This package is designed to import/export the hierarchical gated
cytometry data to and from R (specifically the
openCyto framework) using the
gatingML2.0
and FCS3.0
cytometry data standards. This package makes use of the GatingSet R
object and data model so that imported data can easily be manipulated
and visualized in R using tools like
openCyto and
ggCyto.
CytoML allows you to:
- Import manually gated data into R from Diva, FlowJo and Cytobank.
- Combine manual gating strategies with automated gating strategies in R.
- Export data gated manually, auto-gated, or gated using a combination of manual and automated strategies from R to Diva, FlowJo and Cytobank.
- Share computational flow analyses with users on other platforms.
- Perform comparative analyses between computational and manual gating approaches.
CytoML can be installed in several ways:
For all versions, you must have dependencies installed
library(BiocManager)
# This should pull all dependencies.
BiocManager::install("openCyto")
# Then install latest dependencies from github, using devtools.
install.packages("devtools")
library(devtools) #load it
install_github("RGLab/flowWorkspace", ref="trunk")
install_github("RGLab/openCyto", ref="trunk")Installing from BioConductor.
library(BiocManager)
#this should pull all dependencies.
BiocManager::install("CytoML", version = "devel") library(BiocManager)
#this should pull all dependencies.
BiocManager::install("CytoML", version = "devel") install.packges("devtools")
devtools::install_github("RGLab/CytoML", ref = "trunk")install.packges("devtools")
devtools::install_github("RGLab/CytoML@*release")- A reproducible workflow can be found at the RGLab site, and was prepared with version 1.7.10 of CytoML, R v3.5.0, and dependencies that can be installed by:
# We recomend using R version 3.5.0
devtools::install_github("RGLab/[email protected]")
devtools::install_github("RGLab/[email protected]")
devtools::install_github("RGLab/[email protected]")
devtools::install_github("RGLab/[email protected]")
devtools::install_github("RGLab/[email protected]")
devtools::install_github("RGLab/[email protected]")
devtools::install_github("RGLab/[email protected]")To import data you need the xml workspace and the raw FCS files.
Import gatingML generated from Cytobank.
library(CytoML)
xmlfile <- system.file("extdata/cytotrol_tcell_cytobank.xml", package = "CytoML")
fcsFiles <- list.files(pattern = "CytoTrol", system.file("extdata", package = "flowWorkspaceData"), full.names = T)
gs <- cytobank2GatingSet(xmlfile, fcsFiles)Import a Diva workspace.
ws <- openDiva(system.file('extdata/diva/PE_2.xml', package = "flowWorkspaceData"))
# The path to the FCS files is stored in ws@path.
# It can also be passed in to parseWorksapce via the `path` argument.
gs <- parseWorkspace(ws, name = 2, subset = 1)We need flowWorkspace to interact with the imported data.
library(flowWorkspace)We can visualize the gating tree as follows:
#get the first sample
gh <- gs[[1]]
#plot the hierarchy tree
plot(gh)
For more information see the flowWorkspace package.
We can print all the cell populations defined in the gating tree.
#show all the cell populations(/nodes)
getNodes(gh)## [1] "root" "/P1" "/P1/P2" "/P1/P2/P3"
## [5] "/P1/P2/P3/P4" "/P1/P2/P3/P4/P5"
We can extract the cell population statistics.
#show the population statistics
getPopStats(gh)## openCyto.freq xml.freq openCyto.count xml.count node
## 1: 1.00000000 1.00000000 19090 19090 root
## 2: 0.93609219 0.93776847 17870 17902 P1
## 3: 0.97991046 0.97994637 17511 17543 P2
## 4: 0.70327223 0.70307245 12315 12334 P3
## 5: 0.09378806 0.09404897 1155 1160 P4
## 6: 0.95151515 0.94827586 1099 1100 P5
The openCyto.count column shows the cell counts computed via the
import. The xml.count column shows the cell counts computed by FlowJo
(note not all platforms report cell counts in the workspace). It is
normal for these to differ by a few cells due to numerical differences
in the implementation of data transformations. CytoML and openCyto are
reproducing the data analysis from the raw data based on the
information in the workspace.
We can plot all the gates defined in the workspace.
#plot the gates
plotGate(gh) 
Because CytoML and flowWorkspace reproduce the entire analysis in a workspace in R, we have access to information about which cells are part of which cell popualtions.
flowWorkspace has convenience methods to extract the cells from specific cell populations:
getData(gh,"P3")## flowFrame object '9a1897d7-ebc9-4077-aa34-6d9e1367fa67'
## with 12315 cells and 15 observables:
## name desc range minRange maxRange
## $P1 Time <NA> 262144 0.0000000 262144.0
## $P2 FSC-A <NA> 262144 0.0000000 262144.0
## $P3 FSC-H <NA> 262144 0.0000000 262144.0
## $P4 FSC-W <NA> 262144 0.0000000 262144.0
## $P5 SSC-A <NA> 262144 0.0000000 262144.0
## $P6 SSC-H <NA> 262144 0.0000000 262144.0
## $P7 SSC-W <NA> 262144 0.0000000 262144.0
## $P8 FITC-A <NA> 262144 0.1516347 4.5
## $P9 PE-A CD3 262144 0.2953046 4.5
## $P10 PerCP-Cy5-5-A <NA> 262144 0.4697134 4.5
## $P11 PE-Cy7-A <NA> 262144 0.5638024 4.5
## $P12 APC-A bob 262144 0.7838544 4.5
## $P13 APC-Cy7-A Viab 262144 0.6886181 4.5
## $P14 Bd Horizon V450-A CD44 262144 0.6413334 4.5
## $P15 Pacific Orange-A CD8 262144 0.3376040 4.5
## 231 keywords are stored in the 'description' slot
This returns a flowFrame with the cells in gate P3 (70% of the cells
according to the plot).
The matrix of expression can be extracted from a flowFrame using the
exprs() method:
e <- exprs(getData(gh,"P3"))
class(e)## [1] "matrix"
dim(e)## [1] 12315 15
colnames(e)## [1] "Time" "FSC-A" "FSC-H"
## [4] "FSC-W" "SSC-A" "SSC-H"
## [7] "SSC-W" "FITC-A" "PE-A"
## [10] "PerCP-Cy5-5-A" "PE-Cy7-A" "APC-A"
## [13] "APC-Cy7-A" "Bd Horizon V450-A" "Pacific Orange-A"
#compute the MFI of the fluorescence channels.
colMeans(e[,8:15])## FITC-A PE-A PerCP-Cy5-5-A PE-Cy7-A
## 0.8305544 1.3162145 0.7746655 0.8017132
## APC-A APC-Cy7-A Bd Horizon V450-A Pacific Orange-A
## 1.0482656 1.1636819 2.2960560 1.3684352
In order to export gated data, it must be in GatingSet
format.
Load something to export.
dataDir <- system.file("extdata",package="flowWorkspaceData")
gs <- load_gs(list.files(dataDir, pattern = "gs_manual",full = TRUE))## loading R object...
## loading tree object...
## Done
#Cytobank
outFile <- tempfile(fileext = ".xml")
GatingSet2cytobank(gs, outFile)## Warning in GatingSet2cytobank(gs, outFile): With 'cytobank.default.scale'
## set to 'TRUE', data and gates will be re-transformed with cytobank's
## default scaling settings, which may affect how gates look like.
## [1] "/var/folders/jh/x0h3v3pd4dd497g3gtzsm8500000gn/T//RtmpS4mDCu/file154f72419eb4f.xml"
#flowJo
outFile <- tempfile(fileext = ".wsp")
GatingSet2flowJo(gs, outFile)## [1] "/var/folders/jh/x0h3v3pd4dd497g3gtzsm8500000gn/T//RtmpS4mDCu/file154f774d1c840.wsp"
See the flowWorskspace and
[openCyto](http://www.github.com/RGLab/openCyto] packages to learn
more about what can be done with GatingSet objects.
Please note that this project is released with a Contributor Code of Conduct. By participating in this project you agree to abide by its terms.