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/

Dear Ben, > .... > 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? Nothing wrong on your side. This is a "feature" of makeContrasts(), constrasts must not start with a digit. Put a letter in front of your Target definition. Everything should work fine then. Regards Gerhard