samtools mpileup and overlapping paired-end reads

[alpha2zee]

I am using samtools mpileup to generate a pileup for paired-end RNA sequencing data. I am curious about how samtools handles pair mates whose read mappings overlap. With regard to the simple example below, for positions 7-11, are both pair mates enumerated?

position     1    6    11   16
reference    ATGCATGCATGCATGC
pair mate 1  ATGCATGCATG
pair mate 2'       GCATGCATGC    (reverse complemented)

I am parsing the pileup output to quantify RNA editing at some positions and do not want to count an 'RNA molecule' twice just because the read pair mates 'overlapped.'

Thanks.

[lindenb]

cross posted on http://www.biostars.org/p/87299

[alpha2zee]

cross posted on http://www.biostars.org/p/87299

Sorry, is cross-posting a no-no? I am trying to reach more people and plan to post here a link to any conclusive answer I get elsewhere.

[dariober]

I am using samtools mpileup to generate a pileup for paired-end RNA sequencing data. I am curious about how samtools handles pair mates whose read mappings overlap. With regard to the simple example below, for positions 7-11, are both pair mates enumerated?

Hi- I'm pretty sure mpileup is going to double count overlapping reads. You can clip overlapping mates with, among other tools, clipOverlap .

Dario

[swbarnes2]

Sorry, is cross-posting a no-no? I am trying to reach more people and plan to post here a link to any conclusive answer I get elsewhere.

It's frowned on because it's a waste of time for someone here to compose a good answer for you, when they don't know that you already have a good answer somewhere else.

I think it would have been better to have posted on one site, and if after a couple of days, you got no good answer, then posted the question somewhere else.

[alpha2zee]

Hi- I'm pretty sure mpileup is going to double count overlapping reads. You can clip overlapping mates with, among other tools, clipOverlap .

Dario

Thanks. I got the same answer on my cross-post on biostars.org. And thanks for suggesting clipOverlap.

[owenman]

Note, the above answer is now incorrect, if you're using samtools >= 1.0 (which was released more than 3 years ago).

See my post here: https://www.biostars.org/p/87299/#284421