I have 32 samples with the following conditions:
16 dry season (DS) forms: 4 stages (A, B, C, D) x 4 replicates 16 wet season (WS) forms: 4 stages (A, B, C, D) x 4 replicates
I have two questions: 1. How does 'seasonal form' affect gene expression 2. How does 'stage' affect gene expression.
For Q1, I simply use all 16 DS samples and all 16 WS samples to find DE genes between the two 'forms':
design = ~ Form
For Q2, I take pair-wise comparisons between stages. For example, first I examine DE genes between A and B, I use 8 A samples (4DS+4WS) and 8 B samples (4DS+4WS) and only consider 'stage'.
design = ~Stage
Then I repeat this for B vs C, and C vs D. Then I take the union of the DE genes from the three pair-wise comparisons as DE genes.
Is this a proper design?