jacobpwagner
commented
4 years ago Some aspects of your code don't seem to work in my hands. In particular, the fcs.dir you specify does not exist. I'll construct an example using the FCS files I think you were tryng to steer towards and use it to illustrate the method.
> library(flowCore)
> library(flowStats)
> fcs.dir <- system.file("extdata", "compdata", "data", package="flowCore")
> matchfile <- system.file("extdata", "compdata", "comp_match", package="flowCore")The matchfile is just a simple 2-column csv mapping channel to control file
> writeLines(readLines(matchfile))
filename,channel
060909.001,unstained
060909.002,FL1-H
060909.004,FL4-H
060909.005,FL3-H
060909.003,FL2-HNote you could use lapply with read.FCS as you were (if the paths were correct), but read.flowSet is a bit easier
> frames <- read.flowSet(list.files(fcs.dir, full.names=TRUE))
> sampleNames(frames)
[1] "060909.001" "060909.002" "060909.003" "060909.004" "060909.005"spillover_match updates the sample names of the flowSet using the matchfile. Unfortunately the scatter channels in this example data are -H instead of the default -A, so we need to specify that.
> matched <- spillover_match(frames, fsc="FSC-H", ssc="SSC-H", matchfile=matchfile)
> sampleNames(matched)
[1] "unstained" "FL1-H" "FL2-H" "FL4-H" "FL3-H" So now the spillover matrix calculation method will know which control sample goes with which channel. Note the prematched=TRUE flag here, which will tell the spillover method not to attempt its automated logic to match control files to channels, but use the sample names as they are.
> comp_matrix <- spillover(matched, fsc="FSC-H", ssc="SSC-H", prematched=TRUE)
> comp_matrix
FL1-H FL2-H FL3-H FL4-H
FL1-H 1.0000000000 0.2420222776 0.032083706 0.001127816
FL2-H 0.0077220477 1.0000000000 0.140788232 0.002632689
FL3-H 0.0150806322 0.1755899032 1.000000000 0.229593860
FL4-H 0.0007590319 0.0009620459 0.003218614 1.000000000That compensation matrix could then be used with the compensate method. Note that you don't need to use spillover_match. You could manually reassign the sampleNames of each of the controls in the flowSet yourself, like so:
sampleNames(frames) <- c("unstained", "FL1-H", "FL2-H", "FL4-H", "FL3-H")And then still used the prematched=TRUE flag in spillover. The spillover_match functionality mostly helps for repeated batch processing.
Also, keep in mind that the spillover method is sort of overloaded. If you call spillover on a single flowFrame, it will attempt to extract the spillover matrix included in the FCS file using the appropriate keywords. If you call spillover on a flowSet, it will attempt to calculate the spillover matrix, assuming the set of flowFrames represent a set of controls.
Please let me know if you have any more questions.
vhollek
I'm new to R and openCyto/flowCore etc. For analyzing my FACS data, I first want to compensate them using
spillover_match,spilloverandcompensate. I got all the necessary single stained fcs samples (including unstained) and created a csv file containing filenames and channels. Unfortunately, I get the erroreven though I did library(flowStats) [Version 3.44.0].
My code:
[version R version 3.6.2 (2019-12-12) os Windows 10 x64
system x86_64, mingw32]
Does anybody know what I can do/change to make it work?