SNP calling from pooled RNA-seq data

Question

[thjnant]

Hello,

I want to call SNPs from pooled RNA-seq data that have been mapped to a reference genome. My organism is a bird. Does anyone have any experience with this, which software to use, for example GATK, samtools, FreeBayes, etc?

[Devon Ryan]

At least with GATK you can set the -ploidy option (I've not tried this, but it allegedly works). You'll need a good bit of sequencing per pooled sample to reliably call things in anything but super-high expressing gene, though.

Answer 1

[Ashutosh Pandey]

There are a few papers that talk about reliable identification of variants from RNA-seq data.

• http://www.sciencedirect.com/science/article/pii/S0002929713003832
• http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0058815

Answer 2

[Khader Shameer]

We have developed a method called ESNV-Detect to do this task from human transcriptome data. (ESNV = expressed SNV).

Manuscript is in press; you can download the software here.

Abstract decribing intial results from AACR 2013 meeting is here.

Update:

Manuscript is now published in NAR. Abstract here and full-article here.

ESNV-Detect Workflow: Call expressed SNVs from RNA-Seq data

Answer 3

[fu_entomology]

Hi, I am working on some pooled data as well. I think GATK does support pooled sequencing, as far as you set the ploidy number, check their documents. I used Varscan, and I like it. It generates the standard vcf output, which is very compatible with lots of programs downstream.

Let me know if you have other question.

[thjnant]

Thank you everyone for your very useful comments, I am going to start the SNP calling in the next few days, will let you know about my questions and experience.