Re: [Samtools-help] retrieving base frequencies at specific position

[Tychele T. <ttu...@jh...>]

GATK also has a tool that will do this. It's called DepthOfCoverage. 

http://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_sting_gatk_walkers_annotator_DepthOfCoverage.html

If you use the --printBaseCounts option it should also give you the count of A, C, T, and G at every position and in every individual. There are other options as well so you might also want to look in those. Hope this helps. 

Cheers,

Tychele
________________________________________
From: Hanspeter Niederstrasser [fi...@sn...]
Sent: Tuesday, January 29, 2013 12:42 PM
To: sam...@li...
Subject: [Samtools-help] retrieving base frequencies at specific position

I have a set of 86 aligned bam files from an Illumina targeted
resequencing run.  Because the input samples were not clonal, we are
interested in determining the frequency of bases at each position along
the sequenced amplicon (~3kb).

Basically, at the end of the day, I want to be able to generate a table
that conceptually looks like this (pos 1 had 13 reads in the bam: 6 C
and 7 T):

Pos A C T G
1   0 6 7 0
2   0 0 0 12
3  10 1 1 0
...

Parsing the Illumina provided VCF files doesn't give me the coverage
that I need since many of the variants are of low frequency (< 5%) and
many positions have more than one variant.  The closest I've been able
to get is to modify this script <http://www.biostars.org/p/45384/> to
work with mpileup output, and that gives me output like this:

311     A 40
312     T 39    C 1
313     C 29    T 7

but when I look at my .bam file in IGViewer, the number of reads
actually present is about 2.5 times higher than what the script presents
(eg 86 reads for position 313).

Is there some software solution that already exists that would allow me
individually read and tally bases along a bam? Or can someone point out
why the above script is not giving the accurate counts for each position
from the mpileup output?

Thanks,

Hanspeter

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