Hello all,

I have a quick question regarding the makeContrasts function of the limma package, for which I couldn't find a clear answer online. I am running a differential expression pipeline and I want to explore the effect of 3 mutations, lets call them: mutA, mutB, mutC. I built a design matrix by putting the mutations in a factor variable along with the control. Now the variable has 4 levels (mutA, mutB, mutC, control) and I have set the "control" level as the first level.

How do I make a contrast that detects DE elements that are found in all mutated samples when compared to the control?

Basically I want to make a contrast like this: "(mutA + mutB + mutC)/3 - control"

but "control" doesn't appear as a coefficient in the design matrix since it is the reference level.

Is this:

design <- model.matrix( ~ factor with mutations as levels + some confounders)

con <- makeContrasts("(mutA + mutB + mutC)/3", levels=design)

the correct way to make the contrast I am looking for? Or is this doing something else?

Thanks in advance!

Stefan

Yes, that's the way to make the contrast. Since you set "control" as the first level, all the mut coefficients measure fold-changes vs control: mutA-control, mutB-control etc.