[BioC] Issue with limma and normalization of Agilent data generated with a 20-bit scan
White, Peter
Peter.White at nationwidechildrens.org
Mon Mar 15 22:47:52 CET 2010
Hi Wolfgang,
So with the new scanner from Agilent this data is not saturated. The scanner went from 16-bit (0-65,000) to 20-bit (0-1,048,576). All of these values are well below the new saturation point, yet they are not being normalized.
Thanks,
Peter
> -----Original Message-----
> From: Wolfgang Huber [mailto:whuber at embl.de]
> Sent: Monday, March 15, 2010 5:25 PM
> To: White, Peter
> Cc: 'Gordon K Smyth'; 'Bioconductor mailing list'
> Subject: Re: [BioC] Issue with limma and normalization of Agilent data
> generated with a 20-bit scan
>
>
> Dear Peter
>
> have you tried with different (i.e. smaller) values of the "span"
> parameter for the loess fit?
>
> The data seem badly saturated... I'd prefer avoiding the kind of
> saturation such as seen in the data you posted by better settings of
> the
> scanner, rather than doing post hoc loess normalisation.
>
> Best wishes
> Wolfgang
>
>
> White, Peter scripsit 15/03/10 15:53:
> > Dear Gordon,
> >
> > The plots are visible in the blog view on gmane.org:
> >
> >
> http://permalink.gmane.org/gmane.science.biology.informatics.conductor/
> 27731
> >
> > I thought you may be on to something with the weights but I tried it
> with and without a flag function (also double checked the Agilent file
> and the high intensity spots are not flagged). It really does look like
> the loess is just not fitted beyond for elements with an A value >
> 16??? These 20-bit scans from Agilent are quite new and I suspect most
> folks with just use the Agilent normalized data rather than starting
> with the raw data, so maybe this just hasn't been observed before now?
> >
> > Thanks,
> >
> > Peter
> >
> > Below is the code I used:
> >
> > library(limma)
> > agilentFiles <- list.files(pattern="U")
> > rawObj <- read.maimages(agilentFiles,
> > columns = list(G = "gMedianSignal", Gb = "gBGMedianSignal",
> > R = "rMedianSignal", Rb = "rBGMedianSignal"),
> > annotation= c("ProbeName", "SystematicName","ControlType"))
> > #Remove spike controls and remove background signals
> > bgObj <- rawObj
> > posControls <- grep(T,rawObj$genes$ControlType == 1)
> > bgObj$G[posControls,] <- NA
> > bgObj$R[posControls,] <- NA
> > bgObj$Gb <- bgObj$Rb <- NULL
> > #Loess normalize
> > normObj <- normalizeWithinArrays(bgObj, method="loess",
> weights=NULL)
> > #Plot MvA
> > for (i in 1:ncol(normObj)) {
> > figureName <- paste(i, " MvA Plots")
> > mat <- matrix(c(3,1,2),nrow=3,ncol=1)
> > layout(mat,heights=c(1,10,10))
> > plotMA(rawObj, array=i, main = "Pre-Normalization MvA",
> > ylim=c(-3.5,3.5), zero.weights=TRUE)
> > abline(0,0)
> > plotMA(normObj, array=i, main = "Normalized MvA",
> > ylim=c(-3.5,3.5), zero.weights=TRUE)
> > abline(0,0)
> > layout(1)
> > mtext(figureName, cex=1.25, line=3)
> > savePlot(filename=figureName, type=c("png"), device=dev.cur())
> > }
> >
> >> sessionInfo()
> > R version 2.10.1 (2009-12-14)
> > i386-pc-mingw32
> >
> > locale:
> > [1] LC_COLLATE=English_United States.1252
> > [2] LC_CTYPE=English_United States.1252
> > [3] LC_MONETARY=English_United States.1252
> > [4] LC_NUMERIC=C
> > [5] LC_TIME=English_United States.1252
> >
> > attached base packages:
> > [1] grDevices datasets splines graphics stats tcltk utils
> > [8] methods base
> >
> > other attached packages:
> > [1] limma_3.2.2 svSocket_0.9-48 TinnR_1.0.3 R2HTML_1.59-1
> > [5] Hmisc_3.7-0 survival_2.35-9
> >
> > loaded via a namespace (and not attached):
> > [1] cluster_1.12.1 grid_2.10.1 lattice_0.18-3 svMisc_0.9-56
> tools_2.10.1
> >
> >> -----Original Message-----
> >> From: Gordon K Smyth [mailto:smyth at wehi.EDU.AU]
> >> Sent: Saturday, March 13, 2010 6:39 PM
> >> To: White, Peter
> >> Cc: Bioconductor mailing list
> >> Subject: [BioC] Issue with limma and normalization of Agilent data
> >> generated with a 20-bit scan
> >>
> >> Dear Peter,
> >>
> >> You can't send attachments to the Bioconductor mailing list, so I
> have
> >> not
> >> seen your plots. However I am not aware of any issue such as you
> >> describe. The limma function normalizeWithinArrays includes all
> spots
> >> in
> >> the normalization, regardless of how large the A-value is. You
> haven't
> >> shown us any code, or any problem we can reproduce, so we can't tell
> >> whether or not you're doing something wrong. We don't know whether
> >> you're
> >> using probe weights, whether you've filtered control spots, etc etc.
> >>
> >> Best wishes
> >> Gordon
> >>
> >>> Date: Fri, 12 Mar 2010 10:21:41 -0500
> >>> From: "White, Peter" <Peter.White at nationwidechildrens.org>
> >>> To: "'bioconductor at stat.math.ethz.ch'"
> >>> <bioconductor at stat.math.ethz.ch>
> >>> Subject: [BioC] Issue with limma and normalization of Agilent data
> >>> generated with a 20-bit scan
> >>> Content-Type: text/plain
> >>>
> >>> I have noticed an issue with the limma normalizeWithinArrays
> function
> >>> (and also with marray and maNorm). When normalizing two color data
> >>> generated with an Agilent 20-bt scanner it fails to normalize the
> >> high
> >>> intensity data (i.e. any points with an A value > 16). In our
> dataset
> >> we
> >>> have in excess of 400 elements with red and green intensities
> ranging
> >>> from 65500 to 475100. When we loess normalize the data any points
> >> beyond
> >>> A=16 appear to be untouched by the normalization. If the attached
> >>> figures come through this should be clear - when using maNorm and
> >> maPlot
> >>> it will plot the loess line and you can see it stop at 16.
> >>>
> >>> Is it possible for loess normalization to be extended to this
> higher
> >>> intensity data? Or am I just doing something wrong?
> >>>
> >>> Thanks,
> >>>
> >>> Peter
> >>>
> >>>
> >>> Peter White, Ph.D.
> >>> Director, Biomedical Genomics
> Core<http://genomics.nchresearch.org/>
> >>> Research Assistant Professor of Pediatrics
> >>> The Research Institute at
> >>> Nationwide Children's Hospital and
> >>> The Ohio State University
> >>>
> >>> Mailing Address:
> >>>
> >>> The Research Institute at
> >>> Nationwide Children's Hospital
> >>> 700 Children's Drive, W510
> >>> Columbus, OH 43205
> >>>
> >>> Assistant (Jennifer Neelans): (614) 722-2915
> >>> Office: (614) 355-2671
> >>> Lab: (614) 355-5252
> >>> Fax: (614) 722-2818
> >>> Web: http://genomics.nchresearch.org/
> >>
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> >>
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> >
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>
> --
>
> Best wishes
> Wolfgang
>
>
> --
> Wolfgang Huber
> EMBL
> http://www.embl.de/research/units/genome_biology/huber/contact
>
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