[BioC] Should I skip the eBayes step when using Limma for Affymetrix miRNA v1 chip

Gordon K Smyth smyth at wehi.EDU.AU
Fri Aug 22 12:50:44 CEST 2014

Dear Scott,

> Date: Thu, 21 Aug 2014 04:49:49 -0700 (PDT)
> From: "Scott Robinson [guest]" <guest at bioconductor.org>
> To: bioconductor at r-project.org, scott.robinson at glasgow.ac.uk
> Subject: [BioC] Should I skip the eBayes step when using Limma for
> 	Affymetrix miRNA v1 chip?
> Dear List,
> I am working with Affymetrix's miRNA V1 chip, which uses very different 
> probe sets for different molecule types, e.g. 4 identical probes for one 
> miR, or 11 different probes for a snoRNA.
> I have read that the eBayes step assumes equal error variance between 
> probe sets so it is not suitable for this kind of mixed set of probe set 
> designs.

Having a variances from the same distribution is not the same as having 
the same variance.

> To further complicate matters I am thinking about generating a 
> custom CDF where the miR probe sets would have varied number of probes.
> http://pomelo2.bioinfo.cnio.es/help/pomelo2-help.html#toc10
> Should I look at everything through Limma without the eBayes step 
> (making it equivelant to a normal t-test?),

That would throw the baby out with the bath water.

I doubt that the error variance depends quite as directly on the number of 
probes in a probe-set as you might think.  When we have analysed the miRNA 
Affymetrix chip, we have found that it has major problems from the point 
of view of normalization, while the issue that you raise is relatively 

I could suggest ways to take into account the number of probes per 
probe-set in the eBayes calculations, but I don't think this will be 

Best wishes

PS.  If you have the choice, RNA-seq is cheaper and better.

> or separate into several different analyses for different molecule types 
> and only drop the eBayes step for the miRs (which will have varying 
> sizes of probe sets)?
> Many thanks,
> Scott
> -- output of sessionInfo():
>> sessionInfo()
> R version 3.0.2 (2013-09-25)
> Platform: x86_64-w64-mingw32/x64 (64-bit)
> locale:
> [1] LC_COLLATE=English_United Kingdom.1252
> [2] LC_CTYPE=English_United Kingdom.1252
> [3] LC_MONETARY=English_United Kingdom.1252
> [5] LC_TIME=English_United Kingdom.1252
> attached base packages:
> [1] stats     graphics  grDevices utils     datasets  methods   base
> --
> Sent via the guest posting facility at bioconductor.org.

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