YT:Z:CP" => 1 } }] #}, #{ name => "Fasta paired 4", # ref => [ "AGCATCGATCAAAAACTGA" ], # args => "-s 1 -L 4 -i C,1,0", # # AGCATCGATC # # TCAAAAACTGA # # 0123456789012345678 # fasta1 => "\n\n\r\n>r0\nAGCATCGATC\r\n". # # "\n\n>r1\nTC\r\n". # "\n\n>r2\nTCAGTTTTTGA\r\n", # fasta2 => "\n\n\r\n>r0\nTCAGTTTTTGA\n\n". # # "\n\n\r\n>r1\nAG". # "\n>r2\nAGCATCGATC", # # pairhits => [ { }, { "*,*" => 1 }, { "0,8" => 1 } ], # pairhits => [ { }, { "0,8" => 1 } ], # samoptflags_map => [ # { }, # #{ "*" => { "YT:Z:UP" => 1, "YF:Z:LN" => 1 } }, # { 0 => { "MD:Z:10" => 1, "YT:Z:CP" => 1 }, # 8 => { "MD:Z:11" => 1, "YT:Z:CP" => 1 } }] #}, #{ name => "Raw paired 4", # ref => [ "AGCATCGATCAAAAACTGA" ], # args => "-s 1 -L 4 -i C,1,0", # # AGCATCGATC # # TCAAAAACTGA # # 0123456789012345678 # raw1 => "\n\n\r\nAGCATCGATC\r\n". ## "\n\nTC\r\n". # "\n\nTCAGTTTTTGA\r\n", # raw2 => "\n\n\r\nTCAGTTTTTGA\n\n". # "\n\n\r\nAG". # "\nAGCATCGATC", # pairhits => [ { }, { "*,*" => 1 }, { "0,8" => 1 } ], # pairhits => [ { }, { "0,8" => 1 } ], # samoptflags_map => [ # { }, # { "*" => { "YT:Z:UP" => 1, "YF:Z:LN" => 1 } }, # { 0 => { "MD:Z:10" => 1, "YT:Z:CP" => 1 }, # 8 => { "MD:Z:11" => 1, "YT:Z:CP" => 1 } }] #}, # # Check that skipping of empty reads is handled correctly. A read that is # empty or becomes empty after --trim3/--trim5 are applied should still # count as a first-class read that gets propagated up into the alignment # loop. And it should be counted in the -s/-u totals. # { ref => [ "AGCATCGATCAGTATCTGA" ], reads => [ "", "ATCGATCAGTA" ], args => "-s 1", hits => [ {}, { 3 => 1 }] }, { ref => [ "AGCATCGATCAGTATCTGA" ], mate1s => [ "", "AGCATCGATC" ], mate2s => [ "", "TCAGATACTG" ], args => "-s 1", pairhits => [ {}, { "0,9" => 1 }] }, { ref => [ "AGCATCGATCAGTATCTGA" ], reads => [ "", "ATCGATCAGTA" ], args => "-s 2", hits => [ {}, {} ] }, { ref => [ "AGCATCGATCAGTATCTGA" ], mate1s => [ "", "AGCATCGATC" ], mate2s => [ "", "TCAGATACTG" ], args => "-s 2", pairhits => [ {}, {} ] }, { ref => [ "AGCATCGATCAGTATCTGA" ], reads => [ "", "ATCGATCAGTA", "AGTATCTGA" ], args => "-s 1 -u 1", hits => [ {}, { 3 => 1 }] }, { ref => [ "AGCATCGATCAGTATCTGA" ], reads => [ "AC", "ATCGATCAGTA" ], args => "-s 1 --trim3 2", norc => 1, hits => [ {}, { 3 => 1 }] }, { ref => [ "AGCATCGATCAGTATCTGA" ], reads => [ "AC", "ATCGATCAGTA" ], args => "-s 1 --trim3 2", nofw => 1, hits => [ {}, { 5 => 1 }] }, { ref => [ "AGCATCGATCAGTATCTGA" ], reads => [ "AC", "ATCGATCAGTA" ], args => "-s 1 --trim5 2", nofw => 1, hits => [ {}, { 3 => 1 }] }, { ref => [ "AGCATCGATCAGTATCTGA" ], reads => [ "AC", "ATCGATCAGTA" ], args => "-s 1 --trim5 2", norc => 1, hits => [ {}, { 5 => 1 }] }, # # Alignment with overhang # { ref => [ "TGC" ], reads => [ "ATGC" ], args => "--overhang --policy \"SEED=0,3\\;IVAL=C,1,0\\;NCEIL=L,1,0\"", hits => [ { 0 => 1 } ], cigar => [ "1S3M" ], samoptflags => [ { "AS:i:-1" => 1, "YT:Z:UU" => 1, "MD:Z:3" => 1, "XN:i:1" => 1 } ] }, { ref => [ "TTGTTCGT" ], reads => [ "TTGTTCG" ], args => "--policy \"SEED=0,2\\;IVAL=C,1,0\\;NCEIL=L,2,0\"", hits => [ { 0 => 1 } ], cigar => [ "7M" ], samoptflags => [ { "AS:i:0" => 1, "YT:Z:UU" => 1, "MD:Z:7" => 1 } ] }, { ref => [ "TTGTTCGT" ], reads => [ "TTGTTCG" ], args => "", hits => [ { 0 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:7=" ], cigar => [ "7M" ], samoptflags => [ { "AS:i:0" => 1, "YT:Z:UU" => 1, "MD:Z:7" => 1 } ] }, { ref => [ "TTGTTCGT" ], reads => [ "TGTTCGT", "TTGTTCG" ], args => "--overhang", hits => [ { 1 => 1 }, { 0 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:7=", "XM:0,XP:0,XT:UU,XC:7=" ], cigar => [ "7M", "7M" ], samoptflags => [ { "YT:Z:UU" => 1, "MD:Z:7" => 1 }, { "YT:Z:UU" => 1, "MD:Z:7" => 1 } ] }, { ref => [ "TTGTTCGT" ], reads => [ "TGTTCGT", "TTGTTCG" ], args => "", hits => [ { 1 => 1 }, { 0 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:7=", "XM:0,XP:0,XT:UU,XC:7=" ], cigar => [ "7M", "7M" ], samoptflags => [ { "YT:Z:UU" => 1, "MD:Z:7" => 1 }, { "YT:Z:UU" => 1, "MD:Z:7" => 1 } ] }, # Reads 1 and 2 don't have overhang, reads 3 and 4 overhang opposite ends { ref => [ "TTGTTCGT" ], # TGTTCGT # GTTCGTA # ATTGTTC reads => [ "TGTTCGT", "GTTCGTA", "ATTGTTC" ], args => "--overhang --policy \"SEED=0,2\\;IVAL=C,1,0\\;NCEIL=L,2,0\"", hits => [ { 1 => 1 }, { 2 => 1 }, { 0 => 1 } ], cigar => [ "7M", "6M1S", "1S6M" ], samoptflags => [ { "YT:Z:UU" => 1, "MD:Z:7" => 1 }, { "AS:i:-1" => 1, "XN:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:6" => 1 }, { "AS:i:-1" => 1, "XN:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:6" => 1 } ]}, # Same as previous case but --overhang not specified { ref => [ "TTGTTCGT" ], reads => [ "TGTTCGT", "TTGTTCG", "GTTCGTA", "ATTGTTC" ], args => "--policy \"SEED=0,2\\;IVAL=C,1,0\\;NCEIL=L,2,0\"", hits => [ { 1 => 1 }, { 0 => 1 } ], # only the internal hits cigar => [ "7M", "7M", "*", "*" ], samoptflags => [ { "YT:Z:UU" => 1, "MD:Z:7" => 1 }, { "YT:Z:UU" => 1, "MD:Z:7" => 1 }, { "YT:Z:UU" => 1 }, { "YT:Z:UU" => 1 } ] }, # A simple case that should align with or without overhang, with or without # a special NCEIL setting. { ref => [ "TTGTTCGT" ], reads => [ "TTGTTCG" ], args => "--overhang --policy \"SEED=0,2\\;IVAL=C,1,0\\;NCEIL=L,2,0\"", hits => [ { 0 => 1 } ]}, { ref => [ "TTGTTCGT" ], reads => [ "TTGTTCG" ], args => "--overhang", hits => [ { 0 => 1 } ]}, # # Testing the various -M/-m/-k/-a alignment modes in both unpaired and # paired-end modes. Ensuring that SAM optional flags such as YM:i, YP:i # are set properly in all cases. # # # Paired-end # { name => "P.M.58.G.b Unpaired -M 5 w/ 8 hits global, but mate #1 has just 1", # 0 1 2 3 0 1 2 0 1 2 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 0123456789012345678901234567 0123456789012345678901234567 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGT" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 # 0 1 2 3 4 5 6 7 8 9 # 0 0 0 0 0 0 0 0 0 0 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped args => "-X 1000", report => "-M 5", pairhits => [{ "12,78" => 1, "12,249" => 1, "12,315" => 1, "12,486" => 1, "12,552" => 1, "12,723" => 1, "12,789" => 1, "12,960" => 1 }], hits_are_superset => [ 1 ], cigar_map => [{ 12 => "33M", 78 => "28M", 249 => "28M", 315 => "28M", 486 => "28M", 552 => "28M", 723 => "28M", 789 => "28M", 960 => "28M" }], samoptflags_map => [ { 12 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 78 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 249 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 315 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 486 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 552 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 723 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 789 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 960 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, } ] }, { name => "P.M.58.L.b Unpaired -M 5 w/ 8 hits local, but mate #1 has just 1", # 0 1 2 3 0 1 2 0 1 2 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 0123456789012345678901234567 0123456789012345678901234567 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGT" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 # 0 1 2 3 4 5 6 7 8 9 # 0 0 0 0 0 0 0 0 0 0 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped args => "-X 1000 --local", report => "-M 5", pairhits => [{ "12,78" => 1, "12,249" => 1, "12,315" => 1, "12,486" => 1, "12,552" => 1, "12,723" => 1, "12,789" => 1, "12,960" => 1 }], hits_are_superset => [ 1 ], cigar_map => [{ 12 => "33M", 78 => "28M", 249 => "28M", 315 => "28M", 486 => "28M", 552 => "28M", 723 => "28M", 789 => "28M", 960 => "28M" }], samoptflags_map => [ { 12 => { "AS:i:66" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 78 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 249 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 315 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 486 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 552 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 723 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 789 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 960 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, } ] }, { name => "P.k.58.G.b Unpaired -k 5 w/ 8 hits global, but mate #1 has just 1", # 0 1 2 3 0 1 2 0 1 2 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 0123456789012345678901234567 0123456789012345678901234567 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGT" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 # 0 1 2 3 4 5 6 7 8 9 # 0 0 0 0 0 0 0 0 0 0 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped args => "-X 1000", report => "-k 5", pairhits => [{ "12,78" => 1, "12,249" => 1, "12,315" => 1, "12,486" => 1, "12,552" => 1, "12,723" => 1, "12,789" => 1, "12,960" => 1 }], hits_are_superset => [ 1 ], cigar_map => [{ 12 => "33M", 78 => "28M", 249 => "28M", 315 => "28M", 486 => "28M", 552 => "28M", 723 => "28M", 789 => "28M", 960 => "28M" }], samoptflags_map => [ { 12 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 78 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 249 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 315 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 486 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 552 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 723 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 789 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 960 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, } ] }, { name => "P.k.58.L.b Unpaired -k 5 w/ 8 hits local, but mate #1 has just 1", # 0 1 2 3 0 1 2 0 1 2 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 0123456789012345678901234567 0123456789012345678901234567 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACACACACCCCTATAGCTCGGAGCTGACTGGATCGACGACGT" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 # 0 1 2 3 4 5 6 7 8 9 # 0 0 0 0 0 0 0 0 0 0 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped args => "-X 1000 --local", report => "-k 5", pairhits => [{ "12,78" => 1, "12,249" => 1, "12,315" => 1, "12,486" => 1, "12,552" => 1, "12,723" => 1, "12,789" => 1, "12,960" => 1 }], hits_are_superset => [ 1 ], cigar_map => [{ 12 => "33M", 78 => "28M", 249 => "28M", 315 => "28M", 486 => "28M", 552 => "28M", 723 => "28M", 789 => "28M", 960 => "28M" }], samoptflags_map => [ { 12 => { "AS:i:66" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 78 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 249 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 315 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 486 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 552 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 723 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 789 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 960 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, } ] }, { name => "P.M.22.G. Paired -M 2 w/ 2 paired hit, 2 unpaired hits each, global", # 0 1 2 3 0 1 2 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 # 0 1 2 3 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped mate1fw => 1, mate2fw => 0, args => "-X 150", report => "-M 2", pairhits => [ { "12,78" => 1, "249,315" => 1 } ], cigar_map => [{ 12 => "33M", 249 => "33M", 78 => "28M", 315 => "28M" }], hits_are_superset => [ 1 ], samoptflags_map => [{ 12 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 78 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 249 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 315 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, }] }, { name => "P.M.22.L. Paired -M 2 w/ 2 paired hit, 2 unpaired hits each, local", # 0 1 2 3 0 1 2 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 # 0 1 2 3 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped mate1fw => 1, mate2fw => 0, args => "--local -X 150", report => "-M 2", pairhits => [ { "12,78" => 1, "249,315" => 1 } ], cigar_map => [{ 12 => "33M", 249 => "33M", 78 => "28M", 315 => "28M" }], hits_are_superset => [ 1 ], samoptflags_map => [{ 12 => { "AS:i:66" => 1, "XS:i:66" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 78 => { "AS:i:56" => 1, "XS:i:56" => 1, "MD:Z:28" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 249 => { "AS:i:66" => 1, "XS:i:66" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 315 => { "AS:i:56" => 1, "XS:i:56" => 1, "MD:Z:28" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, }] }, { name => "P.k.2.G. Paired -k 1 w/ 2 paired hit, 2 unpaired hits each, global", # 0 1 2 3 0 1 2 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 # 0 1 2 3 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped mate1fw => 1, mate2fw => 0, args => "-X 150", report => "-k 1", pairhits => [ { "12,78" => 1, "249,315" => 1 } ], hits_are_superset => [ 1 ], cigar_map => [{ 12 => "33M", 249 => "33M", 78 => "28M", 315 => "28M" }], samoptflags_map => [{ 12 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 78 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 249 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 315 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, }] }, { name => "P.k.2.L. Paired -k 1 w/ 2 paired hit, 2 unpaired hits each, local", # 0 1 2 3 0 1 2 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 # 0 1 2 3 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped mate1fw => 1, mate2fw => 0, args => "--local -X 150", report => "-k 1", pairhits => [ { "12,78" => 1, "249,315" => 1 } ], hits_are_superset => [ 1 ], cigar_map => [{ 12 => "33M", 249 => "33M", 78 => "28M", 315 => "28M" }], samoptflags_map => [{ 12 => { "AS:i:66" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 78 => { "AS:i:56" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 249 => { "AS:i:66" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 315 => { "AS:i:56" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, }] }, { name => "P.M.2.G. Paired -M 1 w/ 2 paired hit, 2 unpaired hits each, global", # 0 1 2 3 0 1 2 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 # 0 1 2 3 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped mate1fw => 1, mate2fw => 0, report => "-M 1 -X 150", pairhits => [ { "12,78" => 1, "249,315" => 1 } ], hits_are_superset => [ 1 ], cigar_map => [{ 12 => "33M", 249 => "33M", 78 => "28M", 315 => "28M" }], samoptflags_map => [{ 12 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 78 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 249 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 315 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, }] }, { name => "P.M.2.L. Paired -M 1 w/ 2 paired hit, 2 unpaired hits each, local", # 0 1 2 3 0 1 2 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 # 0 1 2 3 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped mate1fw => 1, mate2fw => 0, report => "-M 1 --local -X 150", pairhits => [ { "12,78" => 1, "249,315" => 1 } ], hits_are_superset => [ 1 ], cigar_map => [{ 12 => "33M", 249 => "33M", 78 => "28M", 315 => "28M" }], samoptflags_map => [{ 12 => { "AS:i:66" => 1, "XS:i:66" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 78 => { "AS:i:56" => 1, "XS:i:56" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 249 => { "AS:i:66" => 1, "XS:i:66" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 315 => { "AS:i:56" => 1, "XS:i:56" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, }] }, { name => "P.k.1.G. Paired -k w/ 1 paired hit, 1 unpaired hit each, global", # 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345 # 0 1 2 3 4 5 6 7 8 9 0 1 2 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped mate1fw => 1, mate2fw => 0, report => "-k 1 -X 150", pairhits => [ { "12,78" => 1 } ], cigar_map => [{ 12 => "33M", 78 => "28M" }], samoptflags_map => [{ 12 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 78 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, }] }, { name => "P.k.1.L. Paired -k 1 w/ 1 paired hit, 1 unpaired hit each, local", # 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345 # 0 1 2 3 4 5 6 7 8 9 0 1 2 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped mate1fw => 1, mate2fw => 0, args => "--local -X 150", report => "-k 1", pairhits => [ { "12,78" => 1 } ], cigar_map => [{ 12 => "33M", 78 => "28M" }], samoptflags_map => [{ 12 => { "AS:i:66" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 78 => { "AS:i:56" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, }] }, { name => "P.M.1.G. Paired -M w/ 1 paired hit, 1 unpaired hit each, global", # 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345 # 0 1 2 3 4 5 6 7 8 9 0 1 2 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped mate1fw => 1, mate2fw => 0, args => "-X 150", report => "-M 1", pairhits => [ { "12,78" => 1 } ], cigar_map => [{ 12 => "33M", 78 => "28M" }], samoptflags_map => [{ 12 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 78 => { "AS:i:0" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, }] }, { name => "P.M.1.L. Paired -M w/ 1 paired hit, 1 unpaired hit each, local", # 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345 # 0 1 2 3 4 5 6 7 8 9 0 1 2 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped mate1fw => 1, mate2fw => 0, args => "-X 150 --local", report => "-M 1", pairhits => [ { "12,78" => 1 } ], cigar_map => [{ 12 => "33M", 78 => "28M" }], samoptflags_map => [{ 12 => { "AS:i:66" => 1, "XS:i:0" => 1, "MD:Z:33" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 78 => { "AS:i:56" => 1, "XS:i:0" => 1, "MD:Z:28" => 1, "YM:i:0" => 1, "YP:i:0" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, }] }, { name => "P.M.58.G. Unpaired -M 5 w/ 8 hits global", # 0 1 2 3 0 1 2 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGA" ], # 012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 # 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped args => "-X 150", report => "-M 5", pairhits => [ { "12,78" => 1, "249,315" => 1, "486,552" => 1, "723,789" => 1, "960,1026" => 1, "1197,1263" => 1, "1434,1500" => 1, "1671,1737" => 1, "1908,1974" => 1, "2145,2211" => 1, "2382,2448" => 1 } ], hits_are_superset => [ 1 ], cigar_map => [{ 12 => "33M", 78 => "28M", 249 => "33M", 315 => "28M", 486 => "33M", 552 => "28M", 723 => "33M", 789 => "28M", 960 => "33M", 1026 => "28M", 1197 => "33M", 1263 => "28M", 1434 => "33M", 1500 => "28M", 1671 => "33M", 1737 => "28M", 1908 => "33M", 1974 => "28M", 2145 => "33M", 2211 => "28M", 2382 => "33M", 2448 => "28M", }], samoptflags_map => [ { 12 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 78 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 249 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 315 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 486 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 552 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 723 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 789 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 960 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 1026 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 1197 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 1263 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 1434 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 1500 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 1671 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 1737 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 1908 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 1974 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 2145 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 2211 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 2382 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, 2448 => { "AS:i:0" => 1, "XS:i:0" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:0" => 1 }, } ] }, { name => "P.M.58.L. Unpaired -M 5 w/ 8 hits local", # 0 1 2 3 0 1 2 0 1 2 3 0 1 2 # 012345678901234567890123456789012 0123456789012345678901234567 012345678901234567890123456789012 0123456789012345678901234567 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG CAGCGTACGGTATCTAGCTATGGGCATCGATCG ACACACCCCTATAGCTCGGAGCTGACTG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGAGCGGTATCTACAGCCACTCATCACACACCCCTATAGCTCGGAGCTGACTGGGTTACTGGGGGGGATGCGTATCGACTATCGACAATATGACGCGTCGGTCACCCCATAATATGCAAAAATTATAGCTCACGACGCGTACTAATAGAAAACGCGCTATCAGCCTCCGACGCGGCGGTATCGAAGACGCAGTC" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 # 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 mate1s => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], mate2s => [ "CAGTCAGCTCCGAGCTATAGGGGTGTGT" ], # rev comped args => "--local -X 150", report => "-M 5", pairhits => [ { "12,78" => 1, "249,315" => 1, "486,552" => 1, "723,789" => 1, "960,1026" => 1, "1197,1263" => 1, "1434,1500" => 1, "1671,1737" => 1 } ], hits_are_superset => [ 1 ], cigar_map => [{ 12 => "33M", 78 => "28M", 249 => "33M", 315 => "28M", 486 => "33M", 552 => "28M", 723 => "33M", 789 => "28M", 960 => "33M", 1026 => "28M", 1197 => "33M", 1263 => "28M", 1434 => "33M", 1500 => "28M", 1671 => "33M", 1737 => "28M" }], samoptflags_map => [ { 12 => { "AS:i:66" => 1, "XS:i:66" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 78 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 249 => { "AS:i:66" => 1, "XS:i:66" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 315 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 486 => { "AS:i:66" => 1, "XS:i:66" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 552 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 723 => { "AS:i:66" => 1, "XS:i:66" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 789 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 960 => { "AS:i:66" => 1, "XS:i:66" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 1026 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 1197 => { "AS:i:66" => 1, "XS:i:66" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 1263 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 1434 => { "AS:i:66" => 1, "XS:i:66" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 1500 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, 1671 => { "AS:i:66" => 1, "XS:i:66" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:33" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:56" => 1 }, 1737 => { "AS:i:56" => 1, "XS:i:56" => 1, "XN:i:0" => 1, "XM:i:0" => 1, "XO:i:0" => 1, "XG:i:0" => 1, "NM:i:0" => 1, "MD:Z:28" => 1, "YM:i:1" => 1, "YP:i:1" => 1, "YT:Z:CP" => 1, "YS:i:66" => 1 }, } ] }, # # Unpaired # { name => "U.M.1.G. Unpaired -M w/ 1 hit global", # 0 1 2 3 # 012345678901234567890123456789012 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG # CAGCGTACGGTATCTAGCTATG # GGTATCTAGCTATGGGCATCGA # AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGA ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGA" ], # 01234567890123456789012345678901234567890123456789012345 # 0 1 2 3 4 5 reads => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], report => "-M 1", hits => [ { 12 => 1 } ], cigar => [ "33M" ], samoptflags => [{ "AS:i:0" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:33" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion }] }, { name => "U.M.1.L. Unpaired -M w/ 1 hit local", # 0 1 2 3 # 012345678901234567890123456789012 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGA" ], # 01234567890123456789012345678901234567890123456789012345 # 0 1 2 3 4 5 reads => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], args => "--local", report => "-M 1", hits => [ { 12 => 1 } ], cigar => [ "33M" ], samoptflags => [{ "AS:i:66" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:33" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion }] }, { name => "U.k.1.G. Unpaired -k 1 w/ 1 hit global", # 0 1 2 3 # 012345678901234567890123456789012 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGA" ], # 01234567890123456789012345678901234567890123456789012345 # 0 1 2 3 4 5 reads => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], report => "-k 1", hits => [ { 12 => 1 } ], cigar => [ "33M" ], samoptflags => [ { "AS:i:0" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:33" => 1, # mismatching positions/bases "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.M.1.L. Unpaired -m w/ 1 hit local", # 0 1 2 3 # 012345678901234567890123456789012 # CAGCGTACGGTATCTAGCTATGGGCATCGATCG ref => [ "AGACGCAGTCACCAGCGTACGGTATCTAGCTATGGGCATCGATCGACGACGTACGA" ], # 01234567890123456789012345678901234567890123456789012345 # 0 1 2 3 4 5 reads => [ "CAGCGTACGGTATCTAGCTATGGGCATCGATCG" ], args => "--local", report => "-k 1", hits => [ { 12 => 1 } ], cigar => [ "33M" ], samoptflags => [ { "AS:i:66" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:33" => 1, # mismatching positions/bases "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.M.2.G. Unpaired -M 1 w/ 2 hit global", # 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234 # 0 1 2 3 4 5 6 7 8 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], args => "", report => "-M 1", hits => [ { 6 => 1, 48 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "30M" ], samoptflags => [ { "AS:i:0" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:1" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.M.2.L. Unpaired -M 1 w/ 2 hit local", # 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234 # 0 1 2 3 4 5 6 7 8 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], args => "--local", report => "-M 1", hits => [ { 6 => 1, 48 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "30M" ], samoptflags => [ { "AS:i:60" => 1, # alignment score "XS:i:60" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:1" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.k.2.G. Unpaired -k 1 w/ 2 hit global", # 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234 # 0 1 2 3 4 5 6 7 8 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], report => "-k 1", hits => [ { 6 => 1, 48 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "30M" ], samoptflags => [ { "AS:i:0" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.k.2.L. Unpaired -k 1 w/ 2 hit local", # 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234 # 0 1 2 3 4 5 6 7 8 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], report => "-k 1", hits => [ { 6 => 1, 48 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "30M" ], samoptflags => [ { "AS:i:0" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.M.22.G. Unpaired -M 2 w/ 2 hit global", # 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234 # 0 1 2 3 4 5 6 7 8 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], report => "-M 2", hits => [ { 6 => 1, 48 => 1 } ], cigar => [ "30M" ], hits_are_superset => [ 1 ], samoptflags => [ { "AS:i:0" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.M.22.L. Unpaired -M 2 w/ 2 hit local", # 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234 # 0 1 2 3 4 5 6 7 8 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], report => "-M 2 --local", hits => [ { 6 => 1, 48 => 1 } ], cigar => [ "30M" ], hits_are_superset => [ 1 ], samoptflags => [ { "AS:i:60" => 1, # alignment score "XS:i:60" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.k.22.G. Unpaired -k 2 w/ 2 hit global", # 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234 # 0 1 2 3 4 5 6 7 8 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], report => "-k 2", hits => [ { 6 => 1, 48 => 1 } ], cigar => [ "30M" ], samoptflags => [ { "AS:i:0" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.k.22.L. Unpaired -k 2 w/ 2 hit local", # 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234 # 0 1 2 3 4 5 6 7 8 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], args => "--local", report => "-k 2", hits => [ { 6 => 1, 48 => 1 } ], cigar => [ "30M" ], samoptflags => [ { "AS:i:60" => 1, # alignment score "XS:i:60" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.M.58.G. Unpaired -M 5 w/ 8 hits global", # 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 # 0 1 2 3 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], args => "-X 150", report => "-M 5", hits => [ { 6 => 1, 48 => 1, 91 => 1, 133 => 1, 176 => 1, 218 => 1, 261 => 1, 303 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "30M" ], samoptflags => [ { "AS:i:0" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:1" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.M.58.L. Unpaired -M 5 w/ 8 hits global", # 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 # 0 1 2 3 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], args => "--local", report => "-M 5", hits => [ { 6 => 1, 48 => 1, 91 => 1, 133 => 1, 176 => 1, 218 => 1, 261 => 1, 303 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "30M" ], samoptflags => [ { "AS:i:60" => 1, # alignment score "XS:i:60" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:1" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.k.58.G. Unpaired -k 5 w/ 8 hits global", # 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 # 0 1 2 3 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], report => "-k 5", hits => [ { 6 => 1, 48 => 1, 91 => 1, 133 => 1, 176 => 1, 218 => 1, 261 => 1, 303 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "30M" ], samoptflags => [ { "AS:i:0" => 1, # alignment score "XS:i:0" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, { name => "U.k.58.L. Unpaired -k 5 w/ 8 hits local", # 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 0 1 2 # 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 012345678901234567890123456789 # AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA AGATTACGGATCTACGATTCGAGTCGGTCA ref => [ "AGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGAAGACGCAGATTACGGATCTACGATTCGAGTCGGTCAGTCACCAGCGTAAGATTACGGATCTACGATTCGAGTCGGTCAAGTGCGA" ], # 0123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789012345678901234567890123456789 # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 # 0 1 2 3 reads => [ "AGATTACGGATCTACGATTCGAGTCGGTCA" ], args => "--local", report => "-k 5", hits => [ { 6 => 1, 48 => 1, 91 => 1, 133 => 1, 176 => 1, 218 => 1, 261 => 1, 303 => 1 } ], hits_are_superset => [ 1 ], cigar => [ "30M" ], samoptflags => [ { "AS:i:60" => 1, # alignment score "XS:i:60" => 1, # suboptimal alignment score "XN:i:0" => 1, # num ambiguous ref bases "XM:i:0" => 1, # num mismatches "XO:i:0" => 1, # num gap opens "XG:i:0" => 1, # num gap extensions "NM:i:0" => 1, # num edits "MD:Z:30" => 1, # mismatching positions/bases "YM:i:0" => 1, # read aligned repetitively in unpaired fashion "YT:Z:UU" => 1, # unpaired read aligned in unpaired fashion } ] }, # Following cases depend on this being the case: # # static const float DEFAULT_CEIL_CONST = 3.0f; # static const float DEFAULT_CEIL_LINEAR = 3.0f; # Just enough budget for hits, so it should align { ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCAT" ], # budget = 3 + 8 * 3 = 27 args => "-L 6 -i C,1,0 --policy \"MMP=C27\\;MIN=L,-3,-3\\;RDG=25,15\\;RFG=25,15\"", # penalty = 27 report => "-a", hits => [ { 0 => 1, 8 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:6=1X1=" ], cigar => [ "8M" ], samoptflags => [ { "AS:i:-27" => 1, "XS:i:-27" => 1, "NM:i:1" => 1, "XM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:6G1" => 1 } ] }, # Not quite enough budget for hits, so it should NOT align { ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCAT" ], # budget = 3 + 8 * 3 = 27 args => "-L 6 -i C,1,0 --policy \"MMP=C28\\;MIN=L,-3,-3\\;RDG=25,15\\;RFG=25,15\"", # penalty = 28 report => "-a", hits => [ { "*" => 1 } ], flags => [ "XM:0,XP:0,XT:UU" ], cigar => [ "*" ], samoptflags => [ { "YT:Z:UU" => 1 } ] }, # Check that using a seed of length 1 with 1-mismatch doesn't crash. # Perhaps we should disallow it though? { ref => [ "AAAAAAAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCCCCCCCC" ], reads => [ "AA", "AA", "AA", "AA", "CC", "CC", "CC", "CC", "AA", "AA", "AA", "AA", "CC", "CC", "CC", "CC" ], names => [ "r1", "r1", "r1", "r1", "r2", "r2", "r2", "r2", "r3", "r3", "r3", "r3", "r4", "r4", "r4", "r4" ], args => "--policy \"SEED=1,1\"", check_random => 1, report => "-k 1" }, # # Gap penalties # # Alignment with 1 read gap { name => "Gap penalties 1", ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCTTTGTT" ], # budget = 3 + 12 * 3 = 39 args => "--policy \"MMP=C30\\;SEED=0,3\\;IVAL=C,1,0\\;RDG=29,10\\;RFG=25,15\\;MIN=L,-3,-3\"", report => "-a", hits => [ { 0 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:6=1D6=" ], cigar => [ "6M1D6M" ], samoptflags => [{ "AS:i:-39" => 1, "NM:i:1" => 1, "XO:i:1" => 1, "XG:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:6^G6" => 1 }] }, # Alignment with 1 read gap, but not enough budget { name => "Gap penalties 2", ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCTTTGTT" ], # budget = 3 + 12 * 3 = 39 args => "--policy \"MMP=C30\\;SEED=0,3\\;IVAL=C,1,0\\;RDG=30,10\\;RFG=25,15\\;MIN=L,-3,-3\"", report => "-a", hits => [ { "*" => 1 } ], flags => [ "XM:0,XP:0,XT:UU" ], cigar => [ "*" ], samoptflags => [{ "YT:Z:UU" => 1 }] }, # Alignment with 1 reference gap { name => "Gap penalties 3", ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCGATTTGTT" ], # budget = 3 + 14 * 3 = 45 args => "--policy \"MMP=C30\\;SEED=0,3\\;IVAL=C,1,0\\;RDG=25,15\\;RFG=30,15\\;MIN=L,-3,-3\"", report => "-a", hits => [ { 0 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:7=1I6=" ], cigar => [ "7M1I6M" ], samoptflags => [{ "AS:i:-45" => 1, "NM:i:1" => 1, "XO:i:1" => 1, "XG:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:13" => 1 }] }, # 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 # T T G T T C G T T T G T T C G T # 0 1 1 0 2 3 1 0 0 1 1 0 2 3 1 # 0 T x # 0 0 x # 1 T x # 1 1 x # 2 G x # 2 1 x # 3 T x # 3 0 x # 4 T x # 4 2 x # 5 C x # 5 3 x # 6 G # 6 2 x # 7 A # 7 3 x # 8 T x # 8 0 x # 9 T x # 9 0 x # 0 T x # 0 1 x # 1 G x # 1 1 x # 2 T x # 2 0 x # 3 T # # Alignment with 1 reference gap, but not enough budget { name => "Gap penalties 4", ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCGATTTGTT" ], # budget = 3 + 14 * 3 = 45 args => "--policy \"MMP=C30\\;SEED=0,3\\;IVAL=C,1,0\\;RDG=25,15\\;RFG=30,16\\;MIN=L,-3,-3\"", report => "-a", hits => [ { "*" => 1 } ], flags => [ "XM:0,XP:0,XT:UU" ], cigar => [ "*" ], samoptflags => [ { "YT:Z:UU" => 1 } ] }, # Alignment with 1 reference gap, but not enough budget { name => "Gap penalties 5", ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCGATTTGTT" ], # budget = 3 + 14 * 3 = 45 args => "--policy \"MMP=C30\\;SEED=0,3\\;IVAL=C,1,0\\;RDG=25,15\\;RFG=31,15\\;MIN=L,-3,-3\"", report => "-a", hits => [ { "*" => 1 } ], flags => [ "XM:0,XP:0,XT:UU" ], cigar => [ "*" ], samoptflags => [ { "YT:Z:UU" => 1 } ] }, # Alignment with 1 reference gap and 1 read gap { name => "Gap penalties 6", ref => [ "ATTGTTCGTTTGTTCGTA" ], reads => [ "ATTGTTGTTTGATTCGTA" ], # budget = 3 + 18 * 3 = 57 args => "--policy \"MMP=C30\\;SEED=0,3\\;IVAL=C,1,0\\;RDG=19,10\\;RFG=18,10\\;MIN=L,-3,-3\"", report => "-a", hits => [ { 0 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:6=1D5=1I6=" ], cigar => [ "6M1D5M1I6M" ] }, # Alignment with 1 reference gap and 1 read gap, but not enough budget { name => "Gap penalties 7", ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTGTTTGATTCGT" ], # budget = 3 + 16 * 3 = 51 args => "--policy \"MMP=C30\\;SEED=0,3\\;IVAL=C,1,0\\;RDG=16,10\\;RFG=16,10\\;MIN=L,-3,-3\"", report => "-a", hits => [ { "*" => 1 } ], flags => [ "XM:0,XP:0,XT:UU" ], cigar => [ "*" ] }, # Experiment with N filtering { name => "N filtering 1", ref => [ "GAGACTTTATACGCATCGAACTATCGCTCTA" ], reads => [ "ATACGCATCGAAC" ], # 0123456789012345678901234567890 # 1 2 3 args => "--policy \"NCEIL=L,0,0\"", report => "-a", hits => [ { 8 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:13=" ] }, { name => "N filtering 2", ref => [ "GAGACTTTATNCGCATCGAACTATCGCTCTA" ], reads => [ "ATACGCATCGAAC" ], # 0123456789012345678901234567890 # 1 2 3 args => "--policy \"NCEIL=L,0,0\"", report => "-a", hits => [ { "*" => 1 } ] }, { name => "N filtering 3", ref => [ "GAGACTTTATACGCATCGAANTATCGCTCTA" ], reads => [ "ATACGCATCGAAC" ], # 0123456789012345678901234567890 # 1 2 3 args => "--policy \"NCEIL=L,0,0\"", report => "-a", hits => [ { "*" => 1 } ] }, { name => "N filtering 4", ref => [ "GAGACTTTNTACGCATCGAACTATCGCTCTA" ], reads => [ "ATACGCATCGAAC" ], # 0123456789012345678901234567890 # 1 2 3 args => "--policy \"NCEIL=L,0,0\"", report => "-a", hits => [ { "*" => 1 } ] }, { name => "N filtering 5", ref => [ "GAGACTTTATNCGCATCGAACTATCGCTCTA" ], reads => [ "ATACGCATCGAAC" ], # 0123456789012345678901234567890 # 1 2 3 args => "--policy \"NCEIL=L,0,0.1\\;SEED=0,10\\;IVAL=C,1,0\"", report => "-a", hits => [ { 8 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:2=1X10=" ] }, { name => "N filtering 6", ref => [ "GAGACTTTNTACGCATCGAANTATCGCTCTA" ], reads => [ "ATACGCATCGAAC" ], # 0123456789012345678901234567890 # 1 2 3 args => "--policy \"NCEIL=L,0,0.1\\;SEED=0,10\\;IVAL=C,1,0\"", report => "-a", hits => [ { "*" => 1 } ] }, # No discordant alignment because one mate is repetitive. # Alignment with 1 reference gap { ref => [ "TTTTGTTCGTTTG" ], reads => [ "TTTTGTTCGATTTG" ], # budget = 3 + 14 * 3 = 45 args => "--policy \"SEED=0,8\\;IVAL=C,1,0\\;MMP=C30\\;RDG=25,15\\;RFG=25,20\\;MIN=L,-3,-3\"", report => "-a", hits => [ { 0 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:9=1I4=" ], cigar => [ "9M1I4M" ], samoptflags => [ { "AS:i:-45" => 1, "NM:i:1" => 1, "XO:i:1" => 1, "XG:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:13" => 1 }, ] }, # TTGTTCGTTTGTT # Tx # T x # G x # T x # T x # C x # G x # A x # T x # T x # T x # G x # T x # T x # Alignment with 1 reference gap { ref => [ "TTGTTCGTTTGTT" ], reads => [ "TTGTTCGATTTGTT" ], # budget = 3 + 14 * 3 = 45 args => "--policy \"SEED=0,3\\;IVAL=C,1,0\\;MMP=C30\\;RDG=25,15\\;RFG=25,20\\;MIN=L,-3,-3\"", report => "-a", hits => [ { 0 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:7=1I6=" ], cigar => [ "7M1I6M" ], samoptflags => [ { "AS:i:-45" => 1, "NM:i:1" => 1, "XO:i:1" => 1, "XG:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:13" => 1 }, ] }, { ref => [ "ACNCA" ], reads => [ "CA" ], args => "", report => "-a --policy \"SEED=0,2\\;IVAL=C,1,0\\;NCEIL=L,0,0\"", hits => [ { 3 => 1 } ], edits => [ ], flags => [ "XM:0,XP:0,XT:UU,XC:2=" ], cigar => [ "2M" ], samoptflags => [ { "YT:Z:UU" => 1, "MD:Z:2" => 1 }, ] }, { name => "N ceil = 0, 2 legit hits (1)", ref => [ "ACNCA" ], reads => [ "AC" ], args => "", report => "-a --policy \"SEED=0,2\\;IVAL=C,1,0\\;NCEIL=L,0,0\"", hits => [ { 0 => 1 } ], edits => [ ], flags => [ ] }, { name => "N ceil = 0, 2 legit hits (2)", ref => [ "ACNCANNNNNNNNCGNNNNNNNNCG" ], # 0123456789012345678901234 # 0 1 2 reads => [ "CG" ], args => "", report => "-a --policy \"SEED=0,2\\;IVAL=C,1,0\\;NCEIL=L,0,0\"", hits => [ { 13 => 2, 23 => 2 } ], edits => [ ], cigar => [ "2M", "2M" ], samoptflags => [ { "YT:Z:UU" => 1, "MD:Z:2" => 1 }, { "YT:Z:UU" => 1, "MD:Z:2" => 1 }, ] }, { ref => [ "ACNCANNNNNNAACGNNNNNNNACGAANNNNCGAAAN" ], # 0123456789012345678901234567890123456 # 0 1 2 3 reads => [ "CG" ], args => "", report => "-a --policy \"SEED=0,2\\;IVAL=C,1,0\\;NCEIL=L,0,0\"", hits => [ { 13 => 2, 23 => 2, 31 => 2 } ], edits => [ ], flags => [ "XM:0,XP:0,XT:UU,XC:2=", "XM:0,XP:0,XT:UU,XC:2=", "XM:0,XP:0,XT:UU,XC:2=" ], cigar => [ "2M", "2M", "2M" ], samoptflags => [ { "YT:Z:UU" => 1, "MD:Z:2" => 1 }, { "YT:Z:UU" => 1, "MD:Z:2" => 1 }, { "YT:Z:UU" => 1, "MD:Z:2" => 1 }, ] }, { ref => [ "ACNCANNNNNNAACGNNNNNNNACGAANNNNCGAAAN" ], # 0123456789012345678901234567890123456 # 0 1 2 3 reads => [ "CG" ], args => "", report => "-a --policy \"SEED=0,1\\;IVAL=C,1,0\\;NCEIL=L,0,0\"", hits => [ { 13 => 2, 23 => 2, 31 => 2 } ], edits => [ ], flags => [ "XM:0,XP:0,XT:UU,XC:2=", "XM:0,XP:0,XT:UU,XC:2=", "XM:0,XP:0,XT:UU,XC:2=" ], cigar => [ "2M", "2M", "2M" ], samoptflags => [ { "YT:Z:UU" => 1, "MD:Z:2" => 1 }, { "YT:Z:UU" => 1, "MD:Z:2" => 1 }, { "YT:Z:UU" => 1, "MD:Z:2" => 1 }, ] }, # # Alignment involving ambiguous reference character # # First read has non-compatible unambiguous charcacter (G for Y), # second read has compatible one { ref => [ "TTGTTYGT" ], reads => [ "TTGTTGGT", "TTGTTCGT" ], args => "", report => "-a --policy \"SEED=0,5\\;IVAL=C,1,0\\;NCEIL=L,2,0\"", hits => [ { 0 => 1 }, { 0 => 1 } ], norc => 1, edits => [ "5:N>G", "5:N>C" ], flags => [ "XM:0,XP:0,XT:UU,XC:5=1X2=", "XM:0,XP:0,XT:UU,XC:5=1X2=" ], cigar => [ "8M", "8M" ], samoptflags => [ { "AS:i:-1" => 1, "NM:i:1" => 1, "XM:i:1" => 1, "XN:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:5N2" => 1 }, { "AS:i:-1" => 1, "NM:i:1" => 1, "XM:i:1" => 1, "XN:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:5N2" => 1 }, ] }, # # Alignment with multi-character read gap # # Relatively small example with a read gap extend { ref => [ "ATAACCTTCG" ], reads => [ "ATAATTCG" ], # 3 * 19 + 3 = 60 # ^ # 4:CC>- args => "", report => "-a --overhang --gbar 3 --policy \"MMP=C30\\;RDG=5,5\\;SEED=0,4\\;IVAL=C,1,0\\;RFG=25,20\\;MIN=L,-3,-3\"", hits => [ { 0 => 1 } ], edits => [ "4:CC>-" ], flags => [ "XM:0,XP:0,XT:UU,XC:4=2D4=" ], cigar => [ "4M2D4M" ], samoptflags => [ { "AS:i:-15" => 1, "NM:i:2" => 1, "XO:i:1" => 1, "XG:i:2" => 3, "YT:Z:UU" => 1, "MD:Z:4^CC4" => 1 } ] }, # Reads 1 and 2 don't have overhang, reads 3 and 4 overhang opposite ends { ref => [ "ATATGCCCCATGCCCCCCTCCG" ], reads => [ "ATATGCCCCCCCCCCTCCG" ], # 3 * 19 + 3 = 60 # ^ # 9:ATG>- args => "--policy \"SEED=0,8\\;IVAL=C,1,0\\;MMP=C30\\;RDG=5,5\\;RFG=25,15\\;MIN=L,-3,-3\"", hits => [ { 0 => 1 } ], edits => [ "9:ATG>-" ], norc => 1, flags => [ "XM:0,XP:0,XT:UU,XC:9=3D10=" ], cigar => [ "9M3D10M" ], samoptflags => [ { "AS:i:-20" => 1, "NM:i:3" => 1, "XO:i:1" => 1, "XG:i:3" => 3, "YT:Z:UU" => 1, "MD:Z:9^ATG10" => 1 } ] }, # Reads 1 and 2 don't have overhang, reads 3 and 4 overhang opposite ends { ref => [ "ATATGCCCCATGCCCCCCTCCG" ], reads => [ "CGGAGGGGGGGGGGCATAT" ], # ATATGCCCCCCCCCCTCCG # ^ # 10:GTA>- args => "", report => "-a --overhang --policy \"SEED=0,8\\;IVAL=C,1,0\\;MMP=C30\\;RDG=5,5\\;RFG=25,20\\;MIN=L,-3,-3\"", hits => [ { 0 => 1 } ], edits => [ "10:GTA>-" ], norc => 1, flags => [ "XM:0,XP:0,XT:UU,XC:9=3D10=" ], cigar => [ "9M3D10M" ], samoptflags => [ { "AS:i:-20" => 1, "NM:i:3" => 1, "XO:i:1" => 1, "XG:i:3" => 3, "YT:Z:UU" => 1, "MD:Z:9^ATG10" => 1 } ] }, # 1 discordant alignment and one concordant alignment. Discordant because # the fragment is too long. { name => "Simple paired-end 13", ref => [ "TTTATAAAAATATTTCCCCCCCCCCCCCCTGTCGCTACCGCCCCCCCCCCC" ], # 012345678901234567890123456789012345678901234567890 # 0 1 2 3 4 5 # ATAAAAATAT GTCGCTACCG # ATAAAAATAT TGTCGCTACC # ATAAAAATAT CTGTCGCTAC # ATAAAAATAT CCTGTCGCTA # TAAAAATATT GTCGCTACCG # TAAAAATATT TGTCGCTACC # TAAAAATATT CTGTCGCTAC # TAAAAATATT CCTGTCGCTA # 012345678901234567890123456789012345678901234567890 # 0 1 2 3 4 5 # ----------------------------------- # 012345678901234567890123456789012345678901234567 # 0 1 2 3 4 mate1s => [ "ATAAAAATAT", "ATAAAAATAT", "ATAAAAATAT", "ATAAAAATAT", "TAAAAATATT", "TAAAAATATT", "TAAAAATATT", "TAAAAATATT", ], mate2s => [ "GTCGCTACCG", "TGTCGCTACC", "CTGTCGCTAC", "CCTGTCGCTA", "GTCGCTACCG", "TGTCGCTACC", "CTGTCGCTAC", "CCTGTCGCTA" ], mate1fw => 1, mate2fw => 1, args => "-I 0 -X 35", # Not really any way to flag an alignment as discordant pairhits => [ { "3,30" => 1 }, { "3,29" => 1 }, { "3,28" => 1 }, { "3,27" => 1 }, { "4,30" => 1 }, { "4,29" => 1 }, { "4,28" => 1 }, { "4,27" => 1 } ], flags => [ "XM:0,XP:0,XT:DP,XC:10=", "XM:0,XP:0,XT:DP,XC:10=", "XM:0,XP:0,XT:CP,XC:10=", "XM:0,XP:0,XT:CP,XC:10=", "XM:0,XP:0,XT:DP,XC:10=", "XM:0,XP:0,XT:CP,XC:10=", "XM:0,XP:0,XT:CP,XC:10=", "XM:0,XP:0,XT:CP,XC:10=" ] }, # 1 discordant alignment and one concordant alignment. Discordant because # the fragment is too long. { name => "Simple paired-end 12", ref => [ "TTTATAAAAATATTTCCCCCCCCCCCCCCGGGCCCGCCCGCCCCCCCCCCC" ], # ATAAAAATAT GGCCCGCCCG # ATAAAAATAT CCGGGCCCGC # 012345678901234567890123456789012345678901234567890 # 0 1 2 3 4 5 # ------------------------------------- # 012345678901234567890123456789012345678901234567 mate1s => [ "ATAAAAATAT", "ATAAAAATAT" ], mate2s => [ "GGCCCGCCCG", "CCGGGCCCGC" ], mate1fw => 1, mate2fw => 1, args => "-I 0 -X 36", # Not really any way to flag an alignment as discordant pairhits => [ { "3,30" => 1 }, { "3,27" => 1 } ], flags => [ "XM:0,XP:0,XT:DP,XC:10=", "XM:0,XP:0,XT:CP,XC:10=" ] }, # 1 discordant alignment. Discordant because the fragment is too long. { name => "Simple paired-end 11", ref => [ "TTTATAAAAATATTTCCCCCCCCCCCCCCCCGATCGCCCGCCCCCCCCCCC" ], # ATAAAAATAT CGATCGCCCG # 012345678901234567890123456789012345678901234567890 # 0 1 2 3 4 5 # ------------------------------------- # 012345678901234567890123456789012345678901234567 mate1s => [ "ATAAAAATAT" ], mate2s => [ "CGATCGCCCG" ], mate1fw => 1, mate2fw => 1, args => "-I 0 -X 36", # Not really any way to flag an alignment as discordant pairhits => [ { "3,30" => 1 } ], flags => [ "XM:0,XP:0,XT:DP,XC:10=" ] }, # 1 discordant alignment. Discordant because the fragment is too short. { name => "Simple paired-end 10", ref => [ "TTTATAAAAATATTTCCCCCCGATCGCCCGCCCCCCCCCCC" ], # ATAAAAATAT CGATCGCCCG # 01234567890123456789012345678901234567890 # 0 1 2 3 4 # --------------------------- # 012345678901234567890123456 mate1s => [ "ATAAAAATAT" ], mate2s => [ "CGATCGCCCG" ], mate1fw => 1, mate2fw => 1, args => "-I 28 -X 80", # Not really any way to flag an alignment as discordant pairhits => [ { "3,20" => 1 } ], flags => [ "XM:0,XP:0,XT:DP,XC:10=" ] }, # Like 6, but with -M limit { name => "Simple paired-end 9", ref => [ "CCCATATATATATCCTCCCATATATATATCCCTCCCCATATATATATCCCTTTTCCTTTCGCGCGCGCGTTTCCCCCCCCC" ], # ATATATATAT ATATATATAT ATATATATAT CGCGCGCGCG # 012345678901234567890123456789012345678901234567890123456789012345678901234567890 # 0 1 2 3 4 5 6 7 8 mate1s => [ "ATATATATAT" ], mate2s => [ "CGCGCGCGCG" ], mate1fw => 1, mate2fw => 0, args => "-I 0 -X 80", report => "-M 2", lines => 2, pairhits => [ { "3,59" => 1, "19,59" => 1, "37,59" => 1 } ], hits_are_superset => [ 1 ], flags => [ "XM:1,XP:1,XT:CP,XC:10=", "XM:1,XP:1,XT:CP,XC:10=" ] }, # Like 6, but without -m limit { name => "Simple paired-end 8", ref => [ "CCCATATATATATCCTCCCATATATATATCCCTTCCCATATATATATCCCTTTTTTTTTCGCGCGCGCGTTTCCCCCCCCC" ], # ATATATATAT ATATATATAT ATATATATAT CGCGCGCGCG # 012345678901234567890123456789012345678901234567890123456789012345678901234567890 # 0 1 2 3 4 5 6 7 8 mate1s => [ "ATATATATAT" ], mate2s => [ "CGCGCGCGCG" ], mate1fw => 1, mate2fw => 0, args => "-I 0 -X 80", pairhits => [ { "3,59" => 1, "19,59" => 1, "37,59" => 1 } ], flags => [ "XM:0,XP:0,XT:CP,XC:10=" ] }, # Paired-end read, but only one mate aligns { name => "Simple paired-end 2; no --no-mixed", ref => [ "CCCATATATATATCCCTTTTTTTCCCCCCCCCCTTCGCGCGCGCGTTTCCCCC" ], # ATATATATAT CGCGCGCGCG # 01234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 mate1s => [ "ATATATATAT" ], mate2s => [ "CCCCCGGGGG" ], mate1fw => 1, mate2fw => 1, args => "-I 0 -X 50 --nofw", nofw => 1, pairhits => [ { "*,3" => 1 } ], flags_map => [ { 3 => "XM:0,XP:0,XT:UP,XC:10=", "*" => "XM:0,XP:0,XT:UP" } ], cigar_map => [{ 3 => "10M", "*" => "*" }], samoptflags_map => [{ 3 => { "MD:Z:10" => 1, # mismatching positions/bases "YT:Z:UP" => 1, # type of alignment (concordant/discordant/etc) }, "*" => { "YT:Z:UP" => 1, # type of alignment (concordant/discordant/etc) } }] }, { name => "Simple paired-end 2; --no-mixed", ref => [ "CCCATATATATATCCCTTTTTTTCCCCCCCCTTTTCGCGCGCGCGTTTCCCCC" ], # ATATATATAT CGCGCGCGCG # 01234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 mate1s => [ "ATATATATAT" ], mate2s => [ "CCCCCGGGGG" ], mate1fw => 1, mate2fw => 1, args => "-I 0 -X 50 --no-mixed", pairhits => [ { "*,*" => 1 } ] }, # Simple paired-end alignment { name => "Simple paired-end 1", ref => [ "CCCATATATATATCCCTTTTTTTCCCCCCCCTTTTCGCGCGCGCGTTTTCCCC" ], # ATATATATAT CGCGCGCGCG # 01234567890123456789012345678901234567890123456789012 # 0 1 2 3 4 5 mate1s => [ "ATATATATAT" ], mate2s => [ "CGCGCGCGCG" ], mate1fw => 1, mate2fw => 1, args => "-I 0 -X 50", pairhits => [ { "3,35" => 1 } ], flags => [ "XM:0,XP:0,XT:CP,XC:10=" ], cigar_map => [{ 3 => "10M", 35 => "10M" }], samoptflags_map => [{ 3 => { "MD:Z:10" => 1, # mismatching positions/bases "YT:Z:CP" => 1, # type of alignment (concordant/discordant/etc) }, 35 => { "MD:Z:10" => 1, # mismatching positions/bases "YT:Z:CP" => 1, # type of alignment (concordant/discordant/etc) } }] }, # Check that pseudo-random generation is always the same for # same-sequence, same-name reads { ref => [ "AAAAAAAAAAAAAAAAAAAAAAAAACCCCCCCCCCCCCCCCCCCCCCCC" ], reads => [ "AA", "AA", "AA", "AA", "CC", "CC", "CC", "CC", "AA", "AA", "AA", "AA", "CC", "CC", "CC", "CC" ], names => [ "r1", "r1", "r1", "r1", "r2", "r2", "r2", "r2", "r3", "r3", "r3", "r3", "r4", "r4", "r4", "r4" ], args => "", check_random => 1, report => "-k 1" }, { ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTGTTCGT" ], report => "-M 1", hits => [ { 0 => 1, 8 => 1 } ], flags => [ "XM:1,XP:0,XT:UU,XC:8=" ], hits_are_superset => [ 1 ], cigar => [ "8M" ], samoptflags => [ { "YM:i:1" => 1, "YT:Z:UU" => 1, "MD:Z:8" => 1, "YM:i:1" => 1 } ], }, # Read 3 overhangs right end { ref => [ "TTGTTCGT" ], reads => [ "GTTCGTA" ], args => "--overhang --policy \"SEED=0,3\\;IVAL=C,1,0\\;NCEIL=L,2,0\"", hits => [ { 2 => 1 } ], flags => [ "XM:0,XP:0,XT:UU,XC:6=1X" ] }, # Mess with arguments # Default should be 1-mismatch, so this shouldn't align { ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTATTAGT" ], args => "", hits => [ { "*" => 1 } ], flags => [ "XM:0,XP:0,XT:UU" ], cigar => [ "*" ], samoptflags => [{ "YT:Z:UU" => 1 }], }, # Shouldn't align with 0 mismatches either { ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTATTAGT" ], args => "--policy SEED=0", hits => [ { "*" => 1 } ], flags => [ "XM:0,XP:0,XT:UU" ], cigar => [ "*" ], samoptflags => [{ "YT:Z:UU" => 1 }], }, # Should align with 0 mismatches if we can wedge a seed into the 2 # matching characters between the two mismatches. Here we fail to # wedge a length-3 seed in (there's no room) { ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTATTAGT" ], args => "--policy \"SEED=0,3\\;IVAL=C,1,0\\;MMP=C1\"", hits => [ { "*" => 1 } ], flags => [ "XM:0,XP:0,XT:UU" ], cigar => [ "*" ], samoptflags => [{ "YT:Z:UU" => 1 }], }, # Should align with 0 mismatches if we can wedge a seed into the 2 # matching characters between the two mismatches. Here we wedge a # length-2 seed in { ref => [ "TTGTTCGTTTGTTCGT" ], reads => [ "TTATTAGT" ], args => "--policy \"SEED=0,2\\;IVAL=C,1,0\\;MMP=C1\"", # # TTGTTCGTTTGTTCGT TTGTTCGTTTGTTCGT TTGTTCGTTTGTTCGT # || || || || | | || || # TTATTAGT TTATTAGT TTATTAGT # # TTGTTCGTTTGTTCGT TTGTTCGTTTGTTCGT TTGTTCGTTTGTTCGT # || | || | || || || # TTATTAGT TTATTAGT TTATTAGT # hits => [ { 0 => 1, 3 => 1, 4 => 1, 5 => 1, 7 => 1, 8 => 1} ], flag_map => [ { 0 => "XM:0,XP:0,XT:UU,XC:2=1X2=1X2=", 3 => "XM:0,XP:0,XT:UU,XC:2=2X1=3X", 4 => "XM:0,XP:0,XT:UU,XC:1=2X2=1X2=", 5 => "XM:0,XP:0,XT:UU,XC:3X2=2X1=", 7 => "XM:0,XP:0,XT:UU,XC:2=2X1=3X", 8 => "XM:0,XP:0,XT:UU,XC:2=1X2=1X2="} ], cigar_map => [ { 0 => "8M", 3 => "8M", 4 => "8M", 5 => "8M", 7 => "8M", 8 => "8M" } ], samoptflags_map => [{ 0 => { "AS:i:-2" => 1, "XS:i:-2" => 1, "NM:i:2" => 1, "XM:i:2" => 1, "YT:Z:UU" => 1, "MD:Z:2G2C2" => 1 }, 3 => { "AS:i:-5" => 1, "XS:i:-2" => 1, "NM:i:5" => 1, "XM:i:5" => 1, "YT:Z:UU" => 1, "MD:Z:2C0G1T0T0G0" => 1 }, 4 => { "AS:i:-3" => 1, "XS:i:-2" => 1, "NM:i:3" => 1, "XM:i:3" => 1, "YT:Z:UU" => 1, "MD:Z:1C0G2T2" => 1 }, 5 => { "AS:i:-5" => 1, "XS:i:-2" => 1, "NM:i:5" => 1, "XM:i:5" => 1, "YT:Z:UU" => 1, "MD:Z:0C0G0T2G0T1" => 1 }, 7 => { "AS:i:-5" => 1, "XS:i:-2" => 1, "NM:i:5" => 1, "XM:i:5" => 1, "YT:Z:UU" => 1, "MD:Z:2T0G1T0C0G0" => 1 }, 8 => { "AS:i:-2" => 1, "XS:i:-2" => 1, "NM:i:2" => 1, "XM:i:2" => 1, "YT:Z:UU" => 1, "MD:Z:2G2C2" => 1 }, }], }, ); ## # Take a list of reference sequences and write them to a temporary # FASTA file of the given name. # sub writeFasta($$) { my ($l, $fa) = @_; open(FA, ">$fa") || die "Could not open $fa for writing"; my $idx = 0; for(my $i = 0; $i < scalar(@$l); $i++) { print FA ">$idx\n".$l->[$i]."\n"; $idx++; } close(FA); } ## # Take a lists of named reads/mates and write them to appropriate # files. # sub writeReads($$$$$$$$$) { my ( $reads, $quals, $mate1s, $qual1s, $mate2s, $qual2s, $names, $fq1, $fq2) = @_; open(FQ1, ">$fq1") || die "Could not open '$fq1' for writing"; open(FQ2, ">$fq2") || die "Could not open '$fq2' for writing"; my $pe = (defined($mate1s) && $mate1s ne ""); if($pe) { for (0..scalar(@$mate1s)-1) { my $m1 = $mate1s->[$_]; my $m2 = $mate2s->[$_]; my $q1 = $qual1s->[$_]; my $q2 = $qual2s->[$_]; my $nm = $names->[$_]; defined($m1) || die; defined($m2) || die; $q1 = $q1 || ("I" x length($m1)); $q2 = $q2 || ("I" x length($m2)); $nm = $nm || "r$_"; print FQ1 "\@$nm/1\n$m1\n+\n$q1\n"; print FQ2 "\@$nm/2\n$m2\n+\n$q2\n"; } } else { for (0..scalar(@$reads)-1) { my $read = $reads->[$_]; defined($read) || die; my $qual = $quals->[$_]; my $nm = $names->[$_]; $qual = $qual || ("I" x length($read)); $nm = $nm || "r$_"; print FQ1 "\@$nm\n$read\n+\n$qual\n"; } } close(FQ1); close(FQ2); } ## # Run bowtie2 with given arguments # sub runbowtie2($$$$$$$$$$$$$$$$$$$$$) { my ( $do_build, $args, $color, $fa, $reportargs, #5 $read_file_format, $read_file, $mate1_file, $mate2_file, $reads, $quals, $mate1s, $qual1s, $mate2s, $qual2s, $names, $ls, $rawls, $header_ls, $raw_header_ls, $should_abort) = @_; $args .= " --quiet"; $reportargs = "-a" unless defined($reportargs); $args .= " -C" if $color; $args .= " $reportargs"; # Write the reference to a fasta file print "References:\n"; open(FA, $fa) || die; while() { print $_; } close(FA); if($do_build) { my $build_args = ($color ? "-C" : ""); my $cmd = "$bowtie2_build --quiet --sanity $build_args $fa .simple_tests.tmp"; print "$cmd\n"; system($cmd); ($? == 0) || die "Bad exitlevel from bowtie2-build: $?"; } my $pe = (defined($mate1s) && $mate1s ne ""); $pe = $pe || (defined($mate1_file)); my $mate1arg; my $mate2arg; my $readarg; my $formatarg = "-c"; my ($readstr, $m1str, $m2str) = (undef, undef, undef); $readstr = join(",", @$reads) if defined($reads); $m1str = join(",", @$mate1s) if defined($mate1s); $m2str = join(",", @$mate2s) if defined($mate2s); if(defined($read_file) || defined($mate1_file)) { defined($read_file_format) || die; my $ext = ""; if($read_file_format eq "fastq") { $formatarg = "-q"; $ext = ".fq"; } elsif($read_file_format eq "tabbed") { $formatarg = "--12"; $ext = ".tab"; } elsif($read_file_format eq "fasta") { $formatarg = "-f"; $ext = ".fa"; } elsif($read_file_format eq "qseq") { $formatarg = "--qseq"; $ext = "_qseq.txt"; } elsif($read_file_format eq "raw") { $formatarg = "-r"; $ext = ".raw"; } else { die "Bad format: $read_file_format"; } if(defined($read_file)) { # Unpaired open(RD, ">.simple_tests$ext") || die; print RD $read_file; close(RD); $readarg = ".simple_tests$ext"; } else { defined($mate1_file) || die; defined($mate2_file) || die; # Paired open(M1, ">.simple_tests.1$ext") || die; print M1 $mate1_file; close(M1); open(M2, ">.simple_tests.2$ext") || die; print M2 $mate2_file; close(M2); $mate1arg = ".simple_tests.1$ext"; $mate2arg = ".simple_tests.2$ext"; } } else { writeReads( $reads, $quals, $mate1s, $qual1s, $mate2s, $qual2s, $names, ".simple_tests.1.fq", ".simple_tests.2.fq"); $mate1arg = ".simple_tests.1.fq"; $mate2arg = ".simple_tests.2.fq"; $formatarg = "-q"; $readarg = $mate1arg; } my $cmd; if($pe) { # Paired-end case $cmd = "$bowtie2 --debug $args .simple_tests.tmp $formatarg -1 $mate1arg -2 $mate2arg"; } else { # Unpaired case $cmd = "$bowtie2 --debug $args .simple_tests.tmp $formatarg $readarg"; } print "$cmd\n"; open(BT, "$cmd |") || die "Could not open pipe '$cmd |'"; while() { print $_; chomp; if(substr($_, 0, 1) eq "@") { push @$header_ls, [ split(/\t/, $_, -1) ]; push @$raw_header_ls, $_; } else { push @$ls, [ split(/\t/, $_, -1) ]; push @$rawls, $_; } } close(BT); ($? == 0 || $should_abort) || die "bowtie2 aborted with exitlevel $?\n"; ($? != 0 || !$should_abort) || die "bowtie2 failed to abort!\n"; } ## # Compare a hash ref of expected SAM flags with a hash ref of observed SAM # flags. # sub matchSamOptionalFlags($$) { my ($flags, $ex_flags) = @_; my %ex = (); for(keys %$ex_flags) { my ($nm, $ty, $vl) = split(/:/, $_); defined($vl) || die "Could not parse optional flag field \"$_\""; ($ex{$nm}{ty}, $ex{$nm}{vl}) = ($ty, $vl); } for(keys %$flags) { my ($ex_ty, $ex_vl); if(defined($ex{$_})) { ($ex_ty, $ex_vl) = ($ex{$_}{ty}, $ex{$_}{vl}); } else { ($ex_ty, $ex_vl) = ("i", "0"); } defined($ex_ty) || die; defined($ex_vl) || die; my ($ty, $vl) = ($flags->{$_}{ty}, $flags->{$_}{vl}); defined($ty) || die; defined($vl) || die; $ex_ty eq $ty || die "Expected SAM optional flag $_ to have type $ex_ty, had $ty"; $ex_vl eq $vl || die "Expected SAM optional flag $_ to have value $ex_vl, had $vl"; } return 1; } my $tmpfafn = ".simple_tests.pl.fa"; my $last_ref = undef; for (my $ci = 0; $ci < scalar(@cases); $ci++) { my $c = $cases[$ci]; last unless defined($c); # If there's any skipping of cases to be done, do it here prior to the # eq_deeply check my $color = 0; $color = $c->{color} if defined($c->{color}); next if ($color && $skipColor); my $do_build = 0; unless(defined($last_ref) && eq_deeply($c->{ref}, $last_ref)) { writeFasta($c->{ref}, $tmpfafn); $do_build = 1; } $last_ref = $c->{ref}; # For each set of arguments... my $case_args = $c->{args}; $case_args = "" unless defined($case_args); my $first = 1; # did we build the index yet? # Forward, then reverse-complemented my $fwlo = ($c->{nofw} ? 1 : 0); my $fwhi = ($c->{norc} ? 0 : 1); for(my $fwi = $fwlo; $fwi <= $fwhi; $fwi++) { my $fw = ($fwi == 0); my $sam = 1; my $reads = $c->{reads}; my $quals = $c->{quals}; my $m1s = $c->{mate1s}; my $q1s = $c->{qual1s}; my $m2s = $c->{mate2s}; my $q2s = $c->{qual2s}; my $read_file = undef; my $mate1_file = undef; my $mate2_file = undef; $read_file = $c->{fastq} if defined($c->{fastq}); $read_file = $c->{tabbed} if defined($c->{tabbed}); $read_file = $c->{fasta} if defined($c->{fasta}); $read_file = $c->{qseq} if defined($c->{qseq}); $read_file = $c->{raw} if defined($c->{raw}); $mate1_file = $c->{fastq1} if defined($c->{fastq1}); $mate1_file = $c->{tabbed1} if defined($c->{tabbed1}); $mate1_file = $c->{fasta1} if defined($c->{fasta1}); $mate1_file = $c->{qseq1} if defined($c->{qseq1}); $mate1_file = $c->{raw1} if defined($c->{raw1}); $mate2_file = $c->{fastq2} if defined($c->{fastq2}); $mate2_file = $c->{tabbed2} if defined($c->{tabbed2}); $mate2_file = $c->{fasta2} if defined($c->{fasta2}); $mate2_file = $c->{qseq2} if defined($c->{qseq2}); $mate2_file = $c->{raw2} if defined($c->{raw2}); my $read_file_format = undef; if(!defined($reads) && !defined($m1s) && !defined($m2s)) { defined($read_file) || defined($mate1_file) || die; $read_file_format = "fastq" if defined($c->{fastq}) || defined($c->{fastq1}); $read_file_format = "tabbed" if defined($c->{tabbed}) || defined($c->{tabbed}); $read_file_format = "fasta" if defined($c->{fasta}) || defined($c->{fasta1}); $read_file_format = "qseq" if defined($c->{qseq}) || defined($c->{qseq1}); $read_file_format = "raw" if defined($c->{raw}) || defined($c->{raw1}); next unless $fw; } # Run bowtie2 my @lines = (); my @rawlines = (); my @header_lines = (); my @header_rawlines = (); print $c->{name}." " if defined($c->{name}); print "(fw:".($fw ? 1 : 0).", sam:$sam)\n"; my $mate1fw = 1; my $mate2fw = 0; $mate1fw = $c->{mate1fw} if defined($c->{mate1fw}); $mate2fw = $c->{mate2fw} if defined($c->{mate2fw}); if(!$fw) { # Reverse-complement the reads my @s = (); @s = @$reads if defined($reads); my @q = (); @q = @$quals if defined($quals); # Reverse-complement mates and switch mate1 with mate2 my @m1 = (); @m1 = @$m1s if defined($m1s); my @m2 = (); @m2 = @$m2s if defined($m2s); my @q1 = (); @q1 = @$q1s if defined($q1s); my @q2 = (); @q2 = @$q2s if defined($q2s); for(0..scalar(@s)-1) { $s[$_] = DNA::revcomp($s[$_], $color); $q[$_] = reverse $q[$_] if $_ < scalar(@q); } if($mate1fw == $mate2fw) { for(0..$#m1) { $m1[$_] = DNA::revcomp($m1[$_], $color); } for(0..$#m2) { $m2[$_] = DNA::revcomp($m2[$_], $color); } for(0..$#q1) { $q1[$_] = reverse $q1[$_]; } for(0..$#q2) { $q2[$_] = reverse $q2[$_]; } } $reads = \@s if defined($reads); $quals = \@q if defined($quals); $m1s = \@m2 if defined($m1s); $q1s = \@q2 if defined($q1s); $m2s = \@m1 if defined($m2s); $q2s = \@q1 if defined($q2s); } my $a = $case_args; if(defined($m2s)) { $a .= " --"; $a .= ($mate1fw ? "f" : "r"); $a .= ($mate2fw ? "f" : "r"); } runbowtie2( $do_build && $first, "$a", $color, $tmpfafn, $c->{report}, $read_file_format, # formate of read/mate files $read_file, # read file $mate1_file, # mate #1 file $mate2_file, # mate #2 file $reads, # read list $quals, # quality list $m1s, # mate #1 sequence list $q1s, # mate #1 quality list $m2s, # mate #2 sequence list $q2s, # mate #2 quality list $c->{names}, \@lines, \@rawlines, \@header_lines, \@header_rawlines, $c->{should_abort}); $first = 0; my $pe = defined($c->{mate1s}) && $c->{mate1s} ne ""; $pe = $pe || defined($mate1_file); $pe = $pe || $c->{paired}; my ($lastchr, $lastoff, $lastoff_orig) = ("", -1, -1); # Keep temporary copies of hits and pairhits so that we can # restore for the next orientation my $hitstmp = []; $hitstmp = clone($c->{hits}) if defined($c->{hits}); my $pairhitstmp = []; $pairhitstmp = clone($c->{pairhits}) if defined($c->{pairhits}); my $pairhits_orig_tmp = []; $pairhits_orig_tmp = clone($c->{pairhits_orig}) if defined($c->{pairhits_orig}); # Record map from already-seen read name, read sequence and # quality to the place on the reference where it's reported. # This allows us to check that the pseudo-random generator # isn't mistakenly yielding different alignments for identical # reads. my %seenNameSeqQual = (); if(defined($c->{lines})) { my $l = scalar(@lines); $l == $c->{lines} || die "Expected $c->{lines} lines, got $l"; } for my $li (0 .. scalar(@lines)-1) { my $l = $lines[$li]; my ($readname, $orient, $chr, $off_orig, $off, $seq, $qual, $mapq, $oms, $editstr, $flagstr, $samflags, $cigar, $rnext, $pnext, $tlen); my %samoptflags = (); if($sam) { scalar(@$l) >= 11 || die "Bad number of fields; expected at least 11 got ". scalar(@$l).":\n$rawlines[$li]\n"; ($readname, $samflags, $chr, $off) = @$l[0..3]; ($seq, $qual) = @$l[9..10]; $orient = ((($samflags >> 4) & 1) == 0) ? "+" : "-"; $mapq = $l->[4]; # mapping quality $cigar = $l->[5]; # CIGAR string $rnext = $l->[6]; # ref seq of next frag in template $pnext = $l->[7]; # position of next frag in template $tlen = $l->[8]; # template length if($pnext == 0) { $pnext = "*"; } else { $pnext--; } for(my $m = 11; $m < scalar(@$l); $m++) { next if $l->[$m] eq ""; my ($nm, $ty, $vl) = split(/:/, $l->[$m]); defined($vl) || die "Could not parse optional flag field $m: ". "\"$l->[$m]\""; $samoptflags{$nm}{ty} = $ty; $samoptflags{$nm}{vl} = $vl; } if($off > 0) { $off--; } else { $off = "*"; } $off_orig = $off; $off = "*" if $cigar eq "*"; } else { scalar(@$l) == 9 || die "Bad number of fields; expected 9 got ". scalar(@$l).":\n$rawlines[$li]\n"; ($readname, $orient, $chr, $off, $seq, $qual, $oms, $editstr, $flagstr) = @$l; $off_orig = $off; } if($c->{check_random}) { my $rsqKey = "$readname\t$orient\t$seq\t$qual"; my $rsqVal = "$chr\t$off"; if(defined($seenNameSeqQual{$rsqKey})) { $seenNameSeqQual{$rsqKey} eq $rsqVal || die "Two hits for read/seq/qual:\n$rsqKey\n". "had different alignments:\n". "$seenNameSeqQual{$rsqKey}\n$rsqVal\n"; } $seenNameSeqQual{$rsqKey} = $rsqVal; } $readname ne "" || die "readname was blank:\n".Dumper($c); my $rdi = $readname; $rdi = substr($rdi, 1) if substr($rdi, 0, 1) eq "r"; my $mate = 0; if($readname =~ /\//) { ($rdi, $mate) = split(/\//, $readname); defined($rdi) || die; } $rdi = $c->{idx_map}{$rdi} if defined($c->{idx_map}{$rdi}); $rdi ne "" || die "rdi was blank:\nreadname=$readname\n".Dumper($c); if($rdi != int($rdi)) { # Read name has non-numeric characters. Figure out # what number it is by scanning the names list. my $found = 0; for(my $i = 0; $i < scalar(@{$c->{names}}); $i++) { if($c->{names}->[$i] eq $readname) { $rdi = $i; $found = 1; last; } } $found || die "No specified name matched reported name $readname"; } # Check that the sequence printed in the alignment is sane if($color) { # It's a decoded nucleotide sequence my $dseq = $c->{dec_seq}->[$rdi]; if(defined($dseq)) { $seq eq $dseq || die "Expected decoded sequence '$seq' from alignment to match '$dseq'"; } my $dqual = $c->{dec_qual}->[$rdi]; if(defined($dqual)) { $qual eq $dqual || die "Expected decoded qualities '$qual' from alignment to match '$dqual'"; } } else { } # Make simply-named copies of some portions of the test case # 'hits' my %hits = (); %hits = %{$c->{hits}->[$rdi]} if defined($c->{hits}->[$rdi]); # 'flags' my $flags = undef; $flags = $c->{flags}->[$rdi] if defined($c->{flags}->[$rdi]); # 'samflags' my $ex_samflags = undef; $ex_samflags = $c->{ex_samflags}->[$rdi] if defined($c->{ex_samflags}->[$rdi]); # 'samflags_map' my $ex_samflags_map = undef; $ex_samflags_map = $c->{samflags_map}->[$rdi] if defined($c->{samflags_map}->[$rdi]); # 'samoptflags' my $ex_samoptflags = undef; $ex_samoptflags = $c->{samoptflags}->[$rdi] if defined($c->{samoptflags}->[$rdi]); # 'cigar' my $ex_cigar = undef; $ex_cigar = $c->{cigar}->[$rdi] if defined($c->{cigar}->[$rdi]); # 'cigar_map' my $ex_cigar_map = undef; $ex_cigar_map = $c->{cigar_map}->[$rdi] if defined($c->{cigar_map}->[$rdi]); # 'mapq_hi' - boolean indicating whether mapq is hi/lo my $ex_mapq_hi = undef; $ex_mapq_hi = $c->{mapq_hi}->[$rdi] if defined($c->{mapq_hi}->[$rdi]); # 'mapq' my $ex_mapq = undef; $ex_mapq = $c->{mapq}->[$rdi] if defined($c->{mapq}->[$rdi]); # 'mapq_map' my $ex_mapq_map = undef; $ex_mapq_map = $c->{mapq_map}->[$rdi] if defined($c->{mapq_map}->[$rdi]); # 'rnext_map' my $ex_rnext_map = undef; $ex_rnext_map = $c->{rnext_map}->[$rdi] if defined($c->{rnext_map}) && defined($c->{rnext_map}->[$rdi]); # 'pnext_map' my $ex_pnext_map = undef; $ex_pnext_map = $c->{pnext_map}->[$rdi] if defined($c->{pnext_map}) && defined($c->{pnext_map}->[$rdi]); # 'tlen_map' my $ex_tlen_map = undef; $ex_tlen_map = $c->{tlen_map}->[$rdi] if defined($c->{tlen_map}) && defined($c->{tlen_map}->[$rdi]); # 'flags_fw' my $flags_fw = undef; $flags_fw = $c->{flags_fw}->[$rdi] if defined($c->{flags_fw}->[$rdi]); # 'flags_rc' my $flags_rc = undef; $flags_rc = $c->{flags_rc}->[$rdi] if defined($c->{flags_rc}->[$rdi]); # 'pairhits' my %pairhits = (); %pairhits = %{$c->{pairhits}->[$rdi]} if defined($c->{pairhits}->[$rdi]); # 'pairhits_orig' my %pairhits_orig = (); %pairhits_orig = %{$c->{pairhits_orig}->[$rdi]} if defined($c->{pairhits_orig}->[$rdi]); # 'pairflags' my %pairflags = (); %pairflags = %{$c->{pairflags}->[$rdi]} if defined($c->{pairflags}->[$rdi]); # 'hits_are_superset' my $hits_are_superset = 0; $hits_are_superset = $c->{hits_are_superset}->[$rdi] if defined($ci); # edits my $ex_edits = undef; $ex_edits = $c->{edits}->[$rdi] if defined($c->{edits}->[$rdi]); if(!$sam) { # Bowtie flags if(defined($flags)) { $flagstr eq $flags || die "Expected flags=\"$flags\", got \"$flagstr\""; } if(defined($flags_fw) && $fw) { $flagstr eq $flags_fw || die "Expected flags=\"$flags_fw\", got \"$flagstr\""; } if(defined($flags_rc) && !$fw) { $flagstr eq $flags_rc || die "Expected flags=\"$flags_rc\", got \"$flagstr\""; } if(defined($c->{flag_map})) { if(defined($c->{flag_map}->[$rdi]->{$off})) { $flagstr eq $c->{flag_map}->[$rdi]->{$off} || die "Expected flags=\"$c->{flag_map}->[$rdi]->{$off}\"". " at offset $off, got \"$flagstr\""; } } } if($sam) { # SAM flags if(defined($ex_samflags)) { $samflags eq $ex_samflags || die "Expected flags $ex_samflags, got $samflags"; } if(defined($ex_samflags_map)) { if(defined($c->{samflags_map}->[$rdi]->{$off})) { my $ex = $c->{samflags_map}->[$rdi]->{$off}; $samflags eq $ex || die "Expected FLAGS value $ex at offset $off, got $samflags" } else { die "Expected to see alignment with offset $off parsing samflags_map"; } } # CIGAR string if(defined($ex_cigar)) { $cigar eq $ex_cigar || die "Expected CIGAR string $ex_cigar, got $cigar"; } if(defined($ex_cigar_map)) { if(defined($c->{cigar_map}->[$rdi]->{$off})) { my $ex = $c->{cigar_map}->[$rdi]->{$off}; $cigar eq $ex || die "Expected CIGAR string $ex at offset $off, got $cigar" } else { die "Expected to see alignment with offset $off parsing cigar_map"; } } # MAPQ if(defined($ex_mapq)) { $mapq eq $ex_mapq || die "Expected MAPQ $ex_mapq, got $mapq"; } if(defined($ex_mapq_map)) { if(defined($c->{mapq_map}->[$rdi]->{$off})) { my $ex = $c->{mapq_map}->[$rdi]->{$off}; $mapq eq $ex || die "Expected MAPQ string $ex at offset $off, got $mapq" } else { die "Expected to see alignment with offset $off parsing mapq_map"; } } # MAPQ if(defined($ex_mapq_hi)) { if($ex_mapq_hi == 0) { $mapq < 20 || die "Expected MAPQ < 20, got $mapq"; } else { $mapq >= 20 || die "Expected MAPQ >= 20, got $mapq"; } } if(defined($ex_mapq_map)) { if(defined($c->{mapq_map}->[$rdi]->{$off})) { my $ex = $c->{mapq_map}->[$rdi]->{$off}; $mapq eq $ex || die "Expected MAPQ string $ex at offset $off, got $mapq" } else { die "Expected to see alignment with offset $off parsing mapq_map"; } } # SAM optional flags if(defined($ex_samoptflags)) { matchSamOptionalFlags(\%samoptflags, $ex_samoptflags); } if(defined($c->{samoptflags_map})) { if(defined($c->{samoptflags_map}->[$rdi]->{$off})) { matchSamOptionalFlags( \%samoptflags, $c->{samoptflags_map}->[$rdi]->{$off}); } else { die "Expected to see alignment with offset $off parsing samoptflags_map"; } } if(defined($c->{samoptflags_flagmap})) { if(defined($c->{samoptflags_flagmap}->[$rdi]->{$samflags})) { matchSamOptionalFlags( \%samoptflags, $c->{samoptflags_flagmap}->[$rdi]->{$samflags}); } else { die "Expected to see alignment with flag $samflags parsing samoptflags_flagmap"; } } # RNEXT map if(defined($c->{rnext_map})) { if(defined($c->{rnext_map}->[$rdi]->{$off})) { my $ex = $c->{rnext_map}->[$rdi]->{$off}; $rnext eq $ex || die "Expected RNEXT '$ex' at offset $off, got '$rnext'" } else { die "Expected to see alignment with offset $off parsing rnext_map".Dumper($c); } } # PNEXT map if(defined($c->{pnext_map})) { if(defined($c->{pnext_map}->[$rdi]->{$off})) { my $ex = $c->{pnext_map}->[$rdi]->{$off}; $pnext eq $ex || die "Expected PNEXT '$ex' at offset $off, got '$pnext'" } else { die "Expected to see alignment with offset $off parsing pnext_map"; } } # TLEN map if(defined($c->{tlen_map})) { if(defined($c->{tlen_map}->[$rdi]->{$off})) { my $ex = $c->{tlen_map}->[$rdi]->{$off}; $tlen eq $ex || die "Expected TLEN '$ex' at offset $off, got '$tlen'" } else { die "Expected to see alignment with offset $off parsing tlen_map"; } } } if($pe && $lastchr ne "") { my $offkey_orig = $lastoff.",".$off_orig; $offkey_orig = $off_orig.",".$lastoff_orig if $off_orig eq "*"; my $offkey = $lastoff.",".$off; $offkey = $off.",".$lastoff if $off eq "*"; if($lastoff ne "*" && $off ne "*") { $offkey = min($lastoff, $off).",".max($lastoff, $off); } if(defined($c->{pairhits}->[$rdi])) { defined($pairhits{$offkey}) || die "No such paired off as $offkey in pairhits list: ".Dumper(\%pairhits)."\n"; $c->{pairhits}->[$rdi]->{$offkey}--; delete $c->{pairhits}->[$rdi]->{$offkey} if $c->{pairhits}->[$rdi]->{$offkey} == 0; %pairhits = %{$c->{pairhits}->[$rdi]}; } if(defined($c->{pairhits_orig}->[$rdi])) { defined($pairhits_orig{$offkey_orig}) || die "No such paired off as $offkey in pairhits_orig list: ".Dumper(\%pairhits_orig)."\n"; $c->{pairhits_orig}->[$rdi]->{$offkey_orig}--; delete $c->{pairhits_orig}->[$rdi]->{$offkey_orig} if $c->{pairhits_orig}->[$rdi]->{$offkey_orig} == 0; %pairhits_orig = %{$c->{pairhits_orig}->[$rdi]}; } ($lastchr, $lastoff, $lastoff_orig) = ("", -1, -1); } elsif($pe) { # Found an unpaired alignment from aligning a pair my $foundSe = defined($c->{pairhits}->[$rdi]) && $c->{pairhits}->[$rdi]->{$off}; if($foundSe) { $c->{pairhits}->[$rdi]->{$off}--; delete $c->{pairhits}->[$rdi]->{$off} if $c->{pairhits}->[$rdi]->{$off} == 0; %pairhits = %{$c->{pairhits}->[$rdi]}; } else { ($lastchr, $lastoff) = ($chr, $off); } # Found an unpaired alignment from aligning a pair $foundSe = defined($c->{pairhits_orig}->[$rdi]) && $c->{pairhits_orig}->[$rdi]->{$off_orig}; if($foundSe) { $c->{pairhits_orig}->[$rdi]->{$off_orig}--; delete $c->{pairhits_orig}->[$rdi]->{$off_orig} if $c->{pairhits_orig}->[$rdi]->{$off_orig} == 0; %pairhits_orig = %{$c->{pairhits_orig}->[$rdi]}; } else { ($lastchr, $lastoff, $lastoff_orig) = ($chr, $off, $off_orig); } } else { if(defined($c->{hits}->[$rdi])) { defined($hits{$off}) || die "No such off as $off in hits list: ".Dumper(\%hits)."\n"; $c->{hits}->[$rdi]->{$off}--; delete $c->{hits}->[$rdi]->{$off} if $c->{hits}->[$rdi]->{$off} == 0; %hits = %{$c->{hits}->[$rdi]}; } } if(!$sam && defined($ex_edits)) { my $eds = $l->[7]; $eds eq $ex_edits || die "For edit string, expected \"$ex_edits\" got \"$eds\"\n"; } } # Go through all the per-read my $klim = 0; $klim = scalar(@{$c->{hits}}) if defined($c->{hits}); $klim = max($klim, scalar(@{$c->{pairhits}})) if defined($c->{pairhits}); for (my $k = 0; $k < $klim; $k++) { # For each read my %hits = %{$c->{hits}->[$k]} if defined($c->{hits}->[$k]); my %pairhits = %{$c->{pairhits}->[$k]} if defined($c->{pairhits}->[$k]); my %pairhits_orig = %{$c->{pairhits_orig}->[$k]} if defined($c->{pairhits_orig}->[$k]); my $hits_are_superset = $c->{hits_are_superset}->[$k]; # Check if there are any hits left over my $hitsLeft = scalar(keys %hits); if($hitsLeft != 0 && !$hits_are_superset) { print Dumper(\%hits); die "Had $hitsLeft hit(s) left over at position $k"; } my $pairhitsLeft = scalar(keys %pairhits); if($pairhitsLeft != 0 && !$hits_are_superset) { print Dumper(\%pairhits); die "Had $pairhitsLeft hit(s) left over at position $k"; } my $pairhits_orig_Left = scalar(keys %pairhits_orig); if($pairhits_orig_Left != 0 && !$hits_are_superset) { print Dumper(\%pairhits_orig); die "Had $pairhits_orig_Left hit(s) left over at position $k"; } } $c->{hits} = $hitstmp; $c->{pairhits} = $pairhitstmp; $c->{pairhits_orig} = $pairhits_orig_tmp; } $last_ref = undef if $first; } print "PASSED\n"; hisat2-2.2.1/scripts/test/simple_tests.sh000066400000000000000000000016361370663763200204460ustar00rootroot00000000000000#!/bin/sh # # Copyright 2011, Ben Langmead # # This file is part of Bowtie 2. # # Bowtie 2 is free software: you can redistribute it and/or modify # it under the terms of the GNU General Public License as published by # the Free Software Foundation, either version 3 of the License, or # (at your option) any later version. # # Bowtie 2 is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # # You should have received a copy of the GNU General Public License # along with Bowtie 2. If not, see . # # simple_tests.sh make $* bowtie2-align bowtie2-align-debug bowtie2-build bowtie2-build-debug && \ perl scripts/test/simple_tests.pl \ --bowtie2=./bowtie2 \ --bowtie2-build=./bowtie2-build-debug hisat2-2.2.1/scripts/validate_repeat.py000077500000000000000000000126341370663763200201260ustar00rootroot00000000000000#!/usr/bin/python import sys, subprocess import re from argparse import ArgumentParser, FileType from collections import defaultdict, Counter flag_include_N = True """ """ def read_genome(genome_file): chr_dic = {} chr_name, sequence = "", "" for line in genome_file: if line.startswith(">"): if chr_name and sequence: chr_dic[chr_name] = sequence chr_name = line.strip().split()[0][1:] sequence = "" else: line = line.strip() if not flag_include_N: # remove N-bases line = line.replace('N', '') sequence += line; if chr_name and sequence: chr_dic[chr_name] = sequence return chr_dic """ """ def reverse_complement(seq): result = "" for nt in seq: base = nt if nt == 'A': base = 'T' elif nt == 'a': base = 't' elif nt == 'C': base = 'G' elif nt == 'c': base = 'g' elif nt == 'G': base = 'C' elif nt == 'g': base = 'c' elif nt == 'T': base = 'A' elif nt == 't': base = 'a' result = base + result return result """ """ def read_snp(snp_file): snps = defaultdict(dict) for line in snp_file: line = line.strip() if not line or line.startswith('#'): continue try: snpID, type, chr, pos, data = line.split('\t') except ValueError: continue assert type in ["single", "deletion", "insertion"] if type == "deletion": data = int(data) snps[chr][snpID] = [snpID, type, int(pos), data]; return snps def indelCount(snp_list, snp_id_list): indel = 0 for snp_id in snp_id_list: snp = snp_list[snp_id] if snp[1] == 'deletion': indel -= int(snp[3]) elif snp[1] == 'insertion': indel += len(snp[3]) return indel def applySNPs(snp_list, ref_sqn, snp_id_list, base_pos): ref_pos = 0 read_pos = 0 read = "" for snp_id in snp_id_list: snp = snp_list[snp_id] pos = snp[2] - base_pos; while ref_pos < pos: read += ref_sqn[ref_pos] ref_pos += 1 if snp[1] == 'single': read += snp[3] ref_pos += 1 elif snp[1] == 'deletion': ref_pos += int(snp[3]) elif snp[1] == 'insertion': read += snp[3] #print snp_id, snp_list[snp_id] while ref_pos < len(ref_sqn): read += ref_sqn[ref_pos] ref_pos += 1 return read def main(genome_file, rpt_name): # load genome sequeuce chr_dic = read_genome(genome_file) rpt_fa_name = rpt_name + ".rep.fa" rpt_info_name = rpt_name + ".rep.info" rpt_snp_name = rpt_name + ".rep.snp" # load repeat sequence fp = open(rpt_fa_name, 'r') rpt_dic = read_genome(fp) fp.close() # load repeat snp fp = open(rpt_snp_name, 'r') rpt_snps = read_snp(fp) fp.close() # Validates # load repeat info fp = open(rpt_info_name, 'r') repeat_sequence = "" repeat_length = 0 snp_cnt = 0 indel = 0 snp_id_list = [] for line in fp: line = line.strip() if line.startswith('>'): line = line[1:] fields = line.split() #print fields name, rpt_seq_name, rpt_pos, rpt_len, pos_cnt, snp_cnt = fields[0:6] snp_cnt = int(snp_cnt) rpt_pos = int(rpt_pos) rpt_len = int(rpt_len) if snp_cnt > 0: snp_id_list = fields[6].split(',') else: snp_id_list = [] #print name, snp_cnt, snp_list # make repeat_sequence (with snp) repeat_sequence = rpt_dic[rpt_seq_name][rpt_pos:rpt_pos + rpt_len] indel = 0 if snp_cnt > 0: # apply snps repeat_sequence = applySNPs(rpt_snps[rpt_seq_name], repeat_sequence, snp_id_list, rpt_pos) # in/del count indel = indelCount(rpt_snps[rpt_seq_name], snp_id_list) #repeat_length = rpt_len + indel repeat_length = len(repeat_sequence) #print repeat_sequence else: coords = line.split() for coord in coords: chr, pos, strand = coord.split(':') pos = int(pos) # get string seq = chr_dic[chr][pos:pos + repeat_length] if strand == '-': seq = reverse_complement(seq) if seq != repeat_sequence: print 'Mismatch', seq, repeat_sequence, snp_cnt, coord, snp_id_list, repeat_length fp.close() if __name__ == '__main__': parser = ArgumentParser( description='Validate repeat files') parser.add_argument('genome_file', nargs='?', type=FileType('r'), help='input genome file (e.g. genome.fa)') parser.add_argument('-r', dest='rpt_name', type=str, help='Repeat Name') args = parser.parse_args() if not args.genome_file or not args.rpt_name: parser.print_help() exit(1) main(args.genome_file, args.rpt_name) hisat2-2.2.1/search_globals.h000066400000000000000000000026421370663763200160500ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef SEARCH_GLOBALS_H_ #define SEARCH_GLOBALS_H_ #include // declared in ebwt_search.cpp extern bool gColor; extern bool gColorExEnds; extern bool gReportOverhangs; extern bool gColorSeq; extern bool gColorEdit; extern bool gColorQual; extern bool gNoMaqRound; extern bool gStrandFix; extern bool gRangeMode; extern int gVerbose; extern int gQuiet; extern bool gNofw; extern bool gNorc; extern bool gMate1fw; extern bool gMate2fw; extern int gMinInsert; extern int gMaxInsert; extern int gTrim5; extern int gTrim3; extern int gGapBarrier; extern int gAllowRedundant; #endif /* SEARCH_GLOBALS_H_ */ hisat2-2.2.1/sequence_io.h000066400000000000000000000072171370663763200154020ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef SEQUENCE_IO_H_ #define SEQUENCE_IO_H_ #include #include #include #include #include "assert_helpers.h" #include "ds.h" #include "filebuf.h" #include "sstring.h" using namespace std; /** * Parse the fasta file 'infile'. Store */ template static void parseFastaLens( const TFnStr& infile, // filename EList& namelens, // destination for fasta name lengths EList& seqlens) // destination for fasta sequence lengths { FILE *in = fopen(sstr_to_cstr(infile), "r"); if(in == NULL) { cerr << "Could not open sequence file" << endl; throw 1; } FileBuf fb(in); while(!fb.eof()) { namelens.expand(); namelens.back() = 0; seqlens.expand(); seqlens.back() = 0; fb.parseFastaRecordLength(namelens.back(), seqlens.back()); if(seqlens.back() == 0) { // Couldn't read a record. We're probably done with this file. namelens.pop_back(); seqlens.pop_back(); continue; } } fb.close(); } /** * Parse the fasta file 'infile'. Store each name record in 'names', each * sequence record in 'seqs', and the lengths of each */ template static void parseFasta( const TFnStr& infile, // filename EList& names, // destination for fasta names EList& namelens, // destination for fasta name lengths EList& seqs, // destination for fasta sequences EList& seqlens) // destination for fasta sequence lengths { assert_eq(namelens.size(), seqlens.size()); assert_eq(names.size(), namelens.size()); assert_eq(seqs.size(), seqlens.size()); size_t cur = namelens.size(); parseFastaLens(infile, namelens, seqlens); FILE *in = fopen(sstr_to_cstr(infile), "r"); if(in == NULL) { cerr << "Could not open sequence file" << endl; throw 1; } FileBuf fb(in); while(!fb.eof()) { // Add a new empty record to the end names.expand(); seqs.expand(); names.back() = new char[namelens[cur]+1]; seqs.back() = new char[seqlens[cur]+1]; fb.parseFastaRecord(names.back(), seqs.back()); if(seqs.back().empty()) { // Couldn't read a record. We're probably done with this file. names.pop_back(); seqs.pop_back(); continue; } } fb.close(); } /** * Read a set of FASTA sequence files of the given format and alphabet type. * Store all of the extracted sequences in vector ss. */ template static void parseFastas( const EList& infiles, // filenames EList& names, // destination for fasta names EList& namelens, // destination for fasta name lengths EList& seqs, // destination for fasta sequences EList& seqlens) // destination for fasta sequence lengths { for(size_t i = 0; i < infiles.size(); i++) { parseFasta( infiles[i], names, namelens, seqs, seqlens); } } #endif /*SEQUENCE_IO_H_*/ hisat2-2.2.1/shmem.cpp000066400000000000000000000024751370663763200145500ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifdef BOWTIE_SHARED_MEM #include #include #include #include #include #include "shmem.h" using namespace std; /** * Notify other users of a shared-memory chunk that the leader has * finished initializing it. */ void notifySharedMem(void *mem, size_t len) { ((volatile uint32_t*)((char*)mem + len))[0] = SHMEM_INIT; } /** * Wait until the leader of a shared-memory chunk has finished * initializing it. */ void waitSharedMem(void *mem, size_t len) { while(((volatile uint32_t*)((char*)mem + len))[0] != SHMEM_INIT) { sleep(1); } } #endif hisat2-2.2.1/shmem.h000066400000000000000000000115561370663763200142150ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef SHMEM_H_ #define SHMEM_H_ #ifdef BOWTIE_SHARED_MEM #include #include #include #include #include #include #include #include "str_util.h" #include "btypes.h" extern void notifySharedMem(void *mem, size_t len); extern void waitSharedMem(void *mem, size_t len); #define ALLOC_SHARED_U allocSharedMem #define ALLOC_SHARED_U8 allocSharedMem #define ALLOC_SHARED_U32 allocSharedMem #define FREE_SHARED shmdt #define NOTIFY_SHARED notifySharedMem #define WAIT_SHARED waitSharedMem #define SHMEM_UNINIT 0xafba4242 #define SHMEM_INIT 0xffaa6161 /** * Tries to allocate a shared-memory chunk for a given file of a given size. */ template bool allocSharedMem(std::string fname, size_t len, T ** dst, const char *memName, bool verbose) { using namespace std; int shmid = -1; // Calculate key given string key_t key = (key_t)hash_string(fname); shmid_ds ds; int ret; // Reserve 4 bytes at the end for silly synchronization size_t shmemLen = len + 4; if(verbose) { cerr << "Reading " << len << "+4 bytes into shared memory for " << memName << endl; } T *ptr = NULL; while(true) { // Create the shrared-memory block if((shmid = shmget(key, shmemLen, IPC_CREAT | 0666)) < 0) { if(errno == ENOMEM) { cerr << "Out of memory allocating shared area " << memName << endl; } else if(errno == EACCES) { cerr << "EACCES" << endl; } else if(errno == EEXIST) { cerr << "EEXIST" << endl; } else if(errno == EINVAL) { cerr << "Warning: shared-memory chunk's segment size doesn't match expected size (" << (shmemLen) << ")" << endl << "Deleteing old shared memory block and trying again." << endl; shmid = shmget(key, 0, 0); if((ret = shmctl(shmid, IPC_RMID, &ds)) < 0) { cerr << "shmctl returned " << ret << " for IPC_RMID, errno is " << errno << ", shmid is " << shmid << endl; throw 1; } else { cerr << "Deleted shared mem chunk with shmid " << shmid << endl; } continue; } else if(errno == ENOENT) { cerr << "ENOENT" << endl; } else if(errno == ENOSPC) { cerr << "ENOSPC" << endl; } else { cerr << "shmget returned " << shmid << " for and errno is " << errno << endl; } throw 1; } ptr = (T*)shmat(shmid, 0, 0); if(ptr == (void*)-1) { cerr << "Failed to attach " << memName << " to shared memory with shmat()." << endl; throw 1; } if(ptr == NULL) { cerr << memName << " pointer returned by shmat() was NULL." << endl; throw 1; } // Did I create it, or did I just attach to one created by // another process? if((ret = shmctl(shmid, IPC_STAT, &ds)) < 0) { cerr << "shmctl returned " << ret << " for IPC_STAT and errno is " << errno << endl; throw 1; } if(ds.shm_segsz != shmemLen) { cerr << "Warning: shared-memory chunk's segment size (" << ds.shm_segsz << ") doesn't match expected size (" << shmemLen << ")" << endl << "Deleteing old shared memory block and trying again." << endl; if((ret = shmctl(shmid, IPC_RMID, &ds)) < 0) { cerr << "shmctl returned " << ret << " for IPC_RMID and errno is " << errno << endl; throw 1; } } else { break; } } // while(true) *dst = ptr; bool initid = (((volatile uint32_t*)((char*)ptr + len))[0] == SHMEM_INIT); if(ds.shm_cpid == getpid() && !initid) { if(verbose) { cerr << " I (pid = " << getpid() << ") created the " << "shared memory for " << memName << endl; } // Set this value just off the end of the chunk to // indicate that the data hasn't been read yet. ((volatile uint32_t*)((char*)ptr + len))[0] = SHMEM_UNINIT; return true; } else { if(verbose) { cerr << " I (pid = " << getpid() << ") did not create the shared memory for " << memName << ". Pid " << ds.shm_cpid << " did." << endl; } return false; } } #else #define ALLOC_SHARED_U(...) 0 #define ALLOC_SHARED_U8(...) 0 #define ALLOC_SHARED_U32(...) 0 #define FREE_SHARED(...) #define NOTIFY_SHARED(...) #define WAIT_SHARED(...) #endif /*BOWTIE_SHARED_MEM*/ #endif /* SHMEM_H_ */ hisat2-2.2.1/simple_func.cpp000066400000000000000000000044611370663763200157400ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include #include "simple_func.h" #include "ds.h" #include "mem_ids.h" int SimpleFunc::parseType(const std::string& otype) { string type = otype; if(type == "C" || type == "Constant") { return SIMPLE_FUNC_CONST; } else if(type == "L" || type == "Linear") { return SIMPLE_FUNC_LINEAR; } else if(type == "S" || type == "Sqrt") { return SIMPLE_FUNC_SQRT; } else if(type == "G" || type == "Log") { return SIMPLE_FUNC_LOG; } std::cerr << "Error: Bad function type '" << otype.c_str() << "'. Should be C (constant), L (linear), " << "S (square root) or G (natural log)." << std::endl; throw 1; } SimpleFunc SimpleFunc::parse( const std::string& s, double defaultConst, double defaultLinear, double defaultMin, double defaultMax) { // Separate value into comma-separated tokens EList ctoks(MISC_CAT); string ctok; istringstream css(s); SimpleFunc fv; while(getline(css, ctok, ',')) { ctoks.push_back(ctok); } if(ctoks.size() >= 1) { fv.setType(parseType(ctoks[0])); } if(ctoks.size() >= 2) { double co; istringstream tmpss(ctoks[1]); tmpss >> co; fv.setConst(co); } else { fv.setConst(defaultConst); } if(ctoks.size() >= 3) { double ce; istringstream tmpss(ctoks[2]); tmpss >> ce; fv.setCoeff(ce); } else { fv.setCoeff(defaultLinear); } if(ctoks.size() >= 4) { double mn; istringstream tmpss(ctoks[3]); tmpss >> mn; fv.setMin(mn); } else { fv.setMin(defaultMin); } if(ctoks.size() >= 5) { double mx; istringstream tmpss(ctoks[4]); tmpss >> mx; fv.setMax(mx); } else { fv.setMax(defaultMax); } return fv; } hisat2-2.2.1/simple_func.h000066400000000000000000000066231370663763200154070ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef SIMPLE_FUNC_H_ #define SIMPLE_FUNC_H_ #include #include #include #include "tokenize.h" #define SIMPLE_FUNC_CONST 1 #define SIMPLE_FUNC_LINEAR 2 #define SIMPLE_FUNC_SQRT 3 #define SIMPLE_FUNC_LOG 4 /** * A simple function of one argument, parmeterized by I, X, C and L: min * value, max value, constant term, and coefficient respectively: * * 1. Constant: f(x) = max(I, min(X, C + L * 0)) * 2. Linear: f(x) = max(I, min(X, C + L * x)) * 3. Square root: f(x) = max(I, min(X, C + L * sqrt(x))) * 4. Log: f(x) = max(I, min(X, C + L * ln(x))) * * Clearly, the return value of the Constant function doesn't depend on x. */ class SimpleFunc { public: SimpleFunc() : type_(0), I_(0.0), X_(0.0), C_(0.0), L_(0.0) { } SimpleFunc(int type, double I, double X, double C, double L) { init(type, I, X, C, L); } void init(int type, double I, double X, double C, double L) { type_ = type; I_ = I; X_ = X; C_ = C; L_ = L; } void init(int type, double C, double L) { type_ = type; C_ = C; L_ = L; I_ = -std::numeric_limits::max(); X_ = std::numeric_limits::max(); } void setType (int type ) { type_ = type; } void setMin (double mn) { I_ = mn; } void setMax (double mx) { X_ = mx; } void setConst(double co) { C_ = co; } void setCoeff(double ce) { L_ = ce; } int getType () const { return type_; } double getMin () const { return I_; } double getMax () const { return X_; } double getConst() const { return C_; } double getCoeff() const { return L_; } void mult(double x) { if(I_ < std::numeric_limits::max()) { I_ *= x; X_ *= x; C_ *= x; L_ *= x; } } bool initialized() const { return type_ != 0; } void reset() { type_ = 0; } template T f(double x) const { assert(type_ >= SIMPLE_FUNC_CONST && type_ <= SIMPLE_FUNC_LOG); double X; if(type_ == SIMPLE_FUNC_CONST) { X = 0.0; } else if(type_ == SIMPLE_FUNC_LINEAR) { X = x; } else if(type_ == SIMPLE_FUNC_SQRT) { X = sqrt(x); } else if(type_ == SIMPLE_FUNC_LOG) { X = log(x); } else { throw 1; } double ret = std::max(I_, std::min(X_, C_ + L_ * X)); if(ret == std::numeric_limits::max()) { return std::numeric_limits::max(); } else if(ret == std::numeric_limits::min()) { return std::numeric_limits::min(); } else { return (T)ret; } } static int parseType(const std::string& otype); static SimpleFunc parse( const std::string& s, double defaultConst = 0.0, double defaultLinear = 0.0, double defaultMin = 0.0, double defaultMax = std::numeric_limits::max()); protected: int type_; double I_, X_, C_, L_; }; #endif /*ndef SIMPLE_FUNC_H_*/ hisat2-2.2.1/splice_site.cpp000066400000000000000000001015411370663763200157340ustar00rootroot00000000000000/* * Copyright 2013, Daehwan Kim * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "edit.h" #include "splice_site.h" #include "aligner_report.h" #include "aligner_result.h" #if defined(NEW_PROB_MODEL) #include "splice_site_mem.h" #else float donor_prob[4][donor_len] = { {0.340f, 0.604f, 0.092f, 0.001f, 0.001f, 0.526f, 0.713f, 0.071f, 0.160f}, {0.363f, 0.129f, 0.033f, 0.001f, 0.001f, 0.028f, 0.076f, 0.055f, 0.165f}, {0.183f, 0.125f, 0.803f, 1.000f, 0.001f, 0.419f, 0.118f, 0.814f, 0.209f}, {0.114f, 0.142f, 0.073f, 0.001f, 1.000f, 0.025f, 0.093f, 0.059f, 0.462f} }; float acceptor_prob[4][acceptor_len] = { {0.090f, 0.084f, 0.075f, 0.068f, 0.076f, 0.080f, 0.097f, 0.092f, 0.076f, 0.078f, 0.237f, 0.042f, 1.000f, 0.001f, 0.239f}, {0.310f, 0.310f, 0.307f, 0.293f, 0.326f, 0.330f, 0.373f, 0.385f, 0.410f, 0.352f, 0.309f, 0.708f, 0.001f, 0.001f, 0.138f}, {0.125f, 0.115f, 0.106f, 0.104f, 0.110f, 0.113f, 0.113f, 0.085f, 0.066f, 0.064f, 0.212f, 0.003f, 0.001f, 1.000f, 0.520f}, {0.463f, 0.440f, 0.470f, 0.494f, 0.471f, 0.463f, 0.408f, 0.429f, 0.445f, 0.504f, 0.240f, 0.246f, 0.001f, 0.001f, 0.104f} }; float donor_prob_sum[1 << (donor_len << 1)]; float acceptor_prob_sum1[1 << (acceptor_len1 << 1)]; float acceptor_prob_sum2[1 << (acceptor_len2 << 1)]; #endif void init_junction_prob() { #if !defined(NEW_PROB_MODEL) for(size_t i = 0; i < donor_len; i++) { ASSERT_ONLY(float sum = 0.0f); for(size_t j = 0; j < 4; j++) { float prob = donor_prob[j][i]; assert_gt(prob, 0.0f); ASSERT_ONLY(sum += prob); donor_prob[j][i] = log(prob / background_prob[j]); } assert_range(0.9f, 1.05f, sum); } for(size_t i = 0; i < acceptor_len; i++) { ASSERT_ONLY(float sum = 0.0f); for(size_t j = 0; j < 4; j++) { float prob = acceptor_prob[j][i]; assert_gt(prob, 0.0f); ASSERT_ONLY(sum += prob); acceptor_prob[j][i] = log(prob / background_prob[j]); } assert_range(0.9f, 1.05f, sum); } const size_t donor_elms = 1 << (donor_len << 1); for(size_t i = 0; i < donor_elms; i++) { float sum = 0.0f; for(size_t j = 0; j < donor_len; j++) { int base = (i >> (j << 1)) & 0x3; sum += donor_prob[base][donor_len - j - 1]; } donor_prob_sum[i] = exp(-sum); } const size_t acceptor_elms1 = 1 << (acceptor_len1 << 1); for(size_t i = 0; i < acceptor_elms1; i++) { float sum = 0.0f; for(size_t j = 0; j < acceptor_len1; j++) { int base = (i >> (j << 1)) & 0x3; sum += acceptor_prob[base][acceptor_len1 - j - 1]; } acceptor_prob_sum1[i] = exp(-sum); } const size_t acceptor_elms2 = 1 << (acceptor_len2 << 1); for(size_t i = 0; i < acceptor_elms2; i++) { float sum = 0.0f; for(size_t j = 0; j < acceptor_len2; j++) { int base = (i >> (j << 1)) & 0x3; sum += acceptor_prob[base][acceptor_len - j - 1]; } acceptor_prob_sum2[i] = exp(-sum); } #endif } ostream& operator<<(ostream& out, const SpliceSite& s) { out << s.ref() << "\t" << s.left() << "\t" << s.right() << "\t"; if(s.splDir() == SPL_FW || s.splDir() == SPL_SEMI_FW) { out << "+"; } else if(s.splDir() == SPL_RC || s.splDir() == SPL_SEMI_RC) { out << "-"; } else { out << "."; } out << endl; return out; } SpliceSiteDB::SpliceSiteDB( const BitPairReference& refs, const EList& refnames, bool threadSafe, bool write, bool read) : _numRefs(refs.numRefs()), _write(write), _read(read), _threadSafe(threadSafe), _empty(true) { for(size_t r = 0; r < refnames.size(); r++) { const string& refname = refnames[r]; _refnames.expand(); size_t i = 0; for(; i < refname.size(); i++) { if(isspace(refname[i])) { break; } } _refnames.back() = refname.substr(0, i); } assert_gt(_numRefs, 0); assert_eq(_numRefs, _refnames.size()); for(uint64_t i = 0; i < _numRefs; i++) { _fwIndex.push_back(new RedBlack(16 << 10, CA_CAT)); _bwIndex.push_back(new RedBlack(16 << 10, CA_CAT)); _pool.expand(); _spliceSites.expand(); _mutex.push_back(MUTEX_T()); } donorstr.resize(donor_exonic_len + donor_intronic_len); acceptorstr.resize(acceptor_intronic_len + acceptor_exonic_len); } SpliceSiteDB::~SpliceSiteDB() { assert_eq(_fwIndex.size(), _bwIndex.size()); assert_eq(_fwIndex.size(), _pool.size()); for(uint64_t i = 0; i < _numRefs; i++) { delete _fwIndex[i]; delete _bwIndex[i]; EList& pool = _pool[i]; for(size_t j = 0; j < pool.size(); j++) { delete pool[j]; } } } size_t SpliceSiteDB::size(uint64_t ref) const { if(!_read) return 0; assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); assert_lt(ref, _fwIndex.size()); assert_eq(_fwIndex.size(), _bwIndex.size()); ThreadSafe t(const_cast(&_mutex[ref]), _threadSafe && _write); return _fwIndex.size(); } bool SpliceSiteDB::empty(uint64_t ref) const { return size(ref) == 0; } bool SpliceSiteDB::addSpliceSite( const Read& rd, const AlnRes& rs, uint32_t minAnchorLen) { if(!_write) return false; if(rs.trimmed5p(true) + rs.trimmed3p(true) > 0) return false; _empty = false; Coord coord = rs.refcoord(); uint64_t ref = coord.ref(); assert_lt(ref, _numRefs); const EList& edits = rs.ned(); if(!coord.orient()) { Edit::invertPoss(const_cast&>(edits), rd.length(), false); } // daehwan - for debugging purposes uint32_t editdist = 0; for(size_t i = 0; i < edits.size(); i++) { const Edit& edit = edits[i]; if(edit.isGap() || edit.isMismatch()) editdist++; } SpliceSitePos ssp; uint32_t refoff = (uint32_t)coord.off(); uint32_t leftAnchorLen = 0, rightAnchorLen = 0; size_t eidx = 0; size_t last_eidx = 0; uint32_t mm = 0; for(size_t i = 0; i < rd.length(); i++, refoff++) { while(eidx < edits.size() && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { refoff++; } else if(edits[eidx].isRefGap()) { assert_gt(refoff, 0); refoff--; } if(edits[eidx].isGap() || edits[eidx].isMismatch()) mm++; if(edits[eidx].isSpliced()) { assert_gt(refoff, 0); if(ssp.inited()) { assert(edits[last_eidx].isSpliced()); assert_lt(edits[last_eidx].pos, edits[eidx].pos); rightAnchorLen = edits[eidx].pos - edits[last_eidx].pos; uint32_t minLeftAnchorLen = minAnchorLen + mm * 2 + (edits[eidx].splDir == SPL_UNKNOWN ? 6 : 0); uint32_t mm2 = 0; for(size_t j = eidx + 1; j < edits.size(); j++) { if(edits[j].isGap() || edits[j].isMismatch()) mm2++; } uint32_t minRightAnchorLen = minAnchorLen + mm2 * 2 + (edits[eidx].splDir == SPL_UNKNOWN ? 6 : 0); if(leftAnchorLen >= minLeftAnchorLen && rightAnchorLen >= minRightAnchorLen) { bool added = false; assert_lt(ref, _mutex.size()); ThreadSafe t(&_mutex[ref], _threadSafe && _write); assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); Node *cur = _fwIndex[ref]->add(pool(ref), ssp, &added); if(added) { assert_lt(ref, _spliceSites.size()); _spliceSites[ref].expand(); _spliceSites[ref].back().init(ssp.ref(), ssp.left(), ssp.right(), ssp.splDir()); _spliceSites[ref].back()._readid = rd.rdid; _spliceSites[ref].back()._leftext = leftAnchorLen; _spliceSites[ref].back()._rightext = rightAnchorLen; _spliceSites[ref].back()._editdist = editdist; _spliceSites[ref].back()._numreads = 1; assert(cur != NULL); cur->payload = (uint32_t)_spliceSites[ref].size() - 1; SpliceSitePos rssp(ssp.ref(), ssp.right(), ssp.left(), ssp.splDir()); assert_lt(ref, _bwIndex.size()); assert(_bwIndex[ref] != NULL); cur = _bwIndex[ref]->add(pool(ref), rssp, &added); assert(added); assert(cur != NULL); cur->payload = (uint32_t)_spliceSites[ref].size() - 1; assert_eq(_fwIndex[ref]->size(), _bwIndex[ref]->size()); } else { assert(cur != NULL); assert_lt(ref, _spliceSites.size()); assert_lt(cur->payload, _spliceSites[ref].size()); if(leftAnchorLen > _spliceSites[ref][cur->payload]._leftext) _spliceSites[ref][cur->payload]._leftext = leftAnchorLen; if(rightAnchorLen > _spliceSites[ref][cur->payload]._rightext) _spliceSites[ref][cur->payload]._rightext = rightAnchorLen; if(editdist < _spliceSites[ref][cur->payload]._editdist) _spliceSites[ref][cur->payload]._editdist = editdist; _spliceSites[ref][cur->payload]._numreads += 1; if(rd.rdid < _spliceSites[ref][cur->payload]._readid) { _spliceSites[ref][cur->payload]._readid = rd.rdid; } } } leftAnchorLen = rightAnchorLen; rightAnchorLen = 0; } else { leftAnchorLen = edits[eidx].pos; } ssp.init((uint32_t)coord.ref(), refoff - 1, refoff + edits[eidx].splLen, edits[eidx].splDir); refoff += edits[eidx].splLen; last_eidx = eidx; } eidx++; } } if(ssp.inited()) { assert(edits[last_eidx].isSpliced()); assert_lt(edits[last_eidx].pos, rd.length()); rightAnchorLen = (uint32_t)(rd.length() - edits[last_eidx].pos); uint32_t minLeftAnchorLen = minAnchorLen + mm * 2 + (edits[last_eidx].splDir == SPL_UNKNOWN ? 6 : 0); uint32_t mm2 = 0; for(size_t j = last_eidx + 1; j < edits.size(); j++) { if(edits[j].isGap() || edits[j].isMismatch()) mm2++; } uint32_t minRightAnchorLen = minAnchorLen + mm2 * 2 + (edits[last_eidx].splDir == SPL_UNKNOWN ? 6 : 0); if(leftAnchorLen >= minLeftAnchorLen && rightAnchorLen >= minRightAnchorLen) { bool added = false; assert_lt(ref, _mutex.size()); ThreadSafe t(&_mutex[ref], _threadSafe && _write); assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); Node *cur = _fwIndex[ref]->add(pool(ref), ssp, &added); if(added) { assert_lt(ref, _spliceSites.size()); _spliceSites[ref].expand(); _spliceSites[ref].back().init(ssp.ref(), ssp.left(), ssp.right(), ssp.splDir()); _spliceSites[ref].back()._readid = rd.rdid; _spliceSites[ref].back()._leftext = leftAnchorLen; _spliceSites[ref].back()._rightext = rightAnchorLen; _spliceSites[ref].back()._editdist = editdist; _spliceSites[ref].back()._numreads = 1; assert(cur != NULL); cur->payload = (uint32_t)_spliceSites[ref].size() - 1; SpliceSitePos rssp(ssp.ref(), ssp.right(), ssp.left(), ssp.splDir()); assert_lt(ref, _bwIndex.size()); assert(_bwIndex[ref] != NULL); cur = _bwIndex[ref]->add(pool(ref), rssp, &added); assert(added); assert(cur != NULL); cur->payload = (uint32_t)_spliceSites[ref].size() - 1; assert_eq(_fwIndex[ref]->size(), _bwIndex[ref]->size()); } else { assert(cur != NULL); assert_lt(ref, _spliceSites.size()); assert_lt(cur->payload, _spliceSites[ref].size()); if(leftAnchorLen > _spliceSites[ref][cur->payload]._leftext) _spliceSites[ref][cur->payload]._leftext = leftAnchorLen; if(rightAnchorLen > _spliceSites[ref][cur->payload]._rightext) _spliceSites[ref][cur->payload]._rightext = rightAnchorLen; if(editdist < _spliceSites[ref][cur->payload]._editdist) _spliceSites[ref][cur->payload]._editdist = editdist; _spliceSites[ref][cur->payload]._numreads += 1; if(rd.rdid < _spliceSites[ref][cur->payload]._readid) { _spliceSites[ref][cur->payload]._readid = rd.rdid; } } } } if(!coord.orient()) { Edit::invertPoss(const_cast&>(edits), rd.length(), false); } return true; } bool SpliceSiteDB::getSpliceSite(SpliceSite& ss) const { if(!_read) return false; uint64_t ref = ss.ref(); assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); ThreadSafe t(const_cast(&_mutex[ref]), _threadSafe && _write); assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); const Node *cur = _fwIndex[ref]->lookup(ss); if(cur == NULL) return false; assert(cur != NULL); assert_lt(ref, _spliceSites.size()); ss = _spliceSites[ref][cur->payload]; return true; } void SpliceSiteDB::getLeftSpliceSites(uint32_t ref, uint32_t left, uint32_t range, EList& spliceSites) const { if(!_read) return; assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); ThreadSafe t(const_cast(&_mutex[ref]), _threadSafe && _write); assert_gt(range, 0); assert_geq(left + 1, range); assert_lt(ref, _bwIndex.size()); assert(_bwIndex[ref] != NULL); const Node *cur = _bwIndex[ref]->root(); if(cur != NULL) getSpliceSites_recur(cur, left + 1 - range, left, spliceSites); } void SpliceSiteDB::getRightSpliceSites(uint32_t ref, uint32_t right, uint32_t range, EList& spliceSites) const { if(!_read) return; assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); ThreadSafe t(const_cast(&_mutex[ref]), _threadSafe && _write); assert_gt(range, 0); assert_gt(right + range, range); assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); const Node *cur = _fwIndex[ref]->root(); if(cur != NULL) getSpliceSites_recur(cur, right, right + range - 1, spliceSites); } void SpliceSiteDB::getSpliceSites_recur( const RedBlackNode *node, uint32_t left, uint32_t right, EList& spliceSites) const { assert(node != NULL); if(node->key.left() >= left && node->left != NULL) { getSpliceSites_recur( node->left, left, right, spliceSites); } if(node->key.left() >= left && node->key.left() <= right) { uint32_t ref = node->key.ref(); assert_lt(ref, _spliceSites.size()); assert_lt(node->payload, _spliceSites[ref].size()); ASSERT_ONLY(const SpliceSite& ss = _spliceSites[ref][node->payload]); assert_eq(ss.ref(), node->key.ref()); assert(ss.left() == node->key.left() || ss.right() == node->key.left()); spliceSites.push_back(_spliceSites[ref][node->payload]); } if(node->key.left() <= right && node->right != NULL) { getSpliceSites_recur( node->right, left, right, spliceSites); } } bool SpliceSiteDB::hasSpliceSites( uint32_t ref, uint32_t left1, uint32_t right1, uint32_t left2, uint32_t right2, bool includeNovel) const { if(!_read) return false; assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); ThreadSafe t(const_cast(&_mutex[ref]), _threadSafe && _write); if(left1 < right1) { assert_lt(ref, _bwIndex.size()); assert(_bwIndex[ref] != NULL); const Node *cur = _bwIndex[ref]->root(); if(cur != NULL) { if(hasSpliceSites_recur(cur, left1, right1, includeNovel)) return true; } } if(left2 < right2) { assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); const Node *cur = _fwIndex[ref]->root(); if(cur != NULL) { return hasSpliceSites_recur(cur, left2, right2, includeNovel); } } return false; } bool SpliceSiteDB::hasSpliceSites_recur( const RedBlackNode *node, uint32_t left, uint32_t right, bool includeNovel) const { assert(node != NULL); if(node->key.left() >= left && node->key.left() <= right) { uint32_t ref = node->key.ref(); assert_lt(ref, _spliceSites.size()); assert_lt(node->payload, _spliceSites[ref].size()); const SpliceSite& ss = _spliceSites[ref][node->payload]; if(includeNovel || ss._known) return true; } if(node->key.left() >= left && node->left != NULL) { if(hasSpliceSites_recur( node->left, left, right, includeNovel)) return true; } if(node->key.left() <= right && node->right != NULL) { if(hasSpliceSites_recur( node->right, left, right, includeNovel)) return true; } return false; } bool SpliceSiteDB::insideExon( uint32_t ref, uint32_t left, uint32_t right) const { if(_exons.empty()) return false; assert_lt(ref, _numRefs); assert_lt(left, right); Exon e(ref, left + 1, 0, true); size_t i = _exons.bsearchLoBound(e); for(; i > 0; i--) { const Exon& e = _exons[i-1]; if(e.right() < left) break; if(e.left() <= left && right <= e.right()) return true; } return false; } void calculate_splicesite_read_dist_impl(const RedBlackNode *node, const EList &spliceSites, EList& splicesite_read_dist) { if(node == NULL) return; calculate_splicesite_read_dist_impl(node->left, spliceSites, splicesite_read_dist); assert_lt(node->payload, spliceSites.size()); const SpliceSite& ss = spliceSites[node->payload]; if(ss.numreads() < splicesite_read_dist.size()) splicesite_read_dist[ss.numreads()] += 1; else splicesite_read_dist.back() += 1; calculate_splicesite_read_dist_impl(node->right, spliceSites, splicesite_read_dist); } uint32_t calculate_splicesite_read_dist(const EList* >& fwIndex, const ELList &spliceSites, EList& splicesite_read_dist) { for(size_t i = 0; i < fwIndex.size(); i++) { assert(fwIndex[i] != NULL); const RedBlackNode *root = fwIndex[i]->root(); assert_lt(i, spliceSites.size()); if(root != NULL) calculate_splicesite_read_dist_impl(root, spliceSites[i], splicesite_read_dist); } for(size_t i = 1; i < splicesite_read_dist.size(); i++) { splicesite_read_dist[i] += splicesite_read_dist[i-1]; } for(size_t i = 0; i < splicesite_read_dist.size(); i++) { float cmf_i = float(splicesite_read_dist[i]) / splicesite_read_dist.back(); if(cmf_i > 0.7) return (uint32_t)i; } return 0; } void SpliceSiteDB::print(ofstream& out) { EList splicesite_read_dist; for(size_t i = 0; i < 100; i++) { splicesite_read_dist.push_back(0); } uint32_t numreads_cutoff = calculate_splicesite_read_dist(_fwIndex, _spliceSites, splicesite_read_dist); size_t numsplicesites = 0; for(size_t i = 0; i < _spliceSites.size(); i++) { numsplicesites += _spliceSites[i].size(); } uint32_t numreads_cutoff2 = (uint32_t)(numsplicesites / 100000); EList ss_list; for(size_t i = 0; i < _fwIndex.size(); i++) { assert(_fwIndex[i] != NULL); const Node *root = _fwIndex[i]->root(); if(root != NULL) print_recur(root, out, numreads_cutoff, numreads_cutoff2, ss_list); } print_impl(out, ss_list); } void SpliceSiteDB::print_recur( const RedBlackNode *node, ofstream& out, const uint32_t numreads_cutoff, const uint32_t numreads_cutoff2, EList& ss_list) { if(node == NULL) return; print_recur(node->left, out, numreads_cutoff, numreads_cutoff2, ss_list); const SpliceSitePos& ssp = node->key; assert_lt(ssp.ref(), _spliceSites.size()); assert_lt(node->payload, _spliceSites[ssp.ref()].size()); const SpliceSite& ss = _spliceSites[ssp.ref()][node->payload]; if(ss.numreads() >= numreads_cutoff || (ss.editdist() == 0 && ss.numreads() >= numreads_cutoff2)) print_impl(out, ss_list, &ss); print_recur(node->right, out, numreads_cutoff, numreads_cutoff2, ss_list); } void SpliceSiteDB::print_impl( ofstream& out, EList& ss_list, const SpliceSite* ss) { size_t i = 0; while(i < ss_list.size()) { const SpliceSite& tmp_ss = ss_list[i]; bool do_print = true; if(ss != NULL) { if(tmp_ss.ref() == ss->ref()) { assert_leq(tmp_ss.left(), ss->left()); if(ss->left() < tmp_ss.left() + 10) { do_print = false; if(abs(((int)ss->left() - (int)tmp_ss.left()) - ((int)ss->right() - (int)tmp_ss.right())) <= 10) { if(tmp_ss.numreads() < ss->numreads()) { ss_list.erase(i); ss_list.push_back(*ss); } return; } } } } if(!do_print) { i++; continue; } assert_lt(tmp_ss.ref(), _refnames.size()); out << _refnames[tmp_ss.ref()] << "\t" << tmp_ss.left() << "\t" << tmp_ss.right() << "\t"; if(tmp_ss.splDir() == SPL_FW || tmp_ss.splDir() == SPL_SEMI_FW) { out << "+"; } else if(tmp_ss.splDir() == SPL_RC || tmp_ss.splDir() == SPL_SEMI_RC) { out << "-"; } else { out << "."; } out << endl; ss_list.erase(i); } if(ss != NULL) ss_list.push_back(*ss); } void SpliceSiteDB::read(const GFM& gfm, const EList >& alts) { EList exons; _empty = false; assert_eq(_numRefs, _refnames.size()); for(size_t i = 0; i < alts.size(); i++) { const ALT& alt = alts[i]; if(!alt.splicesite() && !alt.exon()) continue; if(alt.left > alt.right) continue; TIndexOffU ref = 0, left = 0, tlen = 0; char fw = alt.fw; bool straddled2 = false; gfm.joinedToTextOff( 1, alt.left, ref, left, tlen, true, // reject straddlers? straddled2); // straddled? assert_lt(ref, _spliceSites.size()); TIndexOffU right = left + (alt.right - alt.left); if(alt.splicesite()) { left -= 1; right += 1; _spliceSites[ref].expand(); _spliceSites[ref].back().init(ref, left, right, fw ? SPL_FW : SPL_RC, alt.exon(), true, // from file? true); // known splice site? assert_gt(_spliceSites[ref].size(), 0); bool added = false; assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); Node *cur = _fwIndex[ref]->add(pool(ref), _spliceSites[ref].back(), &added); if(!added) { _spliceSites[ref].pop_back(); continue; } assert(added); assert(cur != NULL); cur->payload = (uint32_t)_spliceSites[ref].size() - 1; added = false; SpliceSitePos rssp(ref, right, left, fw ? SPL_FW : SPL_RC); assert_lt(ref, _bwIndex.size()); assert(_bwIndex[ref] != NULL); cur = _bwIndex[ref]->add(pool(ref), rssp, &added); assert(added); assert(cur != NULL); cur->payload = (uint32_t)_spliceSites[ref].size() - 1; } else { assert(alt.exon()); // Given some relaxation if(left >= 10) left -= 10; else left = 0; if(right + 10 < tlen) right += 10; else right = tlen - 1; exons.expand(); exons.back().init(ref, left, right, fw == '+' ? SPL_FW : SPL_RC); } } if(exons.size() > 0) { _exons.resizeExact(exons.size()); _exons.clear(); _exons.push_back_array(exons.begin(), exons.size()); _exons.sort(); } } void SpliceSiteDB::read(ifstream& in, bool known) { _empty = false; assert_eq(_numRefs, _refnames.size()); while(!in.eof()) { string refname; uint32_t left = 0, right = 0; char fw = 0; in >> refname >> left >> right >> fw; uint32_t ref = 0; for(; ref < _refnames.size(); ref++) { if(_refnames[ref] == refname) break; } if(ref >= _numRefs) continue; assert_lt(ref, _spliceSites.size()); _spliceSites[ref].expand(); _spliceSites[ref].back().init(ref, left, right, fw == '+' ? SPL_FW : SPL_RC, false, // exon? true, // from file? known); // known splice site? assert_gt(_spliceSites[ref].size(), 0); bool added = false; assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); Node *cur = _fwIndex[ref]->add(pool(ref), _spliceSites[ref].back(), &added); if(!added) { _spliceSites[ref].pop_back(); continue; } assert(cur != NULL); cur->payload = (uint32_t)_spliceSites[ref].size() - 1; added = false; SpliceSitePos rssp(ref, right, left, fw == '+' ? SPL_FW : SPL_RC); assert_lt(ref, _bwIndex.size()); assert(_bwIndex[ref] != NULL); cur = _bwIndex[ref]->add(pool(ref), rssp, &added); assert(added); assert(cur != NULL); cur->payload = (uint32_t)_spliceSites[ref].size() - 1; } } Pool& SpliceSiteDB::pool(uint64_t ref) { assert_lt(ref, _numRefs); assert_lt(ref, _pool.size()); EList& pool = _pool[ref]; if(pool.size() <= 0 || pool.back()->full()) { pool.push_back(new Pool(1 << 20 /* 1MB */, 16 << 10 /* 16KB */, CA_CAT)); } assert(pool.back() != NULL); return *pool.back(); } float SpliceSiteDB::probscore( int64_t donor_seq, int64_t acceptor_seq) { float probscore = 0.0f; #if defined(NEW_PROB_MODEL) float donor_probscore = 0.0f; assert_leq(donor_seq, 0x3ffff); int64_t donor_exonic_seq = (donor_seq >> 4) & (~0xff); int64_t donor_intronic_seq = donor_seq & 0xff; int64_t donor_rest_seq = donor_exonic_seq | donor_intronic_seq; int donor_seq3 = (donor_seq >> 10) & 0x3; int donor_seq4 = (donor_seq >> 8) & 0x3; donor_probscore = donor_cons1[donor_seq3] * donor_cons2[donor_seq4] / (background_bp_prob[donor_seq3] * background_bp_prob[donor_seq4]) * donor_me2x5[donor_rest_seq]; float acceptor_probscore = 0.0f; assert_leq(acceptor_seq, 0x3fffffffffff); int64_t acceptor_intronic_seq = (acceptor_seq >> 4) & (~0x3f); int64_t acceptor_exonic_seq = acceptor_seq & 0x3f; int64_t acceptor_rest_seq = acceptor_intronic_seq | acceptor_exonic_seq; int acceptor_seq18 = (acceptor_seq >> 8) & 0x3; int acceptor_seq19 = (acceptor_seq >> 6) & 0x3; acceptor_probscore = acceptor_cons1[acceptor_seq18] * acceptor_cons2[acceptor_seq19] / (background_bp_prob[acceptor_seq18] * background_bp_prob[acceptor_seq19]); int64_t acceptor_seq1 = acceptor_rest_seq >> 28 & 0x3fff; // [0, 7] acceptor_probscore *= acceptor_me2x3acc1[acceptor_seq1]; int64_t acceptor_seq2 = (acceptor_rest_seq >> 14) & 0x3fff; // [7, 7] acceptor_probscore *= acceptor_me2x3acc2[acceptor_seq2]; int64_t acceptor_seq3 = acceptor_rest_seq & 0x3fff; // [14, 7] acceptor_probscore *= acceptor_me2x3acc3[acceptor_seq3]; int64_t acceptor_seq4 = (acceptor_rest_seq >> 20) & 0x3fff; // [4, 7] acceptor_probscore *= acceptor_me2x3acc4[acceptor_seq4]; int64_t acceptor_seq5 = (acceptor_rest_seq >> 6) & 0x3fff; // [11, 7] acceptor_probscore *= acceptor_me2x3acc5[acceptor_seq5]; int64_t acceptor_seq6 = acceptor_seq1 & 0x3f; // [4, 3] acceptor_probscore /= acceptor_me2x3acc6[acceptor_seq6]; int64_t acceptor_seq7 = acceptor_seq4 & 0xff; // [7, 4] acceptor_probscore /= acceptor_me2x3acc7[acceptor_seq7]; int64_t acceptor_seq8 = acceptor_seq2 & 0x3f; // [11, 3] acceptor_probscore /= acceptor_me2x3acc8[acceptor_seq8]; int64_t acceptor_seq9 = acceptor_seq5 & 0xff; // [14, 4] acceptor_probscore /= acceptor_me2x3acc9[acceptor_seq9]; donor_probscore /= (1.0f + donor_probscore); acceptor_probscore /= (1.0f + acceptor_probscore); probscore = (donor_probscore + acceptor_probscore) / 2.0; #else assert_lt(donor_seq, (int)(1 << (donor_len << 1))); probscore = donor_prob_sum[donor_seq]; int acceptor_seq1 = (int)(acceptor_seq >> (acceptor_len2 << 1)); assert_lt(acceptor_seq1, (int)(1 << (acceptor_len1 << 1))); probscore *= acceptor_prob_sum1[acceptor_seq1]; int acceptor_seq2 = acceptor_seq % (1 << (acceptor_len2 << 1)); probscore *= acceptor_prob_sum2[acceptor_seq2]; probscore = 1.0 / (1.0 + probscore); #endif return probscore; } hisat2-2.2.1/splice_site.h000066400000000000000000000431471370663763200154100ustar00rootroot00000000000000/* * Copyright 2013, Daehwan Kim * * This file is part of HISAT. * * HISAT is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT. If not, see . */ #ifndef SPLICE_SITE_H_ #define SPLICE_SITE_H_ #include #include #include #include #include "assert_helpers.h" #include "mem_ids.h" #include "ref_coord.h" #include "ds.h" #include "read.h" #include "reference.h" #include "hier_idx_common.h" #include "gfm.h" #include "alt.h" enum { SPL_UNKNOWN = 1, SPL_FW, SPL_RC, SPL_SEMI_FW, SPL_SEMI_RC, }; using namespace std; // #define NEW_PROB_MODEL // the following parameters are borrowed from Yeo and Burge 2004 in Journal of Computational Biology const size_t donor_exonic_len = 3; const size_t donor_intronic_len = 6; const size_t donor_len = donor_exonic_len + donor_intronic_len; #if defined(NEW_PROB_MODEL) const size_t acceptor_intronic_len = 20; const size_t acceptor_exonic_len = 3; const size_t acceptor_len = acceptor_intronic_len + acceptor_exonic_len; #else // the following parameters are borrowed from Ch 3. in Bioinformatics - From Genomes to Drugs. Volume I: Basic Technologies (Victor Solovyev) const size_t acceptor_intronic_len = 14; const size_t acceptor_exonic_len = 1; const size_t acceptor_len = acceptor_intronic_len + acceptor_exonic_len; const size_t acceptor_len1 = acceptor_len / 2; const size_t acceptor_len2 = acceptor_len - acceptor_len1; // the following parameters are borrowed from Yeo and Burge 2004 in Journal of Computational Biology const float background_prob[4] = {0.27f, 0.23f, 0.23f, 0.27f}; extern float donor_prob[4][donor_len]; extern float acceptor_prob[4][acceptor_len]; extern float donor_prob_sum[1 << (donor_len << 1)]; extern float acceptor_prob_sum1[1 << (acceptor_len1 << 1)]; extern float acceptor_prob_sum2[1 << (acceptor_len2 << 1)]; #endif const size_t intronic_len = max(donor_intronic_len, acceptor_intronic_len); extern void init_junction_prob(); /** * */ class SpliceSitePos { public: SpliceSitePos() { reset(); } SpliceSitePos(const SpliceSitePos& c) { init(c); } SpliceSitePos(uint32_t ref, uint32_t left, uint32_t right, uint8_t splDir, bool exon = false) { init(ref, left, right, splDir, exon); } /** * Copy given fields into this Coord. */ void init(uint32_t ref, uint32_t left, uint32_t right, uint8_t splDir, bool exon = false) { _ref = ref; _left = left; _right = right; _splDir = splDir; _exon = exon; } /** * Copy contents of given Coord into this one. */ void init(const SpliceSitePos& c) { _ref = c._ref; _left = c._left; _right = c._right; _splDir = c._splDir; _exon = c._exon; } /** * Return true iff this Coord is identical to the given Coord. */ bool operator==(const SpliceSitePos& o) const { assert(inited()); assert(o.inited()); return _ref == o._ref && _left == o._left && _right == o._right && _splDir == o._splDir && _exon == o._exon; } /** * Return true iff this Coord is less than the given Coord. One Coord is * less than another if (a) its reference id is less, (b) its orientation is * less, or (c) its offset is less. */ bool operator<(const SpliceSitePos& o) const { if(_ref < o._ref) return true; if(_ref > o._ref) return false; if(_left < o._left) return true; if(_left > o._left) return false; if(_right < o._right) return true; if(_right > o._right) return false; if(_splDir < o._splDir) return true; if(_splDir > o._splDir) return false; if(_exon != o._exon) return _exon; return false; } /** * Return the opposite result from operator<. */ bool operator>=(const SpliceSitePos& o) const { return !((*this) < o); } /** * Return true iff this Coord is greater than the given Coord. One Coord * is greater than another if (a) its reference id is greater, (b) its * orientation is greater, or (c) its offset is greater. */ bool operator>(const SpliceSitePos& o) const { if(_ref > o._ref) return true; if(_ref < o._ref) return false; if(_left > o._left) return true; if(_left < o._left) return false; if(_right > o._right) return true; if(_right < o._right) return false; if(_splDir > o._splDir) return true; if(_splDir < o._splDir) return false; if(_exon != o._exon) return !_exon; return false; } /** * Return the opposite result from operator>. */ bool operator<=(const SpliceSitePos& o) const { return !((*this) > o); } /** * Reset this coord to uninitialized state. */ virtual void reset() { _ref = std::numeric_limits::max(); _left = std::numeric_limits::max(); _right = std::numeric_limits::max(); _splDir = SPL_UNKNOWN; _exon = false; } /** * Return true iff this Coord is initialized (i.e. ref and off have both * been set since the last call to reset()). */ bool inited() const { if(_ref != std::numeric_limits::max() && _left != std::numeric_limits::max() && _right != std::numeric_limits::max()) { return true; } return false; } #ifndef NDEBUG /** * Check that coord is internally consistent. */ bool repOk() const { if(_ref == std::numeric_limits::max() || _left == std::numeric_limits::max() || _right == std::numeric_limits::max()) { return false; } return true; } #endif /** * Check whether an interval defined by this coord and having * length 'len' is contained within an interval defined by * 'inbegin' and 'inend'. */ #if 0 bool within(int64_t len, int64_t inbegin, int64_t inend) const { return off_ >= inbegin && off_ + len <= inend; } #endif uint32_t ref() const { return _ref; } uint32_t left() const { return _left; } uint32_t right() const { return _right; } uint8_t splDir() const { return _splDir; } bool canonical() const { return _splDir == SPL_FW || _splDir == SPL_RC; } uint32_t intron_len() const { return _right - _left - 1; } bool exon() const { return _exon; } protected: uint32_t _ref; // which reference? uint32_t _left; // 0-based offset of the right most base of the left flanking exon uint32_t _right; // 0-based offset of the left most base of the right flanking exon uint8_t _splDir; bool _exon; }; /** * */ class SpliceSite : public SpliceSitePos { public: SpliceSite() { reset(); } SpliceSite(const SpliceSite& c) { init(c); } SpliceSite(uint32_t ref, uint32_t left, uint32_t right, uint8_t splDir, bool exon = false, bool fromFile = false, bool known = false) { init(ref, left, right, splDir, exon, fromFile, known); } /** * Copy given fields into this Coord. */ void init(uint32_t ref, uint32_t left, uint32_t right, uint8_t splDir, bool exon = false, bool fromFile = false, bool known = false) { SpliceSitePos::init(ref, left, right, splDir, exon); // _donordint = 0; // _acceptordint = 0; // _leftseq = 0; // _rightseq = 0; _leftext = 0; _rightext = 0; _numreads = 0; _editdist = 0; // _probscore = 0.0f; _readid = 0; _exon = exon; _fromfile = fromFile; _known = known; } /** * Copy contents of given Coord into this one. */ void init(const SpliceSite& c) { SpliceSitePos::init(c); // _donordint = c._donordint; // _acceptordint = c._acceptordint; // _leftseq = c._leftseq; // _rightseq = c._rightseq; _leftext = c._leftext; _rightext = c._rightext; _numreads = c._numreads; _editdist = c._editdist; // _probscore = c._probscore; _readid = c._readid; _fromfile = c._fromfile; _known = c._known; } /** * Reset this coord to uninitialized state. */ virtual void reset() { SpliceSitePos::reset(); // _donordint = 0; // _acceptordint = 0; // _leftseq = 0; // _rightseq = 0; _leftext = 0; _rightext = 0; _numreads = 0; _editdist = 0; // _probscore = 0.0; _readid = 0; _fromfile = false; _known = false; } // uint8_t donordint() const { return _donordint; } // uint8_t acceptordint() const { return _acceptordint; } // uint64_t leftseq() const { return _leftseq; } // uint64_t rightseq() const { return _rightseq; } uint32_t leftext() const { return _leftext; } uint32_t rightext() const { return _rightext; } uint64_t numreads() const { return _numreads; } uint32_t editdist() const { return _editdist; } // float probscore() const { return _probscore; } public: // uint8_t _donordint; // 3' dinucleotide on Watson strand // uint8_t _acceptordint; // 5' dinucleotide on Watson strand // uint64_t _leftseq; // left 32bp flanking seq on Watson strand // uint64_t _rightseq; // right 32bp flanking seq on Watson strand uint32_t _leftext; uint32_t _rightext; uint64_t _numreads; // number of reads spanning this splice site uint32_t _editdist; // float _probscore; uint64_t _readid; bool _fromfile; bool _known; }; std::ostream& operator<<(std::ostream& out, const SpliceSite& c); /** * */ class Exon { public: Exon() { reset(); } Exon(const Exon& c) { init(c); } Exon(uint32_t ref, uint32_t left, uint32_t right, bool fw) { init(ref, left, right, fw); } /** * Copy given fields into this Coord. */ void init(uint32_t ref, uint32_t left, uint32_t right, bool fw) { _ref = ref; _left = left; _right = right; _fw = fw; } /** * Copy contents of given Coord into this one. */ void init(const Exon& c) { _ref = c._ref; _left = c._left; _right = c._right; _fw = c._fw; } /** * Return true iff this Coord is identical to the given Coord. */ bool operator==(const Exon& o) const { assert(inited()); assert(o.inited()); return _ref == o._ref && _left == o._left && _right == o._right && _fw == o._fw; } /** * Return true iff this Coord is less than the given Coord. One Coord is * less than another if (a) its reference id is less, (b) its orientation is * less, or (c) its offset is less. */ bool operator<(const Exon& o) const { if(_ref < o._ref) return true; if(_ref > o._ref) return false; if(_left < o._left) return true; if(_left > o._left) return false; if(_right < o._right) return true; if(_right > o._right) return false; if(_fw != o._fw) return _fw; return false; } /** * Return the opposite result from operator<. */ bool operator>=(const Exon& o) const { return !((*this) < o); } /** * Return true iff this Coord is greater than the given Coord. One Coord * is greater than another if (a) its reference id is greater, (b) its * orientation is greater, or (c) its offset is greater. */ bool operator>(const Exon& o) const { if(_ref > o._ref) return true; if(_ref < o._ref) return false; if(_left > o._left) return true; if(_left < o._left) return false; if(_right > o._right) return true; if(_right < o._right) return false; if(_fw != o._fw) return !_fw; return false; } /** * Return the opposite result from operator>. */ bool operator<=(const Exon& o) const { return !((*this) > o); } /** * Reset this coord to uninitialized state. */ void reset() { _ref = std::numeric_limits::max(); _left = std::numeric_limits::max(); _right = std::numeric_limits::max(); _fw = true; } /** * Return true iff this Coord is initialized (i.e. ref and off have both * been set since the last call to reset()). */ bool inited() const { if(_ref != std::numeric_limits::max() && _left != std::numeric_limits::max() && _right != std::numeric_limits::max()) { return true; } return false; } #ifndef NDEBUG /** * Check that coord is internally consistent. */ bool repOk() const { if(_ref == std::numeric_limits::max() || _left == std::numeric_limits::max() || _right == std::numeric_limits::max()) { return false; } return true; } #endif uint32_t ref() const { return _ref; } uint32_t left() const { return _left; } uint32_t right() const { return _right; } bool fw() const { return _fw; } protected: uint32_t _ref; // which reference? uint32_t _left; // 0-based offset of the right most base of the left flanking exon uint32_t _right; // 0-based offset of the left most base of the right flanking exon bool _fw; // true -> Watson strand }; class AlnRes; class SpliceSiteDB { public: typedef RedBlackNode Node; public: SpliceSiteDB( const BitPairReference& refs, const EList& refnames, bool threadSafe = true, bool write = false, bool read = false); ~SpliceSiteDB(); bool addSpliceSite( const Read& rd, const AlnRes& rs, uint32_t minAnchorLen = 15); static float probscore( int64_t donor_seq, int64_t acceptor_seq); size_t size(uint64_t ref) const; bool empty(uint64_t ref) const; bool empty() { return _empty; } bool write() const { return _write; } bool read() const { return _read; } bool getSpliceSite(SpliceSite& ss) const; void getLeftSpliceSites(uint32_t ref, uint32_t left, uint32_t range, EList& spliceSites) const; void getRightSpliceSites(uint32_t ref, uint32_t right, uint32_t range, EList& spliceSites) const; bool hasSpliceSites(uint32_t ref, uint32_t left1, uint32_t right1, uint32_t left2, uint32_t right2, bool includeNovel = false) const; bool insideExon(uint32_t ref, uint32_t left, uint32_t right) const; void print(ofstream& out); void read(const GFM& gfm, const EList >& alts); void read(ifstream& in, bool known = false); private: void getSpliceSites_recur( const RedBlackNode *node, uint32_t left, uint32_t right, EList& spliceSites) const; bool hasSpliceSites_recur( const RedBlackNode *node, uint32_t left, uint32_t right, bool includeNovel) const; const RedBlackNode* getSpliceSite_temp(const SpliceSitePos& ssp) const; void print_recur( const RedBlackNode *node, ofstream& out, const uint32_t numreads_cutoff, const uint32_t numreads_cutoff2, EList& ss_list); Pool& pool(uint64_t ref); void print_impl( ofstream& out, EList& ss_list, const SpliceSite* ss = NULL); private: uint64_t _numRefs; EList _refnames; EList* > _fwIndex; EList* > _bwIndex; ELList _spliceSites; ELList _pool; // dispenses memory pages bool _write; bool _read; EList _mutex; bool _threadSafe; SStringExpandable raw_refbuf; ASSERT_ONLY(SStringExpandable destU32); BTDnaString donorstr; BTDnaString acceptorstr; bool _empty; EList _exons; }; #endif /*ifndef SPLICE_SITE_H_*/ hisat2-2.2.1/splice_site_mem.h000066400000000000000000041220471370663763200162470ustar00rootroot00000000000000#ifndef SPLICE_SITE_MEM_H_ #define SPLICE_SITE_MEM_H_ static const float background_bp_prob[4] = {0.27f, 0.23f, 0.23f, 0.27f}; static const float donor_cons1[4] = {0.004f, 0.0032f, 0.9896f, 0.0032f}; static const float donor_cons2[4] = {0.0034f, 0.0039f, 0.0042f, 0.9884f}; static const float acceptor_cons1[4] = {0.9903f, 0.0032f, 0.0034f, 0.0030f}; static const float acceptor_cons2[4] = {0.0027f, 0.0037f, 0.9905f, 0.0030f}; static const float donor_me2x5[16384] = { 0.143596f, 0.204157f, 0.192979f, 1.061763f, 0.231687f, 0.483900f, 0.466345f, 1.279266f, 7.648993f, 10.067296f, 6.140895f, 53.603145f, 0.119893f, 0.098323f, 0.141841f, 0.789030f, 0.017593f, 0.048968f, 0.034133f, 0.188161f, 0.040230f, 0.164498f, 0.116904f, 0.321303f, 1.594035f, 4.107312f, 1.847533f, 16.157861f, 0.022057f, 0.035412f, 0.037672f, 0.209963f, 0.007744f, 0.021649f, 0.006671f, 0.075685f, 0.008285f, 0.034023f, 0.010689f, 0.060461f, 0.786409f, 2.035123f, 0.404707f, 7.283977f, 0.003849f, 0.006207f, 0.002919f, 0.033480f, 0.019657f, 0.048412f, 0.030548f, 0.172960f, 0.023873f, 0.086370f, 0.055565f, 0.156854f, 0.930388f, 2.121214f, 0.863749f, 7.758677f, 0.014753f, 0.020958f, 0.020183f, 0.115538f, 0.000036f, 0.000079f, 0.000033f, 0.000718f, 0.000041f, 0.000132f, 0.000057f, 0.000609f, 0.013171f, 0.027007f, 0.007361f, 0.251065f, 0.000045f, 0.000058f, 0.000037f, 0.000809f, 0.000001f, 0.000004f, 0.000001f, 0.000026f, 0.000001f, 0.000009f, 0.000003f, 0.000032f, 0.000570f, 0.002290f, 0.000460f, 0.015726f, 0.000002f, 0.000004f, 0.000002f, 0.000045f, 0.000001f, 0.000005f, 0.000001f, 0.000029f, 0.000001f, 0.000005f, 0.000001f, 0.000016f, 0.000759f, 0.003059f, 0.000272f, 0.019118f, 0.000001f, 0.000002f, 0.000000f, 0.000019f, 0.000002f, 0.000009f, 0.000003f, 0.000057f, 0.000002f, 0.000011f, 0.000003f, 0.000036f, 0.000778f, 0.002763f, 0.000503f, 0.017645f, 0.000003f, 0.000006f, 0.000003f, 0.000058f, 0.005656f, 0.019340f, 0.010995f, 0.107534f, 0.010056f, 0.050509f, 0.029277f, 0.142761f, 1.189892f, 3.766273f, 1.381765f, 21.439941f, 0.003482f, 0.006868f, 0.005959f, 0.058922f, 0.000416f, 0.002786f, 0.001168f, 0.011444f, 0.001049f, 0.010311f, 0.004407f, 0.021532f, 0.148908f, 0.922725f, 0.249638f, 3.880905f, 0.000385f, 0.001485f, 0.000950f, 0.009416f, 0.000106f, 0.000710f, 0.000132f, 0.002652f, 0.000124f, 0.001229f, 0.000232f, 0.002335f, 0.042330f, 0.263445f, 0.031510f, 1.008098f, 0.000039f, 0.000150f, 0.000042f, 0.000865f, 0.000125f, 0.000743f, 0.000282f, 0.002838f, 0.000168f, 0.001461f, 0.000565f, 0.002836f, 0.023448f, 0.128565f, 0.031487f, 0.502760f, 0.000069f, 0.000237f, 0.000137f, 0.001398f, 0.000345f, 0.000440f, 0.000464f, 0.010163f, 0.000033f, 0.000062f, 0.000067f, 0.000728f, 0.043093f, 0.050775f, 0.034547f, 1.201494f, 0.000144f, 0.000106f, 0.000171f, 0.003780f, 0.000047f, 0.000117f, 0.000091f, 0.002005f, 0.000006f, 0.000023f, 0.000019f, 0.000204f, 0.009996f, 0.023059f, 0.011570f, 0.403146f, 0.000030f, 0.000042f, 0.000050f, 0.001120f, 0.000029f, 0.000072f, 0.000025f, 0.001123f, 0.000002f, 0.000007f, 0.000002f, 0.000053f, 0.006867f, 0.015910f, 0.003529f, 0.253063f, 0.000007f, 0.000010f, 0.000005f, 0.000249f, 0.000066f, 0.000145f, 0.000102f, 0.002301f, 0.000005f, 0.000015f, 0.000011f, 0.000124f, 0.007286f, 0.014872f, 0.006755f, 0.241743f, 0.000025f, 0.000031f, 0.000034f, 0.000769f, 0.009874f, 0.008446f, 0.012943f, 0.086116f, 1.028860f, 1.292943f, 2.020043f, 6.700973f, 13.092332f, 10.367984f, 10.252782f, 108.224270f, 0.271467f, 0.133951f, 0.313275f, 2.107366f, 0.001717f, 0.002876f, 0.003250f, 0.021663f, 0.253596f, 0.623901f, 0.718808f, 2.389038f, 3.872948f, 6.004407f, 4.378581f, 46.307291f, 0.070892f, 0.068483f, 0.118107f, 0.796016f, 0.000362f, 0.000609f, 0.000304f, 0.004171f, 0.024999f, 0.061770f, 0.031462f, 0.215197f, 0.914634f, 1.424157f, 0.459131f, 9.992836f, 0.005922f, 0.005746f, 0.004381f, 0.060761f, 0.001131f, 0.001676f, 0.001715f, 0.011741f, 0.088727f, 0.193148f, 0.201444f, 0.687660f, 1.332838f, 1.828380f, 1.206975f, 13.110603f, 0.027958f, 0.023897f, 0.037309f, 0.258268f, 0.000014f, 0.000019f, 0.000013f, 0.000342f, 0.001058f, 0.002070f, 0.001445f, 0.018734f, 0.132319f, 0.163239f, 0.072129f, 2.975054f, 0.000601f, 0.000462f, 0.000482f, 0.012681f, 0.000001f, 0.000001f, 0.000001f, 0.000018f, 0.000054f, 0.000208f, 0.000107f, 0.001388f, 0.008134f, 0.019645f, 0.006401f, 0.264521f, 0.000033f, 0.000049f, 0.000038f, 0.000995f, 0.000000f, 0.000001f, 0.000000f, 0.000009f, 0.000014f, 0.000055f, 0.000013f, 0.000337f, 0.005180f, 0.012565f, 0.001810f, 0.153938f, 0.000007f, 0.000011f, 0.000004f, 0.000205f, 0.000001f, 0.000002f, 0.000001f, 0.000023f, 0.000044f, 0.000150f, 0.000070f, 0.000933f, 0.006540f, 0.013977f, 0.004123f, 0.174993f, 0.000030f, 0.000040f, 0.000028f, 0.000755f, 0.000424f, 0.000872f, 0.000804f, 0.009508f, 0.048683f, 0.147128f, 0.138255f, 0.815245f, 2.220356f, 4.228586f, 2.515052f, 47.191117f, 0.008596f, 0.010200f, 0.014348f, 0.171564f, 0.000044f, 0.000178f, 0.000121f, 0.001436f, 0.007206f, 0.042633f, 0.029543f, 0.174538f, 0.394424f, 1.470576f, 0.644992f, 12.125531f, 0.001348f, 0.003131f, 0.003248f, 0.038916f, 0.000005f, 0.000022f, 0.000007f, 0.000159f, 0.000409f, 0.002432f, 0.000745f, 0.009059f, 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0.007069f, 0.008523f, 0.028282f, 0.000324f, 0.001519f, 0.000531f, 0.003584f, 0.000200f, 0.001376f, 0.000490f, 0.001649f, 0.119364f, 0.518419f, 0.116840f, 1.251963f, 0.000277f, 0.000750f, 0.000400f, 0.002729f, 0.000383f, 0.001585f, 0.001133f, 0.003820f, 0.000268f, 0.001629f, 0.001188f, 0.001996f, 0.065873f, 0.252053f, 0.116320f, 0.622053f, 0.000495f, 0.001181f, 0.001289f, 0.004393f, 0.001271f, 0.001129f, 0.002242f, 0.016464f, 0.000064f, 0.000083f, 0.000169f, 0.000617f, 0.145690f, 0.119797f, 0.153592f, 1.789021f, 0.001240f, 0.000635f, 0.001926f, 0.014294f, 0.000112f, 0.000194f, 0.000285f, 0.002095f, 0.000008f, 0.000020f, 0.000030f, 0.000111f, 0.021799f, 0.035091f, 0.033177f, 0.387184f, 0.000164f, 0.000164f, 0.000367f, 0.002731f, 0.000072f, 0.000126f, 0.000081f, 0.001232f, 0.000002f, 0.000006f, 0.000004f, 0.000031f, 0.015726f, 0.025426f, 0.010627f, 0.255235f, 0.000042f, 0.000042f, 0.000042f, 0.000637f, 0.000163f, 0.000251f, 0.000333f, 0.002516f, 0.000006f, 0.000014f, 0.000019f, 0.000071f, 0.016623f, 0.023678f, 0.020265f, 0.242909f, 0.000143f, 0.000127f, 0.000257f, 0.001963f, 0.000648f, 0.001211f, 0.001818f, 0.013123f, 0.005855f, 0.016085f, 0.024620f, 0.088612f, 0.559901f, 0.969306f, 0.939052f, 10.754886f, 0.000717f, 0.000773f, 0.001772f, 0.012935f, 0.000015f, 0.000054f, 0.000060f, 0.000435f, 0.000190f, 0.001022f, 0.001153f, 0.004159f, 0.021803f, 0.073897f, 0.052792f, 0.605784f, 0.000025f, 0.000052f, 0.000088f, 0.000643f, 0.000003f, 0.000013f, 0.000006f, 0.000092f, 0.000021f, 0.000111f, 0.000055f, 0.000410f, 0.005641f, 0.019202f, 0.006065f, 0.143215f, 0.000002f, 0.000005f, 0.000004f, 0.000054f, 0.000021f, 0.000067f, 0.000067f, 0.000496f, 0.000140f, 0.000666f, 0.000680f, 0.002520f, 0.015794f, 0.047364f, 0.030631f, 0.361009f, 0.000020f, 0.000038f, 0.000058f, 0.000439f, 0.000000f, 0.000000f, 0.000000f, 0.000002f, 0.000000f, 0.000001f, 0.000001f, 0.000010f, 0.000234f, 0.000632f, 0.000274f, 0.012245f, 0.000000f, 0.000000f, 0.000000f, 0.000003f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000002f, 0.000010f, 0.000003f, 0.000143f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000007f, 0.000001f, 0.000091f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000003f, 0.000015f, 0.000004f, 0.000200f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000023f, 0.000102f, 0.000092f, 0.001176f, 0.000225f, 0.001486f, 0.001368f, 0.008752f, 0.077084f, 0.320928f, 0.187000f, 3.807040f, 0.000018f, 0.000048f, 0.000066f, 0.000855f, 0.000000f, 0.000003f, 0.000002f, 0.000023f, 0.000004f, 0.000057f, 0.000038f, 0.000247f, 0.001803f, 0.014692f, 0.006313f, 0.128770f, 0.000000f, 0.000002f, 0.000002f, 0.000026f, 0.000000f, 0.000000f, 0.000000f, 0.000003f, 0.000000f, 0.000004f, 0.000001f, 0.000014f, 0.000269f, 0.002200f, 0.000418f, 0.017542f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000000f, 0.000001f, 0.000001f, 0.000007f, 0.000001f, 0.000010f, 0.000006f, 0.000040f, 0.000352f, 0.002541f, 0.000988f, 0.020703f, 0.000000f, 0.000000f, 0.000000f, 0.000005f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000011f, 0.000017f, 0.000018f, 0.000837f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000001f, 0.000053f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000000f, 0.000017f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000001f, 0.000039f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, }; static const float acceptor_me2x3acc1[16384] = { 0.194114f, 0.254369f, 0.059224f, 0.479611f, 0.184195f, 0.431590f, 0.364757f, 0.677230f, 0.039156f, 0.074210f, 0.046061f, 0.142616f, 0.263375f, 0.540027f, 0.492918f, 0.741151f, 0.196628f, 0.370974f, 0.085338f, 0.772998f, 0.163797f, 0.552574f, 0.461409f, 0.958220f, 0.116240f, 0.317182f, 0.194511f, 0.673638f, 0.250918f, 0.740738f, 0.668017f, 1.123481f, 0.052415f, 0.117194f, 0.024461f, 0.180371f, 0.066584f, 0.266197f, 0.201679f, 0.340960f, 0.037786f, 0.122190f, 0.067988f, 0.191682f, 0.081306f, 0.284450f, 0.232751f, 0.318665f, 0.304223f, 0.440426f, 0.084923f, 0.930491f, 0.255470f, 0.661310f, 0.462862f, 1.162744f, 0.211373f, 0.442572f, 0.227494f, 0.953032f, 0.335322f, 0.759586f, 0.574182f, 1.168105f, 0.145656f, 0.232551f, 0.041391f, 0.372302f, 0.134074f, 0.382754f, 0.247288f, 0.509960f, 0.039260f, 0.090656f, 0.043015f, 0.147930f, 0.246315f, 0.615336f, 0.429361f, 0.717061f, 0.207366f, 0.476669f, 0.083824f, 0.843342f, 0.167569f, 0.688744f, 0.439648f, 1.014110f, 0.163806f, 0.544582f, 0.255300f, 0.982053f, 0.329812f, 1.186261f, 0.817813f, 1.527686f, 0.121829f, 0.331880f, 0.052954f, 0.433706f, 0.150127f, 0.731262f, 0.423528f, 0.795293f, 0.117358f, 0.462374f, 0.196672f, 0.615874f, 0.235539f, 1.003979f, 0.628002f, 0.955004f, 0.292668f, 0.516223f, 0.076092f, 0.926038f, 0.238406f, 0.751906f, 0.402310f, 1.122523f, 0.271717f, 0.693156f, 0.272375f, 1.267379f, 0.402058f, 1.109644f, 0.641221f, 1.448911f, 0.050828f, 0.081012f, 0.015369f, 0.126731f, 0.072420f, 0.206391f, 0.142132f, 0.268697f, 0.018783f, 0.043298f, 0.021898f, 0.069037f, 0.110917f, 0.276615f, 0.205733f, 0.314974f, 0.067112f, 0.154005f, 0.028867f, 0.266243f, 0.083945f, 0.344441f, 0.234358f, 0.495561f, 0.072683f, 0.241225f, 0.120539f, 0.425059f, 0.137740f, 0.494574f, 0.363432f, 0.622358f, 0.036190f, 0.098419f, 0.016738f, 0.125675f, 0.069030f, 0.335666f, 0.207222f, 0.356712f, 0.047796f, 0.187988f, 0.085231f, 0.244672f, 0.090289f, 0.384196f, 0.256158f, 0.357099f, 0.128070f, 0.225511f, 0.035431f, 0.395288f, 0.161483f, 0.508429f, 0.289965f, 0.741681f, 0.163015f, 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8.218508f, }; static const float acceptor_me2x3acc2[16384] = { 0.028739f, 0.051428f, 0.000313f, 0.073324f, 0.034680f, 0.087951f, 0.021917f, 0.102035f, 0.000252f, 0.000765f, 0.000211f, 0.001303f, 0.086087f, 0.188764f, 0.066268f, 0.215400f, 0.034283f, 0.056280f, 0.000377f, 0.081658f, 0.058641f, 0.136431f, 0.037455f, 0.161069f, 0.023430f, 0.065190f, 0.019763f, 0.112954f, 0.091516f, 0.184088f, 0.071197f, 0.213767f, 0.000407f, 0.000736f, 0.000005f, 0.000961f, 0.000959f, 0.002458f, 0.000722f, 0.002610f, 0.000251f, 0.000770f, 0.000250f, 0.001200f, 0.001973f, 0.004369f, 0.001807f, 0.004564f, 0.056136f, 0.110463f, 0.000789f, 0.143996f, 0.140495f, 0.391797f, 0.114664f, 0.415578f, 0.037582f, 0.125335f, 0.040506f, 0.195116f, 0.152611f, 0.367964f, 0.151713f, 0.383898f, 0.035291f, 0.086949f, 0.000364f, 0.110031f, 0.040326f, 0.140805f, 0.024134f, 0.144985f, 0.000333f, 0.001390f, 0.000263f, 0.002100f, 0.096583f, 0.291575f, 0.070407f, 0.295308f, 0.059092f, 0.133562f, 0.000615f, 0.171997f, 0.095713f, 0.306585f, 0.057892f, 0.321253f, 0.043382f, 0.166183f, 0.034652f, 0.255569f, 0.144117f, 0.399132f, 0.106178f, 0.411368f, 0.026513f, 0.065985f, 0.000325f, 0.076430f, 0.059155f, 0.208640f, 0.042131f, 0.196639f, 0.017575f, 0.074131f, 0.016530f, 0.102541f, 0.117347f, 0.357850f, 0.101800f, 0.331735f, 0.084513f, 0.228965f, 0.001124f, 0.264913f, 0.200288f, 0.768999f, 0.154800f, 0.723963f, 0.060777f, 0.279067f, 0.062035f, 0.385591f, 0.209909f, 0.696825f, 0.197614f, 0.645258f, 0.000302f, 0.001475f, 0.000005f, 0.001613f, 0.000400f, 0.002769f, 0.000416f, 0.002464f, 0.000003f, 0.000028f, 0.000005f, 0.000037f, 0.001070f, 0.006402f, 0.001353f, 0.005604f, 0.000430f, 0.001928f, 0.000008f, 0.002146f, 0.000808f, 0.005130f, 0.000848f, 0.004646f, 0.000376f, 0.002852f, 0.000521f, 0.003791f, 0.001358f, 0.007457f, 0.001737f, 0.006643f, 0.000177f, 0.000874f, 0.000004f, 0.000875f, 0.000458f, 0.003203f, 0.000566f, 0.002609f, 0.000140f, 0.001167f, 0.000228f, 0.001395f, 0.001014f, 0.006133f, 0.001527f, 0.004914f, 0.000826f, 0.004438f, 0.000019f, 0.004438f, 0.002270f, 0.017278f, 0.003045f, 0.014059f, 0.000707f, 0.006432f, 0.001252f, 0.007681f, 0.002656f, 0.017481f, 0.004340f, 0.013991f, 0.091342f, 0.202450f, 0.001093f, 0.214612f, 0.085940f, 0.269942f, 0.059752f, 0.232843f, 0.000775f, 0.002911f, 0.000711f, 0.003685f, 0.256620f, 0.696928f, 0.217328f, 0.591287f, 0.156262f, 0.317725f, 0.001890f, 0.342749f, 0.208397f, 0.600508f, 0.146437f, 0.527108f, 0.103177f, 0.355557f, 0.095746f, 0.458055f, 0.391221f, 0.974695f, 0.334848f, 0.841529f, 0.064571f, 0.144564f, 0.000920f, 0.140269f, 0.118620f, 0.376366f, 0.098146f, 0.297145f, 0.038496f, 0.146072f, 0.042064f, 0.169259f, 0.293377f, 0.804820f, 0.295672f, 0.624993f, 0.163052f, 0.397389f, 0.002520f, 0.385156f, 0.318167f, 1.098934f, 0.285680f, 0.866659f, 0.105462f, 0.435623f, 0.125055f, 0.504214f, 0.415736f, 1.241521f, 0.454686f, 0.963052f, 0.026316f, 0.066875f, 0.000385f, 0.071194f, 0.027083f, 0.097539f, 0.023015f, 0.084491f, 0.000232f, 0.000998f, 0.000260f, 0.001268f, 0.068795f, 0.214218f, 0.071208f, 0.182519f, 0.047793f, 0.111420f, 0.000706f, 0.120706f, 0.069721f, 0.230352f, 0.059878f, 0.203055f, 0.032741f, 0.129366f, 0.037134f, 0.167367f, 0.111340f, 0.318055f, 0.116473f, 0.275767f, 0.000575f, 0.001475f, 0.000010f, 0.001438f, 0.001155f, 0.004202f, 0.001168f, 0.003331f, 0.000356f, 0.001547f, 0.000475f, 0.001800f, 0.002430f, 0.007643f, 0.002993f, 0.005961f, 0.074404f, 0.207915f, 0.001405f, 0.202370f, 0.158812f, 0.628929f, 0.174283f, 0.498102f, 0.049930f, 0.236471f, 0.072362f, 0.274867f, 0.176525f, 0.604427f, 0.235963f, 0.470846f, 0.054146f, 0.189449f, 0.000750f, 0.179006f, 0.052768f, 0.261648f, 0.042464f, 0.201163f, 0.000512f, 0.003036f, 0.000544f, 0.003425f, 0.129323f, 0.554431f, 0.126764f, 0.419274f, 0.138031f, 0.443047f, 0.001932f, 0.426005f, 0.190673f, 0.867340f, 0.155075f, 0.678594f, 0.101575f, 0.552570f, 0.109099f, 0.634505f, 0.293786f, 1.155453f, 0.291040f, 0.889184f, 0.062705f, 0.221615f, 0.001034f, 0.191664f, 0.119315f, 0.597617f, 0.114264f, 0.420552f, 0.041664f, 0.249567f, 0.052693f, 0.257758f, 0.242201f, 1.048876f, 0.282525f, 0.726005f, 0.187688f, 0.722103f, 0.003357f, 0.623820f, 0.379347f, 2.068363f, 0.394237f, 1.453927f, 0.135296f, 0.882213f, 0.185688f, 0.910157f, 0.406828f, 1.917883f, 0.514992f, 1.326040f, 0.016347f, 0.113386f, 0.000393f, 0.092596f, 0.018467f, 0.181521f, 0.025793f, 0.120620f, 0.000184f, 0.002160f, 0.000339f, 0.002107f, 0.050532f, 0.429464f, 0.085970f, 0.280696f, 0.035459f, 0.225629f, 0.000862f, 0.187508f, 0.056779f, 0.512010f, 0.080149f, 0.346225f, 0.031026f, 0.334591f, 0.057839f, 0.332064f, 0.097679f, 0.761570f, 0.167950f, 0.506534f, 0.014776f, 0.103528f, 0.000423f, 0.077385f, 0.032592f, 0.323613f, 0.054173f, 0.196826f, 0.011674f, 0.138621f, 0.025625f, 0.123741f, 0.073868f, 0.634155f, 0.149554f, 0.379377f, 0.064742f, 0.493788f, 0.002010f, 0.368689f, 0.151681f, 1.639505f, 0.273598f, 0.996066f, 0.055491f, 0.717295f, 0.132184f, 0.639588f, 0.181625f, 1.697372f, 0.399048f, 1.014311f, 0.080868f, 0.254532f, 0.001301f, 0.201467f, 0.064889f, 0.289445f, 0.060664f, 0.186416f, 0.000688f, 0.003668f, 0.000849f, 0.003467f, 0.198274f, 0.764682f, 0.225784f, 0.484414f, 0.210619f, 0.608155f, 0.003425f, 0.489853f, 0.239556f, 0.980286f, 0.226344f, 0.642480f, 0.139400f, 0.682192f, 0.173941f, 0.656207f, 0.460188f, 1.628172f, 0.529620f, 1.049605f, 0.088118f, 0.280163f, 0.001687f, 0.202973f, 0.138058f, 0.622059f, 0.153596f, 0.366703f, 0.052660f, 0.283761f, 0.077371f, 0.245506f, 0.349402f, 1.361186f, 0.473493f, 0.789258f, 0.208946f, 0.723173f, 0.004342f, 0.523345f, 0.347723f, 1.705565f, 0.419819f, 1.004315f, 0.135468f, 0.794642f, 0.215996f, 0.686752f, 0.464936f, 1.971734f, 0.683739f, 1.142007f, 0.000383f, 0.000697f, 0.000006f, 0.001236f, 0.000447f, 0.001154f, 0.000387f, 0.001666f, 0.000005f, 0.000014f, 0.000005f, 0.000030f, 0.001259f, 0.002809f, 0.001326f, 0.003989f, 0.000625f, 0.001044f, 0.000009f, 0.001886f, 0.001036f, 0.002452f, 0.000905f, 0.003603f, 0.000582f, 0.001647f, 0.000671f, 0.003551f, 0.001833f, 0.003753f, 0.001952f, 0.005424f, 0.000008f, 0.000015f, 0.000000f, 0.000025f, 0.000019f, 0.000049f, 0.000020f, 0.000065f, 0.000007f, 0.000022f, 0.000009f, 0.000042f, 0.000044f, 0.000100f, 0.000055f, 0.000129f, 0.000883f, 0.001769f, 0.000017f, 0.002870f, 0.002141f, 0.006077f, 0.002392f, 0.008022f, 0.000805f, 0.002732f, 0.001188f, 0.005293f, 0.002638f, 0.006474f, 0.003590f, 0.008406f, 0.000702f, 0.001760f, 0.000010f, 0.002771f, 0.000777f, 0.002760f, 0.000636f, 0.003537f, 0.000009f, 0.000038f, 0.000010f, 0.000072f, 0.002110f, 0.006482f, 0.002105f, 0.008171f, 0.001610f, 0.003703f, 0.000023f, 0.005935f, 0.002526f, 0.008233f, 0.002091f, 0.010736f, 0.001609f, 0.006271f, 0.001759f, 0.012004f, 0.004314f, 0.012158f, 0.004350f, 0.015595f, 0.000808f, 0.002046f, 0.000014f, 0.002949f, 0.001745f, 0.006265f, 0.001702f, 0.007348f, 0.000729f, 0.003128f, 0.000938f, 0.005385f, 0.003928f, 0.012188f, 0.004664f, 0.014062f, 0.001987f, 0.005479f, 0.000036f, 0.007889f, 0.004561f, 0.017820f, 0.004825f, 0.020880f, 0.001945f, 0.009089f, 0.002718f, 0.015629f, 0.005422f, 0.018317f, 0.006987f, 0.021110f, 0.000168f, 0.000834f, 0.000004f, 0.001135f, 0.000215f, 0.001516f, 0.000306f, 0.001679f, 0.000003f, 0.000022f, 0.000005f, 0.000035f, 0.000653f, 0.003977f, 0.001131f, 0.004332f, 0.000328f, 0.001494f, 0.000008f, 0.002069f, 0.000596f, 0.003849f, 0.000856f, 0.004338f, 0.000389f, 0.003008f, 0.000739f, 0.004975f, 0.001136f, 0.006346f, 0.001988f, 0.007036f, 0.000151f, 0.000757f, 0.000004f, 0.000943f, 0.000378f, 0.002687f, 0.000639f, 0.002724f, 0.000162f, 0.001376f, 0.000361f, 0.002047f, 0.000949f, 0.005836f, 0.001955f, 0.005820f, 0.000543f, 0.002967f, 0.000017f, 0.003693f, 0.001444f, 0.011187f, 0.002652f, 0.011329f, 0.000632f, 0.005852f, 0.001532f, 0.008698f, 0.001917f, 0.012839f, 0.004288f, 0.012789f, 0.002140f, 0.004828f, 0.000035f, 0.006369f, 0.001950f, 0.006234f, 0.001856f, 0.006693f, 0.000025f, 0.000094f, 0.000031f, 0.000149f, 0.006606f, 0.018258f, 0.007658f, 0.019279f, 0.005017f, 0.010381f, 0.000083f, 0.013937f, 0.006480f, 0.019001f, 0.006233f, 0.020758f, 0.004509f, 0.015811f, 0.005727f, 0.025351f, 0.013799f, 0.034985f, 0.016166f, 0.037592f, 0.002318f, 0.005281f, 0.000045f, 0.006378f, 0.004124f, 0.013316f, 0.004671f, 0.013084f, 0.001881f, 0.007263f, 0.002813f, 0.010475f, 0.011571f, 0.032301f, 0.015962f, 0.031219f, 0.004518f, 0.011204f, 0.000096f, 0.013515f, 0.008537f, 0.030008f, 0.010493f, 0.029453f, 0.003977f, 0.016717f, 0.006455f, 0.024082f, 0.012655f, 0.038456f, 0.018944f, 0.037126f, 0.051204f, 0.124938f, 0.000859f, 0.177101f, 0.062222f, 0.215159f, 0.060612f, 0.248166f, 0.000471f, 0.001948f, 0.000606f, 0.003298f, 0.120455f, 0.360131f, 0.142925f, 0.408566f, 0.064977f, 0.145444f, 0.001101f, 0.209803f, 0.111920f, 0.355038f, 0.110186f, 0.416723f, 0.046539f, 0.176557f, 0.060508f, 0.304147f, 0.136214f, 0.373602f, 0.163346f, 0.431320f, 0.000826f, 0.002036f, 0.000016f, 0.002641f, 0.001960f, 0.006846f, 0.002272f, 0.007227f, 0.000534f, 0.002231f, 0.000818f, 0.003458f, 0.003143f, 0.009491f, 0.004437f, 0.009855f, 0.097248f, 0.260922f, 0.002106f, 0.338160f, 0.245088f, 0.931919f, 0.308324f, 0.982755f, 0.068231f, 0.310267f, 0.113356f, 0.480209f, 0.207619f, 0.682565f, 0.318142f, 0.707995f, 0.123843f, 0.416040f, 0.001966f, 0.523432f, 0.142502f, 0.678440f, 0.131459f, 0.694533f, 0.001224f, 0.006970f, 0.001491f, 0.010472f, 0.266168f, 1.095634f, 0.299083f, 1.103229f, 0.220589f, 0.679821f, 0.003540f, 0.870383f, 0.359788f, 1.571398f, 0.335440f, 1.637032f, 0.169718f, 0.886471f, 0.208965f, 1.355387f, 0.422487f, 1.595411f, 0.479789f, 1.634793f, 0.105928f, 0.359457f, 0.002002f, 0.413942f, 0.237989f, 1.144517f, 0.261266f, 1.072431f, 0.073587f, 0.423221f, 0.106686f, 0.582025f, 0.368181f, 1.530900f, 0.492330f, 1.410954f, 0.288361f, 1.065216f, 0.005913f, 1.225316f, 0.688157f, 3.602606f, 0.819830f, 3.371965f, 0.217329f, 1.360643f, 0.341926f, 1.869123f, 0.562453f, 2.545868f, 0.816190f, 2.343803f, 0.029090f, 0.193726f, 0.000802f, 0.210656f, 0.038800f, 0.366191f, 0.062123f, 0.324003f, 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11.019880f, 3.072788f, 13.908550f, 4.459000f, 12.804630f, 0.198457f, 1.321657f, 0.005469f, 1.437156f, 0.296421f, 2.797614f, 0.474608f, 2.475305f, 0.002303f, 0.025989f, 0.004869f, 0.033748f, 0.504980f, 4.120724f, 0.984849f, 3.586190f, 0.443259f, 2.708066f, 0.012346f, 2.996637f, 0.938457f, 8.125368f, 1.518596f, 7.315998f, 0.400303f, 4.144920f, 0.855452f, 5.477394f, 1.005107f, 7.524218f, 1.981112f, 6.663628f, 0.113890f, 0.766148f, 0.003735f, 0.762541f, 0.332142f, 3.166501f, 0.632864f, 2.564402f, 0.092868f, 1.058813f, 0.233685f, 1.258500f, 0.468663f, 3.863100f, 1.087715f, 3.077240f, 0.474616f, 3.475632f, 0.016892f, 3.455443f, 1.470235f, 15.258280f, 3.040062f, 12.343300f, 0.419866f, 5.211077f, 1.146528f, 6.187004f, 1.096017f, 9.834592f, 2.760461f, 7.825298f, 1.266897f, 3.828666f, 0.023370f, 4.035146f, 1.344118f, 5.756667f, 1.440496f, 4.936720f, 0.011119f, 0.056949f, 0.015736f, 0.071677f, 2.556927f, 9.468311f, 3.337812f, 7.986541f, 3.397565f, 9.419414f, 0.063342f, 10.102420f, 5.109502f, 20.075310f, 5.534197f, 17.519400f, 2.320977f, 10.905680f, 3.319906f, 13.968100f, 6.110718f, 20.758510f, 8.061894f, 17.818540f, 0.876448f, 2.675523f, 0.019240f, 2.580989f, 1.815600f, 7.854714f, 2.315552f, 6.165431f, 0.540604f, 2.796970f, 0.910526f, 3.222167f, 2.860697f, 10.700460f, 4.444009f, 8.261411f, 1.976665f, 6.568699f, 0.047089f, 6.329595f, 4.349427f, 20.483570f, 6.019714f, 16.060460f, 1.322736f, 7.449810f, 2.417663f, 8.572838f, 3.620580f, 14.742530f, 6.103666f, 11.369550f, }; static const float acceptor_me2x3acc3[16384] = { 0.036350f, 0.044079f, 0.017386f, 0.046962f, 0.031088f, 0.045756f, 0.024587f, 0.034370f, 0.025195f, 0.034486f, 0.017536f, 0.034052f, 0.076383f, 0.099866f, 0.073811f, 0.086068f, 0.047550f, 0.057744f, 0.028229f, 0.064311f, 0.043775f, 0.064522f, 0.042972f, 0.050664f, 0.064784f, 0.088804f, 0.055968f, 0.091663f, 0.081233f, 0.106361f, 0.097434f, 0.095823f, 0.243824f, 0.254641f, 0.117527f, 0.282971f, 0.147646f, 0.187151f, 0.117680f, 0.146631f, 0.160177f, 0.188823f, 0.112355f, 0.194472f, 0.391222f, 0.440520f, 0.380999f, 0.395997f, 0.023217f, 0.027583f, 0.011848f, 0.035175f, 0.032216f, 0.046454f, 0.027185f, 0.041768f, 0.034881f, 0.046775f, 0.025903f, 0.055285f, 0.052301f, 0.066993f, 0.053925f, 0.069110f, 0.845246f, 1.058768f, 0.485224f, 0.886728f, 0.769988f, 1.170633f, 0.730911f, 0.691241f, 0.573267f, 0.810543f, 0.478904f, 0.629150f, 1.393519f, 1.882007f, 1.616261f, 1.275036f, 0.529473f, 0.664187f, 0.377275f, 0.581493f, 0.519190f, 0.790481f, 0.611732f, 0.487938f, 0.705887f, 0.999501f, 0.731951f, 0.811009f, 0.709682f, 0.959845f, 1.021687f, 0.679778f, 2.480488f, 2.675926f, 1.435063f, 2.337585f, 1.599884f, 2.094809f, 1.530535f, 1.290199f, 1.594530f, 1.941649f, 1.342452f, 1.571999f, 3.122639f, 3.632029f, 3.650020f, 2.566577f, 0.344638f, 0.422943f, 0.211095f, 0.423992f, 0.509371f, 0.758703f, 0.515904f, 0.536249f, 0.506656f, 0.701830f, 0.451605f, 0.652073f, 0.609120f, 0.805956f, 0.753799f, 0.653580f, 0.000004f, 0.000005f, 0.000008f, 0.000022f, 0.000003f, 0.000004f, 0.000009f, 0.000013f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000003f, 0.000003f, 0.000011f, 0.000013f, 0.000003f, 0.000004f, 0.000008f, 0.000020f, 0.000002f, 0.000004f, 0.000010f, 0.000013f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000002f, 0.000002f, 0.000009f, 0.000009f, 0.000022f, 0.000023f, 0.000046f, 0.000113f, 0.000011f, 0.000014f, 0.000038f, 0.000048f, 0.000000f, 0.000000f, 0.000001f, 0.000002f, 0.000011f, 0.000012f, 0.000046f, 0.000049f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000000f, 0.000000f, 0.000001f, 0.000001f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.441540f, 0.580346f, 0.242142f, 0.576465f, 0.392887f, 0.626763f, 0.356277f, 0.438944f, 0.287594f, 0.426675f, 0.229515f, 0.392801f, 0.643154f, 0.911428f, 0.712614f, 0.732353f, 0.289517f, 0.381084f, 0.197074f, 0.395705f, 0.277302f, 0.443015f, 0.312126f, 0.324331f, 0.370682f, 0.550742f, 0.367189f, 0.530015f, 0.342855f, 0.486571f, 0.471526f, 0.408705f, 1.929791f, 2.184471f, 1.066560f, 2.263273f, 1.215789f, 1.670371f, 1.111102f, 1.220176f, 1.191355f, 1.522222f, 0.958182f, 1.461696f, 2.146398f, 2.619611f, 2.396761f, 2.195528f, 0.175285f, 0.225716f, 0.102565f, 0.268371f, 0.253054f, 0.395503f, 0.244844f, 0.331544f, 0.247474f, 0.359707f, 0.210726f, 0.396378f, 0.273716f, 0.380021f, 0.323589f, 0.365504f, 0.019208f, 0.024062f, 0.010894f, 0.022668f, 0.014719f, 0.022378f, 0.013803f, 0.014864f, 0.014026f, 0.019833f, 0.011577f, 0.017316f, 0.037260f, 0.050324f, 0.042695f, 0.038350f, 0.016945f, 0.021258f, 0.011929f, 0.020935f, 0.013977f, 0.021281f, 0.016270f, 0.014776f, 0.024323f, 0.034443f, 0.024918f, 0.031436f, 0.026723f, 0.036145f, 0.038009f, 0.028794f, 0.137628f, 0.148482f, 0.078666f, 0.145900f, 0.074667f, 0.097772f, 0.070571f, 0.067735f, 0.095254f, 0.115998f, 0.079231f, 0.105638f, 0.203849f, 0.237119f, 0.235411f, 0.188478f, 0.013467f, 0.016528f, 0.008149f, 0.018637f, 0.016742f, 0.024939f, 0.016753f, 0.019827f, 0.021315f, 0.029529f, 0.018771f, 0.030860f, 0.028004f, 0.037056f, 0.034239f, 0.033802f, 0.439889f, 0.569215f, 0.299447f, 0.421534f, 0.359023f, 0.563863f, 0.404129f, 0.294408f, 0.314317f, 0.459095f, 0.311371f, 0.315099f, 0.669467f, 0.934012f, 0.920758f, 0.559527f, 0.185830f, 0.240811f, 0.157017f, 0.186422f, 0.163258f, 0.256777f, 0.228102f, 0.140151f, 0.261010f, 0.381786f, 0.320939f, 0.273924f, 0.229928f, 0.321250f, 0.392521f, 0.201176f, 1.378923f, 1.536710f, 0.946001f, 1.187005f, 0.796836f, 1.077804f, 0.903945f, 0.586975f, 0.933870f, 1.174734f, 0.932331f, 0.840985f, 1.602435f, 1.925409f, 2.221119f, 1.203082f, 0.196878f, 0.249591f, 0.142997f, 0.221244f, 0.260703f, 0.401141f, 0.313111f, 0.250703f, 0.304928f, 0.436346f, 0.322300f, 0.358478f, 0.321212f, 0.439051f, 0.471370f, 0.314825f, 0.000025f, 0.000032f, 0.000061f, 0.000131f, 0.000016f, 0.000024f, 0.000064f, 0.000070f, 0.000000f, 0.000001f, 0.000001f, 0.000002f, 0.000015f, 0.000020f, 0.000074f, 0.000068f, 0.000014f, 0.000018f, 0.000043f, 0.000077f, 0.000010f, 0.000015f, 0.000048f, 0.000044f, 0.000000f, 0.000001f, 0.000002f, 0.000002f, 0.000007f, 0.000009f, 0.000042f, 0.000033f, 0.000152f, 0.000164f, 0.000372f, 0.000706f, 0.000067f, 0.000089f, 0.000274f, 0.000269f, 0.000002f, 0.000003f, 0.000008f, 0.000011f, 0.000069f, 0.000081f, 0.000344f, 0.000281f, 0.000001f, 0.000001f, 0.000003f, 0.000006f, 0.000001f, 0.000002f, 0.000005f, 0.000006f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000001f, 0.000004f, 0.000004f, 0.425449f, 0.577669f, 0.276672f, 0.507379f, 0.339174f, 0.558950f, 0.364721f, 0.346135f, 0.291949f, 0.447446f, 0.276285f, 0.364236f, 0.572068f, 0.837472f, 0.751632f, 0.595026f, 0.188132f, 0.255814f, 0.151858f, 0.234878f, 0.161443f, 0.266440f, 0.215484f, 0.172479f, 0.253771f, 0.389496f, 0.298090f, 0.331444f, 0.205662f, 0.301513f, 0.335404f, 0.223942f, 1.986234f, 2.322637f, 1.301736f, 2.127841f, 1.121130f, 1.591204f, 1.214981f, 1.027785f, 1.291850f, 1.705156f, 1.232074f, 1.447805f, 2.039324f, 2.571151f, 2.700340f, 1.905447f, 0.185394f, 0.246620f, 0.128638f, 0.259279f, 0.239795f, 0.387161f, 0.275128f, 0.286980f, 0.275760f, 0.414061f, 0.278443f, 0.403453f, 0.267243f, 0.383291f, 0.374643f, 0.325972f, 0.004930f, 0.005477f, 0.002425f, 0.006531f, 0.004481f, 0.006043f, 0.003645f, 0.005080f, 0.003602f, 0.004518f, 0.002579f, 0.004992f, 0.009529f, 0.011416f, 0.009473f, 0.011011f, 0.004830f, 0.005374f, 0.002950f, 0.006698f, 0.004725f, 0.006382f, 0.004772f, 0.005608f, 0.006937f, 0.008713f, 0.006165f, 0.010065f, 0.007590f, 0.009106f, 0.009365f, 0.009181f, 0.023858f, 0.022830f, 0.011830f, 0.028393f, 0.015354f, 0.017832f, 0.012589f, 0.015636f, 0.016524f, 0.017848f, 0.011923f, 0.020572f, 0.035215f, 0.036332f, 0.035279f, 0.036551f, 0.002212f, 0.002408f, 0.001161f, 0.003436f, 0.003262f, 0.004309f, 0.002831f, 0.004336f, 0.003503f, 0.004304f, 0.002676f, 0.005694f, 0.004583f, 0.005379f, 0.004861f, 0.006210f, 0.164161f, 0.188414f, 0.096944f, 0.176597f, 0.158929f, 0.221392f, 0.155193f, 0.146303f, 0.117379f, 0.152066f, 0.100872f, 0.132097f, 0.248967f, 0.308086f, 0.297050f, 0.233591f, 0.077016f, 0.088522f, 0.056453f, 0.086734f, 0.080259f, 0.111965f, 0.097279f, 0.077346f, 0.108247f, 0.140439f, 0.115466f, 0.127530f, 0.094960f, 0.117680f, 0.140633f, 0.093272f, 0.347589f, 0.343578f, 0.206866f, 0.335893f, 0.238258f, 0.285842f, 0.234473f, 0.197025f, 0.235562f, 0.262825f, 0.204015f, 0.238139f, 0.402523f, 0.428984f, 0.484009f, 0.339256f, 0.047018f, 0.052870f, 0.029626f, 0.059315f, 0.073853f, 0.100792f, 0.076947f, 0.079727f, 0.072872f, 0.092492f, 0.066818f, 0.096172f, 0.076444f, 0.092678f, 0.097317f, 0.084110f, 0.000000f, 0.000001f, 0.000001f, 0.000003f, 0.000000f, 0.000000f, 0.000001f, 0.000002f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000001f, 0.000000f, 0.000000f, 0.000001f, 0.000002f, 0.000000f, 0.000000f, 0.000001f, 0.000001f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000001f, 0.000002f, 0.000002f, 0.000004f, 0.000010f, 0.000001f, 0.000001f, 0.000003f, 0.000004f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000001f, 0.000001f, 0.000004f, 0.000004f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.000000f, 0.068159f, 0.082084f, 0.038451f, 0.091249f, 0.064454f, 0.094212f, 0.060126f, 0.073841f, 0.046803f, 0.063623f, 0.038423f, 0.065550f, 0.091328f, 0.118587f, 0.104096f, 0.106639f, 0.033472f, 0.040369f, 0.023438f, 0.046911f, 0.034071f, 0.049874f, 0.039450f, 0.040863f, 0.045180f, 0.061506f, 0.046039f, 0.066243f, 0.036463f, 0.047414f, 0.051587f, 0.044571f, 0.214933f, 0.222926f, 0.122199f, 0.258484f, 0.143906f, 0.181158f, 0.135290f, 0.148098f, 0.139887f, 0.163771f, 0.115737f, 0.175994f, 0.219909f, 0.245919f, 0.252607f, 0.230662f, 0.019007f, 0.022426f, 0.011441f, 0.029841f, 0.029162f, 0.041761f, 0.029025f, 0.039178f, 0.028291f, 0.037677f, 0.024781f, 0.046465f, 0.027303f, 0.034733f, 0.033204f, 0.037386f, 0.022710f, 0.028287f, 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3.673952f, 5.106563f, }; static const float acceptor_me2x3acc4[16384] = { 0.034748f, 0.053536f, 0.000903f, 0.143367f, 0.038723f, 0.115762f, 0.066831f, 0.172192f, 0.000570f, 0.001446f, 0.000683f, 0.004695f, 0.103045f, 0.319651f, 0.153655f, 0.431369f, 0.031342f, 0.057415f, 0.000667f, 0.141258f, 0.049473f, 0.175853f, 0.069935f, 0.240318f, 0.019452f, 0.058702f, 0.019117f, 0.175139f, 0.108159f, 0.398928f, 0.132097f, 0.494599f, 0.001270f, 0.002731f, 0.000050f, 0.006952f, 0.001982f, 0.008271f, 0.005189f, 0.011695f, 0.000587f, 0.002081f, 0.001069f, 0.006425f, 0.005728f, 0.024805f, 0.012958f, 0.031821f, 0.055800f, 0.093433f, 0.001475f, 0.254089f, 0.136888f, 0.444747f, 0.240457f, 0.671810f, 0.045152f, 0.124550f, 0.055143f, 0.410743f, 0.239982f, 0.809049f, 0.364211f, 1.108744f, 0.031725f, 0.047173f, 0.000821f, 0.104089f, 0.043776f, 0.126302f, 0.075239f, 0.154798f, 0.000747f, 0.001829f, 0.000892f, 0.004895f, 0.122122f, 0.365610f, 0.181345f, 0.406533f, 0.035930f, 0.063523f, 0.000761f, 0.128773f, 0.070226f, 0.240910f, 0.098859f, 0.271266f, 0.032022f, 0.093267f, 0.031341f, 0.229277f, 0.160948f, 0.572919f, 0.195754f, 0.585271f, 0.017237f, 0.035780f, 0.000676f, 0.075049f, 0.033314f, 0.134182f, 0.086870f, 0.156333f, 0.011451f, 0.039159f, 0.020760f, 0.099604f, 0.100942f, 0.421881f, 0.227413f, 0.445931f, 0.061330f, 0.099109f, 0.001615f, 0.222078f, 0.186295f, 0.584149f, 0.325885f, 0.727045f, 0.071266f, 0.189723f, 0.086673f, 0.515528f, 0.342379f, 1.113986f, 0.517458f, 1.257883f, 0.004423f, 0.007235f, 0.000101f, 0.018111f, 0.004154f, 0.013185f, 0.006277f, 0.018334f, 0.000114f, 0.000307f, 0.000120f, 0.000931f, 0.015410f, 0.050752f, 0.020118f, 0.064025f, 0.005178f, 0.010071f, 0.000096f, 0.023162f, 0.006889f, 0.025998f, 0.008526f, 0.033212f, 0.005044f, 0.016160f, 0.004340f, 0.045071f, 0.020994f, 0.082213f, 0.022449f, 0.095285f, 0.002654f, 0.006061f, 0.000092f, 0.014423f, 0.003492f, 0.015471f, 0.008005f, 0.020450f, 0.001927f, 0.007249f, 0.003071f, 0.020920f, 0.014068f, 0.064682f, 0.027865f, 0.077567f, 0.011676f, 0.020757f, 0.000270f, 0.052768f, 0.024141f, 0.083276f, 0.037128f, 0.117591f, 0.014828f, 0.043427f, 0.015855f, 0.133877f, 0.058997f, 0.211174f, 0.078393f, 0.270532f, 0.065421f, 0.088787f, 0.001523f, 0.230012f, 0.084845f, 0.223429f, 0.131239f, 0.321508f, 0.001515f, 0.003387f, 0.001629f, 0.010641f, 0.255104f, 0.697073f, 0.340922f, 0.910024f, 0.077558f, 0.125152f, 0.001479f, 0.297870f, 0.142476f, 0.446105f, 0.180505f, 0.589759f, 0.068001f, 0.180770f, 0.059896f, 0.521745f, 0.351936f, 1.143423f, 0.385224f, 1.371411f, 0.051527f, 0.097624f, 0.001820f, 0.240414f, 0.093602f, 0.344106f, 0.219662f, 0.470700f, 0.033676f, 0.105110f, 0.054945f, 0.313901f, 0.305678f, 1.166054f, 0.619776f, 1.447082f, 0.105237f, 0.155220f, 0.002494f, 0.408352f, 0.300451f, 0.859873f, 0.473005f, 1.256520f, 0.120302f, 0.292315f, 0.131674f, 0.932566f, 0.595132f, 1.767350f, 0.809483f, 2.343045f, 0.043054f, 0.125193f, 0.001437f, 0.200584f, 0.037671f, 0.212547f, 0.083547f, 0.189157f, 0.000584f, 0.002795f, 0.000900f, 0.005431f, 0.094915f, 0.555685f, 0.181868f, 0.448661f, 0.040118f, 0.138704f, 0.001097f, 0.204170f, 0.049721f, 0.333559f, 0.090318f, 0.272725f, 0.020586f, 0.117250f, 0.025998f, 0.209295f, 0.102920f, 0.716435f, 0.161523f, 0.531438f, 0.001965f, 0.007975f, 0.000099f, 0.012147f, 0.002408f, 0.018966f, 0.008102f, 0.016045f, 0.000751f, 0.005025f, 0.001758f, 0.009282f, 0.006589f, 0.053856f, 0.019156f, 0.041335f, 0.062594f, 0.197808f, 0.002127f, 0.321844f, 0.120565f, 0.739290f, 0.272143f, 0.668136f, 0.041876f, 0.218012f, 0.065718f, 0.430155f, 0.200121f, 1.273318f, 0.390276f, 1.044022f, 0.051858f, 0.145530f, 0.001724f, 0.192122f, 0.056183f, 0.305933f, 0.124084f, 0.224335f, 0.001009f, 0.004666f, 0.001550f, 0.007470f, 0.148396f, 0.838482f, 0.283164f, 0.557812f, 0.060673f, 0.202451f, 0.001652f, 0.245544f, 0.093110f, 0.602838f, 0.168431f, 0.406123f, 0.044708f, 0.245758f, 0.056227f, 0.361459f, 0.202043f, 1.357374f, 0.315771f, 0.829620f, 0.035189f, 0.137859f, 0.001775f, 0.173005f, 0.053399f, 0.405931f, 0.178929f, 0.282959f, 0.019328f, 0.124745f, 0.045027f, 0.189840f, 0.153194f, 1.208391f, 0.443497f, 0.764190f, 0.090760f, 0.276809f, 0.003071f, 0.371096f, 0.216460f, 1.280997f, 0.486572f, 0.953900f, 0.087195f, 0.438109f, 0.136270f, 0.712247f, 0.376656f, 2.312943f, 0.731505f, 1.562580f, 0.045130f, 0.139328f, 0.001319f, 0.208679f, 0.033281f, 0.199370f, 0.064624f, 0.165862f, 0.000960f, 0.004882f, 0.001296f, 0.008868f, 0.116891f, 0.726591f, 0.196100f, 0.548404f, 0.054583f, 0.200362f, 0.001307f, 0.275703f, 0.057016f, 0.406110f, 0.090679f, 0.310397f, 0.043957f, 0.265821f, 0.048604f, 0.443564f, 0.164518f, 1.215922f, 0.226060f, 0.843146f, 0.033823f, 0.145773f, 0.001500f, 0.207546f, 0.034937f, 0.292173f, 0.102923f, 0.231062f, 0.020304f, 0.144162f, 0.041586f, 0.248904f, 0.133278f, 1.156537f, 0.339224f, 0.829794f, 0.107861f, 0.361900f, 0.003208f, 0.550441f, 0.175103f, 1.139995f, 0.346055f, 0.963106f, 0.113253f, 0.626001f, 0.155610f, 1.154623f, 0.405160f, 2.737056f, 0.691798f, 2.097866f, 0.081419f, 0.208546f, 0.002436f, 0.323237f, 0.082907f, 0.412052f, 0.164791f, 0.354749f, 0.001559f, 0.006578f, 0.002154f, 0.012364f, 0.236017f, 1.217171f, 0.405308f, 0.950699f, 0.099715f, 0.303683f, 0.002443f, 0.432441f, 0.143826f, 0.849924f, 0.234148f, 0.672256f, 0.072285f, 0.362665f, 0.081815f, 0.626259f, 0.336371f, 2.062579f, 0.473123f, 1.480089f, 0.080091f, 0.286386f, 0.003635f, 0.421960f, 0.114234f, 0.792587f, 0.344482f, 0.648657f, 0.043278f, 0.254939f, 0.090735f, 0.455512f, 0.353209f, 2.542926f, 0.920252f, 1.888100f, 0.118573f, 0.330074f, 0.003610f, 0.519534f, 0.265796f, 1.435677f, 0.537707f, 1.255187f, 0.112069f, 0.513936f, 0.157622f, 0.980969f, 0.498480f, 2.793865f, 0.871260f, 2.216052f, 0.004387f, 0.011568f, 0.000139f, 0.027394f, 0.005143f, 0.026314f, 0.010857f, 0.034613f, 0.000060f, 0.000259f, 0.000087f, 0.000742f, 0.010342f, 0.054906f, 0.018863f, 0.065523f, 0.004439f, 0.013917f, 0.000116f, 0.030279f, 0.007371f, 0.044842f, 0.012745f, 0.054190f, 0.002280f, 0.011776f, 0.002741f, 0.031068f, 0.012177f, 0.076868f, 0.018191f, 0.084276f, 0.000190f, 0.000701f, 0.000009f, 0.001577f, 0.000313f, 0.002233f, 0.001001f, 0.002792f, 0.000073f, 0.000442f, 0.000162f, 0.001207f, 0.000683f, 0.005060f, 0.001889f, 0.005740f, 0.007728f, 0.022146f, 0.000250f, 0.053258f, 0.019943f, 0.110897f, 0.042851f, 0.148134f, 0.005175f, 0.024431f, 0.007731f, 0.071249f, 0.026419f, 0.152441f, 0.049045f, 0.184739f, 0.006234f, 0.015866f, 0.000197f, 0.030958f, 0.009050f, 0.044688f, 0.019026f, 0.048434f, 0.000121f, 0.000509f, 0.000178f, 0.001205f, 0.019077f, 0.097751f, 0.034651f, 0.096116f, 0.007921f, 0.023967f, 0.000205f, 0.042964f, 0.016286f, 0.095620f, 0.028043f, 0.095211f, 0.005842f, 0.029122f, 0.006994f, 0.063307f, 0.028204f, 0.171832f, 0.041960f, 0.155227f, 0.004023f, 0.014291f, 0.000193f, 0.026508f, 0.008179f, 0.056383f, 0.026087f, 0.058090f, 0.002212f, 0.012944f, 0.004904f, 0.029116f, 0.018727f, 0.133955f, 0.051606f, 0.125209f, 0.013220f, 0.036565f, 0.000426f, 0.072454f, 0.042246f, 0.226720f, 0.090395f, 0.249532f, 0.012713f, 0.057928f, 0.018913f, 0.139193f, 0.058669f, 0.326712f, 0.108461f, 0.326231f, 0.005292f, 0.014816f, 0.000147f, 0.032799f, 0.005229f, 0.028406f, 0.009665f, 0.034929f, 0.000113f, 0.000520f, 0.000145f, 0.001395f, 0.014657f, 0.082623f, 0.023407f, 0.092171f, 0.006950f, 0.023136f, 0.000158f, 0.047055f, 0.009727f, 0.062831f, 0.014726f, 0.070979f, 0.005603f, 0.030724f, 0.005897f, 0.075776f, 0.022401f, 0.150140f, 0.029300f, 0.153878f, 0.003771f, 0.014740f, 0.000159f, 0.031019f, 0.005219f, 0.039584f, 0.014637f, 0.046269f, 0.002266f, 0.014591f, 0.004418f, 0.037235f, 0.015891f, 0.125054f, 0.038502f, 0.132614f, 0.015325f, 0.046630f, 0.000434f, 0.104826f, 0.033334f, 0.196802f, 0.062709f, 0.245744f, 0.016106f, 0.080735f, 0.021066f, 0.220096f, 0.061557f, 0.377111f, 0.100051f, 0.427214f, 0.012751f, 0.029619f, 0.000363f, 0.067854f, 0.017397f, 0.078410f, 0.032916f, 0.099776f, 0.000244f, 0.000935f, 0.000321f, 0.002598f, 0.039526f, 0.184855f, 0.064613f, 0.213406f, 0.016958f, 0.046835f, 0.000396f, 0.098573f, 0.032772f, 0.175623f, 0.050786f, 0.205314f, 0.012305f, 0.055985f, 0.013257f, 0.142889f, 0.061170f, 0.340149f, 0.081901f, 0.360770f, 0.011927f, 0.038677f, 0.000515f, 0.084227f, 0.022793f, 0.143415f, 0.065429f, 0.173479f, 0.006451f, 0.034462f, 0.012875f, 0.091011f, 0.056247f, 0.367231f, 0.139499f, 0.403008f, 0.022501f, 0.056801f, 0.000652f, 0.132142f, 0.067578f, 0.331019f, 0.130136f, 0.427748f, 0.021287f, 0.088525f, 0.028499f, 0.249745f, 0.101150f, 0.514115f, 0.168290f, 0.602723f, 0.057900f, 0.090182f, 0.001353f, 0.218290f, 0.056994f, 0.172248f, 0.088483f, 0.231589f, 0.000790f, 0.002027f, 0.000853f, 0.005950f, 0.100644f, 0.315616f, 0.134995f, 0.384985f, 0.060025f, 0.111161f, 0.001149f, 0.247203f, 0.083693f, 0.300743f, 0.106422f, 0.371487f, 0.031006f, 0.094596f, 0.027411f, 0.255103f, 0.121416f, 0.452721f, 0.133389f, 0.507343f, 0.002512f, 0.005461f, 0.000089f, 0.012566f, 0.003463f, 0.014611f, 0.008157f, 0.018674f, 0.000967f, 0.003464f, 0.001584f, 0.009666f, 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1.573853f, 3.818569f, 0.345036f, 0.954307f, 0.011112f, 1.890938f, 1.162176f, 6.237056f, 2.486770f, 6.864629f, 0.442195f, 2.014830f, 0.657830f, 4.841386f, 1.310182f, 7.296059f, 2.422132f, 7.285319f, 0.093931f, 0.262980f, 0.002613f, 0.582163f, 0.097829f, 0.531455f, 0.180825f, 0.653487f, 0.002665f, 0.012293f, 0.003425f, 0.033001f, 0.222610f, 1.254843f, 0.355496f, 1.399856f, 0.129905f, 0.432437f, 0.002960f, 0.879491f, 0.191644f, 1.237869f, 0.290132f, 1.398400f, 0.139556f, 0.765323f, 0.146888f, 1.887537f, 0.358262f, 2.401204f, 0.468601f, 2.460984f, 0.058446f, 0.228432f, 0.002467f, 0.480706f, 0.085262f, 0.646615f, 0.239098f, 0.755817f, 0.046803f, 0.301357f, 0.091249f, 0.769034f, 0.210726f, 1.658276f, 0.510553f, 1.758533f, 0.173906f, 0.529147f, 0.004924f, 1.189549f, 0.398722f, 2.354047f, 0.750092f, 2.939472f, 0.243585f, 1.220990f, 0.318589f, 3.328590f, 0.597714f, 3.661743f, 0.971496f, 4.148250f, 0.386848f, 0.898572f, 0.011016f, 2.058525f, 0.556329f, 2.507427f, 1.052606f, 3.190655f, 0.009881f, 0.037810f, 0.012997f, 0.105038f, 1.026063f, 4.798655f, 1.677299f, 5.539796f, 0.541753f, 1.496225f, 0.012636f, 3.149097f, 1.103572f, 5.913980f, 1.710201f, 6.913809f, 0.523883f, 2.383585f, 0.564439f, 6.083619f, 1.672146f, 9.298273f, 2.238840f, 9.861945f, 0.315933f, 1.024474f, 0.013650f, 2.231023f, 0.636401f, 4.004245f, 1.826828f, 4.843643f, 0.227735f, 1.216567f, 0.454498f, 3.212782f, 1.274856f, 8.323376f, 3.161767f, 9.134267f, 0.436422f, 1.101710f, 0.012649f, 2.563031f, 1.381632f, 6.767642f, 2.660623f, 8.745250f, 0.550241f, 2.288311f, 0.736683f, 6.455706f, 1.678743f, 8.532534f, 2.793042f, 10.003120f, 0.409265f, 0.637447f, 0.009563f, 1.542972f, 0.424646f, 1.283367f, 0.659260f, 1.725493f, 0.007442f, 0.019095f, 0.008032f, 0.056049f, 0.608718f, 1.908922f, 0.816484f, 2.328484f, 0.446786f, 0.827413f, 0.008551f, 1.840018f, 0.656644f, 2.359590f, 0.834972f, 2.914640f, 0.307578f, 0.938378f, 0.271915f, 2.530581f, 0.773303f, 2.883402f, 0.849560f, 3.231292f, 0.015501f, 0.033704f, 0.000550f, 0.077553f, 0.022528f, 0.095046f, 0.053062f, 0.121478f, 0.007954f, 0.028493f, 0.013026f, 0.079506f, 0.035075f, 0.153554f, 0.071377f, 0.178052f, 0.488528f, 0.826947f, 0.011618f, 2.032708f, 1.115853f, 3.665042f, 1.763167f, 5.004085f, 0.438489f, 1.222777f, 0.481705f, 3.644910f, 1.053768f, 3.591417f, 1.438579f, 4.448712f, 0.826135f, 1.241844f, 0.019224f, 2.476768f, 1.061378f, 3.095779f, 1.640940f, 3.429545f, 0.021572f, 0.053420f, 0.023187f, 0.129200f, 1.594973f, 4.827273f, 2.130483f, 4.851669f, 1.132410f, 2.023963f, 0.021582f, 3.708572f, 2.060774f, 7.146823f, 2.609549f, 7.273870f, 1.119497f, 3.296267f, 0.985585f, 7.324345f, 2.544162f, 9.155376f, 2.783441f, 8.453790f, 0.465272f, 0.976374f, 0.016426f, 1.851119f, 0.837280f, 3.409287f, 1.963928f, 3.590293f, 0.342869f, 1.185325f, 0.559138f, 2.725196f, 1.366600f, 5.774083f, 2.769482f, 5.516609f, 1.187132f, 1.939383f, 0.028115f, 3.927942f, 3.357474f, 10.642930f, 5.283141f, 11.973220f, 1.530144f, 4.118104f, 1.673971f, 10.114420f, 3.323879f, 10.933080f, 4.518841f, 11.158740f, 0.317742f, 0.525447f, 0.006500f, 1.188950f, 0.277870f, 0.891617f, 0.377698f, 1.120629f, 0.009068f, 0.024703f, 0.008569f, 0.067784f, 0.555250f, 1.848734f, 0.652070f, 2.108049f, 0.450233f, 0.885267f, 0.007544f, 1.840327f, 0.557714f, 2.127805f, 0.620909f, 2.456977f, 0.486455f, 1.575721f, 0.376526f, 3.972306f, 0.915567f, 3.624590f, 0.880660f, 3.797091f, 0.197645f, 0.456282f, 0.006135f, 0.981451f, 0.242102f, 1.084496f, 0.499268f, 1.295720f, 0.159182f, 0.605397f, 0.228226f, 1.579128f, 0.525451f, 2.442370f, 0.936204f, 2.647388f, 0.623513f, 1.120592f, 0.012983f, 2.574932f, 1.200345f, 4.185930f, 1.660607f, 5.342674f, 0.878343f, 2.600558f, 0.844813f, 7.246480f, 1.580166f, 5.717911f, 1.888712f, 6.621050f, 0.953460f, 1.308144f, 0.019967f, 3.063171f, 1.151331f, 3.065045f, 1.601953f, 3.986583f, 0.024493f, 0.055359f, 0.023693f, 0.157198f, 1.864726f, 5.151109f, 2.241646f, 6.078375f, 1.368070f, 2.231744f, 0.023466f, 4.801158f, 2.339988f, 7.406847f, 2.666710f, 8.850816f, 1.330531f, 3.575705f, 1.054201f, 9.328364f, 3.113579f, 10.226530f, 3.065663f, 11.086670f, 0.778443f, 1.490985f, 0.024733f, 3.318859f, 1.316646f, 4.893268f, 2.779400f, 6.050104f, 0.564346f, 1.780705f, 0.828253f, 4.806729f, 2.316176f, 8.932038f, 4.224312f, 10.019290f, 1.140074f, 1.699945f, 0.024300f, 4.042348f, 3.030576f, 8.768203f, 4.291724f, 11.581320f, 1.445651f, 3.551124f, 1.423332f, 10.240170f, 3.233625f, 9.707871f, 3.956386f, 11.633060f, }; static const float acceptor_me2x3acc5[16384] = { 0.008602f, 0.120562f, 0.000004f, 0.088841f, 0.006477f, 0.082328f, 0.000037f, 0.118493f, 0.001347f, 0.025650f, 0.000001f, 0.015608f, 0.008022f, 0.078301f, 0.000010f, 0.090715f, 0.010717f, 0.107252f, 0.000027f, 0.114039f, 0.009805f, 0.088994f, 0.000306f, 0.184821f, 0.014729f, 0.200307f, 0.000034f, 0.175875f, 0.015004f, 0.104573f, 0.000102f, 0.174814f, 0.000185f, 0.002733f, 0.000003f, 0.002511f, 0.000165f, 0.002207f, 0.000029f, 0.003960f, 0.000166f, 0.003334f, 0.000002f, 0.002529f, 0.000169f, 0.001734f, 0.000006f, 0.002506f, 0.016295f, 0.164076f, 0.000076f, 0.163631f, 0.022160f, 0.202370f, 0.001262f, 0.394198f, 0.024095f, 0.329686f, 0.000100f, 0.271508f, 0.044644f, 0.313057f, 0.000552f, 0.490855f, 0.008946f, 0.158159f, 0.000028f, 0.102085f, 0.007099f, 0.113822f, 0.000273f, 0.143496f, 0.001548f, 0.037199f, 0.000004f, 0.019827f, 0.006010f, 0.073990f, 0.000050f, 0.075084f, 0.039041f, 0.492823f, 0.000677f, 0.458992f, 0.037644f, 0.430968f, 0.007995f, 0.783978f, 0.059318f, 1.017524f, 0.000920f, 0.782561f, 0.039372f, 0.346120f, 0.001816f, 0.506814f, 0.005822f, 0.108483f, 0.000565f, 0.087313f, 0.005463f, 0.092327f, 0.006493f, 0.145141f, 0.005778f, 0.146299f, 0.000501f, 0.097233f, 0.003822f, 0.049596f, 0.000987f, 0.062757f, 0.030575f, 0.388328f, 0.000968f, 0.339225f, 0.043822f, 0.504779f, 0.016995f, 0.861261f, 0.049981f, 0.862619f, 0.001415f, 0.622252f, 0.060339f, 0.533702f, 0.005083f, 0.732984f, 0.000359f, 0.005415f, 0.000002f, 0.003942f, 0.000256f, 0.003504f, 0.000022f, 0.004983f, 0.000085f, 0.001739f, 0.000001f, 0.001045f, 0.000352f, 0.003695f, 0.000006f, 0.004230f, 0.000553f, 0.005954f, 0.000021f, 0.006254f, 0.000480f, 0.004682f, 0.000224f, 0.009606f, 0.001147f, 0.016780f, 0.000039f, 0.014555f, 0.000814f, 0.006100f, 0.000083f, 0.010074f, 0.000241f, 0.003826f, 0.000051f, 0.003473f, 0.000203f, 0.002928f, 0.000532f, 0.005191f, 0.000326f, 0.007042f, 0.000062f, 0.005279f, 0.000231f, 0.002551f, 0.000131f, 0.003641f, 0.000820f, 0.008879f, 0.000057f, 0.008748f, 0.001057f, 0.010378f, 0.000902f, 0.019971f, 0.001830f, 0.026922f, 0.000114f, 0.021903f, 0.002361f, 0.017801f, 0.000438f, 0.027573f, 0.021364f, 0.239581f, 0.000096f, 0.128936f, 0.017000f, 0.172901f, 0.000940f, 0.181746f, 0.003462f, 0.052760f, 0.000014f, 0.023447f, 0.022598f, 0.176486f, 0.000271f, 0.149328f, 0.047887f, 0.383459f, 0.001195f, 0.297774f, 0.046302f, 0.336264f, 0.014157f, 0.510028f, 0.068124f, 0.741289f, 0.001521f, 0.475352f, 0.076044f, 0.424067f, 0.005050f, 0.517738f, 0.014583f, 0.172370f, 0.002037f, 0.115672f, 0.013723f, 0.147108f, 0.023477f, 0.192819f, 0.013550f, 0.217648f, 0.001693f, 0.120609f, 0.015074f, 0.124085f, 0.005601f, 0.130917f, 0.098077f, 0.790184f, 0.004471f, 0.575534f, 0.140961f, 1.030003f, 0.078699f, 1.465299f, 0.150112f, 1.643474f, 0.006120f, 0.988473f, 0.304774f, 1.710042f, 0.036958f, 1.958200f, 0.023448f, 0.425815f, 0.000002f, 0.192385f, 0.012998f, 0.214069f, 0.000013f, 0.188907f, 0.002727f, 0.067285f, 0.000000f, 0.025103f, 0.014411f, 0.182256f, 0.000003f, 0.129462f, 0.030761f, 0.398883f, 0.000014f, 0.260041f, 0.020720f, 0.243668f, 0.000114f, 0.310269f, 0.031400f, 0.553300f, 0.000013f, 0.297863f, 0.028383f, 0.256310f, 0.000034f, 0.262706f, 0.000456f, 0.008735f, 0.000001f, 0.004921f, 0.000299f, 0.005193f, 0.000009f, 0.005715f, 0.000304f, 0.007914f, 0.000001f, 0.003682f, 0.000274f, 0.003654f, 0.000002f, 0.003236f, 0.038821f, 0.506482f, 0.000032f, 0.309696f, 0.038868f, 0.459901f, 0.000391f, 0.549263f, 0.042634f, 0.755866f, 0.000031f, 0.381658f, 0.070094f, 0.636866f, 0.000153f, 0.612247f, 0.042977f, 0.984438f, 0.000024f, 0.389587f, 0.025107f, 0.521579f, 0.000170f, 0.403164f, 0.005525f, 0.171969f, 0.000003f, 0.056199f, 0.019026f, 0.303508f, 0.000028f, 0.188840f, 0.197487f, 3.230102f, 0.000605f, 1.844500f, 0.140188f, 2.079541f, 0.005261f, 2.319395f, 0.222858f, 4.953288f, 0.000611f, 2.335689f, 0.131253f, 1.495054f, 0.001070f, 1.342229f, 0.025313f, 0.611129f, 0.000434f, 0.301575f, 0.017488f, 0.382910f, 0.003672f, 0.369067f, 0.018658f, 0.612116f, 0.000286f, 0.249434f, 0.010951f, 0.184127f, 0.000499f, 0.142852f, 0.128371f, 2.112536f, 0.000718f, 1.131464f, 0.135454f, 2.021639f, 0.009282f, 2.114878f, 0.155856f, 3.485356f, 0.000780f, 1.541498f, 0.166957f, 1.913410f, 0.002485f, 1.611210f, 0.012231f, 0.238836f, 0.000015f, 0.106602f, 0.006425f, 0.113788f, 0.000096f, 0.099199f, 0.002146f, 0.056951f, 0.000002f, 0.020990f, 0.007899f, 0.107416f, 0.000026f, 0.075377f, 0.019831f, 0.276523f, 0.000134f, 0.178091f, 0.012659f, 0.160084f, 0.001046f, 0.201374f, 0.030548f, 0.578822f, 0.000184f, 0.307833f, 0.019227f, 0.186706f, 0.000345f, 0.189050f, 0.007420f, 0.152711f, 0.000280f, 0.084993f, 0.004609f, 0.086040f, 0.002131f, 0.093531f, 0.007465f, 0.208790f, 0.000252f, 0.095957f, 0.004683f, 0.067119f, 0.000470f, 0.058730f, 0.024396f, 0.342259f, 0.000300f, 0.206747f, 0.023147f, 0.294523f, 0.003492f, 0.347495f, 0.040430f, 0.770787f, 0.000445f, 0.384484f, 0.046285f, 0.452216f, 0.001517f, 0.429475f, 0.062235f, 0.904301f, 0.000050f, 0.298389f, 0.036458f, 0.480456f, 0.000356f, 0.309648f, 0.007491f, 0.147907f, 0.000005f, 0.040301f, 0.043384f, 0.439010f, 0.000092f, 0.227747f, 0.146893f, 1.524084f, 0.000648f, 0.725647f, 0.104564f, 0.983940f, 0.005649f, 0.915019f, 0.155205f, 2.188273f, 0.000612f, 0.860354f, 0.153727f, 1.110782f, 0.001804f, 0.831482f, 0.038448f, 0.588839f, 0.000949f, 0.242278f, 0.026636f, 0.369973f, 0.008052f, 0.297325f, 0.026534f, 0.552221f, 0.000586f, 0.187624f, 0.026192f, 0.279358f, 0.001720f, 0.180711f, 0.249707f, 2.606741f, 0.002011f, 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0.053125f, 1.345146f, 0.004610f, 0.894011f, 0.037707f, 0.489295f, 0.009733f, 0.619146f, 0.213409f, 2.710455f, 0.006758f, 2.367723f, 0.274438f, 3.161191f, 0.106432f, 5.393669f, 0.283073f, 4.885566f, 0.008017f, 3.524217f, 0.366688f, 3.243352f, 0.030888f, 4.454410f, 0.003764f, 0.056728f, 0.000026f, 0.041297f, 0.002410f, 0.032939f, 0.000204f, 0.046834f, 0.000722f, 0.014778f, 0.000004f, 0.008884f, 0.003211f, 0.033707f, 0.000059f, 0.038578f, 0.003502f, 0.037682f, 0.000134f, 0.039581f, 0.002724f, 0.026586f, 0.001274f, 0.054546f, 0.005893f, 0.086173f, 0.000201f, 0.074747f, 0.004485f, 0.033613f, 0.000455f, 0.055510f, 0.002412f, 0.038319f, 0.000515f, 0.034784f, 0.001826f, 0.026312f, 0.004778f, 0.046651f, 0.002652f, 0.057237f, 0.000507f, 0.042904f, 0.002011f, 0.022250f, 0.001143f, 0.031754f, 0.005059f, 0.054781f, 0.000353f, 0.053971f, 0.005850f, 0.057451f, 0.004995f, 0.110556f, 0.009160f, 0.134784f, 0.000571f, 0.109656f, 0.012681f, 0.095624f, 0.002352f, 0.148119f, 0.196148f, 2.199672f, 0.000885f, 1.183806f, 0.140042f, 1.424320f, 0.007742f, 1.497183f, 0.025793f, 0.393065f, 0.000104f, 0.174681f, 0.180645f, 1.410807f, 0.002169f, 1.193707f, 0.265614f, 2.126933f, 0.006631f, 1.651666f, 0.230432f, 1.673488f, 0.070457f, 2.538258f, 0.306610f, 3.336384f, 0.006846f, 2.139456f, 0.367241f, 2.047961f, 0.024389f, 2.500331f, 0.128018f, 1.513157f, 0.017882f, 1.015436f, 0.108087f, 1.158686f, 0.184913f, 1.518724f, 0.096523f, 1.550349f, 0.012058f, 0.859126f, 0.115216f, 0.948409f, 0.042813f, 1.000625f, 0.530342f, 4.272847f, 0.024175f, 3.112147f, 0.683905f, 4.997288f, 0.381824f, 7.109221f, 0.658653f, 7.211161f, 0.026853f, 4.337175f, 1.434893f, 8.050978f, 0.174002f, 9.219319f, 0.353632f, 6.421863f, 0.000029f, 2.901427f, 0.175878f, 2.896677f, 0.000175f, 2.556204f, 0.033367f, 0.823404f, 0.000002f, 0.307201f, 0.189233f, 2.393181f, 0.000041f, 1.699940f, 0.280271f, 3.634266f, 0.000126f, 2.369261f, 0.169379f, 1.991934f, 0.000933f, 2.536391f, 0.232144f, 4.090576f, 0.000093f, 2.202114f, 0.225155f, 2.033244f, 0.000269f, 2.083979f, 0.006581f, 0.125960f, 0.000017f, 0.070963f, 0.003871f, 0.067190f, 0.000119f, 0.073934f, 0.003560f, 0.092603f, 0.000008f, 0.043080f, 0.003441f, 0.045872f, 0.000023f, 0.040631f, 0.344819f, 4.498723f, 0.000283f, 2.750806f, 0.309757f, 3.665185f, 0.003116f, 4.377351f, 0.307282f, 5.447819f, 0.000226f, 2.750758f, 0.542075f, 4.925219f, 0.001184f, 4.734822f, 0.671521f, 15.381840f, 0.000372f, 6.087302f, 0.351982f, 7.312174f, 0.002389f, 5.652077f, 0.070047f, 2.180331f, 0.000035f, 0.712522f, 0.258837f, 4.128981f, 0.000382f, 2.569017f, 1.864185f, 30.490640f, 0.005712f, 17.411210f, 1.187324f, 17.612630f, 0.044561f, 19.644070f, 1.706993f, 37.939960f, 0.004678f, 17.890330f, 1.078733f, 12.287390f, 0.008794f, 11.031370f, 0.378166f, 9.129998f, 0.006484f, 4.505397f, 0.234410f, 5.132644f, 0.049224f, 4.947074f, 0.226177f, 7.420350f, 0.003468f, 3.023750f, 0.142446f, 2.395010f, 0.006497f, 1.858135f, 1.181333f, 19.440540f, 0.006610f, 10.412270f, 1.118410f, 16.692230f, 0.076643f, 17.462080f, 1.163806f, 26.025820f, 0.005824f, 11.510660f, 1.337706f, 15.330800f, 0.019911f, 12.909490f, 0.168929f, 3.298788f, 0.000206f, 1.472377f, 0.079621f, 1.410125f, 0.001189f, 1.229326f, 0.024053f, 0.638272f, 0.000026f, 0.235250f, 0.094985f, 1.291744f, 0.000308f, 0.906459f, 0.165476f, 2.307363f, 0.001116f, 1.486025f, 0.094772f, 1.198501f, 0.007830f, 1.507626f, 0.206831f, 3.919071f, 0.001248f, 2.084263f, 0.139684f, 1.356426f, 0.002507f, 1.373453f, 0.097983f, 2.016711f, 0.003698f, 1.122416f, 0.054615f, 1.019480f, 0.025247f, 1.108240f, 0.079994f, 2.237352f, 0.002700f, 1.028262f, 0.053840f, 0.771734f, 0.005406f, 0.675282f, 0.198452f, 2.784156f, 0.002444f, 1.681815f, 0.168946f, 2.149633f, 0.025487f, 2.536260f, 0.266870f, 5.087758f, 0.002940f, 2.537875f, 0.327816f, 3.202854f, 0.010742f, 3.041791f, 0.753354f, 10.946590f, 0.000601f, 3.612017f, 0.395976f, 5.218264f, 0.003868f, 3.363114f, 0.073577f, 1.452809f, 0.000052f, 0.395857f, 0.457241f, 4.626934f, 0.000970f, 2.400333f, 1.074228f, 11.145650f, 0.004739f, 5.306669f, 0.686097f, 6.456124f, 0.037069f, 6.003896f, 0.920989f, 12.985270f, 0.003634f, 5.105362f, 0.978815f, 7.072584f, 0.011487f, 5.294220f, 0.445001f, 6.815232f, 0.010984f, 2.804123f, 0.276607f, 3.842021f, 0.083618f, 3.087600f, 0.249197f, 5.186203f, 0.005501f, 1.762077f, 0.263942f, 2.815120f, 0.017331f, 1.821045f, 1.780250f, 18.584400f, 0.014340f, 8.299260f, 1.690122f, 16.001590f, 0.166736f, 13.957210f, 1.642118f, 23.294840f, 0.011830f, 8.590324f, 3.174305f, 23.077270f, 0.068019f, 16.202530f, 0.112643f, 2.155673f, 0.000016f, 1.482782f, 0.073844f, 1.281659f, 0.000126f, 1.721915f, 0.010868f, 0.282629f, 0.000001f, 0.160535f, 0.084949f, 1.132162f, 0.000031f, 1.224364f, 0.096703f, 1.321442f, 0.000074f, 1.311560f, 0.077032f, 0.954679f, 0.000726f, 1.850726f, 0.081903f, 1.520890f, 0.000056f, 1.246513f, 0.109485f, 1.041915f, 0.000224f, 1.625847f, 0.002660f, 0.053659f, 0.000011f, 0.046023f, 0.002062f, 0.037728f, 0.000109f, 0.063204f, 0.001472f, 0.040338f, 0.000006f, 0.028570f, 0.001961f, 0.027540f, 0.000022f, 0.037138f, 0.145283f, 1.997482f, 0.000204f, 1.859504f, 0.172026f, 2.145063f, 0.002962f, 3.900311f, 0.132386f, 2.473420f, 0.000166f, 1.901390f, 0.321880f, 3.081984f, 0.001204f, 4.510785f, 0.106242f, 2.564575f, 0.000101f, 1.545168f, 0.073402f, 1.606957f, 0.000852f, 1.891078f, 0.011332f, 0.371716f, 0.000010f, 0.184940f, 0.057713f, 0.970200f, 0.000146f, 0.919029f, 0.319474f, 5.506598f, 0.001675f, 4.787287f, 0.268205f, 4.192679f, 0.017227f, 7.119386f, 0.299130f, 7.006406f, 0.001403f, 5.029912f, 0.260538f, 3.127430f, 0.003635f, 4.274656f, 0.075928f, 1.931801f, 0.002228f, 1.451337f, 0.062037f, 1.431474f, 0.022295f, 2.100556f, 0.046436f, 1.605453f, 0.001219f, 0.996008f, 0.040307f, 0.714184f, 0.003146f, 0.843575f, 0.247218f, 4.287334f, 0.002367f, 3.495971f, 0.308503f, 4.852257f, 0.036182f, 7.728036f, 0.249041f, 5.869010f, 0.002133f, 3.951886f, 0.394530f, 4.764913f, 0.010050f, 6.108617f, 0.064067f, 1.318432f, 0.000134f, 0.895914f, 0.039803f, 0.742868f, 0.001018f, 0.985972f, 0.009328f, 0.260850f, 0.000017f, 0.146372f, 0.050769f, 0.727597f, 0.000282f, 0.777332f, 0.067980f, 0.998916f, 0.000785f, 0.979452f, 0.051319f, 0.683915f, 0.007256f, 1.309787f, 0.086884f, 1.734913f, 0.000897f, 1.404724f, 0.080873f, 0.827600f, 0.002484f, 1.275798f, 0.047160f, 1.022896f, 0.003046f, 0.866733f, 0.034648f, 0.681581f, 0.027412f, 1.128018f, 0.039369f, 1.160389f, 0.002275f, 0.811927f, 0.036521f, 0.551654f, 0.006276f, 0.734900f, 0.099554f, 1.471867f, 0.002099f, 1.353621f, 0.111713f, 1.497924f, 0.028843f, 2.690687f, 0.136895f, 2.750320f, 0.002581f, 2.088674f, 0.231764f, 2.386291f, 0.012997f, 3.450319f, 0.229625f, 3.516160f, 0.000313f, 1.766375f, 0.159088f, 2.209358f, 0.002660f, 2.167831f, 0.022932f, 0.477177f, 0.000028f, 0.197949f, 0.196415f, 2.094566f, 0.000713f, 1.654306f, 0.354671f, 3.877973f, 0.002678f, 2.811026f, 0.298583f, 2.960885f, 0.027609f, 4.192051f, 0.310932f, 4.619892f, 0.002100f, 2.765356f, 0.455450f, 3.468075f, 0.009147f, 3.952356f, 0.172133f, 2.778143f, 0.007271f, 1.740262f, 0.141032f, 2.064354f, 0.072964f, 2.525743f, 0.098566f, 2.161748f, 0.003724f, 1.118212f, 0.143888f, 1.617268f, 0.016169f, 1.592757f, 0.717747f, 7.896047f, 0.009894f, 5.368392f, 0.898172f, 8.961379f, 0.151645f, 11.900200f, 0.676982f, 10.120510f, 0.008347f, 5.681932f, 1.803643f, 13.818370f, 0.066144f, 14.770620f, 0.540862f, 5.927737f, 0.000042f, 2.994635f, 0.272764f, 2.711236f, 0.000260f, 2.675267f, 0.045113f, 0.671886f, 0.000003f, 0.280292f, 0.287541f, 2.194675f, 0.000059f, 1.743142f, 0.426478f, 3.337560f, 0.000183f, 2.432932f, 0.261347f, 1.854927f, 0.001377f, 2.641025f, 0.312271f, 3.320871f, 0.000120f, 1.998996f, 0.340384f, 1.855106f, 0.000389f, 2.126067f, 0.007354f, 0.084947f, 0.000018f, 0.053512f, 0.004386f, 0.045947f, 0.000129f, 0.056533f, 0.003517f, 0.055207f, 0.000008f, 0.028718f, 0.003821f, 0.030735f, 0.000024f, 0.030440f, 0.465792f, 3.667610f, 0.000366f, 2.507600f, 0.424288f, 3.029905f, 0.004081f, 4.046216f, 0.366939f, 3.926193f, 0.000258f, 2.216694f, 0.727491f, 3.989203f, 0.001520f, 4.288138f, 0.892000f, 12.331220f, 0.000473f, 5.456662f, 0.474095f, 5.944066f, 0.003077f, 5.137474f, 0.082252f, 1.545167f, 0.000039f, 0.564619f, 0.341584f, 3.288573f, 0.000481f, 2.287895f, 2.463646f, 24.319170f, 0.007219f, 15.528010f, 1.591102f, 14.244450f, 0.057104f, 17.764690f, 1.994234f, 26.750640f, 0.005227f, 14.104580f, 1.416348f, 9.736629f, 0.011041f, 9.774239f, 0.367008f, 5.347572f, 0.006017f, 2.950690f, 0.230679f, 3.048361f, 0.046322f, 3.285324f, 0.194042f, 3.842072f, 0.002845f, 1.750618f, 0.137344f, 1.393671f, 0.005990f, 1.209023f, 1.385936f, 13.764870f, 0.007416f, 8.243534f, 1.330488f, 11.984430f, 0.087190f, 14.018560f, 1.206998f, 16.290080f, 0.005776f, 8.056075f, 1.559187f, 10.784380f, 0.022193f, 10.154160f, 0.346666f, 4.085599f, 0.000404f, 2.039036f, 0.165683f, 1.770917f, 0.002367f, 1.726284f, 0.043635f, 0.698815f, 0.000046f, 0.287998f, 0.193657f, 1.589441f, 0.000601f, 1.247156f, 0.337853f, 2.843162f, 0.002179f, 2.047465f, 0.196206f, 1.497488f, 0.015502f, 2.106314f, 0.373305f, 4.268979f, 0.002154f, 2.538621f, 0.283340f, 1.660537f, 0.004862f, 1.880056f, 0.146909f, 1.824875f, 0.005303f, 1.135659f, 0.083032f, 0.935422f, 0.036705f, 1.137018f, 0.106025f, 1.789694f, 0.003423f, 0.919714f, 0.080200f, 0.693783f, 0.007701f, 0.678806f, 0.359691f, 3.045515f, 0.004237f, 2.057075f, 0.310500f, 2.384354f, 0.044794f, 3.145610f, 0.427592f, 4.919815f, 0.004505f, 2.744082f, 0.590298f, 3.480736f, 0.018497f, 3.696305f, 1.111658f, 9.748637f, 0.000848f, 3.596823f, 0.592489f, 4.712271f, 0.005535f, 3.395864f, 0.095977f, 1.143743f, 0.000065f, 0.348468f, 0.670323f, 4.093784f, 0.001360f, 2.374693f, 1.577077f, 9.875405f, 0.006653f, 5.257458f, 1.021365f, 5.800435f, 0.052770f, 6.031514f, 1.195270f, 10.170810f, 0.004510f, 4.471317f, 1.427657f, 6.225787f, 0.016021f, 5.211019f, 0.479757f, 4.434388f, 0.011324f, 2.040115f, 0.302386f, 2.534849f, 0.087414f, 2.277812f, 0.237497f, 2.983031f, 0.005014f, 1.133281f, 0.282707f, 1.819771f, 0.017751f, 1.316270f, 2.320164f, 14.617710f, 0.017872f, 7.299190f, 2.233548f, 12.762430f, 0.210712f, 12.447250f, 1.891897f, 16.197410f, 0.013034f, 6.678823f, 4.110108f, 18.033580f, 0.084220f, 14.157440f, }; static const float acceptor_me2x3acc6[64] = { 0.198561f, 0.337610f, 0.053207f, 0.494507f, 0.281041f, 0.889447f, 0.497835f, 1.051663f, 0.054833f, 0.146701f, 0.059791f, 0.204920f, 0.431595f, 1.165403f, 0.729493f, 1.225165f, 0.318682f, 0.805716f, 0.125296f, 1.247968f, 0.425639f, 2.003059f, 1.106270f, 2.504460f, 0.277875f, 1.105458f, 0.444579f, 1.632886f, 0.703515f, 2.824722f, 1.744707f, 3.140203f, 0.121459f, 0.355438f, 0.052579f, 0.416660f, 0.260773f, 1.420445f, 0.746255f, 1.344131f, 0.122075f, 0.562122f, 0.215047f, 0.628406f, 0.355410f, 1.651741f, 0.970475f, 1.389698f, 0.534367f, 0.995770f, 0.131584f, 1.612177f, 0.696320f, 2.415218f, 1.133477f, 3.156525f, 0.518371f, 1.519946f, 0.519427f, 2.346789f, 1.001111f, 2.962646f, 1.554947f, 3.442660f, }; static const float acceptor_me2x3acc7[256] = { 0.081108f, 0.167573f, 0.002337f, 0.363544f, 0.096275f, 0.397918f, 0.186669f, 0.465951f, 0.001415f, 0.004990f, 0.001895f, 0.012787f, 0.191144f, 0.781838f, 0.312855f, 0.859362f, 0.106486f, 0.277527f, 0.002589f, 0.530254f, 0.178124f, 0.928697f, 0.291412f, 0.957738f, 0.070281f, 0.312724f, 0.079420f, 0.705780f, 0.291511f, 1.504128f, 0.402592f, 1.456027f, 0.003287f, 0.010233f, 0.000151f, 0.019997f, 0.005449f, 0.033928f, 0.016796f, 0.035788f, 0.001596f, 0.008479f, 0.003397f, 0.019573f, 0.011741f, 0.072355f, 0.030554f, 0.071640f, 0.136216f, 0.300112f, 0.003885f, 0.664942f, 0.346218f, 1.525973f, 0.664408f, 1.824904f, 0.115029f, 0.432688f, 0.152476f, 1.132412f, 0.449239f, 1.959528f, 0.727757f, 2.199667f, 0.157231f, 0.313885f, 0.004443f, 0.562003f, 0.229863f, 0.918001f, 0.437029f, 0.887163f, 0.003917f, 0.013349f, 0.005144f, 0.028233f, 0.465016f, 1.837885f, 0.746333f, 1.667212f, 0.267623f, 0.673953f, 0.006381f, 1.062730f, 0.551359f, 2.777670f, 0.884509f, 2.364105f, 0.252278f, 1.084667f, 0.279548f, 2.020313f, 0.919431f, 4.583978f, 1.245120f, 3.662190f, 0.094987f, 0.285687f, 0.004267f, 0.460769f, 0.193895f, 1.166641f, 0.586098f, 1.015600f, 0.065845f, 0.338115f, 0.137479f, 0.644149f, 0.425748f, 2.535123f, 1.086372f, 2.071556f, 0.326828f, 0.695776f, 0.009140f, 1.272283f, 1.023115f, 4.357276f, 1.925271f, 4.300530f, 0.394195f, 1.432757f, 0.512374f, 3.094677f, 1.352704f, 5.701262f, 2.148795f, 5.281901f, 0.057108f, 0.111893f, 0.001336f, 0.257086f, 0.061805f, 0.242256f, 0.097290f, 0.300431f, 0.001612f, 0.005392f, 0.001753f, 0.014634f, 0.151666f, 0.588321f, 0.201536f, 0.684852f, 0.105920f, 0.261792f, 0.002091f, 0.529736f, 0.161543f, 0.798747f, 0.214563f, 0.872378f, 0.113136f, 0.477410f, 0.103795f, 1.141098f, 0.326767f, 1.598953f, 0.366378f, 1.639244f, 0.038181f, 0.112707f, 0.001420f, 0.233266f, 0.057697f, 0.340719f, 0.144395f, 0.380619f, 0.029990f, 0.151144f, 0.051842f, 0.369505f, 0.153674f, 0.898096f, 0.324659f, 0.941737f, 0.187401f, 0.391559f, 0.004339f, 0.918799f, 0.434288f, 1.815277f, 0.676619f, 2.299108f, 0.256113f, 0.913623f, 0.275617f, 2.532323f, 0.696500f, 2.881136f, 0.916035f, 3.425251f, 0.232940f, 0.409618f, 0.005813f, 0.875147f, 0.306761f, 1.079138f, 0.515071f, 1.244432f, 0.005656f, 0.016981f, 0.006560f, 0.042855f, 0.677246f, 2.357764f, 0.959925f, 2.552155f, 0.388069f, 0.860830f, 0.008171f, 1.619737f, 0.720186f, 3.195905f, 1.020324f, 3.245741f, 0.356594f, 1.350495f, 0.348960f, 3.001574f, 1.310623f, 5.755779f, 1.567457f, 5.487020f, 0.197520f, 0.523291f, 0.007836f, 1.007094f, 0.363197f, 1.924930f, 0.969550f, 1.999559f, 0.133470f, 0.603706f, 0.246104f, 1.372401f, 0.870313f, 4.564836f, 1.961226f, 4.450991f, 0.385616f, 0.723116f, 0.009524f, 1.577815f, 1.087389f, 4.079232f, 1.807083f, 4.804181f, 0.453372f, 1.451507f, 0.520424f, 3.741071f, 1.568960f, 5.824824f, 2.201048f, 6.439262f, }; static const float acceptor_me2x3acc8[64] = { 0.134444f, 0.409251f, 0.002027f, 0.474269f, 0.184336f, 0.805301f, 0.161375f, 0.751628f, 0.001443f, 0.007759f, 0.001694f, 0.010525f, 0.386007f, 1.386879f, 0.409270f, 1.281481f, 0.372889f, 1.094436f, 0.005800f, 1.222804f, 0.718837f, 3.027880f, 0.649183f, 2.724678f, 0.308629f, 1.600430f, 0.373846f, 2.093096f, 0.975473f, 3.379250f, 1.066943f, 3.010408f, 0.089765f, 0.290835f, 0.001698f, 0.279494f, 0.240649f, 1.118979f, 0.264289f, 0.866080f, 0.068014f, 0.389342f, 0.100188f, 0.437969f, 0.484599f, 1.853180f, 0.644566f, 1.419979f, 0.379547f, 1.332811f, 0.007810f, 1.355863f, 1.060978f, 5.346963f, 1.267546f, 4.380911f, 0.311496f, 1.932612f, 0.499147f, 2.301325f, 0.956094f, 3.962763f, 1.383397f, 3.214281f, }; static const float acceptor_me2x3acc9[256] = { 0.080864f, 1.061148f, 0.000011f, 0.632647f, 0.047418f, 0.576001f, 0.000076f, 0.661114f, 0.009049f, 0.167492f, 0.000001f, 0.080486f, 0.051563f, 0.472722f, 0.000019f, 0.445638f, 0.091072f, 0.893180f, 0.000061f, 0.745645f, 0.064861f, 0.588840f, 0.000540f, 0.946364f, 0.088873f, 1.229428f, 0.000052f, 0.827251f, 0.086646f, 0.593677f, 0.000166f, 0.783673f, 0.001797f, 0.027297f, 0.000007f, 0.019437f, 0.001261f, 0.017732f, 0.000064f, 0.024308f, 0.001179f, 0.025278f, 0.000004f, 0.014508f, 0.001114f, 0.011825f, 0.000013f, 0.013314f, 0.101349f, 0.983407f, 0.000137f, 0.783197f, 0.107646f, 0.966884f, 0.001807f, 1.482449f, 0.105833f, 1.448493f, 0.000126f, 0.929812f, 0.191886f, 1.300784f, 0.000743f, 1.638076f, 0.154788f, 2.479552f, 0.000105f, 1.241938f, 0.092410f, 1.370311f, 0.000774f, 1.321338f, 0.018062f, 0.408120f, 0.000011f, 0.164761f, 0.066491f, 0.744129f, 0.000128f, 0.589341f, 0.610440f, 7.308212f, 0.002132f, 5.125611f, 0.442626f, 4.905333f, 0.019136f, 6.623243f, 0.621187f, 10.489910f, 0.001904f, 5.929898f, 0.391247f, 3.272414f, 0.003899f, 3.629058f, 0.099005f, 1.836332f, 0.002115f, 1.098519f, 0.070736f, 1.214508f, 0.018706f, 1.398699f, 0.067780f, 1.773278f, 0.001270f, 0.855015f, 0.041357f, 0.535914f, 0.002521f, 0.506924f, 0.339500f, 4.021317f, 0.002391f, 2.690590f, 0.367127f, 4.025388f, 0.032003f, 5.185070f, 0.369690f, 6.176573f, 0.002284f, 3.330946f, 0.433020f, 3.583319f, 0.008701f, 3.791018f, 0.054050f, 0.613302f, 0.000071f, 0.393464f, 0.030405f, 0.319364f, 0.000496f, 0.394443f, 0.008783f, 0.140572f, 0.000010f, 0.072689f, 0.036114f, 0.286286f, 0.000136f, 0.290417f, 0.078085f, 0.662187f, 0.000531f, 0.594866f, 0.053349f, 0.418797f, 0.004490f, 0.724286f, 0.110652f, 1.323586f, 0.000660f, 0.958366f, 0.077845f, 0.461199f, 0.001510f, 0.655115f, 0.033487f, 0.439955f, 0.001393f, 0.337108f, 0.022544f, 0.274172f, 0.011605f, 0.404438f, 0.031925f, 0.591623f, 0.001165f, 0.365381f, 0.021758f, 0.199711f, 0.002582f, 0.241966f, 0.079155f, 0.664122f, 0.001085f, 0.569156f, 0.080653f, 0.626402f, 0.013686f, 1.033485f, 0.120029f, 1.420490f, 0.001444f, 0.981210f, 0.157035f, 0.920483f, 0.006143f, 1.247354f, 0.260466f, 2.581281f, 0.000283f, 1.096237f, 0.154217f, 1.414753f, 0.002073f, 1.156691f, 0.027955f, 0.390769f, 0.000027f, 0.133761f, 0.180139f, 1.247216f, 0.000558f, 0.837533f, 0.515311f, 3.816698f, 0.002889f, 2.269680f, 0.370563f, 2.540647f, 0.025710f, 2.908632f, 0.482302f, 5.038710f, 0.002372f, 2.415110f, 0.531751f, 2.751541f, 0.008505f, 2.587283f, 0.170667f, 1.958368f, 0.005852f, 0.993328f, 0.120930f, 1.284526f, 0.051320f, 1.254322f, 0.107464f, 1.739365f, 0.003232f, 0.711099f, 0.114782f, 0.920173f, 0.011229f, 0.738005f, 0.770290f, 5.644597f, 0.008706f, 3.202243f, 0.826094f, 5.603657f, 0.115564f, 6.120130f, 0.771476f, 7.974125f, 0.007649f, 3.646240f, 1.581806f, 8.098064f, 0.051009f, 7.264300f, }; #endif hisat2-2.2.1/splice_site_new.cpp000066400000000000000000001331721370663763200166120ustar00rootroot00000000000000/* * Copyright 2013, Daehwan Kim * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "edit.h" #include "splice_site.h" #include "aligner_report.h" #include "aligner_result.h" #if defined(NEW_PROB_MODEL) #include "splice_site_mem.h" #else float donor_prob[4][donor_len] = { {0.340f, 0.604f, 0.092f, 0.001f, 0.001f, 0.526f, 0.713f, 0.071f, 0.160f}, {0.363f, 0.129f, 0.033f, 0.001f, 0.001f, 0.028f, 0.076f, 0.055f, 0.165f}, {0.183f, 0.125f, 0.803f, 1.000f, 0.001f, 0.419f, 0.118f, 0.814f, 0.209f}, {0.114f, 0.142f, 0.073f, 0.001f, 1.000f, 0.025f, 0.093f, 0.059f, 0.462f} }; float acceptor_prob[4][acceptor_len] = { {0.090f, 0.084f, 0.075f, 0.068f, 0.076f, 0.080f, 0.097f, 0.092f, 0.076f, 0.078f, 0.237f, 0.042f, 1.000f, 0.001f, 0.239f}, {0.310f, 0.310f, 0.307f, 0.293f, 0.326f, 0.330f, 0.373f, 0.385f, 0.410f, 0.352f, 0.309f, 0.708f, 0.001f, 0.001f, 0.138f}, {0.125f, 0.115f, 0.106f, 0.104f, 0.110f, 0.113f, 0.113f, 0.085f, 0.066f, 0.064f, 0.212f, 0.003f, 0.001f, 1.000f, 0.520f}, {0.463f, 0.440f, 0.470f, 0.494f, 0.471f, 0.463f, 0.408f, 0.429f, 0.445f, 0.504f, 0.240f, 0.246f, 0.001f, 0.001f, 0.104f} }; float donor_prob_sum[1 << (donor_len << 1)]; float acceptor_prob_sum1[1 << (acceptor_len1 << 1)]; float acceptor_prob_sum2[1 << (acceptor_len2 << 1)]; #endif void init_junction_prob() { #if !defined(NEW_PROB_MODEL) for(size_t i = 0; i < donor_len; i++) { ASSERT_ONLY(float sum = 0.0f); for(size_t j = 0; j < 4; j++) { float prob = donor_prob[j][i]; assert_gt(prob, 0.0f); ASSERT_ONLY(sum += prob); donor_prob[j][i] = log(prob / background_prob[j]); } assert_range(0.9f, 1.05f, sum); } for(size_t i = 0; i < acceptor_len; i++) { ASSERT_ONLY(float sum = 0.0f); for(size_t j = 0; j < 4; j++) { float prob = acceptor_prob[j][i]; assert_gt(prob, 0.0f); ASSERT_ONLY(sum += prob); acceptor_prob[j][i] = log(prob / background_prob[j]); } assert_range(0.9f, 1.05f, sum); } const size_t donor_elms = 1 << (donor_len << 1); for(size_t i = 0; i < donor_elms; i++) { float sum = 0.0f; for(size_t j = 0; j < donor_len; j++) { int base = (i >> (j << 1)) & 0x3; sum += donor_prob[base][donor_len - j - 1]; } donor_prob_sum[i] = exp(-sum); } const size_t acceptor_elms1 = 1 << (acceptor_len1 << 1); for(size_t i = 0; i < acceptor_elms1; i++) { float sum = 0.0f; for(size_t j = 0; j < acceptor_len1; j++) { int base = (i >> (j << 1)) & 0x3; sum += acceptor_prob[base][acceptor_len1 - j - 1]; } acceptor_prob_sum1[i] = exp(-sum); } const size_t acceptor_elms2 = 1 << (acceptor_len2 << 1); for(size_t i = 0; i < acceptor_elms2; i++) { float sum = 0.0f; for(size_t j = 0; j < acceptor_len2; j++) { int base = (i >> (j << 1)) & 0x3; sum += acceptor_prob[base][acceptor_len - j - 1]; } acceptor_prob_sum2[i] = exp(-sum); } #endif } ostream& operator<<(ostream& out, const SpliceSite& s) { out << s.ref() << "\t" << s.left() << "\t" << s.right() << "\t" << (s.fw() ? "+" : "-") << endl; return out; } #if defined(SPLIT_DB) static const uint64_t ref_segment_size = 1 << 24; SpliceSiteDB::SpliceSiteDB( const BitPairReference& refs, const EList& refnames, bool threadSafe, bool write, bool read) : _numRefs(refs.numRefs()), _refnames(refnames), _write(write), _read(read), _threadSafe(threadSafe), _empty(true) { assert_gt(_numRefs, 0); assert_eq(_numRefs, _refnames.size()); for(uint64_t i = 0; i < _numRefs; i++) { uint64_t reflen = refs.approxLen(i); assert_gt(reflen, 0); uint64_t numsegs = (reflen + ref_segment_size - 1) / ref_segment_size; assert_gt(numsegs, 0); _fwIndex.expand(); _bwIndex.expand(); for(uint64_t j = 0; j < numsegs; j++) { _fwIndex.back().push_back(new RedBlack(16 << 10, CA_CAT)); _bwIndex.back().push_back(new RedBlack(16 << 10, CA_CAT)); } _pool.expand(); _pool.back().resize(numsegs); _spliceSites.expand(); _spliceSites.back().resize(numsegs); _mutex.expand(); for(uint64_t j = 0; j < numsegs; j++) { _mutex.back().push_back(MUTEX_T()); } } donorstr.resize(donor_exonic_len + donor_intronic_len); acceptorstr.resize(acceptor_intronic_len + acceptor_exonic_len); } SpliceSiteDB::~SpliceSiteDB() { assert_eq(_fwIndex.size(), _bwIndex.size()); assert_eq(_fwIndex.size(), _pool.size()); for(uint64_t i = 0; i < _numRefs; i++) { assert_eq(_fwIndex[i].size(), _bwIndex[i].size()); for(uint64_t j = 0; j < _fwIndex[i].size(); j++) { delete _fwIndex[i][j]; delete _bwIndex[i][j]; } _fwIndex[i].clear(); _bwIndex[i].clear(); ELList& pool = _pool[i]; for(size_t j = 0; j < pool.size(); j++) { for(size_t k = 0; k < pool[j].size(); k++) { delete pool[j][k]; } } } } #if 0 size_t SpliceSiteDB::size(uint64_t ref) const { if(!_read) return 0; assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); assert_lt(ref, _fwIndex.size()); assert_eq(_fwIndex.size(), _bwIndex.size()); ThreadSafe t(const_cast(&_mutex[ref]), _threadSafe && _write); return _fwIndex.size(); } bool SpliceSiteDB::empty(uint64_t ref) const { return size(ref) == 0; } #endif bool SpliceSiteDB::addSpliceSite( const Read& rd, const AlnRes& rs, uint32_t minAnchorLen) { if(!_write) return false; if(rs.trimmed5p(true) + rs.trimmed3p(true) > 0) return false; _empty = false; Coord coord = rs.refcoord(); uint64_t ref = coord.ref(); assert_lt(ref, _numRefs); const EList& edits = rs.ned(); if(!coord.orient()) { Edit::invertPoss(const_cast&>(edits), rd.length(), false); } SpliceSitePos ssp; uint32_t refoff = coord.off(); uint32_t leftAnchorLen = 0, rightAnchorLen = 0; size_t eidx = 0; size_t last_eidx = 0; for(size_t i = 0; i < rd.length(); i++, refoff++) { while(eidx < edits.size() && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { refoff++; } else if(edits[eidx].isRefGap()) { assert_gt(refoff, 0); refoff--; } if(edits[eidx].isSpliced()) { assert_gt(refoff, 0); if(ssp.inited()) { assert(edits[last_eidx].isSpliced()); assert_lt(edits[last_eidx].pos, edits[eidx].pos); rightAnchorLen = edits[eidx].pos - edits[last_eidx].pos; if(leftAnchorLen >= minAnchorLen && rightAnchorLen >= minAnchorLen) { bool added = false; uint64_t ref_seg = ssp.left() / ref_segment_size; uint64_t ref_seg2 = ssp.right() / ref_segment_size; assert_leq(ref_seg, ref_seg2); assert_lt(ref, _mutex.size()); assert_lt(ref_seg, _mutex[ref].size()); assert_lt(ref_seg2, _mutex[ref].size()); ThreadSafe t(&_mutex[ref][ref_seg], _threadSafe && _write); ThreadSafe t2(&_mutex[ref][ref_seg2], _threadSafe && _write && ref_seg != ref_seg2); assert_lt(ref, _fwIndex.size()); assert_lt(ref_seg, _fwIndex[ref].size()); assert(_fwIndex[ref][ref_seg] != NULL); Node *cur = _fwIndex[ref][ref_seg]->add(pool(ref, ref_seg), ssp, &added); if(added) { assert_lt(ref, _spliceSites.size()); assert_lt(ref_seg, _spliceSites[ref].size()); _spliceSites[ref][ref_seg].expand(); _spliceSites[ref][ref_seg].back().init(ssp.ref(), ssp.left(), ssp.right(), ssp.fw(), ssp.canonical()); _spliceSites[ref][ref_seg].back()._readid = rd.rdid; assert(cur != NULL); cur->payload = _spliceSites[ref][ref_seg].size() - 1; SpliceSitePos rssp(ssp.ref(), ssp.right(), ssp.left(), ssp.fw(), ssp.canonical()); assert_lt(ref, _bwIndex.size()); assert_lt(ref_seg2, _bwIndex[ref].size()); assert(_bwIndex[ref][ref_seg2] != NULL); cur = _bwIndex[ref][ref_seg2]->add(pool(ref, ref_seg2), rssp, &added); assert(added); assert(cur != NULL); if(ref_seg != ref_seg2) { _spliceSites[ref][ref_seg2].expand(); _spliceSites[ref][ref_seg2].back().init(ssp.ref(), ssp.left(), ssp.right(), ssp.fw(), ssp.canonical()); _spliceSites[ref][ref_seg2].back()._readid = rd.rdid; } cur->payload = _spliceSites[ref][ref_seg2].size() - 1; } else { assert(cur != NULL); assert_lt(ref, _spliceSites.size()); assert_lt(ref_seg, _spliceSites[ref].size()); assert_lt(cur->payload, _spliceSites[ref][ref_seg].size()); if(rd.rdid < _spliceSites[ref][ref_seg][cur->payload]._readid) { _spliceSites[ref][ref_seg][cur->payload]._readid = rd.rdid; } if(ref_seg != ref_seg2) { SpliceSitePos rssp(ssp.ref(), ssp.right(), ssp.left(), ssp.fw(), ssp.canonical()); cur = _bwIndex[ref][ref_seg2]->add(pool(ref, ref_seg2), rssp, &added); assert(cur != NULL); assert(!added); if(rd.rdid < _spliceSites[ref][ref_seg2][cur->payload]._readid) { _spliceSites[ref][ref_seg2][cur->payload]._readid = rd.rdid; } } } } leftAnchorLen = rightAnchorLen; rightAnchorLen = 0; } else { leftAnchorLen = edits[eidx].pos; } bool fw = (edits[eidx].splDir == EDIT_SPL_FW || edits[eidx].splDir == EDIT_SPL_UNKNOWN); bool canonical = (edits[eidx].splDir != EDIT_SPL_UNKNOWN); ssp.init(coord.ref(), refoff - 1, refoff + edits[eidx].splLen, fw, canonical); refoff += edits[eidx].splLen; last_eidx = eidx; } eidx++; } } if(ssp.inited()) { assert(edits[last_eidx].isSpliced()); assert_lt(edits[last_eidx].pos, rd.length()); rightAnchorLen = rd.length() - edits[last_eidx].pos; if(leftAnchorLen >= minAnchorLen && rightAnchorLen >= minAnchorLen) { bool added = false; uint64_t ref_seg = ssp.left() / ref_segment_size; uint64_t ref_seg2 = ssp.right() / ref_segment_size; assert_leq(ref_seg, ref_seg2); assert_lt(ref, _mutex.size()); assert_lt(ref_seg, _mutex[ref].size()); assert_lt(ref_seg2, _mutex[ref].size()); ThreadSafe t(&_mutex[ref][ref_seg], _threadSafe && _write); ThreadSafe t2(&_mutex[ref][ref_seg2], _threadSafe && _write && ref_seg != ref_seg2); assert_lt(ref, _fwIndex.size()); assert_lt(ref_seg, _fwIndex[ref].size()); assert(_fwIndex[ref][ref_seg] != NULL); Node *cur = _fwIndex[ref][ref_seg]->add(pool(ref, ref_seg), ssp, &added); if(added) { assert_lt(ref, _spliceSites.size()); assert_lt(ref_seg, _spliceSites[ref].size()); _spliceSites[ref][ref_seg].expand(); _spliceSites[ref][ref_seg].back().init(ssp.ref(), ssp.left(), ssp.right(), ssp.fw(), ssp.canonical()); _spliceSites[ref][ref_seg].back()._readid = rd.rdid; assert(cur != NULL); cur->payload = _spliceSites[ref][ref_seg].size() - 1; SpliceSitePos rssp(ssp.ref(), ssp.right(), ssp.left(), ssp.fw(), ssp.canonical()); assert_lt(ref, _bwIndex.size()); assert_lt(ref_seg2, _bwIndex[ref].size()); assert(_bwIndex[ref][ref_seg2] != NULL); cur = _bwIndex[ref][ref_seg2]->add(pool(ref, ref_seg2), rssp, &added); assert(added); assert(cur != NULL); if(ref_seg != ref_seg2) { _spliceSites[ref][ref_seg2].expand(); _spliceSites[ref][ref_seg2].back().init(ssp.ref(), ssp.left(), ssp.right(), ssp.fw(), ssp.canonical()); _spliceSites[ref][ref_seg2].back()._readid = rd.rdid; } cur->payload = _spliceSites[ref][ref_seg2].size() - 1; } else { assert(cur != NULL); assert_lt(ref, _spliceSites.size()); assert_lt(ref_seg, _spliceSites[ref].size()); assert_lt(cur->payload, _spliceSites[ref][ref_seg].size()); if(rd.rdid < _spliceSites[ref][ref_seg][cur->payload]._readid) { _spliceSites[ref][ref_seg][cur->payload]._readid = rd.rdid; } if(ref_seg != ref_seg2) { SpliceSitePos rssp(ssp.ref(), ssp.right(), ssp.left(), ssp.fw(), ssp.canonical()); cur = _bwIndex[ref][ref_seg2]->add(pool(ref, ref_seg2), rssp, &added); assert(cur != NULL); assert(!added); if(rd.rdid < _spliceSites[ref][ref_seg2][cur->payload]._readid) { _spliceSites[ref][ref_seg2][cur->payload]._readid = rd.rdid; } } } } } if(!coord.orient()) { Edit::invertPoss(const_cast&>(edits), rd.length(), false); } return true; } bool SpliceSiteDB::getSpliceSite(SpliceSite& ss) const { if(!_read) return false; uint64_t ref = ss.ref(); uint64_t ref_seg = ss.left() / ref_segment_size; assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); assert_lt(ref_seg, _mutex[ref].size()); ThreadSafe t(const_cast(&_mutex[ref][ref_seg]), _threadSafe && _write); assert_lt(ref, _fwIndex.size()); assert_lt(ref_seg, _fwIndex[ref].size()); assert(_fwIndex[ref][ref_seg] != NULL); const Node *cur = _fwIndex[ref][ref_seg]->lookup(ss); if(cur == NULL) return false; assert(cur != NULL); assert_lt(ref, _spliceSites.size()); assert_lt(ref_seg, _spliceSites[ref].size()); ss = _spliceSites[ref][ref_seg][cur->payload]; return true; } void SpliceSiteDB::getLeftSpliceSites(uint32_t ref, uint32_t left, uint32_t range, EList& spliceSites) const { if(!_read) return; spliceSites.clear(); assert_lt(ref, _numRefs); assert_gt(range, 0); assert_geq(left + 1, range); uint32_t begin = left + 1 - range, end = left; for(uint32_t i = begin / ref_segment_size; i <= end / ref_segment_size; i++) { assert_lt(ref, _mutex.size()); assert_lt(i, _mutex[ref].size()); ThreadSafe t(const_cast(&_mutex[ref][i]), _threadSafe && _write); assert_lt(ref, _bwIndex.size()); assert_lt(i, _bwIndex[ref].size()); assert(_bwIndex[ref][i] != NULL); const Node *cur = _bwIndex[ref][i]->root(); if(cur != NULL) getSpliceSites_recur(cur, _spliceSites[ref][i], ref, begin, end, spliceSites); } } void SpliceSiteDB::getRightSpliceSites(uint32_t ref, uint32_t right, uint32_t range, EList& spliceSites) const { if(!_read) return; spliceSites.clear(); assert_lt(ref, _numRefs); assert_gt(range, 0); assert_gt(right + range, range); uint32_t begin = right, end = right + range - 1; for(uint32_t i = begin / ref_segment_size; i <= end / ref_segment_size; i++) { assert_lt(ref, _mutex.size()); assert_lt(i, _mutex[ref].size()); ThreadSafe t(const_cast(&_mutex[ref][i]), _threadSafe && _write); assert_lt(ref, _fwIndex.size()); assert_lt(i, _fwIndex[ref].size()); assert(_fwIndex[ref][i] != NULL); const Node *cur = _fwIndex[ref][i]->root(); if(cur != NULL) getSpliceSites_recur(cur, _spliceSites[ref][i], ref, begin, end, spliceSites); } } void SpliceSiteDB::getSpliceSites_recur( const RedBlackNode *node, const EList& spliceSites_db, uint32_t ref, uint32_t left, uint32_t right, EList& spliceSites) const { assert(node != NULL); bool goleft = true, goright = true; if((node->key.ref() > ref) || (node->key.ref() == ref && node->key.left() > right)) { goright = false; } if((node->key.ref() < ref) || (node->key.ref() == ref && node->key.left() < left)) { goleft = false; } if(goleft && node->left != NULL) { getSpliceSites_recur( node->left, spliceSites_db, ref, left, right, spliceSites); } if(node->key.ref() == ref && node->key.left() >= left && node->key.left() <= right) { assert_lt(node->payload, spliceSites_db.size()); #ifndef NDEBUG const SpliceSite& ss = spliceSites_db[node->payload]; assert_eq(ss.ref(), node->key.ref()); assert(ss.left() == node->key.left() || ss.right() == node->key.left()); #endif spliceSites.push_back(spliceSites_db[node->payload]); } if(goright && node->right != NULL) { getSpliceSites_recur( node->right, spliceSites_db, ref, left, right, spliceSites); } } void SpliceSiteDB::print(ofstream& out) { EList ss_list; for(uint64_t i = 0; i < _fwIndex.size(); i++) { for(uint64_t j = 0; j < _fwIndex[i].size(); j++) { assert(_fwIndex[i][j] != NULL); const Node *root = _fwIndex[i][j]->root(); if(root != NULL) print_recur(root, out, ss_list); } } print_impl(out, ss_list); } void SpliceSiteDB::print_recur( const RedBlackNode *node, ofstream& out, EList& ss_list) { if(node == NULL) return; print_recur(node->left, out, ss_list); print_impl(out, ss_list, &(node->key)); print_recur(node->right, out, ss_list); } void SpliceSiteDB::print_impl( ofstream& out, EList& ss_list, const SpliceSitePos* ss) { size_t i = 0; while(i < ss_list.size()) { const SpliceSitePos& tmp_ss = ss_list[i]; bool do_print = true; if(ss != NULL) { if(tmp_ss.ref() == ss->ref()) { assert_leq(tmp_ss.left(), ss->left()); if(ss->left() < tmp_ss.left() + 10) { do_print = false; if((int)ss->left() - (int)tmp_ss.left() == (int)ss->right() - (int)tmp_ss.right()) { if(!tmp_ss.canonical() && ss->canonical()) { ss_list.erase(i); ss_list.push_back(*ss); } return; } } } } if(!do_print) { i++; continue; } assert_lt(tmp_ss.ref(), _refnames.size()); out << _refnames[tmp_ss.ref()] << "\t" << tmp_ss.left() << "\t" << tmp_ss.right() << "\t" << (tmp_ss.canonical() ? (tmp_ss.fw() ? "+" : "-") : ".") << endl; ss_list.erase(i); } if(ss != NULL) ss_list.push_back(*ss); } void SpliceSiteDB::read(ifstream& in, bool novel) { _empty = false; assert_eq(_numRefs, _refnames.size()); while(!in.eof()) { string refname; uint32_t left = 0, right = 0; char fw = 0; in >> refname >> left >> right >> fw; uint32_t ref = 0; for(; ref < _refnames.size(); ref++) { if(_refnames[ref] == refname) break; } if(ref >= _numRefs) continue; uint64_t ref_seg = left / ref_segment_size; assert_lt(ref, _spliceSites.size()); assert_lt(ref_seg, _spliceSites[ref].size()); _spliceSites[ref][ref_seg].expand(); _spliceSites[ref][ref_seg].back().init(ref, left, right, fw == '+' || fw == '.', fw != '.'); _spliceSites[ref][ref_seg].back()._fromfile = true; assert_gt(_spliceSites[ref][ref_seg].size(), 0); bool added = false; assert_lt(ref, _fwIndex.size()); assert_lt(ref_seg, _fwIndex[ref].size()); assert(_fwIndex[ref][ref_seg] != NULL); Node *cur = _fwIndex[ref][ref_seg]->add(pool(ref, ref_seg), _spliceSites[ref][ref_seg].back(), &added); assert(added); assert(cur != NULL); cur->payload = _spliceSites[ref][ref_seg].size() - 1; added = false; uint64_t ref_seg2 = right / ref_segment_size; SpliceSitePos rssp(ref, right, left, fw == '+' || fw == '.', fw != '.'); assert_lt(ref, _bwIndex.size()); assert_lt(ref_seg2, _bwIndex.size()); assert(_bwIndex[ref][ref_seg2] != NULL); cur = _bwIndex[ref][ref_seg2]->add(pool(ref, ref_seg2), rssp, &added); assert(added); assert(cur != NULL); if(ref_seg != ref_seg2) { _spliceSites[ref][ref_seg2].expand(); _spliceSites[ref][ref_seg2].back().init(ref, left, right, fw == '+' || fw == '.', fw != '.'); _spliceSites[ref][ref_seg2].back()._fromfile = true; } cur->payload = _spliceSites[ref][ref_seg2].size() - 1; } } Pool& SpliceSiteDB::pool(uint64_t ref, uint64_t ref_seg) { assert_lt(ref, _numRefs); assert_lt(ref, _pool.size()); assert_lt(ref_seg, _pool[ref].size()); if(_pool[ref][ref_seg].size() <= 0 || _pool[ref][ref_seg].back()->full()) { _pool[ref][ref_seg].push_back(new Pool(1 << 18 /* 256KB */, 16 << 10 /* 16KB */, CA_CAT)); } assert(_pool[ref][ref_seg].back() != NULL); return *_pool[ref][ref_seg].back(); } #else SpliceSiteDB::SpliceSiteDB( const BitPairReference& refs, const EList& refnames, bool threadSafe, bool write, bool read) : _numRefs(refs.numRefs()), _refnames(refnames), _write(write), _read(read), _threadSafe(threadSafe), _empty(true) { assert_gt(_numRefs, 0); assert_eq(_numRefs, _refnames.size()); for(uint64_t i = 0; i < _numRefs; i++) { _fwIndex.push_back(new RedBlack(16 << 10, CA_CAT)); _bwIndex.push_back(new RedBlack(16 << 10, CA_CAT)); _pool.expand(); _spliceSites.expand(); _mutex.push_back(MUTEX_T()); } donorstr.resize(donor_exonic_len + donor_intronic_len); acceptorstr.resize(acceptor_intronic_len + acceptor_exonic_len); } SpliceSiteDB::~SpliceSiteDB() { assert_eq(_fwIndex.size(), _bwIndex.size()); assert_eq(_fwIndex.size(), _pool.size()); for(uint64_t i = 0; i < _numRefs; i++) { delete _fwIndex[i]; delete _bwIndex[i]; EList& pool = _pool[i]; for(size_t j = 0; j < pool.size(); j++) { delete pool[j]; } } } size_t SpliceSiteDB::size(uint64_t ref) const { if(!_read) return 0; assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); assert_lt(ref, _fwIndex.size()); assert_eq(_fwIndex.size(), _bwIndex.size()); ThreadSafe t(const_cast(&_mutex[ref]), _threadSafe && _write); return _fwIndex.size(); } bool SpliceSiteDB::empty(uint64_t ref) const { return size(ref) == 0; } bool SpliceSiteDB::addSpliceSite( const Read& rd, const AlnRes& rs, uint32_t minAnchorLen) { if(!_write) return false; if(rs.trimmed5p(true) + rs.trimmed3p(true) > 0) return false; _empty = false; Coord coord = rs.refcoord(); uint64_t ref = coord.ref(); assert_lt(ref, _numRefs); const EList& edits = rs.ned(); if(!coord.orient()) { Edit::invertPoss(const_cast&>(edits), rd.length(), false); } SpliceSitePos ssp; uint32_t refoff = coord.off(); uint32_t leftAnchorLen = 0, rightAnchorLen = 0; size_t eidx = 0; size_t last_eidx = 0; uint32_t mm = 0; for(size_t i = 0; i < rd.length(); i++, refoff++) { while(eidx < edits.size() && edits[eidx].pos == i) { if(edits[eidx].isReadGap()) { refoff++; } else if(edits[eidx].isRefGap()) { assert_gt(refoff, 0); refoff--; } if(edits[eidx].isGap() || edits[eidx].isMismatch()) mm++; if(edits[eidx].isSpliced()) { assert_gt(refoff, 0); if(ssp.inited()) { assert(edits[last_eidx].isSpliced()); assert_lt(edits[last_eidx].pos, edits[eidx].pos); rightAnchorLen = edits[eidx].pos - edits[last_eidx].pos; uint32_t minLeftAnchorLen = minAnchorLen + mm * 2 + (edits[eidx].splDir == EDIT_SPL_UNKNOWN ? 6 : 0); uint32_t mm2 = 0; for(size_t j = eidx + 1; j < edits.size(); j++) { if(edits[j].isGap() || edits[j].isMismatch()) mm2++; } uint32_t minRightAnchorLen = minAnchorLen + mm2 * 2 + (edits[eidx].splDir == EDIT_SPL_UNKNOWN ? 6 : 0); if(leftAnchorLen >= minLeftAnchorLen && rightAnchorLen >= minRightAnchorLen) { bool added = false; assert_lt(ref, _mutex.size()); ThreadSafe t(&_mutex[ref], _threadSafe && _write); assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); Node *cur = _fwIndex[ref]->add(pool(ref), ssp, &added); if(added) { assert_lt(ref, _spliceSites.size()); _spliceSites[ref].expand(); _spliceSites[ref].back().init(ssp.ref(), ssp.left(), ssp.right(), ssp.fw(), ssp.canonical()); _spliceSites[ref].back()._readid = rd.rdid; _spliceSites[ref].back()._leftext = leftAnchorLen; _spliceSites[ref].back()._rightext = rightAnchorLen; _spliceSites[ref].back()._numreads = 1; assert(cur != NULL); cur->payload = _spliceSites[ref].size() - 1; SpliceSitePos rssp(ssp.ref(), ssp.right(), ssp.left(), ssp.fw(), ssp.canonical()); assert_lt(ref, _bwIndex.size()); assert(_bwIndex[ref] != NULL); cur = _bwIndex[ref]->add(pool(ref), rssp, &added); assert(added); assert(cur != NULL); cur->payload = _spliceSites[ref].size() - 1; assert_eq(_fwIndex[ref]->size(), _bwIndex[ref]->size()); } else { assert(cur != NULL); assert_lt(ref, _spliceSites.size()); assert_lt(cur->payload, _spliceSites[ref].size()); if(leftAnchorLen > _spliceSites[ref][cur->payload]._leftext) _spliceSites[ref][cur->payload]._leftext = leftAnchorLen; if(rightAnchorLen > _spliceSites[ref][cur->payload]._rightext) _spliceSites[ref][cur->payload]._rightext = rightAnchorLen; _spliceSites[ref][cur->payload]._numreads += 1; if(rd.rdid < _spliceSites[ref][cur->payload]._readid) { _spliceSites[ref][cur->payload]._readid = rd.rdid; } } } leftAnchorLen = rightAnchorLen; rightAnchorLen = 0; } else { leftAnchorLen = edits[eidx].pos; } bool fw = (edits[eidx].splDir == EDIT_SPL_FW || edits[eidx].splDir == EDIT_SPL_UNKNOWN); bool canonical = (edits[eidx].splDir != EDIT_SPL_UNKNOWN); ssp.init(coord.ref(), refoff - 1, refoff + edits[eidx].splLen, fw, canonical); refoff += edits[eidx].splLen; last_eidx = eidx; } eidx++; } } if(ssp.inited()) { assert(edits[last_eidx].isSpliced()); assert_lt(edits[last_eidx].pos, rd.length()); rightAnchorLen = rd.length() - edits[last_eidx].pos; uint32_t minLeftAnchorLen = minAnchorLen + mm * 2 + (edits[last_eidx].splDir == EDIT_SPL_UNKNOWN ? 6 : 0); uint32_t mm2 = 0; for(size_t j = last_eidx + 1; j < edits.size(); j++) { if(edits[j].isGap() || edits[j].isMismatch()) mm2++; } uint32_t minRightAnchorLen = minAnchorLen + mm2 * 2 + (edits[last_eidx].splDir == EDIT_SPL_UNKNOWN ? 6 : 0); if(leftAnchorLen >= minLeftAnchorLen && rightAnchorLen >= minRightAnchorLen) { bool added = false; assert_lt(ref, _mutex.size()); ThreadSafe t(&_mutex[ref], _threadSafe && _write); assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); Node *cur = _fwIndex[ref]->add(pool(ref), ssp, &added); if(added) { assert_lt(ref, _spliceSites.size()); _spliceSites[ref].expand(); _spliceSites[ref].back().init(ssp.ref(), ssp.left(), ssp.right(), ssp.fw(), ssp.canonical()); _spliceSites[ref].back()._readid = rd.rdid; _spliceSites[ref].back()._leftext = leftAnchorLen; _spliceSites[ref].back()._rightext = rightAnchorLen; _spliceSites[ref].back()._numreads = 1; assert(cur != NULL); cur->payload = _spliceSites[ref].size() - 1; SpliceSitePos rssp(ssp.ref(), ssp.right(), ssp.left(), ssp.fw(), ssp.canonical()); assert_lt(ref, _bwIndex.size()); assert(_bwIndex[ref] != NULL); cur = _bwIndex[ref]->add(pool(ref), rssp, &added); assert(added); assert(cur != NULL); cur->payload = _spliceSites[ref].size() - 1; assert_eq(_fwIndex[ref]->size(), _bwIndex[ref]->size()); } else { assert(cur != NULL); assert_lt(ref, _spliceSites.size()); assert_lt(cur->payload, _spliceSites[ref].size()); if(leftAnchorLen > _spliceSites[ref][cur->payload]._leftext) _spliceSites[ref][cur->payload]._leftext = leftAnchorLen; if(rightAnchorLen > _spliceSites[ref][cur->payload]._rightext) _spliceSites[ref][cur->payload]._rightext = rightAnchorLen; _spliceSites[ref][cur->payload]._numreads += 1; if(rd.rdid < _spliceSites[ref][cur->payload]._readid) { _spliceSites[ref][cur->payload]._readid = rd.rdid; } } } } if(!coord.orient()) { Edit::invertPoss(const_cast&>(edits), rd.length(), false); } return true; } bool SpliceSiteDB::getSpliceSite(SpliceSite& ss) const { if(!_read) return false; uint64_t ref = ss.ref(); assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); ThreadSafe t(const_cast(&_mutex[ref]), _threadSafe && _write); assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); const Node *cur = _fwIndex[ref]->lookup(ss); if(cur == NULL) return false; assert(cur != NULL); assert_lt(ref, _spliceSites.size()); ss = _spliceSites[ref][cur->payload]; return true; } void SpliceSiteDB::getLeftSpliceSites(uint32_t ref, uint32_t left, uint32_t range, EList& spliceSites) const { if(!_read) return; assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); ThreadSafe t(const_cast(&_mutex[ref]), _threadSafe && _write); assert_gt(range, 0); assert_geq(left + 1, range); assert_lt(ref, _bwIndex.size()); assert(_bwIndex[ref] != NULL); const Node *cur = _bwIndex[ref]->root(); if(cur != NULL) getSpliceSites_recur(cur, ref, left + 1 - range, left, spliceSites); } void SpliceSiteDB::getRightSpliceSites(uint32_t ref, uint32_t right, uint32_t range, EList& spliceSites) const { if(!_read) return; assert_lt(ref, _numRefs); assert_lt(ref, _mutex.size()); ThreadSafe t(const_cast(&_mutex[ref]), _threadSafe && _write); assert_gt(range, 0); assert_gt(right + range, range); assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); const Node *cur = _fwIndex[ref]->root(); if(cur != NULL) getSpliceSites_recur(cur, ref, right, right + range - 1, spliceSites); } void SpliceSiteDB::getSpliceSites_recur( const RedBlackNode *node, uint32_t ref, uint32_t left, uint32_t right, EList& spliceSites) const { assert(node != NULL); bool goleft = true, goright = true; if((node->key.ref() > ref) || (node->key.ref() == ref && node->key.left() > right)) { goright = false; } if((node->key.ref() < ref) || (node->key.ref() == ref && node->key.left() < left)) { goleft = false; } if(goleft && node->left != NULL) { getSpliceSites_recur( node->left, ref, left, right, spliceSites); } if(node->key.ref() == ref && node->key.left() >= left && node->key.left() <= right) { assert_lt(ref, _spliceSites.size()); assert_lt(node->payload, _spliceSites[ref].size()); #ifndef NDEBUG const SpliceSite& ss = _spliceSites[ref][node->payload]; assert_eq(ss.ref(), node->key.ref()); assert(ss.left() == node->key.left() || ss.right() == node->key.left()); #endif spliceSites.push_back(_spliceSites[ref][node->payload]); } if(goright && node->right != NULL) { getSpliceSites_recur( node->right, ref, left, right, spliceSites); } } void calculate_splicesite_read_dist_impl(const RedBlackNode *node, const EList &spliceSites, EList& splicesite_read_dist) { if(node == NULL) return; calculate_splicesite_read_dist_impl(node->left, spliceSites, splicesite_read_dist); assert_lt(node->payload, spliceSites.size()); const SpliceSite& ss = spliceSites[node->payload]; if(ss.numreads() < splicesite_read_dist.size()) splicesite_read_dist[ss.numreads()] += 1; else splicesite_read_dist.back() += 1; calculate_splicesite_read_dist_impl(node->right, spliceSites, splicesite_read_dist); } uint32_t calculate_splicesite_read_dist(const EList* >& fwIndex, const ELList &spliceSites, EList& splicesite_read_dist) { for(size_t i = 0; i < fwIndex.size(); i++) { assert(fwIndex[i] != NULL); const RedBlackNode *root = fwIndex[i]->root(); assert_lt(i, spliceSites.size()); if(root != NULL) calculate_splicesite_read_dist_impl(root, spliceSites[i], splicesite_read_dist); } for(size_t i = 1; i < splicesite_read_dist.size(); i++) { splicesite_read_dist[i] += splicesite_read_dist[i-1]; } for(size_t i = 0; i < splicesite_read_dist.size(); i++) { float cmf_i = float(splicesite_read_dist[i]) / splicesite_read_dist.back(); if(cmf_i > 0.7) return i; } return 0; } void SpliceSiteDB::print(ofstream& out) { EList splicesite_read_dist; for(size_t i = 0; i < 100; i++) { splicesite_read_dist.push_back(0); } uint32_t numreads_cutoff = calculate_splicesite_read_dist(_fwIndex, _spliceSites, splicesite_read_dist); EList ss_list; for(size_t i = 0; i < _fwIndex.size(); i++) { assert(_fwIndex[i] != NULL); const Node *root = _fwIndex[i]->root(); if(root != NULL) print_recur(root, out, numreads_cutoff, ss_list); } print_impl(out, ss_list); } void SpliceSiteDB::print_recur( const RedBlackNode *node, ofstream& out, const uint32_t numreads_cutoff, EList& ss_list) { if(node == NULL) return; print_recur(node->left, out, numreads_cutoff, ss_list); const SpliceSitePos& ssp = node->key; assert_lt(ssp.ref(), _spliceSites.size()); assert_lt(node->payload, _spliceSites[ssp.ref()].size()); const SpliceSite& ss = _spliceSites[ssp.ref()][node->payload]; if(ss.numreads() >= numreads_cutoff) print_impl(out, ss_list, &ss); print_recur(node->right, out, numreads_cutoff, ss_list); } void SpliceSiteDB::print_impl( ofstream& out, EList& ss_list, const SpliceSite* ss) { size_t i = 0; while(i < ss_list.size()) { const SpliceSite& tmp_ss = ss_list[i]; bool do_print = true; if(ss != NULL) { if(tmp_ss.ref() == ss->ref()) { assert_leq(tmp_ss.left(), ss->left()); if(ss->left() < tmp_ss.left() + 10) { do_print = false; if(abs(((int)ss->left() - (int)tmp_ss.left()) - ((int)ss->right() - (int)tmp_ss.right())) <= 10) { if(tmp_ss.numreads() < ss->numreads()) { ss_list.erase(i); ss_list.push_back(*ss); } return; } } } } if(!do_print) { i++; continue; } assert_lt(tmp_ss.ref(), _refnames.size()); out << _refnames[tmp_ss.ref()] << "\t" << tmp_ss.left() << "\t" << tmp_ss.right() << "\t" << (tmp_ss.canonical() ? (tmp_ss.fw() ? "+" : "-") : ".") << endl; ss_list.erase(i); } if(ss != NULL) ss_list.push_back(*ss); } void SpliceSiteDB::read(ifstream& in, bool novel) { _empty = false; assert_eq(_numRefs, _refnames.size()); while(!in.eof()) { string refname; uint32_t left = 0, right = 0; char fw = 0; in >> refname >> left >> right >> fw; uint32_t ref = 0; for(; ref < _refnames.size(); ref++) { if(_refnames[ref] == refname) break; } if(ref >= _numRefs) continue; assert_lt(ref, _spliceSites.size()); _spliceSites[ref].expand(); _spliceSites[ref].back().init(ref, left, right, fw == '+' || fw == '.', fw != '.'); _spliceSites[ref].back()._fromfile = true; assert_gt(_spliceSites[ref].size(), 0); bool added = false; assert_lt(ref, _fwIndex.size()); assert(_fwIndex[ref] != NULL); Node *cur = _fwIndex[ref]->add(pool(ref), _spliceSites[ref].back(), &added); assert(added); assert(cur != NULL); cur->payload = _spliceSites[ref].size() - 1; added = false; SpliceSitePos rssp(ref, right, left, fw == '+' || fw == '.', fw != '.'); assert_lt(ref, _bwIndex.size()); assert(_bwIndex[ref] != NULL); cur = _bwIndex[ref]->add(pool(ref), rssp, &added); assert(added); assert(cur != NULL); cur->payload = _spliceSites[ref].size() - 1; } } Pool& SpliceSiteDB::pool(uint64_t ref) { assert_lt(ref, _numRefs); assert_lt(ref, _pool.size()); EList& pool = _pool[ref]; if(pool.size() <= 0 || pool.back()->full()) { pool.push_back(new Pool(1 << 20 /* 1MB */, 16 << 10 /* 16KB */, CA_CAT)); } assert(pool.back() != NULL); return *pool.back(); } #endif float SpliceSiteDB::probscore( int64_t donor_seq, int64_t acceptor_seq) { float probscore = 0.0f; #if defined(NEW_PROB_MODEL) float donor_probscore = 0.0f; assert_leq(donor_seq, 0x3ffff); int64_t donor_exonic_seq = (donor_seq >> 4) & (~0xff); int64_t donor_intronic_seq = donor_seq & 0xff; int64_t donor_rest_seq = donor_exonic_seq | donor_intronic_seq; int donor_seq3 = (donor_seq >> 10) & 0x3; int donor_seq4 = (donor_seq >> 8) & 0x3; donor_probscore = donor_cons1[donor_seq3] * donor_cons2[donor_seq4] / (background_bp_prob[donor_seq3] * background_bp_prob[donor_seq4]) * donor_me2x5[donor_rest_seq]; float acceptor_probscore = 0.0f; assert_leq(acceptor_seq, 0x3fffffffffff); int64_t acceptor_intronic_seq = (acceptor_seq >> 4) & (~0x3f); int64_t acceptor_exonic_seq = acceptor_seq & 0x3f; int64_t acceptor_rest_seq = acceptor_intronic_seq | acceptor_exonic_seq; int acceptor_seq18 = (acceptor_seq >> 8) & 0x3; int acceptor_seq19 = (acceptor_seq >> 6) & 0x3; acceptor_probscore = acceptor_cons1[acceptor_seq18] * acceptor_cons2[acceptor_seq19] / (background_bp_prob[acceptor_seq18] * background_bp_prob[acceptor_seq19]); int64_t acceptor_seq1 = acceptor_rest_seq >> 28 & 0x3fff; // [0, 7] acceptor_probscore *= acceptor_me2x3acc1[acceptor_seq1]; int64_t acceptor_seq2 = (acceptor_rest_seq >> 14) & 0x3fff; // [7, 7] acceptor_probscore *= acceptor_me2x3acc2[acceptor_seq2]; int64_t acceptor_seq3 = acceptor_rest_seq & 0x3fff; // [14, 7] acceptor_probscore *= acceptor_me2x3acc3[acceptor_seq3]; int64_t acceptor_seq4 = (acceptor_rest_seq >> 20) & 0x3fff; // [4, 7] acceptor_probscore *= acceptor_me2x3acc4[acceptor_seq4]; int64_t acceptor_seq5 = (acceptor_rest_seq >> 6) & 0x3fff; // [11, 7] acceptor_probscore *= acceptor_me2x3acc5[acceptor_seq5]; int64_t acceptor_seq6 = acceptor_seq1 & 0x3f; // [4, 3] acceptor_probscore /= acceptor_me2x3acc6[acceptor_seq6]; int64_t acceptor_seq7 = acceptor_seq4 & 0xff; // [7, 4] acceptor_probscore /= acceptor_me2x3acc7[acceptor_seq7]; int64_t acceptor_seq8 = acceptor_seq2 & 0x3f; // [11, 3] acceptor_probscore /= acceptor_me2x3acc8[acceptor_seq8]; int64_t acceptor_seq9 = acceptor_seq5 & 0xff; // [14, 4] acceptor_probscore /= acceptor_me2x3acc9[acceptor_seq9]; donor_probscore /= (1.0f + donor_probscore); acceptor_probscore /= (1.0f + acceptor_probscore); probscore = (donor_probscore + acceptor_probscore) / 2.0; #else assert_lt(donor_seq, (int)(1 << (donor_len << 1))); probscore = donor_prob_sum[donor_seq]; int acceptor_seq1 = acceptor_seq >> (acceptor_len2 << 1); assert_lt(acceptor_seq1, (int)(1 << (acceptor_len1 << 1))); probscore *= acceptor_prob_sum1[acceptor_seq1]; int acceptor_seq2 = acceptor_seq % (1 << (acceptor_len2 << 1)); probscore *= acceptor_prob_sum2[acceptor_seq2]; probscore = 1.0 / (1.0 + probscore); #endif return probscore; } hisat2-2.2.1/spliced_aligner.h000066400000000000000000003422261370663763200162310ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ #ifndef SPLICED_ALIGNER_H_ #define SPLICED_ALIGNER_H_ #include "hi_aligner.h" /** * With a hierarchical indexing, SplicedAligner provides several alignment strategies * , which enable effective alignment of RNA-seq reads */ template class SplicedAligner : public HI_Aligner { public: /** * Initialize with index. */ SplicedAligner( const GFM& gfm, bool anchorStop, uint64_t threads_rids_mindist = 0) : HI_Aligner(gfm, anchorStop, threads_rids_mindist) { } ~SplicedAligner() { } /** * Given a partial alignment of a read, try to further extend * the alignment bidirectionally using a combination of * local search, extension, and global search */ virtual void hybridSearch( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, bool fw, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink); /** * Given a partial alignment of a read, try to further extend * the alignment bidirectionally using a combination of * local search, extension, and global search */ virtual int64_t hybridSearch_recur( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, const GenomeHit& hit, index_t hitoff, index_t hitlen, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink, bool alignMate = false, index_t dep = 0); }; /** * Given a partial alignment of a read, try to further extend * the alignment bidirectionally using a combination of * local search, extension, and global search */ template void SplicedAligner::hybridSearch( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, bool fw, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink) { assert_lt(rdi, 2); assert(this->_rds[rdi] != NULL); him.localatts++; const ReportingParams& rp = sink.reportingParams(); // before further alignment using local search, extend the partial alignments directly // by comparing with the corresponding genomic sequences // this extension is performed without any mismatches allowed for(index_t hi = 0; hi < this->_genomeHits.size(); hi++) { GenomeHit& genomeHit = this->_genomeHits[hi]; index_t leftext = (index_t)INDEX_MAX, rightext = (index_t)INDEX_MAX; genomeHit.extend( *(this->_rds[rdi]), gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, INDEX_MAX, tpol, gpol, leftext, rightext); } // for the candidate alignments, examine the longest (best) one first this->_genomeHits_done.resize(this->_genomeHits.size()); this->_genomeHits_done.fill(false); for(size_t hi = 0; hi < this->_genomeHits.size(); hi++) { index_t hj = 0; for(; hj < this->_genomeHits.size(); hj++) { if(!this->_genomeHits_done[hj]) break; } if(hj >= this->_genomeHits.size()) break; for(index_t hk = hj + 1; hk < this->_genomeHits.size(); hk++) { if(this->_genomeHits_done[hk]) continue; GenomeHit& genomeHit_j = this->_genomeHits[hj]; GenomeHit& genomeHit_k = this->_genomeHits[hk]; if(genomeHit_k.hitcount() > genomeHit_j.hitcount() || (genomeHit_k.hitcount() == genomeHit_j.hitcount() && genomeHit_k.len() > genomeHit_j.len())) { hj = hk; } } // given a candidate partial alignment, extend it bidirectionally him.anchoratts++; GenomeHit& genomeHit = this->_genomeHits[hj]; int64_t maxsc = std::numeric_limits::min(); maxsc = hybridSearch_recur(sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, genomeHit, genomeHit.rdoff(), genomeHit.len(), wlm, prm, swm, him, rnd, sink); if(rp.bowtie2_dp == 2 || (rp.bowtie2_dp == 1 && maxsc < this->_minsc[rdi])) { const Read& rd = *this->_rds[rdi]; // Initialize the aligner with a new read swa.initRead(rd.patFw, // fw version of query rd.patRc, // rc version of query rd.qual, // fw version of qualities rd.qualRev, // rc version of qualities 0, // off of first char in 'rd' to consider rd.length(), // off of last char (excl) in 'rd' to consider sc); // scoring scheme bool found = genomeHit.len() >= rd.length(); if(!found) { DynProgFramer dpframe(false); // trimToRef size_t tlen = ref.approxLen(genomeHit.ref()); size_t readGaps = 10, refGaps = 10, nceil = 0, maxhalf = 10; index_t refoff = genomeHit.refoff() > genomeHit.rdoff() ? genomeHit.refoff() - genomeHit.rdoff() : 0; DPRect rect; dpframe.frameSeedExtensionRect(refoff, // ref offset implied by seed hit assuming no gaps rd.length(), // length of read sequence used in DP table tlen, // length of reference readGaps, // max # of read gaps permitted in opp mate alignment refGaps, // max # of ref gaps permitted in opp mate alignment (size_t)nceil, // # Ns permitted maxhalf, // max width in either direction rect); // DP rectangle assert(rect.repOk()); size_t cminlen = 2000, cpow2 = 4, nwindow = 10, nsInLeftShift = 0; swa.initRef(fw, // whether to align forward or revcomp read genomeHit.ref(), // reference aligned against rect, // DP rectangle ref, // Reference strings tlen, // length of reference sequence sc, // scoring scheme this->_minsc[rdi], // minimum score permitted true, // use 8-bit SSE if possible? cminlen, // minimum length for using checkpointing scheme cpow2, // interval b/t checkpointed diags; 1 << this false, // triangular mini-fills? true, // this is a seed extension - not finding a mate nwindow, nsInLeftShift); // Now fill the dynamic programming matrix and return true iff // there is at least one valid alignment TAlScore bestCell = std::numeric_limits::min(); found = swa.align(rnd, bestCell); if(found) { SwResult res; res.reset(); res.alres.init_raw_edits(&(this->_rawEdits)); found = swa.nextAlignment(res, this->_minsc[rdi], rnd); if(found) { if(!fw) res.alres.invertEdits(); const Coord& coord = res.alres.refcoord(); assert_geq(genomeHit._joinedOff + coord.off(), genomeHit.refoff()); index_t joinedOff = genomeHit._joinedOff + coord.off() - genomeHit.refoff(); genomeHit.init(fw, 0, // rdoff rd.length(), 0, // trim5 0, // trim3 coord.ref(), coord.off(), joinedOff, this->_sharedVars, genomeHit.repeat(), // repeat? &res.alres.ned(), NULL, res.alres.score().score()); genomeHit.replace_edits_with_alts(rd, altdb.alts(), ssdb, sc, this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), (index_t)tpol.minAnchorLen(), (index_t)tpol.minAnchorLen_noncan(), ref); } } } if(found) { hybridSearch_recur(sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, genomeHit, genomeHit.rdoff(), genomeHit.len(), wlm, prm, swm, him, rnd, sink); } } this->_genomeHits_done[hj] = true; } } /** * Given a partial alignment of a read, try to further extend * the alignment bidirectionally using a combination of * local search, extension, and global search */ template int64_t SplicedAligner::hybridSearch_recur( const Scoring& sc, const PairedEndPolicy& pepol, // paired-end policy const TranscriptomePolicy& tpol, const GraphPolicy& gpol, const GFM& gfm, const ALTDB& altdb, const RepeatDB& repeatdb, const BitPairReference& ref, SwAligner& swa, SpliceSiteDB& ssdb, index_t rdi, const GenomeHit& hit, index_t hitoff, index_t hitlen, WalkMetrics& wlm, PerReadMetrics& prm, SwMetrics& swm, HIMetrics& him, RandomSource& rnd, AlnSinkWrap& sink, bool alignMate, index_t dep) { const ReportingParams& rp = sink.reportingParams(); int64_t maxsc = numeric_limits::min(); him.localsearchrecur++; assert_lt(rdi, 2); assert(this->_rds[rdi] != NULL); const Read& rd = *(this->_rds[rdi]); index_t rdlen = (index_t)rd.length(); TAlScore cushion = 0; if(tpol.no_spliced_alignment()) { cushion = alignMate ? rdlen * 0.03 * sc.mm(255) : 0; } if(hit.score() + cushion < this->_minsc[rdi]) return maxsc; if(dep >= 128) return maxsc; // if it's already examined, just return if(hitoff == hit.rdoff() - hit.trim5() && hitlen == hit.len() + hit.trim5() + hit.trim3()) { if(this->isSearched(hit, rdi)) return maxsc; this->addSearched(hit, rdi); } // for effective use of memory allocation and deallocation if(this->_coords.size() <= dep) { this->_coords.expand(); assert_leq(this->_local_genomeHits.size(), dep); this->_local_genomeHits.expand(); assert_leq(this->_spliceSites.size(), dep); this->_spliceSites.expand(); } EList& coords = this->_coords[dep]; EList& spliceSites = this->_spliceSites[dep]; // daehwan - for debugging purposes #if 0 cout << rd.name << "\t" << (hit.fw() ? "+" : "-") << "\t" << hitoff << "\t" << hitoff + hitlen << "\t" << "( " << hit.rdoff() << "\t" << hit.rdoff() + hit.len() << " )" << "\t" << hit.refoff() << "\t" << hit.getRightOff() << "\t" << hit.score() << "\t" << "dep: " << dep << "\t"; Edit::print(cout, hit.edits()); cout << endl; #endif assert_leq(hitoff + hitlen, rdlen); // if this is a full alignment, report it if(hitoff == 0 && hitlen == rdlen) { if(!this->redundant(sink, rdi, hit)) { bool another_spliced = false; if(!ssdb.empty()) { int64_t best_score = hit.score(); this->_local_genomeHits[dep].clear(); this->_anchors_added.clear(); this->_local_genomeHits[dep].expand(); this->_local_genomeHits[dep].back() = hit; this->_anchors_added.push_back(0); index_t fragoff = 0, fraglen = 0, left = 0, right = 0; hit.getLeft(fragoff, fraglen, left); const index_t minMatchLen = (index_t)this->_minK; index_t min_left_anchor = rdlen, min_right_anchor = rdlen; // make use of a list of known or novel splice sites to further align the read if(fraglen >= minMatchLen && left >= minMatchLen && hit.trim5() == 0 && !hit.repeat() && !tpol.no_spliced_alignment()) { spliceSites.clear(); ssdb.getLeftSpliceSites(hit.ref(), left + minMatchLen, minMatchLen, spliceSites); for(size_t si = 0; si < spliceSites.size(); si++) { const SpliceSite& ss = spliceSites[si]; if(!ss._fromfile && ss._readid + this->_thread_rids_mindist > rd.rdid) continue; if(left + fraglen - 1 < ss.right()) continue; index_t frag2off = ss.left() - (ss.right() - left); if(frag2off + 1 < hitoff) continue; GenomeHit tempHit; if(fragoff + ss.right() < left + 1) continue; index_t readoff = fragoff + ss.right() - left - 1; index_t joinedOff = 0; bool success = gfm.textOffToJoined(hit.ref(), ss.left(), joinedOff); if(!success) { continue; } #ifndef NDEBUG index_t debug_tid = 0, debug_toff = 0, debug_tlen = 0; bool debug_straddled = false; gfm.joinedToTextOff(1, // qlen joinedOff, debug_tid, debug_toff, debug_tlen, false, debug_straddled); assert_eq(hit.ref(), debug_tid); assert_eq(ss.left(), debug_toff); #endif tempHit.init(hit.fw(), readoff + 1, // rdoff 0, // len 0, // trim5 0, // trim3 hit.ref(), ss.left() + 1, joinedOff + 1, this->_sharedVars, gfm.repeat()); index_t leftext = readoff + 1, rightext = 0; tempHit.extend(rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext); if(tempHit.len() <= 0) continue; if(!tempHit.compatibleWith( hit, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.no_spliced_alignment())) continue; int64_t minsc = max(this->_minsc[rdi], best_score); bool combined = tempHit.combineWith( hit, rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, sc, minsc, rnd, (index_t)this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), 1, 1, gpol.maxAltsTried(), &ss, tpol.no_spliced_alignment()); if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); index_t leftAnchorLen = 0, nedits = 0; tempHit.getLeftAnchor(leftAnchorLen, nedits); if(combined && tempHit.score() >= minsc && nedits <= leftAnchorLen / 4) { // prevent (short) anchors from having many mismatches if(this->isSearched(tempHit, rdi)) continue; if(!this->redundant(sink, rdi, tempHit)) { another_spliced = true; if(tempHit.score() > best_score) best_score = tempHit.score(); this->_local_genomeHits[dep].expand(); this->_local_genomeHits[dep].back() = tempHit; this->_anchors_added.push_back(1); index_t temp_fragoff = 0, temp_fraglen = 0, temp_left = 0; tempHit.getLeft(temp_fragoff, temp_fraglen, temp_left); if(temp_fraglen < min_left_anchor) min_left_anchor = temp_fraglen; } } } } size_t num_local_genomeHits = this->_local_genomeHits[dep].size(); for(size_t i = 0; i < num_local_genomeHits; i++) { this->_local_genomeHits[dep][i].getRight(fragoff, fraglen, right); if(this->_local_genomeHits[dep][i].score() < best_score) continue; // make use of a list of known or novel splice sites to further align the read if(fraglen >= minMatchLen && this->_local_genomeHits[dep][i].trim3() == 0 && !hit.repeat() && !tpol.no_spliced_alignment()) { spliceSites.clear(); assert_gt(fraglen, 0); ssdb.getRightSpliceSites(this->_local_genomeHits[dep][i].ref(), right + fraglen - minMatchLen, minMatchLen, spliceSites); for(size_t si = 0; si < spliceSites.size(); si++) { const GenomeHit& canHit = this->_local_genomeHits[dep][i]; const SpliceSite& ss = spliceSites[si]; if(!ss._fromfile && ss._readid + this->_thread_rids_mindist > rd.rdid) continue; if(right > ss.left()) continue; GenomeHit tempHit; index_t readoff = fragoff + ss.left() - right + 1; if(readoff >= rdlen) continue; index_t joinedOff = 0; bool success = gfm.textOffToJoined(canHit.ref(), ss.right(), joinedOff); if(!success) { continue; } #ifndef NDEBUG index_t debug_tid = 0, debug_toff = 0, debug_tlen = 0; bool debug_straddled = false; gfm.joinedToTextOff(1, // qlen joinedOff, debug_tid, debug_toff, debug_tlen, false, debug_straddled); assert_eq(canHit.ref(), debug_tid); assert_eq(ss.right(), debug_toff); #endif tempHit.init(canHit.fw(), readoff, 0, // len 0, // trim5 0, // trim3 canHit.ref(), ss.right(), joinedOff, this->_sharedVars, gfm.repeat()); index_t leftext = 0, rightext = rdlen - readoff; tempHit.extend(rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext); if(tempHit.len() <= 0) continue; if(!canHit.compatibleWith(tempHit, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.no_spliced_alignment())) continue; GenomeHit combinedHit = canHit; int64_t minsc = max(this->_minsc[rdi], best_score); bool combined = combinedHit.combineWith( tempHit, rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, sc, minsc, rnd, (index_t)this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), 1, 1, gpol.maxAltsTried(), &ss, tpol.no_spliced_alignment()); if(rdi == 0) minsc = max(minsc, sink.bestUnp1()); else minsc = max(minsc, sink.bestUnp2()); index_t rightAnchorLen = 0, nedits = 0; combinedHit.getRightAnchor(rightAnchorLen, nedits); if(combined && combinedHit.score() >= minsc && nedits <= rightAnchorLen / 4) { // prevent (short) anchors from having many mismatches if(this->isSearched(combinedHit, rdi)) continue; if(!this->redundant(sink, rdi, combinedHit)) { another_spliced = true; if(combinedHit.score() > best_score) best_score = tempHit.score(); this->_local_genomeHits[dep].expand(); this->_local_genomeHits[dep].back() = combinedHit; this->_anchors_added.push_back(this->_anchors_added[i] + 1); index_t temp_fragoff = 0, temp_fraglen = 0, temp_right = 0; combinedHit.getLeft(temp_fragoff, temp_fraglen, temp_right); if(temp_fraglen < min_right_anchor) min_right_anchor = temp_fraglen; } } } } } assert_eq(this->_local_genomeHits[dep].size(), this->_anchors_added.size()); for(size_t i = 0; i < this->_local_genomeHits[dep].size(); i++) { const GenomeHit& canHit = this->_local_genomeHits[dep][i]; if(!rp.secondary && canHit.score() < best_score) continue; // if(min(min_left_anchor, min_right_anchor) <= this->_minK_local) { // daehwan - for debugging purposes // if(this->_anchors_added[i] < this->_anchors_added.back()) continue; //} if(i > 0 && !this->isSearched(canHit, rdi)) { this->addSearched(canHit, rdi); } if(!this->redundant(sink, rdi, canHit)) { this->reportHit(sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, ssdb, sink, rdi, canHit, alignMate); maxsc = max(maxsc, canHit.score()); } } } else { this->reportHit(sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, ssdb, sink, rdi, hit, alignMate); maxsc = max(maxsc, hit.score()); } return maxsc; } } else if(hitoff > 0 && (hitoff + hitlen == rdlen || hitoff + hitoff < rdlen - hitlen)) { // Decide which side to extend first (left or right) if(!ssdb.empty()) { // extend the partial alignment in the left direction index_t fragoff = 0, fraglen = 0, left = 0; hit.getLeft(fragoff, fraglen, left); const index_t minMatchLen = (index_t)this->_minK_local; // make use of a list of known or novel splice sites to further align the read if(fraglen >= minMatchLen && left >= minMatchLen && !hit.repeat() && !tpol.no_spliced_alignment()) { spliceSites.clear(); ssdb.getLeftSpliceSites(hit.ref(), left + minMatchLen, minMatchLen + min(minMatchLen, fragoff), spliceSites); for(size_t si = 0; si < spliceSites.size(); si++) { const SpliceSite& ss = spliceSites[si]; if(!ss._fromfile && ss._readid + this->_thread_rids_mindist > rd.rdid) continue; if(left + fraglen - 1 < ss.right()) continue; if(fragoff + ss.right() < left + 1) continue; index_t readoff = fragoff + ss.right() - left - 1; index_t joinedOff = 0; bool success = gfm.textOffToJoined(hit.ref(), ss.left(), joinedOff); if(!success) { continue; } #ifndef NDEBUG index_t debug_tid = 0, debug_toff = 0, debug_tlen = 0; bool debug_straddled = false; gfm.joinedToTextOff(1, // qlen joinedOff, debug_tid, debug_toff, debug_tlen, false, debug_straddled); assert_eq(hit.ref(), debug_tid); assert_eq(ss.left(), debug_toff); #endif GenomeHit tempHit; tempHit.init(hit.fw(), readoff + 1, // rdoff 0, // len 0, // trim5 0, // trim3 hit.ref(), ss.left() + 1, joinedOff + 1, this->_sharedVars, gfm.repeat()); index_t leftext = readoff + 1, rightext = 0; tempHit.extend(rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext); if(tempHit.len() <= 0) continue; if(!tempHit.compatibleWith(hit, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.no_spliced_alignment())) continue; int64_t minsc = this->_minsc[rdi]; bool combined = tempHit.combineWith( hit, rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, sc, minsc, rnd, (index_t)this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), 1, 1, gpol.maxAltsTried(), &ss, tpol.no_spliced_alignment()); if(!rp.secondary) { if(rdi == 0) minsc = max(minsc, sink.bestUnp1() - cushion); else minsc = max(minsc, sink.bestUnp2() - cushion); } if(combined && tempHit.score() >= minsc && // soft-clipping might be better tempHit.score() + sc.sc(0) * hit.rdoff() >= hit.score()) { assert_eq(tempHit.trim5(), 0); assert_leq(tempHit.rdoff() + tempHit.len() + tempHit.trim3(), rdlen); int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff(), tempHit.len() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } } } } bool use_localindex = true; if(hitoff == hit.rdoff() && hitoff <= this->_minK) { index_t leftext = (index_t)INDEX_MAX, rightext = (index_t)0; GenomeHit tempHit = hit; tempHit.extend( rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext, 1); if(tempHit.rdoff() == 0) { use_localindex = false; } } // Choose a local index based on the genomic location of the partial alignment const HGFM* hGFM = (const HGFM*)(&gfm); const LocalGFM* lGFM = hGFM->getLocalGFM(hit.ref(), hit.refoff()); assert_leq(lGFM->_localOffset, hit.refoff()); bool success = false, first = true; index_t count = 0; // Use at most two local indexes const index_t max_count = 2; int64_t prev_score = hit.score(); this->_local_genomeHits[dep].clear(); while(!success && count++ < max_count && use_localindex) { if(him.localindexatts >= this->max_localindexatts) break; if(first) { first = false; } else { lGFM = hGFM->prevLocalGFM(lGFM); if(lGFM == NULL || lGFM->empty()) break; } // local index search index_t extlen = 0; local_index_t top = (local_index_t)INDEX_MAX, bot = (local_index_t)INDEX_MAX; local_index_t node_top = (local_index_t)INDEX_MAX, node_bot = (local_index_t)INDEX_MAX; index_t extoff = hitoff - 1; if(extoff > 0) extoff -= 1; if(extoff < tpol.minAnchorLen()) { extoff = tpol.minAnchorLen(); } index_t nelt = (index_t)INDEX_MAX; index_t max_nelt = std::max(5, extlen); bool no_extension = false; bool uniqueStop= false; index_t minUniqueLen = (index_t)this->_minK_local; for(; extoff < rdlen; extoff++) { extlen = 0; uniqueStop = true; him.localindexatts++; this->_local_node_iedge_count.clear(); nelt = this->localGFMSearch( *lGFM, // BWT index rd, // read to align sc, // scoring scheme sink.reportingParams(), hit.fw(), extoff, extlen, top, bot, node_top, node_bot, this->_local_node_iedge_count, rnd, uniqueStop, minUniqueLen); if(extoff + 1 - extlen >= hitoff) { no_extension = true; break; } if(nelt <= max_nelt) break; } assert_leq(node_top, node_bot); assert_eq(nelt, (index_t)(node_bot - node_top)); assert_leq(extlen, extoff + 1); if(nelt > 0 && nelt <= max_nelt && extlen >= tpol.minAnchorLen() && !no_extension) { assert_leq(nelt, max_nelt); coords.clear(); bool straddled = false; // get genomic locations for this local search this->getGenomeCoords_local( *lGFM, altdb, ref, rnd, top, bot, node_top, node_bot, this->_local_node_iedge_count, hit.fw(), extoff + 1 - extlen, extlen, coords, wlm, prm, him, true, // reject straddled? straddled); assert_leq(coords.size(), nelt); coords.sort(); for(int ri = (int)coords.size() - 1; ri >= 0; ri--) { const Coord& coord = coords[ri]; GenomeHit tempHit; tempHit.init(coord.orient(), extoff + 1 - extlen, extlen, 0, // trim5 0, // trim3 (index_t)coord.ref(), (index_t)coord.off(), (index_t)coord.joinedOff(), this->_sharedVars, gfm.repeat()); if(!tempHit.adjustWithALT(*this->_rds[rdi], gfm, altdb, ref, gpol)) continue; // check if the partial alignment is compatible with the new alignment using the local index if(!tempHit.compatibleWith(hit, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.no_spliced_alignment())) { if(count == 1) continue; else break; } if(uniqueStop) { assert_eq(coords.size(), 1); index_t leftext = (index_t)INDEX_MAX, rightext = (index_t)0; tempHit.extend( rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext); } // combine the partial alignment and the new alignment int64_t minsc = this->_minsc[rdi]; bool combined = tempHit.combineWith( hit, rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, sc, minsc, rnd, (index_t)this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.minAnchorLen(), tpol.minAnchorLen_noncan(), gpol.maxAltsTried(), NULL, // splice sites tpol.no_spliced_alignment()); if(!rp.secondary) { if(rdi == 0) minsc = max(minsc, sink.bestUnp1() - cushion); else minsc = max(minsc, sink.bestUnp2() - cushion); } if(combined && tempHit.score() >= minsc) { assert_eq(tempHit.trim5(), 0); assert_leq(tempHit.rdoff() + tempHit.len() + tempHit.trim3(), rdlen); if(tempHit.score() >= prev_score - sc.mmpMax) { // extend the new partial alignment recursively int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff(), tempHit.len() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } else { this->_local_genomeHits[dep].push_back(tempHit); } } } } if(maxsc >= prev_score - sc.mmpMax) success = true; if(!success && (him.localindexatts >= this->max_localindexatts || count == max_count || hGFM->prevLocalGFM(lGFM) == NULL)) { for(index_t ti = 0; ti < this->_local_genomeHits[dep].size(); ti++) { GenomeHit& tempHit = this->_local_genomeHits[dep][ti]; int64_t minsc = this->_minsc[rdi]; if(!rp.secondary) { if(rdi == 0) minsc = max(minsc, sink.bestUnp1() - cushion); else minsc = max(minsc, sink.bestUnp2() - cushion); } if(tempHit.score() >= minsc) { int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff(), tempHit.len() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } } } } // while(!success && count++ < 2) if(!success) { if(hitoff > this->_minK && him.localindexatts < this->max_localindexatts) { index_t extlen = 0; index_t top = (index_t)INDEX_MAX, bot = (index_t)INDEX_MAX; index_t node_top = (index_t)INDEX_MAX, node_bot = (index_t)INDEX_MAX; this->_node_iedge_count.clear(); index_t extoff = hitoff - 1; bool uniqueStop = true; // perform global search for long introns index_t nelt = this->globalGFMSearch( gfm, // GFM index rd, // read to align sc, // scoring scheme sink.reportingParams(), hit.fw(), extoff, extlen, top, bot, node_top, node_bot, this->_node_iedge_count, rnd, uniqueStop); if(nelt > 0 && nelt <= 5 && extlen >= this->_minK) { coords.clear(); bool straddled = false; this->getGenomeCoords( gfm, altdb, ref, rnd, top, bot, node_top, node_bot, this->_node_iedge_count, hit.fw(), bot - top, extoff + 1 - extlen, extlen, coords, wlm, prm, him, true, // reject straddled? straddled); assert_leq(coords.size(), nelt); if(coords.size() > 1) coords.sort(); for(int ri = (int)coords.size() - 1; ri >= 0; ri--) { const Coord& coord = coords[ri]; GenomeHit tempHit; tempHit.init(coord.orient(), extoff + 1 - extlen, extlen, 0, // trim5 0, // trim3 (index_t)coord.ref(), (index_t)coord.off(), (index_t)coord.joinedOff(), this->_sharedVars, gfm.repeat()); if(!tempHit.adjustWithALT(*this->_rds[rdi], gfm, altdb, ref, gpol)) continue; if(!tempHit.compatibleWith(hit, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.no_spliced_alignment())) continue; if(uniqueStop) { assert_eq(coords.size(), 1); index_t leftext = (index_t)INDEX_MAX, rightext = (index_t)0; tempHit.extend( rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext); } int64_t minsc = this->_minsc[rdi]; bool combined = tempHit.combineWith( hit, rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, sc, minsc, rnd, (index_t)this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.minAnchorLen(), tpol.minAnchorLen_noncan(), gpol.maxAltsTried(), NULL, // splice sites tpol.no_spliced_alignment()); if(!rp.secondary) { if(rdi == 0) minsc = max(minsc, sink.bestUnp1() - cushion); else minsc = max(minsc, sink.bestUnp2() - cushion); } if(combined && tempHit.score() >= minsc) { assert_eq(tempHit.trim5(), 0); assert_leq(tempHit.rdoff() + tempHit.len() + tempHit.trim3(), rdlen); int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff(), tempHit.len() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } } } } GenomeHit tempHit = hit; index_t trimMax = (index_t)((tempHit.score() - max(maxsc, this->_minsc[rdi])) / sc.sc(0)); if(tempHit.rdoff() < trimMax) { index_t trim5 = tempHit.rdoff(); GenomeHit trimedHit = tempHit; trimedHit.trim5(trim5, rd, ssdb, sc, (index_t)this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.minAnchorLen(), tpol.minAnchorLen_noncan(), ref); assert_leq(trimedHit.len() + trimedHit.trim5() + trimedHit.trim3(), rdlen); int64_t tmp_score = trimedHit.score(); if(tmp_score > maxsc && tmp_score >= this->_minsc[rdi]) { int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, trimedHit, 0, trimedHit.len() + trimedHit.trim5() + trimedHit.trim3(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); // return maxsc; } } // extend the partial alignment directly comparing with the corresponding genomic sequence // with mismatches or a gap allowed int64_t minsc = this->_minsc[rdi]; assert_geq(tempHit.score(), minsc); index_t mm = (index_t)((tempHit.score() - minsc) / sc.mmpMax); index_t leftext = (index_t)INDEX_MAX, rightext = (index_t)0; index_t num_mismatch_allowed = 1; if(hitoff <= this->_minK_local) { num_mismatch_allowed = min(tempHit.rdoff(), mm); } him.localextatts++; tempHit.extend( rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext, num_mismatch_allowed); if(!rp.secondary) { if(rdi == 0) minsc = max(minsc, sink.bestUnp1() - cushion); else minsc = max(minsc, sink.bestUnp2() - cushion); } if(tempHit.score() >= minsc && leftext >= min((index_t)this->_minK_local, hit.rdoff())) { assert_eq(tempHit.trim5(), 0); assert_leq(tempHit.rdoff() + tempHit.len() + tempHit.trim3(), rdlen); int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff(), tempHit.len() + tempHit.trim3(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } else if(hitoff > this->_minK_local) { // skip some bases of a read index_t jumplen = hitoff > this->_minK ? (index_t)this->_minK : (index_t)this->_minK_local; assert_leq(hitoff, hit.rdoff()); int64_t expected_score = hit.score() - (hit.rdoff() - hitoff) / jumplen * sc.mmpMax - sc.mmpMax; if(expected_score >= minsc) { assert_lt(hitlen + jumplen, rdlen); assert_eq(hit.trim5(), 0); assert_leq(hitoff + hitlen, rdlen); int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, hit, hitoff - jumplen, hitlen + jumplen, wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } } } } else { // extend the partial alignment in the right direction assert_lt(hitoff + hitlen, rdlen); if(!ssdb.empty()) { index_t fragoff = 0, fraglen = 0, right = 0; hit.getRight(fragoff, fraglen, right); const index_t minMatchLen = (index_t)this->_minK_local; // make use of a list of known or novel splice sites to further align the read if(fraglen >= minMatchLen && !hit.repeat() && !tpol.no_spliced_alignment()) { spliceSites.clear(); assert_gt(fraglen, 0); assert_leq(fragoff + fraglen, rdlen); index_t right_unmapped_len = rdlen - fragoff - fraglen; ssdb.getRightSpliceSites(hit.ref(), right + fraglen - minMatchLen, minMatchLen + min(minMatchLen, right_unmapped_len), spliceSites); for(size_t si = 0; si < spliceSites.size(); si++) { const SpliceSite& ss = spliceSites[si]; if(!ss._fromfile && ss._readid + this->_thread_rids_mindist > rd.rdid) continue; if(right > ss.left()) continue; GenomeHit tempHit; assert_leq(right, ss.left()); index_t readoff = fragoff + ss.left() - right + 1; if(readoff >= rdlen) continue; index_t joinedOff = 0; bool success = gfm.textOffToJoined(hit.ref(), ss.right(), joinedOff); if(!success) { continue; } #ifndef NDEBUG index_t debug_tid = 0, debug_toff = 0, debug_tlen = 0; bool debug_straddled = false; gfm.joinedToTextOff(1, // qlen joinedOff, debug_tid, debug_toff, debug_tlen, false, debug_straddled); assert_eq(hit.ref(), debug_tid); assert_eq(ss.right(), debug_toff); #endif tempHit.init(hit.fw(), readoff, 0, // len 0, // trim5 0, // trim3 hit.ref(), ss.right(), joinedOff, this->_sharedVars, gfm.repeat()); index_t leftext = 0, rightext = rdlen - readoff; tempHit.extend(rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext); if(tempHit.len() <= 0) continue; if(!hit.compatibleWith(tempHit, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.no_spliced_alignment())) continue; GenomeHit combinedHit = hit; int64_t minsc = this->_minsc[rdi]; bool combined = combinedHit.combineWith( tempHit, rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, sc, minsc, rnd, (index_t)this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), 1, 1, gpol.maxAltsTried(), &ss, tpol.no_spliced_alignment()); if(!rp.secondary) { if(rdi == 0) minsc = max(minsc, sink.bestUnp1() - cushion); else minsc = max(minsc, sink.bestUnp2() - cushion); } if(combined && combinedHit.score() >= minsc && // soft-clipping might be better combinedHit.score() + sc.sc(0) * (rdlen - hit.rdoff() - hit.len() - hit.trim5()) >= hit.score()) { assert_leq(combinedHit.trim5(), combinedHit.rdoff()); int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, combinedHit, combinedHit.rdoff() - combinedHit.trim5(), combinedHit.len() + combinedHit.trim5(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } } } } bool use_localindex = true; if(hit.len() == hitlen && hitoff + hitlen + this->_minK > rdlen) { index_t leftext = (index_t)0, rightext = (index_t)INDEX_MAX; GenomeHit tempHit = hit; tempHit.extend( rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext, 1); if(tempHit.rdoff() + tempHit.len()== rdlen) { use_localindex = false; } } // Choose a local index based on the genomic location of the partial alignment const HGFM* hGFM = (const HGFM*)(&gfm); const LocalGFM* lGFM = hGFM->getLocalGFM(hit.ref(), hit.refoff()); bool success = false, first = true; index_t count = 0; // Use at most two local indexes const index_t max_count = 2; int64_t prev_score = hit.score(); this->_local_genomeHits[dep].clear(); while(!success && count++ < max_count && use_localindex) { if(him.localindexatts >= this->max_localindexatts) break; if(first) { first = false; } else { lGFM = hGFM->nextLocalGFM(lGFM); if(lGFM == NULL || lGFM->empty()) break; } // local index search index_t extlen = 0; local_index_t top = (local_index_t)INDEX_MAX, bot = (local_index_t)INDEX_MAX; local_index_t node_top = (local_index_t)INDEX_MAX, node_bot = (local_index_t)INDEX_MAX; index_t extoff = hitoff + hitlen + (index_t)this->_minK_local; if(extoff + 1 < rdlen) extoff += 1; if(extoff >= rdlen) { extoff = rdlen - 1; } index_t nelt = (index_t)INDEX_MAX; index_t max_nelt = std::max(5, extlen); bool no_extension = false; bool uniqueStop; index_t minUniqueLen = (index_t)this->_minK_local; index_t maxHitLen = max(extoff - hitoff - hitlen, (index_t)this->_minK_local); for(; maxHitLen < extoff + 1 && extoff < rdlen;) { extlen = 0; uniqueStop = false; him.localindexatts++; this->_local_node_iedge_count.clear(); nelt = this->localGFMSearch( *lGFM, // GFM index rd, // read to align sc, // scoring scheme sink.reportingParams(), hit.fw(), extoff, extlen, top, bot, node_top, node_bot, this->_local_node_iedge_count, rnd, uniqueStop, minUniqueLen, maxHitLen); if(extoff < hitoff + hitlen) { no_extension = true; break; } if(nelt <= max_nelt) break; if(extoff + 1 < rdlen) extoff++; else { if(extlen < maxHitLen) break; else maxHitLen++; } } assert_leq(node_top, node_bot); assert_eq(nelt, (index_t)(node_bot - node_top)); assert_leq(extlen, extoff + 1); assert_leq(extoff, rdlen); if(nelt > 0 && nelt <= max_nelt && extlen >= tpol.minAnchorLen() && !no_extension) { assert_leq(nelt, max_nelt); coords.clear(); bool straddled = false; // get genomic locations for this local search this->getGenomeCoords_local( *lGFM, altdb, ref, rnd, top, bot, node_top, node_bot, this->_local_node_iedge_count, hit.fw(), extoff + 1 - extlen, extlen, coords, wlm, prm, him, true, // reject straddled? straddled); assert_leq(coords.size(), nelt); if(coords.size() > 1) coords.sort(); for(index_t ri = 0; ri < coords.size(); ri++) { const Coord& coord = coords[ri]; GenomeHit tempHit; tempHit.init(coord.orient(), extoff + 1 - extlen, extlen, 0, // trim5 0, // trim3 (index_t)coord.ref(), (index_t)coord.off(), (index_t)coord.joinedOff(), this->_sharedVars, gfm.repeat()); if(!tempHit.adjustWithALT(*this->_rds[rdi], gfm, altdb, ref, gpol)) continue; // check if the partial alignment is compatible with the new alignment using the local index if(!hit.compatibleWith(tempHit, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.no_spliced_alignment())) { if(count == 1) continue; else break; } index_t leftext = (index_t)0, rightext = (index_t)INDEX_MAX; tempHit.extend( rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext); GenomeHit combinedHit = hit; int64_t minsc = this->_minsc[rdi]; // combine the partial alignment and the new alignment bool combined = combinedHit.combineWith( tempHit, rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, sc, minsc, rnd, (index_t)this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.minAnchorLen(), tpol.minAnchorLen_noncan(), gpol.maxAltsTried(), NULL, // splice sites tpol.no_spliced_alignment()); if(!rp.secondary) { if(rdi == 0) minsc = max(minsc, sink.bestUnp1() - cushion); else minsc = max(minsc, sink.bestUnp2() - cushion); } if(combined && combinedHit.score() >= minsc) { assert_leq(combinedHit.trim5(), combinedHit.rdoff()); if(combinedHit.score() >= prev_score - sc.mmpMax) { // extend the new partial alignment recursively int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, combinedHit, combinedHit.rdoff() - combinedHit.trim5(), combinedHit.len() + combinedHit.trim5(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } else { this->_local_genomeHits[dep].push_back(combinedHit); } } } } // int64_t minsc = (rdi == 0 ? sink.bestUnp1() : sink.bestUnp2()); if(maxsc >= prev_score - sc.mmpMax) success = true; if(!success && (him.localindexatts >= this->max_localindexatts || count == max_count || hGFM->nextLocalGFM(lGFM) == NULL) ) { for(index_t ti = 0; ti < this->_local_genomeHits[dep].size(); ti++) { GenomeHit& tempHit = this->_local_genomeHits[dep][ti]; int64_t minsc = this->_minsc[rdi]; if(!rp.secondary) { if(rdi == 0) minsc = max(minsc, sink.bestUnp1() - cushion); else minsc = max(minsc, sink.bestUnp2() - cushion); } if(tempHit.score() >= minsc) { int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff() - tempHit.trim5(), tempHit.len() + tempHit.trim5(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } } } } // while(!success && count++ < 2) if(!success) { // perform global search for long introns if(hitoff + hitlen + this->_minK + 1 < rdlen && him.localindexatts < this->max_localindexatts) { index_t extlen = 0; index_t top = (index_t)INDEX_MAX, bot = (index_t)INDEX_MAX; index_t node_top = (index_t)INDEX_MAX, node_bot = (index_t)INDEX_MAX; this->_node_iedge_count.clear(); index_t extoff = hitoff + hitlen + (index_t)this->_minK + 1; bool uniqueStop = true; index_t nelt = this->globalGFMSearch( gfm, // GFM index rd, // read to align sc, // scoring scheme sink.reportingParams(), hit.fw(), extoff, extlen, top, bot, node_top, node_bot, this->_node_iedge_count, rnd, uniqueStop); if(nelt > 0 && nelt <= 5 && extlen >= this->_minK) { coords.clear(); bool straddled = false; this->getGenomeCoords( gfm, altdb, ref, rnd, top, bot, node_top, node_bot, this->_node_iedge_count, hit.fw(), bot - top, extoff + 1 - extlen, extlen, coords, wlm, prm, him, true, // reject straddled straddled); assert_leq(coords.size(), nelt); coords.sort(); for(index_t ri = 0; ri < coords.size(); ri++) { const Coord& coord = coords[ri]; GenomeHit tempHit; tempHit.init(coord.orient(), extoff + 1 - extlen, extlen, 0, // trim5 0, // trim3 (index_t)coord.ref(), (index_t)coord.off(), (index_t)coord.joinedOff(), this->_sharedVars, gfm.repeat()); if(!tempHit.adjustWithALT(*this->_rds[rdi], gfm, altdb, ref, gpol)) continue; if(!hit.compatibleWith(tempHit, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.no_spliced_alignment())) continue; index_t leftext = (index_t)0, rightext = (index_t)INDEX_MAX; tempHit.extend( rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext); GenomeHit combinedHit = hit; int64_t minsc = this->_minsc[rdi]; bool combined = combinedHit.combineWith( tempHit, rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, sc, minsc, rnd, (index_t)this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.minAnchorLen(), tpol.minAnchorLen_noncan(), gpol.maxAltsTried(), NULL, // splice sites tpol.no_spliced_alignment()); if(!rp.secondary) { if(rdi == 0) minsc = max(minsc, sink.bestUnp1() - cushion); else minsc = max(minsc, sink.bestUnp2() - cushion); } if(combined && combinedHit.score() >= minsc) { assert_leq(combinedHit.trim5(), combinedHit.rdoff()); int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, combinedHit, combinedHit.rdoff() - combinedHit.trim5(), combinedHit.len() + combinedHit.trim5(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } } } } GenomeHit tempHit = hit; assert(tempHit.trim5() == 0 || hitoff == 0); index_t trimLen = rdlen - hitoff - tempHit.len() - tempHit.trim5(); index_t trimMax = (index_t)((tempHit.score() - max(maxsc, this->_minsc[rdi])) / sc.sc(0)); if(trimLen < trimMax) { index_t trim3 = rdlen - hitoff - tempHit.len() - tempHit.trim5(); GenomeHit trimedHit = tempHit; trimedHit.trim3(trim3, rd, ssdb, sc, (index_t)this->_minK_local, (index_t)tpol.minIntronLen(), (index_t)tpol.maxIntronLen(), tpol.minAnchorLen(), tpol.minAnchorLen_noncan(), ref); assert_leq(trimedHit.trim5(), trimedHit.rdoff()); assert_leq(trimedHit.len() + trimedHit.trim5() + trimedHit.trim3(), rdlen); int64_t tmp_score = trimedHit.score(); if(tmp_score > maxsc && tmp_score >= this->_minsc[rdi]) { int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, trimedHit, trimedHit.rdoff() - trimedHit.trim5(), trimedHit.len() + trimedHit.trim5() + trimedHit.trim3(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); // return maxsc; } } // extend the partial alignment directly comparing with the corresponding genomic sequence // with mismatches or a gap allowed int64_t minsc = this->_minsc[rdi]; assert_geq(tempHit.score(), minsc); index_t leftext = (index_t)0, rightext = (index_t)INDEX_MAX; index_t mm = (index_t)((tempHit.score() - minsc) / sc.mmpMax); index_t num_mismatch_allowed = 1; if(rdlen - hitoff - hitlen <= this->_minK_local) { num_mismatch_allowed = min(rdlen - tempHit.rdoff() - tempHit.len(), mm); } him.localextatts++; tempHit.extend( rd, gfm, ref, altdb, repeatdb, ssdb, swa, swm, prm, sc, this->_minsc[rdi], rnd, (index_t)this->_minK_local, tpol, gpol, leftext, rightext, num_mismatch_allowed); if(!rp.secondary) { if(rdi == 0) minsc = max(minsc, sink.bestUnp1() - cushion); else minsc = max(minsc, sink.bestUnp2() - cushion); } if(tempHit.score() >= minsc && rightext >= min((index_t)this->_minK_local, rdlen - hit.len() - hit.rdoff())) { assert_eq(tempHit.trim3(), 0); assert_leq(tempHit.trim5(), tempHit.rdoff()); int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, tempHit, tempHit.rdoff() - tempHit.trim5(), tempHit.len() + tempHit.trim5(), wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } else if(hitoff + hitlen + this->_minK_local < rdlen) { // skip some bases of a read index_t jumplen = hitoff + hitlen + this->_minK < rdlen ? (index_t)this->_minK : (index_t)this->_minK_local; assert_lt(hitoff + hitlen + jumplen, rdlen); assert_leq(hit.len(), hitlen); int64_t expected_score = hit.score() - (hitlen - hit.len()) / jumplen * sc.mmpMax - sc.mmpMax; if(expected_score >= minsc) { assert_eq(hit.trim3(), 0); int64_t tmp_maxsc = hybridSearch_recur( sc, pepol, tpol, gpol, gfm, altdb, repeatdb, ref, swa, ssdb, rdi, hit, hitoff, hitlen + jumplen, wlm, prm, swm, him, rnd, sink, alignMate, dep + 1); maxsc = max(maxsc, tmp_maxsc); } } } } return maxsc; } #endif /*SPLICED_ALIGNER_H_*/ hisat2-2.2.1/sse_util.cpp000066400000000000000000000017231370663763200152610ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "sse_util.h" #include "aligner_swsse.h" #include "limit.h" /** * Given a column of filled-in cells, save the checkpointed cells in cs_. */ void Checkpointer::commitCol( __m128i *pvH, __m128i *pvE, __m128i *pvF, size_t coli) { } hisat2-2.2.1/sse_util.h000066400000000000000000000337031370663763200147310ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef SSE_UTIL_H_ #define SSE_UTIL_H_ #include "assert_helpers.h" #include "ds.h" #include "limit.h" #include #include class EList_m128i { public: /** * Allocate initial default of S elements. */ explicit EList_m128i(int cat = 0) : cat_(cat), last_alloc_(NULL), list_(NULL), sz_(0), cur_(0) { assert_geq(cat, 0); } /** * Destructor. */ ~EList_m128i() { free(); } /** * Return number of elements. */ inline size_t size() const { return cur_; } /** * Return number of elements allocated. */ inline size_t capacity() const { return sz_; } /** * Ensure that there is sufficient capacity to expand to include * 'thresh' more elements without having to expand. */ inline void ensure(size_t thresh) { if(list_ == NULL) lazyInit(); expandCopy(cur_ + thresh); } /** * Ensure that there is sufficient capacity to include 'newsz' elements. * If there isn't enough capacity right now, expand capacity to exactly * equal 'newsz'. */ inline void reserveExact(size_t newsz) { if(list_ == NULL) lazyInitExact(newsz); expandCopyExact(newsz); } /** * Return true iff there are no elements. */ inline bool empty() const { return cur_ == 0; } /** * Return true iff list hasn't been initialized yet. */ inline bool null() const { return list_ == NULL; } /** * If size is less than requested size, resize up to at least sz * and set cur_ to requested sz. */ void resize(size_t sz) { if(sz > 0 && list_ == NULL) lazyInit(); if(sz <= cur_) { cur_ = sz; return; } if(sz_ < sz) { expandCopy(sz); } cur_ = sz; } /** * Zero out contents of vector. */ void zero() { if(cur_ > 0) { memset(list_, 0, cur_ * sizeof(__m128i)); } } /** * If size is less than requested size, resize up to at least sz * and set cur_ to requested sz. Do not copy the elements over. */ void resizeNoCopy(size_t sz) { if(sz > 0 && list_ == NULL) lazyInit(); if(sz <= cur_) { cur_ = sz; return; } if(sz_ < sz) { expandNoCopy(sz); } cur_ = sz; } /** * If size is less than requested size, resize up to exactly sz and set * cur_ to requested sz. */ void resizeExact(size_t sz) { if(sz > 0 && list_ == NULL) lazyInitExact(sz); if(sz <= cur_) { cur_ = sz; return; } if(sz_ < sz) expandCopyExact(sz); cur_ = sz; } /** * Make the stack empty. */ void clear() { cur_ = 0; // re-use stack memory // Don't clear heap; re-use it } /** * Return a reference to the ith element. */ inline __m128i& operator[](size_t i) { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline __m128i operator[](size_t i) const { assert_lt(i, cur_); return list_[i]; } /** * Return a reference to the ith element. */ inline __m128i& get(size_t i) { return operator[](i); } /** * Return a reference to the ith element. */ inline __m128i get(size_t i) const { return operator[](i); } /** * Return a pointer to the beginning of the buffer. */ __m128i *ptr() { return list_; } /** * Return a const pointer to the beginning of the buffer. */ const __m128i *ptr() const { return list_; } /** * Return memory category. */ int cat() const { return cat_; } private: /** * Initialize memory for EList. */ void lazyInit() { assert(list_ == NULL); list_ = alloc(sz_); } /** * Initialize exactly the prescribed number of elements for EList. */ void lazyInitExact(size_t sz) { assert_gt(sz, 0); assert(list_ == NULL); sz_ = sz; list_ = alloc(sz); } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ __m128i *alloc(size_t sz) { __m128i* last_alloc_; try { last_alloc_ = new __m128i[sz + 2]; } catch(std::bad_alloc& e) { std::cerr << "Error: Out of memory allocating " << sz << " __m128i's for DP matrix: '" << e.what() << "'" << std::endl; throw e; } __m128i* tmp = last_alloc_; size_t tmpint = (size_t)tmp; // Align it! if((tmpint & 0xf) != 0) { tmpint += 15; tmpint &= (~0xf); tmp = reinterpret_cast<__m128i*>(tmpint); } assert_eq(0, (tmpint & 0xf)); // should be 16-byte aligned assert(tmp != NULL); gMemTally.add(cat_, sz); return tmp; } /** * Allocate a T array of length sz_ and store in list_. Also, * tally into the global memory tally. */ void free() { if(list_ != NULL) { delete[] last_alloc_; gMemTally.del(cat_, sz_); list_ = NULL; sz_ = cur_ = 0; } } /** * Expand the list_ buffer until it has at least 'thresh' elements. Size * increases quadratically with number of expansions. Copy old contents * into new buffer using operator=. */ void expandCopy(size_t thresh) { if(thresh <= sz_) return; size_t newsz = (sz_ * 2)+1; while(newsz < thresh) newsz *= 2; expandCopyExact(newsz); } /** * Expand the list_ buffer until it has exactly 'newsz' elements. Copy * old contents into new buffer using operator=. */ void expandCopyExact(size_t newsz) { if(newsz <= sz_) return; __m128i* tmp = alloc(newsz); assert(tmp != NULL); size_t cur = cur_; if(list_ != NULL) { for(size_t i = 0; i < cur_; i++) { // Note: operator= is used tmp[i] = list_[i]; } free(); } list_ = tmp; sz_ = newsz; cur_ = cur; } /** * Expand the list_ buffer until it has at least 'thresh' elements. * Size increases quadratically with number of expansions. Don't copy old * contents into the new buffer. */ void expandNoCopy(size_t thresh) { assert(list_ != NULL); if(thresh <= sz_) return; size_t newsz = (sz_ * 2)+1; while(newsz < thresh) newsz *= 2; expandNoCopyExact(newsz); } /** * Expand the list_ buffer until it has exactly 'newsz' elements. Don't * copy old contents into the new buffer. */ void expandNoCopyExact(size_t newsz) { assert(list_ != NULL); assert_gt(newsz, 0); free(); __m128i* tmp = alloc(newsz); assert(tmp != NULL); list_ = tmp; sz_ = newsz; assert_gt(sz_, 0); } int cat_; // memory category, for accounting purposes __m128i* last_alloc_; // what new[] originally returns __m128i *list_; // list ptr, aligned version of what new[] returns size_t sz_; // capacity size_t cur_; // occupancy (AKA size) }; struct CpQuad { CpQuad() { reset(); } void reset() { sc[0] = sc[1] = sc[2] = sc[3] = 0; } bool operator==(const CpQuad& o) const { return sc[0] == o.sc[0] && sc[1] == o.sc[1] && sc[2] == o.sc[2] && sc[3] == o.sc[3]; } int16_t sc[4]; }; /** * Encapsulates a collection of checkpoints. Assumes the scheme is to * checkpoint adjacent pairs of anti-diagonals. */ class Checkpointer { public: Checkpointer() { reset(); } /** * Set the checkpointer up for a new rectangle. */ void init( size_t nrow, // # of rows size_t ncol, // # of columns size_t perpow2, // checkpoint every 1 << perpow2 diags (& next) int64_t perfectScore, // what is a perfect score? for sanity checks bool is8, // 8-bit? bool doTri, // triangle shaped? bool local, // is alignment local? for sanity checks bool debug) // gather debug checkpoints? { assert_gt(perpow2, 0); nrow_ = nrow; ncol_ = ncol; perpow2_ = perpow2; per_ = 1 << perpow2; lomask_ = ~(0xffffffff << perpow2); perf_ = perfectScore; local_ = local; ndiag_ = (ncol + nrow - 1 + 1) / per_; locol_ = MAX_SIZE_T; hicol_ = MIN_SIZE_T; // debug_ = debug; debug_ = true; commitMap_.clear(); firstCommit_ = true; size_t perword = (is8 ? 16 : 8); is8_ = is8; niter_ = ((nrow_ + perword - 1) / perword); if(doTri) { // Save a pair of anti-diagonals every per_ anti-diagonals for // backtrace purposes qdiag1s_.resize(ndiag_ * nrow_); qdiag2s_.resize(ndiag_ * nrow_); } else { // Save every per_ columns and rows for backtrace purposes qrows_.resize((nrow_ / per_) * ncol_); qcols_.resize((ncol_ / per_) * (niter_ << 2)); } if(debug_) { // Save all columns for debug purposes qcolsD_.resize(ncol_ * (niter_ << 2)); } } /** * Return true iff we've been collecting debug cells. */ bool debug() const { return debug_; } /** * Check whether the given score matches the saved score at row, col, hef. */ int64_t debugCell(size_t row, size_t col, int hef) const { assert(debug_); const __m128i* ptr = qcolsD_.ptr() + hef; // Fast forward to appropriate column ptr += ((col * niter_) << 2); size_t mod = row % niter_; // which m128i size_t div = row / niter_; // offset into m128i // Fast forward to appropriate word ptr += (mod << 2); // Extract score int16_t sc = (is8_ ? ((uint8_t*)ptr)[div] : ((int16_t*)ptr)[div]); int64_t asc = MIN_I64; // Convert score if(is8_) { if(local_) { asc = sc; } else { if(sc == 0) asc = MIN_I64; else asc = sc - 0xff; } } else { if(local_) { asc = sc + 0x8000; } else { if(sc != MIN_I16) asc = sc - 0x7fff; } } return asc; } /** * Return true iff the given row/col is checkpointed. */ bool isCheckpointed(size_t row, size_t col) const { assert_leq(col, hicol_); assert_geq(col, locol_); size_t mod = (row + col) & lomask_; assert_lt(mod, per_); return mod >= per_ - 2; } /** * Return the checkpointed H, E, or F score from the given cell. */ inline int64_t scoreTriangle(size_t row, size_t col, int hef) const { assert(isCheckpointed(row, col)); bool diag1 = ((row + col) & lomask_) == per_ - 2; size_t off = (row + col) >> perpow2_; if(diag1) { if(qdiag1s_[off * nrow_ + row].sc[hef] == MIN_I16) { return MIN_I64; } else { return qdiag1s_[off * nrow_ + row].sc[hef]; } } else { if(qdiag2s_[off * nrow_ + row].sc[hef] == MIN_I16) { return MIN_I64; } else { return qdiag2s_[off * nrow_ + row].sc[hef]; } } } /** * Return the checkpointed H, E, or F score from the given cell. */ inline int64_t scoreSquare(size_t row, size_t col, int hef) const { // Is it in a checkpointed row? Note that checkpointed rows don't // necessarily have the horizontal contributions calculated, so we want // to use the column info in that case. if((row & lomask_) == lomask_ && hef != 1) { int64_t sc = qrows_[(row >> perpow2_) * ncol_ + col].sc[hef]; if(sc == MIN_I16) return MIN_I64; return sc; } hef--; if(hef == -1) hef = 2; // It must be in a checkpointed column assert_eq(lomask_, (col & lomask_)); // Fast forward to appropriate column const __m128i* ptr = qcols_.ptr() + hef; ptr += (((col >> perpow2_) * niter_) << 2); size_t mod = row % niter_; // which m128i size_t div = row / niter_; // offset into m128i // Fast forward to appropriate word ptr += (mod << 2); // Extract score int16_t sc = (is8_ ? ((uint8_t*)ptr)[div] : ((int16_t*)ptr)[div]); int64_t asc = MIN_I64; // Convert score if(is8_) { if(local_) { asc = sc; } else { if(sc == 0) asc = MIN_I64; else asc = sc - 0xff; } } else { if(local_) { asc = sc + 0x8000; } else { if(sc != MIN_I16) asc = sc - 0x7fff; } } return asc; } /** * Given a column of filled-in cells, save the checkpointed cells in cs_. */ void commitCol(__m128i *pvH, __m128i *pvE, __m128i *pvF, size_t coli); /** * Reset the state of the Checkpointer. */ void reset() { perpow2_ = per_ = lomask_ = nrow_ = ncol_ = 0; local_ = false; niter_ = ndiag_ = locol_ = hicol_ = 0; perf_ = 0; firstCommit_ = true; is8_ = debug_ = false; } /** * Return true iff the Checkpointer has been initialized. */ bool inited() const { return nrow_ > 0; } size_t per() const { return per_; } size_t perpow2() const { return perpow2_; } size_t lomask() const { return lomask_; } size_t locol() const { return locol_; } size_t hicol() const { return hicol_; } size_t nrow() const { return nrow_; } size_t ncol() const { return ncol_; } const CpQuad* qdiag1sPtr() const { return qdiag1s_.ptr(); } const CpQuad* qdiag2sPtr() const { return qdiag2s_.ptr(); } size_t perpow2_; // 1 << perpow2_ - 2 is the # of uncheckpointed // anti-diags between checkpointed anti-diag pairs size_t per_; // 1 << perpow2_ size_t lomask_; // mask for extracting low bits size_t nrow_; // # rows in current rectangle size_t ncol_; // # cols in current rectangle int64_t perf_; // perfect score bool local_; // local alignment? size_t ndiag_; // # of double-diags size_t locol_; // leftmost column committed size_t hicol_; // rightmost column committed // Map for committing scores from vector columns to checkpointed diagonals EList commitMap_; bool firstCommit_; EList qdiag1s_; // checkpoint H/E/F values for diagonal 1 EList qdiag2s_; // checkpoint H/E/F values for diagonal 2 EList qrows_; // checkpoint H/E/F values for rows // We store columns in this way to reduce overhead of populating them bool is8_; // true -> fill used 8-bit cells size_t niter_; // # __m128i words per column EList_m128i qcols_; // checkpoint E/F/H values for select columns bool debug_; // get debug checkpoints? (i.e. fill qcolsD_?) EList_m128i qcolsD_; // checkpoint E/F/H values for all columns (debug) }; #endif hisat2-2.2.1/sstring.cpp000066400000000000000000000125431370663763200151250ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifdef MAIN_SSTRING #include #include #include "ds.h" #include "sstring.h" using namespace std; int main(void) { cerr << "Test inter-class comparison operators..."; { SString s(2); s.set('a', 0); s.set('b', 1); assert(sstr_eq(s, (const char *)"ab")); assert(!sstr_neq(s, (const char *)"ab")); assert(!sstr_lt(s, (const char *)"ab")); assert(!sstr_gt(s, (const char *)"ab")); assert(sstr_leq(s, (const char *)"ab")); assert(sstr_geq(s, (const char *)"ab")); SStringExpandable s2; s2.append('a'); s2.append('b'); assert(sstr_eq(s, s2)); assert(sstr_eq(s2, (const char *)"ab")); assert(!sstr_neq(s, s2)); assert(!sstr_neq(s2, (const char *)"ab")); assert(!sstr_lt(s, s2)); assert(!sstr_lt(s2, (const char *)"ab")); assert(!sstr_gt(s, s2)); assert(!sstr_gt(s2, (const char *)"ab")); assert(sstr_leq(s, s2)); assert(sstr_leq(s2, (const char *)"ab")); assert(sstr_geq(s, s2)); assert(sstr_geq(s2, (const char *)"ab")); SStringFixed s3; s3.append('a'); s3.append('b'); assert(sstr_eq(s, s3)); assert(sstr_eq(s2, s3)); assert(sstr_eq(s3, (const char *)"ab")); assert(!sstr_neq(s, s3)); assert(!sstr_neq(s2, s3)); assert(!sstr_neq(s3, (const char *)"ab")); assert(!sstr_lt(s, s3)); assert(!sstr_lt(s2, s3)); assert(!sstr_lt(s3, (const char *)"ab")); assert(!sstr_gt(s, s3)); assert(!sstr_gt(s2, s3)); assert(!sstr_gt(s3, (const char *)"ab")); assert(sstr_geq(s, s3)); assert(sstr_geq(s2, s3)); assert(sstr_geq(s3, (const char *)"ab")); assert(sstr_leq(s, s3)); assert(sstr_leq(s2, s3)); assert(sstr_leq(s3, (const char *)"ab")); } cerr << "PASSED" << endl; cerr << "Test flag for whether to consider end-of-word < other chars ..."; { SString ss("String"); SString sl("String1"); assert(sstr_lt(ss, sl)); assert(sstr_gt(ss, sl, false)); assert(sstr_leq(ss, sl)); assert(sstr_geq(ss, sl, false)); } cerr << "PASSED" << endl; cerr << "Test toZBuf and toZBufXForm ..."; { SString s(10); for(int i = 0; i < 10; i++) { s[i] = (uint32_t)i; } assert(strcmp(s.toZBufXForm("0123456789"), "0123456789") == 0); } cerr << "PASSED" << endl; cerr << "Test S2bDnaString ..."; { const char *str = "ACGTACGTAC" "ACGTACGTAC" "ACGTACGTAC" "ACGTACGTAC" "ACGTACGTAC" "ACGTACGTAC"; const char *gs = "GGGGGGGGGG" "GGGGGGGGGG" "GGGGGGGGGG" "GGGGGGGGGG" "GGGGGGGGGG" "GGGGGGGGGG"; for(size_t i = 0; i < 60; i++) { S2bDnaString s(str, i, true); S2bDnaString sr; BTDnaString s2(str, i, true); assert(sstr_eq(s, s2)); if(i >= 10) { BTDnaString s3; s.windowGetDna(s3, true, false, 3, 4); assert(sstr_eq(s3.toZBuf(), (const char*)"TACG")); s.windowGetDna(s3, false, false, 3, 4); assert(sstr_eq(s3.toZBuf(), (const char*)"CGTA")); assert_eq('A', s.toChar(0)); assert_eq('G', s.toChar(2)); assert_eq('A', s.toChar(4)); assert_eq('G', s.toChar(6)); assert_eq('A', s.toChar(8)); s.reverseWindow(1, 8); s2.reverseWindow(1, 8); assert_eq('A', s.toChar(1)); assert_eq('T', s.toChar(2)); assert_eq('G', s.toChar(3)); assert_eq('C', s.toChar(4)); assert_eq('A', s.toChar(5)); assert_eq('T', s.toChar(6)); assert_eq('G', s.toChar(7)); assert_eq('C', s.toChar(8)); assert(sstr_eq(s, s2)); s.reverseWindow(1, 8); s2.reverseWindow(1, 8); assert(sstr_eq(s, s2)); } if(i > 1) { s.reverse(); sr.installReverseChars(str, i); s2.reverse(); assert(sstr_eq(s, s2)); assert(sstr_eq(sr, s2)); s.reverse(); sr.reverse(); assert(sstr_neq(s, s2)); assert(sstr_neq(sr, s2)); s.fill(2); s2.reverse(); assert(sstr_leq(s, gs)); assert(sstr_gt(s, s2)); assert(sstr_gt(s, sr)); s2.fill(2); sr.fill(2); assert(sstr_eq(s, s2)); assert(sstr_eq(s, sr)); } } S2bDnaString s(str, true); S2bDnaString sr; BTDnaString s2(str, true); assert(sstr_eq(s2.toZBuf(), str)); assert(sstr_eq(s, s2)); s.reverse(); sr.installReverseChars(str); s2.reverse(); assert(sstr_eq(s, s2)); assert(sstr_eq(sr, s2)); s.reverse(); sr.reverse(); assert(sstr_neq(s, s2)); assert(sstr_neq(sr, s2)); } cerr << "PASSED" << endl; cerr << "Test operator=() ..."; { S2bDnaString s; s.installChars(string("gtcagtca")); assert(sstr_eq(s.toZBuf(), (const char *)"GTCAGTCA")); } cerr << "PASSED" << endl; cerr << "Conversions from string ..."; { SStringExpandable se(string("hello")); EList > sel; sel.push_back(SStringExpandable(string("hello"))); } cerr << "PASSED" << endl; cerr << "PASSED" << endl; } #endif /*def MAIN_SSTRING*/ hisat2-2.2.1/sstring.h000066400000000000000000002264111370663763200145730ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef SSTRING_H_ #define SSTRING_H_ #include #include #include "assert_helpers.h" #include "alphabet.h" #include "random_source.h" /** * Four kinds of strings defined here: * * SString: * A fixed-length string using heap memory with size set at construction time * or when install() member is called. * * S2bDnaString: * Like SString, but stores a list uint32_t words where each word is divided * into 16 2-bit slots interpreted as holding one A/C/G/T nucleotide each. * * TODO: S3bDnaString allowing N. S4bDnaString allowing nucleotide masks. * * SStringExpandable: * A string using heap memory where the size of the backing store is * automatically resized as needed. Supports operations like append, insert, * erase, etc. * * SStringFixed: * A fixed-length string using stack memory where size is set at compile * time. * * All string classes have some extra facilities that make it easy to print the * string, including when the string uses an encoded alphabet. See toZBuf() * and toZBufXForm(). * * Global lt, eq, and gt template functions are supplied. They are capable of * doing lexicographical comparisons between any of the three categories of * strings defined here. */ template class Class_sstr_len { public: static inline size_t sstr_len(const T& s) { return s.length(); } }; template class Class_sstr_len { public: static inline size_t sstr_len(const char s[N]) { return strlen(s); } }; template<> class Class_sstr_len { public: static inline size_t sstr_len(const char *s) { return strlen(s); } }; template<> class Class_sstr_len { public: static inline size_t sstr_len(const unsigned char *s) { return strlen((const char *)s); } }; template static inline bool sstr_eq(const T1& s1, const T2& s2) { size_t len1 = Class_sstr_len::sstr_len(s1); size_t len2 = Class_sstr_len::sstr_len(s2); if(len1 != len2) return false; for(size_t i = 0; i < len1; i++) { if(s1[i] != s2[i]) return false; } return true; } template static inline bool sstr_neq(const T1& s1, const T2& s2) { return !sstr_eq(s1, s2); } /** * Return true iff the given suffix of s1 is equal to the given suffix of s2 up * to upto characters. */ template static inline bool sstr_suf_upto_eq( const T1& s1, size_t suf1, const T2& s2, size_t suf2, size_t upto, bool endlt = true) { assert_leq(suf1, Class_sstr_len::sstr_len(s1)); assert_leq(suf2, Class_sstr_len::sstr_len(s2)); size_t len1 = Class_sstr_len::sstr_len(s1) - suf1; size_t len2 = Class_sstr_len::sstr_len(s2) - suf2; if(len1 > upto) len1 = upto; if(len2 > upto) len2 = upto; if(len1 != len2) return false; for(size_t i = 0; i < len1; i++) { if(s1[suf1+i] != s2[suf2+i]) { return false; } } return true; } /** * Return true iff the given suffix of s1 is equal to the given suffix of s2 up * to upto characters. */ template static inline bool sstr_suf_upto_neq( const T1& s1, size_t suf1, const T2& s2, size_t suf2, size_t upto, bool endlt = true) { return !sstr_suf_upto_eq(s1, suf1, s2, suf2, upto, endlt); } /** * Return true iff s1 is less than s2. */ template static inline bool sstr_lt(const T1& s1, const T2& s2, bool endlt = true) { size_t len1 = Class_sstr_len::sstr_len(s1); size_t len2 = Class_sstr_len::sstr_len(s2); size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[i] < s2[i]) { return true; } else if(s1[i] > s2[i]) { return false; } } if(len1 == len2) return false; return (len1 < len2) == endlt; } /** * Return true iff the given suffix of s1 is less than the given suffix of s2. */ template static inline bool sstr_suf_lt( const T1& s1, size_t suf1, const T2& s2, size_t suf2, bool endlt = true) { assert_leq(suf1, Class_sstr_len::sstr_len(s1)); assert_leq(suf2, Class_sstr_len::sstr_len(s2)); size_t len1 = Class_sstr_len::sstr_len(s1) - suf1; size_t len2 = Class_sstr_len::sstr_len(s2) - suf2; size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[suf1+i] < s2[suf2+i]) { return true; } else if(s1[suf1+i] > s2[suf2+i]) { return false; } } if(len1 == len2) return false; return (len1 < len2) == endlt; } /** * Return true iff the given suffix of s1 is less than the given suffix of s2. * Treat s1 and s2 as though they have lengths len1/len2. */ template static inline bool sstr_suf_lt( const T1& s1, size_t suf1, size_t len1, const T2& s2, size_t suf2, size_t len2, bool endlt = true) { assert_leq(suf1, len1); assert_leq(suf2, len2); size_t left1 = len1 - suf1; size_t left2 = len2 - suf2; size_t minleft = (left1 < left2 ? left1 : left2); for(size_t i = 0; i < minleft; i++) { if(s1[suf1+i] < s2[suf2+i]) { return true; } else if(s1[suf1+i] > s2[suf2+i]) { return false; } } if(left1 == left2) return false; return (left1 < left2) == endlt; } /** * Return true iff the given suffix of s1 is less than the given suffix of s2 * up to upto characters. */ template static inline bool sstr_suf_upto_lt( const T1& s1, size_t suf1, const T2& s2, size_t suf2, size_t upto, bool endlt = true) { assert_leq(suf1, Class_sstr_len::sstr_len(s1)); assert_leq(suf2, Class_sstr_len::sstr_len(s2)); size_t len1 = Class_sstr_len::sstr_len(s1) - suf1; size_t len2 = Class_sstr_len::sstr_len(s2) - suf2; if(len1 > upto) len1 = upto; if(len2 > upto) len2 = upto; size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[suf1+i] < s2[suf2+i]) { return true; } else if(s1[suf1+i] > s2[suf2+i]) { return false; } } if(len1 == len2) return false; return (len1 < len2) == endlt; } /** * Return true iff the given prefix of s1 is less than the given prefix of s2. */ template static inline bool sstr_pre_lt( const T1& s1, size_t pre1, const T2& s2, size_t pre2, bool endlt = true) { assert_leq(pre1, Class_sstr_len::sstr_len(s1)); assert_leq(pre2, Class_sstr_len::sstr_len(s2)); size_t len1 = pre1; size_t len2 = pre2; size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[i] < s2[i]) { return true; } else if(s1[i] > s2[i]) { return false; } } if(len1 == len2) return false; return (len1 < len2) == endlt; } /** * Return true iff s1 is less than or equal to s2. */ template static inline bool sstr_leq(const T1& s1, const T2& s2, bool endlt = true) { size_t len1 = Class_sstr_len::sstr_len(s1); size_t len2 = Class_sstr_len::sstr_len(s2); size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[i] < s2[i]) { return true; } else if(s1[i] > s2[i]) { return false; } } if(len1 == len2) return true; return (len1 < len2) == endlt; } /** * Return true iff the given suffix of s1 is less than or equal to the given * suffix of s2. */ template static inline bool sstr_suf_leq( const T1& s1, size_t suf1, const T2& s2, size_t suf2, bool endlt = true) { assert_leq(suf1, Class_sstr_len::sstr_len(s1)); assert_leq(suf2, Class_sstr_len::sstr_len(s2)); size_t len1 = Class_sstr_len::sstr_len(s1) - suf1; size_t len2 = Class_sstr_len::sstr_len(s2) - suf2; size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[suf1+i] < s2[suf2+i]) { return true; } else if(s1[suf1+i] > s2[suf2+i]) { return false; } } if(len1 == len2) return true; return (len1 < len2) == endlt; } /** * Return true iff the given prefix of s1 is less than or equal to the given * prefix of s2. */ template static inline bool sstr_pre_leq( const T1& s1, size_t pre1, const T2& s2, size_t pre2, bool endlt = true) { assert_leq(pre1, Class_sstr_len::sstr_len(s1)); assert_leq(pre2, Class_sstr_len::sstr_len(s2)); size_t len1 = pre1; size_t len2 = pre2; size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[i] < s2[i]) { return true; } else if(s1[i] > s2[i]) { return false; } } if(len1 == len2) return true; return (len1 < len2) == endlt; } /** * Return true iff s1 is greater than s2. */ template static inline bool sstr_gt(const T1& s1, const T2& s2, bool endlt = true) { size_t len1 = Class_sstr_len::sstr_len(s1); size_t len2 = Class_sstr_len::sstr_len(s2); size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[i] > s2[i]) { return true; } else if(s1[i] < s2[i]) { return false; } } if(len1 == len2) return false; return (len1 > len2) == endlt; } /** * Return true iff the given suffix of s1 is greater than the given suffix of * s2. */ template static inline bool sstr_suf_gt( const T1& s1, size_t suf1, const T2& s2, size_t suf2, bool endlt = true) { assert_leq(suf1, Class_sstr_len::sstr_len(s1)); assert_leq(suf2, Class_sstr_len::sstr_len(s2)); size_t len1 = Class_sstr_len::sstr_len(s1) - suf1; size_t len2 = Class_sstr_len::sstr_len(s2) - suf2; size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[suf1+i] > s2[suf2+i]) { return true; } else if(s1[suf1+i] < s2[suf2+i]) { return false; } } if(len1 == len2) return false; return (len1 > len2) == endlt; } /** * Return true iff the given prefix of s1 is greater than the given prefix of * s2. */ template static inline bool sstr_pre_gt( const T1& s1, size_t pre1, const T2& s2, size_t pre2, bool endlt = true) { assert_leq(pre1, Class_sstr_len::sstr_len(s1)); assert_leq(pre2, Class_sstr_len::sstr_len(s2)); size_t len1 = pre1; size_t len2 = pre2; size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[i] > s2[i]) { return true; } else if(s1[i] < s2[i]) { return false; } } if(len1 == len2) return false; return (len1 > len2) == endlt; } /** * Return true iff s1 is greater than or equal to s2. */ template static inline bool sstr_geq(const T1& s1, const T2& s2, bool endlt = true) { size_t len1 = Class_sstr_len::sstr_len(s1); size_t len2 = Class_sstr_len::sstr_len(s2); size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[i] > s2[i]) { return true; } else if(s1[i] < s2[i]) { return false; } } if(len1 == len2) return true; return (len1 > len2) == endlt; } /** * Return true iff the given suffix of s1 is greater than or equal to the given * suffix of s2. */ template static inline bool sstr_suf_geq( const T1& s1, size_t suf1, const T2& s2, size_t suf2, bool endlt = true) { assert_leq(suf1, Class_sstr_len::sstr_len(s1)); assert_leq(suf2, Class_sstr_len::sstr_len(s2)); size_t len1 = Class_sstr_len::sstr_len(s1) - suf1; size_t len2 = Class_sstr_len::sstr_len(s2) - suf2; size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[suf1+i] > s2[suf2+i]) { return true; } else if(s1[suf1+i] < s2[suf2+i]) { return false; } } if(len1 == len2) return true; return (len1 > len2) == endlt; } /** * Return true iff the given prefix of s1 is greater than or equal to the given * prefix of s2. */ template static inline bool sstr_pre_geq( const T1& s1, size_t pre1, const T2& s2, size_t pre2, bool endlt = true) { assert_leq(pre1, Class_sstr_len::sstr_len(s1)); assert_leq(pre2, Class_sstr_len::sstr_len(s2)); size_t len1 = pre1; size_t len2 = pre2; size_t minlen = (len1 < len2 ? len1 : len2); for(size_t i = 0; i < minlen; i++) { if(s1[i] > s2[i]) { return true; } else if(s1[i] < s2[i]) { return false; } } if(len1 == len2) return true; return (len1 > len2) == endlt; } template static inline const char * sstr_to_cstr(const T& s) { return s.toZBuf(); } template<> inline const char * sstr_to_cstr >( const std::basic_string& s) { return s.c_str(); } /** * Simple string class with backing memory whose size is managed by the user * using the constructor and install() member function. No behind-the-scenes * reallocation or copying takes place. */ template class SString { public: explicit SString() : cs_(NULL), printcs_(NULL), len_(0) { } explicit SString(size_t sz) : cs_(NULL), printcs_(NULL), len_(0) { resize(sz); } /** * Create an SStringExpandable from another SStringExpandable. */ SString(const SString& o) : cs_(NULL), printcs_(NULL), len_(0) { *this = o; } /** * Create an SStringExpandable from a std::basic_string of the * appropriate type. */ explicit SString(const std::basic_string& str) : cs_(NULL), printcs_(NULL), len_(0) { install(str.c_str(), str.length()); } /** * Create an SStringExpandable from an array and size. */ explicit SString(const T* b, size_t sz) : cs_(NULL), printcs_(NULL), len_(0) { install(b, sz); } /** * Create an SStringExpandable from a zero-terminated array. */ explicit SString(const T* b) : cs_(NULL), printcs_(NULL), len_(0) { install(b, strlen(b)); } /** * Destroy the expandable string object. */ virtual ~SString() { if(cs_ != NULL) { delete[] cs_; cs_ = NULL; } if(printcs_ != NULL) { delete[] printcs_; printcs_ = NULL; } len_ = 0; } /** * Assignment to other SString. */ SString& operator=(const SString& o) { install(o.cs_, o.len_); return *this; } /** * Assignment to other SString. */ SString& operator=(const std::basic_string& o) { install(o); return *this; } /** * Resizes the string without preserving its contents. */ void resize(size_t sz) { if(cs_ != NULL) { delete cs_; cs_ = NULL; } if(printcs_ != NULL) { delete printcs_; printcs_ = NULL; } if(sz != 0) { cs_ = new T[sz+1]; } len_ = sz; } /** * Return ith character from the left of either the forward or the * reverse version of the read. */ T windowGet( size_t i, bool fw, size_t depth = 0, size_t len = 0) const { if(len == 0) len = len_; assert_lt(i, len); assert_leq(len, len_ - depth); return fw ? cs_[depth+i] : cs_[depth+len-i-1]; } /** * Return ith character from the left of either the forward or the * reverse-complement version of the read. */ void windowGet( T& ret, bool fw, size_t depth = 0, size_t len = 0) const { if(len == 0) len = len_; assert_leq(len, len_ - depth); ret.resize(len); for(size_t i = 0; i < len; i++) { ret.set(fw ? cs_[depth+i] : cs_[depth+len-i-1], i); } } /** * Set character at index 'idx' to 'c'. */ inline void set(int c, size_t idx) { assert_lt(idx, len_); cs_[idx] = c; } /** * Retrieve constant version of element i. */ inline const T& operator[](size_t i) const { assert_lt(i, len_); return cs_[i]; } /** * Retrieve mutable version of element i. */ inline T& operator[](size_t i) { assert_lt(i, len_); return cs_[i]; } /** * Retrieve constant version of element i. */ inline const T& get(size_t i) const { assert_lt(i, len_); return cs_[i]; } /** * Copy 'sz' bytes from buffer 'b' into this string. memcpy is used, not * operator=. */ virtual void install(const T* b, size_t sz) { if(sz == 0) return; resize(sz); memcpy(cs_, b, sz * sizeof(T)); } /** * Copy 'sz' bytes from buffer 'b' into this string. memcpy is used, not * operator=. */ virtual void install(const std::basic_string& b) { size_t sz = b.length(); if(sz == 0) return; resize(sz); memcpy(cs_, b.c_str(), sz * sizeof(T)); } /** * Copy all bytes from zero-terminated buffer 'b' into this string. */ void install(const T* b) { install(b, strlen(b)); } /** * Copy 'sz' bytes from buffer 'b' into this string, reversing them * in the process. */ void installReverse(const char* b, size_t sz) { if(sz == 0) return; resize(sz); for(size_t i = 0; i < sz; i++) { cs_[i] = b[sz-i-1]; } len_ = sz; } /** * Copy 'sz' bytes from buffer 'b' into this string, reversing them * in the process. */ void installReverse(const SString& b) { installReverse(b.cs_, b.len_); } /** * Return true iff the two strings are equal. */ bool operator==(const SString& o) { return sstr_eq(*this, o); } /** * Return true iff the two strings are not equal. */ bool operator!=(const SString& o) { return sstr_neq(*this, o); } /** * Return true iff this string is less than given string. */ bool operator<(const SString& o) { return sstr_lt(*this, o); } /** * Return true iff this string is greater than given string. */ bool operator>(const SString& o) { return sstr_gt(*this, o); } /** * Return true iff this string is less than or equal to given string. */ bool operator<=(const SString& o) { return sstr_leq(*this, o); } /** * Return true iff this string is greater than or equal to given string. */ bool operator>=(const SString& o) { return sstr_geq(*this, o); } /** * Reverse the buffer in place. */ void reverse() { for(size_t i = 0; i < (len_ >> 1); i++) { T tmp = get(i); set(get(len_-i-1), i); set(tmp, len_-i-1); } } /** * Reverse the buffer in place. */ void reverseComplement(int* rcmap) { size_t mid = len_ >> 1; for(size_t i = 0; i < (len_ >> 1); i++) { T tmp = get(i); set(rcmap[get(len_-i-1)], i); set(rcmap[tmp], len_-i-1); } if (len_ % 2) { set(rcmap[get(mid)], mid); } } /** * Reverse a substring of the buffer in place. */ void reverseWindow(size_t off, size_t len) { assert_leq(off, len_); assert_leq(off + len, len_); size_t mid = len >> 1; for(size_t i = 0; i < mid; i++) { T tmp = get(off+i); set(get(off+len-i-1), off+i); set(tmp, off+len-i-1); } } /** * Set the first len elements of the buffer to el. */ void fill(size_t len, const T& el) { assert_leq(len, len_); for(size_t i = 0; i < len; i++) { set(el, i); } } /** * Set all elements of the buffer to el. */ void fill(const T& el) { fill(len_, el); } /** * Return the length of the string. */ inline size_t length() const { return len_; } /** * Clear the buffer. */ void clear() { len_ = 0; } /** * Return true iff the buffer is empty. */ inline bool empty() const { return len_ == 0; } /** * Put a terminator in the 'len_'th element and then return a * pointer to the buffer. Useful for printing. */ const char* toZBufXForm(const char *xform) const { ASSERT_ONLY(size_t xformElts = strlen(xform)); // Lazily allocate space for print buffer if(printcs_ == NULL) { const_cast(printcs_) = new char[len_+1]; } char* printcs = const_cast(printcs_); assert(printcs != NULL); for(size_t i = 0; i < len_; i++) { assert_lt(cs_[i], (int)xformElts); printcs[i] = xform[cs_[i]]; } printcs[len_] = 0; return printcs_; } /** * Put a terminator in the 'len_'th element and then return a * pointer to the buffer. Useful for printing. */ virtual const T* toZBuf() const { const_cast(cs_)[len_] = 0; return cs_; } /** * Return a const version of the raw buffer. */ const T* buf() const { return cs_; } /** * Return a writeable version of the raw buffer. */ T* wbuf() { return cs_; } protected: T *cs_; // +1 so that we have the option of dropping in a terminating "\0" char *printcs_; // +1 so that we have the option of dropping in a terminating "\0" size_t len_; // # elements }; /** * Simple string class with backing memory whose size is managed by the user * using the constructor and install() member function. No behind-the-scenes * reallocation or copying takes place. */ class S2bDnaString { public: explicit S2bDnaString() : cs_(NULL), printcs_(NULL), len_(0) { } explicit S2bDnaString(size_t sz) : cs_(NULL), printcs_(NULL), len_(0) { resize(sz); } /** * Copy another object of the same class. */ S2bDnaString(const S2bDnaString& o) : cs_(NULL), printcs_(NULL), len_(0) { *this = o; } /** * Create an SStringExpandable from a std::basic_string of the * appropriate type. */ explicit S2bDnaString( const std::basic_string& str, bool chars = false, bool colors = false) : cs_(NULL), printcs_(NULL), len_(0) { if(chars) { if(colors) { installColors(str.c_str(), str.length()); } else { installChars(str.c_str(), str.length()); } } else { install(str.c_str(), str.length()); } } /** * Create an SStringExpandable from an array and size. */ explicit S2bDnaString( const char* b, size_t sz, bool chars = false, bool colors = false) : cs_(NULL), printcs_(NULL), len_(0) { if(chars) { if(colors) { installColors(b, sz); } else { installChars(b, sz); } } else { install(b, sz); } } /** * Create an SStringFixed from a zero-terminated string. */ explicit S2bDnaString( const char* b, bool chars = false, bool colors = false) : cs_(NULL), printcs_(NULL), len_(0) { if(chars) { if(colors) { installColors(b, strlen(b)); } else { installChars(b, strlen(b)); } } else { install(b, strlen(b)); } } /** * Destroy the expandable string object. */ virtual ~S2bDnaString() { if(cs_ != NULL) { delete[] cs_; cs_ = NULL; } if(printcs_ != NULL) { delete[] printcs_; printcs_ = NULL; } len_ = 0; } /** * Assignment to other SString. */ template S2bDnaString& operator=(const T& o) { install(o.c_str(), o.length()); return *this; } /** * Assignment from a std::basic_string */ template S2bDnaString& operator=(const std::basic_string& o) { install(o); return *this; } /** * Resizes the string without preserving its contents. */ void resize(size_t sz) { if(cs_ != NULL) { delete cs_; cs_ = NULL; } if(printcs_ != NULL) { delete printcs_; printcs_ = NULL; } len_ = sz; if(sz != 0) { cs_ = new uint32_t[nwords()]; } } /** * Return DNA character corresponding to element 'idx'. */ char toChar(size_t idx) const { int c = (int)get(idx); assert_range(0, 3, c); return "ACGT"[c]; } /** * Return color character corresponding to element 'idx'. */ char toColor(size_t idx) const { int c = (int)get(idx); assert_range(0, 3, c); return "0123"[c]; } /** * Return ith character from the left of either the forward or the * reverse version of the read. */ char windowGet( size_t i, bool fw, size_t depth = 0, size_t len = 0) const { if(len == 0) len = len_; assert_lt(i, len); assert_leq(len, len_ - depth); return fw ? get(depth+i) : get(depth+len-i-1); } /** * Return ith character from the left of either the forward or the * reverse-complement version of the read. */ template void windowGet( T& ret, bool fw, size_t depth = 0, size_t len = 0) const { if(len == 0) len = len_; assert_leq(len, len_ - depth); ret.resize(len); for(size_t i = 0; i < len; i++) { ret.set((fw ? get(depth+i) : get(depth+len-i-1)), i); } } /** * Return length in 32-bit words. */ size_t nwords() const { return (len_ + 15) >> 4; } /** * Set character at index 'idx' to 'c'. */ void set(int c, size_t idx) { assert_lt(idx, len_); assert_range(0, 3, c); size_t word = idx >> 4; size_t bpoff = (idx & 15) << 1; cs_[word] = cs_[word] & ~(uint32_t)(3 << bpoff); cs_[word] = cs_[word] | (uint32_t)(c << bpoff); } /** * Set character at index 'idx' to DNA char 'c'. */ void setChar(int c, size_t idx) { assert_in(toupper(c), "ACGT"); int bp = asc2dna[c]; set(bp, idx); } /** * Set character at index 'idx' to color char 'c'. */ void setColor(int c, size_t idx) { assert_in(toupper(c), "0123"); int co = asc2col[c]; set(co, idx); } /** * Set the ith 32-bit word to given word. */ void setWord(uint32_t w, size_t i) { assert_lt(i, nwords()); cs_[i] = w; } /** * Retrieve constant version of element i. */ char operator[](size_t i) const { assert_lt(i, len_); return get(i); } /** * Retrieve constant version of element i. */ char get(size_t i) const { assert_lt(i, len_); size_t word = i >> 4; size_t bpoff = (i & 15) << 1; return (char)((cs_[word] >> bpoff) & 3); } /** * Copy packed words from string 'b' into this packed string. */ void install(const uint32_t* b, size_t sz) { if(sz == 0) return; resize(sz); memcpy(cs_, b, sizeof(uint32_t)*nwords()); } /** * Copy 'sz' DNA characters encoded as integers from buffer 'b' into this * packed string. */ void install(const char* b, size_t sz) { if(sz == 0) return; resize(sz); size_t wordi = 0; for(size_t i = 0; i < sz; i += 16) { uint32_t word = 0; for(int j = 0; j < 16 && (size_t)(i+j) < sz; j++) { uint32_t bp = (int)b[i+j]; uint32_t shift = (uint32_t)j << 1; assert_range(0, 3, (int)bp); word |= (bp << shift); } cs_[wordi++] = word; } } /** * Copy 'sz' DNA characters from buffer 'b' into this packed string. */ void installChars(const char* b, size_t sz) { if(sz == 0) return; resize(sz); size_t wordi = 0; for(size_t i = 0; i < sz; i += 16) { uint32_t word = 0; for(int j = 0; j < 16 && (size_t)(i+j) < sz; j++) { char c = b[i+j]; assert_in(toupper(c), "ACGT"); int bp = asc2dna[(int)c]; assert_range(0, 3, (int)bp); uint32_t shift = (uint32_t)j << 1; word |= (bp << shift); } cs_[wordi++] = word; } } /** * Copy 'sz' color characters from buffer 'b' into this packed string. */ void installColors(const char* b, size_t sz) { if(sz == 0) return; resize(sz); size_t wordi = 0; for(size_t i = 0; i < sz; i += 16) { uint32_t word = 0; for(int j = 0; j < 16 && (size_t)(i+j) < sz; j++) { char c = b[i+j]; assert_in(c, "0123"); int bp = asc2col[(int)c]; assert_range(0, 3, (int)bp); uint32_t shift = (uint32_t)j << 1; word |= (bp << shift); } cs_[wordi++] = word; } } /** * Copy 'sz' DNA characters from buffer 'b' into this packed string. */ void install(const char* b) { install(b, strlen(b)); } /** * Copy 'sz' DNA characters from buffer 'b' into this packed string. */ void installChars(const char* b) { installChars(b, strlen(b)); } /** * Copy 'sz' DNA characters from buffer 'b' into this packed string. */ void installColors(const char* b) { installColors(b, strlen(b)); } /** * Copy 'sz' DNA characters from buffer 'b' into this packed string. */ void install(const std::basic_string& b) { install(b.c_str(), b.length()); } /** * Copy 'sz' DNA characters from buffer 'b' into this packed string. */ void installChars(const std::basic_string& b) { installChars(b.c_str(), b.length()); } /** * Copy 'sz' DNA characters from buffer 'b' into this packed string. */ void installColors(const std::basic_string& b) { installColors(b.c_str(), b.length()); } /** * Copy 'sz' bytes from buffer 'b' into this string, reversing them * in the process. */ void installReverse(const char* b, size_t sz) { resize(sz); if(sz == 0) return; size_t wordi = 0; size_t bpi = 0; cs_[0] = 0; for(size_t i =sz; i > 0; i--) { assert_range(0, 3, (int)b[i-1]); cs_[wordi] |= ((int)b[i-1] << (bpi<<1)); if(bpi == 15) { wordi++; cs_[wordi] = 0; bpi = 0; } else bpi++; } } /** * Copy all chars from buffer of DNA characters 'b' into this string, * reversing them in the process. */ void installReverse(const char* b) { installReverse(b, strlen(b)); } /** * Copy 'sz' bytes from buffer of DNA characters 'b' into this string, * reversing them in the process. */ void installReverseChars(const char* b, size_t sz) { resize(sz); if(sz == 0) return; size_t wordi = 0; size_t bpi = 0; cs_[0] = 0; for(size_t i =sz; i > 0; i--) { char c = b[i-1]; assert_in(toupper(c), "ACGT"); int bp = asc2dna[(int)c]; assert_range(0, 3, bp); cs_[wordi] |= (bp << (bpi<<1)); if(bpi == 15) { wordi++; cs_[wordi] = 0; bpi = 0; } else bpi++; } } /** * Copy all chars from buffer of DNA characters 'b' into this string, * reversing them in the process. */ void installReverseChars(const char* b) { installReverseChars(b, strlen(b)); } /** * Copy 'sz' bytes from buffer of color characters 'b' into this string, * reversing them in the process. */ void installReverseColors(const char* b, size_t sz) { resize(sz); if(sz == 0) return; size_t wordi = 0; size_t bpi = 0; cs_[0] = 0; for(size_t i =sz; i > 0; i--) { char c = b[i-1]; assert_in(c, "0123"); int bp = asc2col[(int)c]; assert_range(0, 3, bp); cs_[wordi] |= (bp << (bpi<<1)); if(bpi == 15) { wordi++; cs_[wordi] = 0; bpi = 0; } else bpi++; } } /** * Copy all chars from buffer of color characters 'b' into this string, * reversing them in the process. */ void installReverseColors(const char* b) { installReverseColors(b, strlen(b)); } /** * Copy 'sz' bytes from buffer 'b' into this string, reversing them * in the process. */ void installReverse(const S2bDnaString& b) { resize(b.len_); if(b.len_ == 0) return; size_t wordi = 0; size_t bpi = 0; size_t wordb = b.nwords()-1; size_t bpb = (b.len_-1) & 15; cs_[0] = 0; for(size_t i = b.len_; i > 0; i--) { int bbp = (int)((b[wordb] >> (bpb << 1)) & 3); assert_range(0, 3, bbp); cs_[wordi] |= (bbp << (bpi << 1)); if(bpi == 15) { wordi++; cs_[wordi] = 0; bpi = 0; } else bpi++; if(bpb == 0) { wordb--; bpi = 15; } else bpi--; } } /** * Return true iff the two strings are equal. */ bool operator==(const S2bDnaString& o) { return sstr_eq(*this, o); } /** * Return true iff the two strings are not equal. */ bool operator!=(const S2bDnaString& o) { return sstr_neq(*this, o); } /** * Return true iff this string is less than given string. */ bool operator<(const S2bDnaString& o) { return sstr_lt(*this, o); } /** * Return true iff this string is greater than given string. */ bool operator>(const S2bDnaString& o) { return sstr_gt(*this, o); } /** * Return true iff this string is less than or equal to given string. */ bool operator<=(const S2bDnaString& o) { return sstr_leq(*this, o); } /** * Return true iff this string is greater than or equal to given string. */ bool operator>=(const S2bDnaString& o) { return sstr_geq(*this, o); } /** * Reverse the 2-bit encoded DNA string in-place. */ void reverse() { if(len_ <= 1) return; size_t wordf = nwords()-1; size_t bpf = (len_-1) & 15; size_t wordi = 0; size_t bpi = 0; while(wordf > wordi || (wordf == wordi && bpf > bpi)) { int f = (cs_[wordf] >> (bpf << 1)) & 3; int i = (cs_[wordi] >> (bpi << 1)) & 3; cs_[wordf] &= ~(uint32_t)(3 << (bpf << 1)); cs_[wordi] &= ~(uint32_t)(3 << (bpi << 1)); cs_[wordf] |= (uint32_t)(i << (bpf << 1)); cs_[wordi] |= (uint32_t)(f << (bpi << 1)); if(bpf == 0) { bpf = 15; wordf--; } else bpf--; if(bpi == 15) { bpi = 0; wordi++; } else bpi++; } } /** * Reverse a substring of the buffer in place. */ void reverseWindow(size_t off, size_t len) { assert_leq(off, len_); assert_leq(off+len, len_); if(len <= 1) return; size_t wordf = (off+len-1) >> 4; size_t bpf = (off+len-1) & 15; size_t wordi = (off ) >> 4; size_t bpi = (off ) & 15; while(wordf > wordi || (wordf == wordi && bpf > bpi)) { int f = (cs_[wordf] >> (bpf << 1)) & 3; int i = (cs_[wordi] >> (bpi << 1)) & 3; cs_[wordf] &= ~(uint32_t)(3 << (bpf << 1)); cs_[wordi] &= ~(uint32_t)(3 << (bpi << 1)); cs_[wordf] |= (uint32_t)(i << (bpf << 1)); cs_[wordi] |= (uint32_t)(f << (bpi << 1)); if(bpf == 0) { bpf = 15; wordf--; } else bpf--; if(bpi == 15) { bpi = 0; wordi++; } else bpi++; } } /** * Set the first len elements of the buffer to el. */ void fill(size_t len, char el) { assert_leq(len, len_); assert_range(0, 3, (int)el); size_t word = 0; if(len > 32) { // Copy el throughout block uint32_t bl = (uint32_t)el; bl |= (bl << 2); bl |= (bl << 4); bl |= (bl << 8); bl |= (bl << 16); // Fill with blocks size_t blen = len >> 4; for(; word < blen; word++) { cs_[word] = bl; } len = len & 15; } size_t bp = 0; for(size_t i = 0; i < len; i++) { cs_[word] &= ~(uint32_t)(3 << (bp << 1)); cs_[word] |= (uint32_t)(el << (bp << 1)); if(bp == 15) { word++; bp = 0; } else bp++; } } /** * Set all elements of the buffer to el. */ void fill(char el) { fill(len_, el); } /** * Return the ith character in the window defined by fw, color, depth and * len. */ char windowGetDna( size_t i, bool fw, bool color, size_t depth = 0, size_t len = 0) const { if(len == 0) len = len_; assert_lt(i, len); assert_leq(len, len_ - depth); if(fw) { return get(depth+i); } else { return color ? get(depth+len-i-1) : compDna(get(depth+len-i-1)); } } /** * Fill the given DNA buffer with the substring specified by fw, * color, depth and len. */ template void windowGetDna( T& buf, bool fw, bool color, size_t depth = 0, size_t len = 0) const { if(len == 0) len = len_; assert_leq(len, len_ - depth); buf.resize(len); for(size_t i = 0; i < len; i++) { buf.set( (fw ? get(depth+i) : (color ? get(depth+len-i-1) : compDna(get(depth+len-i-1)))), i); } } /** * Return the length of the string. */ inline size_t length() const { return len_; } /** * Clear the buffer. */ void clear() { len_ = 0; } /** * Return true iff the buffer is empty. */ inline bool empty() const { return len_ == 0; } /** * Return a const version of the raw buffer. */ const uint32_t* buf() const { return cs_; } /** * Return a writeable version of the raw buffer. */ uint32_t* wbuf() { return cs_; } /** * Note: the size of the string once it's stored in the print buffer is 4 * times as large as the string as stored in compact 2-bit-per-char words. */ const char* toZBuf() const { if(printcs_ == NULL) { const_cast(printcs_) = new char[len_+1]; } char *printcs = const_cast(printcs_); size_t word = 0, bp = 0; for(size_t i = 0; i < len_; i++) { int c = (cs_[word] >> (bp << 1)) & 3; printcs[i] = "ACGT"[c]; if(bp == 15) { word++; bp = 0; } else bp++; } printcs[len_] = '\0'; return printcs_; } protected: uint32_t *cs_; // 2-bit packed words char *printcs_; size_t len_; // # elements }; /** * Simple string class with backing memory that automatically expands as needed. */ template class SStringExpandable { public: explicit SStringExpandable() : cs_(NULL), printcs_(NULL), len_(0), sz_(0) { } explicit SStringExpandable(size_t sz) : cs_(NULL), printcs_(NULL), len_(0), sz_(0) { expandNoCopy(sz); } /** * Create an SStringExpandable from another SStringExpandable. */ SStringExpandable(const SStringExpandable& o) : cs_(NULL), printcs_(NULL), len_(0), sz_(0) { *this = o; } /** * Create an SStringExpandable from a std::basic_string of the * appropriate type. */ explicit SStringExpandable(const std::basic_string& str) : cs_(NULL), printcs_(NULL), len_(0), sz_(0) { install(str.c_str(), str.length()); } /** * Create an SStringExpandable from an array and size. */ explicit SStringExpandable(const T* b, size_t sz) : cs_(NULL), printcs_(NULL), len_(0), sz_(0) { install(b, sz); } /** * Create an SStringExpandable from a zero-terminated array. */ explicit SStringExpandable(const T* b) : cs_(NULL), printcs_(NULL), len_(0), sz_(0) { install(b, strlen(b)); } /** * Destroy the expandable string object. */ virtual ~SStringExpandable() { if(cs_ != NULL) { delete[] cs_; cs_ = NULL; } if(printcs_ != NULL) { delete[] printcs_; printcs_ = NULL; } sz_ = len_ = 0; } /** * Return ith character from the left of either the forward or the * reverse-complement version of the read. */ T windowGet( size_t i, bool fw, size_t depth = 0, size_t len = 0) const { if(len == 0) len = len_; assert_lt(i, len); assert_leq(len, len_ - depth); return fw ? cs_[depth+i] : cs_[depth+len-i-1]; } /** * Return ith character from the left of either the forward or the * reverse-complement version of the read. */ void windowGet( T& ret, bool fw, size_t depth = 0, size_t len = 0) const { if(len == 0) len = len_; assert_leq(len, len_ - depth); for(size_t i = 0; i < len; i++) { ret.append(fw ? cs_[depth+i] : cs_[depth+len-i-1]); } } /** * Assignment to other SStringFixed. */ SStringExpandable& operator=(const SStringExpandable& o) { install(o.cs_, o.len_); return *this; } /** * Assignment from a std::basic_string */ SStringExpandable& operator=(const std::basic_string& o) { install(o.c_str(), o.length()); return *this; } /** * Insert char c before position 'idx'; slide subsequent chars down. */ void insert(const T& c, size_t idx) { assert_lt(idx, len_); if(sz_ < len_ + 1) expandCopy((len_ + 1 + S) * M); len_++; // Move everyone down by 1 // len_ is the *new* length for(size_t i = len_; i > idx+1; i--) { cs_[i-1] = cs_[i-2]; } cs_[idx] = c; } /** * Set character at index 'idx' to 'c'. */ void set(int c, size_t idx) { assert_lt(idx, len_); cs_[idx] = c; } /** * Append char c. */ void append(const T& c) { if(sz_ < len_ + 1) expandCopy((len_ + 1 + S) * M); cs_[len_++] = c; } /** * Delete char at position 'idx'; slide subsequent chars up. */ void remove(size_t idx) { assert_lt(idx, len_); assert_gt(len_, 0); for(size_t i = idx; i < len_-1; i++) { cs_[i] = cs_[i+1]; } len_--; } /** * Retrieve constant version of element i. */ const T& operator[](size_t i) const { assert_lt(i, len_); return cs_[i]; } /** * Retrieve mutable version of element i. */ T& operator[](size_t i) { assert_lt(i, len_); return cs_[i]; } /** * Retrieve constant version of element i. */ const T& get(size_t i) const { assert_lt(i, len_); return cs_[i]; } /** * Copy 'sz' bytes from buffer 'b' into this string. */ virtual void install(const T* b, size_t sz) { if(sz_ < sz) expandNoCopy((sz + S) * M); memcpy(cs_, b, sz * sizeof(T)); len_ = sz; } /** * Copy all bytes from zero-terminated buffer 'b' into this string. */ void install(const T* b) { install(b, strlen(b)); } /** * Copy 'sz' bytes from buffer 'b' into this string, reversing them * in the process. */ void installReverse(const char* b, size_t sz) { if(sz_ < sz) expandNoCopy((sz + S) * M); for(size_t i = 0; i < sz; i++) { cs_[i] = b[sz-i-1]; } len_ = sz; } /** * Copy 'sz' bytes from buffer 'b' into this string, reversing them * in the process. */ void installReverse(const SStringExpandable& b) { if(sz_ < b.len_) expandNoCopy((b.len_ + S) * M); for(size_t i = 0; i < b.len_; i++) { cs_[i] = b.cs_[b.len_ - i - 1]; } len_ = b.len_; } /** * Return true iff the two strings are equal. */ bool operator==(const SStringExpandable& o) { return sstr_eq(*this, o); } /** * Return true iff the two strings are not equal. */ bool operator!=(const SStringExpandable& o) { return sstr_neq(*this, o); } /** * Return true iff this string is less than given string. */ bool operator<(const SStringExpandable& o) { return sstr_lt(*this, o); } /** * Return true iff this string is greater than given string. */ bool operator>(const SStringExpandable& o) { return sstr_gt(*this, o); } /** * Return true iff this string is less than or equal to given string. */ bool operator<=(const SStringExpandable& o) { return sstr_leq(*this, o); } /** * Return true iff this string is greater than or equal to given string. */ bool operator>=(const SStringExpandable& o) { return sstr_geq(*this, o); } /** * Reverse the buffer in place. */ void reverse() { for(size_t i = 0; i < (len_ >> 1); i++) { T tmp = get(i); set(get(len_-i-1), i); set(tmp, len_-i-1); } } /** * Reverse a substring of the buffer in place. */ void reverseWindow(size_t off, size_t len) { assert_leq(off, len_); assert_leq(off + len, len_); size_t mid = len >> 1; for(size_t i = 0; i < mid; i++) { T tmp = get(off+i); set(get(off+len-i-1), off+i); set(tmp, off+len-i-1); } } /** * Simply resize the buffer. If the buffer is resized to be * longer, the newly-added elements will contain garbage and should * be initialized immediately. */ void resize(size_t len) { if(sz_ < len) expandCopy((len + S) * M); len_ = len; } /** * Simply resize the buffer. If the buffer is resized to be * longer, new elements will be initialized with 'el'. */ void resize(size_t len, const T& el) { if(sz_ < len) expandCopy((len + S) * M); if(len > len_) { for(size_t i = len_; i < len; i++) { cs_[i] = el; } } len_ = len; } /** * Set the first len elements of the buffer to el. */ void fill(size_t len, const T& el) { assert_leq(len, len_); for(size_t i = 0; i < len; i++) { cs_[i] = el; } } /** * Set all elements of the buffer to el. */ void fill(const T& el) { fill(len_, el); } /** * Trim len characters from the beginning of the string. */ void trimBegin(size_t len) { assert_leq(len, len_); if(len == len_) { len_ = 0; return; } for(size_t i = 0; i < len_-len; i++) { cs_[i] = cs_[i+len]; } len_ -= len; } /** * Trim len characters from the end of the string. */ void trimEnd(size_t len) { if(len >= len_) len_ = 0; else len_ -= len; } /** * Copy 'sz' bytes from buffer 'b' into this string. */ void append(const T* b, size_t sz) { if(sz_ < len_ + sz) expandCopy((len_ + sz + S) * M); memcpy(cs_ + len_, b, sz * sizeof(T)); len_ += sz; } /** * Copy bytes from zero-terminated buffer 'b' into this string. */ void append(const T* b) { append(b, strlen(b)); } /** * Return the length of the string. */ size_t length() const { return len_; } /** * Clear the buffer. */ void clear() { len_ = 0; } /** * Return true iff the buffer is empty. */ bool empty() const { return len_ == 0; } /** * Put a terminator in the 'len_'th element and then return a * pointer to the buffer. Useful for printing. */ const char* toZBufXForm(const char *xform) const { ASSERT_ONLY(size_t xformElts = strlen(xform)); if(empty()) { const_cast(zero_) = 0; return &zero_; } char* printcs = const_cast(printcs_); // Lazily allocate space for print buffer for(size_t i = 0; i < len_; i++) { assert_lt(cs_[i], (int)xformElts); printcs[i] = xform[(int)cs_[i]]; } printcs[len_] = 0; return printcs_; } /** * Put a terminator in the 'len_'th element and then return a * pointer to the buffer. Useful for printing. */ virtual const T* toZBuf() const { if(empty()) { const_cast(zeroT_) = 0; return &zeroT_; } assert_leq(len_, sz_); const_cast(cs_)[len_] = 0; return cs_; } /** * Return true iff this DNA string matches the given nucleotide * character string. */ bool eq(const char *str) const { const char *self = toZBuf(); return strcmp(str, self) == 0; } /** * Return a const version of the raw buffer. */ const T* buf() const { return cs_; } /** * Return a writeable version of the raw buffer. */ T* wbuf() { return cs_; } protected: /** * Allocate new, bigger buffer and copy old contents into it. If * requested size can be accommodated by current buffer, do nothing. */ void expandCopy(size_t sz) { if(sz_ >= sz) return; // done! T *tmp = new T[sz + 1]; char *ptmp = new char[sz + 1]; if(cs_ != NULL) { memcpy(tmp, cs_, sizeof(T)*len_); delete[] cs_; } if(printcs_ != NULL) { memcpy(ptmp, printcs_, sizeof(char)*len_); delete[] printcs_; } cs_ = tmp; printcs_ = ptmp; sz_ = sz; } /** * Allocate new, bigger buffer. If requested size can be * accommodated by current buffer, do nothing. */ void expandNoCopy(size_t sz) { if(sz_ >= sz) return; // done! if(cs_ != NULL) delete[] cs_; if(printcs_ != NULL) delete[] printcs_; cs_ = new T[sz + 1]; printcs_ = new char[sz + 1]; sz_ = sz; } T *cs_; // +1 so that we have the option of dropping in a terminating "\0" char *printcs_; // +1 so that we have the option of dropping in a terminating "\0" char zero_; // 0 terminator for empty string T zeroT_; // 0 terminator for empty string size_t len_; // # filled-in elements size_t sz_; // size capacity of cs_ }; /** * Simple string class with in-object storage. * * All copies induced by, e.g., operator=, the copy constructor, * install() and append(), are shallow (using memcpy/sizeof). If deep * copies are needed, use a different class. * * Reading from an uninitialized element results in an assert as long * as NDEBUG is not defined. If NDEBUG is defined, the result is * undefined. */ template class SStringFixed { public: explicit SStringFixed() : len_(0) { } /** * Create an SStringFixed from another SStringFixed. */ SStringFixed(const SStringFixed& o) { *this = o; } /** * Create an SStringFixed from another SStringFixed. */ explicit SStringFixed(const std::basic_string& str) { install(str.c_str(), str.length()); } /** * Create an SStringFixed from an array and size. */ explicit SStringFixed(const T* b, size_t sz) { install(b, sz); } /** * Create an SStringFixed from a zero-terminated string. */ explicit SStringFixed(const T* b) { install(b, strlen(b)); } virtual ~SStringFixed() { } // C++ needs this /** * Retrieve constant version of element i. */ inline const T& operator[](size_t i) const { return get(i); } /** * Retrieve mutable version of element i. */ inline T& operator[](size_t i) { return get(i); } /** * Retrieve constant version of element i. */ inline const T& get(size_t i) const { assert_lt(i, len_); return cs_[i]; } /** * Retrieve mutable version of element i. */ inline T& get(size_t i) { assert_lt(i, len_); return cs_[i]; } /** * Return ith character from the left of either the forward or the * reverse-complement version of the read. */ T windowGet( size_t i, bool fw, size_t depth = 0, size_t len = 0) const { if(len == 0) len = len_; assert_lt(i, len); assert_leq(len, len_ - depth); return fw ? cs_[depth+i] : cs_[depth+len-i-1]; } /** * Return ith character from the left of either the forward or the * reverse-complement version of the read. */ void windowGet( T& ret, bool fw, size_t depth = 0, size_t len = 0) const { if(len == 0) len = len_; assert_leq(len, len_ - depth); for(size_t i = 0; i < len; i++) { ret.append(fw ? cs_[depth+i] : cs_[depth+len-i-1]); } } /** * Assignment to other SStringFixed. */ SStringFixed& operator=(const SStringFixed& o) { install(o.cs_, o.len_); return *this; } /** * Assignment from a std::basic_string */ SStringFixed& operator=(const std::basic_string& o) { install(o); return *this; } /** * Insert char c before position 'idx'; slide subsequent chars down. */ void insert(const T& c, size_t idx) { assert_lt(len_, S); assert_lt(idx, len_); // Move everyone down by 1 for(int i = len_; i > idx; i--) { cs_[i] = cs_[i-1]; } cs_[idx] = c; len_++; } /** * Set character at index 'idx' to 'c'. */ void set(int c, size_t idx) { assert_lt(idx, len_); cs_[idx] = c; } /** * Append char c. */ void append(const T& c) { assert_lt(len_, S); cs_[len_++] = c; } /** * Delete char at position 'idx'; slide subsequent chars up. */ void remove(size_t idx) { assert_lt(idx, len_); assert_gt(len_, 0); for(size_t i = idx; i < len_-1; i++) { cs_[i] = cs_[i+1]; } len_--; } /** * Copy 'sz' bytes from buffer 'b' into this string. */ virtual void install(const T* b, size_t sz) { assert_leq(sz, S); memcpy(cs_, b, sz * sizeof(T)); len_ = sz; } /** * Copy all bytes from zero-terminated buffer 'b' into this string. */ void install(const T* b) { install(b, strlen(b)); } /** * Copy 'sz' bytes from buffer 'b' into this string, reversing them * in the process. */ void installReverse(const char* b, size_t sz) { assert_leq(sz, S); for(size_t i = 0; i < sz; i++) { cs_[i] = b[sz-i-1]; } len_ = sz; } /** * Copy 'sz' bytes from buffer 'b' into this string, reversing them * in the process. */ void installReverse(const SStringFixed& b) { assert_leq(b.len_, S); for(size_t i = 0; i < b.len_; i++) { cs_[i] = b.cs_[b.len_ - i - 1]; } len_ = b.len_; } /** * Return true iff the two strings are equal. */ bool operator==(const SStringFixed& o) { return sstr_eq(*this, o); } /** * Return true iff the two strings are not equal. */ bool operator!=(const SStringFixed& o) { return sstr_neq(*this, o); } /** * Return true iff this string is less than given string. */ bool operator<(const SStringFixed& o) { return sstr_lt(*this, o); } /** * Return true iff this string is greater than given string. */ bool operator>(const SStringFixed& o) { return sstr_gt(*this, o); } /** * Return true iff this string is less than or equal to given string. */ bool operator<=(const SStringFixed& o) { return sstr_leq(*this, o); } /** * Return true iff this string is greater than or equal to given string. */ bool operator>=(const SStringFixed& o) { return sstr_geq(*this, o); } /** * Reverse the buffer in place. */ void reverse() { for(size_t i = 0; i < (len_ >> 1); i++) { T tmp = get(i); set(get(len_-i-1), i); set(tmp, len_-i-1); } } /** * Reverse a substring of the buffer in place. */ void reverseWindow(size_t off, size_t len) { assert_leq(off, len_); assert_leq(off + len, len_); size_t mid = len >> 1; for(size_t i = 0; i < mid; i++) { T tmp = get(off+i); set(get(off+len-i-1), off+i); set(tmp, off+len-i-1); } } /** * Simply resize the buffer. If the buffer is resized to be * longer, the newly-added elements will contain garbage and should * be initialized immediately. */ void resize(size_t len) { assert_lt(len, S); len_ = len; } /** * Simply resize the buffer. If the buffer is resized to be * longer, new elements will be initialized with 'el'. */ void resize(size_t len, const T& el) { assert_lt(len, S); if(len > len_) { for(size_t i = len_; i < len; i++) { cs_[i] = el; } } len_ = len; } /** * Set the first len elements of the buffer to el. */ void fill(size_t len, const T& el) { assert_leq(len, len_); for(size_t i = 0; i < len; i++) { cs_[i] = el; } } /** * Set all elements of the buffer to el. */ void fill(const T& el) { fill(len_, el); } /** * Trim len characters from the beginning of the string. */ void trimBegin(size_t len) { assert_leq(len, len_); if(len == len_) { len_ = 0; return; } for(size_t i = 0; i < len_-len; i++) { cs_[i] = cs_[i+len]; } len_ -= len; } /** * Trim len characters from the end of the string. */ void trimEnd(size_t len) { if(len >= len_) len_ = 0; else len_ -= len; } /** * Copy 'sz' bytes from buffer 'b' into this string. */ void append(const T* b, size_t sz) { assert_leq(sz + len_, S); memcpy(cs_ + len_, b, sz * sizeof(T)); len_ += sz; } /** * Copy bytes from zero-terminated buffer 'b' into this string. */ void append(const T* b) { append(b, strlen(b)); } /** * Return the length of the string. */ size_t length() const { return len_; } /** * Clear the buffer. */ void clear() { len_ = 0; } /** * Return true iff the buffer is empty. */ bool empty() const { return len_ == 0; } /** * Put a terminator in the 'len_'th element and then return a * pointer to the buffer. Useful for printing. */ virtual const T* toZBuf() const { const_cast(cs_)[len_] = 0; return cs_; } /** * Return true iff this DNA string matches the given nucleotide * character string. */ bool eq(const char *str) const { const char *self = toZBuf(); return strcmp(str, self) == 0; } /** * Put a terminator in the 'len_'th element and then return a * pointer to the buffer. Useful for printing. */ const char* toZBufXForm(const char *xform) const { ASSERT_ONLY(size_t xformElts = strlen(xform)); char* printcs = const_cast(printcs_); for(size_t i = 0; i < len_; i++) { assert_lt(cs_[i], (int)xformElts); printcs[i] = xform[cs_[i]]; } printcs[len_] = 0; return printcs_; } /** * Return a const version of the raw buffer. */ const T* buf() const { return cs_; } /** * Return a writeable version of the raw buffer. */ T* wbuf() { return cs_; } protected: T cs_[S+1]; // +1 so that we have the option of dropping in a terminating "\0" char printcs_[S+1]; // +1 so that we have the option of dropping in a terminating "\0" size_t len_; }; // // Stream put operators // template std::ostream& operator<< (std::ostream& os, const SStringExpandable& str) { os << str.toZBuf(); return os; } template std::ostream& operator<< (std::ostream& os, const SStringFixed& str) { os << str.toZBuf(); return os; } extern uint8_t asc2dna[]; extern uint8_t asc2col[]; /** * Encapsulates a fixed-length DNA string with characters encoded as * chars. Only capable of encoding A, C, G, T and N. The length is * specified via the template parameter S. */ template class SDnaStringFixed : public SStringFixed { public: explicit SDnaStringFixed() : SStringFixed() { } /** * Create an SStringFixed from another SStringFixed. */ SDnaStringFixed(const SDnaStringFixed& o) : SStringFixed(o) { } /** * Create an SStringFixed from a C++ basic_string. */ explicit SDnaStringFixed(const std::basic_string& str) : SStringFixed(str) { } /** * Create an SStringFixed from an array and size. */ explicit SDnaStringFixed(const char* b, size_t sz) : SStringFixed(b, sz) { } /** * Create an SStringFixed from a zero-terminated string. */ explicit SDnaStringFixed( const char* b, bool chars = false, bool colors = false) : SStringFixed() { if(chars) { if(colors) { installColors(b, strlen(b)); } else { installChars(b, strlen(b)); } } else { install(b, strlen(b)); } } virtual ~SDnaStringFixed() { } // C++ needs this /** * Copy 'sz' bytes from buffer 'b' into this string, reverse- * complementing them in the process, assuming an encoding where * 0=A, 1=C, 2=G, 3=T, 4=N. */ void installReverseComp(const char* b, size_t sz) { assert_leq(sz, S); for(size_t i = 0; i < sz; i++) { this->cs_[i] = (b[sz-i-1] == 4 ? 4 : b[sz-i-1] ^ 3); } this->len_ = sz; } /** * Copy 'sz' bytes from buffer 'b' into this string, reverse- * complementing them in the process, assuming an encoding where * 0=A, 1=C, 2=G, 3=T, 4=N. */ void installReverseComp(const SDnaStringFixed& b) { assert_leq(b.len_, S); for(size_t i = 0; i < b.len_; i++) { this->cs_[i] = (b.cs_[b.len_-i-1] == 4 ? 4 : b.cs_[b.len_-i-1] ^ 3); } this->len_ = b.len_; } /** * Either reverse or reverse-complement (depending on "color") this * DNA buffer in-place. */ void reverseComp(bool color = false) { if(color) { this->reverse(); } else { for(size_t i = 0; i < (this->len_ >> 1); i++) { char tmp1 = (this->cs_[i] == 4 ? 4 : this->cs_[i] ^ 3); char tmp2 = (this->cs_[this->len_-i-1] == 4 ? 4 : this->cs_[this->len_-i-1] ^ 3); this->cs_[i] = tmp2; this->cs_[this->len_-i-1] = tmp1; } // Do middle element iff there are an odd number if((this->len_ & 1) != 0) { char tmp = this->cs_[this->len_ >> 1]; tmp = (tmp == 4 ? 4 : tmp ^ 3); this->cs_[this->len_ >> 1] = tmp; } } } /** * Copy 'sz' bytes from buffer 'b' into this string. */ virtual void install(const char* b, size_t sz) { assert_leq(sz, S); memcpy(this->cs_, b, sz); #ifndef NDEBUG for(size_t i = 0; i < sz; i++) { assert_leq(this->cs_[i], 4); assert_geq(this->cs_[i], 0); } #endif this->len_ = sz; } /** * Copy buffer 'b' of ASCII DNA characters into normal DNA * characters. */ virtual void installChars(const char* b, size_t sz) { assert_leq(sz, S); for(size_t i = 0; i < sz; i++) { assert_in(toupper(b[i]), "ACGTN-"); this->cs_[i] = asc2dna[(int)b[i]]; assert_geq(this->cs_[i], 0); assert_leq(this->cs_[i], 4); } this->len_ = sz; } /** * Copy buffer 'b' of ASCII color characters into normal DNA * characters. */ virtual void installColors(const char* b, size_t sz) { assert_leq(sz, S); for(size_t i = 0; i < sz; i++) { assert_in(b[i], "0123."); this->cs_[i] = asc2col[(int)b[i]]; assert_geq(this->cs_[i], 0); assert_leq(this->cs_[i], 4); } this->len_ = sz; } /** * Copy C++ string of ASCII DNA characters into normal DNA * characters. */ virtual void installChars(const std::basic_string& str) { installChars(str.c_str(), str.length()); } /** * Copy C++ string of ASCII color characters into normal DNA * characters. */ virtual void installColors(const std::basic_string& str) { installColors(str.c_str(), str.length()); } /** * Set DNA character at index 'idx' to 'c'. */ void set(int c, size_t idx) { assert_lt(idx, this->len_); assert_leq(c, 4); assert_geq(c, 0); this->cs_[idx] = c; } /** * Append DNA char c. */ void append(const char& c) { assert_lt(this->len_, S); assert_leq(c, 4); assert_geq(c, 0); this->cs_[this->len_++] = c; } /** * Set DNA character at index 'idx' to 'c'. */ void setChar(char c, size_t idx) { assert_lt(idx, this->len_); assert_in(toupper(c), "ACGTN"); this->cs_[idx] = asc2dna[(int)c]; } /** * Append DNA character. */ void appendChar(char c) { assert_lt(this->len_, S); assert_in(toupper(c), "ACGTN"); this->cs_[this->len_++] = asc2dna[(int)c]; } /** * Return DNA character corresponding to element 'idx'. */ char toChar(size_t idx) const { assert_geq((int)this->cs_[idx], 0); assert_leq((int)this->cs_[idx], 4); return "ACGTN"[(int)this->cs_[idx]]; } /** * Retrieve constant version of element i. */ const char& operator[](size_t i) const { return this->get(i); } /** * Retrieve constant version of element i. */ const char& get(size_t i) const { assert_lt(i, this->len_); assert_leq(this->cs_[i], 4); assert_geq(this->cs_[i], 0); return this->cs_[i]; } /** * Return the ith character in the window defined by fw, color, * depth and len. */ char windowGetDna( size_t i, bool fw, bool color, size_t depth = 0, size_t len = 0) const { if(len == 0) len = this->len_; assert_lt(i, len); assert_leq(len, this->len_ - depth); if(fw) return this->cs_[depth+i]; else return color ? this->cs_[depth+len-i-1] : compDna(this->cs_[depth+len-i-1]); } /** * Fill the given DNA buffer with the substring specified by fw, * color, depth and len. */ void windowGetDna( SDnaStringFixed& buf, bool fw, bool color, size_t depth = 0, size_t len = 0) const { if(len == 0) len = this->len_; assert_leq(len, this->len_ - depth); for(size_t i = 0; i < len; i++) { buf.append(fw ? this->cs_[depth+i] : (color ? this->cs_[depth+len-i-1] : compDna(this->cs_[depth+len-i-1]))); } } /** * Put a terminator in the 'len_'th element and then return a * pointer to the buffer. Useful for printing. */ virtual const char* toZBuf() const { return this->toZBufXForm("ACGTN"); } }; /** * Encapsulates a fixed-length DNA string with characters encoded as * chars. Only capable of encoding A, C, G, T and N. The length is * specified via the template parameter S. */ template class SDnaStringExpandable : public SStringExpandable { public: explicit SDnaStringExpandable() : SStringExpandable() { } /** * Create an SStringFixed from another SStringFixed. */ SDnaStringExpandable(const SDnaStringExpandable& o) : SStringExpandable(o) { } /** * Create an SStringFixed from a C++ basic_string. */ explicit SDnaStringExpandable( const std::basic_string& str, bool chars = false, bool colors = false) : SStringExpandable() { if(chars) { if(colors) { installColors(str); } else { installChars(str); } } else { install(str); } } /** * Create an SStringFixed from an array and size. */ explicit SDnaStringExpandable( const char* b, size_t sz, bool chars = false, bool colors = false) : SStringExpandable() { if(chars) { if(colors) { installColors(b, sz); } else { installChars(b, sz); } } else { install(b, sz); } } /** * Create an SStringFixed from a zero-terminated string. */ explicit SDnaStringExpandable( const char* b, bool chars = false, bool colors = false) : SStringExpandable() { install(b, chars, colors); } virtual ~SDnaStringExpandable() { } // C++ needs this /** * Copy 'sz' bytes from buffer 'b' into this string, reverse- * complementing them in the process, assuming an encoding where * 0=A, 1=C, 2=G, 3=T, 4=N. */ void installReverseComp(const char* b, size_t sz) { if(this->sz_ < sz) this->expandCopy((sz + S) * M); for(size_t i = 0; i < sz; i++) { this->cs_[i] = (b[sz-i-1] == 4 ? 4 : b[sz-i-1] ^ 3); } this->len_ = sz; } /** * Copy 'sz' bytes from buffer 'b' into this string, reverse- * complementing them in the process, assuming an encoding where * 0=A, 1=C, 2=G, 3=T, 4=N. */ void installReverseComp(const SDnaStringExpandable& b) { if(this->sz_ < b.len_) this->expandCopy((b.len_ + S) * M); for(size_t i = 0; i < b.len_; i++) { this->cs_[i] = (b.cs_[b.len_-i-1] == 4 ? 4 : b.cs_[b.len_-i-1] ^ 3); } this->len_ = b.len_; } /** * Either reverse or reverse-complement (depending on "color") this * DNA buffer in-place. */ void reverseComp(bool color = false) { if(color) { this->reverse(); } else { for(size_t i = 0; i < (this->len_ >> 1); i++) { char tmp1 = (this->cs_[i] == 4 ? 4 : this->cs_[i] ^ 3); char tmp2 = (this->cs_[this->len_-i-1] == 4 ? 4 : this->cs_[this->len_-i-1] ^ 3); this->cs_[i] = tmp2; this->cs_[this->len_-i-1] = tmp1; } // Do middle element iff there are an odd number if((this->len_ & 1) != 0) { char tmp = this->cs_[this->len_ >> 1]; tmp = (tmp == 4 ? 4 : tmp ^ 3); this->cs_[this->len_ >> 1] = tmp; } } } /** * Copy 'sz' bytes from buffer 'b' into this string. */ virtual void install( const char* b, bool chars = false, bool colors = false) { if(chars) { if(colors) { installColors(b, strlen(b)); } else { installChars(b, strlen(b)); } } else { install(b, strlen(b)); } } /** * Copy 'sz' bytes from buffer 'b' into this string. */ virtual void install(const char* b, size_t sz) { if(this->sz_ < sz) this->expandCopy((sz + S) * M); memcpy(this->cs_, b, sz); #ifndef NDEBUG for(size_t i = 0; i < sz; i++) { assert_range(0, 4, (int)this->cs_[i]); } #endif this->len_ = sz; } /** * Copy buffer 'b' of ASCII DNA characters into normal DNA * characters. */ virtual void installChars(const char* b, size_t sz) { if(this->sz_ < sz) this->expandCopy((sz + S) * M); for(size_t i = 0; i < sz; i++) { assert_in(toupper(b[i]), "ACGTN-"); this->cs_[i] = asc2dna[(int)b[i]]; assert_range(0, 4, (int)this->cs_[i]); } this->len_ = sz; } /** * Copy buffer 'b' of ASCII color characters into normal DNA * characters. */ virtual void installColors(const char* b, size_t sz) { if(this->sz_ < sz) this->expandCopy((sz + S) * M); for(size_t i = 0; i < sz; i++) { assert_in(b[i], "0123."); this->cs_[i] = asc2col[(int)b[i]]; assert_range(0, 4, (int)this->cs_[i]); } this->len_ = sz; } /** * Copy C++ string of ASCII DNA characters into normal DNA * characters. */ virtual void installChars(const std::basic_string& str) { installChars(str.c_str(), str.length()); } /** * Copy C++ string of ASCII color characters into normal DNA * characters. */ virtual void installColors(const std::basic_string& str) { installColors(str.c_str(), str.length()); } /** * Set DNA character at index 'idx' to 'c'. */ void set(int c, size_t idx) { assert_lt(idx, this->len_); assert_range(0, 4, c); this->cs_[idx] = c; } /** * Append DNA char c. */ void append(const char& c) { if(this->sz_ < this->len_ + 1) { this->expandCopy((this->len_ + 1 + S) * M); } assert_range(0, 4, (int)c); this->cs_[this->len_++] = c; } /** * Set DNA character at index 'idx' to 'c'. */ void setChar(char c, size_t idx) { assert_lt(idx, this->len_); assert_in(toupper(c), "ACGTN"); this->cs_[idx] = asc2dna[(int)c]; } /** * Append DNA character. */ void appendChar(char c) { if(this->sz_ < this->len_ + 1) { this->expandCopy((this->len_ + 1 + S) * M); } assert_in(toupper(c), "ACGTN"); this->cs_[this->len_++] = asc2dna[(int)c]; } /** * Return DNA character corresponding to element 'idx'. */ char toChar(size_t idx) const { assert_range(0, 4, (int)this->cs_[idx]); return "ACGTN"[(int)this->cs_[idx]]; } /** * Retrieve constant version of element i. */ inline const char& operator[](size_t i) const { return this->get(i); } /** * Retrieve constant version of element i. */ inline const char& get(size_t i) const { assert_lt(i, this->len_); assert_range(0, 4, (int)this->cs_[i]); return this->cs_[i]; } /** * Return the ith character in the window defined by fw, color, * depth and len. */ char windowGetDna( size_t i, bool fw, bool color, size_t depth = 0, size_t len = 0) const { if(len == 0) len = this->len_; assert_lt(i, len); assert_leq(len, this->len_ - depth); if(fw) return this->cs_[depth+i]; else return color ? this->cs_[depth+len-i-1] : compDna(this->cs_[depth+len-i-1]); } /** * Fill the given DNA buffer with the substring specified by fw, * color, depth and len. */ void windowGetDna( SDnaStringExpandable& buf, bool fw, bool color, size_t depth = 0, size_t len = 0) const { if(len == 0) len = this->len_; assert_leq(len, this->len_ - depth); for(size_t i = 0; i < len; i++) { buf.append(fw ? this->cs_[depth+i] : (color ? this->cs_[depth+len-i-1] : compDna(this->cs_[depth+len-i-1]))); } } /** * Put a terminator in the 'len_'th element and then return a * pointer to the buffer. Useful for printing. */ virtual const char* toZBuf() const { return this->toZBufXForm("ACGTN"); } }; /** * Encapsulates an expandable DNA string with characters encoded as * char-sized masks. Encodes A, C, G, T, and all IUPAC, as well as the * empty mask indicating "matches nothing." */ template class SDnaMaskString : public SStringExpandable { public: explicit SDnaMaskString() : SStringExpandable() { } /** * Create an SStringFixed from another SStringFixed. */ SDnaMaskString(const SDnaMaskString& o) : SStringExpandable(o) { } /** * Create an SStringFixed from a C++ basic_string. */ explicit SDnaMaskString(const std::basic_string& str) : SStringExpandable(str) { } /** * Create an SStringFixed from an array and size. */ explicit SDnaMaskString(const char* b, size_t sz) : SStringExpandable(b, sz) { } /** * Create an SStringFixed from a zero-terminated string. */ explicit SDnaMaskString(const char* b, bool chars = false) : SStringExpandable() { if(chars) { installChars(b, strlen(b)); } else { install(b, strlen(b)); } } virtual ~SDnaMaskString() { } // C++ needs this /** * Copy 'sz' bytes from buffer 'b' into this string, reverse- * complementing them in the process, assuming an encoding where * 0=A, 1=C, 2=G, 3=T, 4=N. */ void installReverseComp(const char* b, size_t sz) { while(this->sz_ < sz) { this->expandNoCopy((sz + S) * M); } for(size_t i = 0; i < sz; i++) { this->cs_[i] = maskcomp[(int)b[sz-i-1]]; } this->len_ = sz; } /** * Copy 'sz' bytes from buffer 'b' into this string, reverse- * complementing them in the process, assuming an encoding where * 0=A, 1=C, 2=G, 3=T, 4=N. */ void installReverseComp(const SDnaMaskString& b) { while(this->sz_ < b.len_) { this->expandNoCopy((b.len_ + S) * M); } for(size_t i = 0; i < b.len_; i++) { this->cs_[i] = maskcomp[(int)b.cs_[b.len_-i-1]]; } this->len_ = b.len_; } /** * Either reverse or reverse-complement (depending on "color") this * DNA buffer in-place. */ void reverseComp(bool color = false) { if(color) { this->reverse(); } else { for(size_t i = 0; i < (this->len_ >> 1); i++) { char tmp1 = maskcomp[(int)this->cs_[i]]; char tmp2 = maskcomp[(int)this->cs_[this->len_-i-1]]; this->cs_[i] = tmp2; this->cs_[this->len_-i-1] = tmp1; } // Do middle element iff there are an odd number if((this->len_ & 1) != 0) { char tmp = this->cs_[this->len_ >> 1]; tmp = maskcomp[(int)tmp]; this->cs_[this->len_ >> 1] = tmp; } } } /** * Copy 'sz' bytes from buffer 'b' into this string. */ virtual void install(const char* b, size_t sz) { while(this->sz_ < sz) { this->expandNoCopy((sz + S) * M); } memcpy(this->cs_, b, sz); #ifndef NDEBUG for(size_t i = 0; i < sz; i++) { assert_range((int)this->cs_[i], 0, 15); } #endif this->len_ = sz; } /** * Copy buffer 'b' of ASCII DNA characters into DNA masks. */ virtual void installChars(const char* b, size_t sz) { while(this->sz_ < sz) { this->expandNoCopy((sz + S) * M); } for(size_t i = 0; i < sz; i++) { assert_in(b[i], iupacs); this->cs_[i] = asc2dnamask[(int)b[i]]; assert_range((int)this->cs_[i], 0, 15); } this->len_ = sz; } /** * Copy C++ string of ASCII DNA characters into normal DNA * characters. */ virtual void installChars(const std::basic_string& str) { installChars(str.c_str(), str.length()); } /** * Set DNA character at index 'idx' to 'c'. */ void set(int c, size_t idx) { assert_lt(idx, this->len_); assert_range(c, 0, 15); this->cs_[idx] = c; } /** * Append DNA char c. */ void append(const char& c) { while(this->sz_ < this->len_+1) { this->expandNoCopy((this->len_ + 1 + S) * M); } assert_range((int)c, 0, 15); this->cs_[this->len_++] = c; } /** * Set DNA character at index 'idx' to 'c'. */ void setChar(char c, size_t idx) { assert_lt(idx, this->len_); assert_in(toupper(c), iupacs); this->cs_[idx] = asc2dnamask[(int)c]; } /** * Append DNA character. */ void appendChar(char c) { while(this->sz_ < this->len_+1) { expandNoCopy((this->len_ + 1 + S) * M); } assert_in(toupper(c), iupacs); this->cs_[this->len_++] = asc2dnamask[(int)c]; } /** * Return DNA character corresponding to element 'idx'. */ char toChar(size_t idx) const { assert_range((int)this->cs_[idx], 0, 15); return mask2iupac[(int)this->cs_[idx]]; } /** * Retrieve constant version of element i. */ const char& operator[](size_t i) const { return this->get(i); } /** * Retrieve mutable version of element i. */ char& operator[](size_t i) { return this->get(i); } /** * Retrieve constant version of element i. */ const char& get(size_t i) const { assert_lt(i, this->len_); assert_range((int)this->cs_[i], 0, 15); return this->cs_[i]; } /** * Retrieve mutable version of element i. */ char& get(size_t i) { assert_lt(i, this->len_); assert_range((int)this->cs_[i], 0, 15); return this->cs_[i]; } /** * Return the ith character in the window defined by fw, color, * depth and len. */ char windowGetDna( size_t i, bool fw, bool color, size_t depth = 0, size_t len = 0) const { if(len == 0) len = this->len_; assert_lt(i, len); assert_leq(len, this->len_ - depth); if(fw) return this->cs_[depth+i]; else return color ? this->cs_[depth+len-i-1] : maskcomp[this->cs_[depth+len-i-1]]; } /** * Fill the given DNA buffer with the substring specified by fw, * color, depth and len. */ void windowGetDna( SDnaStringFixed& buf, bool fw, bool color, size_t depth = 0, size_t len = 0) const { if(len == 0) len = this->len_; assert_leq(len, this->len_ - depth); for(size_t i = 0; i < len; i++) { buf.append(fw ? this->cs_[depth+i] : (color ? this->cs_[depth+len-i-1] : maskcomp[this->cs_[depth+len-i-1]])); } } /** * Sample a random substring of the given length from this DNA * string and install the result in 'dst'. */ template void randSubstr( RandomSource& rnd, // pseudo-random generator T& dst, // put sampled substring here size_t len, // length of substring to extract bool watson = true, // true -> possibly extract from Watson strand bool crick = true) // true -> possibly extract from Crick strand { assert(watson || crick); assert_geq(this->len_, len); size_t poss = this->len_ - len + 1; assert_gt(poss, 0); uint32_t rndoff = (uint32_t)(rnd.nextU32() % poss); bool fw; if (watson && !crick) fw = true; else if(!watson && crick) fw = false; else { fw = rnd.nextBool(); } if(fw) { // Install Watson substring for(size_t i = 0; i < len; i++) { dst[i] = this->cs_[i + rndoff]; } } else { // Install Crick substring for(size_t i = 0; i < len; i++) { dst[i] = maskcomp[(int)this->cs_[i + rndoff + (len - i - 1)]]; } } } /** * Put a terminator in the 'len_'th element and then return a * pointer to the buffer. Useful for printing. */ virtual const char* toZBuf() const { return this->toZBufXForm(iupacs); } }; typedef SStringExpandable BTString; typedef SDnaStringExpandable<1024, 2> BTDnaString; typedef SDnaMaskString<32, 2> BTDnaMask; #endif /* SSTRING_H_ */ hisat2-2.2.1/str_util.h000066400000000000000000000021751370663763200147460ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef STR_UTIL_H_ #define STR_UTIL_H_ #include /** * Given a string, return an int hash for it. */ static inline int hash_string(const std::string& s) { int ret = 0; int a = 63689; int b = 378551; for(size_t i = 0; i < s.length(); i++) { ret = (ret * a) + (int)s[i]; if(a == 0) { a += b; } else { a *= b; } if(a == 0) { a += b; } } return ret; } #endif /* STR_UTIL_H_ */ hisat2-2.2.1/third_party/000077500000000000000000000000001370663763200152545ustar00rootroot00000000000000hisat2-2.2.1/third_party/cpuid.h000066400000000000000000000127251370663763200165400ustar00rootroot00000000000000/* * Copyright (C) 2007, 2008, 2009 Free Software Foundation, Inc. * * This file is free software; you can redistribute it and/or modify it * under the terms of the GNU General Public License as published by the * Free Software Foundation; either version 3, or (at your option) any * later version. * * This file is distributed in the hope that it will be useful, but * WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU * General Public License for more details. * * Under Section 7 of GPL version 3, you are granted additional * permissions described in the GCC Runtime Library Exception, version * 3.1, as published by the Free Software Foundation. * * You should have received a copy of the GNU General Public License and * a copy of the GCC Runtime Library Exception along with this program; * see the files COPYING3 and COPYING.RUNTIME respectively. If not, see * . */ /* %ecx */ #define bit_SSE3 (1 << 0) #define bit_PCLMUL (1 << 1) #define bit_SSSE3 (1 << 9) #define bit_FMA (1 << 12) #define bit_CMPXCHG16B (1 << 13) #define bit_SSE4_1 (1 << 19) #define bit_SSE4_2 (1 << 20) #define bit_MOVBE (1 << 22) #define bit_POPCNT (1 << 23) #define bit_AES (1 << 25) #define bit_XSAVE (1 << 26) #define bit_OSXSAVE (1 << 27) #define bit_AVX (1 << 28) #define bit_F16C (1 << 29) #define bit_RDRND (1 << 30) /* %edx */ #define bit_CMPXCHG8B (1 << 8) #define bit_CMOV (1 << 15) #define bit_MMX (1 << 23) #define bit_FXSAVE (1 << 24) #define bit_SSE (1 << 25) #define bit_SSE2 (1 << 26) /* Extended Features */ /* %ecx */ #define bit_LAHF_LM (1 << 0) #define bit_ABM (1 << 5) #define bit_SSE4a (1 << 6) #define bit_XOP (1 << 11) #define bit_LWP (1 << 15) #define bit_FMA4 (1 << 16) #define bit_TBM (1 << 21) /* %edx */ #define bit_LM (1 << 29) #define bit_3DNOWP (1 << 30) #define bit_3DNOW (1 << 31) /* Extended Features (%eax == 7) */ #define bit_FSGSBASE (1 << 0) #define bit_BMI (1 << 3) #if defined(__i386__) && defined(__PIC__) /* %ebx may be the PIC register. */ #if __GNUC__ >= 3 #define __cpuid(level, a, b, c, d) \ __asm__ ("xchg{l}\t{%%}ebx, %1\n\t" \ "cpuid\n\t" \ "xchg{l}\t{%%}ebx, %1\n\t" \ : "=a" (a), "=r" (b), "=c" (c), "=d" (d) \ : "0" (level)) #define __cpuid_count(level, count, a, b, c, d) \ __asm__ ("xchg{l}\t{%%}ebx, %1\n\t" \ "cpuid\n\t" \ "xchg{l}\t{%%}ebx, %1\n\t" \ : "=a" (a), "=r" (b), "=c" (c), "=d" (d) \ : "0" (level), "2" (count)) #else /* Host GCCs older than 3.0 weren't supporting Intel asm syntax nor alternatives in i386 code. */ #define __cpuid(level, a, b, c, d) \ __asm__ ("xchgl\t%%ebx, %1\n\t" \ "cpuid\n\t" \ "xchgl\t%%ebx, %1\n\t" \ : "=a" (a), "=r" (b), "=c" (c), "=d" (d) \ : "0" (level)) #define __cpuid_count(level, count, a, b, c, d) \ __asm__ ("xchgl\t%%ebx, %1\n\t" \ "cpuid\n\t" \ "xchgl\t%%ebx, %1\n\t" \ : "=a" (a), "=r" (b), "=c" (c), "=d" (d) \ : "0" (level), "2" (count)) #endif #else #define __cpuid(level, a, b, c, d) \ __asm__ ("cpuid\n\t" \ : "=a" (a), "=b" (b), "=c" (c), "=d" (d) \ : "0" (level)) #define __cpuid_count(level, count, a, b, c, d) \ __asm__ ("cpuid\n\t" \ : "=a" (a), "=b" (b), "=c" (c), "=d" (d) \ : "0" (level), "2" (count)) #endif /* Return highest supported input value for cpuid instruction. ext can be either 0x0 or 0x8000000 to return highest supported value for basic or extended cpuid information. Function returns 0 if cpuid is not supported or whatever cpuid returns in eax register. If sig pointer is non-null, then first four bytes of the signature (as found in ebx register) are returned in location pointed by sig. */ static __inline unsigned int __get_cpuid_max (unsigned int __ext, unsigned int *__sig) { unsigned int __eax, __ebx, __ecx, __edx; #ifndef __x86_64__ #if __GNUC__ >= 3 /* See if we can use cpuid. On AMD64 we always can. */ __asm__ ("pushf{l|d}\n\t" "pushf{l|d}\n\t" "pop{l}\t%0\n\t" "mov{l}\t{%0, %1|%1, %0}\n\t" "xor{l}\t{%2, %0|%0, %2}\n\t" "push{l}\t%0\n\t" "popf{l|d}\n\t" "pushf{l|d}\n\t" "pop{l}\t%0\n\t" "popf{l|d}\n\t" : "=&r" (__eax), "=&r" (__ebx) : "i" (0x00200000)); #else /* Host GCCs older than 3.0 weren't supporting Intel asm syntax nor alternatives in i386 code. */ __asm__ ("pushfl\n\t" "pushfl\n\t" "popl\t%0\n\t" "movl\t%0, %1\n\t" "xorl\t%2, %0\n\t" "pushl\t%0\n\t" "popfl\n\t" "pushfl\n\t" "popl\t%0\n\t" "popfl\n\t" : "=&r" (__eax), "=&r" (__ebx) : "i" (0x00200000)); #endif if (!((__eax ^ __ebx) & 0x00200000)) return 0; #endif /* Host supports cpuid. Return highest supported cpuid input value. */ __cpuid (__ext, __eax, __ebx, __ecx, __edx); if (__sig) *__sig = __ebx; return __eax; } /* Return cpuid data for requested cpuid level, as found in returned eax, ebx, ecx and edx registers. The function checks if cpuid is supported and returns 1 for valid cpuid information or 0 for unsupported cpuid level. All pointers are required to be non-null. */ static __inline int __get_cpuid (unsigned int __level, unsigned int *__eax, unsigned int *__ebx, unsigned int *__ecx, unsigned int *__edx) { unsigned int __ext = __level & 0x80000000; if (__get_cpuid_max (__ext, 0) < __level) return 0; __cpuid (__level, *__eax, *__ebx, *__ecx, *__edx); return 1; } hisat2-2.2.1/threading.h000066400000000000000000000025451370663763200150470ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef THREADING_H_ #define THREADING_H_ #include #include "tinythread.h" #include "fast_mutex.h" #ifdef NO_SPINLOCK # define MUTEX_T tthread::mutex #else # define MUTEX_T tthread::fast_mutex #endif /* NO_SPINLOCK */ /** * Wrap a lock; obtain lock upon construction, release upon destruction. */ class ThreadSafe { public: ThreadSafe(MUTEX_T* ptr_mutex, bool locked = true) { if(locked) { this->ptr_mutex = ptr_mutex; ptr_mutex->lock(); } else this->ptr_mutex = NULL; } ~ThreadSafe() { if (ptr_mutex != NULL) ptr_mutex->unlock(); } private: MUTEX_T *ptr_mutex; }; #endif hisat2-2.2.1/timer.h000066400000000000000000000046141370663763200142210ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef TIMER_H_ #define TIMER_H_ #include #include #include #include using namespace std; /** * Use time() call to keep track of elapsed time between creation and * destruction. If verbose is true, Timer will print a message showing * elapsed time to the given output stream upon destruction. */ class Timer { public: Timer(ostream& out = cout, const char *msg = "", bool verbose = true) : _t(time(0)), _out(out), _msg(msg), _verbose(verbose) { } /// Optionally print message ~Timer() { if(_verbose) write(_out); } /// Return elapsed time since Timer object was created time_t elapsed() const { return time(0) - _t; } void write(ostream& out) { time_t passed = elapsed(); // Print the message supplied at construction time followed // by time elapsed formatted HH:MM:SS time_t hours = (passed / 60) / 60; time_t minutes = (passed / 60) % 60; time_t seconds = (passed % 60); std::ostringstream oss; oss << _msg << setfill ('0') << setw (2) << hours << ":" << setfill ('0') << setw (2) << minutes << ":" << setfill ('0') << setw (2) << seconds << endl; out << oss.str().c_str(); } private: time_t _t; ostream& _out; const char *_msg; bool _verbose; }; static inline void logTime(std::ostream& os, bool nl = true) { struct tm *current; time_t now; time(&now); current = localtime(&now); std::ostringstream oss; oss << setfill('0') << setw(2) << current->tm_hour << ":" << setfill('0') << setw(2) << current->tm_min << ":" << setfill('0') << setw(2) << current->tm_sec; if(nl) oss << std::endl; os << oss.str().c_str(); } #endif /*TIMER_H_*/ hisat2-2.2.1/tinythread.cpp000077500000000000000000000217061370663763200156130ustar00rootroot00000000000000/* -*- mode: c++; tab-width: 2; indent-tabs-mode: nil; -*- Copyright (c) 2010-2012 Marcus Geelnard This software is provided 'as-is', without any express or implied warranty. In no event will the authors be held liable for any damages arising from the use of this software. Permission is granted to anyone to use this software for any purpose, including commercial applications, and to alter it and redistribute it freely, subject to the following restrictions: 1. The origin of this software must not be misrepresented; you must not claim that you wrote the original software. If you use this software in a product, an acknowledgment in the product documentation would be appreciated but is not required. 2. Altered source versions must be plainly marked as such, and must not be misrepresented as being the original software. 3. This notice may not be removed or altered from any source distribution. */ #include #include "tinythread.h" #if defined(_TTHREAD_POSIX_) #include #include #elif defined(_TTHREAD_WIN32_) #include #endif namespace tthread { //------------------------------------------------------------------------------ // condition_variable //------------------------------------------------------------------------------ // NOTE 1: The Win32 implementation of the condition_variable class is based on // the corresponding implementation in GLFW, which in turn is based on a // description by Douglas C. Schmidt and Irfan Pyarali: // http://www.cs.wustl.edu/~schmidt/win32-cv-1.html // // NOTE 2: Windows Vista actually has native support for condition variables // (InitializeConditionVariable, WakeConditionVariable, etc), but we want to // be portable with pre-Vista Windows versions, so TinyThread++ does not use // Vista condition variables. //------------------------------------------------------------------------------ #if defined(_TTHREAD_WIN32_) #define _CONDITION_EVENT_ONE 0 #define _CONDITION_EVENT_ALL 1 #endif #if defined(_TTHREAD_WIN32_) condition_variable::condition_variable() : mWaitersCount(0) { mEvents[_CONDITION_EVENT_ONE] = CreateEvent(NULL, FALSE, FALSE, NULL); mEvents[_CONDITION_EVENT_ALL] = CreateEvent(NULL, TRUE, FALSE, NULL); InitializeCriticalSection(&mWaitersCountLock); } #endif #if defined(_TTHREAD_WIN32_) condition_variable::~condition_variable() { CloseHandle(mEvents[_CONDITION_EVENT_ONE]); CloseHandle(mEvents[_CONDITION_EVENT_ALL]); DeleteCriticalSection(&mWaitersCountLock); } #endif #if defined(_TTHREAD_WIN32_) void condition_variable::_wait() { // Wait for either event to become signaled due to notify_one() or // notify_all() being called int result = WaitForMultipleObjects(2, mEvents, FALSE, INFINITE); // Check if we are the last waiter EnterCriticalSection(&mWaitersCountLock); -- mWaitersCount; bool lastWaiter = (result == (WAIT_OBJECT_0 + _CONDITION_EVENT_ALL)) && (mWaitersCount == 0); LeaveCriticalSection(&mWaitersCountLock); // If we are the last waiter to be notified to stop waiting, reset the event if(lastWaiter) ResetEvent(mEvents[_CONDITION_EVENT_ALL]); } #endif #if defined(_TTHREAD_WIN32_) void condition_variable::notify_one() { // Are there any waiters? EnterCriticalSection(&mWaitersCountLock); bool haveWaiters = (mWaitersCount > 0); LeaveCriticalSection(&mWaitersCountLock); // If we have any waiting threads, send them a signal if(haveWaiters) SetEvent(mEvents[_CONDITION_EVENT_ONE]); } #endif #if defined(_TTHREAD_WIN32_) void condition_variable::notify_all() { // Are there any waiters? EnterCriticalSection(&mWaitersCountLock); bool haveWaiters = (mWaitersCount > 0); LeaveCriticalSection(&mWaitersCountLock); // If we have any waiting threads, send them a signal if(haveWaiters) SetEvent(mEvents[_CONDITION_EVENT_ALL]); } #endif //------------------------------------------------------------------------------ // POSIX pthread_t to unique thread::id mapping logic. // Note: Here we use a global thread safe std::map to convert instances of // pthread_t to small thread identifier numbers (unique within one process). // This method should be portable across different POSIX implementations. //------------------------------------------------------------------------------ #if defined(_TTHREAD_POSIX_) static thread::id _pthread_t_to_ID(const pthread_t &aHandle) { static mutex idMapLock; static std::map idMap; static unsigned long int idCount(1); lock_guard guard(idMapLock); if(idMap.find(aHandle) == idMap.end()) idMap[aHandle] = idCount ++; return thread::id(idMap[aHandle]); } #endif // _TTHREAD_POSIX_ //------------------------------------------------------------------------------ // thread //------------------------------------------------------------------------------ /// Information to pass to the new thread (what to run). struct _thread_start_info { void (*mFunction)(void *); ///< Pointer to the function to be executed. void * mArg; ///< Function argument for the thread function. thread * mThread; ///< Pointer to the thread object. }; // Thread wrapper function. #if defined(_TTHREAD_WIN32_) unsigned WINAPI thread::wrapper_function(void * aArg) #elif defined(_TTHREAD_POSIX_) void * thread::wrapper_function(void * aArg) #endif { // Get thread startup information _thread_start_info * ti = (_thread_start_info *) aArg; try { // Call the actual client thread function ti->mFunction(ti->mArg); } catch(...) { // Uncaught exceptions will terminate the application (default behavior // according to C++11) std::terminate(); } // The thread is no longer executing lock_guard guard(ti->mThread->mDataMutex); ti->mThread->mNotAThread = true; // The thread is responsible for freeing the startup information delete ti; return 0; } thread::thread(void (*aFunction)(void *), void * aArg) { // Serialize access to this thread structure lock_guard guard(mDataMutex); // Fill out the thread startup information (passed to the thread wrapper, // which will eventually free it) _thread_start_info * ti = new _thread_start_info; ti->mFunction = aFunction; ti->mArg = aArg; ti->mThread = this; // The thread is now alive mNotAThread = false; // Create the thread #if defined(_TTHREAD_WIN32_) mHandle = (HANDLE) _beginthreadex(0, 0, wrapper_function, (void *) ti, 0, &mWin32ThreadID); #elif defined(_TTHREAD_POSIX_) int err = 0; pthread_attr_t stackSizeAttribute; size_t stackSize = 0; err = pthread_attr_init(&stackSizeAttribute); if(err) throw "Error: pthread_attr_init"; err = pthread_attr_getstacksize(&stackSizeAttribute, &stackSize); if(err) throw "Error: pthread_attr_getstacksize"; size_t REQUIRED_STACK_SIZE = 4 * 1024 * 1024; if(stackSize < REQUIRED_STACK_SIZE) { err = pthread_attr_setstacksize(&stackSizeAttribute, REQUIRED_STACK_SIZE); if(err) throw "Error: pthread_attr_setstacksize"; } err = pthread_attr_getstacksize(&stackSizeAttribute, &stackSize); if(pthread_create(&mHandle, &stackSizeAttribute, wrapper_function, (void *) ti) != 0) mHandle = 0; #endif // Did we fail to create the thread? if(!mHandle) { mNotAThread = true; delete ti; } } thread::~thread() { if(joinable()) std::terminate(); } void thread::join() { if(joinable()) { #if defined(_TTHREAD_WIN32_) WaitForSingleObject(mHandle, INFINITE); CloseHandle(mHandle); #elif defined(_TTHREAD_POSIX_) pthread_join(mHandle, NULL); #endif } } bool thread::joinable() const { mDataMutex.lock(); bool result = !mNotAThread; mDataMutex.unlock(); return result; } void thread::detach() { mDataMutex.lock(); if(!mNotAThread) { #if defined(_TTHREAD_WIN32_) CloseHandle(mHandle); #elif defined(_TTHREAD_POSIX_) pthread_detach(mHandle); #endif mNotAThread = true; } mDataMutex.unlock(); } thread::id thread::get_id() const { if(!joinable()) return id(); #if defined(_TTHREAD_WIN32_) return id((unsigned long int) mWin32ThreadID); #elif defined(_TTHREAD_POSIX_) return _pthread_t_to_ID(mHandle); #endif } unsigned thread::hardware_concurrency() { #if defined(_TTHREAD_WIN32_) SYSTEM_INFO si; GetSystemInfo(&si); return (int) si.dwNumberOfProcessors; #elif defined(_SC_NPROCESSORS_ONLN) return (int) sysconf(_SC_NPROCESSORS_ONLN); #elif defined(_SC_NPROC_ONLN) return (int) sysconf(_SC_NPROC_ONLN); #else // The standard requires this function to return zero if the number of // hardware cores could not be determined. return 0; #endif } //------------------------------------------------------------------------------ // this_thread //------------------------------------------------------------------------------ thread::id this_thread::get_id() { #if defined(_TTHREAD_WIN32_) return thread::id((unsigned long int) GetCurrentThreadId()); #elif defined(_TTHREAD_POSIX_) return _pthread_t_to_ID(pthread_self()); #endif } } hisat2-2.2.1/tinythread.h000077500000000000000000000513441370663763200152610ustar00rootroot00000000000000/* -*- mode: c++; tab-width: 2; indent-tabs-mode: nil; -*- Copyright (c) 2010-2012 Marcus Geelnard This software is provided 'as-is', without any express or implied warranty. In no event will the authors be held liable for any damages arising from the use of this software. Permission is granted to anyone to use this software for any purpose, including commercial applications, and to alter it and redistribute it freely, subject to the following restrictions: 1. The origin of this software must not be misrepresented; you must not claim that you wrote the original software. If you use this software in a product, an acknowledgment in the product documentation would be appreciated but is not required. 2. Altered source versions must be plainly marked as such, and must not be misrepresented as being the original software. 3. This notice may not be removed or altered from any source distribution. */ #ifndef _TINYTHREAD_H_ #define _TINYTHREAD_H_ /// @file /// @mainpage TinyThread++ API Reference /// /// @section intro_sec Introduction /// TinyThread++ is a minimal, portable implementation of basic threading /// classes for C++. /// /// They closely mimic the functionality and naming of the C++11 standard, and /// should be easily replaceable with the corresponding std:: variants. /// /// @section port_sec Portability /// The Win32 variant uses the native Win32 API for implementing the thread /// classes, while for other systems, the POSIX threads API (pthread) is used. /// /// @section class_sec Classes /// In order to mimic the threading API of the C++11 standard, subsets of /// several classes are provided. The fundamental classes are: /// @li tthread::thread /// @li tthread::mutex /// @li tthread::recursive_mutex /// @li tthread::condition_variable /// @li tthread::lock_guard /// @li tthread::fast_mutex /// /// @section misc_sec Miscellaneous /// The following special keywords are available: #thread_local. /// /// For more detailed information (including additional classes), browse the /// different sections of this documentation. A good place to start is: /// tinythread.h. // Which platform are we on? #if !defined(_TTHREAD_PLATFORM_DEFINED_) #if defined(_WIN32) || defined(__WIN32__) || defined(__WINDOWS__) #define _TTHREAD_WIN32_ #else #define _TTHREAD_POSIX_ #endif #define _TTHREAD_PLATFORM_DEFINED_ #endif // Platform specific includes #if defined(_TTHREAD_WIN32_) #ifndef WIN32_LEAN_AND_MEAN #define WIN32_LEAN_AND_MEAN #define __UNDEF_LEAN_AND_MEAN #endif #include #ifdef __UNDEF_LEAN_AND_MEAN #undef WIN32_LEAN_AND_MEAN #undef __UNDEF_LEAN_AND_MEAN #endif #else #include #include #include #include #endif // Generic includes #include /// TinyThread++ version (major number). #define TINYTHREAD_VERSION_MAJOR 1 /// TinyThread++ version (minor number). #define TINYTHREAD_VERSION_MINOR 1 /// TinyThread++ version (full version). #define TINYTHREAD_VERSION (TINYTHREAD_VERSION_MAJOR * 100 + TINYTHREAD_VERSION_MINOR) // Do we have a fully featured C++11 compiler? #if (__cplusplus > 199711L) || (defined(__STDCXX_VERSION__) && (__STDCXX_VERSION__ >= 201001L)) #define _TTHREAD_CPP11_ #endif // ...at least partial C++11? #if defined(_TTHREAD_CPP11_) || defined(__GXX_EXPERIMENTAL_CXX0X__) || defined(__GXX_EXPERIMENTAL_CPP0X__) #define _TTHREAD_CPP11_PARTIAL_ #endif // Macro for disabling assignments of objects. #ifdef _TTHREAD_CPP11_PARTIAL_ #define _TTHREAD_DISABLE_ASSIGNMENT(name) \ name(const name&) = delete; \ name& operator=(const name&) = delete; #else #define _TTHREAD_DISABLE_ASSIGNMENT(name) \ name(const name&); \ name& operator=(const name&); #endif /// @def thread_local /// Thread local storage keyword. /// A variable that is declared with the @c thread_local keyword makes the /// value of the variable local to each thread (known as thread-local storage, /// or TLS). Example usage: /// @code /// // This variable is local to each thread. /// thread_local int variable; /// @endcode /// @note The @c thread_local keyword is a macro that maps to the corresponding /// compiler directive (e.g. @c __declspec(thread)). While the C++11 standard /// allows for non-trivial types (e.g. classes with constructors and /// destructors) to be declared with the @c thread_local keyword, most pre-C++11 /// compilers only allow for trivial types (e.g. @c int). So, to guarantee /// portable code, only use trivial types for thread local storage. /// @note This directive is currently not supported on Mac OS X (it will give /// a compiler error), since compile-time TLS is not supported in the Mac OS X /// executable format. Also, some older versions of MinGW (before GCC 4.x) do /// not support this directive. /// @hideinitializer #if !defined(_TTHREAD_CPP11_) && !defined(thread_local) #if defined(__GNUC__) || defined(__INTEL_COMPILER) || defined(__SUNPRO_CC) || defined(__IBMCPP__) #define thread_local __thread #else #define thread_local __declspec(thread) #endif #endif /// Main name space for TinyThread++. /// This namespace is more or less equivalent to the @c std namespace for the /// C++11 thread classes. For instance, the tthread::mutex class corresponds to /// the std::mutex class. namespace tthread { /// Mutex class. /// This is a mutual exclusion object for synchronizing access to shared /// memory areas for several threads. The mutex is non-recursive (i.e. a /// program may deadlock if the thread that owns a mutex object calls lock() /// on that object). /// @see recursive_mutex class mutex { public: /// Constructor. mutex() #if defined(_TTHREAD_WIN32_) : mAlreadyLocked(false) #endif { #if defined(_TTHREAD_WIN32_) InitializeCriticalSection(&mHandle); #else pthread_mutex_init(&mHandle, NULL); #endif } /// Destructor. ~mutex() { #if defined(_TTHREAD_WIN32_) DeleteCriticalSection(&mHandle); #else pthread_mutex_destroy(&mHandle); #endif } /// Lock the mutex. /// The method will block the calling thread until a lock on the mutex can /// be obtained. The mutex remains locked until @c unlock() is called. /// @see lock_guard inline void lock() { #if defined(_TTHREAD_WIN32_) EnterCriticalSection(&mHandle); while(mAlreadyLocked) Sleep(1000); // Simulate deadlock... mAlreadyLocked = true; #else pthread_mutex_lock(&mHandle); #endif } /// Try to lock the mutex. /// The method will try to lock the mutex. If it fails, the function will /// return immediately (non-blocking). /// @return @c true if the lock was acquired, or @c false if the lock could /// not be acquired. inline bool try_lock() { #if defined(_TTHREAD_WIN32_) bool ret = (TryEnterCriticalSection(&mHandle) ? true : false); if(ret && mAlreadyLocked) { LeaveCriticalSection(&mHandle); ret = false; } return ret; #else return (pthread_mutex_trylock(&mHandle) == 0) ? true : false; #endif } /// Unlock the mutex. /// If any threads are waiting for the lock on this mutex, one of them will /// be unblocked. inline void unlock() { #if defined(_TTHREAD_WIN32_) mAlreadyLocked = false; LeaveCriticalSection(&mHandle); #else pthread_mutex_unlock(&mHandle); #endif } _TTHREAD_DISABLE_ASSIGNMENT(mutex) private: #if defined(_TTHREAD_WIN32_) CRITICAL_SECTION mHandle; bool mAlreadyLocked; #else pthread_mutex_t mHandle; #endif friend class condition_variable; }; /// Recursive mutex class. /// This is a mutual exclusion object for synchronizing access to shared /// memory areas for several threads. The mutex is recursive (i.e. a thread /// may lock the mutex several times, as long as it unlocks the mutex the same /// number of times). /// @see mutex class recursive_mutex { public: /// Constructor. recursive_mutex() { #if defined(_TTHREAD_WIN32_) InitializeCriticalSection(&mHandle); #else pthread_mutexattr_t attr; pthread_mutexattr_init(&attr); pthread_mutexattr_settype(&attr, PTHREAD_MUTEX_RECURSIVE); pthread_mutex_init(&mHandle, &attr); #endif } /// Destructor. ~recursive_mutex() { #if defined(_TTHREAD_WIN32_) DeleteCriticalSection(&mHandle); #else pthread_mutex_destroy(&mHandle); #endif } /// Lock the mutex. /// The method will block the calling thread until a lock on the mutex can /// be obtained. The mutex remains locked until @c unlock() is called. /// @see lock_guard inline void lock() { #if defined(_TTHREAD_WIN32_) EnterCriticalSection(&mHandle); #else pthread_mutex_lock(&mHandle); #endif } /// Try to lock the mutex. /// The method will try to lock the mutex. If it fails, the function will /// return immediately (non-blocking). /// @return @c true if the lock was acquired, or @c false if the lock could /// not be acquired. inline bool try_lock() { #if defined(_TTHREAD_WIN32_) return TryEnterCriticalSection(&mHandle) ? true : false; #else return (pthread_mutex_trylock(&mHandle) == 0) ? true : false; #endif } /// Unlock the mutex. /// If any threads are waiting for the lock on this mutex, one of them will /// be unblocked. inline void unlock() { #if defined(_TTHREAD_WIN32_) LeaveCriticalSection(&mHandle); #else pthread_mutex_unlock(&mHandle); #endif } _TTHREAD_DISABLE_ASSIGNMENT(recursive_mutex) private: #if defined(_TTHREAD_WIN32_) CRITICAL_SECTION mHandle; #else pthread_mutex_t mHandle; #endif friend class condition_variable; }; /// Lock guard class. /// The constructor locks the mutex, and the destructor unlocks the mutex, so /// the mutex will automatically be unlocked when the lock guard goes out of /// scope. Example usage: /// @code /// mutex m; /// int counter; /// /// void increment() /// { /// lock_guard guard(m); /// ++ counter; /// } /// @endcode template class lock_guard { public: typedef T mutex_type; lock_guard() : mMutex(0) {} /// The constructor locks the mutex. explicit lock_guard(mutex_type &aMutex) { mMutex = &aMutex; mMutex->lock(); } /// The destructor unlocks the mutex. ~lock_guard() { if(mMutex) mMutex->unlock(); } private: mutex_type * mMutex; }; /// Condition variable class. /// This is a signalling object for synchronizing the execution flow for /// several threads. Example usage: /// @code /// // Shared data and associated mutex and condition variable objects /// int count; /// mutex m; /// condition_variable cond; /// /// // Wait for the counter to reach a certain number /// void wait_counter(int targetCount) /// { /// lock_guard guard(m); /// while(count < targetCount) /// cond.wait(m); /// } /// /// // Increment the counter, and notify waiting threads /// void increment() /// { /// lock_guard guard(m); /// ++ count; /// cond.notify_all(); /// } /// @endcode class condition_variable { public: /// Constructor. #if defined(_TTHREAD_WIN32_) condition_variable(); #else condition_variable() { pthread_cond_init(&mHandle, NULL); } #endif /// Destructor. #if defined(_TTHREAD_WIN32_) ~condition_variable(); #else ~condition_variable() { pthread_cond_destroy(&mHandle); } #endif /// Wait for the condition. /// The function will block the calling thread until the condition variable /// is woken by @c notify_one(), @c notify_all() or a spurious wake up. /// @param[in] aMutex A mutex that will be unlocked when the wait operation /// starts, an locked again as soon as the wait operation is finished. template inline void wait(_mutexT &aMutex) { #if defined(_TTHREAD_WIN32_) // Increment number of waiters EnterCriticalSection(&mWaitersCountLock); ++ mWaitersCount; LeaveCriticalSection(&mWaitersCountLock); // Release the mutex while waiting for the condition (will decrease // the number of waiters when done)... aMutex.unlock(); _wait(); aMutex.lock(); #else pthread_cond_wait(&mHandle, &aMutex.mHandle); #endif } /// Notify one thread that is waiting for the condition. /// If at least one thread is blocked waiting for this condition variable, /// one will be woken up. /// @note Only threads that started waiting prior to this call will be /// woken up. #if defined(_TTHREAD_WIN32_) void notify_one(); #else inline void notify_one() { pthread_cond_signal(&mHandle); } #endif /// Notify all threads that are waiting for the condition. /// All threads that are blocked waiting for this condition variable will /// be woken up. /// @note Only threads that started waiting prior to this call will be /// woken up. #if defined(_TTHREAD_WIN32_) void notify_all(); #else inline void notify_all() { pthread_cond_broadcast(&mHandle); } #endif _TTHREAD_DISABLE_ASSIGNMENT(condition_variable) private: #if defined(_TTHREAD_WIN32_) void _wait(); HANDLE mEvents[2]; ///< Signal and broadcast event HANDLEs. unsigned int mWaitersCount; ///< Count of the number of waiters. CRITICAL_SECTION mWaitersCountLock; ///< Serialize access to mWaitersCount. #else pthread_cond_t mHandle; #endif }; /// Thread class. class thread { public: #if defined(_TTHREAD_WIN32_) typedef HANDLE native_handle_type; #else typedef pthread_t native_handle_type; #endif class id; /// Default constructor. /// Construct a @c thread object without an associated thread of execution /// (i.e. non-joinable). thread() : mHandle(0), mNotAThread(true) #if defined(_TTHREAD_WIN32_) , mWin32ThreadID(0) #endif {} /// Thread starting constructor. /// Construct a @c thread object with a new thread of execution. /// @param[in] aFunction A function pointer to a function of type: /// void fun(void * arg) /// @param[in] aArg Argument to the thread function. /// @note This constructor is not fully compatible with the standard C++ /// thread class. It is more similar to the pthread_create() (POSIX) and /// CreateThread() (Windows) functions. thread(void (*aFunction)(void *), void * aArg); /// Destructor. /// @note If the thread is joinable upon destruction, @c std::terminate() /// will be called, which terminates the process. It is always wise to do /// @c join() before deleting a thread object. ~thread(); /// Wait for the thread to finish (join execution flows). /// After calling @c join(), the thread object is no longer associated with /// a thread of execution (i.e. it is not joinable, and you may not join /// with it nor detach from it). void join(); /// Check if the thread is joinable. /// A thread object is joinable if it has an associated thread of execution. bool joinable() const; /// Detach from the thread. /// After calling @c detach(), the thread object is no longer assicated with /// a thread of execution (i.e. it is not joinable). The thread continues /// execution without the calling thread blocking, and when the thread /// ends execution, any owned resources are released. void detach(); /// Return the thread ID of a thread object. id get_id() const; /// Get the native handle for this thread. /// @note Under Windows, this is a @c HANDLE, and under POSIX systems, this /// is a @c pthread_t. inline native_handle_type native_handle() { return mHandle; } /// Determine the number of threads which can possibly execute concurrently. /// This function is useful for determining the optimal number of threads to /// use for a task. /// @return The number of hardware thread contexts in the system. /// @note If this value is not defined, the function returns zero (0). static unsigned hardware_concurrency(); _TTHREAD_DISABLE_ASSIGNMENT(thread) private: native_handle_type mHandle; ///< Thread handle. mutable mutex mDataMutex; ///< Serializer for access to the thread private data. bool mNotAThread; ///< True if this object is not a thread of execution. #if defined(_TTHREAD_WIN32_) unsigned int mWin32ThreadID; ///< Unique thread ID (filled out by _beginthreadex). #endif // This is the internal thread wrapper function. #if defined(_TTHREAD_WIN32_) static unsigned WINAPI wrapper_function(void * aArg); #else static void * wrapper_function(void * aArg); #endif }; /// Thread ID. /// The thread ID is a unique identifier for each thread. /// @see thread::get_id() class thread::id { public: /// Default constructor. /// The default constructed ID is that of thread without a thread of /// execution. id() : mId(0) {}; id(unsigned long int aId) : mId(aId) {}; id(const id& aId) : mId(aId.mId) {}; inline id & operator=(const id &aId) { mId = aId.mId; return *this; } inline friend bool operator==(const id &aId1, const id &aId2) { return (aId1.mId == aId2.mId); } inline friend bool operator!=(const id &aId1, const id &aId2) { return (aId1.mId != aId2.mId); } inline friend bool operator<=(const id &aId1, const id &aId2) { return (aId1.mId <= aId2.mId); } inline friend bool operator<(const id &aId1, const id &aId2) { return (aId1.mId < aId2.mId); } inline friend bool operator>=(const id &aId1, const id &aId2) { return (aId1.mId >= aId2.mId); } inline friend bool operator>(const id &aId1, const id &aId2) { return (aId1.mId > aId2.mId); } inline friend std::ostream& operator <<(std::ostream &os, const id &obj) { os << obj.mId; return os; } private: unsigned long int mId; }; // Related to - minimal to be able to support chrono. typedef long long __intmax_t; /// Minimal implementation of the @c ratio class. This class provides enough /// functionality to implement some basic @c chrono classes. template <__intmax_t N, __intmax_t D = 1> class ratio { public: static double _as_double() { return double(N) / double(D); } }; /// Minimal implementation of the @c chrono namespace. /// The @c chrono namespace provides types for specifying time intervals. namespace chrono { /// Duration template class. This class provides enough functionality to /// implement @c this_thread::sleep_for(). template > class duration { private: _Rep rep_; public: typedef _Rep rep; typedef _Period period; /// Construct a duration object with the given duration. template explicit duration(const _Rep2& r) : rep_(r) {}; /// Return the value of the duration object. rep count() const { return rep_; } }; // Standard duration types. typedef duration<__intmax_t, ratio<1, 1000000000> > nanoseconds; ///< Duration with the unit nanoseconds. typedef duration<__intmax_t, ratio<1, 1000000> > microseconds; ///< Duration with the unit microseconds. typedef duration<__intmax_t, ratio<1, 1000> > milliseconds; ///< Duration with the unit milliseconds. typedef duration<__intmax_t> seconds; ///< Duration with the unit seconds. typedef duration<__intmax_t, ratio<60> > minutes; ///< Duration with the unit minutes. typedef duration<__intmax_t, ratio<3600> > hours; ///< Duration with the unit hours. } /// The namespace @c this_thread provides methods for dealing with the /// calling thread. namespace this_thread { /// Return the thread ID of the calling thread. thread::id get_id(); /// Yield execution to another thread. /// Offers the operating system the opportunity to schedule another thread /// that is ready to run on the current processor. inline void yield() { #if defined(_TTHREAD_WIN32_) Sleep(0); #else sched_yield(); #endif } /// Blocks the calling thread for a period of time. /// @param[in] aTime Minimum time to put the thread to sleep. /// Example usage: /// @code /// // Sleep for 100 milliseconds /// this_thread::sleep_for(chrono::milliseconds(100)); /// @endcode /// @note Supported duration types are: nanoseconds, microseconds, /// milliseconds, seconds, minutes and hours. template void sleep_for(const chrono::duration<_Rep, _Period>& aTime) { #if defined(_TTHREAD_WIN32_) Sleep(int(double(aTime.count()) * (1000.0 * _Period::_as_double()) + 0.5)); #else usleep(int(double(aTime.count()) * (1000000.0 * _Period::_as_double()) + 0.5)); #endif } } } // Define/macro cleanup #undef _TTHREAD_DISABLE_ASSIGNMENT #endif // _TINYTHREAD_H_ hisat2-2.2.1/tokenize.h000066400000000000000000000033351370663763200147300ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef TOKENIZE_H_ #define TOKENIZE_H_ #include #include #include using namespace std; /** * Split string s according to given delimiters. Mostly borrowed * from C++ Programming HOWTO 7.3. */ template static inline void tokenize( const string& s, const string& delims, T& ss, size_t max = std::numeric_limits::max()) { //string::size_type lastPos = s.find_first_not_of(delims, 0); string::size_type lastPos = 0; string::size_type pos = s.find_first_of(delims, lastPos); while (string::npos != pos || string::npos != lastPos) { ss.push_back(s.substr(lastPos, pos - lastPos)); lastPos = s.find_first_not_of(delims, pos); pos = s.find_first_of(delims, lastPos); if(ss.size() == (max - 1)) { pos = string::npos; } } } template static inline void tokenize(const std::string& s, char delim, T& ss) { std::string token; std::istringstream iss(s); while(getline(iss, token, delim)) { ss.push_back(token); } } #endif /*TOKENIZE_H_*/ hisat2-2.2.1/tp.h000066400000000000000000000072701370663763200135250ustar00rootroot00000000000000/* * Copyright 2015, Daehwan Kim * * This file is part of HISAT 2. * * HISAT 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * HISAT 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with HISAT 2. If not, see . */ /* * tp.h * */ #ifndef TP_H_ #define TP_H_ #include #include /** * Encapsulates alignment policy for transcriptome */ class TranscriptomePolicy { public: TranscriptomePolicy() { reset(); } TranscriptomePolicy( size_t minIntronLen, size_t maxIntronLen, uint32_t minAnchorLen = 7, uint32_t minAnchorLen_noncan = 14, bool no_spliced_alignment = false, bool transcriptome_mapping_only = false, bool transcriptome_assembly = false, bool xs_only = false, bool avoid_pseudogene = false) { init(minIntronLen, maxIntronLen, minAnchorLen, minAnchorLen_noncan, no_spliced_alignment, transcriptome_mapping_only, transcriptome_assembly, xs_only, avoid_pseudogene); } /** */ void reset() { init(false, false, false); } /** */ void init( size_t minIntronLen, size_t maxIntronLen, uint32_t minAnchorLen = 7, uint32_t minAnchorLen_noncan = 14, bool no_spliced_alignment = false, bool transcriptome_mapping_only = false, bool transcriptome_assembly = false, bool xs_only = false, bool avoid_pseudogene = false) { minIntronLen_ = minIntronLen; maxIntronLen_ = maxIntronLen; minAnchorLen_ = minAnchorLen; minAnchorLen_noncan_ = minAnchorLen_noncan; no_spliced_alignment_ = no_spliced_alignment; transcriptome_mapping_only_ = transcriptome_mapping_only; transcriptome_assembly_ = transcriptome_assembly; xs_only_ = xs_only; avoid_pseudogene_ = avoid_pseudogene; } size_t minIntronLen() const { return minIntronLen_; } size_t maxIntronLen() const { return maxIntronLen_; } uint32_t minAnchorLen() const { return minAnchorLen_; } uint32_t minAnchorLen_noncan() const { return minAnchorLen_noncan_; } bool no_spliced_alignment() const { return no_spliced_alignment_; } bool transcriptome_mapping_only() const { return transcriptome_mapping_only_; } bool transcriptome_assembly() const { return transcriptome_assembly_; } bool xs_only() const { return xs_only_; } bool avoid_pseudogene() const { return avoid_pseudogene_; } private: size_t minIntronLen_; size_t maxIntronLen_; // Minimum anchor length required for canonical splice sites uint32_t minAnchorLen_; // Minimum anchor length required for non-canonical splice sites uint32_t minAnchorLen_noncan_; bool no_spliced_alignment_; bool transcriptome_mapping_only_; bool transcriptome_assembly_; bool xs_only_; bool avoid_pseudogene_; }; #endif /*ndef TP_H_*/ hisat2-2.2.1/unique.cpp000066400000000000000000000045251370663763200147430ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #include "unique.h" using namespace std; // There is no valid second-best alignment and the best alignment has a // perfect score. const TMapq unp_nosec_perf = 44; // There is no valid second-best alignment. We stratify the alignment // score of the best alignment into 10 bins. const TMapq unp_nosec[11] = { 43, 42, 41, 36, 32, 27, 20, 11, 4, 1, 0 }; // The best alignment has a perfect score, and we stratify the distance // between best and second-best alignment scores into 10 bins. const TMapq unp_sec_perf[11] = { 2, 16, 23, 30, 31, 32, 34, 36, 38, 40, 42 }; // The best alignment has a non-perfect score, and we stratify both by best // alignment score (specifically, the maximum score minus the best "best") // and by the distance between the best and second-best alignment scores // ("difference"). Each is stratified into 10 bins. Each row is a // difference (smaller elts = smaller differences) and each column is a // best score (smaller elts = higher best alignment scores). const TMapq unp_sec[11][11] = { { 2, 2, 2, 1, 1, 0, 0, 0, 0, 0, 0}, { 20, 14, 7, 3, 2, 1, 0, 0, 0, 0, 0}, { 20, 16, 10, 6, 3, 1, 0, 0, 0, 0, 0}, { 20, 17, 13, 9, 3, 1, 1, 0, 0, 0, 0}, { 21, 19, 15, 9, 5, 2, 2, 0, 0, 0, 0}, { 22, 21, 16, 11, 10, 5, 0, 0, 0, 0, 0}, { 23, 22, 19, 16, 11, 0, 0, 0, 0, 0, 0}, { 24, 25, 21, 30, 0, 0, 0, 0, 0, 0, 0}, { 30, 26, 29, 0, 0, 0, 0, 0, 0, 0, 0}, { 30, 27, 0, 0, 0, 0, 0, 0, 0, 0, 0}, { 30, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, }; // // Paired mapping quality: // // There is no valid second-best alignment and the best alignment has a // perfect score. const TMapq pair_nosec_perf = 44; hisat2-2.2.1/unique.h000066400000000000000000000346411370663763200144120ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ /* * unique.h * * Encapsulates objects and routines for determining whether and to * what extend the best alignment for a read is "unique." In the * simplest scenario, uniqueness is determined by whether we found only * one alignment. More complex scenarios might assign a uniqueness * score based that is a function of (of a summarized version of): all * the alignments found and their scores. * * Since mapping quality is related to uniqueness, objects and * routings for calculating mapping quality are also included here. */ #ifndef UNIQUE_H_ #define UNIQUE_H_ #include #include "aligner_result.h" #include "simple_func.h" #include "util.h" #include "scoring.h" typedef int64_t TMapq; /** * Class that returns yes-or-no answers to the question of whether a */ class Uniqueness { public: /** * Given an AlnSetSumm, determine if the best alignment is "unique" * according to some definition. */ static bool bestIsUnique( const AlnSetSumm& s, const AlnFlags& flags, bool mate1, size_t rdlen, size_t ordlen, char *inps) { assert(!s.empty()); return !VALID_AL_SCORE(s.secbest(mate1)); } }; /** * Collection of routines for calculating mapping quality. */ class Mapq { public: virtual ~Mapq() { } virtual TMapq mapq( const AlnSetSumm& s, const AlnFlags& flags, bool mate1, size_t rdlen, size_t ordlen, char *inps) const = 0; }; extern const TMapq unp_nosec_perf; extern const TMapq unp_nosec[11]; extern const TMapq unp_sec_perf[11]; extern const TMapq unp_sec[11][11]; extern const TMapq pair_nosec_perf; /** * V3 of the MAPQ calculator */ class BowtieMapq3 : public Mapq { public: BowtieMapq3( const SimpleFunc& scoreMin, const Scoring& sc) : scoreMin_(scoreMin), sc_(sc) { } virtual ~BowtieMapq3() { } /** * Given an AlnSetSumm, return a mapping quality calculated. */ virtual TMapq mapq( const AlnSetSumm& s, const AlnFlags& flags, bool mate1, size_t rdlen, size_t ordlen, char *inps) // put string representation of inputs here const { if(s.paired()) { return pair_nosec_perf; } else { bool hasSecbest = VALID_AL_SCORE(s.secbest(mate1)); if(!flags.canMax() && !s.exhausted(mate1) && !hasSecbest) { return 255; } TAlScore scMax = (TAlScore)sc_.perfectScore(rdlen); TAlScore scMin = scoreMin_.f((float)rdlen); assert_geq(scMax, scMin); TAlScore best = scMax - s.best(mate1).score(); // best score (lower=better) size_t best_bin = (size_t)((double)best * (10.0 / (double)(scMax - scMin)) + 0.5); assert_geq(best_bin, 0); assert_lt(best_bin, 11); if(hasSecbest) { assert_geq(s.best(mate1).score(), s.secbest(mate1).score()); size_t diff = s.best(mate1).score() - s.secbest(mate1).score(); size_t diff_bin = (size_t)((double)diff * (10.0 / (double)(scMax - scMin)) + 0.5); assert_geq(diff_bin, 0); assert_lt(diff_bin, 11); // A valid second-best alignment was found if(best == scMax) { // Best alignment has perfect score return unp_sec_perf[best_bin]; } else { // Best alignment has less than perfect score return unp_sec[diff_bin][best_bin]; } } else { // No valid second-best alignment was found if(best == scMax) { // Best alignment has perfect score return unp_nosec_perf; } else { // Best alignment has less than perfect score return unp_nosec[best_bin]; } } } } protected: SimpleFunc scoreMin_; const Scoring& sc_; }; /** * V2 of the MAPQ calculator */ class BowtieMapq2 : public Mapq { public: BowtieMapq2( const SimpleFunc& scoreMin, const Scoring& sc) : scoreMin_(scoreMin), sc_(sc) { } virtual ~BowtieMapq2() { } /** * Given an AlnSetSumm, return a mapping quality calculated. */ virtual TMapq mapq( const AlnSetSumm& s, const AlnFlags& flags, bool mate1, size_t rdlen, size_t ordlen, char *inps) // put string representation of inputs here const { // Did the read have a second-best alignment? bool hasSecbest = s.paired() ? VALID_AL_SCORE(s.secbestPaired()) : VALID_AL_SCORE(s.secbest(mate1)); // for hisat bool equalSecbest = false; if(hasSecbest) { if(s.paired()) { assert_geq(s.bestPaired(), s.secbestPaired()); equalSecbest = s.bestPaired() == s.secbestPaired(); } else { assert_geq(s.best(mate1), s.secbest(mate1)); equalSecbest = s.best(mate1) == s.secbest(mate1); } } // This corresponds to a scenario where we found one and only one // alignment but didn't really look for a second one if(!flags.canMax() && !s.exhausted(mate1) && (!hasSecbest || !equalSecbest)) { return 60; } // scPer = score of a perfect match TAlScore scPer = (TAlScore)sc_.perfectScore(rdlen); if(s.paired()) { scPer += (TAlScore)sc_.perfectScore(ordlen); } // scMin = score of a just barely valid match TAlScore scMin = scoreMin_.f((float)rdlen); if(s.paired()) { scMin += scoreMin_.f((float)ordlen); } TAlScore secbest = scMin-1; TAlScore diff = (scPer - scMin); // scores can vary by up to this much TMapq ret = 0; TAlScore best = s.paired() ? s.bestPaired().score() : s.best(mate1).score(); // best score but normalized so that 0 = worst valid score TAlScore bestOver = best - scMin; if(sc_.monotone) { // End-to-end alignment if(!hasSecbest) { if (bestOver >= diff * (double)0.8f) ret = 42; else if(bestOver >= diff * (double)0.7f) ret = 40; else if(bestOver >= diff * (double)0.6f) ret = 24; else if(bestOver >= diff * (double)0.5f) ret = 23; else if(bestOver >= diff * (double)0.4f) ret = 8; else if(bestOver >= diff * (double)0.3f) ret = 3; else ret = 0; } else { secbest = s.paired() ? s.secbestPaired().score() : s.secbest(mate1).score(); TAlScore bestdiff = abs(abs(static_cast(best))-abs(static_cast(secbest))); if(bestdiff >= diff * (double)0.9f) { if(bestOver == diff) { ret = 39; } else { ret = 33; } } else if(bestdiff >= diff * (double)0.8f) { if(bestOver == diff) { ret = 38; } else { ret = 27; } } else if(bestdiff >= diff * (double)0.7f) { if(bestOver == diff) { ret = 37; } else { ret = 26; } } else if(bestdiff >= diff * (double)0.6f) { if(bestOver == diff) { ret = 36; } else { ret = 22; } } else if(bestdiff >= diff * (double)0.5f) { // Top third is still pretty good if (bestOver == diff) { ret = 35; } else if(bestOver >= diff * (double)0.84f) { ret = 25; } else if(bestOver >= diff * (double)0.68f) { ret = 16; } else { ret = 5; } } else if(bestdiff >= diff * (double)0.4f) { // Top third is still pretty good if (bestOver == diff) { ret = 34; } else if(bestOver >= diff * (double)0.84f) { ret = 21; } else if(bestOver >= diff * (double)0.68f) { ret = 14; } else { ret = 4; } } else if(bestdiff >= diff * (double)0.3f) { // Top third is still pretty good if (bestOver == diff) { ret = 32; } else if(bestOver >= diff * (double)0.88f) { ret = 18; } else if(bestOver >= diff * (double)0.67f) { ret = 15; } else { ret = 3; } } else if(bestdiff >= diff * (double)0.2f) { // Top third is still pretty good if (bestOver == diff) { ret = 31; } else if(bestOver >= diff * (double)0.88f) { ret = 17; } else if(bestOver >= diff * (double)0.67f) { ret = 11; } else { ret = 0; } } else if(bestdiff >= diff * (double)0.1f) { // Top third is still pretty good if (bestOver == diff) { ret = 30; } else if(bestOver >= diff * (double)0.88f) { ret = 12; } else if(bestOver >= diff * (double)0.67f) { ret = 7; } else { ret = 0; } } else if(bestdiff > 0) { // Top third is still pretty good if(bestOver >= diff * (double)0.67f) { ret = 6; } else { ret = 2; } } else { assert_eq(bestdiff, 0); // Top third is still pretty good if(bestOver >= diff * (double)0.67f) { ret = 1; } else { ret = 0; } } } } else { // Local alignment if(!hasSecbest) { if (bestOver >= diff * (double)0.8f) ret = 44; else if(bestOver >= diff * (double)0.7f) ret = 42; else if(bestOver >= diff * (double)0.6f) ret = 41; else if(bestOver >= diff * (double)0.5f) ret = 36; else if(bestOver >= diff * (double)0.4f) ret = 28; else if(bestOver >= diff * (double)0.3f) ret = 24; else ret = 22; } else { secbest = s.paired() ? s.secbestPaired().score() : s.secbest(mate1).score(); TAlScore bestdiff = abs(abs(static_cast(best))-abs(static_cast(secbest))); if (bestdiff >= diff * (double)0.9f) ret = 40; else if(bestdiff >= diff * (double)0.8f) ret = 39; else if(bestdiff >= diff * (double)0.7f) ret = 38; else if(bestdiff >= diff * (double)0.6f) ret = 37; else if(bestdiff >= diff * (double)0.5f) { if (bestOver == diff) ret = 35; else if(bestOver >= diff * (double)0.50f) ret = 25; else ret = 20; } else if(bestdiff >= diff * (double)0.4f) { if (bestOver == diff) ret = 34; else if(bestOver >= diff * (double)0.50f) ret = 21; else ret = 19; } else if(bestdiff >= diff * (double)0.3f) { if (bestOver == diff) ret = 33; else if(bestOver >= diff * (double)0.5f) ret = 18; else ret = 16; } else if(bestdiff >= diff * (double)0.2f) { if (bestOver == diff) ret = 32; else if(bestOver >= diff * (double)0.5f) ret = 17; else ret = 12; } else if(bestdiff >= diff * (double)0.1f) { if (bestOver == diff) ret = 31; else if(bestOver >= diff * (double)0.5f) ret = 14; else ret = 9; } else if(bestdiff > 0) { if(bestOver >= diff * (double)0.5f) ret = 11; else ret = 2; } else { assert_eq(bestdiff, 0); if(bestOver >= diff * (double)0.5f) ret = 1; else ret = 0; } } } // Note: modifications to inps must be synchronized //if(inps != NULL) { // inps = itoa10(best, inps); // *inps++ = ','; // inps = itoa10(secbest, inps); // *inps++ = ','; // inps = itoa10(ret, inps); //} return ret; } protected: SimpleFunc scoreMin_; const Scoring& sc_; }; /** * TODO: Do BowtieMapq on a per-thread basis prior to the mutex'ed output * function. * * topCoeff :: top_coeff * botCoeff :: bot_coeff * mx :: mapqMax * horiz :: mapqHorizon (sort of) * * sc1 <- tab$sc1 * sc2 <- tab$sc2 * mapq <- rep(mx, length(sc1)) * diff_top <- ifelse(sc1 != best & sc2 != best, abs(best - abs(pmax(sc1, sc2))), 0) * mapq <- mapq - diff_top * top_coeff * diff_bot <- ifelse(sc2 != horiz, abs(abs(sc2) - abs(horiz)), 0) * mapq <- mapq - diff_bot * bot_coeff * mapq <- round(pmax(0, pmin(mx, mapq))) * tab$mapq <- mapq */ class BowtieMapq : public Mapq { public: BowtieMapq( const SimpleFunc& scoreMin, const Scoring& sc) : scoreMin_(scoreMin), sc_(sc) { } virtual ~BowtieMapq() { } /** * Given an AlnSetSumm, return a mapping quality calculated. */ virtual TMapq mapq( const AlnSetSumm& s, const AlnFlags& flags, bool mate1, size_t rdlen, size_t ordlen, char *inps) // put string representation of inputs here const { bool hasSecbest = VALID_AL_SCORE(s.secbest(mate1)); if(!flags.canMax() && !s.exhausted(mate1) && !hasSecbest) { return 255; } TAlScore scPer = (TAlScore)sc_.perfectScore(rdlen); TAlScore scMin = scoreMin_.f((float)rdlen); TAlScore secbest = scMin-1; TAlScore diff = (scPer - scMin); float sixth_2 = (float)(scPer - diff * (double)0.1666f * 2); float sixth_3 = (float)(scPer - diff * (double)0.1666f * 3); TMapq ret = 0; TAlScore best = s.best(mate1).score(); if(!hasSecbest) { // Top third? if(best >= sixth_2) { ret = 37; } // Otherwise in top half? else if(best >= sixth_3) { ret = 25; } // Otherwise has no second-best? else { ret = 10; } } else { secbest = s.secbest(mate1).score(); TAlScore bestdiff = abs(abs(static_cast(best))-abs(static_cast(secbest))); if(bestdiff >= diff * 0.1666 * 5) { ret = 6; } else if(bestdiff >= diff * 0.1666 * 4) { ret = 5; } else if(bestdiff >= diff * 0.1666 * 3) { ret = 4; } else if(bestdiff >= diff * 0.1666 * 2) { ret = 3; } else if(bestdiff >= diff * 0.1666 * 1) { ret = 2; } else { ret = 1; } } // Note: modifications to inps must be synchronized //if(inps != NULL) { // inps = itoa10(best, inps); // *inps++ = ','; // inps = itoa10(secbest, inps); // *inps++ = ','; // inps = itoa10(ret, inps); //} return ret; } protected: SimpleFunc scoreMin_; const Scoring& sc_; }; /** * Create and return new MAPQ calculating object. */ static inline Mapq *new_mapq( int version, const SimpleFunc& scoreMin, const Scoring& sc) { if(version == 3) { return new BowtieMapq3(scoreMin, sc); } else if(version == 2) { return new BowtieMapq2(scoreMin, sc); } else { return new BowtieMapq(scoreMin, sc); } } #endif /*ndef UNIQUE_H_*/ hisat2-2.2.1/util.h000066400000000000000000000027401370663763200140540ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef UTIL_H_ #define UTIL_H_ #include #include /** * C++ version char* style "itoa": */ template char* itoa10(const T& value, char* result) { // Check that base is valid char* out = result; T quotient = value; if(std::numeric_limits::is_signed) { if(quotient <= 0) quotient = 0-quotient; } // Now write each digit from most to least significant do { *out = "0123456789"[quotient % 10]; ++out; quotient /= 10; } while (quotient > 0); // Only apply negative sign for base 10 if(std::numeric_limits::is_signed) { // Avoid compiler warning in cases where T is unsigned if (value <= 0 && value != 0) *out++ = '-'; } reverse( result, out ); *out = 0; // terminator return out; } #endif /*ndef UTIL_H_*/ hisat2-2.2.1/word_io.h000066400000000000000000000201231370663763200145340ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef WORD_IO_H_ #define WORD_IO_H_ #include #include #include #include #include "assert_helpers.h" #include "endian_swap.h" /** * Write a 32-bit unsigned to an output stream being careful to * re-endianize if caller-requested endianness differs from current * host. */ static inline void writeU32(std::ostream& out, uint32_t x, bool toBigEndian) { uint32_t y = endianizeU32(x, toBigEndian); out.write((const char*)&y, 4); } /** * Write a 32-bit unsigned to an output stream using the native * endianness. */ static inline void writeU32(std::ostream& out, uint32_t x) { out.write((const char*)&x, 4); } /** * Write a 32-bit signed int to an output stream being careful to * re-endianize if caller-requested endianness differs from current * host. */ static inline void writeI32(std::ostream& out, int32_t x, bool toBigEndian) { int32_t y = endianizeI32(x, toBigEndian); out.write((const char*)&y, 4); } /** * Write a 32-bit unsigned to an output stream using the native * endianness. */ static inline void writeI32(std::ostream& out, int32_t x) { out.write((const char*)&x, 4); } /** * Write a 16-bit unsigned to an output stream being careful to * re-endianize if caller-requested endianness differs from current * host. */ static inline void writeU16(std::ostream& out, uint16_t x, bool toBigEndian) { uint16_t y = endianizeU16(x, toBigEndian); out.write((const char*)&y, 2); } /** * Write a 16-bit unsigned to an output stream using the native * endianness. */ static inline void writeU16(std::ostream& out, uint16_t x) { out.write((const char*)&x, 2); } /** * Write a 16-bit signed int to an output stream being careful to * re-endianize if caller-requested endianness differs from current * host. */ static inline void writeI16(std::ostream& out, int16_t x, bool toBigEndian) { int16_t y = endianizeI16(x, toBigEndian); out.write((const char*)&y, 2); } /** * Write a 16-bit unsigned to an output stream using the native * endianness. */ static inline void writeI16(std::ostream& out, int16_t x) { out.write((const char*)&x, 2); } /** * Write a 8-bit unsigned to an output stream. */ static inline void writeU8(std::ostream& out, uint8_t x) { out.write((const char*)&x, 1); } /** * Read a 32-bit unsigned from an input stream, inverting endianness * if necessary. */ static inline uint32_t readU32(std::istream& in, bool swap) { uint32_t x; in.read((char *)&x, 4); assert_eq(4, in.gcount()); if(swap) { return endianSwapU32(x); } else { return x; } } /** * Read a 32-bit unsigned from a file descriptor, optionally inverting * endianness. */ #ifdef BOWTIE_MM static inline uint32_t readU32(int in, bool swap) { uint32_t x; if(read(in, (void *)&x, 4) != 4) { assert(false); } if(swap) { return endianSwapU32(x); } else { return x; } } #endif /** * Read a 32-bit unsigned from a FILE*, optionally inverting * endianness. */ static inline uint32_t readU32(FILE* in, bool swap) { uint32_t x; if(fread((void *)&x, 1, 4, in) != 4) { assert(false); } if(swap) { return endianSwapU32(x); } else { return x; } } /** * Read a 32-bit signed from an input stream, inverting endianness * if necessary. */ static inline int32_t readI32(std::istream& in, bool swap) { int32_t x; in.read((char *)&x, 4); assert_eq(4, in.gcount()); if(swap) { return endianSwapI32(x); } else { return x; } } /** * Read a 32-bit unsigned from a file descriptor, optionally inverting * endianness. */ #ifdef BOWTIE_MM static inline uint32_t readI32(int in, bool swap) { int32_t x; if(read(in, (void *)&x, 4) != 4) { assert(false); } if(swap) { return endianSwapI32(x); } else { return x; } } #endif /** * Read a 32-bit unsigned from a FILE*, optionally inverting * endianness. */ static inline uint32_t readI32(FILE* in, bool swap) { int32_t x; if(fread((void *)&x, 1, 4, in) != 4) { assert(false); } if(swap) { return endianSwapI32(x); } else { return x; } } /** * Read a 16-bit unsigned from an input stream, inverting endianness * if necessary. */ static inline uint16_t readU16(std::istream& in, bool swap) { uint16_t x; in.read((char *)&x, 2); assert_eq(2, in.gcount()); if(swap) { return endianSwapU16(x); } else { return x; } } /** * Read a 16-bit unsigned from a file descriptor, optionally inverting * endianness. */ #ifdef BOWTIE_MM static inline uint16_t readU16(int in, bool swap) { uint16_t x; if(read(in, (void *)&x, 2) != 2) { assert(false); } if(swap) { return endianSwapU16(x); } else { return x; } } #endif /** * Read a 16-bit unsigned from a FILE*, optionally inverting * endianness. */ static inline uint16_t readU16(FILE* in, bool swap) { uint16_t x; if(fread((void *)&x, 1, 2, in) != 2) { assert(false); } if(swap) { return endianSwapU32(x); } else { return x; } } /** * Read a 16-bit signed from an input stream, inverting endianness * if necessary. */ static inline int32_t readI16(std::istream& in, bool swap) { int16_t x; in.read((char *)&x, 2); assert_eq(2, in.gcount()); if(swap) { return endianSwapI16(x); } else { return x; } } /** * Read a 16-bit unsigned from a file descriptor, optionally inverting * endianness. */ #ifdef BOWTIE_MM static inline uint16_t readI16(int in, bool swap) { int16_t x; if(read(in, (void *)&x, 2) != 2) { assert(false); } if(swap) { return endianSwapI16(x); } else { return x; } } #endif /** * Read a 16-bit unsigned from a FILE*, optionally inverting * endianness. */ static inline uint16_t readI16(FILE* in, bool swap) { int16_t x; if(fread((void *)&x, 1, 2, in) != 2) { assert(false); } if(swap) { return endianSwapI16(x); } else { return x; } } /** * Read a 8-bit unsigned from an input stream */ static inline uint8_t readU8(std::istream& in) { uint8_t x; in.read((char *)&x, 1); assert_eq(1, in.gcount()); return x; } template void writeIndex(std::ostream& out, index_t x, bool toBigEndian) { index_t y = endianizeIndex(x, toBigEndian); out.write((const char*)&y, sizeof(index_t)); } /** * Read a unsigned from an input stream, inverting endianness * if necessary. */ template static inline index_t readIndex(std::istream& in, bool swap) { index_t x; in.read((char *)&x, sizeof(index_t)); assert_eq(sizeof(index_t), in.gcount()); if(swap) { return endianSwapIndex(x); } else { return x; } } /** * Read a unsigned from a file descriptor, optionally inverting * endianness. */ #ifdef BOWTIE_MM template static inline index_t readIndex(int in, bool swap) { index_t x; if(read(in, (void *)&x, sizeof(index_t)) != sizeof(index_t)) { assert(false); } if(swap) { if(sizeof(index_t) == 8) { assert(false); return 0; } else if(sizeof(index_t) == 4) { return endianSwapU32(x); } else { assert_eq(sizeof(index_t), 2); return endianSwapU16(x); } } else { return x; } } #endif /** * Read a unsigned from a FILE*, optionally inverting * endianness. */ template static inline index_t readIndex(FILE* in, bool swap) { index_t x; if(fread((void *)&x, 1, sizeof(index_t), in) != sizeof(index_t)) { assert(false); } if(swap) { if(sizeof(index_t) == 8) { assert(false); return 0; } else if(sizeof(index_t) == 4) { return endianSwapU32(x); } else { assert_eq(sizeof(index_t), 2); return endianSwapU16(x); } } else { return x; } } #endif /*WORD_IO_H_*/ hisat2-2.2.1/zbox.h000066400000000000000000000052751370663763200140670ustar00rootroot00000000000000/* * Copyright 2011, Ben Langmead * * This file is part of Bowtie 2. * * Bowtie 2 is free software: you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation, either version 3 of the License, or * (at your option) any later version. * * Bowtie 2 is distributed in the hope that it will be useful, * but WITHOUT ANY WARRANTY; without even the implied warranty of * MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the * GNU General Public License for more details. * * You should have received a copy of the GNU General Public License * along with Bowtie 2. If not, see . */ #ifndef ZBOX_H_ #define ZBOX_H_ #include "btypes.h" /** * Fill z with Z-box information for s. String z will not be resized * and will only be filled up to its size cap. This is the linear-time * algorithm from Gusfield. An optional sanity-check uses a naive * algorithm to double-check results. */ template void calcZ(const T& s, TIndexOffU off, EList& z, bool verbose = false, bool sanityCheck = false) { size_t lCur = 0, rCur = 0; size_t zlen = z.size(); size_t slen = s.length(); assert_gt(zlen, 0); assert_eq(z[0], 0); //assert_leq(zlen, slen); for (size_t k = 1; k < zlen && k+off < slen; k++) { assert_lt(lCur, k); assert(z[lCur] == 0 || z[lCur] == rCur - lCur + 1); if(k > rCur) { // compare starting at k with prefix starting at 0 size_t ki = k; while(off+ki < s.length() && s[off+ki] == s[off+ki-k]) ki++; z[k] = (TIndexOffU)(ki - k); assert_lt(off+z[k], slen); if(z[k] > 0) { lCur = k; rCur = k + z[k] - 1; } } else { // position k is contained in a Z-box size_t betaLen = rCur - k + 1; size_t kPrime = k - lCur; assert_eq(s[off+k], s[off+kPrime]); if(z[kPrime] < betaLen) { z[k] = z[kPrime]; assert_lt(off+z[k], slen); // lCur, rCur unchanged } else if (z[kPrime] > 0) { int q = 0; while (off+q+rCur+1 < s.length() && s[off+q+rCur+1] == s[off+betaLen+q]) q++; z[k] = (TIndexOffU)(betaLen + q); assert_lt(off+z[k], slen); rCur = rCur + q; assert_geq(k, lCur); lCur = k; } else { z[k] = 0; assert_lt(off+z[k], slen); // lCur, rCur unchanged } } } #ifndef NDEBUG if(sanityCheck) { // Recalculate Z-boxes using naive quadratic-time algorithm and // compare to linear-time result assert_eq(0, z[0]); for(size_t i = 1; i < z.size(); i++) { size_t j; for(j = i; off+j < s.length(); j++) { if(s[off+j] != s[off+j-i]) break; } assert_eq(j-i, z[i]); } } #endif } #endif /*ZBOX_H_*/

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q'/l>~&.Ww=VB-xNDŦbw8߽@[xz$7C7E^/xb-֔`8>#ڱCȡqZ֕Y]Oܵcp!!").addClass("header-link").attr("href", "#" + id).html('')); } }); }); hisat2-2.2.1/docs/assets/js/script.js000066400000000000000000000021601370663763200174320ustar00rootroot00000000000000$(function () { // focus on search input with '/' key. $("body").on("keyup", function (e) { e.stopPropagation(); var slashKeys = [47, 111, 191]; if (slashKeys.some(function (value) { return e.keyCode == value })) { $("#search").focus(); } }); // add `target="_blank"` into all outer links. var host = document.location.host; $("a[href]").each(function() { var re = new RegExp(host, "g"); if ($(this).attr("href").match(/\/\//) && !$(this).attr("href").match(re)) { $(this).attr("target", "_blank"); } }); // center and linkable all images. var $images = $("article img:not(.emoji, .eye-catch)"); $images.closest("p").css("text-align", "center"); $images.each(function () { var imgUrl = $(this).attr("src"); var $a = $("    ").attr("href", imgUrl).attr("target", "_blank"); $(this).wrap($a); }); // stick aside. var topSpacing = $(".site-aside").css("padding-top").replace(/px/, ""); $(".site-aside .sticky").sticky({ topSpacing: parseInt(topSpacing) }); }); hisat2-2.2.1/docs/assets/js/search.js000066400000000000000000000062261370663763200174020ustar00rootroot00000000000000$(function () { var query = getQuery(["q", "t", "a", "d"]); var targets; switch (query.key) { case "t": targets = ["tags"]; break; case "a": targets = ["author"]; break; case "d": targets = ["date"]; break; case "q": default: targets = ["title", "tags", "author", "url", "date", "content"]; break; } showPosts(query.words, targets); if (query.key == "q") { $("#search").val(query.query).focus(); } }); function getQuery(keys) { var query = ""; var key = ""; var words = []; keys.forEach(function (queryKey) { var regex = RegExp("[?&]" + queryKey + "=([^&]+)", 'i'); var matched; if (matched = window.location.search.match(regex)) { query = decodeURIComponent(matched[1]).replace(/(　| |\+)+/g, ' '); words = query.split(' '); key = queryKey; return false; // break; } return true; // continue; }); return { query: query, key: key, words: words }; } function showPosts(words, targets) { var getJson = function () { var dfd = $.Deferred(); $.ajax({ url: baseurl + "/search.json", dataType: "json", timeout: 3000, // 3 sec success: function (posts) { var matchedPosts = []; posts.forEach(function (post) { // concatenate target fields as a string. var searchee = ""; for (var i = 0; i < targets.length; i++) { var target = post[targets[i]]; var targetString = ""; if (target instanceof Array) { for (var j = 0; j < target.length; j++) { targetString += target[j]; } } else if (typeof target == "object") { for (key in target) { targetString += target[key]; } } else { targetString = target; } searchee += targetString; } // matching. var matched = true; words.forEach(function (word) { var regex = new RegExp(word, 'i'); if (searchee.match(regex) == null) { matched = false; return false; // break; } return true; // continue; }); if (matched) { matchedPosts.push(post); } }); dfd.resolve(matchedPosts); } }); return dfd.promise(); }; getJson().then(function (matchedPosts) { matchedPosts.forEach(function (post) { $("#search-results").find("#" + post.id).show(); }); }); } hisat2-2.2.1/docs/assets/lib/000077500000000000000000000000001370663763200157235ustar00rootroot00000000000000hisat2-2.2.1/docs/assets/lib/garand-sticky/000077500000000000000000000000001370663763200204635ustar00rootroot00000000000000hisat2-2.2.1/docs/assets/lib/garand-sticky/jquery.sticky.js000077500000000000000000000131531370663763200236530ustar00rootroot00000000000000// Sticky Plugin v1.0.0 for jQuery // ============= // Author: Anthony Garand // Improvements by German M. Bravo (Kronuz) and Ruud Kamphuis (ruudk) // Improvements by Leonardo C. Daronco (daronco) // Created: 2/14/2011 // Date: 2/12/2012 // Website: http://labs.anthonygarand.com/sticky // Description: Makes an element on the page stick on the screen as you scroll // It will only set the 'top' and 'position' of your element, you // might need to adjust the width in some cases. (function($) { var defaults = { topSpacing: 0, bottomSpacing: 0, className: 'is-sticky', wrapperClassName: 'sticky-wrapper', center: false, getWidthFrom: '', responsiveWidth: false }, $window = $(window), $document = $(document), sticked = [], windowHeight = $window.height(), scroller = function() { var scrollTop = $window.scrollTop(), documentHeight = $document.height(), dwh = documentHeight - windowHeight, extra = (scrollTop > dwh) ? dwh - scrollTop : 0; for (var i = 0; i < sticked.length; i++) { var s = sticked[i], elementTop = s.stickyWrapper.offset().top, etse = elementTop - s.topSpacing - extra; if (scrollTop <= etse) { if (s.currentTop !== null) { s.stickyElement .css('width', '') .css('position', '') .css('top', ''); s.stickyElement.trigger('sticky-end', [s]).parent().removeClass(s.className); s.currentTop = null; } } else { var newTop = documentHeight - s.stickyElement.outerHeight() - s.topSpacing - s.bottomSpacing - scrollTop - extra; if (newTop < 0) { newTop = newTop + s.topSpacing; } else { newTop = s.topSpacing; } if (s.currentTop != newTop) { s.stickyElement .css('width', s.stickyElement.width()) .css('position', 'fixed') .css('top', newTop); if (typeof s.getWidthFrom !== 'undefined') { s.stickyElement.css('width', $(s.getWidthFrom).width()); } s.stickyElement.trigger('sticky-start', [s]).parent().addClass(s.className); s.currentTop = newTop; } } } }, resizer = function() { windowHeight = $window.height(); for (var i = 0; i < sticked.length; i++) { var s = sticked[i]; if (typeof s.getWidthFrom !== 'undefined' && s.responsiveWidth === true) { s.stickyElement.css('width', $(s.getWidthFrom).width()); } } }, methods = { init: function(options) { var o = $.extend({}, defaults, options); return this.each(function() { var stickyElement = $(this); var stickyId = stickyElement.attr('id'); var wrapperId = stickyId ? stickyId + '-' + defaults.wrapperClassName : defaults.wrapperClassName var wrapper = $('