I have 2 conditions each having 4 replicates. I ran tophat-cuffdiff twice - once using 2 groups of 4 replicates and once excluding one of the replicates for one of the conditions (i.e. one condition had only 3 replicates, the other had the same set of 4 replicates as before).
I was somewhat surprised to find out that the FPKM values differed substantially between these two analytic runs for BOTH conditions even though the data for one of the conditions was the same in both runs.
The only explanation I can think of is that cuffdiff estimates the FPKM values by pooling the data from both conditions. Does anyone know whether this is true?