Illumina beadlevel expression data
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Ina Hoeschele ▴ 110
@ina-hoeschele-2310
Last seen 10.2 years ago
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,imageMa nipulation="sharpen", backgroundSize=17,storeXY=TRUE,metrics=FALSE,backgroundMethod="none",o ffset=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
beadarray beadarray • 2.1k views
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Ina Hoeschele ▴ 110
@ina-hoeschele-2310
Last seen 10.2 years ago
One other question - to create bead summary data using createBeadSummaryData, would you recommend to summarize the unlogged or logged bead intensities? Thanks again, Ina
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Hi Ina, > One other question - to create bead summary data using > createBeadSummaryData, would you recommend to summarize the unlogged or > logged bead intensities? > Thanks again, Ina We find it useful to summarise the data on the log scale - then the means and SEs are on the same scale that you would typically do your differential expression analysis on (assuming you want to use limma, SAM or similar for DE). Note that if you want to use vst (variance stabilising transformation) from lumi, you will need to summarise the data on the original scale before applying this transformation (I believe it models the mean-variance relationship on the original scale. See ?normaliseIllumina in beadarray and ?lumiT and ?lumiN in lumi for more information). Best wishes, Matt
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Matt Ritchie ▴ 460
@matt-ritchie-2048
Last seen 10.2 years ago
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,image Manipulati > 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 > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: > http://news.gmane.org/gmane.science.biology.informatics.conductor
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Thank you, Matt. Matt Ritchie wrote: > 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). > I am using R-2.5.1 (not sure what version of beadarray), which is still the current released version. Do I have to go to R-2.6.0? > 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,] > I have a total of 319952 beads. So I printed the information for the first 10 and for 10 others towards the end. Even for the first 10 beads the GrnX and GrnY are not negative. Here it is. > BLData[[1]][1:10,] ProbeID G Gb GrnX GrnY 1 10008 0.0000000 0 1556.7592 13526.200 2 10008 2.5249867 0 1713.2862 17711.700 3 10008 -0.0359710 0 45.9317 6676.658 4 10008 0.0000000 0 1401.8142 2153.181 5 10008 0.0000000 0 2078.2252 15974.100 6 10008 -6.8111267 0 1193.8842 8654.250 7 10008 0.0000000 0 45.0026 13990.970 8 10008 77.2345222 0 542.1487 14834.780 9 10008 -0.8237067 0 1341.1462 13791.170 10 10008 -0.2324444 0 1312.7072 7549.003 > > BLData[[1]][300000:300010,] ProbeID G Gb GrnX GrnY 300000 2140131 0.00000000 0 1258.9752 17301.580 300001 2140131 -0.24859133 0 398.1137 6108.199 300002 2140131 -0.07388444 0 2094.6222 16298.300 300003 2140131 0.00000000 0 642.9227 2094.740 300004 2140131 11.67171933 0 1352.2322 6294.191 300005 2140131 0.00000000 0 1104.5312 14069.740 300006 2140131 -0.15372500 0 963.8952 9653.909 300007 2140131 0.00000000 0 161.5619 14317.910 300008 2140131 -0.08331556 0 1515.5492 13932.460 300009 2140131 0.61296444 0 1934.0542 15118.710 300010 2140131 0.00000000 0 177.0217 16717.080 > 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. > I'll do that next if you don't have any other suggestions. Many thanks for your time. Ina > 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,imag eManipulati >> 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 >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor at stat.math.ethz.ch >> https://stat.ethz.ch/mailman/listinfo/bioconductor >> Search the archives: >> http://news.gmane.org/gmane.science.biology.informatics.conductor >>
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Dear Ina, I'm sorry to say that I haven't seen values like this before, and the X and Y coordinates don't look unusual. I have 2 further suggestions... 1. Try running readIllumina with useImages=FALSE to see the values BeadScan calculated for each bead. Are they also negative? Note that these will be background corrected, so Gb will be set to 0 in BLData. 2. Upgrade to R 2.6.0 and beadarray 1.6.0 (the C code in readIllumina changed recently). Do you still see strange values? In future emails, it would be helpful to include the output from sessionInfo(). If this doesn't uncover anything, perhaps you can send me the data off list so that I can take a closer look. Best wishes, Matt On 15/11/07 15:10, "Ina Hoeschele" <inah at="" vt.edu=""> wrote: > Thank you, Matt. > > Matt Ritchie wrote: >> 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). >> > I am using R-2.5.1 (not sure what version of beadarray), which is still > the current released version. Do I have to go to R-2.6.0? >> 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,] >> > I have a total of 319952 beads. So I printed the information for the > first 10 and for 10 others towards the end. Even for the first 10 beads > the GrnX and GrnY are not negative. Here it is. >> BLData[[1]][1:10,] > ProbeID G Gb GrnX GrnY > 1 10008 0.0000000 0 1556.7592 13526.200 > 2 10008 2.5249867 0 1713.2862 17711.700 > 3 10008 -0.0359710 0 45.9317 6676.658 > 4 10008 0.0000000 0 1401.8142 2153.181 > 5 10008 0.0000000 0 2078.2252 15974.100 > 6 10008 -6.8111267 0 1193.8842 8654.250 > 7 10008 0.0000000 0 45.0026 13990.970 > 8 10008 77.2345222 0 542.1487 14834.780 > 9 10008 -0.8237067 0 1341.1462 13791.170 > 10 10008 -0.2324444 0 1312.7072 7549.003 >> >> BLData[[1]][300000:300010,] > ProbeID G Gb GrnX GrnY > 300000 2140131 0.00000000 0 1258.9752 17301.580 > 300001 2140131 -0.24859133 0 398.1137 6108.199 > 300002 2140131 -0.07388444 0 2094.6222 16298.300 > 300003 2140131 0.00000000 0 642.9227 2094.740 > 300004 2140131 11.67171933 0 1352.2322 6294.191 > 300005 2140131 0.00000000 0 1104.5312 14069.740 > 300006 2140131 -0.15372500 0 963.8952 9653.909 > 300007 2140131 0.00000000 0 161.5619 14317.910 > 300008 2140131 -0.08331556 0 1515.5492 13932.460 > 300009 2140131 0.61296444 0 1934.0542 15118.710 > 300010 2140131 0.00000000 0 177.0217 16717.080 > >> 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. >> > I'll do that next if you don't have any other suggestions. > Many thanks for your time. > Ina > >> 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,ima geManipula >>> ti >>> on="sharpen", >>> backgroundSize=17,storeXY=TRUE,metrics=FALSE,backgroundMethod="non e",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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Matt Ritchie wrote: > Dear Ina, > > I'm sorry to say that I haven't seen values like this before, and the X and > Y coordinates don't look unusual. I have 2 further suggestions... > > 1. Try running readIllumina with useImages=FALSE to see the values BeadScan > calculated for each bead. Are they also negative? Note that these will be > background corrected, so Gb will be set to 0 in BLData. > those values are different but still look strange to me (were they computed using the subtract or minimum method for background correction?) - here they are: > BLData[[1]][1:10,] ProbeID G Gb GrnX GrnY 1 10008 56 0 1556.7592 13526.200 2 10008 46 0 1713.2862 17711.700 3 10008 4 0 45.9317 6676.658 4 10008 37 0 1401.8142 2153.181 5 10008 48 0 2078.2252 15974.100 6 10008 65 0 1193.8842 8654.250 7 10008 129 0 45.0026 13990.970 8 10008 63 0 542.1487 14834.780 9 10008 24 0 1341.1462 13791.170 10 10008 45 0 1312.7072 7549.003 > BLData[[1]][300000:300010,] ProbeID G Gb GrnX GrnY 300000 2140131 4 0 1258.9752 17301.580 300001 2140131 4 0 398.1137 6108.199 300002 2140131 0 0 2094.6222 16298.300 300003 2140131 3 0 642.9227 2094.740 300004 2140131 0 0 1352.2322 6294.191 300005 2140131 -1 0 1104.5312 14069.740 300006 2140131 22 0 963.8952 9653.909 300007 2140131 2 0 161.5619 14317.910 300008 2140131 0 0 1515.5492 13932.460 300009 2140131 0 0 1934.0542 15118.710 300010 2140131 -1 0 177.0217 16717.080 > > 2. Upgrade to R 2.6.0 and beadarray 1.6.0 (the C code in readIllumina > changed recently). Do you still see strange values? In future emails, it > would be helpful to include the output from sessionInfo(). > I did upgrade to R-2.6.0 and beadarray 1.6.0 and I am getting the exact same strange values! Here is the output from sessionInfo(). > sessionInfo() R version 2.6.0 (2007-10-03) i386-pc-mingw32 locale: LC_COLLATE=English_United States.1252;LC_CTYPE=English_United States.1252;LC_MONETARY=English_United States.1252;LC_NUMERIC=C;LC_TIME=English_United States.1252 attached base packages: [1] tools stats graphics grDevices utils datasets methods [8] base other attached packages: [1] beadarray_1.6.0 affy_1.16.0 preprocessCore_1.0.0 [4] affyio_1.6.1 geneplotter_1.16.0 lattice_0.16-5 [7] annotate_1.16.1 xtable_1.5-2 AnnotationDbi_1.0.6 [10] RSQLite_0.6-4 DBI_0.2-4 Biobase_1.16.1 [13] limma_2.12.0 loaded via a namespace (and not attached): [1] grid_2.6.0 KernSmooth_2.22-21 RColorBrewer_1.0-2 > If this doesn't uncover anything, perhaps you can send me the data off list > so that I can take a closer look. > > Best wishes, > > Matt > > On 15/11/07 15:10, "Ina Hoeschele" <inah at="" vt.edu=""> wrote: > > >> Thank you, Matt. >> >> Matt Ritchie wrote: >> >>> 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). >>> >>> >> I am using R-2.5.1 (not sure what version of beadarray), which is still >> the current released version. Do I have to go to R-2.6.0? >> >>> 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,] >>> >>> >> I have a total of 319952 beads. So I printed the information for the >> first 10 and for 10 others towards the end. Even for the first 10 beads >> the GrnX and GrnY are not negative. Here it is. >> >>> BLData[[1]][1:10,] >>> >> ProbeID G Gb GrnX GrnY >> 1 10008 0.0000000 0 1556.7592 13526.200 >> 2 10008 2.5249867 0 1713.2862 17711.700 >> 3 10008 -0.0359710 0 45.9317 6676.658 >> 4 10008 0.0000000 0 1401.8142 2153.181 >> 5 10008 0.0000000 0 2078.2252 15974.100 >> 6 10008 -6.8111267 0 1193.8842 8654.250 >> 7 10008 0.0000000 0 45.0026 13990.970 >> 8 10008 77.2345222 0 542.1487 14834.780 >> 9 10008 -0.8237067 0 1341.1462 13791.170 >> 10 10008 -0.2324444 0 1312.7072 7549.003 >> >>> BLData[[1]][300000:300010,] >>> >> ProbeID G Gb GrnX GrnY >> 300000 2140131 0.00000000 0 1258.9752 17301.580 >> 300001 2140131 -0.24859133 0 398.1137 6108.199 >> 300002 2140131 -0.07388444 0 2094.6222 16298.300 >> 300003 2140131 0.00000000 0 642.9227 2094.740 >> 300004 2140131 11.67171933 0 1352.2322 6294.191 >> 300005 2140131 0.00000000 0 1104.5312 14069.740 >> 300006 2140131 -0.15372500 0 963.8952 9653.909 >> 300007 2140131 0.00000000 0 161.5619 14317.910 >> 300008 2140131 -0.08331556 0 1515.5492 13932.460 >> 300009 2140131 0.61296444 0 1934.0542 15118.710 >> 300010 2140131 0.00000000 0 177.0217 16717.080 >> >> >>> 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. >>> >>> >> I'll do that next if you don't have any other suggestions. >> Many thanks for your time. >> Ina >> >> >>> 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,im ageManipula >>>> ti >>>> on="sharpen", >>>> backgroundSize=17,storeXY=TRUE,metrics=FALSE,backgroundMethod="no ne",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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Dear Ina, I'd suggest that you use the values in the text files (useImages=FALSE) as these look reasonable, whereas the ones obtained from redoing the image analysis look strange. The values in the text file are already background corrected (foreground - background). As I mentioned in previous emails, if you send me the data off-list, I can take a closer look to try and work out what is going on. Best wishes, Matt On 15/11/07 23:39, "Ina Hoeschele" <inah at="" vt.edu=""> wrote: > Matt Ritchie wrote: >> Dear Ina, >> >> I'm sorry to say that I haven't seen values like this before, and the X and >> Y coordinates don't look unusual. I have 2 further suggestions... >> >> 1. Try running readIllumina with useImages=FALSE to see the values BeadScan >> calculated for each bead. Are they also negative? Note that these will be >> background corrected, so Gb will be set to 0 in BLData. >> > those values are different but still look strange to me (were they > computed using the subtract or minimum method for background > correction?) - here they are: >> BLData[[1]][1:10,] > ProbeID G Gb GrnX GrnY > 1 10008 56 0 1556.7592 13526.200 > 2 10008 46 0 1713.2862 17711.700 > 3 10008 4 0 45.9317 6676.658 > 4 10008 37 0 1401.8142 2153.181 > 5 10008 48 0 2078.2252 15974.100 > 6 10008 65 0 1193.8842 8654.250 > 7 10008 129 0 45.0026 13990.970 > 8 10008 63 0 542.1487 14834.780 > 9 10008 24 0 1341.1462 13791.170 > 10 10008 45 0 1312.7072 7549.003 >> BLData[[1]][300000:300010,] > ProbeID G Gb GrnX GrnY > 300000 2140131 4 0 1258.9752 17301.580 > 300001 2140131 4 0 398.1137 6108.199 > 300002 2140131 0 0 2094.6222 16298.300 > 300003 2140131 3 0 642.9227 2094.740 > 300004 2140131 0 0 1352.2322 6294.191 > 300005 2140131 -1 0 1104.5312 14069.740 > 300006 2140131 22 0 963.8952 9653.909 > 300007 2140131 2 0 161.5619 14317.910 > 300008 2140131 0 0 1515.5492 13932.460 > 300009 2140131 0 0 1934.0542 15118.710 > 300010 2140131 -1 0 177.0217 16717.080 >> > >> 2. Upgrade to R 2.6.0 and beadarray 1.6.0 (the C code in readIllumina >> changed recently). Do you still see strange values? In future emails, it >> would be helpful to include the output from sessionInfo(). >> > I did upgrade to R-2.6.0 and beadarray 1.6.0 and I am getting the exact > same strange values! Here is the output from sessionInfo(). >> sessionInfo() > R version 2.6.0 (2007-10-03) > i386-pc-mingw32 > > locale: > LC_COLLATE=English_United States.1252;LC_CTYPE=English_United > States.1252;LC_MONETARY=English_United > States.1252;LC_NUMERIC=C;LC_TIME=English_United States.1252 > > attached base packages: > [1] tools stats graphics grDevices utils datasets methods > [8] base > > other attached packages: > [1] beadarray_1.6.0 affy_1.16.0 preprocessCore_1.0.0 > [4] affyio_1.6.1 geneplotter_1.16.0 lattice_0.16-5 > [7] annotate_1.16.1 xtable_1.5-2 AnnotationDbi_1.0.6 > [10] RSQLite_0.6-4 DBI_0.2-4 Biobase_1.16.1 > [13] limma_2.12.0 > > loaded via a namespace (and not attached): > [1] grid_2.6.0 KernSmooth_2.22-21 RColorBrewer_1.0-2 > > > > > > >> If this doesn't uncover anything, perhaps you can send me the data off list >> so that I can take a closer look. >> >> Best wishes, >> >> Matt >> >> On 15/11/07 15:10, "Ina Hoeschele" <inah at="" vt.edu=""> wrote: >> >> >>> Thank you, Matt. >>> >>> Matt Ritchie wrote: >>> >>>> 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). >>>> >>>> >>> I am using R-2.5.1 (not sure what version of beadarray), which is still >>> the current released version. Do I have to go to R-2.6.0? >>> >>>> 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,] >>>> >>>> >>> I have a total of 319952 beads. So I printed the information for the >>> first 10 and for 10 others towards the end. Even for the first 10 beads >>> the GrnX and GrnY are not negative. Here it is. >>> >>>> BLData[[1]][1:10,] >>>> >>> ProbeID G Gb GrnX GrnY >>> 1 10008 0.0000000 0 1556.7592 13526.200 >>> 2 10008 2.5249867 0 1713.2862 17711.700 >>> 3 10008 -0.0359710 0 45.9317 6676.658 >>> 4 10008 0.0000000 0 1401.8142 2153.181 >>> 5 10008 0.0000000 0 2078.2252 15974.100 >>> 6 10008 -6.8111267 0 1193.8842 8654.250 >>> 7 10008 0.0000000 0 45.0026 13990.970 >>> 8 10008 77.2345222 0 542.1487 14834.780 >>> 9 10008 -0.8237067 0 1341.1462 13791.170 >>> 10 10008 -0.2324444 0 1312.7072 7549.003 >>> >>>> BLData[[1]][300000:300010,] >>>> >>> ProbeID G Gb GrnX GrnY >>> 300000 2140131 0.00000000 0 1258.9752 17301.580 >>> 300001 2140131 -0.24859133 0 398.1137 6108.199 >>> 300002 2140131 -0.07388444 0 2094.6222 16298.300 >>> 300003 2140131 0.00000000 0 642.9227 2094.740 >>> 300004 2140131 11.67171933 0 1352.2322 6294.191 >>> 300005 2140131 0.00000000 0 1104.5312 14069.740 >>> 300006 2140131 -0.15372500 0 963.8952 9653.909 >>> 300007 2140131 0.00000000 0 161.5619 14317.910 >>> 300008 2140131 -0.08331556 0 1515.5492 13932.460 >>> 300009 2140131 0.61296444 0 1934.0542 15118.710 >>> 300010 2140131 0.00000000 0 177.0217 16717.080 >>> >>> >>>> 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. >>>> >>>> >>> I'll do that next if you don't have any other suggestions. >>> Many thanks for your time. >>> Ina >>> >>> >>>> 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,i mageManipu >>>>> la >>>>> ti >>>>> on="sharpen", >>>>> backgroundSize=17,storeXY=TRUE,metrics=FALSE,backgroundMethod="n one",offse >>>>> t= >>>>> 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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Entering edit mode
Matt Ritchie wrote: > Dear Ina, > > I'd suggest that you use the values in the text files (useImages=FALSE) as > these look reasonable, whereas the ones obtained from redoing the image > analysis look strange. The values in the text file are already background > corrected (foreground - background). > yes, although that way I am getting quite a few (actually a lot) of negative G values, then my only choices are to apply the 'minimum' or 'half' methods myself (but not others such as RMA that I wanted to try). > As I mentioned in previous emails, if you send me the data off-list, I can > take a closer look to try and work out what is going on. > the folder containing all files for the 12 arrays of one Beadchip are at http://staff.vbi.vt.edu/inah/1814647013/ Thanks, Matt! Ina > Best wishes, > > Matt > > On 15/11/07 23:39, "Ina Hoeschele" <inah at="" vt.edu=""> wrote: > > >> Matt Ritchie wrote: >> >>> Dear Ina, >>> >>> I'm sorry to say that I haven't seen values like this before, and the X and >>> Y coordinates don't look unusual. I have 2 further suggestions... >>> >>> 1. Try running readIllumina with useImages=FALSE to see the values BeadScan >>> calculated for each bead. Are they also negative? Note that these will be >>> background corrected, so Gb will be set to 0 in BLData. >>> >>> >> those values are different but still look strange to me (were they >> computed using the subtract or minimum method for background >> correction?) - here they are: >> >>> BLData[[1]][1:10,] >>> >> ProbeID G Gb GrnX GrnY >> 1 10008 56 0 1556.7592 13526.200 >> 2 10008 46 0 1713.2862 17711.700 >> 3 10008 4 0 45.9317 6676.658 >> 4 10008 37 0 1401.8142 2153.181 >> 5 10008 48 0 2078.2252 15974.100 >> 6 10008 65 0 1193.8842 8654.250 >> 7 10008 129 0 45.0026 13990.970 >> 8 10008 63 0 542.1487 14834.780 >> 9 10008 24 0 1341.1462 13791.170 >> 10 10008 45 0 1312.7072 7549.003 >> >>> BLData[[1]][300000:300010,] >>> >> ProbeID G Gb GrnX GrnY >> 300000 2140131 4 0 1258.9752 17301.580 >> 300001 2140131 4 0 398.1137 6108.199 >> 300002 2140131 0 0 2094.6222 16298.300 >> 300003 2140131 3 0 642.9227 2094.740 >> 300004 2140131 0 0 1352.2322 6294.191 >> 300005 2140131 -1 0 1104.5312 14069.740 >> 300006 2140131 22 0 963.8952 9653.909 >> 300007 2140131 2 0 161.5619 14317.910 >> 300008 2140131 0 0 1515.5492 13932.460 >> 300009 2140131 0 0 1934.0542 15118.710 >> 300010 2140131 -1 0 177.0217 16717.080 >> >>> 2. Upgrade to R 2.6.0 and beadarray 1.6.0 (the C code in readIllumina >>> changed recently). Do you still see strange values? In future emails, it >>> would be helpful to include the output from sessionInfo(). >>> >>> >> I did upgrade to R-2.6.0 and beadarray 1.6.0 and I am getting the exact >> same strange values! Here is the output from sessionInfo(). >> >>> sessionInfo() >>> >> R version 2.6.0 (2007-10-03) >> i386-pc-mingw32 >> >> locale: >> LC_COLLATE=English_United States.1252;LC_CTYPE=English_United >> States.1252;LC_MONETARY=English_United >> States.1252;LC_NUMERIC=C;LC_TIME=English_United States.1252 >> >> attached base packages: >> [1] tools stats graphics grDevices utils datasets methods >> [8] base >> >> other attached packages: >> [1] beadarray_1.6.0 affy_1.16.0 preprocessCore_1.0.0 >> [4] affyio_1.6.1 geneplotter_1.16.0 lattice_0.16-5 >> [7] annotate_1.16.1 xtable_1.5-2 AnnotationDbi_1.0.6 >> [10] RSQLite_0.6-4 DBI_0.2-4 Biobase_1.16.1 >> [13] limma_2.12.0 >> >> loaded via a namespace (and not attached): >> [1] grid_2.6.0 KernSmooth_2.22-21 RColorBrewer_1.0-2 >> >> >> >> >> >> >> >>> If this doesn't uncover anything, perhaps you can send me the data off list >>> so that I can take a closer look. >>> >>> Best wishes, >>> >>> Matt >>> >>> On 15/11/07 15:10, "Ina Hoeschele" <inah at="" vt.edu=""> wrote: >>> >>> >>> >>>> Thank you, Matt. >>>> >>>> Matt Ritchie wrote: >>>> >>>> >>>>> 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). >>>>> >>>>> >>>>> >>>> I am using R-2.5.1 (not sure what version of beadarray), which is still >>>> the current released version. Do I have to go to R-2.6.0? >>>> >>>> >>>>> 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,] >>>>> >>>>> >>>>> >>>> I have a total of 319952 beads. So I printed the information for the >>>> first 10 and for 10 others towards the end. Even for the first 10 beads >>>> the GrnX and GrnY are not negative. Here it is. >>>> >>>> >>>>> BLData[[1]][1:10,] >>>>> >>>>> >>>> ProbeID G Gb GrnX GrnY >>>> 1 10008 0.0000000 0 1556.7592 13526.200 >>>> 2 10008 2.5249867 0 1713.2862 17711.700 >>>> 3 10008 -0.0359710 0 45.9317 6676.658 >>>> 4 10008 0.0000000 0 1401.8142 2153.181 >>>> 5 10008 0.0000000 0 2078.2252 15974.100 >>>> 6 10008 -6.8111267 0 1193.8842 8654.250 >>>> 7 10008 0.0000000 0 45.0026 13990.970 >>>> 8 10008 77.2345222 0 542.1487 14834.780 >>>> 9 10008 -0.8237067 0 1341.1462 13791.170 >>>> 10 10008 -0.2324444 0 1312.7072 7549.003 >>>> >>>> >>>>> BLData[[1]][300000:300010,] >>>>> >>>>> >>>> ProbeID G Gb GrnX GrnY >>>> 300000 2140131 0.00000000 0 1258.9752 17301.580 >>>> 300001 2140131 -0.24859133 0 398.1137 6108.199 >>>> 300002 2140131 -0.07388444 0 2094.6222 16298.300 >>>> 300003 2140131 0.00000000 0 642.9227 2094.740 >>>> 300004 2140131 11.67171933 0 1352.2322 6294.191 >>>> 300005 2140131 0.00000000 0 1104.5312 14069.740 >>>> 300006 2140131 -0.15372500 0 963.8952 9653.909 >>>> 300007 2140131 0.00000000 0 161.5619 14317.910 >>>> 300008 2140131 -0.08331556 0 1515.5492 13932.460 >>>> 300009 2140131 0.61296444 0 1934.0542 15118.710 >>>> 300010 2140131 0.00000000 0 177.0217 16717.080 >>>> >>>> >>>> >>>>> 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. >>>>> >>>>> >>>>> >>>> I'll do that next if you don't have any other suggestions. >>>> Many thanks for your time. >>>> Ina >>>> >>>> >>>> >>>>> 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, imageManipu >>>>>> la >>>>>> ti >>>>>> on="sharpen", >>>>>> backgroundSize=17,storeXY=TRUE,metrics=FALSE,backgroundMethod=" none",offse >>>>>> t= >>>>>> 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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Entering edit mode
Dear Ina, Thanks for the data. There appears to be a quality problem with these arrays that occurred during scanning. The registration scores in the Metrics.txt file indicates that the images have not registered properly (the RegGrn values should be around 1 for a good scan - in your case they are around 0.14). As a result, many beads have zero intensities. Please check with the people who generated this data to determine what went wrong. Best wishes, Matt On 16/11/07 14:16, "Ina Hoeschele" <inah at="" vt.edu=""> wrote: > Matt Ritchie wrote: >> Dear Ina, >> >> I'd suggest that you use the values in the text files (useImages=FALSE) as >> these look reasonable, whereas the ones obtained from redoing the image >> analysis look strange. The values in the text file are already background >> corrected (foreground - background). >> > yes, although that way I am getting quite a few (actually a lot) of > negative G values, then my only choices are to apply the 'minimum' or > 'half' methods myself (but not others such as RMA that I wanted to try). >> As I mentioned in previous emails, if you send me the data off- list, I can >> take a closer look to try and work out what is going on. >> > the folder containing all files for the 12 arrays of one Beadchip are at > http://staff.vbi.vt.edu/inah/1814647013/ > > Thanks, Matt! > Ina >> Best wishes, >> >> Matt >> >> On 15/11/07 23:39, "Ina Hoeschele" <inah at="" vt.edu=""> wrote: >> >> >>> Matt Ritchie wrote: >>> >>>> Dear Ina, >>>> >>>> I'm sorry to say that I haven't seen values like this before, and the X and >>>> Y coordinates don't look unusual. I have 2 further suggestions... >>>> >>>> 1. Try running readIllumina with useImages=FALSE to see the values >>>> BeadScan >>>> calculated for each bead. Are they also negative? Note that these will be >>>> background corrected, so Gb will be set to 0 in BLData. >>>> >>>> >>> those values are different but still look strange to me (were they >>> computed using the subtract or minimum method for background >>> correction?) - here they are: >>> >>>> BLData[[1]][1:10,] >>>> >>> ProbeID G Gb GrnX GrnY >>> 1 10008 56 0 1556.7592 13526.200 >>> 2 10008 46 0 1713.2862 17711.700 >>> 3 10008 4 0 45.9317 6676.658 >>> 4 10008 37 0 1401.8142 2153.181 >>> 5 10008 48 0 2078.2252 15974.100 >>> 6 10008 65 0 1193.8842 8654.250 >>> 7 10008 129 0 45.0026 13990.970 >>> 8 10008 63 0 542.1487 14834.780 >>> 9 10008 24 0 1341.1462 13791.170 >>> 10 10008 45 0 1312.7072 7549.003 >>> >>>> BLData[[1]][300000:300010,] >>>> >>> ProbeID G Gb GrnX GrnY >>> 300000 2140131 4 0 1258.9752 17301.580 >>> 300001 2140131 4 0 398.1137 6108.199 >>> 300002 2140131 0 0 2094.6222 16298.300 >>> 300003 2140131 3 0 642.9227 2094.740 >>> 300004 2140131 0 0 1352.2322 6294.191 >>> 300005 2140131 -1 0 1104.5312 14069.740 >>> 300006 2140131 22 0 963.8952 9653.909 >>> 300007 2140131 2 0 161.5619 14317.910 >>> 300008 2140131 0 0 1515.5492 13932.460 >>> 300009 2140131 0 0 1934.0542 15118.710 >>> 300010 2140131 -1 0 177.0217 16717.080 >>> >>>> 2. Upgrade to R 2.6.0 and beadarray 1.6.0 (the C code in readIllumina >>>> changed recently). Do you still see strange values? In future emails, it >>>> would be helpful to include the output from sessionInfo(). >>>> >>>> >>> I did upgrade to R-2.6.0 and beadarray 1.6.0 and I am getting the exact >>> same strange values! Here is the output from sessionInfo(). >>> >>>> sessionInfo() >>>> >>> R version 2.6.0 (2007-10-03) >>> i386-pc-mingw32 >>> >>> locale: >>> LC_COLLATE=English_United States.1252;LC_CTYPE=English_United >>> States.1252;LC_MONETARY=English_United >>> States.1252;LC_NUMERIC=C;LC_TIME=English_United States.1252 >>> >>> attached base packages: >>> [1] tools stats graphics grDevices utils datasets methods >>> [8] base >>> >>> other attached packages: >>> [1] beadarray_1.6.0 affy_1.16.0 preprocessCore_1.0.0 >>> [4] affyio_1.6.1 geneplotter_1.16.0 lattice_0.16-5 >>> [7] annotate_1.16.1 xtable_1.5-2 AnnotationDbi_1.0.6 >>> [10] RSQLite_0.6-4 DBI_0.2-4 Biobase_1.16.1 >>> [13] limma_2.12.0 >>> >>> loaded via a namespace (and not attached): >>> [1] grid_2.6.0 KernSmooth_2.22-21 RColorBrewer_1.0-2 >>> >>> >>> >>> >>> >>> >>> >>>> If this doesn't uncover anything, perhaps you can send me the data off list >>>> so that I can take a closer look. >>>> >>>> Best wishes, >>>> >>>> Matt >>>> >>>> On 15/11/07 15:10, "Ina Hoeschele" <inah at="" vt.edu=""> wrote: >>>> >>>> >>>> >>>>> Thank you, Matt. >>>>> >>>>> Matt Ritchie wrote: >>>>> >>>>> >>>>>> 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). >>>>>> >>>>>> >>>>>> >>>>> I am using R-2.5.1 (not sure what version of beadarray), which is still >>>>> the current released version. Do I have to go to R-2.6.0? >>>>> >>>>> >>>>>> 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,] >>>>>> >>>>>> >>>>>> >>>>> I have a total of 319952 beads. So I printed the information for the >>>>> first 10 and for 10 others towards the end. Even for the first 10 beads >>>>> the GrnX and GrnY are not negative. Here it is. >>>>> >>>>> >>>>>> BLData[[1]][1:10,] >>>>>> >>>>>> >>>>> ProbeID G Gb GrnX GrnY >>>>> 1 10008 0.0000000 0 1556.7592 13526.200 >>>>> 2 10008 2.5249867 0 1713.2862 17711.700 >>>>> 3 10008 -0.0359710 0 45.9317 6676.658 >>>>> 4 10008 0.0000000 0 1401.8142 2153.181 >>>>> 5 10008 0.0000000 0 2078.2252 15974.100 >>>>> 6 10008 -6.8111267 0 1193.8842 8654.250 >>>>> 7 10008 0.0000000 0 45.0026 13990.970 >>>>> 8 10008 77.2345222 0 542.1487 14834.780 >>>>> 9 10008 -0.8237067 0 1341.1462 13791.170 >>>>> 10 10008 -0.2324444 0 1312.7072 7549.003 >>>>> >>>>> >>>>>> BLData[[1]][300000:300010,] >>>>>> >>>>>> >>>>> ProbeID G Gb GrnX GrnY >>>>> 300000 2140131 0.00000000 0 1258.9752 17301.580 >>>>> 300001 2140131 -0.24859133 0 398.1137 6108.199 >>>>> 300002 2140131 -0.07388444 0 2094.6222 16298.300 >>>>> 300003 2140131 0.00000000 0 642.9227 2094.740 >>>>> 300004 2140131 11.67171933 0 1352.2322 6294.191 >>>>> 300005 2140131 0.00000000 0 1104.5312 14069.740 >>>>> 300006 2140131 -0.15372500 0 963.8952 9653.909 >>>>> 300007 2140131 0.00000000 0 161.5619 14317.910 >>>>> 300008 2140131 -0.08331556 0 1515.5492 13932.460 >>>>> 300009 2140131 0.61296444 0 1934.0542 15118.710 >>>>> 300010 2140131 0.00000000 0 177.0217 16717.080 >>>>> >>>>> >>>>> >>>>>> 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. >>>>>> >>>>>> >>>>>> >>>>> I'll do that next if you don't have any other suggestions. >>>>> Many thanks for your time. >>>>> Ina >>>>> >>>>> >>>>> >>>>>> 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 ,imageMani >>>>>>> pu >>>>>>> la >>>>>>> ti >>>>>>> on="sharpen", >>>>>>> backgroundSize=17,storeXY=TRUE,metrics=FALSE,backgroundMethod= "none",off >>>>>>> se >>>>>>> t= >>>>>>> 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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Thanks for writing, Wei. Sorry about that, I did get the wrong number. For the total number of beads I get length(BLData[[1]][,1])= 1,035,907. Please let me know if this does not look right. Ina Wei Shi wrote: > Hi Ina: > > It surprised me that you only got around 320,000 beads. You should > see at least 700,000 beads for each strip since there are about 24,000 > bead types in each strip and each bead type have 30 or more beads. And > we actually saw about 1 million beads for each strip for our data. I > suspect you did not retrieve bead level data correctly. > > Cheers, > Wei > > Ina Hoeschele wrote: >> I have a total of 319952 beads. So I printed the information for the >> first 10 and for 10 others towards the end. Even for the first 10 beads >> the GrnX and GrnY are not negative. Here it is. >> >>> BLData[[1]][1:10,] >>> >> ProbeID G Gb GrnX GrnY >> 1 10008 0.0000000 0 1556.7592 13526.200 >> 2 10008 2.5249867 0 1713.2862 17711.700 >> 3 10008 -0.0359710 0 45.9317 6676.658 >> 4 10008 0.0000000 0 1401.8142 2153.181 >> 5 10008 0.0000000 0 2078.2252 15974.100 >> 6 10008 -6.8111267 0 1193.8842 8654.250 >> 7 10008 0.0000000 0 45.0026 13990.970 >> 8 10008 77.2345222 0 542.1487 14834.780 >> 9 10008 -0.8237067 0 1341.1462 13791.170 >> 10 10008 -0.2324444 0 1312.7072 7549.003 >> >>> BLData[[1]][300000:300010,] >>> >> ProbeID G Gb GrnX GrnY >> 300000 2140131 0.00000000 0 1258.9752 17301.580 >> 300001 2140131 -0.24859133 0 398.1137 6108.199 >> 300002 2140131 -0.07388444 0 2094.6222 16298.300 >> 300003 2140131 0.00000000 0 642.9227 2094.740 >> 300004 2140131 11.67171933 0 1352.2322 6294.191 >> 300005 2140131 0.00000000 0 1104.5312 14069.740 >> 300006 2140131 -0.15372500 0 963.8952 9653.909 >> 300007 2140131 0.00000000 0 161.5619 14317.910 >> 300008 2140131 -0.08331556 0 1515.5492 13932.460 >> 300009 2140131 0.61296444 0 1934.0542 15118.710 >> 300010 2140131 0.00000000 0 177.0217 16717.080 >> >> >>
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