Hi, I am trying to learn about DEG and Deseq2 and using the Beginner's guide

1) does running dds <- DESeq(dds) automatically apply "Normal" shrinkage and also apply rlog ?

2) in the beginners guide section5, it says to transform using rld <- rlog( dds ) .. is this just showing what dds has already done? Or does this need doing each time?

3) I attempted to perform deseq2 on my data - on my volcano plot, gene PALB2 has a strong pval, but only modest LFC, and on the volcano plot it only shows small/moderate expression. how can this be explained?

many thanks

1) No. See the vignette. Fold change shrinkage is done via the lfcShrink function and rlog is not part of differential testing. It's an optional transformation for downstream analysis.

2) Not sure I understand. rlog as said is a transformation method, and has nothing to do with the DESeq function.

3) There is no "strong" p-values in your plots. -log10(padj) of 3 is just a FDR of 0.001, and that is not low for RNA-seq or OMICS in general. It is simply below the typical (and arbitrary) 0.05 cutoff. There is simply not terribly much evidence for differential expression.