Fwd: Question about normalizing one color Agilent array using loess
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@eugene-bolotin-2112
Last seen 8.4 years ago
Dear Bioconductor, I am running a protein binding microarray experiment(PBM). In this experiment protein is bound to double stranded microarray and visualized using antibody (red fluorescence). Basically this gives you a one color Agilent (gpr) microarray file with an additional gal file. I tried using loess and other normalizations on this setup using bioconductor, limma and marray package. I have run into some problems. One is the importing problem, which I solved using dummy column for green spectrum. The other problem is that all the normalization schemes in Limma and marray require a "two color" array. Does anyone know how to run normalization with just one color. My main problem is uneven fluorescence across the array. One side seems noticeably brighter than the other, and I want to even it out somehow, loess is supposed to do it. Thanks a lot -- Eugene Bolotin Ph.D. candidate Genetics Genomics and Bioinformatics University of California Riverside ybolo001 at student.ucr.edu Dr. Frances Sladek Lab
Microarray Genetics Normalization limma marray Microarray Genetics Normalization limma • 1.0k views
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@wolfgang-huber-3550
Last seen 2 days ago
EMBL European Molecular Biology Laborat…
Dear Eugene, marray's and limma's so-called loess normalisation aim to adjust for trends in the log-ratios (M) as a function of the log geometric mean intensity (A), but are not designed to deal with spatial trends on your arrays. You will need to decide whether the data is worth trying to adjust for such spatial trends computationally, by some other means, or whether you call it a data quality problem and address it by improved experimental procedures. Other people may be better qualified to advise on either of these. Best wishes Wolfgang ------------------------------------------------------------------ Wolfgang Huber EBI/EMBL Cambridge UK http://www.ebi.ac.uk/huber 13/05/2008 22:32 Eugene Bolotin a ?crit > Dear Bioconductor, > I am running a protein binding microarray experiment(PBM). In this > experiment protein is bound to double stranded microarray and > visualized using antibody (red fluorescence). Basically this gives you > a one color Agilent (gpr) microarray file with an additional gal file. > I tried using loess and other normalizations on this setup using > bioconductor, limma and marray package. I have run into some problems. > One is the importing problem, which I solved using dummy column for > green spectrum. The other problem is that all the normalization > schemes in Limma and marray require a "two color" array. Does anyone > know how to run normalization with just one color. My main problem is > uneven fluorescence across the array. One side seems noticeably > brighter than the other, and I want to even it out somehow, loess is > supposed to do it. > Thanks a lot > -- > Eugene Bolotin > Ph.D. candidate > Genetics Genomics and Bioinformatics > University of California Riverside > ybolo001 at student.ucr.edu > Dr. Frances Sladek Lab > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor
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Dear Eugene, I think you might be able to find some useful functions that work on one-channel arrays in the package aroma.light (e.g. normalizeQuantileSpline). You also should have no problem narmalizing your data with vsn. Also, some of the functions in the limma package demand two channels but they seem to treat the different channels independently (e.g. backgroundCorrect and normalizeForPrintorder). I made a few changes to some of these functions and got them to operate on single channel data. I am not a skilled or experienced programmer but these were minor changes and the functions seem to be working properly. I would be glad to send them to you to use at your own risk. :-) Cheers, Artur Veloso On Thu, May 15, 2008 at 6:19 AM, Wolfgang Huber <huber@ebi.ac.uk> wrote: > > > Dear Eugene, > > marray's and limma's so-called loess normalisation aim to adjust for > trends in the log-ratios (M) as a function of the log geometric mean > intensity (A), but are not designed to deal with spatial trends on your > arrays. > > You will need to decide whether the data is worth trying to adjust for > such spatial trends computationally, by some other means, or whether you > call it a data quality problem and address it by improved experimental > procedures. Other people may be better qualified to advise on either of > these. > > Best wishes > Wolfgang > > ------------------------------------------------------------------ > Wolfgang Huber EBI/EMBL Cambridge UK http://www.ebi.ac.uk/huber > > > 13/05/2008 22:32 Eugene Bolotin a écrit > > Dear Bioconductor, > > I am running a protein binding microarray experiment(PBM). In this > > experiment protein is bound to double stranded microarray and > > visualized using antibody (red fluorescence). Basically this gives you > > a one color Agilent (gpr) microarray file with an additional gal file. > > I tried using loess and other normalizations on this setup using > > bioconductor, limma and marray package. I have run into some problems. > > One is the importing problem, which I solved using dummy column for > > green spectrum. The other problem is that all the normalization > > schemes in Limma and marray require a "two color" array. Does anyone > > know how to run normalization with just one color. My main problem is > > uneven fluorescence across the array. One side seems noticeably > > brighter than the other, and I want to even it out somehow, loess is > > supposed to do it. > > Thanks a lot > > -- > > Eugene Bolotin > > Ph.D. candidate > > Genetics Genomics and Bioinformatics > > University of California Riverside > > ybolo001@student.ucr.edu > > Dr. Frances Sladek Lab > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@stat.math.ethz.ch > > https://stat.ethz.ch/mailman/listinfo/bioconductor > > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > > _______________________________________________ > Bioconductor mailing list > Bioconductor@stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor > [[alternative HTML version deleted]]
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