I have 4 RNA-seq samples A,B,C,D in triplicate. They're aligned with STAR and counted with summarizeOverlaps, following the Bioconductor RNA-seq workflow. I'm comparing A vs B and C vs D (other comparisons later but these for now). I'm getting different and odd patterns in the MA-plot depending on whether I first subset the summarizedExperiment (se) into 2 sets (A+B, C+D) or keep everything together:
se1 <- se[,1:6] se2 <- se[,7:12] dds <- DESeqDataSet(se, design = ~sample) dds1 <- DESeqDataSet(se1, design = ~sample) dds2 <- DESeqDataSet(se2, design = ~sample) dds <- DESeq(dds) dds1 <- DESeq(dds1) dds2 <- DESeq(dds2) resultsa <- results(dds, contrast=c("sample","B","A")) results1a <- results(dds1, contrast=c("sample","B","A")) resultsb <- results(dds, contrast=c("sample","D","C")) results2b <- results(dds2, contrast=c("sample","D","C")) plotMA(resultsa, ylim = c(-5, 5)) plotMA(results1a, ylim = c(-5, 5)) plotMA(resultsb, ylim = c(-5, 5)) plotMA(results2b, ylim = c(-5, 5))
I'm wondering about the patterns on the left side of each image, I think the separate ones look normal but the together ones don't, is that correct? What could be causing the difference?
Yup, and to point you to the code for LFC shrinkage, it's here:
https://bioconductor.org/packages/release/bioc/vignettes/DESeq2/inst/doc/DESeq2.html#lfcShrink