> ------ > > Message: 22 > Date: Fri, 13 Apr 2012 01:22:21 +0100 > From: Nikolas <n.pontikos at="" gmail.com=""> > To: bioconductor at r-project.org > Subject: [BioC] flowWorkspace transformation on FCS 3 data > Message-ID: > <cakxttxp5kflogh4im1uhfmqftomoxz1-g-vwlxqo3uzkth0krg at="" mail.gmail.com=""> > Content-Type: text/plain; charset=UTF-8 > > Hello, > > I am bit puzzled as to how flowWorkspace transforms FCS 3 data (see code below). > I would expect fcs.data.comp and fcs.data.1 to contain similar (if not > identical) data yet the range is completely different. They should not be identical. fcs.data.1 contains compensated and transformed data, while fcs.data.comp has only compensated data. > Some transform has taken place. Yes. > Does this have anything to do with flowWorkspace trying to simulate > the binning process that FlowJo does on FCS 3 data? That binning process is flowJo's way of transforming the data for gating. You have to reproduce the transformation in order to accurately reproduce the gating because the transformation is non- linear and thus gate boundaries on the untransformed (or some inappropriately transformed) scale would also be non-linear. > Someone at TreeStar told me that FlowJo does binning with 4096 bins > which is possibly what is going on here? Yes that's why the range of the transformed data goes from 1...4096. flowWorkspace stores a mapping back to the raw scale so using plotGate to visualize the data will give you appropriate axes on the raw scale. > Many thanks for you help, > > Niko. > > > > > library(flowWorkspace) > library(flowCore) > > #CAD61.xml is a flowjo workspace file which references CAD61.fcs > openWorkspace("CAD61.xml") -> wsp > try(parseWorkspace(wsp, execute=TRUE, isNcdf=FALSE, path=".", > name="All Samples")) -> G > G at set[[1]]->g > > getData(g, 1)->fcs.data.1 > > read.FCS('CAD61.fcs')->fcs.data > compensate(fcs.data, fcs.data at description[["SPILL"]])->fcs.data.comp > >> fcs.data.comp > flowFrame object '71d0bf04-ddb1-4284-ad2b-af44322ecdfb' > with 300195 cells and 9 observables: > name desc range minRange maxRange > $P1 FSC-A <na> 262144 0.00 262143 > $P2 SSC-A <na> 262144 -111.00 262143 > $P3 Alexa-488-A cd127 262144 -111.00 262143 > $P4 PE-Cy7-A hladr 262144 -111.00 262143 > $P5 APC-A cd25_ma251+2a3 262144 -108.00 262143 > $P6 PE-A cd101 262144 -111.00 262143 > $P7 Alexa-700-A cd4 262144 -96.00 262143 > $P8 Pacific Blue-A cd45ra 262144 -91.64 262143 > $P9 Time <na> 262144 0.00 262143 > 162 keywords are stored in the 'description' slot > >> fcs.data.1 > flowFrame object '71d0bf04-ddb1-4284-ad2b-af44322ecdfb' > with 300195 cells and 9 observables: > name desc range minRange maxRange > $P1 FSC-A <na> 3641.837 455.000000 4096.837 > $P2 SSC-A <na> 262254.000 -111.000000 262143.000 > $P3 <alexa-488-a> cd127 4098.176 -1.339711 4096.837 > $P4 <pe-cy7-a> hladr 4098.176 -1.339711 4096.837 > $P5 <apc-a> cd25_ma251+2a3 4088.140 8.696338 4096.837 > $P6 <pe-a> cd101 4098.176 -1.339711 4096.837 > $P7 <alexa-700-a> cd4 4046.566 50.270760 4096.837 > $P8 <pacific blue-a=""> cd45ra 4030.885 65.951978 4096.837 > $P9 Time <na> 21737.000 0.000000 21737.000 > 162 keywords are stored in the 'description' slot > > >

> Yes that's why the range of the transformed data goes from 1...4096. flowWorkspace stores a mapping back to the raw scale so using plotGate to visualize the data will give you appropriate axes on the raw scale. How to access this mapping back to the raw scale? On 13 April 2012 17:38, Greg Finak <gfinak at="" fhcrc.org=""> wrote: >> ------ >> >> Message: 22 >> Date: Fri, 13 Apr 2012 01:22:21 +0100 >> From: Nikolas <n.pontikos at="" gmail.com=""> >> To: bioconductor at r-project.org >> Subject: [BioC] flowWorkspace transformation on FCS 3 data >> Message-ID: >> ? ? ? <cakxttxp5kflogh4im1uhfmqftomoxz1-g-vwlxqo3uzkth0krg at="" mail.gmail.com=""> >> Content-Type: text/plain; charset=UTF-8 >> >> Hello, >> >> I am bit puzzled as to how flowWorkspace transforms FCS 3 data (see code below). >> I would expect fcs.data.comp and fcs.data.1 to contain similar (if not >> identical) data yet the range is completely different. > They should not be identical. fcs.data.1 contains compensated and transformed data, while fcs.data.comp has only compensated data. > >> Some transform has taken place. > Yes. >> Does this have anything to do with flowWorkspace trying to simulate >> the binning process that FlowJo does on FCS 3 data? > That binning process is flowJo's way of transforming the data for gating. You have to reproduce the transformation in order to accurately reproduce the gating because the transformation is non- linear and thus gate boundaries on the untransformed (or some inappropriately transformed) scale would also be non-linear. > >> Someone at TreeStar told me that FlowJo does binning with 4096 bins >> which is possibly what is going on here? > Yes that's why the range of the transformed data goes from 1...4096. flowWorkspace stores a mapping back to the raw scale so using plotGate to visualize the data will give you appropriate axes on the raw scale. > >> Many thanks for you help, >> >> Niko. >> >> >> >> >> library(flowWorkspace) >> library(flowCore) >> >> #CAD61.xml is a flowjo workspace file which references CAD61.fcs >> openWorkspace("CAD61.xml") -> wsp >> try(parseWorkspace(wsp, execute=TRUE, isNcdf=FALSE, path=".", >> name="All Samples")) -> G >> G at set[[1]]->g >> >> getData(g, 1)->fcs.data.1 >> >> read.FCS('CAD61.fcs')->fcs.data >> compensate(fcs.data, fcs.data at description[["SPILL"]])->fcs.data.comp >> >>> fcs.data.comp >> flowFrame object '71d0bf04-ddb1-4284-ad2b-af44322ecdfb' >> with 300195 cells and 9 observables: >> ? ? ? ? ? ? ?name ? ? ? ? ? desc ?range minRange maxRange >> $P1 ? ? ? ? ?FSC-A ? ? ? ? ? <na> 262144 ? ? 0.00 ? 262143 >> $P2 ? ? ? ? ?SSC-A ? ? ? ? ? <na> 262144 ?-111.00 ? 262143 >> $P3 ? ?Alexa-488-A ? ? ? ? ?cd127 262144 ?-111.00 ? 262143 >> $P4 ? ? ? PE-Cy7-A ? ? ? ? ?hladr 262144 ?-111.00 ? 262143 >> $P5 ? ? ? ? ?APC-A cd25_ma251+2a3 262144 ?-108.00 ? 262143 >> $P6 ? ? ? ? ? PE-A ? ? ? ? ?cd101 262144 ?-111.00 ? 262143 >> $P7 ? ?Alexa-700-A ? ? ? ? ? ?cd4 262144 ? -96.00 ? 262143 >> $P8 Pacific Blue-A ? ? ? ? cd45ra 262144 ? -91.64 262143 >> $P9 ? ? ? ? ? Time ? ? ? ? ? <na> 262144 ? ? 0.00 ? 262143 >> 162 keywords are stored in the 'description' slot >> >>> fcs.data.1 >> flowFrame object '71d0bf04-ddb1-4284-ad2b-af44322ecdfb' >> with 300195 cells and 9 observables: >> ? ? ? ? ? ? ? ?name ? ? ? ? ? desc ? ? ?range ? ?minRange ? maxRange >> $P1 ? ? ? ? ? ?FSC-A ? ? ? ? ? <na> ? 3641.837 ?455.000000 ? 4096.837 >> $P2 ? ? ? ? ? ?SSC-A ? ? ? ? ? <na> 262254.000 -111.000000 262143.000 >> $P3 ? ?<alexa-488-a> ? ? ? ? ?cd127 ? 4098.176 ? -1.339711 ? 4096.837 >> $P4 ? ? ? <pe-cy7-a> ? ? ? ? ?hladr ? 4098.176 ? -1.339711 ? 4096.837 >> $P5 ? ? ? ? ?<apc-a> cd25_ma251+2a3 ? 4088.140 ? ?8.696338 ? 4096.837 >> $P6 ? ? ? ? ? <pe-a> ? ? ? ? ?cd101 ? 4098.176 ? -1.339711 ? 4096.837 >> $P7 ? ?<alexa-700-a> ? ? ? ? ? ?cd4 ? 4046.566 ? 50.270760 ? 4096.837 >> $P8 <pacific blue-a=""> ? ? ? ? cd45ra ? 4030.885 ? 65.951978 ? 4096.837 >> $P9 ? ? ? ? ? ? Time ? ? ? ? ? <na> ?21737.000 ? ?0.000000 ?21737.000 >> 162 keywords are stored in the 'description' slot >> >> >> > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor