vsn in limma or marray/arrayMagic
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@aeschiellumcnl-1083
Last seen 8.6 years ago
Dear all, I do have a probably simple question, is running "vsn" in limma (normalizeBetweenArrays) the same as running "vsn" in marray or arrayMagic? My Boxplots suggest that there is a difference and I was wondering if someone can give me a hint why that might be. My particular problem is that I would like to use the fact that my probes are spotted in duplicates but not the spacing limma expects. I therefore want to first normalize all my arrays (22), then export the data to a text file and resort that file to get the right spacing. If I am right I should be able to read such a text file with read.maimages into limma and then continue with my analysis. Therefore I would like to know if I need to do my normalization with marray (or arrayMagic) or if I can use vsn in limma? Many thanks in advance, Anja [[alternative HTML version deleted]]
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@gordon-smyth
Last seen 25 minutes ago
WEHI, Melbourne, Australia
> Date: Tue, 25 Jan 2005 11:30:43 +0100 > From: <a.e.schiel@lumc.nl> > Subject: [BioC] vsn in limma or marray/arrayMagic > To: <bioconductor@stat.math.ethz.ch> > Message-ID: <8A6C92C9EC1DD74AA66886D8DC3A38CE15545C@mailb.lumc.nl> > Content-Type: text/plain > > Dear all, > I do have a probably simple question, is running "vsn" in limma (normalizeBetweenArrays) the same > as running "vsn" in marray or arrayMagic? Running "vsn" in limma is exactly the same as as running "vsn" in vsn, in fact the vsn package is simply used as a plug-in. As far as I know, you can't run vsn from marray. What do you mean? If arrayMagic doesn't agree with limma and hence with vsn, then you need to give precise code to show the discrepancy. Perhap you have not used the code correctly. > My Boxplots suggest that there is a difference and I was wondering if someone can give me a hint > why that might be. > My particular problem is that I would like to use the fact that my probes are spotted in > duplicates but not the spacing limma expects. I therefore want to first normalize all my arrays > (22), then export the data to a text file and resort that file to get the right spacing. If I am > right I should be able to read such a text file with read.maimages into limma and then continue > with my analysis. Therefore I would like to know if I need to do my normalization with marray (or > arrayMagic) or if I can use vsn in limma? I am puzzled why you would think there is a need to write out to a text file at any stage. Gordon > Many thanks in advance, > Anja
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Entering edit mode
@aeschiellumcnl-1083
Last seen 8.6 years ago
I am not sure you got my last mail so I sent it again, Thanks, Anja ----Original Message----- From: Gordon K Smyth [mailto:smyth@wehi.EDU.AU] Sent: Tuesday, January 25, 2005 12:51 PM To: Schiel, A.E. (HKG) Cc: bioconductor@stat.math.ethz.ch Subject: [BioC] vsn in limma or marray/arrayMagic > Date: Tue, 25 Jan 2005 11:30:43 +0100 > From: <a.e.schiel@lumc.nl> > Subject: [BioC] vsn in limma or marray/arrayMagic > To: <bioconductor@stat.math.ethz.ch> > Message-ID: <8A6C92C9EC1DD74AA66886D8DC3A38CE15545C@mailb.lumc.nl> > Content-Type: text/plain > > Dear all, > I do have a probably simple question, is running "vsn" in limma (normalizeBetweenArrays) the same > as running "vsn" in marray or arrayMagic? Running "vsn" in limma is exactly the same as as running "vsn" in vsn, in fact the vsn package is simply used as a plug-in. >>That's good to know, I was a bit concerned because you call vsn in limma with the BetweenArray call instead of the WithinArray >>command and I had the feeling that that call does normalize in a different way. If it is ecaxtly the same than I can do my resorting of >>genes with the MA object I create with the normalization and that saves a lot of time! As far as I know, you can't run vsn from marray. What do you mean? >>I was actually running vsn from arrayMagic (sorry if I didn't make that clear) and was just trying to figure out how to do that in marray >>(but if you say it doesn't work than I can probably stop!) If arrayMagic doesn't agree with limma and hence with vsn, then you need to give precise code to show the discrepancy. Perhap you have not used the code correctly. >>For arrayMagic I used >>description <- readpDataSlides(fileName, loadPath=loadPath, deleteBlanks=TRUE, verbose=TRUE) >>aD <- readIntensities(description, fileNameColumn ="fileName", slideNameColumn="slideName", loadPath=loadPath, >>+ type="GenePix") >>norm <- normalise (aD, subtractBackground=TRUE, method="vsn") >>in limma I used >>RG <- read.maimages(targets$fileName, source="genepix") >>RG$genes <- readGAL("sigmamousec_020603.gal") >>RG$printer <- getLayout(RG$genes) >>spottypes <- readSpotTypes("SpotTypes.txt") >>RG$genes$Status <- controlStatus(spottypes, RG) >>MA <- normalizeWithinArrays(RG) >>MAvsn <-normalizeBetweenArrays(RG, method="vsn") >>in both cases a defined targets before these functions. The Boxplot generated with the quality-function in arrayMagic looks less "nice" >>than the one I got in limma with >>boxplot(MAvsn$M~col(MAvsn$M), names=colnames(MAvsn$M), main="vsn data", col="darkgreen") >>but in fact I do not know how the plots are actually generated in arrayMagic!? Maybe the boxplot definition is not the default one. > My Boxplots suggest that there is a difference and I was wondering if someone can give me a hint > why that might be. > My particular problem is that I would like to use the fact that my probes are spotted in > duplicates but not the spacing limma expects. I therefore want to first normalize all my arrays > (22), then export the data to a text file and resort that file to get the right spacing. If I am > right I should be able to read such a text file with read.maimages into limma and then continue > with my analysis. Therefore I would like to know if I need to do my normalization with marray (or > arrayMagic) or if I can use vsn in limma? I am puzzled why you would think there is a need to write out to a text file at any stage. >> Well I have sorting command lines by know which will transform my data into the recommended form of duplicate >>spots next to each other but I was not sure if I can use that on the MAList objects in limma directly. But I tried this now and it worked >>dataset <- (Ma) >>ngenes <- nrow(dataset) >>ncolumns <-ncol(dataset) >>ind.gene <-rep(1:(ngenes/24),24) >>ii.order <- order(ind.gene) >>n.dataset <-dataset[ii.order,] >>then continue with the limma analysis >>Thanks, you remarks were very helpful (I am just starting with R so I lack a few skills sorry for that!) >> Anja Gordon > Many thanks in advance, > Anja Anja E. Schiel, Ph.D. Departments of General Internal Medicine and Human Genetics Leiden University Medical Center PO Box 9503 2300 RA Leiden The Netherlands tel: -31-(0)71-5276067 fax: -31-(0)71-5276075 [[alternative HTML version deleted]] ADD COMMENT 0 Entering edit mode vsn in limma vs. vsn in arrayMagic The major difference in your code example which can explain the difference in the result is to subtract the background in case of arrayMagic but not in limma. Secondly, arrayMagic does by default use the "Median" columns like "F532.Median" instead of "F532.Mean" - both are given in the GenePix file. You may force arrayMagic to use the "Mean" as well. In your example this would be: dataColumns <- c("F532.Mean", "B532.Mean", "F635.Mean", "B635.Mean") names(dataColumns) <- c("greenForeground", "greenBackground", "redForeground", "redBackground") aD <- readIntensities(description, loadPath=loadPath, type="GenePix", dataColumns=dataColumns) Best Regards Andreas A.E.Schiel@lumc.nl wrote: > I am not sure you got my last mail so I sent it again, > Thanks, Anja > > ----Original Message----- > From: Gordon K Smyth [mailto:smyth@wehi.EDU.AU] > Sent: Tuesday, January 25, 2005 12:51 PM > To: Schiel, A.E. (HKG) > Cc: bioconductor@stat.math.ethz.ch > Subject: [BioC] vsn in limma or marray/arrayMagic > > > >>Date: Tue, 25 Jan 2005 11:30:43 +0100 >>From: <a.e.schiel@lumc.nl> >>Subject: [BioC] vsn in limma or marray/arrayMagic >>To: <bioconductor@stat.math.ethz.ch> >>Message-ID: <8A6C92C9EC1DD74AA66886D8DC3A38CE15545C@mailb.lumc.nl> >>Content-Type: text/plain >> >>Dear all, >>I do have a probably simple question, is running "vsn" in limma (normalizeBetweenArrays) the same >>as running "vsn" in marray or arrayMagic? > > > Running "vsn" in limma is exactly the same as as running "vsn" in vsn, in fact the vsn package is > simply used as a plug-in. > > >>>That's good to know, I was a bit concerned because you call vsn in limma with the BetweenArray call instead of the WithinArray >>>command and I had the feeling that that call does normalize in a different way. If it is ecaxtly the same than I can do my resorting of >>>genes with the MA object I create with the normalization and that saves a lot of time! > > > As far as I know, you can't run vsn from marray. What do you mean? > > >>>I was actually running vsn from arrayMagic (sorry if I didn't make that clear) and was just trying to figure out how to do that in marray >>>(but if you say it doesn't work than I can probably stop!) > > > If arrayMagic doesn't agree with limma and hence with vsn, then you need to give precise code to > show the discrepancy. Perhap you have not used the code correctly. > > >>>For arrayMagic I used > > >>>description <- readpDataSlides(fileName, loadPath=loadPath, deleteBlanks=TRUE, verbose=TRUE) > > >>>aD <- readIntensities(description, fileNameColumn ="fileName", slideNameColumn="slideName", loadPath=loadPath, >>>+ type="GenePix") >>>norm <- normalise (aD, subtractBackground=TRUE, method="vsn") > > >>>in limma I used > > >>>RG <- read.maimages(targets$fileName, source="genepix") >>>RG$genes <- readGAL("sigmamousec_020603.gal") >>>RG$printer <- getLayout(RG$genes) >>>spottypes <- readSpotTypes("SpotTypes.txt") >>>RG$genes$Status <- controlStatus(spottypes, RG) > > >>>MA <- normalizeWithinArrays(RG) >>>MAvsn <-normalizeBetweenArrays(RG, method="vsn") > > >>>in both cases a defined targets before these functions. The Boxplot generated with the quality-function in arrayMagic looks less "nice" >>>than the one I got in limma with >>>boxplot(MAvsn$M~col(MAvsn$M), names=colnames(MAvsn$M), main="vsn data", col="darkgreen") > > >>>but in fact I do not know how the plots are actually generated in arrayMagic!? Maybe the boxplot definition is not the default one. > > >>My Boxplots suggest that there is a difference and I was wondering if someone can give me a hint >>why that might be. >>My particular problem is that I would like to use the fact that my probes are spotted in >>duplicates but not the spacing limma expects. I therefore want to first normalize all my arrays >>(22), then export the data to a text file and resort that file to get the right spacing. If I am >>right I should be able to read such a text file with read.maimages into limma and then continue >>with my analysis. Therefore I would like to know if I need to do my normalization with marray (or >>arrayMagic) or if I can use vsn in limma? > > > I am puzzled why you would think there is a need to write out to a text file at any stage. > > >>>Well I have sorting command lines by know which will transform my data into the recommended form of duplicate >>>spots next to each other but I was not sure if I can use that on the MAList objects in limma directly. But I tried this now and it worked > > >>>dataset <- (Ma) >>>ngenes <- nrow(dataset) >>>ncolumns <-ncol(dataset) >>>ind.gene <-rep(1:(ngenes/24),24) >>>ii.order <- order(ind.gene) >>>n.dataset <-dataset[ii.order,] >>>then continue with the limma analysis > > >>>Thanks, you remarks were very helpful (I am just starting with R so I lack a few skills sorry for that!) >>>Anja > > > > > Gordon > > >>Many thanks in advance, >>Anja > > > Anja E. Schiel, Ph.D. > Departments of General Internal Medicine and Human Genetics > Leiden University Medical Center > PO Box 9503 > 2300 RA Leiden > The Netherlands > tel: -31-(0)71-5276067 > fax: -31-(0)71-5276075 > > > > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor -- -------------------------------------------------------------- Andreas Buness Molecular Genome Analysis (B050) German Cancer Research Center room TP3 2.109 Im Neuenheimer Feld 580 phone ++49 6221 42-4716 D-69120 Heidelberg Germany ----------- http://www.dkfz.de/abt0840/home/buness/WWW/ ------