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scRNA-seq_analysis
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veghp
commit
82cc2d191e
188 changed files with 146184 additions and 0 deletions
330
pipelines/11_plot_dr/plot_dr.R
Executable file
330
pipelines/11_plot_dr/plot_dr.R
Executable file
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args = commandArgs(trailingOnly=T)
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args = paste(args, collapse = "")
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args = unlist(strsplit(args, ";"))
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arguments.list = "
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seurat.addr.arg = args[1]
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plot.by.arg = args[2]
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type.to.colours.arg = args[3]
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runDiffusionMap.arg = args[4]
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runAGA.arg = args[5]
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overlay.data.arg = args[6]
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overlay.data.ordered.arg = args[7]
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"
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plotW = 8
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plotH = 8
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expected_arguments = unlist(strsplit(arguments.list, "\n"))
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expected_arguments = expected_arguments[!(expected_arguments == "")]
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if(length(args) != length(expected_arguments)){
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error.msg = sprintf('This pipeline requires %s parameters', as.character(length(expected_arguments)))
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expected_arguments = paste(unlist(lapply(strsplit(expected_arguments, ".arg"), "[", 1)), collapse = "\n")
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stop(sprintf('This pipeline requires %s parameters: '))
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}
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eval(parse(text = arguments.list))
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for(n in 1:length(expected_arguments)){
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argument = expected_arguments[n]
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argument = gsub(pattern=" ", replacement="", x=argument)
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argument.name = unlist(strsplit(argument, "="))[1]
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variable.name = gsub(pattern=".arg", replacement="", argument.name)
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argument.content = eval(parse(text = argument.name))
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eval(parse(text = argument.content))
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if (!exists(variable.name)){
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stop(sprintf("Argument %s not passed. Stopping ... ", variable.name))
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}
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}
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# create required folders for output and work material
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output_folder = paste("11_plot_dr", seurat.addr, sep = "_")
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c.time = Sys.time()
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c.time = gsub(pattern=" BST", replacement="", x=c.time)
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c.time = gsub(pattern=":", replacement="", x=c.time)
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c.time = gsub(pattern=" ", replacement="", x=c.time)
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c.time = gsub(pattern="-", replacement="", x=c.time)
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c.time = substr(x=c.time, start=3, stop=nchar(c.time))
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output_folder = paste(output_folder, c.time, sep = "_")
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output_folder = file.path("../../output", output_folder)
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dir.create(output_folder)
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output_folder_material = file.path(output_folder, "material")
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dir.create(output_folder_material)
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seurat.addr = file.path("../../data", seurat.addr)
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source("../../tools/bunddle_utils.R")
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library(Seurat)
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library(RColorBrewer)
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library(plyr)
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library(dplyr)
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library(destiny)
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#######################################################################################################
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# load data
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print("loading data ... ")
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seurat.obj = readRDS(seurat.addr)
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print("Data loaded.")
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# save raw data to disk
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raw_data = seurat.obj@raw.data
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raw_data = raw_data[rownames(seurat.obj@data), colnames(seurat.obj@data)]
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# save gene names
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gene_names = rownames(raw_data)
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write.csv(data.frame(Genes = gene_names), file.path(output_folder_material, "genenames.csv"))
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# save cell names
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cell_names = colnames(raw_data)
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write.csv(data.frame(Cells = cell_names), file.path(output_folder_material, "cellnames.csv"))
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# write cell labels to disk
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write.csv(data.frame(Cells = names(seurat.obj@ident), Labels = seurat.obj@ident), file.path(output_folder_material, "cell_labels.csv"), row.names = F)
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# run the diffusion map
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if(runDiffusionMap){
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print("Writing .mtx file for diffusion map ...")
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writeMM(raw_data, file.path(output_folder_material, "raw_data.mtx"))
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print("Running the diffusion map ... ")
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command = sprintf("%s ./dm.py %s", python.addr, output_folder_material)
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system(command, wait = T)
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# load dm from disk
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dm = read.csv(file.path(output_folder_material, "dm.csv"), row.names = 1, header = F)
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#dm = DiffusionMap(seurat.obj@dr$pca@cell.embeddings, k = 100, density_norm=F, n_eigs = 20)
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#dm = data.frame(DC1 = dm$DC1, DC2 = dm$DC2, DC2 = dm$DC3)
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}
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print("Computing FDG limits ...")
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fdg.x = seurat.obj@dr$fdg@cell.embeddings[, 1]
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fdg.y = seurat.obj@dr$fdg@cell.embeddings[, 2]
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fdg.limits = 1.15 * c(quantile(fdg.x, c(.01)), quantile(fdg.x, c(.99)), quantile(fdg.y, c(.01)), quantile(fdg.y, c(.99)))
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print("Making the plots ...")
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for (index in 1:length(plot.by)){
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caty = plot.by[index]
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seurat.obj = SetAllIdent(object=seurat.obj, id=caty)
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if (!is.na(type.to.colours[index])){
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type.to.colour = read.csv(file.path("../../resources", type.to.colours[index]))
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filter.key = type.to.colour$CellTypes %in% as.vector(unique(seurat.obj@ident))
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cell.labels = as.vector(type.to.colour$CellTypes[filter.key])
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cell.colours = as.vector(type.to.colour$Colours[filter.key])
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all_celltypes_missing <- levels(factor(seurat.obj@ident))[!levels(factor(seurat.obj@ident)) %in% levels(factor(type.to.colour$CellTypes))]
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all_colours_missing <- levels(factor(type.to.colour$CellTypes))[!levels(factor(type.to.colour$CellTypes)) %in% levels(factor(seurat.obj@ident))]
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if (length(all_celltypes_missing)>0){
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cat(all_celltypes_missing, "have not been found in your type.to.cols .csv \n", sep="\n")
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}
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if (length(all_colours_missing)>0){
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cat(all_colours_missing, "have not been found in your selected metadata column", sep="\n")
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}
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if (length(all_colours_missing)==0|length(all_colours_missing)==0){
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print("All colours and annotations match")
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}
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}else{
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cell.labels = sort(as.vector(unique(seurat.obj@ident)))
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cell.colours = sample(colorRampPalette(brewer.pal(12, "Paired"))(length(cell.labels)))
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}
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caty = gsub(pattern="\\.", replacement="_", caty)
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# file paths for annotated graphs
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tsne.file.name = file.path(output_folder, paste("tsne", paste(caty, "pdf", sep = "."), sep = "_"))
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umap.file.name = file.path(output_folder, paste("umap", paste(caty, "pdf", sep = "."), sep = "_"))
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fdg.file.name = file.path(output_folder, paste("fdg", paste(caty, "pdf", sep = "."), sep = "_"))
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legend.file.name = file.path(output_folder, paste("legend", paste(caty, "pdf", sep = "."), sep = "_"))
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AGA.file.name = file.path(output_folder, paste("AGA", paste(caty, "pdf", sep = "."), sep = "_"))
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# file paths for unannotated plots
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tsne.file.name_unlabeled = file.path(output_folder, paste("tsne_unlabeled", paste(caty, "pdf", sep = "."), sep = "_"))
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umap.file.name_unlabeled = file.path(output_folder, paste("umap_unlabeled", paste(caty, "pdf", sep = "."), sep = "_"))
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fdg.file.name_unlabeled = file.path(output_folder, paste("fdg_unlabeled", paste(caty, "pdf", sep = "."), sep = "_"))
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# preparing data frame
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if(!is.na(overlay.data)){
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df = data.frame(CellType = as.vector(seurat.obj@ident),
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tSNEx = seurat.obj@dr$tsne@cell.embeddings[, 1],
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tSNEy = seurat.obj@dr$tsne@cell.embeddings[, 2],
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UMAPx = seurat.obj@dr$umap@cell.embeddings[, 1],
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UMAPy = seurat.obj@dr$umap@cell.embeddings[, 2],
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FDGx = seurat.obj@dr$fdg@cell.embeddings[, 1],
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FDGy = seurat.obj@dr$fdg@cell.embeddings[, 2],
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overlay.data = seurat.obj@meta.data[overlay.data])
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colnames(df)[8] <- "overlay.data"
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}
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else
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{
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df = data.frame(CellType = as.vector(seurat.obj@ident),
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tSNEx = seurat.obj@dr$tsne@cell.embeddings[, 1],
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tSNEy = seurat.obj@dr$tsne@cell.embeddings[, 2],
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UMAPx = seurat.obj@dr$umap@cell.embeddings[, 1],
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UMAPy = seurat.obj@dr$umap@cell.embeddings[, 2],
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FDGx = seurat.obj@dr$fdg@cell.embeddings[, 1],
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FDGy = seurat.obj@dr$fdg@cell.embeddings[, 2])
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}
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print("printing header of df made at beginning od r script")
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print(head(df))
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interactive_plot_df = data.frame(X = seurat.obj@dr$tsne@cell.embeddings[, 1],
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Y = seurat.obj@dr$tsne@cell.embeddings[, 2])
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interactive_plot_df$Labels = factor(seurat.obj@ident, levels = cell.labels)
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interactive_plot_df$Colours = mapvalues(x = interactive_plot_df$Labels, from = cell.labels, to = cell.colours)
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# make interartive tsne
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interactive_tsne_filename = file.path(output_folder, paste(paste("Interactive_tSNE", caty, sep = "_"), "html", sep = "."))
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make_2D_interactive_page(data_frame_2D=interactive_plot_df, tool_addr=tool_addr, python.addr=python.addr, save.to=interactive_tsne_filename)
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# make interactive UMAP
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interactive_plot_df$X = seurat.obj@dr$umap@cell.embeddings[, 1]
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interactive_plot_df$Y = seurat.obj@dr$umap@cell.embeddings[, 2]
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interactive_umap_filename = file.path(output_folder, paste(paste("Interactive_UMAP", caty, sep = "_"), "html", sep = "."))
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make_2D_interactive_page(data_frame_2D=interactive_plot_df, tool_addr=tool_addr, python.addr=python.addr, save.to=interactive_umap_filename)
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# make interactive FDG
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interactive_plot_df$X = seurat.obj@dr$fdg@cell.embeddings[, 1]
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interactive_plot_df$Y = seurat.obj@dr$fdg@cell.embeddings[, 2]
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interactive_fdg_filename = file.path(output_folder, paste(paste("Interactive_FDG", caty, sep = "_"), "html", sep = "."))
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make_2D_interactive_page(data_frame_2D=interactive_plot_df, tool_addr=tool_addr, python.addr=python.addr, save.to=interactive_fdg_filename)
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n.cols = min(2, length(cell.labels))
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n.rows = ceiling(length(cell.labels) / n.cols)
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# making the plots
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print("making the plots")
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# annotated plots
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print("making annotated plots")
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plot.tsne = dr.plot(point.labels=df$CellType, dr1=df$tSNEx, dr2=df$tSNEy, dr1.name="tSNE-x", dr2.name="tSNE-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=cell.colours, use.labels=cell.labels)
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plot.umap = dr.plot(point.labels=df$CellType, dr1=df$UMAPx, dr2=df$UMAPy, dr1.name="UMAP-x", dr2.name="UMAP-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=cell.colours, use.labels=cell.labels)
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plot.fdg = dr.plot(point.labels=df$CellType, dr1=df$FDGx, dr2=df$FDGy, dr1.name="FDG-x", dr2.name="FDG-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=cell.colours, limits=fdg.limits, use.labels=cell.labels)
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plot.legend = plot.indexed.legend(label.vector=cell.labels, color.vector=cell.colours, ncols=n.cols, left.limit=.2, symbol.size=10, text.size=6, padH=.6, padV=.6)
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# unannotated plots
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print("making un-annotated plots")
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plot.tsne_unlabeled = dr.plot(point.labels=df$CellType, dr1=df$tSNEx, dr2=df$tSNEy, dr1.name="tSNE-x", dr2.name="tSNE-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=cell.colours, use.labels=cell.labels, annotate.plot = F)
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plot.umap_unlabeled = dr.plot(point.labels=df$CellType, dr1=df$UMAPx, dr2=df$UMAPy, dr1.name="UMAP-x", dr2.name="UMAP-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=cell.colours, use.labels=cell.labels, annotate.plot = F)
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plot.fdg_unlabeled = dr.plot(point.labels=df$CellType, dr1=df$FDGx, dr2=df$FDGy, dr1.name="FDG-x", dr2.name="FDG-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=cell.colours, limits=fdg.limits, use.labels=cell.labels, annotate.plot = F, pt.size=1)
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# print the annotated plots
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print("printing annotated plots")
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pdf(tsne.file.name, width = plotW, height = plotH)
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print(plot.tsne)
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dev.off()
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pdf(umap.file.name, width = plotW, height = plotH)
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print(plot.umap)
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dev.off()
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pdf(fdg.file.name, width = plotW, height = plotH)
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print(plot.fdg)
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dev.off()
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pdf(legend.file.name, width = 1.5 + .15 * n.cols * max(unlist(lapply(cell.labels, nchar))), height = .5 + n.rows * .35)
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print(plot.legend)
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dev.off()
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# print the unannotated plots
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print("printing un-annotated plots")
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pdf(tsne.file.name_unlabeled, width = plotW, height = plotH)
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print(plot.tsne_unlabeled)
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dev.off()
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pdf(umap.file.name_unlabeled, width = plotW, height = plotH)
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print(plot.umap_unlabeled)
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dev.off()
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pdf(fdg.file.name_unlabeled, width = plotW, height = plotH)
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print(plot.fdg_unlabeled)
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dev.off()
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# run diffusion map
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if(runDiffusionMap){
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df = as.data.frame(dm[, 1:3])
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df$Labels = factor(seurat.obj@ident, levels = cell.labels)
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df$Colours = mapvalues(x = df$Labels, from = cell.labels, to = cell.colours)
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dm.file.name = file.path(output_folder_material, paste(paste("dm_data", caty, sep="_"), "csv", sep = "."))
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write.csv(df, dm.file.name, row.names = F)
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dm.file.name = file.path(output_folder, paste(paste("DiffusionMap_3D", caty, sep = "_"), "html", sep = "."))
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make_3D_interactive_page(data_frame_3D=df, tool_addr=tool_addr, python.addr=python.addr, save.to=dm.file.name)
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}
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if(runAGA){
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if(runDiffusionMap==F){
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print("Writing .mtx file for AGA map ...")
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writeMM(raw_data, file.path(output_folder_material, "raw_data.mtx"))
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}
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print("running AGA ...")
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AGA_folder = file.path(output_folder, "AGA_folder")
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dir.create(AGA_folder)
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# write cell labels to disk
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write.csv(data.frame(Cells = names(seurat.obj@ident), Labels = seurat.obj@ident), file.path(output_folder_material, "cell_labels.csv"), row.names = F)
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# running AGA
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command =file.path(tool_addr, "AGA/AGA_from_Seurat.py")
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command = paste(paste(python.addr, command, sep = " "), output_folder, sep = " ")
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command = paste(command, caty, sep =" ")
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system(command, wait = T)
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# read the AGA output
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coordinates = read.csv(file.path(AGA_folder, "coordinates.csv"), row.names = 1)
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connectivities = read.csv(file.path(AGA_folder, "connectivities.csv"), row.names = 1)
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# plot AGA
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coordinates = coordinates[cell.labels, ]
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coordinates$Colours = cell.colours
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label.order = match(cell.labels, rownames(connectivities))
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connectivities = connectivities[label.order, label.order]
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plot.obj = ggplot(data=coordinates, aes(x = X, y = Y))
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plot.obj = plot.obj + theme_void() + theme(legend.position="none")
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xi = c(); xf = c(); yi = c(); yf = c(); vs = c();
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for(i in 1:dim(connectivities)[1]){
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for(j in i:dim(connectivities)[2]){
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v = connectivities[i, j]
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if(v > 0){
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xi = c(xi, coordinates$X[i])
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xf = c(xf, coordinates$X[j])
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yi = c(yi, coordinates$Y[i])
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yf = c(yf, coordinates$Y[j])
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vs = c(vs, v)
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}
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}
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}
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lineDF = data.frame(Xi = xi, Yi = yi, Xf = xf, Yf = yf, Vs = vs)
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plot.obj = plot.obj + geom_segment(data = lineDF, aes(x = Xi, y = Yi, xend = Xf, yend = Yf), color = "black", size = 3 * lineDF$Vs)
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plot.obj = plot.obj + geom_point(size = 2 * log(coordinates$Size), color = coordinates$Colours)
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plot.obj = plot.obj + annotate("text", x=coordinates$X, y=coordinates$Y, label = 1:dim(coordinates)[1])
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pdf(AGA.file.name, width = 10, height = 10)
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print(plot.obj)
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dev.off()
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######## now make the interactive AGA app
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#########################################
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print("Making the AGA app ... ")
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# save colours
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colours.df = data.frame(CellTypes = cell.labels, Colours = cell.colours)
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write.csv(colours.df, file.path(AGA_folder, "colours.csv"), row.names = F)
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# run python to built the AGA app
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command = sprintf("%s make_AGA_app.py %s %s", python.addr, output_folder, caty)
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system(command, wait = T)
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}
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}
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# cleaning garbage folders
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unlink(output_folder_material, recursive=T, force=T)
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if (runAGA){
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unlink(AGA_folder, recursive=T, force=T)
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unlink(file.path(output_folder, 'figures'), recursive=T, force=T)
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}
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print("Ended beautifully ... ")
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