I have what may seem like as really simple questions, but being new to using R in general and very new to DEseq2 I just can not figure it out reading the manual (which is great and very informative for a biologist, thanks!)
I have a simple design of 3 donors (A, B and C) and 3 conditions (unit,eth and vitd).
My basic question is how can I do a contrast with a multifactorial design? (IU may be getting these terms mixed up and therein may lie the problem.
If I simply import the "eth" and "vitd" data and follow the manual for MF design I can run through and get great results (with 85 DE genes):
> dds <- DESeqDataSetFromMatrix(countData = cts, colData = coldata, design = ~ condition)
> dds <- dds[ rowSums(counts(dds)) > 1, ]
> ddsMF <- dds
> design(ddsMF) <- formula(~ donor + condition)
> ddsMF <- DESeq(ddsMF)
However when I try and do the same thing but also importing the "uns" sample (so now I have "uns", and the two previous "eth" + "vitd") and try and follow the contrast I get 75 DE genes. So same procedure as before but this time
> res.vitd.eth <- vitdresults(ddsMF, contrast=c("condition","vitd","eth"))
with the ddsMF run as before.
I'm sure I am doing something so basic wrongs here.
Thank you in advance,