[BioC] Illumina BeadChips and beadarray

[Ina Hoeschele]

Hi,
  I am working on low-level analysis of bead-level expression data from Illumina BeadChips using the beadarray package of Bioconductor. Each chip has 6 arrays with 2 strips. For each of the 12 strips per chip, there is a _.csv, _.txt, _Grn.tif and _Grn.locs file (and _Grn.xml). There is also a _Grn.idat file for each of the 6 arrays per chip. My .csv files do not contain bead-level information but rather just probe-id, number of beads, mean Grn and dev Grn (I assume Grn is foreground intensity). My .txt files contain bead-level information with probe-id,  foreground intensity Grn and bead location GrnX, GrnY (bead center coordinates). Therefore, for beadarray analysis I use the .txt (not .csv) and .tif files.    
I read the bead-level data for one chip as follows:

for background correction method subtract or normexp:

BLData.sharpen.subtract.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="subtract/normexp", normalizeMethod="none")

Then my first problem is that the background intensities, which I look at using 
an <- arrayNames(BLData.sharpen.txt.tif.013)
BLData.sharpen.subtract/normexp.txt.tif.013 at beadData[[an[1]]]$Gb
are ALL zero. If I specify a small value for backgroundSize (e.g. 4), then a few of the Gb values are small but nonzero. I did not expect to find all Gb values to be zero for the default backgroundSize = 17, so what is going on here???

When I read the data without background correction as follows (only changing the backgroundMethods option)

BLData.sharpen.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="none", normalizeMethod="none")

then I get the same $G values as for backgroundMethod="subtract" which also does not make sense to me (the values for backgroundMethod="normexp" are different) ???

Of course I can retrieve the not-background adjusted bead intensities from the .txt files by setting
useImages=FALSE), but the above with useImages=TRUE and backgroundMethod="none" should produce the same G values, right?, but it does not. Instead, backgroundMethod="subtract" and backgroundMethod="none" produce the same values!

My last problem is that the bead center coordinates GrnX and GrnY that I find in the .txt files and those from beadarray, BLData.sharpen.subtract/normexp.txt.tif.013 at beadData[[an[1]]]$GrnX/Y, are NOT the same, which worries me!

Any comments are very much appreciated.
Thanks, Ina Hoeschele