[BioC] flowWorkspace transformation on FCS 3 data (Nikolas)

[Nikolas]

> 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
>>
>>
>>
>
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