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...


To make the comparisons you want, your sample data should include a column like cellline.treatment, with values like A.treat, A.untreat.

You make one dds object with cellline.treatment as the design with all the samples, and call results 4 times, each time specifying which cellline.treatment to compare to which.