[BioC] limma makeContrasts

[Ben Nota]

Dear BioC,

I'm trying to make use of the "makeContrasts" function in "limma", but I 
am getting errors. Where do I go wrong? First a little background info:

I have 3 different ages (18, 25 and 29 days) en they are all treated (j) 
and non-treated (n). I used 6 Agilent custom arrays (~5000 genes in 
duplicate). My targets, after using the "targetsA2C" function, looks 
like this:

           channel.col X SlideNumber     FileName      Date Target
Array01A.1           1 1          1A Array01A.txt 2006/7/20    18j
Array01A.2           2 1          1A Array01A.txt 2006/7/20    25n
Array01B.1           1 2          1B Array01B.txt 2006/7/20    18n
Array01B.2           2 2          1B Array01B.txt 2006/7/20    29j
Array02A.1           1 3          2A Array02A.txt 2006/8/17    25j
Array02A.2           2 3          2A Array02A.txt 2006/8/17    29n
Array02B.1           1 4          2B Array02B.txt 2006/8/17    25n
Array02B.2           2 4          2B Array02B.txt 2006/8/17    18j
Array03A.1           1 5          3A Array03A.txt 2006/8/17    29j
Array03A.2           2 5          3A Array03A.txt 2006/8/17    18n
Array03B.1           1 6          3B Array03B.txt 2006/8/17    29n
Array03B.2           2 6          3B Array03B.txt 2006/8/17    25j

This means I have on Slide 1: 18j (Cy3) vs 25n (Cy5), Slide 2: 18n (Cy3) 
vs 29j (Cy5), etc. This is an unconnected design. So I tried to use 
Chapter 9 (Separate Channel Analysis of Two-color Data) of the limma 
User's manual. When I try to make a contrast, I am getting an error:

 > Cont.18<-makeContrasts(18j-18n,levels=design)
Error: syntax error in "Cont.18<-makeContrasts(18j"

When I try to use "", I am getting also an error:

 > Cont.18<-makeContrasts("18j-18n",levels=design)
Error in parse(file, n, text, prompt) : syntax error in "18j"

Does anybody knows where the error occurs? What am I doing wrong?

Thanks in advance!

Regards, Ben.

N.B. I will put my whole script here, in case I made an error somewhere 
before:

#load library
library(limma)

#Read target file
targets<-readTargets("SampleFile2.txt")

#Create raw data from target files
RG<-read.maimages(targets$FileName, source="agilent")

#Missing data omzeilen
RGb<-backgroundCorrect(RG, method="normexp",offset=50)

#Normalize arrays with global loess
MARGb<-normalizeWithinArrays(RGb,method="loess")

#Normalize between the arrays with Aquantile
MARGb2<-normalizeBetweenArrays(MARGb,method="Aquantile")

#Convert targets frame to be channel orientated
targets2<-targetsA2C(targets)

#Make design matrix, I used limma user' guide, chapter 9 ("Separate 
Channel Analysis of Two-Color Data")
targets2
u<-unique(targets2$Target)
f<-factor(targets2$Target,levels=u)
design<-model.matrix(~0+f)
colnames(design)<-u

#Estimate correlation and fit linear model for individual channels of 
two-color data
corfit<-intraspotCorrelation(MARGb2,design)
fit<-lmscFit(MARGb2,design,correlation=corfit$consensus)

Cont.18<-makeContrasts("18.j-18.n",levels=design)


-- 
Benjamin Nota
Vrije Universiteit
Department of Animal Ecology
De Boelelaan 1085
1081 HV AMSTERDAM, The Netherlands
Tel:  +31 (0)20-5987217
Fax: +31 (0)20-5987123
ben.nota at ecology.falw.vu.nl

http://www.bio.vu.nl/do/