
Dear Friends,

I am working on single channel normalization for Agilent 244K chip data using Limma package. I refered the communication happend between Gordon and Abhilash about single channel normalization. I able perform it. But, not able to get the gene list soon after the normalization as we get in dual channel analysis.

I am able to get the gene list in topTable stage. But I do not want gene list at the end of the analysis. Because I want remove the control spots soon after the normalization as we do for dual color.

Following are the steps which I followed to perform single channel normalization,
>library(limma)
> target<-readTargets("/home/mlscrh2/MData/target.txt")
> RG<-read.maimages(target$FileName, source="agilent", path="/home/mlscrh2/PrakrathiData", columns = list(G="gMeanSignal", Gb="gBGMeanSignal",R="gMedianSignal",Rb="gBGMedianSignal"),annotation= c("Row", "Col", "ProbeUID","ProbeName", "GeneName"))
> Ggene<-backgroundCorrect(RG$G,method='normexp')
> MA.q <- normalizeBetweenArrays(Ggene, method="quantile")
> design = c(1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1,1)
> fit = lmFit(MA.q,design)> ebFit <- eBayes(fit)
> a <- topTable(ebFit,genelist = RG$genes,adjust="fdr",n=300000)

I tried to incorporate genelist in the stage of background correction and normalization, but ends up with an error.

So can you please suggest me, How to remove control spots soon after normalization in single channel analysis?

Thanking you in anticipation.

Regards,

Prashantha

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