## ----setup, include=FALSE----------------------------------------------------- knitr::opts_chunk$set(echo = TRUE, results = "markup", message = FALSE) ## ----bcell,fig.show='asis',fig.keep='all'------------------------------------- library(flowCore) library(flowDensity) data_dir <- system.file("extdata", package = "flowDensity") load(list.files(pattern = 'sampleFCS_1', data_dir, full = TRUE)) f sngl <- flowDensity(f,channels = c("FSC-A","FSC-H"),position = c(F,F), percentile =c(.99999,.99999),use.percentile = c(T,T), ellip.gate = T,scale = .99 ) ## ----------------------------------------------------------------------------- plotDens(f,c(1,2)) lines(sngl@filter,type="l") ## ----plot--------------------------------------------------------------------- plot(f,sngl) ## ----plasma------------------------------------------------------------------- data_dir <- system.file("extdata", package = "flowDensity") load(list.files(pattern = 'sampleFCS_1', data_dir, full = TRUE)) #bcell <- flowDensity(f,channels = c(4,9),position = c(NA,T)) CD19pCD20n <- flowDensity(obj=f, channels=c(8, 6), position=c(T,F)) plasmablasts <- flowDensity(obj=CD19pCD20n, channels=c(5, 12), position=c(T, T)) ## ----plot2-------------------------------------------------------------------- plotDens(CD19pCD20n@flow.frame, plasmablasts@channels, pch=19) points(plasmablasts@filter, type='l', col=2, lwd=2) ## ----twsteps------------------------------------------------------------------ library(flowCore) library(flowDensity) data_dir <- system.file("extdata", package = "flowDensity") load(list.files(pattern = 'sampleFCS_2', data_dir, full = TRUE)) f2 channels <- c("V500-A", "SSC-A") # First call to flowDensity tmp.cp1 <- flowDensity(obj=f2, channels=channels, position=c(TRUE, FALSE), percentile=c(0.25, NA)) # Second call to flowDensity tmp.cp2 <- flowDensity(obj=tmp.cp1, channels=channels, position=c(TRUE, FALSE), gates=c(FALSE, NA), percentile=c(NA, 0.85)) # Final call to flowDensity lymph <- flowDensity(obj=f2, channels=channels, position=c(TRUE, FALSE), gates=c(tmp.cp1@gates[1], tmp.cp2@gates[2]), ellip.gate=TRUE, scale=.99) plot(f2, tmp.cp1) plot(f2, tmp.cp2) ## ----plot3-------------------------------------------------------------------- par(mfrow=c(1,1)) plotDens(f2, channels=channels,axes=T) lines(lymph@filter, type="l", col=2, lwd=2) legend("topleft",legend = paste0("count: ",lymph@cell.count),bty = "n") ## ----frame-------------------------------------------------------------------- getflowFrame(lymph) ## ----control,fig.keep='all'--------------------------------------------------- load(list.files(pattern = 'sampleFCS_3.Rdata', data_dir, full = TRUE)) f3 load(list.files(pattern = 'sampleFCS_3_FMO', data_dir, full = TRUE)) f3.fmo f3.gated <- flowDensity(obj=f3, channels=c('BV421-A', 'FSC-A'), position = c(TRUE, NA),use.control = c(TRUE, F) , control = c(f3.fmo, NA),verbose=F) f3.fmo.gated <- flowDensity(obj=f3.fmo, channels=c('BV421-A', 'FSC-A'), position=c(TRUE, NA), gates=c(f3.gated@gates[1], NA),verbose=F) plot(f3.fmo, f3.fmo.gated) plot(f3, f3.gated) ## ----control2,fig.keep='all'-------------------------------------------------- f3.gated.98p <- flowDensity(obj=f3, channels=c('BV421-A', 'FSC-A'), position = c(TRUE, NA),use.percentile = c(TRUE, NA), percentile = 0.98, use.control = c(TRUE, FALSE), control = c(f3.fmo, NA)) f3.fmo.gated.98p <- flowDensity(obj=f3.fmo, channels=c('BV421-A', 'FSC-A'), position = c(TRUE, NA), gates=c(f3.gated.98p@gates[1], NA)) plot(f3.fmo, f3.fmo.gated.98p) plot(f3, f3.gated.98p) ## ----deGate------------------------------------------------------------------- load(list.files(pattern = 'sampleFCS_2', data_dir, full = TRUE)) thresholds <- deGate(obj = f2,channel = 9) #Percentile default is .95, which can be changed thresholds.prcnt <- deGate(f2,channel = 9,use.percentile=T,percentile=.3) thresholds.lo <- deGate(f2,channel = 9,use.upper=T,upper=F,alpha = .9) thresholds.hi <- deGate(f2,channel = 9,use.upper=T,upper=T,alpha = .9) plotDens(f2,c(9,12)) abline(v=c(thresholds,thresholds.prcnt,thresholds.lo,thresholds.hi),col=c(1,2,3,4)) ## ----nutsub------------------------------------------------------------------- cd19.gate <- deGate(f, channel = 8) cd20.gate <- deGate(f,channel = 9) cd20.neg <- notSubFrame(f, channels = c(8,9),position = c(F,F),gates=c(cd19.gate,cd20.gate)) plotDens(f,c(8,9),axes=T) lines(cd20.neg@filter, type="l") ## ----plo4--------------------------------------------------------------------- plotDens(cd20.neg,c(8,9),main="Not CD19-CD20-") ## ----peaks-------------------------------------------------------------------- load(list.files(pattern = 'sampleFCS_2', data_dir, full = TRUE)) getPeaks(f2,channel = 9) ## ----------------------------------------------------------------------------- load(list.files(pattern = 'sampleFCS_2', data_dir, full = TRUE)) plotDens(f2, channels = c(9,12),density.overlay = c(T,T))