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gnomit.pl
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gnomit.pl
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#!/usr/bin/perl
use warnings;
use strict;
use Getopt::Long;
my $version = "0.0.23";
# version update
# 0.0.23
# -added option to require a minimum level of evidence for sample level genotyping
#
# 0.0.22
# -updated version to be consistent with dinumt package
#
# 0.0.16
# -fixed program with too many supporting reads crashing GL calculation
# -updated output to include more original INFO tag information
#
# 0.0.15
# -added option to turn off EM iterations
#
# 0.0.14
# -added MT mapping check for clipped sequences at potential breakpoints
#
# 0.0.13
# -fixed error with likelihood calculation
# -added support for by_chr_dir genotyping
#
# 0.0.12
# -added per-read mapQ errors with likelihood calculation
#
# 0.0.11
# -updated supporting read determination
#
# 0.0.10
# -integrated RP and SR liklihoods, when available
#
# 0.0.9
# -incorporated EM algorithm for implementing allele frequency priors
#
# 0.0.8
# -updated support counting around breakpoints
#
# 0.0.7
# -modified scoring to implement bayesian scoring of evidence
# -incorporated insert length cutoff for alternative allele determination
#
# 0.0.6
# -fixed bug where altSR was not being initialized correctly
# -removed max_read_cov filter in report()
#
# 0.0.5
# -added --chr option for by-chromosome analysis
#
# 0.0.4
# -bug fixes
#
# 0.0.3
# -modified scoring function to better assess clipping in breakpoint determination and scoring
# -modified rp scoring to not consider reads clipped at breakpoint as reference
#
# 0.0.2
# -modified input parameters to expect VCF format
#
# 0.0.1
# -initial version
my %opts = ();
$opts{len_cluster_include} = 600;
$opts{len_cluster_link} = 800;
$opts{min_reads_cluster} = 1;
$opts{min_clipped_seq} = 5;
$opts{clipped_flank} = 50;
$opts{max_num_clipped} = 5;
$opts{include_mask} = 0;
$opts{min_evidence} = 3;
$opts{min_map_qual} = 10;
$opts{filter_qual} = 13;
$opts{filter_depth} = 5;
$opts{max_read_cov} = 200;
$opts{use_priors} = 0;
$opts{mask_filename} = "/home2/remills/projects/numts/numtS.bed";
$opts{info_filename} = "/scratch/remills_flux/remills/sampleInfo.txt";
$opts{reference} = "/nfs/remills-scratch/reference/hs37d5/hs37d5.fa";
$opts{mt_filename} = "/nfs/remills-scratch/reference/GRCh37/Sequence/Chromosomes/MT.fa";
$opts{samtools} = "/home2/remills/bin/samtools";
$opts{exonerate} = "/home2/remills/apps/exonerate-2.2.0/bin/exonerate";
$opts{dir_tmp} = "/tmp";
$opts{prefix} = "numt";
$opts{len_mt} = 16596; #eventually should be read in by BAM header
$opts{ploidy} = 2;
my $optResult = GetOptions(
"input_filename=s" => \$opts{input_filename},
"output_filename=s" => \$opts{output_filename},
"mask_filename=s" => \$opts{mask_filename},
"info_filename=s" => \$opts{info_filename},
"mt_filename=s" => \$opts{mt_filename},
"dir_tmp=s" => \$opts{dir_tmp},
"chr=s" => \$opts{chr},
"include_mask" => \$opts{include_mask},
"len_cluster_include=i" => \$opts{len_cluster_include},
"len_cluster_link=i" => \$opts{len_cluster_link},
"min_reads_cluster=i" => \$opts{min_reads_cluster},
"min_evidence=i" => \$opts{min_evidence},
"min_clipped_seq=i" => \$opts{min_clipped_seq},
"max_num_clipped=i" => \$opts{max_num_clipped},
"min_map_qual=i" => \$opts{min_map_qual},
"max_read_cov=i" => \$opts{max_read_cov},
"read_groups" => \$opts{read_groups},
"breakpoint" => \$opts{breakpoint},
"reference=s" => \$opts{reference},
"samtools=s" => \$opts{samtools},
"exonerate=s" => \$opts{exonerate},
"use_priors" => \$opts{use_priors},
"by_chr_dir" => \$opts{by_chr_dir},
"prefix=s" => \$opts{prefix},
"ucsc" => \$opts{ucsc},
"help" => \$opts{help},
"verbose" => \$opts{verbose}
);
#checkOptions( $optResult, \%opts, $version );
my $seq_num = 0;
my %seq_hash = ();
my %sorted_hash = ();
my $i = 1;
my %sample_hash = ();
my %infile_hash = ();
my %group_hash = ();
my %outfile_hash = ();
my %mask_hash = ();
my %data_hash = ();
getInput( \%infile_hash, \%mask_hash, \%sample_hash );
getData( \%infile_hash, \%mask_hash, \%sample_hash, \%data_hash );
assessBreaks( \%infile_hash, \%sample_hash, \%data_hash );
refineData( \%infile_hash, \%mask_hash, \%sample_hash, \%data_hash );
scoreData( \%data_hash, \%sample_hash );
report( \%infile_hash, \%data_hash );
################################################################################################################
sub mappedClippedSeq {
my ($clipSeq) = @_;
if ( $clipSeq =~ /N/ ) { return 0; }
my $random = int( rand(100000) );
my @timeData = localtime(time);
my $timestamp = join( '', @timeData );
my $fileClp = "$opts{dir_tmp}/clp$timestamp$random.fa";
open( CLP, ">$fileClp" );
print CLP ">clp\n$clipSeq\n";
close CLP;
my $results = `$opts{exonerate} --model affine:local --exhaustive yes --percent 70 --showvulgar no --showalignment no --showcigar yes $fileClp $opts{mt_filename} 2> /dev/null`;
my ( $query_id, $query_start, $query_end, $query_strand, $target_id, $target_start, $target_end, $target_strand, $score, $cigar ) = $results =~ /cigar\:\s+(\S+)\s+(\d+)\s+(\d+)\s+(\+|-)\s+(\S+)\s+(\d+)\s+(\d+)\s+(\+|-)\s+(\d+)\s+(.*?)\n/;
`rm $fileClp`;
if ($cigar) { return 1; }
else { return 0; }
}
sub assessBreaks {
my ( $infile_hash, $sample_hash, $data_hash ) = @_;
print "Entering assessBreaks()\n" if $opts{verbose};
foreach my $var ( keys %$data_hash ) {
print "-variant $var\n" if $opts{verbose};
my %clippedPos = ();
my %clippedSeq = ();
my $sumClipped = 0;
my $winStart = 1e10;
my $winEnd = 0;
my $maxFirst = 0;
my $maxSecond = 0;
foreach my $sample ( keys %{ $data_hash{$var} } ) {
foreach my $cPos ( keys %{ $data_hash{$var}{$sample}{clipPos} } ) {
next if $cPos == -1;
if ( defined( $data_hash{$var}{$sample}{clipPos}{$cPos}{1} ) ) {
if ( mappedClippedSeq( $data_hash{$var}{$sample}{clipSeq}{$cPos} ) ) {
$clippedPos{$cPos} += $data_hash{$var}{$sample}{clipPos}{$cPos}{1};
$sumClipped += $data_hash{$var}{$sample}{clipPos}{$cPos}{1};
#if (!defined($clippedSeq{$cPos} || length($data_hash{$var}{$sample}{clipSeq}{$cPos}) > $clippedSeq{$cPos})) { $clippedSeq{$cPos} = $data_hash{$var}{$sample}{clipSeq}{$cPos}; }
}
}
}
}
my $winLen = scalar keys %clippedPos;
my @sorted = sort { $clippedPos{$b} <=> $clippedPos{$a} } keys %clippedPos;
if ( $winLen == 0 ) { next; }
elsif ( $winLen == 1 ) {
$infile_hash{$var}{leftBkpt} = $sorted[0];
$infile_hash{$var}{rightBkpt} = $sorted[0] + 1;
}
elsif ( $winLen == 2 ) {
$infile_hash{$var}{leftBkpt} = $sorted[0];
$infile_hash{$var}{rightBkpt} = $sorted[0] + 1;
if ( $sorted[1] > $sorted[0] ) {
$infile_hash{$var}{rightBkpt} = $sorted[1];
}
else {
$infile_hash{$var}{leftBkpt} = $sorted[1];
$infile_hash{$var}{rightBkpt}--;
}
}
else {
my @sorted = sort { $clippedPos{$b} <=> $clippedPos{$a} } keys %clippedPos;
my $meanClipped = $sumClipped / $winLen;
my $sumSquares = 0;
print "\tmean clipped: $meanClipped\n" if $opts{verbose};
#for ( my $p = $winStart ; $p <= $winEnd ; $p++ ) {
# if ( !defined( $clippedPos{$p} ) ) { $clippedPos{$p} = 0; }
# $sumSquares += ( $clippedPos{$p} - $meanClipped )**2;
#}
foreach my $cPos ( keys %clippedPos ) {
$sumSquares += ( $clippedPos{$cPos} - $meanClipped )**2;
}
my $sdClipped = sqrt( $sumSquares / ( $winLen - 1 ) );
print "\tsd clipped: $sdClipped\n" if $opts{verbose};
print "\t1st clipped: $clippedPos{ $sorted[0] }\n" if defined( $sorted[0] ) && $opts{verbose};
print "\t2nd clipped: $clippedPos{ $sorted[1] }\n" if defined( $sorted[1] ) && $opts{verbose};
if ( defined( $sorted[0] ) && $clippedPos{ $sorted[0] } > $meanClipped + 1 * $sdClipped ) {
print "\t*updating to 1st\n" if $opts{verbose};
$infile_hash{$var}{leftBkpt} = $sorted[0];
$infile_hash{$var}{rightBkpt} = $sorted[0] + 1;
}
if ( defined( $sorted[1] ) && $clippedPos{ $sorted[1] } > $meanClipped + 1 * $sdClipped ) {
print "\t*updating to 1st and 2nd\n" if $opts{verbose};
if ( $sorted[1] > $sorted[0] ) {
$infile_hash{$var}{rightBkpt} = $sorted[1];
}
else {
$infile_hash{$var}{leftBkpt} = $sorted[1];
$infile_hash{$var}{rightBkpt}--;
}
}
}
print "-variant $var completed\n" if $opts{verbose};
}
print "Exiting assessBreaks()\n\n" if $opts{verbose};
}
sub scoreData {
my ( $data_hash, $sample_hash ) = @_;
print "Entering scoreData()\n" if $opts{verbose};
foreach my $var ( keys %$data_hash ) {
print "Variant: $var\n" if $opts{verbose};
my %priors = ();
my %genoFreq = ();
foreach my $geno ( 0 .. $opts{ploidy} ) {
#start with uniform priors
$priors{$geno} = 1 / ( $opts{ploidy} + 1 );
$genoFreq{$geno}{old} = 0;
$genoFreq{$geno}{new} = 0;
}
my $numIteration = 0;
my $sumGenoFreq = 0;
while ( $numIteration < 10 ) {
$numIteration++;
foreach my $sample ( keys %{$sample_hash} ) {
print "\tSample: $sample\n" if $opts{verbose};
#zero out alternative supporting evidence if below threshold
if ($$data_hash{$var}{$sample}{numAltRP} + $$data_hash{$var}{$sample}{numAltSR} < $opts{min_evidence}) {
$$data_hash{$var}{$sample}{numAltRP} = 0;
$$data_hash{$var}{$sample}{numAltSR} = 0;
$$data_hash{$var}{$sample}{qualAltRP} = ();
$$data_hash{$var}{$sample}{qualAltSR} = ();
}
my $numRefRP = $$data_hash{$var}{$sample}{numRefRP};
my $numAltRP = $$data_hash{$var}{$sample}{numAltRP};
my $numRefSR = $$data_hash{$var}{$sample}{numRefSR};
my $numAltSR = $$data_hash{$var}{$sample}{numAltSR};
foreach my $geno ( 0 .. $opts{ploidy} ) {
$$data_hash{$var}{$sample}{pl}{$geno} = 0;
$$data_hash{$var}{$sample}{gl}{$geno} = 0;
$$data_hash{$var}{$sample}{gl0}{$geno} = 0;
}
$$data_hash{$var}{$sample}{gq} = 0;
$$data_hash{$var}{$sample}{gt} = "./.";
$$data_hash{$var}{$sample}{ft} = "LowQual";
if ( $numAltRP + $numRefRP + $numAltSR + $numRefSR == 0 ) { next; }
if ( $$data_hash{$var}{$sample}{avgQ} <= 0 || $$data_hash{$var}{$sample}{avgQ} > 1 ) { $$data_hash{$var}{$sample}{avgQ} = 0.999999; }
$$data_hash{$var}{$sample}{avgQ} = 0.00001;
foreach my $g ( 0 .. $opts{ploidy} ) {
my $geno = $opts{ploidy} - $g; #need to reverse as calculation is reference allele based
if ( $numAltRP + $numRefRP > 0 && 1 / $opts{ploidy} ** ($numAltRP + $numRefRP) > 0) {
$$data_hash{$var}{$sample}{gl0}{$geno} += calcGl( $opts{ploidy}, $g, $numAltRP + $numRefRP, $numRefRP, $$data_hash{$var}{$sample}{qualRefRP}, $$data_hash{$var}{$sample}{qualAltRP} );
}
if ( $numAltSR + $numRefSR > 0 && $opts{breakpoint} && 1 / $opts{ploidy} ** ($numAltSR + $numRefSR) > 0) {
$$data_hash{$var}{$sample}{gl0}{$geno} += calcGl( $opts{ploidy}, $g, $numAltSR + $numRefSR, $numRefSR, $$data_hash{$var}{$sample}{qualRefSR}, $$data_hash{$var}{$sample}{qualAltSR} );
}
print "\tgl0 returned from calcGL() for geno $geno: $$data_hash{$var}{$sample}{gl0}{$geno}\n" if $opts{verbose};
if ( $$data_hash{$var}{$sample}{gl0}{$geno} < -255 ) { $$data_hash{$var}{$sample}{gl0}{$geno} = -255; } #capped
$$data_hash{$var}{$sample}{gl}{$geno} = log10( $priors{$geno} ) + $$data_hash{$var}{$sample}{gl0}{$geno}; #adjust with population inferred prior
}
my @sortedGeno = sort { $$data_hash{$var}{$sample}{gl}{$b} <=> $$data_hash{$var}{$sample}{gl}{$a} } keys %{ $$data_hash{$var}{$sample}{gl} };
#calculate PL from GL
foreach my $geno ( 0 .. $opts{ploidy} ) {
print "\tcalculating pl from geno ($$data_hash{$var}{$sample}{gl}{$geno})\n" if $opts{verbose};
$$data_hash{$var}{$sample}{pl}{$geno} = int( -10 * $$data_hash{$var}{$sample}{gl}{$geno} );
if ( $$data_hash{$var}{$sample}{pl}{$geno} > 255 ) { $$data_hash{$var}{$sample}{pl}{$geno} = 255; }
print "\t...$$data_hash{$var}{$sample}{pl}{$geno}\n" if $opts{verbose};
}
#normalize PL to most likely genotype
foreach my $geno ( 0 .. $opts{ploidy} ) {
$$data_hash{$var}{$sample}{pl}{$geno} -= $$data_hash{$var}{$sample}{pl}{ $sortedGeno[0] };
}
#determine genotype quality
$$data_hash{$var}{$sample}{gq} = int( 10 * ( $$data_hash{$var}{$sample}{gl}{ $sortedGeno[0] } - $$data_hash{$var}{$sample}{gl}{ $sortedGeno[1] } ) );
print "\t...$$data_hash{$var}{$sample}{gq}\n" if $opts{verbose};
my $gt = "0/0";
if ( $sortedGeno[0] == 1 ) { $gt = "0/1"; }
elsif ( $sortedGeno[0] == 2 ) { $gt = "1/1"; }
$$data_hash{$var}{$sample}{gt} = $gt;
if ( $numAltRP + $numRefRP + $numAltSR + $numRefSR < $opts{filter_depth} || $$data_hash{$var}{$sample}{gq} < $opts{filter_qual} ) { $$data_hash{$var}{$sample}{ft} = "LowQual"; }
else { $$data_hash{$var}{$sample}{ft} = "PASS"; }
$genoFreq{ $sortedGeno[0] }{new}++;
$sumGenoFreq++;
}
my $isConverged = 1;
if ( $sumGenoFreq == 0 ) { warn "no genotypes passing filters\n"; last; }
foreach my $geno ( 0 .. $opts{ploidy} ) {
if ( $genoFreq{$geno}{new} == 0 ) {
$priors{$geno} = 1 / $sumGenoFreq;
}
else {
$priors{$geno} = $genoFreq{$geno}{new} / $sumGenoFreq;
}
if ( $genoFreq{$geno}{old} != $genoFreq{$geno}{new} ) { $isConverged = 0; }
$genoFreq{$geno}{old} = $genoFreq{$geno}{new};
$genoFreq{$geno}{new} = 0;
}
if ($isConverged || !$opts{use_priors}) { last; }
}
}
print "Exiting scoreData()\n\n" if $opts{verbose};
}
sub getDate {
my ( $second, $minute, $hour, $dayOfMonth, $month, $yearOffset, $dayOfWeek, $dayOfYear, $daylightSavings ) = localtime();
my $year = 1900 + $yearOffset;
$month++;
my $fmonth = sprintf( "%.2d", $month );
my $fday = sprintf( "%.2d", $dayOfMonth );
return "$year$fmonth$fday";
}
sub report {
my ( $infile_hash, $data_hash ) = @_;
print "Entering report()\n" if $opts{verbose};
#open output file
if ( defined( $opts{output_filename} ) ) {
open( foutname1, ">$opts{output_filename}" ) or die("error opening file $opts{output_filename}\n");
}
else {
open( foutname1, ">&", \*STDOUT ) or die;
}
my $filedate = getDate();
print foutname1 <<HEADER;
##fileformat=VCFv4.1
##ALT=<ID=DEL,Description="Deletion">
##ALT=<ID=INS:MT,Description="Nuclear Mitochondrial Insertion">
##FILTER=<ID=LowQual,Description="No PASS calls in any sample after merging">
##FORMAT=<ID=CN,Number=1,Type=Integer,Description="Copy number genotype for imprecise events">
##FORMAT=<ID=CNL,Number=.,Type=Float,Description="Copy number likelihoods">
##FORMAT=<ID=CNL0,Number=.,Type=Float,Description="Copy number likelihoods with no frequency prior">
##FORMAT=<ID=CNQ,Number=1,Type=Float,Description="Copy number genotype quality for imprecise events">
##FORMAT=<ID=FT,Number=1,Type=String,Description="Per-sample genotype filter">
##FORMAT=<ID=GL0,Number=G,Type=Float,Description="Genotype likelihoods with no frequency prior">
##FORMAT=<ID=GQ,Number=1,Type=Integer,Description="Genotype Quality">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=PL,Number=G,Type=Integer,Description="Normalized, Phred-scaled likelihoods for genotypes as defined in the VCF specification">
##INFO=<ID=CIEND,Number=2,Type=Integer,Description="Confidence interval around END for imprecise variants">
##INFO=<ID=CIPOS,Number=2,Type=Integer,Description="Confidence interval around POS for imprecise variants">
##INFO=<ID=END,Number=1,Type=Integer,Description="End coordinate of this variant">
##INFO=<ID=HOMLEN,Number=.,Type=Integer,Description="Length of base pair identical micro-homology at event breakpoints">
##INFO=<ID=HOMSEQ,Number=.,Type=String,Description="Sequence of base pair identical micro-homology at event breakpoints">
##INFO=<ID=MSTART,Number=1,Type=Flag,Description="Mitochondrial start coordinate of inserted sequence">
##INFO=<ID=MEND,Number=1,Type=Flag,Description="Mitochondrial end coordinate of inserted sequence">
##INFO=<ID=MLEN,Number=1,Type=Flag,Description="Estimated length of mitochondrial insert">
##INFO=<ID=IMPRECISE,Number=0,Type=Flag,Description="Imprecise structural variation">
##INFO=<ID=NOVEL,Number=0,Type=Flag,Description="Indicates a novel structural variation">
##INFO=<ID=SAMPLES,Number=.,Type=String,Description="Sample(s) in which site was originally discovered">
##INFO=<ID=SVLEN,Number=.,Type=Integer,Description="Difference in length between REF and ALT alleles">
##INFO=<ID=SVTYPE,Number=1,Type=String,Description="Type of structural variant">
##contig=<ID=1,length=249250621,assembly=b37>
##contig=<ID=2,length=243199373,assembly=b37>
##contig=<ID=3,length=198022430,assembly=b37>
##contig=<ID=4,length=191154276,assembly=b37>
##contig=<ID=5,length=180915260,assembly=b37>
##contig=<ID=6,length=171115067,assembly=b37>
##contig=<ID=7,length=159138663,assembly=b37>
##contig=<ID=8,length=146364022,assembly=b37>
##contig=<ID=9,length=141213431,assembly=b37>
##contig=<ID=10,length=135534747,assembly=b37>
##contig=<ID=11,length=135006516,assembly=b37>
##contig=<ID=12,length=133851895,assembly=b37>
##contig=<ID=13,length=115169878,assembly=b37>
##contig=<ID=14,length=107349540,assembly=b37>
##contig=<ID=15,length=102531392,assembly=b37>
##contig=<ID=16,length=90354753,assembly=b37>
##contig=<ID=17,length=81195210,assembly=b37>
##contig=<ID=18,length=78077248,assembly=b37>
##contig=<ID=19,length=59128983,assembly=b37>
##contig=<ID=20,length=63025520,assembly=b37>
##contig=<ID=21,length=48129895,assembly=b37>
##contig=<ID=22,length=51304566,assembly=b37>
##contig=<ID=X,length=155270560,assembly=b37>
##contig=<ID=Y,length=59373566,assembly=b37>
##contig=<ID=MT,length=16569,assembly=b37>
##contig=<ID=GL000207.1,length=4262,assembly=b37>
##contig=<ID=GL000226.1,length=15008,assembly=b37>
##contig=<ID=GL000229.1,length=19913,assembly=b37>
##contig=<ID=GL000231.1,length=27386,assembly=b37>
##contig=<ID=GL000210.1,length=27682,assembly=b37>
##contig=<ID=GL000239.1,length=33824,assembly=b37>
##contig=<ID=GL000235.1,length=34474,assembly=b37>
##contig=<ID=GL000201.1,length=36148,assembly=b37>
##contig=<ID=GL000247.1,length=36422,assembly=b37>
##contig=<ID=GL000245.1,length=36651,assembly=b37>
##contig=<ID=GL000197.1,length=37175,assembly=b37>
##contig=<ID=GL000203.1,length=37498,assembly=b37>
##contig=<ID=GL000246.1,length=38154,assembly=b37>
##contig=<ID=GL000249.1,length=38502,assembly=b37>
##contig=<ID=GL000196.1,length=38914,assembly=b37>
##contig=<ID=GL000248.1,length=39786,assembly=b37>
##contig=<ID=GL000244.1,length=39929,assembly=b37>
##contig=<ID=GL000238.1,length=39939,assembly=b37>
##contig=<ID=GL000202.1,length=40103,assembly=b37>
##contig=<ID=GL000234.1,length=40531,assembly=b37>
##contig=<ID=GL000232.1,length=40652,assembly=b37>
##contig=<ID=GL000206.1,length=41001,assembly=b37>
##contig=<ID=GL000240.1,length=41933,assembly=b37>
##contig=<ID=GL000236.1,length=41934,assembly=b37>
##contig=<ID=GL000241.1,length=42152,assembly=b37>
##contig=<ID=GL000243.1,length=43341,assembly=b37>
##contig=<ID=GL000242.1,length=43523,assembly=b37>
##contig=<ID=GL000230.1,length=43691,assembly=b37>
##contig=<ID=GL000237.1,length=45867,assembly=b37>
##contig=<ID=GL000233.1,length=45941,assembly=b37>
##contig=<ID=GL000204.1,length=81310,assembly=b37>
##contig=<ID=GL000198.1,length=90085,assembly=b37>
##contig=<ID=GL000208.1,length=92689,assembly=b37>
##contig=<ID=GL000191.1,length=106433,assembly=b37>
##contig=<ID=GL000227.1,length=128374,assembly=b37>
##contig=<ID=GL000228.1,length=129120,assembly=b37>
##contig=<ID=GL000214.1,length=137718,assembly=b37>
##contig=<ID=GL000221.1,length=155397,assembly=b37>
##contig=<ID=GL000209.1,length=159169,assembly=b37>
##contig=<ID=GL000218.1,length=161147,assembly=b37>
##contig=<ID=GL000220.1,length=161802,assembly=b37>
##contig=<ID=GL000213.1,length=164239,assembly=b37>
##contig=<ID=GL000211.1,length=166566,assembly=b37>
##contig=<ID=GL000199.1,length=169874,assembly=b37>
##contig=<ID=GL000217.1,length=172149,assembly=b37>
##contig=<ID=GL000216.1,length=172294,assembly=b37>
##contig=<ID=GL000215.1,length=172545,assembly=b37>
##contig=<ID=GL000205.1,length=174588,assembly=b37>
##contig=<ID=GL000219.1,length=179198,assembly=b37>
##contig=<ID=GL000224.1,length=179693,assembly=b37>
##contig=<ID=GL000223.1,length=180455,assembly=b37>
##contig=<ID=GL000195.1,length=182896,assembly=b37>
##contig=<ID=GL000212.1,length=186858,assembly=b37>
##contig=<ID=GL000222.1,length=186861,assembly=b37>
##contig=<ID=GL000200.1,length=187035,assembly=b37>
##contig=<ID=GL000193.1,length=189789,assembly=b37>
##contig=<ID=GL000194.1,length=191469,assembly=b37>
##contig=<ID=GL000225.1,length=211173,assembly=b37>
##contig=<ID=GL000192.1,length=547496,assembly=b37>
##contig=<ID=NC_007605,length=171823,assembly=b37>
##contig=<ID=hs37d5,length=35477943,assembly=b37>
##fileDate=$filedate
##reference=hs37d5
##source=gnomit-$version
HEADER
my @vars = sort { $$infile_hash{$a}{chr} cmp $$infile_hash{$b}{chr} || $$infile_hash{$a}{start} <=> $$infile_hash{$b}{start} } keys %$infile_hash;
my @samples = sort { $a cmp $b } keys %{ $data_hash{ $vars[0] } };
print foutname1 "#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT";
foreach my $sample (@samples) {
print foutname1 "\t$sample";
}
print foutname1 "\n";
my $index = 1;
foreach my $var (@vars) {
my $chrom = $$infile_hash{$var}{chr};
my $id = $$infile_hash{$var}{id};
my $alt = "<INS:MT>";
my $qual = $$infile_hash{$var}{qual};
my $filter = $$infile_hash{$var}{filter};
my %info = ();
my $pos;
my $end;
my $ciDelta;
if ( defined( $$infile_hash{$var}{leftBkpt} ) ) {
$pos = $$infile_hash{$var}{leftBkpt} - 1;
$end = $$infile_hash{$var}{rightBkpt};
$ciDelta = $end - $pos + 1;
}
else {
$pos = $$infile_hash{$var}{start} - 1;
$end = $$infile_hash{$var}{end};
$ciDelta = $end - $pos + 1;
$info{IMPRECISE} = undef;
}
if ( defined($$infile_hash{$var}{mlen}) ){
$info{MLEN} = $$infile_hash{$var}{mlen};
$info{MSTART} = $$infile_hash{$var}{mstart};
$info{MEND} = $$infile_hash{$var}{mend};
}
$info{SAMPLES} = $$infile_hash{$var}{samples};
$info{CIPOS} = "0,$ciDelta";
$info{CIEND} = "-$ciDelta,0";
$info{END} = $end;
$info{SVTYPE} = "INS";
my $refline = `$opts{samtools} faidx $opts{reference} $chrom:$pos-$pos`;
my $ref = ( split( /\n/, $refline ) )[1];
if ( !defined($ref) ) { $ref = "N"; }
my $format = "GT:FT:GL0:GQ:PL";
my $info = "";
my @sKeys = sort { $a cmp $b } keys %info;
for ( my $i = 0 ; $i <= $#sKeys ; $i++ ) {
if ( $i > 0 ) { $info .= ";"; }
if ( defined( $info{ $sKeys[$i] } ) ) {
$info .= "$sKeys[$i]=$info{$sKeys[$i]}";
}
else {
$info .= "$sKeys[$i]";
}
}
print foutname1 "$chrom\t$pos\t$id\t$ref\t$alt\t$qual\t$filter\t$info\t$format";
foreach my $sample (@samples) {
my @gls = ();
my @pls = ();
foreach my $geno ( 0 .. $opts{ploidy} ) {
push @gls, sprintf( "%.2f", $$data_hash{$var}{$sample}{gl0}{$geno} );
push @pls, $$data_hash{$var}{$sample}{pl}{$geno};
}
my $gl = join( ",", @gls );
my $pl = join( ",", @pls );
print foutname1 "\t$$data_hash{$var}{$sample}{gt}:$$data_hash{$var}{$sample}{ft}:$gl:$$data_hash{$var}{$sample}{gq}:$pl";
}
print foutname1 "\n";
$index++;
}
close(foutname1);
print "Exiting report()\n\n" if $opts{verbose};
}
sub calcGl {
my ( $m, $g, $k, $l, $er, $ea ) = @_;
print "in calcGl():\n" if $opts{verbose};
print "\t$m\t$g\t$k\t$l\t$er\t$ea\n" if $opts{verbose};
if ( 1 / $m**$k <= 0 ) { die "problem in calcGL 1, \t$m\t$g\t$k\t$l\t$er\t$ea\n"; }
my $gl = log10( 1 / ( $m**$k ) );
foreach my $e ( @{$er} ) {
if ( ( ( $m - $g ) * $e ) + ( ( 1 - $e ) * $g ) <= 0 ) { die "problem in calcGL 2, \t$m\t$g\t$k\t$l\t$e\n"; }
$gl += log10( ( ( $m - $g ) * $e ) + ( ( 1 - $e ) * $g ) );
}
foreach my $e ( @{$ea} ) {
if ( ( $m - $g ) * ( 1 - $e ) + ( $g * $e ) <= 0 ) { die "problem in calcGL 3, \t$m\t$g\t$k\t$l\t$e\n"; }
$gl += log10( ( $m - $g ) * ( 1 - $e ) + ( $g * $e ) );
}
return $gl;
}
sub log10 {
my $n = shift;
return log($n) / log(10);
}
sub getInput {
my ( $infile_hash, $mask_hash, $sample_hash ) = @_;
print "Entering getInput()\n" if $opts{verbose};
#input sample information
open( INFO, $opts{info_filename} ) || die "error opening file $opts{info_filename}, $!\n";
my @header = ();
my %fields = ();
while (<INFO>) {
chomp;
if (/^sample/) { @header = split(/\t/); next; }
my @row = split(/\t/);
for ( my $i = 0 ; $i <= $#header ; $i++ ) {
$fields{ $header[$i] } = $row[$i];
}
my $sample = $fields{sample};
$$sample_hash{$sample}{pop} = $fields{pop};
$$sample_hash{$sample}{filename} = $fields{filename};
if ( $fields{median_insert_size} ne "NA" ) {
$$sample_hash{$sample}{winlen} = $fields{median_insert_size} + 3 * $fields{median_absolute_deviation};
$$sample_hash{$sample}{median_insert_size} = $fields{median_insert_size};
$$sample_hash{$sample}{median_absolute_deviation} = $fields{median_absolute_deviation};
}
else {
$$sample_hash{$sample}{winlen} = 500;
$$sample_hash{$sample}{median_insert_size} = 400;
$$sample_hash{$sample}{median_absolute_deviation} = 40;
}
$$sample_hash{$sample}{meancoverage} = $fields{mean_coverage};
if ( defined( $fields{read_groups} ) ) {
my @rgs = split( /,/, $fields{read_groups} );
%{ $$sample_hash{$sample}{read_groups} } = map { $_, 1 } @rgs;
}
}
close INFO;
#input mask coordinates
open( MASK, $opts{mask_filename} ) || die "Could not open $opts{mask_filename} for input, $!\n";
while (<MASK>) {
chomp;
my ( $chr, $start, $end, $id ) = split(/\t/);
$chr =~ s/chr//g;
$$mask_hash{$chr}{$start} = $end;
}
close MASK;
#input variant coordinates
if ( $opts{input_filename} =~ /\.gz$/ ) {
open( VARS, "zcat $opts{input_filename} |" ) || die "Could not open $opts{input_filename} for input, $!\n";
}
else {
open( VARS, $opts{input_filename} ) || die "Could not open $opts{input_filename} for input, $!\n";
}
my $varnum = 1;
#BED FORMAT
#while (<VARS>) {
# chomp;
# my ( $chr, $start, $end ) = split(/\t/);
# $chr =~ s/chr//g;
# $$infile_hash{$varnum}{chr} = $chr;
# $$infile_hash{$varnum}{start} = $start;
# $$infile_hash{$varnum}{end} = $end;
# $varnum++;
#}
#VCF FORMAT
while (<VARS>) {
next if /^#/;
chomp;
my ( $chr, $pos, $id, $ref, $alt, $qual, $filter, $info ) = split(/\t/);
$chr =~ s/chr//g;
if ( defined( $opts{chr} ) ) {
if ( $chr ne $opts{chr} ) { next; }
}
$infile_hash{$varnum}{chr} = $chr;
$infile_hash{$varnum}{id} = $id;
$infile_hash{$varnum}{start} = $pos + 1;
my ($end) = $info =~ /END=(\d+)/;
my ($mstart) = $info =~ /MSTART=(\d+)/;
my ($mend) = $info =~ /MEND=(\d+)/;
my ($mlen) = $info =~ /MLEN=(\d+)/;
my ($samples) = $info =~ /SAMPLES=(\w+?);/;
if (defined($mlen)) {
$infile_hash{$varnum}{mlen} = $mlen;
$infile_hash{$varnum}{mstart} = $mstart;
$infile_hash{$varnum}{mend} = $mend;
}
$infile_hash{$varnum}{samples} = $samples;
$infile_hash{$varnum}{end} = $end;
$infile_hash{$varnum}{filter} = $filter;
$infile_hash{$varnum}{qual} = $qual;
$varnum++;
}
close VARS;
print "Exiting getInput()\n\n" if $opts{verbose};
}
sub getMateInfo {
my ( $qname, $rnext, $pnext, $readgroup_hash, $filename ) = @_;
my $command = "";
if ( $opts{by_chr_dir} ) {
if ( $opts{ucsc} ) {
$command = "$opts{samtools} view $filename" . "chr$rnext.*bam chr$rnext:$pnext-$pnext |";
}
else {
$command = "$opts{samtools} view $filename$rnext.*bam $rnext:$pnext-$pnext |";
}
}
else {
if ( $opts{ucsc} ) {
$command = "$opts{samtools} view $filename chr$rnext:$pnext-$pnext |";
}
else {
$command = "$opts{samtools} view $filename $rnext:$pnext-$pnext |";
}
}
open( MCI, "$command" ) || die "Could not open $filename, $!\n";
my $cFlag = 0;
my $cPos = -1;
my $clipside = "n";
my $clipsize = -1;
my $clipseq = "";
my $seqLen = 0;
my $seq = "";
my $matchLen = 0;
while (<MCI>) {
chomp;
my ( $m_qname, $m_flag, $m_rname, $m_pos, $m_mapq, $m_cigar, $m_rnext, $m_pnext, $m_tlen, $m_seq, $m_qual, $opt ) = split(/\t/);
if ( $m_qname ne $qname ) { next; }
my ($read_group) = $_ =~ /RG:Z:(\S+)/;
if ( $opts{read_groups} && !defined($read_group) ) { next; }
elsif ( $opts{read_groups} && !defined( $$readgroup_hash{$read_group} ) ) { next; }
( $cFlag, $cPos, $clipside, $clipsize, $clipseq ) = getSoftClipInfo( $m_pos, $m_cigar, $m_qual, $m_seq );
$seqLen = 0;
$matchLen = 0;
$seq = $m_seq;
while ( $m_cigar =~ /(\d+)M/g ) {
$seqLen += $1;
$matchLen += $1;
}
while ( $m_cigar =~ /(\d+)N/g ) {
$seqLen += $1;
}
while ( $m_cigar =~ /(\d+)D/g ) {
$seqLen += $1;
}
}
close MCI;
return ( $cFlag, $cPos, $clipside, $clipsize, $seqLen, $matchLen, $seq );
}
sub refineData {
my ( $infile_hash, $mask_hash, $sample_hash, $data_hash ) = @_;
print "Entering refineData()\n" if $opts{verbose};
my $numSamp = 0;
foreach my $var ( keys %{$infile_hash} ) {
if ( !defined( $$infile_hash{$var}{leftBkpt} ) ) { next; }
foreach my $sample ( keys %{$sample_hash} ) {
$numSamp++;
#last if $numSamp > 50;
#currently basing coordinates off of ONLY start position, may need to revisit
my $chr = $$infile_hash{$var}{chr};
my $l_start = $$infile_hash{$var}{leftBkpt} - $$sample_hash{$sample}{winlen};
my $l_end = $$infile_hash{$var}{leftBkpt};
my $r_start = $$infile_hash{$var}{rightBkpt};
my $r_end = $$infile_hash{$var}{rightBkpt} + $$sample_hash{$sample}{winlen};
my $command = "";
my $chrM = "";
print "REFINED: $var\t$chr\t$l_start\t$l_end\t$r_start\t$r_end\n" if $opts{verbose};
if ( $opts{by_chr_dir} ) {
if ( $opts{ucsc} ) {
$command = "$opts{samtools} view $$sample_hash{$sample}{filename}chr$chr.*bam chr$chr:$l_start-$r_end |";
$chrM = "M";
}
else {
$command = "$opts{samtools} view $$sample_hash{$sample}{filename}$chr.*bam $chr:$l_start-$r_end |";
$chrM = "MT";
}
}
else {
if ( $opts{ucsc} ) {
$command = "$opts{samtools} view $$sample_hash{$sample}{filename} chr$chr:$l_start-$r_end |";
$chrM = "M";
}
else {
$command = "$opts{samtools} view $$sample_hash{$sample}{filename} $chr:$l_start-$r_end |";
$chrM = "MT";
}
}
$$data_hash{$var}{$sample}{numRefRP} = 0;
$$data_hash{$var}{$sample}{numAltRP} = 0;
$$data_hash{$var}{$sample}{numRefSR} = 0;
$$data_hash{$var}{$sample}{numAltSR} = 0;
$$data_hash{$var}{$sample}{qualRefRP} = ();
$$data_hash{$var}{$sample}{qualAltRP} = ();
$$data_hash{$var}{$sample}{qualRefSR} = ();
$$data_hash{$var}{$sample}{qualAltSR} = ();
$$data_hash{$var}{$sample}{avgQ} = 0;
my %found = ();
#print "command: $command\n" if $opts{verbose};
open( SAM, $command ) || die "error in opening file, $!\n";
while (<SAM>) {
chomp;
my ( $qname, $flag, $rname, $pos, $mapq, $cigar, $rnext, $pnext, $tlen, $seq, $qual ) = split(/\t/);
$rname =~ s/chr//g;
$rnext =~ s/chr//g;
my $mapE = 10**( -1 * $mapq / 10 );
my ($read_group) = $_ =~ /RG:Z:(\S+)/;
if ( $opts{read_groups} && !defined($read_group) ) { next; }
elsif ( $opts{read_groups} && !defined( $$sample_hash{$sample}{read_groups}{$read_group} ) ) { next; }
if ( $mapq < $opts{min_map_qual} ) { next; }
my $dir = 0; #F
if ( $flag & 16 ) { $dir = 1; } #R
my $dnext = 0; #mate F
if ( $flag & 32 ) { $dnext = 1; } #mate R
my $seqLen = 0;
my $matchLen = 0;
while ( $cigar =~ /(\d+)M/g ) {
$matchLen += $1;
$seqLen += $1;
}
while ( $cigar =~ /(\d+)N/g ) {
$seqLen += $1;
}
while ( $cigar =~ /(\d+)D/g ) {
$seqLen += $1;
}
my ( $cFlag, $cPos, $clipside, $clipsize, $clipseq ) = getSoftClipInfo( $pos, $cigar, $qual, $seq );
my ( $m_cFlag, $m_cPos, $m_clipside, $m_clipsize, $m_seqLen, $m_matchLen, $m_seq ) = getMateInfo( $qname, $rname, $pnext, $$sample_hash{$sample}{read_groups}, $$sample_hash{$sample}{filename} );
if ( $cFlag == 1 && ( abs( $cPos - $$infile_hash{$var}{leftBkpt} ) <= $opts{min_clipped_seq} || abs( $cPos - $$infile_hash{$var}{rightBkpt} ) <= $opts{min_clipped_seq} ) ) {
$$data_hash{$var}{$sample}{numAltSR}++;
push @{ $$data_hash{$var}{$sample}{qualAltSR} }, $mapE;
}
elsif ( $matchLen >= 0.95 * length($seq) && ( ( $pos < $$infile_hash{$var}{leftBkpt} && $pos + $seqLen - 1 > $$infile_hash{$var}{leftBkpt} && abs( $$infile_hash{$var}{leftBkpt} - $pos ) > $opts{min_clipped_seq} && abs( $$infile_hash{$var}{leftBkpt} - ( $pos + $seqLen - 1 ) ) > $opts{min_clipped_seq} ) || ( $pos < $$infile_hash{$var}{rightBkpt} && $pos + $seqLen - 1 > $$infile_hash{$var}{rightBkpt} && abs( $$infile_hash{$var}{rightBkpt} - $pos ) > $opts{min_clipped_seq} && abs( $$infile_hash{$var}{rightBkpt} - ( $pos + $seqLen - 1 ) ) > $opts{min_clipped_seq} ) ) ) {
#non-clipped reads with slight overlap with breakpoint may have mismatches instead of clipping, so skip
#also require at least 95% of read be 'matched' to reference, as the presence of insertion can cause some wacky mappings
$$data_hash{$var}{$sample}{numRefSR}++;
push @{ $$data_hash{$var}{$sample}{qualRefSR} }, $mapE;
}
if ( $rnext eq $chrM || checkMaskOverlap( $chr, $pos, $mask_hash ) ) {
#mate mapped to mt sequence
if ( ( $dir == 0 && $pos >= $l_start && $pos <= $l_end ) || ( $dir == 1 && $pos >= $r_start && $pos <= $r_end ) ) { $$data_hash{$var}{$sample}{numAltRP}++; push @{ $$data_hash{$var}{$sample}{qualAltRP} }, $mapE; }
}
elsif ( $dir == 0 && $dnext == 1 && $rnext eq "=" && !$found{$qname} ) {
$found{$qname} = 1;
if ( abs($tlen) > $$sample_hash{$sample}{median_insert_size} + 3 * $$sample_hash{$sample}{median_absolute_deviation} ) { next; } #insert length of potential reference supporting allele out of upper bounds
if ( abs($tlen) < $$sample_hash{$sample}{median_insert_size} - 3 * $$sample_hash{$sample}{median_absolute_deviation} ) { next; } #insert length of potential reference supporting allele out of lower bounds
#allow some flexibility for clipped positions around breakpoint due to alignment artifacts (later may want to inplement a realignment step)
if ( $cPos > -1 && abs( $cPos - $l_end ) <= $opts{min_clipped_seq} && $cFlag == 1 ) { $$data_hash{$var}{$sample}{numAltRP}++; push @{ $$data_hash{$var}{$sample}{qualAltRP} }, $mapE; }
elsif ( $m_cPos > -1 && abs( $m_cPos - $l_end ) <= $opts{min_clipped_seq} && $m_cFlag == 1 ) { $$data_hash{$var}{$sample}{numAltRP}++; push @{ $$data_hash{$var}{$sample}{qualAltRP} }, $mapE; }
elsif ( $cPos > -1 && abs( $cPos - $r_start ) <= $opts{min_clipped_seq} && $cFlag == 1 ) { $$data_hash{$var}{$sample}{numAltRP}++; push @{ $$data_hash{$var}{$sample}{qualAltRP} }, $mapE; }
elsif ( $m_cPos > -1 && abs( $m_cPos - $r_start ) <= $opts{min_clipped_seq} && $m_cFlag == 1 ) { $$data_hash{$var}{$sample}{numAltRP}++; push @{ $$data_hash{$var}{$sample}{qualAltRP} }, $mapE; }
elsif ( ( $pos <= $l_end && $pnext + $m_seqLen >= $l_end ) || ( $pos <= $r_start && $pnext + $m_seqLen >= $r_start ) ) {
#non-clipped reads with slight overlap with breakpoint may have mismatches instead of clipping
if ( abs( $pos - $l_end ) > $opts{min_clipped_seq}
&& abs( $pos + $seqLen - 1 - $l_end ) > $opts{min_clipped_seq}
&& abs( $pnext - $l_end ) > $opts{min_clipped_seq}
&& abs( $pnext + $m_seqLen - 1 - $l_end ) > $opts{min_clipped_seq}
&& abs( $pos - $r_start ) > $opts{min_clipped_seq}
&& abs( $pos + $seqLen - 1 - $r_start ) > $opts{min_clipped_seq}
&& abs( $pnext - $r_start ) > $opts{min_clipped_seq}
&& abs( $pnext + $m_seqLen - 1 - $r_start ) > $opts{min_clipped_seq}
&& $matchLen >= 0.95 * length($seq)
&& $m_matchLen >= 0.95 * length($m_seq) )
{
$data_hash{$var}{$sample}{numRefRP}++;
push @{ $$data_hash{$var}{$sample}{qualRefRP} }, $mapE;
}
}
}
$$data_hash{$var}{$sample}{avgQ} += $mapE;
}
close SAM;
if ( $$data_hash{$var}{$sample}{numRefRP} + $$data_hash{$var}{$sample}{numAltRP} > 0 ) {
$$data_hash{$var}{$sample}{avgQ} /= ( $$data_hash{$var}{$sample}{numRefRP} + $$data_hash{$var}{$sample}{numAltRP} );
}
else {
$data_hash{$var}{$sample}{avgQ} = 0.00001;
}
print "$sample\t$var\t$$data_hash{$var}{$sample}{numRefRP}\t$$data_hash{$var}{$sample}{numRefSR}\t$$data_hash{$var}{$sample}{numAltRP}\t$$data_hash{$var}{$sample}{numAltSR}\t$$data_hash{$var}{$sample}{avgQ}\n" if $opts{verbose};
}
}
print "Exiting refineData()\n\n" if $opts{verbose};
}
sub getData {
my ( $infile_hash, $mask_hash, $sample_hash, $data_hash ) = @_;
print "Entering getData()\n" if $opts{verbose};
my $numSamp = 0;
foreach my $var ( keys %{$infile_hash} ) {
foreach my $sample ( keys %{$sample_hash} ) {
$numSamp++;
my $chr = $$infile_hash{$var}{chr};
my $l_start = $$infile_hash{$var}{start} - $$sample_hash{$sample}{winlen};
my $l_end = $$infile_hash{$var}{start};
my $r_start = $$infile_hash{$var}{end};
my $r_end = $$infile_hash{$var}{end} + $$sample_hash{$sample}{winlen};
my $command = "";
my $chrM = "";
#print "$var\t$chr\t$l_start\t$l_end\t$r_start\t$r_end\n" if $opts{verbose};
if ( $opts{by_chr_dir} ) {
if ( $opts{ucsc} ) {
$command = "$opts{samtools} view $$sample_hash{$sample}{filename}chr$chr.*bam chr$chr:$l_start-$r_end |";
$chrM = "M";
}
else {
$command = "$opts{samtools} view $$sample_hash{$sample}{filename}$chr.*bam $chr:$l_start-$r_end |";
$chrM = "MT";
}
}
else {
if ( $opts{ucsc} ) {
$command = "$opts{samtools} view $$sample_hash{$sample}{filename} chr$chr:$l_start-$r_end |";
$chrM = "M";
}
else {
$command = "$opts{samtools} view $$sample_hash{$sample}{filename} $chr:$l_start-$r_end |";
$chrM = "MT";
}
}
$$data_hash{$var}{$sample}{numRefRP} = 0;
$$data_hash{$var}{$sample}{numAltRP} = 0;
$$data_hash{$var}{$sample}{numRefSR} = 0;
$$data_hash{$var}{$sample}{numAltSR} = 0;
$$data_hash{$var}{$sample}{qualRefRP} = ();
$$data_hash{$var}{$sample}{qualAltRP} = ();
$$data_hash{$var}{$sample}{qualRefSR} = ();
$$data_hash{$var}{$sample}{qualAltSR} = ();
$$data_hash{$var}{$sample}{avgQ} = 0.000001;
#print "command: $command\n" if $opts{verbose};
open( SAM, $command ) || die "error in opening file, $!\n";
while (<SAM>) {
chomp;
my ( $qname, $flag, $rname, $pos, $mapq, $cigar, $rnext, $pnext, $tlen, $seq, $qual ) = split(/\t/);
$rname =~ s/chr//g;
$rnext =~ s/chr//g;
my $mapE = 10**( -1 * $mapq / 10 );
my ($read_group) = $_ =~ /RG:Z:(\S+)/;
if ( $opts{read_groups} && !defined($read_group) ) { next; }
elsif ( $opts{read_groups} && !defined( $$sample_hash{$sample}{read_groups}{$read_group} ) ) { next; }
if ( $mapq < $opts{min_map_qual} ) { next; }
my $dir = 0; #F
if ( $flag & 16 ) { $dir = 1; } #R
my $dnext = 0; #mate F
if ( $flag & 32 ) { $dnext = 1; } #mate R
my ( $cFlag, $cPos, $clipside, $clipsize, $clipseq ) = getSoftClipInfo( $pos, $cigar, $qual, $seq );
if ( $cPos > -1 && $cFlag == 1 && $cPos >= $$infile_hash{$var}{start} - $opts{clipped_flank} && $cPos <= $$infile_hash{$var}{end} + $opts{clipped_flank} ) {
#potential breakpoints must be at or between previous breakpoint bounds
$$data_hash{$var}{$sample}{clipPos}{$cPos}{$cFlag}++;
if ( !defined( $$data_hash{$var}{$sample}{clipSeq}{$cPos} ) || length($clipseq) > length($$data_hash{$var}{$sample}{clipSeq}{$cPos}) ) {
$$data_hash{$var}{$sample}{clipSeq}{$cPos} = $clipseq;
}
}
if ( $rnext eq $chrM || checkMaskOverlap( $chr, $pos, $mask_hash ) ) {
#mate mapped to mt sequence
if ( ( $dir == 0 && $pos >= $l_start && $pos <= $l_end ) || ( $dir == 1 && $pos >= $r_start && $pos <= $r_end ) ) { $$data_hash{$var}{$sample}{numAltRP}++; push @{ $$data_hash{$var}{$sample}{qualAltRP} }, $mapE; }
}
elsif ( $dir == 0 && $dnext == 1 && $rnext eq "=" ) {
if ( abs($tlen) > $$sample_hash{$sample}{median_insert_size} + 3 * $$sample_hash{$sample}{median_absolute_deviation} ) { next; } #insert length of potential reference supporting allele out of upper bounds
if ( abs($tlen) < $$sample_hash{$sample}{median_insert_size} - 3 * $$sample_hash{$sample}{median_absolute_deviation} ) { next; } #insert length of potential reference supporting allele out of lower bounds
#"normal" reads which clip at the breakpoint are -not- reference supporting!
# --------->..............<-------L------ or <------L///////