Uneven PCR in RNA-seq Preparation
Is it normal practice to prepare mRNA-seq libraries with uneven PCR to compensate for low RNA input ? and if so how is it treated in analysis and communicated in article ?
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biostar.anon •
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Can you clarify where this question is originating from? Generally kit manufacturers will have very specific instructions on input requirements/number of PCR cycles one can use with low RNA inputs (an example here).
Yes while it is true that there's a protocole, I am currently in contact with a sequencing facility which states their standard method for if a DNA converted mRNA has too little molarity/ [] in QC is to amplify 5 supplementary cycle. Has anyone heard any similar protocol ?
As long as they are treating all of your samples identically it may be ok. You will need to be mindful of PCR duplicates though they would be hard to completely eliminate if you don't have UMI's.
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