Hi! I'm a beginner in limma and bioconductor, and I'd like to ask a few basic questions. I wrote the following script : library(limma) targets <- readTargets("Targets1.txt") RG <- read.maimages(targets$FileName, columns=list(R="CtrlVol", G="DataVol", Rb="CtrlBg", Gb="DataBg"), annotation=c("ID","Name","Rep")) RG$genes <- readGAL("maize.gal") RG$printer <- getLayout(RG$genes) spottypes <- readSpotTypes() RG$genes$Status <- controlStatus(spottypes, RG) bgCorr <- backgroundCorrect(RG, method="movingmin") nWithin <-normalizeWithinArrays(bgCorr, method="loess") nBetween <- normalizeBetweenArrays(nWithin, method="Aquantile") design <- c(1,1,1) isGene <- nBetween$genes$Status == "cDNA" fit <- lmFit(nBetween[isGene, ], design) fit <- eBayes(fit) res <- topTable(fit, number=1000) write.table(res, file="results24top1000.txt", sep="+++") First, limma didn't recognize the ArrayVision format, and I had to parse the raw data and define the columns myself. Is it correct to pass the CtrlVol to R, DataVol to G, etc? The other question is that I'm not sure about the design. Cy3 was the treated, Cy5 the control, but after I used the read.maimages function and defined the values myself, this design should be OK. Am I right? Thanks for any comment and help. Endre Sebestyen -- Agricultural Research Institute of the Hungarian Academy of Sciences Department of Applied Genomics H-2462 Martonv?s?r, Brunszvik street 2.

Again : Hi! I'm a beginner in limma and bioconductor, and I'd like to ask a few basic questions. I wrote the following script : library(limma) targets <- readTargets("Targets1.txt") RG <- read.maimages(targets$FileName, columns=list(R="CtrlVol", G="DataVol", Rb="CtrlBg", Gb="DataBg"), annotation=c("ID","Name","Rep")) RG$genes <- readGAL("maize.gal") RG$printer <- getLayout(RG$genes) spottypes <- readSpotTypes() RG$genes$Status <- controlStatus(spottypes, RG) bgCorr <- backgroundCorrect(RG, method="movingmin") nWithin <-normalizeWithinArrays(bgCorr, method="loess") nBetween <- normalizeBetweenArrays(nWithin, method="Aquantile") design <- c(1,1,1) isGene <- nBetween$genes$Status == "cDNA" fit <- lmFit(nBetween[isGene, ], design) fit <- eBayes(fit) res <- topTable(fit, number=1000) write.table(res, file="results24top1000.txt", sep="+++") First, limma didn't recognize the ArrayVision format, and I had to parse the raw data and define the columns myself. Is it correct to pass the CtrlVol to R, DataVol to G, etc? The other question is that I'm not sure about the design. Cy3 was the treated, Cy5 the control, but after I used the read.maimages function and defined the values myself, this design should be OK. Am I right? Last question : how can I combine the replicates on a chip? I have some genes with 2,3,etc replicates, but not all. This is the 46k maize array from www.maizearray.org Thanks for any comment and help. Endre Sebestyen -- Agricultural Research Institute of the Hungarian Academy of Sciences Department of Applied Genomics H-2462 Martonv?s?r, Brunszvik street 2.