Question: rRNA removal needed for salmon / DESeq2 analysis
1
gravatar for mark.dunning
23 months ago by
mark.dunning210
The University of Sheffield
mark.dunning210 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,

contamination salmon deseq2 rrna • 1.1k views
ADD COMMENTlink modified 10 months ago by ATpoint39k • written 23 months ago by mark.dunning210

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 REPLYlink written 23 months ago by mark.dunning210
3
gravatar for Devon Ryan
23 months ago by
Devon Ryan96k
Freiburg, Germany
Devon Ryan96k 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 23 months ago by Devon Ryan96k

Thanks! That seems like sensible advice.

ADD REPLYlink written 23 months ago by mark.dunning210

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

ADD REPLYlink written 22 months ago by grp200940

Probably, search pubmed for them.

ADD REPLYlink written 22 months ago by Devon Ryan96k
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