I'm using edgeR to analyze a dataset with two treatment groups, each with three replicates. I've followed the canonical glm functionality pipeline. Below is the code thus far. The problem lies in finding differentially expressed genes. According to the 'decideTests' and the MD plot, all the genes are extremely down-regulated, which doesn't seem like a correct output. The MD plot is extremely wonky.
I'm thinking the problem may be in the set-up of the design matrix. Some examples I've seen include a '0+group' and some do not. Not sure which approach would be best here. The output of my 'design' is below. Changing this seems to have drastic impacts on the DE genes.
Any thoughts or advice is appreciated!
library(edgeR) setwd("myWorkingDirectory") data <- read.delim("myData") eyeCondition <- factor(c(rep("normal",3),rep("AMD",3))) eyeCondition y <- DGEList(counts = data[,3:8], group = eyeCondition, genes = data[,1:2]) keep <- rowSums(cpm(y) > 0.5) >= 3 table(keep) y <- y[keep, , keep.lib.sizes = FALSE] y <- calcNormFactors(y) y$samples design <- model.matrix(~0+eyeCondition) rownames(design) <- colnames(y) colnames(design) <- levels(eyeCondition) design AMD normal macula4 0 1 macula8 0 1 macula13 0 1 AMD.macula17 1 0 AMD.macula18 1 0 AMD.macula19 1 0 attr(,"assign")  1 1 attr(,"contrasts") attr(,"contrasts")$eyeCondition  "contr.treatment" y <- estimateDisp(y, design, robust = TRUE) plotBCV(y) fit <- glmQLFit(y, design, robust = TRUE) plotQLDisp(fit) qlf <- glmQLFTest(fit) topTags(qlf, n = 15) summary(decideTests(qlf)) # below is troublesome bit > summary(decideTests(qlf)) normal Down 18656 NotSig 0 Up 0 > sessionInfo() R version 4.0.5 (2021-03-31) Platform: x86_64-w64-mingw32/x64 (64-bit) Running under: Windows 10 x64 (build 19042) Matrix products: default locale:  LC_COLLATE=English_United States.1252 LC_CTYPE=English_United States.1252  LC_MONETARY=English_United States.1252 LC_NUMERIC=C  LC_TIME=English_United States.1252 attached base packages:  stats graphics grDevices utils datasets methods base other attached packages:  edgeR_3.32.1 limma_3.46.0 loaded via a namespace (and not attached):  BiocManager_1.30.15 compiler_4.0.5 tools_4.0.5  Rcpp_1.0.6 tinytex_0.31 splines_4.0.5  grid_4.0.5 locfit_1.5-9.4 xfun_0.22  statmod_1.4.36 lattice_0.20-41