Hello, I'm performing a diifferential expression analysis on RNAseq data and I'd like to adjust my design to remove the effect of unwanted variation by RUVSeq package. The problem is that I have two important phenotypes in my data, let's say: Pheno_1, Pheno_2; Condition_A and Condition_B, so that my samples can be labelled (by aggregating the two variables) as: Pheno_1-Condition_A, Pheno_1-Condition_B, Pheno_2-Condition_A and Pheno_2-Condition_B.
Now, I'm wondering what is the best way to estimate the "latent variables". Should I aggregate the two variables and perform RUVg or RUVr methods for this aggregated variable with four levels or there is something else I can do?
Thank you very much in advance.