I am analyzing an RNA seq experiments using limma in which I have 4 different RNA species A,B,C,D and each has three replicates. I am using the follwing (assuming data_counts is my count matrix)
dge <- DGEList(counts=data_counts1,lib.size=libsizee) dge<-calcNormFactors(dge,lib.size=libsizee) v <- voom(data_counts1,lib.size=libsizee,design,plot=FALSE) fit<-lmFit(v,design) contrast.matrix1 <- makeContrasts(contrasts=A+B-C-D,levels=design) fit1 <-contrasts.fit(fitted_data_b,contrast.matrix1) fit1<-treat(fit1,lfc=2,robust=TRUE)
As you see in the above code I want to test whether the sum A+B-C-D is equal to zero or not. However, when I play with the lfc argument in the treat function I get different results. I do not know what the log fold change corresponds in my case?
Any help is appreciated