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"
Dr Krystyna A Kelly (Krys)
Department of Pathology
University of Cambridge, Tennis Court Road, Cambridge CB2 1QP
Tel: 01223 333331
and
MRC Biostatistics Unit
Institute of Public Health, Robinson Way, Cambridge CB2 0SR
Tel: 01223 767408
Email: kak28 at cam.ac.uk
Quoting Krys Kelly <kak28 at="" cam.ac.uk="">:
>
> 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
Just a quick comment...
you are converting a jpeg to a tiff, for your quantitation, right?
is the jpeg 16-bit?
Jpegs are *usually* 8-bit, and I'd be surprised if yours is 16-bit,
but they can be. If it's 8-bit, you're only getting 256 different
intensities (2^8) rather than 65536 (2^16)... so I wonder if this may
explain what you're seeing (you'd essentially get what looks like an
extremely weak hyb, if the software reading the raw data expects a
range between 1-65536 but the maximum is 256).
I am not familiar at all with beadarray, but your conversion concerns
me. You'd better check that first.
Jose
--
Dr. Jose I. de las Heras Email: J.delasHeras at
ed.ac.uk
The Wellcome Trust Centre for Cell Biology Phone: +44 (0)131
6513374
Institute for Cell & Molecular Biology Fax: +44 (0)131
6507360
Swann Building, Mayfield Road
University of Edinburgh
Edinburgh EH9 3JR
UK
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"
