abacas-1.3.1/0000755000265600020320000000000011541611714012053 5ustar tilleaadminabacas-1.3.1/style.css0000644000265600020320000000411711203241313013715 0ustar tilleaadmin/* Generated by KompoZer */ body { margin: 0; padding: 0; background-repeat: repeat-x; background-attachment: scroll; background-position: center top; font-family: verdana,arial,sans-serif; font-style: normal; font-variant: normal; font-weight: normal; font-size: 8pt; line-height: 13pt; font-size-adjust: none; font-stretch: normal; background-color: #f9f9f9; } #wrapper { margin: 0 auto; padding: 0; width: 800px; text-align: left; } #top { background: transparent url(images/bgtop2.png) no-repeat scroll center top; width: 800px; height: 78px; } #content { padding: 0px 17px; background: transparent url(images/bgmiddle2.png) repeat-y scroll center; width: 766px; height: 100%; } #header { margin: 0px 0px 4px; padding: 60px 0px 0px 20px; background: transparent url(images/bgtop2.png) no-repeat scroll center top; width: 766px; height: 56px; color: black; font-size: 16px; } #header h1 { padding: 0px 190px 0px 125px; font-size: 18px; font-weight: normal; } #menu { width: 200px; height: 100%; margin-left: 10px; float: left; text-align: left; } #menu li a { voice-family: inherit; height: 29px; text-decoration: none; text-align: left; } #menu li a:link, #menu li a:visited { padding: 8px 0 0 10px; background: transparent url(images/off2.png) no-repeat scroll center top; color: navy; display: block; height: 29px; text-align: left; } #menu li a:hover { padding: 8px 0 0 10px; background: transparent url(images/on.png) no-repeat scroll center top; color: blue; height: 29px; text-align: left; } ul { margin: 0; padding: 0; list-style-type: none; list-style-image: none; list-style-position: outside; text-align: left; } #stuff { border: none; margin: 0px 0px 0px 220px; background-color: transparent; background-repeat: no-repeat; background-attachment: scroll; background-position: left top; width: 520px; padding-top: 55px; } img { border: none; } #bottom { background: transparent url(images/bgbottom3.png) no-repeat scroll center bottom; width: 800px; height: 50px; } abacas-1.3.1/abacas0000644000265600020320000017662311541611714013227 0ustar tilleaadmin#!/usr/bin/perl # Copyright (C) 2008-10 Genome Research Limited. All Rights Reserved. # # 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 # of the License, 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., 59 Temple Place - Suite 330, Boston, MA 02111-1307, USA. #ABACAS.1.3.1 #-------------------------------- #Please report bugs to: #sa4@sanger.ac.uk & tdo@sanger.ac.uk use strict; use warnings; use POSIX qw(ceil floor); use Getopt::Std; our $version="1.3.1"; #------------------------------------------------------------------------------- if (@ARGV < 1) { usage();} my ($help, $reference, $query_file, $choice, $sen, $seed, $mlfas, $fasta_bin, $avoid_Ns, $tbx, $min_id, $min_cov, $diff_cov, $min_len,$add_bin_2ps, $pick_primer, $flank, $chk_uniq,$redoMummer, $is_circular,$escapeToPrimers, $debug, $gaps_2file, $prefix,$optionsLog) =checkUserInput( @ARGV ); my $ref_inline; if ($escapeToPrimers ==1) { pickPrimers ($reference, $query_file, $flank, $chk_uniq); exit; } #BEGIN #------------------------------------------------------------------------------- print_header(); print $optionsLog; $ref_inline = Ref_Inline($reference); #Get length of the reference sequence my $ref_len = length($ref_inline); ################### # Running MUMmer # ################### my ($path_dir, $run_mum, $path_toPass); if ($debug) { print "the seed is $seed \n"; print "RedoMummer= ",$redoMummer."\n"; } my @do_mum_return; my $mummer_tiling; if ($redoMummer==0) { print "PREPARING DATA FOR $choice \n"; @do_mum_return = doMummer($reference, $query_file, $choice,$sen,$seed,$min_id, $min_cov, $diff_cov,$min_len, $debug, $is_circular) or die "Couldn't run MUMmer\n"; $mummer_tiling = $do_mum_return[0]; $path_dir = $do_mum_return[2]; $path_toPass = $do_mum_return[1]; } elsif ($redoMummer ==1) { print "not doing mummer\n"; my @check = checkProg ($choice); $mummer_tiling = '$choice.tiling'; $path_dir = $check[2]; $path_toPass = $check[1]; } else { print "Unknown option for -R\n"; exit; } #################################### # Processing tiling output # #################################### if ($debug) { print "Do tiling...\n"; } #-------------------------------- ################## #Do Tiling #------------------------------------------- doTiling ($mummer_tiling, $path_toPass, $path_dir,$reference, $query_file, $choice, $prefix,$mlfas, $fasta_bin, $avoid_Ns, $ref_len, $gaps_2file, $ref_inline, $add_bin_2ps, $pick_primer, $flank, $chk_uniq, $tbx); ############################################## SUB ROUTINES for CONTIG ORDERING and PRIMER DESIGN ########################################################## # Put in one file for ease of downloading. They could be placed in separate packages. #---------------------------------------------------------------------------------------------------------- #################################Contig ordering ########################################################## ######## sub help { die < -q -p [OPTIONS] -r reference sequence in a single fasta file -q contigs in multi-fasta format -p MUMmer program to use: 'nucmer' or 'promer' OR abacas -r -q -e OPTIONS -h print usage -d use default nucmer/promer parameters -s int minimum length of exact matching word (nucmer default = 12, promer default = 4) -m print ordered contigs to file in multifasta format -b print contigs in bin to file -N print a pseudomolecule without "N"s -i int mimimum percent identity [default 40] -v int mimimum contig coverage [default 40] -V int minimum contig coverage difference [default 1] -l int minimum contig length [default 1] -t run tblastx on contigs that are not mapped -g string (file name) print uncovered regions (gaps) on reference to file name -a append contigs in bin to the pseudomolecule -o prefix output files will have this prefix -P pick primer sets to close gaps -f int number of flanking bases on either side of a gap for primer design (default 350) -R int Run mummer [default 1, use -R 0 to avoid running mummer] -e Escape contig ordering i.e. go to primer design -c Reference sequence is circular EOF } ######## sub usage { die < -q -p [OPTIONS] -r reference sequence in a single fasta file -q contigs in multi-fasta format -p MUMmer program to use: 'nucmer' or 'promer' for contig ordering and primer design OR abacas -r -q -e to escape contig ordering and go directly to primer design OR abacas -h for help EOF } ######## ################## sub print_header { print " *********************************************************************************** * ABACAS: Algorithm Based Automatic Contiguation of Assembled Sequences * * * * * * Copyright (C) 2008-10 The Wellcome Trust Sanger Institute, Cambridge, UK. * * All Rights Reserved. * * * *********************************************************************************** \n"; } ######################################### sub checkUserInput{ my %options; getopts('hr:q:p:ds:mbNi:v:V:l:tg:ao:Pf:Ru:ecD', \%options); my $optionsLog="# Checking user options:"; my ($help, $reference, $query_file, $choice, $sen, $seed, $mlfas, $fasta_bin, $avoid_Ns, $tbx, $min_id, $min_cov, $diff_cov, $min_len,$add_bin_2ps, $pick_primer, $flank, $chk_uniq,$redoMummer, $is_circular,$escapeToPrimers, $debug, $gaps_2file, $prefix); if($options{h}) { $help = $options{h}; help(); } if ($options{r} && $options{q} ){ ($reference, $query_file) = ($options{r},$options{q}); $optionsLog.="\n#\t-r Reference=$reference\n#\t-q Query=$query_file\n"; }else{ usage() unless $options{e}; } if ($options{p}){ $choice = $options{p}; $optionsLog.="#\t-p $choice\n"; unless ($choice eq "nucmer" || $choice eq "promer"){ print "Unknown MuMmer function\n Please use nucmer or promer\n"; exit; } }else{ usage() unless $options{e}; } if ($options{e}){ #$escapeToPrimers) print_header(); print "Primer design selected,... escaping contig ordering\n"; $escapeToPrimers = 1; $chk_uniq = "nucmer"; $choice = ""; $optionsLog.="#\t-e Primer design selected,... escaping contig ordering\n"; }else{ $escapeToPrimers = 0; } if ($options{d}) { $sen =1; $optionsLog.="#\t-d use default setting i.e. --mumreference in $choice\n"; } else { $sen =0; $optionsLog.="#\t-d 0 use sensitive mapping in $choice i.e. --maxmatch\n"; } #print $sen , " ---sen\n"; #print $options{t}, "\n"; exit; if($options{t}) {$tbx = 1;} else {$tbx = 0;} #print $tbx, " ---tbx\n"; # if ($options{s}){ $seed = $options{s}; $optionsLog.="#\t-s seed=$seed\n"; } else{ if ($choice eq "nucmer"){ $seed = 12; } else{ $seed =4; } } if ($options{m}){ $mlfas =1; $optionsLog.="#\t-m print multifasta file of ordered contigs\n" } else { $mlfas =0; } #print $mlfas , " ---mlfasta\n"; if ($options{b}) { $fasta_bin =1; $optionsLog.="#\t-b print multifasta file of contigs in bin to file\n" } else {$fasta_bin =0;} if ($options {N}){ $avoid_Ns =1; $optionsLog.="#\t-N don't print Ns in pseudo-molecule\n" } else {$avoid_Ns=0;} if($options{i}){ $min_id=$options{i}; $optionsLog.="#\t-i $min_id is the minimum identity cutoff\n"; } else{$min_id =40; # $optionsLog.="#\t-i not defined: $min_id is the default minimum identity cutoff\n"; } if ($options{v}){ $min_cov = $options{v}; $optionsLog.="#\t-v $min_cov is the minimum contig-coverage cutoff\n"; } else{ $min_cov =40; #$optionsLog.="#\t-v not defined $min_id is the default contig-coverage cutoff\n"; } if($options{V}){ $diff_cov = $options{V}; $optionsLog.="#\t-V $diff_cov\n"; } else {$diff_cov =1; # $optionsLog.="#\t-V $diff_cov using the default value\n"; } if ($options {l}){ $min_len = $options {l}; $optionsLog.="#\t-l $min_len is the minimum length of contigs to be ordered\n"; } else{$min_len = 1; # $optionsLog.="#\t-l not defined: using 1 as the default minimum length of contigs to be ordered\n"; } if ($options{a}) {$add_bin_2ps = 1; }else {$add_bin_2ps =0;} if ($options{P}) {$pick_primer=1;} else {$pick_primer =0;} if ($options{f}) {$flank = $options{f};} else {$flank = 1000;} if ($options{u}) {$chk_uniq = $options{u}; } else {$chk_uniq = "nucmer";} #unless ($options{R}) {$re} if($options{R}) {$redoMummer = 1; } else {$redoMummer = 0;} if($options{c}) {$is_circular = 1;}else {$is_circular =0;} if ($options{g}) {$gaps_2file = $options{g};} else {$gaps_2file ="";} if($options{o}) {$prefix = $options{o};} else {$prefix = "";} if($options{D}){ $debug=1; $optionsLog.="#\t-D debug\n"; } else {$debug =0}; if ($tbx ==1 && $fasta_bin !=1) { print "ERROR: Please use -t -b if you want to run tblastx on contigs in bin\n"; exit; } # print $redoMummer , "\n"; exit; $optionsLog.="#\tInput checking done!!\n"; #print $optionsLog; return ($help, $reference, $query_file, $choice, $sen, $seed, $mlfas, $fasta_bin, $avoid_Ns, $tbx, $min_id, $min_cov, $diff_cov, $min_len,$add_bin_2ps, $pick_primer, $flank, $chk_uniq,$redoMummer, $is_circular,$escapeToPrimers, $debug, $gaps_2file, $prefix,$optionsLog); } ## end of checkUserInput ############# ## get the reference sequence in one line #-------------------------------------------------- sub Ref_Inline { my $ref = shift; open (refFH, $ref) or die "Could not open file $ref\n"; my $seq =""; my @r = ; my $num_chr =0; foreach(@r){ if ($_ =~ /\>/){ $num_chr +=1; } } if ($num_chr > 1){ print "\nERROR: Please use a single fasta reference file. You can simply merge chromosomes in to a union fasta file.\n\n"; exit; } shift @r; foreach(@r){ chomp; $seq = $seq.$_; } return $seq; } ################ # Run mummer #-------------------------------------------- sub doMummer { my ($reference, $query_file, $choice, $sen,$seed,$min_id, $min_cov, $diff_cov,$min_len, $debug, $is_circular ) = @_; my $df = 'delta-filter'; my $st = 'show-tiling'; my $ask = 'which'; my ($path_toPass, $run_mum); # params to return... my ($command, $Path, $dir) = checkProg($choice); my ($run_df, $df_path, $df_dir) = checkProg($df); my ($run_st, $st_path, $st_dir) = checkProg($st); my (@running, @deltaRes, @coordsRes); if ($choice eq "nucmer") { if ($sen ==0) { @running = `$command --maxmatch -l $seed -p $choice $reference $query_file &> /dev/null`; @deltaRes = `$run_df -q $choice.delta >$choice.filtered.delta`; if ($is_circular == 1) { @coordsRes = `$run_st -c -i $min_id -v $min_cov -V $diff_cov -l $min_len -R -u unused_contigs.out $choice.filtered.delta > $choice.tiling`; } else { @coordsRes = `$run_st -i $min_id -v $min_cov -V $diff_cov -l $min_len -R -u unused_contigs.out $choice.filtered.delta > $choice.tiling`; } } else { @running = `$command -p $choice $reference $query_file &> /dev/null`; @deltaRes = `$run_df -q $choice.delta >$choice.filtered.delta`; if ($is_circular ==1) {@coordsRes = `$run_st -c -i $min_id -v $min_cov -V $diff_cov -l $min_len -R -u unused_contigs.out $choice.filtered.delta > $choice.tiling`;} else { @coordsRes = `$run_st -i $min_id -v $min_cov -V $diff_cov -l $min_len -R -u unused_contigs.out $choice.filtered.delta > $choice.tiling`;} } } else { if ($sen ==0) { @running = `$command --maxmatch -l $seed -x 1 -p $choice $reference $query_file &> /dev/null`; @deltaRes = `$run_df -q $choice.delta >$choice.filtered.delta`; if ($is_circular == 1) { @coordsRes= `$run_st -c -i $min_id -v $min_cov -V $diff_cov -l $min_len -R -u unused_contigs.out $choice.filtered.delta > $choice.tiling`; } else { @coordsRes= `$run_st -i $min_id -v $min_cov -V $diff_cov -l $min_len -R -u unused_contigs.out $choice.filtered.delta > $choice.tiling`; } } else { @running = `$command -l $seed -p $choice $reference $query_file &> /dev/null`; @deltaRes = `$run_df -q $choice.delta >$choice.filtered.delta`; if ($is_circular == 1) { @coordsRes= `$run_st -c -i $min_id -v $min_cov -V $diff_cov -l $min_len -R -u unused_contigs.out $choice.filtered.delta > $choice.tiling`; } else { @coordsRes= `$run_st -i $min_id -v $min_cov -V $diff_cov -l $min_len -R -u unused_contigs.out $choice.filtered.delta > $choice.tiling`; } } } my $Coordsfull= "$choice.tiling"; return ($Coordsfull,$Path, $dir); } ## ############################################################################# sub checkProg{ #checks if a given excutable is in the path... my $prog = shift; my $ask = 'which'; my @check_prog = `$ask $prog`; my $path_toPass; my $path_dir; my $command; if (defined $check_prog[0] && $check_prog[0] =~ /$prog$/) { $path_toPass = $prog; $command = $prog; } else { print "\nENTER the directory for your ", $prog, " executables [default ./]: "; my $path=; chomp $path; $path_dir = $path; if ($path_dir =~/\/$/) { $path_dir = $path_dir; } else { $path_dir = $path_dir."/"; } my @final_check = `$ask $command`; if (exists $final_check[0] && $final_check[0] =~ /$prog$/) { $command = $path_dir.$prog; $path_toPass = $command; } else { print "ERROR: Could not run ", $prog, ", please check if it is installed in your path\n or provide the directory \n"; exit; } } return ($command, $path_toPass, $path_dir); } ############################## # converts a fasta file to an ordered single line #-------------------------------------------------- sub Fasta2ordered { if( @_ != 1 ) { print "Usage: Fasta2ordered \n"; exit; } my $fast = shift; #print $fast; exit; my @fasta = split (/\n/, $fast); if ($fasta[0] =~ /\>/ ) #remove chromosome name if exists in the onput sequence. { my $ch_name = $fasta[0]; shift @fasta; } #print $fasta[0]; exit; foreach(@fasta){chomp;} my $num_lines = scalar(@fasta); my $dna = ''; for(my $i=0; $i< $num_lines; $i+=1) { $dna = $dna.$fasta[$i]; } my $ordered_dna = $dna; return $ordered_dna; } ############################################################## # Hash input contigs #------------------------------------------------ sub hash_contigs { if( @_ != 1 ) { print "Usage: hash_contigs contigs_file"; exit; } my $contigs_file = shift; if( $contigs_file =~ /\.gz$/ ) { open(CONTIGS, $contigs_file, "gunzip -c $contigs_file |" ) or die "Cant open contigs file: $!\n"; } else { open( CONTIGS, $contigs_file) or die "Cant open contigs file: $!\n"; } my %contigs_hash; # hash to store contig names and sequences my $contigName; while () ##add error checking... { if (/^>(\S+)/) { $contigName=$1; } else { chomp; $contigs_hash{$contigName} .= $_; } } close(CONTIGS); #tdo ## check if qual exists my %contigs_qual_hash; if (-r "$contigs_file.qual" or -r "$contigs_file.qual.gz") { if( -r "$contigs_file.qual.gz" ) { open(CONTIGS, "$contigs_file.qual.gz", "gunzip -c $contigs_file.qual.gz |" ) or die "Cant open contigs file: $!\n"; } else { open( CONTIGS, "$contigs_file.qual" ) or die "Cant open contigs file: $!\n"; } while () { if (/^>(\S+)/) { $contigName=$1; } else { chomp; $contigs_qual_hash{$contigName} .= $_; } } } # end tdo # end if exist return (\%contigs_hash,\%contigs_qual_hash); } ####################### ############################## ### it gets a delta name sub getMummerComparison{ my $deltaName = shift; ### transform the delta file to coords my $call ="show-coords -H -T -q $deltaName > $deltaName.coords "; !system(" $call") or die "Problems doing the show-coords comparison: $call $!\n"; ### willh old results my %h; ### has as index the postion with the max hits my %h_max; my $tmp=0; my $tmp_index; my $key=''; my $is_promer =0; if ($deltaName =~/^promer/) { $is_promer =1; } open (F,"$deltaName.coords") or die "Problem in getComparisonFile to open file $deltaName.coords\n"; my @File=; my @a; @a=split(/\s+/,$File[0]); $tmp=$a[5]; $tmp_index=$a[0]; if ($is_promer ==1) { $key=$a[12]; ## nucmer: $key = $a[8] } else { $key = $a[8]; } foreach (@File) { @a=split(/\s+/); if ($is_promer ==1) { push @{ $h{$a[12]}}, "$a[12]\t$a[11]\t$a[7]\t$a[5]\t0\t0\t$a[2]\t$a[3]\t$a[0]\t$a[1]\t1\t$a[5]\n"; if ($key eq $a[12] and $a[5]>$tmp) { $tmp=$a[5]; # length $tmp_index=$a[0]; # position reference } elsif ($key ne $a[12]) { ### here possible bugg... $h_max{$tmp_index}=$key; $key=$a[12]; $tmp=$a[5]; # length $tmp_index=$a[0]; # position reference } } #end if #else i.e. if nucmer else { push @{ $h{$a[8]}}, "$a[8]\t$a[6]\t$a[6]\t$a[5]\t0\t0\t$a[2]\t$a[3]\t$a[0]\t$a[1]\t1\t$a[5]\n"; if ($key eq $a[8] and $a[5]>$tmp) { $tmp=$a[5]; # length $tmp_index=$a[0]; # position reference } elsif ($key ne $a[8]) { ### here possible bugg... $h_max{$tmp_index}=$key; $key=$a[8]; $tmp=$a[5]; # length $tmp_index=$a[0]; # position reference } } } $h_max{$tmp_index}=$key; # print Dumper %h_max; return (\%h,\%h_max); } ################################## ########################### sub writeBinContigs2Ref{ my $nameBin = shift; my $name = shift; open (F, "$nameBin") or die "Couldn't find file $nameBin: $!\n"; my @ar; my $count=0; while () { push @ar, $_; $count++; } #### sa4: added error checking:- if file is empty if (scalar(@ar) < 1) { print "No contigs in unusedcontigs file\n"; $count = 0; } else { open (F, "> $name.notMapped.contigs.tab") or die "Couldn't write file $name.tab: $!\n"; print F doArt(\@ar); close(F); } return $count; } ############################## sub doArt{ my ($ref) = @_; ## hash of array with all positions of the contig my %Pos; ## Hash with note of result line of nucmer my %lines; foreach (@$ref) { chomp; my @ar=split(/\t/); push @{ $Pos{$ar[12]}}, "$ar[0]..$ar[1]"; $lines{$ar[12]} .= "FT $_\n"; } my $res; foreach my $contig (keys %lines) { if (scalar(@{ $Pos{$contig} } >1)) { my $tmp; foreach (@{ $Pos{$contig} }) { $tmp.="$_,"; } $tmp =~ s/,$//g; # get away last comma $res .= "FT contig join($tmp)\n"; } else { $res .= "FT contig $Pos{$contig}[0]\n"; } $res .= "FT /systematic_id=\"$contig\"\n"; $res .= "FT /note=\"Contig $contig couldn't map perfectly.\n"; $res .= $lines{$contig}; $res .= "FT \"\n"; } return $res; } ########################################################################## #--------------------------- sub makeN #creates a string of Ns { my $n = shift; my $Ns= "N"; for (my $i =1; $i < $n; $i+=1) { $Ns = $Ns."N"; } return $Ns; } ########################################################################### ## reverse complement a sequence #--------------------------------------------- sub revComp { my $dna = shift; my $revcomp = reverse($dna); $revcomp =~ tr/ACGTacgt/TGCAtgca/; return $revcomp; } ################################################################################ ### function to visualize rep. regions in Reference genome. sub findRepeats { my $reference = shift; my $name = shift; my $path_prog = shift; # get path my ($path_coords) = $path_prog; $path_coords =~ s/nucmer/show-coords/; my $call = "$path_prog --maxmatch -c 100 -b 40 -p $name.repeats -l 25 $reference $reference &> /dev/null "; !system("$call") or die "Problems doing the nucmer comparison: $call $!\n"; $call ="$path_coords -r -c -l $name.repeats.delta > $name.repeats.coords "; !system(" $call") or die "Problems doing the show-coords comparison: $call $!\n"; my @Res; open (F, "$name.repeats.coords" ) or die "Problems to open file $reference.repeats.coords: Is MUMmer installed correctly and inserted in the PATH environment variable? ($!)\n"; $_=; $_=; $_=; $_=;$_=; while () { my @ar = split(/\s+/); if (!($ar[1] == $ar[4] or $ar[2] == $ar[5] or $ar[7] > 100000)) { # to exclude self alignment foreach ($ar[1]..$ar[2]) { $Res[($_-1)]++; } } } ### write the result to the plot file my $res; foreach (@Res){ if (defined($_)) { $res .= "$_\n"; } else { $res .= "0\n"; } } open (F, "> $name.Repeats.plot") or die "Couldn't open file $name.plot to write: $! \n"; print F $res; close(F); ### delete files unlink("$name.repeats.delta"); unlink("$name.repeats.coords"); unlink("$name.repeats.cluster"); } ################################################################################ # reverse a list of qualities #------------------------------ sub reverseQual{ my $str = shift; $str =~ s/\s+$//g; my @ar = split(/\s/,$str); my $res; for (my $i=(scalar(@ar)-1);$i>=0;$i--) { $res.="$ar[$i] "; } return $res; } ############################################################################## # ---------------- sub getPosCoords{ my $ref_ar = shift; my $contig = shift; my $offset = shift; my $res; # print Dumper $$ref_ar{$contig}; foreach (@{$$ref_ar{$contig}}) { print "in getPos Coords: $_\n";; my @ar=split(/\t/); $ar[6]+=$offset; $ar[7]+=$offset; $res .= join("\t",@ar);; } return $res; } ############################################################################# # ----------------------------- sub getPosCoordsTurn{ my $ref_ar = shift; my $contig = shift; my $offset = shift; my $res; # print Dumper $$ref_ar{$contig}; foreach (@{$$ref_ar{$contig}}) { my @ar=split(/\t/); my $tmp_8=$ar[8]; my $tmp_9=$ar[9]; $ar[8]=$ar[6]+$offset; $ar[9]=$ar[7]+$offset; $ar[6]=$tmp_8; $ar[7]=$tmp_9; ## change query subject $res .= join("\t",@ar);; } return $res; } ############################################################################ #------------------------ sub printStats{ my ($num_fortillingcontigs,$num_notsetTilling,$num_mapped, $num_contigs, $num_inComparisoncontigs, $ref_len, $total_bases_mpd) = @_; $num_fortillingcontigs=$num_notsetTilling+$num_mapped; my $res; $res.= "Short statistics of run.\n"; $res.= "$num_contigs\t\tcontigs entered to be mapped against the reference.\n"; $res.= sprintf("$num_inComparisoncontigs\t%0.2f \%\tmade a hit against the reference to the given parameter (-s -d etc)\n",($num_inComparisoncontigs*100/$num_contigs)); $res.= sprintf("$num_fortillingcontigs\t%0.2f \%\twere considered for the tilling graph (-s -d etc)\n",($num_fortillingcontigs*100/$num_contigs)); $res.= sprintf("$num_mapped\t%0.2f \%\tare mapped in the tilling graph (-s -d etc)\n",($num_mapped*100/$num_contigs)); $res.= sprintf("\nCoverage: The reference is $ref_len long. Up to $total_bases_mpd bp (%0.2f \%) are covered by the contigs (This doesn't mean that these regions are really similar...)\n",($total_bases_mpd*100/$ref_len)); print $res; } ################################################## #### Do Tiling #---------------------------------------------------- sub doTiling { my ($mummer_tiling, $path_toPass, $path_dir,$reference, $query_file, $choice, $prefix,$mlfas, $fasta_bin, $avoid_Ns, $ref_len, $gaps_2file, $ref_inline, $add_bin_2ps, $pick_primer, $flank, $chk_uniq, $run_blast) = @_; ### these are also defined in the main script.... to be changed!! my ($num_contigs , $num_inbincontigs, $avg_cont_size,$num_overlaps , $num_gaps, $num_mapped,$total_bases_mpd,$p_ref_covered,$num_ambigus,$num_inComparisoncontigs, $num_fortillingcontigs,$num_notsetTilling)= (0,0,0,0,0,0,0,0,0,0,0,0); my ($href, $ref_contigs_qual) = hash_contigs($query_file); my $qualAvailable=0; my %contigs_hash = %{$href}; my @c_keys = keys %contigs_hash; $num_contigs = scalar(@c_keys); my @cont_lens; my (@ids ,$id, $id_len); my (@Rs, @Re, @G, @Len, @cov, @pid, @orient, @Cid); my (@Ps, @Pe); my ($total); my $g; #define gap size between contigs my $tiling_gap; #gap size from tiling graph open (TIL, $mummer_tiling) or die "Could not open $mummer_tiling: $!"; while () { chomp; if ($_ =~/^>/) { my $line = substr $_, 1; my @splits = split /\s+/, $line; $id = $splits[0]; push @ids, $id; $id_len= $splits[1]; } else { my @splits = split /\s+/, $_; push @Rs, $splits[0]; push @Re, $splits[1]; push @G, $splits[2]; push @Len, $splits[3]; push @cov, $splits[4]; push @pid, $splits[5]; push @orient, $splits[6]; push @Cid, $splits[7]; } } close (TIL); if (scalar(@Rs) != scalar(@Re)) { print "ERROR: unequal array size\n"; exit; } else { $total = scalar(@Rs); $num_mapped = scalar(@Rs); } my $ref_loc = $reference; # get locations of reference and query files my $qry_loc = $query_file; my $dif_dir =0; #assume query and reference are in the working directory my @splits_reference = split (/\//, $reference); my $new_reference_file = $splits_reference[(scalar(@splits_reference)-1)]; my @splits_query = split (/\//, $query_file); my $new_query_file = $splits_query[(scalar(@splits_query)-1)]; if ($prefix eq "") { $prefix = $new_query_file."_".$new_reference_file; } #------------------------------------------------------------------- #define file handles for output files and open files to write output #------------------------------------------------------------------- my ($seqFH,$tabFH,$binFH,$crunchFH, $gapFH, $gapFHT, $mlFH, $dbinFH, $avoidNFH,$ref_gapsFH); open ($seqFH, '>', $prefix . '.fasta') or die "Could not open file $prefix.fasta for write: $!\n"; open ($tabFH, '>', $prefix . '.tab') or die "Could not open file $prefix.tab for write: $!\n"; open ($binFH, '>', $prefix . '.bin') or die "Could not open file $prefix.bin for write: $!\n"; open ($crunchFH, '>', $prefix . '.crunch') or die "Could not open file $prefix.crunch for write: $!\n"; open ($gapFH, '>', $prefix . '.gaps') or die "Could not open file $prefix.gaps for write: $!\n"; open ($gapFHT, '>', $prefix . '.gaps.tab') or die "Could not open file $prefix.gaps.tab for write: $!\n"; if ($mlfas ==1) { open ($mlFH, '>', $prefix . '.MULTIFASTA.fa') or die "Could not open file $prefix.contigs.fas for write: $!\n"; } if ($fasta_bin ==1) { open ($dbinFH, '>', $prefix . '.contigsInbin.fas') or die "Could not open file $prefix.contigsInbin.fas for write: $!\n"; } if ($avoid_Ns ==1) { open ($avoidNFH, '>', $prefix .'.NoNs.fasta') or die "Could not open file $prefix.NoNs.fasta for write: $!\n"; } if ($gaps_2file ne "") { open ($ref_gapsFH, '>', $gaps_2file.'.Gaps_onRef') or die "Could not open file $gaps_2file.Gaps_onRef for write: $!\n"; } #------------------------------------------------------------------------- # Writing tiling graph and generating a pseudomolecule # Note use use ps for pseudomolecule #@Ps = start of ps, and @Pe = end of ps my $ps_start =1; $Ps[0] = 1; $Pe[0] = $Ps[0] + $Len[0] -1; my $tmp_qual; my $tmp_nqual; my $tmp_seq =""; my $tmp_nseq =""; print "Total contigs = $total \n"; #------------------------------------------------------------ # The 'for loop' loops over each contig in the Tiling output #Writing to file is done for each contig to speed up the process #This part could potentially be a separate subroutine print $tabFH "ID ",$id, "\n"; print $seqFH ">", "ordered_", $id, "\n"; print $gapFHT "ID ",$id, "\n"; for (my $i=1; $i <= $total; $i+=1) { my $covv =sprintf("%.0f",$cov[$i -1]); #ROUNDING my $pidd = sprintf("%.0f", $pid[$i -1]); my ($contig_coord, $color, $contig_seq); my $contig_qual=''; $tiling_gap = $G[$i -1]; if ($tiling_gap <= 1){ #insert 100Ns for overlaps and gaps of size less than or equal to one base $g = 99; # default gap size to } else{ $g = $tiling_gap; } if (defined($Len[$i])) { $Ps[$i] = $Pe[$i-1] +$g +1; $Pe[$i] = $Ps[$i] + $Len[$i] -1; $total_bases_mpd+=$Len[$i]; } if ($Rs[$i -1] <0) #check if a reference starting position is less than 0 { $Rs[$i -1] =1; } if($orient[$i-1] eq "+") { $contig_coord = $Ps[$i -1]."..".$Pe[$i-1]; $color = 4; $contig_seq = $contigs_hash{$Cid[$i-1]}; } else { $contig_coord = "complement(".$Ps[$i -1]."..".$Pe[$i-1].")"; $color =3; $contig_seq = revComp($contigs_hash{$Cid[$i-1]}); #REVERSE COMPLEMENT A SEQUENCE } push (@cont_lens, length($contig_seq)); # tdo if (defined($$ref_contigs_qual{$Cid[$i-1]})) { ## flag to know, that the qual exists $qualAvailable=1; $contig_qual = $$ref_contigs_qual{$Cid[$i-1]}; } $tmp_qual .= $contig_qual; $tmp_seq .= $contig_seq; if ($avoid_Ns ==1) { $tmp_nseq.= $contig_seq; #tdo $tmp_nqual .= $contig_qual; } if ($mlfas ==1) { my $multifasta_seq = write_Fasta ($contig_seq); print $mlFH ">", $Cid[$i-1], "\n", $multifasta_seq; } if ($Re[$i -1] > $ref_len) { $Re[$i -1] = $ref_len -1; } if ($Pe[$i -1] > length($tmp_seq)) { $Pe[$i -1] = length($tmp_seq); } #----------------------------------------- print $crunchFH $covv, " ", $pidd, " ", $Ps[$i -1], " ", $Pe[$i -1], " ", $Cid[$i -1], " ", $Rs[$i -1], " ", $Re[$i-1], " ", "unknown NONE\n"; #WRITE FEATURE FILE print $tabFH "FT contig ",$contig_coord, "\n"; print $tabFH "FT ", "/systematic_id=\"", $Cid[$i-1],"\"","\n"; print $tabFH "FT ", "/method=\"", "mummer\"", "\n"; print $tabFH "FT ", "/Contig_coverage=\"",$cov[$i -1], "\"", "\n"; print $tabFH "FT ", "/Percent_identity=\"",$pid[$i -1], "\"", "\n"; my ($gap_coord, $gapCol,$gap_start,$gap_end,$ref_start, $ref_end) ; $gap_start = $Pe[$i -1] +1; if (defined $Ps[$i]) { $gap_end = $Ps[$i] -1; } else { $gap_end = $gap_start + ($g) -1 ; ############ ...... check..... } $ref_start = $Re[$i -1] +1; if (defined $Rs[$i]) { $ref_end =$Rs[$i]-1; } else { $ref_end = "END"; } $gap_coord = $gap_start."..".$gap_end; my $ov =""; if ($tiling_gap > 1) #WRITE GAP LOCATIONS AND SIZE TO FILE { print $gapFH "Gap\t",$tiling_gap, "\t", $gap_start, "\t", $gap_end, "\t", $ref_start, "\t", $ref_end,"\tNON-Overlapping\n"; $ov = "NO"; $gapCol = 8; if ($gaps_2file ne "" && $ref_start < $ref_len) { my $gapOnref = substr ($ref_inline, $ref_start, $g); print $ref_gapsFH ">StartOnRef_",$ref_start, " [Gap=",$g,"]\n"; my $file_toPrint = write_Fasta ($gapOnref); print $ref_gapsFH $file_toPrint; } } else { $color = 5; $gapCol =9; $ov ="YES"; print $gapFH "Gap\t",$g, "\t", $gap_start, "\t", $gap_end, "\t", $ref_start, "\t", $ref_end,"\tOverlapping\n"; print $tabFH "FT ", "/Overlapping=\"", "YES\"", "\n"; } print $gapFHT "FT GAP ",$gap_coord, "\n"; print $gapFHT "FT ", "/SIZE=\"", $g,"\"","\n"; print $gapFHT "FT ", "/Overlapping=\"", $ov, "\"","\n"; print $gapFHT "FT ", "/colour=\"",$gapCol, "\"", "\n"; print $tabFH "FT ", "/colour=\"",$color, "\"", "\n"; my $ns = makeN($g); $tmp_seq = $tmp_seq.$ns; #tdo for (1..length($ns)) { $tmp_qual .= "0 "; } } #------------------------------------------------------------------ #tdo my @Quality_Array; if ($qualAvailable) { @Quality_Array = split(/\s/,$tmp_qual); my $res; foreach (@Quality_Array) { $res .= "$_\n"; } ## get name my @splits_query = split (/\//, $query_file); $new_query_file = $splits_query[(scalar(@splits_query)-1)]; open (F,"> $new_query_file.qual.plot") or die "problems\n"; print F $res; close(F); } ##WRITE PSEUDOMOLECULE WITHOUT 'N's #-------------------------------------- if ($avoid_Ns ==1) { print $avoidNFH ">", "ordered_", $id, "without 'N's","\n"; my $toWrite = write_Fasta ($tmp_nseq); print $avoidNFH $toWrite; } #################################### #WRITE CONTIGS WITH NO HIT TO FILE # ################################# my %Cids; foreach(@Cid) { chomp; $Cids{$_} = 1; } my @contigs_2bin = (); my %h_contigs_2bin; foreach (@c_keys) { push(@contigs_2bin, $_) unless exists $Cids{$_}; } foreach(@contigs_2bin) { $h_contigs_2bin{$_}=1; print $binFH "$_ \n"; } $num_inbincontigs= scalar(@contigs_2bin); ######## # WRITE PSEUDOMOLECULE TO FILE #---------------------------------- my $new_seq = $tmp_seq; my $prev_len = length($tmp_seq); my $total_len = $prev_len; foreach (@contigs_2bin) { chomp; #my $binseq = $contigs_hash{$contigs_2bin[$i]}; my $l = length ($contigs_hash{$_}); $total_len +=$l; } if ($add_bin_2ps ==1) #appending unmapped contigs to pseudomolecule { for (my $i =0; $i < scalar(@contigs_2bin); $i+=1) { my $binseq = $contigs_hash{$contigs_2bin[$i]}; $new_seq .=$contigs_hash{$contigs_2bin[$i]}; my $len_current_contig = length($binseq); my $start = $prev_len +1; my $end = $start + $len_current_contig -1; my $col = 7; if ($start > $total_len) { $start = $total_len; } if ($end >$total_len) { $end = $total_len; } my $co_cord = $start."..".$end; my $note = "NO_HIT"; print $tabFH "FT contig ",$co_cord, "\n"; print $tabFH "FT ", "/systematic_id=\"", $contigs_2bin[$i],"\"","\n"; print $tabFH "FT ", "/method=\"", "mummer\"", "\n"; print $tabFH "FT ", "/colour=\"",$col, "\"", "\n"; print $tabFH "FT ", "/", $note, "\n"; $prev_len= $end; } } my $to_write = write_Fasta ($new_seq); print $seqFH $to_write; ######## #WRITE CONTIGS IN BIN TO FILE # #------------------------------------------------------ if ($fasta_bin ==1) { foreach(@contigs_2bin) { print $dbinFH ">", $_, "\n"; my $to_write = write_Fasta($contigs_hash{$_}); print $dbinFH $to_write; } } #unlink ("$choice.delta"); #unlink ("$choice.filtered.delta"); #unlink ("$choice.cluster"); #unlink ("$choice.tiling"); #PRINT FINAL MESSAGE print " FINISHED CONTIG ORDERING\n"; print "\nTo view your results in ACT\n\t\t Sequence file 1: $new_reference_file\n\t\t Comparison file 1: $prefix.crunch\n\t\t Sequence file 2: $prefix.fasta\n \t\tACT feature file is: $prefix.tab\n \t\tContigs bin file is: $prefix.bin\n \t\tGaps in pseudomolecule are in: $prefix.gaps\n\n"; #Run tblastx.... if ($run_blast ==1) { print "Running tblastx on contigs in bin...\nThis may take several minutes ...\n"; my $formatdb = 'formatdb -p F -i' ; # my @formating = ` !system("$formatdb $new_reference_file") or die "ERROR: Could not find 'formatdb' for blast\n"; my $blast_opt = 'blastall -m 9 -p tblastx -d '; my $contigs_inBin = $prefix.'.contigsInbin.fas'; # my @bigger_b = ` !system("$blast_opt $new_reference_file -i $contigs_inBin -o blast.out") or die "ERROR: Could not find 'blastall' , please install blast in your working path (other dir==0)\n$blast_opt $new_reference_file -i $contigs_inBin -o blast.out\n \n"; } if ($pick_primer == 1) { print " DESIGNING PRIMERS FOR GAP CLOSURE...\n"; my $qq = "$prefix.fasta"; pickPrimers($qq, $reference, $flank, $path_toPass, $chk_uniq,$qualAvailable,@Quality_Array); } } #------------------------------ sub write_Fasta { my $sequence = shift; my $fasta_seq =""; my $length = length($sequence); if ($length <= 60) { $fasta_seq = $sequence."\n"; } elsif ($length> 60 ) { for (my $i =0; $i < $length; $i+=60) { my $tmp_s = substr $sequence, $i, 60; $fasta_seq .= $tmp_s."\n"; } } return $fasta_seq; } #---------------------------------------------- END OF CONTIG ORDERING SUBROUTINES---------------------------------------------------- #----------------------------------------------- PRIMER DESIGN --------------------------------------------------- sub pickPrimers { #$ps = pseudo molecule,$rf = reference, $flan = flanking region size my ($ps,$rf, $flan, $passed_path, $chk_uniq,$qualAvailable, @Quality_Array); if (@_==4){ ($rf,$ps, $flan, $chk_uniq) = @_; print "Primers without ordering..\n"; print $rf; $passed_path = "nucmer"; $qualAvailable =0; @Quality_Array = []; } else #(@_ == 7) { ($ps,$rf, $flan, $passed_path, $chk_uniq, $qualAvailable, @Quality_Array) = @_; } my $dna=''; my @gappedSeq; my $records=''; my @sequence; my $input=''; #tdo my @gappedQual; #my $quality=''; my $path_toPass = $passed_path; my @fasta; my $query=''; my @exc_regions; my $ch_name; #my $flank = $flan; open (FH, $rf) or die "Could not open reference file\n"; open (FH2, $ps) or die "Could not open query/pseudomolecule file\n"; my $ref; #print ".... ", $rf; exit; my @r = ; my @qry = ; my $dn = join ("", @qry); $ref = join ("", @r); $dna = Fasta2ordered ($dn); #check if primer3 is installed my $pr3 = "primer3_core"; my ($pr3_path, $pr3_Path, $pr3_path_dir) = checkProg ($pr3); #my @check_prog = `which primer3_core`; open (PRI, '>primer3.summary.out') or die "Could not open file for write\n"; # #print $ref; exit; #PARSING FOR PRIMER3 INPUT my ($opt,$min,$max,$optTemp,$minTemp,$maxTemp,$flank,$lowRange,$maxRange,$gcMin,$gcOpt,$gcMax,$gclamp,$exclude,$quality) = getPoptions($qualAvailable); my ($gap_position,@positions, %seq_hash); my $exc1 = $flank -$exclude; #start of left exclude print "Please wait... extracting target regions ...\n"; #regular expression extracts dna sequence before and after gaps in sequence (defined by N) while($dna=~ /([atgc]{$flank,$flank}N+[atgc]{$flank,$flank})/gi) { $records= $1; push (@gappedSeq, $records); $gap_position = index($dna, $records); push @positions, $gap_position; $seq_hash{$gap_position}=$records; #dna if ($qualAvailable) { my $res; for (my $nn=($gap_position-1); $nn <= ($gap_position-1+length($records)-1); $nn++) { $res.="$Quality_Array[$nn] "; } push @gappedQual, $res; } } #loop prints out primer targets into a file format accepted by primer3 my $count=1; my $identify=''; my $seq_num = scalar @gappedSeq; my $name= " "; my ($totalp, @left_name, @right_name, @left_seq, @right_seq); my ($leftP_names, $rightP_names, $leftP_seqs, $rightP_seqs, $leftP_start, $leftP_lens, $rightP_ends, $rightP_lens,$left_Exclude,$right_Exclude, $primers_toExc, $prod_size)= ("","","","","","","","","","", "", ""); print $seq_num, " gaps found in target sequence\n"; print "Please wait...\nLooking for primers...\n"; print "Running Primer3 and checking uniquness of primers...\nCounting left and right primer hits from a nucmer mapping (-c 15 -l 15)\n"; for (my $i=0; $i<$seq_num; $i+=1) { $identify = $count++; if (defined $ch_name) { $name = $ch_name; } my $len = length($gappedSeq[$i]); my $exc2 = $len - $flank; open(FILE, '>data') or die "Could not open file\n"; #tdo my $qual=''; if ($qualAvailable) { $qual="PRIMER_SEQUENCE_QUALITY=$gappedQual[$i]\nPRIMER_MIN_QUALITY=$quality\n"; } #WARNING: indenting the following lines may cause problems in primer3 print FILE "PRIMER_SEQUENCE_ID=Starting_Pos $positions[$i] SEQUENCE=$gappedSeq[$i] PRIMER_OPT_SIZE=$opt PRIMER_MIN_SIZE=$min PRIMER_MAX_SIZE=$max PRIMER_OPT_TM=$optTemp PRIMER_MIN_TM=$minTemp PRIMER_MAX_TM=$maxTemp PRIMER_NUM_NS_ACCEPTED=1 PRIMER_PRODUCT_SIZE_RANGE=$lowRange-$maxRange PRIMER_MIN_GC=$gcMin PRIMER_GC_CLAMP =$gclamp PRIMER_OPT_GC_PERCENT=$gcOpt PRIMER_MAX_GC=$gcMax PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION=$exc1,$exclude $exc2,$exclude ".$qual."Number To Return=1 =\n"; close FILE; #runs primer3 from commandline ################# NOTE: PRIMER3 SHOULD BE IN YOUR WORKING PATH ######### my @Pr3_output = `$pr3_path -format_output ./left_query'); # open a file for left primers print QRY_1 ">left_01\n"; # print QRY_1 $left_primer,"\n"; open (QRY_2, '>./right_query'); print QRY_2 ">right_01\n"; print QRY_2 $right_primer,"\n"; my ($left_count, $right_count); #if ($chk_uniq eq "nucmer") #{ my $options = "-c 15 --coords -l 15 "; my $rq = "right_query"; my $lq = "left_query"; my (@right_ps, @left_ps); # print $path_toPass, "\t", $options, "\n"; my @Rrun = `$path_toPass $options -p R $rf $rq &> /dev/null`; print "."; my $f1 = "R.coords"; open (RP, $f1) or die "Could not open file $f1 while checking uniqueness of right primer\n"; while () { chomp; if ($_ =~ /right_01$/) { push @right_ps, $_; } } close (RP); my @Lrun = `$path_toPass $options -p L $rf $lq &> /dev/null`; print "."; my $f2 = "L.coords"; open (LQ, $f2) or die "Could not open file $f2\n"; while () { chomp; if ($_ =~ /left_01$/) { push @left_ps, $_; } } close (LQ); $right_count = scalar (@right_ps); $left_count = scalar(@left_ps); #check if a primer is not in the excluded region:: my $primer_NearEnd =0; if ($left_st > $exc1 || $right_st < $exc2) { $primer_NearEnd = 1; } if ($left_count < 2 && $right_count<2 && $primer_NearEnd ==0) { $string.=$left_count."\t".$Pr3_output[5]."\n"; $string.=$right_count."\t".$Pr3_output[6]."\n"; $string.="***************************** PRIMER3 OUTPUT **************************\n"; foreach (@Pr3_output) {$string.=$_;} my @prod_size_split = split /\s+/, $Pr3_output[10]; $product_size = substr($prod_size_split[2], 0, -1); $left_name = $position; $right_name = $position; my $lp_uc = uc ($left_primer); my $rp_uc = uc($right_primer); #print $left_count, "..", $right_count, "\t"; $left_seq = $lp_uc; $right_seq= $rp_uc; $left_start= $left_st; $left_len = $left_length; $right_end = $right_st; $right_len = $right_length; $left_exclude = $exc1; $right_exclude =$exc2; $uniq_primer =1; } else { if ($primer_NearEnd ==1) { $string.="One of the oligos is near the end of a contig\n"; } else { $string.="Primer set not unique\n"; } $primers_toExclude.=">L.".$position."\n".$left_primer."\n"; $primers_toExclude.=">R.".$position."\n".$right_primer."\n"; } } else { $string.="No Primers found\n"; } } return ($uniq_primer, $string,$left_name,$right_name,$left_seq, $right_seq,$left_start,$left_len, $right_end,$right_len,$primers_toExclude, $product_size); } ###------------------------------------ # Writes primers and their regions to file sub write_Primers { my ($leftP_names, $rightP_names, $leftP_seqs, $rightP_seqs, $leftP_start, $leftP_lens, $rightP_ends, $rightP_lens,$primers_toExclude,$left_Exclude,$right_Exclude, $product_sizes) = @_; my (@left_name, @right_name, @left_seq, @right_seq, @left_start, @left_len, @right_end, @right_len, @left_exclude, @right_exclude, @product_size); #open files to read @left_name = split /\n/, $leftP_names; @right_name= split /\n/, $rightP_names; @left_seq = split /\n/, $leftP_seqs; @right_seq = split /\n/, $rightP_seqs; @left_start = split /\n/, $leftP_start; @left_len = split /\n/, $leftP_lens; @right_end = split/\n/, $rightP_ends; @right_len = split /\n/, $rightP_lens; @left_exclude = split /\n/, $left_Exclude; @right_exclude = split /\n/,$right_Exclude; @product_size = split /\n/, $product_sizes; my $primers_withSize =""; open (SEN, '>sense_primers.out') or die "Could not open file for write\n"; open (ASEN, '>antiSense_primers.out') or die "Could not open file for write\n"; open (REG_1, '>sense_regions.out') or die "Could not open file for write\n"; open (REG_2, '>antiSense_regions.out') or die "Could not open file for write\n"; if ($primers_toExclude ne "") { open (PEX, '>primers_toExclude.out') or die "Could not open file for write\n"; print PEX $primers_toExclude; } my $totalp = scalar (@left_name); #print $totalp, "\n"; exit; my $well_pos; my $max_plates = ceil($totalp/96); #print "MAX Ps ", $max_plates, "\n"; my $plate=1; my $sen =""; my $asen =""; my $plate_counter =0; my $wells = 96; for (my $index =0; $index < $totalp; $index += $wells) { my $do = $index; my $upper_bound= $index + $wells; if ($upper_bound > $totalp) { $upper_bound = $totalp; } for (my $j=$index; $j <= ($upper_bound-1); $j+=1) { my $i = $j; if ($j < 96) { $well_pos = get_WellPosition ($j); } else { $well_pos = get_WellPosition ($j - $wells) } #$primers_withSize.=$product_size[$i]."\t"."Plate_".$plate. "\t\tS.".$i."\tS.".$left_name[$i]."\t". print SEN "Plate_".$plate, "\t\t","S.", $i, "\tS.", $left_name[$i], "\t", $left_seq[$i], "\t\t+", "\t", $well_pos, "\n"; print ASEN "Plate_".$plate, "\t\t","AS.", $i, "\tAS.", $right_name[$i], "\t", $right_seq[$i], "\t\t-","\t", $well_pos,"\n"; print REG_1 "Plate_".$plate, "\t\t","S.", $i, "\t", $left_start[$i], "\t", $left_len[$i], "\n"; print REG_2 "Plate_".$plate, "\t\t","AS.", $i, "\t", $right_end[$i], "\t",$right_len[$i], "\n"; } $plate +=1; } #delete tmp. files #my $rm = "rm -f"; system ("rm -f data left_query right_query R.delta R.cluster R.coords L.delta L.cluster L.coords"); print "\nPRIMER DESIGN DONE\n\n"; # end of primer design program }#// ##### # returns a well position for oligos sub get_WellPosition{ my $j = shift; my $well_pos; if ($j < 12) { $well_pos = "a".($j+1); } elsif ($j>11 && $j<24) { $well_pos = "b". (($j+1) -12); } elsif ($j>23 && $j<36) { $well_pos = "c". (($j+1) -24); } elsif ($j>35 && $j<48) { $well_pos = "d". (($j+1) - 36); } elsif($j>47 && $j<60) { $well_pos = "e". (($j+1) -48); } elsif ($j>59 && $j<72) { $well_pos = "f". (($j+1) - 60); } elsif ($j>71 && $j< 84) { $well_pos = "g". (($j+1) - 72); } elsif ($j>83 && $j<96) { $well_pos = "h". (($j+1) - 84); } return $well_pos; } #################################################################### #get options for primer design #---------------------- sub getPoptions{ my $qualAvailable = shift; #### USER INPUTS ########## #ask for optimum primer size print "\nEnter Optimum Primer size (default 20 bases):"; my $opt=; chomp $opt; if($opt eq '') { $opt = 20; } #ask for minimum primer size print "\nEnter Minimum Primer size (default 18 bases):"; my $min=; chomp $min; if($min eq '') { $min = 18; } #ask for maximum primer size print "\nEnter Maximum Primer size (default 27 bases):"; my $max= ; chomp $max; if($max eq '') { $max= 27; } #ask for optimum primer temperature print "\nEnter Optimum melting temperature (Celcius) for a primer oligo (default 60.0C):"; my $optTemp=; chomp $optTemp; if($optTemp eq '') { $optTemp = 60.0; } #ask for minimum primer temperature print "\nEnter Minimum melting temperature (Celcius) for a primer oligo (default 57.0C):"; my $minTemp=; chomp $minTemp; if($minTemp eq '') { $minTemp = 57.0; } #ask for maximum primer temperature print "\nEnter Maximum melting temperature (Celcius) for a primer oligo (default 63.0C):"; my $maxTemp=; chomp $maxTemp; if($maxTemp eq '') { $maxTemp = 63.0; } print "\nEnter flanking region size (default 1000 bases): "; my $flank=; chomp $flank; if ($flank eq '') { $flank = 1000; } #ask for primer product range print "\nEnter minimum product size produced by primers (default =flanking size):"; my $lowRange=; chomp $lowRange; if($lowRange eq '') { $lowRange = $flank; } print "\nEnter maxmimum product size produced by primers (default 7000):"; my $maxRange=; chomp $maxRange; if($maxRange eq '') { $maxRange = 7000; } #ask for minimum GC content in primers print "\nEnter minimum GC content in primers (default 20%):"; my $gcMin=; chomp $gcMin; if($gcMin eq '') { $gcMin = 20.0; } #ask for optimum GC content in primers print "\nEnter optimum GC content in primers (default 50%):"; my $gcOpt=; chomp $gcOpt; if($gcOpt eq '') { $gcOpt = 50.0; } #ask for maximum GC content in primers print "\nEnter maximum GC content in primers (default 80%):"; my $gcMax=; chomp $gcMax; if($gcMax eq '') { $gcMax = 80.0; } print "\nEnter GC clamp (default 1):"; my $gclamp=; chomp $gclamp; if($gclamp eq '') { $gclamp = 1; } print "\nEnter size of region to exclude at the end of contigs (default 100 bases):"; my $exclude=; chomp $exclude; if ($exclude eq '') { $exclude = 100; } #tdo my $quality=''; if ($qualAvailable) { print "\nEnter minimum quality for primer pick (default 40):"; $quality=; chomp $quality; if($quality eq '') { $quality = 40; } } return ($opt,$min,$max,$optTemp,$minTemp,$maxTemp,$flank,$lowRange,$maxRange,$gcMin,$gcOpt,$gcMax,$gclamp,$exclude, $quality); } ############### #-----------------------------------------------------END of PRIMER DESIGN ---------------------------------------------------------------- #-----------------------------------------------------END OF ABACAS ----------------------------------------------------------------------- abacas-1.3.1/Manual.html0000644000265600020320000006321611541611714014166 0ustar tilleaadmin ABACAS

  1. Overview
  2. Instructions for use
  3. Requirement
  4. Usage
  5. Options
  6. Input files
  7. Default output files
  8. Optional output files
  9. Colour code
  10. Explanation of output files
  11. Contact 
  12. Test dataset

1. Overview


ABACAS is intended to rapidly contiguate (align, order, orientate) , visualize and design primers to close gaps on shotgun assembled contigs based on a reference sequence. It uses MUMmer to find alignment positions and identify syntenies of assembly contigs against the reference. The output is then processed to generate a pseudomolecule taking overlaping contigs and gaps in to account. MUMmer's alignment generating programs, Nucmer and Promer are used followed by the 'delta-filter' utility function. Users could also run tblastx on contigs that are not used to generate the pseudomolecule.

If the blast search or consecutive MUMmer alignments result in mapping of extra contigs, finishers can use the Arthemis Comparision Tool (ACT) to easily modify ordering of contigs on the pseudomolecule by dragging and droping contigs to the desired location. Overlapping contigs and gaps in the pseudololecule are represented by "N"s. Overlapping contigs are often due to low quality contig ends and low complexity regions. ABACAS could automatically extract gaps on the pseudomolecule and generate primer oligos for gap closure using Primer3. Uniqueness of primer sets is checked by running a sensitive NUCmer alignment. If a quality file (contig_name.qual) exists in the working directory, it will be used during the primer design step.

2. Instructions for use

  1. Download ABACAS from the download page
  2. Run: abacas -r <reference> -q <contigs> -p <nucmer|promer>
    NOTE: If ABACAS cannot find MUMmer from the default path - it will prompt the user to enter the location of MUMer
  3. ABACAS may take several minutes to run for large genomes/chromosomes and will produce a number of different output files in the working directory
  4. Start ACT and load the sequence and comparison files as printed out by ABACAS.
  5. In ACT, load the contig names by going to 'File', <query>, 'Read an Entry', and select the file <query>_<reference>.tab
  6. You can also load the repeat plot for the reference which tells whether or not gaps are due to repetitive sequence. You can load this by going to 'Graph', '<reference>', 'Add User Plot', and select the file '<reference>.Repeats.plot'
  7. The file '<query>.bin' contains the names of the contigs that were not mapped and mapped multiple times to the reference.

3. Requirement

ABACAS requires MUMmer to be installed in the working path for ordering and orienting of contigs. If MUMmer is not found in the working path, users will be asked to provide a valid path for MUMmer. The Arthemis Comparision Tool (ACT) should be downloaded for visualizing scaffolding of contigs. Primer design requires Primer3. Optionally, BLASTALL is required in order to run tblastx on the contigs that are not mapped using Nucmer or Promer.

4. Usage

 

abacas -r <reference file: single fasta> -q <query sequence file: fasta> -p <nucmer/promer> [Options]

    for contig ordering and primer design

OR
abacas -r <reference file: single fasta> -q <pseudomolecule/ordered file: fasta> -e

    to escape contig ordering and go directly to primer design

OR

abacas -h for help

5. Options

-d         use default NUCmer or PROmer parameters

            *ABACAS uses --maxmatch to increase mapping sensitivity during mapping. The option  -d could be useful while dealing with larger genomes or when a higher sensitivity is  not required.

-m         print ordered and orientated contigs to file

            *This option is helpful if users want to further investigate the ordering using other alignment algorithms such as blast.

-b         print contigs in the bin file to multi-fasta file

            *contigs that are not used in generating the pseudomolecule will be placed in a '.bin' file. Since this file only contains contig names, the -b option could be used to print these contigs to a file for further analysis. Note that this option is required if users are interested in running a blast search.

-N         generate a pseudomolecule without 'N's

            *ABACAS produces a pseudomolecule ('.fasta' file) and fills gaps with 'N's. It also puts  100 'N's between overlapping contigs. This option will produce another pseudomolecule without padding (.NoNs.fasta).

-i         default 40

            *minimum percent identity could vary from 0 to 100 depending on the closeness of the two genomes. Choosing a smaller value will pull in more contigs and vice versa

-v         default 40

            *minimum contig coverage: set a value between 0 and 100

-V         default 1

        *minimum contig coverage difference. Use -V 0 to place contigs randomly to one of the positions (in cases where a contig maps to multiple places)

-l         default 100

        * contigs below this cutoff will not be used

-t         run tblastx on contigs that are not used to generate the pseudomolecule

            *-t will run blastall on a fasta file of contigs in the .bin file. The option -b should be used to generate this file

-g         file_name

            * will print sequences of the reference that correspond with gaps on the pseudomolecule in a multi-fasta format

-a         append contigs in the .bin file to the end of the pseudomolecule

            *Contigs could then be easily manipulated and re-ordered using ACT's graphica interface

-o         prefix (string)

            *output files will have this prefix

-P         pick primer oligos to close gaps

-f         default 1000

            *number of flanking bases on either side of a gap for primer design (default 1000bp )

-R         avoid running mummer

-e         Escape contig ordering i.e. go to primer design

-c         Reference sequence is circular


6. Input files

Two fasta files containing the reference and query (contigs) sequences are required. The reference file should be in a single fasta format for speedy contig ordering and orientation.


7. Default output files

Running ABACAS with default options will generate the following files:

  1. Ordered and orientated sequence file (reference_query.fasta or prefix.fasta)

  2. Feature file (reference_query.tab or prefix.tab)

  3. Bin file that contains contigs that are not used in ordering (reference_query.bin or prefix.bin)

  4. Comparison file (reference_query.crunch or prefix.crunch)

  5. Gap information (reference_query.gaps, prefix.gaps)

  6. Information on contigs that have a mapping information but could not be used in the ordering (unused_contigs.out)

  7. Feature file to view contigs with ambiguous mapping (reference.notMapped.contigs.tab). This file should be uploaded on the reference side of ACT view.

  8. A file that shows how repetitive the reference genome is (reference.Repeats.plot).

Files 7 & 8 should be uploaded on the reference side of ACT view.

Please note that contigs in the '.fasta' file will be reverse complemented if they are found to map on the reverse strand. However, the ACT view shows the initial orientation of these contigs i.e. they will be shown on the reverse strand. If you write a fasta file of the pseudomolecule from ACT, the resulting sequence will be a set of ordered contigs (the orientation will not change). It is therefore recommended to use the '.fasta' pseudomolecule file automatically generated for further investigation.


8. Optional output files

It is also possible to generate additional files including:

  1. A list of ordered and orientated contigs in a multi-fasta format (-m ) .

  2. A pseudomolecule with all unmapepd contigs appended to the end for reordering (-a ) .

  3. A pseudomolecule where the gaps are not padded with N (-N )

  4. A multi-fasta file of all unmapped contigs (-b )

  5. A multi-fasta file of regions on the reference that correspond to gaps on pseudomolecule (-g file_name)

  6. A list of sense and antisense primer sets in separate files

  7. A list of locations where sense and antisense primers are found in two separate files

  8. Non-unique primers and primers near contig ends will be printed to a file (primers to exclud)

  9. A standard primer3 output summary file with a detailed information on oligos.


9. Colour code

The feature (file 2 from default output section) file has the following colour codes:

Dark blue (4): contigs with forward orientation

Dark green (3): contigs with reverse orientations

Sky blue (5): contigs that overlap with the next contig

Yellow (7): contigs that have no hit (only added to the pseudomolecule if '-a ' is used)


10. Explanation of output files

Comparison file (.crunch file)

72 100 1 23328 contig00198 1 16690 unknown NONE

100 100 29503 29782 contig00002 22865 23144 unknown NONE

100 100 29952 52948 contig00087 23314 46310 unknown NONE

100 100 52986 63111 contig00243 46348 56473 unknown NONE

100 94 63118 63576 contig00217 56480 56938 unknown NONE

100 100 63775 63932 contig00224 57137 57294 unknown NONE

100 100 63933 64216 contig00250 57295 57578 unknown NONE

The first seven columns of the comparison file represent coverage, percent identity, start on pseudomolecule, end on pseudomolecule, contig ID, start on reference and end on reference.

Gap file (.gaps)

Gap 6174 23329 29502 16691 22864

Gap 6 63112 63117 56474 56479

Gap 198 63577 63774 56939 57136

Columns 2-6 represent gap size, start on pseudomolecule, end on pseudomolecule, start on reference and end on reference.

Bin file (.bin)

A list of contig names that could not be used in generating a pseudomolecule.

Fasta file (.fasta)

This is the pseudomolecule generated from ordered and orientated contigs. Overlapping contigs are separated by 100 'N's. Gaps are also represented by 'N's.


11. Contact

Please email sa4 {at} sanger.ac.uk if you have any problems or comments.

12. Test dataset

We have provided a test dataset from Streptococcus suis which consists of a set of 454 contigs and the reference genome.
454 Contigs download
Reference download

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