I have run my aligned samples through the Cufflinks app in Illumina BaseSpace a couple of different ways. When I do Classic FPKM Normalization + Pairwise comparisons, I get a small list of genes. This analysis seems stringent. When I do Classic FPKM Normalization + No Pairwise comparison, I get a very long list of genes significantly upregulated when I do t-tests on my own. But there is very little overlap between the two lists. If the Pairwise expression tests are more stringent than a t-test of FPKM values, most of the pairwise comparison genes should show up on the t-test list, right?
So my question is: How exactly is the Test statistic, p-value, and q-value calculated on the Pairwise comparisons in Cufflinks Differential Expression? I’m trying to re-create this statistical analysis on my raw FPKM value datasets but I have not been able to.