I am trying to identify novel transcripts across two developmental stages. We'll call them dev1 and dev2. I have the RNA-seq for dev1 and dev2 which I performed the following:
Align via TopHat
Assemble via Cufflinks
Merged the two sets of transcripts via Cuffcompare using refFlat as a reference.
Determine differential expression via CuffDiff from CuffCompare (gtf) with dev1 and dev2.
Join my CuffDiff file with my CuffCompare transcript tracking file to be able to identify transcripts by their CufflinksID.
This has lead to a few questions:
A. Looking at the transcript differential expression testing on CuffDiff, an FPKM is given to a particular transcript in for both dev1 and dev2, even when the transcript does not appear in assembled transcripts for dev2.
B. The FPKM for Cufflinks and Cuffdiff are different. I've seen other people with this question. But still, what's up with that?
C. Essentially the opposite of question A where, looking at my CuffCompare transcript tracking file, I'll have an identified transcript TCONS_xxx but it won't have any values in my cuffdiff file.
Any insight would be greatly appreciated, especially for question A which I have a sneaking suspicion would give insight to the rest of the questions.