Dear all: I have a couple of questions about the ratios calculated by Cuffdiff, in particular those at the isoform level. I notice that a number of isoforms have very high fold change due to the fact that either "q1" or "q2" has been assigned a zero value. The ratio is therefore an extremely high number, which is also given in as the "test_stat" value. Some but not all of these fold changes are considered significant changes by CuffDiff, and when I inspect the ratios with CummeRbund, I see how a given isoform was not expressed at all (or to extremely low levels) in one sample, compare to the other. Can anybody comment on this?
A second related question: What is the standard FPKM cutoff when considering low abundant transcripts? Should one trust the fact that Cufflinks/CuffDiff, assigned a confidence value to a transcript, even if its read intensity is < 1 FPKM as an example? An example is: I have a gene composed by 3 isoforms. Two of these isoforms have a 1:1 ratio. A third one is overexpressed, with an extreme value, with one sample having no reads, like I mention above. This third isoform is a lot less abundant than the other 2, having an intensity lower than 5 FPKM. Cuffdiff tells me, this change is significant? should I consider it as such?
Many thanks in advance. G