beadarray package: problem with bead level data
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Krys Kelly ▴ 270
@krys-kelly-1768
Last seen 7.2 years ago
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
geneplotter affy affyio beadarray quantsmooth beadarraySNP geneplotter affy affyio • 1.1k views
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@jdelasherasedacuk-1189
Last seen 6.8 years ago
United Kingdom
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
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Matt Ritchie ▴ 460
@matt-ritchie-2048
Last seen 7.7 years ago
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"
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Mike Smith ▴ 40
@mike-smith-2291
Last seen 7.7 years ago
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