The analysis of differentially expressed genes (DEGs) is performed with the glm method of the edgeR package (Robinson et al., 2010). The sample comparisons used by this analysis are defined in the header lines of the targets.txt file starting with <CMP>.

Run edgeR

countDF <- read.delim("results/countDFeByg.xls", row.names=1, check.names=FALSE) 
targets <- read.delim("targets.txt", comment="#")
cmp <- readComp(file="targets.txt", format="matrix", delim="-")
edgeDF <- run_edgeR(countDF=countDF, targets=targets, cmp=cmp[[1]], independent=FALSE, mdsplot="")

Add gene descriptions

m <- useMart("plants_mart", dataset="athaliana_eg_gene", host="")
desc <- getBM(attributes=c("tair_locus", "description"), mart=m)
desc <- desc[!duplicated(desc[,1]),]
descv <- as.character(desc[,2]); names(descv) <- as.character(desc[,1])
edgeDF <- data.frame(edgeDF, Desc=descv[rownames(edgeDF)], check.names=FALSE)
write.table(edgeDF, "./results/edgeRglm_allcomp.xls", quote=FALSE, sep="\t", col.names = NA)

Plot DEG results

Filter and plot DEG results for up and down regulated genes. The definition of up and down is given in the corresponding help file. To open it, type ?filterDEGs in the R console.

edgeDF <- read.delim("results/edgeRglm_allcomp.xls", row.names=1, check.names=FALSE) 
png("./results/DEGcounts.png", height=10, width=10, units="in", res=72)
DEG_list <- filterDEGs(degDF=edgeDF, filter=c(Fold=2, FDR=20))
write.table(DEG_list$Summary, "./results/DEGcounts.xls", quote=FALSE, sep="\t", row.names=FALSE)

Figure 3: Up and down regulated DEGs with FDR of 1%

Venn diagrams of DEG sets

The overLapper function can compute Venn intersects for large numbers of sample sets (up to 20 or more) and plots 2-5 way Venn diagrams. A useful feature is the possiblity to combine the counts from several Venn comparisons with the same number of sample sets in a single Venn diagram (here for 4 up and down DEG sets).

vennsetup <- overLapper(DEG_list$Up[6:9], type="vennsets")
vennsetdown <- overLapper(DEG_list$Down[6:9], type="vennsets")
vennPlot(list(vennsetup, vennsetdown), mymain="", mysub="", colmode=2, ccol=c("blue", "red"))

Figure 4: Venn Diagram for 4 Up and Down DEG Sets