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Question: RobustSpline normalization method applied to agilent data
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gravatar for Priscila Grynberg
5.7 years ago by
Priscila Grynberg380 wrote:
Dear BioCs, I received some agilent data to analyse using Limma, and I'm facing a problem when I tried to apply robustspline normalization within arrays method. ##Reading the files: RG = read.maimages(targets,,source="agilent", columns = list(G = "gMedianSignal", Gb = "gBGMedianSignal", R = "rMedianSignal", Rb = "rBGMedianSignal"), annotation = c("Row", "Col","FeatureNum", "ControlType","ProbeName","SystematicName")) ##Background correction: RGn <- backgroundCorrect (RG, method="normexp", offset=50) #Normalization step MA <- normalizeWithinArrays(RGn, method="robustspline") Error in normalizeWithinArrays(RGn, method = "robustspline") : Layout argument not specified ##Setting the printer information: nr <- length(unique(RG$genes$Row)) nc <- length(unique(RG$genes$Col)) RG$printer <- list(ngrid.r=1,ngrid.c=1,nspot.r=nr,nspot.c=nc) ## Trying again MA <- normalizeWithinArrays(RGn, RG$printer, method="robustspline") Error in X[O, ] <- ns(A[O], df = df, intercept = TRUE) : number of items to replace is not a multiple of replacement length I don't have the gal file like for this agilent slides. Can this be exactly the problem? Do you have any sugestions? Cheers, Priscila -- Priscila Grynberg, D.Sc.- Bioinformatics Laboratório de Genética Bioquímica Universidade Federal de Minas Gerais Tel: +55 31 3409-2628 CV: http://lattes.cnpq.br/8808643075395963 [[alternative HTML version deleted]]
ADD COMMENTlink modified 5.7 years ago by Gordon Smyth32k • written 5.7 years ago by Priscila Grynberg380
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gravatar for Heidi Dvinge
5.7 years ago by
Heidi Dvinge2.0k
Heidi Dvinge2.0k wrote:
Hi Priscilla, > Dear BioCs, > > I received some agilent data to analyse using Limma, and I'm facing a > problem when I tried to apply robustspline normalization within arrays > method. > > ##Reading the files: > > RG = read.maimages(targets,,source="agilent", columns = list(G = > "gMedianSignal", Gb = "gBGMedianSignal", R = "rMedianSignal", Rb = > "rBGMedianSignal"), annotation = c("Row", "Col","FeatureNum", > "ControlType","ProbeName","SystematicName")) > > ##Background correction: > > RGn <- backgroundCorrect (RG, method="normexp", offset=50) > > #Normalization step > > MA <- normalizeWithinArrays(RGn, method="robustspline") > > Error in normalizeWithinArrays(RGn, method = "robustspline") : Layout > argument not specified > > ##Setting the printer information: > > nr <- length(unique(RG$genes$Row)) > > nc <- length(unique(RG$genes$Col)) > Does the number of spots in your file actually equal nr*nc? Sometimes GAL files for Agilent doesn't contain entries for spots on the array where nothing has been printed. If you look at the combinations of RG$genes$Row and RG$genes$Col in your file, you may find that some of them are missing. If so, you will perhaps need to add some dummy values manually in order to use robustspline. HTH \Heidi > RG$printer <- list(ngrid.r=1,ngrid.c=1,nspot.rr,nspot.cc) > > ## Trying again > > MA <- normalizeWithinArrays(RGn, RG$printer, method="robustspline") > > Error in X[O, ] <- ns(A[O], df = df, intercept = TRUE) : number of items > to > replace is not a multiple of replacement length > > I don't have the gal file like for this agilent slides. Can this be > exactly > the problem? Do you have any sugestions? > > > Cheers, > > Priscila > > > -- > Priscila Grynberg, D.Sc.- Bioinformatics > Laborat?rio de Gen?tica Bioqu?mica > Universidade Federal de Minas Gerais > Tel: +55 31 3409-2628 > CV: http://lattes.cnpq.br/8808643075395963 > > [[alternative HTML version deleted]] > > _______________________________________________ > 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
ADD COMMENTlink written 5.7 years ago by Heidi Dvinge2.0k
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gravatar for Gordon Smyth
5.7 years ago by
Gordon Smyth32k
Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Gordon Smyth32k wrote:
Dear Priscila, You can't apply robust-spline normalization to an Agilent array. Robust-spline in limma is intended to moderate the loess curves over different print-tip groups. But Agilent arrays don't have print-tips. For an Agilent arrays, only global loess normalization is meaningful, and that does not require the layout. Best wishes Gordon > Date: Wed, 7 Mar 2012 09:16:22 -0300 > From: Priscila Grynberg <priscilag at="" gmail.com=""> > To: <bioconductor at="" stat.math.ethz.ch=""> > Subject: [BioC] RobustSpline normalization method applied to agilent > data > > Dear BioCs, > > I received some agilent data to analyse using Limma, and I'm facing a > problem when I tried to apply robustspline normalization within arrays > method. > > ##Reading the files: > > RG = read.maimages(targets,,source="agilent", columns = list(G = > "gMedianSignal", Gb = "gBGMedianSignal", R = "rMedianSignal", Rb = > "rBGMedianSignal"), annotation = c("Row", "Col","FeatureNum", > "ControlType","ProbeName","SystematicName")) > > ##Background correction: > > RGn <- backgroundCorrect (RG, method="normexp", offset=50) > > #Normalization step > > MA <- normalizeWithinArrays(RGn, method="robustspline") > > Error in normalizeWithinArrays(RGn, method = "robustspline") : Layout > argument not specified > > ##Setting the printer information: > > nr <- length(unique(RG$genes$Row)) > > nc <- length(unique(RG$genes$Col)) > > RG$printer <- list(ngrid.r=1,ngrid.c=1,nspot.r=nr,nspot.c=nc) > > ## Trying again > > MA <- normalizeWithinArrays(RGn, RG$printer, method="robustspline") > > Error in X[O, ] <- ns(A[O], df = df, intercept = TRUE) : number of items to > replace is not a multiple of replacement length > > I don't have the gal file like for this agilent slides. Can this be exactly > the problem? Do you have any sugestions? > > > Cheers, > > Priscila > > > -- > Priscila Grynberg, D.Sc.- Bioinformatics > Laborat?rio de Gen?tica Bioqu?mica > Universidade Federal de Minas Gerais > Tel: +55 31 3409-2628 > CV: http://lattes.cnpq.br/8808643075395963 ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:5}}
ADD COMMENTlink written 5.7 years ago by Gordon Smyth32k
Dear Gordon, Thanks for the explanation. I didnt realize robust spline was dependent of print-tips. Sincerely, Priscila On Thursday, March 8, 2012, Gordon K Smyth <smyth@wehi.edu.au> wrote: > Dear Priscila, > > You can't apply robust-spline normalization to an Agilent array. Robust-spline in limma is intended to moderate the loess curves over different print-tip groups. But Agilent arrays don't have print-tips. For an Agilent arrays, only global loess normalization is meaningful, and that does not require the layout. > > Best wishes > Gordon > >> Date: Wed, 7 Mar 2012 09:16:22 -0300 >> From: Priscila Grynberg <priscilag@gmail.com> >> To: <bioconductor@stat.math.ethz.ch> >> Subject: [BioC] RobustSpline normalization method applied to agilent >> data >> >> Dear BioCs, >> >> I received some agilent data to analyse using Limma, and I'm facing a >> problem when I tried to apply robustspline normalization within arrays >> method. >> >> ##Reading the files: >> >> RG = read.maimages(targets,,source="agilent", columns = list(G = >> "gMedianSignal", Gb = "gBGMedianSignal", R = "rMedianSignal", Rb = >> "rBGMedianSignal"), annotation = c("Row", "Col","FeatureNum", >> "ControlType","ProbeName","SystematicName")) >> >> ##Background correction: >> >> RGn <- backgroundCorrect (RG, method="normexp", offset=50) >> >> #Normalization step >> >> MA <- normalizeWithinArrays(RGn, method="robustspline") >> >> Error in normalizeWithinArrays(RGn, method = "robustspline") : Layout >> argument not specified >> >> ##Setting the printer information: >> >> nr <- length(unique(RG$genes$Row)) >> >> nc <- length(unique(RG$genes$Col)) >> >> RG$printer <- list(ngrid.r=1,ngrid.c=1,nspot.r=nr,nspot.c=nc) >> >> ## Trying again >> >> MA <- normalizeWithinArrays(RGn, RG$printer, method="robustspline") >> >> Error in X[O, ] <- ns(A[O], df = df, intercept = TRUE) : number of items to >> replace is not a multiple of replacement length >> >> I don't have the gal file like for this agilent slides. Can this be exactly >> the problem? Do you have any sugestions? >> >> >> Cheers, >> >> Priscila >> >> >> -- >> Priscila Grynberg, D.Sc.- Bioinformatics >> Laboratório de Genética Bioquímica >> Universidade Federal de Minas Gerais >> Tel: +55 31 3409-2628 >> CV: http://lattes.cnpq.br/8808643075395963 > > ______________________________________________________________________ > The information in this email is confidential and inte...{{dropped:12}}
ADD REPLYlink written 5.7 years ago by Priscila Grynberg380
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