I am analyzing some RNA-Seq data involving three genotypes and 5 time points.
I am particularly interested in those genes for whom time does not present the same effect in all genotypes, that is, genes with a significant interaction genotype:time.
Please find here below a small code summarizing what I have attempted (for info, genotypes are "Col", "KO" and "TAP")
I find the results rather puzzling cause:
1-Only a few genes are significant for the interaction Col:KO or Col:TAP
2-IF I have a closer look at certain genes, I cannot see (a) why either the interaction effect has not been called as significant (e.g. the upper gene in the attached file) or (b) why it is significant in Col (blue line) vs KO (red), but not vs TAP (green) on the bottom gene.
I would be very grateful if you could provide me with any feedback on this matter.
dge <- DGEList(counts=counts.F, group=design.factor)
dge <- calcNormFactors(dge)
dge <- estimateGLMCommonDisp(dge,design,verbose=TRUE)
dge <- estimateGLMTrendedDisp(dge,design)
dge <- estimateGLMTagwiseDisp(dge,design)
fit <- glmFit (dge,design)
names(my.contrasts) <- c("KO","TAP","t","it.KO","it.TAP")
my.contrasts[] <- 2 # KO genotype vs Col genotype at t=0
my.contrasts[] <- 3 # TAP genotype vs Col genotype at t=0
my.contrasts[] <- c(4:7) # Time effect, at any time point, on Col genotype
my.contrasts[] <- c(8,10,12,14) # The interaction effect of time in KO gtype, as compared to time in Col genotype.
my.contrasts[] <- c(9,11,13,15) # The interaction effect of time in TAP gtype, as compared to time in Col genotype.
for (i in 1:length(my.contrasts))
lrt[[i]] <- glmLRT(fit, coef=my.contrasts[[i]])