[BioC] Illumina BeadChips and beadarray

[Matt Ritchie]

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

Matt

> 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