Question: What is the best software for SNP calling from RNA-seq (100 bp single-end) data of a +ssRNA virus population?
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gravatar for wld28
5.6 years ago by
wld280
United States
wld280 wrote:

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

virus snp rna-seq • 1.9k views
ADD COMMENTlink modified 3.7 years ago by Biostar ♦♦ 20 • written 5.6 years ago by wld280

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.

ADD REPLYlink written 5.6 years ago by wld280

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.

ADD REPLYlink written 5.6 years ago by pld4.8k
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gravatar for pld
5.6 years ago by
pld4.8k
United States
pld4.8k wrote:

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.

ADD COMMENTlink written 5.6 years ago by pld4.8k
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