62 lines
2.2 KiB
Perl
62 lines
2.2 KiB
Perl
#!/usr/bin/perl -w
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use strict;
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use Parallel::ForkManager;
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# this script creats one file with UMI unique reads and one with UMI duplicated reads
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# as input you need aligned sorted by coordinate bam file
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my @torun = ();
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foreach my $file ( <*Aligned.sortedByCoord.out.bam> ) {
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push @torun, $file;
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}
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my $pm = Parallel::ForkManager->new( 12 );
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foreach my $file ( @torun ) {
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my $sample = $file;
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$sample =~ s/Aligned\.sortedByCoord\.out\.bam$//;
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next if -s $sample.'_uniq.bam';
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warn "Parsing $sample\n";
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$pm->start and next;
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my %seen = ();
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my %duplicates = ();
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my ( $uniqs, $dups ) = ( 0, 0 );
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open F, 'samtools view -h '.$file.' |';
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open F1, '| samtools view -bS - >'.$sample.'_uniq.bam';
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open F2, '| samtools view -bS - >'.$sample.'_dups.bam';
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while ( <F> ) {
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if ( m/^\@/ ) {
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print F1;
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print F2;
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next;
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}
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my ( $id, $flag, $chr, $pos, $mapq, $cigar, $chr2, $pos2, $tlen ) = split /\t/;
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next if $flag & 256 or $flag & 512 or $flag & 1024; #skip if the read is not primary alignment/read fails platform/vendor quality checks/read is PCR or optical duplicate
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# foreach ( 256, 512, 1024 ) { $flag-=$_ if $flag&$_ }
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my ( $bc ) = $id =~ m/\:([GATCN\d]+)$/; #extract UMI barcode
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my $uniq = join( ':', $chr, $pos, $flag, $tlen, $bc );
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my $pos_ = $pos-1;
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while ( $cigar =~ m/(\d+)([SHMDIN=])/g ) {
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$pos_+=$1 if $2 eq 'M' or $2 eq '=' or $2 eq 'D' or $2 eq 'N'; # find position for minus strand
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}
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my $uniq2 = join( ':', $chr, $pos_, $flag, $tlen, $bc );
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if ( exists($duplicates{$id}) or # already marked as duplicate
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( not($flag&16) and ++$seen{ $uniq } > 1 ) or # plus strand
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( $flag&16 and ++$seen{ $uniq2 } > 1 ) #minus strand
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) {
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print F2;
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$dups++;
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$duplicates{$id} = 1;
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}
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else {
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print F1;
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$uniqs++;
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}
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}
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close F;
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close F1;
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close F2;
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warn "Unique: $uniqs (", 100*$uniqs/($uniqs+$dups), ")\n";
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system( 'samtools', 'index', $sample.'_uniq.bam' );
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# last;
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$pm->finish;
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}
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$pm->wait_all_children;
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