Bioperl samtools: problem with reference

[Ross Chapman]

Hi all

I am having problems processing a BAM file using Bio:B::Sam.

I want to examine variants in a BAM file using code developed from the example offered by Lincoln Stein on the CPAN Bio:B::Sam page. When I run my code (below) I get errors saying:

Use of uninitialized value in transliteration (tr///) at myScript.pl line 22

and also

Use of uninitialized value $refbase in concatenation (.) or string at myScript.pl line 24

It seems that my code is not reading or accessing the reference genome correctly, can any one point me towards a solution?

Thanks

Ross

#!usr/bin/perl -w

use Bio:B::Sam;

use strict;

my $bamFile="myBamFile.bam";

my $bam = Bio:B::Sam->new(-bam => $bamFile,
-fasta=>"MyReference.fasta",
-autoindex => 1);

my @targets = $bam->seq_ids;

my @SNPs; # this will be list of SNPs
my $snp_caller = sub {
my ($seqid,$pos,$p) = @_;

my $segment=$bam->segment($seqid);
my $refbase = $bam->segment($seqid,$pos,$pos)->dna;

$refbase=~tr/a-z/A-Z/;

print $refbase." \n";

};

foreach my $chr (@targets){
print $chr."\n";
$bam->pileup($chr,$snp_caller);
}