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  • Bio-SamTools pileup method

    Hello
    Im trying to use Bio:: DB::Sam to get list of reads covering certain position .Sam file using pileup method using this code
    Code:
    use strict;
    use warnings;
    
    use Bio::DB::Sam;
    
    `samtools view -Sb $ARGV[0].sam > $ARGV[0].bam`;
    `samtools sort $ARGV[0].bam $ARGV[0].sorted`;
    `mv $ARGV[0].sorted.bam $ARGV[0].bam`;
    `samtools index $ARGV[0].bam`;
    
    my $sam = Bio::DB::Sam->new(
            -bam   => "$ARGV[0].bam",
            -fasta => "$ARGV[1]",
            );
    
    my $callback = sub {
            my ($seqid,$pos,$p) = @_;
            if ($pos eq "1240") {
                    for my $pileup (@$p) {
                            my $b   = $pileup->alignment;
                            print $b->qname,"\n";
                            }
                    }
            };
    
    $sam->pileup("GBA",$callback);
    exit;
    So this code only get names of reads which cover position 1240 in reference from $ARGV[0].sam file. The problem is that the output list of read neames is definitely not complete. I.e. pileup ignores some reads.
    Trying to find the root of problem i analysed reads which alignment starts from prosition 1209 (just for example) and found that only the first N of such reads assessed by puleup and the last M of reads are ignored. But after those not ignored N reads it doesnt ignore all reads starting from other positions (i.e. 1150,1048,1231 and any others). So as i understand the problem must be in some kind of sorting, but i havent found any information about sorting should be done in manual and i actually do sort .bam file.
    Also i found that using $sam->pileup("GBA:1239-1241",$callback) instead of $sam->pileup("GBA",$callback) makes another list of reads, which differs from the previous, but i could found any logic in these differrences
    So what's wrong?


    Here are link to some test data: Sam file - https://cloud.mail.ru/public/d9e9de482926/In.sam
    Reference - https://cloud.mail.ru/public/4394275..._gla_ids.fasta
    $: perl test.pl In gaa_gba_gla_ids.fasta > out
    Last edited by m4merg; 07-31-2014, 08:19 AM.

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