## ---- echo = FALSE, message = FALSE------------------------------------------- knitr::opts_chunk$set(collapse = TRUE, comment = "#>", tidy = TRUE) library(flowCore) library(flowTime) library(ggplot2) library(dplyr) ## ----------------------------------------------------------------------------- data <- read.flowSet(path=system.file("extdata", "ss_example", package = "flowTime"), alter.names = TRUE) annotation <- read.csv(system.file("extdata", "ss_example.csv", package = "flowTime")) adat <- annotateFlowSet(yourFlowSet = data, annotation_df = annotation, mergeBy = 'name') ## ----------------------------------------------------------------------------- #library(BiocManager) #BiocManager::install("openCyto") #BiocManager::install("ggcyto") library("openCyto") library("ggcyto") library("flowClust") #vignette("flowWorkspace-Introduction", "flowWorkspace") #vignette('HowToAutoGating', package = "openCyto") ## ----------------------------------------------------------------------------- autoplot(data[21:24], x = "FSC-A", y = "SSC-A") ## ----------------------------------------------------------------------------- Debris <- gate_quantile(fr = data[[3]], channel = "FSC.A", probs = 0.99, filterId = "Debris") autoplot(data[[3]], x = "FSC-A") + geom_gate(Debris) ## ----------------------------------------------------------------------------- autoplot(data[21:24], x = "FSC-A", "SSC-A") + geom_gate(Debris) toTable(summary(filter(data[c(3,21:24)], !Debris))) ## ----------------------------------------------------------------------------- #Initialize the single frame data.1frame <- data[[1]] #fill the single frame with the exprs data from each frame # in the flow set exprs(data.1frame) <- fsApply(data, function(x) { x <- exprs(x) return(x) }) autoplot(data.1frame, x = "FSC-A", "SSC-A") ## ----------------------------------------------------------------------------- autoplot(data.1frame, x = "FSC-A", "SSC-A") + scale_x_logicle() + scale_y_logicle() ## ----------------------------------------------------------------------------- chnls <- c("FSC.A", "SSC.A", "FSC.H", "SSC.H") trans <- estimateLogicle(data.1frame, channels = chnls) inv.trans <- inverseLogicleTransform(trans) data.1frame <- transform(data.1frame, trans) autoplot(data.1frame, x = "FSC-A", "SSC-A") ## ----------------------------------------------------------------------------- yeast <- gate_flowClust_2d(data.1frame, xChannel = "FSC.A", yChannel = "SSC.A", K = 1, quantile = .95, min = c(0,0)) autoplot(data.1frame, x = "FSC-A", y = "SSC-A") + geom_gate(yeast) ## ----------------------------------------------------------------------------- yeast <- transform(yeast, inv.trans) data.1frame <- transform(data.1frame, inv.trans) ## ----------------------------------------------------------------------------- autoplot(data[c(1, 8, 16, 24, 32)], "FSC.A","SSC.A") + geom_gate(yeast) #invisible(capture.output( # we have to use this to prevent summary from printing f<- summary(filter(data, yeast))#)) # Now we can print our summary as a table toTable(f) ## ----------------------------------------------------------------------------- autoplot(Subset(data.1frame, yeast), "FSC-A", "FSC-H") library(flowStats) chnl <- c("FSC-A", "FSC-H") singlets <- gate_singlet(x = Subset(data.1frame, yeast), area = "FSC.A", height = "FSC.H", prediction_level = 0.999, maxit = 20) autoplot(Subset(data.1frame, yeast), "FSC-A", "FSC-H") + geom_gate(singlets) ## ----------------------------------------------------------------------------- autoplot(data[c(1:4, 29:32)], x = "FSC-A", y = "FSC-H") + geom_gate(singlets) + facet_wrap("name", ncol = 4) autoplot(Subset(data[c(1:4, 29:32)], yeast & singlets), x = "FL1-A") + facet_wrap("name", ncol = 4) ## ----------------------------------------------------------------------------- invisible(capture.output( d <- summary(filter(data, yeast & singlets)))) (e <- toTable(d)) e <- left_join(e, pData(data), by = c("sample" = "name")) ggplot(data = e, mapping = aes(x = as.factor(sample), y = percent)) + geom_point() ## ---- eval = FALSE------------------------------------------------------------ # data <- Subset(data, yeast & singlets) # data_sum <- summarizeFlow(data, channel = c("FL1.A", "FL4.A"), gated = TRUE) ## ---- eval=FALSE-------------------------------------------------------------- # saveGates(yeastGate = yeast, dipsingletGate = singlets, fileName = "PSB_Accuri_W303.RData") # loadGates(gatesFile = "PSB_Accuri_W303.RData") # data_sum <- summarizeFlow(data, channel = c("FL1.A", "FL4.A"), ploidy = "diploid", only = "singlets")