RNASeq differential expression masked by pathways disregulation

RNASeq differential expression masked by pathways disregulation

1

I am working on a 20 sample dataset. I need to isentify De genes. 10 samples were collected in the collection center (CC) 1 and 10 samples in the CC2. Each group has 5 samples with both condition1 and condition2. Unfortunately, when I tested DE CC1 vs CC2 only, there are several survival pathways upregulated in CC2 samples. This is due to the fact that CC2 send us samples later respect to CC1 (in terms of days). When I calculated DE among condition1 vs condition2 (using CC as covariate) I cannot observe DE genes (FDR 0.05). From PCA I see that differences in CC are far stronger than condition1 vs 2. To my knowledge, both Combat and remoBatch (limma) destroy biologic variability so I am not confident of using them.
My question is: what to do in this setup?


normalization


Rnaseq


batch

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