Hello, I am using Limma (version 3.12.0) to read 2-channel dye-swap files from an Agilent image analysis scanner, where each sample has 2 files - one for cy3 and one for cy5. However, the only reference to such a 2-file input in the limma user guide is to "ImaGene". When I try to read the files and the target file using the following commands: RG = read.maimages(file_names, ...) targets = readTargets("targets.txt") files = targets[,c("FileNameCy3","FileNameCy5")]; RG = read.maimages(files,source="imagene"); I get the following error message: Error in read.imagene(files = files, path = path, ext = ext, names = names, : Can't find Field Dimensions in ImaGene header In addition: Warning message: In readImaGeneHeader(fullname) : End of file encountered before End Header When I try to use source="Agilent" instead of "imagene", the command doesn't seem to understand that I have 2 file names, and gives the following error message: Error in read.maimages(files, source = "agilent") : targets frame doesn't contain FileName column I tried every single option for "source" that was listed in the help of "read.maimages" but it seems like "imagene" is the only one that is able to digest the two headers for the file names. I am stuck and would appreciate any help. Thank you, Neta.

Hi Neta On page 16 and 17 of the limma vignette (25 March 2012) there is an explanation about column names in the data files. You should check what the header names of your Agilent files are and then change the read.maimages() command accordingly. For example: > RG <- read.maimages(files, + columns=list(R="F635 Mean",G="F532 Mean",Rb="B635 Median",Gb="B532 Median")) The default in limma for Imagine is to extract the signal mean, but for Agilent it is the signal median. You probably need to specify that you want to extract the medians. Hope this helps. Cheers, Belinda -----Original Message----- From: bioconductor-bounces@r-project.org [mailto:bioconductor-bounces at r-project.org] On Behalf Of Neta Sent: Thursday, 14 June 2012 9:48 AM To: bioconductor at stat.math.ethz.ch Subject: [BioC] using Limma to read 2-channel dye-swap in Agilent scanner Hello, I am using Limma (version 3.12.0) to read 2-channel dye-swap files from an Agilent image analysis scanner, where each sample has 2 files - one for cy3 and one for cy5. However, the only reference to such a 2-file input in the limma user guide is to "ImaGene". When I try to read the files and the target file using the following commands: RG = read.maimages(file_names, ...) targets = readTargets("targets.txt") files = targets[,c("FileNameCy3","FileNameCy5")]; RG = read.maimages(files,source="imagene"); I get the following error message: Error in read.imagene(files = files, path = path, ext = ext, names = names, : Can't find Field Dimensions in ImaGene header In addition: Warning message: In readImaGeneHeader(fullname) : End of file encountered before End Header When I try to use source="Agilent" instead of "imagene", the command doesn't seem to understand that I have 2 file names, and gives the following error message: Error in read.maimages(files, source = "agilent") : targets frame doesn't contain FileName column I tried every single option for "source" that was listed in the help of "read.maimages" but it seems like "imagene" is the only one that is able to digest the two headers for the file names. I am stuck and would appreciate any help. Thank you, Neta. _______________________________________________ Bioconductor mailing list Bioconductor at r-project.org https://stat.ethz.ch/mailman/listinfo/bioconductor Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:4}}