I am used to analyse RNA-seq data with the very useful and well-documented DESeq2 package. I have analysed an RNA-seq dataset containing 2 conditions (control and transgenic mice) with 3 replicates for the control condition and only 2 replicates for the transgenic one (we initially sequenced 3 transgenic samples but the quality of one of the sample was not sufficient and we therefore have to exclude this sample from the analysis). We submitted a manuscript containing these analyses but one of the reviewer wrote that “the RNA-seq performed is a 2 against 3 experiment and therefore the statistical analysis applied is not valid“.
As in the DESeq2 Genome Biology article: « experimental design with as little as two or three replicates are common and reasonable » I think this is valid to use DESeq2 with this number of replicates. Moreover as the pasilla dataset used in the DESeq2 vignette contains different number of replicates for each condition I also assume that this is valid to use DESeq2 on an unbalanced experimental design.
I am aware that the power of the analysis would have been better with more replicates per condition and a balanced experimental design, but I just want to have a confirmation that applying DESeq2 on such an experimental design is valid.
Thank you in advance for your answer.