Hi Galaxy Developers,

This is regarding the wrapper for cuffdiff and the –c parameter for “Min alignment count”.  We noted that Galaxy’s default value is set to 1000.  In our experience, setting this parameter that high yields drastically different results from the ones obtained with the author’s original default value of c = 10.

The –c parameter is defined as:

-c/--min-alignment-count <int> The minimum number of alignments in a locus for needed to conduct significance testing on changes in that locus observed between samples. If no testing is performed, changes in the locus are deemed not signficant, and the locus' observed changes don't contribute to correction for multiple testing. The default is 10 fragment alignments.

We have found that the value entered will require that at least this number of fragment alignments be found for *each* sample (or at least each condition) in order for the locus to be tested for differential expression. Although one could save themselves a lot of locus comparisons (and hence take a much smaller hit for multiple hypothesis testing) if he/she were to raise this threshold, this will screen out the most desirable differentially expressed genes – specifically those that are not expressed at all in one condition but highly expressed in the other condition.

The attached image has a real example of a gene that was counted as differentially expressed (as it should have been) with ‘10’ but was not even tested at ‘100’ nor ‘1000’.

-Kevin Silverstein