I am working with edgeR to perform some differential expression work. EdgeR has been a dream to work with and I have quickly reached the point where I am trying to ask some more complicated questions.
My dataset consists of the following:
I have a human cell line, which is either infected or uninfected with a pathogen. I also have two different drug treatments (A and B for simplicity). Therefore I have 6 conditions (with triplicate reps). I have successfully made the simple comparisons like: what genes are changing when uninfected cells are treated with drug A, and what genes are changing when cells are infected with the pathogen?
My more complicated questions take the form:
What gene changes are induced when infected cells are treated with drug A vs when uninfected cells are treated with drug A?
Basically, the hypothesis is that the drugs affect the pathogens, causing them to act differently and we want to find the host gene changes caused by the changed pathogen behavior.
Here is what my samples/targets look like:
Pathogen Treatment Group No_Pathogen-No_drug-rep1 negative negative negative.negative No_Pathogen-No_drug-rep2 negative negative negative.negative No_Pathogen-No_drug-rep3 negative negative negative.negative No_Pathogen-Drug_A-rep1 negative Drug_A negative.Drug_A No_Pathogen-Drug_A-rep2 negative Drug_A negative.Drug_A No_Pathogen-Drug_A-rep3 negative Drug_A negative.Drug_A No_Pathogen-Drug_B-rep1 negative Drug_B negative.Drug_B No_Pathogen-Drug_B-rep2 negative Drug_B negative.Drug_B No_Pathogen-Drug_B-rep3 negative Drug_B negative.Drug_B Pathogen-No_drug-rep1 positive negative positive.negative Pathogen-No_drug-rep2 positive negative positive.negative Pathogen-No_drug-rep3 positive negative positive.negative Pathogen-Drug_A-rep1 positive Drug_A positive.Drug_A Pathogen-Drug_A-rep2 positive Drug_A positive.Drug_A Pathogen-Drug_A-rep3 positive Drug_A positive.Drug_A Pathogen-Drug_B-rep1 positive Drug_B positive.Drug_B Pathogen-Drug_B-rep2 positive Drug_B positive.Drug_B Pathogen-Drug_B-rep3 positive Drug_B positive.Drug_B
I am using the makeContrasts method to set up the comparisons. I am wondering specifically about something like this contrast:
Pathogen_induced_changes_with_drug_A = (positive.DrugA) - (negative.DrugA - negative.negative)
My intention with this contrast is to have the baseline be changes in pathogen negative cells when DrugA is added, and then compare that to changes in pathogen positive cells with DrugA to find the gene changes I mentioned above.
I am wondering if I shouldn't do this instead:
Pathogen_induced_changes_with_drug_A = (positive.DrugA - positive.negative) - (negative.DrugA - negative.negative)
To also remove changes which come from simply the presence of the pathogen.
I would really appreciate any tips / pointers on this.