Dear Endre, I suspect the reason you didn't get a rsponse to your original posting is that your questions are not very specific. Assuming that you have three replicate arrays comparing a control to a treatment, your analysis looks fine. I can't give you any response to the problem reading ArrayVision format because you haven't shown us the error you received or the structure of your data files. Therefore we can't see if your files really are in ArrayVision format or diagnose what the read problem is. I can't tell you if the column names are correct, because ArrayVision columns are used-defined. Your interpretation appears reasonable. You ask if your design is correct, but you don't give any information about your experiment, eg the targets information. I have to assume that you have three replicate arrays. In my opinion, there is no good way to combine irregular numbers of within-array gene replicates, especially if the probes are not indentical. Hope this helps Gordon > Date: Fri, 28 Mar 2008 12:51:39 +0100 > From: "Endre Sebestyen" <endre.sebestyen at="" gmail.com=""> > Subject: [BioC] basic limma questions > To: Bioconductor <bioconductor at="" stat.math.ethz.ch=""> > > 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.

Thanks for your answer. A little more details : I had a control and a treatment, with 3 technical replicates and no dye-swaps. Cy3 was the treated, Cy5 the untreated. The ArrayVision result file looks like this : Ctrl Ctrl Ctrl Data Data Data Spot labels VOL - MDC Bkgd sVOL VOL - MDC Bkgd sVOL Ratio (sVOL): Data / Ctrl Diff (sVOL): Data - Ctrl MZ00023554 - TC248295 (1) 87573.5998 29969.817 57603.783 51217.7619 15349.622 35868.140 0.623 -21735.643 MZ00023408 - TC248006 (1) 29389.2252 29831.153 0.000 15896.2700 15349.622 546.648 1.0000e+100 546.648 I extracted the ID, Ctrl VOL, Ctrl Bkgd, Data VOL, Data Bkgd columns. When I used the read.maimages function, Ctrl VOL became R, Data VOL became G, Ctrl Bkgd became Rb, DataBkgd became Gb. Endre On Mon, Mar 31, 2008 at 5:36 AM, Gordon K Smyth <smyth at="" wehi.edu.au=""> wrote: > Dear Endre, > > I suspect the reason you didn't get a rsponse to your original posting is > that your questions are not very specific. > > Assuming that you have three replicate arrays comparing a control to a > treatment, your analysis looks fine. > > I can't give you any response to the problem reading ArrayVision format > because you haven't shown us the error you received or the structure of > your data files. Therefore we can't see if your files really are in > ArrayVision format or diagnose what the read problem is. > > I can't tell you if the column names are correct, because ArrayVision > columns are used-defined. Your interpretation appears reasonable. > > You ask if your design is correct, but you don't give any information > about your experiment, eg the targets information. I have to assume that > you have three replicate arrays. > > In my opinion, there is no good way to combine irregular numbers of > within-array gene replicates, especially if the probes are not indentical. > > Hope this helps > Gordon > > > Date: Fri, 28 Mar 2008 12:51:39 +0100 > > From: "Endre Sebestyen" <endre.sebestyen at="" gmail.com=""> > > Subject: [BioC] basic limma questions > > To: Bioconductor <bioconductor at="" stat.math.ethz.ch=""> > > > > 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. > -- Agricultural Research Institute of the Hungarian Academy of Sciences Department of Applied Genomics H-2462 Martonv?s?r, Brunszvik street 2.