Hello, 

When using DESeq2, does it matter the order of the groups of samples we feed to this package?

From some preliminary test runs, if I test Group1 against Group2, and then Group2 against Group1, I can see that the base means are the same for all genes, but the log2FoldChange has its sign inverted.  This behavior makes sense to me, but I would like to know the mathematical/statistical rationale for this outcome using this package.

Thanks,
Marcelo

 

hi Marcelo,

This is something particular to the DESeq2 Galaxy wrapper. It is choosing which of the groups should be in the denominator of the log fold change based on the order you provided the files.

If you run DESeq2 in R, and do not specify which should be the "reference level" explicitly, it is chosen alphabetically. 

From the DESeq2 vignette:

"Note on factor levels

By default, R will choose a reference level for factors based on alphabetical order. Then, if you never tell the DESeq2 functions which level you want to compare against (e.g. which level represents the control group), the comparisons will be based on the alphabetical order of the levels. There are two solutions: you can either explicitly tell results which comparison to make using the contrast argument (this will be shown later), or you can explicitly set the factors levels. "