Hello,

I am trying to make a smear plot with edgeR to look at and RNA-seq data set that has no statistically significant differentially expressed genes. When I plot this, I get a few points that are orange, and I can't find anywhere what this is indicative of. I know red is differentially expressed, but unsure what orange is referencing

y2 <- DGEList(counts=data2,group=group)
keep <- rowSums(cpm(y2)>1) >=6
y2 <- y2[keep, ,keep.lib.sizes=FALSE]
y2 <- calcNormFactors(y2)
y2 <- estimateCommonDisp(y2)
y2 <- estimateTagwiseDisp(y2)
et2 <- exactTest(y2, pair = c("WT","KI"))
topTags_sig_genes<- topTags(et2, n=10000, adjust.method = "BH", sort.by = "PValue", p.value = 0.05)
is.de <- decideTestsDGE(et2, p.value=0.05)
summary(is.de)

op <- par(mar = c(4,4,4,4))
par(bty="l")
plotSmear(et2, de.tags=rownames(et2)[is.de!=0], xlab = "Average log CPM", ylab = "log2 RNA (QKI/WT)",
   ylim = c(-2.5,2.5),cex.lab = 1.25, cex.axis = 1.25)
abline(h=c(-1, 1), col="blue")

Note the orange dot around (3,-1.5).

We are no longer regularly using the plotSmear() function and this might be a bug. The orange color is intended for the smeared points (see ?maPlot). Can I suggest that you try the newer function

plotMD(et2, status=is.de)

instead?