Dear community,

I'd need some help in the analysis of my RNAseq data using DEseq2.

I'm trying to characterize the effect of the over expression of three transcription factors (x,y,z) on the transcriptome a cell line.

I have the following groups and each contains 5 replicates.

1) Ctrl

2) x

3) y

4) z

5) x,y,z

As far as I understood I can set up my analysis as a "single factor" with "multiple levels" (as in explained in the manual 3.2 paragraph).

So, if I understood correctly, in order to obtain the transcriptional changes induced by each transcription factor I would need to run the following code:

condition<-c("x","x","x","x","x","y","y","y","y","y","z","z","z","z","z","xyz","xyz","xyz","xyz","xyz","ctrl","ctrl","ctrl","ctrl","ctrl") #declare my conditions
ddsHTSeq <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable, directory = directory,design= ~ condition) #building the dds object
ddsHTSeqfilt <- ddsHTSeq[ rowSums(counts(ddsHTSeq)) > 1, ] #filtering 0 values
ddsHTSeqfilt$condition <- relevel(ddsHTSeqfilt$condition, ref="ctrl") # specifying to use ctrl as reference
library("BiocParallel")
register(SnowParam(2))
dds <- DESeq(ddsHTSeqfilt)
DEx<-results(dds, contrast=c("condition","ctrl","x")) #extracting the differentially expressed genes between ctrl and "x"

Can you please confirm that what I'm obtaining in this way are the differentially expressed genes between "x" and "ctrl" groups?

Thanks in advance for your help.

Best,

Sebastiano