Hi, I was processing my RNAseq data, and after the STAR alignment, I proceeded with getting the counts with HTseq. Since I knew that my sequencing was stranded I said yes to that option and continued with my analysis, shortly after I noticed that I should have done it in reverse mode. My question is the following: after doing it wrong I still got some genes that were DEG. I attributed that to the 0.05 p-value cutoff since they were around 5% of the total. However, after performing it correctly using the reverse mode quite some of those genes appeared again as significant, even with a significant adj p-value. I'm trying to understand what happened, why those genes appeared beforehand in the wrongly made analysis, and if I can still trust them in the new one. Thanks a lot
Thanks a lot! I will