Hello everyone,
In the past few days I have been analyzing my RNAseq data using the Tuxedo suit (Tophat-Cuffdiff). This is my first time using this tools, Im new in this RNAseq field. In my experiments I am comparing the expreesion of a bacteria (B) in 6 time points. My question is regarding to the significance results (yes or no statment in the last column). In some of my genes, virB3, vir B7 and Vir D3 the significance result is NO (comparing D0 and D1), however, when I dig into the FPKM values for the triplicates of this genes, it doesn't seem to be such a big difference between the three values (VirB3-Day0=533.684, 660.256 and 361.781) and (VirB3-Day1=35935.1, 52565.8, 37286.3) but yes, there is a big difference between the raw values and average values for Day 0 compared to Day 1 (The values won't overlap to each other, I think the values are significantly different between both days). For the rest of the vir genes (22 in total), the calculations appear to be as expected. Only this 3 genes doesn't seem to give me concordant results. Is there a way to recalculate this values and check if there is effectively significant difference between them.
I appreciate in advance any help or explanation over this matter,
Evelyn Zuniga
Good, don't get too attached, because you should know that the old 'Tuxedo' pipeline of Tophat and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. (If you can't get access to that publication, let me know and I'll -cough- help you.) There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using kallisto or salmon.
Thanks for your response. I got the paper -cough, cough-, I will give it a good read and try to run it. If I succed, I will compare with my previous results. Great help, Evelyn
I wonder if there is any paper where I could find how this calculations are done?? Im still curious about the fact that it did not work only for those three genes...