What is the best software for SNP calling from RNA-seq (100 bp single-end) data of a +ssRNA virus population?

Question

[wld28]

What is the best software for SNP calling from RNA-seq (100 bp single-end) data of a +ssRNA virus population?

[wld28]

Hi Joe,

My virus is from infected potato plants (PVY). It has the polyA tail, which I use for library preparation - using oligodt magnetic beads for capturing of the mRNAs.

I started using VarsCan for SNP calling, but I notice that I was missing lots of low frequence SNPs. Now, I switch to LoFreq, which seems to do a better job in terms of calling SNPs.

Which protocol do you use for library preparation?

I am getting very low reads depth from infected potato tubers - due to low virus titer in that organ.

I am thinking about using specific probes for the viral genome instead of oligodt beads as a way of enriching for viral genome over the host genome. In average, 99% of the reads in my samples are from potato.

Do you or anyone else have experience with this?

Thanks a million.

[pld]

My situation is a little different, Nidoviruses (Coronaviruses and Arteriviruses specifically) have polyA genomes and make polyA subgenomic RNAs and our titers can get into the 10^6 range.

This might be something you could use: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262991/

You could combine both approaches, using oligodt to capture polyA mRNA, rRNA deplete and then use your genome specific probes. This really isn't my area though.

Answer 1

[pld]

I've used samtools for 454 data on coronaviruses, it mirrored what the Roche Newbler software called for the most part and what I'd seen in papers showing data for similar things. I also played around with FreeBayes.

You might be interested in this: http://bioinformatics.oxfordjournals.org/content/31/1/94.long

Also see this post: Snp Detection In Viruses: Does A Haploid Caller Like Freebayes Need To Be Used Instead Of Something Like Samtools/Bcftools?

Was this virus from infected cells, supernatant or a concentrated (e.g. sucrose cushioned) stock? Some (+)ssRNA viruses polyA their genome and their mRNAs, is this the case with you? I'd be interested in seeing how things turn out, I'm working on a similar project.