Dear Michael,

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!

-Dustin

DESeq2 calculates a single dispersion value for each gene. This means that if one group has a higher dispersion value than the other, the gene-wise estimate will be somewhere in the middle. Remember, then that in the last step, information is shared across all genes to moderate dispersion estimates toward the trend for genes with similar mean (see DESeq2 paper).

I would guess that having the dispersion value in the middle is not a big issue for sensitivity and specificity. There may be a small gain for modeling each condition with its own dispersion, but the big gain in performance for DE methods comes from sharing information about dispersion across genes.

Thanks so much for confirming this, and for responding so quickly! I will do comparisons between the different methods you suggested to see what I am working with.