Mercurial > repos > priti > vdt_sequence
diff SoftSearch.pl @ 10:e741e2f9ad03 draft
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| author | priti |
|---|---|
| date | Fri, 06 Jun 2014 01:53:32 -0400 |
| parents | |
| children |
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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/SoftSearch.pl Fri Jun 06 01:53:32 2014 -0400 @@ -0,0 +1,1193 @@ +#!/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 lib "/home/plus91/2.4/lib" ; + +use Getopt::Long; +use strict; +use warnings; +#use Data::Dumper; +use LevD; +use File::Basename; + +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 $cmd = ""; + +#Declare variables +GetOptions( + 'b=s' => \$INPUT_BAM, + 'f=s' => \$INPUT_FASTA, + 'o:s' => \$OUTPUT_FILE, + 'm:i' => \$minRP, + 'l:i' => \$minSoft, + 'r:i' => \$minSoftReads, + 't:i' => \$temp_output, + 's:s' => \$num_sd, + 'd:i' => \$dist_To_Soft, + 'q:i' => \$MapQ, + 'c:s' => \$chrom, + 'u:s' => \$unmated_pairs, + 'x:s' => \$minBQ, + 'p' => \$pair_only, + 'g' => \$disable_RP_only, + 'j:s' => \$levD_local_threshold, + 'k:s' => \$levD_distl_threshold, + 'a:s' => \$pe_upper_limit, + 'e:s' => \$high_qual, + 'L' => \$sv_only, + 'v' => \$verbose, + 'blacklist:s' => \$blacklist, + 'genome_file:s' => \$genome_file, + "help|h|?" => \&usage); + +unless($sv_only){$sv_only=""}; +if(defined($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"} + +### 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. In other words, don't try to find soft-clipping events!\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)}'`; + +my $metrics=`samtools view -q $MapQ -f2 $INPUT_BAM $chrom|head -10000|cut -f9|awk '{if (\$1<0){\$1=-\$1}else{\$1=\$1} sum+=\$1; sumsq+=\$1*\$1} END {print sum/NR, sqrt(sumsq/NR - (sum/NR)^2)}'`; +#my ($mean,$stdev)=split(/ /,$metrics); +my ($mean,$stdev)=split(/\s/,$metrics); +$stdev=~s/\n//; +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"; + $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)){ + $random_file_sc=join ("",$random_name,".sc.sam"); + $command=join ("","samtools view -q $MapQ -F 1024 $INPUT_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}' > ", $random_file_sc); + + print "Making SAM file of soft-clipped reads\n"; +if($verbose){ print "$command\n";} + system("$command"); + + ############################################################# + # Find areas that have deep enough soft-clip coverage + print "Identifying soft-clipped regions that are at least $minSoft bp long \n"; + open (FILE,"$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\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); + + +my $random_file_disc="$INPUT_BAM"; +### +# +###################################################### +# 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"} + 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","|egrep \";|,\"","|awk '{OFS=\"\t\"}(NF==4)'",">",$outfile); + +print "$command\n" if ($verbose); +system("$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("",$random_name,".sc.fixed.merge.bed"); + +open (INFILE,$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+=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 +############################################################### +open (OUT,">$OUTNAME")||die "Can't write files here!\n"; +&print_header(); +close OUT; + +############################################################### +############################################################### +#### DO the bulk of the work +############################################################### +use List::Util qw(min max); +open (FILE,"$tmp3")|| die "Can't open file\n"; +open (OUT,">>$OUTNAME")|| die "Can't open file\n"; + +#print "\nusing $tmp3 and writing to $OUTPUT_FILE \n"; +while (<FILE>){ + #If left clip {+- or -- or -+ }{+- are uninformative b/c they go upstream} + #If right clip {++ or -- or +-} + chomp $_; + my $line = $_; + my @info = split(/\t/, $_); + + if($info[5] eq "left") { + bulk_work("left", $line, $random_file_disc); + + } elsif ($info[5] eq "right") { + bulk_work("right", $line, $random_file_disc); + } +#if($. ==6){print "THIS IS LINE 6\n$_\n";die} +print "Completed line $.\n" if ($verbose); +} +close FILE; +close OUT; + +############################################################################### +############################################################################### +#### 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 + + + + +############################################################# +##May not need this anymore since filtering on left and right +############################################################# +#my $tmpout=$OUTNAME; +#$tmpout.=".tmp"; +#use FindBin qw($Bin); +##my $path=$Bin; +#$command="perl ".$path."/Extract_nSC.pl $OUTNAME -q nSC > $tmpout"; +##print "Command=$command\n"; +#system($command); +#$command="perl ".$path."/reduce_redundancy.pl $tmpout $upper_limit |cut -f1-10 > $OUTNAME"; +##print "$command\n"; +#system($command); +#system("rm $tmpout"); +######################################################## + + + + +print "Analysis Completed\n\nYou did it!!!\n"; +print "Finish Time : " . &spGetCurDateTime() . "\n"; +$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"> +##INFO=<ID=MATEID,Number=.,Type=String,Description="ID of mate breakends"> +##INFO=<ID=ISIZE,Number=.,Type=String,Description="Size of the SV"> +##INFO=<ID=END,Number=1,Type=Integer,Description="End position of the variant described in this record"> +##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=TDUP,Number=1,Type=Integer,Description="Number of reads supporting a tandem duplication"> +##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 $header; +} + + +############################################################################### +sub bulk_work { +print "#####################################@_\n" if ($verbose); + 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 $INPUT_BAM $target_drp`; + } else { + $num_unmapped_pairs=`samtools view $new_blacklist -q $MapQ -f24 -F 1536 -c $INPUT_BAM $target_drp`; + } +if($verbose){print "samtools view $new_blacklist -q $MapQ -f24 -F 1536 -c $INPUT_BAM $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} = $h->{info}; + } +#print Dumper($counts); + + 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 Summary\n"; + #### remove last colon ":" from + $summary =~ s/:$//; + if (($minRP > $max)&&(!$disable_RP_only )){if ($verbose){print "FAILED BECAUSE ($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 = $plus_Reads[2]; + } + if((!$new_start)||(!$new_end)||($new_start<0)){print "FAILED AT ((!$new_start)||(!$new_end)||($new_start<0))\n";return}; + + $del_seq=getBases($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]}; + print OUT join ("\t", $plus_Reads[0], $start_pos, $BND1_name, $REF2_base, $REF1_base, $QUAL, "PASS", $INFO_1,$FORMAT,$SAMPLE, "\n"); + if ($verbose){print "No Softclipped reads here!\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) || ($mate_chr =~ /NA/) ){ + #PRINT UNKNOWN + if ($nRP =~ /na/i){print "Can't find SC reads\n" if ($verbose);return}; + if ($verbose){print "There is an unknown\nNRP=$nRP Mate_CHR=$mate_chr minRP=$minRP\n"} + $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"; + $SAMPLE=~s/NA/0/g; + #### 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,$SAMPLE, "\n"); + return; + + } + #### end if there is no mate info or nRP+uRP<minRP + if (($nRP<$minRP)&&($unmated_pairs > ($num_unmapped_pairs+$nRP))){ + print "Something failed here\nif (($nRP<$minRP)&&($unmated_pairs > ($num_unmapped_pairs+$nRP)))\n"; + return} + + ################################################################################## + # 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' 'NF==3' | intersectBed -a stdin -b $filename | head -1}; +print "$cmd\n" if $verbose; + $mate_soft = `$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=$event_type","END=$mate_start", "ISIZE=$isize","MATEID=$BND2_name"); + $INFO_2=join(";", "$sv_type", "EVENT=$event_type","END=$plus_Reads[1]", "ISIZE=$isize","MATEID=$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; + $MATE_SAMPLE=~s/NA/0/g; + $SAMPLE=~s/NA/0/g; + + if($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"); + } +} + +############################################################################### +############################################################################### +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}}'}; + $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$4>\$8)&&(\$9<0)&& (\$9>$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 16 -F 1568 $file $target}; +# $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$9<-$upL )){end=\$8+1;print \$3,\$8,end}}'}; + $cmd .= qq{ | awk '{OFS="\\t"}{if((\$7 ~ /=/)&&(\$4<\$8)&&(\$9>0)&&(\$9>$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`; + } + } 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}); +}