**70**wrote:

Hi,

I am running some RNASeq analysis with two experimental factors, namely time (two-level factor) and genotype (three-level factor). In cases like this, I like using edgeR cause, among other reasons, it makeContrasts feature is very handy. Thus, I would create a design factor combining all levels of time and genotype. This would allow me to easily look, for instance, for interactions between Genotype 2 and 1 over time by doing. Like this:

design.factor <- rep (c("Gtype1.Time1","Gtype1.Time2", "Gtype2.Time3","Gtype2.Time2", "Gtype3.Time1","Gtype3.Time2"), each=4) design.1 <- model.matrix(~0+design.factor) colnames(design.1)=sub("design.factor","",colnames(design.1)) my.contrasts=makeContrasts( Inter = (Gtype2.Time2 – Gtype2.Time1) – (Gtype1.Time2 - Gtype1.Time1), Levels=design.1)

However, in this particular experiment there is a strong replicate effect. Biological replicates where carried out over time in four independent experiments. I would like to correct this effect, but this is where things get a little bit complicated when I run the code here below:

reps <- rep(c("R1","R2","R3","R4"),6) design.factor <- rep (c("Gtype1.Time1","Gtype1.Time2", "Gtype2.Time3","Gtype2.Time2", "Gtype3.Time1","Gtype3.Time2"), each=4) design.2 <- model.matrix(~0+reps+design.factor) colnames(design.2)=sub("reps|design.factor","",colnames(design.2)) colnames(design.1) [1] “R1” “R2” “R3” “R4” “ colnames(design.1) [1] "R1” "R2" "R3" "R4" "Gtype1.Time2" "Gty pe2.Time2" "Gtype2.Time3" "Gtype3.Time1" "Gtype3.Time2"

As you cans see, my “Gtype1.Time1” level has disappeared due, I guess, to the way the degrees of freedom have been handled. Then my questions are:

1-Does all that mean that, for instance, the "Gtype1.Time2" coefficient will be in fact the difference "Gtype1.Time2" - "Gtype1.Time1".

2-Can I run interaction-type contrasts as described above? An dif this is the case, how?

3-Should I try tools such as SVASeq instead?

I thank you in advance for any help on this.

Best,

David

**12k**• written 2.1 years ago by David Rengel •

**70**