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- library(tidyverse)
- library(flowWorkspace)
- library(Biobase)
- library(flowGate)
- source("functionsCyto.R")
-
- files<-list.files("Files", pattern = ".LMD", full.names = T)
-
- LMD2FCS(files)
-
- fs<-read.ncdfFlowSet(files=list.files("Files",".fcs", full.names = T), readonly = F)
- gs <- GatingSet(fs)
-
- comp<-as.matrix(read.csv("CompMatrix.csv", check.names = F, row.names = 1))
- gs<-compensate(gs, comp)
-
- trans_params<-transform_gs(gs)
- saveRDS(trans_params, "trans_params.rds")
- trans_params<-readRDS("trans_params.rds")
- trans_apply(gs, trans_params = trans_params)
-
-
- colnames(gs)[c(4,6,11)]<-c("CD16","CD56","CD107a")
- sampleNames(gs)<-gsub("\\s[0-9]*.fcs","",sampleNames(gs))
- sampleNames(gs)<-gsub(".*\\s","",sampleNames(gs))
- pData(gs)$name<-rownames(pData(gs))
-
- gates<-list()
- gs_gate_interactive(gs,
- filterId = "Leukocytes",
- dims = list("FS-A", "SS-A"))
- gates[["Leukocytes"]]<-gs_pop_get_gate(gs, "Leukocytes")
-
- gs_gate_interactive(gs,
- subset = "Leukocytes",
- filterId = "NKdim",
- dims = list("CD16", "CD56"))
- gates[["NKdim"]]<-gs_pop_get_gate(gs, "NKdim")
-
- gs_gate_interactive(gs[["NKs-K562"]],
- subset = "NKdim",
- filterId = "CD107a+",
- dims = list("CD107a", "CD56"), overlayGates = "CD107a+")
- gates[["CD107a+"]]<-gs_pop_get_gate(gs, "CD107a+")
-
- gates<-gates_save(gs, file = "gates.rds")
- gates<-readRDS("gates.rds")
- gs<-gates_apply(gs, gates)
-
- autoplot(gs, "CD107a+")
- ggcyto_trans(gs, "CD107a", "CD56", subset="NKdim")
- autoplot(gs[[5]], bins=0)
- ggcyto_trans_all(gs, index = 5, ncol=1)
-
- stats<-gs_pop_get_stats(gs, nodes="CD107a+", type="perc")
-
- ggpubr::ggarrange(
- ggcyto_trans_all(gs, nrow=1),
- ggcyto::as.ggplot(ggcyto_trans(gs, "CD107a", "CD56", subset="NKdim")+
- facet_grid(.~name)),
- ggplot(stats, aes(sample, percent))+
- geom_bar(stat="identity", color="black", fill="grey70"),
- ncol=1)
- ggsave("Analysis.jpg")
-
- save_gs(gs, "gs_analysis")
- gs<-load_gs("gs_analysis/")
-
- ## For a new experiment with same settings
-
- files<-list.files("Files", pattern = ".LMD", full.names = T)
- LMD2FCS(files)
-
- fs<-read.ncdfFlowSet(files=list.files("Files",".fcs", full.names = T), readonly = F)
- gs <- GatingSet(fs)
-
- comp<-as.matrix(read.csv("CompMatrix.csv", check.names = F, row.names = 1))
- gs<-compensate(gs, comp)
-
- trans_params<-readRDS("trans_params.rds")
- trans_apply(gs, trans_params = trans_params)
-
- colnames(gs)[c(4,6,11)]<-c("CD16","CD56","CD107a")
- sampleNames(gs)<-gsub("\\s[0-9]*.fcs","",sampleNames(gs))
- sampleNames(gs)<-gsub(".*\\s","",sampleNames(gs))
- pData(gs)$name<-rownames(pData(gs))
-
- gates<-readRDS("gates.rds")
- gs<-gates_apply(gs, gates) #if not the same gates, apply a general with "exact=F"
-
-
- stats<-gs_pop_get_stats(gs, nodes="CD107a+", type="perc")
-
- ggpubr::ggarrange(
- ggcyto_trans_all(gs, nrow=1),
- ggcyto::as.ggplot(ggcyto_trans(gs, "CD107a", "CD56", subset="NKdim")+
- facet_grid(.~name)),
- ggplot(stats, aes(sample, percent))+
- geom_bar(stat="identity", color="black", fill="grey70"),
- ncol=1)
- ggsave("Analysis.jpg")
-
- save_gs(gs, "gs_analysis")
- gs<-load_gs("gs_analysis/")
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