[BioC] RMA/QuantileNormalization results difference between oligo and aroma.affymetrix for Hugene

[Mikhail Pachkov]

Dear All,

I am new in microarray analysis and need your expertise.
I need to develop procedure for producing expression values from CEL
files. Data should processed with RMA and quantile normalized. I have
tried two packages - oligo and aroma.affymetrix. Obtained results are
quite different form my point of view. Moreover
aroma.affymetrix::QuantileNormalization function produce dta which do
not look like they were quantile normalized.
 I have made density plots of data after RMA and after quantile
normalization which are available here
http://test.swissregulon.unibas.ch/bioc/index.html There are also
links to two CEL files I have used.

I have a few questions:
Why RMA results are so different?
Which RMA implementation is correct?
Why does quantile normalization in aroma.affymetrics produce two
different distributions?

Thank you in advance!

Here are R scripts I have used:

################################
#aroma.affymetrix
library(aroma.affymetrix);
verbose <- Arguments$getVerbose(-8, timestamp=TRUE);

# read files
cdf <- AffymetrixCdfFile('annotationData/chipTypes/HuGene-1_0-st-v1/HuGene-1_0-st-v1.cdf');
cs <- AffymetrixCelSet$byPath("rawData/mine/HuGene-1_0-st-v1/");

# RMA
bc <- RmaBackgroundCorrection(cs);
csBC <- process(bc,verbose=verbose);

# QuantileNormalization
qn <- QuantileNormalization(csBC, typesToUpdate="pm");
csN <- process(qn);

# Plots
image_file <- ("aroma.affymetrix.RMA.png");
png(image_file,width=1028,height=768);
plotDensity(csBC);
title("aroma.affymetrix RMA data");
dev.off();

image_file <- ("aroma.affymetrix.QN.png");
png(image_file,width=1028,height=768);
plotDensity(csN);
title("aroma.affymetrix QN data");
dev.off()
################################

################################
# oligo
library(oligo);
rawdata=read.celfiles(c("rawData/mine/HuGene-1_0-st-v1/sample1.CEL","rawData/mine/HuGene-1_0-st-v1/sample2.CEL"));
rmadata=rma(rawdata);
qndata=normalize.quantiles(exprs(rmadata))

library(affy)
# Plots
image_file <- ("oligo.RMA.png");
png(image_file,width=1028,height=768);
plotDensity(exprs(rmadata));
title("oligo RMA data");
dev.off();

image_file <- ("oligo.QN.png");
png(image_file,width=1028,height=768);
plotDensity(qndata);
title("oligo QN data");
dev.off()
###############################

Kind regards,

Mikhail Pachkov