I am trying to use limma roast, as advised in a previous post A: Compare groups of different RNAseq sets. However, I have doubts about if it is correct what I am doing, since the barcodeplot gives a different picture of my results. I have an RNA-seq experiment analyzed with limma trend, and want to see if a signature of 48 genes (from another experiment) is enriched herein. Here some essential parts of the code:
design <- model.matrix(~0 + targets$Cell + targets$Donor) fit <- lmFit(logCPM, design) contr <- makeContrasts( hivslo = hi - lo, hivsint = hi - int, intvslo = int - lo, levels = design) fit2 <- eBayes(contrasts.fit(fit, contr), trend=TRUE) results <- decideTests(fit2, method="separate") summary(results) hivslo hivsint intvslo Down 2461 1179 47 NotSig 11245 13746 15486 Up 1891 672 64 Signature <- "X:/names.txt" signature <- scan(Signature, what = "character") set.seed(12) roast_hi_lo_signature <- roast(logCPM, index = signature, design = design, contrast = contr[,1], trend = T) roast_hi_lo_signature Active.Prop P.Value Down 0.2317073 0.8189095 Up 0.2926829 0.1815908 UpOrDown 0.2926829 0.3630000 Mixed 0.5243902 0.0070000 index.vector <- All_genes %in% signature barcodeplot(fit2$t[,1], index = index.vector)
So here my question, in the results only the mixed group has significant enrichment (p < 0.05), however the barcode plot suggest a enrichment in down? So I think I am doing something wrong? Did I do the roast analysis wrong or the barcode?