Samtools Pileup Parser

[Graham Etherington]

Hi,
I'm trying to parse the Samtools Pileup format in Perl.

I've created a paired-end assembly using BWA (aln, sampe) and then used Samtools to create a pileup (using import - sort - index - pileup).

What I want to do is to open up the pileup (which is obviously in the Samtools Pileup format) and iterate over each position of the pileup, examining differences in base-calls.

Does anyone know of anything available, or have and sample code they could post?
Many thanks.

[Pepe]

You cannot open a file in Pileup format with Bio:B::sam

You should convert your SAM file to BAM format (samtools view) and sort it (samtools sort), maybe index it too? I'm not sure (samtools index).

Then follow the instructions in the CPAN website, which are very, veryhelpful. Basically:

use Bio:B::Sam;
my $sam = Bio:B::Sam->new(-bam => "my_sorted_bam_file.bam",
-fasta => "my_ref.fasta");
my @targets = $sam->seq_ids;
foreach my $chr (@targets){
$sam->pileup($chr,$my_subroutine);
}

where $my_subroutine is a subroutine that you'll need to get to do what you want: snp calling, coverage calculation...

[Graham Etherington]

Originally Posted by Pepe

You cannot open a file in Pileup format with Bio::DB::sam

You should convert your SAM file to BAM format (samtools view) and sort it (samtools sort), maybe index it too? I'm not sure (samtools index).

Then follow the instructions in the CPAN website, which are very, veryhelpful. Basically:

use Bio::DB::Sam;
my $sam = Bio::DB::Sam->new(-bam => "my_sorted_bam_file.bam",
-fasta => "my_ref.fasta");
my @targets = $sam->seq_ids;
foreach my $chr (@targets){
$sam->pileup($chr,$my_subroutine);
}

where $my_subroutine is a subroutine that you'll need to get to do what you want: snp calling, coverage calculation...

Hi,
Thanks for your answer. I'd realised that Bio::DB:Sam couldn't parse the pileup, so this is why I am asking if anyone has, or knows about, anything that could parse it, before I spent time writing a Samtools parser myself.
I have a 'Parser.pm' module that parses various pileup formats (maq, bowtie, novoalign, etc), but I haven't added one for Samtools format yet.
Cheers,
Graham

[nekrut]

Galaxy's (http://usegalaxy.org) pileup parser might do the trick:
http://bit.ly/cXtqD9

[Graham Etherington]

Originally Posted by nekrut

Galaxy's (http://usegalaxy.org) pileup parser might do the trick:
http://bit.ly/cXtqD9

Thanks. That should give me enough to get started with.
Best wishes,
Graham

[cschu]

I noticed that the referenced Galaxy code does not check for the '>' and '<' characters. Can anybody please provide some help how to process those? I.e., I don't really understand what "reference skip" means and would think that such a symbol means that the read does not cover the the respective position. Any help would be appreciated, thanks!