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view 2.4/src/SoftSearch.multi.pl @ 16:8eb7d93f7e58 draft
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| author | plus91-technologies-pvt-ltd |
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| date | Sat, 31 May 2014 11:23:36 -0400 |
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#!/usr/bin/perl #### #### Usage: SoftSearch.pl [-lqrmsd] -b <BAM> -f <Genome.fa> -sam <samtools path> -bed <bedtools path> #### Created 1-30-2012 by Steven Hart, PhD #### hart.steven@mayo.edu #### Required bedtools & samtools to be in path #use FindBin; #use lib "$FindBin::Bin/lib"; use lib "/home/plus91/shed_tools/toolshed.g2.bx.psu.edu/repos/plus91-technologies-pvt-ltd/softsearch/e3609c8714fb/softsearch/2.4/lib" ; use Getopt::Long; use strict; use warnings; use Data::Dumper; use LevD; use File::Basename; use List::Util qw(min max); my (@INPUT_BAM,$INPUT_FASTA,$OUTPUT_FILE,$minSoft,$minSoftReads,$dist_To_Soft,$bedtools,$samtools); my ($minRP, $temp_output, $num_sd, $MapQ, $chrom, $unmated_pairs, $minBQ, $pair_only, $disable_RP_only); my ($levD_local_threshold, $levD_distl_threshold,$pe_upper_limit,$high_qual,$sv_only,$blacklist,$genome_file,$verbose); my $cmd = ""; #Declare variables GetOptions( 'b=s{1,}' => \@INPUT_BAM, 'f=s' => \$INPUT_FASTA, 'o:s' => \$OUTPUT_FILE, 'm:i' => \$minRP, 'l:i' => \$minSoft, 'r:i' => \$minSoftReads, 't:i' => \$temp_output, 's:s' => \$num_sd, 'd:i' => \$dist_To_Soft, 'q:i' => \$MapQ, 'c:s' => \$chrom, 'u:s' => \$unmated_pairs, 'x:s' => \$minBQ, 'p' => \$pair_only, 'g' => \$disable_RP_only, #ignore softclips 'j:s' => \$levD_local_threshold, 'k:s' => \$levD_distl_threshold, 'a:s' => \$pe_upper_limit, 'e:s' => \$high_qual, 'L' => \$sv_only, 'v' => \$verbose, 'blacklist:s' => \$blacklist, 'genome_file:s' => \$genome_file, "help|h|?" => \&usage); #print "Using @INPUT_BAM as INPUT_BAM\n"; unless($sv_only){$sv_only=""}; my $INPUT_BAM=$INPUT_BAM[0]; #print "MY NEW INPUT BAM=$INPUT_BAM[0]\n\n";die; if(defined($INPUT_BAM)){$INPUT_BAM=$INPUT_BAM} else {print usage();die "Where is the BAM file?\n\n"} if(defined($INPUT_FASTA)){$INPUT_FASTA=$INPUT_FASTA} else {print usage();die "Where is the fasta file?\n\n"} my ($fn,$pathname) = fileparse($INPUT_BAM,".bam"); #my $index=`ls $pathname/$fn*bai|head -1`; #my $index =`ls \${INPUT_BAM%.bam}*bai`; #print "INDEX=$index\n"; #if(!$index){die "\n\nERROR: you need index your BAM file\n\n"} my $index=""; ### get current time print "Start Time : " . &spGetCurDateTime() . "\n"; my $now = time; #if(defined($OUTPUT_FILE)){$OUTPUT_FILE=$OUTPUT_FILE} else {$OUTPUT_FILE="output.vcf"; print "\nNo outfile specified. Using output.vcf as default\n\n"} if(defined($minSoft)){$minSoft=$minSoft} else {$minSoft=5} if(defined($minRP)){$minRP=$minRP} else {$minRP=5} if(defined($minSoftReads)){$minSoftReads=$minSoftReads} else {$minSoftReads=5} if(defined($dist_To_Soft)){$dist_To_Soft=$dist_To_Soft} else {$dist_To_Soft=300} if(defined($num_sd)){$num_sd=$num_sd} else {$num_sd=6} if(defined($MapQ)){$MapQ=$MapQ} else {$MapQ=20} unless (defined $pe_upper_limit) { $pe_upper_limit = 10000; } unless (defined $levD_local_threshold) { $levD_local_threshold = 0.05; } unless (defined $levD_distl_threshold) { $levD_distl_threshold = 0.05; } #Get sample name if available my $SAMPLE_NAME=""; my $OUTNAME =""; $SAMPLE_NAME=`samtools view -f2 -H $INPUT_BAM|awk '{if(\$1~/^\@RG/){sub("ID:","",\$2);name=\$2;print name}}'|head -1`; $SAMPLE_NAME=~s/\n//g; if (!$OUTPUT_FILE){ if($SAMPLE_NAME ne ""){$OUTNAME=$SAMPLE_NAME.".vcf"} else {$OUTNAME="output.vcf"} } else{$OUTNAME=$OUTPUT_FILE} print "Writing results to $OUTNAME\n"; ##Make sure if submitting on SGE, to prepned the "chr". Not all referecne FAST files require "chr", so we shouldn't force the issue. if(!defined($chrom)){$chrom=""} if(!defined($unmated_pairs)){$unmated_pairs=0} my $badQualValue=chr($MapQ); if(defined($minBQ)){ $badQualValue=chr($minBQ); } if($badQualValue eq "#"){$badQualValue="\#"} # adding and cheking for samtools and bedtools in the PATh ## check for bedtools and samtools in the path $bedtools=`which intersectBed` ; if(!defined($bedtools)){die "\nError:\n\tno bedtools. Please install bedtools and add to the path\n";} #$samtools=`samtools 2>&1`; $samtools=`which samtools`; if($samtools !~ /(samtools)/i){die "\nError:\n\tno samtools. Please install samtools and add to the path\n";} print "Usage = SoftSearch.pl -l $minSoft -q $MapQ -r $minSoftReads -d $dist_To_Soft -m $minRP -s $num_sd -c $chrom -b @INPUT_BAM -f $INPUT_FASTA -o $OUTNAME \n\n"; sub usage { print "\nusage: SoftSearch.pl [-cqlrmsd] -b <BAM> -f <Genome.fa> \n"; print "\t-q\t\tMinimum mapping quality [20]\n"; print "\t-l\t\tMinimum length of soft-clipped segment [5]\n"; print "\t-r\t\tMinimum depth of soft-clipped reads at position [5]\n"; print "\t-m\t\tMinimum number of discordant read pairs [5]\n"; print "\t-s\t\tNumber of sd away from mean to be considered discordant [6]\n"; print "\t-u\t\tNumber of unmated pairs [0]\n"; print "\t-d\t\tMax distance between soft-clipped segments and discordant read pairs [300]\n"; print "\t-o\t\tOutput file name [output.vcf]\n"; print "\t-t\t\tPrint temp files for debugging [no|yes]\n"; print "\t-c\t\tuse only this chrom or chr:pos1-pos2\n"; print "\t-p\t\tuse paired-end mode only \n"; print "\t-g\t\tEnable paired-only seach. This will look for discordant read pairs even without soft clips.\n"; print "\t-a\t\tset the minimum distance for a discordant read pair without soft-clipping info [10000]\n"; print "\t-L\t\tFlag to print out even small deletions (low quality)\n"; print "\t-e\t\tdisable strict quality filtering of base qualities in soft-clipped reads [no]\n"; print "\t-blacklist\tareas of the genome to skip calling. Requires -genome_file\n"; print "\t-genome_file\ttab seperated value of chromosome name and length. Only used with -blacklist option\n\n"; exit 1; } ############################################################# # create temporary variable name ############################################################# srand (time ^ $$ ^ unpack "%L*", `ps axww | gzip -f`); our $random_name=join "", map { ("a".."z")[rand 26] } 1..8; ############################################################# ## create green list ############################################################## # my $new_blacklist=""; if($blacklist){ if(!$genome_file){die "if using a blacklist, you must also specify the location of a genome_file The format of the genome_file should be chrom size chr1 249250621 chr2 243199373 ... If using hg19, you can ge the genome file by mysql --user=genome --host=genome-mysql.cse.ucsc.edu -A -e \"select chrom, size from hg19.chromInfo\" > hg19.genome";} $cmd=join("","complementBed -i $blacklist -g $genome_file >",$random_name,".bed") ; system ($cmd); $new_blacklist=join(""," -L ",$random_name,".bed "); } if($verbose){print "CMD=$cmd\nBlacklist is $new_blacklist\n";} ############################################################# # Calcualte insert size distribution of properly mated reads ############################################################# #Change for compatability with other operating systems #my $metrics=`samtools view -q $MapQ -f2 $INPUT_BAM $chrom|cut -f9|head -10000|awk '{if (\$1<0){\$1=-\$1}else{\$1=\$1} sum+=\$1; sumsq+=\$1*\$1} END {print sum/NR, sqrt(sumsq/NR - (sum/NR)**2)}'`; #print "samtools view -q $MapQ -f2 $INPUT_BAM $chrom|cut -f9|head -10000|awk '{if (\$1<0){\$1=-\$1}else{\$1=\$1} sum+=\$1; sumsq+=\$1*\$1} END {print sum/NR, sqrt(sumsq/NR - (sum/NR)^2)}'\n"; my $metrics=`samtools view -q $MapQ -f2 $INPUT_BAM $chrom|cut -f9|head -10000|awk '{if (\$1<0){\$1=-\$1}else{\$1=\$1} sum+=\$1; sumsq+=\$1*\$1} END {print sum/NR, sqrt(sumsq/NR - (sum/NR)^2)}'`; #my ($mean,$stdev)=split(/ /,$metrics); my ($mean,$stdev)=split(/\s/,$metrics); $stdev=~s/\n//; #print "MEAN=$mean\tSTDEV=$stdev\n\n"; my $upper_limit=int($mean+($num_sd*$stdev)); my $lower_limit=int($mean-($num_sd*$stdev)); die if (!$mean); print qq{The mean insert size is $mean +/- $stdev (sd) The upper limit = $upper_limit The lower limit = $lower_limit\n }; if($lower_limit<0){ print "Warning!! Given this insert size distribution, we can not call small indels. No other data will be affected\n\n"; $lower_limit=1; } my $tmp_name=join ("",$random_name,".tmp.bam"); my $random_file_sc = ""; my $command = ""; ############################################################# # Make sam file that has soft clipped reads ############################################################# #give file a name if(!defined($pair_only)){ foreach my $bam(@INPUT_BAM){ $random_file_sc=join ("",$random_name,".sc.sam"); $command=join ("","samtools view -q $MapQ -F 1024 $bam $chrom $new_blacklist| awk '{OFS=\"\\t\"}{c=0;if(\$6~/S/){++c};if(c == 1){print}}' | perl -ane '\$TR=(\@F[10]=~tr/\#//);if(\$TR<2){print}' >> ", $random_file_sc); print "Making SAM file of soft-clipped reads from $bam\n"; if($verbose){ print "$command\n";} system("$command"); } ############################################################# # Find areas that have deep enough soft-clip coverage print "Identifying soft-clipped regions that are at least $minSoft bp long iin $random_file_sc\n"; open (FILE,"$random_file_sc")||die "Can't open soft-clipped sam file $random_file_sc\n"; my $tmpfile=join("",$random_file_sc,".sc.passfilter"); open (OUT,">$tmpfile")||die "Can't write files here!\n"; while(<FILE>){ @_ = split(/\t/, $_); #### parse CIGAR string and create a hash of array of each operation my @CIGAR = split(/([0-9]+[SMIDNHXP])/, $_[5]); my $hash; map { push(@{$hash->{$2}}, $1) if (/(\d+)([SMIDNHXP])/) } @CIGAR; #for ($i=0; $i<=$#softclip_pos; $i++) { foreach my $softclip (@{$hash->{S}}) { #if ($CIGAR[$softclip_pos[$i]] > $minSoft){ if ($softclip > $minSoft){ ###############Make sure base qualities don't have more than 2 bad marks my $qual=$_[10]; my $TR=($qual=~tr/$badQualValue//); if($badQualValue eq "#"){ $TR=($qual=~tr/\#//); } #Skip the soft clip if there is more than 2 bad qual values #next if($TR > 2); # if (!$high_qual){next if($TR > 2);} print OUT; last; } } } close FILE; close OUT; $command=join(" ","mv",$tmpfile,$random_file_sc); if($verbose){ print "$command\n";} system("$command"); } ######################################################### #Stack up SoftClips ######################################################### my $random_file=join("",$random_name,".sc.direction.bed"); if(!defined($pair_only)){ open (FILE,"$random_file_sc")|| die "Can't open sam file\n"; #$random_file=join("",$random_name,".sc.direction"); print "Calling sides of soft-clips from $random_file_sc\n"; #\nTMPOUT=$random_file\tINPUT=$random_file_sc\n\n"; open (TMPOUT,">$random_file")|| die "Can't create tmp file\n"; while (<FILE>){ @_ = split(/\t/, $_); #### parse CIGAR string and create a hash of array of each operation my @CIGAR = split(/([0-9]+[SMIDNHXP])/, $_[5]); my $hash; map { push(@{$hash->{$2}}, $1) if (/(\d+)([SMIDNHXP])/) } @CIGAR; #### next if softclips on each end next if ($_[5] =~ /^[0-9]+S.*S$/); #### next softclip occurs in the middle next if ($_[5] =~ /^[0-9]+[^S][0-9].*S.+$/); my $softclip = $hash->{S}[0]; my $end1 = 0; my $end2 = 0; my $softBases = ""; my $right_corrected="";my $left_corrected=""; if ($softclip > $minSoft) { ####If the soft clip occurs at end of read and its on the minus strand, then it's a right clip if ($_[5] =~ /^.*S$/) { $end1=$_[3]+length($_[9])-$softclip-1; $end2=$end1+1; next if ($end1<0); #RIGHT clip on Minus $softBases=substr($_[9], length($_[9])-$softclip, length($_[9])); #Right clips don't always get clipped correctly, so fix that # Check to see if sc base matches ref $right_corrected=baseCheck($_[2],$end2,"right",$softBases); print TMPOUT "$right_corrected\n" } else { #### Begins with S (left clip) $end1=$_[3]-$softclip; next if ($end1<0); $softBases=substr($_[9], 0,$softclip);#print "TMP=$softBases\n"; $left_corrected=baseCheck($_[2],$end1,"left",$softBases); if(!$left_corrected){print "baseCheck($_[2],$end1,left,$softBases)\n";next} print TMPOUT "$left_corrected\n"; #print "\nSEQ=$_[9]\t\n"; } } } close FILE; close TMPOUT; } sub baseCheck{ my ($chrom,$pos,$direction,$softBases)=@_; #skip if position is less than 0, which is caused by MT DNA return if ($pos<0); my $exit=""; while(!$exit){ if($direction=~/right/){ my $refBase=getSeq($chrom,$pos,$INPUT_FASTA); my $softBase=substr($softBases,0,1); if ($softBase !~ /$refBase/){ my $value=join("\t",$chrom,$pos,$pos+1,join("|",$softBases,$direction)); $exit="STOP"; return $value; } else{ $pos=$pos+1; $softBases=substr($softBases, 1,length($softBases)); } } else{ my $refBase=getSeq($chrom,$pos+1,$INPUT_FASTA); my $softBase=substr($softBases,-1,1); if ($softBase !~ /$refBase/){ $pos=$pos-1+length($softBases); my $value=join("\t",$chrom,$pos-1,$pos,join("|",$softBases,$direction)); $exit="STOP"; return $value; } else{ $pos=$pos-1; $softBases=substr($softBases, 0, -1); #print "Trying again $softBases\n"; } } } } #Remove SAM files to conserve space unlink($random_file_sc); ### # ###################################################### # Transform Read pair groups into softclip equivalents ###################################################### # # # my $v=""; #if($disable_RP_only){ print "Running Bam2pair.pl\n"; print "Looking for discordant read pairs without requiring soft-clipping information\n"; use FindBin qw($Bin); my $path=$Bin; # print"\n\nPATH=$path\n\n"; if($verbose){$v="-v"} foreach my $random_file_disc(@INPUT_BAM){ my $tmp_out=join("",$random_name,"RP.out"); $command=join("","perl ",$path,"/Bam2pair.pl -b $random_file_disc -o $tmp_out -isize $pe_upper_limit -winsize $dist_To_Soft -min $minRP -chrom $chrom -prefix $random_name -q $MapQ -blacklist $random_name.bed $v"); if($verbose){ print "$command\n"}; system("$command"); $command=join("","perl -ane '\$end1=\@F[1];\$end2=\@F[3];print join(\"\\t\",\@F[0..1],\$end1,\"unknown|left\");print \"\\n\";print join(\"\\t\",\@F[2..3],\$end2,\"unknown|left\");print \"\\n\"' ", $tmp_out," >> ",$random_file); if($verbose){print "$command\n"}; system($command); unlink($tmp_out); #} } ###################################################### unlink("$random_file","$tmp_name","$random_file","$index","$random_name","$new_blacklist") if (-z $random_file || ! -e $random_file) ; if (-z $random_file || ! -e $random_file){ print "Softclipped file is empty($random_file).\nNo soft clipping found using desired paramters\n\n"; open (OUT,">$OUTNAME")||die "Can't write files here!\n"; &print_header(); close OUT; exit; } ############################################################# # Make sure there are enough soft-clippped supporting reads ############################################################# my $outfile=join("",$random_file,".sc.merge.bed"); #sortbed -i .sc.direction | mergeBed -nms -d 25 -i stdin > .sc.merge.bed $command=join(" ","sortBed -i",$random_file," | mergeBed -nms -i stdin","|grep \";\"","|awk '{OFS=\"\t\"}(NF==4)'",">",$outfile); #print "$command\n"; system("$command"); if (-z $outfile || ! -e $outfile){ unlink("$tmp_name","$random_file","$outfile","$index","$random_name","$new_blacklist"); print "mergeBed file is empty.\nNo strucutral variants found\n\n" ; open (OUT,">$OUTNAME")||die "Can't write files here!\n"; &print_header(); close OUT; exit; } print "completed mergeBed\n"; ############################################################### # If left and right are on the same line, make into 2 lines ############################################################### open (INFILE,$outfile)||die "couldn't open temp file : $. \n\n"; my $tmp2=join("",$random_name,".sc.fixed.merge.bed"); #print "INFILE=$outfile\tOUTFILE=$tmp2\n\n"; #INPUT FORMAT=chr9\t131467\t131473\tATGCTTATTAAAA|left;TTATTAAAAGCATA|left open (OUTFILE,">$tmp2")||die "couldn't create temp file : $. \n\n"; while(<INFILE>){ chomp $_; my $l = $_; my @a = split(/\t/, $l); my $info = $a[3]; my @info_arr = split(/\;/, $info); my @left_arr=(); my @right_arr=(); @left_arr = grep(/left/, @info_arr); @right_arr = grep(/right/, @info_arr); #New my $left = join(";", @left_arr); my $right = join(";", @right_arr); $info = join(";", @info_arr); if((@left_arr) && (@right_arr)){ print OUTFILE "$a[0]\t$a[1]\t$a[2]\t$left\n$a[0]\t$a[1]\t$a[2]\t$right\n"; } else{ my $all=join("\t",@a[0..2],$info); print OUTFILE "$all\n"; } } # make sure output file name is $outfile $command=join(" ","sed -e '/ /s//\t/g'", $tmp2,"|awk 'BEGIN{OFS=\"\\t\"}(NF==4)'", "|perl -pne 's/ /\t/g'>",$outfile); system("$command"); if($verbose){print "$command\n"}; unlink("$tmp_name","$random_file","$tmp2","$outfile","$index","random_name","$new_blacklist") if (-z $outfile || ! -e $outfile) ; if (-z $outfile || ! -e $outfile){ print "Fixed mergeBed file is empty($outfile).\nNo strucutral variants found\n\n"; open (OUT,">$OUTNAME")||die "Can't write files here!\n"; &print_header(); close OUT; exit; } print "completed fixing mergeBed\n\n"; ############################################################### # Seperate directions of soft clips ############################################################### my $left_sc = join("", "left", $tmp2); my $right_sc = join("", "right", $tmp2); use FindBin qw($Bin); #my $path=$Bin; $command=join("","grep left ", $tmp2, " |sed -e '/left /s//left\;/g' | sed -e '/ /s//\t/g'|perl ".$path."/direction_filter.pl - >",$left_sc); system("$command"); #print "$command\n"; $command=join("","grep right ", $tmp2, " |sed -e '/right /s//right\;/g' | sed -e '/ /s//\t/g'|perl ".$path."/direction_filter.pl - >",$right_sc); #$command=join(" ","grep right ", $tmp2, " |sed -e '/right /s//right\;/g' | sed -e '/ /s//\t/g' >",$right_sc); system("$command"); #print "$command\n"; #die "CHECK $right_sc\n"; ############################################################### # Count the number and identify directions of soft clips ############################################################### print "Count the number and identify directions of soft clips\n"; #print "looking in $outfile\n"; $outfile=join("",$random_name,".sc.fixed.merge.bed"); #system("ls -lhrt"); open (INFILE,$outfile)||die "couldn't open temp file\n\n"; my $tmp3 = join("", $random_file, "predSV"); open (OUTFILE, ">$tmp3")||die "couldn't create temp file\n\n"; while(<INFILE>){ chomp; @_=split(/\t/,$_); my $count=tr/\;//; $count=$count+1; my $left=0; my $right=0; while ($_ =~ /left/g) { $left++ } # count number of right clips while ($_ =~ /right/g) { $right++ } # count number of left clips ############################################################### if ($count >= $minSoftReads){ ####get longets soft-clipped read my @clips=split(/\;|\|/,$_[3]); my ($max, $temp, $temp2, $temp3, $dir, $maxSclip) = (0) x 6; for (my $i=0; $i<$count; $i++) { my $plus1=$i+1; $temp=length($clips[$i]); $temp2=$clips[$plus1]; $temp3=$clips[$i]; if ($temp > $max){ $maxSclip=$temp3; $max =$temp; $dir=$temp2; } else { $max=$max; $dir=$dir; $maxSclip=$maxSclip; } $i++; } my $order2 = join("|", $left, $right); #print join ("\t",@_[0..2],$maxSclip,$max,$dir,$count,$order2) . "\n"; print OUTFILE join ("\t",@_[0..2],$maxSclip,$max,$dir,$count,$order2) . "\n"; } elsif($_=~/unknown/){ print OUTFILE join ("\t",@_[0..2],"NA","NA","left","NA","NA|NA") . "\n"; print OUTFILE join ("\t",@_[0..2],"NA","NA","right","NA","NA|NA") . "\n"; } ####Format is Chrom,start, end,longest Soft-clip,length of longest Soft-clip, direction of longest soft-clip,#supporting softclips,#right Sclips|#left Sclips } close INFILE; close OUTFILE; unlink("$tmp2","$tmp_name","$random_file","$tmp3","$outfile","$index","$random_name","$right_sc","$left_sc","$new_blacklist") if (-z $tmp3 || !-e $tmp3) ; if (-z $tmp3 || !-e $tmp3){ print "No structural variants found while Counting the number and identify directions of soft clips.\n" ; # open (OUT,">$OUTNAME")||die "Can't write files here!\n"; # &print_header(); # close OUT; exit; } print "Done counting Softclipped reads\n"; ############################################################### #### Print header information ############################################################### foreach my $random_file_disc(@INPUT_BAM){ print "Making the header for $random_file_disc\n"; $SAMPLE_NAME=`samtools view -f2 -H $random_file_disc|awk '{if(\$1~/^\@RG/){sub("ID:","",\$2);name=\$2;print name}}'|head -1`; $SAMPLE_NAME=~s/\n//g; if($chrom){$SAMPLE_NAME.=".".$chrom} $SAMPLE_NAME.=".vcf"; open (OUT,">$SAMPLE_NAME")||die "Can't write files here!\n"; &print_header(); # DO the bulk of the work open (FILE,"$tmp3")|| die "Can't open file\n"; while (<FILE>){ #If left clip {+- or -- or -+ }{+- are uninformative b/c they go upstream} #If right clip {++ or -- or +-} chomp $_; my @res=();my $res; my $line = $_; my @info = split(/\t/, $_); my $i=0; my $basename=basename($random_file_disc);$i=0; if($info[5] eq "left") { $res=bulk_work("left", $line, $random_file_disc); if(!$res){$res=join("\t",".",".",".",".",".",".",".",".",".",".")}; $i++; } elsif ($info[5] eq "right") { $res=bulk_work("right", $line, $random_file_disc); if(!$res){$res=join("\t",".",".",".",".",".",".",".",".",".",".")}; $i++; } if($res){@res=split("\t",$res); print OUT join("\t",@res)."\n"; }} close FILE; close OUT; print "Done with $random_file_disc\n\n"; } ############################################################################### ############################################################################### #### Delete temp files my $meregedBed=join("",$random_name,".sc.direction.bed.sc.merge.bed"); if(defined($temp_output)){$temp_output=$temp_output} else {$temp_output="no"} if ($temp_output eq "no"){ unlink("$tmp_name","$random_file","$tmp2",,"$tmp3","$outfile","$index","$random_name","$right_sc","$left_sc","$meregedBed","$random_name.bed"); } ####Sort VCF #my $tmp=join(".",$random_name,"tmp"); #Get header #$cmd="grep \"#\" $OUTNAME > $tmp"; #system($cmd); #sort results #$cmd="grep -v \"#\" $OUTNAME|perl -pne 's/chr//'|sort -k1,1n -k2,2n|perl -ne 'print \"chr\".\$_' >>$tmp"; #system($cmd); #$cmd="mv $tmp $OUTNAME"; #system($cmd); #remove entries next to each other print "Analysis Completed\n\nYou did it!!!\n"; print "Finish Time : " . &spGetCurDateTime() . "\n"; $now = time - $now; printf("\n\nTotal running time: %02d:%02d:%02d\n\n", int($now / 3600), int(($now % 3600) / 60), int($now % 60)); exit; ############################################################################### sub rev_comp { my $dna = shift; my $revcomp = reverse($dna); $revcomp =~ tr/ACGTacgt/TGCAtgca/; return $revcomp; } ############################################################################### #### to get reference base sub getSeq{ my ($chr,$pos,$fasta)=@_; #don't require chr #if($chr !~ /^chr/){die "$chr is not correct\n";} # die "$pos is not a number\n" if ($pos <0); my @result=(); if ($pos <0){print "$pos is not a valid position (likely caused by circular MT chromosome)\n";return;} @result = `samtools faidx $fasta $chr:$pos-$pos`; if($result[1]){chomp($result[1]); return uc($result[1]); } return("NA"); #### after return will not be printed ####print "RESULTS=@result\n"; } sub getBases{ my ($chr,$pos1,$pos2,$fasta)=@_; #don't require chr #if($chr !~ /^chr/){die "$chr is not correct\n";} my @result=(); if ($pos1 <0){print "$pos1 is not a valid position (likely caused by circular MT chromosome)\n";return;}; @result = `samtools faidx $fasta $chr:$pos1-$pos2`; if(!$result[1]){$result[1]="NA"}; chomp($result[1]); return uc($result[1]); #### after return will not be printed ####print "RESULTS=@result\n"; } ############################################################################### #### to get time sub spGetCurDateTime { my ($sec, $min, $hour, $mday, $mon, $year) = localtime(); my $curDateTime = sprintf "%4d-%02d-%02d %02d:%02d:%02d", $year+1900, $mon+1, $mday, $hour, $min, $sec; return ($curDateTime); } ############################################################################### #### print header sub print_header { my $date=&spGetCurDateTime(); my $header = qq{##fileformat=VCFv4.1 ##fileDate=$date ##source=SoftSearch.pl ##reference=$INPUT_FASTA ##Usage= SoftSearch.pl -l $minSoft -q $MapQ -r $minSoftReads -d $dist_To_Soft -m $minRP -u $unmated_pairs -s $num_sd -b @INPUT_BAM -f $INPUT_FASTA -o $OUTNAME ##INFO=<ID=SVTYPE,Number=1,Type=String,Description="Type of structural variant"> ##INFO=<ID=EVENT,Number=1,Type=String,Description="ID of event associated to breakend"> ##FORMAT=<ID=lSC,Number=1,Type=Integer,Description="Length of the longest soft clips supporting the BND"> ##FORMAT=<ID=nSC,Number=1,Type=Integer,Description="Number of supporting soft-clips\"> ##FORMAT=<ID=uRP,Number=1,Type=Integer,Description="Number of unmated read pairs nearby Soft-Clips"> ##FORMAT=<ID=levD_local,Number=1,Type=Float,Description="Levenstein distance between soft-clipped bases and the area around the original soft-clipped site"> ##FORMAT=<ID=levD_distl,Number=1,Type=Float,Description="Levenstein distance between the soft-clipped bases and mate location"> ##FORMAT=<ID=CTX,Number=1,Type=Integer,Description="Number of chromosomal translocations"> ##FORMAT=<ID=DEL,Number=1,Type=Integer,Description="Number of reads supporting Large Deletions"> ##FORMAT=<ID=INS,Number=1,Type=Integer,Description="Number of reads supporting Large insertions"> ##FORMAT=<ID=NOV_INS,Number=1,Type=Integer,Description="Number of reads supporting novel sequence insertion"> ##FORMAT=<ID=INV,Number=1,Type=Integer,Description="Number of reads supporting inversions"> ##FORMAT=<ID=sDEL,Number=1,Type=Integer,Description="Number of reads supporting novel sequence insertion"> ##INFO=<ID=NO_MATE_SC,Number=1,Type=Flag,Description="When there is no softclipping of the mate read location, an appromiate position is used"> ##FORMAT=<ID=GT,Number=1,Type=String,Description="Dummy value for maintaining VCF-Spec"> #CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT\t$SAMPLE_NAME\n}; print OUT $header; } ############################################################################### sub bulk_work { my ($side, $line, $file) = @_; my $local_levD = 0; my $distl_levD = 0; #my @info = split(/\t/, $line); my @plus_Reads = split(/\t/, $line); $plus_Reads[7] =~ s/\n//g; #### softclip length and softclip size. my $lSC = $plus_Reads[4]; my $nSC = $plus_Reads[6]; #Get all types of compatible reads #Get improperly paired reads (@ max distance) #### default value for left SIDE. #If left-clip, then look downstream for match of softclipped reads to define a deletion, but look for DRPs upstream my $sv_type = "SVTYPE=BND"; my $start_local=0; my $end_local=0;my $target_local="";my $target_drp="";my $start_drp="";my $end_drp=""; if ($side =~ /left/) { $start_local = $plus_Reads[1]-$dist_To_Soft; $end_local = $plus_Reads[2]; $start_drp = $plus_Reads[1]; $end_drp = $plus_Reads[1]+$dist_To_Soft; } else{ $start_local = $plus_Reads[1]; $end_local = $plus_Reads[1]+$dist_To_Soft; $start_drp = $plus_Reads[1]-$dist_To_Soft; $end_drp = $plus_Reads[1]; } $target_local=join("", $plus_Reads[0], ":", $start_local, "-", $end_local); $target_drp=join("", $plus_Reads[0], ":", $start_drp, "-", $end_drp); my $num_unmapped_pairs=""; if ($side =~ /right/) { $num_unmapped_pairs=`samtools view $new_blacklist -q $MapQ -f8 -F 1536 -c $file $target_drp`; } else { $num_unmapped_pairs=`samtools view $new_blacklist -q $MapQ -f24 -F 1536 -c $file $target_drp`; } if($verbose){print "samtools view $new_blacklist -q $MapQ -f24 -F 1536 -c $file $target_drp\n";} $num_unmapped_pairs=~s/\n//; if($verbose){print "NUM UNMAPPED PAIRS= $num_unmapped_pairs\n";} my $REF1_base = ""; my $REF2_base = ""; my $INFO_1 = ""; my $INFO_2 = ""; my $ALT_1 = ""; my $ALT_2 = ""; my $isize = 0; my $QUAL = ""; my $FORMAT = "GT:"; #### get 8 bit rand id my $BND1_name = join "", map { ("a".."z")[rand 26] } 1..8; my $BND2_name = join "", map { ("a".."z")[rand 26] } 1..8; $BND1_name=join "_","BND",$BND1_name; $BND2_name=join "_","BND",$BND2_name; my $counts = {CTX => 0, DEL => 0, INS => 0, INV => 0, TDUP => 0, NOV_INS => 0 }; my $event_mate_info = {CTX => "", DEL => "", INS => "", INV => "", TDUP => "", NOV_INS => "" }; #### get mate pair info and counts per event foreach my $e (sort keys %{$counts}) { my $h = get_counts_n_info($e, $side, $MapQ, $file, $dist_To_Soft, $target_drp, $upper_limit, $lower_limit); $counts->{$e} = $h->{count}; $event_mate_info->{$e} = $h->{info}; } my $max = 0; my $type = "UNKNOWN"; my $nRP = 0; my $mate_info = "NA\tNA\tNA\tNA"; my $summary = "GT:"; #### find max count of events and set type, nRP and info to corresponding #### max count event. #### also create a summary string of all counts to be added to VCF file. foreach my $e (sort keys %{$counts}){ # if ($counts->{$e} >=i $max){ if ($counts->{$e} > $max){ $type = $e .",". $counts->{$e}; $nRP = $counts->{$e}; $max = $counts->{$e}; if (length($event_mate_info->{$e})) { $mate_info = $event_mate_info->{$e}; } } $summary .= $e .",". $counts->{$e} .":"; } #print "done with Summaryi=$summary\n"; #### remove last colon ":" from $summary =~ s/:$//; if (($minRP > $max)&&(!$disable_RP_only )){return}; #### Run Levenstein distance on softclip in target region to find out if its a small deletion/insetion #### passing 1: clip_seq, 2: chr, 3: start, 4: end, 5: ref file. my $levD = new LevD; ######################################################## ######################################################## ######################################################## #### redefine start and end location for LevD calc. # $start = $plus_Reads[1]-$dist_To_Soft; # $end = $plus_Reads[2]; my $num_bases_to_loc=0; my $new_start=0; my $new_end=0; my $del_seq=""; my $start = $start_local; my $end = $end_local; if ($lSC=~/NA/){$lSC=0} if ($side =~ /right/) { $levD->search($plus_Reads[3], $plus_Reads[0], $start, $end, $INPUT_FASTA); $local_levD = sprintf("%.2f", $levD->{relative_edit_dist}); $num_bases_to_loc=$levD->{index}; $new_start = $plus_Reads[2]; if ($plus_Reads[2]=~/^[0-9]/){$new_end=$plus_Reads[2]+$lSC}; } else{ $levD->search($plus_Reads[3], $plus_Reads[0], $start, $end, $INPUT_FASTA); $local_levD = sprintf("%.2f", $levD->{relative_edit_dist}); $num_bases_to_loc=$levD->{index}; if ($plus_Reads[2]=~/^[0-9]/){$new_start=$plus_Reads[2]-$lSC}; $new_end = $plus_Reads[2]; } return if((!$new_start)||(!$new_end)); return if ($new_start<0); $del_seq=getBases($plus_Reads[0], $new_start,$new_end,$INPUT_FASTA); ############################################################################## # #If there is a match, where is the start position of the match? # ############################################################################## #if $plus_Reads[3] eq "NA", then it was found without soft-clipped reads if($plus_Reads[3] !~ /NA/){ if (($local_levD < $levD_local_threshold)) { return if (!$sv_only); #### add value to summary to be written to vcf file. $summary = "GT:sDel," . $plus_Reads[6]; $type = "sDEL"; ########################################################################### ##### Printing output ######################################### ##### Get DNA info ######################################### #$REF1_base = getSeq($plus_Reads[0], $plus_Reads[1], $INPUT_FASTA); $REF1_base = substr($del_seq, 0, 1); #### this is alt ref. for softclip its $plus_Reads[3] $REF2_base = $del_seq; $QUAL = 1/($local_levD + 0.001); $QUAL = sprintf("%.2f",$QUAL); $isize = length($del_seq); #### svtype = none for sDEL #### isize = length($info[3]); #### nRP = NA #### mate_id = NA #### CTX,:DEL,:....sDEL,## $INFO_1=join(";", "SVTYPE=NA", "EVENT=$type", "ISIZE=$isize"); #Add Sample infomration my $FORMAT="GT:sDEL"; $FORMAT .= ":lSC:nSC:uRP:levD_local"; my $SAMPLE= "0/1:"; $SAMPLE .= "$plus_Reads[6]:$lSC:$nSC:$num_unmapped_pairs:$local_levD"; #### remove any white spaces. $INFO_1=~s/\s//g; $INFO_2=~s/\s//g; $BND1_name =~ s/^BND/LEVD/; # If left, then the start position is plus_Reads[1]-isize my $start_pos=0; #Make sure Ref1 and Ref2 bases are different if($REF2_base eq $REF1_base){$REF1_base="NA"} if($side=~/left/){$start_pos=$plus_Reads[1]-$isize}else{$start_pos=$plus_Reads[1]}; my $var=join("\t", $plus_Reads[0], $start_pos, $BND1_name, $REF2_base, $REF1_base, $QUAL, "PASS", $INFO_1,$FORMAT,$SAMPLE); return $var; #print OUT join ("\t", $plus_Reads[0], $start_pos, $BND1_name, $REF2_base, $REF1_base, $QUAL, "PASS", $INFO_1,$FORMAT,$SAMPLE, "\n"); # return; } } #### Otherwise, look for DRP mate info #if($nRP=~/NA/){print "MATE_INFO=$mate_info\tSide=$side\tline=$line\n";} my @mate_info_arr = split(/\t/, $mate_info); $nRP = $mate_info_arr[3]; my $mate_chr=$mate_info_arr[0]; if((! defined $nRP) || ($nRP =~ /na/i) || ($mate_chr =~ /NA/) ){ #PRINT UNKNOWN return if ($nRP =~ /na/i); #print "There is an unknown\nNRP=$nRP Mate_CHR=$mate_chr minRP=$minRP\n";die; $summary .= ":unknown," . $plus_Reads[6]; $type = "unknown"; $REF1_base = getSeq($plus_Reads[0], $plus_Reads[1], $INPUT_FASTA); $REF2_base = $plus_Reads[3]; $BND1_name =~ s/^BND/UNKNOWN/; $QUAL = 1/($local_levD + 0.001); $QUAL = sprintf("%.2f",$QUAL); $INFO_1=join(";", "SVTYPE=unknown", "EVENT=unknown", "ISIZE=unknown"); #Add Sample infomration my $FORMAT="GT:sDEL"; $FORMAT .= ":lSC:nSC:uRP:levD_local"; my $SAMPLE = "0/1:"; $SAMPLE .= "$plus_Reads[6]:$lSC:$nSC:$num_unmapped_pairs:$local_levD"; #### remove any white spaces. $INFO_1=~s/\s//g; #print join ("\t", $plus_Reads[0], $plus_Reads[1], $REF2_base, $REF1_base, $QUAL, "PASS", $INFO_1,$FORMAT,$SAMPLE, "\n"); #print OUT join ("\t", $plus_Reads[0], $plus_Reads[1], $BND1_name, $REF1_base, $REF2_base, $QUAL, "PASS", $INFO_1,$FORMAT,$SAMPLE, "\n"); my $var=join("\t", $plus_Reads[0], $plus_Reads[1], $BND1_name, $REF1_base, $REF2_base, $QUAL, "PASS", $INFO_1,$FORMAT,$SAMPLE); return $var; } #### end if there is no mate info or nRP+uRP<minRP return if (($nRP<$minRP)&&($unmated_pairs > ($num_unmapped_pairs+$nRP))); ################################################################################## # Find out if mates have nearby soft-clips (to refine the breakpoints) ################################################################################## #Look for evidence of soft-clipping near mate my @mate_soft_arr = (); my $mate_start = 0; my $mate_soft = ""; @mate_info_arr = split(/\t/, $mate_info); #### mate start and end locations. my $filename = $right_sc; $start = $mate_info_arr[1] - $dist_To_Soft; $end = $mate_info_arr[1]; if ($side =~ /right/) { $start = $mate_info_arr[2]; $end = $mate_info_arr[2] + $dist_To_Soft; $filename = $left_sc; } #### add levenstein distance to Summary #print "Calc distal Levd\n"; $levD->search(rev_comp($plus_Reads[3]), $mate_info_arr[0], $start, $end, $INPUT_FASTA); $distl_levD = sprintf("%.2f", $levD->{relative_edit_dist}); $distl_levD = "NA" if($plus_Reads[3] =~ /NA/); #If there is no softclips to string match, then give 0 as quality value if ($plus_Reads[3] !~ /NA/){ $QUAL=1/($distl_levD + 0.001); } else { $QUAL=0; }; $QUAL=sprintf("%.2f",$QUAL); #### looking for softclips to refine break point #### if left look in right and vice-versa. $cmd = qq{echo -e "$mate_info_arr[0]\t$start\t$end"}; $cmd .= qq{ | awk -F'\t' 'NR==3' | intersectBed -a stdin -b $filename | head -1}; $mate_soft = `$cmd`; $mate_soft =~ s/\n//g; @mate_soft_arr = split(/\s/, $mate_soft); my $NO_MATE_SC=""; if(@mate_soft_arr){ $mate_chr = $mate_soft_arr[0]; $mate_start = $mate_soft_arr[1]; $NO_MATE_SC="APPROXIMATE"; } else{ @mate_info_arr = split(/\s/,$mate_info); $mate_chr = $mate_info_arr[0]; $mate_start = $mate_info_arr[1]; } #end if there is no mate info return if ($mate_chr eq ""); #end if there is no mate info and !disable_RP_only return if (($lSC =~/NA/)&&(!$disable_RP_only)); ########################################################################### ##### Printing output ######################################### # Get DNA info ######################################### #print "PLUS_READS=$plus_Reads[0],$plus_Reads[1]\nMATE=$mate_chr,$mate_start,$INPUT_FASTA\n"; $REF1_base = getSeq($plus_Reads[0], $plus_Reads[1], $INPUT_FASTA); ### this is alt ref. for softclip its $plus_Reads[3] $REF2_base = getSeq($mate_chr, $mate_start, $INPUT_FASTA); ######################################### # print in VCF format ######################################### #### abs value to account for left and right reads. $isize = abs($plus_Reads[1]-$mate_start); my $event_type=$type; $event_type=~ s/,|[0-9]//g; $INFO_1=join(";", "$sv_type", "EVENT=$event_type", "ISIZE=$isize","MATE_ID=$BND2_name"); $INFO_2=join(";", "$sv_type", "EVENT=$event_type", "ISIZE=$isize","MATE_ID=$BND1_name"); #### remove any white spaces. #### ask: did you mean to remove space from ends? eg. trim() $INFO_1=~s/\s//g; $INFO_2=~s/\s//g; $FORMAT=$summary; $FORMAT=~ s/,|[0-9]//g; $FORMAT .= ":lSC:nSC:uRP:distl_levD"; if($NO_MATE_SC){$INFO_2 .= ":NO_MATE_SC"} my $SAMPLE="0/1:"; $SAMPLE .=$summary; # if($NO_MATE_SC){$SAMPLE.= ":$NO_MATE_SC"} $SAMPLE=~s/[A-Z|,|_]//g; my $MATE_SAMPLE=$SAMPLE; $SAMPLE .= ":$lSC:$nSC:$num_unmapped_pairs:$distl_levD"; $MATE_SAMPLE .=":NA:NA:NA:NA"; $SAMPLE=~s/::/:/g; $MATE_SAMPLE=~s/::/:/g; if($type !~ /INV/){ $ALT_1 = join("","]",$mate_chr,":",$mate_start,"]",$REF1_base); $ALT_2 = join("",$REF2_base,"[",$plus_Reads[0],":",$plus_Reads[1],"["); # 2 321682 bnd_V T ]13:123456]T 6 PASS SVTYPE=BND # 13 123456 bnd_U C C[2:321682[ 6 PASS SVTYPE=BND } else { $ALT_1 = join("", "]", $plus_Reads[0], ":", $plus_Reads[1], "]", $REF2_base); $ALT_2 = join("", $REF1_base, "[", $mate_chr, ":", $mate_start, "["); } if(($mate_chr) && ($plus_Reads[0])){ # print OUT join ("\t", $plus_Reads[0], $plus_Reads[1], $BND1_name, $REF1_base, $ALT_1, $QUAL,"PASS", $INFO_1, $FORMAT,$SAMPLE,"\n"); # print OUT join ("\t", $mate_chr, $mate_start, $BND2_name, $REF2_base, $ALT_2, $QUAL, "PASS", $INFO_2, $FORMAT,$MATE_SAMPLE,"\n"); my $var=join ("\t", $plus_Reads[0], $plus_Reads[1], $BND1_name, $REF1_base, $ALT_1, $QUAL,"PASS", $INFO_1, $FORMAT,$SAMPLE); return $var; } } ############################################################################### ############################################################################### sub get_counts_n_info { my ($event, $side, $mapQ, $file, $dist, $target, $upL, $lwL) = @_; my $mate_info = ""; my $cmd = ""; if ($event =~ /^CTX$/i) { #print "CTX side $side\n"; if ($side =~ /right/i) { $cmd = qq{samtools view $new_blacklist -q $mapQ -f 32 -F 1536 $file $target}; $cmd .= qq{ | perl -ane 'if(\$F[6] ne "="){\$end=\$F[7]+1; print join ("\\t",\$F[6],\$F[7],\$end,"\\n")}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info=`$cmd`; } else { $cmd = qq{ samtools view $new_blacklist -q $mapQ -f 16 -F 1536 $file $target}; $cmd .= qq{ | perl -ane 'if(\$F[6] ne "="){\$end=\$F[7]+1; print join ("\\t",\$F[6],\$F[7],\$end,"\\n")}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info=`$cmd`; } } elsif ($event =~ /^DEL$/i) { #print "DEL side $side\n"; if ($side =~ /right/i) { $cmd = qq{samtools view $new_blacklist -q $mapQ -f 32 -F 1552 $file $target}; $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9>$upL)){end=\$8+1;print \$3,\$8,end}}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info=`$cmd`; } else { $cmd = qq{samtools view $new_blacklist -q $mapQ -F 1568 -f 16 $file $target}; $cmd .= qq{ | awk '{OFS="\\t"} {if((\$7 ~ /=/)&&(\$9<-$upL)){end=\$8+1;print \$3,\$8,end}}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist_To_Soft -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info=`$cmd`; } } elsif ($event =~ /^INS$/i) { #print "INS side $side\n"; if ($side =~ /right/i) { $cmd = qq{samtools view $new_blacklist -q $mapQ -f 32 -F 1552 $file $target}; $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9<$lwL && \$9 > 0 )){end=\$8+1;print \$3,\$8,end}}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info = `$cmd`; } else { $cmd = qq {samtools view $new_blacklist -q $mapQ -f 16 -F 1568 $file $target}; $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9>-$lwL && \$9 < 0 )){end=\$8+1;print \$3,\$8,end}}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info = `$cmd`; } } elsif ($event =~ /^INV$/i) { #print "INV side $side\n"; if ($side =~ /right/i) { $cmd = qq{samtools view $new_blacklist -q $mapQ -F 1596 $file $target}; $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)){end=\$8+1;print \$3,\$8,end}}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist_To_Soft -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info = `$cmd`; } else { $cmd = qq{samtools view $new_blacklist -q $mapQ -f 48 -F 1548 $file $target}; $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)){end=\$8+1;print \$3,\$8,end}}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info = `$cmd`; } } elsif ($event =~ /^TDUP$/i) { #print "TDUP side $side\n"; if ($side =~ /right/i) { $cmd = qq{samtools view $new_blacklist -q $mapQ -f 32 -F 1552 $file $target}; # $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9>$upL)){end=\$8+1;print \$3,\$8,end}}'}; $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$4>\$8)&&(\$9<0)){end=\$8+1;print \$3,\$8,end}}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info = `$cmd`; } else { $cmd = qq{samtools view $new_blacklist -q $mapQ -f 16 -F 1568 $file $target}; # $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9<-$upL )){end=\$8+1;print \$3,\$8,end}}'}; $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$4<\$8)&&(\$9>0)){end=\$8+1;print \$3,\$8,end}}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info = `$cmd`; } } elsif ($event =~ /^NOV_INS$/i) { #print "NOV_INS side $side\n"; if ($side =~ /right/i) { $cmd = qq{samtools view $new_blacklist -q $mapQ -f 8 -F 1552 $file $target}; $cmd .= qq{ | awk '{OFS="\\t"}{end=\$8+1;print \$3,\$8,end}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info = `$cmd`; } else { $cmd = qq{samtools view $new_blacklist -q $mapQ -f 24 -F 1536 $file $target}; $cmd .= qq{ | awk '{OFS="\\t"}{end=\$8+1;print \$3,\$8,end}'}; $cmd .= qq{ | sortBed | mergeBed -d $dist -n | sort -k4nr | head -1}; if($verbose){print "$cmd\n"} $mate_info = `$cmd`; } } $mate_info=~s/\n//g; my @tmp=split(/\t/, $mate_info); my $counts = 0; if (defined $tmp[3]) { $tmp[3] =~ s/\n//g; $counts = $tmp[3] if (length($tmp[3])); } return ({count=>$counts, info=>$mate_info}); }