Hello,

I'm analyzing a time series RNA-Seq data with repeated measures on six different time points corresponding to pre-treatment, on-treatment and post-treatment phases, using edgeR:

Timepoint1          Pretreat

Timepoint2          Pretreat

Timepoint3          Pretreat

Timepoint4          Ontreat

Timepoint5          Ontreat

Timepoint6          Posttreat

And my hypothesis test is to look for DEGs comparing Ontreat VS Pretreat, and Posttreat VS Ontreat.

There are two options I can think of to do this:

1). Include the timepoint variable into the glm model (include subject as well since it's repeated measure data) and setting the contrast as:

design<-model.matrix(~0+timepoint+subject)

mycontrast<-makeContrasts(OnvsPre=(timepoint4+timepoint5)/2-(timepoint1+timepoint2+timepoint3)/3, PostvsOn=timepoint6-(timepoint4+timepoint5)/2, levels=design)

2). Include the treatment phase variable into the model (which essentially combines different timepoints within the same treatment into one group):

design<-model.matrix(~0+treatment+subject)
mycontrast<-makeContrasts(OnvsPre=Ontreat-Pretreat,PostvsOn=Posttreat-OnTreat,levels=design)

Since I am really new to RNA-Seq analysis and ignorant in statistics, my questions are:

1). For the first method, am I setting the contrast in the right way?

2). For the second method, is it justified to combine different timepoints into one group, or will it fall into the issue of repeated measures?

Thanks very much for any help here!

Use the first model. If there are differences between time points in the same treatment category, this will lead to inflation of the dispersion estimates when you try to treat those time points as "replicates" in the second model.

The contrasts for the first model look fine to me.