[BioC] spillover::flowCore Help Needed

[Kevin Schiesser [guest]]

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 

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