Bioinformatics Asked by marilu on January 2, 2021
I am trying to understand library normalization in DESeq2. I would like to ask the following: I know that some samples have been run 15 cycles and some others 20, can I give this information to DESeq2, would it be useful? I mean, I know that DESEQ2 uses an algorithm to normalize library size, but since I have this piece of information, I wonder if it would be useful to provide it somehow to DESEQ2. If it is not, would it make sense to incorporate this information in the design matrix, something like: design(~cycle+condition)? Thank you a lot!
You can include that info in the design, though you'd have to decide whether to include it as a number of just a factor. I've never heard of anyone including PCR cycles. I've heard of people including RIN scores, though I've never done it.
If the PCA looks alright, I wouldn't worry about. I've got tons of examples of the lab people saying "I did a few more cycles on these few samples" and then you can't tell any difference in the PCA. I think needing to do more cycles likely has more to do with variance in the prep than a real biological difference.
Answered by swbarnes2 on January 2, 2021
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