Dear Mitch, You don't say what instructions you are trying to follow here. I think you may be trying to use code which was intended for other data sets. I suspect that there may be more than one problem. Firstly, why do you need to use readGAL()? This is only needed with SPOT data. Your RG object from read.maimages() will already contain annotation information from the Agilent output files. Look at names(RG$genes) to see what you have. Secondly, does your GAL file match your data files? Type dim(RG) and gal <- readGAL() dim(gal) Do the row numbers agree? I am guessing they may have different numbers of rows. BTW, do you need to use "normexp"? I've found the AgilentFE background estimator is already pretty good, and doesn't produce negative intensities anyway. Best wishes Gordon >Date: Mon, 9 Apr 2007 12:21:57 +0200 >From: "Mitch Levesque" <mitch.levesque at="" tuebingen.mpg.de=""> >Subject: [BioC] duplicate correlation on Agilent 4x44 arrays >To: <bioconductor at="" stat.math.ethz.ch=""> > >Hi Bioconductors, > >I am using R 2.4.1 and limma to analyze the new Agilent 4x44 array design >and am having trouble with the duplicate correlation function using the >following script: > > >library(limma) >targets <- readTargets("Targets.txt") >RG <- read.maimages(targets$FileName, source="agilent") >RG$genes<-readGAL() >RG$printer<-getLayout(RG$genes) >RG <- backgroundCorrect(RG, method="normexp", offset=50) >MA <- normalizeWithinArrays(RG, method="loess") >MA <- MA[order(RG$genes[,"ID"]),] > >I get the following error: > >Error in `[.MAList`(MA, order(RG$genes[, "ID"]), ) : > subscript out of bounds > >I would like to treat the duplicate probes on each array as a technical >replicate, but since the spacing is not consistent for each gene, I must >first order the list by reference number. Are there any suggestions about >how I may do this? > >Mitch

Gordon, Thanks for the reply. I am not using any particular instruction set, just what I have put together from the User Guide. You were right about the file dimensions, they are different: > dim(RG) [1] 44407 4 > gal <- readGAL() > dim(gal) [1] 180880 10 Is it possible to read the duplicate positions directly off of the gal file? I tried: layout <- getLayout(gal, guessdups=TRUE) and I get the following: $ngrid.r [1] 1 $ngrid.c [1] 4 $nspot.r [1] 170 $nspot.c [1] 266 $ndups [1] 8 $spacing [1] NA attr(,"class") [1] "PrintLayout" I haven't tried without the normexp, but I will test it. Thanks again. Mitch -----Original Message----- From: Gordon Smyth [mailto:smyth@wehi.EDU.AU] Sent: Tuesday, April 10, 2007 1:03 PM To: Mitch Levesque Cc: bioconductor at stat.math.ethz.ch Subject: [BioC] duplicate correlation on Agilent 4x44 arrays Dear Mitch, You don't say what instructions you are trying to follow here. I think you may be trying to use code which was intended for other data sets. I suspect that there may be more than one problem. Firstly, why do you need to use readGAL()? This is only needed with SPOT data. Your RG object from read.maimages() will already contain annotation information from the Agilent output files. Look at names(RG$genes) to see what you have. Secondly, does your GAL file match your data files? Type dim(RG) and gal <- readGAL() dim(gal) Do the row numbers agree? I am guessing they may have different numbers of rows. BTW, do you need to use "normexp"? I've found the AgilentFE background estimator is already pretty good, and doesn't produce negative intensities anyway. Best wishes Gordon >Date: Mon, 9 Apr 2007 12:21:57 +0200 >From: "Mitch Levesque" <mitch.levesque at="" tuebingen.mpg.de=""> >Subject: [BioC] duplicate correlation on Agilent 4x44 arrays >To: <bioconductor at="" stat.math.ethz.ch=""> > >Hi Bioconductors, > >I am using R 2.4.1 and limma to analyze the new Agilent 4x44 array design >and am having trouble with the duplicate correlation function using the >following script: > > >library(limma) >targets <- readTargets("Targets.txt") >RG <- read.maimages(targets$FileName, source="agilent") >RG$genes<-readGAL() >RG$printer<-getLayout(RG$genes) >RG <- backgroundCorrect(RG, method="normexp", offset=50) >MA <- normalizeWithinArrays(RG, method="loess") >MA <- MA[order(RG$genes[,"ID"]),] > >I get the following error: > >Error in `[.MAList`(MA, order(RG$genes[, "ID"]), ) : > subscript out of bounds > >I would like to treat the duplicate probes on each array as a technical >replicate, but since the spacing is not consistent for each gene, I must >first order the list by reference number. Are there any suggestions about >how I may do this? > >Mitch