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vardict.pl
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#!/usr/bin/env perl
# Parse a list of refseq and check CDS coverage
use warnings;
use Getopt::Std;
use strict;
our ($opt_h, $opt_H, $opt_b, $opt_D, $opt_d, $opt_s, $opt_c, $opt_S, $opt_E, $opt_n, $opt_N, $opt_e, $opt_g, $opt_x, $opt_f, $opt_r, $opt_B, $opt_z, $opt_v, $opt_p, $opt_F, $opt_C, $opt_m, $opt_Q, $opt_T, $opt_q, $opt_Z, $opt_X, $opt_P, $opt_3, $opt_k, $opt_R, $opt_G, $opt_a, $opt_o, $opt_O, $opt_V, $opt_t, $opt_y, $opt_I, $opt_i, $opt_M, $opt_J);
unless( getopts( 'hHvtzypDC3iF:d:b:s:e:S:E:n:c:g:x:f:r:B:N:Q:m:T:q:Z:X:P:k:R:G:a:o:O:V:I:M:J:' )) {
USAGE();
}
USAGE() if ( $opt_H );
USAGE() unless ( $opt_b );
my $BAM = $opt_b; # the bam file
my $sample = $1 if ( $BAM =~ /([^\/\._]+).sorted[^\/]*.bam/ );
my $samplem = "";
if ( $opt_n ) {
$sample = $1 if ( $BAM =~ /$opt_n/ );
}
$sample = $opt_N if ( $opt_N );
unless($sample) { $sample = $1 if ( $BAM =~ /([^\/]+)[_\.][^\/]*bam/ ); }
$opt_d = "\t" unless( $opt_d );
my $c_col = $opt_c ? $opt_c - 1 : 2;
my $S_col = $opt_S ? $opt_S - 1 : 6;
my $E_col = $opt_E ? $opt_E - 1 : 7;
my $s_col = $opt_s ? $opt_s - 1 : 9;
my $e_col = $opt_e ? $opt_e - 1 : 10;
my $g_col = $opt_g ? $opt_g - 1 : 12;
$opt_m = $opt_m ? $opt_m : 8;
$s_col = $S_col if ( $opt_S && (!$opt_s) );
$e_col = $E_col if ( $opt_E && (!$opt_e) );
$opt_F = defined($opt_F) ? $opt_F : "0x500";
my $VEXT = defined($opt_X) ? $opt_X : 3; # the extension of deletion and insertion for complex variants
$opt_P = defined($opt_P) ? $opt_P : 5;
$opt_k = defined($opt_k) ? $opt_k : 1; # Whether to perform local realignment. Set to 0 to disable.
my $fasta = $opt_G ? $opt_G : "/ngs/reference_data/genomes/Hsapiens/hg19/seq/hg19.fa";
my %CHRS; # Key: chr Value: chr_len
my $EXT = defined($opt_x) ? $opt_x : 0;
my $FREQ = $opt_f ? $opt_f : 0.05;
my $QRATIO = $opt_o ? $opt_o : 1.5; # The Qratio
my $BIAS = 0.05; # The cutoff to decide whether a positin has read strand bias
my $MINB = $opt_B ? $opt_B : 2; # The minimum reads for bias calculation
my $MINR = $opt_r ? $opt_r : 2; # The minimum reads for variance allele
my $GOODQ = defined($opt_q) ? $opt_q : 25; # The phred score in fastq to be considered as good base call
$opt_O = defined($opt_O) ? $opt_O : 0; # The minimun mean mapping quality to be considered
$opt_V = defined($opt_V) ? $opt_V : 0.05; # The minimun alelle frequency allowed in normal for a somatic mutation
my $RLEN = 0; # The read length
my $BUFFER = 200;
my $LOWQUAL = 10;
my $INDELSIZE = $opt_I ? $opt_I : 120;
my $MINMATCH = defined($opt_M) ? $opt_M : 25;
my %SPLICE;
if ( $opt_p ) {
$FREQ = -1;
$MINR = 0;
}
if ( $opt_h ) {
print join("\t", qw(Sample Gene Chr Start End Ref Alt Depth AltDepth RefFwdReads RefRevReads AltFwdReads AltRevReads Genotype AF Bias PMean PStd QMean QStd 5pFlankSeq 3pFlankSeq)), "\n";
}
my ($BAM1, $BAM2) = split(/\|/, $BAM);
my @BAMS = split(/:/, $BAM1);
open(BAMH, "samtools view -H $BAMS[0] |");
while(<BAMH>) {
if ( /^\@SQ/ ) {
my $chr = $1 if ( /\s+SN:(\S+)/ );
$CHRS{ $chr } = $1 if( /\sLN:(\d+)/ );
}
}
close(BAMH);
$fasta = $CHRS{ 1 } ? "/ngs/reference_data/genomes/Hsapiens/GRCh37/seq/GRCh37.fa" : "/ngs/reference_data/genomes/Hsapiens/hg19/seq/hg19.fa" unless( $opt_G );
my @SEGS = ();
if ( $opt_R ) {
my ($chr, $reg, $gene) = split(/:/, $opt_R);
$chr =~ /^chr/ ? ( $chr =~ s/^chr// ) : ($chr = "chr$chr") unless( $CHRS{ $chr } );
$gene = $chr unless( $gene );
my ($start, $end) = split(/-/, $reg);
$end = $start unless( $end );
$start =~ s/,//g;
$end =~ s/,//g;
$start -= $EXT;
$end += $EXT;
$start++ if ( $opt_z && $start < $end );
$start = $end if ( $start > $end );
push(@SEGS, [[$chr, $start, $end, $gene]]);
} else {
my @segraw = ();
while( <> ) {
chomp;
next if ( /^#/ || /^browser/ || /^track/ );
unless($opt_a) {
my @a = split(/$opt_d/);
if ((! $opt_a) && @a == 8 && $a[6] =~ /^\d+$/ && $a[7] =~ /^\d+$/ && $a[6] >= $a[1] && $a[7] <= $a[2]) {
$opt_a = "10:0.95";
$opt_z = 1 unless( defined($opt_z) );
}
}
push(@segraw, $_);
}
if ($opt_a) {
my ($pchr, $pend) = (0, 0);
my $SI = 0;
my %tsegs = ();
foreach(@segraw) {
my ($chr, $start, $end, $gene, $score, $strand, $istart, $iend) = split(/$opt_d/);
$chr =~ /^chr/ ? ( $chr =~ s/^chr// ) : ($chr = "chr$chr") unless( $CHRS{ $chr } );
$start++ && $istart++ if ( $opt_z && $start < $end );
push( @{ $tsegs{ $chr } }, [$chr, $start, $end, $gene, $istart, $iend] );
}
while(my ($chr, $sv) = each %tsegs ) {
my @tmp = sort { $a->[4] <=> $b->[4]; } @$sv;
foreach my $tv (@tmp) {
my ($chr, $start, $end, $gene, $istart, $iend) = @$tv;
$SI++ if ( $pend && ($chr ne $pchr || $istart > $pend) );
push(@{$SEGS[$SI]}, [$chr, $start, $end, $gene, $istart, $iend]);
$pchr = $chr;
$pend = $iend;
}
}
ampVardict();
exit(0);
} else {
foreach(@segraw) {
my @A = split(/$opt_d/);
if ( (! $opt_c) && @A == 4 && $A[1] =~ /^\d+$/ && $A[2] =~ /^\d+$/ && $A[1] <= $A[2] ) {
($c_col, $S_col, $E_col, $g_col, $s_col, $e_col) = (0, 1, 2, 3, 1, 2);
$opt_z = 1 unless( defined($opt_z) ); # only set if it's a 4 column BED file
}
my ($chr, $cdss, $cdse, $gene) = @A[$c_col, $S_col, $E_col, $g_col];
$chr =~ /^chr/ ? ( $chr =~ s/^chr// ) : ($chr = "chr$chr") unless( $CHRS{ $chr } );
my @starts = split(/,/, $A[$s_col]);
my @ends = split(/,/, $A[$e_col]);
my @CDS = ();
$gene = $chr unless( $gene );
for(my $i = 0; $i < @starts; $i++) {
my ($s, $e) = ($starts[$i], $ends[$i]);
next if ( $cdss > $e ); # not a coding exon
last if ( $cdse < $s ); # No more coding exon
$s = $cdss if ( $s < $cdss );
$e = $cdse if ( $e > $cdse );
$s -= $EXT; # unless ( $s == $cdss );
$e += $EXT; # unless ( $e == $cdse );
$s++ if ( $opt_z && $s < $e );
push(@CDS, [$chr, $s, $e, $gene]);
}
push(@SEGS, \@CDS);
}
}
}
for(my $i = 0; $i < @SEGS; $i++) {
for(my $j = 0; $j < @{ $SEGS[$i] }; $j++) {
my $REF = getREF(@{ $SEGS[$i]->[$j] }[0..2]);
if ( $BAM2 ) { # pair mode
if ( $opt_n ) {
$sample = $1 if ( $BAM1 =~ /$opt_n/ );
$samplem = $1 if ( $BAM2 =~ /$opt_n/ );
}
($sample, $samplem) = split(/\|/, $opt_N) if ( $opt_N );
$samplem = $sample . "_match" unless( $samplem );
somdict($SEGS[$i]->[$j], toVars(@{ $SEGS[$i]->[$j] }[0..2], $BAM1, $REF), toVars(@{ $SEGS[$i]->[$j] }[0..2], $BAM2, $REF));
} else {
vardict($SEGS[$i]->[$j], toVars(@{ $SEGS[$i]->[$j] }[0..2], $BAM1, $REF));
}
}
}
sub ampVardict {
for(my $i = 0; $i < @SEGS; $i++) {
my @vars = ();
my %pos = ();
my ($chr, $start, $end, $gene, $istart, $iend);
for(my $j = 0; $j < @{$SEGS[$i]}; $j++) {
($chr, $start, $end, $gene, $istart, $iend) = @{$SEGS[$i]->[$j]};
my $REF = getREF($chr, $start, $end);
push(@vars, toVars($chr, $start, $end, $BAM1, $REF));
for(my $p = $istart; $p <= $iend; $p++) {
push(@{ $pos{ $p } }, [$j, $chr, $start, $end, $istart, $iend]);
}
}
while(my ($p, $v) = each %pos) {
my @gvs = (); # Good variants
my @ref = (); # reference
my $nt;
my $maxaf = 0;
my $vartype = "SNV";
my $flag = 0;
my $vref;
my $nocov=0; #
my $maxcov=0;
my %goodamp;
my @vcovs = ();
foreach my $amps (@$v) {
my ($amp, $chr, $S, $E) = @$amps;
if ( $vars[$amp]->{ $p }->{ VAR }->[0] ) {
my $tv = $vars[$amp]->{ $p }->{ VAR }->[0];
push(@vcovs, $tv->{ tcov });
$maxcov = $tv->{ tcov } if ( $tv->{ tcov } > $maxcov );
$vartype = varType($tv->{ refallele }, $tv->{ varallele });
if ( isGoodVar($tv, $vars[$amp]->{ $p }->{ REF }, $vartype) ) {
push(@gvs, [$tv, "$chr:$S-$E"]);
if ( $nt && $tv->{ n } ne $nt ) {
$flag = 1;
}
if ($tv->{ freq } > $maxaf ) {
($maxaf, $nt, $vref) = ($tv->{ freq }, $tv->{ n }, $tv);
}
$goodamp{ "$amp-$tv->{ refallele }-$tv->{ varallele }" } = 1;
}
} elsif ( $vars[$amp]->{ $p }->{ REF } ) {
push(@vcovs, $vars[$amp]->{ $p }->{ REF }->{ tcov });
} else {
push(@vcovs, 0);
}
push(@ref, $vars[$amp]->{ $p }->{ REF }) if ( $vars[$amp]->{ $p }->{ REF } );
}
foreach my $t (@vcovs) {
$nocov++ if ( $t < $maxcov/50 ); # The amplicon that has depth less than 1/50 of the max depth will be considered not working and thus not used.
}
@gvs = sort { $b->[0]->{ freq } <=> $a->[0]->{ freq } } @gvs if ( @gvs > 1 );
@ref = sort { $b->{ tcov } <=> $a->{ tcov } } @ref if ( @ref > 1 );
if ( @gvs < 1 ) { # Only referenece
if ( $opt_p ) {
if ( @ref ) {
$vref = $ref[0];
} else {
print join("\t", $sample, $gene, $chr, $p, $p, "", "", 0, 0, 0, 0, 0, 0, "", 0, "0;0", 0, 0, 0, 0, 0, "", 0, 0, 0, 0, 0, 0, "", "", 0, 0, "$chr:$p-$p", "", 0, 0, 0, 0), "\n";
next;
}
} else {
next;
}
} else {
$vref = $gvs[0]->[0];
}
if ( $flag ) { # different good variants detected in different amplicons
#next if ($gvs[0]->[0]->{ freq }/$gvs[1]->[0]->{ freq } < 10); # need 10 times difference to overwrite it.
my $gdnt = $gvs[0]->[0]->{ n };
my @gcnt = ();
foreach my $amps (@$v) {
my ($amp, $chr, $S, $E) = @$amps;
push(@gcnt, [$vars[$amp]->{ $p }->{ VARN }->{ $gdnt }, "$chr:$S-$E"]) if ( $vars[$amp]->{ $p }->{ VARN }->{ $gdnt } && isGoodVar( $vars[$amp]->{ $p }->{ VARN }->{ $gdnt }, $vars[$amp]->{ $p }->{ REF }) );
}
$flag = 0 if ( @gcnt+0 == @gvs+0 );
@gvs = sort { $b->[0]->{ freq } <=> $a->[0]->{ freq } } @gcnt;
}
my @badv = ();
my $gvscnt = @gvs+0;
unless ( $gvscnt == @$v + 0 && (! $flag) ) {
foreach my $amps (@$v) {
my ($amp, $chr, $S, $E, $iS, $iE) = @$amps;
next if ( $goodamp{"$amp-$vref->{ refallele }-$vref->{ varallele }"} );
my $tref = $vars[$amp]->{ $p }->{ VAR }->[0];
if ( $vref->{ sp } >= $iS && $vref->{ ep } <= $iE ) {
if ( $vars[$amp]->{ $p }->{ VAR } ) {
push( @badv, [$vars[$amp]->{ $p }->{ VAR }->[0], "$chr:$S-$E"] );
} elsif ( $vars[$amp]->{ $p }->{ REF } ) {
push( @badv, [$vars[$amp]->{ $p }->{ REF }, "$chr:$S-$E"] );
} else {
push( @badv, [undef, "$chr:$S-$E"] );
}
} elsif ( ($vref->{ sp } < $iE && $iE < $vref->{ ep }) || ($vref->{ sp } < $iS && $iS < $vref->{ ep }) ) { # the variant overlap with amplicon's primer
#print STDERR "$iS $iE $vref->{sp} $vref->{ep} $vref->{n} $gvscnt\n";
$gvscnt-- if ( $gvscnt > 1 );
}
}
}
$flag = 0 if ( $flag && $gvscnt < @gvs+0 );
my @hds = qw(sp ep refallele varallele tcov cov rfc rrc fwd rev genotype freq bias pmean pstd qual qstd mapq qratio hifreq extrafreq shift3 msi msint nm hicnt hicov leftseq rightseq);
my @hds2 = qw(tcov cov rfc rrc fwd rev genotype freq bias pmean pstd qual qstd mapq qratio hifreq extrafreq);
$vartype = varType($vref->{ refallele }, $vref->{ varallele });
adjComplex($vref) if ( $vartype eq "Complex" );
print join("\t", $sample, $gene, $chr, (map { $vref->{ $_ } ? $vref->{ $_ } : 0; } @hds), $gvs[0]->[1] ? $gvs[0]->[1] : "", $vartype, $gvscnt, $gvscnt+@badv+0, $nocov, $flag);
print "\t", $vref->{ DEBUG } if ( $opt_D );
for(my $gvi = 1; $gvi < @gvs; $gvi++) {
print "\tGood$gvi ", join(" ", (map {$gvs[$gvi]->[0]->{ $_ }; } @hds2), $gvs[$gvi]->[1]) if ( $opt_D );
}
for(my $bvi = 0; $bvi < @badv; $bvi++) {
print "\tBad$bvi ", join(" ", (map {$badv[$bvi]->[0]->{ $_ } ? $badv[$bvi]->[0]->{ $_ } : 0; } @hds2), $badv[$bvi]->[1]) if ( $opt_D );
}
print "\n";
}
}
}
sub varType {
my ($ref, $var) = @_;
if ( length($ref) == 1 && length($var) == 1 ) {
return "SNV";
} elsif ( substr($ref, 0, 1) ne substr($var, 0, 1) ) {
return "Complex";
} elsif ( length($ref) == 1 && length($var) > 1 && $var =~ /^$ref/ ) {
return "Insertion";
} elsif (length($ref) > 1 && length($var) == 1 && $ref =~ /^$var/ ) {
return "Deletion";
}
return "Complex";
}
sub vardict {
my ($seg, $vars) = @_;
my ($chr, $S, $E, $G) = @{ $seg };
my @hds = qw(sp ep refallele varallele tcov cov rfc rrc fwd rev genotype freq bias pmean pstd qual qstd mapq qratio hifreq extrafreq shift3 msi msint nm hicnt hicov leftseq rightseq);
push(@hds, "crispr") if ( $opt_J );
for(my $p = $S; $p <= $E; $p++) {
my @vts = ();
my @vrefs = ();
unless( $vars->{ $p }->{ VAR } ) {
next unless( $opt_p );
my $vref = $vars->{ $p }->{ REF };
unless($vref) {
print join("\t", $sample, $G, $chr, $p, $p, "", "", 0, 0, 0, 0, 0, 0, "", 0, "0;0", 0, 0, 0, 0, 0, "", 0, 0, 0, 0, 0, 0, "", "", 0, 0, "$chr:$S-$E", ""), "\n";
next;
}
push(@vts, "");
push(@vrefs, $vref);
} else {
for(my $i = 0; $i < @{ $vars->{$p}->{VAR} }; $i++) {
my $vref = $vars->{ $p }->{ VAR }->[$i];
last if ( $vref->{refallele} =~ /N/ );
my $vartype = varType($vref->{refallele}, $vref->{varallele});
unless( isGoodVar( $vars->{ $p }->{ VAR }->[$i], $vars->{ $p }->{ REF }, $vartype ) ) {
next unless( $opt_p );
}
push(@vts, $vartype);
push(@vrefs, $vref);
}
}
for(my $vi = 0; $vi < @vts; $vi++) {
my ($vartype, $vref) = ($vts[$vi], $vrefs[$vi]);
adjComplex($vref) if ( $vartype eq "Complex" );
print join("\t", $sample, $G, $chr, (map { $vref->{ $_ } ? $vref->{ $_ } : 0; } @hds), "$chr:$S-$E", $vartype);
print "\t", $vref->{ DEBUG } if ( $opt_D );
print "\n";
}
}
}
# Adjust the complex var
sub adjComplex {
my $vref = shift;
my ($refnt, $varnt) = ($vref->{ refallele }, $vref->{ varallele });
my $n = 0;
$n++ while( length($refnt) - $n > 1 && length($varnt) - $n > 1 && substr($refnt, $n, 1) eq substr($varnt, $n, 1) );
if ( $n ) {
$vref->{ sp } += $n;
$vref->{ refallele } = substr($refnt, $n);
$vref->{ varallele } = substr($varnt, $n);
$vref->{ leftseq } .= substr($refnt, 0, $n);
$vref->{ leftseq } = substr($vref->{ leftseq }, $n);
}
($refnt, $varnt) = ($vref->{ refallele }, $vref->{ varallele });
$n = 1;
$n++ while( length($refnt) - $n > 0 && length($varnt) - $n > 0 && substr($refnt, -$n, 1) eq substr($varnt, -$n, 1) );
if ( $n > 1 ) {
$vref->{ ep } -= $n - 1;
$vref->{ refallele } = substr($refnt, 0, -$n+1);
$vref->{ varallele } = substr($varnt, 0, -$n+1);
$vref->{ rightseq } = substr($refnt, -($n-1), $n-1) . substr($vref->{ rightseq }, 0, -$n+1);
}
}
sub somdict {
my ($seg, $vars1, $vars2) = @_;
my ($chr, $S, $E, $G) = @{ $seg };
my @hdrs = qw(tcov cov rfc rrc fwd rev genotype freq bias pmean pstd qual qstd mapq qratio hifreq extrafreq nm);
my @hd1 = ("sp", "ep", "refallele", "varallele");
my @hd2 = ("shift3", "msi", "msint", "leftseq", "rightseq");
my $FISHERP = 0.01;
for(my $p = $S; $p <= $E; $p++) {
next unless($vars1->{$p} || $vars2->{$p}); # both samples have no coverage
my $vartype = "";
if( ! $vars1->{ $p } ) { # no coverage for sample 1
$vartype = varType($vars2->{$p}->{ VAR }->[0]->{ refallele }, $vars2->{$p}->{ VAR }->[0]->{ varallele }) if ($vars2->{$p}->{ VAR });
next unless( $vars2->{$p}->{ VAR } && isGoodVar($vars2->{$p}->{ VAR }->[0], $vars2->{$p}->{REF}, $vartype) );
adjComplex($vars2->{$p}->{ VAR }->[0]) if ( $vartype eq "Complex" );
print join("\t", $sample, $G, $chr, (map{ $vars2->{$p}->{ VAR }->[0]->{ $_ }; } @hd1), (map{ 0; } @hdrs), (map { $vars2->{$p}->{ VAR }->[0]->{ $_ }; } (@hdrs, @hd2)), "$chr:$S-$E", "Deletion", $vartype), "\n";
} elsif( ! $vars2->{ $p } ) { # no coverage for sample 2
$vartype = varType($vars1->{$p}->{ VAR }->[0]->{ refallele }, $vars1->{$p}->{ VAR }->[0]->{ varallele }) if ($vars1->{$p}->{ VAR });
next unless( $vars1->{$p}->{ VAR } && isGoodVar($vars1->{$p}->{ VAR }->[0], $vars1->{$p}->{REF}, $vartype) );
adjComplex($vars1->{$p}->{ VAR }->[0]) if ( $vartype eq "Complex" );
print join("\t", $sample, $G, $chr, (map { $vars1->{$p}->{ VAR }->[0]->{ $_ }; } (@hd1, @hdrs)), (map { 0; } @hdrs), (map { $vars1->{$p}->{ VAR }->[0]->{$_};} @hd2), "$chr:$S-$E", "SampleSpecific", $vartype), "\n";
} else { # both samples have coverage
my ($v1, $v2) = ($vars1->{$p}, $vars2->{$p});
next unless ( $v1->{ VAR } || $v2->{ VAR } );
if ( $v1->{ VAR } ) {
my $N = 0;
while( $v1->{ VAR }->[$N] && isGoodVar($v1->{ VAR }->[$N], $v1->{ REF }, varType($v1->{ VAR }->[$N]->{ refallele }, $v1->{ VAR }->[$N]->{ varallele }))) {
my $VREF = $v1->{ VAR }->[$N];
my $nt = $VREF->{ n };
#if ( length($nt) > 1 && length($VREF->{ refallele }) == length($VREF->{ varallele }) && (! isGoodVar($v2->{ VARN }->{ $nt })) && $VREF->{ genotype } !~ /-/ && $VREF->{ genotype } !~ /m/ && $VREF->{ genotype } !~ /i/ ) {
# my $fnt = substr($nt, 0, -1); $fnt =~ s/&$//;
# my $lnt = substr($nt, 1); $lnt =~ s/^&//; substr($lnt, 1, 0) = "&" if ( length($lnt) > 1 );
# if ( $v2->{ VARN }->{ $fnt } && isGoodVar($v2->{ VARN }->{ $fnt }, $v2->{ REF })) {
# $VREF->{ sp } += length($VREF->{ refallele }) - 1;
# $VREF->{ refallele } = substr($VREF->{ refallele }, -1 );
# $VREF->{ varallele } = substr($VREF->{ varallele }, -1 );
# } elsif ( $vars2->{ $p + length($nt) -2 }->{ VARN }->{ $lnt } && isGoodVar($vars2->{ $p + length($nt) -2 }->{ VARN }->{ $lnt }, $vars2->{$p+length($nt)-2}->{REF})) {
# $VREF->{ ep } += length($VREF->{ refallele }) - 1;
# $VREF->{ refallele } = substr($VREF->{ refallele }, 0, -1 );
# $VREF->{ varallele } = substr($VREF->{ varallele }, 0, -1 );
# }
# }
$vartype = varType($VREF->{ refallele }, $VREF->{ varallele });
adjComplex($VREF) if ( $vartype eq "Complex" );
if ( $v2->{ VARN }->{ $nt } ) {
#my $type = isGoodVar( $v2->{ VARN }->{ $nt }, $v2->{ REF } ) ? "Germline" : ($v2->{ VARN }->{ $nt }->{ freq } < $opt_V || $v2->{ VARN }->{ $nt }->{ cov } <= 1 ? "LikelySomatic" : "AFDiff");
my $type = isGoodVar( $v2->{ VARN }->{ $nt }, $v2->{ REF }, $vartype ) ? ($VREF->{ freq } > (1-$opt_V) && $v2->{ VARN }->{ $nt }->{ freq } < 0.8 && $v2->{ VARN }->{ $nt }->{ freq } > 0.2 ? "LikelyLOH" : ($v2->{ VARN }->{ $nt }->{ freq } < $opt_V || $v2->{ VARN }->{ $nt }->{ cov } <= 1 ? "LikelySomatic" : "Germline")) : ($v2->{ VARN }->{ $nt }->{ freq } < $opt_V || $v2->{ VARN }->{ $nt }->{ cov } <= 1 ? "LikelySomatic" : "AFDiff");
$type = "StrongSomatic" if ( $vartype eq "SNV" && isNoise($v2->{ VARN }->{ $nt }) );
#if ($type =~ /Somatic/) {
# my $newtype = combineAnalysis($VREF, $v2->{ VARN }->{ $nt }, $chr, $p, $nt);
# if ( $newtype eq "FALSE" ) {$N++; next;}
# $type = $newtype if ( $newtype );
#}
#$type = "StrongSomatic" if ( isNoise($v2->{ VARN }->{ $nt }) );
print join("\t", $sample, $G, $chr, (map { $VREF->{ $_ }; } (@hd1, @hdrs)), (map { $v2->{ VARN }->{ $nt }->{ $_ }; } (@hdrs, @hd2)), "$chr:$S-$E", $type, $vartype), "\n";
} else { # sample 1 only, should be strong somatic
my @tvf = $v2->{ REF } ? (map { $v2->{ REF }->{ $_ } ? $v2->{ REF }->{ $_ } : 0; } @hdrs) : ($v2->{ VAR }->[0]->{ tcov } ? $v2->{ VAR }->[0]->{ tcov } : 0, map { 0; } (1..17));
my $type = "StrongSomatic";
#if ($vartype ne "SNV") {
$v2->{ VARN }->{ $nt }->{ cov } = 0; # Ensure it's initialized before passing to combineAnalysis
my $newtype = $VREF->{ cov } < $MINR + 3 ? combineAnalysis($VREF, $v2->{ VARN }->{ $nt }, $chr, $p, $nt) : "";
if ( $newtype eq "FALSE" ) {$N++; next;}
$type = $newtype if ( $newtype );
#}
if ( $type ne "StrongSomatic" ) {
print join("\t", $sample, $G, $chr, (map { $VREF->{ $_ }; } (@hd1, @hdrs)), (map { $v2->{ VARN }->{ $nt }->{ $_ }; } @hdrs),(map { $VREF->{$_}; } @hd2), "$chr:$S-$E", $type, $vartype), "\n";
} else {
print join("\t", $sample, $G, $chr, (map { $VREF->{ $_ }; } (@hd1, @hdrs)), @tvf, (map { $VREF->{$_};} @hd2), "$chr:$S-$E", "StrongSomatic", $vartype), "\n";
}
}
$N++;
}
unless($N) {
next unless( $v2->{ VAR } );
$vartype = varType($v2->{ VAR }->[0]->{ refallele }, $v2->{ VAR }->[0]->{ varallele });
next unless( isGoodVar( $v2->{ VAR }->[0], $v2->{ REF }, $vartype ) );
# potentail LOH
my $nt = $v2->{ VAR }->[0]->{ n };
if ( $v1->{ VARN }->{ $nt } ) {
my $type = $v1->{ VARN }->{ $nt }->{ freq } < $opt_V ? "LikelyLOH" : "Germline";
adjComplex($v1->{ VARN }->{ $nt }) if ( $vartype eq "Complex" );
print join("\t", $sample, $G, $chr, (map { $v1->{ VARN }->{ $nt }->{ $_ }; } (@hd1, @hdrs)), (map { $v2->{ VAR }->[0]->{ $_ }; } (@hdrs, @hd2)), "$chr:$S-$E", $type, varType($v1->{ VARN }->{ $nt }->{ refallele }, $v1->{ VARN }->{ $nt }->{ varallele })), "\n";
} else {
my @th1 = $v1->{ REF } ? (map { $v1->{ REF }->{ $_ } } @hdrs) : ($v1->{ VAR }->[0]->{ tcov }, (map { 0; } @hdrs[1..$#hdrs]));
adjComplex($v2->{ VAR }->[0]) if ( $vartype eq "Complex" );
print join("\t", $sample, $G, $chr, (map { $v2->{ VAR }->[0]->{ $_ }; } @hd1), @th1, (map { $v2->{ VAR }->[0]->{ $_ }; } (@hdrs, @hd2)), "$chr:$S-$E", "StrongLOH", $vartype), "\n";
}
}
} elsif ( $v2->{ VAR } ) { # sample 1 has only reference
$vartype = varType($v2->{ VAR }->[0]->{ refallele }, $v2->{ VAR }->[0]->{ varallele });
next unless( isGoodVar( $v2->{ VAR }->[0], $v2->{ REF }, $vartype ) );
# potential LOH
my $nt = $v2->{ VAR }->[0]->{ n };
my $type = "StrongLOH";
$v1->{ VARN }->{ $nt }->{ cov } = 0;
my $newtype = $v2->{ VARN }->{ $nt }->{ cov } < $MINR + 3 ? combineAnalysis($v2->{ VARN }->{ $nt }, $v1->{ VARN }->{ $nt }, $chr, $p, $nt) : "";
next if ( $newtype eq "FALSE" );
$type = $newtype if ( $newtype );
my @th1 = $newtype ? (map { $v1->{ VARN }->{ $nt }->{ $_ } ? $v1->{ VARN }->{ $nt }->{ $_ } : 0; } @hdrs) : (map { $v1->{ REF }->{ $_ } ? $v1->{ REF }->{ $_ } : 0 } @hdrs);
adjComplex($v2->{ VAR }->[0]) if ( $vartype eq "Complex" );
print join("\t", $sample, $G, $chr, (map { $v2->{ VAR }->[0]->{ $_ } ? $v2->{ VAR }->[0]->{ $_ } : 0; } @hd1), @th1, (map { $v2->{ VAR }->[0]->{ $_ } ? $v2->{ VAR }->[0]->{ $_ } : 0; } (@hdrs, @hd2)), "$chr:$S-$E", $type, $vartype), "\n";
}
}
}
}
# Taken a likely somatic indels and see whether combine two bam files still support somatic status. This is mainly
# for Indels that softclipping overhang is too short to positively being called in one bam file, but can be called
# in the other bam file, thus creating false positives
sub combineAnalysis {
my ($var1, $var2, $chr, $p, $nt) = @_;
my ($bam1, $bam2) = split(/\|/, $BAM);
print STDERR "Start Combine $p $nt\n" if ( $opt_y );
my $REF = getREF($chr, $var1->{ sp } - $RLEN, $var1->{ ep } + $RLEN);
my $vars = toVars($chr, $var1->{ sp } - $RLEN, $var1->{ ep } + $RLEN, "$bam1:$bam2", $REF);
if ( $vars->{ $p }->{ VARN }->{ $nt } ) {
my $vref = $vars->{ $p }->{ VARN }->{ $nt };
print STDERR "Combine: 1: $var1->{ cov } comb: $vref->{ cov }\n" if ( $opt_y );
if ( $vref->{ cov } - $var1->{ cov } >= $MINR ) {
map { $var2->{ $_ } = $vref->{ $_ } - $var1->{ $_ } > 0 ? $vref->{ $_ } - $var1->{ $_ } : 0; } qw(tcov cov rfc rrc fwd rev);
map { $var2->{ $_ } = sprintf("%.3f", ($vref->{ $_ }*$vref->{ cov } - $var1->{ $_ }*$var1->{ cov })/$var2->{ cov }); } qw(pmean qual mapq hifreq extrafreq nm);
map { $var2->{ $_ } = 1; } qw(pstd qstd);
return "FALSE" if ( $var2->{ tcov } <= 0 );
my $freq2 = sprintf("%.3f", $var2->{ cov }/$var2->{ tcov });
$var2->{ freq } = $freq2;
$var2->{ qratio } = $var1->{ qratio }; # Can't back calculate and should be inaccurate
$var2->{ genotype } = $vref->{ genotype };
$var2->{ bias } = strandBias($var2->{rfc}, $var2->{rrc}) . ";" . strandBias($var2->{fwd}, $var2->{rev});
return "Germline";
} elsif ($vref->{ cov } < $var1->{ cov } - 2) {
print STDERR "Combine produce less: $chr $p $nt $vref->{ cov } $var1->{ cov }\n" if ( $opt_y );
return "FALSE";
} else {
return "";
}
} else {
return "FALSE";
}
return "";
}
# A variance is considered noise if the quality is below $GOODQ and there're no more than 3 reads
sub isNoise {
my $vref = shift;
if ( (($vref->{ qual } < 4.5 || ($vref->{ qual } < 12 && $vref->{ qstd } == 0)) && $vref->{ cov } <= 3) || ($vref->{ qual } < $GOODQ && $vref->{ freq } < 2*$opt_V && $vref->{ cov } <= 1) ) {
$vref->{ tcov } -= $vref->{ cov };
$vref->{ cov } = 0;
$vref->{ fwd } = 0;
$vref->{ rev } = 0;
$vref->{ freq } = 0;
$vref->{ hifreq } = 0;
return 1;
}
return 0;
}
# Determine whether a variant meet specified criteria
sub isGoodVar {
my ($vref, $rref, $type) = @_;
return 0 unless($vref && $vref->{ refallele });
$type = varType($vref->{ refallele }, $vref->{ varallele }) unless( $type );
return 0 if ( $vref->{ freq } < $FREQ );
return 0 if ( $vref->{ hicnt } < $MINR );
return 0 if ( $vref->{ pmean } < $opt_P );
return 0 if ( $vref->{ qual } < $GOODQ );
#print STDERR "$vref->{sp} $vref->{ cov } $vref->{varallele} $type\n";
#return 0 if ( $vref->{ pstd } == 0 ); # Leave it to var2vcf, especially for targeted PCR, where pstd = 0 might be expected.
if ( $rref && $rref->{ hicnt } > $MINR && $vref->{ freq } < 0.25 ) {
#The reference allele has much better mean mapq than var allele, thus likely false positives
return 0 if ( ($vref->{ mapq } + (length($vref->{ refallele }) + length($vref->{ varallele })-2) < 5 && $rref->{ mapq } > 20) || ( 1 + ($vref->{ mapq } + (length($vref->{ refallele }) + length($vref->{ varallele }))))/($rref->{ mapq } + 1) < 0.25 );
#return 0 if ( $type && $type eq "SNV" && (($vref->{ mapq } < 5 && $rref->{ mapq } > 20) || $rref->{ mapq } - $vref->{ mapq } > 30 ));
}
return 0 if ( $type eq "Deletion" && $SPLICE{ $vref->{sp} . "-" . $vref->{ep} } );
return 0 if ( $vref->{ qratio } < $QRATIO );
return 1 if ( $vref->{ freq } > 0.35 );
return 0 if ( $vref->{ mapq } < $opt_O );
return 0 if ( $vref->{ msi } >= 13 && $vref->{ freq } <= 0.275 && $vref->{ msint } == 1);
return 0 if ( $vref->{ msi } >= 8 && $vref->{ freq } <= 0.2 && $vref->{ msint } > 1 );
if ( $vref->{ bias } eq "2;1" && $vref->{ freq } < 0.25 ) {
return 0 unless($type && $type ne "SNV" && (length($vref->{refallele}) > 3 || length($vref->{varallele}) > 3));
}
return 1;
}
sub addCnt {
my ($vref, $dir, $rp, $q, $Q, $nm) = @_; # ref dir read_position quality
$vref->{ cnt }++;
$vref->{ $dir }++;
$vref->{ pmean } += $rp;
$vref->{ qmean } += $q;
$vref->{ Qmean } += $Q;
$vref->{ nm } += $nm;
$q >= $GOODQ ? $vref->{ hicnt }++ : $vref->{ locnt }++;
}
# Given a region, return the REF
sub getREF {
my ($chr, $START, $END) = @_;
my %REF;
my $s_start = $START - $EXT - 700 < 1 ? 1 : $START - $EXT - 700;
my $s_end = $END + $EXT + 700 > $CHRS{ $chr } ? $CHRS{ $chr } : $END + $EXT + 700;
my ($header, $exon) = split(/\n/, `samtools faidx $fasta $chr:$s_start-$s_end`, 2);
$exon =~ s/\s+//g;
for(my $i = $s_start; $i <= $s_start + length($exon); $i++) {
$REF{ $i } = uc(substr( $exon, $i - ($s_start), 1 ));
}
return \%REF;
}
# Add deletion structural variant
sub addSVDel {
my ($sdref, $s, $e, $ms, $me, $dir, $rlen, $mlen) = @_;
$sdref->{ cnt }++;
$sdref->{ dir } = $dir;
$sdref->{ start } = $s unless( $sdref->{ start } && $sdref->{ start } < $s );
$sdref->{ end } = $e unless( $sdref->{ end } && $sdref->{ end } > $e );
push(@{ $sdref->{ mates } }, [$ms, $me, $mlen]);
$sdref->{ mstart } = $ms unless( $sdref->{ mstart } && $sdref->{ mstart } < $ms );
$sdref->{ mend } = $ms+$rlen unless( $sdref->{ mend } && $sdref->{ mend } > $me );
}
# Construct a variant structure given a segment and BAM files.
sub parseSAM {
my ($chr, $START, $END, $bam, $REF) = @_;
my %cov;
my %var;
my %hash;
my %dels5;
my %ins;
my %mnp; # Keep track of MNPs
my %sclip3; # soft clipped at 3'
my %sclip5; # soft clipped at 5'
my @bams = split(/:/, $bam);
$chr =~ s/^chr// if ( $opt_C );
$REF = getREF($chr, $START, $END);
foreach my $bami (@bams) {
#my $tsamcnt = `samtools view $bami $chr:$START-$END | head -1`;
#next unless( $tsamcnt || $opt_p ); # to avoid too much IO for exome and targeted while the BED is whole genome
# Get the reference sequence
my $SAMFILTER = $opt_F ? "-F $opt_F" : "";
open(SAM, "samtools view $SAMFILTER $bami $chr:$START-$END |");
my %DUP = ();
my $DUPP = 0;
while( <SAM> ) {
if ( $opt_Z ) {
next if ( rand() <= $opt_Z );
}
my @a = split(/\t/);
next if ( defined($opt_Q) && $a[4] < $opt_Q ); # ignore low mapping quality reads
#next if ( $opt_F && ($a[1] & 0x200 || $a[1] & 0x100) ); # ignore "non-primary alignment", or quality failed reads.
#next if ( $opt_F && ($a[1] & 0x100) ); # ignore "non-primary alignment"
if ( $a[1] & 0x100 ) {
next if ($opt_F); # -F 0 to turn it off
}
next if ( $a[9] eq "*" );
# filter duplicated reads if option -t is set
if ( $opt_t ) {
%DUP = () if ( $a[3] ne $DUPP );
if ( $a[7] =~ /\d/ ) {
next if ( $DUP{ "$a[3]-$a[6]-$a[7]" } );
$DUP{ "$a[3]-$a[6]-$a[7]" } = 1;
$DUPP = $a[3];
} elsif ( $a[1] & 0x8 ) { # mate not mapped
next if ( $DUP{ "$a[3]-$a[5]" } );
$DUP{ "$a[3]-$a[5]" } = 1;
$DUPP = $a[3];
}
}
my $nm = 0;
my @segid = $a[5] =~ /(\d+)[ID]/g; # For total indels
my $idlen = 0; $idlen += $_ foreach(@segid);
if ( /NM:i:(\d+)/i ) { # number of mismatches. Don't use NM since it includes gaps, which can be from indels
$nm = $1 - $idlen;
next if ( $opt_m && $nm > $opt_m ); # edit distance - indels is the # of mismatches
} else {
print STDERR "No NM tag for mismatches. $_\n" if ( $opt_y && $a[5] ne "*" );
#next;
}
my $n = 0; # keep track the read position, including softclipped
my $p = 0; # keep track the position in the alignment, excluding softclipped
my $dir = $a[1] & 0x10 ? "-" : "+";
# Amplicon based calling
if ( $opt_a ) {
my ($dis, $ovlp) = split( /:/, $opt_a );
($dis, $ovlp) = (10, 0.95) unless($dis && $ovlp);
my $rlen3= 0; $rlen3 += $1 while( $a[5] =~ /(\d+)[MD]/g ); # The total aligned length, excluding soft-clipped bases and insertions
my ($segstart, $segend) = ($a[3], $a[3]+$rlen3-1);
if ( $a[5] =~ /^(\d+)S/ ) {
my $ts1 = $segstart > $START ? $segstart : $START;
my $te1 = $segend < $END ? $segend : $END;
next unless( abs($ts1-$te1)/($segend-$segstart) > $ovlp);
} elsif ($a[5] =~ /(\d+)S$/ ) {
my $ts1 = $segstart > $START ? $segstart : $START;
my $te1 = $segend < $END ? $segend : $END;
next unless( abs($te1-$ts1)/($segend-$segstart) > $ovlp);
} else {
if ($a[6] eq "=" && $a[8]) {
($segstart, $segend) = $a[8] > 0 ? ($segstart, $segstart+$a[8]-1) : ($a[7], $a[7]-$a[8]-1);
}
# No segment overlapping test since samtools should take care of it
my $ts1 = $segstart > $START ? $segstart : $START;
my $te1 = $segend < $END ? $segend : $END;
next unless( (abs($segstart - $START) <= $dis && abs($segend - $END) <= $dis ) && abs(($ts1-$te1)/($segend-$segstart)) > $ovlp);
}
}
if ( $a[1] & 0x8 ) { # Mate unmapped, potential insertion
# to be implemented
} else {
if ( $a[6] eq "=" ) {
if ( /\tSA:Z:(\S+)/ ) {
if ( $a[1] & 0x800 ) { # the supplementary alignment
next; # Ignore the supplmentary for now so that it won't skew the coverage
}
}
}
}
# Modify the CIGAR for potential mis-alignment for indels at the end of reads to softclipping and let VarDict's algorithm to figure out indels
my $offset = 0;
if( $a[5] =~ s/^(\d+)D// ) { $a[3] += $1; }
$a[5] =~ s/\d+D$//;
$a[5] =~ s/^(\d+)I/$1S/;
$a[5] =~ s/(\d+)I$/$1S/;
while( $idlen > 0 && $opt_k ) {
my $flag = 0;
if ($a[5] =~ /^(\d+)S(\d+)([ID])/) {
my $tslen = $1 + ($3 eq "I" ? $2 : 0);
$tslen .= "S";
$a[3] += $3 eq "D" ? $2 : 0;
$a[5] =~ s/^\d+S\d+[ID]/$tslen/;
$flag = 1;
}
if ($a[5] =~ /(\d+)([ID])(\d+)S$/) {
my $tslen = $3 + ($2 eq "I" ? $1 : 0);
$tslen .= "S";
$a[5] =~ s/\d+[ID]\d+S$/$tslen/;
$flag = 1;
}
if ($a[5] =~ /^(\d+)S(\d+)M(\d+)([ID])/) {
my $tslen = $1 + $2 + ($4 eq "I" ? $3 : 0);
my $tmid = $2;
$tslen .= "S";
if ( $tmid <= 10 ) {
$a[3] += $2 + ($4 eq "D" ? $3 : 0);
$a[5] =~ s/^\d+S\d+M\d+[ID]/$tslen/;
$flag = 1;
}
}
if ($a[5] =~ /(\d+)([ID])(\d+)M(\d+)S$/) {
my $tslen = $4 + $3 + ($2 eq "I" ? $1 : 0);
my $tmid = $3;
$tslen .= "S";
if ( $tmid <= 10 ) {
$a[5] =~ s/\d+[ID]\d+M\d+S$/$tslen/;
$flag = 1;
}
}
# The following two clauses to make indels at the end of reads as softly
# clipped reads and let VarDict's algorithm identify indels
if ($a[5] =~ /^(\d)M(\d+)([ID])(\d+)M/) {
my $tslen = $1 + ($3 eq "I" ? $2 : 0);
my $mlen = $4;
if ( $1 <= 8 ) {
$a[3] += $1 + ($3 eq "D" ? $2 : 0);
my $n = 0;
$n++ while( $n < $mlen && $REF->{ $a[3]+$n } && $REF->{ $a[3]+$n } ne substr($a[9], $tslen+$n, 1));
$tslen += $n;
$mlen -= $n;
$a[3] += $n;
$a[5] =~ s/^\dM\d+[ID]\d+M/${tslen}S${mlen}M/;
$flag = 1;
}
}
if ($a[5] =~ /(\d+)([ID])(\d)M$/) {
my $tslen = $3 + ($2 eq "I" ? $1 : 0);
$tslen .= "S";
if ( $3 <= 8 ) {
$a[5] =~ s/\d+[ID]\d+M$/$tslen/;
$flag = 1;
}
}
# Combine two deletions and insertion into one complex if they are close
if ($a[5] =~ /^(.*?)(\d+)M(\d+)D(\d)M(\d+)I(\d)M(\d+)D(\d+)M/) {
my $mid = $4 + $6;
if ( $mid <= 15 ) {
my $tslen = $4 + $5 + $6;
my $dlen = $3 + $4 + $6 + $7;
my $ov5 = $1;
my $refoff = $a[3] + $2;
my $rdoff = $2;
my $RDOFF = $2;
my $rm = $8;
if ( $ov5 ) {
my @rdp = $ov5 =~ /(\d+)[MIS]/g; # read position
my @rfp = $ov5 =~ /(\d+)[MD]/g; # reference position
$refoff += $_ foreach(@rfp);
$rdoff += $_ foreach(@rdp);
}
my $rn = 0;
$rn++ while( $REF->{ $refoff + $rn } && $REF->{ $refoff + $rn } eq substr($a[9], $rdoff + $rn, 1) );
$RDOFF += $rn;
$dlen -= $rn;
$tslen -= $rn;
if ( $tslen <= 0 ) {
$dlen -= $tslen;
$rm += $tslen;
$tslen = $dlen ."D" . $rm ."M";
} else {
$tslen = "${dlen}D${tslen}I${rm}M";
}
#print STDERR "B: $rn $RDOFF $dlen $tslen $a[5]\n";
$a[5] =~ s/\d+M\d+D\dM\d+I\dM\d+D\d+M/${RDOFF}M$tslen/;
$flag = 1;
}
}
# Combine two close deletions (<10bp) into one
if ($a[5] =~ /(\d+)D(\d)M(\d+)([DI])(\d+I)?/ ) {
my $dlen = $1 + $2 + ($4 eq "D" ? $3 : 0);
my $ilen = $2 + ($4 eq "I" ? $3 : 0);
my $istr = $5;
if ( $istr && $4 eq "D") {
$ilen += $1 if ( $istr =~ /(\d+)I/ );
}
$a[5] =~ s/\d+D\dM\d+[DI](\d+I)?/${dlen}D${ilen}I/;
$flag = 1;
}
# Combine two close indels (<10bp) into one
if ($a[5] =~ /(\d+)I(\d)M(\d+)([DI])(\d+I)?/ ) {
my $dlen = $2 + ($4 eq "D" ? $3 : 0);
my $ilen = $1 + $2 + ($4 eq "I" ? $3 : 0);
my $istr = $5;
if ( $istr && $4 eq "D" ) {
$ilen += $1 if ( $istr =~ /(\d+)I/ );
}
$a[5] =~ s/\d+I\dM\d+[DI](\d+I)?/${dlen}D${ilen}I/;
$flag = 1;
}
last unless ( $flag );
}
# The following two clauses to capture sometimes mis-softly clipped reads by aligner
if ($a[5] =~ /^(.*?)(\d+)M(\d+)S$/) {
my $ov5 = $1;
my $mch = $2;
my $soft = $3;
my $refoff = $a[3] + $2;
my $rdoff = $2;
if ( $ov5 ) {
my @rdp = $ov5 =~ /(\d+)[MIS]/g; # read position
my @rfp = $ov5 =~ /(\d+)[MD]/g; # reference position
$refoff += $_ foreach(@rfp);
$rdoff += $_ foreach(@rdp);
}
my $rn = 0;
my %RN = ();
while( $rn + 1 < $soft && $REF->{ $refoff + $rn + 1} && $REF->{ $refoff + $rn + 1} eq substr($a[9], $rdoff + $rn + 1, 1) && ord(substr($a[10], $rdoff + $rn + 1, 1))-33 > $LOWQUAL ) {
$rn++;
$RN{ $REF->{ $refoff + $rn + 1} }++;
}
my @rn_nt = keys %RN; # don't adjust if homopolymer
if ( ($rn > 3 && @rn_nt > 1) || ($REF->{ $refoff } && $REF->{ $refoff } eq substr($a[9], $rdoff, 1) )) {
$mch += $rn + 1;
$soft -= $rn + 1;
if ( $soft > 0 ) {
$a[5] =~ s/\d+M\d+S$/${mch}M${soft}S/;
} else {
$a[5] =~ s/\d+M\d+S$/${mch}M/;
}
}
if ( $rn == 0 ) {
$rn++ while( $rn < $mch && $REF->{ $refoff - $rn - 1 } && $REF->{ $refoff - $rn - 1 } ne substr($a[9], $rdoff - $rn - 1, 1) );
if ( $rn > 0 && $rn < $mch ) {
$soft += $rn;
$mch -= $rn;
$a[5] =~ s/\d+M\d+S$/${mch}M${soft}S/;
}
}
}
if ($a[5] =~ /^(\d+)S(\d+)M/ ) {
my $mch = $2;
my $soft = $1;
my $rn = 0;
my %RN = ();
while( $rn + 1 < $soft && $REF->{ $a[3] - $rn - 2} && $REF->{ $a[3] - $rn - 2} eq substr($a[9], $soft - $rn - 2, 1) && ord(substr($a[10], $soft - $rn - 2, 1))-33 > $LOWQUAL ) {
$rn++;
$RN{ $REF->{ $a[3] - $rn - 2} }++;
}
my @rn_nt = keys %RN; # don't adjust if homopolymer
if ( ($rn > 3 && @rn_nt > 1) || ($REF->{ $a[3] - 1 } && $REF->{ $a[3] - 1 } eq substr($a[9], $soft - 1, 1))) {
$mch += $rn + 1;
$soft -= $rn + 1;
if ( $soft > 0 ) {
$a[5] =~ s/^\d+S\d+M/${soft}S${mch}M/;
} else {
$a[5] =~ s/^\d+S\d+M/${mch}M/;
}
$a[3] -= $rn + 1;
$rn++;
}
if ( $rn == 0 ) {
$rn++ while( $rn < $mch && $REF->{ $a[3] + $rn } && $REF->{ $a[3] + $rn } ne substr($a[9], $soft + $rn, 1));
if( $rn > 0 && $rn < $mch ) {
$soft += $rn;
$mch -= $rn;
$a[5] =~ s/^\d+S\d+M/${soft}S${mch}M/;
$a[3] += $rn;
}
}
}
my $start = $a[3];
my @cigar = $a[5] =~ /(\d+)([A-Z])/g;
my @segs = $a[5] =~ /(\d+)[MI]/g; # Only match and insertion counts toward read length
my @segs2 = $a[5] =~ /(\d+)[MIS]/g; # For total length, including soft-clipped bases
my $rlen = 0; $rlen += $_ foreach(@segs); #$stat->sum(\@segs); # The read length for matched bases
next if ( $opt_M && $rlen < $MINMATCH );
my $rlen2= 0; $rlen2 += $_ foreach(@segs2); #$stat->sum(\@segs2); # The total length, including soft-clipped bases
$RLEN = $rlen2 if ($rlen2 > $RLEN); # Determine the read length
for(my $ci = 0; $ci < @cigar; $ci += 2) {
my $m = $cigar[$ci];
my $C = $cigar[$ci+1];
$C = "S" if ( ($ci == 0 || $ci == @cigar - 2) && $C eq "I" ); # Treat insertions at the edge as softclipping
if ( $C eq "N" ) {
$SPLICE{ ($start-1) . "-" . ($start+$m-1) }++;
$start += $m;
$offset = 0;
next;
} elsif ( $C eq "S" ) {
if ( $ci == 0 ) { # 5' soft clipped
# align softclipped but matched sequences due to mis-softclipping
while( $m-1 >= 0 && $start - 1 > 0 && $start - 1 <= $CHRS{ $chr } && $REF->{ $start-1 } && $REF->{ $start-1 } eq substr($a[9], $m-1, 1) && ord(substr($a[10],$m-1, 1))-33 > 10) {
$hash{ $start - 1 }->{ $REF->{ $start - 1 } }->{ cnt } = 0 unless( $hash{ $start - 1 }->{ $REF->{ $start - 1 } }->{ cnt } );
addCnt($hash{ $start - 1 }->{ $REF->{ $start - 1 } }, $dir, $m, ord(substr($a[10],$m-1, 1))-33, $a[4], $nm);
$cov{ $start - 1 }++;
$start--; $m--;
}
if ( $m > 0 ) {
my $q = 0;
my $qn = 0;
my $lowqcnt = 0;
for(my $si = $m-1; $si >= 0; $si--) {
last if ( substr($a[9], $si, 1) eq "N" );
my $tq = ord(substr($a[10], $si, 1))-33;
$lowqcnt++ if ($tq <= 12);
last if ( $lowqcnt > 1 );
$q += $tq;
$qn++;
}
if ( $qn >= 1 && $qn > $lowqcnt && $start >= $START - $BUFFER && $start <= $END + $BUFFER ) {
for(my $si = $m-1; $m - $si <= $qn; $si--) {
$sclip5{ $start }->{ nt }->[$m-1-$si]->{ substr($a[9], $si, 1) }++;
$sclip5{ $start }->{ seq }->[$m-1-$si]->{ substr($a[9], $si, 1) }->{ cnt } = 0 unless( $sclip5{ $start }->{ seq }->[$m-1-$si]->{ substr($a[9], $si, 1) }->{ cnt });
addCnt($sclip5{ $start }->{ seq }->[$m-1-$si]->{ substr($a[9], $si, 1) }, $dir, $si - ($m-$qn), ord(substr($a[10], $si, 1))-33, $a[4], $nm);
}
$sclip5{ $start }->{ cnt } = 0 unless( $sclip5{ $start }->{ cnt } );
addCnt( $sclip5{ $start }, $dir, $m, $q/$qn, $a[4], $nm);
}
}
$m = $cigar[$ci];
} elsif ( $ci == @cigar - 2 ) { # 3' soft clipped
while( $n < length($a[9]) && $REF->{ $start } && $REF->{ $start } eq substr($a[9], $n, 1) && ord(substr($a[10], $n, 1))-33 > 10) {
$hash{ $start }->{ $REF->{ $start } }->{ cnt } = 0 unless( $hash{ $start }->{ $REF->{ $start } }->{ cnt } );
addCnt($hash{ $start }->{ $REF->{ $start } }, $dir, $rlen2-$p, ord(substr($a[10], $n, 1))-33, $a[4], $nm);
$cov{$start}++;
$n++; $start++; $m--; $p++;
}
if ( length($a[9]) - $n > 0 ) {
my $q = 0;
my $qn = 0;
my $lowqcnt = 0;
for(my $si = 0; $si < $m; $si++) {
last if ( substr($a[9], $n+$si, 1) eq "N" );
my $tq = ord(substr($a[10], $n+$si, 1))-33;
$lowqcnt++ if ($tq <= 12);
last if ( $lowqcnt > 1 );
$q += $tq;
$qn++;
}
if ( $qn >= 1 && $qn > $lowqcnt && $start >= $START - $BUFFER && $start <= $END + $BUFFER ) {
for(my $si = 0; $si < $qn; $si++) {
$sclip3{ $start }->{ nt }->[$si]->{ substr($a[9], $n+$si, 1) }++;
$sclip3{ $start }->{ seq }->[$si]->{ substr($a[9], $n+$si, 1) }->{ cnt } = 0 unless( $sclip3{ $start }->{ seq }->[$si]->{ substr($a[9], $n+$si, 1) }->{ cnt } );
addCnt($sclip3{ $start }->{ seq }->[$si]->{ substr($a[9], $n+$si, 1) }, $dir, $qn - $si, ord(substr($a[10], $n+$si, 1))-33, $a[4], $nm);
}
$sclip3{ $start }->{ cnt } = 0 unless( $sclip3{ $start }->{ cnt } );
addCnt( $sclip3{ $start }, $dir, $m, $q/$qn, $a[4], $nm);
}
}
}
$n += $m;
$offset = 0;
$start = $a[3]; # had to reset the start due to softclipping adjustment
next;
} elsif ( $C eq "H" ) {
$offset = 0;
next;
} elsif ( $C eq "I" ) {
$offset = 0;
my $s = substr($a[9], $n, $m);
my $q = substr($a[10], $n, $m);
my $ss = "";
my ($multoffs, $multoffp, $nmoff) = (0, 0, 0); # For multiple indels within 10bp
if ( $opt_k && $cigar[$ci+2] && $cigar[$ci+2] <= $VEXT && $cigar[$ci+3] =~ /M/ && $cigar[$ci+5] && $cigar[$ci+5] =~ /[ID]/ ) {
$s .= "#" . substr($a[9], $n+$m, $cigar[$ci+2]);
$q .= substr($a[10], $n+$m, $cigar[$ci+2]);
$s .= $cigar[$ci+5] eq "I" ? ("^" . substr($a[9], $n+$m+$cigar[$ci+2], $cigar[$ci+4])) : ("^" . $cigar[$ci+4]);
$q .= $cigar[$ci+5] eq "I" ? substr($a[10], $n+$m+$cigar[$ci+2], $cigar[$ci+4]) : substr($a[10], $n+$m+$cigar[$ci+2], 1);
$multoffs += $cigar[$ci+2] + ($cigar[$ci+5] eq "D" ? $cigar[$ci+4] : 0);
$multoffp += $cigar[$ci+2] + ($cigar[$ci+5] eq "I" ? $cigar[$ci+4] : 0);
if ( $cigar[$ci+6] && $cigar[$ci+7] eq "M" ) {
my ($toffset, $tss, $tq) = findOffset($start+$multoffs, $n+$m+$multoffp, $cigar[$ci+6], \$a[9], \$a[10], $REF, \%cov);
($offset, $ss) = ($toffset, $tss);
$q .= $tq;
}
$ci += 4;
} else {
if ( $opt_k && $cigar[$ci+3] && $cigar[$ci+3] =~ /M/ ) {
my $vsn = 0;
for(my $vi = 0; $vsn <= $VEXT && $vi < $cigar[$ci+2]; $vi++) {
last if ( substr($a[9], $n+$m+$vi, 1) eq "N" );
last if ( ord(substr($a[10], $n+$m+$vi, 1))-33 < $GOODQ );
if ($REF->{ $start+$vi } && substr($a[9], $n+$m+$vi, 1) ne $REF->{ $start+$vi }) {
$offset = $vi+1;
$nmoff++;
$vsn = 0;
} elsif ($REF->{ $start+$vi } && substr($a[9], $n+$m+$vi, 1) eq $REF->{ $start+$vi }) {
$vsn++;
}
}