[BioC] Separate channel analysis of two color micro arrays: avereps and intraspotCorrelation functions

Rob [guest] guest at bioconductor.org
Wed Nov 13 16:51:40 CET 2013

Dear limma users and experts,

I'm working with two color Agilent GE microarrays with a common reference set-up.
Here, I'm using the avereps function to average multiple probes over a specific gene before fitting a linear model to the data.

However, I'm getting low FDR corrected p-values over my whole data resulting in almost no differential expression.
Trying to circumvent this, I've read that by applying the intraspotCorrelation function and performing a separate channel analysis, 
you can get better corrected p-values if the corr < 0.5 (as you are now also incorporating the information available in the A-values). 
I've done this and this is resolving the problem.

My question specifically is now if I should do the intraspotCorrelation on the MA object after averaging the probes over a specific gene (on which the linear model will be based), or on the MA object before averaging?

I think I should do the intraspotCorrelation function on the same MA object as the linear model will be fitted, but not sure.

The two scenarios:

1. Using MA object after averaging over gene ID

MA_norm<-normalizeBetweenArrays(MA_norm, method=between)
MA_ave<-avereps(MA_norm, ID=MA_norm$genes$ID)

corfit_Ave<-intraspotCorrelation(MA_ave, design)
fit_ave<-lmscFit(MA_ave, design, correlation=corfit_Ave$consensus)

2. Using MA object before averaging over gene ID and using two different MA objects for the correlation estimate and linear model

MA_norm<-normalizeBetweenArrays(MA_norm, method=between)
MA_ave<-avereps(MA_norm, ID=MA_norm$genes$ID)

corfit<-intraspotCorrelation(MA_norm, design)
fit<-lmscFit(MA_ave, design, correlation=corfit$consensus)

Any advice on this would be very welcome,

thanks in advance,

 -- output of sessionInfo(): 

R version 3.0.1 (2013-05-16)
Platform: x86_64-w64-mingw32/x64 (64-bit)

[1] LC_COLLATE=Dutch_Belgium.1252  LC_CTYPE=Dutch_Belgium.1252   
[3] LC_MONETARY=Dutch_Belgium.1252 LC_NUMERIC=C                  
[5] LC_TIME=Dutch_Belgium.1252    

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

loaded via a namespace (and not attached):
[1] tools_3.0.1

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