Dear List, I am very sorry for the previous emails, I do not know what happened, so I trying to use another email. I am looking for help to account for duplication in analysis using lmFit in limma of data from a two- channel microarray. The experiment is comparing differences between breeds (A and N) using a dye-swap labelling. The array has the following layout: RG$printer $ngrid.r [1] 12 $ngrid.c [1] 4 $nspot.r [1] 19 $nspot.c [1] 19 attr(,"class") [1] "PrintLayout" The array has been duplicated on the top half and the bottom half of the slide (spots on the tip 1 are duplicated on the tip 48, spots on the tip 2 are duplicated on the tip 47, and so on). Moreover, the slide has control spots with different number of duplicates, which I have been attributed weights=0, so I am using only spots with 2 replicates ("genes"). I have been successful accounting for duplicated spots using the following commands: myfun <- function(x) as.numeric(x$Flags ==0) targets1 <- as.matrix(read.table("targets.txt", header = TRUE)); targets1 SlideNumber Name FileName Cy5 Cy3 [1,] "1" "Treatment1" "N16_A11.gpr" "A" "N" [2,] "2" "Treatment2" "A08_N15.gpr" "N" "A" [3,] "3" "Treatment3" "N12_A06.gpr" "A" "N" [4,] "4" "Treatment4" "A02_N07.gpr" "N" "A" filenames <- matrix (c(targets1[,3]),nrow=4,ncol=1); filenames [,1] [1,] "N16_A11.gpr" [2,] "A08_N15.gpr" [3,] "N12_A06.gpr" [4,] "A02_N07.gpr" RG <- read.maimages(filenames, source="genepix", wt.fun=myfun) MA <- normalizeWithinArrays(RG, method="loess", bc.method="none") MA1 <-MA[order(RG$genes[,4]),] design = matrix(cbind(Dye = 1, c(-1,1, -1,1)), nrow=4, ncol=2,dimnames=list(c("N16_A11", "A08_N15", "N12_A06", "A02_N07"), c("Dye","Treatment"))) dupcor <-duplicateCorrelation(MA1, design, ndups=2, spacing=1) fit <- lmFit(MA1, design, ndups=2, spacing=1, correlation=dupcor$consensus.correlation) fit2 <- eBayes(fit) topTable (fit2, coef = "Treatment", adjust="BH", sort.by="P") However, if the duplicateCorrelation is used to estimate spatial correlation in the slide, I am not sure if makes sense to rearrange MA by GeneID and then apply the duplicateCorrelation with spacing=1, so I have tried to use (unsucessful) the argument spacing="topbottom". dupcor1 <-duplicateCorrelation(MA, design, ndups=2, spacing="topbottom") fit.topbottom <- lmFit(MA, design, ndups=2, spacing="topbottom", correlation=dupcor1.teste$consensus.correlation) Error in nspots/ndups/spacing : non-numeric argument to binary operator sessionInfo() R version 2.5.0 (2007-04-23) i386-pc-mingw32 locale: LC_COLLATE=English_United States.1252;LC_CTYPE=English_United States.1252;LC_MONETARY=English_United States.1252;LC_NUMERIC=C;LC_TIME=English_United States.1252 attached base packages: [1] "stats" "graphics" "grDevices" "utils" "datasets" "methods" "base" other attached packages: statmod limma "1.3.0" "2.10.0" Is it correct what I am doing? Could anyone give me some suggestions with this problem? Thank you a lot for your help! Regards, Leonardo