Dear limma users and developers, At our laboratory we analyze our MA data using limma. Currently we have created a dedicated array on which the same set of genes is spotted three times on one slide. Actually it is one design spotted three times. In Imagene you can now create a META gid for (in this case) the 4*4 grids. This grid you call grid A.. Then you can copy it several times (thereby creating metagrid B and C). You then end up after quantification with a large imagene file having the information of the three metagrids: (its like having three slides on one slide!) e.g. Field GridRow GridCol SpotRow SpotCol ID A 1 1 1 1 Pp11 A 1 1 1 2 Pp12 .............. A 4 4 17 17 P.. B 1 1 1 1 Pp11 B 1 1 1 2 Pp12 .............. B 4 4 17 17 P.. C 1 1 1 1 Pp11 C 1 1 1 2 Pp12 .............. C 4 4 17 17 P.. 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 ? 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. 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. 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). I hope that the developers can implement these suggestions/workarounds for these problems ? 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/

Hi, we routinely analyze Imagene files with BioC, so perhaps I can answer some of your problems. First, you have to define one metagrid for all your spots, BioC won't understand fields. We have a 4x6 grid spotted twice on the slides, so we have defined a 4x12 metagrid. I guess you can modify your imagene output text files in e.g. excel to cope with the grid problem, but I find that tedious. So define your grid in Imagene once and for all. readTargets: I do it exactly the way you describe, first readTargets and the matrix. Is that a problem? It's just two lines of code... Spottypes: Be sure that the heading of your ID column in the spottypes file is "Gene ID" (exact spelling but without quotes), then controlStatus (which I assume you meant when you wrote controlTypes) will match to the right column in RG (RG@genes@Gene ID). Hope this helps Steen -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces@stat.math.ethz.ch] On Behalf Of Dr. Ir. B. van Breukelen Sent: 1. december 2004 13:23 To: bioconductor@stat.math.ethz.ch Subject: [BioC] Limma and limmaGUI with Imagene files, problems/bugs report Dear limma users and developers, At our laboratory we analyze our MA data using limma. Currently we have created a dedicated array on which the same set of genes is spotted three times on one slide. Actually it is one design spotted three times. In Imagene you can now create a META gid for (in this case) the 4*4 grids. This grid you call grid A.. Then you can copy it several times (thereby creating metagrid B and C). You then end up after quantification with a large imagene file having the information of the three metagrids: (its like having three slides on one slide!) e.g. Field GridRow GridCol SpotRow SpotCol ID A 1 1 1 1 Pp11 A 1 1 1 2 Pp12 .............. A 4 4 17 17 P.. B 1 1 1 1 Pp11 B 1 1 1 2 Pp12 .............. B 4 4 17 17 P.. C 1 1 1 1 Pp11 C 1 1 1 2 Pp12 .............. C 4 4 17 17 P.. 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 ? 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. 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. 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). I hope that the developers can implement these suggestions/workarounds for these problems ? 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/ _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://stat.ethz.ch/mailman/listinfo/bioconductor

> Date: Wed, 1 Dec 2004 13:22:53 +0100 > From: "Dr. Ir. B. van Breukelen" <b.vanbreukelen@bio.uu.nl> > Subject: [BioC] Limma and limmaGUI with Imagene files, problems/bugs > report > To: <bioconductor@stat.math.ethz.ch> > Message-ID: <solis102nqptgfryilo000000c9@solis102.soliscom.uu.nl> > Content-Type: text/plain; charset="us-ascii" > > Dear limma users and developers, > > At our laboratory we analyze our MA data using limma. Currently we have > created a dedicated array on which the same set of genes is spotted three > times on one slide. Actually it is one design spotted three times. In > Imagene you can now create a META gid for (in this case) the 4*4 grids. This > grid you call grid A.. Then you can copy it several times (thereby creating > metagrid B and C). You then end up after quantification with a large imagene > file having the information of the three metagrids: (its like having three > slides on one slide!) > e.g. > Field GridRow GridCol SpotRow SpotCol ID > A 1 1 1 1 Pp11 > A 1 1 1 2 Pp12 > .............. > A 4 4 17 17 P.. > B 1 1 1 1 Pp11 > B 1 1 1 2 Pp12 > .............. > B 4 4 17 17 P.. > C 1 1 1 1 Pp11 > C 1 1 1 2 Pp12 > .............. > C 4 4 17 17 P.. > > 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. 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? > 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". > 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.) Gordon > I hope that the developers can implement these suggestions/workarounds for > these problems ? > > 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/ > > > > ------------------------------ > > Message: 3 > Date: Wed, 1 Dec 2004 13:54:46 +0100 > From: "STKH \(Steen Krogsgaard\)" <stkh@novozymes.com> > Subject: RE: [BioC] Limma and limmaGUI with Imagene files, > problems/bugs report > To: <bioconductor@stat.math.ethz.ch> > Message-ID: > <934F95E71B6C9347A873C42AE3C196190213CBAF@NZT0004E.dknz.nzcorp.net> > Content-Type: text/plain; charset="us-ascii" > > Hi, > > we routinely analyze Imagene files with BioC, so perhaps I can answer > some of your problems. > > First, you have to define one metagrid for all your spots, BioC won't > understand fields. We have a 4x6 grid spotted twice on the slides, so we > have defined a 4x12 metagrid. I guess you can modify your imagene output > text files in e.g. excel to cope with the grid problem, but I find that > tedious. So define your grid in Imagene once and for all. > > readTargets: I do it exactly the way you describe, first readTargets and > the matrix. Is that a problem? It's just two lines of code... > > Spottypes: Be sure that the heading of your ID column in the spottypes > file is "Gene ID" (exact spelling but without quotes), then > controlStatus (which I assume you meant when you wrote controlTypes) > will match to the right column in RG (RG@genes@Gene ID). > > Hope this helps > Steen