Question: Is it possible to use a self made txt doc containing normalised FPKM values in cuffdiff - for the purpose of multiple testing (calculate FDR / q value)
I have used the Tuxedo pipeline to analyse my reads (RNASeq) for differential expression.
The design of my experiment is:
5 conditiona (3 gene variants in an overexpression plasmid, empty vector control, and untransfected) replicated twice in two donors. The cells are primary human cells.
After running cuffdiff I do not see many genes that meet FDR of 5% and very few that that are 10%. This may be due to donor (inter and intra) variation.
One suggestion made was to normalise the data to the untransfected/ media control which I have done in excel. I'd like to know if there is a programme that I can use to carry out multiple testing on this data/ or if I can use this file in cuffdiff (for FDR/ to generate q values) so that I can interpret my data with confidence.
An alternative pipeline (htseqcount_DESEQ) has been suggested which I am also following up. I've been advised that the model used in DESEQ may be better suited to my experimental design compared to Cuffdiff.
4.9 years ago by