Hello

I'm trying to use Bio:: DB::Sam to get list of reads covering certain position .Sam file using pileup method using this 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 names is definitely not complete. I.e. pileup ignores some reads.

Trying to find the root of problem I analyzed reads which alignment starts from position 1209 (just for example) and found that only the first N of such reads assessed by pileup and the last M of reads are ignored. But after those not ignored N reads it doesn't 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 haven't 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 differences

So what's wrong?