I'm attempting to use EdgeR to analyze a two or three factor (Virus [virus | no virus]], Treatment [treatment | no treatment], and Family [3 family groups]) RNAseq experiment. I'm not interested in Family except to account for any variation it introduces, perhaps in a separate model (block design?). Here is a breakdown of the targets:
Although I've gathered some vital clues in the excellent EdgeR tutorial, I cannot quite wrap my head around how exactly to go about answering the following two questions:
1) what genes are differentially expressed differently between the three TreatGoups (Virus.Treat, Virus.Control, and Control.Treat) while accounting for the control (Control.Control)? In the tutorial is seemed a nested design was the best bet, but I'm having trouble matching this question to the right contrast(s), as there are subtle differences between my experiment and the examples given (e.g. I'm interested mostly in how the virus.treatment combo group is different from the other two, but I'd like to get the other two groups perspective on different genes as well).
2) what genes are differentially expressed differently in a synergistic fashion between treatgroup 'Virus.Treat' and the other two treatgroups (Virus.Control and Control.Treat), all relative to Control.Control? I realize that the answer to this question will partly overlap with question 1, but I'm thinking they are not necessarily identical. A synergistic response to the interaction of virus and treatment (imagine it has strong side-effects) incudes the "very different" gene responses from question 1, but also can include genes responding much more strongly (but in the same direction) than a simple additive effect would account for. I imagine an interaction coeffeciant would be needed in the full model to make the proper contrast for this, but I'm not sure how to go about it.
3) if there is a strong family effect (large variation due to family), can the block design described in the tutorial be used to account for this variation while still answering the above two questions? I've already run an MDS plot and run the correlation used in example 4.2 and they seem to indicate some pretty strong variation.
Thanks in advance for any help offered!