# about two group comparison of three group data in DESeq2 package

When we have three groups samples (A,B,C) with 25000 genes and the main interest is A vs B, should we limit samples only A and B for normalization to perform DEG analysis? Or better to include all samples (A,B, and C) for normalization and specify the comparison group within three groups?

res <- results(dds, contrast=c("tissu_type","A","B"))

• No gold standard for this. If you probably are going to need C at some point then include it. If not then leave it out. If C is very different from A and B it might negatively influence normalization and dispersion estimation. Depends on your analysis goal. I would probably include it because if you ever need C you have to repeat the analysis and results from A/B would change. Jun 14 '20 at 9:39
• I guess it makes more sense to include A and B. Imagine you are writing a paper, and it simply does not make sense if you include C, which you never talk about in the paper. But there is no absolutely correct answer for this. I also believe that the results are going to be very similar. If the results using two methods are so different, then there might be something wrong. Jun 14 '20 at 19:31
• Thank you so much for your all comments! Jun 14 '20 at 22:17