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Flow/Mass Cytometry Gating via Spatial Kernel Density Estimation
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lance-waller-lab/gateR
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Date repository last updated: January 23, 2024
ThegateR package is a suite ofR functions to identify significant spatial clustering of flow and mass cytometry data used in immunological investigations. For a two-group comparison, we detect clusters using the kernel-based spatial relative risk function estimated using thesparr package. The tests are conducted in a two-dimensional space comprised of two fluorescent markers.
Examples of a single condition with two groups:
- Disease case vs. Healthy control
- Time 2 vs. Time 1 (baseline)
For a two-group comparison of two conditions, we estimate two relative risk surfaces for one condition and then a ratio of the relative risks. For example:
- Estimate a relative risk surface for:
- Condition 2B vs. Condition 2A
- Condition 1B vs. Condition 1A
- Estimate the relative risk surface for the ratio:
Within areas where the relative risk exceeds an asymptotic normal assumption, thegateR package has the functionality to examine the features of these cells. Basic visualization is also supported.
To install the release version from CRAN:
install.packages("gateR")To install the development version from GitHub:
devtools::install_github("lance-waller-lab/gateR")| Function | Description |
|---|---|
gating | Main function. Conduct a gating strategy for flow and mass cytometry data. |
rrs | Called withingating, one condition comparison. |
lotrrs | Called withingating, two condition comparison. |
pval_correct | Called withinrrs andlotrrs, calculates various multiple testing corrections for the alpha level. Five methods account for (spatially) dependent, multiple testing. |
lrr_plot | Called withinrrs andlotrrs, provides functionality for basic visualization of a log relative risk surface. |
pval_plot | Called withinrrs andlotrrs, provides functionality for basic visualization of a significant p-value surface. |
The repository also includes the code and resources to create the project hexagon sticker.
| Data | Description |
|---|---|
randCyto | A sample dataset containing information about flow cytometry data with two binary conditions and four markers. The data are a random subset of the 'extdata' data in theflowWorkspaceData package found onBioconductor and formatted for `gateR` input. |
- Ian D. Buller -Social & Scientific Systems, Inc., a division of DLH Corporation, Silver Spring, Maryland (current) -Occupational and Environmental Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Rockville, Maryland (former) -Environmental Health Sciences, James T. Laney School of Graduate Studies, Emory University, Atlanta, Georgia. (original) -GitHub -ORCID
See also the list ofcontributors who participated in this project. Main contributors include:
- Elena Hsieh -Immunology & Microbiology and Pediatrics, University of Colorado Anschutz School of Medicine -GitHub -ORCID
- Debashis Ghosh -Biostatistics & Informatics, Colorado School of Public Health, Aurora, Colorado -GitHub -ORCID
- Lance A. Waller -Biostatistics and Bioinformatics, Emory University, Atlanta, Georgia -GitHub -ORCID
set.seed(1234)# for reproducibility# ------------------ ## Necessary packages ## ------------------ #library(gateR)library(dplyr)library(flowWorkspaceData)library(ncdfFlow)library(stats)# ---------------- ## Data preparation ## ---------------- ## Use 'extdata' from the {flowWorkspaceData} packageflowDataPath<- system.file("extdata",package="flowWorkspaceData")fcsFiles<- list.files(pattern="CytoTrol",flowDataPath,full=TRUE)ncfs<-ncdfFlow::read.ncdfFlowSet(fcsFiles)fr1<-ncfs[[1]]fr2<-ncfs[[2]]## Comparison of two samples (single condition) "g1"## Two gates (four markers) "CD4", "CD38", "CD8", and "CD3"## Arcsinh Transformation for all markers## Remove cells with NA and Inf values# First sampleobs_dat1<-data.frame("id"= seq(1, nrow(fr1@exprs),1),"g1"= rep(1, nrow(fr1@exprs)),"arcsinh_CD4"= asinh(fr1@exprs[ ,5]),"arcsinh_CD38"= asinh(fr1@exprs[ ,6]),"arcsinh_CD8"= asinh(fr1@exprs[ ,7]),"arcsinh_CD3"= asinh(fr1@exprs[ ,8]))# Second sampleobs_dat2<-data.frame("id"= seq(1, nrow(fr2@exprs),1),"g1"= rep(2, nrow(fr2@exprs)),"arcsinh_CD4"= asinh(fr2@exprs[ ,5]),"arcsinh_CD38"= asinh(fr2@exprs[ ,6]),"arcsinh_CD8"= asinh(fr2@exprs[ ,7]),"arcsinh_CD3"= asinh(fr2@exprs[ ,8]))# Full setobs_dat<- rbind(obs_dat1,obs_dat2)obs_dat<-obs_dat[complete.cases(obs_dat), ]# remove NAsobs_dat<-obs_dat[is.finite(rowSums(obs_dat)), ]# remove Infsobs_dat$g1<- as.factor(obs_dat$g1)# set "g1" as binary factor## Create a second condition (randomly split the data)## In practice, use data with a measured second conditiong2<-stats::rbinom(nrow(obs_dat),1,0.5)obs_dat$g2<- as.factor(g2)obs_dat<-obs_dat[ , c(1:2,7,3:6)]# Export 'randCyto' data for CRAN examplesrandCyto<-dplyr::sample_frac(obs_dat,size=0.1)# random subsample# ---------------------------- ## Run gateR with one condition ## ---------------------------- ## Single condition## A p-value uncorrected for multiple testingtest_gating<-gateR::gating(dat=obs_dat,vars= c("arcsinh_CD4","arcsinh_CD38","arcsinh_CD8","arcsinh_CD3"),n_condition=1,plot_gate=TRUE,upper_lrr=1,lower_lrr=-1)# -------------------- ## Post-gate assessment ## -------------------- ## Density of arcsinh-transformed CD4 post-gatinggraphics::plot(stats::density(test_gating$obs[test_gating$obs$g1==1,4]),main="arcsinh CD4",lty=2)graphics::lines(stats::density(test_gating$obs[test_gating$obs$g1==2,4]),lty=3)graphics::legend("topright",legend= c("Sample 1","Sample 2"),lty= c(2,3),bty="n")
# ----------------------------- ## Run gateR with two conditions ## ----------------------------- ### A p-value uncorrected for multiple testingtest_gating2<-gateR::gating(dat=obs_dat,vars= c("arcsinh_CD4","arcsinh_CD38","arcsinh_CD8","arcsinh_CD3"),n_condition=2)# --------------------------------------------- ## Perform a single gate without data extraction ## --------------------------------------------- ## Single condition## A p-value uncorrected for multiple testing## For "arcsinh_CD4" and "arcsinh_CD38"test_rrs<-gateR::rrs(dat=obs_dat[ ,-7:-6])# Two conditions## A p-value uncorrected for multiple testing## For "arcsinh_CD8" and "arcsinh_CD3"test_lotrrs<-gateR::lotrrs(dat=obs_dat[ ,-5:-4])# ------------------------------------------ ## Run gateR with multiple testing correction ## ------------------------------------------ ### False Discovery Ratetest_gating_fdr<-gateR::gating(dat=obs_dat,vars= c("arcsinh_CD4","arcsinh_CD38","arcsinh_CD8","arcsinh_CD3"),n_condition=1,p_correct="FDR")
This package was developed while the author was originally a doctoral student at in theEnvironmental Health Sciences doctoral program atEmory University and later as a postdoctoral fellow supported by theCancer Prevention Fellowship Program at theNational Cancer Institute. Any modifications since December 05, 2022 were made while the author was an employee of Social & Scientific Systems, Inc., a division ofDLH Corporation.
When citing this package for publication, please follow:
citation("gateR")For questions about the package, please contact the maintainerDr. Ian D. Buller orsubmit a new issue.
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Flow/Mass Cytometry Gating via Spatial Kernel Density Estimation