I am hoping to use DESeq2 to analyze the sequencing results of a Multiply Parallel Reporter Assay that I performed. I was wondering if you might be able to help me answer a question that's been stumping me?
In this assay, we transfect cells with a diverse library of plasmids and then perform RNA-seq to assess the expression of each plasmid. My ultimate goal is to determine if a given sequence takes up a greater (or lesser) fraction in the RNA-seq library than it takes up in the plasmid library (and thus was differentially expressed).
I performed 6 biological replicates of RNA-seq (independent transfections), and 6 replicates of sequencing library preparation from the plasmid library that was used for transfection. The issue I'm running into now is that the dispersion in the RNA-seq replicates will be much higher than the dispersion in the plasmid library prep replicates. I know that DESeq originally calculated dispersion for each condition separately. My question is:
Does DESeq2 still calculate dispersion for each condition separately, such that the low dispersion of my plasmid reps will not artificially lower the overall dispersion?
I would greatly appreciate any help you might be able to provide. Thanks a bunch!