Hi all,

I've been struggling with my 2-way ANOVA analysis. I have 4 genotypes, lets name them A, B, C and D, and 3 time points: 0, 1 and 3. I need to test for time specific and genotype specific responses in my RNAseq data. I am quite new to bioinformatic field. How do I specify groups, do I use normalized counts or row counts? And how to actually do 2-way ANOVA analysis? All I found so far, was one-way...

Any help will be appreciated.

Martyna

A 2-factor analysis is really just the same as a one-way analysis. The easiest way is to make a variable with 12 levels (A.t0, A.t1, A.t3, B.t0, B.t1 ...) and then make contrasts between the levels in the usual edgeR way. For example, to compare C to D at time 3, form the contrast D.t3 - C.73).

There is no need to change the matrix of counts in any way -- edgeR will do the computations for you.

The edgeR workflow provides an example of 2-factor analysis (cell type x pregnancy status):

https://www.bioconductor.org/packages/RnaSeqGeneEdgeRQL

(Click on the "html" link).