I am new to the RNA-seq analysis. I am trying to analyze DE genes using DESeq. I have three conditions and each with triplicates.
What would be the best way to find pair wise comparison of the DE genes.
The way i go about it is make a data matrix with all the replicates and samples (3x3) and specify the groups for DEseq. Following which i perform pairwise contrast. However, when my data replicate columns are not organized I get different result compared to when the replicate are placed in columns next to each other.
This i believe is strange as the order of the columns/replicates should not matter as the column head would define the replicates.
Also, would it be better to perform DESEQ separately for the pairwise comparisons rather than all at once.
Thanks in advance for the help.