Nothing
## ----setup, include = FALSE----------------------------------------------
knitr::opts_chunk$set(
collapse = TRUE,
comment = "#>",
results = "hide",
message = F,
warning = F,
eval = F
)
library(robustSingleCell)
## ----download_data-------------------------------------------------------
# library(robustSingleCell)
# download_LCMV()
## ----initialize----------------------------------------------------------
# LCMV1 <- initialize.project(datasets = "LCMV1",
# origins = "CD44+ cells",
# experiments = "Rep1",
# data.path = file.path(tempdir(), "LCMV"),
# work.path = file.path(tempdir(), "LCMV/LCMV_analysis"))
## ----read_LCMV1----------------------------------------------------------
# LCMV1 <- read.data(LCMV1, subsample = 500)
## ----preprocess----------------------------------------------------------
# LCMV1 <- get.variable.genes(LCMV1)
# exhaustion_markers <- c('Pdcd1', 'Cd244', 'Havcr2', 'Ctla4', 'Cd160', 'Lag3', 'Tigit', 'Cd96')
# LCMV1 <- add.confounder.variables(LCMV1,
# ribosomal.score = ribosomal.score(LCMV1),
# mitochondrial.score = mitochondrial.score(LCMV1),
# cell.cycle.score = cell.cycle.score(LCMV1),
# Exhaustion = controlled.mean.score(LCMV1, exhaustion_markers))
## ----PCA-----------------------------------------------------------------
# LCMV1 <- PCA(LCMV1, local = T)
## ----cluster-------------------------------------------------------------
# LCMV1 <- cluster.analysis(LCMV1, local = T)
## ----annotation----------------------------------------------------------
# types = rbind(
# data.frame(type='Tfh',gene=c('Tcf7','Cxcr5','Bcl6')),
# data.frame(type='Th1',gene=c('Cxcr6','Ifng','Tbx21')),
# data.frame(type='Tcmp',gene=c('Ccr7','Bcl2','Tcf7')),
# data.frame(type='Treg',gene=c('Foxp3','Il2ra')),
# data.frame(type='Tmem',gene=c('Il7r','Ccr7')),
# data.frame(type='CD8',gene=c('Cd8a')),
# data.frame(type='CD4', gene = c("Cd4")),
# data.frame(type='Cycle',gene=c('Mki67','Top2a','Birc5'))
# )
# summarize(LCMV1, local = T)
# LCMV1_cluster_names <- get.cluster.names(LCMV1, types, min.fold = 1.0, max.Qval = 0.01)
# LCMV1 <- set.cluster.names(LCMV1, names = LCMV1_cluster_names)
# summarize(LCMV1, local = T)
## ----plotLCMV1-----------------------------------------------------------
# canonical_genes <- c("Cd8a", "Cd4", "Mki67", "Foxp3", "Il2ra", "Bcl6",
# "Cxcr5", "Cxcr6", "Ifng", "Tbx21", "Id2", "Rora",
# "Cxcr3", "Tcf7", "Ccr7", "Cxcr4", "Pdcd1", "Ctla4")
# plot_simple_heatmap(LCMV1, name = "canonical", markers = canonical_genes, main = "Expression of marker genes")
## ----LCMV_2--------------------------------------------------------------
# LCMV2 <- initialize.project(datasets = "LCMV2",
# origins = "CD44+ cells",
# experiments = "Rep2",
# data.path = file.path(tempdir(), "LCMV"),
# work.path = file.path(tempdir(), "LCMV/LCMV_analysis"))
# LCMV2 <- read.data(LCMV2, subsample = 500)
# LCMV2 <- get.variable.genes(LCMV2)
# LCMV2 <- add.confounder.variables(
# LCMV2,
# ribosomal.score = ribosomal.score(LCMV2),
# mitochondrial.score = mitochondrial.score(LCMV2),
# cell.cycle.score = cell.cycle.score(LCMV2),
# Exhaustion = controlled.mean.score(LCMV2, exhaustion_markers))
#
# LCMV2 <- PCA(LCMV2, local = T)
# LCMV2 <- cluster.analysis(LCMV2, local = T) # 0.05 for KNN ratio
# summarize(LCMV2, local = T)
# LCMV2_cluster_names <- get.cluster.names(LCMV2, types, min.fold = 1.0, max.Qval = 0.01)
# LCMV2 <- set.cluster.names(LCMV2, names = LCMV2_cluster_names)
# summarize(LCMV2, local = T)
# plot_simple_heatmap(LCMV2, name = "canonical", markers = canonical_genes, main = "Expression of marker genes")
## ----initialize_pooled---------------------------------------------------
# pooled_env <- initialize.project(datasets = c("LCMV1", "LCMV2"),
# origins = c("CD44+ cells", "CD44+ cells"),
# experiments = c("Rep1", "Rep2"),
# data.path = file.path(tempdir(), "LCMV"),
# work.path = file.path(tempdir(), "LCMV/LCMV_analysis"))
# pooled_env <- read.preclustered.datasets(pooled_env)
# pooled_env <- add.confounder.variables(
# pooled_env,
# ribosomal.score = ribosomal.score(pooled_env),
# mitochondrial.score = mitochondrial.score(pooled_env),
# cell.cycle.score = cell.cycle.score(pooled_env),
# Exhaustion = controlled.mean.score(pooled_env, exhaustion_markers))
# pooled_env <- PCA(pooled_env, clear.previously.calculated.clustering = F, local = T)
# summarize(pooled_env, contrast = "datasets", local = T)
## ----pooled--------------------------------------------------------------
# cluster.similarity <- assess.cluster.similarity(pooled_env)
# similarity <- cluster.similarity$similarity
# map <- cluster.similarity$map
# filtered.similarity <- get.robust.cluster.similarity(
# pooled_env, similarity, min.sd = qnorm(.9), max.q.val = 0.01, rerun = F
# )
# robust.clusters <- sort(unique(c(filtered.similarity$cluster1,
# filtered.similarity$cluster2)))
# visualize.cluster.cors.heatmaps(pooled_env, pooled_env$work.path,
# filtered.similarity)
## ----summary-------------------------------------------------------------
# similarity <- filtered.similarity
# visualize.cluster.similarity.stats(pooled_env, similarity)
## ----robust_markers------------------------------------------------------
# differential.expression.statistics = get.robust.markers(
# pooled_env, cluster_group1 = c('LCMV2_Tfh_CD4', 'LCMV2_Tfh_Tcmp_CD4'),
# cluster_group2 = c('LCMV2_CD8_1', 'LCMV2_CD8_2'),
# group1_label = 'CD4 T Cells', group2_label = 'CD8 T Cells')
## ----tSNE_overlay--------------------------------------------------------
# plot_contour_overlay_tSNE(pooled_env, genes = c('Cd4','Cd8a'))
## ------------------------------------------------------------------------
# plot_pair_scatter(pooled_env, gene1 = 'Cd4', gene2 = 'Cd8a',
# cluster_group1 = c('LCMV2_Tfh_CD4', 'LCMV2_Tfh_Tcmp_CD4'),
# cluster_group2 = c('LCMV2_CD8_1','LCMV2_CD8_2'),
# group1_label = 'CD4 T Cells', group2_label = 'CD8 T Cells')
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