[BioC] Illumina beadlevel expression data

[Matt Ritchie]

Hi Ina,

What version of beadarray are you using?  In older versions funny background
values have been reported on some operating systems - this should be fixed
on the release version though (beadarray 1.6.0 available on R-2.6.0).

The argument backgroundSize specifies the size of the square (e.g.
backgroundSize=17 uses a 17 x 17 square) around each bead center used for
calculation of the local background.  The average of the 5 minimum pixels
inside this window is the Gb value stored in BLData.  I'd recommend you
stick to the default value (17) which is what BeadScan uses.

The first beads in BLData are typically ones which could not be decoded
properly (ProbeIDs 0).  If these beads are too close to the edge of an
image, or off the image, then zero foreground or background intensities may
occur.  Check the X (GrnX) and Y (GrnY) coordinates for these beads - any
with negative values are off the image, and cannot be quantified.

BLData[[1]][1:10,]

If this doesn't shed any light, perhaps you can put the .tif and .txt files
from the strip online so that I can take a closer look.

Best wishes,

Matt

> Hi,
>    I am new to the analysis of Illumina beadlevel expression data. I
> have data from a collaborator and have started to work with the data on
> the first Beadchip (with 12 arrays or strips).  These are single channel
> data. I am using the beadarray package.  I read the data in as follows:
> BLData <- 
> readIllumina(textType=".txt",useImages=TRUE,singleChannel=TRUE,imageManipulati
> on="sharpen",
> backgroundSize=17,storeXY=TRUE,metrics=FALSE,backgroundMethod="none",offset=0,
> normalizeMethod="none")
> Note: above I'm doing NO background correction and note that I use
> backgroundSize=17.
> 
> Then I looked at the data:
> 
> an <- arrayNames(BLData)
> BLData at beadData[[an[1]]]$G[1:10]
>  [1]  0.0000000  2.5249867 -0.0359710  0.0000000  0.0000000 -6.8111267
>  [7]  0.0000000 77.2345222 -0.8237067 -0.2324444
> BLData at beadData[[an[1]]]$Gb[1:10]
>  [1] 0 0 0 0 0 0 0 0 0 0
> 
> so I get quite a few G intensities that are zero and ALL of my
> background intensities are zero - why is that ?????
> 
> Then I created BLData with backgroundSize=4 and backgroundSize=40 (not
> knowing what value I should be using ?????). For backgroundSize=4, I
> find that
> BLData at beadData[[an[1]]]$Gb[1:100]
>   [1] -1  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0
> 0  0  0
>  [26]  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  2  0
> 0  0  0
>  [51]  0  0 24  2  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0  0
> 0  0  0
>  [76]  0  0  0  0  0  0  0  0  0  0 19  0  0  0  0  0  0  0  0  0  0  0
> 0  0  0
> so now I still have many zero Gb values, but also some nonzero ones and
> even negative (-1)???
> But when I use backgroundSize=40, then R crashes (repeatedly).
> 
> And when I create BLData with backgroundMethod="minimum" and
> backgroundSize=17, then I find the following G intensities:
> BLData at beadData[[an[1]]]$G[1:10]
>  [1] 2.425286e-12 2.524987e+00 2.425286e-12 2.425286e-12 2.425286e-12
> 2.425286e-12 2.425286e-12 7.723452e+01 2.425286e-12
> [10] 2.425286e-12
> 
> These G and Gb values all look suspect to me - can someone please help?
> 
> Many thanks, Ina
> 
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