Ribosomal depletion for RNA seq

[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 (TruSeq RNA kit) without depletion 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.

[GenoMax]

For reference cross-posted: https://www.biostars.org/p/176059/

[nucacidhunter]

More than 90% of total RNA is rRNA, so without further fractionation you will be sequencing mostly rRNA. Two options:

1- mRNA-Seq on polyA selected RNA that does not require depletion. This option also requires less sequencing.

2- Total RNA-Seq that requires Ribo-Zero or other rRNA depletion reagent and library includes both polyA and non-polyA RNA (total RNA without rRNA). This option cost more because of depletion reagents and also more sequencing requirement.

In both method up to 3% of reads can be from rRNA.

[blancha]

I don't understand your post.
You either do ribo-depletion or poly-A enrichment, not both.
You have to pick one or the other, or you will be sequencing mainly rRNA, which is not very useful.

[LOH]

You choose either rRNA depletion (using RNase H) or poly-A enrichment (using poly-A beads) or rRNA exclusion (using specific primers).

We are professional at all three RNA Sequencing Service.