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@giovanni-coppola-893
Last seen 8.1 years ago
Hello Lynn, I am trying to analyze Illumina (Sentrix BeadChip Mouse Ref8) data with the beadarray package. All I have is: 1) a geneprofile.csv (matrix with expression values) 2) for each array strip, three files (.idat, .jpg, .xml) 3) one .sdf file 4) metrix.txt 5) the illumina CD with beadmap files 6) one copy of the BeadStudio software I assume I can't use the beadarray package, is that correct? Can I use the BeadStudio software to obtain bead level text files? Thanks Giovanni On Feb 8, 2006, at 10:18 AM, Lynn Amon wrote: > One possibly helpful note about Illumina arrays is that you need to > make > a change to the Settings.xml file in the ProgramFiles/Illumina/ > BeadScan > directory in order to get summary or bead-level text files which can > then be read in using read.table or with the beadarray package. > > Change the line: <savetextfiles>false</savetextfiles> to > <savetextfiles>true</savetextfiles> > and add the following line if you want bead-level data: > <saveperbeadfiles>true</saveperbeadfiles> > > Keep in mind that the bead-level files are large especially for the > whole genome chips. > > Lynn Amon > Research Scientist > Department of Pathology > University of Washington > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor
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Mark Dunning ▴ 320
@mark-dunning-1634
Last seen 8.1 years ago
Hi Giovanni, You will be able to analyse this data using 'beadarray'. The package is able to read bead-summary data which has been processed by BeadStudio (a single averaged value for each bead type on an array) or the bead-level data. If you make the modifications that Lynn suggests you will able to extract intensities of each bead using the scanning software. It does not sound like you have these files though. However, to make full use of the beadarray package you would also require files which give the coordinates of each bead on each array. Unfortunately, Illumina seem quite reluctant to give out the bead centre coordinates at the present time. We are however pressing them to make this information available. I think the best way for you to proceed would be to read the file geneprofile.csv into beadarray. The quickest way to do this is to put the file in a directory on it's own, change the R working directory to point to this and use the "readBeadSummaryData" command from beadarray. You will probably need to use the columns parameter to specify the names of the columns that are being used in the file. I believe the following command will work for the standard output from BeadStudio BSData = readBeadSummaryData(columns=list(ProbeID="TargetID", AvgSig="AVG_Signal", Nobeads="Avg_NBEADS", BeadStDev="BEAD_STDEV", Detection ="Detection")) This will then create a list object similar to the RGList object used in 'limma', ie the expression values are in the $R matrix with rows representing genes and columns representing arrays. This expression matrix can be used for clustering, pca, linear modelling (via limma) or normalisation. So far we provide median, quantile or qspline normalisation - although methods within the 'affy' package could also be used. Additionally, the 'plotMAXY' function will give scatter and MA plots for multiple arrays and is a useful way of judging array quality. btw do you know how the data in geneprofile.csv was normalised by BeadStudio? Illumina recommend a rank invariant normalisation plus background correction, but that seems like a very bad idea to me looking at some of the data it produces! Please let me know if you have any further questions or comments. The package is still in the development stage, so feedback is very much appreciated :) Regards, Mark ------------- Mark Dunning PhD Student Computational Biology Group Hutchison / MRC Research Centre Department of Oncology University of Cambridge Hills Rd, Cambridge CB2 2XZ -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces at stat.math.ethz.ch]On Behalf Of Giovanni Coppola Sent: 04 March 2006 16:54 To: Lynn Amon Cc: Bioconductor Subject: Re: [BioC] info su illumina Hello Lynn, I am trying to analyze Illumina (Sentrix BeadChip Mouse Ref8) data with the beadarray package. All I have is: 1) a geneprofile.csv (matrix with expression values) 2) for each array strip, three files (.idat, .jpg, .xml) 3) one .sdf file 4) metrix.txt 5) the illumina CD with beadmap files 6) one copy of the BeadStudio software I assume I can't use the beadarray package, is that correct? Can I use the BeadStudio software to obtain bead level text files? Thanks Giovanni On Feb 8, 2006, at 10:18 AM, Lynn Amon wrote: > One possibly helpful note about Illumina arrays is that you need to > make > a change to the Settings.xml file in the ProgramFiles/Illumina/ > BeadScan > directory in order to get summary or bead-level text files which can > then be read in using read.table or with the beadarray package. > > Change the line: <savetextfiles>false</savetextfiles> to > <savetextfiles>true</savetextfiles> > and add the following line if you want bead-level data: > <saveperbeadfiles>true</saveperbeadfiles> > > Keep in mind that the bead-level files are large especially for the > whole genome chips. > > Lynn Amon > Research Scientist > Department of Pathology > University of Washington > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor _______________________________________________ Bioconductor mailing list Bioconductor at stat.math.ethz.ch https://stat.ethz.ch/mailman/listinfo/bioconductor
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Hello Mark, thank you very much for your email. The output from BeadStudio is without normalization. I am analyzing these arrays building the expression matrix, and normalizing between arrays (quantile) with limma. After linear modeling, the results are biologically sound, but I usually do an extensive low-level analysis (eg consistency of replicates across the array, distribution of controls etc), which apparently I can't have now. My only quality measures right now are: boxplot of detection scores, BeadStDev, and AvgNBeads scatterplot of (log) signal vs bead SD, showing a direct correlation. Do you have other suggestions? I will try plotMAXY. Thanks Giovanni On Mar 4, 2006, at 10:58 AM, Mark Dunning wrote: > Hi Giovanni, > > You will be able to analyse this data using 'beadarray'. The > package is able > to read bead-summary data which has been processed by BeadStudio (a > single > averaged value for each bead type on an array) or the bead-level > data. If > you make the modifications that Lynn suggests you will able to extract > intensities of each bead using the scanning software. It does not > sound like > you have these files though. However, to make full use of the > beadarray > package you would also require files which give the coordinates of > each bead > on each array. Unfortunately, Illumina seem quite reluctant to give > out the > bead centre coordinates at the present time. We are however > pressing them to > make this information available. > > I think the best way for you to proceed would be to read the file > geneprofile.csv into beadarray. The quickest way to do this is to > put the > file in a directory on it's own, change the R working directory to > point to > this and use the "readBeadSummaryData" command from beadarray. You > will > probably need to use the columns parameter to specify the names of the > columns that are being used in the file. I believe the following > command > will work for the standard output from BeadStudio > > BSData = readBeadSummaryData(columns=list(ProbeID="TargetID", > AvgSig="AVG_Signal", Nobeads="Avg_NBEADS", BeadStDev="BEAD_STDEV", > Detection > ="Detection")) > > This will then create a list object similar to the RGList object > used in > 'limma', ie the expression values are in the $R matrix with rows > representing genes and columns representing arrays. This expression > matrix > can be used for clustering, pca, linear modelling (via limma) or > normalisation. So far we provide median, quantile or qspline > normalisation - > although methods within the 'affy' package could also be used. > Additionally, > the 'plotMAXY' function will give scatter and MA plots for multiple > arrays > and is a useful way of judging array quality. > > btw do you know how the data in geneprofile.csv was normalised by > BeadStudio? Illumina recommend a rank invariant normalisation plus > background correction, but that seems like a very bad idea to me > looking at > some of the data it produces! > > Please let me know if you have any further questions or comments. The > package is still in the development stage, so feedback is very much > appreciated :) > > Regards, > > Mark > ------------- > Mark Dunning > PhD Student > Computational Biology Group > Hutchison / MRC Research Centre > Department of Oncology > University of Cambridge > Hills Rd, Cambridge CB2 2XZ > > -----Original Message----- > From: bioconductor-bounces at stat.math.ethz.ch > [mailto:bioconductor-bounces at stat.math.ethz.ch]On Behalf Of Giovanni > Coppola > Sent: 04 March 2006 16:54 > To: Lynn Amon > Cc: Bioconductor > Subject: Re: [BioC] info su illumina > > > Hello Lynn, > I am trying to analyze Illumina (Sentrix BeadChip Mouse Ref8) data > with the beadarray package. > All I have is: > > 1) a geneprofile.csv (matrix with expression values) > 2) for each array strip, three files (.idat, .jpg, .xml) > 3) one .sdf file > 4) metrix.txt > 5) the illumina CD with beadmap files > 6) one copy of the BeadStudio software > > I assume I can't use the beadarray package, is that correct? > Can I use the BeadStudio software to obtain bead level text files? > Thanks > Giovanni > > On Feb 8, 2006, at 10:18 AM, Lynn Amon wrote: > >> One possibly helpful note about Illumina arrays is that you need to >> make >> a change to the Settings.xml file in the ProgramFiles/Illumina/ >> BeadScan >> directory in order to get summary or bead-level text files which can >> then be read in using read.table or with the beadarray package. >> >> Change the line: <savetextfiles>false</savetextfiles> to >> <savetextfiles>true</savetextfiles> >> and add the following line if you want bead-level data: >> <saveperbeadfiles>true</saveperbeadfiles> >> >> Keep in mind that the bead-level files are large especially for the >> whole genome chips. >> >> Lynn Amon >> Research Scientist >> Department of Pathology >> University of Washington >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor at stat.math.ethz.ch >> https://stat.ethz.ch/mailman/listinfo/bioconductor > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor >
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On 3/4/06 1:58 PM, "Mark Dunning" <md392 at="" cam.ac.uk=""> wrote: > btw do you know how the data in geneprofile.csv was normalised by > BeadStudio? Illumina recommend a rank invariant normalisation plus > background correction, but that seems like a very bad idea to me looking at > some of the data it produces! Mark, I'd be curious to hear what you have found to be the best way to normalize these arrays. We have also seen some problems with the invariant set method, but haven't come up with a good alternative, given that a majority of the data on the array isn't even measured (according to the detection p-values). Sean
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Sean, It seems to me that the problem with normalising via BeadStudio is that all of the normalisation methods they implement (cubic spline, average scaling, rank invariant etc...) are automatically accompanied by a "background normalisation" which is a subtraction using an average of negative controls. The subtraction is done on the unlogged scale so that lower intensity genes get affected much more so we see a characteristic fanning effect. I don't recall seeing an explanation of why Illumina prefer this method. However, in the BeadStudio manual they do say "Applying the technique [of background normalisation] allows for more quantitative assessments of fold-change differences, especially for genes with dim signals". Note that this background normalisation is in addition to a local background correction which is done to obtain the bead level intensities. Within BeadStudio it is possible to output data without any normalisation (this is the only way to avoid this background subtraction normalisation that Illumina does) and this can be read into R using beadarray or other methods. The non-normalised data is generally high quality and we see good reproducibility between arrays. So far I have used quantile and qspline methods (on both bead level and bead summary data) and I don't think there is a need for anything more complicated. Of course, normalisation on bead level data is probably going to be preferable, but I don't know what affect this actually has (yet!). I haven't yet incorporated the detection scores into the analysis anywhere. Regards, Mark On 6 Mar 2006, at 13:03, Sean Davis wrote: > > > > On 3/4/06 1:58 PM, "Mark Dunning" <md392 at="" cam.ac.uk=""> wrote: > >> btw do you know how the data in geneprofile.csv was normalised by >> BeadStudio? Illumina recommend a rank invariant normalisation plus >> background correction, but that seems like a very bad idea to me >> looking at >> some of the data it produces! > > Mark, > > I'd be curious to hear what you have found to be the best way to > normalize > these arrays. We have also seen some problems with the invariant set > method, but haven't come up with a good alternative, given that a > majority > of the data on the array isn't even measured (according to the > detection > p-values). > > Sean > >
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Hello Mark and Giovanni, Mark is correct that you need to make the changes to the Settings.xml file BEFORE you scan the beadchip. So you might want to do it the next time just to have the data should you decide to use it. It would be nice to normalize at the bead-level but I have had trouble with R and Windows and memory limits when I try to do much with beadlevel data and more than a few arrays. Right now, I just read in the bead-level data, summarize it by probe and then throw away the bead-level stuff. Does anyone have any suggestions on how to get around these memory issues? I have the same problem with Affy data unless I use RMA. Lynn Lynn Amon Research Scientist Department of Pathology University of Washington Mark Dunning wrote: > Hi Giovanni, > > You will be able to analyse this data using 'beadarray'. The package is able > to read bead-summary data which has been processed by BeadStudio (a single > averaged value for each bead type on an array) or the bead-level data. If > you make the modifications that Lynn suggests you will able to extract > intensities of each bead using the scanning software. It does not sound like > you have these files though. However, to make full use of the beadarray > package you would also require files which give the coordinates of each bead > on each array. Unfortunately, Illumina seem quite reluctant to give out the > bead centre coordinates at the present time. We are however pressing them to > make this information available. > > I think the best way for you to proceed would be to read the file > geneprofile.csv into beadarray. The quickest way to do this is to put the > file in a directory on it's own, change the R working directory to point to > this and use the "readBeadSummaryData" command from beadarray. You will > probably need to use the columns parameter to specify the names of the > columns that are being used in the file. I believe the following command > will work for the standard output from BeadStudio > > BSData = readBeadSummaryData(columns=list(ProbeID="TargetID", > AvgSig="AVG_Signal", Nobeads="Avg_NBEADS", BeadStDev="BEAD_STDEV", Detection > ="Detection")) > > This will then create a list object similar to the RGList object used in > 'limma', ie the expression values are in the $R matrix with rows > representing genes and columns representing arrays. This expression matrix > can be used for clustering, pca, linear modelling (via limma) or > normalisation. So far we provide median, quantile or qspline normalisation - > although methods within the 'affy' package could also be used. Additionally, > the 'plotMAXY' function will give scatter and MA plots for multiple arrays > and is a useful way of judging array quality. > > btw do you know how the data in geneprofile.csv was normalised by > BeadStudio? Illumina recommend a rank invariant normalisation plus > background correction, but that seems like a very bad idea to me looking at > some of the data it produces! > > Please let me know if you have any further questions or comments. The > package is still in the development stage, so feedback is very much > appreciated :) > > Regards, > > Mark > ------------- > Mark Dunning > PhD Student > Computational Biology Group > Hutchison / MRC Research Centre > Department of Oncology > University of Cambridge > Hills Rd, Cambridge CB2 2XZ > > -----Original Message----- > From: bioconductor-bounces at stat.math.ethz.ch > [mailto:bioconductor-bounces at stat.math.ethz.ch]On Behalf Of Giovanni > Coppola > Sent: 04 March 2006 16:54 > To: Lynn Amon > Cc: Bioconductor > Subject: Re: [BioC] info su illumina > > > Hello Lynn, > I am trying to analyze Illumina (Sentrix BeadChip Mouse Ref8) data > with the beadarray package. > All I have is: > > 1) a geneprofile.csv (matrix with expression values) > 2) for each array strip, three files (.idat, .jpg, .xml) > 3) one .sdf file > 4) metrix.txt > 5) the illumina CD with beadmap files > 6) one copy of the BeadStudio software > > I assume I can't use the beadarray package, is that correct? > Can I use the BeadStudio software to obtain bead level text files? > Thanks > Giovanni > > On Feb 8, 2006, at 10:18 AM, Lynn Amon wrote: > > >>One possibly helpful note about Illumina arrays is that you need to >>make >>a change to the Settings.xml file in the ProgramFiles/Illumina/ >>BeadScan >>directory in order to get summary or bead-level text files which can >>then be read in using read.table or with the beadarray package. >> >>Change the line: <savetextfiles>false</savetextfiles> to >><savetextfiles>true</savetextfiles> >>and add the following line if you want bead-level data: >><saveperbeadfiles>true</saveperbeadfiles> >> >>Keep in mind that the bead-level files are large especially for the >>whole genome chips. >> >>Lynn Amon >>Research Scientist >>Department of Pathology >>University of Washington >> >>_______________________________________________ >>Bioconductor mailing list >>Bioconductor at stat.math.ethz.ch >>https://stat.ethz.ch/mailman/listinfo/bioconductor > > > _______________________________________________ > Bioconductor mailing list > Bioconductor at stat.math.ethz.ch > https://stat.ethz.ch/mailman/listinfo/bioconductor >
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