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)
gravatar for sangita_b
4.9 years ago by
United Kingdom
sangita_b80 wrote:
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.
rna-seq • 1.1k views
ADD COMMENTlink written 4.9 years ago by sangita_b80

In order to do this with cuffdiff you'd probably have to make your own CXB files, which would require writing a program to do that. You're life will be easier with DESeq2 or edgeR or limma/voom. Having said that, whether even these will work depends a bit on exactly what sort of normalization you're trying to do. Providing some details there might allow better feedback.

ADD REPLYlink written 4.9 years ago by Devon Ryan94k

sorry how did you exclude the over expression effect from the plasmid?

ADD REPLYlink written 3.5 years ago by maria0
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