I am a bit confused about the normalization performed by the DESeq2 varianceStabilizingTransformation() and vst() functions in addition to the actual variance stabilization. My understanding is that the normalization by division by size factors (which are automatically calculated?) corrects for both library size and library composition. But the reference manual specifically states that it corrects for library size, while nothing is mentioned about library composition. Is there something I'm missing here? The use of the variance stabilized data is PCA and heatmap plotting.
Finally, am I correct in assuming the design parameter only affects the variance stabilization in the vst() function, not the additional normalization? The subsetting and stabilization happen first, then the data is normalized as in varianceStabilizingTransformation?