args = commandArgs(trailingOnly=T) args = paste(args, collapse = "") args = unlist(strsplit(args, ";")) arguments.list = " seurat.addr.arg = args[1] set.ident.arg = args[2] n_clusters.arg = args[3] type.to.colour.arg = args[4] " expected_arguments = unlist(strsplit(arguments.list, "\n")) expected_arguments = expected_arguments[!(expected_arguments == "")] if(length(args) != length(expected_arguments)){ error.msg = sprintf('This pipeline requires %s parameters', as.character(length(expected_arguments))) expected_arguments = paste(unlist(lapply(strsplit(expected_arguments, ".arg"), "[", 1)), collapse = "\n") stop(sprintf('This pipeline requires %s parameters: ')) } eval(parse(text = arguments.list)) for(n in 1:length(expected_arguments)){ argument = expected_arguments[n] argument = gsub(pattern=" ", replacement="", x=argument) argument.name = unlist(strsplit(argument, "="))[1] variable.name = gsub(pattern=".arg", replacement="", argument.name) argument.content = eval(parse(text = argument.name)) eval(parse(text = argument.content)) if (!exists(variable.name)){ stop(sprintf("Argument %s not passed. Stopping ... ", variable.name)) } } # create required folders for output and work material output_folder = gsub(pattern="^\\d+_", replacement="", x=basename(getwd())) output_folder = paste(output_folder, seurat.addr, sep = "_") c.time = Sys.time() c.time = gsub(pattern=" BST", replacement="", x=c.time) c.time = gsub(pattern=":", replacement="", x=c.time) c.time = gsub(pattern=" ", replacement="", x=c.time) c.time = gsub(pattern="-", replacement="", x=c.time) c.time = substr(x=c.time, start=3, stop=nchar(c.time)) output_folder = paste(output_folder, c.time, sep = "_") output_folder = file.path("../../output", output_folder) dir.create(output_folder) output_folder_material = file.path(output_folder, "material") dir.create(output_folder_material) seurat.addr = file.path("../../data", seurat.addr) n_clusters = as.numeric(n_clusters) source("../../tools/bunddle_utils.R") library(Seurat) library(plyr) library(dplyr) library(reshape2) library(RColorBrewer) ####################################################################################################### # load data print("loading data ... ") seurat.obj = readRDS(seurat.addr) print("Data loaded.") seurat.obj = SetAllIdent(seurat.obj, id=set.ident) # saving pca data to disk pca.df = seurat.obj@dr$pca@cell.embeddings pca.df = cbind(pca.df, data.frame(Ident = as.vector(seurat.obj@ident))) pca.fname = file.path(output_folder_material, 'pca.csv') write.csv(pca.df, pca.fname) print("Performing clustering ... ") command = sprintf("%s ./clustering.py %s %s", python.addr, output_folder, n_clusters) system(command, wait = T) agg.clusters = read.csv(file.path(output_folder_material, 'agglomerative_clustering.csv')) colnames(agg.clusters) = c('CellNames', 'ClusterIdx') gau.clusters = read.csv(file.path(output_folder_material, 'gaussian_mixture.csv')) colnames(gau.clusters) = c('CellNames', 'ClusterIdx') metrics_file = file.path(output_folder_material, 'agreement_measures.txt') metrics_file = file(metrics_file) metrics = readLines(metrics_file) close(metrics_file) metrics = round(as.numeric(metrics), digits=2) metrics = matrix(metrics, nrow=2) colnames(metrics) = c('Rand Index', 'Adj. Mutual Information') rownames(metrics) = c('Louvain vs Agglomerative', 'Louvain vs Gaussian Mixture') metrics = as.table(metrics) library(gridExtra) library(grid) grid.fname = file.path(output_folder, 'metrics.pdf') pdf(grid.fname, width = 6, height = 1.5) grid.table(metrics) dev.off() seurat.obj@meta.data$gmm = gau.clusters$ClusterIdx seurat.obj@meta.data$agg = agg.clusters$ClusterIdx if (!is.na(type.to.colour)){ type.to.colour = read.csv(file.path("../../resources", type.to.colour)) filter.key = type.to.colour$CellTypes %in% as.vector(unique(seurat.obj@ident)) cell.labels = as.vector(type.to.colour$CellTypes[filter.key]) cell.colours = as.vector(type.to.colour$Colours[filter.key]) }else{ cell.labels = sort(as.vector(unique(seurat.obj@ident))) cell.colours = sample(colorRampPalette(brewer.pal(12, "Paired"))(length(cell.labels))) } louvain_idx = as.vector(seurat.obj@ident) gmm_idx = as.vector(seurat.obj@meta.data$gmm) agg_idx = as.vector(seurat.obj@meta.data$agg) umap_data = as.data.frame(seurat.obj@dr$umap@cell.embeddings) plot.umap.louvain = dr.plot(point.labels=louvain_idx, dr1=umap_data$umap1, dr2=umap_data$umap2, dr1.name="UMAP-x", dr2.name="UMAP-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=cell.colours, use.labels=cell.labels) plot.umap.louvain = plot.umap.louvain + ggtitle('Cell types from\nLouvain clustering') gmm_types = as.vector(unique(gmm_idx)) gmm_types = cell.labels[cell.labels %in% gmm_types] gmm_colours = mapvalues(x=gmm_types, from=cell.labels, to=cell.colours) gmm_index = seq(1, length(cell.labels))[cell.labels %in% gmm_types] plot.umap.gm = dr.plot(point.labels=gmm_idx, dr1=umap_data$umap1, dr2=umap_data$umap2, dr1.name="UMAP-x", dr2.name="UMAP-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=gmm_colours, use.labels=gmm_types, index.map=gmm_index) plot.umap.gm = plot.umap.gm + ggtitle('Cell types from\nGaussian Mixture clustering') agg_types = as.vector(unique(agg_idx)) agg_types = cell.labels[cell.labels %in% agg_types] agg_colours = mapvalues(x=agg_types, from=cell.labels, to=cell.colours) agg_index = seq(1, length(cell.labels))[cell.labels %in% agg_types] plot.umap.agg = dr.plot(point.labels=agg_idx, dr1=umap_data$umap1, dr2=umap_data$umap2, dr1.name="UMAP-x", dr2.name="UMAP-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=agg_colours, use.labels=agg_types, index.map=agg_index) plot.umap.agg = plot.umap.agg + ggtitle('Cell types from\nAgglomerative clustering') dm.fname = file.path(output_folder, 'clustering_comparisons.pdf') pdf(dm.fname, width = 18, height = 5) plot_grid(plot.umap.louvain, plot.umap.gm, plot.umap.agg, nrow=1) dev.off() n.cols = min(2, length(cell.labels)) n.rows = ceiling(length(cell.labels) / n.cols) plot.legend = plot.indexed.legend(label.vector=cell.labels, color.vector=cell.colours, ncols=n.cols, left.limit=.2, symbol.size=10, text.size=6, padH=.6, padV=.6) legend.file.name = file.path(output_folder, 'legend.pdf') pdf(legend.file.name, width = 1.5 + .15 * n.cols * max(unlist(lapply(cell.labels, nchar))), height = .5 + n.rows * .35) print(plot.legend) dev.off() plot.umap.louvain = dr.plot(point.labels=louvain_idx, dr1=umap_data$umap1, dr2=umap_data$umap2, dr1.name="UMAP-x", dr2.name="UMAP-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=cell.colours, use.labels=cell.labels, annotate.plot=F) plot.umap.louvain = plot.umap.louvain + ggtitle('Cell types from\nLouvain clustering') gmm_types = as.vector(unique(gmm_idx)) gmm_types = cell.labels[cell.labels %in% gmm_types] gmm_colours = mapvalues(x=gmm_types, from=cell.labels, to=cell.colours) gmm_index = seq(1, length(cell.labels))[cell.labels %in% gmm_types] plot.umap.gm = dr.plot(point.labels=gmm_idx, dr1=umap_data$umap1, dr2=umap_data$umap2, dr1.name="UMAP-x", dr2.name="UMAP-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=gmm_colours, use.labels=gmm_types, index.map=gmm_index, annotate.plot=F) plot.umap.gm = plot.umap.gm + ggtitle('Cell types from\nGaussian Mixture clustering') agg_types = as.vector(unique(agg_idx)) agg_types = cell.labels[cell.labels %in% agg_types] agg_colours = mapvalues(x=agg_types, from=cell.labels, to=cell.colours) agg_index = seq(1, length(cell.labels))[cell.labels %in% agg_types] plot.umap.agg = dr.plot(point.labels=agg_idx, dr1=umap_data$umap1, dr2=umap_data$umap2, dr1.name="UMAP-x", dr2.name="UMAP-y", no.legend=T, plt.lb.sz=7, txt.lb.size=4, use.cols=agg_colours, use.labels=agg_types, index.map=agg_index, annotate.plot=F) plot.umap.agg = plot.umap.agg + ggtitle('Cell types from\nAgglomerative clustering') dm.fname = file.path(output_folder, 'clustering_comparisons_no_dots.pdf') pdf(dm.fname, width = 18, height = 5) plot_grid(plot.umap.louvain, plot.umap.gm, plot.umap.agg, nrow=1) dev.off() unlink(output_folder_material, recursive=T, force=T) print("Ended beautifully ... ")