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vardict.pl
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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 Getopt::Long qw{ :config bundling no_ignore_case no_auto_abbrev};
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_p, $opt_F,
$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_L, $opt_U, $opt_w, $opt_W, $opt_A, $opt_J, $opt_j, $opt_u, $opt_mfreq, $opt_nmfreq);
#unless( getopts( 'hHtzypD3iUF: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:L:w:W:A:J:j:' )) {
# USAGE();
#}
our $VERSION = "1.8.2";
my @adaptor;
my $CHIMERIC;
my $REFEXT;
GetOptions( "h|header" => \$opt_h,
"H|?" => \$opt_H,
"dedup|t" => \$opt_t,
"z" => \$opt_z,
"verbose|y" => \$opt_y,
"chimeric" => \$CHIMERIC,
"p" => \$opt_p,
"u" => \$opt_u,
"debug|D" => \$opt_D,
"3" => \$opt_3,
"splice|i" => \$opt_i,
"nosv|U" => \$opt_U,
"F=s" => \$opt_F,
"d=s" => \$opt_d,
"b=s" => \$opt_b,
"s=i" => \$opt_s,
"e=i" => \$opt_e,
"S=i" => \$opt_S,
"E=i" => \$opt_E,
"c=i" => \$opt_c,
"g=i" => \$opt_g,
"n=s" => \$opt_n,
"N=s" => \$opt_N,
"x=i" => \$opt_x,
"X=i" => \$opt_X,
"f=f" => \$opt_f,
"r=i" => \$opt_r,
"B=i" => \$opt_B,
"Q=f" => \$opt_Q,
"m=i" => \$opt_m,
"T|trim=i" => \$opt_T,
"q=f" => \$opt_q,
"Z|downsample=f" => \$opt_Z,
"P=i" => \$opt_P,
"k=i" => \$opt_k,
"R=s" => \$opt_R,
"a|amplicon=s" => \$opt_a,
"o=f" => \$opt_o,
"O=f" => \$opt_O,
"G=s" => \$opt_G,
"V=f" => \$opt_V,
"I=i" => \$opt_I,
"M=i" => \$opt_M,
"L=i" => \$opt_L,
"w|insert-size=i" => \$opt_w,
"W|insert-std=i" => \$opt_W,
"A=f" => \$opt_A,
"adaptor=s" => \@adaptor,
"nmfreq=f" => \$opt_nmfreq,
"mfreq=f" => \$opt_mfreq,
"Y|ref-extension=i" => \$REFEXT,
"J|crispr=i" => \$opt_J,
"j=i" => \$opt_j ) || Usage();
USAGE() if ( $opt_H );
USAGE() unless ( $opt_b );
my %adaptor;
my %adaptor_rev;
my $ADSEED = 6; # adaptor seed
if ( @adaptor ) {
foreach my $seq (@adaptor) {
for(my $i = 0; $i <= 6 && $i + $ADSEED < length($seq); $i++) {
my $seed = substr($seq, $i, $ADSEED);
my $rseed = reverse($seed);
$rseed =~ y/ATGC/TACG/;
$adaptor{ $seed } = $i + 1;
$adaptor_rev{ $rseed } = $i + 1;
}
}
}
my $BAM = $opt_b; # the bam file
my $sample = $1 if ( $BAM =~ /([^\/\._]+).sorted[^\/]*.bam/ );
my $samplem = "";
my $EXTENSION = 5000;
$REFEXT = 1200 unless( $REFEXT ); # extension of reference sequences
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;
$opt_m = defined($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 : "0x504";
my $VEXT = defined($opt_X) ? $opt_X : 2; # 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 %GENOMES = ( hg19 => "/ngs/reference_data/genomes/Hsapiens/hg19/seq/hg19.fa",
hg38 => "/ngs/reference_data/genomes/Hsapiens/hg38/seq/hg38.fa",
mm10 => "/ngs/reference_data/genomes/Mmusculus/mm10/seq/mm10.fa" );
my $fasta = $opt_G ? ($GENOMES{ $opt_G } ? $GENOMES{ $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 : 0.01;
my $QRATIO = $opt_o ? $opt_o : 1.5; # The Qratio
my $NMFREQ = $opt_nmfreq ? $opt_nmfreq : 0.1; # frequency threshold for non-monomer MSI
my $MFREQ = $opt_mfreq ? $opt_mfreq : 0.25; # frequency threshold for monomer MSI
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 : 22.5; # 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 $SVMINLEN = $opt_L ? $opt_L : 1000; # The minimum structural variant length to be presented using <DEL> <DUP> <INV> <INS>, etc.
my $RLEN = 0; # The read length
my $LOWQUAL = 10; # any base with quality <= 10 will be consider low quality in soft-clipped seq and extention will stop
my $SEED1 = 17; # The larger seed size
my $SEED2 = 12; # The smaller seed size
my $DISCPAIRQUAL = 35; # The minimum mapping quality when structural variant is only supported by discordant pairs
my $INDELSIZE = $opt_I ? $opt_I : 50;
my $MINMATCH = defined($opt_M) ? $opt_M : 25;
my %SPLICE;
my $INSSIZE = $opt_w ? $opt_w : 300; # Mean Insert size
my $INSSTD = $opt_W ? $opt_W : 100; # Insert std
my $INSSTDAMT = $opt_A ? $opt_A : 4; # Insert std amount
my $SVMAXLEN = 150000; # Max Structure variant size to be called in realignment step
my %REVCOMP = ( A => "T", T => "A", C => "G", G => "C" );
my $SVFLANK = 50; # the flanking sequence length for SV
my $MINSVCDIST = 1.5; # the minimum distance between two SV clusters in term of read length
my $MINMAPBASE = 15; # the minimum position for mapping quality in SV analysis
my $MINSVPOS= 25; # the minimum distance between start position and end of SV structure in inter-chr translocation
my %SOFTP2SV;
my %IUPAC_AMBIGUITY_CODES = (M => "A", R => "A", W => "A", S => "C", Y => "C", K => "G", V => "A",
H => "A", D => "A", B => "C");
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 MQ Sig_Noise HiAF ExtraAF shift3 MSI MSI_NT NM HiCnt HiCov 5pFlankSeq 3pFlankSeq Seg VarType Duprate SV_info));
print $opt_J ? "\tCRISPR\n" : "\n";
}
my ($BAM1, $BAM2) = split(/\|/, $BAM);
my @BAMS = $BAM1 =~ /^http/ ? ($BAM1) : 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) {
s/\r//g;
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/, $_, -1);
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);
}
}
}
my @CURSEG = (); # the current processing segment (chr, start, end)
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 ( $var =~ /<(...)>/ ) {
return $1;
} elsif ( substr($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) = @{ $seg };
my @hds = qw(sp ep refallele varallele tcov cov rfc rrc fwd rev genotype freq bias pmean pstd qual qstd mapq qratio hifreq extrafreq shift3 msi msint nm hicnt hicov leftseq rightseq);
while( my ($p, $pv) = each %$vars ) {
unless( $pv->{ SV } ) {
next unless( $p >= $S && $p <= $E );
}
my @vts = ();
my @vrefs = ();
unless( $pv->{ VAR } ) {
next unless( $opt_p );
my $vref = $pv->{ REF };
unless($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", "", 0, 0), "\n";
next;
}
push(@vts, "");
push(@vrefs, $vref);
} else {
for(my $i = 0; $i < @{ $pv->{VAR} }; $i++) {
my $vref = $pv->{ VAR }->[$i];
last if ( $vref->{refallele} =~ /N/ );
if ($vref->{ refallele } eq $vref->{ varallele }) {
next unless( $opt_p );
}
if ($vref->{sp} != $p && $opt_p && @{$pv->{ VAR }}== 1 ) {
my $vref2 = $pv->{ REF };
unless($vref2) {
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", "", 0, 0), "\n";
}
push(@vts, "");
push(@vrefs, $vref2);
}
my $vartype = varType($vref->{refallele}, $vref->{varallele});
unless( isGoodVar( $pv->{ VAR }->[$i], $pv->{ 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" );
my $duprate = $vref->{ duprate } ? $vref->{ duprate } : 0;
my $crispr = $opt_J ? ($vref->{ crispr } ? $vref->{ crispr } : 0) : "";
print join("\t", $sample, $G, $chr, (map { $vref->{ $_ } ? $vref->{ $_ } : 0; } @hds), "$chr:$S-$E", $vartype, $duprate, $pv->{ SV } ? $pv->{ SV } : 0);
print "\t$crispr" if ( $opt_J );
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 });
next if ( $varnt =~ /^</ );
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) = @{ $seg };
#print STDERR "$chr:$S-$E\n";
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
foreach my $vref2 (@{$vars2->{$p}->{ VAR }}) {
next if ($vref2->{ refallele } eq $vref2->{ varallele });
$vartype = varType($vref2->{ refallele }, $vref2->{ varallele });
next unless(isGoodVar($vref2, $vars2->{$p}->{REF}, $vartype) );
adjComplex($vref2) if ( $vartype eq "Complex" );
print join("\t", $sample, $G, $chr, (map{ $vref2->{ $_ }; } @hd1), (map{ 0; } @hdrs), (map { $vref2->{ $_ }; } (@hdrs, @hd2)), "$chr:$S-$E", "Deletion", $vartype, "0", "0", $vref2->{ duprate } ? $vref2->{ duprate } : 0, $vars2->{$p}->{ SV } ? $vars2->{$p}->{ SV } : 0 ), "\n";
}
} elsif( ! $vars2->{ $p } ) { # no coverage for sample 2
foreach my $vref1 (@{$vars1->{$p}->{ VAR }}) {
next if ($vref1->{ refallele } eq $vref1->{ varallele });
$vartype = varType($vref1->{ refallele }, $vref1->{ varallele });
next unless(isGoodVar($vref1, $vars1->{$p}->{REF}, $vartype) );
adjComplex($vref1) if ( $vartype eq "Complex" );
print join("\t", $sample, $G, $chr, (map { $vref1->{ $_ }; } (@hd1, @hdrs)), (map { 0; } @hdrs), (map { $vref1->{$_};} @hd2), "$chr:$S-$E", "SampleSpecific", $vartype, $vref1->{ duprate } ? $vref1->{ duprate } : 0, $vars1->{$p}->{ SV } ? $vars1->{$p}->{ SV } : 0, 0, 0 ), "\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 = $VREF->{ n };
if ($VREF->{ refallele } eq $VREF->{ varallele }) {
$N++;
next;
}
#if ( length($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");
$type = "StrongSomatic" if ( isNoise($v2->{ VARN }->{ $nt }) && $vartype eq "SNV" );
#if ($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, $VREF->{ duprate } ? $VREF->{ duprate } : 0, $v1->{ SV } ? $v1->{ SV } : 0, $v2->{ VARN }->{ $nt }->{ duprate } ? $v2->{ VARN }->{ $nt }->{ duprate } : 0, $v2->{ SV } ? $v2->{ SV } : 0), "\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 @tvf = ();
if ( $v2->{ VAR } ) {
my $v2r = $v2->{ VAR }->[0];
@tvf = ($v2r->{ tcov }, 0, $v2r->{ rfc }, $v2r->{ rrc }, map { 0; } (1..14));
} elsif ( $v2->{ REF } ) {
@tvf = (map { $v2->{ REF }->{ $_ } ? $v2->{ REF }->{ $_ } : 0; } @hdrs) ;
} else {
@tvf = (map { 0; } (1..18));
}
my $type = "StrongSomatic";
if ($vartype ne "SNV" && (length($nt) > 10 || $nt =~ /-\d\d/)) {
$v2->{ VARN }->{ $nt }->{ cov } = 0; # Ensure it's initialized before passing to combineAnalysis
my $newtype = $VREF->{ cov } < $MINR + 3 && $nt !~ /</ ? combineAnalysis($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, $VREF->{ duprate } ? $VREF->{ duprate } : 0, $v1->{ SV } ? $v1->{ SV } : 0, $v2->{ VARN }->{ $nt }->{ duprate } ? $v2->{ VARN }->{ $nt }->{ duprate } : 0, $v2->{ SV } ? $v2->{ SV } : 0), "\n";
} else {
print join("\t", $sample, $G, $chr, (map { $VREF->{ $_ }; } (@hd1, @hdrs)), @tvf, (map { $VREF->{$_};} @hd2), "$chr:$S-$E", "StrongSomatic", $vartype, $VREF->{ duprate } ? $VREF->{ duprate } : 0, $v1->{ SV } ? $v1->{ SV } : 0, $v2->{ VARN }->{ $nt }->{ duprate } ? $v2->{ VARN }->{ $nt }->{ duprate } : 0, $v2->{ SV } ? $v2->{ SV } : 0), "\n";
}
}
$N++;
}
unless($N) {
next unless( $v2->{ VAR } );
foreach my $v2var (@{$v2->{ VAR }}) {
$vartype = varType($v2var->{ refallele }, $v2var->{ varallele }) ;
next unless( isGoodVar( $v2var, $v2->{ REF }, $vartype ) );
# potentail LOH
my $nt = $v2var->{ n };
if ( $v1->{ VARN }->{ $nt } ) {
next if ($v1->{ VARN }->{ $nt }->{ refallele } eq $v1->{ VARN }->{ $nt }->{ varallele });
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 { $v2var ->{ $_ }; } (@hdrs, @hd2)), "$chr:$S-$E", $type, varType($v1->{ VARN }->{ $nt }->{ refallele }, $v1->{ VARN }->{ $nt }->{ varallele }), $v1->{ VARN }->{ $nt }->{ duprate } ? $v1->{ VARN }->{ $nt }->{ duprate } : 0, $v1->{ SV } ? $v1->{ SV } : 0, $v2var ->{ duprate } ? $v2var ->{ duprate } : 0, $v2->{ SV } ? $v2->{ SV } : 0), "\n";
} else {
next if ($v2var->{ refallele } eq $v2var->{ varallele });
my @th1 = ($v1->{ VAR } ? $v1->{ VAR }->[0]->{ tcov } : 0, 0, $v1->{ REF } ? (map { $v1->{ REF }->{ $_ } } ("fwd", "rev")) : (0, 0), 0, 0);
push(@th1, $v1->{ VAR } ? $v1->{ VAR }->[0]->{ genotype } : ($v1->{ REF } ? "$v1->{ REF }->{ n }/$v1->{ REF }->{ n }" : "N/N"));
adjComplex($v2var) if ( $vartype eq "Complex" );
push(@th1, map { 0; } @hdrs[7..$#hdrs]);
print join("\t", $sample, $G, $chr, (map { $v2var->{ $_ }; } @hd1), @th1, (map { $v2var->{ $_ }; } (@hdrs, @hd2)), "$chr:$S-$E", "StrongLOH", $vartype, 0, 0, $v2var->{ duprate } ? $v2var->{ duprate } : 0, $v2->{ SV } ? $v2->{ SV } : 0), "\n";
}
}
}
} elsif ( $v2->{ VAR } ) { # sample 1 has only reference
foreach my $v2var (@{$v2->{ VAR }}) {
next if ($v2var->{ refallele } eq $v2var->{ varallele });
$vartype = varType($v2var->{ refallele }, $v2var->{ varallele });
next unless( isGoodVar( $v2var, $v2->{ REF }, $vartype ) );
# potential LOH
my $nt = $v2var->{ n };
my $type = "StrongLOH";
$v1->{ VARN }->{ $nt }->{ cov } = 0;
my $newtype = $v2->{ VARN }->{ $nt }->{ cov } < $MINR + 3 && $nt !~ /</ && (length($nt) > 10 || $nt =~ /-\d\d/ ) ? combineAnalysis($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 }->{ $_ } : 0 } @hdrs);
adjComplex($v2var) if ( $vartype eq "Complex" );
print join("\t", $sample, $G, $chr, (map { $v2var->{ $_ } ? $v2var->{ $_ } : 0; } @hd1), @th1, (map { $v2var->{ $_ } ? $v2var->{ $_ } : 0; } (@hdrs, @hd2)), "$chr:$S-$E", $type, $vartype, 0, 0, $v2var->{ duprate } ? $v2var->{ duprate } : 0, $v2->{ SV } ? $v2->{ SV } : 0), "\n";
}
}
}
}
}
# Taken a likely somatic indels and see whether combine two bam files still support somatic status. This is mainly
# for Indels that softclipping overhang is too short to positively being called in one bam file, but can be called
# in the other bam file, thus creating false positives
sub combineAnalysis {
my ($var1, $var2, $chr, $p, $nt) = @_;
my ($bam1, $bam2) = split(/\|/, $BAM);
print STDERR "Start Combine $p $nt\n" if ( $opt_y );
return "" if ( $var1->{ ep } - $var1->{ sp } > $SVMINLEN ); # Don't do it for structural variants
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);
$var2->{ pmean } = $var2->{ cov } > 0 ? sprintf("%.1f", ($vref->{ pmean } * $vref->{ cov } - $var1->{ pmean } * $var1->{ cov })/$var2->{ cov }) : 0;
$var2->{ qual } = $var2->{ cov } > 0 ? sprintf("%.1f", ($vref->{ qual } * $vref->{ cov } - $var1->{ qual }*$var1->{ cov })/$var2->{ cov }) : 0;
$var2->{ mapq } = $var2->{ cov } > 0 ? sprintf("%.1f", ($vref->{ mapq }*$vref->{ cov } - $var1->{ mapq }*$var1->{ cov })/$var2->{ cov }) : 0;
$var2->{ hifreq } = $var2->{ cov } > 0 ? sprintf("%.4f", ($vref->{ hifreq }*$vref->{ cov } - $var1->{ hifreq }*$var1->{ cov })/$var2->{ cov }) : 0;
$var2->{ extrafreq } = $var2->{ cov } > 0 ? sprintf("%.4f", ($vref->{ extrafreq }*$vref->{ cov } - $var1->{ extrafreq }*$var1->{ cov })/$var2->{ cov }) :0;
$var2->{ nm } = $var2->{ cov } > 0 ? sprintf("%.1f", ($vref->{ nm }*$vref->{ cov } - $var1->{ nm }*$var1->{ cov })/$var2->{ cov }) : 0;
$var2->{ nm } = $var2->{ nm } > 0 ? $var2->{ nm } : 0;
map { $var2->{ $_ } = 1; } qw(pstd qstd);
return "FALSE" if ( $var2->{ tcov } <= 0 );
my $freq2 = sprintf("%.4f", $var2->{ cov }/$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}, $var2->{rev});
roundingAlmostZeros($var2);
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 } > 0.30 );
return 0 if ( $vref->{ mapq } < $opt_O );
return 0 if ( $vref->{ msi } >= 15 && $vref->{ freq } <= $MFREQ && $vref->{ msint } == 1);
return 0 if ( $vref->{ msi } >= 12 && $vref->{ freq } <= $NMFREQ && $vref->{ msint } > 1 );
if ( $vref->{ bias } eq "2;1" && $vref->{ freq } < 0.20 ) {
return 0 unless($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, $ref, $ext) = @_;
my %REF = ();
$ref = $ref ? $ref : \%REF;
my $extension = $ext ? $ext : $REFEXT;
my $s_start = $START - $EXT - $extension < 1 ? 1 : $START - $EXT - $extension;
my $s_end = $END + $EXT + $extension > $CHRS{ $chr } ? $CHRS{ $chr } : $END + $EXT + $extension;
print STDERR "TIME: Getting REF: ", time(), "\n" if ( $opt_y );
my ($header, $exon) = split(/\n/, `samtools faidx $fasta $chr:$s_start-$s_end`, 2);
return $ref if ( isLoaded($chr, $s_start, $s_end, $ref) );
push(@{ $ref->{ loaded } }, [$chr, $s_start, $s_end]);
$exon =~ s/\s+//g;
$exon = uc($exon);
my $siteEnd = $CHRS{ $chr } == $s_end ? length($exon) : length($exon) - $SEED1;
for(my $i = 0; $i < $siteEnd; $i++) {
next if ( $ref->{ $i + $s_start } ); # don't process it more than once
$ref->{ $i + $s_start } = substr( $exon, $i, 1 );
# Do not create adaptor sequences for the very end of chromosome
next if ($CHRS{ $chr } == $s_end && $i > length($exon) - $SEED1);
push( @{ $ref->{ substr( $exon, $i, $SEED1) } }, $i + $s_start );
push( @{ $ref->{ substr( $exon, $i, $SEED2) } }, $i + $s_start );
}
print STDERR "TIME: Got REF.", time(), "\n" if ( $opt_y );
return $ref;
}
# Check whether a region is alreday loaded in reference
sub isLoaded {
my ($chr, $s, $e, $ref) = @_;
return 0 unless( $ref->{ loaded } );
foreach my $r (@{ $ref->{ loaded } }) {
return 1 if ( $chr eq $r->[0] && $s >= $r->[1] && $e <= $r->[2] );
}
return 0;
}
# Add deletion structural variant
sub addSV {
my ($sdref, $s, $e, $ms, $me, $dir, $rlen, $mlen, $softp, $rp, $q, $Q, $nm) = @_;
$sdref->{ cnt }++;
$sdref->{ dir } = $dir;
$sdref->{ $dir }++;
#$sdref->{ pmean } += $rp;
#$sdref->{ qmean } += $q;
#$sdref->{ Qmean } += $Q;
#$sdref->{ nm } += $nm;
$q >= $GOODQ ? $sdref->{ hicnt }++ : $sdref->{ locnt }++;
$sdref->{ start } = $s unless( $sdref->{ start } && $sdref->{ start } < $s );
$sdref->{ end } = $e unless( $sdref->{ end } && $sdref->{ end } > $e );
push(@{ $sdref->{ mates } }, [$ms, $me, $mlen, $s, $e, $rp, $q, $Q, $nm]);
$sdref->{ mstart } = $ms unless( $sdref->{ mstart } && $sdref->{ mstart } < $ms );
$sdref->{ mend } = $ms+$rlen unless( $sdref->{ mend } && $sdref->{ mend } > $me );
if ($softp) {
if ( $dir == 1 ) {
$sdref->{ soft }->{ $softp }++ if ( abs($softp - $sdref->{end}) < 10 );
} else {
$sdref->{ soft }->{ $softp }++ if ( abs($softp - $sdref->{start}) < 10 );
}
}
}
# Construct a variant structure given a segment and BAM files.
sub parseSAM {
my ($chr, $START, $END, $bams, $REF, $hash, $cov, $sclip5, $sclip3, $svflag) = @_;
my %COV = ();
$cov = $cov ? $cov : \%COV;
my %HASH = ();
$hash = $hash ? $hash : \%HASH;
my %dels5;
my %ins;
my %mnp; # Keep track of MNPs
my %SCLIP3 = (); # soft clipped at 3'
my %SCLIP5 = (); # soft clipped at 5'
$sclip3 = $sclip3 ? $sclip3 : \%SCLIP3;
$sclip5 = $sclip5 ? $sclip5 : \%SCLIP5;
%SPLICE = ();
my @svfdel;
my @svrdel;
my ($svdelfend, $svdelrend) = (0, 0); # for strutral variant: Deletion
my @svfdup;
my @svrdup;
my ($svdupfend, $svduprend) = (0, 0); # for strutral variant: Dupplication
my @svfinv3;
my @svfinv5;
my @svrinv3;
my @svrinv5;
my ($svinvfend3, $svinvrend3) = (0, 0); # for strutral variant: Inversion
my ($svinvfend5, $svinvrend5) = (0, 0); # for strutral variant: Inversion
my %svffus;
my %svrfus;
my %svfusfend;
my %svfusrend;
my @svfins;
my @svrins;
my ($svinsfend, $svinsrend) = (0, 0); # for strutral variant: Insertion
#$REF = getREF($chr, $START, $END);
my ($totalreads, $dupreads, $disc) = (0, 0, 0);
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" : "";
print STDERR "TIME: Start parsing SAM: ", time(), " $bami $chr:$START-$END\n" if ( $opt_y );
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/, $_, 12);
next if ( defined($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
$totalreads++;
if ( $opt_t ) {
%DUP = () if ( $a[3] ne $DUPP );
if ( $a[7] =~ /^\d/o ) {
if ( $DUP{ "$a[3]-$a[6]-$a[7]" } ) {
$dupreads++;
next;
}
$DUP{ "$a[3]-$a[6]-$a[7]" } = 1;
$DUPP = $a[3];
} elsif ( $a[1] & 0x8 ) { # mate not mapped
if ( $DUP{ "$a[3]-$a[5]" } ) {
$dupreads++;
next;
}
$DUP{ "$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);
# Number of mismatches from NM tag (or nM for STAR).
if ( $a[11] && $a[11] =~ /NM:i:(\d+)/io ) { # 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. $a[0] $a[2] $a[3] $a[11]\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 ? -1 : 1;
# 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);
}
}
# 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/;
if ( $a[5] =~ /^(\d\d+)S/ ) {
my $m = $1;
if ( (! $CHIMERIC) && $m >= $SEED2 ) {
my $sseq = substr($a[9], 0, $m);
$sseq = reverse($sseq);
$sseq =~ y/ATGC/TACG/;
if ( $REF->{ substr($sseq, 0, $SEED2) } && @{ $REF->{ substr($sseq, 0, $SEED2) } } == 1 && abs($a[3] - $REF->{ substr($sseq, 0, $SEED2) }->[0]) < 2*$RLEN ) {
$a[5] =~ s/^\d+S//;
$a[9] = substr($a[9], $m);
$a[10] = substr($a[10], $m);
print STDERR "$sseq at 5' is a chimeric at $a[3] by SEED $SEED2\n" if ( $opt_y );
}
}
} elsif ( $a[5] =~ /(\d\d+)S$/ ) {
my $m = $1;
if ( (! $CHIMERIC) && $m >= $SEED2 ) {
my $sseq = substr($a[9], -$m, $m);
$sseq = reverse($sseq);
$sseq =~ y/ATGC/TACG/;
if ( $REF->{ substr($sseq, -$SEED2, $SEED2) } && @{ $REF->{ substr($sseq, -$SEED2, $SEED2) } } == 1 && abs($a[3] - $REF->{ substr($sseq, -$SEED2, $SEED2) }->[0]) < 2*$RLEN ) {
$a[5] =~ s/\d\d+S$//;
substr($a[9], -$m) = "";
substr($a[10], -$m) = "";
print STDERR "$sseq at 3' is a chimeric at $a[3] by SEED $SEED2\n" if ( $opt_y );
}
}
}
while( $idlen > 0 && $opt_k ) {
my $flag = 0;
if ($a[5] =~ /^(\d+)S(\d+)([ID])/o) {
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$/o) {
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])/o) {
if ( $2 <= 10 ) {
my $tslen = $1 + $2 + ($4 eq "I" ? $3 : 0);
$tslen .= "S";
$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$/o) {
if ( $3 <= 10 ) {
my $tslen = $4 + $3 + ($2 eq "I" ? $1 : 0);
$tslen .= "S";
$a[5] =~ s/\d+[ID]\d+M\d+S$/$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/o) {
my $tslen = $1 + ($3 eq "I" ? $2 : 0);
my $mlen = $4;
$a[3] += $1 + ($3 eq "D" ? $2 : 0);
my $tn = 0;
$tn++ while( $tn < $mlen && $REF->{ $a[3]+$tn } && $REF->{ $a[3]+$tn } ne substr($a[9], $tslen+$tn, 1));
$tslen += $tn;
$mlen -= $tn;
$a[3] += $tn;
$a[5] =~ s/^\dM\d+[ID]\d+M/${tslen}S${mlen}M/;
$flag = 1;
}
if ($a[5] =~ /(\d+)([ID])(\d)M$/o) {
my $tslen = $3 + ($2 eq "I" ? $1 : 0);
$tslen .= "S";
$a[5] =~ s/\d+[ID]\d+M$/$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/o) {
my $tslen = $4 + $5 + $6;
my $dlen = $3 + $4 + $6 + $7;
my $mid = $4 + $6;
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+)[MND]/g; # reference position
$refoff += $_ foreach(@rfp);
$rdoff += $_ foreach(@rdp);