discrepancy between BeadStudio images and beadarray imageplot
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@luis-f-menezes-1651
Last seen 9.6 years ago
Hello. I have been using beadarray (v. 1.10.0) to analyze some Illumina mouse ref8v2 arrays. When I import the images and expression data using readIllumina() and plot them using imageplot() [either before or after background subtraction], I get an unusual pattern that would suggest some sort of artifact in the array, with "white streaks" across it. When I questioned the core who had done the microarray experiment, they said the BeadStudio image did not show any of the odd patterns. Using outlierPlot() or BASH, some areas were flagged for masking, but they did not match the "white streak areas" (I assume that is because the outliers would in general have increased expression levels, as opposed to the white areas with almost no signal). My questions are: 1) is there any reason why the images generated by BeadStudio and beadarray should be different? 2) why are the streaks with almost no signal not flagged as regions that should be masked? 3) should I be concerned about these low signal streaks? Thank you. Best regards, Luis. > sessionInfo() R version 2.8.0 Patched (2008-10-22 r46776) i386-apple-darwin9.5.0 locale: en_US.UTF-8/en_US.UTF-8/C/C/en_US.UTF-8/en_US.UTF-8 attached base packages: [1] tools stats graphics grDevices utils datasets methods base other attached packages: [1] beadarray_1.10.0 sma_0.5.15 hwriter_0.93 geneplotter_1.20.0 annotate_1.20.0 xtable_1.5-4 AnnotationDbi_1.4.1 [8] lattice_0.17-15 Biobase_2.2.0 limma_2.16.2 loaded via a namespace (and not attached): [1] DBI_0.2-4 KernSmooth_2.22-22 RColorBrewer_1.0-2 RSQLite_0.7-0 grid_2.8.0
Microarray beadarray Microarray beadarray • 866 views
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@cei-abreu-goodger-4433
Last seen 9.1 years ago
Mexico
Hi Luis, I do hope you'll get a better response, but I can tell you that I found similar issues when analyzing mouse beadarrays. I think that the authors haven't tested the package as extensively for mouse as for human, and thus there a probably some problems in displaying the correct layout for these chips. So, it is my "guess" that the white streaks are artifacts generated by the package and do not reflect real artifacts on the array. Cheers, Cei Luis Fernando Menezes wrote: > Hello. > > I have been using beadarray (v. 1.10.0) to analyze some Illumina mouse > ref8v2 arrays. > When I import the images and expression data using readIllumina() and > plot them using imageplot() [either before or after background > subtraction], I get an unusual pattern that would suggest some sort of > artifact in the array, with "white streaks" across it. > When I questioned the core who had done the microarray experiment, they > said the BeadStudio image did not show any of the odd patterns. > Using outlierPlot() or BASH, some areas were flagged for masking, but > they did not match the "white streak areas" (I assume that is because > the outliers would in general have increased expression levels, as > opposed to the white areas with almost no signal). > > My questions are: > 1) is there any reason why the images generated by BeadStudio and > beadarray should be different? > 2) why are the streaks with almost no signal not flagged as regions that > should be masked? > 3) should I be concerned about these low signal streaks? > > Thank you. Best regards, > > Luis. > > > sessionInfo() > R version 2.8.0 Patched (2008-10-22 r46776) > i386-apple-darwin9.5.0 > > locale: > en_US.UTF-8/en_US.UTF-8/C/C/en_US.UTF-8/en_US.UTF-8 > > attached base packages: > [1] tools stats graphics grDevices utils datasets > methods base > > other attached packages: > [1] beadarray_1.10.0 sma_0.5.15 hwriter_0.93 > geneplotter_1.20.0 annotate_1.20.0 xtable_1.5-4 > AnnotationDbi_1.4.1 > [8] lattice_0.17-15 Biobase_2.2.0 limma_2.16.2 > > loaded via a namespace (and not attached): > [1] DBI_0.2-4 KernSmooth_2.22-22 RColorBrewer_1.0-2 > RSQLite_0.7-0 grid_2.8.0 > > _______________________________________________ > 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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Mark Dunning ★ 1.1k
@mark-dunning-3319
Last seen 12 months ago
Sheffield, Uk
Hi Luis, The white streaks you see on the imageplots are because of missing data on the array. This occurs when the Illumina scanner is unable to decode the identity of beads on the array. Information about these undecoded beads is not reported in the output that comes out from the scanner and therefore we cannot use them in any analysis, including BASH. The white streaks would not show up in the BeadStudio images or the TIFF images generated by the scanner as these are just the raw pixel intensities and do not take bead identity into account. Hope this helps Regards, Mark On Thu, Mar 5, 2009 at 2:13 AM, Luis Fernando Menezes <lmenezes at="" jhmi.edu=""> wrote: > Hello. > > I have been using beadarray (v. 1.10.0) ?to analyze some Illumina mouse > ref8v2 arrays. > When I import the images and expression data using readIllumina() and plot > them using imageplot() [either before or after background subtraction], I > get an unusual pattern that would suggest some sort of artifact in the > array, with "white streaks" across it. > When I questioned the core who had done the microarray experiment, they said > the BeadStudio image did not show any of the odd patterns. > Using outlierPlot() or BASH, some areas were flagged for masking, but they > did not match the "white streak areas" (I assume that is because the > outliers would in general have increased expression levels, as opposed to > the white areas with almost no signal). > > My questions are: > 1) is there any reason why the images generated by BeadStudio and beadarray > should be different? > 2) why are the streaks with almost no signal not flagged as regions that > should be masked? > 3) should I be concerned about these low signal streaks? > > Thank you. Best regards, > > Luis. > >> sessionInfo() > R version 2.8.0 Patched (2008-10-22 r46776) > i386-apple-darwin9.5.0 > > locale: > en_US.UTF-8/en_US.UTF-8/C/C/en_US.UTF-8/en_US.UTF-8 > > attached base packages: > [1] tools ? ? stats ? ? graphics ?grDevices utils ? ? datasets ?methods > base > > other attached packages: > ?[1] beadarray_1.10.0 ? ?sma_0.5.15 ? ? ? ? ?hwriter_0.93 > ?geneplotter_1.20.0 ?annotate_1.20.0 ? ? xtable_1.5-4 > ?AnnotationDbi_1.4.1 > ?[8] lattice_0.17-15 ? ? Biobase_2.2.0 ? ? ? limma_2.16.2 > > loaded via a namespace (and not attached): > [1] DBI_0.2-4 ? ? ? ? ?KernSmooth_2.22-22 RColorBrewer_1.0-2 RSQLite_0.7-0 > ? ?grid_2.8.0 > > _______________________________________________ > 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 for you comments, Mark and Cei. Just another quick question: the number of beads per probe that are detected in the different arrays of the 8-array slide is slightly lower in the the array with the white streaks compared with a "normal" array in the same slide (median:36 vs. 41; min: 9 vs. 13). Could this potentially bias the analysis (I'm using lumi) in such a way that I should ask Illumina to replace the array? And, if so, is this a common enough problem with Illumina to raise concerns about the reliability/ reproducibility of this platform? Thanks again, Luis. On Mar 5, 2009, at 5:28 AM, Mark Dunning wrote: > Hi Luis, > > The white streaks you see on the imageplots are because of missing > data on the array. This occurs when the Illumina scanner is unable to > decode the identity of beads on the array. Information about these > undecoded beads is not reported in the output that comes out from the > scanner and therefore we cannot use them in any analysis, including > BASH. > > The white streaks would not show up in the BeadStudio images or the > TIFF images generated by the scanner as these are just the raw pixel > intensities and do not take bead identity into account. > > Hope this helps > > Regards, > > Mark > > On Thu, Mar 5, 2009 at 2:13 AM, Luis Fernando Menezes <lmenezes at="" jhmi.edu=""> > wrote: >> Hello. >> >> I have been using beadarray (v. 1.10.0) to analyze some Illumina >> mouse >> ref8v2 arrays. >> When I import the images and expression data using readIllumina() >> and plot >> them using imageplot() [either before or after background >> subtraction], I >> get an unusual pattern that would suggest some sort of artifact in >> the >> array, with "white streaks" across it. >> When I questioned the core who had done the microarray experiment, >> they said >> the BeadStudio image did not show any of the odd patterns. >> Using outlierPlot() or BASH, some areas were flagged for masking, >> but they >> did not match the "white streak areas" (I assume that is because the >> outliers would in general have increased expression levels, as >> opposed to >> the white areas with almost no signal). >> >> My questions are: >> 1) is there any reason why the images generated by BeadStudio and >> beadarray >> should be different? >> 2) why are the streaks with almost no signal not flagged as regions >> that >> should be masked? >> 3) should I be concerned about these low signal streaks? >> >> Thank you. Best regards, >> >> Luis. >> >>> sessionInfo() >> R version 2.8.0 Patched (2008-10-22 r46776) >> i386-apple-darwin9.5.0 >> >> locale: >> en_US.UTF-8/en_US.UTF-8/C/C/en_US.UTF-8/en_US.UTF-8 >> >> attached base packages: >> [1] tools stats graphics grDevices utils datasets >> methods >> base >> >> other attached packages: >> [1] beadarray_1.10.0 sma_0.5.15 hwriter_0.93 >> geneplotter_1.20.0 annotate_1.20.0 xtable_1.5-4 >> AnnotationDbi_1.4.1 >> [8] lattice_0.17-15 Biobase_2.2.0 limma_2.16.2 >> >> loaded via a namespace (and not attached): >> [1] DBI_0.2-4 KernSmooth_2.22-22 RColorBrewer_1.0-2 >> RSQLite_0.7-0 >> grid_2.8.0 >> >> _______________________________________________ >> 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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