I have two groups with four biological replicates per group. I tried comparing group 1 to group 2 using cuffdiff. I got 544 significantly differentially expressed genes as my result. My lab mate ran the exact same differential expression analysis with the exact same settings and got ~1500 differentially expressed genes. The only difference between our cuffdiff runs was the order in which the bam files were selected under the "Condition" fields. To confirm that the order was what is causing the difference in our results. My lab mate ran cuffdiff using the exact same order of bam files as me, and got 544 significantly differentially expressed genes. We compared log2 fold change, and found these values to be virtually identical when the order is identical, but not when the order is not identical, even in overlapping genes between the two different results.
My question is why does the order of bam file (which should be biological replicates) cause such a big difference in the number of differentially expressed genes? Is there something we can do to minimize these differences?
Here is a screenshot of what I mean by "bam file order" : http://imgur.com/a/jG8Z3 So basically the order in that picture is JD10, S14, JD11, S15 for the first condition, and JD8, S10, S9, JD9 for the second condition. If I switch it up to S14, JD10, JD11, S15 and S10, JD8, S9 JD9 for example, I get completely different results.
