[BioC] Issue with limma and normalization of Agilent data generated with a 20-bit scan

Gordon K Smyth smyth at wehi.EDU.AU
Sun Mar 14 00:39:27 CET 2010

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

> 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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