Hi all,

I have two different RNA-seq data from mouse; 1) paired-end read (40 bp) data of neonatal cardiac myocytes were dissociated from whole mouse hearts at 0, 4 and 7 days of birth with two replicates and 2) paired-end read (100 bp) data from the ventricular myocardium of mouse treated with factor X at 1th and 7 th days after birth with corresponding control with two replicates. I would like to get the differentially expressed genes between group 1 and group 2. Here is the MDS plot showing two distinct groups 1 and 2 at the right and left end of the graph:

Considering such a batch effect, could you please kindly tell me how I can include this batch effect in edgeR analysis?

Thank you in advance

If I understand your experiment correctly, you want to compare neonatal cardiac myocytes (vp) and ventricular myocardium (vd), but all the vp samples were sequenced to 40bp whereas all the vd samples were sequenced in a different batch at 100bp.

The design of this experiment has fundamental flaw in that the batches are entirely confounded with vp vs vd. That means it is impossible to remove the batch effect in edgeR.

You can still compare different times (vp7 vs vp0) or test for a treatment effect (vdr7 vs vds7), and you can interact such comparisons between vp and vd (for example vp7vsvp0 - vd7vsvd0), but you cannot make any direct comparisons of vp with vd, because you cannot separate the biological difference from the batch effect.