Dear Mr Smyth, >> When loading these files in either limma and limmaGUI only the A >> Field is read. Not the values belonging to the B and C metagrid >> (which are the replicates). >> Is there a simple way to solve this problem ? Or do I have to cut >> these files into three parts ? >Yes, this is a known inadequacy of the limma read.imagenes() function >-- it >reads only data for the first ImaGene field. This problem has >persisted partly because we >haven't had time to fix it, and partly >because we are genuinely unsure how the multiple fields should >be >treated. For example, how should be multiple fields be treated in >print-tip-loess >normalisation, or in an imageplot? The multiple >fields mean that the concept of PrinterLayout >which is used for other types of arrays is no longer adequate. We are waiting for advise from >ImaGene users as to how the data should be treated. As a ImaGene user myself I'm always willing to share ideas ofcourse. Currently I'm treating this data as three different slides, thereby I have automatically triplos (good for the statistics). Ofcourse the idea of Steen has been considered and used here too. I just wondered why this other approach wasn't possible. For the pratical use it is easier in Imagene to do it the way as I wrote before. Maybe for a inbetween solution limma could automatically cut the file into multiple separate arrays (1.a 1.b 1.ect) . I think for normalization (printip and even inbetween) this should work fine. Actually as mentioned above already treat my slide as if it were three slides and the results are ok. (also compared to other software). >In the meantime, the solution suggested by Steen is the most elegant. >>Otherwise, you might have to read in your data manually using read.table() or similar. >> Secondly, using limma and the targets <- readTargets function you >> cannot use the targets object directly in the read.imagene function. >> I solved this to create matrix with ncol=2 and nrow same as in the >> targets object. Then this can be used directly in read.imagene. >I'm not sure what you see as the problem here. When I read ImaGene >data, I >include columns >FileNameCy3 and FileNameCy5 in the targets file, and I use >read.maimages() >with > files = targets[, c("FileNameCy3","FileNameCy5")] >This seems neat enough. Did you something else in mind as to how you >thought limma should work? This is the way I do this: targets <- readTargets("hybridization_file.txt") Then I get the targets array If I use this targets objects like this: RG <- read.imagene(targets) I get an error message. So I put the files indeed like you do above in an matrix. My question is why not use automatically the FileNameCy3 and FileNameCy5 columns in the targets object ? If you have the read.imagene function you could assume that the targets object has the following columms: Slidenumber|FileNameCy3|FileNameCy5|Cy3|Cy5 since this is also what you show in the manual. >> Next you would like to add color to your MA plots using controlTypes >> and Spottypes. This doesn't work either because when reading the >> imagene data files no columns RG$genes$Name and RG$genes$ID are >> created (actually onlu RG$genes[['Gene ID']] exists.. I solved this >> by : RG$genes$Name <- RG$genes[['Gene ID']] and same for ID. Then you >> will be able to use the conrol types and colors for the colored MA plots. >Actually this is not a limitation of limma. You can already use >arbitrary >column names, including "Gene ID", in your spot-types file >to assign colors and other plotting >attributes using controlStatus(). There is no need to created new columns with the names >"Name" and "ID". Ok, my mistake, I must have overlooked this option. >> The same problem applies for limmaGUI. If you do not have a GAL file >> and load the imagene files directly into limmaGUI you cannot assign >> colors >>using spotTypes either. (Error messages appear already after loading the files). >Yes, limmaGUI is a bit behind limma in this respect. (It is a whole >lot >more work to update a menu-driven package than a command line.) I understand. I hope in the future this will be added ? (I understand this has not high priority) >Gordon Thank you for your answers, Yours Bas van Breukelen ================================= Dr. Ir. B. van Breukelen PostDoc, Bioinformatics, Molecular genetics Dept. of Biology. Room N407: H.R. Kruytgebouw Padualaan 8 3574 CH Utrecht Tel: +31(0)30 253 3355 Mobile: +31(0)6 24 996046 e-mail: b.vanbreukelen@bio.uu.nl website: http://genomics.bio.uu.nl/ ================================= [[alternative HTML version deleted]]