Hi, I am analysing RNASeq data from an experiment with complex design.

The design is ~ drug + isolate

each has 3 values: isolate1, isolate2, isolate3; control, drug1, drug2

The experiment has been 3X3 nested design, namely RNA Seq dataset coming from 3 independent cell isolates (isolate1, isolate2, isolate3), each given 3 different treatments (control, drug1, drug2)

I am trying the following plots in Glimma

GlimmaMA(dds, groups = dds$drug)

However, due to the complex design, we have several different contrast types, which is represented in the results

res <-results(dds, contrast = c("drug","control","drug1"))

However, due to the fact that GlimmaMA can only act on dds rather than res, it is impossible to define which contrast we are looking for. I found out that the contrast is set to be the default one which is comparing isolate1 vs isolate2.

Could you please help with

1. How I can define which contrast to use in the GlimmaMA
2. How to visualise the un-used parameter (not subjected to "groups") on the expression plot? i.e, when I select groups=dds$drug, on the expression plot it only shows which drug corresponds to which sample, but I cannot visualise which isolates they are from.

Thank you very much!