Normalization by variance stabilizing transformation VST

Normalization by variance stabilizing transformation VST

1

@6c372dab

Last seen 8 hours ago

Sweden

Hello!

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?

thanks!
Kris


library


composition


vst


DESeq2


Normalization

• 84 views

@mikelove

Last seen 56 minutes ago

United States

Can you say what you mean by library composition? Can you give an example of what you want to control for?

The design is only used for estimating the parameters of the transformation (the design is needed to assess the amount of within-group dispersion) but then afterwards the same transformation is applied to all the samples, so in that way it is not using the sample grouping in applying the transformation. Sample group information is also not used in the size factor calculation.


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