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I ran DESeq2 with KO and WT samples(triplicated from each) and found high padj value for KO gene. The target gene KO was validated in many different ways i.e. gPCR, RT-qPCR, and even WB. The p-value for the KO gene is 0.003, but the p.adj value is > 0.05 (0.123). In this case, how can I set up the threshold? I wonder its really a reliable result or not.
Thank you for the answer, Michael. Do you think the significantly low KO gene count number can affect the weight during normalization? If so, can I expect better contrast if I remove the target gene's raw count number from both WT and KO samples before running DESeq? Many thank you in advance!
No you wouldn't remove the target gene ever.
What is the sequencing depth per sample (roughly) and what are the counts for this gene?
Thank you for the reply. Here is the raw count numbers. WT(971, 899, 713) vs KO(493, 655, 567). Actually, We expected complete zero count number in KO samples. Exon 2-4 region was targeted (bi-allelic) in KO cell line. Prob. the numbers from different isotype. I have no idea. If I remember correctly, it was 30x.