[BioC] Illumina BeadChips and beadarray

Ina Hoeschele inah at vbi.vt.edu
Tue Aug 26 21:24:36 CEST 2008

  when I work with the Grn values from the .txt files (i.e. I read the data into beadarray with useImages=FALSE), everything looks good. Only when I read from the .tif files (useImages=TRUE), then I have a problem. What I am unclear about it whether the Grn values in the .txt files are foreground intensities or already background corrected. I posed this question to our Illumina representative but his answer is not clear to me (see below).
"The value "Grn" is in fact the foreground signal intensity of that
particular bead.  There has not been a global background subtraction
applied at this step.  There is a local pixel level correction that is
done to generate this bead level intensity value, but this image
pre-processing cannot be avoided."

So I am wondering whether I can simply work with the Grn values from the .txt files and still perform background subtraction, using useImages=FALSE and backgroundMethod=normexp? I.e., should this produce the same result as setting useImages=TRUE and backgroundMethod=normexp?

Thanks, Ina

----- Original Message -----
From: "Matt Ritchie" <Matt.Ritchie at cancer.org.uk>
To: "Ina Hoeschele" <inah at vbi.vt.edu>
Cc: bioconductor at stat.math.ethz.ch
Sent: Monday, August 25, 2008 11:16:02 PM GMT -05:00 US/Canada Eastern
Subject: Re: [BioC] Illumina BeadChips and beadarray

Dear Ina,

I can't see anything wrong with your commands (although you might find

cor(exprs(BSData.Illumina.txt.013), method="pearson")

is an easier way of getting the correlation between pairs of arrays).

Are there any quality issues with this data set that could be driving this
unusual result?  Try plotting the raw data from BLData.Illumina.txt.013 and
BLData.sharpen.normexp.txt.tif.013.  You could also try running

summary(getArrayData(BLData.Illumina.txt.013, what="G", array=1, log=FALSE))
summary(getArrayData(BLData.sharpen.normexp.txt.tif.013, what="G", array=1,
summary(exprs( BSData.sharpen.normexp.txt.tif.013))

to check that both the raw and summarised intensities are sensible (they
should be on mostly positive and on the range 0 - 80000 (or so) if you have
run getArrayData() and createBeadSummaryData() with log=FALSE).  If this
doesn't turn up anything, perhaps you can send me a few .txt and .tif files
from this experiment off list so that I can take a closer look at what is
going on.

Best wishes,


> Hi Matt and others,
>      I am working with Illumina beadlevel expression data.  When I read the
> foreground intensities as provided by Illumina in the .txt files (Grn column),
> then perform beadsummary and then calculate Pearson correlation coefficients
> between the six samples on one chip, I get correlations between .68 and .96.
> However, when I read from the .tif files and use backgroundMethod = normexp or
> subtract, then perform beadsummary and again calculate Pearson correlation
> coefficients, then I only get values that are essentially zero!
> Here is the code that I use:
> For reading from .txt files:
> BLData.Illumina.txt.013 = readIllumina(textType=".txt",
> arrayNames = c("1814647013_A_1","1814647013_A_2","1814647013_B_1",
> "1814647013_B_2","1814647013_C_1","1814647013_C_2","1814647013_D_1",
> "1814647013_D_2","1814647013_E_1","1814647013_E_2","1814647013_F_1",
> "1814647013_F_2"), singleChannel=TRUE, useImages=FALSE,
> normalizeMethod="none", backgroundMethod="none")
> BSData.Illumina.txt.013 <-
> createBeadSummaryData(BLData.Illumina.txt.013,log=FALSE,n=3,imagesPerArray=2,w
> hat="G",method="illumina")
> Gvec1.13 <- NULL
> Gvec1.13 <- exprs(BSData.Illumina.txt.013)[,1]
> Gvec2.13 <- NULL
> Gvec2.13 <- exprs(BSData.Illumina.txt.013)[,2]
> Gvec3.13 <- NULL
> Gvec3.13 <- exprs(BSData.Illumina.txt.013)[,3]
> Gvec4.13 <- NULL
> Gvec4.13 <- exprs(BSData.Illumina.txt.013)[,4]
> Gvec5.13 <- NULL
> Gvec5.13 <- exprs(BSData.Illumina.txt.013)[,5]
> Gvec6.13 <- NULL
> Gvec6.13 <- exprs(BSData.Illumina.txt.013)[,6]
> Gvec13 <- cbind(Gvec1.13,Gvec2.13,Gvec3.13,Gvec4.13,Gvec5.13,Gvec6.13)
> cor(Gvec13,method="pearson")
> For reading from .tif files:
> BLData.sharpen.normexp.txt.tif.013 = readIllumina(textType=".txt",
> arrayNames = c("1814647013_A_1","1814647013_A_2","1814647013_B_1",
> "1814647013_B_2","1814647013_C_1","1814647013_C_2","1814647013_D_1",
> "1814647013_D_2","1814647013_E_1","1814647013_E_2","1814647013_F_1",
> "1814647013_F_2"), singleChannel=TRUE, useImages=TRUE,
> beadInfo=NULL, targets=NULL, storeXY=TRUE,
> imageManipulation="sharpen", metrics=TRUE, metricsFile="Metrics.txt",
> backgroundMethod="normexp", normalizeMethod="none")
> BSData.sharpen.normexp.txt.tif.013 <-
> createBeadSummaryData(BLData.sharpen.normexp.txt.tif.013,log=FALSE,n=3,imagesP
> erArray=2,what="G",method="illumina")
> Š as above
> What am I doing wrong with the reading/processing of the .tif files?
> Thanks again, Ina

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