Dear Davide, thanks for the quick answer, I will try and elaborate the experimental design.
||number of individual / nbr of technical replicates
|D. fuchsii (diploid)
||4, 1 ind with tech rep
|D. incarnata (diploid)
||5, 0 ind with tech rep
|D. majalis (polyploid)
||8, 4 ind with tech rep
|D. traunsteineri (polyploid)
||11, 6 ind with tech rep
||28, 11 ind with tech rep
I am doing two-by-two comparaison between these species. I observe a clear batch effect between the polyploid species
I believe (at least for the polyploid comparaisons) that the unwanted batch effect (W unwanted factor) and the biological variance of interest (difference between species) are not colinear. I have samples of both species in both batches.
When I compare the polyploid to the diploid on the other hand, none of the diploid samples are in either of the batches for which I observe a batch effect in the polyploids. I still correct, using RUVs, the batch effect in the polyploid. After finding DE genes, I get a strange MA plot
I hope the "design" is a bit clearer.
I am interested in differences between species (which happen to have different polyploidy levels). Could RUV be affected by these levels of polyploidy (e.g. if I look at DE genes overlaps between the comparaison diplo.vs.poly and poly.vs.poly, are these groups comparable after normalisation?)? Should I look into removing different numbers of factors of unwanted variance (k) when doing diplo.vs.poly, poly.vs.poly or diplo.vs.diplo comparaisons? I am not sure...
Thanks again for your help, cheers