Thanks for the advice Mike. I have tried changing the fsc/ssc calls around as you suggested, and get the same error. On the bright side, I was able to use spillover() successfully with the sample data in flowCore/extdata/compdata/data. I reckon my flowSet needs some cleaning up yet. -k On 11/06/2012 04:06 PM, Mike wrote: > Hi,Kevin, > > Your spillover call should go like this: > spill.mat<- spillover(comp.data,..., fsc="FSC-A", ssc="SSC-A") > > Regarding to importing flowJo xml to R,you can try "flowWorkspace" package. Hope this helps. > > > Mike Jiang,PhD > > System Analyst > > PS Statistics, Vaccine and Infectious Disease Division > > Fred Hutchinson Cancer Research Center > > (206)667-1905 <tel:%28206%29667-1905> > > > On 11/06/2012 03:06 PM, Kevin Schiesser [guest] wrote: >> Hello there, >> I am using Bioconductor to look at flow cytometry data. To begin, I would like to generate and apply a compensation matrix to the experimental samples. Below is the work flow, as well as more general information on the flowSet and the R version that I use. Any recommendations would be splendid. I am thinking I might have to figure on how to import FlowJo 10 xml files into bionconductor, a step that I prefer to avoid. >> >> Much thanks, >> Kevin >> >> PS I have tried many permutations of the spillover() method including patt=..., useNormFilt=TRUE/FALSE, changing the unstained = index around, etc... And also, I cannot seem to get system.file("compC", package="flowCore") to return a non-empty value, maybe that would make compensation easier. Just a thought. -k >> >> ~~~~~~~~~~~~~~~~~~~~~~~~~~~ >> >> ##import the flowSet >> >> library(flowCore) >> >> compData<- read.flowSet(path = ".../compC/", transformation = FALSE) >> >> ##trim the time column off (I know there are faster ways, but to be very explicit...) >> >> comp.data<- compData[c(1,2,3,4,5,6,7,8,9,10,11,12),c("FSC-A", "SSC-A", "FITC-A", "PerCP-A", "Pacific Blue-A", "AARD-A", "QDot605-A", "APC-A", "Alexa700-A", "APC-Cy7-A", "PE-A", "ECD-A", "PE-Cy7-A")] >> >> ##generate compensation matrix >> >> spill.mat<- spillover(comp.data,unstained = sampleNames(compData)[12], fsc=compData[,1], ssc=compData[,2]) >> >>> traceback() >> 3: .local(x, ...) : subscript out of bounds >> 2: spillover(comp.data, unstained<- sampleNames(compData)[12], fsc = compData[,1], ssc = compData[, 2]) >> 1: spillover(comp.data, unstained<- sampleNames(compData)[12], fsc = compData[,1], ssc = compData[, 2]) >> >>> compData[[12]] >> flowFrame object 'Compensation Controls_Unstained Control.fcs' >> with 10000 cells and 14 observables: >> name desc range minRange maxRange >> $P1 FSC-A NA 262144 0.00 262143 >> $P2 SSC-A NA 262144 0.00 262143 >> $P3 FITC-A NA 262144 -106.22 262143 >> $P4 PerCP-A NA 262144 -111.00 262143 >> $P5 Pacific Blue-A NA 262144 -111.00 262143 >> $P6 AARD-A NA 262144 -111.00 262143 >> $P7 QDot605-A NA 262144 -111.00 262143 >> $P8 APC-A NA 262144 -29.05 262143 >> $P9 Alexa700-A NA 262144 -82.17 262143 >> $P10 APC-Cy7-A NA 262144 -111.00 262143 >> $P11 PE-A NA 262144 -87.69 262143 >> $P12 ECD-A NA 262144 -63.27 262143 >> $P13 PE-Cy7-A NA 262144 -25.53 262143 >> $P14 Time NA 262144 0.00 262143 >> 182 keywords are stored in the 'description' slot >> >> -- output of sessionInfo(): >> >>> sessionInfo() >> R version 2.15.1 (2012-06-22) >> Platform: x86_64-pc-linux-gnu (64-bit) >> >> locale: >> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C >> [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 >> [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8 >> [7] LC_PAPER=C LC_NAME=C >> [9] LC_ADDRESS=C LC_TELEPHONE=C >> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C >> >> attached base packages: >> [1] stats graphics grDevices utils datasets methods base >> >> other attached packages: >> [1] flowCore_1.24.0 rrcov_1.3-02 pcaPP_1.9-48 mvtnorm_0.9-9993 >> [5] robustbase_0.9-4 Biobase_2.18.0 BiocGenerics_0.4.0 >> >> loaded via a namespace (and not attached): >> [1] feature_1.2.8 graph_1.36.0 ks_1.8.10 MASS_7.3-22 stats4_2.15.1 >> [6] tools_2.15.1 >> >> -- >> Sent via the guest posting facility at bioconductor.org. > [[alternative HTML version deleted]]

> mat.cfs.clean <- spillover(cfs.clean[,(1:12)], 12, fsc="FSC-A", ssc="SSC-A", useNormFilt=TRUE, method="mean") Error in .local(x, ...) : subscript out of bounds > traceback() 3: .local(x, ...) 2: spillover(cfs.clean[, (1:12)], 12, fsc = "FSC-A", ssc = "SSC-A", useNormFilt = TRUE, method = "mean") 1: spillover(cfs.clean[, (1:12)], 12, fsc = "FSC-A", ssc = "SSC-A", useNormFilt = TRUE, method = "mean") On 11/08/2012 10:28 AM, Greg Finak wrote: > Kevin, could you provide a traceback() after this error occurs? > > ---------------------------------------------------------- > Greg Finak, Ph.D > Staff Scientist > Vaccine and Infectious Disease Division. > Fred Hutchinson Cancer Research Center > Seattle, WA > (206)667-3116 > gfinak@fhcrc.org <mailto:gfinak@fhcrc.org> > > On 2012-11-08, at 10:17 AM, Kevin Schiesser wrote: > >> After a couple days of troubleshooting, I am able to generate an >> /incomplete/ compensation matrix. It's a bit baffling but here goes. If: >> >> useNormFilt =TRUE and method = "mean" in spillover() >> >> Then: the spillover() function returns a non-empty value /only /when >> the stain+fsc+ssc parameters are limited to 11 or less. For instance: >> >> > mat.cfs.clean <- spillover(cfs.clean[,(1:11)], 12, fsc="FSC-A", >> ssc="SSC-A", method="mean", useNormFilt=TRUE) >> > mat.cfs.clean >> FITC-A PerCP-A Pacific Blue-A >> AARD-A QDot605-A APC-A Alexa700-A APC-Cy7-A PE-A >> FITC-A 1.000000e+00 0.0358395804 0.0008531686 1.993349e-02 >> 6.022913e-03 0.0013364573 0.000000e+00 0.0000000000 5.264458e-03 >> PerCP-A 0.000000e+00 1.0000000000 0.0008722239 0.000000e+00 >> 1.286419e-05 0.0242856076 3.460406e-02 0.0059878924 0.000000e+00 >> Pacific Blue-A 4.631962e-05 0.0009085721 1.0000000000 1.179575e-01 >> 1.169263e-02 0.0021385373 1.742831e-05 0.0000000000 0.000000e+00 >> AARD-A 1.008167e-01 0.2910952827 0.2913268220 1.000000e+00 >> 5.124343e-01 0.0000000000 8.682088e-02 0.0507779717 3.867331e-02 >> QDot605-A 6.119886e-04 0.0040399152 0.0005064663 0.000000e+00 >> 1.000000e+00 0.0000000000 0.000000e+00 0.0000000000 9.767048e-03 >> APC-A 5.944180e-05 0.0151967641 0.0001962633 0.000000e+00 >> 6.093479e-05 1.0000000000 3.447000e-01 0.0676159928 1.971772e-05 >> Alexa700-A 1.465875e-03 0.0150411274 0.0009918405 0.000000e+00 >> 0.000000e+00 0.0058617085 1.000000e+00 0.1992198495 3.531374e-04 >> APC-Cy7-A 1.169540e-04 0.0063815758 0.0014514320 6.223971e-05 >> 9.907666e-06 0.2403238884 1.825396e-01 1.0000000000 8.540711e-05 >> PE-A 4.780649e-02 0.2291811583 0.0001488972 0.000000e+00 >> 6.747392e-02 0.0001477967 2.612027e-04 0.0002589873 1.000000e+00 >> >> > mat.cfs.clean <- spillover(cfs.clean[,(1:13)], 12, fsc="FSC-A", >> ssc="SSC-A", method="mean", useNormFilt=TRUE) >> Error in .local(x, ...) : subscript out of bounds >> >> Alternatively >> >> > mat.cfs.clean <- spillover(cfs.clean[,-(3:4)], 12, fsc="FSC-A", >> ssc="SSC-A", method="mean", useNormFilt=TRUE) >> completes >> > mat.cfs.clean <- spillover(cfs.clean[,-(3)], 12, fsc="FSC-A", >> ssc="SSC-A", method="mean", useNormFilt=TRUE) >> Error in .local(x, ...) : subscript out of bounds >> >> Any thoughts? I has also occurred to me that I might first gate the >> stained/unstained beads before calculating the spillover matrix. Has >> anyone tried this before? -k >> >> On 11/07/2012 06:14 PM, Kevin Schiesser wrote: >>> Could the 'script out of bounds' error be arising because the some >>> of the florescent intensity measurements are negative? Do you >>> transform your compensation measurements before generating the >>> compensation matrix? >>> >>> Thanks much, >>> Kevin >>> >>> On 11/06/2012 04:10 PM, Kevin Schiesser wrote: >>>> Thanks for the advice Mike. I have tried changing the fsc/ssc calls >>>> around as you suggested, and get the same error. On the bright >>>> side, I was able to use spillover() successfully with the sample >>>> data in flowCore/extdata/compdata/data. I reckon my flowSet needs >>>> some cleaning up yet. -k >>>> >>>> On 11/06/2012 04:06 PM, Mike wrote: >>>>> Hi,Kevin, >>>>> >>>>> Your spillover call should go like this: >>>>> spill.mat<- spillover(comp.data,..., fsc="FSC-A", ssc="SSC-A") >>>>> >>>>> Regarding to importing flowJo xml to R,you can try "flowWorkspace" package. Hope this helps. >>>>> >>>>> >>>>> Mike Jiang,PhD >>>>> >>>>> System Analyst >>>>> >>>>> PS Statistics, Vaccine and Infectious Disease Division >>>>> >>>>> Fred Hutchinson Cancer Research Center >>>>> >>>>> (206)667-1905 <tel:%28206%29667-1905> >>>>> >>>>> >>>>> On 11/06/2012 03:06 PM, Kevin Schiesser [guest] wrote: >>>>>> Hello there, >>>>>> I am using Bioconductor to look at flow cytometry data. To begin, I would like to generate and apply a compensation matrix to the experimental samples. Below is the work flow, as well as more general information on the flowSet and the R version that I use. Any recommendations would be splendid. I am thinking I might have to figure on how to import FlowJo 10 xml files into bionconductor, a step that I prefer to avoid. >>>>>> >>>>>> Much thanks, >>>>>> Kevin >>>>>> >>>>>> PS I have tried many permutations of the spillover() method including patt=..., useNormFilt=TRUE/FALSE, changing the unstained = index around, etc... And also, I cannot seem to get system.file("compC", package="flowCore") to return a non-empty value, maybe that would make compensation easier. Just a thought. -k >>>>>> >>>>>> ~~~~~~~~~~~~~~~~~~~~~~~~~~~ >>>>>> >>>>>> ##import the flowSet >>>>>> >>>>>> library(flowCore) >>>>>> >>>>>> compData<- read.flowSet(path = ".../compC/", transformation = FALSE) >>>>>> >>>>>> ##trim the time column off (I know there are faster ways, but to be very explicit...) >>>>>> >>>>>> comp.data<- compData[c(1,2,3,4,5,6,7,8,9,10,11,12),c("FSC-A", "SSC-A", "FITC-A", "PerCP-A", "Pacific Blue-A", "AARD-A", "QDot605-A", "APC-A", "Alexa700-A", "APC-Cy7-A", "PE-A", "ECD-A", "PE-Cy7-A")] >>>>>> >>>>>> ##generate compensation matrix >>>>>> >>>>>> spill.mat<- spillover(comp.data,unstained = sampleNames(compData)[12], fsc=compData[,1], ssc=compData[,2]) >>>>>> >>>>>>> traceback() >>>>>> 3: .local(x, ...) : subscript out of bounds >>>>>> 2: spillover(comp.data, unstained<- sampleNames(compData)[12], fsc = compData[,1], ssc = compData[, 2]) >>>>>> 1: spillover(comp.data, unstained<- sampleNames(compData)[12], fsc = compData[,1], ssc = compData[, 2]) >>>>>> >>>>>>> compData[[12]] >>>>>> flowFrame object 'Compensation Controls_Unstained Control.fcs' >>>>>> with 10000 cells and 14 observables: >>>>>> name desc range minRange maxRange >>>>>> $P1 FSC-A NA 262144 0.00 262143 >>>>>> $P2 SSC-A NA 262144 0.00 262143 >>>>>> $P3 FITC-A NA 262144 -106.22 262143 >>>>>> $P4 PerCP-A NA 262144 -111.00 262143 >>>>>> $P5 Pacific Blue-A NA 262144 -111.00 262143 >>>>>> $P6 AARD-A NA 262144 -111.00 262143 >>>>>> $P7 QDot605-A NA 262144 -111.00 262143 >>>>>> $P8 APC-A NA 262144 -29.05 262143 >>>>>> $P9 Alexa700-A NA 262144 -82.17 262143 >>>>>> $P10 APC-Cy7-A NA 262144 -111.00 262143 >>>>>> $P11 PE-A NA 262144 -87.69 262143 >>>>>> $P12 ECD-A NA 262144 -63.27 262143 >>>>>> $P13 PE-Cy7-A NA 262144 -25.53 262143 >>>>>> $P14 Time NA 262144 0.00 262143 >>>>>> 182 keywords are stored in the 'description' slot >>>>>> >>>>>> -- output of sessionInfo(): >>>>>> >>>>>>> sessionInfo() >>>>>> R version 2.15.1 (2012-06-22) >>>>>> Platform: x86_64-pc-linux-gnu (64-bit) >>>>>> >>>>>> locale: >>>>>> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C >>>>>> [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8 >>>>>> [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8 >>>>>> [7] LC_PAPER=C LC_NAME=C >>>>>> [9] LC_ADDRESS=C LC_TELEPHONE=C >>>>>> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C >>>>>> >>>>>> attached base packages: >>>>>> [1] stats graphics grDevices utils datasets methods base >>>>>> >>>>>> other attached packages: >>>>>> [1] flowCore_1.24.0 rrcov_1.3-02 pcaPP_1.9-48 mvtnorm_0.9-9993 >>>>>> [5] robustbase_0.9-4 Biobase_2.18.0 BiocGenerics_0.4.0 >>>>>> >>>>>> loaded via a namespace (and not attached): >>>>>> [1] feature_1.2.8 graph_1.36.0 ks_1.8.10 MASS_7.3-22 stats4_2.15.1 >>>>>> [6] tools_2.15.1 >>>>>> >>>>>> -- >>>>>> Sent via the guest posting facility atbioconductor.org <http: bioconductor.org="">. >>>>> >>>> >>> >> > [[alternative HTML version deleted]]