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Nimblegen and Ringo package

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João Fadista ▴ 500

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ADD COMMENT • link updated 17.2 years ago by Todd Richmond ▴ 140 • written 17.2 years ago by João Fadista ▴ 500

score 0 · Answer 1 · 2007-08-16

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Todd Richmond ▴ 140

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Hi Jo?o, NimbleGen does use the qspline method (normalize.qspline from the affy package) for normalizing CGH data. The tukey.biweight mean is used to scale ChIP-chip ratio gff files so that the data is centered on zero. We do not use qspline, or indeed any other normalization method, for ChIP-chip data, since we have not found a method that does not adversely affect downstream data quality. Since Ringo is a package for analyzing ChIP-chip data, I'm assuming that's why that's there. I'm curious, though, why you are trying to use it for CGH data, when there are a number of other packages specifically mean for CGH analysis. Todd > -----Original Message----- > From: bioconductor-bounces at stat.math.ethz.ch [mailto:bioconductor- > bounces at stat.math.ethz.ch] On Behalf Of Jo?o Fadista > Sent: Thursday, August 16, 2007 2:46 AM > To: bioconductor at stat.math.ethz.ch > Subject: [BioC] Nimblegen and Ringo package > > Dear all, > > I am using the Ringo package to analyse some demo CGH data from Nimblegen > (ftp://ftp.nimblegen.com/pub/demo_data/). > > 1 - In the NimbleScan User?s Guide version 2.3 it is written that they use > the qspline normalization method (Christopher Workman, et al. "A new non- > linear normalization method for reducing variability in dna microarray > experiments". Genome Biology 2002) which I believe is the one implemented > in the affy package, right? Despite of this, the Ringo package has a > supposed nimblegen normalization method related to the tukey.biweight > function also appearing in the affy package. > Maybe I am wrong, but why did the Ringo package named a supposed nimblegen > normalization method to a method which as far as I know is not from > nimblegen? > > 2 - Another question is related to the probeAnno.rda file which is needed > in some function implemented in the Ringo package. Since I don?t have > these kind of files I was wondering if I have the start/end positions and > the indices of the probes is there any easy way to make the probeAnno.rda > files? P.S. If it helps, In the demo data I have .pos, .ndf, .pair and > .gff files. > > Best regards > > Jo?o Fadista > Ph.d. student > > > > UNIVERSITY OF AARHUS > Faculty of Agricultural Sciences > Dept. of Genetics and Biotechnology > Blichers All? 20, P.O. BOX 50 > DK-8830 Tjele > > Phone: +45 8999 1900 > Direct: +45 8999 1900 > E-mail: Joao.Fadista at agrsci.dk <mailto:joao.fadista at="" agrsci.dk=""> > Web: www.agrsci.org <http: www.agrsci.org=""/> -- Todd Richmond NimbleGen Systems, Inc. Manager of Research Informatics One Science Court Madison, WI 53711 1-608-218-7600

ADD COMMENT • link 17.2 years ago Todd Richmond ▴ 140

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Hi Joern and Todd, Thanks for the replies. Indeed, after the reply from Joern I found in the Nimblegen website that the the qspline is for normalizing CGH data while the tukey.biweight is for scaling ChIP-chip data. I am analysing the demo Nimblegen CGH data with Ringo package because I think is the only bioconductor package that can convert the .pair raw files from nimblegen into RGList, right? After I get the RGList then I can use the packages suitable for CGH analysis. By the way, I came up with another question. As I read through the CGH NimbleScan User?s Guide I didn?t find a file that describes the statistics of the fluorescent spots for each probe, like the GenePix software does (SD of the pixel intensity, Signal to noise ratio, a score for each spot, etc.). Can you generate that kind of files? If not, is it possible to transform a .ndf into a .gal file (http://www.m oleculardevices.com/pages/software/gn_genepix_file_formats.html#gal) in order to be able to analyse the nimblegen .tiff images in GenePix? Best regards, Jo?o Fadista -----Original Message----- From: Todd Richmond [mailto:todd@nimblegen.com] Sent: Thursday, August 16, 2007 3:53 PM To: Jo?o Fadista; bioconductor at stat.math.ethz.ch Subject: RE: [BioC] Nimblegen and Ringo package Hi Jo?o, NimbleGen does use the qspline method (normalize.qspline from the affy package) for normalizing CGH data. The tukey.biweight mean is used to scale ChIP-chip ratio gff files so that the data is centered on zero. We do not use qspline, or indeed any other normalization method, for ChIP-chip data, since we have not found a method that does not adversely affect downstream data quality. Since Ringo is a package for analyzing ChIP-chip data, I'm assuming that's why that's there. I'm curious, though, why you are trying to use it for CGH data, when there are a number of other packages specifically mean for CGH analysis. Todd > -----Original Message----- > From: bioconductor-bounces at stat.math.ethz.ch [mailto:bioconductor- > bounces at stat.math.ethz.ch] On Behalf Of Jo?o Fadista > Sent: Thursday, August 16, 2007 2:46 AM > To: bioconductor at stat.math.ethz.ch > Subject: [BioC] Nimblegen and Ringo package > > Dear all, > > I am using the Ringo package to analyse some demo CGH data from > Nimblegen (ftp://ftp.nimblegen.com/pub/demo_data/). > > 1 - In the NimbleScan User?s Guide version 2.3 it is written that they > use the qspline normalization method (Christopher Workman, et al. "A > new non- linear normalization method for reducing variability in dna > microarray experiments". Genome Biology 2002) which I believe is the > one implemented in the affy package, right? Despite of this, the Ringo > package has a supposed nimblegen normalization method related to the > tukey.biweight function also appearing in the affy package. > Maybe I am wrong, but why did the Ringo package named a supposed > nimblegen normalization method to a method which as far as I know is > not from nimblegen? > > 2 - Another question is related to the probeAnno.rda file which is > needed in some function implemented in the Ringo package. Since I > don?t have these kind of files I was wondering if I have the start/end > positions and the indices of the probes is there any easy way to make > the probeAnno.rda files? P.S. If it helps, In the demo data I have > .pos, .ndf, .pair and .gff files. > > Best regards > > Jo?o Fadista > Ph.d. student > > > > UNIVERSITY OF AARHUS > Faculty of Agricultural Sciences > Dept. of Genetics and Biotechnology > Blichers All? 20, P.O. BOX 50 > DK-8830 Tjele > > Phone: +45 8999 1900 > Direct: +45 8999 1900 > E-mail: Joao.Fadista at agrsci.dk <mailto:joao.fadista at="" agrsci.dk=""> > Web: www.agrsci.org <http: www.agrsci.org=""/> -- Todd Richmond NimbleGen Systems, Inc. Manager of Research Informatics One Science Court Madison, WI 53711 1-608-218-7600

ADD REPLY • link 17.2 years ago João Fadista ▴ 500

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> -----Original Message----- > From: Jo?o Fadista [mailto:Joao.Fadista at agrsci.dk] > Sent: Thursday, August 16, 2007 9:49 AM > To: Todd Richmond; bioconductor at stat.math.ethz.ch; toedling at ebi.ac.uk > Subject: RE: [BioC] Nimblegen and Ringo package > > I am analysing the demo Nimblegen CGH data with Ringo package because I > think is the only bioconductor package that can convert the .pair raw > files from nimblegen into RGList, right? After I get the RGList then I can > use the packages suitable for CGH analysis. Steven McKinney posted some scripts to get NimbleGen data into snapCGH awhile ago (do a search for NimbleGen + snapCGH). snapCGH will then give you access to at least 3 different algorithms. > By the way, I came up with another question. As I read through the CGH > NimbleScan User?s Guide I didn?t find a file that describes the statistics > of the fluorescent spots for each probe, like the GenePix software does > (SD of the pixel intensity, Signal to noise ratio, a score for each spot, > etc.). Can you generate that kind of files? > NimbleScan can generate a feature report, which has the mean and SD of the pixel intensities comprising the feature, but that's about it. Our features are only 3x3 pixels, in a checkerboard arrangement, so there's not really enough real estate surrounding each feature to get a reasonable estimate of local background. > If not, is it possible to transform a .ndf into a .gal file > (http://www.moleculardevices.com/pages/software/gn_genepix_file_form ats.ht > ml#gal) in order to be able to analyse the nimblegen .tiff images in > GenePix? It's possible to use GenePix 6.0 to extract the .tif images, since it does support square features and rectangular positioning. But when I've tried that, it appears to only quantify a 2x2 pixel feature with no background information - probably due to the close packing of the features. You might be able to make a custom .gal file which would only quantify the "on" positions of the checkboard, thus letting GenePix make some background calculation. But we have no tools to convert a .ndf file to a .gal file... Regards, Todd -- Todd Richmond NimbleGen Systems, Inc. Manager of Research Informatics One Science Court Madison, WI 53711 1-608-218-7600

ADD REPLY • link 17.2 years ago Todd Richmond ▴ 140

score 0 · Answer 2 · 2007-08-16

Hello Jo?o, Jo?o Fadista wrote: > Dear all, > > I am using the Ringo package to analyse some demo CGH data from Nimblegen (ftp://ftp.nimblegen.com/pub/demo_data/). > > 1 - In the NimbleScan User?s Guide version 2.3 it is written that they use the qspline normalization method (Christopher Workman, et al. "A new non-linear normalization method for reducing variability in dna microarray experiments". Genome Biology 2002) which I believe is the one implemented in the affy package, right? Despite of this, the Ringo package has a supposed nimblegen normalization method related to the tukey.biweight function also appearing in the affy package. > Maybe I am wrong, but why did the Ringo package named a supposed nimblegen normalization method to a method which as far as I know is not from nimblegen? > I am not totally sure if it is still the case, but NimbleGen have used the tukey-biweight scaling to produce the data *.gff files from the scanned two-channel intensities. See http://www.nimblegen.com/products/chip/index.html and scroll down to the FAQ and show all topics. Various manuals mention it as well. I did not find this scaling terribly useful and did mainly include if for sake of completeness. > > 2 - Another question is related to the probeAnno.rda file which is needed in some function implemented in the Ringo package. Since I don?t have these kind of files I was wondering if I have the start/end positions and the indices of the probes is there any easy way to make the probeAnno.rda files? P.S. If it helps, In the demo data I have .pos, .ndf, .pair and .gff files. > Yes, there is. Have a look in the scripts directory of the installed Ringo package. In there you will find an R script called "makeProbeAnno.R" that shows how to produce a probeAnno environment from NimbleGen's POS files. Please get back to me, if you encounter any problems with this script. I would also suggest to download and install the current development version of Ringo (version > 1.1.8) from the Bioconductor web site at http://www.bioconductor.org/packages/2.1/bioc/html/Ringo.html since I streamlined that script considerably after the original release. Best regards, Joern -- Joern Toedling EMBL - European Bioinformatics Institute Wellcome Trust Genome Campus Hinxton, Cambridge CB10 1SD United Kingdom Phone +44(0)1223 492566 Email toedling at ebi.ac.uk