Dear all,

I would like to make a differential analysis with DESeq2 for RNA-seq data.

I wanted to watch the effect of the normalization. You can find below, my R code. 

dds <- DESeq(ddsHTSeq)

dds <- estimateSizeFactors(dds)

countNorm <- counts(dds, normalized=TRUE)

countUnnorm <- counts(dds, normalized=FALSE)

res1 <- results(dds, independentFiltering=FALSE, normalized = TRUE)

res2 <- results(dds, independentFiltering=FALSE, normalized = FALSE)

I made 2 boxplots with countNorm and countUnnorm. On boxplots, we can see that the normalization is working.

But when I made 2 volcano plots with res1 and res2 both volcanos are identical. Furthermore when I have a look on res1 and res2, both are identical (I mean same values for baseMean, log2FoldChance, lfcSE, stat, pvalue).

I would like to know why res1 (with normalization) and res2 (without normalization) are identical ?

Could you please help me ?

Thank in advance,

Regards,

Julien

Check ?results. “normalized” is not one of its arguments. If you ever have questions about the behavior of a function relative to its arguments, the function help page is a good first place to check.