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down_and_perm_ttest.R
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down_and_perm_ttest.R
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library("dplyr")
library("Matrix")
library("Seurat")
library("stringr")
geneCap <- function(gene, gene_names) {
# Gene the gene name in the right format
gene_lower <- tolower(gene)
gene_upper <- toupper(gene)
gene_title <- str_to_title(gene)
error <- FALSE
if (gene_lower %in% gene_names) {
gene <- gene_lower
} else if (gene_upper %in% gene_names) {
gene <- gene_upper
} else if (gene_title %in% gene_names) {
gene <- gene_title
} else {
error <- TRUE
}
return(c(gene, error))
}
validGenes <- function(genes, gene_names) {
valid_genes <- c()
for (gene in genes) {
result <- geneCap(gene, gene_names)
gene <- result[1]
error <- as.logical(result[2])
if (! error) {
valid_genes <- c(valid_genes, gene)
}
} # end gene for
return(valid_genes)
} # end validGenes function
downsample <- function(combined, marker_genes, run) {
set.seed(run)
min_trans <- min(combined$nCount_RNA)
gene_names <- rownames(combined@assays$RNA@counts)
# new_matrix <- matrix(, nrow = nrow(combined@assays$RNA@counts), ncol = ncol(combined@assays$RNA@counts), dimnames = list(gene_names, colnames(combined@assays$RNA@counts)))
# new_new_matrix <- matrix(, nrow=nrow(combined@assays$RNA@counts))
marker_matrix <- matrix(, nrow=length(marker_genes), ncol = ncol(combined@assays$RNA@counts), dimnames = list(marker_genes, colnames(combined@assays$RNA@counts)))
i <- 0
for (cell in colnames(combined@assays$RNA@counts)) {
# i <- i + 1
# if (i%%500 == 1) {
# print(cell)
# }
# start.time <- Sys.time()
trans_names <- rep(gene_names, combined@assays$RNA@counts[,cell])
ran_trans_names <- sample(trans_names, min_trans)
ran_trans_names <- ran_trans_names[which(ran_trans_names %in% marker_genes)]
ran_df <- as.data.frame(table(ran_trans_names))
zero_gene_names <- marker_genes[which(! marker_genes %in% ran_trans_names)]
zero_df <- setNames(data.frame(zero_gene_names <- zero_gene_names, Freq <- rep(0, length(zero_gene_names))), c("ran_trans_names", "Freq"))
ran_df <- rbind(ran_df, zero_df)
rownames(ran_df) <- ran_df$ran_trans_names
ran_df <- ran_df[marker_genes,2]
# new_matrix[,cell] <-as.matrix(ran_df)
marker_matrix[,cell] <- as.matrix(ran_df)
# new_new_matrix <- cbind(new_new_matrix, as.matrix(ran_df))
# end.time <- Sys.time()
# time.taken <- end.time - start.time
# print(time.taken)
}
return(marker_matrix)
}
## END FUNCTIONS ##
# rna_path <- "C:/Users/miles/Downloads/brain/"
rna_path <- "~/scratch/brain/"
combined <- readRDS(paste(rna_path, "/brain_scripts/brain_shiny/data/combined.rds", sep = ""))
marker_path <- paste(rna_path, "data/markers/", sep="")
marker_files <- dir(marker_path, pattern =paste("*.txt", sep=""))
markers <- data.frame(gene <- c(), bio <- c())
for (i in 1:length(marker_files)) {
file <- read.table(paste(marker_path, marker_files[i], sep=""), header=FALSE, sep="\t", stringsAsFactors=FALSE)
file[,1] <- toupper(file[,1])
markers <- rbind(markers, file[,1:2])
}
colnames(markers) <- c("gene", "bio")
bio <- "ROCK_SAND"
markers <- markers[which(markers$bio == bio),]
print("Before gene_names")
gene_names <- rownames(combined@assays$RNA)
print("After gene_names")
marker_genes <- unique(validGenes(markers$gene, gene_names))
valid_genes <- marker_genes
num_clusters <- as.numeric(tail(levels([email protected]$seurat_clusters), n=1))
down_avg_avg_gene <- rep(0, num_clusters+1)
run_num <- 50
# No Perm, Bootstrap
down_genes_per_cell <- lapply(0:num_clusters, function(x) c())
for (run in 1:run_num) {
cat(paste("no_perm", run, "\n"))
mat <- downsample(combined, marker_genes, run)
mat[which(mat[,] > 1)] <- 1
cells_per_cluster <- c()
genes_per_cluster <- c()
for (i in 0:num_clusters) {
this_cells <- WhichCells(combined, idents = i)
# genes_per_cluster <- c(genes_per_cluster, length(which(as.vector(combined@assays$RNA@counts[ran_markers,this_cells]) != 0))) # genes
down_genes_per_cell[[i+1]] <- c(down_genes_per_cell[[i+1]], colSums(mat[,this_cells])) # genes
cells_per_cluster <- c(cells_per_cluster, length(this_cells))
}
}
# Perm, Bootstrap
backup_ids <- [email protected]$seurat_clusters
perm_down_avg_gene <- lapply(0:num_clusters, function(x) c())
for (run in (run_num+1):(run_num+run_num)) {
cat(paste("perm", run, "\n"))
set.seed(run)
shuffled <- sample(backup_ids)
mat <- downsample(combined, marker_genes, run)
mat[which(mat[,] > 1)] <- 1
Idents(object = combined) <- shuffled
num_clusters <- as.numeric(tail(levels([email protected]$seurat_clusters), n=1))
gene_names <- rownames(combined@assays$RNA)
cells_per_cluster <- c()
genes_per_cluster <- c()
for (i in 0:num_clusters) {
this_cells <- WhichCells(combined, idents = i)
perm_down_avg_gene[[i+1]] <- c(perm_down_avg_gene[[i+1]], colSums(mat[,this_cells]))
}
}
# Compare empirical data to 97.5th percentile of the permutated data on a PER CLUSTER basis
df <- data.frame()
for (i in 0:num_clusters) {
mean_down <- mean(down_genes_per_cell[[i+1]])
mean_perm <- mean(perm_down_avg_gene[[i+1]])
p <- t.test(down_genes_per_cell[[i+1]], perm_down_avg_gene[[i+1]])$p.value
p_sig_enrich <- p < 0.05 & mean_down > mean_perm
df <- rbind(df, t(c(i, mean_down, mean_perm, p, p_sig_enrich)))
}
colnames(df) <- c("cluster", "mean_down", "mean_down_perm", "p", "p_sig_enrich")
df$q <- p.adjust(df$p)
df$q_sig_enrich <- df$q < 0.05 & df$mean_down > df$mean_down_perm
write.table(df, file = paste(rna_path, "/results/down_and_perm_ttest_", bio, ".tsv", sep=""), sep = "\t", row.names = FALSE, quote=FALSE)