[BioC] Illumina BeadChips and beadarray
Ina Hoeschele
inah at vbi.vt.edu
Mon Aug 25 22:54:19 CEST 2008
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
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