DESeq 'stabilazing' transformations usage

[Daniel1977]

Hi,

I´m a little puzzled about the 'Dierential analysis of count data : the
DESeq2 package' protocol

I was trying it with (only) two samples and got the DE results, then I tried the suggested transformations:

(being 'des' my previous results, just as it appears in the 'manual')

dseBlind <- dse
design(dseBlind) <- formula(~ 1)
dseBlind <- estimateDispersions(dseBlind)

rld <- rlogTransformation(dseBlind)
vsd <- varianceStabilizingTransformation(dseBlind)

At this point I had assumed that the 'rld' and 'vsd' objects are like 'dse' but with the transformations, however when I try to retrieve the results I get:

> Prueba.rld.res <- results(rld)
Error in tail(all.vars(design(object)), 1) :
error in evaluating the argument 'x' in selecting a method for function 'tail': Error in (function (classes, fdef, mtable) :
unable to find an inherited method for function ‘design’ for signature ‘"SummarizedExperiment"’

Am I missing something, should I instead use the 'rld' or 'vsd' objects with my DE analysis somehow???

many many thanks!!

[Wolfgang Huber]

Dear Daniel

the error message means that you are calling a method (results) on an object from a class (SummarizedExperiment) for which no such method is defined.

The class SummarizedExperiment stores matrices of data (here: the transformed counts), with rich metadata for the matrix columns and rows. The accessors of this class are described in the documentation of the GenomicRanges package, where the class is defined.

The class DESeqSummarizedExperiment is derived from that class, but contains additional data - the results from a differential expression analysis - which is accessed with the results method. See e.g. the man page of DESeq function in the DESeq2 package. However, since rlogTransformation and varianceStabilizingTransformation do not do any differential expression analysis, and just perform data transformations, they produce no "results".

Perhaps a good starting point would be to state what it is it that you actually want to achieve?

I hope this helps - the package documentation is always good place to check, no need to just rely on your assumptions

Best wishes
Wolfgang