Hello,

I have two covariates in addition to condition, let's call them A and B. So I set the design as formula(~ A + B + condition) and I contrast on the level of condition.

Contrast can only distinguish between two factors (e.g WT v KO, single-read v paired-end). What if my covariate "B" has four factors (a,b,c,d).

So if I call

dds <- DESeqDataSetFromMatrix(countData = countdata, colData = colData, design = ~ A + B + condition)

Does DESeq and results take into account the four factors of B? Or is this overlooked?

If the case is the latter, how can I use all four?

Thanks

"Does DESeq and results take into account the four factors of B?"

Yes, DESeq2 takes into account all the levels of the factors in the design.

You can see that this is the case by looking at the coefficients produced, by calling resultsNames(dds).

That the results() function can be used to contrast pairs (or more) of the coefficients doesn't change this fact that all levels are used in the model.