Forum:ribosomal depletion for RNA seq

Question

[montenegro]

We are currently embarking on an RNAseq project using a sequencing service provider but have a few questions concerning the ribosomal RNA depletion step (Ribo Zero) that would markedly add to the cost of the project.

Our initial quotation without depletion (TruSeq RNA kit) is quite competitive. Is it necessary to carry out the depletion step if the RNA is to be further fractionated via a polyA step? Does anyone have an idea how much ribosomal RNA would carry over past the polyA isolation? Could this ribosomal RNA be depleted via bioinformatics? We realize that its presence would reduce the proportion of sequences of interest but are unsure as to how much it would do so relative to the added cost.

We would appreciate anyone's thoughts on this and thank-you in advance.

Answer 1

[Devon Ryan]

There's no need to do ribo depletion if you're already doing polyA selection. The levels of rRNA remaining after polyA selection are vastly lower than what the rRNA kits produce. The only question is whether you need to look at non-coding RNA. If you just want the normal protein coding genes then absolutely do not bother with rRNA depletion, it'd be a complete waste of money.

For what it's worth, I routinely control the RNAseq datasets we produce (we have multiple machines) and commonly see rRNA levels in the low double digits after rRNA depletion. rRNA in polyA-selected samples is typically <<1 %.

[montenegro]

Thank you very much for your explanation. I assume that the low percentage of rRNA that is still left can be removed by bioinformatics. What software would you recommend for this? I will appreciate your advice

[Devon Ryan]

There's no need to remove it, it's too low to affect anything :)