Software advice needed regarding SNP calling

[Burger]

Hi all

I would like your input on software to use for SNP calling from a pooled transcriptome sample, I have a whole genome (Eucalyptus grandis).

The problem is the incorporation of splice junctions into my pipeline.
-What should I map with? (so far I'm thinking Tophat)
-What can I use for calling of SNPs? (As Tophat produces a BAM file this should be easy)

All advice welcome. PS I'm working with Illumina paired end reads

[cedance]

Tophat does great job of mapping. So it should be alright.

As for SNP calling, there are plenty of softwares out there, but most of them are tuned not for RNA-seq I believe. Having said that, you could use samtools to call for variants and then supply that to GATK and then obtain SNPs from GATK pipeline by performing local realignment, quality recalibration etc... The GATK nature article is also a very interested read.

Over the last few days, I have been reading about local realignment by SRMA and it seems impressive. So, I'd like to try that as well. I've seen many of them who have tried GATK realignment and SRMA preferring SRMA for local realignment.

There are of course many other tools that are equally good. You could find a few in this seq-wiki

There is also a Nature review on comparison of some of the tools here. I still think there are many things that needs improvement though.

[Burger]

I was thinking of going the SAMtools and GATK route, thank you for the articles. Will have a read.

[paolo.kunder]

I am using samtools pileup with the alignment output of tophat (bam)

Then analyze the output with VarScan.v2.2.11 pileup2snp, results are quite nice and straightforward,

pk