[BioC] Limma warnings and how to extract normalized CY5, CY3 intensities

[Christian Brière]

Le 30/11/2011 14:48, asas asasa a écrit :
> Hello limma people,
>
> I used Limma on genePix data .I have two questions and will
> be appreciated your help:
>
> 1) Since in my case the compared conditions are not paired on same slides,
> I decided to use separate channel analysis for my two color data (Limma
> manual oct 2011, chapter 9). The script I used is shown bellow, and though
> it worked and gave nice biological results, there were 21 warnings
> during lmscFit() run ("reml: Max iterations exceeded"). Is it a problem ?
>
> 2) wondering how to extract normalized CY5,CY3 intensities after background
> correction, loess and quantile. The problem is that the normalized data is
> stored as  M and A, and not as absolute quantities of CY3, CY5. I need it
> in order to use this data in other programs.
If M = CY3 - CY5 and A = (CY3+CY5)/2 , after normalisation the following 
formula should give back normalized intensities:

CY3  = (2*A + M) / 2
CY5 = (2*A - M) / 2

Hope this helps.
Christian

>
>
> Best,
> Assaf
>
>
> SCRIPT USED:
>
> library(limma)
> d<- "/home/home/gprs/"
> targets<- readTargets(paste(d,"targets1_.txt",sep=""))
> RG<- read.maimages(targets, source="genepix",
> path=d,wt.fun=wtflags(weight=0,cutoff=-50))
> RGb<- backgroundCorrect(RG, method="normexp", offset=50)
> MA<- normalizeWithinArrays(RGb)
> MA2<- normalizeBetweenArrays(MA, method="Aquantile")
> #  "Cy5","Cy3" columns exit in the targets table
> targets2<- targetsA2C(targets, grep=FALSE)
> targets2
> u<- unique(targets2$Target)
> f<- factor(targets2$Target, levels=u)
> design<- model.matrix(~0+f)
> colnames(design)<- u
> design
> corfit<- intraspotCorrelation(MA2, design)
> fit<- lmscFit(MA2, design, correlation=corfit$consensus)  # got 21
> warnings here: reml: Max iterations exceeded
> cont.matrix<- makeContrasts("x-y",levels=design)
> fit2<- contrasts.fit(fit, cont.matrix)
> fit3<- eBayes(fit2)
> topTable(fit3, adjust="BH")
> write.fit(fit3, results=NULL, "x-y.txt", adjust="BH", sep="\t", digits=15)
>
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>
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-- 

Christian Brière
"Cytosolic and nuclear calcium signalling" group
UMR CNRS/UPS 5546
BP42617 Auzeville
F-31326 Castanet-Tolosan (France)

Tel: +33(0)5 34 32 38 90
Fax: +33(0)5 34 32 38 02
http://www.lrsv.ups-tlse.fr