[BioC] beadarray package: problem with bead level data

[Matt Ritchie]

Hi Krys,

The values you are getting do look weird, which may have something to do
with the conversion from jpg to tiff.

To re-do the image analysis in R using beadarray, you need the original
tiffs output by the Illumina scanning software (BeadScan), which are
generally around 80Mb each.  How big are your converted tiffs?

Alternatively, if you upgrade to a newer version of beadarray (1.5.x) you
can bypass the images and use the intensity values stored in the text files
(these have been background corrected)

BLData <- readIllumina(textType=".txt", useImages=FALSE)

Best wishes,

Matt

> I am having a problem with bead level data.
> 
> I have .txt files containing over 900,000 lines with four columns: Code,
> Grn, GrnX and GrnY.
> 
> The image files are .jpg rather than .tif. I have converted the .jpg images
> to .tif using Adobe Photoshop (trying several of the conversion options).
> They always look like a beautiful starry night sky, which I think is what is
> expected.
> 
> However, the data in the beadarray object is not what I expected.  In G
> there are a lot of negative and zero values and all the Gb values are zero.
> 
> On Wednesday, I downloaded the latest version of R, beadarray and the
> packages it depends on (see sessionInfo below).
> 
> Is the problem with my .tif files, or is there a problem of incompatibility
> with the new version of R and the dependencies, or something else?
> 
> Thanks for any light you can shed.
> 
> Krys
> 
> Here is my code and output:
> 
>> BLData <- readIllumina(textType=".txt")
> Found 2 arrays 
> Reading pixels of 1863191039_A_1_Grn.tif
> Calculating background
> Sharpening Image
> Calculating foregound
> Background correcting: method = none
> Reading pixels of 1863191039_A_2_Grn.tif
> Calculating background
> Sharpening Image
> Calculating foregound
> Background correcting: method = none
>> an <- arrayNames(BLData)
>> summary(BLData at beadData[[an[1]]]$G)
>    Min. 1st Qu.  Median    Mean 3rd Qu.    Max.
> -9547.0     0.0     0.0   371.1   267.5 30110.0
>> summary(BLData at beadData[[an[1]]]$Gb)
>    Min. 1st Qu.  Median    Mean 3rd Qu.    Max.
>       0       0       0       0       0       0
>> 
>> lengthG <- length(BLData at beadData[[an[1]]]$G)
>> lengthG
> [1] 904963
>> myNegatives <-
> length(BLData at beadData[[an[1]]]$G[BLData at beadData[[an[1]]]$G<0])
>> myNegatives
> [1] 169302
>> myZeros <-
> length(BLData at beadData[[an[1]]]$G[BLData at beadData[[an[1]]]$G==0])
>> myZeros
> [1] 360331
>> myPositives <-
> length(BLData at beadData[[an[1]]]$G[BLData at beadData[[an[1]]]$G>0])
>> myPositives
> [1] 375330
>> 
> 
> Here is my sessionInfo:
> 
> R version 2.5.1 (2007-06-27)
> i386-pc-mingw32 
> 
> locale:
> LC_COLLATE=English_United Kingdom.1252;LC_CTYPE=English_United
> Kingdom.1252;LC_MONETARY=English_United
> Kingdom.1252;LC_NUMERIC=C;LC_TIME=English_United Kingdom.1252
> 
> attached base packages:
> [1] "grid"      "tools"     "stats"     "graphics"  "grDevices" "utils"
> [7] "datasets"  "methods"   "base"
> 
> other attached packages:
>    beadarray beadarraySNP  quantsmooth      lodplot     quantreg
> SparseM 
>      "1.4.0"      "1.2.0"      "1.2.0"        "1.1"       "4.08"
> "0.73" 
>         affy       affyio  geneplotter      lattice     annotate
> Biobase 
>     "1.14.2"      "1.4.1"     "1.14.0"    "0.15-11"     "1.14.1"
> "1.14.1" 
>        limma 
>     "2.10.5"