Hi,

I'll preface my concern by saying that I'm a novice to Cufflinks. Back in September, I performed a Cuffdiff analysis comparing a wild-type and mutant condition. The analysis returned ~800 transcripts differentially regulated between the two with statistical significance. Recently, I've rerun the Cuffdiff analysis - using exactly the same files stored in Galaxy for all inputs, and with all the same parameters - and only get a few dozen statistically significant hits. However, all of the data besides the p and q values are essentially identical between these two runs, so I am really unclear as to what is causing the difference. Here is just one clear example:

From run 1:

YFR026C FPKM 1 = 17.2434 FPKM 2 = 196.735 log2(fold change) = 3.51214 p = 1.64E-8 q = 7.33E-6 significant = yes

From run 2:

YFR026C FPKM 1 = 14.4489 FPKM 2 = 144.939 log2(fold change) = 3.32641 p = 0.000170034 q = 0.0719964 significant = no

The second Cuffdiff analysis shows there is still a ~10-fold difference between conditions, but this is not statistically significant. Has the version of Cuffdiff on Galaxy been updated such that some parameters have changed, that could explain this difference? Or, is there some setting I am missing that would cause very large changes to fail statistical significance testing? Any help or input would be appreciated, I am really at a loss for why executing what should be exactly the same task is giving vastly different results. I could just be overlooking something very fundamental that is obvious to someone with more experience with this program. Thanks.

-Jenna Smith