SNP discovey in 454 (RNA-seq) data

[pravee1216]

Hi All,

I've a query regarding mutation analysis on 454 RNA data.I've RNA of specific human gene, say PRMT5 (isolated from 100 individuals,pooled and sequenced in 454) of avg read length 400bp.

I used BWA to map reads to mRNA sequence and used GATK for variant calls, but it did not detect any variants. the coverage was ~ 3000x. We're expecting few variants on the sample, but don't know the optimal pipeline for this.

I want to know:
1) Optimal strategy and combination of tools (for mapping, SNP calling, annotation for 454 RNA-seq) to use to achieve this?
2) Do I used mRNA or chromosome sequence to map against? I want to annotate SNPs at later stage.
3) Since this is a pooled sample, is duplicate removal recommended?

Look forward to any suggestions and guidelines

Thanks

Raj