>> I have 3 groups of accepted hits, each group has 3 replicates. When I add 3 
>> groups with corresponding replicates, the cuffdiff output only gave 2 groups 
>> comparison of differential expression, Please help me here


Cuffdiff does only pairwise differential expression testing, so each DE test 
will consider two groups, not three or more. However, Cuffdiff does iterate 
through and provide data for all possible group pairings. E.g.

A vs B
A vs C
B vs C


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