[BioC] summarizing probe intensites before or after normalization- 1. how to do with RMA 2. Opinions?

[k. brand]

Dear All,

I compared two normalization approaches for an experiment using twelve 
affy 430-2.0 chips. (histogram plot comparing bith methods forwarded on 
request).

#1. RMA
library(affy)
data <- ReadAffy()
datarma <- rma(data)
exprs2excel(datarma, file="dataRMA.csv")

Plotting histograms of the output shows arrays NOT perfectly aligning at 
the means and spreads.

I used a custom script to effect a quantile normalization on MAS5 
preprocessed but unnormalized data-

#2. Mas5 sans interchip normalization
library(affy)
data <- ReadAffy()
datamas5sannorm <- mas5(data, normalize=FALSE)
exprs2excel(datamas5sannorm, file="datamas5sannorm.csv")
f.qnorm <- function(x,qinit=0.75,perc=100)  {...

The means and spreads of this normalization approach do align perfectly.

THUS- summarizing probe intensites before or after normalization does 
appear to make a noticeable difference, as may be expected.

My questions/requests-

1. Help to effect Bolstad normalization of the RMA preprocessed and 
summarized data. Whilst I succeed in generating unnormalized RMA 
preprocessed data with-

library(affy)
data <- ReadAffy()
datarma <- rma(data, normalize=FALSE)

As a result of my limited R experience, I failed in finding a method to 
effect Bolstad (quantile) normalization on this output.

2. Thoughts/comments on the benefits/caveats of normalizing before or 
after summarizing probe intensities.

I look forward to any thoughts, advice & suggestions from users.

thanks in advance,

Karl


===========================================

   > sessionInfo()
Version 2.3.0 (2006-04-24)
i386-pc-mingw32

attached base packages:
[1] "tools"     "methods"   "stats"     "graphics"  "grDevices" "utils"
       "datasets"  "base"

other attached packages:
       affy   affyio  Biobase
"1.10.0"  "1.0.0" "1.10.0"

-- 
Karl Brand <k.brand at erasmusmc.nl>
Department of Cell Biology and Genetics
Erasmus MC
Dr Molewaterplein 50
3015 GE Rotterdam
lab +31 (0)10 408 7409 fax +31 (0)10 408 9468