I have RNA-Seq, time series data from six conditions and five time points (i.e. 1h, 2h, 12h,24h, 48h). The conditions are the following:

WT (as Control)

Mut1

Mut2

Mut3

Mut1+2

Mut1+3

I want to run a differential analysis per time point using limma, using the WT as a control in each respective time point.

My design matrix is specified by

design <- model.matrix(~ 0 + fac)

where fac is a factor combining Mutant_Timepoint (Mut1_1h, Mut1_2h, ... , Mut1+3_24h, Mut1+3_48h)

Would there be any difference between using limma/voom with time series and the Time course section in limma's users guide? If yes, which one would be the most appropriate?

Thank you in advance for the help.

You are free to make whichever comparisons are most of interest to your scientific study. Obviously, comparing conditions for each respective time will give different results than you would get by comparing time points for each condition, but it depends on what is of interest to you. You could even do both.