HNRNPH1 drives glioblastoma progression by regulating the splicing of cell cycle genes.
The 2 matches
- [1] § Materials and Methods › Analysis of spatial transcriptomics data ↔ Module1_MP.R, lines 56 to 99 · score 0.91 · FindVariableFeatures, NormalizeData, ScaleData, RunPCA, Seurat, assignment · Report a mismatch
- [2] § Materials and Methods › Analysis of single-cell RNA sequencing data ↔ Module1_MP.R, lines 56 to 99 · score 0.76 · FindClusters, FindNeighbors, RunPCA, Seurat, variable, log · Report a mismatch
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The authors' code
R · 650 lines · 32 KB · no license · 2 matches
- Sys.setenv(RETICULATE_PYTHON = "/.conda/envs/leiden/bin/python")
- Sys.setenv(RETICULATE_PYTHON = "/.conda/envs/leiden/bin/python")
- library(reticulate)
- library(Seurat)
- library(scalop)
- library(leiden)
- library(ggplot2)
- library(RColorBrewer)
- library(viridis)
- library(scales)
- library(reshape2)
- library(reshape2)
- library(scales)
- library(NMF)
- library(MetBrewer)
- library(colorspace)
- library(tibble)
- library(dplyr)
- library(data.table)
- library(stringr)
- library(readr)
- library(Matrix)
- library(bigmemory)
- library(doMC)
- library(patchwork)
- ###### Per sample Leiden clustering##############################
- samples_names <- (read.delim("general/GBM_samples.txt", header = FALSE, sep = "\t"))$V1
- # set parameters
- complexity_filter <- 1000
- mitochondrial_filter <- 20
- genes_filter <- 7000
- n_dim <- 20
- dim_filter <- 5^(-15)
- res_param <- 1
- sig_th <- .005
- mp_num <- 13
- distinct16_pal<-c("#11A579","#F2B701","#66C5CC","#80BA5A","#F6CF71","#7F3C8D","#CF1C90","#3969AC","#f97b72","#E73F74","#4b4b8f","#ACA4E2","#31C53F","#B95FBB","#D4D915","#28CECA")
- dim2use_list <- c(8,20,19,9,12,14,16,10,10,14,8,10,12,16,7,5,14,16,4,12,6,6,8,12,13,10) # set by jackstraw
- leiden_clustering <- sapply(c(1:length(samples_names)), function(i){
- print(samples_names[i])
- # load spatial data
- unfilt_obj<-Load10X_Spatial(data.dir = paste("general/GBM_data/",samples_names[i],"/outs", sep = ""),
- filename = "filtered_feature_bc_matrix.h5",
- assay = "Spatial",
- slice = "detected_tissue_image.jpg",
- filter.matrix = TRUE,
- to.upper = FALSE
- )
- # filtering
- exp_obj <- subset(unfilt_obj, subset = nCount_Spatial > complexity_filter) # filter out spots with less than #complexity_filter UMIs
- exp_obj[["percent.mt"]] <- PercentageFeatureSet(exp_obj, pattern = "^MT-") # filter spots with high percent (mitochondrial_filter) mitochondrial genes
- exp_obj <- subset(exp_obj, subset = percent.mt<mitochondrial_filter)
- # normalization and centering
- exp_obj <- NormalizeData(exp_obj, assay = "Spatial", normalization.method="LogNormalize", scale.factor=10000)
- exp_obj <- FindVariableFeatures(exp_obj, selection.method = "vst", nfeatures = genes_filter) # use only #genes_filter most variable genes
- exp_obj <- ScaleData(exp_obj)
- # PCA
- exp_obj <- RunPCA(exp_obj, features = VariableFeatures(object = exp_obj))
- #PCA dims to use determined by Jackstraw
- #exp_obj <- JackStraw(exp_obj, num.replicate = 100)
- #exp_obj <- ScoreJackStraw(exp_obj, dims = 1:n_dim)
- #js_scores <- exp_obj@reductions$pca@jackstraw$overall.p.values
- #dim2use <- max(js_scores[,"PC"][js_scores[,"Score"] < dim_filter])
- dim2use <- dim2use_list[i]
- # clustering
- exp_obj <- FindNeighbors(exp_obj, dims = 1:dim2use)
- exp_obj <- FindClusters(exp_obj, algorithm=4, resolution = res_param) # leiden based clustering
- spatial_sample_programs <- FindAllMarkers(exp_obj, only.pos = TRUE, return.thresh = sig_th)
- genes_num <- table(spatial_sample_programs$cluster)
- spots_clusters <- [email hidden][,"seurat_clusters"]
- names(spots_clusters) <- row.names([email hidden])
- cluster_num <- table([email hidden]$seurat_clusters)
- return(list(spatial_sample_programs,genes_num,spots_clusters,cluster_num)) # output: #genes per cluster, #spots per cluster, clusters genes sig, spots assignment to cluster
- })
- # clusters quality
- genes_num <- c()
- spots_num <- c()
- sapply(c(1:length(samples_names)), function(i){
- genes_num <<- c(genes_num,as.numeric(leiden_clustering[2,i][[1]]))
- spots_num <<- c(spots_num,as.numeric(leiden_clustering[4,i][[1]]))
- })
- hist(genes_num, breaks = 30)
- hist(spots_num, breaks = 30)
- # create genes sig table for save
- genes_sig <- leiden_clustering[1,1][[1]]
- genes_sig$sample <- rep(samples_names[1],dim(genes_sig)[1])
- sapply(c(2:length(samples_names)),function(i){
- genes_sig_temp <- leiden_clustering[1,i][[1]]
- genes_sig_temp$sample <- rep(samples_names[i],dim(genes_sig_temp)[1])
- genes_sig <<- rbind(genes_sig,genes_sig_temp)
- })
- # create clusters assignment table for save
- spots_assign <- as.data.frame(leiden_clustering[3,1][[1]])
- spots_assign$sample <- rep(samples_names[1],dim(spots_assign)[1])
- row.names(spots_assign) <- paste(row.names(spots_assign), "_", samples_names[1], sep = "")
- colnames(spots_assign) <- c("cluster","sample")
- sapply(c(2:length(samples_names)),function(i){
- spots_assign_temp <- as.data.frame(leiden_clustering[3,i][[1]])
- spots_assign_temp$sample <- rep(samples_names[i],dim(spots_assign_temp)[1])
- row.names(spots_assign_temp) <- paste(row.names(spots_assign_temp), "_", samples_names[i], sep = "")
- colnames(spots_assign_temp) <- c("cluster","sample")
- spots_assign <<- rbind(spots_assign,spots_assign_temp)
- })
- # set gene signatures list in the right format
- genes_list <- lapply(c(1:length(samples_names)), function(i){
- sample_table <- leiden_clustering[1,i][[1]]
- sample_genes <- lapply(c(1:length(unique(sample_table$cluster))),function(j){
- cluster_table <- sample_table[sample_table$cluster == j,]
- genes <- cluster_table$gene[order(cluster_table$avg_log2FC, decreasing = TRUE)][1:min(50,nrow(cluster_table))]
- return(genes)
- })
- return(sample_genes)
- })
- genes_list <- unlist(genes_list, recursive = FALSE)
- names(genes_list) <- unlist(sapply(c(1:length(samples_names)),function(i){
- paste(samples_names[i],c(1:length(leiden_clustering[4,i][[1]])), sep = "_")
- }))
- samples_list <- unlist(sapply(c(1:length(samples_names)),function(i){
- rep(samples_names[i],length(leiden_clustering[4,i][[1]]))
- }))
- #saveRDS(genes_list,"/home/labs/tirosh/spatial_glioma/results/leiden_MPs/all_leiden_programs_v2.rds")
- # generate an ordered jaccard matrix and clusters
- jac_mat = Jaccard(genes_list)
- jac_matord = scalop::hca_reorder(jac_mat)
- jac_plot <- melt(jac_matord)
- prog_ordered <- colnames(jac_matord)
- clusters <- scalop::hca_groups(jac_matord,
- cor.method="none",
- k=mp_num,
- min.size=4,
- max.size=0.5)
- clusterCut <- sapply(names(clusters), function(i){
- new_vec <- rep(i,length(clusters[[i]]))
- names(new_vec) <- clusters[[i]]
- return(new_vec)
- })
- names(clusterCut) <- NULL
- clusterCut <- unlist(clusterCut)
- # plot jaccard
- jac_plot$value[jac_plot$value >= 0.35] <- 0.35
- p1 <- ggplot(data = jac_plot, aes(x = Var1, y = Var2, fill = value)) + geom_raster() +
- scale_fill_gradientn(colours = colorspace::sequential_hcl(9, palette="YlOrRd", rev=T), limits = c(0,0.35), name = "Jaccard\nIndex") +
- theme(axis.ticks = element_blank(), panel.background = element_rect(fill = "white"), axis.line = element_blank(), legend.text.align = 0.5) +
- scale_x_discrete(name = "\nPrograms", labels = element_blank(), breaks = element_blank()) + scale_y_discrete(name = "\nPrograms", labels = element_blank(), breaks = element_blank())
- metaprog_df <- data.frame(row.names = prog_ordered,
- cluster = as.factor(clusterCut[prog_ordered]),
- samples = stringr::str_replace(prog_ordered, "_.*",""),
- cells = prog_ordered)
- samples_annotation_plot <- ggplot(metaprog_df, aes(x = factor(cells, levels = cells), y = 1)) +
- geom_raster(aes(fill = samples)) + theme(legend.position = "none",axis.ticks = element_blank(), panel.background = element_rect(fill = "white"), axis.text = element_blank(), axis.line = element_blank(), axis.text.x = element_blank()) +
- labs(x = "", y = "samples", fill = "")
- metaprog_annotation_plot <- ggplot(metaprog_df, aes(x = factor(cells, levels = cells), y = 1)) +
- geom_raster(aes(fill = cluster)) + theme(legend.position = "none", axis.ticks = element_blank(), panel.background = element_rect(fill = "white"), axis.text = element_blank(), axis.line = element_blank(), axis.text.x = element_blank()) +
- labs(x = "", y = "", fill = "")
- MP_plot <- egg::ggarrange(p1, metaprog_annotation_plot, samples_annotation_plot, ncol = 1, nrow = 3, heights = c(40, 2, 1))
- #Define meta-programs from program clusters, Sort by frequency across programs in cluster (>= 3)
- cut_off <- table(metaprog_df$cluster)*0.25
- cut_off[cut_off < 3] <- 3
- mp_freqs = sapply(clusters, function(k) sort(table(unlist(genes_list[k])),decreasing = T),simplify = F)
- metaprograms = sapply(names(mp_freqs), function(c) head(names(mp_freqs[[c]])[mp_freqs[[c]] >= as.numeric(cut_off[c])], 50), simplify = F)
- names(metaprograms)<-c("vascular","neuron","hypoxia","hypoxia.immune","oligo","AC.immune","low.quality","NPC2","macrophage","MES.stromal","OPC.AC","metabolism","MES1.RGL")
- metaprog_df$samp_cluster <- str_split(metaprog_df$cells, "_", simplify = T)[,2]
- metaprog_df$anno_cluster <- metaprog_df$cluster
- class(metaprog_df$anno_cluster)
- levels(metaprog_df$anno_cluster) <- c("vascular","MES.stromal","OPC.AC","metabolism","MES1.RGL","neuron","hypoxia","hypoxia.immune","oligo","AC.immune","low.quality","NPC2","macrophage")
- sapply(c(1:26), function(i){
- print(samples_names[i])
- spots_gen_assign <- as.data.frame(leiden_clustering[3,i][[1]])
- spots_gen_assign$generalized <- apply(spots_gen_assign,1,function(r){
- return(metaprog_df$anno_cluster[metaprog_df$samples == samples_names[i] & metaprog_df$samp_cluster == r])
- })
- spots_gen_assign$barcodes <- row.names(spots_gen_assign)
- spots_gen_assign <- spots_gen_assign[,c("barcodes", "generalized")]
- })
- ######generating input matrices for NMF
- # vector of file paths
- file_paths <- list.files(path ="/general/exp_mats_GBM/", pattern = "\\.rds", full.names = TRUE)#directory with counts mats
- sample_ls <- gsub(pattern = "\\.rds$", replacement = "", x = basename(file_paths))
- per_sample_mat <- lapply(file_paths, readRDS)
- for (i in seq_along(per_sample_mat)){
- m <- as.matrix(per_sample_mat[[i]])
- if(min(colSums(m)) == 0){m <- m[, colSums(m) != 0]}
- scaling_factor <- 1000000/colSums(m)
- m_CPM <- sweep(m, MARGIN = 2, STATS = scaling_factor, FUN = "*")
- m_loged <- log2(1 + (m_CPM/10))
- # removing genes with zero variance across all cells
- var_filter <- apply(m_loged, 1, var)
- m_proc <- m_loged[var_filter != 0, ]
- # filtering out lowly expressed genes
- exp_genes <- rownames(m_proc)[(rowMeans(m_proc) > 0.4)]
- m_proc <- m_proc[exp_genes, ]
- # centering data gene-wise
- count_mat_cent<- m_proc - rowMeans(m_proc)
- #nmf preprocessing
- count_mat_nmf<- count_mat_cent
- count_mat_nmf[count_mat_nmf<0] <- 0 # negative values should be set to 0 in initial matrix
- # output to a list of gene expression profiles (GEP)
- per_sample_mat[[i]] <- count_mat_nmf
- names(per_sample_mat)[i] <- sample_ls[[i]]
- rm(m,m_loged, var_filter, exp_genes, m_proc,count_mat_cent)
- #saveRDS(count_mat_nmf, paste("MP/NMF/NMF_mats_GBM/", samples_names[i], ".rds", sep =""))
- }
- ## Extract sample names for sample list file:
- #samples <- list.files("MP/NMF/NMF_mats_GBM/") %>% substri(., pos = 1) %>% str_c(., collapse = "\n")
- #write_lines(samples, ("/samples.txt"), sep = "\n")
- ###### NMF (Previously run by server. Possible to run per sample below)---------------------------------------------------------------------
- #!/usr/bin/env Rscript
- #args = commandArgs(trailingOnly = TRUE)
- sname <- "MGH258"# enter sample name here
- rank_lb <- 2
- rank_ub <- 11
- m <- readRDS(paste0("MP/NMF/NMF_mats_GBM/", sname, ".rds"))
- m <- as.matrix(m)
- res <- NMF::nmf(x = m, rank = rank_lb:rank_ub, nrun = 5, method = "snmf/r", .opt = list(debug=F, parallel=F, shared.memory=F, verbose=T))
- ###### Generation of NMF + Leiden GBM spatial metaprograms#########
- #
- #robust_nmf_programs function
- robust_nmf_programs <- function(nmf_programs, intra_min = 30, intra_max = 10, inter_filter=T, inter_min = 10) {
- # Select NMF programs based on the minimum overlap with other NMF programs from the same sample
- intra_intersect <- lapply(nmf_programs, function(z) apply(z, 2, function(x) apply(z, 2, function(y) length(intersect(x,y)))))
- intra_intersect_max <- lapply(intra_intersect, function(x) apply(x, 2, function(y) sort(y, decreasing = T)[2]))
- nmf_sel <- lapply(names(nmf_programs), function(x) nmf_programs[[x]][,intra_intersect_max[[x]]>=intra_min])
- names(nmf_sel) <- names(nmf_programs)
- # Select NMF programs based on i) the maximum overlap with other NMF programs from the same sample and
- # ii) the minimum overlap with programs from another sample
- nmf_sel_unlist <- do.call(cbind, nmf_sel)
- inter_intersect <- apply(nmf_sel_unlist , 2, function(x) apply(nmf_sel_unlist , 2, function(y) length(intersect(x,y)))) ## calculating intersection between all programs
- final_filter <- NULL
- for(i in names(nmf_sel)) {
- a <- inter_intersect[grep(i, colnames(inter_intersect), invert = T),grep(i, colnames(inter_intersect))]
- b <- sort(apply(a, 2, max), decreasing = T) # for each sample, ranks programs based on their maximum overlap with programs of other samples
- if(inter_filter==T) b <- b[b>=inter_min] # selects programs with a maximum intersection of at least 10
- if(length(b) > 1) {
- c <- names(b[1])
- for(y in 2:length(b)) {
- if(max(inter_intersect[c,names(b[y])]) <= intra_max) c <- c(c,names(b[y])) # selects programs iteratively from top-down. Only selects programs that have a intersection smaller than 10 with a previously selected programs
- }
- final_filter <- c(final_filter, c)
- } else {
- final_filter <- c(final_filter, names(b))
- }
- }
- return(final_filter)
- }
- # Custom color palette
- custom_magma <- c(colorRampPalette(c("white", rev(magma(323, begin = 0.15))[1]))(10), rev(magma(323, begin = 0.18)))
- ## Create list of NMF matrics where each sample is an entry
- path <- "MP/NMF/out_dir/"
- sample_ls <- list.files(path)
- ## Create list of NMF matrics where each sample is an entry
- prog_genes_ls <- list()
- for(i in seq_along(sample_ls)) {
- nmf_obj <- readRDS(paste(path, sample_ls[[i]], sep = "/"))
- samp_name <- stringr::str_split(sample_ls[[i]], pattern = "_")[[1]][[1]]
- nmf_mats <- c()
- for(j in names(nmf_obj$fit)) {
- get_basis <- basis(nmf_obj$fit[[j]])
- colnames(get_basis) <- paste0(samp_name, ".", j, ".", 1:j)
- nmf_mats <- cbind(nmf_mats, get_basis)
- }
- prog_genes_ls[[i]] <- nmf_mats
- names(prog_genes_ls)[i] <- paste0(samp_name)
- rm(nmf_obj, nmf_mats, get_basis)
- }
- Genes_nmf_w_basis <- do.call(c, list(prog_genes_ls))
- # Find robust NMFs
- # get gene programs (top 50 genes by NMF score)
- nmf_programs_sig <- lapply(prog_genes_ls, function(x) apply(x, 2, function(y) names(sort(y, decreasing = T))[1:50]))
- # for each sample, select robust NMF programs (i.e. observed using different ranks in the same sample), remove redundancy due to multiple ranks, and apply a filter based on the similarity to programs from other samples.
- nmf_filter_all <- robust_nmf_programs(nmf_programs_sig, intra_min = 35, intra_max = 10, inter_filter=T, inter_min = 10)
- nmf_programs_sig <- lapply(nmf_programs_sig, function(x) x[, is.element(colnames(x), nmf_filter_all),drop=F])
- nmf_programs_sig <- do.call(cbind, nmf_programs_sig)
- #leiden clusters
- all_leiden_programs<-readRDS("MP/all_leiden_programs_v2.rds")
- all_leiden_programs <- do.call(cbind, all_leiden_programs)
- nmf_programs_sig<-cbind(nmf_programs_sig,all_leiden_programs)
- # calculate similarity between programs
- nmf_intersect <- apply(nmf_programs_sig , 2, function(x) apply(nmf_programs_sig , 2, function(y) length(intersect(x,y))))
- # hierarchical clustering of the similarity matrix
- nmf_intersect_hc_all <- hclust(as.dist(50-nmf_intersect), method="average")
- nmf_intersect_hc_all <- reorder(as.dendrogram(nmf_intersect_hc_all), colMeans(nmf_intersect))
- nmf_intersect <- nmf_intersect[order.dendrogram(nmf_intersect_hc_all), order.dendrogram(nmf_intersect_hc_all)]
- nmf_intersect<-readRDS("MP/NMF/nmf_intersect_124.RDS")
- nmf_programs_sig<-readRDS("MP/NMF/nmf_programs_sig_124.RDS")
- ### use a clustering approach that updates MPs in each iteration
- nmf_intersect_KEEP <- nmf_intersect
- nmf_programs_sig_KEEP <- nmf_programs_sig
- ### Parameters (later change to function form)v1-keep!
- Min_intersect_initial <- 12 # the minimal intersection cutoff for defining the Founder NMF program of a cluster
- Min_intersect_cluster <- 12 # the minimal intersection cuttof for adding a new NMF to the forming cluster
- Min_group_size <- 4 # the minimal group size to consider for defining the Founder_NMF of a MP
- Sorted_intersection <- sort(apply(nmf_intersect , 2, function(x) (length(which(x>=Min_intersect_initial))-1) ) , decreasing = TRUE)
- Cluster_list <- list() ### Every entry contains the NMFs of a chosec cluster
- k <- 1
- Curr_cluster <- c()
- MP_list <- list()
- while (Sorted_intersection[1]>Min_group_size) {
- Curr_cluster <- c(Curr_cluster , names(Sorted_intersection[1]))
- ### intersection between all remaining NMFs and Genes in MP
- Genes_MP <- nmf_programs_sig[,names(Sorted_intersection[1])] # initial genes are those in the first NMF. Genes_MP always has only 50 genes consisting of the current MP
- nmf_programs_sig <- nmf_programs_sig[,-match(names(Sorted_intersection[1]) , colnames(nmf_programs_sig))] # remove selected NMF
- Intersection_with_Genes_MP <- sort(apply(nmf_programs_sig, 2, function(x) length(intersect(Genes_MP,x))) , decreasing = TRUE) # intersection between all other NMFs and Genes_MP
- NMF_history <- Genes_MP # has all genes in all NMFs in the current cluster, for newly defining Genes_MP after adding a new NMF
- ### Create gene list - composed of intersecting genes in descending order. Update Curr_cluster each time.
- while ( Intersection_with_Genes_MP[1] >= Min_intersect_cluster) { ### Define current cluster
- Curr_cluster <- c(Curr_cluster , names(Intersection_with_Genes_MP)[1])
- Genes_MP_temp <- sort(table(c(NMF_history , nmf_programs_sig[,names(Intersection_with_Genes_MP)[1]])), decreasing = TRUE) ## Genes_MP is newly defined each time according to all NMFs in the current cluster
- Genes_at_border <- Genes_MP_temp[which(Genes_MP_temp == Genes_MP_temp[50])] ### genes with overlap equal to the 50th gene
- if (length(Genes_at_border)>1){
- ### Sort last genes in Genes_at_border according to maximal NMF gene scores
- ### Run over all NMF programs in Curr_cluster and extract NMF scores for each gene
- Genes_curr_NMF_score <- c()
- for (i in Curr_cluster) {
- curr_study <- strsplit(i, "[.]")[[1]][[1]]
- Q <- Genes_nmf_w_basis[[curr_study]][ match(names(Genes_at_border),toupper(rownames(Genes_nmf_w_basis[[curr_study]])))[!is.na(match(names(Genes_at_border),toupper(rownames(Genes_nmf_w_basis[[curr_study]]))))] ,i]
- #names(Q) <- names(Genes_at_border[!is.na(match(names(Genes_at_border),toupper(rownames(Genes_nmf_w_basis[[curr_study]]))))]) ### sometimes when adding genes the names do not appear
- Genes_curr_NMF_score <- c(Genes_curr_NMF_score, Q )
- }
- Genes_curr_NMF_score_sort <- sort(Genes_curr_NMF_score , decreasing = TRUE)
- Genes_curr_NMF_score_sort <- Genes_curr_NMF_score_sort[unique(names(Genes_curr_NMF_score_sort))] ## take only the maximal score of each gene - which is the first entry after sorting
- Genes_MP_temp <- c(names(Genes_MP_temp[which(Genes_MP_temp > Genes_MP_temp[50])]) , names(Genes_curr_NMF_score_sort))
- } else {
- Genes_MP_temp <- names(Genes_MP_temp)[1:50]
- }
- NMF_history <- c(NMF_history , nmf_programs_sig[,names(Intersection_with_Genes_MP)[1]])
- Genes_MP <- Genes_MP_temp[1:50]
- nmf_programs_sig <- nmf_programs_sig[,-match(names(Intersection_with_Genes_MP)[1] , colnames(nmf_programs_sig))] # remove selected NMF
- Intersection_with_Genes_MP <- sort(apply(nmf_programs_sig, 2, function(x) length(intersect(Genes_MP,x))) , decreasing = TRUE) # intersection between all other NMFs and Genes_MP
- }
- Cluster_list[[paste0("Cluster_",k)]] <- Curr_cluster
- MP_list[[paste0("MP_",k)]] <- Genes_MP
- k <- k+1
- nmf_intersect <- nmf_intersect[-match(Curr_cluster,rownames(nmf_intersect) ) , -match(Curr_cluster,colnames(nmf_intersect) ) ] # remove current chosen cluster
- Sorted_intersection <- sort(apply(nmf_intersect , 2, function(x) (length(which(x>=Min_intersect_initial))-1) ) , decreasing = TRUE) ### Sort intersection of remaining NMFs not included in any of the previous clusters
- Curr_cluster <- c()
- print(dim(nmf_intersect)[2])
- }
- #### ***** Sort Jaccard similarity plot according to new clusters:
- inds_sorted <- c()
- for (j in 1:length(Cluster_list)){
- inds_sorted <- c(inds_sorted , match(Cluster_list[[j]] , colnames(nmf_intersect_KEEP)))
- }
- inds_new <- c(inds_sorted , which(is.na( match(1:dim(nmf_intersect_KEEP)[2],inds_sorted)))) ### combine inds in clusters with inds without clusters
- # plot re-ordered similarity matrix heatmap
- nmf_intersect_meltI_NEW <- reshape2::melt(nmf_intersect_KEEP[inds_new,inds_new])
- custom_magma <- c(colorRampPalette(c("white", rev(magma(323, begin = 0.15))[1]))(10), rev(magma(323, begin = 0.18)))
- ggplot(data = nmf_intersect_meltI_NEW, aes(x=Var1, y=Var2, fill=100*value/(100-value), color=100*value/(100-value))) +
- geom_tile() +
- scale_color_gradient2(limits=c(2,25), low=custom_magma[1:111], mid =custom_magma[112:222], high = custom_magma[223:333], midpoint = 13.5, oob=squish, name="Similarity\n(Jaccard index)") +
- scale_fill_gradient2(limits=c(2,25), low=custom_magma[1:111], mid =custom_magma[112:222], high = custom_magma[223:333], midpoint = 13.5, oob=squish, name="Similarity\n(Jaccard index)") +
- theme( axis.ticks = element_blank(), panel.border = element_rect(fill=F), panel.background = element_blank(), axis.line = element_blank(), axis.text = element_text(size = 11), axis.title = element_text(size = 12), legend.title = element_text(size=11), legend.text = element_text(size = 10), legend.text.align = 0.5, legend.justification = "bottom") +
- theme(axis.title.x=element_blank(), axis.text.x=element_blank(), axis.ticks.x=element_blank()) +
- theme(axis.title.y=element_blank(), axis.text.y=element_blank(), axis.ticks.y=element_blank()) +
- guides(fill = guide_colourbar(barheight = 4, barwidth = 1))
- ggplot(data = nmf_intersect_meltI_NEW, aes(x=Var1, y=Var2, fill=100*value/(100-value), color=100*value/(100-value))) +
- geom_tile() +
- scale_color_gradient2(limits=c(2,30), low=custom_magma[1:111], mid =custom_magma[112:222], high = custom_magma[223:333], midpoint = 16, oob=squish, name="Similarity\n(Jaccard index)") +
- scale_fill_gradient2(limits=c(2,30), low=custom_magma[1:111], mid =custom_magma[112:222], high = custom_magma[223:333], midpoint = 16, oob=squish, name="Similarity\n(Jaccard index)") +
- theme( axis.ticks = element_blank(), panel.border = element_rect(fill=F), panel.background = element_blank(), axis.line = element_blank(), axis.text = element_text(size = 11), axis.title = element_text(size = 12), legend.title = element_text(size=11), legend.text = element_text(size = 10), legend.text.align = 0.5, legend.justification = "bottom") +
- theme(axis.title.x=element_blank(), axis.text.x=element_blank(), axis.ticks.x=element_blank()) +
- theme(axis.title.y=element_blank(), axis.text.y=element_blank(), axis.ticks.y=element_blank()) +
- guides(fill = guide_colourbar(barheight = 4, barwidth = 1))
- MP <- do.call(cbind, MP_list)
- names(MP_list)=c("Neuron","Vasc","MES.Hyp","Mac","OPC.AC","Oligo","LQ.Chromatin.reg","MES","Prolif.Metab","MES.Ast","Reactive.Ast","NPC","Inflammatory.Mac")
- ###### RECLUSTERING/HEIRARCHICAL CLUSTERING, EXTENDED MPs, AND SUBCLUSTER PROGRAMS ################
- programs_ls<-readRDS("MP/NMF/programs_ls_124.rds") #this is "nmf_programs_sig"
- Cluster_list<-readRDS("MP/NMF/Cluster_list_124.rds")
- str(Cluster_list)
- MP_list<-readRDS("MP/NMF/combined_gbm_metaprograms_raw_124.rds")
- #reclustering OPC.AC into 2 clusters
- opc.ac <- Cluster_list[[5]]
- mat2list <- function(x) {
- stopifnot(is.matrix(x))
- lapply(seq_len(ncol(x)), function(i) x[, i])
- }
- program_ls<-mat2list(programs_ls)
- names(program_ls)<-colnames(programs_ls)
- opc.ac_mat <- program_ls[names(program_ls) %in% opc.ac]
- jac_mat <- scalop::jaccard(opc.ac_mat)
- hc <- hclust(dist(1 - jac_mat), method = "average")
- jac_plot <- melt(jac_mat[hc$order, hc$order])
- #summary(jac_plot$value)
- p1 <- ggplot(data = jac_plot, aes(x = Var1, y = Var2, fill = value)) + geom_raster() +
- scale_fill_gradient2(limits = c(0, 0.4), low = rev(magma(323, begin = 0.15)), mid = "antiquewhite", high = rev(magma(323, begin = 0.18)), midpoint = 0 , oob = squish, name = "Pearson\nCorrelation") +
- theme(axis.ticks = element_blank(), panel.background = element_rect(fill = "white"), axis.line = element_blank(),axis.title = element_text(size=24), legend.text.align = 0.5) +
- scale_x_discrete(name = "\nPrograms", breaks = colnames(jac_mat)[hc$order][seq(5, ncol(jac_mat), by = 5)], labels = seq(5, ncol(jac_mat), by = 5)) +
- scale_y_discrete(name = "\nPrograms", breaks = colnames(jac_mat)[hc$order][seq(5, ncol(jac_mat), by = 5)], labels = seq(5, ncol(jac_mat), by = 5))
- prog_ordered <- colnames(jac_mat[hc$order, hc$order])
- plot(hc)
- rect.hclust(hc , k = 2, border = 2:6)
- clusterCut <- stats::cutree(tree = hc, k = 2)
- metaprog_df <- data.frame(row.names = prog_ordered)
- metaprog_df$cluster <- (clusterCut[prog_ordered])
- metaprog_df$cells <- prog_ordered
- metaprog_df$cluster <- as.factor(metaprog_df$cluster)
- metaprog_annotation_plot <- ggplot(metaprog_df, aes(x = factor(cells, levels = cells), y = 1)) +
- geom_raster(aes(fill = cluster)) + theme(legend.position = "bottom", axis.ticks = element_blank(),axis.title = element_text(size=24), panel.background = element_rect(fill = "white"), axis.text = element_blank(), axis.line = element_blank(), axis.text.x = element_blank()) +
- labs(x = "", y = "", fill = "")
- egg::ggarrange(p1, metaprog_annotation_plot, ncol = 1, nrow = 2, heights = c(40, 2))
- clust_list_ac_opc <- list(names(clusterCut[clusterCut == 1]), names(clusterCut[clusterCut == 2])) # use this ordering to reorder cluster 5 in main MP heatmap
- mp_freqs <- sapply(clust_list_ac_opc, function(k) sort(table(unlist(opc.ac_mat[k])), decreasing = T), simplify = F)
- opc.ac_metaprograms <- sapply(mp_freqs, function(tab) head(names(tab)[tab >= 3], 50), simplify = F)
- names(opc.ac_metaprograms)<-c("AC","OPC")
- #dividing low quality from chromatin.reg cluster
- chromatin <- Cluster_list[[7]]
- mat2list <- function(x) {
- stopifnot(is.matrix(x))
- lapply(seq_len(ncol(x)), function(i) x[, i])
- }
- program_ls<-mat2list(programs_ls)
- names(program_ls)<-colnames(programs_ls)
- chromatin_mat <- program_ls[names(program_ls) %in% chromatin]
- jac_mat <- scalop::jaccard(chromatin_mat)
- hc <- hclust(dist(1 - jac_mat), method = "average")
- jac_plot <- melt(jac_mat[hc$order, hc$order])
- #summary(jac_plot$value)
- p1 <- ggplot(data = jac_plot, aes(x = Var1, y = Var2, fill = value)) + geom_raster() +
- scale_fill_gradient2(limits = c(0, 0.4), low = rev(magma(323, begin = 0.15)), mid = "antiquewhite", high = rev(magma(323, begin = 0.18)), midpoint = 0 , oob = squish, name = "Pearson\nCorrelation") +
- theme(axis.ticks = element_blank(), panel.background = element_rect(fill = "white"), axis.line = element_blank(),axis.title = element_text(size=24), legend.text.align = 0.5) +
- scale_x_discrete(name = "\nPrograms", breaks = colnames(jac_mat)[hc$order][seq(5, ncol(jac_mat), by = 5)], labels = seq(5, ncol(jac_mat), by = 5)) +
- scale_y_discrete(name = "\nPrograms", breaks = colnames(jac_mat)[hc$order][seq(5, ncol(jac_mat), by = 5)], labels = seq(5, ncol(jac_mat), by = 5))
- prog_ordered <- colnames(jac_mat[hc$order, hc$order])
- plot(hc)
- rect.hclust(hc , k = 3, border = 2:6)
- clusterCut <- stats::cutree(tree = hc, k = 3)
- metaprog_df <- data.frame(row.names = prog_ordered)
- metaprog_df$cluster <- (clusterCut[prog_ordered])
- metaprog_df$cells <- prog_ordered
- metaprog_df$cluster <- as.factor(metaprog_df$cluster)
- metaprog_annotation_plot <- ggplot(metaprog_df, aes(x = factor(cells, levels = cells), y = 1)) +
- geom_raster(aes(fill = cluster)) + theme(legend.position = "bottom", axis.ticks = element_blank(),panel.background = element_rect(fill = "white"), axis.text = element_blank(), axis.line = element_blank(), axis.text.x = element_blank(),axis.title = element_text(size=16)) +
- labs(x = "", y = "", fill = "")
- egg::ggarrange(p1, metaprog_annotation_plot, ncol = 1, nrow = 2, heights = c(40, 2))
- clust_list <- list(names(clusterCut[clusterCut == 1]), names(clusterCut[clusterCut == 2]),names(clusterCut[clusterCut == 3]))
- mp_freqs <- sapply(clust_list, function(k) sort(table(unlist(chromatin_mat[k])), decreasing = T), simplify = F)
- chromatin_metaprograms <- sapply(mp_freqs, function(tab) head(names(tab)[tab >= 3], 50), simplify = F)
- names(chromatin_metaprograms)<-c("LQ1","chromatin_clean","LQ2")
- chromatin_metaprogram_clean<-chromatin_metaprograms$chromatin_clean
- clust_list2 <- list(names(clusterCut[clusterCut == 1]), names(clusterCut[clusterCut == 3]),names(clusterCut[clusterCut == 2]))
- #this is the order to use for the final MP heatmap
- clust_list_chromatin <- c(names(clusterCut[clusterCut == 1]), names(clusterCut[clusterCut == 2]),names(clusterCut[clusterCut == 3]))
- ###### MPs after OP/AC split and LQ/chromatin.reg split#######
- MP_list2<-MP_list[-c(5,7)]
- MP_list2[["chromatin.reg"]] <- chromatin_metaprogram_clean
- MP_list2[["OPC"]] <- opc.ac_metaprograms$OPC
- MP_list2[["AC"]] <- opc.ac_metaprograms$AC
- ###### SPOT ASSIGNMENTS TO CLEANED METAPROGRAMS##########
- # generate normalized exp matrices
- file_paths <- list.files(path ="general/exp_mats_GBM", pattern = "\\.rds", full.names = TRUE)
- sample_ls <- gsub(pattern = "\\.rds$", replacement = "", x = basename(file_paths))
- sample_ls->samples_names
- sample_ls->samples
- per_sample_mat <- lapply(file_paths, readRDS)
- for (i in seq_along(per_sample_mat)){
- m <- as.matrix(per_sample_mat[[i]])
- m <- m[-grep("^MT-|^RPL|^RPS", rownames(m)), ]
- if(min(colSums(m)) == 0){m <- m[, colSums(m) != 0]}
- scaling_factor <- 1000000/colSums(m)
- m_CPM <- sweep(m, MARGIN = 2, STATS = scaling_factor, FUN = "*")
- m_loged <- log2(1 + (m_CPM/10))
- # removing genes with zero variance across all cells
- var_filter <- apply(m_loged, 1, var)
- m_proc <- m_loged[var_filter != 0, ]
- # filtering out lowly expressed genes
- exp_genes <- rownames(m_proc)[(rowMeans(m_proc) > 0.4)]
- m_proc <- m_proc[exp_genes, ]
- # output to a list of gene expression profiles (GEP)
- per_sample_mat[[i]] <- m_proc
- names(per_sample_mat)[i] <- sample_ls[[i]]
- rm(m,m_loged, var_filter, exp_genes, m_proc)
- }
- #generate a list with the filtered exp matrix for each sample, i.e. m_proc<-(per_sample_mat[[1]])
- score_mat <- lapply(c(1:length(per_sample_mat)), function(i){
- m_proc<-per_sample_mat[[i]]
- metaprograms_gene_list <- readRDS("MP/clean_spatial_gbm_metaprograms_124.rds")
- signatures <- scalop::sigScores(m_proc, metaprograms_gene_list, expr.center = TRUE, conserved.genes = 0.5)
- spot_scores <- data.frame(spot_names = rownames(signatures))
- spot_scores$Neuron <- signatures$Neuron
- spot_scores$Vasc<- signatures$Vasc
- spot_scores$MES.Hyp <- signatures$MES.Hyp
- spot_scores$Mac <- signatures$Mac
- spot_scores$Oligo <- signatures$Oligo
- spot_scores$MES <- signatures$MES
- spot_scores$Prolif.Metab <- signatures$Prolif.Metab
- spot_scores$MES.Ast <- signatures$MES.Ast
- spot_scores$Reactive.Ast <- signatures$Reactive.Ast
- spot_scores$NPC <- signatures$NPC
- spot_scores$Inflammatory.Mac <- signatures$Inflammatory.Mac
- spot_scores$chromatin.reg <- signatures$chromatin.reg
- spot_scores$OPC <- signatures$OPC
- spot_scores$AC <- signatures$AC
- return(spot_scores)
- })
- names(score_mat) <- sample_ls
- for (i in seq_along(score_mat)){
- score_df <- as.data.frame(score_mat[[i]])
- score_df <-column_to_rownames(score_df, 'spot_names')
- maxcol_meta<-maxcol_strict(score_df)
- maxcol_meta<-stack(maxcol_meta)
- setnames(maxcol_meta,2,"spot_type_meta_new")
- setnames(maxcol_meta,1,"SpotID")
- }
Module1_MP.R at commit f38f9b6, no license · at the source
Overview
- Harvey W. Cushing Neuro-oncology Laboratories, Department of Neurosurgery, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA USA
- Harvard Medical School, Boston, MA USA
- Faculty of Medical Sciences, UM6P Hospitals, Mohammed VI Polytechnic University, Benguerir, Morocco
- Carle Illinois College of Medicine, Urbana, IL USA
Abstract
Although glioblastoma (GBM) harbors multiple genetic abnormalities leading to cell cycle deregulation, a functional mitotic checkpoint is essential to prevent mitotic catastrophe and tumor cell death. Here, we identify the RNA-binding protein HNRNPH1 as a key post-transcriptional modulator of G2/
Reproduced under the paper's license (CC BY), from the paper cited above.
Repository
Its files are read in the Code ↔ Paper reader above, with 2 matches between paragraphs and lines of code.
tiroshlab/Spatial_Glioma
f38f9b60ddbeefd9739e1483cbc66b2650f931d3, 6 January 2025Availability: 1 check, the latest on 30 September 2026: the link answers
- 30 September 2026: the link answers
7 files
- Module1_MP.R, R, 650 lines, 2 matches
- Module2_spatial_coherenc
e_org_zones.R , R, 1,051 lines - Module3_spatial_associat
ions.R , R, 1,385 lines - Module4_consensus_intera
ctions.R , R, 305 lines - Module5_CNA.R, R, 798 lines
- Module6_CODEX.R, R, 944 lines
- README.md, Text, 42 lines
Tracing map
Proposed by the machine: these links were found in the paper and verified at the source, without human review. The map will receive a Zenodo DOI once one of the paper's authors has validated it with their ORCID.
What the map holds:
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- 2 matches between paragraphs of the paper and lines of the code (method lexical-v1);
- neither the text of the paper nor the code itself.
Its JSON (tracing-map.json) is deposited on Zenodo with its DOI once the map is validated.
Data
Datasets cited
- geo:GSE131928, at NCBI GEO; found in the text, “Data collection”
Data availability
The datasets generated and/
Reproduced under the paper's license (CC BY), from the paper cited above.
Versions
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Version 1, 30 September 2026: the first record
Recorded: type, language, journal, volume, issue, pages, dates, 13 authors, 2 keywords, 14 MeSH terms, 4 funders, 41 references.
Cite
This paper
Villa, G. R., Alimonti, P., Toker, J. S., Piranlioglu, R., Karkoski, M. A., Mazzetti, D., Ben Mrid, R., El Guendouzi, S., Lauinger, A., Chiocca, A. N., El Fatimy, R., Chiocca, E. A., & Mineo, M. (2026). HNRNPH1 drives glioblastoma progression by regulating the splicing of cell cycle genes. Cell death & disease, 17(1), 352. https://
BibTeX
@article{villa2026hnrnph
author = {Villa, Genaro R and Alimonti, Paolo and Toker, Joseph S and Piranlioglu, Raziye and Karkoski, Mikayla A and Mazzetti, Debora and Ben Mrid, Reda and El Guendouzi, Sara and Lauinger, Alexa and Chiocca, Andrew N and El Fatimy, Rachid and Chiocca, E Antonio and Mineo, Marco},
title = {{HNRNPH1 drives glioblastoma progression by regulating the splicing of cell cycle genes}},
journal = {Cell death \& disease},
year = {2026},
month = mar,
volume = {17},
number = {1},
pages = {352},
publisher = {Nature Publishing Group},
issn = {2041-4889},
doi = {10.1038/
url = {https://
pmid = {41872153},
pmcid = {PMC13039110}
}
RIS
TY - JOUR
AU - Villa, Genaro R
AU - Alimonti, Paolo
AU - Toker, Joseph S
AU - Piranlioglu, Raziye
AU - Karkoski, Mikayla A
AU - Mazzetti, Debora
AU - Ben Mrid, Reda
AU - El Guendouzi, Sara
AU - Lauinger, Alexa
AU - Chiocca, Andrew N
AU - El Fatimy, Rachid
AU - Chiocca, E Antonio
AU - Mineo, Marco
TI - HNRNPH1 drives glioblastoma progression by regulating the splicing of cell cycle genes
T2 - Cell death & disease
J2 - Cell Death Dis
PY - 2026
DA - 2026/
VL - 17
IS - 1
SP - 352
SN - 2041-4889
PB - Nature Publishing Group
DO - 10.1038/
UR - https://
LA - en
ER -
CSL-JSON
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"family": "Villa",
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"URL": "https://
"language": "en",
"issued": {
"date-parts": [
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}
}
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