[BioC] Illumina BeadChips and beadarray

[Ina Hoeschele]

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,what="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,imagesPerArray=2,what="G",method="illumina")
… as above

What am I doing wrong with the reading/processing of the .tif files?

Thanks again, Ina