After substantial performance testing, we have determined that the false discovery rate of Jensen-Shannon-based tests (differential splicing, CDS switching, and promoter switching) can be unnacceptably high when used with fewer than three replicates in the conditions being compared. Cuffdiff now refrains from performing significance tests when one of the conditions involved has fewer than three replicates. You can change this behavior with the new --min-reps-for-js-test option. Cuffdiff still produces splicing.diff, cds.diff, andpromoters.diff regardless of how many replicates you have. These files will include the JS distance scores, but none of the genes will be marked significant if you have fewer than the required number of replicates.