Hello,

I have what might be a simple question but I would really like to get this right and despite all my readings it is still not clear to me what the best option is. 

I am using DESeq2 to analyse an RNAseq experiment. The design of the experiment is as follow (each sample is in triplicate):

 - cell line A - untrt
 - cell line A - trt
 - cell line B - untrt
 - cell line B - trt

The goal is to peform the following contrats:

 - cell line A - untrt **versus**  cell line B - untrt
 - cell line A - trt **versus**  cell line B - trt
 - cell line A - untrt **versus**  cell line A - trt
 - cell line B - untrt **versus**  cell line B - trt

My question is, in order to do things "correctly", should I create a separate DESeq object for each contrast using only the samples I will be using for each contrast, or should I have just a single object from which I do the contrasts that I need?

For your information, the difference between these cell lines is that "cell line A" is overexpressing gene X, while "cell line B" is overexpressing genes X and Y.

Any help would be very much appreciated...


"should I create a separate DESeq object for each contrast using only the samples"

Create one object. 

and then for comparison, 

do this - 
res, comparison1<-results(dds, contrast = c("A.untreated", "B.treated"))
 and so on. 

You can list the contrast names in the object by doing resultNames(object).
