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| Original file line number | Diff line number | Diff line change |
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| library(data.table) | ||
| library(tidyverse) | ||
| library(Seurat) | ||
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| outdir <- "/directflow/SCCGGroupShare/projects/DrewNeavin/iPSC_Village/output/Expression_Boxplots/pluri_degs/" | ||
| dir.create(outdir, recursive = TRUE) | ||
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| pluri_genes <- fread("/directflow/SCCGGroupShare/projects/DrewNeavin/iPSC_Village/data/pluripotency_genes.tsv") | ||
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| seurat <- readRDS("/directflow/SCCGGroupShare/projects/DrewNeavin/iPSC_Village/output/All_data_integrated_remove_bad/seurat_integrated_all_times_clustered.rds") | ||
| seurat@meta.data$Location <- gsub("_.+", "", seurat@meta.data$Location) | ||
| seurat@meta.data$Cryopreserved <- ifelse(grepl("Thawed", seurat@meta.data$Location_Time), "Cryopreserved", "Fresh") | ||
| seurat@meta.data$Location_Cryopreserved_Line <- paste0(seurat@meta.data$Location, "_", seurat@meta.data$Cryopreserved, "_", seurat@meta.data$Final_Assignment) | ||
| seurat@meta.data$Village <- gsub("Baseline", "Uni-Culture", seurat@meta.data$Time) %>% | ||
| gsub("Thawed Village Day 0", "Uni-Culture", .) %>% | ||
| gsub("Thawed Village Day 7", "Village", .) %>% | ||
| gsub("Village Day 4", "Village", .) | ||
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| ##### Combine replicates from same groups together ##### | ||
| seurat_list <- lapply(unique(seurat@meta.data$Location_Cryopreserved_Line), function(x){ | ||
| subset(seurat, subset = Location_Cryopreserved_Line == x) | ||
| }) | ||
| names(seurat_list) <- unique(seurat@meta.data$Location_Cryopreserved_Line) | ||
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| seurat_list <- lapply(seurat_list, function(x){ | ||
| Idents(x) <- "Village" | ||
| return(x) | ||
| }) | ||
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| seurat_list <- lapply(seurat_list, function(x){ | ||
| SCTransform(x, vars.to.regress = c("scores.G1", "scores.S", "scores.G2M", "percent.mt", "percent.rb"), return.only.var.genes = FALSE) | ||
| }) | ||
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| seurat_list <- lapply(seurat_list, function(x){ | ||
| PrepSCTFindMarkers(x) | ||
| }) | ||
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| saveRDS(seurat_list, paste0(outdir, "sct_location_cryo_line.rds")) | ||
| seurat_list <- readRDS(paste0(outdir, "sct_location_cryo_line.rds")) | ||
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| ##### Combine Village and Baselines together ##### | ||
| ### Try with logistic MAST ### | ||
| MAST_DEGs <- lapply(seurat_list, function(x){ | ||
| FindMarkers(x, ident.1 = "Uni-Culture", ident.2 = "Village", latent.vars = "MULTI_classification", test.use = "MAST", logfc.threshold = 0, assay = "RNA") | ||
| }) | ||
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| saveRDS(MAST_DEGs, paste0(outdir, "MAST_DEGs.rds")) | ||
| MAST_DEGs <- readRDS(paste0(outdir, "MAST_DEGs.rds")) | ||
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| ### Update multiple testing for all sites ### | ||
| MAST_DEGs_nrow_list <- lapply(MAST_DEGs, function(x) data.table(nrow(x))) | ||
| MAST_DEGs_nrow <- sum(do.call(rbind, MAST_DEGs_nrow_list)$V1) | ||
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| MAST_DEGs <- lapply(MAST_DEGs, function(x){ | ||
| x$p_val_adj_updated <- p.adjust(x$p_val, method = "bonferroni", n = MAST_DEGs_nrow) | ||
| return(x) | ||
| }) | ||
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| MAST_DEGs_pluri <- lapply(MAST_DEGs, function(x){ | ||
| x$ENSG <- rownames(x) | ||
| x <- data.table(x) | ||
| x <- x[pluri_genes, on = c("ENSG")] | ||
| x <- x[!is.na(p_val) & p_val_adj_updated < 0.05] | ||
| return(x) | ||
| }) | ||
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| MAST_DEGs_pluri <- lapply(MAST_DEGs, function(x){ | ||
| x$ENSG <- rownames(x) | ||
| x <- data.table(x) | ||
| x <- x[pluri_genes, on = c("ENSG")] | ||
| x <- x[!is.na(p_val) & p_val_adj < 0.05] | ||
| return(x) | ||
| }) | ||
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| MAST_DEGs_pluri_4 <- lapply(MAST_DEGs_pluri, function(x){ | ||
| x[GeneID %in% c("MYC", "NANOG", "POU5F1", "SOX2")] | ||
| }) | ||
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| ### Try with logistic regression ### | ||
| LR_DEGs <- lapply(seurat_list, function(x){ | ||
| FindMarkers(x, ident.1 = "Uni-Culture", ident.2 = "Village", latent.vars = "MULTI_classification", test.use = "LR", logfc.threshold = 0) | ||
| }) | ||
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| saveRDS(LR_DEGs, paste0(outdir, "LR_DEGs.rds")) | ||
| ##### Will use LR instead of MAST - MAST finds more DEG and lower p-values which I'm not sure is legitimate ##### | ||
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| ### Update multiple testing for all sites ### | ||
| LR_DEGs_nrow_list <- lapply(LR_DEGs, function(x) data.table(nrow(x))) | ||
| LR_DEGs_nrow <- sum(do.call(rbind, LR_DEGs_nrow_list)$V1) | ||
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| LR_DEGs <- lapply(LR_DEGs, function(x){ | ||
| x$p_val_adj_updated <- p.adjust(x$p_val, method = "bonferroni", n = LR_DEGs_nrow) | ||
| return(x) | ||
| }) | ||
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| LR_DEGs_pluri <- lapply(LR_DEGs, function(x){ | ||
| x$ENSG <- rownames(x) | ||
| x <- data.table(x) | ||
| x <- x[pluri_genes, on = c("ENSG")] | ||
| x <- x[!is.na(p_val) & p_val_adj_updated < 0.05] | ||
| return(x) | ||
| }) | ||
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| LR_DEGs_pluri_4 <- lapply(LR_DEGs_pluri, function(x){ | ||
| x[GeneID %in% c("MYC", "NANOG", "POU5F1", "SOX2")] | ||
| }) | ||
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| saveRDS(LR_DEGs_pluri_4, paste0(outdir, "LR_DEGs_4pluri_genes.rds")) | ||
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