Discovery of RNA viruses from metagenome and metatranscriptome datasets? a good protocol?

Guillermo Domínguez Huerta @Guillermo-Dominguez-Huerta

23 July 2018 1 4K Report

Hi all,

this one is a (I guess) tricky question...

RNA virus discovery from metagenome/metatranscriptome dataset (overall from environmental samples) is particularly difficult because of their VERY DIVERGENT genome sequences, with poor relationship with what is available in reference sequence databases.

Can you recommend a "typical" protocol for this?

I found 2 "versions" by now:

**#FIRST PROTOCOL#**

- Assemble reads with Trinity or metaSPAdes.

- Do tBLASTn with the generated contigs/scaffolds against a database made of RNA virus proteins (ssRNA and dsRNA viruses). Use an e-value cutoff of

Darren J Obbard

I would start by translating the RNA to obtain putative protein sequences - just concatenate all of the translations to make a protein pseudo-sequence that captures the (potential) protein content of your RNA. This will be faster to search with, and is usually more sensitive than blastx or tblastn.

I would strongly recommend using the virus proteins from nr rather than refseq, as refseq only has (near)complete genomes, and divergent (especially) segmented viruses will gain a lot by using all rather than refseq proteins. In my experience this will find viruses with short regions of ~30%+ identitiy to known viruses.

An HMM will be more sensitive - and is probably the best option - but for transcriptome-scale data might prove too slow. I would recommend diamond (method blastp) as being much much faster, and only a little less sensitive than blastp itself (default options).

Having identified possible viruses in this way, *then* blast (or diamond) against refseq (as DNA and protein) to exclude those that are much closer to cellular organisms than viruses, even if they hit viruses initially

Be aware that many DNA virus 'hits' will turn out to be transposable elements from the host. This reflects the presence of many closely-related transposable elements in very large DNA viruses that are not well sampled from their eukaryotic hosts

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