Hi everyone,

I have met a problem with RNA-seq analysis.

My data: 4 experimental conditions with each of 3 biological replicates, total 12 samples.

When used cuffdiff, I got a p-value and q-value for every gene (the algorithm seems to be a beta-negative binomial distribution).

And then, I used R packages genefilter::rowFtest to calculate the welch t-test p-value of every gene based on their FPKM values.

In the end, every gene had two different p-values from cuffdiff and R::genefilter.

The numbers of differentially expressed genes from cuffdiff and genefilter are quite different.

My question is that:

Which one could I believe?

Is the welch t-test fit for calculation of RNA-seq differentially expressed genes?

When I deal with FPKM values from cufflinks for other analysis, just like for R:package::genefilter, should I perform a normalization for the matrix of the FPKM values?

Thank you very much

My Best.

Junfeng Shi

Thank you very much. I have learned a lot from your answer. I need to re-consider my methods currently used in my analysis. Thanks a lot.