Question: rRNA removal needed for salmon / DESeq2 analysis
0
gravatar for mark.dunning
5 weeks ago by
mark.dunning200
The University of Sheffield
mark.dunning200 wrote:

Hi all,

I am re-analysing some RNA-seq data that I'm told have high levels of rRNA contamination (not sure how high yet). My usual workflow is salmon -> tximport -> DESeq2

I've seen recommendations that any reads matching rRNA need to removed prior to analysis. Is this still the case with the salmon (alignment-free) protocol? if the rRNA are not in my gtf file, then they will not be quantified and appear in the analysis. Obviously I'll get smaller counts than expected, but should be Ok to proceed with the counts for all the other genes?

Thanks,

ADD COMMENTlink modified 4 weeks ago • written 5 weeks ago by mark.dunning200
2
gravatar for Devon Ryan
5 weeks ago by
Devon Ryan86k
Freiburg, Germany
Devon Ryan86k wrote:

Unless rRNA is present in your transcriptome fasta file there's no reason to remove it (likewise, there's no reason to remove it in other cases unless it's actually annotated). What you likely should do is quantify how variable rRNA levels are. Sometimes rRNA level ends up being a nice surrogate for other issues (e.g., RNA degradation) with a given sample and you might need to use it for reliably excluding samples.

ADD COMMENTlink written 5 weeks ago by Devon Ryan86k

Thanks! That seems like sensible advice.

ADD REPLYlink written 4 weeks ago by mark.dunning200

Are there any papers that study these issues (rRNA level as surrogate for RNA degradation, etc.)?

ADD REPLYlink written 4 weeks ago by grp200920

Probably, search pubmed for them.

ADD REPLYlink written 4 weeks ago by Devon Ryan86k
0
gravatar for mark.dunning
4 weeks ago by
mark.dunning200
The University of Sheffield
mark.dunning200 wrote:

So the transcript file I was planning to use from Gencode does include rRNA sequences that I will have to remove before calling salmon

ADD COMMENTlink written 4 weeks ago by mark.dunning200
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