[BioC] Normalize background on marray Agilent object

James W. MacDonald jmacdon at uw.edu
Fri Jun 21 16:18:40 CEST 2013

Hi Guillermo,

On 6/21/2013 3:06 AM, Guillermo Marco Puche wrote:
> Dear Gordon,
> Thank you for your answer. I'll look further into Agilent array image
> files with limma.
> As I said the problem is that i'm not currently reading image from
> Agilent array, but the text data file with marray library and loading it
> into a maData object like this:

Please note that the read.maimages function doesn't read image files - 
it reads in the same text files you are reading with read.Agilent.

Your original question had to do with the 'correct' background 
correction to use for your Agilent array data. Gordon has therefore 
suggested that you use the 'normexp' method in limma. This does of 
course require you to switch to a different package, but limma tends to 
get better support than marray, so you might be wise to make the switch.

But to your original point, you are asking a question that might not 
have a definitive answer. There is no 'best' way to do a background 
correction. There are methods that seem to do a reasonable job over a 
range of experiments, and if I understand correctly, this is why Gordon 
is suggesting you use normexp. But which method might be best for your 
particular situation will be difficult for anybody to predict.



> maData = read.Agilent(fnames=input , path=NULL, name.Gf = "gMedianSignal", name.Gb = "gBGMedianSignal", name.Rf = "rMedianSignal", name.Rb = "rBGMedianSignal", name.W= NULL, layout = NULL, gnames = NULL, targets = NULL, notes=NULL, skip=NULL, sep="\t", quote="\"", DEBUG=FALSE, info.id=NULL)
>> On 06/20/2013 01:11 PM, Gordon K Smyth wrote:
>>> Dera Guillermo,
>>> The usual process is to (1) background correct the foreground
>>> intensities with respect to the background, then (2) normalize the
>>> M-values (log-ratios).
>>> For an Agilent two colour array, I do this by:
>>>    library(limma)
>>>    RG<- read.maimages(files, source="agilent")
>>>    RGb<- backgroundCorrect(RG, method="normexp")
>>>    MA<- normalizeWithinArrays(RGb, method="loess")
>>> although it is sometimes a good idea to remove positive control
>>> probes before the normalization step.
>>> A recent example using this pipeline is:
>>> http://www.biomedcentral.com/1471-2105/14/165
>>> Best wishes
>>> Gordon
>>>> Date: Wed, 19 Jun 2013 22:38:34 +0200
>>>> From: Guillermo Marco Puche<guillermo.marco at sistemasgenomicos.com>
>>>> To: "bioconductor at r-project.org"<bioconductor at r-project.org>
>>>> Subject: [BioC] Normalize background on marray Agilent object
>>>> Hello,
>>>> I'm currently trying to normalize rBG values for a marray object.
>>>> Data origin is Agilent dual channel array. I've loaded information with
>>>> readAgilent() function.
>>>> What's the correct way to normalize the data? I would like to normalize
>>>> background information first maNorm function manual isn't very
>>>> clarifying for me.
>>>> Thanks !
>>>> Best regards,
>>>> Guillermo.
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James W. MacDonald, M.S.
University of Washington
Environmental and Occupational Health Sciences
4225 Roosevelt Way NE, # 100
Seattle WA 98105-6099

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