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

[White, Peter]

Here is a plot made from data normalized with the limma package.

From: White, Peter
Sent: Friday, March 12, 2010 10:22 AM
To: 'bioconductor at stat.math.ethz.ch'
Subject: Issue with limma and normalization of Agilent data generated with a 20-bit scan

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

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