I am analyzing data from RNA-seq. I have six groups: group1 (batch1), group2 (batch 1 + batch2), group3 (batch2), group4 (batch1), group5 (batch2) and group6 (batch1+batch2). My main interest is in detecting differential expression genes between groups using limma R package. Unfortunately, my batch effect is confounded with the biological groups.
I have a strong batch effect in my data. I have tried to eliminate it using comBat. However, when I plot batch corrected data using PCA/heatmap the set effect is still there although it has been reduced.
Do you have any ideas how to diluted more the batch effect? I am detecting differential expressed genes but I think that most of them can be just because of the batch effect.
Thanks a lot for your help in advance!